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Proceeding
ASEAN COSaT 2014
The Proceeding of
ASEAN Conference on Science and Technology 2014
– 9th ASEAN Science and Technology Week (ASTW-9)

Innovation for better ASEAN Community:


Science and Technology Innovation in Food, Energy,
Water and Related Topics for ASEAN Development.

Editor in Chief
Prof. Dr. Estiko Rijanto

LIPI Press
© 2014 Indonesian Institute of Sciences (LIPI)

Cataloging in Publication Data


Proceeding of ASEAN Conference on Science and Technology 2014/Estiko Rijanto (Ed.). –
Jakarta: LIPI Press, 2014.
pp. xvi + pp. 562; 17,6 x 25 cm
ISBN 978-979-799-812-7
1. ASEAN COSAT 2. Science and Technology
600

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First Edition : December 2014


Abstracts available online at penerbit.lipi.go.id

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JAIF

Japan-ASEAN Cooperation

iv
Contents
Editorial Foreword............................................................................................................. xi
The Remarks by the Indonesia National ASEAN COST Chair.........................................xiii
Preface by Minister of Research, Technology and Higher Education, the Republic of
­Indonesia............................................................................................................................ xv

SCB (14)
Gamma Radiation Induced Changes of Molecular and Phytochemi-
cal Profiles on Mutants of Andrographis paniculata (Burm.F.) Wal-
lich Ex Ness
(J. I. Royani, et al.)............................................................................................................3
The Chemical Constituent and Antioxidant Activity of the (-)-Epi-
cathecin From AnEndophytic Fungus Mycoleptodiscus indicus
(P. C. Mawarda, T. Ernawati and Y. Srikandace)..............................................................19
Potency of Hibiscus (Hibiscus Rosa-Sinensis L.) As Antioxidant To Re-
duce Carbon Tetra Chloride Compounds In Red Blood Cells
(D. Priadi and Kusmiati).................................................................................................29
Purification of Bioactive Peptide with Proteases Inhibitory Activi-
ties from Streptomyces misionensis
(J. M. Yusoff, K. Simaran and Z. Alias)...........................................................................37
Isolation, Identification, and Screening of Locally Isolated Xan-
thomonas sp.
(N. I. S. Bokhari, K. Simarani and M. S. M. Annuar).....................................................43
Ultraviolet Irradiation Effect of Penicillium chrysogenum on Peni-
cillin Production
(D. Hardianto, et al.).......................................................................................................49
Isolation and Cloning of Partial Her-2 Gene from Indonesian
Breast Cancer Patients for DNA Vaccine Development
(Desriani and L. Triratna)................................................................................................57
Heterologous Expression of Recombinant Plantaricin Ws34 in Esch-
erichia coli
(A. S. Putri, et al.)............................................................................................................65
Evaluation of Low Temperature Induced Mutant of Soybean Mosaic
Virus for Cross Protection in Soybean
(W. R. Andayanie and P. G. Adinurani.)..........................................................................73
Process Design of Peptone Production from Peanut Meal as By-
Product of Peanut Oil Industry Using Crude Papain
(M. Rahayuningsih and N.G. Wiranti)............................................................................85

v
Potency Of IAA Hormone Produced By Endophytes Bacteria Isolated
From Shorea Selanica On Supporting The Growth Of Paraserinthes
Falcataria
(T. Widowati, et al.)........................................................................................................93
Production And Characterization Of The Biosurfactant By The
Formation Of Glycolipid Isolated From Pseudozyma Hubeiensis
Y10bs025
(M. Sari, F. Afiati and W. Kusharyoto)...........................................................................103
Application Of Marker Assisted Selection And Sensory Test For
Selecting Aroma On F2 Progeny Of Rice Derived From Crossing Be-
tween Ciherang X Basmati
(S. Sari, et al.)................................................................................................................111
Development Of Rice Lines Resistant To Brown Planthopper With
Aromatic Traits: Selection Based On Molecular Marker
(A. P. Asri, N. Carsono and S. Amien)...........................................................................122

SCSER (12)
Assessment of E-Waste Recovery Facilities in Selangor and Kuala
Lumpur, Malaysia
(N. A. M. Nordin and P. Agamuthu).............................................................................129
Public Perception on Current Waste Management System: A Malay-
sian Case Study
(S. H. Fauziah and S. F. Ser)..........................................................................................135
Biomass Flow and Carbon Sequestration in An Organic Farm
(L. H. Yeng and P. Agamuthu).......................................................................................147
Biomass Gasification for Power Generation Using Dual Chamber
Circulating Fluidized Bed Reactor
(H. Wahyu, et al.)..........................................................................................................153
Roof Mounted Micro-Wind Turbine For Power Generation In Coast-
al Housing In Semarang, Indonesia
(D. P. Sari).....................................................................................................................161
Performance Of A Radial Turbine For Small Organic Rankine Cycle
Power Generation System
(M. Arifin, B. Wahono and A. D. Pasek).......................................................................169
Effect Of Reaction Time And Cellulase Loading On Dilute Alkali
Pretreatment Of Sugarcane Bagasse To Produce Fermentable Sug-
ars For Bioethanol Production
(T. Fajriutami dan Rizky Rissa Bella).............................................................................177
Performance Of Microbes Consortium On Single-Chamber Micro-
bial Fuel Cell As Electricity Generation
(D. Rahayuningwulan, D. Permana dan H. E. Putra)....................................................187

vi
Heat Release Analysis Of A Two Cylinders in Diesel Engine Fuelled
With Ethanol-Diesel Blends
(Y. Putrasari, et al.)........................................................................................................199
An Evaluation For Enzymatic Saccharification of Fast-Growing
Tree Species From Secondary Forest in West Kalimantan
(L. Risanto, et al.)..........................................................................................................209
A Novel Microwave-Biological Pretreatment Effect On Cellulose
And Lignin Changes Of Betung Bamboo (Dendrocalamus Asper)
(W. Fatriasari, et al.)......................................................................................................219
The Emergence of Biogas Technology For Reducing Rural Poverty:
Empirical Studies In Java Island
(L. Ariana).....................................................................................................................231

SCMG (9)
Assessment Of Erosion Potentials On Various Cropping Patterns Us-
ing Usle: Case Of Subang Region, West Java
(R. I. Sholihah, et al.)....................................................................................................243
New Stage Of International Collaboration On Climatological Ob-
servation
(M. D. Yamanaka) .......................................................................................................253
Simultaneous Correlation Analysis Of Australian Summer Monsoon
Index (Ausmi) Against Rainfall In Bali Region
(S. Mujiasih and I. G. A. Purbawa)................................................................................261
Developing Strategy For Monitoring And Decision Support System
For Smoke Haze Trans-Boundary Problem Within Asean Region
(S. D. A. Kusumaningtyas)............................................................................................275
The Potential Impact Of Carbon Monoxide Emission To The Commu-
nity Health In The Vicinity Of Baranangsiang Toll Gates
(Y. V. Paramitadevi, A. S. Yuwono, and M. Widyarti)....................................................289
The Mechanism Of Dry Mid-Atmosphere In The Western Maritime
Continent During Rainy Season IN 2014
(Supari, et al.)................................................................................................................299
Design Of Automatic Measurement Instrument For Water Dis-
charge On Drainage Monitoring System
(R. T. Wahyuni).............................................................................................................311
Acehseis, A Local Seismic Experiment In Bener Meriah And Central
Aceh
(M. Muzli, et al.)...........................................................................................................319
Selection Of Global Gmpes Models For Seismic Hazards Assessments
In Indonesia (Case Study Sumatra-Java Area)
(A. Rudyanto, et al.)......................................................................................................329

vii
SCMIT (7)
Led-Based Spectrometer For Advanced Chemistry Laboratory Ex-
periments
(M. A. Alagao, et al.).....................................................................................................343
Introduction Investigation: Executive Information System For
University
(S. Warnars, Sasmoko and N. Susianna) ......................................................................353
Design Of Implementation Delay Tolerant At Wireless Mesh Net-
works Using IBR-DTN and Batman-Adv
(H. Yuliandoko, S. Sukaridhoto and M. U. H. A. Rasyid).............................................365
Development Of A Programmable Multipurpose Forced Convection
Type Dryer
(E. C. Guevarra)............................................................................................................377
A Study Of Network Speech Recognition Using Tcp
(A. Jarin, K. Ramli and Suryadi)....................................................................................389
Aural And Photonic Spectrum Based Digital Pest Controller For
Oryza Sativa (Rice)
(I. A. P. Banlawe)...........................................................................................................401
Development Of Programmable Logic Controller (Plc)-Based Cof-
fee Pulper For Wet Process
(M. R. Perena)...............................................................................................................413

SCMST (13)
Effect Of Sintering Temperature Rate On Physical Properties Of Po-
rous Tricalcium Phosphate (Tcp) Ceramics
(A. Fadli, A. Rasyid and R. Firmansyah)........................................................................427
Study Of Kinetics And Thermodynamics As Well As The Effect Of
The Presence Of Co-Ions In Influencing Adsorption Cu2+ Ion By Coal
Fly Ash Adsorbent
(A. Zakaria, et al.)..........................................................................................................433
Organophosphorus (Ops) In The Environment: Effects Of Repeated
Application Of Chlorpyrifos On Agricultural Soil
(C. Carol and S. H. Fauziah).........................................................................................443
Bonded Prfeb Magnet: Fabrication And Characterization
(D. Aryanto, et al.)........................................................................................................449
Effect Of Sintering Temperature On Dielectric Constant Of Silica
Prepared From Rice Husk Ash
(Qudratun, et al.)..........................................................................................................457
Synthesis And Characterization Of Al-Doped Lithium Titanate Li-
4
ti5o12 As Anode Material For Li-Ion Battery
(S. Priyono, et al.)..........................................................................................................463

viii
Cellulose Fibers From Oil Palm Fronds Reinforced Polylactic Acidb
Composite
(F. A. Syamani, Y. D. Kurniawan and L. Suryanegara)...................................................473
Isolation And Characterization Of Lignin From Alkaline Pretreat-
ment Black Liquor Of Oil Palm Empty Fruit Bunch And Sugarcane
Bagasse
(M. A. R. Lubis, et al.)...................................................................................................483
Changes In The Cellulose Crystallinity During Different Phases Of
Distilled Vetiver Root Cellulose Fibers Development
(F. A. Syamani, et al.).....................................................................................................493
Bioethanol Production Using Saccharomyces Cerevisiae Immobil-
ised On Fresh And Modified Sugarcane Bagasse
(S. H. Anita, et al.)........................................................................................................503
Effect Of Ph On Extraction Efficiency And Distribution In Nickel
Ion Separation Using Solvent Extraction
(A. Maimulyanti and A. R. Prihadi)...............................................................................515
Ethoxylated Glycerol Monooleate: Palm Oil Based Nonionic Sur-
factant For Oil-In-Water Emulsion Systems
(I. B. Adilina, et al.).......................................................................................................523
Conversion Of Citronella Oil And Its Derivatives To Menthol Over
Bifunctional Nickel Zeolite Catalysts
(I. B. Adilina, et al)........................................................................................................531

APPENDIX
Editorial Board................................................................................................................ 541
Authors Index................................................................................................................... 543­

ix
Editorial Foreword

The ASEAN Conference on Science and Technology 2014 was held in Bogor -
Indonesia on August 18th–19th, 2014. The conference was a part of activities of
the 9th ASEAN Science and Technology Week (ASTW) event under the theme
“Innovations from the Most Dynamic Region on Earth” which was held on August
18th–28th 2014. It is a triennial event of the ASEAN Committee on Science and
Technology (COST). Innovation for better ASEAN Community was selected as
the sub-theme for the Conference. The Conference covered all subcommittees
and flagships in a focused topic, i.e. Science and Technology Innovation in food,
energy, water and related topics for ASEAN development.
The Proceeding of ASEAN Conference on Science and Technology 2014
– 9th ASEAN Science and Technology Week (ASTW-9) contains papers which
had been presented and discussed in the parallel sessions during the Conference
and have undergone review process. The authors come from Indonesia, Malaysia,
Thailand, Philippines and Japan.
This proceeding is an excellent result of collaborative work. We would like to
express our gratitude to international editorial board members, reviewers, authors
and the publisher for the quality of their works. We would also convey our sincere
appreciation to ASEAN Secretariat, The Ministry of Research, Technology and
Higher Education of the Republic of Indonesia and JAIF for their continued
support.
We hope this proceeding could be a constructive contribution in development
of science and technology, especially among ASEAN member states.

Jakarta, 20 November 2014


Editor in Chief

Prof. Dr. Estiko Rijanto

xi
The Remarks by the Indonesia National ASEAN
COST Chair

The 9th ASEAN Science and Technology Week (the 9th ASTW) was successfully
organized by the Ministry of Research and Technology, Republic of Indonesia at
Bogor, West Java from 18th to 28th August 2014. ASTW event is a flagship project
of the ASEAN Committee on Science and Technology (ASEAN COST) which
is aimed at demonstrating major achievements made and exploring potential of
S&T generated both in ASEAN Member Countries and Dialogue Partners. The
ASTW is conducted triennially on a rotational basis amongst ASEAN Member
Countries. This forum is expected to be used as a media for opening windows of
opportunities for the triple helix plus societal communities (ABG-S) to promote
networking and to expand their S&T co-operations, not only amongst the
ASEANs, but also with ASEAN Dialogue Partners. As the ASTW is deemed as
an important event; therefore, Indonesia has been very pleased to host the ASTW
twice, in 2005 (7th ASTW) and in 2014 (9th ASTW). As a reference other member
countries holding the previous ASTW are: Malaysia (1st, 1986), Philippine (2nd
in 1989 and 8th in 2008), Singapore (3rd in 1992), Thailand (4th in 1995), Viet
Nam (5th in 1998), and Brunei Darussalam (6th in 2001).
The 9th ASTW was proceeded by the 4th ASEAN Science Congress and
Sub Committee Conferences or known as the ASEAN Science and Technology
Conference 2014 (ASEAN COSAT 2014). It was opened by former Minister
of Research and Technology the Republic of Indonesia, Prof. Dr. Ir. Gusti
Muhammad Hatta, M.Si on August 18th 2014. The ASEAN COSAT 2014 was
organized in two days with the following agendas: (1) Convener by Organizing
Committee Chairman, Prof. Dr. Estiko Rijanto; (2) Opening Remarks by His
Excellency Prof. Dr. Ir. Gusti Muhammad Hatta; (3) Plenary session Day 1 by
four keynote speakers: Innovation and Entrepreneurship in Sweden by Dr. Sven
Thore Holm (Sweden), Innovation and Entrepreneurship in ASEAN: Challenge
and Opportunities by Dr. Warsito P. Taruno (Indonesia), ASEAN as a driving force
of global innovation by Dr. Haruo Takeda from Hitachi Ltd (Japan), Science and
Technology Innovation for Food Security by Prof. Ram Rajasekhran (India); (4)
Oral Parallel session Day 1 (7 Sub Committees in 7 Rooms) and Poster session

xiii
Day 1; (5)Plenary session Day 2 by five keynote speakers: Blue Economy for Small
Island Developing State by Mr. Nico Barito (Ambassador, Republic of Seychelles),
Human environmental security in ASEAN: Water –Energy-Food Nexus by Prof.
Robert Delinom (LIPI, Indonesia), Basic material research for renewable energy
applications by Dr. Nicola Seriani (The Abdus Salam ICTP, Italy), An Application
of Synchrotron-based X-ray Absorption Spectroscopy on Materials by Dr. Pinit
Kidkhunthod (Thailand), and China-ASEAN Technology Transfer Center: a Great
Boost to China-ASEAN Science and Technology Cooperation and Technology
Innovation by Dr. Ye Bo (People Republic of China); (6)Oral Parallel sessions
Day 2 (7 Sub Committees in 7 Rooms) and Poster session Day 2.
We are pleased to see the completion of The Proceeding of ASEAN Conference
on Science and Technology 2014 – 9th ASEAN Science and Technology Week
(ASTW-9). As a scientific documentation it will contribute to the development
of science and technology especially among ASEAN member states.
Finally we would like to convey our gratitude to the international edito-
rial board and reviewers who have dedicated up to the end in completing this
proceeding.

Jakarta, 20 November 2014


Indonesia National ASEAN COST Chair

Dr. Agus R. Hoetman

xiv
Preface
The Minister of Research, Technology and
Higher Education,
The Republic of Indonesia

First, I would like to congratulate all of the ASEAN Science, Technology and
Innovation (STI) policy makers and scientists, for the success of the 9th ASEAN
Science and Technology Week (9th ASTW) spectacular event, which was conducted
in Bogor, 18–27 August 2014. I acknowledged that out of 15 activities, the 4th
ASEAN Science and Technology Congress and Conferences (18–19 August
2014), is one of a very important activities, which aims to expose all of S&T
achievements in the past 6 years, since the 8th ASTW in Manila (2008). Therefore,
special gratitude was also delivered to the ASEAN joint committe for this congress
and conference.
Secondly, I would like to take this opportunity to inform you that as of
October 20, 2014, the former Indonesian Ministry of Research and Technology
(RISTEK) has been transformed into the Ministry of Reserach, Technology and
Higher Education (RISTEK-DIKTI). With regard to this development, I am
convinced that Indonesian commitment in the ASEAN Committe on Science and
Technology (ASEAN COST) would be more firmed and continued, especially in
supporting the implementation of ASEAN Plan of Action on Science, Technology
and Innovation (APASTI) 2015–2020, with its vision on “A Science, Technology
and Innovation-enabled ASEAN which is innovative, vibrant, sustainable and
economically integrated”.
APASTI 2015–2020 has several goals, among others are to encourage active
collaboration between public and private sectors, and to improve human resources,
through capacity building and talent mobility. All of these goals are in line with
RISTEK-DIKTI Ministry in the next five years. Therefore, I am expecting that
most of ‘research results presented in this ASEAN congtess and conference’
could be ‘leveraged up’ into prototypes (lab and industrial scales), which then the
‘useful and valuable products’ could be massively produced to fulfill the ASEAN

xv
and world markets. In addition, I also believe that both of the academic papers
and their commercial transformations into the valuable reserach products would
encourage more numbers of techno-entrepreneurs in ASEAN countries, as one
solution to the global challanges that we should face together.
Ending my remarks, I would like to emphasize that developing a proceeding
after the congress and conference is surely very useful to empower Indonesia as
the ‘knowledge-based society’country, as well as to expose the scientific results
and achievements to the users, industries and societies. Regarding this, I would
like to express my appreciation to the editorial board and reviewers for the efforts
and commitments of the International editorial boards and reviewers, as well as
the publishers, who have been working hard to the completion of the 4th ASEAN
Conference on Science and Technology (ASEAN COSAT 2014). I am sure this
proceeding would lead to STI product commercializations in the future, which
benefit all of the ASEAN and world community.

Jakarta, 20 November 2014


Minister of Research, Technology and Higher Education,
The Republic of Indonesia

Prof. Dr. M. Nasir

xvi
SCB

1
GAMMA RADIATION INDUCED CHANGES OF
MOLECULAR AND PHYTOCHEMICAL PROFILES ON
MUTANTS OF Andrographis paniculata (Burm.f.)
Wallich Ex Ness
J. I. Royania,*, A. Purwitob, W. Sumaryonoa, D. Hardiantoa and A. Mahsunaha
The Agency for the Assessment and Application of Technology
a

630 Bld PUSPIPTEK Area, Serpong, Tangerang


b
Departement of Agronomy and Horticulture-Faculty of Agriculture, Bogor Agriculture
University,
Darmaga, Bogor

Abstract
Sambiloto (Andrographis paniculata (Burm.f.) Wallich Ex Ness is a medicinal plant that became
the pre-eminent national and prospective to be developed in Indonesia. Self pollination and
habitual inbreeder from sambiloto affected the low of genetic variation. Quality improvement
of the plants is one strategy that can be used to increase the genetic diversity and improve the
content of the active compounds in medicinal plants. One method to improve genetic diversity
and the content of active compounds is by using radiation. The aim of this research was to find
out changes in mutant characters of sambiloto plants irradiated with gamma rays on molecular
and phytochemicals profiles. Sambiloto seeds were irradiated using gamma rays Cobalt 60 and
grown vegetatively with ex vitro propagation from M1V1 to M1V4 generations. Molecular analysis
of mutant by 10 primers of ISSR marker was used to obtain DNA profiles. Determination of
phytochemical profiles of mutant at M1V4 generation was done by using HPLC method. The
results showed that 5 primers out of 10 primers could distinguish changes of DNA profile, 4
primers showed the same number and size bands and 1 primer could not amplified. Analysis
of genetic similarity obtained 6 groups with genetic distance 0.79–1.00. HPLC analysis with a
wavelength of 230 nm showed the variation of the profiles and contents of phytochemical mutants
of sambiloto. The contents of andrographolide varied in the range between 6.5%–10.9%. Highest
content of andrographolide was found in DK300 mutant, while the lowest one was found in
control. In DB60 mutant, andrographolide content had the same content as control; however,
chromatogram revealed the presence of 4 peaks compared to the control that just had 2 peaks.
Key words: Andrographis paniculata, Gamma radiation, DNA profiles, Phytochemical profiles

i. Introduction
Andrographis paniculata (Burm.f.) Wallich Ex Ness, which is also called “King
Bitter”, is an annual herbaceous plant that comes from Peninsular India and Sri
Langka (Lattoo et al., 2006, Mishra et al., 2007; Jarukamjorn & Nemoto, 2008).

* Corresponding author. Email: idhajr@yahoo.com

3
4 Proceeding ASEAN COSAT 2014

These plants grow naturally in Southeast Asia (India and Srilanka), Pakistan and
Indonesia, but extensively cultivated in China and Thailand, eastern and western
of India, and Mauritius (Mishra et al., 2007). In Indonesia, A. paniculata is
well known as sambiloto, and eventhough A. Paniculata is not originally from
Indonesia, this plant became the pre-eminent national and prospective to be
developed in Indonesia. Subramanian et al. (2012) also said that A. paniculata
is a bitter plant with a sweet future to describe how important this plant is for
medicinal.
Lattoo et al. (2006) reports that A. paniculata is hermaphroditic, self-
compatible, habitual inbreeder and obligate autonomous selfing in the species.
These reasons were affected by the low genetic variation of A. paniculata. Research
generated by Pandey and Mandal (2010) found that there is no variation of A.
paniculata genotype at the phenotypic level from 5 different location in India.
Wijarat et al. (2012) also reinforce this research that could not detect genetic
variation in 58 A. paniculata accessions from Thailand which were evaluated by
SSR, AFLP and RAPD markers. On the other side, the level of active compound
of A. paniculata, namely andrographolide, without any treatment were very low
too. Many researcher reported that the level of andrographolide is between 0.1–2%
(Sabu et al., 2001; Raina et al., 2007; Sharma et al., 2009).
Improvement of quality of the plants is one strategy that can be used to
increase the genetic diversity and improve the content of the active compounds
in medicinal plants. Mutation breeding is a breeding technique that creates
variability in the mutated population through heritable changes in the genotypic
and phenotypic utilized for effective selection of particular traits (Tah et al., 2008).
Induced mutation is the easier method to create genetic variability compared
with other breeding methods (Minn et al., 2008). Mutation by using ionizing
irradiation is one of the most widely used method to generate mutant. Gamma
rays is a mutagen that has a high energy irradiation that can cause damage to
the covalent bonding or hydrogen bonding molecules/biomolecules in cells that
result in damage to chromosomes, genes and ends on cell death (Xiang et al.,
2002). The biological effect of gamma-rays is based on the interaction with atoms
or molecules in the cell, particularly water, to produce free radicals (Borzouie et
al., 2010). These radicals can damage or modify important components of plant
cells and have been reported to affect differentially the morphology, anatomy,
biochemistry and physiology of plants including changes in the plant cellular
structure and metabolism depending on the radiation dose.
Gene mutation without phenotypic expression is usually unrecognized. To
recognize gene mutation in mutants, various methods has been applied to detect
the effect of mutagen in plants. Different methods are available to investigate the
Innovation for Better ASEAN Community 5

effect of mutagens on plants. In medicinal plants, the change of mutant characters


could be detected in morphology, physiology, molecular and phytochemical level.
The aim of this research was to find out changes in mutant characters of A.
paniculata plants that irradiated with gamma rays on molecular and pytochemical
profiles.

ii. MATERIAL AND METHODS


Materials used for this research were A. paniculata seeds from Kanigoro Karan-
ganyar accession. The seeds were obtained from Indonesia Research Institute
for Medicinal and Aromatic Plants (BALITTRO).

A. Irradiation treatments, plantation and subculture of mutants


For the exposure of gamma irradiation on the seeds, 60Co had been used. Seeds
of A. paniculata in clean plastic were irradiated in Cobalt 60 Gamma Chamber
machine at National Nuclear Energy Agency with treatment dose of: 0, 10, 20,
30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275 and 300 Gy.
After irradiation, seeds were planted in pot trays and germination for 6 weeks.
Viability of seed germination from each treatments were calculated to determine
the LD50 value with CurveExpert 1.3 software.
After 8 weeks of planting, shoots from M1V0 generation were cut approxi-
mately 10 cm to subculture to M1V1 until M1V4 generation is obtained with
ex vitro propagation method.

B. Determination of molecular profiles of mutants


1) Extraction of DNA genome
Total genomic DNA of A. paniculata in M1V2 generation was isolated using
CTAB modification method. Fresh leaves (200 mg) were well grounded in
extraction buffer solution with mortar and pestle to fine paste thoroughly.
DNA pellet obtained was dried, then 200 ul TE buffer solution was added
and kept at freezer (-20°C) for subsequent use. DNA quality was checked
visually using electrophoresis machine on 0,8% agarose at 100 volt for 30
minutes. DNA bands are exposed by UV-ray illuminator and documented
using GELDOC. Nanodrop machine was used to determine the isolated
DNA quantity.
2) Amplification of DNA using ISSR primers
Ten primers of ISSR marker were used to amplify 31 genomic DNAs of
A. paniculata mutants. ISSR primers used were: SBLT2 {(AG)8T}, SBLT3
{(AG)8C}, SBLT5 {(GA)8C}, SBLT8 {(CT)8G}, SBLT13 {(GAA)6}, SBLT14
{(GACA)4}, SBLT15 {(GA)8}, SBLT17 {(TG)8G}, SBLT18 {(CCCT)4} and
6 Proceeding ASEAN COSAT 2014

SBLT19 {(GATA)2(GACA)2}. ISSR amplification was carried out with 10


primers using 200-300 ng of genomic DNA. PCR reactions consisted of DNA
template (1 µl extract of DNA genome added by DNA free water), 1.5 mM
MgCl2, 0.2 mM ISSR primer, 0.2 mM dNTP, 1x PCR buffer, 1 U Taq DNA
polymerase (dream taq-fermentas) and add free DNA/RNAse water up to total
volume of 20 µl. The initial condition was: Pre-PCR heating at 94 °C for 5
minutes, then followed by subsequent steps as followed: 40 cycles consisting
of denaturation at 94 °C for 45 seconds, annealing at 50 °C up to 58°C for
45 seconds and extension at 72 °C for 2 minutes. After 40 cycles finished,
post-PCR extension was at 72 °C for 5 minutes. Modification of annealing
temperature of the PCR was conducted to optimize the ISSR primer reaction.
3) Visualization of amplified DNA and Molecular analysis
Amplified DNA products were analyzed through electrophoresis on 1.5%
agarose gel containing 1xTBE (tris borate EDTA) and syber safe to stain the
DNA. Gel running at 100 volts for 30 minutes and DNA bands were exposed
under UV illuminator and documented using GELDOC.
Polymorphic products from ISSR analyses were scored qualitatively for
presence or absence of the amplified products for each primer. If a product was
present as bands, it was designated as 1, and the absence of it was designated
as 0. The cluster analysis was done using NTSYS software version 2.02 and
Dice similarity coefficient was utilized to generate dendrogram by means of
Unweighted Pair Group Method of Arithmetic Means (UPGMA) employing
the Sequential, Agglomerative, Hierarchical and Nested Clustering (SAHN)
from NTSYS program. The dendrogram obtained was used to evaluate the
genetic distance of mutants compared with control.

C. Determination of pytochemical profiles


1) Extraction of A. paniculata leaves
Freshly collected plant leaves from 4 mutants of A. paniculata that exposed
to gamma radiation and 1 leaves from control were washed thoroughly and
dried in the oven at 60 °C for 4 hour to obtain dried material. Dried materials
were weighed for 0.05 g and grounded to fine powder. The fine powder of
A. paniculata leaves were placed into 15 mL falcon tube and then extracted
with 2.5 mL methanol solution as solvent extraction. Dried materials with
methanol into falcon were shaken horizontally at 300 rpm for 30 minutes.
Further extracts were centrifuged at 5,000 rpm for 10 minutes. Superna-
tant was collected and re-extracted crude similar as in the above manner.
Afterwards, supernatant collected from 2 times extraction was concentrated
using centrifugal concentrator for 2 hours until dried. Then dry extracts were
Innovation for Better ASEAN Community 7

dissolved in 1 mL methanol solution and prepared to inject into the HPLC


instrument.
2) Analysis of pytochemical profiles by HPLC
Determination of changes of phytochemical profiles from A. paniculata mu-
tants performed by HPLC using Photodiode Array Detector. For comparison
to phytochemical profiles, andrographolide (Sigma-Aldrich) was used as
standard. Calibration curve for linear regression was made based on peaks
area using standard solutions of andrographolide. Stationary phase using a
Symmetry C18 column (150 x 4.6 mm) size 5 μm. The mobile phase was
water with pH 3.0 (formic acid): acetonitrile in the ratio (70:30 v/v). Column
equilibrated with the mobile phase for an hour. Isocratic elution was carried
out at room temperature and at the pump at a flow rate of 1 mL/min. The
detection was made at a wavelength of 230 nm. The solution was injected as
much as 10 mL. The evaluation was based on the retention time and peak area
formed by linear regression. Chromatogram of standard was compared with
chromatograms from mutants and control of A. paniculata plant. Calculated
levels of andrographolide were formed from each mutant.

III. RESULTS AND DISCUSSIONS


1) The effect of irradiation on seeds germination and LD50
Sensitivity of radiation on A. paniculata seeds can be seen from germination
of seeds in the range of irradiation doses as compared to control. The result
for irradiation sensitivity based on survival percentage of irradiated and
control seeds demonstrated that significant reduction in survival percentage
was observed with increasing gamma dosage.
Dhakshanamoorthy et al. (2011) states that the decrease in germination
at higher doses of the mutagens may be attributed to disturbances at cellular
level (caused either at physiological or physical level) including chromosomal
damages. Based on germination rate, CurveExpert 1.3 analysis was obtained
LD50 from the seeds of irradiation with dose at 140.363 Gy (Figure 1). In
this study, the function of the Gaussian model was the best model to describe
the population mortality of A. paniculata with r = 0.954.
2) Molecular profiles of A. paniculata mutants in M1V2 generation
The PCR amplification using 10 primers of ISSR gave rise to reproducible
amplification products. ISSR primers produced different numbers of DNA
bands, depending upon their simple sequence repeat motifs. The result showed
that 31 mutants based on different morphology were observed in M1V2
generation. These 31 mutants and 1 control were then analyzed using ISSR
molecular marker.
8 Proceeding ASEAN COSAT 2014

S = 7.12167613
r = 0.95458304
0
90.0
0 y=78.4*exp((-(-22.14-x)^2)/(2*171.3^2))
80.0
Seeds germination (%)
benih berkecambah (%)

0
70.0
0
60.0
140.363 Gy
0
50.0
0
40.0
0
30.0
0
20.0
0
10.0
0.0 30.0 60.0 90.0 120.0 150.0 180.0 210.0 240.0 270.0 300.0 330.0

Dosis (Gy)
Dosoge (Gy)
Figure 1. LD50 of seeds A. paniculata irradiated with Cobalt 60 Gamma rays

Molecular analysis showed that genomic DNA of A. paniculata mutants


produced multiple bands using 10 primers of ISSR. Of 10 primers used,
9 primers were amplifying the genomic DNA and 1 primer could not
be amplified. Based on electroforegram, 5 primers, i.e.: SBLT2, SBLT3,
SBLT13, SBLT15 and SBLT18 can distinguish changes in the DNA profiles
of mutants compared with control and 4 primers, i.e.: SBLT5, SBLT8,
SBLT14 and SBLT19 shows the same number and size bands with control.
The total number of amplifield DNA bands yields was 1.450 bands, out of
which 152 bands (10.48%) were polymorphic and 1.298 (89.52%) were
monomorphic. Fragments ranged in size from 1.000–8.000 bp (Table 1).
The number of fragments produced by various ISSR primers ranged from
2–12 with an average of 161 bands per primer. The largest number of bands
was identified in primer SBLT3 and SBLT18 (11 bands) and the smallest in
primers SBLT15 (1 band).
The qualitative data from electroforegram showed that there were 3 types
of DNA profiles of A. paniculata mutant amplified by 10 primers of ISSR.
The first type was DNA profile of mutants with the same DNA bands with
wild type. It can be seen in 4 primers that were used, i.e.: SBLT5, SBLT8,
SBLT14 and SBLT19. DNA profile with this type was monomorphic. The
second type was DNA profile with reduced DNA bands compared with wild
type plant. These type were shown in 5 primers, i.e. : SBLT2, SBLT3, SBLT13,
dan SBLT15. While the third type was DNA profile that had additional
bands compared with wild type. In this research, primer of SBLT18 had this
Innovation for Better ASEAN Community 9

characteristic. The second and third type of DNA profiles were polymorphic
bands. Copra (2005) states that polymorphism of amplified bands is caused
by: (i) base substitutions or deletion in the priming sites, (ii) insertions that
render priming sites too distant to support amplification or (iii) insertions
or deletions that change the size of the amplified fragment. Appearance of
new bands in this research can be explained as the results of different DNA
structure (breaks, transpositions, deletion, etc). The polymorphism revealed
by ISSR due to deletion and or addition may be caused by variation in DNA
binding patterns by gamma rays.
Based on these polymorphic bands, a similarity coefficient matrix was
calculated and a similarity dendrogram was obtained using a UPGMA cluster
analysis (Figure 2). A cophenetic correlation of r = 0.89 was obtained, which
indicates a good fit between the original similarity matrix and the resulting
clustering analysis (Rohlf, 1997). On the basis of the similarity dendrogram,
from 32 irradiated A. paniculata plants, they could be classified into six major
clusters at a Nei’s genetic distance with each group divided into sub-clusters.
This grouping tendency does not refer to a single character or single dose only,
but spreads on each character and the dose. Jaccard’s similarity coefficient
showed genetic distance of A. paniculata ranged from 0.79 to 1.00. Furthest
genetic distance obtained in the DK300, DG275 and DG70 compared with
controls (DN). These coefficient similarities showed that A. paniculata, even
through treatment with gamma rays mutagen, has genetic variation, still
remains low.
The genetic alterations are produced by ionizing radiation due to ionization
and excitations of the DNA molecule. There are two effects of ionizing irradia-
tion on the heredity material: gene mutations and chromosome breaks (Atak
et al., 2004). Irradiation by this physical mutagenic agent leads DNA break
formation via direct and indirect detrimental effects. In direct interactions, the
radiation energy is transferred to the targets and in indirect interactions energy
is absorbed by the water present in the external medium. After hydrolysis of
water, secondary messenger molecules affect the biomolecules (Esnault et
al., 2010). They react with most of the biomolecules including DNA and
scavenger electrons from them. The oxidation of biomolecules by the radicles
damage their structure and biological activity. It is worth, genetic alterations
occur on the DNA molecules. This is the cause of mutations that depends
on radiation (Selvi et al., 2007).
According to Dhakshanamoorthy et al. (2011), the disappearance of normal
bands (loss of bands) maybe related to the occurrence of DNA damage (e.g.
single and double-strand breaks, modified bases, abasic sits, oxidized bases,
bulky adducts), DNA-protein cross links, point mutation and/or complex
10 Proceeding ASEAN COSAT 2014

DN
DK250
DB50
DG200
D3125
DB70
DG60
DG125
D390
D3200
DK90
DB90
DK175
DG40
DKR275
D3250
D3300
DB60
DK150
D4275
DK275
DC3150
DK200
DG150
DB275
DK225
D3100
DG300
D3150
DK300
DG275
DG70
0.79 0.84 0.89 0.95 1.00
Koefisien Kemiripan
Figure 2. Dendogram constructed based on UPGMA model calculated from genetic distance of 32
irradiated A. paniculata by ISSR marker

chromosomal rearrangements induced by gamma radiation. While the addi-


tion of the DNA bands may reveal a change on some oligonucleotide priming
sites due to mutations (new annealing event), large deletions (bringing to
preexisting annealing site closer) and homologous recombination. Atienzar et
al. (2006) reported that mutations can only be responsible for the appearance
of new bands if they occur at same locus in a sufficient number of cells to be
amplified by PCR. The new bands could be attributed to mutation, while the
disappearance of bands could be attributed to DNA damage.
3) Phytochemical profiles of A. paniculata mutants in M1V4 generation
We used 4 leaves of mutants and 1 from control to determine the changes
of pytochemical profiles. The reason for 4 mutants used for comparison of
this pytochemical profiles based of dendrogram data (Figure 2). We used
mutans plant with farthest of genetic distance from control (DG70, DG275
and DK300 mutants) and plant from the middle of the dendrogram (DB60
mutants) in M1V4 generation. Andrographolide standard used for this
research in the range of retention time of 4.98 ± 0.01 with equation of a
linear regression of standard was Y = 18606X +76026 and the correlation
Innovation for Better ASEAN Community 11

coefficient was R2 = 0.993, which indicated a good linear regression for


analysis of andrographolide.
Analysis using HPLC with a wavelength of 230 nm resulted in the variation
of the profiles and contents of phytochemical mutants of A. paniculata. The
contents of andrographolide varied in the range between 6.5%–10.9% (Figure
5). Highest content of andrographolide found in DK300 mutant while the
lowest content found in control (DN). The higher dose of irradiation given
made the increasing levels of andrographolide produced. There were increased
levels of andrographolide up to 1.66% in DK300 mutant compared to control.
In control, the normal of andrographolide content amounted to 2.01%
(Data from BALITTRO, not shown), with the condition if A. paniculata
grown using seeds until harvest in 120 days. In this research, propagation
of A. paniculata was done by ex vitro method and the result showed that
andrographolide content increased up to 4% with 60 days of harvest period.
This can be explained that on conditions of propagation by cutting, parts of
such plants were subjected to stress. Reported by Datta et al. (2011), stress
of plants caused by external factors such as radiation not only causes chro-
mosomal changes but also the pathway disruption of some chemical reaction
linked to abnormal of plant growth and mechanisms of defense of the cell.
This stress will stimulated the endogenous hormone to produce plant growth
regulators such as cytokinin or GA3 to maintain metabolic and physiological
systems and stimulate the growth of shoots. It is known that the effect of
GA3 also change the number of metabolites in cells by regulating the activity
of key enzymes such as DXS and HMGR. Both of these enzymes play a role
in terpenoid biosynthesis pathway either through MVA (HMGR enzyme)
or DXP (DXS enzyme) pathways that produces IPP and DMAPP, which are
precursors for the synthesis of diterpene (Jha et al., 2011). The increase of
andrographolide content on ex vitro plants of A. paniculata and irradiated
plants on M1V4 generation indicated also caused by this. These contents of
andrographolide were very high compared to the contents of andrographolide
isolated from several different locations in Indonesia, with the average ranges
between 0.95%–2% dry weight (Royani et al., 2012).
Research conducted by Koobkokkruad et al. (2008), with mutant of
Artemisia annua that irradiated by gamma rays at 8 Gy, have been able to
increase the levels of artemisinin content from 0.18% to 0.70% or an increase
by approximately 3.89%. Aryanti (2011) also found artemisin in Artemisia
cina mutant was increased from 40 mg/100g to 2103 mg/100g than parent
or an increase of approximately 52%. Likewise, research by Moghaddam et
al. (2011) found content of flavonoid was increased from 2.64 ± 0.02 mg/g
to 8.94 ± 0.04 mg/g or an increase of 3.38% in Centella asiatica mutant. In
12 Proceeding ASEAN COSAT 2014

Lithospermum erythrorhizon mutant (Chung et al., 2006), shikonin production


increased (71.0 mg/l) at 16 Gy was almost 4-fold amounts to that of control
cultures (17.7 mg/l).
Chromatograms peaks generated by HPLC in control plant obtained two
peaks with retention times of 2.823 dan 4.818, the last digit was the peak of
andrographolide. In DB60 mutant, andrographolide content had almost the
same content as control but in chromatogram, it had four peaks. They were
2.837, 5.014, 6.806 and 9.379 with andrographolide peak obtained in 5.014.
The addition of the peak in 6.806 and 9.379 were indicated the addition of
different phytochemical profiles than control as a result of irradiation.
In DG70 mutant, profiles of chromatogram obtained 3 peaks in the
range of 5.011, 6.703 and 9.331. These profiles were also different from those
seen in the control where the peak with 2.823 value was lost in the mutant
DG70, while the value of 6.703 and 9.331 were indicated the same profile
with DB60 peak. Andrographolide content on DG70 mutant was also higher
than the control, it was 7.8%.

10.9
12 9.9
Andrographolide content (%)

10 7.8
6.5 6.8
8
6
4
2
0 Figure 3. The contents of androgra-
DN DB60 DG70 DG275 DK300 pholide from A. paniculata mutants

In DG275 mutant, profiles of chromatogram were identified to also have 3


peaks, namely 2.863, 5.017 and 6.875. These profiles were different from control
because there was an additional peak in 6.875 and peak of 5.017 indicated the
peak of andrographolide. The addition of 6.875 peak is similar to that found
in DB60 and DG70 mutants. Andrographolide levels of mutant DG275 also
increased 9.9% compared to the control with 6.5%.
In DK300 mutant, chromatogram peaks indicated the presence of 2 peaks
at 2.882 and 5.023. Phytochemical profiles in DK300 mutant generated had
the same profile as control but the level of andrographolide was higher than 3
mutants and control, which was at 10.9% level.
Innovation for Better ASEAN Community 13

Of the five chromatograms that formed by gamma rays, the effects of


phytochemical profiles were clearly visible on the different mutants than control.
These differences of phytochemical profiles from DB60, DG70 and DG275
mutants possibility generating new compounds when its compared to control.
It can be explained that irradiation by gamma rays causes oxidative stress and
the impact on the biomolecules that cause conformational changes, oxidative,
breaking of covalent bonds and the formation of free radicals (Lee et al 2005).
The hydroxyl radical (HO-) and superoxide anion (O2-) formed by the radiation
can modify the properties of the protein and lipid molecules that cause oxidative
modification of peroksidase protein and lipid.
The location of the new peaks that formed from phytochemical profiles is
likely that the compound was not much different from the andrographolide
compound in basic structure but in a different kind like neo-andrographolide,
deo-xyandrographolide, or dehydroandrographolide. This possibility was because
the standard used for phytochemical profile was just andrographolide so the other
peaks would not be ascertained.
Gamma rays can cause different levels of damage to the cells. Biological
damage occurs most direct and mediated by reactive oxygen species (ROS) such
as hydrogen radical (HO), superoxide radical (O2), hydrogen peroxide (H2O2),
and singlet oxygen formed from the radiolysis of water (El-Beltagi et al., 2011).
The presence of water radiolysis by gamma radiation causes changes of
chemical from protein that caused by fragmentation, cross-linking, aggregation
and oxidative caused by oxygen radicals were formed (Lee et al., 2005). ROS are
highly reactive in membram lipids, proteins and DNA. ROS was known by its
ability to activate signal nitrogenmonoksida (NO) and NADPH oxidase (Zhang
& Bjorn, 2009). ROS was also believed to be the main cause of stress injuries
and rapid cellular damage (El-Beltagi et al., 2011). Added by Kume and Matsuda
(1995) that the radiation causes behind changes in protein conformation at the
molecular level by means of break covalent bonds from polipetida chains.
In DK300 mutant, phytochemical profile of the mutant with the control
obtained two peaks with the same retention time but contains andrographolide
higher levels than control. This can be explained hypothetically that the role of
these enzymes in the biocatalyst of biosynthesis of the other compounds non
andrographolide were blocked and resulting the optimization of the biosynthesis
of the andrographolide or an increase enzymes that play important role in the
synthesis of the andrographolide.
Allegedly, an increase in the enzyme 3-hydroxy-3-methylglutaryl-coenzyme
A reductase (hmgr) which is one of the key enzymes that play a role in the
accumulation and chlorophyll content of andrographolide on A. paniculata
14 Proceeding ASEAN COSAT 2014

1.00
andrographolide

2.823
0.50
AU

4.818
0.00
2.00 4.00 6.00 8.00 10.00
Minutes

1.00
andrographolide

6.807
2.837

9.379
0.50
AU

0.00
5.014
2.00 4.00 6.00 8.00 10.00
Minutes

1.00
andrographolide
6.704

9.331
0.50
5.011
AU

0.00
2.00 4.00 6.00 8.00 10.00
Minutes

1.00 andrographolide
5.017
2.864

6.876

0.50
AU

0.00
2.00 4.00 6.00 8.00 10.00
Minutes

1.00 andrographolide
5.024
2.882

0.50
AU

0.00
2.00 4.00 6.00 8.00 10.00
Minutes
Figure 4. The results of HPLC chromatograms in control and A. paniculata mutants
Innovation for Better ASEAN Community 15

(Jha et al., 2011). Increased of shikonin compounds by gamma-rays on the


plant Lithospermum erythrorhizon was also due to the increase of enzyme activity
geranyltransferase PHB, as a precursor of shikonin, which resulted in increased
accumulation of shikonin (Chung et al., 2006). Another possibility is that the
expression of genes that involved in the synthesis of andrographolide to increase.
The study conducted by Jha et al. (2011) about the profile of expression of A.
paniculata plants with elicitor GA3 and jasmonic acid can alter expression of
HMGR gene. In this study, the gamma rays were also thought to be elicitor to
increase expression of HMGR gene in A. paniculata plants. Similar research was
also reported in maize plants exposed by UV-B rays, where the expression of genes
that associated with photosynthesis in maize decreased and genes that associated
with antioxidant increased (Casati & Walbot, 2003).
The similarity of the number of peaks at the phytochemical profiles of control
and DK300 mutant compared with the other mutants (DB60, DG70 and
DG275) that have more peaks can be explained that mutation by irradiation was
random. So that the possibility of the mutant had the addition peak of more than
control due to a modulation in the pattern of proteins that induce the presence
or loss of some protein bands (Hegazi & Hamideldin, 2010) which resulted in a
conformational protein change in the making up of the compound.

IV. Conclusion
Genetic distance of A. paniculata ranged from 0.79 to 1.00 indicating that the
genetic variation was still remained low even though it was treated with gamma
radiation mutagen. Futher study such as the structure elucidation of coumpounds
need to be done in order to see what will come up in the A. paniculata mutant
DB60, DG70 and DG275.

V. R eferences
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Andrographolide. Journal of Health Science, 54 (4), 370–381.
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4) Subramanian, R., Asmawi, M. Z. and A. Sadikun. (2012). A bitter plant


with a sweet future? A comprehensive review of an oriental medicinal plant:
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Innovation for Better ASEAN Community 17

15) Abd El-Twab, M. H. and F. A. Zahran. (2010). RAPS, ISSR and RFLP
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The Chemical Constituent and Antioxidant
Activity of The (-)-Epicathecin from an
Endophytic Fungus Mycoleptodiscus indicus

Panji Cahya Mawardaa,*, Teni Ernawatib and Yoice Srikandacea


a
Research Center for Chemistry, Indonesian Institute of Sciences, Jalan Cisitu Sangkuriang-
Bandung, Indonesia
b
Research Center for Chemistry, Indonesian Institute of Sciences, Kawasan Puspitek Serpong-
Tangerang, Indonesia

Abstract
The current study was to investigate the chemical constituent and antioxidant activity of
(-)-epicathecin from an endophytic fungus Mycoleptodiscus indicus. The fungus was isolated from
Indonesian medicinal plant Thyponium flageliiforme. The endophytic fungus was fermented in
Potato Dextrose Broth and extracted with ethyl acetate. Ethyl acetate extract was fractionated using
column chromatography techniques to obtain flavones. The structure of flavones was elucidated
through spectral data based on 1H-NMR, 13C-NMR, and LC-MS spectrometer. The compound
was identified as (-)-epicatechin with molecular weight of 290.11 g/mol. The compound was
tested with DPPH radical scavenging method and it showed strong antioxidative activity with
IC50 0.6 µg/mL. At the first time, (-)-epicathecin from M. indicus Indonesia was reported.
Key words: Mycoleptodiscus indicus, (-)-epicathecin, Antioxidant activity

I. Introduction
Oxidant and free radical are continuously produced as by-product in normal cel-
lular metabolism. The uncontrollable production of free radical leads to oxidative
stress and becomes a main trigger of human degenerative diseases development
[11,21,18]. The harm characteristics of free radical can be well controlled by
antioxidant because it inhibits the oxidation processes on substrates. Therefore, it
is essentially needed to develop a drug that overcomes free radical problems from
organisms which have antioxidant compound. As one of the richest countries
at biodiversity, Indonesia has medical herbs and microbes that can be utilized as
drugs resources [22]. It potentially could be explored for bioactive compound
discovery that serves as antioxidant.
One of the microbe that is promising to be developed as a new source for
bioactive antioxidant compound is endophytic microbes. These microbes live
symbiotically in plant tissue at certain period and survive by forming a colony
on that particular place. Every medicinal plant could possibly have some differ-

* Corresponding author. Phone: +6285711749663. Email: panji.cahya@gmail.com

19
20 Proceeding ASEAN COSAT 2014

ent kind of endophytics that are capable at producing bioactive compound or


secondary metabolites which are hypothesized as a result of coevolution or genetic
transfer from its host plant in to endophytic microbes [17].
The ability of endophytics for producing the same secondary metabolites
with its host plant is a great and reliable. It could be used to produce the same
secondary metabolites that are isolated from its host plant. Then, if the endophytic
microbes from medical herbs are able to produce flavones or the same secondary
metabolites with its host, we do not have to chop the plant down for obtaining
its symplisia which possibly needs a dozen of years to be harvested. Some of the
endophytics have been successfully isolated and cultivated in a proper media,
likewise, its secondary metabolites which have been successfully isolated, purified,
and structurally elucidated [13]. More than 300,000 plants species spreading
around the globe are associated with one or more endophytic microbes [16].
The endophytic microbes usually live in medicinal plants. Typhonium flagelli-
forme is one of medicinal plant which has endophytic microbes. The plant has been
known to contain Ribosome Inacting Protein (RIP) and antioxidant compound.
According to previous study, one of endophytic fungi that has been isolated from
Typhonium flagelliforme is Mycoleptodiscus indicus. It potentially is able to produce
various active secondary metabolites as antioxidant. This endophytic fungus is
remarkably believed to produce the same secondary metabolites with Typhonium
flagelliforme. Mycoleptodicin A and Mycoleptodicin B have been reportedly pro-
duced by Mycoleptodiscus indicus that lives in Desmotesin comparabilis [7]. Whereas,
Mycoleptone A,B,C, austdiol, eugnitin, 6-metoksieugenin, 9-hydroxyieugenin
have been reportedly produced by Mycoleptodiscus indicus who lives in Borreria
verticilata [19].
There are also a significant major compound of flavonoids in other endophytic
fungi. The fungal endophytics which associated with a medicinal plant, Nerium
oleander L. (Apocynaceae) showed flavonoids, phenolic acids and aliphatic com-
pounds [23]. The objective of this research was to isolate the chemical compound
and to evaluate its antioxidant activity from Mycoleptodiscus indicus who lives in
rodent tuber. The antioxidant activity from the isolated compound was determined
by 1,1-diphenyl-2-picrilhydrazil (DPPH-radical) method.
Innovation for Better ASEAN Community 21

II. MATERIAL AND METHOD


A. Chemicals and Materials
Mycoleptodiscus indicus isolate, PDA, PDB, eluent (100% Hexane, ethyl acetate
100%, 100% methanol), MeOH for DPPH Analysis p.a grade, DPPH for sigma.

B. Instruments
Silica gel with a column length of 35 cm; diameter of 4.5 cm and a length of 55
cm; diameter of 2.5 cm; Liquid-Mass Spectrometry chromatography Mariner
Biospectrometry: UVJVis. Simadzu Detector (Perkin Elmer Series 200), LC MS
Mariner, Phenomenex column 2 x 150 mm containing C18; NMR spectrometer
Jeol Inova Unity Plus (500 MHz and 250 MHz), Incubator.

C. Method
1) Fermentation and Extraction
The endophytic fungus M.indicus was cultivated on Potato Dextrose Agar
plates at 25 °C for 7–14 days. Three pieces (0.5 X 0.5 cm) of mycelia agar
plugs were inoculated into 10L PDB and incubated at room temperature
under agitation 150 rpm for 20 days. After this, the media of the fungus
was separated from mycelia by vacuum filtration. The media was extracted
with ethyl acetate and evaporated using rotary vaccum evaporator at 40°C
to obtain the crude extract.
2) Isolation and Identification
Isolation of the crude extract used modification of standard purification
methods for natural products. Initial fractionation by dry flask chromatog-
raphy techniques and distribution components differences in of a mixture
between the two phases. Stationary phase silica gel 60 (E. Merck 7734) and
the mobile phase n-hexane; n-hexane: ethyl acetate (4:1, 3:1, 2:1, 1:1); ethyl
acetate; ethyl acetate: methanol (4:1, 3:1, 2:1, 1:1); and methanol, using
a variety of comparison with a range of 0–100%. The first fractionation
produced 140 fractions. The analysis process of chromatogram for each frac-
tion used silica gel aluminum plates GF254. Spots were observed under UV
light with a wavelength of 254 nm and 366 nm. The fractions that showed
similar chromatogram then combined into 26 fractions (A-Z). The fraction
R (75–79) showed a specific spot and a large number of isolates, so it’s carried
for the purification process using preparative TLC techniques. The weight of
fraction R was 125.7 mg.
Isolation of the fraction R (75–79) was performed by preparative TLC
techniques with ethyl acetate mobile phase. TLC results were observed
22 Proceeding ASEAN COSAT 2014

and marked in UV light with a wavelength of 366 nm. The marked part
was extracted, filtered, and evaporated to obtain 20.3 mg pure fractions.
Spectroscopic Analysis was conducted by LC-MS, ‘H-NMR and 13C-NMR
spectrometer, using NMR (500 MHz and 250 MHz, CD30D) with CD3OD
solvent.
3) Inhibition Assay for DPPH Radical Scavenging Activity
Heading the DPPH radical scavenging activity was performed using estab-
lished procedure [24]. The 1.5 mg DPPH was dissolved in 50 mL methanol.
The mixture was vigorously shaken and incubated in the dark for 30 minutes
at room temperature. The absorbance was measured at 517 nm. Ascorbic acid
was taken as a positive control, DPPH was taken as negative control, and
methanol was taken as blanko. The scavenging ability was calculated as: DPPH
radical scavenging activity (% inhibition) = [1-(A1-A2)/A0] x 100, where A0 is
the absorbance in the lack of the test compound, A1 is the absorbance in the
presence of the test compound and DPPH, and A2 is the absorbance in the
lack of DPPH. The IC50 values (the concentration of the antioxidant required
to scavenge 50% of DPPH present in the test solution) were obtained from
linear regression analysis of the concentration-response curves plotted for
tested compound.

III. R esult and Discussion


The ability of endophytic microbes at producing the same secondary metabolites
as its host plant is a fundamental reason why we did the research. It was a great
and reliable opportunity to produce secondary metabolites from Typhonium flagel-
liforme without chopping the plant down since the endophytic fungus that live on
that plant could possibly produce the same secondary metabolites. The isolated
compound that has been successfully purified and identified is (-)-epicathecin.
The ethyl acetate extract of Mycoleptodiscus indicus fermentation was fractioned
by column chromatography and was purified by preparative TLC. The used
isolation method was the modification method of standard purification for natural
products and chromatogram analysis [10, 14, and 15]. This method has been
efficiently proved at clustering compound group that has similar chromatogram
pattern from each fraction. The fractionation yielded 140 fractions which were
combined in to 26 fractions based on similarity of chromatogram pattern.
The purified compound that has been isolated from 26 fractions was from
Fraction R1 (75–79). The mass of the purified fraction, a brown powder, was 20.3
mg and has molecular formula C15H14O6 from LC-MS spectrum with [M+] ion
at 291.11. The rendement of this compound was 1.01%. It was then analysed by
NMR Spectrometer. The structure of the purified fraction was elucidated on the
Innovation for Better ASEAN Community 23

basis of its 1H NMR and 13C NMR analyses (Table 1) and closely similar to those
of (-)-epicatechin. Based on characteristic signals of δ H 4.82 (1H, br s, H-2), δ
H 4.17 (1H, m, H-3), δ H 2.86 (1H, dd, J = 4.8, 16.8 Hz, Ha-4) and δ H 2.73
(1H, dd, J = 2.8, 16.8 Hz, Hb-4), it showed C-ring protons of flavan-3-ol. The
data of δ H 5.93 (1H, d, J = 2.4 Hz, H-6) and δ H 5.91 (1H, d, J = 2.4 Hz,
H-8) showed the meta-coupled aromatic proton signals which also revealed the
presence of a flavan-3-ol skeleton. The ABX-type aromatic signals at δ H 6.97

Figure 1. (-)-epicatechin molecular structure

Table 1 1H and 13C NMR spectral data of fraction R1


Position δH δC
2 4.82 (1H, br s) 79.97
3 4.17 (1H, m) 67.58
4 2.86 (1H, dd, J = 4.8, 16.8 Hz) 29.37
2.73 (1H, dd, J = 2.8, 16.8 Hz)
5 157.77
6 5.93 (1H, d, J = 2.4 Hz) 96.44
7 158.11
8 5.91 (1H, d, J = 2.4 Hz) 95.95
9 157.46
10 100.14
11 132.38
12 6.97 (1H, d, J = 1.6 Hz) 115.40
13 6.75 (1H, d, J = 8.0 Hz) 146.03
14 145.87
15 115.96
16 6.79 (1H, dd, J = 2.0, 8.4 Hz) 119.47
1
H and 13C NMR spectra were recorded in methanol-d4 at 400 MHz and 100 MHz, respectively. Chemi-
cal shifts (δ) were given in ppm versus TMS.
24 Proceeding ASEAN COSAT 2014

(1H, d, J = 1.6 Hz, H-12), δ H 6.75 (1H, d, J = 8.0 Hz, H-13) and δ H 6.79
(1H, dd, J = 2.0, 8.4 Hz, H-16) corresponding to a B-ring were recognized.
The basic structure was therefore deduced as (-)-epicatechin. Furthermore, the
stereochemistry of (-)-epicatechin was suggested by the proton signal of H-2,
which appeared as a broad singlet at δ H 4.82 [3,12].
The data of 13C-NMR showed 15 signals for 15 carbons (Table 1). Those
signals were δC 79.97 (C-2), 67.58 (C-3), 29.37 (C-4), 157.77 (C-5), 96.44
(C-6), 158.11 (C-7), 95.95 (C-8), 157.46 (C-9), 100.14 (C-10), 132.38 (C-11),
115.40 (C-12), 146.03 (C-13), 145.87 (C-14), 115.96 (C-15) and 119.47 (C-16).
The 1H NMR and 13C NMR data of this fraction were similar with those reported
in the literatures for (-)-epicatechin [4,5,9]. Epicatechin has various biological
activities as follows antimicrobial [2], antipasmodic, broncodilator, vasodilator,
and effectively is used for gingitivis patient. This compound is also popular for
cosmetic and has been tested as antiaging, antiacne, and help to maintain the
weight loss [6].
The antioxidant activity of this compound was evaluated according to the
method previously described. The DPPH is free radical compound that gives
purple colour solution in methanol. Its purple colour is reduced to yellowish
solution in methanol by presence of antioxidant compound. The principle of
this method is the ability of a molecule at donating a hydrogen atom to a radical.
The critical factor in free radical scavenging is the propensity of the hydrogen
donation [1]. The DPPH radical scavenging activities of the purified compound
are presented in Figure 2. The compound was made in to 5 final concentrations
as follows: 3.125 ppm, 1.563 ppm, 0.781 ppm, 0.391 ppm, and 0.195 ppm.
Each concentration exhibits different percentage of inhibition.This compound
exhibited significant inhibition activity with IC50 0.6 µg/mL.

Figure 2. Antioxidant activity of (-)-epictechin


Innovation for Better ASEAN Community 25

IV. Conclusion
Based on 1H NMR and 13C NMR spectra and compared to previous spectral data
that has been reported, the compound of Fraction R1 from Mycoleptodiscus indicus
fermentation extract was identified as (-)-epicatechin. This compound showed
significant activity in the DPPH radical scavenging with IC500.6 µg/mL. To our
knowledge, this is the first report on antioxidative activity of (-)-epicatechin from
Mycoleptodiscus indicus. It was indicated that this endophytic fungus could be
considered as potential source for antioxidant bioactive compound.

V. Acknowledgement
This research has been funded by the Indonesian Government Budget for
Indonesian Institute of Sciences (LIPI). We would also like to thank Dr. Linar
Zalinarudin dan Dra. Puspa Dewi N. Lotulung, M.Eng.

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Potency of Hibiscus (Hibiscus rosa-sinensis
L.) as Antioxidant to Reduce Carbon
Tetrachloride Compounds in Red Blood
Cells
Dody Priadi and Kusmiati*
Research Centre for Biotehnology-LIPI
Jl. Raya Bogor Km.46, Cibinong 16911, Indonesia

Abstract
Hibiscus (Hibiscus rosa-sinensis L.) is a species of flowering plant that is easily found and used for
traditional medicine. Hibiscus flower decoction is used to treat various diseases, such as urinary
tract infections, coughing, whooping cough, dysentery, bronchitis, tuberculosis and various
degenerative diseases. These properties are related to the content of compounds as antioxidants
such as anomuricin and polyphenols. Antioxidant compounds may reduce the risk due to free
radical oxidation process to prevent degenerative diseases. Hibiscus flowers were extracted by
maceration method with ethanol and ethyl acetate solvents. The extract containing antioxidant
of 10, 20, 30, and 40 ppm was tested against sheep red blood cell that induced with CCl4 as
oxidant. Parameters measured were malondialdehyde (MDA) content and activity of superoxide
dismutase (SOD) and catalase enzymes. Data was analyzed using Kruskal-Wallis test. Results
showed that the highest reduction in MDA content (0.7679 nmol/ml) was obtained by the
treatment of ethyl acetate extract at the concentration of 10 ppm. The highest activities of the
SOD and catalase enzymes were 0.8874 U/ml and 460.655 U/ml respectively, obtained by the
treatment of 40 ppm of the extract containing antioxidants.
Key words: Hibiscus (Hibiscus rosa-sinensis L.), Antioxidants, MDA, SOD, Catalase

I. Introduction
An excessive oxidation reactions in the body can trigger the formation of a
highly active free radicals which damage the structure and function of cells, and
unconsciously, the body is constantly form free radicals, either through normal
cellular metabolism, inflammation, malnutrition, and due to the response against
influences from outside the body, such as environmental pollution, ultraviolet
(UV) radiation, cigarette smoke and others [1].
Antioxidants are substances that inhibit oxidative stress in the molecule.
Antioxidants can be categorized into enzymatic antioxidants (enzymes) such as
superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase.
Meanwhile, non-enzymatic antioxidant (extra-cellular) includes vitamin E,

* Corresponding author. Phone: +62-21-8754587. Email: kusmiati02@yahoo.com

29
30 Proceeding ASEAN COSAT 2014

vitamin C, β-carotene, glutathione, ceruloplasmin, albumin, uric acid and


selenium [2].
Hibiscus (Hibiscus rosa-sinensis L.) is one of the ornamental plants that are
widely used as traditional medicine in Indonesia in the form of fresh flowers or a
stew. Herb or decoction of hibiscus flowers is proven to be able to cope with various
diseases, such as gonorrhea, bronchitis and tuberculosis due to chemical efficacy
of antioxidants containing polyphenols such as flavonoids anomuricin reducing
the risk oxidation process by free radicals causing several of degenerative diseases
[3]. Hibiscus rosa-sinensis flower extract exhibited antioxidant, antihyperglycemic
and antihyperlipidemic activities mainly at the dose of 250 mg/kg bwt without
showing any toxic effects on experimental rats [4]. Leaves of this plant species
inhibited the process of spermatogenesis of ddy strain mice. Therefore, it can be
developed into a male contraception [5].
The objective of the study was to identify antioxidant activity of Hibiscus
flower extracts in vitro in sheep red blood cells induced by carbon tetrachloride,
determining the optimal solvent concentration to reduce MDA levels and to
increase the activity of SOD and catalase, and determining the optimal concentra-
tion of hibiscus extract containing antioxidants.

II. Method/M aterial


Preparation hibiscus flower extract
Hibiscus (Hibiscus rosa-sinensis L.) was obtained from Research Institute for
Medicinal and Aromatic Plants, Bogor. Petals of the approximately 2 days-
blooming flowers were cleaned and dehydrated in room temperature for 3 days
and were then powdered using a blender. The powder was stored in a clean and
sealed container. A total of 20 grams of powdered hibiscus flower were macerated
with ethanol andethyl acetate (200 ml) separately, and soaked for 24 hours at
room temperature while stirring using an orbital shaker. The filtrate was separated
using a filter paper.

Figure 1. Hibiscus flower


Innovation for Better ASEAN Community 31

Phytochemical screening of the powdered flowers of hibiscus was conducted


at the Laboratory of Bioprocess 3 Research Centre for Biotechnology-LIPI
Cibinong. Sheep blood was obtained from the Microbiology Division of Faculty
of Medicine, University of Indonesia. The red blood of sheep was separated into
blood plasma and cells by centrifugation at 3.000 rpm at 5oC for 5 minutes.
Blood plasma was used for analysis of MDA (malondialdehyde) content. MDA
content was analyzed according to [6], a volume of each 0.5 ml ethanol and ethyl
acetate extract of hibiscus flower (10, 20, 30 and 40 ppm) were added into a half
milliliter of plasma and then incubated at room temperature for 15 minutes. The
solution was induced with carbon tetrachloride (CCl4) solution and incubated
at room temperature for 15 minutes, centrifuged at 3000 rpm for 5 minutes. A
number of the supernatant was treated with trichloroacetic acid (TCA) 20% and
thiobarbituric acid (TBA) 0.67%, and homogenized. The mixture was heated for
30 minutes in a waterbath at 100 °C and then cooled. Absorption of the solution
was measured at λ 532 nm. Tetra ethoxy propane (TEP) was used as a standard.
The blood cell was washed 3 times with phosphate buffered saline solution
(PBS) and centrifuged at 3.000 rpm at 5 °C for 5 minutes. The red blood cells
(RBC) were used for the study of superoxide dismutase (SOD) and catalase
enzymes activity. The extract of hibiscus flower was added into red blood cells
and incubated at room temperature for 15 minutes. Oxidant of CCl4 was then
added prior to incubation at room temperature for 15 minutes. The mixture was
diluted with distilled water and centrifuged for 5 minutes at 3.000 rpm. Details
of the treatment are presented in Table 1.
Activity of SOD enzyme was analyzed according to [7]; one volume of RBC
supernatant was diluted with distilled water and extracted with one volume
of chloroform-ethanol (3:5), was shaken and centrifuged at 2.500 rpm for 10
minutes. A volume at 50 µl the aqueous phase was added into carbonate buffer
pH 10.2 and 50 µl, of 0.02 M epinephrine solution. Absorption was measured
at λ 480 nm at 30 °C.
Activity of catalase enzyme was analyzed according to [8]; a volume of 100 µl
RBC supernatant was added into H2O2 0.059 M and 1.9 ml 0.05 M phosphate
buffer pH 7. Absorption of enzymatic reaction was measured at λ 240 nm.
Data were analyzed using a non-parametric method of Kruskal-Wallis and
Mann-Whitney for those not normal and not homogeneous. The data were
processed with SPSS 16 statistical software.
Outline of the method above is described in Figure 2.
32 Proceeding ASEAN COSAT 2014

Table 1. Group of treatments


Group Treatments
I Normal Control (RBC)
II Negative Control (RBC + CCl4)
III Positive Control (RBC + CCl4 + Vit C 20 ppm)
IV RBC + CCl4 + Ethanol extract of Hibiscus flower (10 ppm)
V RBC + CCl4 + Ethanol extract of Hibiscus flower (20 ppm)
VI RBC + CCl4 + Ethanol extract of Hibiscus flower (30 ppm)
VII RBC + CCl4 + Ethanol extract of Hibiscus flower (40 ppm)
VIII RBC + CCl4 + Ethyl acetate extract of Hibiscus flower (10 ppm)
IX RBC + CCl4 + Ethyl acetate extract of Hibiscus flower (20 ppm)
X RBC + CCl4 + Ethyl acetate extract of Hibiscus flower (30 ppm)
XI RBC + CCl4 + Ethyl acetate extract of Hibiscus flower (40 ppm)

Figure 2. Outline of the method of study of hibiscus flower as antioxidant


Innovation for Better ASEAN Community 33

III. R esult and Discussion


A. Phytochemical Screening Test
Results of phytochemical screening showed that hibiscus flower powder extracted
with ethanol solvent contains alkaloids, flavonoids, saponins, tannins and trit-
erpenoids, whereas those extracted with ethylacetate solvent contains alkaloids,
flavonoids, and tannins. Flavonoid was obtained in considerable number from
hibiscus flower. Flavonoids test using magnesium and hydrochloric acid gives a
positive result indicated with formation of orange color in amil alcohol solution
(Table 2).
Table 2. Result of fitochemical test of hibiscus flower
No. Compound Powder Ethanol extract Ethyl acetate extract
1 Alkaloid + + +
2 Flavonoid + + +
3 Saponin + + -
4 Tannin + + +
5 Triterpenoid + + -
6 Steroid - - -

Note: + = available; - = not available

B. MDA Content
Free radicals were detected by the presence of lipid peroxidation, which
will produce secondary products, such as malondialdehyde (MDA).
Levels of MDA were detected by UV-VIS spectrophotometer at λ max 532 nm.
MDA content in group II (negative control) was 1.2591 nmol/ml that
increased by 23.33% of the normal control. This suggests that under normal
conditions, the content of lipid peroxidation in the body is low. Increasing of
free radicals in the blood caused the lipid peroxidation process which increase
formation of MDA. This was associated with oxidation increase of unsaturated
fats as a result of the addition of carbon tetrachloride. Group III, IV, V, VI, VII,
VIII, IX, X and XI showed a decrease in MDA levels of 50.38%, 25.75%, 11.52%,
7.60%, 4.11%, 39.01%, 20.05%, 14.35%, and 6.63% respectively compared
to the negative control. The treatments of hibiscus flower extract and vitamin C
(positive control) showed that it potentially inhibits free radicals. It seems that
the ethanol extract of hibiscus flower was more effective as antioxidants (Figure
3). Result of statistical analysis showed that there was a significantly different
result (p<0.05) between the groups of analysis.
34 Proceeding ASEAN COSAT 2014

Figure 3. MDA content in red blood


cells of sheep induced by carbon
tetrachloride.

C. SOD Enzyme Activity


Table 3 showed that SOD enzyme activity was increased with the addition of
vitamin C (Group III) as well as group VIII and XI compared with group I and
II. SOD enzyme activity in group III, IV, V, VI, VII, VIII, IX, X, XI i.e. 66.75%,
17.67%, 41.95%, 51.51%, 60.37%, 6.18%, 26.04%, 45.75% and 53.95%
respectively from the negative control (Figure 4).
Ethanol is the best solvent for hibiscus flower extraction in enhancing activity
of SOD compared with ethylacetate. The low concentration of SOD in the blood
leading to high reactive oxygen (O2*-) plays an important role in inflammation.
Low concentration of SOD caused the release of prostaglandins as an important
factor of inflammation. There is a possibility of the ethanol extract of hibiscus
flowers acted as anti-inflammatory, because it can prevent the decrease in SOD
enzyme activity in the blood [9].
Study conducted by [10] showed that the activity of SOD and the concentra-
tion of MDA were highest in the group of patients with the lowest success of the
hypo-osmotic swelling (HOS) test. The assessment of the antioxidant enzymes
and MDA in addition to the semen analysis and the HOS test may be greatly
useful in diagnosing infertility in men having oxidative stress in their etiology.

Figure 4. SOD enzyme activity in


red blood cells of sheep induced by
carbon tetrachloride.
Innovation for Better ASEAN Community 35

D. Catalase Enzyme Activity


Figure 2 showed that catalase enzyme activity in group II was lower than group I
due to the red blood cells in group II already induced by carbon tetrachloride. In
group II, the catalase enzyme in red blood cells has been used in part to neutralize
free radicals of carbon tetrachloride. This caused reduction of catalase enzyme
activity in red blood cells with the result that the ability to neutralize peroxidation
of hydrogen peroxide was decreased.
The addition of carbon tetrachloride and vitamin C in group III as well as
group IV, V, VI, VII, X and XI with that addition of hibiscus flower extract caused
a higher activity of catalase enzyme compared to group I and II. An increase in
catalase activity in group III and VII shows the antioxidant effects of the ethanol
extract of the hibiscus flower. Meanwhile, group VIII and IX did not show any
antioxidant activity of ethylacetate. Ethanol extract at a concentration of 40 ppm
was the most effective dose to reduce carbon tetrachloride oxidation for up to
38.06%. Increased activity of catalase enzyme in group III, IV, V, VI, X, and IX
were 86.29%, 7.43%, 23.69%, 4.82% and 6.36% respectively from the negative
control (Figure 5).
Results of statistical analysis showed that there were significantly different
(p<0.05) result between the groups of analysis.

Figure 5. Catalase enzyme activity


in red blood cells of sheep induced
by carbon tetrachloride.

IV. Conclusion
Treatments of ethanol and ethyl acetate extract of Hibicus flower reduced MDA
content in blood plasma as well as increased activity of SOD and catalase enzyme
in red blood cells induced by carbon tetrachloride.
The highest reduction of MDA content (0.7679 nmol/ml) in red blood plasma
was obtained by ethyl acetate extract of Hibiscus flower (10 ppm). The highest
activities of SOD and catalase enzyme were 0.8874 U/ml and 460.655 U/ml
36 Proceeding ASEAN COSAT 2014

respectively, obtained by ethanol extract of hibiscus flower (40 ppm). Ethanol is


a polar compound so that the secondary metabolites in the hibiscus flower can
be extracted perfectly. Further study has to be done using experimental animal
to obtain more informative result.

V. R eferences
1) Ahmad, S. A., Hakim, E. H. and L. Makmur. (2009). Ilmu Kimia dan
Kegunaan Tumbuh-tumbuhan Obat Indonesia (p. 120). Bandung: ITB.
2) Halliwel, B. and J. M. C. Gutteridge. (1989). Free Radicals in Biology and
Medicine (pp. 196–200) (2nd ed.). New York: Oxford University Press.
3) Mursito, B. (2002). Tanaman Hias Berkhasiat Obat (p. 33). Jakarta: Penebar
Swadaya.
4) Sankaran, M. and A. Vadivel. (2011). Antioxidant and antidiabetic effect of
hibiscus rosasinensis flower extract on streptozotocin induced experimental
rats-a dose response study. Notulae Scientia Biologicae, 3 (4), 13–21.
5) Mawuntu, M. (2008). The extract of “shoe flower” (Hibiscus rosasinensis
L.) leaves inhibit the spermatogenesis of ddy strain mice. Medical Journal of
Indonesia, 17, 157–62.
6) Tüközkan, N., Erdamar, H. and I. Seven. (2006). Measurement of total
malondialdehyde in plasma and tissues by high-performance liquid chroma-
tography and thiobarbituric acid assay. Fırat Tıp Dergisi, 11 (2), 88–92.
7) Achuba, I. Fidelis. (2005). Effect of vitamins C and E intake on blood lipid
concentration, lippid peroxidation, superoxide dismutase and catalase activi-
ties in petroleum-contaminated diet fed rabbit. European Journal of Scientific,
12 (1), 1–8.
8) Aebi, H. (2974). Catalase. In Bergmeyer (Ed,), Methods in Enzymatic Analysis
(pp. 674–684). New York: Academic Press.
9) Alfonso, V. and R. Chumpy. (2007). Reactive oxygen spesies and superoxide
dismutases: Role in joint disease, Joint Bone Spine. Science Direct Journal,
74, 324–329.
10) Zelen, I., Mitrović, M., Jurišić-Škevin, A. and S. Arsenijević. (2010). Activity
of superoxide dismutase and catalase and content of malondialdehyde in
seminal plasma of infertile patients. Medicinski pregled, 63 (9-10), 624–629.
PURIFICATION OF BIOACTIVE PEPTIDE WITH
PROTEASES INHIBITORY ACTIVITIES FROM
STREPTOMYCES MISIONENSIS
Juwaini Mohd Yusoff, Khanom Simarani* and Zazali Alias
Institute of Biological Sciences, Faculty of Science, University of Malaya,
50603 Kuala Lumpur, Malaysia
Phone: +603 79675843

Abstract
Actinomycetes are the major source of the biologically active compounds. Extensive bioactive
compounds are yet to be screened from these known vast producers of antimicrobials for
future benefits. During the screening of over 400 soil actinomycete isolates for the production
of epsilon poly-L-lysine (ε-PL), a potentially bioactive protein was discovered. The molecular
weight of the protein was estimated as 6 kDa by using Sodium Dodecyl Sulfate Polyacrylamide
Gel Electrophoresis (SDS-PAGE). Cation Exchange Chromatography revealed that the protein
sample was positively charged and thus subjected to proteases activity. The putative protein
exhibited an enhanced activity towards the well-characterized serine proteases; trypsin and
α-chymotrypsin, while inhibiting the activity of α-amylase, suggesting an interesting finding
particularly in enzyme modulation aspect. Furthermore, it showed antagonistic activity against
some pathogens including Gram negative Xanthomonas campestris, Ralstonia and Erwinia, as well
as Gram positive B. cereus. Subsequently, the isolate was identified as Streptomyces misionensis
based on its 16S rRNA gene sequence.

Key words: Actinomycetes, Peptide, Serine Proteases

I. Introduction
Soil microbes represent an important source of biologically active compounds.
Most of the chemically diverse compounds with biological activities were
discovered from actinomycetes. Actinomycetes have provided nearly 80% of the
world’s antibiotics, prominently from the genera Streptomyces and Micromonospora,
and the search for new bioactive compounds continues until today [1].
Enzymes are biomacromolecules (usually proteins), which have important
roles in regulating most of the chemical reactions involved in various biological
processes of living organisms. Besides in vivo functions of enzymes, they are also
widely used in pharmaceutical, medical, food, environmental and industrial fields
as well as in life science studies. With those diverse commercial applications, the
regulation of enzyme activity and stability has always becomes the main issue that

* Corresponding author. Phone: +603 79675843. Email: hanom_ss@um.edu.my

37
38 Proceeding ASEAN COSAT 2014

attracts great attention. Many enzyme regulators have been discovered, ranging
from proteins, peptides and synthetic organic molecules, and the studies on that
aspect continue until now [2].

II. M aterials and Methods


A. Isolation, Screening and Identification of Bacterial Strain
Actinomycetes used in this study were isolated from various types of soil samples
including forest, agricultural, rhizospheric and polluted soils. The isolation proce-
dures were followed as previously described by Hirohara [3]. Purified colonies were
maintained and subcultured on ISP 2 medium prior to use. Actinomycete strains
selected by appearances on the agar plate were subjected to two-stage cultivation
as described by Hirohara [3] for the production of the bioactive metabolites. The
potential isolate was identified based on its 16S rRNA gene sequence.

B. Protein Separation and Concentration


The production broth of actinomycete cultures was filtered using membrane filter
to remove the cells and was subjected to acetone precipitation and centrifugation
using protein concentrator prior to SDS-PAGE, enzymatic assays and antibacterial
testing. The protein concentration was determined by method of Bradford [4]
using bovine serum albumin (BSA) as a standard.

C. Enzymatic Activity Assay


Serine proteases; trypsin and chymotrypsin activity was performed as described by
Jennings [5] using the substrates sodium-benzoyl-DL-arginine ρ-nitroanilide for
trypsin and, N-benzoyl-L-tyrosine ρ-nitroanilide for chymotrypsin. For α-amylase
inhibitory assay, the method was adapted as outlined by Feng [6].

D. Antimicrobial Activity
The antibacterial activity was determined using agar well diffusion method [1]. B.
cereus, X. campestris, Ralstonia and Erwinia strain obtained from UM Microbiology
Culture Collection were used as antimicrobial test strains.

III. R esults and Discussion


Various types of soil samples, including forest, garden, agricultural, rhizospheric
and polluted soils were collected from different places as to increase the chances
of getting different actinomycete strains.
Pure homogenous preparation of peptide using Tricine SDS-PAGE indicated a
single band of low molecular weight (MW) protein approximately 6 kDa (Figure
Innovation for Better ASEAN Community 39

1) after protein separation by acetone precipitation method. As the samples was


from crude part, that means they were of homogenous proteins, allowing for the
thick clearly visualized band on SDS-PAGE. This has become the interesting part
of the study; checking whether the protein possesses biological activities. Thus,
we attempted to measure the activity of the isolated peptide towards the selected
enzymes particularly serine proteases. Two well-characterized serine proteases;
trypsin and chymotrypsin which have important biomedical and industrial ap-
plications were opted for the next stage of the study along with α-amylase enzyme.
Trypsin and chymotrypsin activity assays revealed an interesting finding that
the locally isolated peptide has the capability to significantly enhance the activity
of both of the serine proteases (Table 1). To the best of our knowledge, the
improvement of the proteases activity was rarely documented, in fact this is the
first report that described the ability of the potential peptide from an actinomycete
which could enhance the activity of proteases. Similarly, the study done by Jin
[2] reported the specific enhancement in substrate-dependent trypsin digestion
of phosphoproteins by PEGlated GO nanosheets.
Other than having an effect on the protease activities, the isolated peptide
was shown to exhibit antibacterial property against several selected bacteria when
qualitatively determined by the well diffusion method (Table 2). A clear zone of
inhibition was observed towards some Gram negative plant pathogens including
X. campestris, Ralstonia and Erwinia as well as Gram positive B. cereus. To correlate
this data with the obtained MW of the locally isolated protein, we believed that
it might be the antimicrobial peptide (AMP) as its MW falls within the range
for the AMP. Subsequently, the potential isolate was identified as Streptomyces
misionesis by 16S rRNA gene sequencing.
For our best knowledge, this is the first study that reported the isolation of
AMP with biological activities from Streptomyces misionesis, proposing that this
research could be a basis for constructing new natural antimicrobial compounds
with higher specific activity and broader range of action to serve as a solution
for the never-ending antimicrobial resistance issues as well as to reduce the side
effect problems of using synthetic antibiotics.
40 Proceeding ASEAN COSAT 2014

Figure 1. SDS-PAGE of pure homogenous


preparation of peptide. Lane 1 shows the
benchmark standard marker (Invitrogen).
Lane 2 shows the presence of single band
protein with MW estimated around 6 kDa.
Gel was stained with colloidal Coomassie blue.

Table 1. Enzymatic activity assays


Assay % Activity Activity
α-amylase 78.87 Inhibited
Trypsin 67.18 Enhanced
Chymotrypsin 329.32 Enhanced

Table 2. Antibacterial property of


peptide III. Conclusion
Pathogen Inhibition A locally isolated actinomycete strain,
B. cereus positive identified as Streptomyces misionensis, was
X.campestris positive found to produce a peptide with a molecular
Ralstonia positive weight of 6 kDa which is within a range
Erwinia positive for MW of AMP. The peptide was capable
of enhancing the activity of serine protease
enzymes; trypsin and chymotrypsin, while
inhibiting the activity of α-amylase and exhibited antagonistic action against X.
campestris, Ralstonia, Erwinia and B. cereus. Despite the preliminary data obtained,
this research serves as a fundamental for future works on elucidating enzymes
regulation and stability. Further research on proteases activity using selection of
more substrates can be done to study the effects of substrates on the activity in
details. Besides that, enzyme kinetics of the bioassays should be performed to
particularly elucidate the mode of action as well as the concentration at which
the peptide activity is optimum. To understand the chemical structure of the
newly-isolated peptide, it is best to obtain the amino acid sequence of it.
Innovation for Better ASEAN Community 41

IV. Acknowledgement
Authors would like to thank University of Malaya for providing the following
research funds: PG057-2013A, MOHE FP020-2013A, and RG048-11BIO.

V. R eferences
1) Pandey, A., Ali, I., Butola, K. S., Chatterji, T. and V. Singh. (2011). Isolation
and characterization of actinomycetes from soil and evaluation of antibacterial
activities of actinomycetes against pathogen. International Journal of Applied
Biology and Pharmaceutical Technology, 2 (4), 384–391.
2) Jin, L., Yang, K., Zhang, S., Tao, H., Lee, S.-T., Liu, Z. and R. Peng. (2012).
Functionalized Graphene Oxide in Enzyme Engineering: A Selective Modula-
tor for Enzyme Activity And Thermostability. ACS Nano, 6, 4864–4875.
3) Hihohara, H., Takehara, M., Saimura, M., Masayuki, A. and M. Miyamoto.
(2006). Biosynthesis of poly (ε-L-lysine)s in two newly isolated strains of
Streptomyces sp. Applied Microbiology and Biotechnology, 73, 321–331.
4) Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of
microgram quantities of protein utilizing the principle of protein dye binding.
Analytical Biochemistry, 72, 248–254.
5) Jennings, C., West, J., Walne, C., Cralk, D. and M. Anderson. (2001).
Biosynthesis and insecticidal properties of plant cyclotides: The cyclic knotted
from Oldenlandia affinis. PNAS, 98, 10614–10619.
6) Feng, G. H., Chen, M. S., Kramer, K. J. and G. R. Reeck. (1991).
Reversed-Phase High Performance Liquid Chromatographic Separation
Wheat Proteinaceous Inhibitors of Insect and Mammalian α-amylase. Cereal
Chemistry, 68, 95–99.
7) Young, G. O. (1964). Synthetic structure. In J. Peters (Ed.), Plastics, 2nd ed.,
vol. 3 (pp. 15–64). New York: McGraw-Hill.
ISOLATION, IDENTIFICATION AND SCREENING OF
LOCALLY ISOLATED Xanthomonas sp.
Nur Izlin Shafinaz Bokhari, Khanom Simarani** and Mohamad Suffian
Mohamad Annuar
Institute of Biological Sciences, University of Malaya
50603, Kuala Lumpur, Malaysia
Phone: +603 79674371

Abstract
Xanthan gum is a water soluble, complex exopolysaccharide which have great commercial values
produced industrially by pytopathogen called Xanthomonas sp. Commercially, because of its
rheological properties, it is widely used as a thickener, viscosifier and stabilizer, as well as emulsifier
in both food and non-food industries including dairy products, bakeries product, beverages,
cosmetics, textiles, paper miling and pharmaceutical product. Samples were isolated from diseased
vegetables, and were subjected to set of simple phenotypic. The strains were further identified
by BIOLOG. All positive isolates were compared by fermention in shake flask, under controlled
conditions, and the production of xanthan gum were compared to Xanthomonas campestris pv.
Campestris strain from ATCC culture collection (ATCC33913). 411 bacteria were isolated, and
55 isolates were presumptively identified as Xanthomonas spp. based on the biochemical test.
Thirteen of the presumptive isolates were identified as Xanthomonas campestris pv. Campestris,
Xanthomonas campestris pv. Raphani, Xanthomonas campestris pv. Begonia A, Rhizobium vitis,
Acinetobacter johnsonii, and Microbacterium maritypicum using BIOLOG. Strain C206 has
productivity (0.1074) compare to others as well as control strain, ATCC.
Key words: Xanthan gum, Xanthomonas campestris, Optimization

I. INTRODUCTION
Xanthan gum is a complex extracellular polysaccharide which have great
commercial significance [6] secreted by Xanthomonas campestris [1] during its
normal life cycle. These plant pathogens caused black rot in family members of
the Brassicaceae (Cruciferae) which include cabbage, broccoli and cauliflower.
The subsequent infection of these bacteria will cause defoliation, resulting in
loss of weight and deterioration of its quality [5]. This disease can be accounted
as the major disease constraint in the tomato production all over the world and
in the cabbages plantation in Africa [5]. X. campestris pv. Campestris is the main
producer of xanthan gum, where it thrives well in warm and humid climates [9].

* Corresponding aAuthor. PhoneTel: : +603 79675843. Email: hanom_ss@um.edu.my

43
44 Proceeding ASEAN COSAT 2014

The growth of Xanthomonas sp. and the production of the xanthan gum are
influenced by various types of factor that need to be controlled during the process
of xanthan gum synthesis. The factors include the types of bioreactor, medium
composition, type of strain used and also other factors, such as temperature,
pH and dissolved oxygenconcentration [4,8,3]. The synthesis of xanthan gum
involved an aerobic, submerged fermentation process where the bacteria that have
been purified will be cultured in a well-areated medium containing inexpensive
substrates and nutrient including glucose, nitrogen and other trace elements.
Glucose and sucrose are most frequently used as the carbon sources [8]. Other
types of substrates also might be used such as sucrose, barley, hydrolysed rice,
sugar cane molasses and coconut juice [3].
Due to its superior properties, xanthan gum is applied in different area of
industries. In pharmaceutical, cosmetic, paper, paint textile and oil industries,
xanthan gum is mainly used as gelling and suspending agent, as flocculants or
used for viscosity control [1]. For example, the application of xanthan gum in
toothpaste allows easy extrusion from the toothpaste tube. In food industry, it
was widely used as a thickening and suspending agent in dairy product, bakery
products, beverages and also in pet foods. On the other hand, xanthan gum
functions as a stabilizer and thickener in salad dressings [1], and gelling and
emulsifying agent in food industries besides being used as an inhibitor of ice
crystal formation control [1]. In this study, xanthan gum production, recovery
and properties will be studied by a locally isolated culture of Xanthomonas, which
are isolated from cabbages and lettuces.

II. MATERIAL AND METHODS


A. Bacterial isolation and identification
Samples of diseased vegetables that show a typical spot symptom of Xanthomonas
infection was randomly collected. Bacterial isolation was done based on proce-
dures described by [5]. The presumptive colonies were identified according to
morphological, physiological and biochemical and BIOLOG.

B. Screening among selected strains


Ten isolated strains that were identified as Xanthomonas campestris were screened.
Yield and viscosity of the polymer synthesized in conventional medium were
analysed.

C. Cell production
Xanthan production was done in a batch fermentation method, in the orbital
shaker as have been describe by [12]. In this study, Yeast Malt Media (YM) was
Innovation for Better ASEAN Community 45

used as the inoculums medium that consist of (g/l); glucose (10), malt extract
(3), yeast extract (3) and peptone (5).

D. Xanthan production
The batch fermentations were carried out in a 250 mL conical flask with working
volume of 100 ml containing 10% of inoculums concentration [5]. A complex
medium containing (g/l); glucose (30), yeast extract (3), K2HPO4 (2) and MgSO4.
H2O (0.1) was used as the fermentation medium. Samples were harvested after
72 hours and centrifuged at 13,000 rpm for 30 min to separate the cell mass and
xanthan gum.

E. Viscosity
The viscosity of 3% aqueous solution of the polymer synthesis by the ten strains
were compared with viscosity of xanthan gum synthesized by Xanthomonas
campestris pv. Campestris strain from ATCC culture collection (ATCC33913).

F. Analytical methods
The cell biomass was determined using dry cell weight method. Xanthan gum
was precipitated by adding cell-free supernatant with three times volumes of
acetone.

III. RESULTS AND DISCUSSIONS


A. Isolation and Identification
411 bacteria were isolated from both sampling site. 51 isolates were presumptively
identified as Xanthomonas sp. based on the biochemical test. Table 1 shows the
biochemical characteristic of Xanthomonas sp. Twelve presumptive isolates were
further identified using BIOLOG microbial identification system. Results of
the BIOLOG are as in table 2. According to literature [7], Xanthomonas have
smooth, yellow and mucoid colony. All of twelve isolates have these appearances.

Table 1. Biochemical characteristic of Xanthomonas sp.


Characteristic of Xanthomonas sp.
Gram staining Gram negative
3% KOH Positive
Oxidase Negative
Catalase Positive
46 Proceeding ASEAN COSAT 2014

Table 2. Results of BIOLOG after 24 hours of incubation


Isolates Identification
C85 Acenetobacter johnsonii
C195 No ID
C205 No ID (xanthomonas campestris pv campestris)
C206 No ID (xanthomonas campestris pv campestris)
C253 Xanthomonas campestris pv raphani
C254 Xanthomonas campestris
C256 No ID ( Stenotrophomonas maltophilia)
C272 Xanthomonas campestris pv raphani
C279 xanthomonas campestris pv campestris
C295 xanthomonas campestris pv campestris
C298 xanthomonas campestris pv campestris
C316 Microbacterium maritypicum

B. Screening among selected strains


Ten isolates that were identified as Xanthomonas campestris were further screened by
evaluating the viscosity of exopolymer produced and its productivity. The analysis
shows that xanthan production was influenced by the strains. The productivity
(g h-1) varies from 0.0747 to 0.1074. Figure 1 shows the productivity among
isolates. Strain C206 has the highest productivity (0.1074) compare to others as
well as control strain, ATCC. Xanthan gum synthesized by strain C194 presented
the highest viscosity. Figure 2 shows the comparison of viscosity of polymer
produced among all isolates. Results of viscosity show that the viscosity produced
was dependent on the strains.

Figure 1. Productivity by different strains of newly


isolated X. campestris.
Innovation for Better ASEAN Community 47

Figure 2. Viscosity of polymers synthesized by different strains of newly


isolated X. campestris.

IV. CONCLUSION
The production of xanthan gum and viscosity of the polymers was influenced by
strains and culture conditions. From the comparative studies, it was concluded
that isolates C279 has the highest productivity, but the yield obtain by all strains
was not appropriate for industrial production which are 10 to 20gl-1 according
to literature. The culture condition should be optimized and revalued in attempt
to increase the production.

V. Acknowledgement
The author would like to thank University of Malaya for the sponsorship and
research grant (RG048-11B10) and Institute of Research Management and
Consultancy, University of Malaya for providing Postgraduate Research Grant
(PG057-2013A) and also Ministry of High Education (MOHE) for providing
a research grant (FP020-2013A).

VI. REFERENCES
1) Becker, A., Katzen, F., Puhler, A. and L. Ielpi. (1998). Xanthan gum
biosynthesis and application: a biochemical/genetic perspective. Applied
Microbiology and Biotechnology, 50, 145–152.
2) Sharma, B. R., Naresh, L. and N. Dhuldhoya. (2006). Xanthan gum- A Boon
to Food Industry. Food Promotion Cronicle, 5, 27–30.
48 Proceeding ASEAN COSAT 2014

3) Faria, S., Viera, P., and M. M. Resende. (2010). Application of a model using
the phenomenological approach for detection of growth and xanthan gum
production with sugar cane broth in a batch culture. LWT-Food Science and
Technology, 43, 498–506.
4) Hsu, C. H. and Y. Lo. (2003). Characterization of xanthan gum biosynthesis
in a centrifugal, paxked-bed reactor using metabolic flux analysis. Process
Biochemistry, 38, 1617–1625.
5) Shenge, K. C., Mabagala, R. B. and C. N. Mortensen. (2007). Identification
and characterization of strains of Xanthomonas campestris pv Vesicatoria from
Tanzania by Biolog system and sensitivity to antibiotics. African Journal of
Biotechnology, 6, 15–22.
6) Ochoa, F. G., Santos, V. and Alcon. (1995). Xanthan gum production: an
unstructured kinetic model. Enzyme and Microbial Technology, 17, 206–217.
7) Ochoa, F. G., Santos, V., Casas, J. and E. Gomez. (2000). Xanthan gum:
production, recovery and properties. Biotechnology Advance, 18, 549–579.
8) Rosalam. S., Krishnaiah, D. and A. Bono. (2008). Cell free xanthan gum
using continous recycled packed fibrous-bed bioreactor-membrane. Malaysian
Journal of Microbiology, 4, 1–5.
9) Soudi, M., Alimadadi, N. and P. Ghadam. (2011). Minimal phenotypic test
for simple differentiation of Xanthomonas campestris from other yellow-
pigmented bacteria isolated from soil. Iranian Journal of Microbiology, 84-91.
10) Psomas, S. K. and Liakopoulou-Kyriakides. (2007). Optimization study of
xanthan gum production using response surface methodology. Biochemical
Engineering Journal, 35, 273–280.
11) Massomo, S. M. S., Nielsen, H., Mabagala, R. B., Mansfeld-Giese, K., Hock-
enhull, J. and C. N. Mortensen. (2003). Identification and characterization of
Xanthomonas campestris pv campestris strains from Tanzania by pathogenicity
test, Biolog, rep-PCR and fatty acid methyl ester analysis. European Journal
of Plant Pathology, 109, 775–789.
12) Salah, R. B., Chaari, K., Besbes, S., Ktari, N., Blecker, C., Deroanne, C. and
A. Hammadi. (2010). optimisation of xanthan gum production by palm date
juice by-products using response surface methodology(RSM). Food Chemistry,
121, 627–633.
ULTRAVIOLET IRRADIATION EFFECT OF
Penicillium chrysogenum ON PENICILLIN
PRODUCTION
Dudi Hardianto*, Uli Julia, Eka Siska, Diana Dewi, Suyanto, Erwahyuni E.
Prabandari, Lira Windriawati and Danang Waluyo
Biotech Center, Agency for the Assessment and Application of Technology, Puspiptek Area
Serpong 15314, Indonesia

Abstract
Penicillin is the oldest β-Lactam antibiotic that is produced by the filamentous fungus Penicillium
chrysogenum. Since the discovery of penicillin by Fleming, much effort has been invested to
improve productivity of Penicillium chrysogenum. Strain improvement to increase the penicillin
production can use random mutation with physical and chemical mutagents. In this research,
UV irradiation was used to obtain Penicillium chrysogenum mutant. Penicillin production was
determined by HPLC, and productivity of Penicillium chrysogenum mutants were compared
to wild type. The penicillin yield of mutants are varied, and mutant M12 produced 1.23 fold
compared to wild type.
Key words: Penicillin, Penicillium chrysogenum, Ultraviolet, Mutation

I. Introduction
Penicillin is the first antibiotic discovered and used for treatment of bacterial
infections. The structure of β-lactam penicillin consists of a bicyclic nucleus
formed by a β-lactam ring and a thiazolidine ring containing a sulfur atom
and an acyl side chain bound to the amino group present at C-6 [1]. Although
some bacteria are resistant to penicillin, this antibiotic is still widely used today.
Penicillin is used in treatment, many gram-positive bacterial infections, such as
Staphylococcus pyogenes (strep throat) and Streptococcus pneumoniae (respiratory
tract infection, otitis media) [2,3]. Bacterial cell wall synthesis is inhibited by
penicillin. Penicillin binds the enzyme transpeptidase that links the peptidoglycan
molecules in bacterial cell wall.
Penicillin is industrially produced by the filamentous fungus Penicillium
chrysogenum and commercial production of penicillin began in 1941 [4]. The
production of penicillin by the submerged fed-batch fermentation in stainless
tank reactors of 30,000–100,000 galon capacity is important for several decades

* Corresponding author. Email: dudihd@gmail.com

49
50 Proceeding ASEAN COSAT 2014

[5] and optimization of penicillin production is very important for the penicillin
companies [6]. Since the discovery of the production of antibiotics by Fleming
in 1929, much effort has been invested in the selection and synthesis of strains
with improved productivity [7]. Classical strain improvement with random
mutation and screening has been used to obtain overproducing strains. The UV
irradiation is one of the techniques for strain improvement and its technique is
very effective for mutation because the bases DNA absorb the UV irradiation.
The effect of the absorbed UV irradiation is the formation of thymine dimers and
cross links in the same strand [8]. Mutants are unstable, and mutagenic events
are random and do not necessarily affect only the genes involved in antibiotic
synthesis. Re-isolation of mutants, since prolonged storage of high producing
strains is needed to know stability of mutants.
The increase in penicillin fermentation productivity and high recovery yield
(>90%) has led to significant cost reduction, despite increasing labor, energy and
raw materials costs. In 1953, the bulk cost for penicillin production was ~$300/
kg. In 1980, the bulk price for penicillin was ~$35/kg. In the late 1990s, bulk
penicillin cost ranged from $10 to 20/kg and bulk marketed costs for 6-APA
have been estimated to range from $35 to 40/kg [5].
The aim of this research was obtaining Penicillium chrysogenum mutant that
produces higher amount of penicillin than the wild type.

II. METHOD/MATERIAL
Microorganism. Strain of Penicillium chrysogenum in this study was obtained from
Biotech Center Culture Collection, Agency for the Assessment and Application
of Technology.
Mutation by UV Irradiation. Czapek Dox Agar was used to select Penicil-
lium chrysogenum mutants. A conidial from 10 days old culture of Penicillium
chrysogenum was suspended in distilled water and adjusted to about 103 conidia/
ml. Conidial suspension was irradiated by short wave UV irradiation of 254 nm
and the time of irradiation varied from 5 to 30 minutes with 5 minutes interval.
50 µl conidial suspension of the growth of Penicillium chrysogenum mutants were
inoculated, spreaded into Czapek Dox Agar plates, and the inoculated plates were
incubated for 10 days at 28oC.
Isolation and Selection Mutants. After 10 days, mutant colonies were separated
by inoculation each mutant colony on Czapek Dox Agar plates. The inoculated
plates were incubated for 10 days at 28oC.
Production of Penicillin. One of the mutant colony was inoculated in seed
medium and incubated in shaker for 36 hours at 28oC. 10% Suspension inoculum
Innovation for Better ASEAN Community 51

was inoculated in fermentation medium and incubated in shaker for 10 days at


28 oC.
Penicillin Analysis in Broth of Fermentation. Broth of fermentation was
centrifuged at 15,000 g, and supernatant was filtered through 0.45 µm filter.
10 µl sample was injected to HPLC mechine from Waters with C18 column
(Symmetry), mobile phase: 5 mM KH2PO4 and 6 mM H3PO4: Acetonitrile (60
: 40), flow rate 1.0 mL/min, and ultraviolet detector λmax: 210 nm.

III. result and discussion


P. chrysogenum wild type was irradiated by UV from 5–30 minutes. In this
research, effect of UV irradiation on P. chrysogenum was evaluated by survival
curve (Figure 1). The increase in time of UV exposure on P. chrysogenum caused
a gradual decrease of survivals. DNA bases absorb UV light induce the formation
covalent crosslinking of thymine in the same strand DNA or thymine dimers.
The thymine dimers do not have base pairing and when the DNA replicate, the
wrong base may be inserted [9,10]. The lethal of P. chrysogenum may be caused by
disruption of replication, alteration of gene that inhibit vegetation, or alteration
of the biochemical structure of the molecules that are essential to P. chrysogenum’s
survival (membrane or enzymes to metabolism).

Figure 1. Effect of UV exposure time on P. chrysogenum


52 Proceeding ASEAN COSAT 2014

The survivals of P. chrysogenum mutants were selected and tested for penicil-
lin production. Penicillin yield of mutants varied from 1.635 to 4.594 ppm.
Mutant M12 produced 1.23 fold (4.594 ppm) compared to wild type (3.305
ppm) (Table 1). The hyperproduction of mutant may cause mutation of one
or more biosynthesis of penicillin gene or the disrupsion of the lys2 gene (gene
for lysine biosynthesis). The mutation of biosynthesis of penicillin gene may
increase enzymes activity for penicillin production or disruption of the lys2 gene
increase aminoadipic acid (precursor for penicillin biosynthesis). In Penicillium
chrysogenum, the biosynthesis of penicillin and lysine have several steps in

Figure 2. HPLC chromatogram of lovastatin standard

Figure 3. HPLC chromatogram of lovastatin from fermentation broth of P. chrysogenum wild type
Innovation for Better ASEAN Community 53

Figure 4. HPLC chromatogram of lovastatin from fermentation broth of P. chrysogenum mutant

Table 1. Penicillin production of mutants


Penicilium chrysogenum Concentration of Penicillin (ppm)
Wild type 3711
M1 2534
M2 3193
M3 2674
M4 2068
M5 2482
M6 2638
M7 3193
M8 2859
M9 2650
M10 1635
M11 2087
M12 4594
M13 3067
M14 2548
M15 4404
M16 4216
M17 3663
M18 4138
M19 4272
54 Proceeding ASEAN COSAT 2014

Figure 5. Biosynthesis pathway of penicillin and lysine in P. chrysogenum (Casqueiro


et al., 1999)

common (Figure 5). It should be possible to increase penicillin production by


the disrupsion of lys2 gene [11]. The poor result of penicillin of mutants may be
caused by mutation in penicillin biosynthesis genes. The mutation of biosynthesis
of penicillin gene may cause a decrease enzymes activity for penicillin production.

IV. conclusion
The penicillin yield of mutants varied from 1.635 to 4.594 ppm and mutant
M12 from 30 minutes-irradiated produced 1.23 fold compared to wild type.
Innovation for Better ASEAN Community 55

V. references
1) Liras, P. and J. F. Martin. (2006). Gene cluster for β-lactam antibiotics and
control of their expression: Why have clusters evolved, and from where did
they originate? International Microbiology, 9, 9–19.
2) Christensen, B., Thykaer, J. and J. Nielsen. (2000). Metabolic characteriza-
tion of high- and low-yielding strains of Penicillium chrysogenum. Applied
Microbiology and Biotechnology, 54 (2), 212–217.
3) Rayamajhi, N., Cha, S. B. and H. S. Yoo. (2010). Antibiotics resistances:
Past, present and future. Journal of Biomedical Research, 11 (2), 65–80.
4) Newbert, R. W., Barton, B., Greaves, P., Harper, J. and G. Turner. (1997).
Analysis of a commercially improved Penicillium chrysogenum strain series:
Involvement of recombinogenic regions in amplification and deletion of the
penicillin biosynthesis gene cluster. Journal of Industrial Microbiology and
Biotechnology, 19, 18–27.
5) Elander, R. P. (2003). Industrial production of β-lactam antibiotics. Applied
Microbiology and Biotechnology, 61, 385–392.
6) Thykaer, J. and J. Nielsen. (2003). Metabolic engineering of β-lactam
production. Metabolic Engineering, 5 (1), 56–69.
7) Harris, D. M., van der Krogt, Z. A., Klaassen, P., Raamsdonk, L. M., Hage,
S., van den Berg, M. A., Bovenberg, R. A. L., Pronk, J. T. and J. Daran.
(2009). Exploring and dissecting genome-wide gene expression responses of
Penicillium chrysogenum to phenylacetic acid consumption and penicillinG
production. BMC Genomics, 10, 75.
8) Parekh, S., Vinci, V. A. and R. J. Strobel. (2000). Improvement of microbial
strain and fermentation processes. Applied Microbiology and Biotechnology,
54, 287–301.
9) Irum, W. and T. Anjum. (2012). Production enhancement of cyclosporin ‘A’
by Aspergillus terreus through mutation. African Journal of Biotechnology, 11
(7), 1736–1743.
10) Ravalat, J., Douki, T. and J. Cadet. (2001). Direct and indirect effect of
UV radiation on DNA and its components. Journal of Photochemistry and
Photobiology B: Biology, 63, 88–102.
11) Casqueiro, J., Guteirrez, S., Banuelos, O., Hijarrubia, M. J. and J. F. Martin.
(1999). Gene targeting in Penicillium chrysogenum: disruption of the lys2 gene
leads to penicillin overproduction. Journal of Bacteriology, 181 (4), 1181–1188.
Isolation and Cloning of Partial HER-2 Gene
from Indonesian Breast Cancer Patients for
DNA Vaccine Development
Desriani* and Lita Triratna
Research Center for Biotechnology-Indonesian Institute of Sciences
Jl. Raya Bogor KM 46, Cibinong Bogor, Indonesia

Abstract
Over-expression of proto-oncogene HER-2 occurs in 20–30% of the total breast cancer cases
associated with increased metastatic potential and poor prognosis. It consists of a 620 aa
extracellular domain, followed by a 23 aa trans-membrane domain and a 490 aa intracellular
domain with tyrosine kinase activity. Treatment using the monoclonal antibody, trastuzumab, was
an expensive treatment with an incidence rate of resistance in single use reaches a high percentage of
66-88%. Until now, there are continuous efforts for materials or methods improvement for HER-
2 breast cancer treatment. Vaccination is an attractive alternative approach to provide protective
immunity. Several reports show that a DNA vaccine encoding full-length or truncated HER-2
was immunogenic since it may generate protective immunity. Furthermore, HER-2 engineered
epitope can also be used as a vaccine active agent. Here in this study, we were successful in isolating
and cloning partial of HER-2 gene which include extracellular and trans-membrane domain with
total size of 2.127 base pairs (bp) into pGEMT-easy. This gene material will allow us to develop
new vaccine candidate in the future works.
Key words: HER-2, Proto-oncogene, DNA vaccine, Immunogenic, Vaccination

I. Introduction
Breast cancer cases in the world tend to increase. In 1975, there were about
500,000 cases. After 30 years, nearly 1.4 million cases of breast cancer were
diagnosed across the world in 2008. In 2012, nearly 1.7 million new cases were
diagnosed (second most common cancer overall). This represents about 11%
of all new cancer cases and 23% of all female cancers. It is predicted that the
number of cases will rise to 2.1 million by 2030 (World Cancer Research Fund
International, 2013).
There are 3 types of breast cancer: ER/PR positive breast cancer, ER/PR nega-
tive breast cancer, and HER-2/neu positive breast cancer, with the percentage of
65–75%, 5–10%, and 20-25%, respectively. Although HER-2/neu breast cancer
is only one quarter of all breast cancer cases in the world, this breast cancer tends
to be much more aggressive and fast-growing.

* Corresponding author. Phone: +62-21-8754587. Email: gerodes@yahoo.com

57
58 Proceeding ASEAN COSAT 2014

HER-2/neu, also called HER-2 (human epidermal growth factor receptor 2), is
ERBB2 gene. It is one such gene that can play a role in the development of breast
cancer. The HER-2 gene makes HER-2 proteins. HER-2 proteins are receptors on
breast cells. Normally, HER-2 receptors help control how a healthy breast cells
grows, divides or do proliferation, and repairs itself. But in about 25% of breast
cancers, the HER-2 gene doesn’t work correctly and makes too many copies of
itself (known as HER-2 gene amplification). All these extra HER-2 genes tell
breast cells to make too many HER-2 receptors (HER-2 protein overexpression).
This makes breast cells grow and divide in an uncontrolled way (Clifford and
Hudis, 2007).
The drug trastuzumab is a humanized monoclonal antibody targeted
against the extracellular portion of HER-2. Genentech Company (South San
Francisco, CA) has been developing and producing trastuzumab with the trade
name Herceptin. Since released in 1998, trastuzumab has become an important
treatment for patients with invasive breast cancer (Ross et al., 2004). This is the
first HER-2 targeted agent to be approved by the united States Food and Drug
Administration (FDA) for the treatment of both early stage and metastatic
HER-2 overexpressing. Although HER-2 targeted therapies have had a significant
impact on patient outcomes, resistance to these agents reaches a high percentage
of 66–88% (Pohlman et al., 2009). Furthermore, trastuzumab is also associated
with an increased risk of cardiac dysfunction (Seidman et al., 2002).
Vaccination is an attractive alternative approach to provide protective
immunity against tumor that overexpress HER-2 oncogen product (Hurvitz
et al., 2013). Several reports show that a DNA vaccine encoding full-length or
truncated HER-2 was immunogenic since it may be effective in inducing protective
antitumor immunity (Rovero et al., 2000; Jasnska et al., 2003; Jacob et al., 2006;
Dimitriadis et al., 2009). Chen et al. (1998) reported that co-injection of DNA
vaccine encoding full length or truncated HER-2/neu co-injection together with
a plasmid vector that encoded IL-2 could enhance the ability of HER-2 DNA
vaccine to induce protective antitumor immunity. Norell et al. (2010) reported
that HER-2-pDNA vaccination in conjunction with GM-CSF and IL-2 was well
tolerated and can induce long lasting cellular and humoral immune responses
against HER-2 patients. HER-2 epitope, 597-626 sequence is reported as a
potential vaccine candidate to reduce tumor (Garrett et al., 2006). Until now,
the most effective vaccine has not been found, although there are many studies
to developed DNA Vaccine HER-2.
In this study, we were successful in isolating and cloning partial of HER-2
gene which includes extracellular domain (ECD) and trans-membrane domain
with total size of 2.127 base pairs (bp) into pGEMT-easy. This gene material will
allow us to develop new vaccine candidate in the future works.
Innovation for Better ASEAN Community 59

II. Method/M aterial


2.1. Tissue Collection
Breast cancer tissues from patients in Muhammad Djamil Padang Hospital and
Dharmais Hospital Jakarta were collected and saved at -80°C.

2.2. Total RNA Isolation


The total RNA from breast cancer tissue was isolated by using Purelink RNA
mini kit (Invitrogen/Ambion).The total RNA was confirmed to 1% agarose
electrophoresis with 1X TBE (Tris Boris EDTA) Buffer. Then, these total RNA
can be stored at -80°C for more than a month.

2.3. One Step PCR (Polymerase Chain Reaction)


The pure total RNA was used as a template to synthesis cDNA and to amplify
ECD and trans-membrane domain by using Superscript III One-Step RT-PCR
system with platinum Taq High Fidelity; two specific primers (forward and reverse),
dNTPs, and ddH2O. All these reagent would be transfered into PCR tube (200
microLiter). The PCR product confirmed in 1% Agarose, electrophoresis with
1X TBE buffer.

2.4. Sequencing
PCR product was sequenced with ABI machine and then identified with Basic
Local Alignment Search tool (BLAST) software.

2.5. Cloning
The pure DNA was inserted into the plasmid cloning, pGEM-Teasy, by using
ligase. They were transformed into Escherichia coli DH5alpha by heat shock
technique. This E.coli recombinant was incubated in shaker incubator at 150 rpm,
for 16 hours or overnight, at 37°C. PCR colony and second by using restricted
plasmid isolation were used in order to confirm the success of cloning.

III. R esult and Discussion


3.1 Tissue collection
The research was conducted using ethical clearance issued by Ministry of Health
Efforts, Dharmais Cancer Hospital National Cancer Center. We have managed
to collect 110 breast cancer tissues. The fresh tissue samples were stored at
-80°C or in liquid nitrogen. Breast cancer tissue identified as HER-2 + through
immunohistochemistry screening then was used as a RNA isolation source.
60 Proceeding ASEAN COSAT 2014

3.2. Total RNA Isolation


Total RNA from breast cancer tissue have been successfully isolated (picture 1).
Figure 1 shows that there are 28S RNA and 18S RNA. The size were 2.8 kilobase
pairs (kb) and 1.8 kb, respectively. 1 kb DNA ladder was used as the marker (left
side).

3.3. One Step PCR


The extracellular domain (ECD) and trans-membrane domain from HER-2 gene
was successfully isolated with total size of 2,127 base pairs (bp) (Figure 2).

3.4 Cloning
Cloning of the gene encoding the extracellular domain (ECD) and trans-
membrane domain was successful. The size of pGEM-Teasy, as a plasmid or vector
cloning, is 3.015 bp, while the size of the gene is 2,127 base pairs (bp). In total,
the size of plasmid and insert gene is 5.477 bp (Figure 3).

Figure 1. Total RNA Isolation


Result.

(a) (b) (c)


Figure 2. One step PCR Result. (a) and (b) are the unpurified results; (c) is the
purified result.
Innovation for Better ASEAN Community 61

PCR colonies and enzyme restriction site analysis of targeted plasmid isolation
were done to confirm the cloning results (Figure 4). DNA sequencing methods
was also done in order to confirm DNA bases constituents (data was not shown).
Many approaches for the genetic immunization of tumor have been investi-
gated. Injection of tumor with DNA plasmid designed to promote an immune
response to the tumor resulting the minimal efficiency (only affecting the injected
tumor) and minimal systemic immunologic impact. The most potential part of
DNA, as the candidate of DNA vaccine for immunotheraphy agent, need to be
examined more. (Kirkwood et al., 2012). Not only DNA vaccine, DNA delivery
system and ageing could also affect the effectiveness of immune response (Smorlesi
et al., 2006; Provincialli et al., 2003).
Here in this study, we were succeeded in the isolating and cloning partial of
HER-2 gene which include extracellular and trans-membrane domain with the
total size of 2,127 base pairs (bp) into pGEMT-easy. This gene material will allow
us to develop new vaccine candidate in the future works.

IV. Conclusion
The gene encoding extracellular membrane and trans-membrane HER-2 protein
has been successfully isolated and cloned into plasmid cloning, pGEM-Teasy,
and transformed into E.coli DH5alpha. These genetic materials can be stored for
long term and potentially used in the development of DNA vaccine for HER-2
positive breast cancer.

Figure 3. pGEM-Teasy and insert gene encoding ECD and trans-


membrane domain.
62 Proceeding ASEAN COSAT 2014

Figure 4. (A) PCR colonies results. (B)


Plasmid isolation results.

V. Acknowledgments
Thank you to the competitive program from Indonesian Institute of Science
(LIPI) as the funder. We would like to thank Dharmais Hospital in Jakarta and
M. Djamil Hospital in Padang, for their cooperation in this research in providing
the breast cancer tissue. To Neneng Hasanah, Rivai, M. Ali Warisman, and Wiwit
Amrinola, thank you very much for making this research possible.

VI. R efference
1) Chen, Y. et al. (1998). DNA vaccines encoding full-length or truncated neu
induce protective immunity against neu-expressing mammary tumors. Cancer
Research, 58, 1965-1971.
2) Clifford, A. and M. D. Hudis. (2007). Trastuzumab-mechanism of action
and use in clinical practice. New England Journal of Medicine, 357, 39-51.
3) Dimitriadis, A. et al. (2009). The mannosylated extracellular domain of Her-2/
neu produced in P. pastoris induces protective antitumor immunity. BMC
Cancer, 1-9.
4) Hurvitz, S. A. et al. (2013). Current approaches and future directions in the
treatment of HER2 positive breast cancer. Cancer Treatments Review, 39,
219-229.
5) Garret, J. T. et al. (2010). Novel engineered trastuzumab conformational
epitopes demonstrate in vitro and in vivo antitumor properties against HER-2/
neu. The Journal of Immunology, 8, 1720-1730.
Innovation for Better ASEAN Community 63

6) Jacob, J. et al. (2006). Activity of DNA vaccines encoding self or heteroulogous


Her-2/neu in Her-2 or neu transgenic mice. Cellular Immunology, 240,
96-106.
7) Jasnska, J. et al. (2003). Inhibition of tumor cell growth by antibodies induced
after vaccination with peptides derived from the extracellular domain of Her-2/
neu. International Journal of Cancer, 107, 976-983.
8) Kirkwood, J. M. et al. (2012). Immunotherapy of cancer in 2012. CA: A
Cancer Journal for Clinicians, 62, 309-336.
9) Norell, H. et al. (2010). Vaccination with a plasmid DNA encoding HER-2/
neu together with low doses of GM-CSF and IL-2 in patients with metastatic
breast carcinoma: A pilot clinical trial. Journal of Translational Medicine, 8,
53-64.
10) Pohlman, P. R., Mayer, I. A. and R Mernaugh. (2009). Resistance to
trastuzumab in breast cancer. Clinical Cancer Research, 24, 7479-7491.
11) Provincialli, M. et al. (2003). Low effectiveness of DNA vaccination against
HER-2/neu in ageing. Vaccine, 21, 843-848.
12) Ross, J. S., Fletcher, J. A., Bloom, K. J., Linette, G. P., Stec, J., Symmans,
W. F., Pusztai, L. and G. N. Hortobagyi. (2004). Targeted therapy in breast
cancer. Molecular and Cellular Proteomics, 3 (4), 379-398.
13) Rovero, S. et al. (2000). DNA vaccination against rat Her-2/neu p185 more
effectively inhibits carcinogenesis than transplantable carcinomas in transgenis
BALB/c mice. Journal of Immunology, 165, 5133-5142.
14) Seidman, A. et al. (2002). Cardiac dysfunction in the trastuzumab clinical
trials experience. Journal of Clinical Oncology, 20, 1215-1221.
15) Smorlesi, A. et al. (2006). Evaluation of different plasmid DNA delivery
systems for immunization against HER-2/neu in a transgenic murine model
of mammary carcinoma. Vaccine, 24, 1766-1775.
16) Tai, W., Mahato, R. and K. Cheng. (2012). The role of HER-2 in cancer
therapy and targeted drug delivery. Journal of Controlled Release, 146 (3),
264-275.
64 Proceeding ASEAN COSAT 2014
Heterologous Expression of Recombinant
Plantaricin WS34 in Escherichia coli

Andini Setyanti Putria, Rifqiyah Nur Umamib, Apon Zaenal Mustopab,*


and Hasim Danuria
a
Graduate student of Biochemistry Department, Bogor Agricultural University
Jl. Raya Darmaga Kampus IPB Darmaga, Bogor 16680, Phone/fax: +62-251-8423267
b
Research Center for Biotechnology, Indonesian Institute of Sciences (LIPI)
Jl. Raya Jakarta-Bogor Km 46, Cibinong, Bogor 16911,
Phone: +62 (021) 8754587 Fax: +62 (021) 8754588

Abstract
Plantaricin W is a two peptide lantibiotic produced by Lactobacillus plantarum S34 isolated from
bekasam, a traditional fermented food of meat products from Waykanan, Lampung. Plantaricin
W is a potential antimicrobial peptide. However, the expression of recombinant plantaricin W
has not been reported. To express a recombinant plantaricin WS34, we constructed and expressed
the gene encoding plantaricin W in pET-32a by using the T7 RNA polymerase promoter with
approximately 33 kDa in size in Escherichia coli BL21 (DE3)(pLysS). The result revealed that the
plantaricin WS34 peptide was expressed as a translational fusion protein with thioredoxin and
His-tag. When expressed in E. coli, recombinant plantaricin W was found to be accumulated in
the cell cytoplasm but forming an inclusion body. In this study, two solubilization strategies were
done and showed that inclusion bodies were solubilized at alkaline condition in the presence of
urea solution under mild condition and the expression of plantaricin WS34 has been confirmed
with western blot. The expression of plantaricin WS34-pET32a was succesfully expressed in E. coli.
Key words: Inclusion body, Plantaricin W, Protein expression

I. Introduction
Bacteriocin are bactericidal compounds, act as a suppressor of competitor species.
Bacteriocins kill bacteria by disrupting membrane integrity so it does not induce
resistance [1]. Based on its properties, bacteriocin can be one of the solutions
for antibiotic resistance problems, and it can be used as alternative antibiotic.
Bacteriocin produced by lactic acid bacteria (LAB) was classified into four class [2].
The class I bacteriocin (lantibiotics) defined as ribosomally synthesized peptides
containing the thioether amino acids lanthionine (Lan) and 3-methyl-lanthionine
(meLan) [3]. Plantaricin W is a new family of two-peptide lantibiotics. According
to [3] and [4], plantaricin W was able to inhibit a large number of Gram-positive
and Gram-negative bacteria, such as E. coli, B. cereus, S. typhimurium and S. aureus.

* Corresponding author. Email: azmustopa@yahoo.com

65
66 Proceeding ASEAN COSAT 2014

Lantibiotics plantaricin W is produced from Lactobacillus plantarum [3]. In


this study, we isolated Lactobacillus plantarum S34 from bekasam, a traditional
fermented food of meat products from Waykanan, Lampung. The use of plan-
taricin W as alternative antibiotic required extraction technique from natural
resources in a large scale that is difficult to purified in high yield. One approach
that can be done is making the recombinant plantaricin. According to [5], this
technique allows to produce the target protein abundantly, not expensive, and
more stable. Bacteriocin production systems are currently utilized in expression
from native strain [6], recombinant expression system by using Escherichia coli,
recombinant expression in LAB [7], and chemical synthesis.
There are several advantages of using tag systems, such as a maltose-binding
protein (MBP), His-tag, or glutation-S-transferase. In this study, Plantaricin
W was expressed as a translational fusion protein with thioredoxin, S-tag and
His-tag. Polyhistidine-tag (his-tag) is a short affinity tag consisting polyhistidine
residues, widely used to purify recombinant protein utiliz immobilized metalaf-
finity chromatography. Immobilized metal affinity chromatography is based on
interaction between a transition metal ion (Co2+, Ni2+, Cu2+, Zn2+) immobilized
on a matrix and specific amino acid chains [8]. Histidine is the amino acid that
show the strongest interaction with immobilized metal ion matrix. Plantaricin W
also contains polypeptide that specifically recognized by enterokinase to remove
the tag from protein [8].
Expression of genetically engineered protein in E. coli often results in the
accumulation of the protein product in inactive insoluble form that deposits
inside the cells, called inclusion bodies [9]. This protein can be expressed as
inclusion bodies because of the protein interest is toxic or lethal to the host cell,
then inclusion body expression may be the best available production method [9].
We used several strategies to recover active protein from inclusion bodies, to take
advantage of the high expression levels of inclusion body protein.
Heterologous expression of bacteriocin has been widely studied in recent
years [10,11,12,13], but the expression of plantaricin W has not been reported.
In this study, we have expressed plantaricin W in E. coli to enhance the yield
with solubilization and produce an active plantaricin W as antimicrobial peptide.

II. M aterials and methods


A. Bacterial Strains and Media
Transformant Escherichia coli BL21(DE3) pLysS were grown in Luria Bertani
broth medium (Oxoid, Hampshire, UK) at 37°C with shaking. Salmonella thypi
P2KIM Collection, Escherichia coli NBRC 14237, and Staphylococcus aureus
ATCC 6538 were obtained from Biotechnology Research Center, Cibinong
Innovation for Better ASEAN Community 67

collection, grown in nutrient broth medium (Oxoid) at 37 °C with shaking.


When appropriate, 100 mg/ml ampicillin was added for E. coli.

B. Expression Plantaricin WS34


Expression of recombinant plantaricin WS34, furtherly we name it PlnWS34-p32,
was referred to [14] with modification at the amount of starter culture. Bacterial
cultures of E. coli BL21 transformant (DE3) pLysS carrying the gene Plantaricin
W (Pln W) were grown in Luria Bertani medium (LB), 20 mL has been added
with 100 mg/mL of ampicillin. Bacterial cultures were incubated at 37°C for 16
hours, shaked at 150 rpm. The expression of PlnWS34-p32 was performed by
adding 20 mL of culture into 200 mL of LB medium with 100 mg/mL ampicillin.
The cultures were incubated with shaker at 150 rpm, 37°C for 2 hours or until
the OD600 reached 0.6 (mid log phase), and then the culture were inducted with
0.5 mM IPTG (Thermo Scientific). After inducted, the culture were incubated
at 22 °C for 5 hours with shaking at 150 rpm. After 5 hours, the culture was
centrifuged at 8,000 g, 4°C, for 10 minutes. Removed supernatant (media) and
pellet (medium free cell) are stored at -20°C.

C. Protein Solubilization
We studied two solubilization buffers at low urea and high urea. Solubilization
of inclusion bodies reffered to [15] and [16]. After sonication, insoluble fraction
as inclusion bodies was separated from soluble protein by sentrifugation for 30
min with centrifugation speed at 17,000 g. The cells were washed with washing
buffer (50 mM Tris HCl pH 8, 100 mM NaCl, 2 M Urea, 1% Triton X-100)
and centrifuged at 12,000 g for 5 min. The washed pellet was resuspended in
solubilizing buffer. Two solubilizing buffers were studied. Buffer I that contained
high Urea [14] and buffer II that contained lower urea but high pH [15] had
been optimized to solubilize recombinant protein that cloned in E. coli. Buffer I
contained 8 M Urea, 50 mM Tris HCl pH 8.0, 800 mM mercaptoethanol and
buffer II contained 100 mM Tris HCl pH 12.5 and 2 M urea. Pellet were incubated
over night at mild condition with gentle agitation. Subsequently, solubilized pellet
was centrifuged at 17,000 g for 15 min, 4 °C. Protein was analyzed using SDS-
PAGE. We had observed these solubilization buffers to compare solubilization
ability on inclusion bodies and improving the yield of bioactive protein.

D. Protein Refolding
Protein refolding method was referred to [15]. Dialysis was performed to refold
PlnWS34-p32 after solubilization step. Two dialysis step were applied, first step
was to remove denaturing agent using denaturing removal buffer (50 mM Tris
HCl pH 8, 0.2 mM EDTA). The supernatant of solubilization was dialyzed
68 Proceeding ASEAN COSAT 2014

overnight in dialyzed tube (MWCO 11 463, Sigma) with 500 mL denaturing


removal buffer at 4 °C with gentle agitation. Secondly, the fraction was dialyzed
to renature and to retain native like secondary structure of PlnWS34-p32. To
renature PlnWS34-p32, the protein was dialyzed in 500 mL refolding buffer (0.4
M urea, 0.2 mM EDTA, 50 mM Tris HCl pH 8.0, 0.25 mM mercaptoethanol)
over night at 4 °C with gentle agitation.

E. Western Blot Analysis


Western blot was performed to detect the presence of the target protein by
using imunoaffinity (Invitrogen, R931-25) [17]. The process of western
blot performed several stages, first electrophoresis with SDS-PAGE without
staining, protein transfer to nitrocellulose membrane (blotting), blocking with
5% skim milk on a nitrocellulose membrane to prevent non specific binding
to the antibodies. These protein has his-tag fusion, probing is done by using
antibodies (anti-his-HRP-Cterm) so that the antibody would bind to proteins
that containing his-tag. Detection is done by adding the HRP substrate that is
TMB (3,3’,5,5’-Tetramethylbenzidine) (Invitrogen) that will react with the HRP
and give colour.

F. Inhibition Activity
The antimicrobial activity of each sample was determined by agar diffusion
method [4] with modification in kind of the medium agar used. A 50 mL
samples were applied to the well (5 mm diameter). Agar containing 108 CFU/
mL of indicator microorganisms (Salmonella typhi and S. aureus). Petri stored at
4 °C for 1-2 hours so that the sample could diffuse into agar before incubated at
optimum temperature. Then incubated at 37°C for 18 hours. Then the diameter
of inhibition zones were measured.

III. R esults and Discussion


The SDS-PAGE profile of the solubilize PlnWS34-p32 by two buffers is shown
in Figure 1. The purity of PlnWS34-p32 that was solubilize by buffer II (100
mM Tris pH 12.5 and 2 M urea) is better than buffer I (50 mM Tris HCl pH 8,
8 M urea, 800 mM 2-mercaptoethanol). Buffer II can solubilize 48.72%±4.44 of
wet pellet when 500 ml buffer solubilization was added for each 0.1 g wet pellet.
The antimicrobial activity of solubilized PlnWS34-p32 is shown in Table 1
and Figure 2. Based on Table 1, solubilization with buffer II has antimicrobial
activity against bacteria Gram positive and negative compare to solubilization
with buffer I.
Innovation for Better ASEAN Community 69

The best solubilization method that produced PlnWS34-p32 biologically


active was observed in 100 mM Tris buffer at pH 12.5 containing 2 M urea.
Solubilization PlnWS34-p32 inclusion bodies in the presence of low concentra-
tion of urea helped in retaining the existing native-like secondary structures of
PlnWS34-p32 [16]. Since PlnWS34 is class I A bacteriocin, their mode of action
is by pore formation to kill target cells [18].
Escherichia coli has an inducible expression system that strongly suppresses
the expression of the cloned gene in the presence of the expression inducer
(isopropyl-β-d-thiogalactopyranoside-IPTG) [21]. We also observed the expres-
sion when induced with IPTG and not induced (Figure 3). Based on Figure 3,
the expression of PlnWS34-p32 is higher in the presence of IPTG. The expression
of PlnWS34-p32 was confirmed with western blot (Figure 4). Based on Figure 3
and Figure 4, PlnWS34-p32 (~33 KDa ) was successfully expressed.
In the present work, we isolated and constructed recombinant PlnWS34 in
pET 32a. The plantaricin of WS34 belong to class I (lantibiotic) of bacteriocin
produced by Lactobacillus plantarum. Lantibiotic is a peptide containing the
unusual post-tranlationally modified residues lanthionine, 3-methyllanthionine,
and other modified residues. Lantibiotics are devided into type A and B. The
type A are elongated peptides with seven net positive charges. These peptides kill
target cells by pore formation to permeabilizing their membranes. The type B
are globular peptide and have either a low net positive charge, no net charge, or
a net negative charge. Their mode of action is inhibition of enzymatic activity
[18]. According to [3], Plantaricin W are indeed lantibiotic class A.
Plantaricin W peptide was expressed as a translational fusion protein with
thioredoxin, S-tag, and His-tag in E. coli. E. coli is common employed as the
host for vectors expressing recombinant proteins, because of its ease of growth
and generally low cost to cultivate. But sometimes, it is quite common that
overproduced recombinant protein is expressed into insoluble inclussion bodies
[19].
His-tags are the most widely used affinity tags. Purification of his-tagged
protein is based on metal affinity, by using chelated metal ions as affinity ligand.
This is possible to purification of the desired protein from the crude extract cell
of the host cells in a single step [20].
S-tag sequence is a fusion-peptide composed of four cationic, three anionic,
three uncharged polar, and five non-polar residues. This composition makes the
S-tag soluble [8]. Thioredoxin (Trx) is hydrophilic tag that can increase solubility
[8]. Altough Pln W has fused with several tags that could increase solubility,
PlnWS34 was found in a insoluble form. According to [9], this protein can be
expressed as inclusion bodies because of the protein is toxic or lethal to the host
cell, then inclusion body expression may be the best available production method.
70 Proceeding ASEAN COSAT 2014

M 1 2 Figure 1. Solubilization optimization. Lane 1: PlnWS34-p32


210 solubilized with buffer 1 (8 M Urea, 50 mM Tris HCl pH 8,
800 mM mercaptoethanol), lane 2: Solubilization with buffer
78 II (100 mM Tris pH 12.5 and 2 M urea).
55

45
Table 1. Activities of solubilization PlnWS34-p32 towards
34 different indicator strains
Clear zone (mm)
23 Gram
Indicator Strain Buffer Buffer
(+/-)
I II
16
EPEC K.1.1 - - 2 mm
  S. aureus ATCC 6539 + - 4 mm
E. coli NBRC 14237 - - 5 mm
7
  S. typhi P2KIM col- - - 2 mm
4
lection

1 2 K

Figure 2. Activities Pln WS34 towards S. typhi P2KIM collection. 1. PlnWS34 (buffer I),
2. PlnWS34-p32 (buffer II), K (negative control, buffer without PlnWS34-p32)

M 1 2 Figure 3. Differentiation be-


210 tween expression PlnWS34-p32
78
55 that is induced with IPTG (lane
45 1) and non induced with IPTG
(lane 2).
34

23

Figure 4. Western Blot of


16
PlnWS34. Lane 1: Solubiliza-
  tion of PlnWS34-p32 (mem-
7 brane nitrocellulose), lane 2:
4 Solubilization of PlnWS34
(gel SDS-PAGE).
Innovation for Better ASEAN Community 71

IV. Conclusion
Solubilization of the recombinant Plantaricin WS34 inclusion bodies in the
presence of low urea improving the yield of bioactive protein during refolding.
The biologically active of recombinant Plantaricin WS34 was succesfully expressed
in E. coli with the size of ~33 KDa.

V. Acknowledgement
This research was funded by Competitive Program Indonesian Institute of Science
2014 granted to Apon Zaenal Mustopa.

VI. R eferences
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Plantaricin W from Lactobacillus plantarum belongs to a new family of
two-peptide lantibiotics. Microbiology, 147, 643–651.
4) Arief, I., Jakaria, Suryati, T., Wulandari, Z. and E. Andreas. (2013). Isolation
and characterization of plantaricin produced by Lactobacillus plantarum strains
(IIA-1A5, IIA-1B1, IIA-2B2). Media Peternakan, 36 (2), 91–100.
5) Ma, J., Pascal, M. W. and C. Paul. (2003). The production of recombinant
pharmaceutical proteins in plants [review]. Nature, 4, 794–805.
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119–126.
8) Terpe, K. (2003). Overview of tag protein fusions: from molecular and
biochemical fundamentals to commercial systems. Applied Microbiology and
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10) Pal, G. and S. Srivastava. (2013). Cloning and heterologous expression of plnE,
-F, -J and –K genes derived from soil metagenome and purification of active
plantaricin peptides. Applied Microbiology and Biotechnology, 98, 1441–1446.
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Heterologous expression and purification of active divercin V41, a class IIa
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186 (13), 4276–-4284.
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for large-scale production and purification of recombinant class IIa bacteriocins
and its application to piscicolin 126. Applied and Environmental Microbiology,
70 (6), 3292–3297.
14) Utama, A., Shimizu, A., Morikawa, S., Hasebe, F., Morita, K., Igarashi, A., Hatsu,
M., Takamizawa, K. and T. Miyamura. (2000). Identification and characterization
of the RNA helicase activity of Japanese encephalitis virus NS3 protein. FEBS
Letters, 465, 74–78.
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human interferon a2b soluble protein overproduction and primary recovery of
its inclusion bodies. Microbiology Indonesia, 5, 27–32.
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K. Panda. (2000). Optimization of inclusion body solubilization and renaturation
of recombinant human growth hormone from Escherichia coli. Protein Expression
and Purification, 18 (2), 182–192.
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antibody (R930-25, R931-25). Invitrogen. Carlsbad. USA. (Online). Available:
https://www. invitrogen.com.
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Plantaricin F, one of the peptides constituining the two-peptide bacteriocin Plantaricin
EF (thesis). Oslo: University of Oslo.
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A strategy for the expression of recombinant proteins traditionally hard to purify.
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to bacteria. Genome Research, 22, 802–809.
Innovation for Better ASEAN Community 73

Evaluation of Low Temperature Induced


Mutant of Soybean mosaic virus for Cross
Protection in Soybean
Wuye Ria Andayanie* and Praptiningsih Gamawati Adinurani
Faculty of Agriculture, Merdeka University
Serayu, Madiun, Indonesia

Abstract
Early natural infection by soybean mosaic virus (SMV) can reduce seed production in soybean.
Cross protection enables the production of SMV free seed and is a mechanism which can
signficantly reduce the impact of SMV. We proposed that attenuated isolates of SMV obtained
by cold temperature treatments are able to produce SMV free seed in soybean [Glycine max
(L.)] Merr. plants. We inoculated the cotyledons with SMV infected plants, incubated at low
temperature, mechanically inoculated seedlings with virus were subjected to cold and finally
transplanted into pot and then into the field. We examined plants with different symptoms.
Serological assays, RT-PCR analysis and electron micrography did not distinguish between the
very mild mosaic symptom of attenuated isolate and the original virulent isolate. However, the
mutant of SMV did not give rise to local lesions on Chenopodium amaranticolor. Our results suggest
that attenuated isolate of SMV is potentially useful for reducing the impact of SMV infection.
When plants are inoculated with the attenuated isolate at 8 days after planting, symptoms of the
disease do not develop.
Key words: Attenuated isolate, Cross protection, Induced mutant, soybean, soybean mosaic virus (SMV)

I. Introduction
Soybean is a valuable economic commodity in Indonesia with great potential for
production in the future. Imports of soybean reached 1.6 million of tons in 2010.
Soybean mosaic disease is a major problem caused by soybean mosaic virus
(SMV), a seedborne and aphid-borne disease. This viral disease reduces seed
quality, decreases oil content and nodulation [1]. Soybean mosaic virus is com-
monly transmitted by aphids and through seed. Mottling of soybean seeds does
not indicate that plants grown from such seeds will contain SMV or whether seed
produced by plants grown from mottled seeds will themselves produce mottled
seed [2,3].
The increase of soybean production was absolutely needed to meet national
needs and security of foreign exchange. Mosaic disease caused by cowpea mild

* Corresponding author. Phone: +62-81335401517. Email: wuye_andayanie@yahoo.com


74 Proceeding ASEAN COSAT 2014

mottle virus (CMMV) and soybean mosaic virus (SMV) are commonly found in
soybean [Glycine max (L.) Merr.] in East Java Province, Indonesia, but there was
no efficient controlling measure to SMV. Etiology of the viral disease of soybean
crops, prevalent in the soybean growing regions of Ngawi District in East Java
Province, clearly indicates the dominance of SMV. Infection by SMV, especially
at an early stage of cropping, has a serious impact on seed production in soybean.
Early natural infection by SMV has been found to reduce seed production by
13–30% in soybean [4,5]. Therefore, a control strategy that delays SMV infection
improves soybean production. This can be achieved by the use of SMV free seeds
or seedlings to minimize the primary inoculum source that is spread by aphids.
However, it is difficult to obtain SMV free seed from field grown soybean plants
under high disease pressure [6].
The induced resistance of plant is that the inducting factors induce the plant
to resist pathogeny microorganism. The cross protection is induced by attenuated
virus to protecting the plant infected by virus. The effectiveness of the use of
attenuated isolates of SMV to protect soybean plants from the seed transmitted
mosaic disease caused SMV.
Cross protection is described as “a specific symptom, a single virus”, that
is the presence of one virus inhibits the accumulation of second and produces
a specific symptomatology. Cross protection is a phenomenon whereby plants
infected with one strain of the virus do not develop additional symptoms when
inoculated with another strain of the same virus. Cross protection interprets as
a symbiotic union between the virulent and avirulent srains and consider it an
important source of physiological immunity. The coat protein of the mild strain
inhibits the uncoating of the challenging virus and the presence of the mild strain
blocks the systemic movement of the challenge virus [7,8].
Cross protection with a mild or avirulent strain could offer an alternative
route to control by protecting soybean plants from subsequent infection by a
virulent strain. Deployment of such a strain could be similar to using virus-free
seed. If a field is virus free, even if the vector population is high, no infection
will take place because the virus is non-persistent and will not be present in the
vector. Attenuated isolates exploited for cross protection have been obtained by
the treatment of virus with heat, nitrous acid and UV light [9,10]. Heat or cold
treatment may also yield mild isolates [11].
The most common limiting factor hindering the acceptance of cross protection
being used commercially is the difficulty involved in large number of plants
with the mild strain, especially when the virus is non-mechanically transmis-
sible. Another unpredictable drawback is that the mild strain might associate
Innovation for Better ASEAN Community 75

synergistically with an unrelated virus which can then infect hosts under field
conditions [12,13].
Succes in virus control by cross protection depends on whether the avirulent
virus can invade and replace the virulent virus and whether the virulent virus is
prevented from reestablishing. Ideally, a protective strain should induce no or
very mild symptoms that: will not reduce the market value of the crop; will not
inflict disease on other crops that are not targets for cross protection; will be
completely systemic affecting all host tissues; will be genetically stable, will not
revert to the severe form; will be non-transmissible by vectors in order to limit
non-intentional spread to other crops or fields; will provide protection towards
the widest possible range of severe strains. Also, the inoculum should be easy to
produce, to apply and needs to be very stable, during the process of transportation
and utilization [14].
There is a dire need to investigate the effect of this protection on the yield
parameters and also study should be planed to study the behaviour of these isolates
under natural field conditions, especially their effect on other prevalent crops and
their interaction with the prevalent viruses in the area, before allowing the use of
this isolate for commercial control at field level.
We proposed that attenuated isolates of SMV obtained by cold temperature
treatments may be able to produce SMV free seed on soybean plants. The objec-
tives of this study were therefore (i) to investigate the cross protection offered
by a mild mutant isolate of SMV, induced at low temperature, against a severe
strain and (ii) to detect very mild symptoms of the mutant isolate of SMV in
soybean crops.

II. Method/M aterial


The SMV was isolated from soybean samples collected at Ngawi, East Java
Province, Indonesia in 2011 and was maintained on mechanically inoculated
plants of C. amaranticolor in insect proof green house at Merdeka Madiun
University, Indonesia.

A. Inoculation of Soybean Seedling Leaves with SMV


The heavily infected SMV isolate originated from soybean samples collected in
Ngawi District. The presence of SMV was confirmed by local lesion assay, serology
assay, RT-PCR and electron micrograph [5,15].
To produce SMV free seeds, we determined the SMV to protect seedling
plants and detect with Enzyme Linked Immnusorbent Assay (ELISA). Healthy
seeds of soybean were germinated in soil contained in 12 plastic pots, each pot
had two seeds. After germination thinning was done to reduce the number of
76 Proceeding ASEAN COSAT 2014

seedling to one in each pot. The soybean seedlings were mechanically inoculated
with the severe SMV isolate. Mechanical inoculation was done at the first true leaf
stage by grinding 1 g of infected leaf tissue in 50 ml of 0.05 potassium phosphate
buffer (pH 7.3) and rubbing the inoculum on carborundum (600 mesh) dusted
healthy leaves.

B. Incubation Under Low Temperature


Immediately after inoculation, the plants were cut off at the base of the hypocotyl,
and the stems were immersed in distilled water in vials. The cuttings were then
transferred into different growth cabinets, maintained at one of the following
temperatures: 10oC, 15oC, 20oC and 25°C as a control in a 14 hr photoperiode,
respectively. The cuttings were incubated for periods of 8 days, 14 days or 20 days.
The cotyledons of 8 day old seedling were inoculated with leaf extracts from the
cuttings subjected to incubation at different temperatures to evaluate of SMV
mutants obtained by exposure to cold.

C. Testing Cold Induced SMV Mutantsin the Field


To evaluate of cross protection strategy, soybean seedling was inoculated with the
attenuated SMV isolate. The seedlings (7 days after inoculation with attenuated
isolate) were planted into the field under conditions of natural infection.
The experimental design comprised plots with seedlings inoculated with the
cold induced SMV mutant and non-inoculated (control) plots. Inoculated and
non-inoculated seedlings were planted in the plots at intervals of 0.5 m in 2
rows; 40 plants (5 plants in each row and 4 replication in each row), respectively.

D. Examination of Plant Symptoms


Symptoms were observed and diseased plants were counted for 10 days, 20 days
and 30 days after transplanting into the field. Plants were scored for disease
severity according to the following scale as reported earlier by Ilyas et al., (l992)
as: 0 = no visible syptoms (plants apparently healthy); 1 = very mild mosaic
(mild mosaic mild mosaic on few leaves/plants); 2 = moderate mosaic (mosaic
on many leaves/plant and vein clearing); 3 = severe mosaic (severe mosaic and
mild mottling); 4 = severe mosaic and severe mottling); 5 = severe mosaic plus
severe mottling plus necrosis and occasinally death of plants. To check for the
presence of the attenuated SMV isolate, leaf samples with no visible symptoms
or only very mild symptoms were tested for the ability of their extracts to cause
local lesion on Chenopodium amaranticolor Coste et. Reyn.
Virus inoculum was prepared by grinding systemically infected leaves with a
mortar and pestle at an approximate dilution of 1:10 (w/v) with 0.05 M potassium
Innovation for Better ASEAN Community 77

phosphate buffer, pH 7.2. Inoculations were performed by rubbing the inoculum


onto C. amaranticolor leaves that had been previously dusted with carborundum,
using a pestle dipped in the inoculum (one dip per leaflet, approximately 100
ul inoculum per leaflet) [5] in an attempt to find out any reasonable local lesion
hosts for mild strain seection. Leaf samples were also tested with ELISA, RT-PCR,
and observed under electron microscopy.

E. Total RNA extraction and RT- PCR


The sensitivity of RT-PCR enabled the detection of SMV from plants in which
the number of virus particles was too low to be detected by ELISA. Total RNA
was extracted from leaf samples grown in an insect proof greenhouse using
Nucleon Phytopure plant DNA extraction kit (Amersham pharmacia biotech,
Buckinghamshire, England).
Samples were obtained from plants with no visible symptom or very mild
symptom and was detected by RT-PCR. First Strand cDNAs Synthesis Kit: Am-
ersham Bioscience, Backinghamshire, Uk) and primer 3093R (5΄-ATGCTCTTC-
GCATGTACTCG-3΄;2.5 pmol). A pair of oligonucleotides amplifying a 1385 bp
fragment at posisition from 4,176 to 5,560 including the CI coding region was
designed based on the sequence published by Jayaram et al. (l992). The forward
primer, CÍ (5΄-GCATTCAACTGTGCGCTTAAAGAAT-3΄), was homologous
to nucleotides 4,176 to 4,200 and the reverse primer, CI3΄ (5΄-TTGAGCT-
GCAAAAATTTACTCACTT-3΄), was complementary to nucleotides 5,536
to 5,560 of SMV strain G2. These primers were used for amplification. PCR
was performed in a reaction mixture containing the following: 0.5 µl of cDNA
solution, 2.5 µl of 10 x PCR buffer (KOD plus Neo), 2.5 µl of 2 dNTP mix,
1.5 of 25 mM MgSO4, 0,5 µl of KOD plus Neo, 0.5 of each primer (50 pmol),
of each forward and reverse, 16.5 µl of sterile distilated water.
Thermocycling was programmed as follows: cDNA synthesis at 42°C for 60
min and 95°C for 5 min, and RNA/cDNA/primer denaturation at 94°C for 2
min, followed by 40 cycles for template denaturation at 94°C for 10 s, primer
annealing at 47 °C for 30 s, and extention at 68 °C for 1 min, and a final extention
at 68°C for 1 min. The amplified products were separated by 1.2% agarose gel
electrophoresis.

F. Electron Microscopy
Electron microscopy was also done for the selected samples to confirm the results
of serological assay. Sap from soybean leaf samples infected with attenuated
isolate was coated on carbon-formvar (400 mesh) grids, negatively stained with
2% phosphotungstate (PTA) pH 6.5 and were examined with JEOL 100 S x
78 Proceeding ASEAN COSAT 2014

electron microscope.

G. Data Analysis
The percentage of diseased plant was calculated from the number of plants
showing leaf roll and rugose symptom characteristic of the virulent SMV infect.
A randomized complete block design (RCBD) was used for agriculture traits.
Each treatment was performed four times. Disease severity scores associated with
SMV mutant were arcsine-transformed prior to analysis of variance. Analysis of
variance were performed by SAS [17]. Mean separation was done by calculating
the least significant difference (LSD) at P ≤ 0.05.

III. R esult and Discussion


A. Cross Protection Using Attenuated Isolates in the Field
Disease severity of SMV was significantly lower (P ≤ 0.05, by chi-square test of
independence) in the field where seedlings from the SMV mutant obtained by
cold treatment (at 10°C for 8 days or 14 days) were grown (0–5%), compared
to other treatments. None of the symptomatic or very mild symptom plants in
the field when inoculated with the attenuated isolate produced by the treatment
temperature of 10°C for 14 days. In this study, no difference in the percentage
disease plant were found treatment at 10°C for 8 days or 14 days. An isolate of
virulent SMV obtained from field was subjected to low temperatures and mutants
strain induced; mutant isolates of the virus resulting from treatment at 10°C for
8 days or 14 days caused very mild and mild symptom, respectively.
These results showed that plants had the ability to cross-protect plants from
SMV infection in endemic field. Hence, plants were finally selected for the
attenuated isolate selection. The inoculated seedlings were kept in a green house
with they were transplanted to endemic area.
In contrast, SMV in endemic area used as the challenge virus was detected in
soybean plants that had previously been inoculated with other treatments. These
result indicated that the severity of viral pathogenicity to induce symptoms might
be not associated with cross-protective ability.

B. Serological Assay
Preinoculated soybean healthy samples with SMV maintained at 10°C, 15°C,
20°C, and 25 °C for 8 days, 14 days, 20 days, respectively gave a positive reaction
when tested with the antibodies of SMV (Table 2). Although soybean plants
have very mild and mild symptoms with SMV maintained at 10 °C for 8 days
and 14 days.

C. Differentiation of Attenuated Isolates by RT-PCR


Innovation for Better ASEAN Community 79

Table 1. Disease Severity of SMV in Soybean Plants Inoculated with the SMV Mutant
Treatments SMV maintained at % Disease plants a
Preinoculated 10 °C 8 days 5 f(2)
14 days 0 f (1)
20 days 25 c (1,2)
15 °C 8 days 55 d (1,2,3)
14 days 85 b (2,3)
20 days 100 a (3,4)
20 °C 8 days 80 c(3,4)
14 days 95 a (3,4)
20 days 100a(4)
25 °C 8 days 95 a(4)
14 days 100 a(3,4)
20 days 100 a (4)
Noninoculated 100 a (3,4)
LSD 5.34

Similar letters in each column indicate no significant difference at 5% level


a
Symptom severity on diseases plants at 30 days after the challenge inoculation: 1 = very mild;
2 = mild; 3 = mosaic; 4 = severe mosaic and stunting

Table 2. Detection of SMV Maintained in Soybean Plants by Indirect ELISA

Treatments SMV maintained at Absorbance value (A405 nm) /


ELISA reaction
Preinoculated 10 °C 8 days 0.364/+
14 days 0.388/+
20 days 0.357/+
15 ° C 8 days 0.358/+
14 days 0.356/+
20 days 0.351/+
20 °C 8 days 0.356/+
14 days 0.351/+
20 days 0.354/+
25 °C 8 days 0.359/+
14 days 0.364/+
20 days 0.361/+
Noninoculated 0.357/+
Healthyleaves 0.117/−
Buffer 0.066/−

Remarks: Sample is considered positive if the absorbance value at 405 nm is up to three times
the value of the negative control (healthy leaves)
80 Proceeding ASEAN COSAT 2014

1 2 3 4

Figure 1. Specific detection of attenuated isolate by RT-PCR analy-


sis of total extracts using primer CI (lower lanes) which amplify a
1385 bp product. Lane 1 = healthy soybean leaves (negative con-
trol); lane 2 = very mild symptom of soybean leaves; lane 3 = mild
symptom of soybean leaves; lane M = 1 kb DNA ladder

RT-PCR analysis using primers CI yielded a DNA fragment of the expected size
1.385 bp from soybean leaves with very mild and mild symptoms.

D. Electron Microscopy
When checked under electron microscope, virus particles could be detected in
six samples from leaf tissue with very mild symptom. Filamentous particles of
ca. 13 x 750 nm were present in the leaf dip preparation examined by electron
microscopy.
The diagnosis of attenuated isolates showed that serological assay, RT-PCR
analysis and electron micrograph showing the results of very mild mosaic symptom
of attenuated isolate similar to original isolate.

E. Local Lesion Host Assay


No satisfactory local lesion host could be found in the assay with the SMV mutant;
however, the isolate SMV that it originated from caused chlorotic lesions on C.
amaranticolor.
Detection of the mild strain of SMV and its differentiation from severe SMV
is very important in order to prove its efficacy as a succesful cross protectant
[11,10]. Protective inoculation with SMV mutant (temperature 10°C for 8 days
or 14 days) has been effective for control of SMV in endemic areas. Artificial
mutants are being used, with no apparent problem for control of SMV. Those
plants not having been infected at the time of protective inoculation with SMV
mutant to a severe strain of SMV. This efforts could be directed to induce mild
strain, to help farmer controlling the severe virus under field conditions or at least
minimizing the detrimental effects to economic benefecial level. There is the real
possibility of the mild strain mutating to a severe form.
Innovation for Better ASEAN Community 81

Since polyclonal antibody can detect the presence of virus coat protein from
either infectious or non-infectious, positive reaction with polyclonal antibodies
against SMV was caused by low antibody specificity so that these antibodies
still react with other viruses and strain viruses or SMV mutant. Although not
all samples were tested, a reverse transriptase polymerase chain reaction detection
test showed promise and detected both mild and severe SMV detection. Further
molecular studies are required to differentiate the mild strain of SMV from the
severe strain. In our study, variant mutants that may have been obtained with
cold treatment were not investigated but such an approach might give rise to a
more easily identifiable mutant SMV isolate. However, it is known that SMV
came into the region where the seedling were transplanted via infected seed and
vector (Aphis glycines Matsumura) [5,15].

Figure 2. Electron micrograph of soybean


Figure 3. Symptoms on C.
leaf sample infected with attenuated isolate,
amaranticolor plant infected
showing of SMV viral particles. The bar in
with mutant of SMV andorigi-
the electron micrographs is 100 nm
nal isolate of SMV

IV. Conclusion
Soybean plants with very mild mosaic symptoms were found in SMV mutant
seedlings transplanted from pots after cold treatment. The mutant isolate of SMV
obtained by cold treatment provided effective protection against field spread of
the virulent SMV. Although SMV was aphidborne, the attenuated virus was not
aphid transmitted in soybean field growing conditions. Immunity or tolerance is
established by infection of the attenuated virus and plants showed mild symptom.
This may be explained: 1) plants were effectively protected against the virulent
SMV as it was transmitted in the field; 2) plants were not showing symptoms
when observed at an early stage. After the application of cross protection in the
field, there was a trend for soybean yield to increase because early SMV infection
was reduced. The very mild symptom did not give rise to local lesion on C.
amaranticolor.
82 Proceeding ASEAN COSAT 2014

V. Acknowledgement
The author gratefully acknowledges Directorate General of Higher Education
for providing financial support by grant competition.

VI. R eferences
1) Sudaryanto, T. and D. K. S. Swastika. (2010). Ekonomi kedelai di Indonesia
(pp. 1–30). Badan Penelitian dan Pengembangan Pertanian, Kementerian
Pertanian: PT Balai Pustaka.
2) Li, D., Chen, P., Shi, A., Shakiba, E., Gergerich, R. and Y. Chen. (2009).
Temperature effects expression of symptoms induced by soybean mosaic
virus in homozygous and heterozygous plants. Journal of Heredity, 100 (3),
348–354.
3) Domier, L. L., Hobbs, H. A., Coppin, Mc. N. K., Bowen, C. R., Steinlage, T.
A., Chang, S., Wang Y. and G. L. Hartman. (2011). Multiple loci condition
seed transmission of Soybean mosaic virus (SMV) and SMV induced seed
coat mottling in soybean. Phytopathology, 101, 750–756.
4) Gunduz, I. (2000). Genetic analysis of Soybean mosaic virus resistance in
soybean. In partial fulfillment of the requirements for the degree of Doctor
of Phylosophy in Crop and Soil Enviromental Science, Virginia Polytechnic
Institute and Institute and State University.
5) Andayanie, W. R., Sumardiyono, Y. B., Hartono, S. and P. Yudono. (2011).
Incidence of soybean mosaic disease in East Java Province. Journal of Agrivita
Science, 33 (1), 15–22.[6]Andayanie, W. R., Sumardiyono, Y. B., Hartono, S.
and P. Yudono. (2011). Identification and seed transmission of Soybean mosaic
virus management. In partial fulfillment of the requirements for the degree
of Doctor of Phytopatology Science, Gadjah Mada University. [7]Freitas,
D. M. S. (2008). Protection between strains of Papaya ringspot virus-type
W in Zuchini squash involves competition for viral replicatin sites. Scientia
Agricola, 65 (2), 183-189.
6) Ziebell, H. and P. C. John. (2009). Effects of dicer-like endoribonucleases
2 and 4 on infection of Arabidopsis thaliana by Cucumber mosaic virus and a
mutant virus lacking the 2 b counter-defence protein gene. Journal of General
Virology, 90, 2288–2292.
7) Hooks, C. R. R. and A. Fereres. (2006). Protecting crop from non persistently
aphid transmitted viruses; a review on the use of barrier plants as a manage-
ment tool. Virus Research, 120, 1–16.
Innovation for Better ASEAN Community 83

8) Nishiguchi, M. and K. Kobayashi. (2011). Attenuated plant viruses: prevent-


ing virus diseases and understanding the molecular mechanism. Journal of
General Plant Pathology, 77 (4), 221–229.
9) Kosaka, Y. and T. Fukunishi. (1994). Application of cross protection to the
control of black soybean mosaic disease. Plant Disease, 78, 339–341.
10) Untiveros, M. and S. Fuentes. (2007). Synergistic interaction of Sweet
potato chlorotic stunt virus (Crinivirus) with Carla-, Cucumo-, Ipomo-, and
Potyvirus infecting sweet potato. Plant Disease, 91, 669–676.
11) Hwang, T. Y. (2011). Intra host competition and interaction between Soybean
mosaic virus (SMV) Strain in mixed infected soybean. Australian Journal of
Crop Science, 5 (11), 1379–1387.
12) Gal-on, A. and Y. H. Shiboleth. (2006). Cross protection. In G. Loebenstein
and J. P. Carr (Eds.), Natural resistance mechanisms of plant to viruses (pp.
261-288). Springer.
13) Andayanie, W. R. (2012). Diagnosis of soybean seed transmission. Journal of
Tropical Plant Pests and Diseases, 12 (2), 185–191.
14) Konig, G. R. (1981). Indirect ELISA methods for the broad specificity
detection of plant viruses. Journal of General Virology, 55, 53–62.
15) SAS. (2003). SAS/STAT User’s Guide. Release 9.1. edn. SAS Institute Inc.
Cary NC, USA.
Process Design of PeptoneProduction from
Peanut Meal as byproduct of Peanut Oil
Industry Using Crude Papain
Mulyorini Rahayuningsih* and Ninuk Gilang Wiranti
Departement of Agroindustrial Technology, Faculty of Agricultural Technology, Bogor
Agricultural University
Jl. Puspa, Kampus IPB Darmaga, Bogor, Indonesia
Contact author: +628128534505
Email: mulyorinir@yahoo.com

Abstract
Peptone is an important component in the microbial growth media which has function as the
source of amino acids. Up until now, Indonesia still imports peptone which reaches $17.84
milion per year in the last five years. That fact is the reason to develop the research in peptone
production by using protein source material that is available in Indonesia, such as peanut meal
that can be obtained as by product of peanut oil industry. The objectives of this research was to
design the production process of peanut peptone using enzymatic hydrolysis by crude papain.
Design process was performed by determination of the best condition (time of hydrolysis, crude
papain concentration and temperature of hydrolysis. Peanut peptone produced was characterized
on its amino acids content and applied to be used as microbial growth media and compared with
a commercial product. Crude papain powder was obtained from the sap of papaya fruit, through
drying and milling process. The activity of crude papain used in this experiment was 5057.47
U/g min. Hydrolysis process was conducted on peanut meal that have been diluted in water
with ratio of 1:2. The research showed that the best hydrolysis condition of peanut peptone was
obtained by using 0.4% of crude papain for 4 h at temperature of 55 °C. The peptone product
has appearance of brownish yellow color in liquid form. The yield peptone produced was 40.8%.
The analysis of amino acids content showed that peanut peptone produced had high glutamic
acid aspartic acid and arginine. Those 3 amino acids are very important for microbial growth.
The result of growth test of Escherichia coli and Staphylococcus aureus by determining optical
density (OD620nm) and Total Plate Count showed that peanut peptone produced has similar
performance with commercial peptone.
Key words: Enzymatic hydrolysis, Peanut meal, Crude papain, Peanut peptone

I. Introduction
Peptone is a protein hydrolisate product which is common to be used as nitrogen
source in growth media of microorganisms. Up until now, Indonesia still imports
peptone to fulfill its demand due to there is no peptone industry available in

* Corresponding author. Email: mulyorinir@yahoo.com

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86 Proceeding ASEAN COSAT 2014

Indonesia. Imported peptone within the last two years increase by 3,296 tonnes
with the value of US $12,15 million in 2012 and become 5,102 tonnes with the
value of US $20,76 million in 2013 [1]. This facts lead to the need of preliminary
research of development of peptone industry in Indonesia using raw materials
which are available such as protein from plants.
Peanut is one of the protein source plants with the potential to be used as
peptone raw material. Usually, peanut is used as materials for snacks, peanut
butter and peanut oil. On the production of peanut oil, peanut meal will be
produced as by products which contain 45–50% crude protein that can be
utilized as raw material of peptone. Usually, by products of peanut oil industry
is used for feed or traditional food, such as oncom which have low added value.
By conducting this research, peanut meal is utilized as peptone raw material and
applied to substitute imported peptone as microbial growth media. This research
was aimed to design the production process of peanut peptone using enzymatic
hydrolysis by crude papain. Design process was performed by determination of
hydrolysis time, enzyme concentration and hydrolysis temperature and compared
the peptone produced with commercial peptone as growth media of Escherichia
coli and Staphylococcus aureus.

II. M aterials and Methods


A. Materials
Materials that were used were peanut meal, papaya sap, Sodium metabilsulphite,
NaCL, yeast extract, BactoTMpeptone from BD (Beckton Dickinson), BactoAgar
(BD), aquadest, Escherichia coli, Staphylococcus aureus and Nutrient Broth (BD).

B. Methods
Enzyme Preparation
Enzyme preparation was obtained by mixing papaya sap with 1.4% sodium
metabilsulphite and 0.3% NaCl in proportion of 1:1. The mixture was then
gently stirred, filtered and dried in 50–55°C, and milled. The crude enzyme was
characterized by analyzing its enzyme activity using casein as substrates. Yield of
papain enzyme obtained was 19.8% with protease activity of 5,057.47 U/g min.

Determination of Hydrolysis Time and Enzyme Concentration


Peanut meal was added with aquadest of 1:2 (b/v) and papain enzyme of 0.2%;
0.4%; and 0.6%. The mixture was mixed well and hydrolyzed for 4, 6 and 8 h.
Hydrolisis was performed at temperature of 60°C in incubator. This step was
stopped by enzyme inactivation by heating the samples at 85°C for 15 minutes
in water bath. The samples were then filtered in 225 meshes and centrifuged at
Innovation for Better ASEAN Community 87

4,000 rpm for 15 minutes to obtain soluble fraction. Hydrolisis performance was
determined by analyzing ratio of Total of Soluble Nitrogen to Nitrogen Total of
material using Kjedhal method.

Determination of Hydrolisis Temperature


After hydrolysis, time and enzyme concentration were obtained and the research
was continued by determining hydrolysis temperature. Hydrolysis was carried
out in temperature of 50, 55 and 60°C. Performance of hydrolysis was measured
by determining ratio of Total of Soluble Nitrogen to Nitrogen Total of material
using Kjedhal method.

Determination of Amino Acid Content in Peptone Produced


The peptone produced was analyzed by HPLC to determine the amino acids
content.

Application of Peptone Produced as Bacterial Growth Media


The peptone produced was analyzed to be used as growth media of bacteria and
compared with commercial peptone (BactoTMpeptone, BD). The bacteria used
were Escherichia coli and Staphylococcus aureus.
Liquid medium for growing bacteria was prepared by mixing 0.5% yeast
extract, 1% peptone, and 1% NaCl. The using of peptone samples that were
produced from peanut meal hydrolysate was set up by equalizing the total of
nitrogen samples with total of nitrogen commercial peptone. Medium was
sterilized at 121°C for 15 minutes. Bacteria were inoculated to 10 ml of medium
containing peptone produced and then incubated at 37°C for 24 h and the optical
density (OD 620 nm) was measured as indicator of bacterial growth.
Bacterial growth as Total Plate Count was also measured by plating bacteria
on agar plate medium using peptone produced and compared with commercial
peptone. The composition of media was the same as liquid medium but it was
added by 1.5% Bacto Agar. After inoculation, the agar plates were incubated
at 37°C for 48 h. The number of colony was counted by using Colony Counter
Quebec.

Experimental Design
Experimental design used in this research was Randomized Factorial Design
which performed in two steps namely Randomized Factorial Design two factors to
determine hydrolysis time and enzyme concentration; and Randomized Factorial
Design one factor to determine the effect of hydrolysis temperature. The results
that showed significant different value was analyzed further using Duncan test.
88 Proceeding ASEAN COSAT 2014

III. R esults and Discussion


Hydrolysis Time and Enzyme Concentration
The best hydrolysis condition was determined based on ratio of Total of Soluble
Nitrogen (NTT) to Nitrogen Total of Material (NTB). Higher Ratio of NTT/
NTB meant that hydrolysis process was optimally occurred [2].
Figure 1 showed the effect of hydrolysis time and enzyme concentration on
ratio of NTT/NTB. Based on statistical analysis, it was determined that hydrolysis
time and enzyme concentration significantly affected ratio of NTT/NTB (p <
0.05). The highest ratio of NTT/NTB was obtained at 4 h of hydrolysis time with
enzyme concentration of 0.4%. Duncan test result showed that 4 h hydrolysis
time was significantly different with 6 h and 8 h of hydrolysis time. Enzyme
concentration of 0.4% was significantly different with enzyme concentration of
0.2% but not significantly different with enzyme concentration of 0.6%.
Hydrolisis time is one factor that affects the enzyme stability, which tends
to decrease along with hydrolysis time [3]. In addition to that, [4] stated that
hydrolysis rate and nitrogen recovery will increase along with the increasing of
enzyme concentration. Ratio of NTT/NTB which relatively low with increasing

Figure 1. The effect of hydrolysis time and enzyme concentration on


ratio of NTT/NTB. Different superscript indicated significant differ-
ence (p < 0.05).
Innovation for Better ASEAN Community 89

hydrolysis time can be affected by decreasing enzyme stability. Besides that, [5]
stated that enzymatic hydrolysis rate decrease and reaches stationary phase when
there is no more hydrolysis process occured. Soluble protein hydrolysate will be
obtained in early stage of hydrolysis, but although an amount of enzyme is added
on stationary stage of hydrolysis process, there will be no increase of hydrolysate
yield. On that condition, product inhibition will be occurred. More over, the
decrease of enzymatic reaction rate can be occurred by decreasing specific peptide
bond, enzyme inactivation and competition between substrates with peptide
obtained from hydrolysis process [6].

Hydrolysis Temperature
Figure 2 showed that hydrolysis temperature affected ratio of NTT/NTB. The
highest ratio of NTT/NTB was obtained on hydrolysis temperature of 55°C.
Statistical analyzes followed by Duncan test showed that hydrolysis temperature
of 55°C was significantly different with hydrolysis temperature of 50°C and
was not significantly different with hydrolysis temperature of 60°C. Therefore,
hydrolysis temperature of 55°C was decided as the best temperature for peanut
meal peptone production using crude papain concentration of 0.4% for 4 h.
The increase of temperature affects chemical reaction rate due to the
increase of kinetic energy between reactant. This phenomenon also occurred in
enzymatic reaction which involves substratesand enzymes. However, because
an enzyme is protein, the increase of enzymatic reaction will be performed on
certain temperature. If hydrolysis temperature is too high, protein enzyme can be
denaturated, tertiary enzyme structure can be disrupted and the enzyme catalytic
activity will decrease [7].

Figure 2. The effect of hydrolysis temperature on Ratio


of NTT/NTB. Different superscript indicated significant
difference (p < 0.05).
90 Proceeding ASEAN COSAT 2014

Amino Acid Content in Peptone Produced


The concentration of amino acids in peptone produced was different with
commercial peptone (BactoTMpeptone). Peanut peptone contained three highest
amino acids, namely 1.63% glutamic acid, 0.87% aspartic acid and 0.78%
arginine while the lowest amino acid was methionine (0.06%). Commercial
peptone contained the highest amino acid namely glycine amounted 2.03%.
The different of amino acid content is caused by the difference of raw material
used in peptone production. [8] stated that BactoTMpeptone was produced by
using of animal protein as raw material and hydrolyzed by pancreatic enzyme.

Application of Peptone Produced as Bacterial Growth Media


Escherichia coli
Testing result of peptone produced on formulation of liquid media showed
that peanut peptone produced had the higher ability for growing of E. coli than
BactoTMpeptone. This can be showed by the results of turbidity determination
of the culture (OD620 nm) which relatively higher than commercial peptone
(Figure 3a).
Statistical analysis showed that OD620 nm of culture broth using peanut
peptone produced was significantly different with commercial peptone. Different
with liquid media, application of peanut peptone produced in solid media showed
that the growth ability was not significantly different with commercial peptone
(Figure 3b).
According to [9], amino acid serine, aspartic acid, and glutamic acid are amino
acids which are very important for bacterial growth. Peanut peptone produced
contained higher glutamic acid and aspartic acid than commercial peptone, so
those could support better cells growth of bacteria.

Staphylococcus aureus
Figure 4a showed testing result of peanut peptone produced for growing of
Staphylococcus aureus. The result showed that peptone produced had higher ability
to support growth of S. auerus than commercial peptone. Statistical analysis
showed that the growth response of peanut peptone produced was significantly
different with commercial peptone.
The same with its response to E. coli, application of peanut peptone in solid
media for growing Staphylococcus aureus showed that they were not significantly
different (Figure 4b). By testing the peanut peptone produced for growing of
E. coli and S. aureus both in solid and liquid media, generally, peanut peptone
produced has similar ability with commercial peptone BactoTMpeptone from BD.
Innovation for Better ASEAN Community 91

*Peptone on hydrolysis temperature: 50°C (S50); 55°C (S55); 60°C (S60); and commercial
peptone (C)
(a) (b)
Figure 3. Growth for Escherichia coli in (a) liquid media and (b) solid media. Different superscript
indicated significant difference (p < 0.05).

*Peptone on hydrolysis temperature: 50oC (S50); 55oC


(S55); 60oC (S60); and commercial peptone(C)

Figure 4a. Growth of Staphylococcus aureus in liquid


media. Different superscript indicated significant
difference (p < 0.05).

IV. CONCLUSION
Peanut peptone can be produced by hydrolysis of peanut meal using crude papain
enzyme. Production process of the peptone can be performed by hydrolysis
condition using 0.4% of crude papain for 4 hat temperature of 55 oC. The yield
peptone produced was 40.8%. Amino acids content in peanut peptone that was
obtained had high glutamic acid, aspartic acid and arginine. Generally, peanut
peptone produced has similar ability with commercial peptone BactoTMpeptone
for growing of E. coli and Staphylococcus aureus in liquid and solid media.

V. ACKNOWLEDGMENT
This research was funded by Directorate General of Higher Education, Ministry
of National Education from the scheme of Student Creativity Programme 2014.
92 Proceeding ASEAN COSAT 2014

*Peptone on hydrolysis temperature:


50 °C (S50); 55 °C (S55); 60 °C
(S60); and commercial peptone (C)

Figure 4b. Growth of Staphylococcus


aureus in solid media. Different super-
script indicated significant difference
(p < 0.05).

VI. REFERENCES
1) Badan Pusat Statistik. (2014). Tabel impor menurut komoditi. Retrieved from
http://www.bps.go.id/exim-frame.php?kat=2&id_subyek=08&notab=50
2) Wijayanti, A. T. (2009). Kajian penyaringan dan lama penyimpanan dalam
pembuatan fish peptone dari ikan selar kuning (Caranx leptolepis) (Master’s
thesis). Bogor, Indonesia: Institut Pertanian Bogor.
3) Rose, A. H. (1980). Micobial Enzyme and Bioconversion (Economics
Microbiology Vol. 5). New York, US: Academic Press.
4) Benjakul, S. and M. Morrisey. (1997). Protein hydrolysates from pasific whit-
ing solid wastes. Journal of Agricultural and Food Chemistry, 45, 3423–3430.
5) Shahidi, F., Xio-Qing, H. and J. Synowiecki. (1995). Production and
characteristics of protein hydrolysates from capelin (Mallotus villosus). Food
Chemistry, 53, 285–293.
6) Netto, F. M. and M. A. M. Galeazzi. (1998). Production and characterization
of enzymatic hydrolysates from soy protein isolate. Lebensmittel-Wissenschaft
& Technologie, 31 (8), 624–631.
7) Thenawidjaja, M. (1984). Pengantar Kinetika Enzim. Bogor, Indonesia:
Institut Pertanian Bogor.
8) Becton, Dickinson and Company. (2009). DifcoTM & BBLTM Manual of
Microbiological Culture Media 2nd ed. Sparks, Maryland: BD Diagnostics.
9) Selvarasu, S., Wei Ow, D. S., Lee, S. Y., Lee, M. M, Weng Oh, S. K., Karimi,
I. A. and D. Y. Lee. (2008). Characterizing Escherichia coli DH5α growth
and metabolism in a complex medium using genome-scale flux analysis.
Biotechnology and Bioengineering, 102, 923–934.
Potency of IAA Hormone Produced by
Endophytes Bacteria Isolated from Shorea
selanica on Supporting the Growth of
Paraserinthes falcataria

Tiwit Widowati*, Sylvia J. R. Lekatompessy, Nuriyanah and Harmastini Sukiman


Research Center for Biotechnology-LIPI
Jl. Raya Bogor KM 46, Cibinong, Bogor, Indonesia

Abstract
Indole acetic acid is a key hormone for various aspects of plant growth and development. The
objectives of this study were to screen endophytes bacteria from Shorea selanica that are able
produce IAA hormone and determine the effect of IAA hormone on the growth of Paraserianthes
falcataria. Eight isolates of endophytes were studied by growing them in Nutrient Broth medium
supplemented with Tryptophan. Isolates SSBt1 and SSBt2 had ability to produce the highest IAA
with concentration about 40 µg/ml. Both isolates were studied on producing IAA at different pH
for 120 hours and were also selected for determining their capability on supporting the growth of
P. falcataria in semi solid media. The results indicated that IAA produced by endophytes bacteria
isolated from S. selanica are able to induce elongation of primary root and stem, numerous of
lateral and sub lateral roots of P. falcataria.
Key words: IAA, Endophytes bacteria, Paraserianthes falcataria

I. INTRODUCTION
Shorea selanica is a Dipterocarp family, which has a high economic value. It is
valuable for joinery, furniture, paneling, flooring and plywood manufacture [1].
The resin, called damar, is used in traditional medicine, while the bark is used for
tanning. Dipterocarpaceae is one of forest plants which have suffered a reduction
in population mainly because of timber exploitation. The conservation efforts for
these species have been initiated in Indonesia.
Shorea is one of featured trees that recommended by Indonesian government
for rehabilitation and management of natural and industry forest. The aims of
forest management are to establish a healthy, prospective and sustainable forest
[2]. One of the components that were developed in the forest management
program is the use of microbes to support the growth of seedlings. A healthy
forest ecosystem inhabited by beneficial organisms such as microbes producing
plant growth regulators [3]. It is reported that the rhizosphere of soil ecosystems
* Corresponding author. Phone: +62-21-8754587. Email: tiwitwidowati@yahoo.com

93
94 Proceeding ASEAN COSAT 2014

will be inhabited by beneficial organisms, including microbes producing plant


growth regulators. Microbe,s mainly bacteria, can live symbiotically with plants,
free-living in the soil or around plant roots (rhizosphere) [4].
Endophytic bacteria can be defined as those bacteria that colonize the internal
tissue of the plant without harming the host plant. These bacteria play a role in
supporting the growth of plants in several ways, such as through the secretion
of plant growth regulators, phosphate solubilizing, siderophore production and
supply of nitrogen [5]. Growth hormones, such as auxin, cytokinins, gibberellins,
ethylene and absisic acid are organic compound in low concentration that affect
the growth, differentiation and development of plants [6].
Auxin is a plant hormone that regulates several physiological processes, such
as stimulates cell division and elongation, root initiation, seed germination and
seedling growth. IAA is an endogenous auxin hormone that is synthesized in stem
and roots. L-tryptophan is an amino acid as precursors for biosynthesis of auxins
in plants and microbes [7]. Biosynthesis of IAA-producing microbes in the soil
can be stimulated by the tryptophan derived from root exudates. This precursor
contains active compounds that stimulate the growth of microbes in producing
secondary metabolites.
The utilization of endophytes bacteria as phytohormone that isolated from
forest plant in the tropics is still limited. It is necessary to study the application of
IAA-producing bacteria on stimulating plant growth. In this study, the bacteria
were selected to determine their capability on producing IAA hormone and
compatibility on supporting the growth of Paraserianthes falcataria root.

II. MATERIALS AND METHODS


Isolation and screening of IAA production
Shorea selanica samples were obtained from Forest Research of Mount Dahu,
Bogor. Isolation of endophytes bacteria from the stems and leaves of Shorea
selanica was done according to [8]. Isolation and screening of endophytic bacteria
was performed in the Laboratory of Plant Symbiotic Microbes, Biotechnology
Research Center of LIPI. Bacteria isolates were selected for IAA production in vitro
by Dey et al. [9] method. These pure cultures were grown on 10 ml of Nutrient
Broth (NB) containing 0.5 mM L-tryptophan. These cultures were incubated at
28°C with shaking at 150 rpm for 24 hours and then harvested by centrifugation
at 10,000 rpm for 10 minutes. IAA production was tested by colorimetric using
a reagent Salkowsky [10]. Reagent Salkowsky contained 150 ml H2SO4, 250
ml aquades, 7,5 ml FeCl3.6H2O 0.5M. Two ml of supernatant was mixed with
2 ml of reagent Salkowsky. The suspension was incubated at room temperature
in dark for 60 minutes and then Optical Density (OD) was measured with a
Innovation for Better ASEAN Community 95

spectrophotometer at wavelength of 530 nm. The appearance of a pink color


indicated IAA production. The value of IAA produced was compared with the
IAA standard.

Culture optimization for the production of IAA


The isolates, which produce the highest IAA, was studied to identify the optimal
condition for IAA production. IAA concentration was measured using different
parameters. The effect of incubation time on IAA production was studied by
cultivating the isolates in NB media containing 0,5 mM L-tryptophan. Samples
were observed every 24 hours for 5 days. The effect of pH on IAA production
was studied by cultivating the isolates in NB containing 0,5 mM L-tryptophan
at different pH levels ranging from 4.0–7.0 for 3 days.

Biological Test on the Growth of P. falcataria


Another experiment was done to see if IAA producing Shorea selanica can induce
rooting in another plant species. Isolates which produce the highest of IAA were
grown on NB medium and incubated for 24 hours. Seedlings of Paraserianthes
falcataria were soaked in 10 ml of bacterial suspension for 30 minutes. Each
seedling was inserted into tube containing 25 ml of agar 1%, then added 1
ml of bacterial suspension in each tube. On the control treatment, seedlings
of P. falcataria were added with NB. Each treatment consisted of 5 replicates.
Measurements of growth were done after 2 months planting, consisted of stem
length, root length, number of lateral and sub lateral roots.

III. RESULT AND DISCUSSION


Eight endophytic bacteria have been obtained from the stems and leaves of S.
selanica. Morphologically, there is a difference round colony size ranging 1–3
mm and color variations (white and pink) (Table 1).
Table 1. Colony color of endophyte bacteria from Shorea selanica
No Code of isolate Color of colony
1 SSBt1 White
2 SSBt2 White
3 SSKK2 White
4 SSKK4 White
5 SSKK6 White
6 SSDt1 Pink
7 SLBt1 White
8 SLBt2 Pink
96 Proceeding ASEAN COSAT 2014

Differences of shapes, sizes, color and the amount of endophytic bacteria


obtained, related to the type and age of plant, the type of infected tissue, sampling
time and environment. Environment where the plant grows also affects the
composition and structure of microbial species that colonize the roots, stems,
twigs and leaves [11].
All of the isolates were selected capability to produce the IAA hormone. There
are 3 isolates that able to produce IAA ranged from 15–43 µg/ml, whereas the
other isolates could not produce IAA.
Table 2. IAA production of endophyte bacteria from S. selanica
No Code of isolate IAA production (µg/ml)
1 SSBt1 39,89041
2 SSBt2 43,00685
3 SSKK2 -0,4863
4 SSKK4 -2,69863
5 SSKK6 5,061644
6 SSDt1 -3,59589
7 SLBt1 -3,4863
8 SLBt2 -5,31507
Isolates that were able to produce IAA indicates that it can synthesize trypto-
phan contained in the media to become IAA. The difference of IAA concentration
produced by bacteria might be caused by differences of bacteria ability to utilize
tryptophan. These differences are influenced by differences of indole pyruvate
decarboxylase enzyme activity. The concentration of IAA produced also depends
on the activity and the number of cells, nutrients and substrate availability of
tryptophan in the medium. The presence of tryptophan in the medium causing
microbes can produce IAA and other compounds in large quantities [12].
Optimization of the production of IAA from isolates SSBt1 and SSBt2 that
produce the highest IAA, started after 24 hours and reached a maximum after 72
hours and then decreased slowly (Figure 1). IAA is a secondary metabolite that is
excreted by the microorganisms nearing the end of the growth phase or during
the stationary phase. According to Lestari et al. (2007) [13], at the beginning of
growth, there are many nutrients so that IAA production is high and continues to
increase, but not significantly and consistently until the end of the growth phase.
According to Kresnawaty et al. (2008) [14], at 24-hours incubation time,
production of IAA was still minimal because it is still in the logarithmic phase
and the content of enzymes to convert tryptophan is still low. At 72-hours
incubation time, the highest IAA was produced because isolate was at the end
of logarithmic phase and the enzymes used to synthesize tryptophan into IAA is
quite high and active in line with the growth rate. The enzyme involved in the
Innovation for Better ASEAN Community 97

Figure 1. Effect of incubation time on IAA production from isolates SSBt1 and SSBt2

synthesis of IAA including tryptophan monooxygenase, IAM hydrolase, indole-


pyruvate decarboxylase and IAAld dehydrogenase. Declining of IAA production
after 72-hours incubation time caused the isolates to enter the death phase. This
decline is caused by release of IAA degrading enzyme like IAA peroxidase and
IAA oxidase [15].
The maximum production of IAA was at pH 5 (Figure 2). There was significant
relationship between the growth of bacteria and IAA production where pH
influenced the function of enzyme system and dissolves the important compounds
for bacteria growth. pH is too acidic or alkaline and this is not suitable for IAA
production.
Biological test showed that the growth of P. falcataria treated with inoculants
was higher than control. The height of plant and length of roots (Figure 3) looked
significantly different.
IAA produced by SSBt1 and SSBt2 isolates showed the ability of bacteria
to stimulate the primary roots growth of P. falcataria, where the roots that were
treated by endophyte bacteria was 65% longer compared to control. Plant heights
of treatment were 45% higher than control. There was a significant relationship
between auxin produced endophyte bacteria by in vitro with the growth of P.
falcataria seeds. This indicated that auxin was produced both of isolates causing
improvement of root system to resulted greater biomass.
Seeds of P. falcataria were inoculated with SSBt1 and SSBt2 isolates stimulated
the growth of lateral roots number compared to controls (Figure 4). The ability
of bacteria to produce high IAA may be related to the high level of IAA in the
roots. Endophyte change IAA on the roots that contribute in the development
98 Proceeding ASEAN COSAT 2014

Figure 2. Effect of pH on IAA production of isolates SSBt1 and SSBt2

Figure 3. Plant height and root length of P. falcataria

of root hairs and root number in plants. Interaction between IAA-producing


bacteria and plants resulted in plant growth promoter [16].
Auxins were synthesized naturally by plants from amino acid tryptophan.
The ability to synthesize IAA spread on soil bacteria and bacteria associated with
plants [17]. IAA was secreted by plant growth promoter bacteria directly through
Innovation for Better ASEAN Community 99

Figure 4. Number of lateral and sublateral root of P. falcataria

stimulation of plant cell elongation or cell division or indirectly influenced by


ACC deaminase enzyme activity. ACC deaminase in bacteria was involved in
root elongation of seed [10]. The synthesis of plant growth regulator microbe is
an important factor for soil fertilization.

IV. CONCLUSION
This study showed that bacteria SSBt1 and SSBt2 isolated from stem of Shorea
selanica had the capability to produced IAA on medium containing tryptophan.
Both isolates were also effective and compatible to stimulate elongation of P.
falcataria roots.

V. REFERENCES
1) Sakai, C., Subiakto, A., Heriansyah, I. and H. S. Nuroniah. (1999). Reha-
bilitation of degraded forest with Shorea leprosula and Shorea selanica. In S.
Kobayashi, J. W. Turnbull, T. Toma, T. Mori, and N. M. N. A. Majid (Eds.),
Rehabilitation of Degraded Tropical Forest Ecosystems. Workshop Proceedings
2-4 November (pp. 191–195). Bogor, Indonesia: CIFOR.
2) Soekotjo. (2007). Komponen silvikultur intensif dalam rangka membangun
hutan yang sehat, prospektif dan lestari. Jakarta: Dirjen Bina Produksi
Kehutanan, Departemen Kehutanan RI.
3) Hindersah, R. and T. Simarmata. (2004). Potensi rhizobakteri Azotobacter
dalam meningkatkan kesehatan tanah. Jurnal Natur Indonesia, 5, 127–133.
100 Proceeding ASEAN COSAT 2014

4) Glick, B. R. (1995). The enhancement of plant growth by free-living bacteria.


Canadian Journal of Microbiology, 41, 109–117.
5) Bandara, W. M. M. S., Seneveratne, G. and S. A. Kulasooriya. (2006).
Interactions among endophytic bacteria and fungi: effects and potentials.
Journal of Biosciences, 31, 645–650.
6) Kiba, T., Kudo, T., Kojima, M. and H. Sakakibara. (2010). Hormonal control
of nitrogen acquisition: roles of auxin, abscisic acid and cytokinin. Journal of
Experimental Botany, 62, 1399–1409.
7) Tarabily, K., Nassar, A. H. and K. Sivasithamparam. (2003). Promotion of
plant growth by an auxin producing isolate of the yeast Williopsis saturnus
endophytic in maize roots. Paper presented at the Sixth U. A. E. University
Research Conference, 2003.
8) Tomita, F. (2003). Endophytes in Southeast Asia and Japan: their taxonomic
diversity and potential applications. Fungal Diversity, 14, 187–204.
9) Dey, R., Pal, K. K., Bhat, D. M. and S. M. Chauhan. (2004). Growth promo-
tion and yield enhancement of peanut (Arachis hypogeal L.) by application
of plant growth promotion rhizobacteria. Microbiological Research, 159,
371–394.
10) Patten, C. L. and B. R. Glick. (2002). Role of Pseudomonas putida indoleacetic
acid in development of the host plant root system. Applied and Environmental
Microbiology, 68, 3795–3801.
11) Araujo, W. L., Marcon, J., Maccheroni, W., Elsas, J. D., Vuurde J. W. L. and
J. L. Azevedo. (2002). Diversity of bacterial populations and their interaction
with Xylella fastidiosa in citrus plant. Applied and Environmental Microbiology,
68, 4906–4914.
12) Lee, S., Encarnation, M. F., Zentalla, M. C., Flores, L. G., Escamilla, J. E.
and C. Kennedy. (2004). Indole-3 acetic acid biosynthesis is deficient in
Gluconacetobacter diazotrophicus strains with mutations in cytochrome c
biogenesis genes. Journal of Bacteriology, 186, 5384–5391.
13) Lestari, P., Susilowati, D. N. and R. I. Riyanti. (2007). Pengaruh hormon
asam indol asetat yang dihasilkan oleh Azospirillum sp terhadap perkembangan
akar padi. Jurnal AgroBiogen, 3, 66–71.
14) Kresnawaty, I., Andawarih, S., Suharyanto and T. Panji. (2008). Optimization
and purification of iaa produced by Rhizobium sp. in latex serum media
supplemented with tryptophan from chicken manure. Balai Penelitian
Bioteknologi Perkebunan, 76, 74–82.
Innovation for Better ASEAN Community 101

15) Datta, C. and P. S. Basu. (2000). Indole acetic acid production by a Rhizobium
species from root nodules of leguminous shrub, Cajanus cajan. Microbiological
Research, 155, 123–127.
16) Spaepen, S., Fanderleyden, J. and R. Remans. (2007). Indole acetic acid in
microbial and microorganism-plant signaling. FEMS Microbiology Reviews,
31, 425–448.
17) Verma, S. C., Ladha, J. K. and A. K. Tripathi. (2001). Evaluation of plant
growth promoting and colonization ability of endophytes diazotrophs from
deep water rice. Journal of Biotechnology, 91, 127–141.
Production and characterization of
the Biosurfactant by the formation of
glycolipid isolated from Pseudozyma
hubeiensis Y10BS025

Martha Sari*, Fifi Afiati and Wien Kusharyoto


Research Center for Biotechnology-Indonesian Institute of Sciences (LIPI)
Jalan Raya Jakarta-Bogor Km. 46, Cibinong, Indonesia

Abstract
The present study demonstrates the production and properties of a biosurfactant isolated from
P. hubeiensis Y10BS025. Biosurfactants are produced by a variety microorganism to produce
renewable resources which have a unique properties. P. hubeiensis was cultivated in a medium
containing glucose and soybean oil to induce and intensify biosurfactant (bioemulsifier) synthesis.
To confirm the ability of isolate in glycolipid biosurfactant production, TLC pattern, emulsification
test and stability of the biosurfactant was investigated. The strain Y10BS025 produce bioemulsifier
and exihibit emulsification index (E24) of 47–72% with oils tested and resultant emulsion
found more stable compare to chemically made surfactant. This strain is a potential candidate
for microbial enchanced oil recovery.
Key words: P. hubeiensis, Biosurfactant, Glycolipid, TLC, Emulsifier

I. Introduction
There is a considerable interest in the development of biobased materials, such
as biosurfactants molecule from the global environmental. Microbes are well
known for their varied bioactive properties, include production of secondary
metabolites and bioactive compounds [7]. Biosurfactant are a structurally diverse
group of surface active molecules synthesized by various microbial genera like
bacteria, fungi and yeast [1]. Biosurfactants can aggregate at interfaces between
fluids having different polarities, such as water and oil, leading to the reduction
of interfacial tension, therefore become a good candidates as enhanced oil
recovery [6]. Due to these superior characteristics, biosurfactants have potential
use in pharmaceutical industries, cosmetics, food and energy saving technology
[10]. Several type of biosurfactant have been isolated and characterized, based
on glycolipids, phospholipids, lipopeptides, natural lipids, fatty acids and
lipopolysacharides properties. The principle screening of biosurfactant production

* Corresponding author. Phone: +628754587. Email: martha.biotek@gmail.com

103
104 Proceeding ASEAN COSAT 2014

is finding new structures with strong interfacial activity, high emulsion stability,
and good solubility [4].
Yeast glycolipids biosurfactant, mannosylerythritol lipids (MELs) are one of
the most promising biosurfactants known and are abundantly produced from
vegetable oils by Pseudozyma [8]. The strain showed the ability to produce
assayable biosurfactant activity [9]. Sesame oil is the best substrate for glycolipid
biosurfactant production; however, the production and recovery processes are
very complicated. Therefore, we used soybean oil as raw material for producing
glycolipid biosurfactant using P. hubeiensis instead of sesame oil. The purification
was done using different solvents, and it determined the active emulsion properties
of the biosurfactant within 30 days stability compared with chemicals surfactant,
Triton X at room temperature.

II. Method/material
A. Biosurfactant production
P. hubeiensis YB10BS025 in this study was obtained from Indonesian Institute of
Sciences (LIPI). Standard procedure for the production of glycolipid by fermenta-
tion was adopted from Sari et al., 2013. Seed culture were prepared by growing
cells in medium (40 g glucose/l, 3 g NaNO3/l, 0.3 g Mg SO4/l, 0.3 KH2PO4/l, 1 g
yeast extract/l) at 30°C on a reciprocal shaker (200 rpm) for 2 days. Seed culture
(0.1 ml) were transferred to Erlenmeyer flask containing 100 ml basal medium
using 40 g soybean oil/l as substrate. Cultivation was performed at 30°C, for 8
days using agitation at 150 rpm. After cultivation. The biosurfactant was harvested
from the supernatant, after the cells were separated by centrifugation at 6,000
rpm for 20 min and washed twice with distilled water under sterile conditions.

B. Isolation of glycolipid biosurfactant


The glycolipid biosurfactant, MELs produced were extracted from the culture
medium with an equal amount of ethyl acetate (1:1, v/v) for 1 h [5]. The organic
layer was separated and evaporated. Its volume was measured and concentrated
using vacuum evaporation. The concentrate of the glycolipid was washed with
300 ml n-hexane-methanol-water (1:6:3) to remove the remaining oil and fatty
acids (top phase) and then purified by silica gel (Wako gel C-200) column
chromatography using a gradient elution of chloroform/acetone (10:0-7:3,
v/v) mixture as solvent systems [2]. A separation of residual fatty acid was
achieved by using methanol and hexane. The extracts were analyzed by thin layer
chromatography (TLC) on a silica gel plate G-60 (Merck) with a solvent system
consisting of chloroform-MeOH-NH4OH (65:15:2, v:v:v). The compounds on
Innovation for Better ASEAN Community 105

the plates were located by charring at 110°C for 5 min after spraying an anthrone
reagent as previously reported [3].

C. Assessment of bioemulsification index (E24) & Stability of emulsion


Bioemulsification index of culture sample was measured by adding 2.0 ml of
various hydrocarbon (sesame oil, canola oil, olive oil, crude oil, and soya oil)
to the same amount of culture, vortex for 30 min in individual tubes, and kept
at room temperature. Emulsification index (E24) was calculated after 24 h, by
measuring the height of emulsion layer with respect to original volume and
expressed as percentage.
For the assessment of stability of emulsion, extract purified obtained from
experiments are kept at room temperature for the period of 0–30 d. Sample
exhibiting nilphase separation were considered as high stable emulsion [7].

III. R esults and discussion


In this study, P. hubeiensis Y10BS025 was tested for ability to produce glycolipids
biosurfactant using soybean oil as substrate. We found that glycolipid biosurfactant
are abundantly produced by P. hubeiensis Y10BS025 with wide variety of ratio of
eluent in isolation systems. The mixture glycolipids were extracted from the total
culture suspension of P. hubeiensis Y10BS025, and purified by solvent diversifica-
tion system. The key components of the major glycolipid were checked into five
main fractions eluted base on the solvent mixture (Figure 1). As shown in Figure
1, the partial glycolipid contains those five fractions, named A, B, C, D, and E.
On TLC, the major glycolipid spot from the strain P. hubeiensis Y10BS025
was selected based on the anthrone reagent positive spots as those in the brown
bead of silica gel plate. The fraction of glycolipid was eluted mainly with fraction
chloroform/acetone (B). This result indicated that P. hubeiensis Y10BS025 is able
to use glucose as the carbon and soybean oil as substrate to provide glycolipid
biosurfactant. We also detected some glycolipid spot that eluted together with
residual oils in the chloroform/acetone fraction (C) when analyzed on TLC
(blue beads). In the extract from the broth culture with combination glucose and
soybean oil, by product of the residual oil were separated by using methanol (D)
with subsequent threefold extraction by n-hexane.
The varied solvent extraction is the most commonly used technique for isola-
tion and purification of biosurfactant spot on silica gel plate. The choice of method
for the purification of a particular biosurfactant depends on its ionic charge, its
solubility in water, and on whether the product is cell bound or extracellular [3].
Yeast strain belonging to genus Pseudozyma showed potential emulsification
activity. Figure 2 shows that different emulsification indexes of the crude
106 Proceeding ASEAN COSAT 2014

Figure 1. TLC pattern of the extracellular products from


soybean oil by P. hubeiensis Y10BS025 under different
fractions (A) crude ethyl acetate, (B) fraction chloroform/
acetone 10:0, (C) fraction chloroform/acetone 7:3, (D)
methanol fraction, (E) hexane fraction.

A B C D E

biosurfactant produced by P. hubeiensis strain Y10BS025 in a comparison with


Triton X using different oils. The crude biosurfactant was found to be able to form
stable emulsion between water and various oils (soybean oil, sesame oil, crude
oil, canola oil). Results showed that soybean oil has maximum activity against
all of test oils. The emulsifying index of the biosurfactant ranged from 47–72%.
In all oils tested, supernatant supplemented with soybean oil gave a better yield
was achieved 72% of emulsification index compared to synthetic supernatant,
Triton X (57%). On other hand, crude oil were found to be good substrates for
emulsification by the glycolipid biosurfactant from P. aeruginosa strain SP4 [6].
With regard to stability of emulsions, we observed emulsion (without emulsi-
fiers, with bioemulsifiers of Y10BS025, and synthetic emulsifiers). Amongst the
three bioemulsifier tested, the Y10BS025 bioemulsifier exhibits the good stability
emulsion and equivalent with synthetic surfactant without any phase separation
(Figure 3). However, Radhakrishnan et al., 2011 reported stability of emulsion
formation with crude oil up to 90 d. In general, stability of emulsion depends
on various physical and biochemical factors, include distribution of emulsion,
size of particals, external force, ion strength, structure, molecular weight of the
emulsifier, pH, metal ions, and finally the temperature.

IV. CONCLUSION
In this study, P. hubeiensis Y10BS025 was found to produce glycolipid biosurfac-
tant from a fermentation medium containing soybean oil as substrate. The major
product was identified on the fraction chloroform/acetone that appears as positive
brown bead spot in silica plate. The crude biosurfactant showed comparable
physicochemical properties in term of the emulsification index compared to
synthetic surfactants (Triton X). The emulsion found stable without any phase
separation till 30 days. This findings indicated that the present strain have great
potential for microbial enchanced oil recovery (MEOR) applications.
Innovation for Better ASEAN Community 107

Figure 2. Emulsifying index of the bioemulsifiers of yeast


strain with various oils, compared with chemical emulsifier,
Triton X.

Figure 3. Comparison of emulsion stability of (a) without emulsifier,


(b) with bioemulsifiers Y10BS025, and (c) with synthetic emulsifiers,
observed before and after 30 days of incubation.
108 Proceeding ASEAN COSAT 2014

V. Acknowledgement
We would like to thank Dr. Atit Kanti (Indonesian Institute of Science) for
provision of isolate in this study.

VI. R eferences
1) Khopade, A., Ren, B., Liu, X. Y., Mahadik, K., Zhang, L. and C. Kokare.
(2012). Production and characterization of biosurfactant from marine
Streptomyces species B3. Journal of Colloid and Interface Science, 367, 311–318.
2) Konishi, M., Nagahama, T., Fukuoka, T., Morita, T., Imura, T., Kitamoto,
D. and Y. Hatada. (2011). Yeast extract stimulates production of glycolipid
biosurfactants, mannosylerythritol lipids, by Pseudozyma hubeiensis SY62.
Journal of Bioscience and Bioengineering, 111 (6), 702–705.
3) Liu, Y., Koh, C. M. J. and L. Ji. (2011). Bioconversion of crude glycerol to
glycolipids in Ustilago maydis. Bioresources Technology, 102, 3927–3933.
4) Morita, T., Konishi, M., Fukuoka, T., Imura, T., Yamamoto, S., Kitagawa,
M. and D. Kitamoto. (2009). Production of a novel glycolipid biosurfactants,
mannosylerythritol lipids, by Pseudozyma parantarctica and their interfacial
properties. Journal of Bioscience and Bioengineering, 105 (5), 493–502.
5) Morita, T., Ogura, Y., Takashima, M., Hirose, N., Fukuoka, T., Imura, T.,
Kondo, Y. and D. Kitamoto. (2011). Isolation of Pseudozyma churashimaensis
sp, a novel ustilaginomycetous yeast species as a producer of glycolipid
biosurfactant, mannosylerythritol lipids. Journal of Bioscience and Bioengineering,
112 (2), 137–144.
6) Pornsunthorntawee, O., Wongpanit, P., Chavadej, S., Abe, M. and R.
Rujiravanit. (2008). Structural and physicochemical characterization of
crude biosurfactant produced by Pseudomonas aeruginosa SP4 isolated from
petroleum contaminated soil. Bioresource Technology, 99, 1589–1595.
7) Radhakrishnan, N., Kavitha, V., Madhavacharyulu, E., Gnanamani, A.
and A. B. Mandal. (2011). Isolation, production and characterization of
bioemulsifiers of marine bacteria of coastal Tamil Nadu. Indian Journal of
Geo-marine Science, 40, 76–82.
8) Sari, M., Kanti, A., Artika, M. and W. Kusharyoto. (2013). Identification
of Pseudozyma hubeiensis Y10BS025 as a potent producer of glycolipid
biosurfactant mannosylerythritol lipids. American Journal of Biochemistry and
Biotechnology, 9 (4), 430–437.
Innovation for Better ASEAN Community 109

9) Sari, M., Kusharyoto, W. and M. Artika. (2014). Screening for biosurfactant


producing yeast: Confirmation of biosurfactant production. Biotechnology,
13, 106–111.
10) Takahashi, M., Morita, T., Wada, K., Hirose, N., Fukuoka, T., Imura, T. and
D. Kitamoto. (2011). Production of sophorolipid glycolipid biosurfactant
from sugarcane molasses using Starmerella bombicola NBRC 10243. Journal
of Oleo Science, 60 (5), 267–273.
APPLICATION OF MARKER ASSISTED SELECTION
AND SENSORY TEST FOR SELECTING AROMA ON
F2 PROGENY OF RICE DERIVED FROM CROSSING
BETWEEN CIHERANG X BASMATI
Santika Saria, Nono Carsonob,*, Murdaningsih Haerumanb and Farida Damayantib
Graduated Student, Master Program in Plant Breeding
a

Faculty of Agriculture, Universitas Padjadjaran, Jatinangor, Sumedang, Indonesia


Phone/Fax +62227796316
b
Laboratory of Plant Biotechnology and Breeding
Faculty of Agriculture, Universitas Padjadjaran, Jatinangor, Sumedang, Indonesia
Phone/Fax +62227796316

Abstract
Aroma is one of the characters that influences consumer acceptance in the market. Tracing
aromatic rice genotype on F2 segregated population by applying marker assisted selection
(MAS) in combination with phenotypic assessment (sensory test) is highly demanded since it
will effectively assist in finding desired genotypes. This experiment aimed to select individual
plant of F2 progeny from Ciherang (high productivity) with Basmati (aromatic rice), based
on the molecular markers and sensory test. Two hundred and thirty-three F2 rice plants were
screened. Molecular marker of Bradbury and RM223 were applied to detect fragrance gene (fgr).
The fgr gene corresponds to the BADH2 (betaine aldehyde dehydrogenase) that is responsible
for aroma metabolism in fragrant rice varieties. One percent of KOH test was used to detect
2-acetyl-1-pyrroline aromatic compound. The experiments were conducted at the Laboratory of
Plant Biotechnology and Breeding, Faculty of Agriculture, Universitas Padjadjaran. Twenty-one
genotypes were detected by molecular markers; meanwhile, fifteen genotypes were identified by
sensory test. The calculation of Spearman’s correlation resulted a value of r = 0.51 and r = 0.49
for RM223-sensory test and Bradbury’s primer-sensory test, respectively, indicated a moderate
relationship between molecular marker and sensory test. Genotype #7, 37, 49, 66, 89, 175, 206,
and 212 were confirmed by both molecular marker as well as sensory test. These genotypes are
recommended for further experiment in developing aromatic rice.
Key words: Aroma, Correlation, MAS, Rice, Sensory test

I. INTRODUCTION
The fragrance of aromatic rice is preferred by consumers all over the world. This
type of rice is highly demanded and it has a premium price in both domestic
and international markets. Rice grain aroma results from the production of many
biochemical compounds [1], but the most important compounds is 2-acetyl-1-
* Corresponding author. Phone: +6281395567514. Email: ncarsono@mail.unpad.ac.id

111
112 Proceeding ASEAN COSAT 2014

pyrroline (2AP), which is considered to be a potent aroma constituent [2]. A


single recessive gene located on chromosome 8 has been identified as the gene
responsible for the aroma trait [3,4], because it has a key role in the synthesis of
2AP [3]. The identified gene has been named differently by different groups as
BAD2 [3,5], BADH2 [6], and Os2AP [4]. The 2AP is actually detected in all parts
of the rice plant, except in the roots [7,8]. The following sensory methods have
been applied to determine the aroma in rice: chewing several seeds or cooking a
sample of seed from individual plants [9], heating leaf tissue in water or eluting
the aroma from leaf tissue with diluted potassium hydroxide (KOH) [10,11] and
heating several half-cut seeds in fresh water [12]. Chemical methods are available
which involves smelling leaf tissue or grains with solution of KOH or I2KI [10],
but these can cause damage to the nasal passages. A panel of analysts is required
to distinguish between fragrant and non-fragrant samples. However, those sensory
evaluation methods are not consistent or reliable because the aroma is subjected
to human preference.
Molecular markers, especially functional marker, which linked to economically
important traits, have been extensively exploited to assist breeders in selecting
individuals with target traits and these markers are useful with respect to the
aromatic trait in rice breeding programs. By using these markers, the difficulty in
evaluating the aroma phenotype is minimized and accuracy of selecting desired
plants is increased. Markers associated with the fgr locus could be used to assist
in distinguishing aromatic and non-aromatic rice varieties for marker assisted
selection (MAS) [13,14]. A number of marker were identified that are closely
linked to fgr [14,15] and Bradbury et al. [5] suggested that a gene encoding
putative betaine aldehyde dehydrogenase (BAD2) is most likely to be the fgr gene.
The objective of this study was to select the F2 progeny of rice plants from
crosses between Ciherang (high-yielding cultivar) with Basmati (aromatic rice),
having valuable traits: fragrance which assessed by molecular and sensory test.

II. MATERIALS AND METHODS


A. Plant materials
Two hundred and thirty three of F2 individuals derived from a cross between
Ciherang (high-yielding cultivar) and Basmati (aromatic rice) and their parents:
Ciherang and Basmati, as check, were used in this experiment. Dehulled F3 seeds
were subjected to sensory test.

B. DNA preparation and molecular analysis


The genomic DNA was isolated from young leaves according to the protocol
[16]. Concentration of DNA was estimated using a spectrophotometer (Rayleigh
Innovation for Better ASEAN Community 113

UV-9200) at a wavelength of 260 nm and 280 nm. Molecular marker for detecting
aromatic trait was carried out using RM223 [17,18] and Bradbury’s marker [19].
Termocycler PCR machine (Eppendorf ) was used. Total volume of PCR mixture
was 25 µL, consisted of 9,5 µL Nuclease-free water, 12,5 µL Go Taq® Green
Master Mix (Taq DNA polymerase, 400 µM dATP, 400 µM dGTP, 400 µM
dCTP, 400 µM dTTP, and 3 mM MgCl2), 1 µL Forward Primer, 1 µL Reverse
Primer, and 1 µL Template (DNA) 20 ng. PCR profile RM223 was 95°C for
5 min, followed by 35 cycles of 94°C for 60 seconds, 55°C for 30 seconds and
72°C for 60 seconds, and finally by 5 min at 72°C for final extention. Bradbury’s
primer was performed at 95°C for 5 min (initial denaturation); then for 35 cycles
of 95°C for 1 min; 58°C for 30 sec; 72°C for 1 min followed by 72°C for 5 min.
The amplification products were separated on 1.5%–2% Agarose gel. Gels were
then run for 75 minutes at 70V in 0.5 x TBE buffer. Agarose gel was stained
with ethidium bromide solution for 30 minutes. DNA fragments were visualized
under UV light and photographed using gel doc (Syngene Inc.).

C. Sensory test
Aromatic characteristic of rice varieties was identified by testing individual grains
or cooked rice. For the sensory test of grains, 1% KOH solution was applied to
the tissue [10]. One milligram of rice flour from single grain was soaked in 1.5
ml tube with 50 µl of 1% KOH solution at room temperature. After 30 minutes,
the tube was opened and immediately smelled. The presence or absence of aroma
was scored. Each individual sampel was evaluated by two persons.

D. Data analysis
Molecular data in terms of PCR band derived from image visualization system
using Geldoc under UV transilluminator (captured by Genesnap software pro-
gram) was interpreted. The images were then analyzed using a GeneTool software
(Syngene, UK) for ensuring the differences in size of each band. Molecular data
were scored as presence (1) and absence (0) according to band pattern of parent.
Sensory test for aroma was done as aroma presence (1) or aroma absence (0) for
each marker and data was entered in binary data matrix as discrete variables.
Selection was done on individual as detected by both methods. Spearman’s
Rank correlation coefficient was applied to identify and to test the strength of a
relationship between two sets of data.

III. RESULT AND DISCUSSION


In this study, the detection and identification of specific markers for the aroma trait
in rice was aimed attempts were made to identify a specific band of DNA amplified
114 Proceeding ASEAN COSAT 2014

product which would enable to distinguish aromatic rice from non-aromatic


ones. Bradbury’s primers were designed for allele specific amplification. External
primers produced a fragment of approximately 580 bp as a positive control for each
sample. Internal and corresponding external primers generate a 355 bp fragment
from a non-aromatic allele and a 257 bp fragment from an aromatic allele. Three
possible outcomes were obtained from the application of those four primers. In
all cases, a positive control band that approximately 580 bp was produced. In the
first case, it was indicated that individual homozygous non-fragrant had 355 bp
band. In the second case, it was indicated that individual homozygous fragrant
had 257 bp band. Meanwhile, in the last case, the occurrences of both bands
(355 bp and 257 bp) indicated that the individual was heterozygous non-fragrant.

Figure 1. PCR product visualization of several individuals from F2 population


using primer Bradbury et al. (a) and RM223 (b) to detect frg gene (NF: non-
fragrance, F: fragrance).
Innovation for Better ASEAN Community 115

RM223 is one simple sequence repeats (SSR) or microsatellite marker. The


fragments amplified ranged from 90 to 190 bp [19]. This polymorphic marker
is used to distinguish between the fgr gene and its allelomorph conferring non-
aromatic property. The 120 bp band corresponds to an allele fragrance and the
160 bp band corresponds to non-fragrance allele.
Those two primers were screened for DNA polymorphism among parent. The
primers were successfully amplifying the target locus of fgr gene from Ciherang,
Basmati, and were able to distinguish aromatic from non-aromatic genotypes
(Figure 1). Among 233 F2 individuals that scored based on the Bradbury’s marker,
21 plants were found to be homozygous aromatic lines, 209 plants and 1 plant
were found to be homozygous and heterozygous non-aromatic, respectively (Table
1). Based on RM223 marker, 20 plants were found to be homozygous aromatic
line, 209 plants and 7 plants were found to be homozygous and heterozygous
Table 1. Comparison on detection of aroma using molecular markers and sensory test for 233
genotypes derived from a cross between Ciherang x Basmati

Parent Genotype (F2 and F3) Number


Markers Recurent Donor Homozygote Heterozygote Homozygote of
Ciherang Basmati Non-Fragrance Non-Fragrance Fragrance plant
Primer Bradbury - + 209 1 21 231
PrimerRM223 - + 204 7 20 231
Sensory test (1% KOH) - + 216 0 15 231
Note : (-) non-fragrance, (+) fragrance, F2 used for molecular analysis, F3 used for sensory test

non-aromatic, respectively (Table 1). This suggests that aromatic is controlled


by a single recessive [7,10].
The presence or absence of aroma in the rice grain was assessed for 233 F3
grains derived from a cross between Ciherang x Basmati, including both parents
identified by the sensory test using 1% KOH solution. In Table 1, fifteen genotypes
of F3 have been identified having aroma trait. The concentration of 2AP in rice
is affected by environmental conditions [20]. It means that the aroma strength
in aromatic rice may decrease and/or disappear when rice grows in unfavorable
conditions. In this study, grains of F3 progeny were tested for aroma in order
to avoid the confusion due to segregation among grains in the same panicle.
Meanwhile, there was a considerable variation among the panelists in the etima-
tion of the strength of aroma. In the present study, the focus was only on the
presence or absence of aroma and no quantitative determination was performed.
Moreover, sensory assessment of aroma in rice is still favorable method applied
in some studies.
The two molecular data sets were significantly correlated (r = 0.72) according to
Spearman’s Rank correlation coefficient. The molecular data were also significantly
116 Proceeding ASEAN COSAT 2014

correlated to the phenotypic data (sensory test) (r = 0.51 for RM223-sensory test
and r = 0.49 for Bradbury’s primer-sensory test, p-value < 0.0000). These results
suggested that molecular markers and sensory test can be applied in selection of
aromatic rice. This study confirmed that the sensory test using single grain and
1% KOH solution is a suitable method for rapid identification of aroma in rice.
At the early development stages of rice plant, a small amount of leaf tissue could
be assessed before maturity by the molecular markers for earlier estimation of
aroma and for reducing the number and segregation of seed samples. Genotype
#7, 37, 49, 66, 89, 175, 206 and 212 were selected by both molecular marker as
well as sensory test. These selected genotypes will be recommended as a promosing
genotypes and utilized for backcrossing and pyramiding breeding programs.
In this study, we followed both sensory tests and molecular marker methods to
detect the presence or absence of aroma in F2 and F3. The F2 and F3 individuals
which were classified having the aroma alleles also showed presence of aroma in
sensory tests. Although they had aroma alleles and showed presence of aroma
in sensory tests, they varied from light aroma to strong aroma in leaf and grain.
However, some of the F3 individuals did not exhibit aroma in grain aromatic
test, although F2 had aroma alleles. In another cases, those F2 were classified as
absent of aroma alleles, surprisingly produced aroma in grain aromatic tests at
F3. Therefore, there were only less than 50% F2 that classified as aromatic or non-
aromatic rice by using molecular method was well agreed with sensory methods in
grain. In Hien et al. [21] report, they detected a coincidence among conventional
methods, which is 1.7% KOH sensory test, and molecular marker analysis in the
classification between aromatic and non-aromatic rice, but sometimes molecular
markers could not classify heterozygous and homozygous genotypes due to
molecular nature. However, in a research by Bounphanousay et al. [22], they
used the chemical analysis (detect 2AP) with molecular marker analysis. They
reported that the molecular markers are well agreed with the chemical analysis
in most of the rice varieties, except some contrasting results such as in a local
aromatic rice variety, Kai Noi Leuang. It was detected producing aroma but
was identified as homozygous non-aromatic by molecular marker analysis. They
claimed that different gene location might be responsible for the observed aroma
in rice or would be the presence of another major aromatic compound. In Myint
Yi et al. [23] investigation, they mentioned that the variation in the sensory score
may cause by minor genes or environmental factors or some rice varieties may
carry the minor QTLs which would have an influence on rice aroma. As sensory
quality has always been an important consideration in rice improvement (Paule
and Powers, 1989), [24] integration of sensory methods and molecular markers
is reliable, fast and cost-effective ways essential for rice breeders to evaluate rice
aroma in the breeding programs.
Innovation for Better ASEAN Community 117

Different aroma score from light to strong which were found in F2 and F3,
are homozygous aromatic, were reported in our experiments. The aroma in rice
might also be controlled by minor genes or minor QTLs [23]. Lorieux et al. [8]
reported that aroma is a quantitative trait. A major gene on chromosome 8 and
two QTLs on chromosomes 4 and 12 regulated the formation of aroma in the
rice cultivars Azucena. However, more experiments need to be carried out to
confirm the minor genes effect and their location.
Environmental factors might be another important role in determining the
aroma in rice. In our observation, it was expected that F2 individuals detected
with aroma gene (BADH2) supposed to produce aroma in grain at F3 seed.
This might be due to high temperature during their grain filling and ripening
stage [25]. During the period of grain filling to ripening, the temperature of the
planting field is around 22–29°C at day time. High temperature may also affect the
accumulation of 2-acetyl-1-pyrroline. The concentration may be very low when
the grain exposed under high temperature for a long time. Further experiments
can be carried out by controlling the temperature during the maturity period to
confirm the accumulation of 2-acetyl-1-pyrroline in rice grains. Aroma is mainly
controlled by a major gene, but it is easily influenced by environmental conditions
such as soil type, cultural practices, and temperature during the grain filling stage,
storage conditions, as well as storage time [26,27]. During our observation in rice
field, we found some F2 plants emitted pleasant aroma from the flowering organs,
especially in the morning from 8 a.m. to 10 a.m. However, pleasant aroma which
we smelled in field during flowering was a result of a large number of compounds
present in a specific proportion. In aromatic varieties, pleasant aroma is not only
associated with cooked rice. It also means that aroma emit at different stages of
rice plant growing. Whether volatile aromatic compounds released in field at the
time of flowering differ from those released after cooking is an important question
to be answered in further investigation.

IV. CONCLUSION
Based on two methods in detecting aroma trait (molecular marker and sensory
test), genotype #7, 37, 49, 66, 89, 175, 206 and 212 were selected for fgr gene.

V. ACKNOWLEDGEMENTS
Authors wish to thank Directorate General of Higher Education, Ministry of
Education and Culture in providing a research grant (Hibah Stranas) that was
awarded to Farida Damayanti.
118 Proceeding ASEAN COSAT 2014

VI. REFERENCES
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Kamolsukyunyong. (2008). Transgenic rice plants with reduced expression
of Os2AP and elevated levels of 2-acetyl-1-pyrroline. US patent 7,319,181.
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(1996). Aroma in rice: genetic analysis of quantitative trait. Theoretical Applied
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rice varieties. Plant Production Science, 9, 294-297.
Innovation for Better ASEAN Community 119

13) Wanchana, S., Kamolsukyunyong, W., Ruengphayak, S., Toojinda, T.,


Tragoonrung, S. and A. Vanavichit. (2005). A rapid construction of a physical
contig across a 4.5 cM region for rice grain aroma facilities marker enrichment
for positional cloning. Science Asia, 31, 299–306.
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PCR-based molecular markers for the fragrance gene in rice (Oryza sativa
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(2005). A perfect marker for fragrance genotyping in rice. Molecular Breeding,
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KOH method, molecular markers and measurement of 2-acetyl-1-pyrroline
concentration. Japanese Journal of Tropical Agricultural, 50 (4), 190–198.
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Hamilton. (2008). Chemical and molecular characterization of fragrance in
black glutinous rice from Loa PDR. Asian Journal of Plant Sciences, 1, 1–7.
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(2009). Marker assisted backcross breeding to improve cooking quality traits
in Myanmar. Field Crops Research, 113, 178–186.
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Variation of 2-acetyl-1-pyrroline concentration in aromatic rice grains
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affecting the concentration of the aroma compound 2-acetyl-1-pyrroline in
two fragrant rice cultivars grown in South China. Frontiers of Agriculture in
China, 4 (1), 1–9.
Development of Rice Lines Resistant to
Brown Planthopper with Aromatic Traits:
Selection Based on Molecular Marker
Anggia Puspa Asria, Nono Carsonob,* and Suseno Amienb
a
Student, Master Program in Agronomy, Universitas Padjadjaran
Jl. Raya Jatinangor KM 21 45363, Sumedang, Phone/Fax. ( +6222) 7796316, Indonesia
b
Associate Professor, Faculty of Agriculture, Universitas Padjadjaran
Jl. Raya Jatinangor KM 21 45363, Sumedang,Phone/Fax. ( +6222) 7796316, Indonesia

Abstract
Development of rice lines that are resistant to BPH is an alternative pest management. However,
most of resistant varieties is unfavorable, whereas aroma, flavor and texture are actually major
factors that consumer preferred. Selection for two different characters at the early generation (F2)
is time consuming; therefore, molecular assisted selection needs to be performed to speed up the
selection process. The objective was to obtain F2 progeny derived from Sintanur, aromatic rice,
as a recurrent parent and PTB-33, brown planthopper resistant rice, as a donor parent by using
brown planthopper resistant and aromatic markers. This experiment was done at the Laboratory
of Plant Analysis and Biotechnology, Faculty of Agriculture, Universitas Padjadjaran. A number of
261 rice plants of F2 progeny were analysed using RM589 and RM8213 for brown planthopper
resistance, while aromatic was detected by Bradburry’s primer. There were 125 genotypes which
were selected by brown planthopper resistance markers, and eight of them were selected by using
RM589, RM8213, and Bradburry’s primer. Eight selected genotypes i.e. SP35, SP55, SP84,
SP87, SP141, SP151, SP237, and SP261 are recommended for the next generation for brown
planthopper resistant and aromatic rice breeding program.
Key words: Aromatic, Brown planthopper, Molecular marker, Rice, Selection

I. Introduction
Brown planthopper/BPH (Nilapavarta lugens Stal) is one of the most destructive
pests, which causes significant losses of rice yield [1]. Development of rice lines
that are resistant to BPH is an alternative pest management. However, most
of resistant varieties is unfavorable, e.g. IR74 [2], whereas, consumer prefer to
choose rice which have good aromatic, flavor, and texture [3]. Because of that,
rice breeding and followed by selection of rice which are resistant to BPH with
aromatic trait are needed.
PTB-33 is a rice variety from India and has several Bph resistant genes such
as Bph3 and bph4 [4] and quantitative Bph (QBph). Meanwhile, Sintanur is

* Corresponding author. Phone: +6281395567514. Email: ncarsono@mail.unpad.ac.id

121
122 Proceeding ASEAN COSAT 2014

high yielding aromatic rice that could reach up to 7 ton.ha-1 from Indonesia
[5]. Combining two different characters by hybridization followed by selection
of desired genotypes are difficult and time consuming, so the right and fast
breeding methods are needed. Molecular marker offers an easy way to overcome
such problem since it speed up the selection process with high accuracy, and not
influenced by the environment [6] and development stage of plant.
Simple sequence repeat (SSR) markers that are associated with resistant to
BPH are RM589 and RM586 (linkage to Bph3 and bph4 genes) [7,8], RM8213
and RM5953 which are associated to Qbph4 and Bph17(t) [9]. For detecting
aromatic trait, Bradburry’s specific marker is widely applied. Its marker contains
of Internal Fragrant Antisense Primer (IFAP), External Sense Primer (ESP) for
detecting aromatic and Internal Non-fragrant Sense Primer (INSP), External
Antisense Primer (EAP) for detecting non-aromatic [10]. The objective was to
obtain F2 progeny derived from Sintanur, aromatic rice, as a recurrent parent
and PTB-33, brown planthopper resistant rice, as a donor parent by using brown
planthopper resistant and aromatic markers.

II. M aterial and Methods


A. Plant material
Two hundred sixty one plants from a cross between Sintanur (recipient) and
PTB-33 (donor) were used in this research. This experiment was done at
the Laboratory of Plant Analysis and Biotechnology, Faculty of Agriculture,
Universitas Padjadjaran.

B. Molecular Marker Analysis


Total genomic DNA was extracted according to Dellaporta’s method [11] with a
slight modification. PCR amplification was perfomed using DNAs from 261 F2
plant genotypes and each parent. Component reactions for PCR amplification
containing 1 µl 20 ng DNA template, 9.5 µl KAPA2GTM Fast ReadyMix (DNA
Taq polymerase (0.25 U), dNTPs (0.2 mM), MgCl2 (1.5 mM)), 1 µl each primer.
For detecting resistant to BPH, SSR primer RM589 and RM8213 were used
and Bradburry’s primer was used for detecting aromatic trait. The PCR program
for RM589 and RM8213 primers was performed at 94°C for 5 min (initial
denaturation); then for 36 cycles of 94°C for 1 min; 55°C for 1 min; 72°C for 1
min followed by 72°C for 7 min. Different from SSR primers, Bradburry’s primer
was performed at 95°C for 5 min (initial denaturation); then for 35 cycles of 95°C
for 1 min; 58°C for 30 second; 72°C for 1 min followed by 72°C for 5 min. PCR
products were separated on gel electrophoresis in 3.0% agarose gel at 75 volt for
90 min (SSR primers) and 1.5% agarose gel at 70 volt for 60 min (Bradburry’s
Innovation for Better ASEAN Community 123

primer). Visualization was done by staining on 0.2 µl/ml Ethidium-Bromide for


30 min and used gel documentation system (G-Box from Syngene) for analyzing
DNA banding pattern.

C. Data Analysis
DNA banding pattern of examined genotypes was then compared to its DNA
banding pattern of PTB-33 (for RM589 and RM8213) and of Sintanur for
aromatic trait (Bradburry’s primer). When the DNA banding pattern matched
with their parents, marked them by plus (+) and vice versa.

D. Results and Discussion


Visualization of DNA banding pattern was prior conducted by brown planthopper
resistant markers to select F2 plant that supposed to be resistant to BPH. F2 that
had DNA banding pattern same as PTB-33 were selected. There were two F2 plants
that were selected by RM589 (a) and five plants that were selected by RM8213
(b) (Figure 1). PCR product size of RM5889 (detected Bph3 and bph4) was 186
bp [8] and 177 bp for RM8213 (detected Qbph4 and Bph17(t)) [9]. Plant that
had Bph3 and bph6 genes was resistant to BPH biotype 4 [12] and PTB-33 had
resistance to BPH biotype Indian or biotype 4 [13]. Using PTB-33 as the donor
parent is the best choice for developing rice lines that are resistant to BPH.

(a)

(b)
Figure 1. DNA banding pattern visualization by RM589 (a) and RM8213 (b); (respectively,
from left to right of Ladder 100 bp (L): Sintanur (SN), PTB-33, nine number of F2 progeny
plant, and Ladder)
124 Proceeding ASEAN COSAT 2014

Figure 2. DNA banding pattern visualization by Bradburry’s primer;


(respectively, from left to right of Ladder 1kb (L), Sintanur (SN), PTB-
33, 23 number of F2 progeny plant)

Based on DNA pattern visualization, there were 125 genotypes of F2 progeny


plant that were selected by RM589 and RM8213, but just 27 genotypes that were
selected by both primers. Then, the number of 125 genotypes were analyzed by
Bradburry’s primer. Similarity of Sintanur DNA banding pattern that appeared
on progeny were selected. PCR product size of aromatic trait was 257 bp and
355 bp for non-aromatic [10]. In Figure 2, Sintanur was 257 bp (aromatic) and
PTB-33 was 355 bp (non-aromatic). On F2 progeny plant, there were three types
of band i.e. DNA banding pattern same as Sintanur, PTB-33, and have both of
them. DNA banding pattern that had two bands were heterozygous non-aromatic
[10], so it was not selected for aromatic trait. Sintanur had aromatic trait, but it
is susceptible variety to BPH so it would be better when it combined with other
resistant varieties.
There were eight selected genotypes based on molecular markers (RM589,
RM8213, and Bradburry’s primer) i.e. SP35, SP55, SP84, SP87, SP141, SP151,
SP237, and SP261. All of the selected genotype are promising lines that could
be further examined for brown planthopper resistant and aromatic rice breeding
program and also could be parents for pyramiding.

III. Conclusion
Selection based on molecular marker method would be an alternative approach for
rice breeding program on early generation especially on segregating populations.
Developing rice lines that are resistant to BPH is one of the concern of rice breeder.
Resistant rice lines to BPH that have another special character, like aromatic, have
to be developed in order to answer consumer needs.

IV. Acknowledgement
Authors wish to thank Directorate General of Higher Education, Ministry of
Education and Culture in providing research grant (Hibah Stranas) that is awarded
to Nono Carsono, Ph.D.
Innovation for Better ASEAN Community 125

V. R eferences
1) Baehaki, S. E. and I. N. Widiarta. (2009). Hama wereng dan cara pengenda-
liannya pada tanaman padi. In Padi: Inovasi Teknologi Produksi. Buku 2 (pp.
347–3833). Jakarta: LIPI Press.
2) Baehaki, S. E. (2007, August). Perkembangan wereng coklat biotipe 4. Tabloid
Sinar Tani.
3) Rohaeni, W. R., Anna, S. and M. I. Ishaq. (2012). Preferensi responden
terhadap keragaan tanaman dan kualitas produk beberapa varietas unggul
baru padi. Informatika Pertanian, 21 (2), 107–115.
4) Jairin, J., Phengrat, K., Teangdeerith, S., Vanavichit, A. and T. Toojinda.
(2007). Mapping of a broad-spectrum brown planthopper resistance gene,
Bph3, on rice chromosome 6. Molecular Breeding, 19, 35–44.
5) Suprihatno, B., Daradjat, A., Satoto, S. E., Suprihanto, Indrasari, S. D. and
W. I. Putu. (2010). Deskripsi Varietas Padi. Subang: Balai Besar Penelitian
Tanaman Padi.
6) Lakshmikumaran, M., Das, S. and P. S. Srivastava. (2003). Application of
molecular markers in Brassica coenospecies: comparative mapping and tagging
(abstract). Biotechnology in Agriculture and Forestry, 52, 37–68.
7) Jena, K. K. and D. J. Mackill. (2008). Molecular markers and their use in
marker-assisted selection in rice. Crop Science, 48, 1266–1276.
8) Jairin, J., Teangdeerith, S., Leelagud, P., Phengrat, K., Vanavhicit, A. and T.
Toojinda. (2007). Detection of brown planthopper resistance genes from
different rice mapping populations in the same genomic location. Science
Asia, 33, 347–352.
9) Sun, L., Su, C., Wang, C., Zhai, H. and J. Wan. (2005). Mapping of a major
resistance gene to the brown planthopper in the rice cultivar Rathu Heenati.
Breeding Science, 55, 391–396.
10) Bradbury, L. M. T., Henry, R. J., Jin, Q., Reinke, R. F. and D. L. E. Waters.
(2005). A perfect marker for fragrance genotyping in rice. Molecular Breeding,
16 (4), 279–283.
11) Dellaporta, S. L., Wood J. and J. B. Hicks. (1983). A plant DNA miniprepara-
tion: version II. Plant Molecular Biology, 1 (4), 19–21.
12) Baehaki, S. E. and D. Munawar. (2008). Uji biotipe wereng coklat, Nilaparvata
lugens STAL. di sentra produksi padi. Seminar Nasional Padi. 347–359.
13) Santhanalakshmi, S., Saikumar, S. and S. Rao. (2010). Mapping genetic lokus
linked to brown planthopper linked to rice Oryza sativa L. International Jounal
of Plant Breeding and Genetics, 4 (1), 13–22.
SCSER
Assessment of E-waste Recovery Facilities in
Selangor and Kuala Lumpur, Malaysia

Nurul Ain Mohd Nordin* and P. Agamuthu


Institute of Biological Sciences, University of Malaya, 50603 Kuala Lumpur, Malaysia

Abstract
The fast development of the world has increased the use of electrical and electronic equipments
(EEE). EEE is often being upgraded to more sophisticated device and users keep changing their
out of date equipment to a new product and increase EEE waste (e-waste) generation. The
implication of the increment of e-waste and its improper disposal will lead to major issues to
health and environment. Material recovery facility (MRF) of e-waste is the most critical element
in e-waste industry. This article attempts to assess e-waste recovery facilities in Selangor and Kuala
Lumpur, Malaysia. A total of 15 questionnaires were sent to MRF through email and interviews.
The results show that many challenges are faced by recovery facilities to be in e-waste industry.
E-waste recycling in Malaysia is still at infancy as there is lack of specific regulation on e-waste,
lack of MRF that carry out complete recycling process and poor e-waste disposal by public. At
the same time, this research helps us to understand various activities going on within MRF and
thus, encourage the formulation for a proper e-waste management strategy in Malaysia
Key words: E-waste, Material recovery facilities, Management, Malaysia

I. Introduction
In this era of modernization, electrical and electronic equipments (EEE) have
made our daily life more convenient. However, one of the consequences of the
rising of EEE is the generation of its waste. Waste of electric and electronic
equipments (WEEE) or e-waste is electric and electronic products that meet
their end of useful life (Sthiannopkao & Wong, 2012). According to Herat &
Agamuthu (2012), the world generates around 20–50 million tonnes of e-waste
annually and most of them are from Asian countries.

A. E-waste in Malaysia
Globally, e-waste issue has become an emerging problem that should be placed
under scrutiny (Widmer et al., 2005). This situation has affected many developing
countries, including Malaysia (Babington et al., 2010). Malaysia has became
an importer and exporter of e-waste as the geographic location of the country
is located in the middle of international e-waste trade route that makes it an

* Corresponding author. Phone: +6014-8029382. Email: nurulain.nordin90@gmail.com

129
130 Proceeding ASEAN COSAT 2014

attractive target for e-waste smugglers (Tengku-Hamzah, 2011). According to


Puckett (2005), Malaysia is listed as one of the countries that receive e-waste
from United States. The major issue of e-waste in Malaysia is its high amount
and will continue to increase exponentially (Afroz et al., 2013). Department
of Environment (DOE) Malaysia recorded 78,278.05 metric ton of e-waste in
2012. Malaysian citizens are not aware of the proper disposal of e-waste. This
lack of awareness will lead to leaking of hazardous content in e-waste and thus
harming the environment. Malaysia also suffers from an increasing volume of
illegal e-waste recycling by irresponsible party.

B. Material Recovery Facility


Material recovery facility (MRF) is a critical element within e-waste recycling
industry. At MRF, collected e-waste will be more valuable as marketable output
products including reusable systems/components (working and repairable elec-
tronics) and secondary scraps (plastics, metals and glass) (Kang & Schoenung,
2006). There are also precious metals, such as gold, silver and copper that can
be recovered from e-waste. DOE, Malaysia has encouraged the establishment of
e-waste recovery facilities and 147 recovery facilities have been established so far.

II. Methodology
The main methods for this research were survey questionnaire and interview
among managers of e-waste recovery facilities. It was followed by site visit and
observation to the recovery facilities. This study focused in Klang Valley, which
includes Malaysia capital city, Kuala Lumpur, and the most populous state in
Malaysia, which is Selangor.

III. R esults and Discussion


In Malaysia, recovery facilities are classified into “full and partial”. Full recovery
facilities are those with capacity to recycles all part of e-waste they receive, while
partial recovery facilities are those with limited capability to recycle all parts of
e-waste (Babington et al., 2010). There are five full recovery facilities and ten
partial recovery facilities involved in this study.

A. Type of e-waste collected at recovery facilities


Figure 1 shows that Klang Valley generates various type of e-waste. The result
shows that the most generated e-waste is PC. Nowadays, most electronic users
use laptops/notebooks as another alternative to PCs. Therefore, their unused PCs
were eventually becoming wastes. Most organizations and institutions change their
old PC model to more sophisticated PC that is evolving today. From the results,
Innovation for Better ASEAN Community 131

it is indicated that the majority of recovery facilities collect their e-waste from
commercial organizations like corporate companies and government agencies
(see Figure 2).

B. Source of e-waste collected at recovery facilities


Source of e-waste collected at recovery facilities are shown in Figure 2. Corporate
companies and government agencies contribute the most e-waste thrown to
recovery facilities with 30% each. Both sources provide a huge supply of e-waste
to recovery facilities as they are big scale organisations, in parallel to its high
usage of electronic devices. Electrical and electronic factories produce 20% of
e-waste, while smaller amount are from household and public. Poor collection
from household and public indicates that large amount of e-waste is stored at
household level without being collected. There might be a possibility that most
public just throw away their e-waste with other municipal waste.

Figure 1. Type of e-waste collected at re- Figure 2. Source of e-waste collected at recov-
covery facilities ery facilities

C. Residues generated by recovery facilities


There are six different types of residues generated by recovery facilities, which are
plastics, metals, glasses, PVC, fibre and dust/other waste (Figure 3). Plastics, metals
and dust/other waste are the highest types of residue generated which contribute
to 23% each. Usually plastics and metals will be sold to plastic factory and metal
scrap company, respectively. Dust and other waste will be sent to Kualiti Alam,
the only company in Malaysia that is licensed to dispose hazardous waste.
132 Proceeding ASEAN COSAT 2014

Figure 3. Residues generated by recovery facili- Figure 4. Challenges faced by recovery facilities
ties

D. Challenges faced by recovery facilities


The challenges faced by recovery facilities are shown in Figure 4. The most
prominent challenge cited are “high price of e-waste to be collected” and “low
amount of e-waste that can be collected”. Most e-waste generator demand a high
price for their e-waste when sold to recovery facilities. The recovery facilities feel
burdened by this situation as they might not obtain profitable income at the end.
Furthermore, the amount of e-waste that can be collected was quite low. This is
parallel with the other challenge, which is “lack of awareness from public”. In
reality, the generation of e-waste was higher than expected. However, the public
did not have enough knowledge to properly dispose their e-waste and eventually
the e-waste will end up in municipal landfill.

E. Economic Value of E-waste


From the survey conducted towards the e-waste contractors, the estimated best
market price of e-waste is RM 3.00/kg depending on the condition of e-waste.
Based on the best price scenario, the total value of e-waste collected by 15 recovery
facilities within Klang Valley is RM 529,890.00. If estimation has been made that
the average value of e-waste collected by one recovery facility per month is RM
35,326.00 (total value/no. of recovery facilities involved), then the estimated total
value of e-waste that is collected by whole recovery facilities within Klang Valley
(30 recovery facilities) is RM 1,059,788.57. This value of e-waste only applies to
e-waste generators that send their e-waste to recovery facilities. There are many
other generators that do not send their e-waste to recyclers. Therefore, imagine
how much money is lost when e-waste is dumped at the landfill.
Innovation for Better ASEAN Community 133

IV. Conclusion
MRF is the main element in e-waste industry; their management is still at infancy
stage. The Department of Environment (DOE) Malaysia must have a proper
scrutiny on the individual contractors before issuing license. DOE must also
make a strict regulation, not only to the large organization, but also to the public
on disposing e-waste. The study suggests that e-waste collection should include
household instead of focusing on industries and organizations alone. By having
a connection between e-waste recovery facilities and municipal council, e-waste
collection around household level can be significantly improved.

V. Acknowledgement
I would like to express my greatest appreciation towards my supervisor, Prof.
Agamuthu Pariatamby, for spending his precious time, ideas and efforts in helping
me in doing this research. I am grateful to all respondents from material recovery
facilities and the officer from DOE Malaysia. I am also grateful to Universiti of
Malaya. This study was sponsored by UM grant Postgraduate Research Fund
P0005-2013B. Furthermore, I would also like to thank the Solid Waste Lab
members for giving me guidance and support. Their kindness is greatly appreci-
ated. Consideration and supports from family and friends are always remembered.

VI. R eferences
1) Sthiannopkao, S. and M. H. Wong. (2012). Handling e-waste in developed
and developing countries: initiatives, practices, and consequences. Science of
The Total Environment, 463–464, 1147–1153.
2) Herat, S. and P. Agamuthu. (2012). E-waste: a problem or an opportunity?
Review of issues, challenges and solutions in Asian countries. Waste Manage-
ment and Research, 30, 1113–1129.
3) Widmer, R., Oswald-Krapf, H., Sinha-Khetriwal, D., Schnellmann, M.
and H. Böni. (2005). Global perspectives on e-waste. Environmental Impact
Assessment Review, 25, 436–458.
4) Babington, J., Siwar, C. and M. Ahmad Fariz. (2010). Bridging the Gaps: An
E-waste management and recycling assessment of material recycling facilities
in Selangor and Penang. International Journal of Environmental Science, 1 (3),
383–389.
5) Tengku-Hamzah, T. A. A. (2011). Making Sense of Environmental Governance:
A study of E-waste in Malaysia (Doctoral’s thesis, Durham University.
6) Puckett, J. (2005). The Digital Dump: Exporting Re-use and Abuse to Africa.
Seattle: Basel Action Network.
134 Proceeding ASEAN COSAT 2014

7) Afroz, R., Masud, M. M., Akhtar, R. and J. B. Duasa. (2013). Survey and
analysis of public knowledge, awareness and willingness to pay in Kuala
Lumpur, Malaysia–a case study on household WEEE management. Journal
of Cleaner Production, 52, 185–193.
8) Department of Environment. (2012). Malaysia Environmental Quality
Report.
9) Kang, H. Y. and J. M. Schoenung. (2006). Economic analysis of electronic
waste recycling: modeling the cost and revenue of a materials recovery facility
in California. Environmental Science and Technology, 40, 1672–1680.
Public Perception on Current Waste
Management System: A Malaysian case study

S. H. Fauziah* and S. F. Ser


Institute of Biological Sciences, Faculty of Science, University of Malaya
50603 Kuala Lumpur, Malaysia

Abstract
The fact that public should be socially and financially prepared is undeniable to ensure an effective
implementation of waste management system in a country. This paper deliberates on public
perceptions towards the efforts to improve a waste management, including pay-as-you-throw
scheme. The study was conducted through distribution of questionnaires on issues related to
current waste practices and public understanding on various concepts in waste management
hierarchy to 759 randomly selected respondents in Peninsular Malaysia. Statistical analysis was
conducted using SPSS to correlate the socio-economic background of the respondents with their
perception and level of awareness. With 96% confidence level, this study indicates that 50% of
the respondents were in agreement that individual environmental awareness dictates personal
environmental behaviour, including waste generation and participation in recycling activities. As
for the legislations related to waste management in Malaysia, majority (68%) was not aware of
the existence of such regulations and provision. This is followed by 19% that felt that the lack of
enforcement is the causal factor towards ineffective waste management system. On the statement
that people become more attentive towards the environment only when they are threatened by
the negative impacts are supported by 86% of the respondents. This indicates that in general,
public tends to be ignorance of various environmental issues if there is no direct implication to
them. The study concludes that understanding on issues related to environment in general, and
waste management in particular, is highly influenced by the education level and attentiveness
of the community.
Key words: Recycling, Public participation, Pay-as-you-throw scheme, Solid Waste Management
Act 2007

I. Introduction
Fast progress in technology has resulted with many countries in the world
benefiting the consumer-based lifestyle [1]. Consumption patterns and choices
in technology influenced the impact on environment [2]. Development resulting
from industrial revolution causes waste management to be a crucial issue globally.
An increase of 1% GDP is estimated to increase municipal solid waste generation
by 0.69% [2].

* Corresponding author. Phone: +603 7967 6739. Email: fauziahsh@um.edu.my

135
136 Proceeding ASEAN COSAT 2014

Urbanization and economic development makes the waste management a


critical concern due to the fact that the increase in the quantity of waste will lead to
health problems, portraying poor sanitation as well as downgrading aesthetic value
[3]. Therefore, it is imperative to manage waste properly to prevent detrimental
impacts to the environment.
Most of the developing nations in the world are facing huge challenges in
improving the country’s waste management system. Among the most critical
challenges are the rapid increase in waste generation and the absence of appropri-
ate and effective waste management policy. Daily waste generation which had
exceeded 3.5 million tonnes in 2010 might double up by 2050 should no drastic
actions are taken [4].
It is reported that human population worldwide can produce thrice the
amount of waste today by 2100 [5]. The amount of waste produced by the
human population is undeniably on the rise with very limited portion undergoing
transformational changes of materials [5]. The waste issue will get more serious
particularly in developing countries where the cataclysm of dumping sites might
outbalance proper waste management strategies. Mexico City and Shanghai was
reported to generate more than 10,000 tonnes of waste everyday and this will
continue to increase due to lack of initiatives on waste reduction [5]. This is evident
in many parts of Asian countries where the population is expanding rapidly and
the needs for more dumping grounds are unavoidable [6].
While developing nations struggle to cope with the countries’ waste manage-
ment, cities in many developed nations had established various strategies to tackle
waste issues in which waste management is programmed to generate income rather
than consuming the profit of waste managers. San Francisco city for example,
create “Zero Waste” program via appropriate policy implementation to eliminate
waste from landfills, while 565,000 tonnes of waste are diverted from industries
in Kawasaki, Japan [5,7]. This is possible only with the implementation of an
integrated solid waste management with the aid of suitable policy framework,
proper available technologies, ability and capacity to establish a system and
appropriate financial assistance from the authorities [8].
The 3R concept (Reduce, Reuse and Recycle) has been practiced throughout
the globe in order to achieve sustainable waste management [9,10]. However, it
can be very challenging to developing countries where environmental concern
is of less priority than that of economic development. As a result, many of these
nations resort to the cheapest waste disposal method available, landfilling [11].
Landfilling is known to be the simplest waste disposal option, but with the
least preference. With expanding human population, land scarcity made landfilling
unfavourable due to the competition of landuse. However, landfill is still necessary
Innovation for Better ASEAN Community 137

since regardless of type of treatment used, there would still be unwanted residues
that need disposal [11]. Thus, the need for landfill is undeniable. However, the
matter of concern is the impacts of landfilling, as well as improper waste disposal,
towards the health of the environment. Improper managed disposal sites have been
constantly reported over the years to cause environmental pollution [12,13,14].
Landfill leachate, namely the effluent generated from waste disposal
activities, was reported to contain pollutants like monocyclic hydrocarbons and
organopesticides, and heavy metals which undoubtedly can negatively impact the
aquatic life [15]. Therefore, it is highly imperative that waste disposal sites are
properly managed in a sustainable manner to prevent any form of environmental
pollutions. Yet, it can be a real challenge if a country is not prepared to embrace
the technology. In order to move towards sustainable waste management practices,
it is necessary to highlight the appropriate driving factors.
Environmnetal drives, human drives, institutional drives and economic drives
are among the highlighted factors in many developed nations with approriate
mechanism of sustainable waste management [9]. These factors had been proven
to drive the country’s waste management system towards a more sustainable
practice [9].
Regardless the types of driving challenges existed, public plays an up most
important role in ensuring the success of an implemented strategies. The fact
that public should be socially and financially prepared is undeniable to enable a
proposed waste management strategy to be effective. Therefore, it is important
that planning of an effective waste management system should incorporate
the importance of the general public as the main stakeholders since this is the
group that initiates the waste generation process. It is necessary to understand
the behavior, the belief and the practices of the general public, so any planning
will take into consideration the possible need and expected responses after the
implementation of any waste management scheme.
In many developing nations in particular, the lack of information about the
public perspective on various environmental issues including waste management
has led to the failure of many environmental programmes, such as recycling
program [11]. Uncertainties in solid waste data, including outdated or inaccurately
estimated using regional averages, lack of a global database on waste production
and non-transparency among the stakeholders give rise to the unsuccessful
planning strategies [16].
Similar to many developing countries in the world, Malaysia too has been
struggling to improve the country’s waste management system. The introduction of
3R program in early 1980s and again in early 2000 has only increased recycling rate
to 5%, indicating the lack of public participation in the voluntary program [11].
138 Proceeding ASEAN COSAT 2014

This is mainly due to the absence of appropriate rules and regulations pertaining
to waste management in the country. As a result, a ‘not-bothered’ attitude plays a
major deciding role that public was not responsive. Yet, in 2007, contamination
of landfill leachate into the water catchment area in Klang Valley had caused
a huge public uproar that Solid Waste and Public Cleansing Management Act
2007 was passed by the Parliament [11]. The chain reaction from the event in
2007 has created a more environmental conscious public which pushed the
government to continuously improve the waste management system via a major
step of federalizing waste management sector in the country. Drastic improvement
from the management point of view has been recorded [11]. However, it is
somewhat hampered when it involves the participation from the general public.
Among the most glaring issue is the failure to improve the 5% recycling rate in
the country. In addition to that, the passed Act 2007 also includes the clause
on the implementation of Pay-As-You-Throw (PAYT) scheme. However, it is
yet to be implemented that the outcome is still uncertain. Undeniably, public
plays a crucial role in ensuring the success of any waste management program.
Therefore, it is important that their perception is taken into consideration prior
to the implementation of a program in a country. This paper aimed to investigate
the public perceptions towards efforts to improve a waste management including
PAYT scheme in particular and other waste related-environmental issues in
Malaysia.

II. R esearch Methodology


The study was conducted through dissemination of questionnaires on issues
related to current waste practices and public understanding on various concepts
in waste management hierarchy.
The questionnaires were divided into four sections with 55 questions in total.
The sections include background information, enquiries on level of knowledge
on various issues related to environment, awareness and knowledge on the past
and current programs run by the government in waste management and finally
on their level of satisfaction on the current waste management system in their
respective areas.
Reliability tests were conducted prior to actual distribution of questionnaires to
determine its reliability and coherency. The reliability test involved 30 respondents
which were selected randomly within Klang Valley. Cronbach’s Alpha was utilized
to determine the reliability of the set questionnaires.
For the actual survey, stratified random approach was adopted which identified
the number of respondent in each state of Peninsular Malaysia according to the
population distribution. Selangor, which represent the most densely populated
Innovation for Better ASEAN Community 139

state (24.4%), has the largest number of respondents for this study, followed by
14.7% in Johor, 10.3% in Perak and other states accordingly.
A total of 759 respondents were randomly selected from cities in Peninsular
Malaysia to attempt the questionnaires given. Statistical analysis using SPSS were
conducted to correlate the socio-economic background of the respondents with
their perception and level of awareness.

III. R esults and Discussions


With 96% confidence level, this study indicates that 50% of the respondents
were in agreement that individual environmental awareness dictates personal
environmental behaviour including waste generation and participation in recycling
activities. Others were either unsure or disagreed. Majority of the respondents
believed that they are active participants of recycling if they practice any form
of recycling. The survey revealed that individual believed that they are recycling
regardless the volume involved. Many of the respondents indicated that they
recycled newspaper or aluminum cans only.
As for the legislations related to waste management in Malaysia, majority
(68%) was not aware of the existence of such regulations and provision. As a
result to that, many failed to understand that it is their responsibility to reduce
the generation of waste, and to reduce the total waste to be sent to landfill for
disposal. More than 50% of the respondents think that environmental awareness
plays a major role in determining the generation of waste by individuals. Figure
1 shows the likely factors which can influence the waste generation habit among
Malaysians.

Figure 1. Public opinion on the contributing factors to waste


generation among individuals in Malaysia
140 Proceeding ASEAN COSAT 2014

The survey exposed that only 32% of Malaysian public can correctly identify
their waste collection service provider. This low percentage is probably contributed
by the lack of dissemination of information by the waste collection service provid-
ers. In Peninsular Malaysia, three main consortia, namely Alam Flora Pvt Ltd.,
SWM Pvt Ltd., and E-Idaman Pvt Ltd., have been awarded with the consession
to manage the municipal solid waste in Peninsular Malaysia.
Since all residential areas are to be serviced efficiently, many sub-contractors are
hired to cater the need of the population, under the three consortia. As a result,
many small contractors run the operation under the appointment of the consortia.
This could also be the contributing factor to the public confusion on the correct
waste consortia responsible in their area. In fact, the study showed that 68%
failed to identify the waste collection service provider in their neighbourhood.
Additionally, this negligence is probably resulting from the ‘not bothered’ attitude
since there is no direct financial implication involved.
As for quality of services provided, 79% indicated that current waste
management system in the country is satisfactory. This generally is due to the
strict key performance index (KPI) imposed by the regulating bodies to ensure
that the waste managers execute their responsibility appropriately. Significant
improvements have been reported since the passing of Solid Waste and Public
Cleansing Management Act 2007 [11]. This is because the regulating body namely
the Minsitry of Urban Wellbeing, Housing and Local Government has the right
to revoke the license of any waste consortia should they fail to deliver the required
KPI. Thus, to ensure smooth renewing of waste management concession, the waste
consortia has been diligently improving their services to the public, particularly
for waste collection. For example, many of the consortia tend to upgrade their
garbage truck to generate a good image to the general public.
Since main complaints from the general public revolve around the untidiness
of garbage truck, leakage of leachate from garbage trucks and inconsistent
frequency of collection due to garbage truck break-down, replacement with
tip-top condition garbage trucks resolved the problem. However, the method of
waste treatment and disposal is given less priority since it does not involve the
public directly. As a result, pollution from disposal sites including landfill persists.
Landfilling being the main method of disposal in Malaysia is not a well known
fact by the majority of Malaysians. Only 37% of the respondents know that the
waste collected from their premises will be disposed off into landfills. Yet, the
ignorance of 73% of the respondents probably is attributed to the fact that public
is less concern about the fate of the waste they disposed. The “out of sight and
out of mind” probably is the reason that the majority is oblivious of the disposal
Innovation for Better ASEAN Community 141

Figure 2. Knowledge on waste disposal method practiced in Malaysia among


respondents in Peninsular Malaysia

method practiced in Malaysia. Figure 2 depicts the public knowledge on waste


disposal methods in Malaysia.
Approximately, 75% actually believed that incineration is being practiced. In
reality, incinerators in Malaysia mainly cater for the disposal of hazardous waste
and very small volume of municipal solid waste in small islands, while the bigger
incinerators for municipal solid waste is still at its planning stage.
On issues related to factors that contribute to waste management problem in
Malaysia, 42% admitted that public attitude is the main cause. This is followed by
19% that felt that the lack of enforcement is the causal factor towards ineffective
waste management system. Figure 3 illustrates the public perception on the
contributing factors for waste management problem in Malaysia.
Indifferent attitude among the general public has always been the main reason
that lead to the failure of a program. Since public is the main generator of waste, it
is very important that they play a crucial role in any waste management program.
This includes recycling, waste reduction strategies and many more. Reduction on
the use of plastic bags (‘No-Plastic Bags’ on Saturday), for example has managed
to reduce the use of plastics bags by consumers. Rather than because consumers
are resorted to use alternative bags during shopping, the reduction was actually
due to avoidance among public to purchase the bags (RM 0.20 is charged for
each plastic bags on Saturday) or avoidance to do any shopping on Saturday. In
other words, instead of the reason to save the environment, the reduction on
plastic bags usage is heavily due to economic concern among the consumers.
142 Proceeding ASEAN COSAT 2014

Figure 3. Public perception on the contributing factors to waste management


problems in Malaysia

This is because for the majority of the public, economy is of higher priority than
environmental justification [11].
Only 12% feel that lack of education among Malaysian is the reason why
problem in waste management emerged. This probably is because ignorance
from lack of knowledge among the public can lead to lack of attentiveness and
refusal to embrace improvement. On the other hand, 9% blamed insufficient
number of facility being the factors that lead to problem in waste management.
It is also true based on the fact that waste generators are not able to participate
in waste management program if facilities provided are insufficient. This can be
supported by many cases of indiscriminate waste dumping when existing waste
bins had been filled up to the rim.
As for statements that people become more attentive towards the environment
only when they are threatened by the negative impacts are supported by 86%
of the respondents. This indicates that in general, public tend to be ignorance
of various environmental issues if there is no direct implication to them. Thus,
people in the ‘comfort zones’ are less susceptive to environmental issues in general.
This lead to the reason why the ‘Not In My Back Yard’ or NIMBY syndrome are
very strong among Malaysians.
Many developed nations had charged the public the service fees to manage
the waste generated based on the amount produced. Pay-as-you-throw (PAYT)
scheme has been reported to be very effective Japan, Korea and many parts of
EU. However, it is an unfamiliar concept in Malaysia where the cost of waste
management is embedded in the annual assessment fee paid by premise owners
to the local municipalities. As a matter of the fact, many of the general public
is oblivious of this situation. This study had ruled out that approximately 59%
Innovation for Better ASEAN Community 143

Figure 4. Public acceptance on PAYT system in Malaysia

of the respondents believed that they are not charged for the waste management
services. To make matter worse, 93% of the general public is clueless of the cost
incurred to manage the waste they generate everyday. This is probably contributed
to the absence of information on the actual process of waste management and the
cost involved to deal with its collection, treatment and disposal. Thus, it is highly
recommended that the local government and non-governmental organization
(NGOs) to step in to provide the information to the general public on related
issues in waste management. Not only it will be an eye-opener to the public but
also allow them to consider carefully how to deal with their wastes.
Only 47% of the respondents thought that PAYT can be implemented in
Malaysia, while the remaining 53% were skeptical of this scheme. This is likely
due to the fact that Malaysians can be very fussy when it involved their finance.
Therefore, the need to pay for the amount of waste generated may not be appealing
since similar assessment such as sewage treatment service in the past 15 years has
been very problematic in its implementation. 43% of the respondents also think
that PAYT is not fair. Figure 4 depicts the public acceptance on PAYT system
in Malaysia.
The results obtained from this study indicated the importance of public
understanding and awareness to mould the genuine belief towards environmental
conscious society.

IV. Conclusions
The study concluded that public understanding on various issues related to
environment in general, and waste management in particular is highly influenced
by the attentiveness of the community. PAYT scheme generally is not well accepted
among the Malaysians. Nevertheless, many believed that laws and regulations can
144 Proceeding ASEAN COSAT 2014

assist in moulding the right attitude of the general public towards the improvement
in waste management in the country.

V. Acknowledgement
The authors would like express their gratitudes to respondents that participated
in this project. Appreciation also goes to the National Department of Solid Waste
(JPSPN) under the Ministry of Urban Wellbeing, Housing and Local Government
for the provision of token for the respondents in the survey. Finally, special thanks
go to Institute of Biological Sciences, Faculty of Science, University of Malaya,
for the financial assistance given to complete this study.

VI. R eferences
1) Bhada-Tata, P. and D. Hoornweg. (2012). What a waste: A global review of
solid waste management. World Bank Report.
2) Satterthwaite, D. (2009). The implications of population growth and urbaniza-
tion for climate change. Environment and Urbanization, 21 (2), 545–567.
3) Lau, V. L. (2004). Case study on the management of waste materials in
Malaysia. Forum der Geookologie, 15 (2), 7–14.
4) Hoornweg, D., Bhada-Tata, P. and C. Kennedy. (2013). Waste production
must peak this century. Nature, 502, 615–617.
5) World Bank. (2013). Global Waste on Pace to triple by 2100. Retrieved from
http://www.worldbank.org/en/news/feature/2013/10/30/global-waste-on-
the-pace-to-triple.
6) Hoornweg, D. and P. Bhada-Tata. (2012). What a Waste: A global review of
solid waste management. World Bank Report.
7) SF Environment. (2014). Zero Waste. Retrieved from http://www.sfenviron-
ment.org/zero-waste
8) UNEP. (2009). Developing Integrated Solid Waste Management Plan: Assessment
of Current Waste Management System and Gaps Therein. United Nations
Environment Programme.
9) Agamuthu, P., Khidzir, K. M. and S. H. Fauziah. (2009). Drivers of sustainable
waste management in Asia. Waste Management and Research, 27 (7), 625–633.
10) Agamuthu, P., Fauziah, S. H., Khidzir, K. M. and L. P. Chong. (2008). Issues
and challenges of 3Rs in the Asia and Pacific regions”. Proceedings of the Asian
Productivity Organization Conference 2008. Japan, 6–9 October 2008.
11) Fauziah, S. H. and P. Agamuthu. (2012). Trends in Sustainable Landfilling
in Malaysia, a developing country. Waste Management and Research, 30 (7),
656–663.
Innovation for Better ASEAN Community 145

12) Palmiotto, M., Fattore, E., Paiano, V., Celeste, G., Colombo, A. and E.
Davoli. (2014). Influence of a municipal solid waste landfill in the surrounding
environment: Toxicological risk and odor nuisance effects. Environmental
International, 68, 16–24.
13) Nguyen, N. S., Satoshi, S., Ishigaki, T. and M. Ike. (2012). Microorganisms
in landfill bioreactors for accelerated stabilization of solid wastes. Journal of
Bioscience and Bioengineering, 114 (3), 243–250.
14) Magda, M., El-Salam, A., Gaber, I. and Abu-Zuid. (2014). Impact of
landfill leachate on the groundwater quality: A case study in Egypt. Journal
of Advanced Research,( In Press).
15) Fauziah, S.H., Emenike, C. U. and P. Agamuthu. (2013). Leachate risk
and identification of accumulated heavy metals in Pangasius suitchi. Waste
Management & Research, 31 (10), 75–80.
16) Tchobanoglous, G. and S. E. Vergara. (2012). Municipal solid waste and
the environment: A global perspective. Annual Review of Environment and
Resources, 37, 277.
Biomass Flow and Carbon Sequestration
in an Organic Farm

L. Hong Yeng* and P. Agamuthu


Institute of Biological Sciences, Faculty Science, University of Malaya
50603 Kuala Lumpur, Malaysia

Abstract
Biomass is believed to improve soil fertility and carbon sequestration by enhancing soil organic
matter (SOM) content. Organic farm uses large amount of biomass input to replace chemical
fertilizer. Thus, it is considered a sustainable option for greenhouse gas (GHG) mitigation in
agriculture. There are inconsistent findings on the increase in soil carbon concentrations in
organically managed soil and this has become a hotly debated issue. In this paper, biomass carbon
flow of an organic farm was evaluated with material/substance flow analysis (MFA/SFA) for
evidence of carbon stock. Annually, there was 3,046 ton ha-1 y-1 of biomass input, which accounts
for 73% of total farm input. The only biomass output from organic farm was harvested vegetables
which was 112 ton ha-1 y-1. The substance flow shows a total of 156 ton ha-1 y-1 of carbon stock
within the system and this indicates that the farm system was playing a role as carbon sink. In
addition, carbon dioxide and carbon monoxide emission is also one of the major culprits of
carbon outflow from farm system which comprised a total of 172 ton ha-1 y-1 of carbon per year.
Moreover, the soil carbon concentration of the organic farm increased 29% during the study
period. Despite the carbon outflow, results indicate that organic farm management practices
focused large amount of biomass input had increased soil carbon concentration.
Key words: Organic, Vegetable farm, Material/substance flow analysis, STAN

I. Introduction
Biomass is believed to improve soil fertility and carbon sequestration by enhancing
soil organic matter (SOM) content and terrestrial ecosystem is an ideal reservoir for
carbon sequestration that could offset the CO2 emission due to human activities
[1]. Thus, IPCC has identified biomass application in soil as the promising tool
to capture and store carbon at terrestrial reservoir [2]. Crop residue, bio-solid,
fertilizer and manure are the common biomass applied at farm which contributes
to soil carbon sequestration [3]. Organic farm is believed to be carbon sequester
because biomass application is a common practices there.
Several reports discussed on the benefit of conversion from conventional farm
to organic farm in regards to carbon sequestration [4,5,6]. However, none draw a

* Corresponding author. Phone: +6012-6538938. Email: nicolelhy5134@gmail.com

147
148 Proceeding ASEAN COSAT 2014

definite conclusion. There are reports reveal that soil carbon sequestration through
conversion from conventional farm to organic farm is a temporary solution [7].
This article presents a case study of carbon flow in an organic farm. The farm
level analysis was chosen for three reasons. First, farm management plays a major
role in carbon sequestration. Proper farm management can create carbon sink
by encouraging carbon sequester practices [8]. Second, it provides information
about the flow and stocks of material and substances within farm system. Third,
it provides fundamental information for the development of farm management
strategies at farm level.
This analysis was conducted based on laboratory results done which present
the onsite situation for this particular study. The goal of this article are to (1)
identify key process and pathway, (2) determine the carbon stocks, (3) develop
recommendation for goal-oriented farm management in order to ensure maximum
carbon stock and minimize carbon output from the system.

II. M aterial and Methods


This study utilized the combination of field sample analysis, onsite survey and
material/substance flow analysis (MFA/SFA) method to achieve objectives.
Material/substance flow modeling was performed using modeling software STAN
2.5 (subSTance flow ANalysis) based on the field data collected [9].

A. Material/Substance Flow Analysis


The basic theory of MFA/SFA is that output is derived from input which also
knows as mass balance (Eq. 1, 2 and 3) [11,12].
Balance equation:
Σ inputs = Σ outputs + change in stock (1)
Transfer coefficient equation:
Output = transfer coefficient output x Σ inputs (2)
Stock equation:
Stock Period i+1 = stock Period i+change in stock Period i (3)

MFA/SFA is a dynamic approach that analyses material/substance flows or


any stock accumulation over a period of time based on mathematical probabilistic
distributions. MFA/SFA identifies the associations between input, output, process,
inventory and stocks. Five fundamental steps of MFA process has been followed
in this study [10,11]: (1) Determination and selection of research objective and
monitoring parameters; (2) Identification of system boundaries, scope and time
Innovation for Better ASEAN Community 149

frame; (3) Identification of key pathways, processes and stocks; (4) Material flow
modelling; (5) Mass balance.

B. Farm Description
The system boundary is within an organic farm (2°56’56.59”N, 101°53’25.69”E)
which located at area with an elevation of 75 meter. The major soil groups there
are Typic Hapludult. It is managed in accordance to the standards enacted
by the regulation of Malaysia Organic Scheme based on Malaysian Standard
MS1529:2001 (the production, processing, labelling and marketing of plant
based organically produced foods).

C. Sample Analysis
Composite samples of soil, vegetable, compost, fertilizer, manure, organic waste
and water were sampled from the field through random sampling method and
analysed at the laboratory [12,13]. Solid samples were dried at 65 °C for a period
of 72 hour and sieved (1 mm mesh), and were analysed with Perkin Elmer
CHNS/O Series II 2400 for carbon content [14]. The carbon content of water
samples were analysed HACH DR/4000 (HACH procedure method 10128) and
gran alkalinity method [15].
Carbon dioxide and carbon monoxide emission was measured with 2 mm
acrylic static chamber (32cm x 22cm x 22cm) [16].

III. RESULTS AND DISCUSSION


A. Material Flow Model
Three processes have been defined: Farm Land; Vegetable Processing and
Packaging; and Husbandry. The process Farm Land is the pre-harvest stage which
involves planting activities while the process Vegetable Processing and Packaging
is the post-harvest stage. The process Husbandry does not participated in farm
production but was included in the model because organic waste was recycled as
animal feed. Based on the biomass input and output identified, a biomass balance
was established (Eq. 4).
Biomass Balance = (Bokashi Compost + Compost + Peat Moss + Vermi-
compost) – Harvested Crop (4)

Figure 1 shows the compost (2,757 ton ha-1 y-1) and Bokashi compost (276
ton ha-1 y-1) were the major biomass input in the farm system while harvested
vegetable (112 ton ha-1 y-1) was the main biomass output. The MFA model shows
that the farm recycled organic wastes generated from the post-harvest processing
as animal feed (6.15 ton ha-1 y-1).
150 Proceeding ASEAN COSAT 2014

Figure 1. Material flow model for organic farm, 2011–2013

B. Carbon Flow Analysis


The STAN model shows the biomass input has contributed to 366 ton ha−1 y−1
of total carbon into the farm system. There are reports questioning the benefit of
carbon input in carbon sequestration. This is because the carbon input increases
soil carbon but in the same time, encourages microbial decomposition activity
which leads to higher carbon emission [4]. Thus, the STAN model (Figure 2)
included carbon gaseous emission in the model to evaluate the effect of gaseous
emission in carbon sequestration. According to the STAN model, the largest
carbon flows out from the farm system was through carbon monoxide and carbon

Figure 2. Carbon flow analysis of organic farm, 2011–2013


Innovation for Better ASEAN Community 151

dioxide gaseous emission which accounts for 81% of the total carbon output from
the farm system. The rest of the carbon output was vegetable sold to the consumer.
Each year, around 156 ton ha−1 y−1 (dstock) of carbon has stocked into the
farm system. This indicates the farm has the potential to be carbon sequester due
to high input and low output of biomass. This result agrees with the carbon flux
modelling by Vleeshouwers and Verhagen (2002) where the usage of organic
matter input increases carbon input and resulted to 1.50 ton ha−1 y−1 of carbon
flux [8]. To further investigate, the soil carbon concentration during the study
period was measured and the results shows the increment of 29% in terms of soil
carbon concentration. This result is higher than the result reported by Leifeld and
Fuhrer (2010) where 2.2% increase in soil carbon concentration [5].

IV. Conclusion
The carbon flow model in this study implies that the organic farm has the potential
to be carbon sink even when carbon emissions were taken into account for the
carbon flow analysis. Further research shall be conducted at other organic farm
located at different region to generate regional data.

V. Acknowledgement
The authors are grateful to University of Malaya (IPPP Project No: PV009-2012A)
for providing the financial support for executing the work. Special thanks to
Department of Agriculture Malaysia (Permanent Food Production Areas, TKPM-
Ulu Yam), SPC Organic farm, Green Oasis Sdn. Bhd., Mr. Zulklifi, Mr. David,
Mr. Thiam Kong Seng, Mr. Teo Wai Kit, and Ministry of Higher Education
Malaysia (MyPhD programme).

VI. R eferences
1) Luo, Z., Wang, E. and O. J. Sun. (2010). Soil carbon change and its responses
to agricultural practices in Australian agro-ecosystems: a review and synthesis.
Geoderma, 155 (3), 211–223.
2) Sims, R. E. H., Schock, R. N. and A. Adegbululgbe. (2007). IPCC Fourth
Assessment Report: Climate Change 2007 Working Group III: Mitigation of
Climate Change, Chapter 4: Energy Supply. IPCC.
3) Paustian, K., Parton, W. J. and J. Persson. (1992). Modeling soil organic
matter in organic-amended and nitrogen-fertilized long-term plots. Soil Science
Society of America Journal, 56 (2), 476–488.
4) Janzen, H. H. (2006). The soil carbon dilemma: Shall we hoard it or use it?
Soil Biology and Biochemistry, 38 (3), 419–424.
152 Proceeding ASEAN COSAT 2014

5) Leifeld, J. and J. Fuhrer. (2010). Organic farming and soil carbon sequestra-
tion: what do we really know about the benefits? Ambio, 39 (8), 585–599.
6) Liu, R., Xu, J. M. and C. E. Clapp. (2013). Carbon Sequestration in Organic
Farming. In Functions of Natural Organic Matter in Changing Environment
(pp. 377–379). Netherlands: Springer.
7) Smith, P. (2004). Carbon sequestration in croplands: the potential in Europe
and the global context. European Journal of Agronomy, 20 (3), 229–236.
8) Vleeshouwers, L. M. and A. Verhagen. (2002). Carbon emission and
sequestration by agricultural land use: a model study for Europe. Global
Change Biology, 8, 519–530.
9) Cencic, O. and H. Rechberger. (2008). Material flow analysis with software
STAN. Journal of Environmental Engineering and Management, 18 (1), 3–7.
10) Baccini, P. and P. H. Brunner. (2012). Metabolism of the Anthroposphere:
Analysis, Evaluation, Design, 2nd ed. (pp. 15–40). Cambridge, MA, USA: The
MIT Press.
11) Brunner, P. H. and H. Rechberger. (2004). Practical handbook of material
flow analysis. The International Journal of Life Cycle Assessment, 9 (5), 337–338.
12) Voll, M. and O. Roots. (1999). Soil water sample collector. Environmental
Monitoring and Assessment, 54 (3), 283–287.
13) Migliaccio, K. W., Li, Y. C., Trafford, H. and E. Evans. (2006). A simple
lysimeter for soil water sampling in south Florida [Online]. Retrieved from
http://trec.ifas.ufl.edu/kwm/files/pdf/publications/edis_ABE361.pdf.
14) Pereira, M. G., Espindula, A. Jr., Valladares, G. S., Cunha dos Anjos, L. H.,
de Melo Benites, V. and N. Schultz. (2007). Comparison of total nitrogen
methods applied for Histosols and soil horizons with high organic matter
content. Communications in Soil Science and Plant Analysis, 37 (7–8), 939–943.
15) Wetzel, R. G. and G. E. Likens. (2010). The Inorganic Carbon Complex:
Alkalinity, Acidity, CO2, pH, Total Inorganic Carbon, Hardness, Aluminum.
In Limnological Analyses, 3rd ed. (pp. 113–135). New York: Springer.
16) Parkin, T. B. and R. T. Venterea. (2010). USDA-ARS GRACEnet Project
Protocols Chapter 3. Chamber-Based Trace Gas Flux Measurements 4.
Sampling Protocols. USDA-ARS, Fort Collins, CO, 3-1. [Online]. Retrieved
from: http://www.usmarc.usda.gov/SP2UserFiles/person/31831/2011%20
Parkin%20and%20Venterea%20Trace%20Gas%20Protocol%20Revi-
sion%20Final.pdf
Biomass Gasification for Power Generation
Using Dual Chamber Circulating Fluidized
Bed Reactor
Haifa Wahyu*, Imam Djunaedi, M. Affendi and Sugiyatno
Research Centre for Physics, Indonesian Institute of Sciences
Jln. Cisitu-Sangkuriang 21/154D, Bandung 40135 Indonesia

Abstract
This paper presents an investigation on using dual chamber circulating fluidized bed reactor for
biomass gasification in power generation. Gasification process has been around for many years for
charcoal production from biomass or gas production from biomass or coal. The technology varies
from simple downdraft reactor or more complex system, such as circulating fluidized bed and
combined cycle. Gasification technology is an alternative solution to conventional steam power
plant to generate electricity from biomass waste. Its use can be combined with diesel generator
or gas engine where the gas products from biomass gasification system can replace up to 70%
of diesel oil while the gas can be used as 100% fuel in a gas engine. This will help reduce the
electricity cost generated from diesel engine. A major problem that still occurs in a gasification
reaction is the formation of tar. The most effective way to eliminate tar is by complete burning.
In circulating fluidized bed, tar can be captured using sand media. The tar covered sand will be
brought to the second chamber to be completely burned to remove the tar. This way, the plant
can be operated continuously without interruption. For a commercial power plant, it is important
that the plant operational is reliable. A circulating fluidized bed can be used to perform this task.
In this work, a dual chamber reactor is developed so that tar removal can be done continuously.
We have constructed a test plant with the capacity of 30 kg biomass per hour. This work includes
the gasification of several biomass types, that is wood sawdust, coffee husk, empty fruit bunch,
rice husk and vertiver root. On average, the plant is able to produce fuel gas with the composition
of H2 around 4%, CO between 10 to 12%, CO2 around 12% and CH4 around 2%. The tar
produced is less than 50 mg. Most biomass produced similar gas composition, except for the
rice husk. The rice husk must be mixed with other type of biomass to enable the production of
fuel gas satisfactorily.
Key words: Biomass gasification, Circulating fluidized bed, Dual fuel power generation

I. Introduction
Biomass gasification is an important process that can be an alternative to direct
combustion. In some cases, lignocellulosic material is hard to burn in a conven-
tional furnace, therefore it must be processed by thermal cracking to produce

* Corresponding author. Phone: +62-22-2507771. Email: haifa.wahyu@gmail.com, haifa.wahyu@lipi.


go.id

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154 Proceeding ASEAN COSAT 2014

gas and liquid. Gaseous product consists of reactive substances mainly of carbon
monoxide and hydrogen, which is called synthesis gas (syngas). Syngas can be
used as gas fuel in diesel engine or processed further as raw material for chemical
productions. Biomass, such as rice husk, has been used as fuel in gasification
process, especially in countries where rice fields are abundant as in China [1].
Gasification of rice husk is usually done in a downdraft gasification reactor
[1,2]. Other type of reactor was also studied for gasification of rice husk, such as
fluidized bed [3], a dual distributor fluidized bed [4] and a cyclone gasifier [5].
Each type of reactor has the advantages and disadvantages. Downdraft gasifier is
more simple and easier to operate although less efficient, while fluidized bed and
cyclone gasifier are more complicated but more efficient. However, in all types
of reactors, the formation of tar is always a problem. The most effective way to
remove the tar is by complete burning. In order to remove the tar in the downdraft
gasifier, Affendi et al. [2] cleaned the equipment manually and burned the tar.
For a continuous application, downdraft gasification is a little cumbersome
since tar is cleaned when the system is shut off and the downstream components
must be disassembled. Furthermore, other type of fuel may not suitable to be used
in a downdraft gasifier. An example is wood sawdust gasification which provides
better results in a fluidized bed reactor [6]. This also applies for other material,
such as coal gasification, where an integrated gasification combined cycle has
been developed in New Zealand [7] and Austria [8] to cope with large capacity
power producing system. Circulating fluidized bed which is based on entrained
flow has the advantage of producing clean tar free gas continuously, while the
ash is produced in the form of inert slag [9].
The present work aims to investigate the use of a circulating fluidized
bed system using atmospheric air as a reacting agent to gasify several types of
biomass. We have developed a dual chamber circulating fluidized bed for biomass
gasification [10]. The system is expected to deal with the elimination of tar
and the production of gas continuously. This paper provides the results of the
gasification of sawdust, coffee husk, rice husk and empty fruit bunch. The actual
gas composition was analyzed using Orsat gas analyzer.

II. Method
The experiment was conducted in a dual chamber circulating fluidized with the
capacity of 30 kg/hour (Figure 1). The reactors are divided into two chambers
gasification chamber and combustion chamber. Gasification chamber is painted
in blue color while combustion chamber is painted in orange. Gasification
process is held in the blue chamber using sand as the heating media. The sand is
also functioning to capture the tar formed during the gasification process. The
Innovation for Better ASEAN Community 155

combustion chamber is used to burn sand coated tar to completely convert the
tar into flue gas. Each reactor is equipped with dust cyclone to separate the gas
from the sand and dust. The sand is recirculated in the system, clean sand from
the combustion chamber is returned to the gasifier to perform the next cycle of
gasification process. Biomass enters the gasifier through the biomass feeder and
brought into the chamber using screw feeder.
Table 1. Design parameter of the dual chamber circulating fluidized bed
Parameter Value
Reactor diameter, mm 20
Reactor height, mm 2,250
Minimum fluidization velocity, m/s 0.5-0.8
Biomass flowrate, kg/hour 15-25
Sand flowrate, kg/hour 15

Figure 1. Dual chamber circulating fluidized bed for


biomass gasification

The following table contains the design parameter of the CFB reactor.
In this experiment, we used biomass that is common in agricultural industries
in Indonesia, such as rice husk, wood sawdust, empty fruit bunch and coffee
husk. The following table shows the calorific value of each type of the biomass.
156 Proceeding ASEAN COSAT 2014

Table 2 Calorific value of biomass


Types of biomass (ar) CV (kJ/kg)
Empty fruit bunch 18,960
Rice husk 15,719
Vertiver root 18,422
Wood sawdust 18,036
Coffee husks 18,422

Table 3 contains the properties of the sand used as the heating media.

Table 3 Sand properties


Properties Sand
Type Quartz
Average particle diameter (µm) 385
Density (kg.m-3) 2,650
Porosity 0.46
Sphericity 0.78

The results are shown using data on reaction temperature and syngas analysis.
Orsat gas analyzer was used to record the gas composition, while K-type ther-
mocouple was used to measure reaction temperature. The reaction temperature
during the experiment was recorded at several places. The reaction temperature
was taken at the inlet, in the middle of the reactor and at the syngas outlet.
The reactor was heated up until about 400 °C before the biomass entered the
gasification reactor.

III. R esults A nd Discussions


The results of the experiments show the composition of combustible gas for
each type of biomass measured using Orsat gas analyzer. The experiments were
compared with the theoretical results whose gas composition are as follows: CO
12%, H2 4.0% and CH4 3.0%.
The experimental results of the syngas composition are slightly different from
that of the theoretical values. The percentage of CO is about 2% lower during
the experiment than that of the theory. The percentage of CH4 is higher during
the experiment than that of the theory, about twice. The production of hydrogen
gas is the same for all the biomass, except in wood sawdust. The hydrogen
production is as twice as much for wood sawdust. CO2 and CnHm production
varies slightly among the biomass. Although the percentages are different, the
whole results indicate the ability of the equipment to produce CO and H2 gases
which is crucial in biomass gasification. To prove the flammability of the gas, the
syngas was burned at the outlet of the reactor. The flame was quite stable during
the experiment.
Innovation for Better ASEAN Community 157

The following picture shows the burning of the syngas at the outlet.
Figure 4 shows the temperature profile in the reactor. The figure shows the
reaction temperature taken progressively from the middle of the reactor during
the experiment. The temperature shows the gradient on hourly basis. It shows
that the reaction temperatures are similar for all biomass.

Figure 3. The flaring of syngas at the outlet


Figure 2. Syngas composition compared with
theoretical values
158 Proceeding ASEAN COSAT 2014

Figure 4. The profile of the reaction temperature in the middle of the


gasifier taken after heating up until up to 4 hour reaction

IV. Conclusion
The results of the gasification experiments on several types biomass in the dual
chamber circulating fluidized bed are in agreement with the theoretical values.
Syngas can be produced successfully in this type of reactor indicated by the
composition of the gas and the burning capability of the gas products. Based on
the experimentals results, it can be concluded that a dual chamber circulating
fluidized bed can be used to produce gas fuel that can be applied in diesel
generators for electricity production.

V. Acknowledgement
The authors would like to thank the organizer of the Conference for the invita-
tion to present this paper. The authors would also like to express their thanks
to colleagues at the Research Centre of Physics LIPI for the support provided.

VI. R eferences
1) Lin, K. S., Wang, H. P., Lin, C. J. and C. I. Juch. (1998). A process develop-
ment for gasification of rice husk. Fuel Processing Technology, 55, 185–192.
2) Affendi, M., Sugiyatno, Suhartono and I. Djunaedi. (2009). Kajian tekno-
ekonomi pengoperasian PLTD-sekam 100 KW Di Haurgeulis, Indramayu.
Laporan Akhir Tahun 2009, Kegiatan Program Kompetitif LIPI. Pusat
Penelitian Fisika- LIPI.
3) Ramírez, J. J., Martínez, J. D. and S. L. Petro. (2007). Basic design of a
fluidized bed gasifier for rice husk on a pilot scale. Latin American Applied
Research, 37, 299–306.
4) Mansaray, K. G, Ghaly, A. E., Al-Taweel, A. M., Hamdullahpur, F. and V.
I. Ugursal. (1999). Air gasification of rice husk in a dual distributor type
fluidized bed gasifier. Biomass and Bioenergy, 17, 315–332.
5) Zhao, Y., Sun, S., Che, H., Guo, Y. and C. Gao. (2012). Characteristics of
cyclone gasification of rice husk. International Journal of Hydrogen Energy,
37, 6962–6966.
6) Kumaran, V. (2007). Design of a low cost fluidized bed gasifier for sawdust
gasification in rural China. Energy from Biomass and Wastes, 421–825.
7) Brown, J. (2006). Biomass Gasification: Fast Internal Circulating Fluidised
Bed Gasifier Characterisation and Comparison (ME Thesis). New Zealand:
University of Canterbury.
8) Institute of Chemical Engineering TU Wien Austria. (2011). The FICFB-
gasification system. Retrieved from http://www.ficfb.at/
9) Higman C. and M. van der Burgt. (2008). Gasification, Second Edition.
Amsterdam: Elsevier.
10) Wahyu, H., Djunaedi, I., Affendi, M. and Sugiyatno. (2013). Design,
Simulation and Experiment of Circulating Fluidized Bed Reactor for
Biomass Gasification. Proceedings of the International Seminar on Biorenewable
Resources Utilization for Energy and Chemicals 2013. Bandung, 9–11 October
(p. 243). Bandung, Indonesia.
Roof Mounted Micro-Wind Turbine for
Power Generation in Coastal Housing in
Semarang, Indonesia

Dany Perwita Sari*


Research Center for Biomaterials, Indonesian Institute of Scienes (LIPI)
Jl. Raya Bogor km.46, Cibinong, Bogor 1691, Indonesia

Abstract
Mounted micro-wind turbines have the potential power saving in coastal housing. In this paper,
the meteorological data of five year wind velocity and wind direction of Semarang, Indonesia,
were used to find out the wind energy potential. From wind direction evaluation at a height of 10
m above the ground level, it was found that the highest wind power potential is on north wind.
This research aims to present the result of Computational Fluid Dynamics (CFD) simulations
for identifying wind velocity and wind flow of two kind roof profiles: gable roof and hip roof.
Result shows that the wind flows are strongly dependent on the profile of the roof. It was also
concluded that roof mounted micro-wind turbine is suitable for electric wind application, which
can reach 20% more than wind velocity approach.
Key words: Micro-wind turbine, Wind velocity, CFD, Wind energy, Semarang, Indonesia

I. Introduction
The government of Indonesia commits to reduce fossil fuel consumption up to
26% by the year 2020 [1]. The increasing interest in renewable energy has led
to a desire to explore wind energy. Since the majority of Indonesia’s population
lives in coastal areas, implementing micro-wind turbine for housing has the
potential to make a significant contribution to government’s targets. Semarang
that has coastal housing which is famous with the traditional roof design has been
presently chosen for the case study. Semarang forecasting within 5 years period
(2008–2012) [2] shows that coastal housing where is located along northern area
of Semarang has huge potential for power generation.
With high mean wind velocity and low levels of turbulence (coastal area),
the micro-wind turbine installation will become one of the potentially low-cost
renewable sources of energy. However, wind flow and turbulence intensity at the
roof level strongly depend on the roof profile [3]. Otherwise, if a turbine is situated
in the wrong location on the roof; possibly the power output is not significant [4].
Roof shape is one of the main factors affecting the installation of roof mounted
* Corresponding author. Phone: +62-8132-5693808. Email: dany.perwitasari@gmail.com

161
162 Proceeding ASEAN COSAT 2014

wind direction wind direction


building position 0° building position 0°

wind direction
wind direction
building position 0°
building position 0°

Figure 1. Roof profile, from left to right: gable roof, building position 0°;
gable roof, building position 90°; hip roof, building position 0°; hip roof,
building position 90°

wind turbine [5]. Coastal housing in Semarang has a unique roof profile, such as
gable roof and hip roof. Based on this condition, this paper attempts to address
one of the main factors for building integrated micro-wind turbine which is
wind velocity around the roof. Computational Fluid Dynamics (CFD) is used
to analyze air flow and wind velocity over the roof profile to ascertain the most
power productive location for micro-wind turbine.

II. CFD Simulations


CFD was used to simulate wind flow above gable roof and hip roof, which
represent the basic shapes of traditional coastal housing in Semarang (Figure 1).
This modeled was assumed as residential houses 45 m2 (4 m x 9 m). Simulations
were undertaken with two different wind direction (Figure 1), with direction 0°
parallel to the roof profile and 90° cross to the roof profile.
Innovation for Better ASEAN Community 163

Table 1. Percentage of wind speed and wind direction in Semarang, Indonesia (2008-2012) [6]
Wind Speed Wind Direction To-tal
(km/hr) N E SE S SW NW (%)
7-9 13,3 11,7 6,67 3,33 5 8,333 48,33
10-12 10 5 6,67 3,33 1,67 10 36,67
13-15 - - - 1,67 - 1,667 3,333
16-18 - 1,67 - - 1,67 6,667 10
19-20 - - - - - 1,667 1,667
Total (%) 23,33 18,33 13,33 8,333 8,333 28,33 100

From Table 1, wind direction evaluation at a height of 10 m above ground


level was found that the highest wind power potential is on north wind (7–9 km/
hr). The roughness classification is using ASCE (1999) [7] with smooth terrain
roughness. Wind profile for CFD analysis in Semarang can be expressed as:

(1)
Where U is wind velocity (m/s), Uref is wind velocity reference (m/s), Z is the
gradient height (m), Zref is the gradient height reference (m) and α is power law
exponent. From the equation, velocity profile could be configured (Table 2). The
wind speed profile measured in the CFD using the power law of an exponent of
0.125. The mean wind speed was 5 m/s at 10 m above the sea level.

III. Wind Power Density of Semarang City, Indonesia


Wind power density is a measure of the energy available in the wind at some
region. Wind turbine power of a wind generator [11] can be expressed as:
(2)

where:
Pturbine = the wind turbine power,
Ρ = the air density (kg/m3)
Cp = the coefficient of performance,
A = the swept area of the blades (m2),
V = free wind speed (m/s).
Two different micro-wind turbines, D400 Stealth Gen (swept area 1.1 m)
and Renewable Devices Swift Turbine (swept area 2.1 m), have been carried out
to investigate wind power density. The wind power density resulted from two
different micro-wind turbines that were given in Table 2. As shown, the majority
164 Proceeding ASEAN COSAT 2014

Table 2. Velocity profile and wind power density using D400 Stealth and Renewable Device
Swift micro-wind turbine.
Height Avarage Velocity Wind Power Density (W/m2)
(m) (m/s) D400 Stealth Gen Renewable Devices Swift Turbine
(d=1.1 m) (d=2.1 m)
1 3,749471 35,37414 67,53245
2 4,088827 45,87459 87,57876
3 4,301403 53,40801 101,9607
4 4,458898 59,49198 113,5756
5 4,58502 64,68442 123,4884
6 4,690714 69,26162 132,2267
7 4,781975 73,38338 140,0955
8 4,862462 77,15155 147,2893
9 4,934581 80,63561 153,9407
10 5 83,88531 160,1447
11 5,059925 86,93771 165,972
12 5,115259 89,82121 171,4769
13 5,166696 92,55815 176,7019
14 5,21478 95,16647 181,6814
15 5,259948 97,66077 186,4433

of wind power density in Semarang is categorized as fair wind resources [8]. This
is largely due to the inlet wind velocity. Wind power density function of the cube
(third power) of the wind speed (2). If the wind speed is doubled, power in the
wind increases by a factor of eight. This relationship means that small differences
in wind velocity lead to large differences in wind power density.

IV. R esult and Discussion


Specifying the optimum wind power density of roof mounted wind turbine
depends on the wind velocity. CFD is used for predicting wind velocity and wind
flow above the roof profiles. Figure 3a shows the average wind velocity result for
gable roof in 0° position. The simulation is expressed the wind velocity increase
in the height 1m above roof surface (7 m/s to 8 m/s) then stable in the height
10 m above the roof surface. No increase wind velocity after passing gable roof.
Hip roof in 0° position result (Figure 3b) records similar with gable roof in 0°
position result. Case study model two changes building position from 0° to 90°
(Figure 1). Figure 4a shows the average wind velocity contour of gable roof. The
wind velocities are stable and almost similar with wind velocity approach. This
contrasts with hip roof with 90° position (Figure 4b), which wind velocity increase
20% more than wind velocity approach (8 m/s up to 8.50 m/s).
Innovation for Better ASEAN Community 165

Figure 3a. Average wind velocity contour for gable roof at 0° position

Figure 3b. Average wind velocity contour for hip roof at 0° position
166 Proceeding ASEAN COSAT 2014

V. Conclusion
Coastal area has the potential for wind energy. It has been found that wind velocity
depends on the roof profiles as well as the wind direction. From meteorological
data at Semarang, Indonesia, it was found that the highest wind power potential
is on north wind with average wind velocity 5 m/s in the height 10 m from the
ground surface. A comparison between two traditional roof profile, gable roof and
hip roof, suggest that hip roof is more suitable in Semarang. The wind velocity
above gable roofs has lower velocity compared to hip roof profile. Based on the
velocity above the roof, hip roof with 90° position is the most favorable shape in
Semarang, Indonesia which could increase wind velocity 20% bigger than wind
velocity approach (wind profile). Future studies of wind velocity above other roof
profiles will be covering different building shapes and places.

VI. Acknowledgement
This paper was developed and delivered with data support from Meteorological
Department (BMKG) Semarang City and Indonesian Institute of Sciences (LIPI).

VII. R eferences
1) National Standarization Agency of Indonesia (BSN). (2011). Presidential
Regulation (Peraturan Presiden) No. 6/2011: National plan to reduce
greenhouse gas emission.
2) Badan Meteorologi Klimatologi dan Geofisika (BMKG) Stasiun Klimatologi
Semarang. (2013). Wind speed, solar radiation intensity, temperathure and
humidity in Semarang, Central Java, Indonesia years 2008–2012.
3) Johansen, O. (2011). The spatial diffusion of green building technologies: the
case of Leadership in Energy and Environment Design (LEED) in the United
States. International Journal of Technology Management and Sustainable, 10
(3), 251–266.
4) Ayhan, D. and S. Saglam. (2012). A technical review of Building-mounted
wind power systems and a sample simulation model. Renewable and Sustain-
able Energy Reviews, 16, 1040–1049.
5) Abohela, I., Hamza, N. and S. Dudek. (2013). Effect of Roof Shape, Wind
Direction, Building Height and Urban Configuration on The Energy Yield
and Positioning of roof Mounted Wind Turbine. Renewable Energy, 50,
1106–1118.
Innovation for Better ASEAN Community 167

6) Sari, D. P., and W. B. Kusumaningrum. (2014). A Technical Review of


Building Integrated Wind Turbine System and a Sample Simulation Model
in Central Java, Indonesia. Energy Procedia, 47, 29–36.
7) American Wind Energy Association (AWEA). Wind Classes [Online].
Retrieved from www.awea.org/fag/basicwr.html.
8) Soeripno, S. (2012). Wind energy potential and development in Indonesia.
In Proceeding of The 2nd Clean Energy Power Asia Denpasar-Bali. 14–15 May
2012.
PERFORMANCE OF A RADIAL TURBINE FOR SMALL
ORGANIC RANKINE CYCLE POWER GENERATION
SYSTEM
Maulana Arifina,*, Bambang Wahonoa and Ari Darmawan Pasekb
a
Research Center for Electric Power and Mechatronics – LIPI
Komp. LIPI Gd. 20, Jl. Cisitu No.21/154D, Bandung 4015, Jawa Barat
Phone: 022 2503055, Fax: 022 2504773
b
Faculty of Mechanical and Aerospace Engineering – Bandung Institute of Technology
Jl. Ganesha No.10 Taman Sari, Bandung 41032, Jawa Barat
Phone: 022 2504243, Fax: 022 2534099

Abstract
Coresponding to global environmental problems and energy crisis in recent years, a method to
choose the optimal working fluids which improves performance of power generation with Organic
Rankine Cycles (ORC) is required. This paper investigates the optimal working fluids for ORC
with focus on thermo-fluid. In the ORC system, radial turbine component is highly influential
in resulting high and low performance. This paper discusses comparative study of radial turbine
using R134a and R123 as working fluids. Comparative study consisted of numerical analysis to
determine the performance of radial turbine for ORC system. Numerical study was carried out in
area of fluid flow turbo-expander rotor radial with R134a and R123 as the working fluids. Analysis
was performed using two turbulence models, the k-epsilon and SST (shear stress transport). The
results shows analysis with grid of 250000 (fine grid), turbulence model SST at steady state, mass
flow rate of 0.4 kg/s, torque of 15000 rpm, inlet pressure of 5 bar, inlet temperature of 373 K,
and working fluid of R134a produces power of 6,7 kW whereas R123 produces power of 5,5 kW.
Key words: Organic Rankine Cycle, Radial turbine, Shear Stress Transport

I. Introduction
Renewable energy technologies use natural resources, such as sunlight, wind, rain,
tides and geothermal heat, all of which are naturally replenished. Climate change
concerns coupled with high oil prices are driving research and development on
renewable energies. The ORC uses organic fluid as the working fluid to provide
higher thermal cycle efficiency compared to the conventional steam Rankine cycle
at resource temperatures below 300 °C. ORC has been studied as the utilization
of waste heat recovery [1,2], solar energy [3], the combination of heat and power
(CHP) [4], geothermal [5] and heat recovery from the exhaust gases from the
engine [6]. The results of experimental studies show that the small-scale units

* Corresponding author. Phone: +6222 2503055. Email: mfilzah@gmail.com, maul004@lipi.go.id

169
170 Proceeding ASEAN COSAT 2014

ORC showed a promising performance for small-scale power generation especially


in remote areas, because the ORC has reliability, a wide output power range, the
availability of large component parts, as well as the reduced number of rotating
parts, so that ORC’s construction is more compact and smaller compared to
conventional power plants.
Most studies have focused on the study of the thermodynamic cycle of the
ORC and the selection of the working fluid, with particular attention to the ef-
ficiency of power generation. On the other hand, there are relatively few published
works on the design and optimization of turbomachinery equipment. Basically,
the relevant power range (5–5000 kW) two options are proposed: turbine axial
or radial turbines (often called turbo-expanders). The latter option is considered
more attractive, because it allows better performance at a lower scale. Therefore,
knowledge of radial turbine in ORC systems required further study.

II. M aterial and methods


The performance characteristic of the radial turbine for small organic rankine
cycle was evaluated using ANSYS by analyzing the torque and power output on
relationship between mach number and rotational speed of rotor radial turbine
with R134a and R123 as working fluid. The main components of radial turbine
are rotor, shown in Figure 1. Table 1 shows operational condition for parameter
input on analysis CFD with ANSYS.

Figure 1. Geometry radial rotor turbo-expander for small organic


rankine cycle system
Innovation for Better ASEAN Community 171

Table 1. Parameter input on analysis CFD


Parameter Value
Temperature inlet (K) 353-423
Mass flow (kg/s) 0.1-1.0
Pressure outlet (atm) 1
Temperature outlet (K) 313

A. Simulation with Different Grid


In simulation with different number of grids, the objective is to know how to
influence the amount of output generated grid. Effect of grid number is the
expected outputs of the model with a certain number of grid which can be more
accurate and the selected model is used as a model for subsequent simulations.
Figure 2 shows grid model for analysis CFD and Table 2 shows grid number.

B. Simulation with two turbulence models


ANSYS uses an element-based finite volume method linked to pressure-based
coupled solver for solving the hydrodynamic equations (u,v,w,p) [7]. A second-
order discretization scheme has been applied to these equations while a first-order
upwind advection scheme has been used for turbulence equations. The turbulence
intensity has been initialised at a medium level of 5%. This value has also been
used at the inlet of the rotor.
Numerical study is carried out in area of fluid flow rotor radial turbine
with R134a and R123 as the working fluid. Analysis was performed using two
turbulence models, the k-epsilon and SST (shear stress transport).

mainstream

downstream

upstream

Figure 2. Grid model for CFD computations


172 Proceeding ASEAN COSAT 2014

III. R esults and discussion


A. Simulation with Different Grid
Simulation with different grid showing the output results of numerical simulation
using the finite volume method represent a power of radial turbine as a function
of rotational speed. Figure 3 shows three model grid flow field predictions for
radial turbine using R123 as working fluid with rotational speed of 20000 rpm.
In model 3 (250,000 grid), a strong vortex can be seen forming on the pressure
surface soon after the leading edge, separating flow from the hub surface and
moving it up the blade toward the tip. After calculation process are solved by
ANSYS, torque and power radial tubine will be generated from the results of
calculations and represented in Table 2.

Model 1 Model 2 Model 3


Figure 3. Three model grid flow field predictions for radial turbine using R123 as working fluid
with rotational speed of 20,000 rpm

Table 2. Power and efficiency for rotor radial turbo-expander with different grid number
Model 1 Model 2 Model 3
Number Grid 20000 100000 250000
Fluid : R123
20,000 rpm
Torque (Nm) 0,32 0,35 0,36
Power (kW) 6,56 7,08 7,21
Efficiency 0,63 0,64 0,65
Time consumed 6 minute 30 second 11 minute 56 second 28 minute 05 second

Fluida R134a
20,000 rpm
Torque (Nm) 0,44 0,47 0,48
Power (kW) 7,94 8,60 8,53
Efficiency 0,63 0,64 0,65
Time consumed 6 minute 21 second 11 minute 54 second 27 minute 30 second
Innovation for Better ASEAN Community 173

Table 3. Time computation, iteration number on turbulence models for radial turbine small
ORC with R123, 20,000 rpm
Model Time consumed Iteration Power (kW)
k-epsilon (k-ɛ) 28 minute 200 6,1
SST 28 minute 200 7,2

Figure 4. Velocity contour for turbulence models (a) k-epsilon and (b) SST

0 rpm 15000 rpm

20000 rpm 30000 rpm

Figure 4. Velocity contour for steady flow radial turbine with R123 as working fluid on various rotational
speed
174 Proceeding ASEAN COSAT 2014

0 rpm 15000 rpm

20000 rpm 30000 rpm

Figure 5. Velocity contour for steady flow radial turbine with R134a as working fluid
on various rotational speed

B. Simulation with two turbulence models


Table 3 shows the computation time and the number of iterations in the k-epsilon
and SST turbulence models. In Figure 4 and Figure 5, models with SST have
good swirl flow prediction on blade passage and the significant power generated
differences occurred.
In the k-epsilon turbulence model, the power is generated at 6.1 kW while
the SST turbulence models generated power of 7.2 kW. The increase in power
for the k-epsilon model of turbulence model to SST reaches 18%. It is to be
considered for the selection of turbulence models.

IV. Conclusion
Performance of radial turbine for small Organic Rankine Cycle system has been
evaluated using 3D flow field analysis method. Grid element analysis yields 3
models, which the 250,000 grid has good results. The analysis used two turbulence
models, and SST models has good results with mass flow rate of 0.4 kg/s, 15,000
rpm, 5 bar inlet pressure, and 373 K inlet temperature. Working fluid R134a
produces power of 6,7 kW, with total efficiency-to-static (ηts) 0,71, and R123
produces power of 5,5 kW, with total efficiency-to-static (ηts) 0,66.
Innovation for Better ASEAN Community 175

Figure 6. Rpm vs Power of working fluid R134a and R123 performance

V. Acknowledgement
Authors would like to thank all the members of the Research Centre for Electrical
Power and Mechatronics and members of Thermodynamics Laboratory of
Mechanical Engineering Faculty, Bandung Institute of Technology, for any
assistance that has been given.

VI. R eferences
1) Hung, T.-C. (2001). Waste heat recovery of organic Rankine cycle using dry
fluids. Energy Conversion and Management, 42 (5), 539-553.
2) Gnutek, Z. and A. Bryszewska-Mazurek. (2001). The thermodynamic analysis
of multicycle ORC engine. Energy, 26 (12), 1075–1082.
3) Manolakos, D., Papadakis, G., Kyritsis, S. and K. Bouzianas. (2007). Experi-
mental evaluation of an autonomous low-temperature solar Rankine cycle
system for reverse osmosis desalination. Desalination, 203 (1-3), 366–374.
4) Schuster, A., Karellas, S., Kakaras, E. and H. Spliethoff. (2009). Energetic
and economic investigation of Organic Rankine Cycle applications. Applied
Thermal Engineering, 29 (8-9), 1809–1817.
5) Kanoglu, M. (2002). Exergy analysis of a dual-level binary geothermal power
plant. Geothermics, 31 (6), 709–724.
176 Proceeding ASEAN COSAT 2014

6) Talbi, M. and B. Agnew. (2002). Energy recovery from diesel engine exhaust
gases for performance enhancement and air conditioning. Applied Thermal
Engineering, 22 (6), 693–702.
7) Ventura, C. A. M., Jacobs, P. A., Rowlands, A. S., Petrie-Repar, P. and E.
Sauret. (2012). Preliminary Design and Performance Estimation of Radial
Inflow Turbines: An Automated Approach. Journal of Fluids Engineering, 134,
031102.
Effect of Reaction Time and Cellulase
Loading on Dilute Alkali Pretreatment of
Sugarcane Bagasse to Produce Fermentable
Sugars for Bioethanol Production

Triyani Fajriutamia,* and Rizky Rissa Bellab


Research Center for Biomaterials, Indonesian Institute of Sciences (LIPI)
a

Jl Raya Bogor KM 46, Bogor, Indonesia


b
Bina Putera Nusantara Vocational High School
Jl Sukarindik 63A, Tasikmalaya, Indonesia

Abstract
Lignocellulosic materials, which consist mainly of cellulose, hemicellulose and lignin, are among
the most promising renewable feedstocks for the production of bioethanol. Its production typically
involves a hydrolysis-fermentation route, which has three main steps: pretreatment and hydrolysis
to get fermentable sugars, fermentation to produce bioethanol and a separation process to obtain
highly concentrated bioethanol. The pretreatment step has been recognized as a technological
bottleneck for the cost-effective development of bioprocesses from lignocellulosic materials. In
this work, we analyze the potential of dilute alkali pretreatment of sugarcane bagasse, followed by
enzymatic hydrolysis for fermentable sugars production when using 1:20 ratio between substrate
and 1% NaOH. Alkali pretreatment of sugarcane bagasse at 121 °C and reaction time of 7.5, 15,
30, 60 and 90 minutes were investigated. Furthermore, the enzymatic hydrolysis using cellulase 10
and 20 FPU/g substrate was examined. The result shows that the highest lignin loss was 74.95%
when sugarcane bagasse was pretreated for 60 minutes. The enzymatic hydrolysis of pretreated
sugarcane bagasse for 48 hours and 20 FPU/g of cellulase loading produced the highest yield of
fermentable sugars of 49.11%.
Key words: Lignocellulosic, Sugarcane bagasse, Alkali pretreatment, Cellulase hydrolysis, Ferment-
able sugars

I. Introduction
Lignocellulosic materials have been recently considered as promising sources of
bioethanol [1]. An advantage of lignocellulosic materials is the avoidance of the
competition with food production. Furthermore, there are abundant source for
lignocellulosic materials including waste materials. Lignocellulosic materials are
characterized by three components, which are cellulose, hemicelluloses and lignin.
These components are complex polymers. Lignin acts as a wall covering hemicel-
lulose and cellulose; hemicellulose do not have a defined form, and cellulose

* Corresponding author. Phone: +62-2187914509. Email: triyani@biomaterial.lipi.go.id

177
178 Proceeding ASEAN COSAT 2014

is the most difficult component to access. Among the available lignocellulosic


materials, sugarcane bagasse has been considered as a high-potential candidate
for bioethanol production in Indonesia because it is available in abundance in
sugarcane mills [2].
Three principal steps to convert lignocellulosic materials into bioethanol are
pretreatment, hydrolysis and fermentation. Pretreatment of lignocellulosic materi-
als to remove lignin and hemicellulose can significantly enhance the hydrolysis of
cellulose [3]. According to the literature [4,5,6,7], pretreatment is the bottleneck
into the development of bioprocess from lignocellulosic materials.
One of the major aspects in bioethanol production is the energy consumption,
so the pretreatment method should be as energy efficient as possible to contribute
in the success of the overall process. Alkaline pretreatment methods are receiving
much attention due to relatively cheap, less energy intensive and effectiveness
towards many feedstocks rather than other pretreatment methods [8,9]. Alkaline
pretreatments lead to delignification, disruption of structural linkages, decrystal-
lization of cellulose and depolymerization of the carbohydrates [10,11].
Enzymatic hydrolysis and fermentation are known to be environmentally
friendly processes for converting lignocellulose to ethanol [12]. Enzymatic pro-
cesses under development are supposed to have roughly equal costs today, but
the costs can be decreased further. Therefore, most studies focus on enzymatic
hydrolysis [13]. The fermentation step, on its turn, does not yet convert all sugars
with equal success. Future overall performance depends strongly on development
of cheaper and more efficient microorganisms and enzymes for fermentation.
In this study, we investigated the effect of reaction time at 121°C from dilute
sodium hydroxide pretreatment of sugarcane bagasse followed by the effect of
cellulase loading on hydrolysis process for its reducing sugar production.

II. Method
A. Raw Material
Sugarcane bagasse from sugar factory in Subang, West Java-Indonesia was milled
and sieved to get particles of 40–60 mesh, then it was stored in sealed plastic bag
at room temperature until it was ready to be used for pretreatment. The sugarcane
bagasse was measured for its ash and moisture content by gravimetric method
and extractives content by Soxhlet extraction as well as its lignin, cellulose and
hemicellulose content.

B. Pretreatment
Sodium hydroxide (NaOH) at concentration of 1% (w/v) was used to pretreat 5
g milled sugarcane bagasse samples at 1:20 ratio (substrate: NaOH solution) in
Innovation for Better ASEAN Community 179

250 mL erlenmeyer flask. Treatments were performed in an autoclave at 121°C


with reaction time of 7.5, 15, 30, 60 and 90 minutes. Water is used to substitute
the alkaline solution as a control. After pretreatment reaction, the samples were
filtered to separate the solid from the liquid (pretreated hydrolysate). Pretreated
solids were washed with water until the filtrate registered a neutral pH and sealed
in plastic bags to retain moisture. Then some wet samples were refrigerated for
further Scanning Electron Microscope (SEM) analysis using Phenom ProX
Desktop SEM and subjected to enzymatic hydrolysis. The other ones were dried
at 60°C and determined for its lignin contents after pretreatment. Lignin loss
was also calculated.

C. Enzymatic Hydrolysis
Enzymatic hydrolysis of pretreated sugarcane bagasse was carried out using a
commercial cellulase (Meicellase from Meiji Seika, Japan). Vial bottles (20 ml)
containing 0.1 g (dry weight) delignified sugarcane bagasse was mixed with
cellulase solutions (10 and 20 FPU/g), 0.1 ml of sodium azide (20 mg/ml) to
inhibit microbial contamination, and weight was made to 10 g mark with 0.05 M
citrate buffer of pH 5. The vials were incubated at 50°C in orbital shaker at 150
rpm for 48 h. The vials were placed horizontal in shaking incubator to increase
surface area contact between substrate and enzyme. Reducing sugar concentration
was determined by Nelson-Somogyi method.

III. R esult and Discussion


A. Characterization of Sugarcane Bagasse
Sugarcane bagasse is a polydisperse particulate. Most of the bagasse weight is in the
form of the so-called fiber and rind particles, which have high length-width ratios
(lengths up to a few centimeters) and correspond mostly to stalk fibrovascular
bundles [14]. The sugarcane bagasse in this work was milled and sieved before
chemical analysis because of the heterogeneous and fibrous nature of sugarcane
bagasse. Milling is an important step in the development of conversion routes
for production of cellulosic biofuels and co-products [14].
Characterization of sugarcane bagasse was carried out to determine its major
of principal components. The chemical composition of initial sugarcane bagasse
(before pretreatment) used in this study is presented in Table 1. The carbohydrate
fraction (holocellulose fraction) of sugarcane bagasse was 61.96% of the total
biomass, which consist of 34.48% alpha-cellulose and 27.48% hemicellulose.
The major component of sugarcane bagasse was alpha-cellulose, a polymer of
glucose, which is very potential as a sugar source for ethanol production. The lignin
content of sugarcane bagasse was 22.45%, comparable to the lignin content of
180 Proceeding ASEAN COSAT 2014

Table 1. Chemical contents of the raw sugarcane bagasse


Components Percentage (%)a
Ash content 2.13 ± 0.3756
Extractive in EtOH-Benzene 1.58 ± 0.1983
Klason lignin 22.45 ± 0.0095
Holocellulose:
Alpha-cellulose 34.48 ± 0.2032
Hemicellulose 27.48 ± 0.2032
composition percentages are on dry-weight basis
a

hardwoods (18–25 %) [15]. This high lignin content is the main reason of alkali
(sodium hydroxide) pretreatment which would be applied to sugarcane bagasse.
The main effect of alkali pretreatment on lignocellulosic biomass is delignification
by breaking ester bonds cross-link lignin and xylan, thus increasing the porosity
of the biomass [16].

B. Effect of Reaction Time of Pretreatment


Alkali delignification of sugarcane bagasse is to expose cellulose polymers for
hydrolysis and bioethanol production. Pretreatment refers to complete or partial
degradation of lignocellulosic biomass to expose cellulose polymers for convenient
cellulose hydrolysis into sugars [7]. Alkali treatments were used for delignification
of sugarcane bagasse before hydrolysis by cellulase.
Concentration of NaOH used in this study was 1% (w/v) for pretreatment/
delignification of sugarcane bagasse for different reaction time. Lignin contents
of treated bagasse were determined. The degradation of lignin in the pretreated
sugarcane bagasse is represented as the component loss of lignin, shown in Figure
1. Figure 1 shows that the increase of reaction time affected the loss of lignin
from 7.5 to 60 minutes. Maximum delignification (74.95%) was caused by 1%
NaOH treatment for 60 minutes (Figure 1). Varga et al. [17] reported 95%
reduction in lignin content as a result of pretreatment of corn stover with 10%
NaOH for 1 hour in the autoclave. The higher reduction level than this work
result may be attributed to a higher NaOH concentration of 10%, which in this
study was limited to 1%.
SEM data reveal difference between the raw and pretreated samples. Morpho-
logical changes of raw and alkali pretreated sugarcane bagasse were examined by
SEM to evaluate the structural modification of the surface. Figure 2 shows the
scanning electron micrographs of raw and alkali pretreated sugarcane bagasse. The
raw samples have a compact rigid structure and highly ordered fibrils, while the
pretreated samples showed a distorted structure. Moreover, the microfibrils were
separated from the initial connected structure and fully exposed, thus increasing
the external surface area and the porosity of the sugarcane bagasse. By hand-touch
Innovation for Better ASEAN Community 181

Figure 1. Lignin loss after NaOH 1% pretreatment of sugarcane bagasse at 121 °C

Figure 2. SEM of sugarcane bagasse (a) before and after 1% NaOH pretreatment
at 121 °C for (b) 7.5 minutes, (c) 15 minutes, (d) 30 minutes, (e) 60 minutes, (f )
90 minutes

of the material, we also felt that pretreated sugarcane bagasse was much softer than
the untreated one. Zhang and Cai [18] treated rice straw with 2% NaOH and
reported reduction in lignin content of before and pretreated straw from 14.9%
to 9.5% respectively and also mentioned that after treatment with NaOH the
basic tissue become severely shrank.
182 Proceeding ASEAN COSAT 2014

The effectiveness of different pre-treatment methods including alkali, acid and


chlorite pretreatment of lignocellulosic feedstock’s for improving the hydrolysis of
cellulose has been evaluated by Gupta et al. [19]. Mild alkali treatment conditions
caused complete conversion of cellulose I to cellulose II, which is a more stable
form with antiparallel chain structure in NaOH solution [20]. In another recent
study, Liu et al. [21] reported that an alkaline pretreatment can improve the
cellulose hydrolysis by modifying the cellulose crystalline structure.

C. Effect of Cellulase Loading on Hydrolysis


The delignified bagasse were then treated for 48 h at 50 °C with cellulase enzyme
solution, at enzyme loadings of 10 and 20 FPU/g. Cellulase is a mixture of several
enzymes. There are at least three major groups of cellulases that involved in the
hydrolysis process: 1) endoglucanase, which attacks regions of low crystallinity in
the cellulose fiber, creating free chain-ends; 2) exoglucanase or cellobiohydrolase,
which degrades the molecule further by removing cellobiose units from the free
chain-ends; and 3) β- glucosidase, which hydrolyzes cellobiose to produce glucose
[16].
The effect of enzyme concentration on the hydrolysis of alkali-pretreated
sugarcane bagasse is shown in Table 2 and Figure 3. Reducing sugar concentration
is shown in Table 2 and reducing sugar yield is shown in Figure 3. The reducing
sugar concentration is amount of reducing sugar found on the 1 L of liquid
hydrolysate after the hydrolysis. Meanwhile, the reducing sugar yield is the total
amount of reducing sugar produced when 100 g pulp of pretreated sugarcane
bagasse was hydrolyzed.
Cellulase concentration of 10 FPU/g resulted yield of fermentable sugars,
which are 40.30%, 41.21%, 41.69%, 45.90%, 43.23% for 7.5, 15, 30, 60,
90 minutes of reaction time of pretreatment, respectively. Meanwhile, cellulase
concentration of 20 FPU/g resulted yield of fermentable sugars, which are 41.85%,
46.29%, 41.69%, 49.11%, 45.10% for 7.5, 15, 30, 60, 90 minutes of reaction
time of pretreatment, respectively. Based on these results, during 48 hours of
hydrolysis of 60 minutes pretreatment, the 20 FPU/g of cellulase loading resulted
Table 2. Reducing sugar concentration of hydrolysate after cellulase hydrolysis of pretreated
sugarcane bagasse
Reaction Time Reducing Sugar Concentration (g/L)
(minutes) 10 FPU/g 20 FPU/g
7.5 4.99 5.18
15 4.41 4.95
30 5.03 5.03
60 4.09 4.37
90 4.92 5.13
Innovation for Better ASEAN Community 183

Figure 3. Reducing sugar yield after cellulase hydrolysis of pretreated sugarcane bagasse

in highest yield of fermentable sugars of 49.11%. It is revealed that longer reaction


time of pretreatment at 121°C to 90 minutes did not increase the yield of sugars.
Generally, enzymatic hydrolysis efficiency of cellulose correspondingly
increased with pretreatment temperature, reaction time and alkali concentration.
McIntosh and Vancov [6] reported that temperature had a significant impact with
121°C being more acquiescent to cellulose hydrolysis than 60°C. Comparable
glucose yields were also observed with a pretreatment time of 60 minutes.
Moreover, when alkali strengths were below 2%, no advantage was observed
when reaction times increased to 90 minutes.

IV. Conclusion
As expected, higher enzyme loading results in higher yield of sugar. In the present
study, the highest hydrolysis was obtained at enzyme concentration 20 FPU/g.
The dilute alkali pretreatment resulted in a high sugar yield of sugarcane bagasse
associated with the high reduction in lignin and the increase in cellulose content.
There may be opportunities for further process optimization, finding the right
pretreatment condition and/or enzyme combinations and dosages. Considering
its abundance and high sugar potential, sugarcane bagasse is an excellent feedstock
for ethanol production.

V. Acknowledgement
We gratefully acknowledge the financial support provided by DIPA LIPI 2013
for this work. We express our gratitude to Mr. Raden Permana Budi Laksana and
Mr. Sudarmanto for their technical assistance.
184 Proceeding ASEAN COSAT 2014

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carbpol.2012.03.058
Performance of Microbes Consortium On
Single-Chamber Microbial Fuel Cell as
Electricity Generation
Diana Rahayuningwulana,*, Dani Permanaa and Herlian Eriska Putraa
Research Center for Chemistry, Indonesian Institutes of Sciences,
a

LIPI Campus Cisitu, Bandung, Indonesia

Abstract
Microbial Fuel Cell (MFC) systems use microbes to convert organic compounds, as in food
wastewater treatment, and could produce direct current. Single microbe has showed their
performance as good biocatalysts on previous researches, both on synthetic media or wastewater.
This research studied 1-liter single-chamber MFC (SCMFC) using microbes as consortium on
tofu wastewater, since it has high organic pollutant value (COD as O2). Variation of consortium
concentration consists of three single microbe, Saccaromyces cereviceae, Saccaromycopsis fibuligera,
and Escherichia coli that previously acclimated with the wastewater. Result shows that tofu
wastewater, as substrate for the consortium decreases 76% COD value compared to the blank on
variation 1. This SCMFC system also produced maximum current at 0.25 mA with consorsium
SF2SC1:EC1 during 40hours.
Key words: SCMFC, Microbes, Consortium, Tofu wastewater, Current

I. Introduction
Microbial fuel cell would be an alternative of renewable energy source, where
bacteria as biocatalyst source on oxidizing organic and/or inorganic matter and
produce electricty. Electron produced by bacteria activity from its substrate would
be transferred into anode (negative pole) to cathode (positive pole) by conductor
and resistor.
In MFC, bacteria catalyse oxidation process from reducted substrate release
electrons from respiration cell to anode, which flows by external circuit loop to
cathode chamber and produce current. Every electron produced, a proton could be
transferred via electrolyte (liquid phase) to maintain current continuity. Electron
and proton react with oxygen in cathode chamber, which catalyzed by common
catalyst, as platinum, to form water.
Based on previous research [1], electrons can be transferred to the anode by
electron mediators or shuttles, direct membrane associated electron transfer, or

* Corresponding author. Phone: +62-222503051. Email: dian009@lipi.go.id

187
188 Proceeding ASEAN COSAT 2014

so-called nanowires produced by the bacteria, or undiscovered means. Bacteria


gain the energy by electron transfer from substrate reduction on low potential
(as glucose) to electron acceptor on higher potential (as oxygen).
Basic and economic design consists of two chambers MFC of H-shape, i.e.
two bottles connected completed by separator, e.g. cation exchange membrane or
simple salt bridge. Main component consists of anode, cathode, and electrolyte.
Factors that influence MFC operation system are temperature, pH, electron
acceptor, surface area, reactor dimension and operation time. Substrates also
determine the performance of MFC, for example acetate, glucose, biomass
lignocellulose, beer wastewater, starch, cellulose and chitin [2,3]. Most of MFC
operating on neutral pH to maintain growth condition of microbes on current
production [4]. Ionic strength also influences the conductivity of suspension on
MFC that impacts to internal resistance and its performance [4].
Wastewater could be functioned as electron donor, to be placed in anode
chamber, and have large potential energy, for example domestic wastewater
with–32.80 kJ/eq [5].
Based on recent research using different types of wastewater and inoculum
source [6], current density showed below 2 mA/cm2. High current density resulted
from two-chamber MFC using chocolate wastewater and graphite as electrodes, ie.
0.302 mA/cm2 [7], which COD 1,459 mg/L and using activated sludge inoculum.
Single inoculum of microbe has been studied and showed their result on
previous research. S. cereviceae on double chamber MFC using glucose yeast
extract medium with riboflavin mediator [8], S. cereviceae on salt-brigde MFC
using rice-rinsing wastewater [9], E. coli on salt bridge MFC on glucose and
brewey wastewater [10]. S. fibuligera application on decomposed amylum and
produced amylase had been studied [11], and potential as biocatalyst on MFC.
Since mixed culture gives better organic degradation in wastewater treatment
performance [12,13], this research aim to determine the influence of consortium
microbes variation to current production and organic removal on aerobic single-
chamber MFC using tofu industry wastewater. Tofu industry as one of traditional
food industry in Indonesia produced 15–20 liter wastewater/kg soybean with
BOD dan COD concentration approximately 65 gr and 130 gr/kg soybean
[Potter, C.S., M and Gani A., 1994 in [14].

II. Method/M aterial


A. Reactor
This research used MFC reactor consists of single 1 liter-glass tube, which anode
chamber filled with 900 mL wastewater and 100 mL isolate microbes suspension
and using air cathode. Tofu industry wastewater was from process PT X, Bandung
Innovation for Better ASEAN Community 189

Figure 1. Scheme of Single Chamber MFC

with COD concentration 7,600–10,000 mg/L (pH 4.0–5.6 and conductivity


9.6 mS).
Batch reactor operated aerobically on temperature 25+0.1°C. Electrodes
connected to multimeter SANWA and data collected every 4 hours as shown
Figure 1, with COD sample collected every 12 hours.
Carbon plate 5 mm of 4 cm x 7 cm was placed as the anode and cathode, as
their good performance and inexpensive material electrode for MFC [15].
Salt-bridge, as cation exchange, was clamped between anode and cathode
chamber. Salt bridge was prepared from 11.6 gram sodium chloride added on 5%
(b/v) commercial agar powder and diluted in 100ml aquadest, and then boiled
and stirred homogeniously. Then, agar solution was poured to the 3 cm diameter
pipe and allowed to solidify.

B. Microbes
Microbes were consortium of S. cereviceae, S. fibuligera, and E. Coli, obtained
from Biochemistry Laboratory, University of Padjadjaran, Bandung, Indonesia.
Enrichment of microbes consortium used standard methods [16]. Before operat-
ing, 100% wastewater on MFC, activation and acclimatization was conducted
for each microbes with comparison from 25, 50, until 75% of tofu wastewater
to MFC medium.
190 Proceeding ASEAN COSAT 2014

The medium and inoculum medium were used for S. cereviceae, S. fibuligera,
and E. coli cultivation was YEPD (Yeast Extract Potato Dextrose) medium.
Liquid YEPD medium placed in closed Erlenmeyer. It was sterilized at 121 °C of
temperature and 15 psi of pressure for 15 minutes with an autoclave (Hirayama
HL36 AE, Japan) [17].
Composition of YEPD (yeast extract peptone dextrose) medium for MFC
medium and inoculums medium were 0.5% (w/v) of yeast extract (Becto and
Dickinson), 0.5% (w/v) of bacteriological peptone (Becto and Dickinson),
0.3% (w/v) of ammonium sulfate (Merck), 0.3% (w/v) of potassium dihydrogen
phosphate (Merck), 2% (w/v) of glucose (Sygma-Aldrich), and 1.5% (w/v) of
agar (Becto and Dickinson) [17].
Inoculum of S. cereviceae, S. fibuligera, and E. coli was made by took a loop
of S. cereviceae, S. fibuligera, and E. coli pure culture from agar slant, and then
inoculated into sterile YEPD inoculum medium with 25 mL of volume. Then,
inoculum medium incubated for 18 hours at 30°C and shaken at 150 rpm in a
shaking incubator (Certomat B Braun). The ratio of Erlenmeyer size of to the
volume of the culture volume was maintained at 4:1 to maintain the availability
of dissolved oxygen. The entire MFCs inoculum was transferred to the medium
of MFCs. The volume of MFCs inoculum that added to MFCs medium was
10 % (v/v) of 1000 mL of MFCs medium. Concentration of microbes was 106
CFU/mL.
Since consortium concentration might influenced the electricity generation,
microbe of S. fibuligera (SF), S. cereviceae (SC), and E. coli (EC), was made on
consortium ratio between 1) SF2 : SC1 : EC1, and 2) SF1 : SC2 : EC1. Those
consortiums were tested on 100% tofu wastewater on single chamber MFC and
compared to control reactor without consortium addition.

C. Analysis
Data collection of Optical Density (OD) was set every 4 hours and 12 hours
for Chemical Oxygen Demand (COD) analysis sample. Microbes growth was
approached by detecting its optical density, where the measurement of OD (λ 560
nm) using spektrofotometer LW Scientific V325XS. COD sample was analized
using closed reflux titrimetry SNI 6989.2:2009 method.
Collected data was analized for microbes performance on acclimatization and
running MFC, COD removal, current, and voltage measurement.

III. R esult and Discussion


A. Acclimatization
On acclimatization, microbes were adapted step by step into wastewater char-
acteristic, in order to prevent any shock loading. This 0% wastewater, increased
Innovation for Better ASEAN Community 191

into 25%, 50%, and 75% wastewater was done until OD concentration showed
constant graph, where indicated their growth not significantly different between
each microbes, as shown in Figure 2. On 0% wastewater ratio, S. cereviceae was
the easiest adaptable microbe than two other microbes. It reached its maximum
growth in exponential phase on 20 hours, where the microbe growth, the cell
division and increasing cell numbers run very fast, and also cell biomass increased.
Although suddenly decreased into death phase.
At 75% wastewater ratio, those three microbes need only 12 hours for
exponential phase before entered the stationary phase, as the indicator of limited
nutrient and demand of substrate supply [18]. This phase also indicate that
microbes consortium ready for wastewater substrate.
S. cereviceae also reached maximum growth better than S. fibuligera and E.coli,
either on ratio 25% wastewater or 50%. On 75% ratio, E. coli and S. fibuligera
showed their performance approached to S. cereviceae.

(a) (b)

(c) (d)
Figure 2. Growth Curves in Acclimatization Stage of (a) 0% ; (b) 25%; (c) 50%; (d) 75%
wastewater to substrate
192 Proceeding ASEAN COSAT 2014

S. cerevisiae or Baker’s yeast was able to produce bioethanol in medium of


acclimatization and medium of MFC. As we know, ethanol is toxic for micro-
organism. During ethanol fermentations, yeast cells suffer from various stresses.
Not only yeast cells suffered various stresses, but also bacteria cells like E. coli.
S. cerevisiae have ethanol tolerance and can survive in medium that ethanol
existed. However, high ethanol concentrations cause a problem, in that the
fermentative microorganisms have limited ethanol tolerance. S. cerevisiae can
survive in maximum concentration of ethanol of 18 % (v/v) [19]. Growth curve
and cells amount of S. cerevisiae are higher than E. coli and S. fibuligera. It is
possibly caused by the ability of S. cerevisiae to growth in ethanol stress but E.
coli and S. fibuligera were not.

B. MFC Operation
At the end of acclimatization stage, the consortium microbes were tested into
single-chamber MFC with 100% tofu wastewater. Organic matter in wastewater
was oxidized by microbes, the electron released and transferred to electrode (anode)
[20]. This electron was transferred (determined as current), and open circuit
voltage (OCV) was compared with control reactor without microbes applied.
Current production on both variations was shown in Figure 3. Compared to the
control reactor, there was a significant difference and variation with SF1:SC2:EC1
gave better result in current production. Control reactor produced 0.11 mA
current at the maximum performance.

Figure 3. Current Production


Innovation for Better ASEAN Community 193

In this study, compared to control reactor, consorsium addition increased


current into 0.15 mA for SF2:SC1:EC1 and into 0.25 mA for SF1:SC2:EC1 in
16 hours. The consortium addition influenced current production, almost 127%.
Mix culture as biocatalyst sinergy treated on the substrate. Utilization consortium
culture consists of S. fibuligera and E.coli in tofu wastewater as substrate, which
resulted maximum current 0.23 mA, and it had increased from 0.19 mA maximum
current when using E.coli only [21], this study gave higher result. The advantages
of using microbes consorsium are microbes could degrade the substrate sequently,
consortium can increase the rate of subsrate degradation overall, and consortium
allow microbes find the easiest thermodynamic path or process [22].
S.cereviceae is a facultative microbe that could respirated under aerob and
anaerob condition, and could use media containing high glucose and protein, as
in tofu wastewater, as source of its metabolism.
Based on Figure 4, maximum voltage 0.432 volt and 0.505 volt were resulted
by SF2:SC1:EC1 and SF1:SC2:EC1 variation respectively, in 16 hours. Compared
to double chamber using potassium fericyanate (Rohan, 2013), maximum voltage
using E. coli reached 0.534 V in 2 days.
Consortium allows microbes to find the easiest thermodynamic path or
process [22], so that consortium could oxidize the wastewater substrate faster
than single microbes.
Other fuel cell application, i.e. hydrogen fuel cell and dan Direct Ethanol Fuel
Cell (DEFC) resulted 0.95 volt and 0.6 volt, respectively [23]. Base on that fuel
cell, MFC system could be developed by optimatization of reactor configuration,
microbes performance, and electrode material.
To calculate the electricity energy produced during 40 hours, average of
current and voltage in Joule’s law multiplied by time during MFC operation P
(mW-hour) = V(volt) x I_(mA) x time (hour). In control reactor yielded 0.60
mWh, in SF2:SC1:EC1 and SF1:SC2:EC1 are 1.14 and 2.03 mWh, respectively.

C. COD Removal
As the current generated during reactor running, the tofu wastewater as substrate
tend to decrease on its organic concentration by the time, expressed as COD. This
advantage of MFC system was applied as one of wastewater biological treatment.
According to growth curve during acclimatization stage, in first 12 hours the
consortium entered growth phase rapidly so that the degradation of substrate
was maximum. After that, rate of cell decay and divide tend to similar, indicated
stationer phase had began. The substrate was utilized by microbes for their
metabolism. Since available substrate was limited, it caused the tendency of
microbes to decay, so that the COD value became fluctuated.
194 Proceeding ASEAN COSAT 2014

Figure 4. Voltage Measurement during Reaction

Overall COD removal efficiency resulted from SF2:SC1:EC1 and


SF1:SC2:EC1 variations were 76% and 58% respectively, gave better result than
51% on control reactor during 48 hours, as shown in Figure 4.
There is still challenge to increase the current and voltage production by
single chamber MFC system, either by effective proton exchange, reactor design
and reactor configuration or stacking, considering the volume of tofu wastewater
that produced every day.

IV. Conclusion
Performance of microbes consortium, S. cereviceae, S. fibuligera and E. coli, on
single-chamber microbial fuel cell as electricity generation with tofu wastewater
as substrate has been studied. Acclimatization was an important stage to adapt the
microbes to wastewater in order to increase the overall removal efficiency. Overall
COD removal efficiency with SF2:SC1:EC1 variations were 76%; it gave better
result than 51% on control reactor during 48 hours. Addition of SF2:SC1:EC1
into substrate resulted maximum current 0.25 mA in 16 hours.

V. Acknowledgement
We would like to thank to Djaenudin for his valuable outlook, as well as Oman
Rohman and Mahyar Ependi for wastewater sampling.
Innovation for Better ASEAN Community 195

Figure 5. COD Removal

VI. R eferences
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Aelterman, P., Verstraete, W. and K. Rabaey. (2006a). Microbial Fuel Cells:
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20) Kim, In S., Chae, K. J., Choi, M. J. and W. Verstraete. (2008). Microbial
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Heat Release Analysis of a Two Cylinders IN
Diesel Engine Fuelled with Ethanol-Diesel
Blends

Yanuandri Putrasari*, Arifin Nur, Achmad Praptijanto and Aam Muharam


Research Centre for Electrical Power and Mechatronics, Indonesian Institute of Sciences
Komplek LIPI, Jl. Cisitu No. 154D/21 Gd. 20, Bandung 40135, Jawa Barat, Indonesia

Abstract
An experiment on the application of ethanol-diesel blends as fuel in diesel engine was carried
out at various engine loads and ethanol percentages. The experiments were performed using neat
diesel fuel of 2.5%, 5%, and 7.5% ethanol-diesel blends with 1,500 rpm of engine working speed
at three different loads namely 0, 10 and 40 Nm. Several engine parameters data, such as torque,
fuel consumption, cylinder pressure, air intake flow, radioator coolant temperature, the exhaust
gas temperature, lubricating oil temperature and exhaust emission were collected. The combustion
or heat release of the engine then was calculated and analyzed. The results show several interesting
features from heat release phenomena in every combustion process from different fuel blends.
The results indicate that the increase of ethanol fraction in the ethanol-diesel blends causes the
maximum values of cylinder pressure and heat release value to increase as well.
Key words: Heat release, Combustion, Ethanol, Diesel

I. Introduction
Diesel engine has a high thermal efficiency and produces higher power that can
save more fuel compared to gasoline engine due to its high compression ratio.
Therefore, diesel engines are commonly preferred used on large buses, trucks, heavy
duty equipment, agricultural equipment and industrial machinery. However,
diesel engines also produce, gaseous pollutants, such as carbon monoxide (CO),
carbon dioxide (CO2), sulphur oxides (SOx), nitrogen oxides (NOx), unburned
hydrocarbons (HC) and particulate matter (PM) when using the common diesel
fuel that derived from fossil or living matter of a previous geologic time. Beside
produces high emission, the existence of fossil fuel in the world is estimated
to decrease from year to year. Due to this, condition the usage of renewable
alternative fuels are needed to replace and reduce the exhaust gas pollutants and
fossil-based fuel consumption.
Nowadays, ethanol becomes popular as a potential alternative fuel for vehicles
because it is a renewable and oxygenated biofuel. Ethanol can be produced from

* Corresponding author. Phone: +62-82120136976 Email: yanu001@lipi.go.id

199
200 Proceeding ASEAN COSAT 2014

sugar cane and others renewable energy sources. The other advantage of ethanol
as engine fuel for vehicles is that ethanol has greater power density than all others
alternative fuels.
However, due to its properties that different from normal gasoline or neat
diesel fuel, it is necessary to make engine modification in the utilization of ethanol
as fuel both for spark ignition or compressed ignition engine. According to Hansen
[1], the addition of ethanol to diesel fuel affects certain key properties, such as
blend stability, viscosity and lubricity, energy content and cetane number. The
two mentioned later are suspected mainly to influence to combustion process in
the combustion chamber of the diesel engine. The simple way to utilize ethanol
as fuel for existing diesel engine without modification is by blending with main
diesel fuel.
Recently, with the advancement of technology, many researchers conducted
experiment and investigation on ethanol as fuel for diesel engine by modifying
the engine or manipulating the ethanol as ethanol-diesel blends [2-12]. Among
them there was Xing-cai et al. [12] that investigated the effect of cetane number
improver of ethanol diesel blends on heat release rate and emissions of high
speed diesel engine. Different percentages of cetane number improver (0, 0.2,
and 0.4%) were added into the blends. The results show that the ignition delay
prolonged, and the total combustion duration shortened for ethanol-diesel blends
fuels when compared to normal diesel fuel. Meanwhile, Rakopoulos et al. [2]
studied about combustion heat release analysis of ethanol or n-butanol diesel fuel
blends in heavy-duty DI diesel engine. The study used ethanol in proportions
of 5% and 10% or n-butanol in 8% and 16% (by vol.) with the engine working
at three loads and at engine speeds of 1,200 and 1,500 rpm. It can be reported
from the study that the ignition delay is increased, maximum cylinder pressure
are slightly reduced and cylinder temperatures are reduced during the first part
of combustion.
Related to this research, previous author’s study [13] reported about perfor-
mance and emission characteristic on a two cylinder DI diesel engine fuelled with
ethanol-diesel blends. The report shows that the engine performance increased
and emission of CO, HC and smoke were decreased. Based on this background,
the aim of this study is to determine the heat release analysis of the two cylinder
direct injection diesel engine fuelled with ethanol-diesel blends.
In this study, determination of the combustion process in the diesel engine can
be conducted by using heat release analysis. Combustion process stated as heat
release at every crank angle position. The heat release can be calculated during
compression stroke and combustion stroke. In this condition, both of intake and
exhaust valves are closed. The heat release analysis estimates the amount of heat
Innovation for Better ASEAN Community 201

or energy that added to the combustion chamber to obtain the pressure variation
in the combustion chamber. The heat release formula according to the first law
of thermodynamics single zone models [14] is:

(1)

Where γ is heat specific ratio, cp/cv. The appropriate range of γ for the heat
release analysis of diesel engine is 1.3 to 1.35 [14]. p is combustion chamber
pressure, and V is cylinder volume (volume clearance of top death centre plus
stroke volume). Cylinder volume based on crank angle position can be obtained
using the following formula:

(2)
where r is crank throw that equals to a half of piston stroke, l is long of
connecting rod, Vc is clearance volume, B cylinder diameter and θ is crank angle
position.

II. Methodology
A. Fuel Preparation
Normal diesel fuel and three ethanol-diesel blends on volume base namely
2.5%, 5%, and 7.5% then called E2.5, E5, E7.5 respectively, were used in this
study. Ther raw material of neat diesel fuel purchased from Indonesian national
oil corporation, while 99.6% purified ethanol and “SPAN 80” sorbytan methyl
ester surfactant as blends stabilizer were obtained from local supplier. Additional
information about properties of diesel fuel and ethanol are presented in Table 1
according to Rakopoulos et al. [15].
Ethanol-diesel blends were prepared by using a blender machine in a certain
desired dose for 15 minutes at 250 rpm to obtain the homogeneity of the blends.
To maintain the stability of the blends, 1% of surfactant, measured from total
blends was added during the blending process. The fuel preparation was conducted
just before the test started to run, to make sure the blends in stable condition.

B. Engine and Instruments Installation


A two cylinder direct injection stationary diesel engine was selected for this study.
The engine specifications data are presented in Table 2. The study was carried
out by setting-up the engine on an engine test bed. Fuel balance, emission meter,
smoke meter, pressure sensor crank angle sensor and temperature sensor for
intake and exhaust manifold were also installed. To manage the rpm and engine
202 Proceeding ASEAN COSAT 2014

Table 1. Properties of diesel fuel and ethanol [15]


Properties of diesel fuel and ethanol Diesel Ethanol
Density 20 °C, kg/m3 837 788
Cetane number 50 5-8
Kinematic viscosity at 40 °C, mm2/s 2,6 1,2
Surface tension at 20 °C, N/m 0,023 0,015
Lower calorific value, MJ/kg 43 26,8
Specific heat capacity, J/kg°C 1850 2100
Boiling point 180-360 78
Oxygen, % weight 0 34,8
Latent heat of evaporation, kJ/kg 250 840
Bulk modulus of elasticity, bar 16000 13200
Stoichiometric air-fuel ratio 15,0 9,0
Molecular weight 170 46

loads the engine was coupled with eddy current dynamometer. Fuel balance
used for fuel consumption measurement, and flow of air intake was measured
using hotwire anemometer. Meanwhile, the pressure sensor and crank angle
sensor were combined using engine indicating system to measure the pressure of
the combustion chamber and crank angle position. The schematic diagram for
experimental set up is presented in Figure 1.

Figure 1. Schematic diagram of experimental se-tup


Innovation for Better ASEAN Community 203

Table 2. Engine basic data


Engine parameters Basic data
Model and type Fujikawa 295D, diesel four stroke
Number of valve 4
Air charge system Naturally aspirated
Cylinder / type 2 / Vertical
Volume (cc) 1630 cc
Diameter x stroke 95 x 115 mm
Compression ratio 19:1
Maximum torque 96.9 Nm at 1500 rpm
Maximum power 13.5 kW at 1500 rpm
Fuel system Direct Injection 195 bar

C. Engine Testing Procedure and Heat Release Analysis


To obtain heat release value of diesel engine, the pressure and volume data of
combustion chamber for every crank angle position is required. The data of
combustion chamber pressure and crank angle position can be obtained by engine
testing. The engine was operated using prepared fuels at 1,500 rpm. The loads
were set on 0, 10 and 40 Nm using dynamometer panel control. The parameter,
such as fuel consumption, air intake consumption, oil engine temperature, air
intake and exhaust temperature, coolant temperature both intake and outlet of
radiator, and emission of CO, HC and smoke were measured. The pressure of
combustion chamber was measured every 1° crank angel position and recorded
during 36 cycles. The main required data specifically combustion chamber pressure
and crank angle position then collected from engine indicating system. The
amount of pressure data from indicating system is about 720 data. To calculate
the heat release, it was used the mean pressure data from 36 cycles recorded data.
Meanwhile, the volume of combustion chamber in every crank angle position
was also calculated manually using Formula 2 aided by Microsoft Excel software.
The γ value is assumed constant during the combustion process. To fulfill all
requirements data, the measurement of bore, stroke, compression ratio, length
of connecting rod, and the gap between piston and cylinder liner were also
conducted. The heat release value then calculated and analyzed.

III. R esults and Discussion


A. Cylinder Pressure
Figure 2 shows the cylinder pressure of diesel engine using E0, E2.5, E5 and
E7.5% (a) with 0 Nm of load, (b) with 10 Nm of load and (c) with 40 Nm of
load. It can be seen from the Figure 2 (a), (b) and (c), although the engine was
run in different load, for all the peaks, value of cylinder pressure increases due to
204 Proceeding ASEAN COSAT 2014

the increase of ethanol fraction in the blends. The significant influence of ethanol
fraction to the cylinder pressure of diesel engine occurred on the E5 and E7.5%,
while E2.5% caused no significant influences.
This is happened on all of loading variation namely 0, 10 and 40 Nm. The
increasing of ethanol fraction caused the graph seen a little bit shifted to the right.
It means that ingnition delay increase and start of combustion postphoned. This
condition suspected caused by the addition of ethanol that will increase the cetane
number of the blends, then increase the ignition delay which lead the increasing
the amount of fuel burned in the premixed burning phase. In detail, when the
engine run without load as seen in Figure 2 (a), the highest pressure peak is E

(a)

(b)

(c)
Figure 2. Cylinder pressure at (a) 0, (b) 10 and (c) 40 Nm
Innovation for Better ASEAN Community 205

5% followed by E 7.5%, E 2.5% and the lowest is E0. Meanwhile, in the diesel
engine when run with 10 Nm of load, the value of cylinder pressure for E 7.5%
and E 5% are almost same, higher compared with the others. Lastly, in the diesel
engine that was run in 40 Nm of load, the highest pressure cylinder value is for
E7.5 followed by E5%, then followed by E2.5 and E0.

B. Heat Release
Figure 3 gives the heat release curves of diesel engine fuelled with neat diesel and
ethanol-diesel blends at (a) 0, (b) 10 and (c) 40 Nm of loading variation. From
of all the graph it can be seen the most distinguished features that the beginning
stage of heat release process is delayed and the maximum value of heat release
increases with increase in the ethanol fraction of ethanol-diesel blends. The
increasing of ethanol fraction in the ethanol-diesel blends caused the decreases
of cetane number of the fuels. The decreasing of cetane number influnce to
the increasing of ignition delay period which will involve to the enhancement
amount of combustible mixture during the ignition delay period. This condition
will results the increasing of the fuel burned in the premixed burning phase and
produces higher maximum cylinder pressure that involves increasing of heat
release. Furthermore, the high volatility of ethanol causes the fueld more evaporate
to be, therefore this will also increase the amount of combustible mixture during
the ignition delay period. The higher the ethanol fraction of ethanol-diesel blends,
the larger the amount of combustible mixture occurred.

(a) (b)

Figure 3. Heat release at (a) 0, (b) 10


and (c) 40 Nm

(c)
206 Proceeding ASEAN COSAT 2014

IV. Conclusions
From the complete experiment and heat release analysis in this study, several
conclusions can be drawn as follows:
1) The value of cylinder pressure increases due to the increase of ethanol fraction
in the blends.
2) The increasing of ethanol fraction caused the cylinder pressure graph seen a
little bit shifted to the right. It means that ignition delay increase and start
of combustion postponed.
3) The beginning stage of heat release process is delayed and the maximum value
of heat release increases with increase in the ethanol fraction of ethanol-diesel
blends.

V. Acknowledgement
This project is financially supported by Indonesian Institute of Sciences-Research
Centre for Electrical Power and Mechatronics through the Kompetitif LIPI
2011/2012 research project. The assistance of Mr. Ahmad Dimyani, ST. and
Mr. Mulia Pratama, ST. in conducting the experiments in internal combustion
engine laboratory is also highly appreciated.

VI. R eferences
1) Hansen, A. C. et al. (2004). Ethanol diesel fuel blends a review. Bioresource
Technology, 96, 277–285.
2) Rakopoulos, D. C. et al. (2011). Combustion heat release analysis of ethanol
or n-butanol diesel fuel blends in heavy-duty DI diesel engine. Fuel, 90,
1855–1867.
3) Song, C. et al. (2010). Carbonyl compound emissions from a heavy-duty
diesel engine fueled with diesel fuel and ethanol–diesel blend. Chemosphere,
79, 1033–1039.
4) Lee, W.-J. et al. (2011). Assessment of energy performance and air pol-
lutant emissions in a diesel engine generator fueled with water-containing
ethanol–biodiesel–diesel blend of fuels. Energy, 36, 5591–5599.
5) Lei, J. et al. (2012). A novel emulsifier for ethanol–diesel blends and its effect
on performance and emissions of diesel engine. Fuel, 93, 305–311.
6) Armas, O. et al. (2011). Effect of an ethanol–biodiesel–diesel blend on a
common rail injection system. Fuel Processing Technology, 92, 2145–2153.
Innovation for Better ASEAN Community 207

7) Sayin, C. and M. Canakci. (2009). Effects of injection timing on the engine


performance and exhaust emissions of a dual-fuel diesel engine. Energy
Conversion and Management, 50, 203–213.
8) Çetin, M. et al. (2009). Emission characteristics of a converted diesel engine
using ethanol as fuel. Energy for Sustainable Development, 13, 250–254.
9) Pidol, L. et al. (2012). Ethanol–biodiesel–Diesel fuel blends Performances and
emissions in conventional Diesel and advanced Low Temperature Combus-
tions. Fuel, 93, 329–338.
10) Park, S. H. et al. (2011). Influence of ethanol blends on the combustion
performance and exhaust emission characteristics of a four-cylinder diesel
engine at various engine loads and injection timings. Fuel, 90, 748–755.
11) Hulwan, D. B. and S. V. Joshi. (2011). Performance, emission and combustion
characteristic of a multicylinder DI diesel engine running on diesel–ethanol–
biodiesel blends of high ethanol content. Applied Energy, 88, 5042–5055.
12) Xing-cai, L. et al. (2004). Effect of cetane number improver on heat release
rate and emissions of high speed diesel engine fueled with ethanol–diesel
blend fuel. Fuel, 83, 2013–2020.
13) Putrasari, Y. et al., (2013). Performance and emission characteristic on a two
cylinder DI diesel engine fuelled with ethanol-diesel blends. Energy Procedia,
32, 21–30.
14) Heywood, J. B. (1988). Internal combustion engine fundamental. McGraw-Hill.
15) Rakopoulos, D. C. et al. (2008). Effects of ethanol–diesel fuel blends on the
performance and exhaust emissions of heavy duty DI diesel engine. Energy
Conversion and Management, 49, 3155–3162.
An Evaluation For Enzymatic
Saccharification of Fast-growing Tree
Species from Secondary Forest in West
Kalimantan

Lucky Risantoa,*, Danang Sudarwoko Adia, Masakasu Kanekob, Yosuke


Kurosakib, Deden Girmansyahc, Ruliyana Susantic, Euis Hermiatia and
Takashi Watanabeb
Research Center for Biomaterials, Indonesian Institute of Sciences,
a

Jl. Raya Bogor Km 46, Cibinong, Bogor 16911, Indonesia


b
Laboratory of Biomass Conversion, Research Institute for Sustainable Humanosphere
(RISH),
Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan
c
Research Center for Biology, Indonesian Institute of Sciences,
Jl. Raya Bogor Km 46, Cibinong, Bogor 16911, Indonesia

Abstract
Fast growing tree species can be considered suitable raw material for the production of second
generation bioethanol due to their promising biomass sustainability. However, until now, there is
less information regarding the bioethanol utilization from these species. In this study, we examined
the enzymatic hydrolysis of potential fast growing tree species that have been previously prescreened
from fifteen species. Fast growing tree species were taken from secondary forest concession area
of PT. Sari Bumi Kusuma in West Kalimantan and Eucalytus globulus was used as a standard
wood. The experimental work was carried out using hydrothermal, diluted sodium hydroxide
(NaOH), ammonia (NH4OH) and maleic acid (MA) pretreatment with 30 minutes of heating
duration, and several conditions of temperature and concentration. Based on the results of the
saccharification after hydrotermolysis pretreatment, Ilex cissoides has the highest yield of sugars
among the other fast growing tree species, from pulp saccharification was obtained 8.16 g/100g-
biomass when pretreated at 190 °C, and sugars from the filtrate saccharification was obtained 5.30
g/100 g-biomass, thus making total yield of sugars 13.46 g/100 g-biomass. When I. cissoides was
pretreated with diluted NaOH, NH4OH and MA, the yield of sugars were increased. The highest
yield of sugars and saccharification level for I. cissoides was obtained 18.04 g/100 g-biomass and
29.50 g/100 g-pulp after pretreated with 0.5% MA at 180 °C. I. cissoides have a potential as raw
material for bioethanol production if we used pretreatment using diluted MA, due to a similar
saccharification result when compared with E. globulus as standard.
Key words: Bioetanol, Enzymatic saccharification, Fast growing species, Lignocelluloses, Pretreatment

* Corresponding author. Phone: +62-21-87914511. Email: Lrisanto@biomaterial.lipi.go.id

209
210 Proceeding ASEAN COSAT 2014

I. Introduction
The search for alternative energy sources fuel oil is currently the world’s attention
related to declining availability of energy resources of petroleum fuels [1]. One
of the most potential renewable energies that have attracted worldwide attention
is bioethanol.
Bioethanol can be produced from a variety of raw materials and through a
number of ways. Basically, bioethanol production is processed from conversion
of carbohydrates into sugars and fermentation of these sugars to ethanol. In ad-
dition, bioethanol can also be produced from plant materials containing complex
cellulose. Cellulose is the most abundant source of carbohydrates on earth, which
is the main component of the plant structure, such as wood. Consequently,
cellulose has the potential to be one of the alternative raw materials because it is
more abundant and less expensive than food crops, especially from fast growing
tree species [2].
Fast-growing tree species have been well-known for plantation forest, thus the
sustainability of these species is propitious. They can grow easily in plantation
forest, such as the forest concession area of PT. Sari Bumi Kusuma in West
Kalimantan. The benefits of these species are faster growth than the wood species
from natural forest and they also have a short period of harvesting. On the other
hand, imperfect wood quality makes them are less interest to be further explored.
Therefore, the study on these species is necessary to give more information and
it will enhance the use of the woods [3].
Woody biomass is seen as a promising raw material for future feed stock
for bioethanol. Apperently, one of the main challenges for the production of
bioethanol from woody biomass is to develop of an efficient pretreatment. The
purposes of the pretreatment are to remove lignin and hemicellulose, reduce
cellulose crystallinity, and increase the porosity of the materials [4].
There are several woody biomass investigations for the renewable fuel,
such as Eucalyptus globulus [5-7], pine [8], and Japanese cedar [9]. Romani
et. al. [5] reported that up to 94% of carbohydrates were converted mono- or
oligo-saccharides in the hydrolysis media for E. globulus after pretreated using
hydrothermal at 220°C. However, the utilization of these species has been limited
to pulp and paper making.
In previous study, Ilex cissoides, Tristaniopsis whiteana, Evodia latifolia,
Horsfieldia crassifolia, and Adinandra collina were obtained potential species from
the saccharification result among the 15 species of topical fast growing trees
from PT. PT. Sari Bumi Kusuma [10]. Considering to lower saccharification
result achieved from the screening methods, we tried another pretreatment
methods. In this study, we examined again these potential fast growing trees using
Innovation for Better ASEAN Community 211

hydrothermolysis pretreatment and we compared it with E. globulus as a standard


wood. Furthermore, we treated promising wood species with microwave assisted
pretreatement using diluted base and acid.

II. M aterials and Methods


A. Biomass Collection and Preparation
The wood samples were collected from Sari Bumi Kusuma (SBK) concession area,
Central Kalimantan, Indonesia. The minimum diameter of wood is greater than
10 cm. The wood samples were cut, chipped and air-dried at room temperature.
After that, the wood chips are grinded to past a 32-mesh screen; finally the wood
meal that holds at 40-mesh screen was collected. Eucalytus globulus was used as
a standard wood.

B. Chemical Composition
The chemical analyses of raw material were conducted using two replicates per
sample. The ash content was measured by combusting the wood meal at 525°C
base on TAPPI Standard T 211 om-02 [11]. The wood meal was extracted in the
Soxhlet apparatus with a mixture of ethanol and benzene (1:2) according to the
TAPPI Standard T 204 cm-97 [12]. The extractive-free wood meal was oven-dried
at 60 °C for overnight. Acid insoluble lignin (AIL) and acid soluble lignin (ASL)
content were determined according to the National Renewable Energy Laboratory
(NREL) Chemical Analysis and Testing Task Laboratory Analytical Procedures
#003 [13] and #004 [14], repectively. Holocellulose content was analyzed ac-
cording to Wise et al. [15] and the alpha-cellulose content by Rowell et al. [16].

C. Hydrotermolysis Pretreatment
Wood meal around 1.1 g (air-dried basis) was placed in microwave vial, and then
added with water until 20 g in total. The sample was degassed in the vacuum
desicator for 5 min. The pretreatment was performing using a 2.45 MHz
microwave reactor (Initiator ver. 2.0, Biotage Co. Ltd.) with a 400 W magnetron,
stirring rate at 900 rpm and pre-stirring for 30 s. The sample was exposed to
microwave irradiation at 180 & 190°C for 30 min of holding time, and then
cooled until room temperature. After that, the pretreated sample was filtered to
get pulp fraction and the filtrate fraction for xylose sacch. The pulp fraction was
washed with distilled water and the yield of pulp was calculated. Each pretreatment
condition was repeated three times. Two promising wood species that resulted
higher sugar yield were further pretreated with diluted base and acid.
212 Proceeding ASEAN COSAT 2014

D. Microwave Assisted Pretreatment with Diluted Sodium Hydroxide,


Ammonia Solution, and Maleic Acid
In this steps, the wood powder was pretreated using diluted base and acid to
increase the sugar yield from the woods, such as sodium hydroxide (NaOH)
(0.5 wt%), ammonia solution (NH4OH) (7 and 14 wt%), and maleic acid (MA)
(0.5; 1.0; and 1.5 wt%). The wood samples that we used were sample No. 31
and 38; also E. globulus as standard. All pretreatment conditions were the same
as hydrotermal pretreatment, except the degassing step was eliminated and the
reaction temperature were 170 °C (NH4OH) and 180°C (NaOH and MA). Each
pretreatment condition was repeated two times.

E. Pulp Saccharification
The pulp fraction was hydrolysed with CTEC2. The cellulase enzyme loading
was 5 FPU/g-substrate. Enzymatic hydrolysis was performed at a substrate
concentration of 2 wt% in 0.05 M sodium succinate buffer pH 5.0 at 50 °C on
a rotary shaker (NTS-4000C, Rikakikai, Japan) at 140 rpm for 48 h.

F. Filtrate Saccharification
Ten gram of filtrate fraction was hydrolysed using HTEC2. The endoxylanase
enzyme loading was 3 mg/g substrate, then added with 0.05 M sodium succinate
buffer 0.05 M; pH 5.0; until 20 g in total. Enzymatic hydrolysis was performed
on a waterbath shaker at 50 °C; 140 rpm; for 48 h.
Total sugar yield was measured using HPLC base on the weight of original
wood; except for MA pretreatment was determined by Somoygi-Nelson method
[17].

III. R esult and Discussion


A. Chemical Composition of Raw Material
Table 1 presents major chemical compositions of woody biomass; it showed
that the holocellulose content of potential fast growing trees were 71.42, 71.98,
68.45, 69.12, and 71.91% for I. cissoides, T. whiteana, E. latifolia, H. crassifolia
and A. collina, respectively.

B. Hydrotermolysis Pretreatment
The hydrothermolysis pretreatment for the woody biomass are eco-friendly
pretreatment methods. Sample was contact with water at elevated temperature
and pressure. Hydrotermolysis can cause breakdown of lignocellulose structure,
this due acetic acid from hemicelluloses can improve delignification and break
the lignin-cellulose matrix. Higher temperature would increase of weight loss due
Innovation for Better ASEAN Community 213

to degradation of hemicellulose and lignin. From figure 1, the highest weight


loss was obtained 25.66% from sample I. cissoides 190°C, but still lower than E.
globulus (37.68%) as standard.
Generally, all the wood samples that pretreated with hydrotermolysis at 190
°C showed increasing in saccharification level compare with wood samples that
pretreated at 180°C. The highest yield of sugars from pulp saccharification was
obtained 8.16 g/100g-biomass from sample I. cissoides using 190 °C, and sugars
from the xylose saccharification was obtained 5.30 g/100g-biomass, thus making
total sugars 13.46 g/100g-biomass. Meanwhile yield of sugars from E. globulus
as standard at the same conditions from pulp and xylose saccharification were
obtained 21.92 and 10.48 g/100 g-biomass, making total sugars 32.40 g/100
g-biomass. It seems that recalcitrant of E. globulus is more easy to reduce than
the other wood samples when using hydrotermolysis.

C. Effect Microwave asissted pretreatment with maleic acid, ammonia


solution, and sodium hydroxide
As we can see from hydrotermolysis pretreatment, I. cissoides and E. latifolia have
potential to be raw material than the other wood samples. So in this step we added
diluted NaOH, NH4OH, and MA to increase the sugar yield from the woods.
Microwave-assisted pretreatment with maleic acid (conc. 0.5%; 180 °C) of all
the wood samples have a highest weight loss (Figure 3) than ammonia solution
(conc. 14%; 170°C) and sodium hydroxide (conc. 0.5%; 180 °C). This due dilute
acid pretreatment can accelerating solubilization of the hemicellulose, and also
have an advantage for conversion of the solubilized hemicellulose into sugars.
Meanwhile, alkali pretreatment can increases the swelling of cellulose and the
solubilization of lignin [18].
Table 1. Chemical composition (wt% of oven dry) of biomass
E. globu- I. cis- T. E. latifo- H. crassi- A. collina
Composition lus [wt%] soides whiteana lia [wt%] folia [wt%]
[wt%] [wt%] [wt%]
Ash Content 0.66 0.97 1.15 0.82 0.96 0.76
Extractive in EtOH- 2.34 1.83 1.98 1.76 1.87 1.61
Benzene
Lignin:
AIL 23.72 26.45 25.74 28.34 27.34 23.93
ASL 1.34 1.56 1.45 1.87 1.94 1.27
Holocellulose: 72.72 71.42 71.98 68.45 69.12 71.91
α-cellulose 42.89 40.97 41.87 40.28 41.19 42.52
Hemicellulose 29.83 30.45 30.11 28.17 27.93 29.39
214 Proceeding ASEAN COSAT 2014

45

40

35

30

25

20

15

10

0
180°C 190°C 180°C 190°C 180°C 190°C 180°C 190°C 180°C 190°C 180°C 190°C

globulus
E. globulus I. cissoides
cissoides T. whiteana
T. whiteana E. latifolia
E. latifolia H.crassifolia
H. crassifolia A. collina
A.

Sugar Yield from Pulp Sacch. [g/100 g-biomass]


Sugar Yield from Filtrate Sacch. [g/100 g-biomass]
Total Sugar Yield from Pulp and Filtrate Sacch. [g/100 g-biomass]
Weight Loss [wt%]

Figure 2. Effect of temperature to the yield of sugars determined by HPLC (a) and to the
yield of reducing sugars determined by Somoygi-Nelson Methods (b) after enzymatic of
wood samples hydrolysis using hydrotermolysis pretreatment.
The highest yield of sugars was obtained 18.04 g/100 g-biomass and sac-
charification level was 29.50 g/100 g-pulp from I. cissoides when pretreated with
maleic acid (conc. 0.5%; 180°C) and have similar level with E. globulus as standard,
were obtained 18.54 g/100 g-biomass and 30.06 g/100 g-pulp for the yield of
sugars and saccharification level. It seems that recalcitrant of I. cissoides can be
easier to reduce with addition of reagent such as MA. I. cissoides also gave similar
level when compare with E. globulus after pretreated with NH4OH (Figure 3).
45
40
35
30
25
20
15
10
5
0
E. globulus I. cissoides E. latifolia E. globulus I. cissoides E. latifolia E. globulus I. cissoides E. latifolia
E. globulus I. cissoides E. latifolia E. globulus I. cissoides E. latifolia E. globulus I. cissoides E. latifolia

170°C; 14% NH4OH.


NH4OH 180°C; 0.5% NaOH 180°C; 0.5% M.A.

Total sugar from Pulp Sacch. [g/100 g-pulp]


Total sugar from Pulp Sacch. [g/100 g-biomass]
Weight Loss [wt%]

Figure 3. Effect of microwave-assisted pretreatment with diluted base and acid to the yield
of sugars after enzymatic of wood samples.
Innovation for Better ASEAN Community 215

30

25

20

15

10

0
170°C;7%;
170°C; 7% 170°C; 14%;
170°C; 14% 170°C;
170°C;7%;
7% 170°C; 14%;
170°C; 14% 170°C;
170°C;7%;
7% 170°C; 14%;
170°C; 14%
NH4OH
NH 4 OH NH4OH
NH4 OH NH4OH
NH 4 OH NH4OH
NH4 OH NH4OH
NH 4 OH NH4OH
NH4 OH
E. globulus I. cissoides E. latifolia
E. globulus I. cissoides E. latifolia

Total sugar from Pulp Sacch. [g/100 g-pulp]


Total sugar from Pulp Sacch. [g/100 g-biomass]
Weight Loss [wt%]

Figure 4. Effect of microwave-assisted pretreatment with different concentration of ammonia


solution (NH4OH) to the yield of sugars after enzymatic of wood samples.

50
45
40
35
30
25
20
15
10
5
0
180°C; 0.5% 180°C; 1.0% 180°C; 1.5% 180°C; 0.5% 180°C; 1.0% 180°C; 1.5% 180°C; 0.5% 180°C; 1.0% 180°C; 1.5%
M.A. M.A. M.A. M.A. M.A. M.A. M.A. M.A. M.A.

E.g.
E. globulus 31
I. cissoides 38
E. latifolia

Reducing Sugar from Pulp Sacch. [g/100g-pulp]


Reducing Sugar from Pulp Sacch. [g/100g-biomass]
Reducing Sugar from Filtrate Fraction after MW Assisted [g/100g-biomass]
Reducing Sugar from Filtrate Sacch. [g/100g-biomass]
Total Reducing Sugar from Pulp and Filtrate Sacch. [g/100 g-biomass]
weight loss [wt%]

Figure 5. Effect of microwave-assisted pretreatment with different concentration of maleic


acid (MA) to the yield of reducing sugars after enzymatic of wood samples.
216 Proceeding ASEAN COSAT 2014

D. Effect Microwave-asissted pretreatment with different concentration


maleic acid
The organic acids, such as MA can be an alternative for increasing the sugar yield
from the woods. In general, there is no significant different for the saccharification
between when using 0.5; 1.0; and 1.5% of maleic acid for each wood samples.
The highest yield of sugars and saccharification level was obtained 18.04 g/100
g-biomass and 29.50 g/100g-pulp from I. cissoides when using 0.5% MA, mean-
while the yield of sugars and saccharification level from E. globulus was obtained
19.20 g/100 g-biomass and 32.11 g/100 g-pulp, respectively. An increament of
the concentration would increase the saccharification level, but the sugar yield
did not raise (Figure 5), this is due to the increase in weight loss.

E. Effect Microwave-asissted pretreatment with different concentration


ammonia solution
The benefits to use NH4OH are swelling of the lignocellulose material, selective
to remove lignin, low interaction with carbohydrates, and easy to remove
because NH4OH was very volatile. The saccharification after microwave assisted
pretreatment with 7% NH4OH was higher than pretreated with 14% NH4OH
(Figure 4). The highest yield of sugars and saccharification level were obtained
15.42 g/100 g-biomass and 20.84 g/100g-pulp from I. cissoides, meanwhile yield
of sugars and saccharification level from E. globulus was obtained 18.48 g/100
g-biomass and 23.40 g/100 g-pulp, respectively.

IV. Conclusion
Based on the results of the saccharification after hydrotermolysis and microwave
assisted pretreatement diluted base and acid, it can be concluded that I. cissoides
have a potential as raw material for bioethanol production if we used microwave
assisted pretreatement using diluted MA, due a similar saccharification result
when compared with E. globulus as standard.

V. Acknowledgement
The guidelines for citing electronic information as offered below are a modified
illustration of the adaptation by the International Standards Organization (ISO)
documentation system and the American Psychological Association (APA) style
and finalized in Information for IEEE Transactions, Journals, and Letters Authors.
Innovation for Better ASEAN Community 217

VI. R eferences
1) Liu, S. and C. Lin. (2009). Development and perspective of promising
energy plants for bioethanol production in Taiwan. Renewable Energy, 34
(8), 1902–1907.
2) Limayem, A. and S. C. Ricke. (2012). Lignocellulosic biomass for bioethanol
production: Current perspectives, potential issues and future prospects.
Progress in Energy and Combustion Science, 38 (4), 449–467.
3) Adi, D. S., Risanto, L., Damayanti, R., Rullyati, S., Dewi, L. M., Susanti,
R., Dwianto, W., Hermiati, E. and T. Watanabe. (2014). Exploration of
Unutilized Fast Growing Wood Species from Secondary Forest in Central
Kalimantan: Study on the Fiber Characteristic and Wood Density. Procedia
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a Novel Microwave-Biological Pretreatment
Effect on Cellulose and Lignin Changes of Betung
Bamboo (Dendrocalamus Asper)

Widya Fatriasaria,b,*, Wasrin Syafiic, Nyoman J. Wistarab, Khaswar Syamsud,


Bambang Prasetyae and Muhammad Adly R. Lubisa
a
Research Center for Biomaterials, Indonesian Institute of Sciences (LIPI)
,Jl. Raya Bogor KM 46, Cibinong, Bogor 16911, Indonesia
b
Departement of Forest Product Technology, Faculty of Forestry, Bogor Agricultural University
(IPB)
PO Box 168, Bogor 16001, Indonesia
cDepartement of Forest Product Technology, Faculty of Forestry, Bogor Agricultural
University (IPB) PO Box 168, Bogor 16001, Indonesia
d
Departement of Agroindustrial Technology, Faculty of Agricultural Engineering and
Technology, Bogor Agricultural University, IPB
PO Box 220, Bogor 16002, Indonesia
e
Institute of National Standarization, Manggala Wanabakti Building Blok IV
Jl. Gatot Subroto Senayan, Jakarta, Indonesia

Abstract
This study was to evaluate the effect of microwave-biological pretreatment of betung bamboo
on the characteristic changes of cellulose and lignin. Chemical component analysis, FT IR
spectroscopy, X-Ray diffraction, SEM-EDX analysis was used to analyze the characteristic changes
after pretreatment. The microwave-biological pretreatment caused the weight loss, lignin and
hemicellulose removal. FT IR spectra indicated that the microwave pretreatment for 12.5 min
(330 W) with 5% inoculum loading caused loss of the absorbed O-H and conjugated C-O. This
treatment also affected C-H2 scissoring motion lost in 5 and 10 min (330 W) with 5% inoculum
loading. Aromatic skeletal of lignin (1605 cm-1) did not appear in microwave pretreatment for 5
and 10 min (330 W) and 10% inoculum loading. The lowest absorbance of lignin (1512 cm-1)
in 5% inoculum loading was founded in pretreatment for 5 min (330 W). Absorbance Syringil
propane units were lower than guiacyl moties which indicates that syringyl was more soluble
than that of guiacyl. The crystallinity of cellulose in 5% of inoculum loading tends to decrease,
while the 10% of inoculum loading is since versa. SEM images illustrates that the pretreatment
disrupted the fiber structure (more porous and soften structure). EDX analysis shows that there
was lost in minor element constituent of pretreated bamboo. Crystalline allomorph of 5, 10 and
12.5 min (330 W) with 5% inoculum loading shows Iα (triclinic) structure.
Key words: Betung bamboo, Microwave-fungal pretreatment, Crystalline allomorph, Functional
groups, Crystallinity index

* Corresponding Author. Phone: +21-87914511. Email: widya_fatriasari@yahoo.com

219
220 Proceeding ASEAN COSAT 2014

I. Introduction
To improve sugar monomer production from lignocellusic materials (LM), it
is required to break down the lignin and crystalline system as major inhibitor.
The combination of pretreatments is attractive method to enhance reducing
sugar yield. One of pretreatment kinds is microwave-biological pretreatment in
which this pretreatment included in the environmental friendly pretreatment
sources. Microwave-assisted alkali pretreatment on rice straw had been reported
previously [1]. The combination microwave with acid [2] and hydrogen peroxide
[3] pretreatment had been also conducted before.
Bamboos belonging have high potency as bioenergy source. These plants
have high biomass production and grow mainly in Asia [4]. This production
is higher than that of the other energy crops [5,6]. One of the most important
bamboo species in Indonesia is betung bamboo [7] with high fiber morphological
characteristic and good chemical component content [8].
The microwave pretreatment facilitates to increase the substrate porosity,
surface area and soften the substrate. This heating derived from microwave source
causes the vibration of polar molecules and creates hot spot with inhomogeneous
materials lead to improve the substrate structure [9,10]. Application of biological
pretreatment using white rot fungi such as Trametes versicolor to depolymerize the
lignin polymer and then solubilize it. Lignin degrading enzyme can be secreted by
fungus to degradde on the LM [13]. The positive effect of microwave-biological
pretreatment can increase the enzymatic accesibility due to more opened substrate.
In our paralel study on biological and microwave pretreatment of betung
bamboo showed that the lignin and hemicellulose degradation was reported
[15,16].These changes improved the reducing sugar yield compared to control
[17,18]. Therefore, this research was foccused to evaluate the characteristic changes
after combination of microwave-biological pretreatment. The detail characteristic
analysis of pretreatment combination can used to understand the difference effect
single and combination pretreatment.

II. M aterial /methods


A. Material Preparation
Fresh and barkless 2 year-old bamboo betung (Dendrocalamus asper) produced
from bamboo garden of Research Center for Biomaterials LIPI Cibinong, Bogor,
Indonesia was used as samples. It was cut into chip and then processed by drum
chipper, ring flaker, hammermil and disk mill to obtain bamboo powder size of
40–60 mesh.
Innovation for Better ASEAN Community 221

B. Microwave Pretreatment
Oven microwave SHARP P-360J (S) set at 2,450 MHz and power output of
1,100 Watt was used in this treatment. Previously, as much as 1 g of oven dried
powder was inserted into the teflon tube, then added distilled water to obtain
a solid-to-liquid ratio (SLR) 1:30 (w/v) and then was stirred for 15 minutes.
Subsequently, the samples were transferred and irradiated for irradiation time of
5, 10 and 12.5 minutes at 330 watt and 5 minutes at 770 watt. After microwaving
finished, the pulp removed and immediately ice water cooled for 15–20 minutes
and then was filtered to separate solid residue out.

C. Biological Pretreatment
The solid fraction resulted from microwave pretreatment was subjected to
biological pretreatment following previously method described by Fatriasari et
al.[12]. The samples were incubated at 27°C up to 30 day with 5 and 10% of
inoculum loading (IL). They were then washed with distilled water for removing
the fungus. The residues of these samples were analyzed the structure characteristic
changes after microwave-biological pretreatment.

D. Cellulose, Lignin and Morphological Characteristic Changes


The characteristic changes after microwave-biological pretreatment was evaluated
following method described by Fatriasari et al. [12]. These characteristics consisted
of chemical component changes, X-ray analysis Diffraction (XRD) (crystalline
allomorph and crystallinity index), Fourier Transform Infrared Spectrometry
(FT IR) analysis (determination of functional groups), and morphological
characteristic (SEM images) and Energy Dispersive X-ray Spectroscopy (EDX)
analysis.

E. Data analysis
All the experiments were conducted in triplicate and the results were presented
as mean ± standard deviation.

III. RESULT AND DISCUSSION


A. Chemical Component Changes
The chemical component loss of pretreated bamboo was presented in Table 1.
The microwave-biological pretreatment caused the weight loss of the samples.
It might be related with heating of microwave caused by vibration of polar
molecules in solution. The lignin polymer attacking due to enzyme degrading
lignin in the biological pretreatment is also affected this loss. All treatments
decreased the lignin content, inwhich lignin loss of 10% IL was higher than
222 Proceeding ASEAN COSAT 2014

Table 1. Chemical component loss after pretreatment


Component loss ([wt%)
MV-Bio Pretreatment
Weight alpha cellulose Hemicellulose Klason lignin
7.68± (3.21)± 3.14± 13.078±
A 1.80 0.84 0.96 2.74
13.79± 8.32± 12.94± 23.46±
B 0.13 4.42 3.95 4.0
15.27± 6.89± 9.22± 24.59±
C 0.65 0.00 1.75 1.19
7.84± (5.52)± 3.23± 14.08±
D 0.06 2.44 0.21 4.81
4.37± (8.57)± 3.77± 43.39±
E 0.31 1.83 1.14 0.10
7.42± (7.08)± 1.88± 44.65±
F 1.11 1.37 0.14 2.92
6.25± (7.06)± 44.21±
G 2.23 2.05 12.72±4.24 5.46
3.85± (15.79)± 38.51±
H 1.64 4.26 5.17±1.39 1.62

MV: Microwave, Bio: Biological, A: 5 min (330W) with 5% IL, B: 10 min (330 W) with
5% IL, C: 12.5 min (330W) with 5% IL, D: 5 min (770 W) with 5% inc.loading, E: 5 min
(330W) with 10% IL, B: 10 min (330 W) with 10% IL, C: 12.5 min (330W) with 10% IL,
D: 5 min (770 W) with 10% IL, W: Weight, A: Alphacellulose, H: Hemicellulose, KL: Klason
lignin

that of 5%. It means that the combination pretreatment with higher IL is more
effective to degrade lignin. Pretreatment also caused hemicellulose removal.
It related with the deconstruction of carbohydrate-lignin complex as result of
the partial removal of lignin and hemicellulose accessing the disruption of the
hydrogen bond between cellulose [16].
In most treatment condition, the treatment caused increase in cellulose
contents. It suggested with volumetric heating allows greater volume of bamboo
contacts directly. The delignification effect of lignin was also increase more removal
of amorphous part and left cellulose rich. Furthermore, the beneficial aspect of
microwave irradiation could enhance the lignin degradation and provide the
potential of exposing cellulose and increasing cellulose contents [1].

B. Carbohydrate Changes after Microwave Irradiation of Bamboo


The infrared spectra (IR) in the 800–4,000 cm-1 region for pretreated bamboo
was shown in Figure 1A and B. A strong broad absorption at 3,441–3,286 cm-1 is
observed to H-bonded OH group derived cellulose I. The microwave-biological
pretreatment caused broadening of OH group band associating the O-H group
weaknesses [17].
Innovation for Better ASEAN Community 223

Figure 1. FTIR spectra of bamboo pretreated Microwave-biological pretreatment (A) 5% and


(B) 10% inoculum loading

The microwave energy delivery to polysaccaride via molecular interactions with


electromagnetic field [18] was responsible it. Moreover, the polar molecul vibration
and ion movement generate heat and extensive collisions accelerating chemical,
biological and physical processes [19]. The saponification of intermolecular
ester bonds cross-linking xylan and other components and decreasing of O-H
band intensity caused by its consumption in this reaction was also contributed
it [23]. Lignin structure can be observed in the region of 1,589, 1,511, 1,462,
and 1,420 cm-1 [9].
There are no changes in functional groups and the treatment only shifted the
wavenumber of identified peaks. This phenomenon was also reported previously
in biological pretreatment [12]. A hydrogen bonded (O-H) stretching absorption
224 Proceeding ASEAN COSAT 2014

was identified at 3,420 cm-1 (1) and C-H stretching absorption was seen at 2,920
cm-1 (2). The finger print peaks were 1,736 cm-1 (3) for unconjugated C=O in
xylans, 1,635 cm-1(4) for absorbed O-H and conjugated C-O, aromatic skeletal
in lignin at 1,605 cm-1(5) and 1512 cm-1 (6), C-H deformation at 1,462 cm-1 (7),
1,426 cm-1 (8) for C-H2 scissoring in cellulose, 1,378 cm-1 (9) for C-H deformation
in cellulose and hemicellulose, 1,328 cm-1 (10) for C-H vibration in cellulose
and C1-O vibration in syringyl derivatives, 1,246 cm-1 (11) for guaiacyl ring and
C-O stretch in lignin and xylan, 1,164 cm-1 (12) for C-O-C vibration in cellulose
and hemicellulose, 1,110 cm-1 (13) for aromatic skeletal and C-O stretch, 1,053
cm-1 (14) for C-O stretch in cellulose and hemicellulose, the band at 897 cm-1
(15) for C-O-C stretching at the β-glycosidic linkage characteristics in cellulose
[21], and 833 cm-1 (16) for C-H vibration in lignin [22].
The microwave pretreatment 12.5 min (330 W) and 5% IL caused the
absorbed O-H and conjugated C-O lost from spectra. This treatment also affected
C-H2 scissoring lost in 5 and 10 min (330 W) with 5% IL. Aromatic skeletal
of lignin did not appear in microwave pretreatment for 5 and 10 min (330 W)
inoculated with 10% IL.
The decrease in intensity at 1,328 cm-1 (deformation combination of syringyl
and xylan) was lower than at 1,246 cm-1 (guaiacyl of lignin) after combination
pretreatment. It means that syringyl moties are more reactive than that of guaiacyl
ones. It is caused by much metoxyl group content in syringyl and syringyl was
more degradable to microorganisme compared to guiacyl. More over, syringyl
has degree of polymerization, thus it is easier to be relocated. The higher syringyl
to guiacyl (S/G) ratio means greater delignification rate which can improve
enzymatic hydrolysis [16].

C.Morphological Characteristics of Pretreated Bamboo


The morphological structural changes on the surface of pretreated bamboo were
demonstrated in Figure 2. The pretreatment destroyed the bamboo structure
demonstrated by any holes, cracks, rugged and broken faced as well. The structure
is more soften and porous, thus it will increase the possibility contact between
substrate and enzyme in hydrolysis process. The partial lignin depolimerization
and hemicellulose removal in cell wall caused the disorganized morphology, with
greater exposure of the fibers. This finding was in line with the morphological
observation after microwave pretreatment on bagasse [9] and biological pretreat-
ment on bamboo [12].
Innovation for Better ASEAN Community 225

Figure 2. SEM images of microwave-biological pretreated bamboo (A) 5%


and (B) 10% inoculum loading
226 Proceeding ASEAN COSAT 2014

The changes of element constituent in weight after microwave-biological


pretreatnent based on EDX analysis was listed in Table 2A and B. As structural
component of cell wall, O was dominated compared to C. Beside that, LM also
consist of minor element constituent as inorganic constituent in ash content.
Fe, P, Ca, Na, Si was founded in pretreated bamboo. N was still founded in all
treatments using 5% IL.
Pretreatment with 10% inoculum loading gave more effect to remove minor
element.
Table 2A. Element constituent of pretreated bamboo with 5% inoculum loading
MV
A B C D
Element w/w %
BP
5% Inoculum loading
C (carbon) 16.9±0.4 13.8±0.7 13.1±0.5 12.4±1.2
O (oxygen) 73.5±0.6 71.6±0.8 79.2±0.5 72.7±1.5
Fe (ferro) - - - 6.4±3.8
Na (Natrium) 9.3±1.5 7.9±2.2 7.6±1.8 7.5±4.2
Si (Silikon) 0.4±4.8 4.6±1.2 - -
Ca (Calsium) - 1.2±3.1 - -
P (Phospor) - 0.9±4.2 - -
Total 100 100 100 100

Table 2B Element constituent of pretreated bamboo with 10% inoculum loading


MV
E F G H
Element %
BP
10% inoculum loading
C (carbon) 11.4±1.2 25.5±1.5 12.3±1.0 14.6±0.8
O (oxygen) 81.1±1.3 66.5±2.5 80.4±1.1 77.9±1.0
Fe (ferro) - - - -
Na (Natrium) 7.5±3.6 - 7.3±3.3 7.5±3.1
Total 100 100 100 100

D. Crystalline Allomorph of Pretreated Bamboo


Table 3 presented crystalline allomorph of pretreated bamboo analyzed by XRD.
To determine cellulose allomorph was used by Z-discriminant, in which
the Iβ (monoclinic) and Iα (triclinic) type were characterized as Z < 0 and Z > 0
[23]. Eventhough, microwave-biological pretreatment with 10% IL gave higher
lignin loss than that of 5% IL; however monoclinic structure was founded in this
treatment. The transformation in triclinic phase was founded in pretreatment
Innovation for Better ASEAN Community 227

Table 3. Crystalline allomorph of pretreated bamboo


Crystallite allomorph
Crystal al-
MV-Bio Pretreatment D d
z lomorph
(101) nm (10-1) nm
A 0.612 0.525 13.44 Iα
B 0.639 0.550 38.27 Iα
C 0.574 0.552 -73.83 Iβ
D 0.656 0.553 62.29 Iα
E 0.581 0.512 -27.75 Iβ
F 0.589 0.546 -44.55 Iβ
G 0.589 0.522 -20.78 Iβ
H 0.598 0.516 -1,14 Iβ

A, B and D. Improvement a better effect on hydrolysis performance of Iα phase


was expected in this treatment. This phase was more degradable than Iβ [24]
meta-stable and also more reactive than Iβ [25].

E. Crystallinity Index (CI) of pretreated bamboo


The crystallinity during microwave-biological pretreatment was presented in
Table 4. The enzymatic hydrolysis is mostly affected by CI [26]. To enhance sugar
release in the enzymatic hydrolysis, the crystalline system of LM should be opened
through pretreatment process. Disruption the hydrogen bond by increasing
splitting effect on crystalline region and maximize amorphous expansion can be
conducted by microwave pretreatment. This effect can be improved by delignifica-
tion on the lignin polymer by biological process. The lignin and hemicellulose
loss (Table 1) described it. In biomass, hemicellulose and lignin are amorphous
while cellulose is crystalline. It means that pretreatment give more effect on the
amorphous region than that of crystalline ones. Microwave irradiation for up to
5 min resulted higher CI both on 5 and 10% IL. It might be related with the
severe pretreatment tend to remove more amorphous fraction. Thus it makes the
increase of the crystalline proportion.
228 Proceeding ASEAN COSAT 2014

Table 4. Crystallinity index of pretreated bamboo


CI
MV-Bio Pretreatment
Crystal (Fc) Amorphous (Fa) CI
A 1.347 1.858 42.02
B 0.774 1.172 39.77
C 0.996 1.544 39.20
D 1.096 1.572 41.07
E 0.898 1.354 39.89
F 0.638 0.898 41.55
G 1.175 1.640 41.74
H 1.255 1.444 46.50

IV. Conclusion
Microwave-biological pretreatment on bamboo caused the structural modification
demonstarted by the absorbed O-H and conjugated C-O loss from spectra in
pretreatment for 12.5 min (330 W) with 5% IL. Aromatic skeletal of lignin did
not appear in microwave pretreatment for 5 and 10 min (330 W) inoculated with
10% IL. The lowest absorbance of lignin in 5% IL was founded in pretreatment
for 5 min (330 W). The higher reactivity of syringyl moties caused syringyl was
more soluble than that of guiacyl ones. The CI of cellulose in 5% IL tended to
decrease while the 10% IL was since versa. The pretreatment disrupted the fiber
confirmed by SEM image. Moreover, it founded that 10% IL more affected to
remove minor element constituent. Monoclinic structure has transformed into
triclinic structure in the 5, 10 12.5 min (330 W) with 5% IL.

V. Acknowledgement
The research was financially supported by Ministry of Research and Technology
(RISTEK) for PhD scholarship. The author expresses the gratitude to PT. MTI
for capturing the SEM image.

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The emergence of biogas technology for
reducing rural poverty: Empirical studies in
java island

Lutfah Ariana*
Center for Science and Technology Development Studies, Indonesian Institute of Sciences
Gedung A PDII, 4th floor, Jl. Gatot Soebroto Kav 10 Jakarta, Indonesia

Abstract
Many people in Indonesia, like in many developing countries, have lack of access to economical
and convenient energy sources. For various reasons, energy services provided by the government
or the private sector are difficult to access by those living in remote areas. When accessible, the
communities–mostly the poor–are burdened by the expensive price of the services, leading to an
even more economically vulnerable state. Although sustainable energy services will not solve the
underlying cause of poverty, its limited availability will hinder the pathway to prosperity. This
paper analyzes the arising development programs of biogas technology in some rural regions in
Java as one of the solution. These programs are mainly developed in a wide range of affordable
and appropriate technologies to address energy poverty. One of the actors in this program is the
Indonesia Domestic Biogas Programme, or commonly called BIRU. However, successful project
types and technologies have often faced barriers that prevent them from scaling up and becoming
widely disseminated. This paper briefly describes some of the internal and external obstacles faced
by biogas user in rural areas, characteristics of successful users and some empowerment programs
and strategies undertaken by various stakeholders in the development of biogas technology in
Indonesia.
Key words: Biogas, Rural, Program, Cost efficiency, Energy

I. Introduction
Indonesia is to be known as one of oil producer in the world; however, depletion
of oil reserve and decretion of environmental quality, has caused some rural people
to utilize renewable energy as alternative energy source. Biogas is one of some
renewable energy sources, that is produced during anaerobic digestion of organic
substrates, such as manure, sewage sludge, liquid manure of hens, cattle, pig,
organic waste from market and so on [5]. Biogas production enables a sustainable
agriculture with renewable and environmental friendly process system. It ranges
several benefits, such as eliminating greenhouse gas, reduction of odor, betterment
of fertilizer and production of heat and power.

* Corresponding author. Phone: +6281578723901 Email: juffrow@yahoo.com, lutf001@lipi.go.id.

231
232 Proceeding ASEAN COSAT 2014

Production and utilization of biogas has several environmental advantages


such as produces renewable energy source, reduces the release of methane to the
atmosphere, and can be used as a substitute for fossil fuels. It is also known as a
high quality digestate that can be used as a fertilizer.
In India and China, biogas technology development has been running fastly.
In 1998, India had 12 million units biogas processing instalation for generating
1,7000 MW electricity. While China had 9 million units with estimation gas
production of 6,2000–1,45000 million m3. Indonesia has potential of biogas
energy 684.83 MW with installed capacity of 0.06 MW or only 0.009% was
utilized [1,6].
Actually, biogas has long been known in Indonesia. Since 1970s, the Indone-
sian people have known biogas. Nonetheless, dissemination and demonstration
of biogas is still needs to be done in various parts of Indonesia so that people get
to know and have interest in biogas. This paper considers the development of
biogas in Indonesia is slower in the appeal of other countries in Asia or Africa.
The availability of fuel wood and fossil energy sources is abundant in the first
period to make slow progress of biogas in Indonesia.
However, in Indonesia, biogas technology development has a good prospect
because there are a lot of agribusiness regions in rural areas, such as special region
for animal husbandry, integration region of palm oil and cattle, and agropolitan
that has main commodity of animal husbandry [2]. Although the technical
viability of small-scale biogas technology has repeatedly been proven in several
Asian countries, mass dissemination of this technology has not been accomplished
in Indonesia.
Some impediments related to develop biogas are its availability, security of
supplies, price, ease of handling and ease of use. In addition, other factors like
technological development, introduction of subsidies, environmental constraints
and legislation play the role bringing its development [4].
Many studies emphasized the case of biogas because its strategic position as
alternative energy resources in current years. In addition, the possible benefits
and risks associated with the implementation of biogas require further analysis
especially related to its potential to alleviate poverty in rural regions. A study
need to be conducted to identify the current condition of biogas implementation
in different rural communities and it should be focused on its efficiency of the
digesters produced. This paper provides information about the material inputs
of some practices of biogas development which will allow an social analysis of
potential costs and benefits of using biogas digesters.
Innovation for Better ASEAN Community 233

II. Method
This study is a kind of social research that emphasized the qualitative approach
in collecting the data. By interviewing several key informants in rural region,
this study identify the arising condition in initiating biogas development, and
examine the influential factors that determine the sustainable development of
such technology. In addition, this study conducted three case studies of different
regions for showing the variety of different success factors. Multiple case studies
differed in term of their historical and environmental condition in adopting
biogas technology. The other characteristics emerged among the rurals because
of the availability of ownership of cattle and financial support. However, some
impediments are found in compelling the data since the justification of the
validity are quite difficult. Therefore, triangulation is considered to be preferable in
explaining the depth of information and the breadth of data that will be analysed.

III. Biogas for rural development


The benefit of biogas is actually a lot. In terms of technology, biogas may reduce
the risk of environmental pollution by reducing the use of fossil energy. From
the economic side, the presence of biogas makes the availability of energy sources
that can reduce daily energy costs. Biogas can also be used to run lights (similar
lamps strongking), a fuel stove for cooking and others.
Biogas is built specifically for households owning cattle. Every household
should be able to provide at least 30 kg of cow dung. The cow dung as a source
is to be converted into gas. The amount of dirt as much as it should at least be
provided by 3 cows. So each household must have at least 3 cows.
By cooperating with the Indonesian Ministry of Energy and Mineral
Resources and SNV Netherlands Development Organisation through Hivos has
implemented Indonesian Domestic Biogas Program (IDBP), also known as the
BIRU programme. The first phase (2009–2012) has been funded by the Dutch
embassy in Indonesia. This programme seeks to distribute 8,000 biogas digesters
as a local sustainable energy source by developing a commercial, market-oriented
sector that also provides job and business opportunities for masons and partner
organisations in construction. The IDBP focuses on implementation through a
multi-stakeholder sector development approach, creating a market-based biogas
Table 1. Production of Biogas by the Type of The Slurry
Type of the slurry Volume of production
Cow/buffalo, m3/kg 0.023 – 0.040
Pig, m3/kg 0.040 – 0.059
Chickens/birds, m3/kg 0.065 – 0.116

Source: Sulaeman, dkk, (2009)


234 Proceeding ASEAN COSAT 2014

Table 2. Equivalence Value of Various Types of Energy Compared to Biogas


Type of Energy Equivalency Value compared to 1 m3 Biogas
LPG 0,46 kg
Kerosene 0,62 liter
Solar 0,52 liter
Gasoline 0,80 liter
Dried woods 3,50 kg

Source: Sulaeman, dkk, (2009)

sector, involving locally trained contractors and masons who are supported by
vocational training institutions, in this case is Rural Development Institute of
Technology or known as Lembaga Pengembangan Teknologi Pedesaan (LPTP)
Solo. LPTP Solo is an extension agencies and manufacture biogas reactor in Yogya,
Boyolali and Klaten. This institution received funds from Germany (Burda), the
Netherlands (HIVOS), CORDEC (Netherlands), Ministry of Environment,
Ministry of Energy and Mineral Resources and the local Government. LPTP
focuses on rural technology provider in the fields of energy, food, agriculture and
environmental management and have a basic strategy ‘to build future projects’.
This LPTP perform activities such as sustainable agriculture (biogas), waste
management, economy, organic wastewater treatment (from industry, hospitals,
households) and the engineering workshop. In addition, LPTP is a center of
sustainable energy development, which consists of biogas, firewood saving stoves,
biodiesel and coal briquettes.
In order to produce biogas, it needs a digester. One digester could reduce
methane gas emission by decomposition of organic matter from agriculture and
animal husbandry sectors. The raw material for producing biogas is cow dung
that has been fermented and then resulted methane gas.
A farmer who invests in biogas can have his investment back within around
three years if the household only uses the biogas, and even within two years if the
bioslurry is applied appropriately (leading to higher yields and lower dependency
on chemical fertiliser). A digester can serve its owner for 15 to 20 years with
minimum maintenance costs.

IV. R esult and Discussion


A. Biogas for Remote People in Cepogo, Boyolali
The village that has already implemented biogas technology is located in District
Cepogo, Boyolali, Central Java. This village consists of 18 hamlet (at the Pillars
of Citizens). One hamlet average at least have 30 cows. The average production
of milk cows in the village between 12 to 20 liters per day with medium quality.
Innovation for Better ASEAN Community 235

The history of the formation of biogas user community began in 1992, by


introduction of biogas program BIRU, grant financing in the form of subsidies
from the German GTZ; Rp700,000 to build a biogas digester household with size
6 m3. The grant was distributed to farmers who have a minimum of 3 cows. In
addition, they should contribute to build biogas as much as Rp400,000 and the
rest of the cost would be supported by the Dutch Non Governmental Organization
named Borda, which partnered with LPTP Boyolali to build a biogas reactor
at the farmer’s yard. With a capital of Rp1,100,000 (1992), farmers received a
biogas reactor completed with the installation and the stove. The amount of the
reactor is equivalent to buy one adult cows at that time.
In 2011, there were about 40 families (KK) with the amount of as much as
10 pieces of biogas reactor. At the beginning of the year of manufacture of 1991,
there were 13 pieces of the reactor. There were three biogas reactors that could
not be used due to several things, including ranchers moved house, owned cattle
ranchers sold, and changed jobs into tofu and tempeh producers.
Of the 10 existing biogas reactors currently used almost all of them just for the
sake of cooking and some have been used as an additional means of illumination
with the use of patromak electricity if dead. No biogas is converted into electricity
in this village. Reactors were built by various capacity ranging from 3 m3, 6 m3,
9 m3 and 13 m3 depending on the number of animals owned. For farmers who
have 8 cows, the amount is 13 m3 reactor.

Figure 1. Biogas for Rural Energy


236 Proceeding ASEAN COSAT 2014

Figure 2. Identity for Owning Biogas for Rural Household

Since 2010, household biogas program (BIRU) with a subsidy of Rp2 million
of Hivos was introduced by LPTP (Hivos partners in Boyolali) to the village
community Cepogo. Until 2011, there were only 3 families who had build biogas
reactor. Requirements to obtain subsidies from Hivos biogas among other breeders
must have at least 2 cows.

B. Biogas in Merapi Erruption Area, Balerante, Klaten


The first biogas made ​​of concrete built in 2003 with the assistance of PT. Sari
Husada at the home of a breeder who is on of the board of Sarana Makmur
Cooperative. After the post-eruption of Mount Merapi (2010), the biogas
digester could still operate. Until 2011, Balerante has about twelve biogas
digester that has been operating, including four digesters located in the hamlet
of Balerante, namely one comes from grants Cordaid and Hivos three with
subsidies. The other three digesters located in the hamlet of Kaligompyong,
with details of one of the grants Cordaid, one from the subsidy of Hivos and
one from the grant of Ministry of Environment. Four digesters located in the
hamlet Sidorejo, comprised three digesters with subsidies from Hivos and one
from Cordaid’s grant. The only one digester located in the hamlet Kendalsari
was supported by the grants from Cordaid. The total population of about 136
families Balerante village, and about 90% of that amount has livelihood as
Innovation for Better ASEAN Community 237

Figure 3. Biogas for Energy Substitution (LPG)

farmers, with an average ownership of cattle around 2–7 cows per family.
After the eruption of Mount Merapi mid-year 2010, the village of Balerante
received several programs to improve the environment and economy of central and
local government, the program known as “Early Recovery”. Biogas development
program included as one of the activities.
Biogas is already operating in Balerante almost entirely used for the benefit of
cooking. Average savings obtained is equivalent to the use of four LPG cylinders
(3 kg) every week for owners with a large family (5–8 people) and about 4 LPG
cylinders every month for a family of very small (2–3 people). In that time(2011),
in Balerante the average price of 3 kg LPG tube is Rp18,000. Minimal savings
made ​​by a small family in a month is Rp72,000.

C. Bio-waste Energy in Pesantren Al-Hikmah, Gunung Kidul


Biogas in rural region namely Sumber Rejo located in Pesantren Al Hikmah was
built by Kyai H. Harun Al Rashid. In 2011, this Pesantren has 400 students, and
in 2010, build a wastewater treatment plant (WWTP) with the size of biogas
digester 9 m3 and the raw materials of human sanitary waste. Development
funding mechanism came from the Ministry of Public Works project which
granted for Rp100 million, the Department of Public Works District of Rp200
million, and the boarding schools provide Rp4 million. The assistants construction
of this project is LPTP.
238 Proceeding ASEAN COSAT 2014

Biogas in this boarding school used as cooking fuel in the kitchen. The benefits
of this biogas is a portion of the cost savings to purchase firewood Rp1,500,000
per month (50% of total demand). Currently, the kitchen boarding, in addition
to using biogas, is still using firewood at a cost of Rp1,500,000.
In addition, with the biogas and wastewater treatment program, the cost to
suck human waste holding tank (“safety tank”) is reduced. Before, there was this
program every 3 months must pay about Rp900,000 to suck or dispose of waste.
But this time the cost is no more.

D. Biogas for Ranchers in Nongkojajar, Pasuruan


Nongkojajar located in Pasuruan regency of East Java. Nongkojajar divided into
12 villages and an agricultural and dairy cattle breeding. A total of 11 villages are
producing dairy cow and a village is bull breeders and vegetable farmers. First
development of biogas is started in 1987 by the Department of Agriculture of
East Java Province. Until 2011, there are about 40 biogas built on the initiative
of the citizens with the assistance of several grants for government projects, or
with their own capital in the form of loans from the cooperative. In 2009, Hivos
collaborated with KPSP Setia Kawan developed biogas in this area. Hivos trained
around 22 biogas technicians certified and successfully educate approximately
60 energy companion. Hivos program, providing subsidies worth Rp2 million
to build a biogas digester and the rest is around Rp4 million to be met by
developing breeder reactors which will measure approximately 6 m3 of biogas.
If the size of biogas digester to be built is larger, the funds required will also be
greater. Many ranchers who enjoy Hivos’ subsidy also take benefit from the loan
of cooperatives in developing biogas digester. In further, the cooperative worked
with KLH GEF Program run by Bank Syariah Mandiri provide assistance with
low interest rate (9% per year) for the farmers. Farmers pay off the debt (for
example the size of digester 8 m3) for biogas development for Rp42,000 per
10 days. These mechanisms is considered is more appropriate in facilitating the
farmers to develop biogas [3].
In this case, Hariyanto, a head of a household in this rural, has 4 oxen with
13 liters of cow milk production per day. Cow dung produced per day is 30 kg /
day / cow. With size 6 m3 of biogas, the gas produced was channeled to the two
houses. The biogas is used for cooking, lighting and water heaters.
Before using biogas, the family was using 2 tubes size 12 kg LPG and kerosene
by 15 liters for cooking. A tube 12 kg LPG used for heating water for 3 months.
After using biogas, the family was able to make savings of Rp300,000 for cooking
and Rp25,000 for energy water heater. Waste (slurry) from waste digester of 100
Innovation for Better ASEAN Community 239

kg per day can be used for garden fertilizer. Estimated savings to buy fertilizer
can be pressed up to 30%. Price manure slurry if sold at around Rp300 per kg.

V. Conclusion
Utilization of alternative energy sources biogas from livestock animals and
other impurities can reduce the cost of electricity consumption in remote areas.
Utilization of alternative energy is able to create energy independence for rural
communities so that in the long run it can reduce poverty for people who are
vulnerable to energy availability.
Development program of biogas technology can improve active participation
of various stakeholders ranging from government, donors, development agencies,
technology, and society. However, the sustainability of the development of biogas
technology is still impeded by affordability level of capital for early initiation
of construction of the digester, the technical aspects of waste treatment is still
insufficient demand, and scale up technologies that require a huge cost, especially
for the needs of the industry.
The Indonesian Ministry of Energy and Mineral Resources is looking into
options for faster upscaling by providing higher subsidies or at least with a
segmented market, in which certain target groups have higher subsidies. This will
provide accelerated access to renewable energy for many farming households, but
will limit the possibilities of developing a market-based biogas sector. Intensive
discussions about the way forward are ongoing.The main conclusions of the study
may be presented in a short Conclusions section, which may stand alone or form
a subsection of a Discussion or Results and Discussion section. The conclusion
section should lead the reader to important matter of the paper. It also can be
followed by suggestion or recommendation related to further research.

VI. Acknowledgement
This study is part of a research funded by Ministry of Research and Technology
through “Program Peningkatan Peneliti dan Perekaya (PKPP)”, or namely Incentive
Program in 2011. The author are deeply appreciated to the research team (coordi-
nated by Wati Hermawati) for the support and teamwork. The data is mainly sourced
from Research Report entitled “R & D Funding Pattern and Implementation
of New Energy-Renewable in Indonesia Case Studies: Microhydro power and
Biogas”.
240 Proceeding ASEAN COSAT 2014

VII. R eferences
1) Department of Energy and Mineral Resources. (2004). The International
Workshop on Biomass and Clean Fossil Fuel Power Plant Technology. Sustainable
Energy Development and CDM. Jakarta, January 13–14, 2004
2) Ditjen Pengembangan Peternakan, Ditjen Bina Produksi Peternakan,
Departemen Pertanian. (2003a). Pengembangan Kawasan Agribisnis Berbasis
Peternakan.
3) Hermawati, W. et al. (2011). Pola Pembiayaan Litbang dan Implementasi EBT
di Indonesia, Kasus: PLTMH dan Biogas. Laporan Penelitian Program PKPP
Ristek 2011.
4) Koopmans, A. (1998). Trend in Energy Use. Expert Consultation on Wood
Energy, Climate and Health. Phuket, Thailand, 7–9 October, 1998.
5) Widodo, T. W. and A. Hendriadi. (2005). Development of Biogas for Small
Scale Cattle Farm in Indonesia. Paper presented in the International Seminar
on Biogas technology for poverty reduction and sustainable development,
Beijing, China, 18-20 October 2005.
6) Widodo, T. W. and A. Nurhasanah. (2004). Study on Biogas Technology
and its development potency in Indonesia. Proceeding of National Seminar
on Agricultural Mechanization. Bogor, 5 August 2004.
SCMG
Assessment of Erosion Potentials on Various
Cropping Patterns Using USLE: Case of Subang
Region, West Java

R. I’anatus Sholihah*, Dyah R. Panuju, Enni D. Wahjunie, Bambang H.


Trisasongko
Department of Soil Science and Land Resource, Bogor Agricultural University
Jl. Meranti, Darmaga Campus, Bogor 16680, Indonesia

Abstract
Degraded lands in Subang regency have been increasing within this decade. Land inventory
conducted by the government shows that degraded areas expanded from 7,785 ha in 2011 up
to 9,581 ha in a year. Improvements of this type of land, primarily due to erosion, are necessity
in order to support intensive agriculture. This research aims to study erosion in various cropping
pattern based on land units in four districts of Subang region, West Java, which is important for
conservation planning. The research indicates that there are 19 land units formed by combining
land capability classes and land use types. The result shows that erosion in the test site varies from
0.30 to 133.19 ton ha-1y-1. Actual erosion from USLE is found higher than tolerable soil loss,
meaning that conservation is urgent to maintain land productivity.
Key words: Erosion, Land unit, Soil and Water conservation, USLE

I. Introduction
Agricultural sector in Indonesia has laid the base for the nation’s economy.
Population increase escalates the demand for land to maintain food production.
Nonetheless, land resource is fixed and limited with competing interest to other
utilizations, such as industries and residences. Throughout the nation, land use
changes have been reported to give significant impact on soil erosion, because
it serves the interaction between natural environment and human activities [1].
Inevitable alteration urges the uses of marginal or unsuitable lands for agriculture,
or alternatively the intensification of agricultural fields. The main challenge of
the latter is uncontrolled soil erosion, which in turn ignites further problems
including pollution.
Since the last century, soil erosion has been considered a serious environmental
problem. Majority of pasture and agricultural fields in the world faces problems
of erosion [2]. Humid tropic climate in Indonesia triggers high amount of
erosion, especially in intensive agricultural fields. Despite its importance, soil
* Corresponding Author. Tel: +62-251-8422325. E-mail: rizqiianatus_99@yahoo.com

243
244 Proceeding ASEAN COSAT 2014

erosion considering local environmental settings has yet been thoroughly studied.
Estimation of the loss due to soil erosion is often difficult to quantify since it
involves many factors, including climate, land cover, soil, topography and human
activities [1].
Erosion potential is often assessed using USLE [3], which is one of the
simplest methods to date. This paper explores the model to analyze various
cropping patterns based on land units, which in turn be able to construct suitable
conservation strategies for agriculture sustainability.

II. Method/material
A. Study Area
Subang is situated in West Java, surrounded by Indramayu and Sumedang regen-
cies in the East, while Purwakarta and Karawang are in the West border. Java sea
lies in the north, and emerging industrial regency of Bandung Barat in the south.
Geographically, Subang is located between 6°11’-6°49’S and 107°31’-107°54’E.
To be able to focus on the problem, only four districts are considered, namely
Cipeundeuy, Kalijati, Pabuaran, and Patokbeusi (Figure 1). These districts are
located in wavy landform with various cropping pattern, at the altitude between
50–500 m above sea level.

Figure 1. The Study area: Subang region


Innovation for Better ASEAN Community 245

B. Method
Various data are utilized for this analysis. Each data processing is taken in sequence
as follows.

1) Determining Land Unit


Land unit is formed by combining land capability and land use maps through
boolean overlay. In this research, land use types consisting of rice field (sawah),
mixed garden (kebun campuran) and dry land agriculture (tegalan) are considered.
As a result, polygons containing land use types for each land capability classes are
constructed, which will serve as the primary unit for further analysis.

2) Erosion Soil Analysis


Soil erosion potentials using USLE, firstly communicated by Wischmeier and
Smith [4], is evaluated in this paper. The model, consisting of six factors, can be
expressed by the following equation:

(1)
Where A is the amount of soil erosion in ton ha-1 y-1, R is rainfall erosivity, K
is soil erodibility, LS is topographic factor, comprises of slope steepness and slope
length, C is cropping management and P is soil conservation practice.
a) Rainfall erosivity (R)
Rainfall is one of driving forces of soil erosion. Its erosivity and temporal rainfall
pattern deliver a direct impact to soil erosion [5]. In this study, we used Bols
method by considering available data to calculate the rainfall erosivity. Bols
equation proposed for Indonesia is shown below [6]:
R = 6.119 x Rf1.21x Rn-0.47 x Rm0.53 (2)
Where R is monthly erosivity, Rf is total monthly rainfall, Rn is sum of rainy
day per month and Rm is the maximum rainfall during 24 hour in the observed
month.
The R-factor for each land unit was then spatially estimated through interpola-
tion using Inverse Distance Weighting (IDW) technique.
b) Soil erodibility (K)
Erodibility is susceptibility of soil to erosion that influenced by many factors, such
as chemical, mineralogical, biological properties of soil as well as physical terrain
attributes, hydrological and climatic factors [7]. The value of K was computed
using the following equation:
246 Proceeding ASEAN COSAT 2014

K = [( 2.1 x M 1.14 x 10-4) x (12-a)) + (3.25

(b-2) + (2.5(c-3)) x 1.293% (3)


Where M is (Svf+St)(100-Cf ), a is percentage of soil organic matter, b is
structural code of the soil, c is permeability class code of the soil, Svf, St, and Cf
are percentage very fine sand, silt, and clay fraction respectively.
Physical soil characteristics which represent K-factor value need to be
considered. In this study, we used soil texture, percentage of soil organic matter,
and permeability class code of the soil to estimate K-factor.
c) Topographic factor (LS)
Topography influences surface runoff rate, therefore it is essential to quantify LS
properly [8]. Slope steepness and its length are measured in the field for some
locations, while the rest are calculated from existing slope map, provided by the
Citarum-Ciliwung River Basin Management Agency. Slope length and steepness
were then calculated using Wischmeier-Smith equation:

LS = l1/2 (0.0139+0.0965 S+0.00139S2) (4)


Where l is slope length in m, S is the slope steepness in percent.
d) Cropping management and soil conservation practice (CP)
The C- and P-factors are derived from Abdurachman et al. [9] study according to
actual land use. In this research, C-factor was weighed by considering cropping
period in a year. C-factor from cropping pattern in dryland (tegalan) was computed
by considering monthly R-factor, and then was spatially modeled using IDW.
The equation to calculate C-factor from various cropping pattern in dryland
agriculture (tegalan) is:

(6)
Where Ci is the average of C-factor of certain cropping pattern, Ri is monthly
R-factor, Ci is C-factor [9].

3) Assessing Tolerable Soil Loss (TSL)


Tolerable soil loss was computed using Hammer equation [10]:
TSL = ED/T (7)
Where TSL is tolerable soil loss in ton mm/yr, ED is equivalent depth in mm,
T is soil usage (400 years).
Innovation for Better ASEAN Community 247

Figure 2. Land use types in Subang region

III. R esult and Discussion


A. Description of Land Cover and Land Units
Result of visual image interpretation from ALOS AVNIR-2 is summarized in
Figure 2. Delineation of land cover considered image interpretation keys such as
color, texture, association, shape, etc. Verification by ground survey indicates that
there are seven land use types, i.e. water body, forest, mixed garden, real estate,
plantation, rice field and dryland agriculture.
Figure 2 shows that rice field (sawah) dominates research site of about
19313.73 ha or 53% of total area. Irrigated rice fields are located in Patokbeusi,
while Kalijati and Cipeundeuy districts are dominated by rain-fed rice fields.
Pabuaran district has both of them equally. Mixed garden and dryland fields are
often utilized by local people for cash crops, fruits, softwood timber and others.
Rice and dryland fields as well as mixed garden are the main source of income.
Table 1. Land capability classes in research area.
Acreage
No Land capability
Ha %
1 IIe 329.92 0.92
2 IIIc 13044.11 36.27
3 IIIe 9944.18 27.65
4 IIIw 1514.40 4.21
5 IVe 1843.97 5.13
6 IVw 6414.81 17.84
7 VIe 2874.09 7.99
248 Proceeding ASEAN COSAT 2014

Table 2. Distribution of land units.


Land units code Land units Acreage (ha)
IIe-KC IIe-Mixed Garden 108.22
IIe-SW IIe-Rice Field 88.40
IIIc-KC IIIc-Mixed Garden 1709.93
IIIc-SW IIIc-Rice Field 1577.93
IIIc-TG IIIc-Dryland 78.81
IIIe-KC IIIe-Mixed Garden 173.50
IIIe-SW IIIe-Rice Field 2726.49
IIIe-TG IIIe-Dryland 67.44
IIIw-KC IIIw-Mixed Garden 235.18
IIIw-SW IIIw-Rice Field 456.90
IIIw-TG IIIw-Dryland 39.35
IVe-KC IVe-Mixed Garden 182.22
IVe-SW IVe-Rice Field 338.28
IVw-TG IVw-Dryland 7.30
IVw-KC IVw-Mixed Garden 127.61
IVw-SW IVw-Rice Field 2466.90
VIe-KC VIe-Mixed Garden 148.34
VIe-SW VIe-Rice Field 154.24
VIe-TG VIe-Dryland 7.05
Total   10694.09

Land management should conform to land capability to maintain land


productivity. In this study, land capability data were generated by combining
slope and soil type maps. Land capability in this region consists of seven classes
(Table 1).
The research area is dominated by land capability IIIc class about 13,044.11
ha in undulating terrain and the soil type is Latosol. The main characteristics
of land capability IIIc class are medium to high permeability with fairly high
climate resistance.
Land use and land capability maps designates land unit in research area. Land
units arranged at district level are presented in Table 2.
Table 2 indicates that there are 19 land units available in this test site. Land
unit IIIe-Rice Field is the highest area about 2,726.49 ha, consisting of rice field
with land capability IIIe class. This land unit is distributed on almost all districts,
mostly located in Pabuaran. This is probably due to the fact that this district is
situated between flat and undulating landform. In addition, majority of soil type
is podsolic, which is sensitive to erosion. Many farmers utilize the land for rice
field because Pabuaran relief is rather flat, while water has been sufficient.
Innovation for Better ASEAN Community 249

Land unit VIe-Dryland (VIe-TG) is the smallest area covered in the test site,
i.e. about 7.05 ha. This land unit is located in steep, undulating terrain, hence it
is seldom utilized for agriculture.
Table 3. USLE individual factors
Land units R K LS C P
IIe-KC 3224.77 0.22 0.05 0.25 1.00
IIe-SW 3482.25 0.22 0.05 0.01 1.00
IIIc-KC 2513.08 0.04 0.43 0.41 0.65
IIIc-SW 2130.91 0.04 0.44 0.04 0.70
IIIc-TG 2259.17 0.04 0.47 0.50 0.70
IIIe-KC 1839.04 0.36 0.40 0.41 0.56
IIIe-SW 1784.96 0.43 0.32 0.01 0.81
IIIe-TG 1937.13 0.54 0.43 0.42 0.50
IIIw-KC 2295.83 0.26 0.26 0.28 0.55
IIIw-SW 2314.91 0.25 0.25 0.01 0.82
IIIw-TG 2230.39 0.25 0.47 0.65 0.60
IVe-KC 3463.29 0.04 1.55 0.41 0.60
IVe-SW 3422.90 0.15 1.57 0.01 0.83
IVw-KC 1732.07 0.27 0.09 0.29 0.65
IVw-SW 1468.17 0.27 0.09 0.01 0.88
IVw-TG 1601.97 0.04 1.55 0.65 0.35
VIe-KC 2704.89 0.09 2.88 0.31 0.73
VIe-SW 2877.96 0.07 2.76 0.01 0.85
VIe-TG 2632.15 0.04 2.27 0.77 0.80

B. Soil Erosion in Subang Region


Preparation of soil conservation plan requires erosion data, which is often
measured in-situ. In addition, conservation planning also needs to consider soil
tolerable loss [11]. Prior assessing TSL, soil erosion needs to be calculated. Factors
influencing soil erosion are presented in Table 3.
Values shown in Table 3 are then applicable to estimate soil erosion potentials
for each land unit. Erosion potentials and their tolerable soil loss quantifications
are presented in Table 4.
Tolerable soil loss in the research area spans about 14 up to 40.50 ton ha-1y-1.
This indicates that high annual erosion rate applies in the research area was still
250 Proceeding ASEAN COSAT 2014

Table 4. Erosion potentials and tolerable soil loss (TSL) in ton ha-1y-1 for each land unit

Land units Erosion TSL

IIe-KC 8.67 14.00


IIe-SW 0.37 14.00
IIIc-KC 10.96 39.46
IIIc-SW 0.77 40.50
IIIc-TG 13.19 39.00
IIIe-KC 57.11 30.75
IIIe-SW 1.78 29.75
IIIe-TG 91.03 28.00
IIIw-KC 34.84 21.68
IIIw-SW 1.04 21.77
IIIw-TG 103.01 19.68
IVe-KC 48.04 36.00

IVe-SW 6.83 24.40


IVw-KC 7.33 29.10
IVw-SW 0.30 29.79
IVw-TG 20.31 36.00
VIe-KC 123.21 30.60
VIe-SW 5.70 32.47
VIe-TG 133.19 36.00

tolerable. This result signifies that recent situation allows reasonable support for
plant growth [12].
Table 3 also shows the average of actual erosion in the study area, which varies
from 0.30 up to 133.19 ton ha-1y-1. Land unit type IVw-SW has the lowest soil
erosion mainly because of very low erosivity of rainfall at gentle slope region.
The highest erosion rates based on Table 3 is in land unit type VIe-TG. This
land unit is located in Jalupang village, Kalijati district, which has wavy to hilly
terrain. Land use type of this land unit is dryland agriculture (tegalan) which is
often cultivated in steep slope (26–40%), with high rainfall. Based on information
presented in Table 3, there are seven land units which have soil erosion level of
more than their tolerable soil loss. In these cases, soil conservation is urgently
Innovation for Better ASEAN Community 251

needed. Soil erosion has positive correlation with slope steepness, cropping
condition and conservation practice, which is in line with previous research [13].
Land use without proper conservation practice in VIe-TG is likely to expose
high-level of erosion.
In contrary, twelve land units have actual erosion lower than tolerable soil
loss. Hence, these areas require further maintenance to sustain current condition.
Seven land units, i.e. IIIe-KC, IIIe-TG, IIIw-KC, IIIw-TG, IVe-KC, VIe-KC,
and VIe-TG have actual soil erosion prediction exceeding tolerable soil loss
(A>TSL). Soil and water conservation planning is therefore a necessity, probably
by considering alternative crop management and soil conservation techniques.
Soil and water conservation specifically planned to each land units is important
for region with actual erosion higher than soil tolerable loss. Options for soil and
water conservation include planting cover crops, constructing multilevel canopies
of different plants as well as terrace making [14].

IV. Conclusion
Assessment of erosion potentials using USLE in Subang region shows that some
land units have actual erosion exceeding tolerable soil loss value. Therefore, soil
and water conservation strategies such as crop management are critical to this
site. Various cropping patterns on cultivated field have a greater influence on
actual soil erosion or soil loss. This is due to its direct relationship with human
activities which are responsible in land use alteration. The research summarizes
that USLE is simple model which can be used to provide information on soil
erosion. Utilization of this information, in turn, leads to a better soil and water
management planning.

V. Acknowledgments
We would like to thank the Citarum Ciliwung River Basin Management Agency
and Agricultural Land Resources Agency for the data sets used in this research.
Finally, we thank to anonymous reviewers of which their comments significantly
improve the paper.

VI. R eferences
1) Alkharabsheh, M.M., Alexandrilis, T.K., Bilas, G., Misopolinos. N, and
Silleos. N. (2013). Impact of land cover change on soil erosion hazard in
northern Jordan using remote sensing and GIS. Procedia Environmental
Sciences, 912–921.
252 Proceeding ASEAN COSAT 2014

2) Pimentel, D., Harvey, C., Resosudarmo, P., Sinclair, K., Kurz, D., McNair,
M., S. Crist, L. Shpritz, L. Fitton, R. Saffouri, and R. Blair. (Feb, 1995).
Environmental and economic costs of soil erosion and conservation benefits.
Science, 267, 5201, 1117-1123.
3) Sonneveld, B.G.J.S. and M.A. Nearing. (2003). A nonparametric/parametric
analysis of the Universal Soil Loss Equation. Catena, 52, 9-21.
4) Wischmeier, W.H., and D.D. Smith. (1978). Predicting Rainfall Erosion
Losses: A Guide to Conservation Planning, Washington: United States Depart-
ment of Agriculture, 58.
5) L. Liu and X.H. Liu. (2010). Sensitivity analysis of soil erosion in the Northern
Loess Plateau. in Procedia Environmental Sciences, 134-148.
6) Aflizar, Afrizal, R., and T. Masunaga. (2013). Asessment erosion 3D hazard
with USLE and surfer tool: a case study of Sumani Watershed in West Sumatra
Indonesia. Tropical Soils Journal, 18, 81-92.
7) Perez-Rodriguez, R., Marques, M.J., and R. Bienes. (2007). Spatially vari-
ability of the soil erodibility parameters and their relation with the soil map
at subgroup level. Science of the Total Environment, 378, 166-173.
8) Beskow, S., Mello, C.R., Norton, L.D., Curi, N., Viola, M.R., and J.C.
Avanzi. (2009). Soil erosion prediction in the Grande River Basin, Brazil
using distributed modeling. Catena, 79, 49-59.
9) Abdurachman A, S. Abuyamin, and U. Kurnia. (1984). Soil and Crop
Management to Conservation. Bogor: PPT Bogor.
10) Hammer, M.J., and K.A. Mac Kichan. (1981). Hydrology and Quality of Water
Resources. New York: John Willey and Sons.
11) Dariah, A., Rachman, A., and U. Kurnia. (2004). Erosion and dry land
degradation in Indonesia. Soil Conservation Technology on Dry Land Slope,
Bogor: Research Center and Soil and Agroklimat Research, 1-8.
12) Arsyad, S. (1989). Soil and Water Conservation. Bogor: IPB Press.
13) Halim, R., Clemente, R.S., Routray, J.K., and R.P. Shresta. (2007). Integration
of biophysical and socio-economic factors to assess soil erosion hazard in the
Upper Kaligarang Watershed, Indonesia. Land Degradation and Development,
4, 453-469.
14) Dewi, I.G.A.S.U., Trigunasih, N.M., and T. Kusmawati. (Jul, 2012). Erosion
prediction and soil and water conservation planning in Saba River Basin.
E-Journal Tropica Agrotechnology, 1, 12-23.
New Stage of International Collaboration
on Climatological Observation

Manabu D. Yamanakaa,b,*
a
Japan Agency for Marine-Earth Science and Technology (JAMSTEC), Yokosuka, Japan
b
Graduate School of Science, Kobe University, Kobe, Japan

Abstract
Based on collaborations with Japanese scientists (principal institution: JAMSTEC) for three
decades, Indonesian Government (principal agency: BPPT) has installed the Maritime Continent
Center of Excellence (MCCOE) which operates weather and wind-profiling radars, develops/
installs climate buoys, and informs/investigates local/global climatic variations. We have been
clarifying various scientific results on the multiple-scale climate variability which is triggered in this
Maritime Continent region and is governing the global climate. This type of truly international
high-level collaboration is necessary to watch/understand to complex climatological issues.
Key words: Climatological observation, Multiple scales, International collaboration

I. Introduction-Climate of the L and-Sea Coexsisting


Planet E arth
The climate of a planet is maintained by an energy balance between the solar
heating and the planetary infrared cooling [1][2]. The solar heating is dependent
on the distance from the sun, and the infrared cooling is dependent on the
planetary temperature. Thus, the planetary temperature is basically determined
by the solar distance, and the Earth is only one habitable planet with liquid
water. The solar heating on the Earth is with annual and diurnal cycles and
latitudinal dependence, and it is decreased by reflection by earth’s surface and
clouds (parasol effect). The Earth’s infrared cooling is partially returned to the
Earth (greenhouse effect), of which the anthropogenic increase is being watched
with critical interest by international community as so-called global climatic
change. The balance mentioned above does not hold locally, with over-heating
and -cooling in the equatorial and polar regions, respectively, and the equatorial
over-heating is also due to hydrological cycles (latent heating) associated with
a peak 2,000 mm/year of rainfall produced by clouds with spatially/temporally
small scales. These imbalances are compensated by large-scale atmospheric and
oceanic circulations transporting heat poleward, through which the climate and

* Corresponding Author E-mail: mdy@jamstec.go.jp. URL: http://aoe.scitec.kobe-u.ac.jp/~mdy/index-e.


html

253
254 Proceeding ASEAN COSAT 2014

its variability in tropics affect substantially those in extratropics and therefore the
whole Earth’s climate.
The Indonesian maritime continent (IMC) [3] is a miniature of our land-sea
coexisting planet Earth. Firstly, without an interior activity, the Earth becomes
an even-surfaced “aqua-planet” with both atmosphere and ocean flowing almost
zonally, and solar differential heating generates (global thermal tides and) Hadley’s
meridional circulations with ITCZ along the equator as observed actually over
open (Indian and Pacific) oceans in the both sides of IMC. ITCZ involves
intraseasonal variations or super cloud clusters moving eastward [4][5].
Secondly, the lands and seas over the actual Earth have been keeping the area
ratio of 3:7 (similar to that islands and inland/surrounding seas in IMC), but
their displacements have produced IMC near the equator, which turns equatorial
Pacific easterly current northward (Kuroshio) and reflects equatorial oceanic
waves inducing coupled ocean-atmosphere interannual variations, such as El
Niño-southern oscillation (ENSO) [6] and Indian-Ocean dipole mode (IOD)
[7], or displacements of Walker’s zonal circulations. These are correlated with
climate variations over IMC [8][9][10][11] (see Figure 1).

Figure 1. Correlatons with ENSO and IOD (left) and simultaneous correlation with SST (right)
for rainfall averaged for 9 stations in around Jakarta [11].

Thirdly, because IMC consists of many large/small islands with very long
coastlines, many narrow straits become a dam for the global (Pacific to Indian)
ocean circulation, and the land-sea heat capacity contrasts along the coastlines
generate the world’s largest rainfall with diurnal cycles (sea-land breeze circula-
tions) [12][13][14][15][16][17][18][19] (see Figure 2). The diurnal cycles
are dominant in the rainy season (austral summer in Java and Bali) because
rainfall-induced sprinkler-like land cooling reverses the trans-coastal temperature
gradient before sunrise, and subsequent clear sky on land until around noon
provides solar heating depending on season [8][9]. These processes lead to rapid
land/hydrosphere-atmosphere water exchange [20], local air pollutant washout,
Innovation for Better ASEAN Community 255

Figure 2. Geographical distributions of power-spectral intensities for cloud-top temperature variation


components with 6 hour-2 year periods (left) and morning-evening rainfall difference (right top) [16],
and a mechanism of diurnmal cycle over IMC [19].

and transequatorial boreal winter monsoon (cold surge) [21][22][23]. In El


Niño years, the cooler sea-surface temperature suppresses the morning coastal-sea
rainfall, and often induces serious smog over IMC.

II. Observation Strategy


Based on climatological features mentioned in the previous section, high-resolution
observations/models covering both over islands and seas are necessary. During
FY2005-09, observation networks have been installed both over the land and
ocean of IMC during these two decades under collaborations by Japan Agency
for Marine-Earth Science and Technology (JAMSTEC) and Kyoto University
in the Japanese side and the Agency for the Assessment and Application of
Technology (BPPT), the Indonesian Institute for Space and Aeronautics (LAPAN)
and the Meteorological, Climatological and Geophysical Agency (BMKG) in
the Indonesian side. On land of IMC, based on several foregoing projects, a
radar-profiler network observation project called Hydrometeorological Array
256 Proceeding ASEAN COSAT 2014

for Interaseasonal Variation-Monsoon Automonitoring (HARIMAU) have been


carried out started [24]. Over the Indonesian exclusive economic zone (EEZ)
surrounding IMC, collaborations of installation and maintenance of buoys, as
well as research vessel observations, have been continued as a part of the global
network of Tropical Ocean Climate Study (TOCS) [25]. (see Fig. 3)
During FY 2009-2013, another 5 year international (Japan-Indonesia)
project entitled “Climate Variability Study and Societal Application through
Indonesian-Japan Maritime Continent COE: Radar-Buoy Network Optimization
for Rainfall Prediction (SATREPS-MCCOE project) has been carried out as
a Science and Technology Research Partnership for Sustainable Development
(SATREPS), supported financially by Japan Science and Technology Agency (JST)
and Japan International Cooperation Agency (JICA). The project is carried out
by JAMSTEC, under collaborations with Kyoto University and Kobe University
in Japanese side and with BPPT, BMKG and LAPAN in Indonesian side.
In order to keep observations of this important region permanently, it must
be promoted independently by Indonesia which becomes a G20 country now.
For this purpose the SATREPS-MCCOE project has promoted establishment
of an international research center (MCCOE) by the Indonesian Government
(Output 1). The MCCOE has three functions: observations by radars (Output 2)
and buoys (Output 3), data management (Output 4), and researches on regional

Figure 3. The Maritime Continent Center of Excellence (MCCOE) established under a


SATREPS project, which is operating the HARIMAU radar-profiler network [24] and an
InaTRITON station of the TOCS buoy network [25].
Innovation for Better ASEAN Community 257

Figure 4. An example of observation networks proposed for the Year of Maritime Continent
(YMC) [26]. .

rainfall (Output 5) and global climate (Output 6). A building was constructed
at the National Research and Technology District (PUSPIPTEK) in Serpong (20
km southwest from the central Jakarta) completed by the Indonesian Government
and society under organization between BPPT, BMKG and LAPAN, as well as
researchers in universities and other agencies. The MCCOE has been opened in
November 2013 and a homepage has been constructed at BPPT: http://neonet.
bppt.go.id/satreps/

III. Conclusion-Future Scopes


As being promoted at MCCOE, the climatological observations in the next
step should be independent and more completed by each country under more
international (not bilateral) framework. A typical example is the project called
“Year of Maritime Continent”, which is now being proposed for 2017–2008 [26]
(see Figure 4). Through such activities in the next step, science and technology
specialized for equatorial/tropical climate with conditions/characteristics different
from extratropics (such as electronic parts invulnerable for very humid/hot envi-
ronment, description on non-annual periodicities, and so on) must be developed/
established by Indonesia and surrounding ASEAN countries. Completing them
and covering the whole tropical/equatorial region with a half of earth surface
will lead to a breakthrough for understanding/perdiction of the global climate.

IV. Acknowledgment
The author acknowledges many colleagues of Japan and Indonesia for collabora-
tions described here. In particular Dr. Fadli Syamsudin of BPPT has provided
comments from Indonesian side and Dr. Kunio Yoneyama of JAMSTEC has
provided information on YMC. The author’s participation at this meeting was
supported by JST.
258 Proceeding ASEAN COSAT 2014

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21) Hattori, M., Mori, S., and Matsumoto, J. (2011). The cross-equatorial
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Simultaneous Correlation Analysis
of Australian Summer Monsoon Index (AUSMI)
against Rainfall in Bali Region
Subekti Mujiasiha)* and I Gede Agus Purbawab)
a
Balai Besar MKG Wilayah III Denpasar
Jl. Raya Tuban, Kuta 80362, Kabupaten Badung, Bali,Indonesia,
b
Banyuwangi Meteorogical Station
Jl.Jaksa Agung Suprapto No.152, East Java, Indonesia

Abstract
Rainfall is one of the most important parameters of weather and climate which needs to be studied
in depth because it has a large degree of variability. Its pattern and characteristics in Bali region
are influenced by the Asian and Australian Monsoon. Furthermore, monsoon is considered as
the most significant phenomena in affecting rainfall in Indonesia region. From previous studies,
Australian Monsoon Index (AUSMI), one indicator of the Asian-Australian monsoon activity,
can capture rainfall variability in Australia and the Indonesia maritime continent. In this study
we analyzed the relationship between AUSMI and rainfall in Bali region using simultaneous
correlation method. The used data was monthly averaged rainfall from 36 rainfall post station
during 30 years since 1979–2008. With expectations, we can know how strong influence of
AUSMI against rainfall, so that we can use AUSMI as one of predictors to predict rainfall. The
result shows that the AUSMI greatly affects rainfall in this region. The degree of influence varies
in range 0.5–0.8 of correlation, coefficient, spatially and temporally.
Key words : AUSMI, Simultaneous correlation, Predictor, Bali

I. Introduction
Indonesia is a maritime region. This is indicated by the composition of its territory
which consists of a vast ocean with interspersed islands large and small and also
the position is right around the equator. That type of climate is generally known
as the continental maritime climate. Continental maritime climate which is
owned by Indonesia actually has a comparative advantage than other country,
including the year-round warm climate, rainfall variability between regions
and rich in solar radiation with irradiation time range from 3–10.5 hours and
solar radiation intensity 235–535 cal/cm2/day [1]. Variability of rainfall as one
characteristic of the continental maritime climate, can also be used to determine
the time of the rainy season and dry season, apart from the wind circulation
pattern [2]. Climatology Meteorology and Geophysics Agency (BMKG) defines

* Corresponding Author.Tel: +62-85313131831. E-mail: subekti.mujiasih@bmkg.go.id.

261
262 Proceeding ASEAN COSAT 2014

the average monthly rainfall of 150 mm/month for wet season and the average
monthly rainfall less than 150 mm/ month for dry season [3]. Based on rainfall
pattern in Indonesia which has three types such as monsoon, equatorial and local
types, then Bali has a monsoon rain pattern [4]. Furthermore, by using double
correlation method, rainfall pattern in Bali is included in monsoon region [5].
According to BMKG, Bali is included in the category of regions receiving the
highest rainfall over 3,000 mm/year [6]. Based on research on average rainfall
Indonesia for 32 years (1961–1993), the rainfall pattern in Bali increased in
period of November to March and decreased in April to October [5]. Generally,
rainfall in wet season in Bali depends on atmospheric phenomena activity both
globally and regionally.
Global scale factor includes the occurrence of ENSO (La Nina or El Nino),
Dipole Mode, and the Madden Julian Oscillation (MJO). Regional scale factor
includes monsoon activity, including Asian Cold Surge, Tropical Disturbance
such as a vortex or a tropical storm, the ITCZ ​​and so on. Whereas, local factors
include Orographic shape, land-sea breeze, mountain-valley winds, convectivity
and atmospheric stability [7].
The influence of regional scale atmospheric happens when cold surge activity
from Asia becomes the cross-equatorial flow in Indonesia region in south of the
equator, or when MJO is active in Indonesian territory or when tropical cyclones
are formed around the western and northern Australia. These phenomenons
cause rain duration tends to occur in a long and sustained period in Bali. Even
in extreme case, rain events can get all day with moderate to heavy intensity and
strong wind. However, when the global and regional phenomenon are decreasing,
rain events tend to be dominated by local factors. One of local factors causing
Bali has different climate of each region, although composed of the island and
surrounded by the sea, is orographic of each region. It means that climate pat-
terns in the highlands tend to be different from the area near the coast. In these
circumstances, it usually occurs in the intensity of light rain and sunny to cloudy
weather conditions during the day in most of the region. Rain around the coastal
areas generally occurs at night until in the morning. Whereas, in mountainous
areas or terrain, rainfall occurs in the late morning or in the afternoon.
In relation to the pattern of monsoon rainfall, rainfall variability in Australia
and the Indonesian Maritime Continent, can be detected through AUSMI.
AUSMI is dynamic monsoon index, which is based on 850 hPa zonal wind
averaged over the area (5°S–15°S, 110°E–130°E) [8]. According to AUSMI Data,
1948–2009 shows the highest monsoon index happened in peak of wet season
month (DJF), whereas the lowest index happened in peak of dry season month
(JJA)[9]. AUSMI research for the Indonesian region has already been done using
TRMM [10] especially in Tabing, West Sumatra [11]. Related to the various
Innovation for Better ASEAN Community 263

climate patterns over Bali, high rainfall and AUSMI ability to recognize rainfall
variability, the authors intend to explore the relationship between AUSMI and
rainfall in Bali by using correlation method. This method is used to determine the
relationship between two or more variables and magnitude of the relationship [12]
.This method has also been used to examine the influence of ENSO on Pacific
Basin precipitation [13]. Hopefully, the results of this study can be considered in
predicting rain in Bali and surroundings by using AUSMI as one of the predictors.
Furthermore, determination of wet and dry seasons preliminary might be more
accurate by considering AUSMI as indicator of monsoon.

II. Data and Methodology


A. Rainfall Dataset
The data used is monthly rainfall observation rate of 36 rain gauge stations. Data
periods are during 30 years for 1979–2008. Then, data was preprocessed by table
and categorized by month and year.

B. Australian Summer Monsoon Indices (AUSMI) Dataset


Broad-scale Australian monsoon index (AUSMI) describing multi-time scale
variations is defined by using 850 hPa zonal wind averaged over the area (5 °S–15
°S, 110 °E–130 °E). This circulation index reflects monsoonal rainfall variability
over Northern Australia and maritime continent. The index can be used to
depict the seasonal cycle (for instance the onset) and measure the intraseasonal,
interannual and interdecadal variations of the Australian monsoon [8]. AUSMI
data set is monthly rate during 30 years for 1948–2008. AUSMI dataset can be
downloaded freely from Asia Pacific Data Research Center website [14].
AUSM Index = U850(110-130E,15S-5S) (1)[15]

Figure 1 AUSMI coverage area [8]


264 Proceeding ASEAN COSAT 2014

From two dataset above, we obtained monthly rainfall rate and monthly
AUSMI rate. Next step, these data were processed using correlation method.
Where, R (x,y) is correlation coefficient between X (AUSMI) and Y (Rainfall). x is
monthly AUSMI rate and y is monthly rainfall rate (see Equation 2). The criteria
for the correlation coefficient is as follows: if the price of r (x,y) approaches +1,
it means the relationship between the two variables is stronger and proportional
nature. If the price of r (x,y) close to -1, it means that the relationship between
the two variabel is stronger and inversely nature. If the price of r (x, y) ≥ +0.5 or
≤ -0.5, it means the relationship between the two variables is strong enough. If
the price of r (x, y) ≤ ≥ +0.5 or -0.5, it means the relationship between the two
variables is weak. Finally, the calculated correlation of data series was mapped
both series (averaged 30 years) and monthly.

III. R esult and Discussion


In this research, authors give 3 results. They are Series Correlation Map (figure
2) Monthly Correlation Map and Linear Regression for Tejakula and Ngurah
Rai. We displayed monthly correlation map such as March (figure 3) and July
(figure 4). Author compared the two types against observation monthly rainfall
in 2012 (figure 5) and 2013 (figure 6) and Ausmi Analysis 2012–2013 (figure 7).

Figure 2. Series Correlation Map


Innovation for Better ASEAN Community 265

Figure 3. March correlation map.

Figure 4. July Correlation map.


266 Proceeding ASEAN COSAT 2014

Figure 5. Observation rainfall Pos Station in Bali Region - 2012

Figure 6. Observation rainfall Pos Station in Bali Region - 2013


Innovation for Better ASEAN Community 267

Figure 7. Ausmi Analysis August of 2012 - 2013

A. Series Correlation Map


Series Correlation map (figure 2) shows that all Bali regions have correlation
coefficient above 0.5. It means influence of monsoon is strong enough and linear
with rainfall. It causes rainfall increasing in December, January, February (DJF)
and decreasing in June, July and Auguts (JJA).
Generally, this pattern happened in Bali region for observation rainfall in
2012 (figure 5) and 2013 (figure 6). This is similar with pattern of AUSMI
Analysis of 2012–2013 (figure 7). The AUSMI pattern shows highest monsoon
index happened in months of peak of wet season (DJF), whereas lowest index
happened in months of peak of dry season (JJA)[9]. And also, based on research
on average rainfall Indonesia for 32 years (1961–1993), the pattern of rainfall
in Bali increased in the period in November–March and decreased in April to
October [5].

B. Linear Regression
Beside correlation, this research also has resulted series of regression equation for
each region. In this report, we displayed Ngurah Rai and Tejakula Raingauge
Pos Station. These stations have a positive linear relation with AUSMI. Linear
regression equation for Ngurah Rai is Rainfall is equal to 30.11*AUSMI+223.53
268 Proceeding ASEAN COSAT 2014

with Determination coefficient 0.57. For Tejakula, Rainfal is equal to


28.3*AUSMI+179.42, with Determination coefficient 0.62.

C. Monthly Correlation Map


Beside Series Correlation Map, we also created monthly correlation map from
series data. In this research we displayed only two maps of March and July since
they have anomaly. The first anomaly is March in 2012. First, March Monthly
correlation map (figure 3) shows Karang Asem has strong enough correlation
(0.5–0.7). It means these areas may be affected by AUSMI. It means the area
should have decreased monthly observation rainfall in March as AUSMI pattern.
But, Observation in Karang (figure 8) does not show it. It means, March correla-
tion map is not suitable for Karang Asem.

Figure 8. Karang Asem - March Anomaly 2012

Second, March monthly correlation map (figure 3) shows Badung has strong
enough correlation (0.5-0.7). It means these areas may be affected by AUSMI.
It means the area should have decreased monthly observation rainfall in March
as AUSMI pattern. However, observation in Badung (figure 9) does not show it.
It means, March correlation map is not suitable for Badung.
Third, In other hand, it shows different result for Buleleng. It has weak
correlation (0–0.3) in figure 3. It means Buleleng may not be affected by AUSMI.
It means the area should have increased monthly observation rainfall in March as
Innovation for Better ASEAN Community 269

Figure 9. Badung - March Anomaly 2012

Figure 10. Buleleng - March Anomaly 2012

AUSMI pattern. Buleleng observation result (figure 10) shows March is higher
than February. It means March correlation map is suitable for Buleleng.
Fourth, as the literature, The AUSMI pattern shows highest monsoon index
happened in months of peak wet season (DJF), whereas lowest index happened
270 Proceeding ASEAN COSAT 2014

Figure 11. Badung - July Anomaly 2013

in months of peak of dry season (JJA)[9] and AUSMI 2012-2013 (figure 7).
However, for monthly observation rainfall in 2012, March is peak of wet season.
The rainfall trend of DJF and MAM period should be decreasing in March.
However, it is not decreasing in March. The March anomaly happened at 18
locations (81%) from 22 locations of observation in 2012. These are some
examples, such as Karang Asem (Selat Duda), Badung (Ngurah Rai), Buleleng
(Gerokgak, Sukasada, Tangguwisia). The areas got March monthly rainfall higher
than February. This is not similar with AUSMI pattern that March should have
lower than February.
The Other anomaly is July monthly rainfall in 2013. First, Badung has weak
correlation (0–0.3) in figure 4. It means the area may not be affected by AUSMI.
It means the area should have increased monthly observation rainfall in July.
Badung observation result (Figure 11) shows July is higher than June. It means
July correlation map is suitable for Badung.
Second, other area also gets July anomaly 2013 is Jembrana. It has weak
correlation (0–0.3) in figure 4. It means the area may not be affected by AUSMI.
It means the area should have increased monthly observation rainfall in July.
Jembrana observation result (Figure 12) shows July is higher than June. It means
July correlation map is suitable for Jembrana.
Third, other area of anomaly July monthly rainfall in 2013 is Tabanan. It has
weak correlation (0–0.3) in figure 4. It means the area may not be affected by
AUSMI. It means the area should have increased monthly observation rainfall
Innovation for Better ASEAN Community 271

Figure 12. Jembrana – July Anomaly 2013

Figure 13. Tabanan – July Anomaly 2013


272 Proceeding ASEAN COSAT 2014

in July. Tabanan observation result (figure 13) shows July is higher than June. It
means July correlation map is suitable for Tabanan.
Fourth, as literature, July should have lowest monthly rainfall. But, the
observation in July shows different result. There is an increasing monthly rainfall
in July. The July anomaly happened in 32 locations (70%) from 41 locations
of observation in 2013. These are some examples such as Badung, Jembrana
(Nursasari), Bangli, and Gianyar (Kemenuh).
From the above explanation, there are some different result in some areas
between monthly correlation map, observation and AUSMI pattern. It may
be influenced not only regional factor like monsoon but also orographic factor
(local factor).

IV. Conclusion
Australian Summer Monsun (AUSMI) affecting rainfall in Bali, has various
influences in spatially and temporally with strong positive correlation (r > 0.5).
There is some different result in some areas between monthly correlation map
and observation. It may be influenced not only regional factor like monsoon but
also orographic factor (local factor). AUSMI can be used as one of predictor
for predicting rainfall and season in Bali. It is necessary long series data for each
season to get more accurate influences of Monsoon to rainfall. In the future, it is
better if research involving other factors for rainfall predicting such as sea surface
temperature in Bali.

V. R eferences
1) Syahbuddin, H. (2005). Jangan Lupa Swasembada Pangan. Majalah Inovasi
Online, 4, XII.
2) Ramage C. (1971). Monsoon Meteorology. International Geophysics Series, 15.
San Diego, CA: Academic Press.
3) BMG.(2000). Guidance of Season prediction and Analysis.
4) Tjasyono, B. (2004). Klimatologi. Penerbit ITB.
5) Aldrian, E. and Susanto, RD. (2003). Identification of three dominant
rainfall regions within Indonesia and their relationship to sea surface
temperature. International Journal of Climatology, 23, 1435–1452.
6) BMG. (2006). Peningkatan Pemahaman Informasi Iklim. BMG. Jakarta.
7) Pusdiklat BMKG. (2012). Bahan Ajar Diklat Teknis Analisa Cuaca permukaan.
8) Kajikawa, Wang, Y. B., and J. Yang. (2010). A Multi-time scale Australian
Monsoon Index. International Journal of Climatology.
Innovation for Better ASEAN Community 273

9) Link http://apdrc.soest.hawaii.edu/projects/monsoon/ausmidx/index.html.
Accessed on 01 January 2014
10) Nuryanto, DE. 2012. Relationship between Indo-Australia Monsoon with
Seasonal rainfall Variability Indonesia Maritime Component Spatially based
on analysis Result of TRMM Satellite Data. Journal of Meteorology and
Geophysics, 13, 2, 91-102
11) Link https://www.academia.edu/7473108/Full_Paper. Accessed on 10
August 2014
12) Hernowo, B. 1997. Metode Korelasi Akademi Meteorologi dan Geofisika.
Jakarta
13) Link http://www.cpc.noaa.gov/pacdir/cont_chp6.html. Accessed on 01
January 2014
14) Link http://iprc.soest.hawaii.edu/users/ykaji/monsoon/seasonal-monidx.
html. Accessed on 01 January 2014
15) Link http://iprc.soest.hawaii.edu/users/ykaji/monsoon/definition.
html#ausm. Accessed on 01 January 2014
Developing Strategy for Monitoring and
Decision Support System for Smoke Haze
Trans-boundary Problem within ASEAN Region

Sheila Dewi Ayu Kusumaningtyas*


*
Agency for Meteorology, Climatology, and Geophysics (BMKG)
Jl. Angkasa I, No. 2, Kemayoran, Jakarta, Indonesia

Abstract
Land clearing for plantation and the subsequent simple approaches of biomass burning which
occur in many provinces in Indonesia, especially in Riau province increases during the past decades
and reduces the quality of air in the region. The associated trans-boundary haze pollution issue
has been a political debate and creates tensions among neighboring countries. When biomass
burns, certain aerosol pollutant is emitted to the atmosphere. The present study aims to describe a
concerted effort in monitoring the challenge of smoke haze distributions in the region to improve
early warning and minimizing the unprecedented impact on economy, health and environment.
The use of satellite remote sensing and in-situ measurement from Aerosol Robotic Network
(AERONET) Program in determining trans-boundary haze pollution in South East Asia will be
shown. Other political and operational field instruments to support the mechanism to put out
fires are applied in the region. The haze pollution that is detected from observation of aerosol
loading in Singapore during dry period in June 2013 is used as case study. Necessary data of
air quality and remote sensing are analyzed to provide comprehensive picture of the problem.
Hysplit Trajectory Model has also been applied to study the dispersion of smoke haze pollution.
According to the remote sensing satellite, smoke haze originate from Riau during fire period of
June 2013 had affected neighboring countries such as Singapore, Malaysia, and floated up to the
southwestern edge of the Philippines. This study suggests a further extension of such a project
in order to monitor further the disturbing low quality of air due to smoke fire. Ultimately, a
decision support system within the ASEAN countries is needed to have data exchange and join
analyses in detecting, monitoring and minimizing the risk of the problem.
Key words: Vegetation fires, Trans-boundary haze pollution, AERONET, Hysplit Model

I. Introduction
Over the last 20 years, fires have risen not only global attention but also Indonesia
since causing serious damage on environment and economy especially following
the 1997/1998 El Nino Southern Oscillation (ENSO) event which devastated
up to 25 million hectares of land worldwide. Fires are considered a potential
threat to sustainable development because of their direct effect to ecosystem, their
contribution to carbon emission and their impact to biodiversity [1].
* Corresponding Author.Tel: +6281381430023.E-mail: sheila_bmg@yahoo.com

275
276 Proceeding ASEAN COSAT 2014

Vegetation fires are common phenomena during dry periods in Indonesia,


especially in Riau. However, it is getting wors in line with the increasing of rapid
deforestation and land clearing. According to Anderson and Bowen [2], over the
last 5 years some 30% of the fires detected in Sumatra have been in Riau province.
Riau in all probability routinely has the greatest frequency of vegetation fires of
any province in Indonesia in terms of number of fires per square kilometers. The
province is quickly and rampantly logging its remaining forests. The dry land
forests are now nearly exhausted and attention has already turned to the swamp
forests. Like the dry areas, the land is then converted to other purposes, mainly
oil palm. Conversion is a major and planned component of Riau’s development
strategy.
The use of fire as a tool to clear land for agriculture is a long established
practice in Indonesia. When this tool is used by farmers in years of average or
above average rainfall, the practice poses little threat to the environment. This
kind of tool is considered cheap, easy, and effective and has been done not only by
small-holder farmers but also estate crop companies to clear thousands of hectares,
in the main for oil palm, is a much more worrying problem [3]. Anderson and
Bowen [2], also wrote that there is a strong correlation between fire numbers /
area burnt and the land clearing activities of oil palm companies and Sumatra
has been hard-hit when compared to Kalimantan and elsewhere in Indonesia.
Agency for Meteorology, Climatology, and Geophysics of Indonesia (BMKG)
records a considerable number of fires occur in Riau almost every year and
increasing during some dry periods. In June 2013, through observation from
Terra and Aqua MODIS Satellite, BMKG recorded up to 3,400 hotspots occurred
in Riau Pro-vince [4]. A number of hotspot as an indicator of fires has given
negative contribution to the environment, social, economy, and health as well as
trans-boundary haze pollution. Great and massive fires which occurred in several
countries like Indonesia, Brunei Darussalam, Malaysia, Philippine, Singapore,
and Thailand in 1997–1998 have destroyed more than 9 million hectares of
land in which 6,6 hectares was biomass [1]. Periods of smoke haze pollution in
Indonesia, some of it spreading to neighboring countries now regular events and
are likely to become more severe in the immediate future. In Southeast Asia, the
concern with the impacts of fires is particularly significant, as exemplified by
the signing of the Agreement on Trans-boundary Haze Pollution by the country
members of the Association of Southeast Asian Nations (ASEAN) in June 2002
in Kuala Lumpur.
According to Kunii et al., [5], the south west monsoon wind contributes
to the occurrence of cross-equatorial transport of smoke haze originate from
Indonesia to neighboring countries causing regional pollution which contain
high concentration of aerosol. When biomass burns, certain pollutant, such as
Innovation for Better ASEAN Community 277

greenhouse gasses, hydrocarbon substances, and aerosol mixture contain black


carbon, organic compound, sulfate and nitrate, are emitted to the atmosphere.
According to Yokelsen et al., [6], in a large cloud of smoke caused by biomass
burning identified the presence of very high concentrations of PM10. This is
supported by research of Holben et.al., [7] which states that biomass burning in
the tropics produces high carbon aerosol pollution especially in the dry season
in August and September. A similar study by Artaxo et al., [8] which measures
the increase in the concentration of fine particulate matter (diameter <2.0 µm)
during dry season as a result of the biomass burning and allows the transport of
pollutants. The high concentration of aerosols can reduce visibility and increase
the risk of disruption of health.
Atmospheric aerosols absorb incoming solar radiation and scatter infrared
radiation emitted from the earth. The influence of atmospheric aerosols on solar
radiation is very dependent on the distribution size, shape, concentration, and
optical properties [9]. In this case, the aerosol optical thickness or commonly
referred to as aerosol optical depth (AOD) is one of the important parameters
to study the presence of aerosols in the atmosphere and its impact on air quality,
health and environment, as well as to monitor the vegetation fires.
Considering remarkable impact of vegetation fires to environment and to
overcome smoke haze pollution within ASEAN region, strategy for monitoring
and decision making system is required to build. The present study aims to describe
a concerted effort in monitoring the challenge of smoke haze distributions in
the region to improve early warning and minimizing the unprecedented impact
on economy, health and environment. The use of satellite remote sensing and
in-situ measurement from Aerosol Robotic Network (AERONET) Program in
determining trans-boundary haze pollution in South East Asia will be discussed.
The haze pollution that is detected from observation of aerosol loading in
Singapore during dry period in June 2013 is used as case study. Necessary data
of air quality and remote sensing are analyzed to provide comprehensive picture
of the problem. Moreover, this paper also discusses policy responses addressing
on trans-boundary haze pollution.

II. Method/M aterial


To study the smoke haze episode in Riau, analyses on the optical properties of
smoke haze with the aid of an AERONET Sun Photometer was conducted. The
AERONET program is a federation of ground-based remote sensing aerosol
networks established by NASA. To study events such as the June 2013 trans-
boundary smoke haze episode, the atmospheric radiation data used in this paper
have been taken from Singapore site. The AERONET Singapore site is located
278 Proceeding ASEAN COSAT 2014

at the National University of Singapore (1.30° N, 103.77° E, 79 m above mean


sea level) [10]. This atmospheric site was established as part of the cooperative
framework of the Seven South East Asian Studies (7-SEAS) mission created in
2007 [11]. The purpose of this frame-work is to engage the participating countries
on a regional multi-years campaign set to study the regional aerosol, cloud and
radiation environment [12]. Since this site is situated off the southern part of the
Malay Peninsula and north of the Indonesian Archipelago, it is ideally positioned
for monitoring regional pollution events, such as trans-boundary biomass burning
emissions, clouds, local anthropogenic emissions and climate variability [10].
Sun photometer instrument CIMEL Electro-nique CE-318A measures the
intensity of solar radiation at the wavelengths 340, 380, 440, 500, 675, 870, 1,020,
and 1,640 nm and then the results are used to calculate the value of AOD. To
analyse impact of regional smoke haze pollution, necessary data of air quality and
visibility parameter in Singapore are needed. Concentration of PM2,5 is taken
from National Environment Agency of Singapore, while visibility data is obtained
from Meteorological Service Singapore. Hotspot parameter as an indication of
biomass burning was derived from Terra satellite and Aqua MODIS originate
from NASA and were collected by BMKG. While Hysplit Trajectory Model has
been applied to study the dispersion of smoke haze pollution.

III. R esult and Discussion


Period of this study is focused in June 2013 in Riau Province, since it was the
month with the highest number of hotspots throughout the year and was suspected
causing regional air pollution to neighboring countries.

3.1 Fire Activity


Active fire (i.e. hotspot) detection from remote sensing satellites is based on
the detection of the thermal infrared radiation emitted by fires. This method
is considered as the most suitable and effective way to detect spatio temporal
distribution of fire activity given the large spatial coverage of remote sensing
satellites. In this work, hotspots detection by Terra satellite and Aqua MODIS
originated from NASA and were taken from BMKG’s database. Figure 1 shows
hotspot detected during June 2013. As can be seen from that, detected hotspots
were mostly concentrated at the province of Riau. Based on data collection, there
were a total of about 3,487 hotspots observed from satellites Aqua and Terra
MODIS with most events on June 19, 2013 with the number of fires reaching
1393 locations.
The concurrent plot of hotspot together with the AOD (Figure. 2) shows that
the increase in number of fires followed by a rise in the value of AOD (showed by
Innovation for Better ASEAN Community 279

Figure 1. June 19th, 2013: Hotspots distribution map over Sumatera observed
by Terra and Aqua MODIS Satellite as collected by BMKG. The color dots
represent the level of confidence.

trend line). As stated by Artaxo, et al., [8] that an increase in the concentration of
fine particulate matter (diameter <2.0 m) during the dry season as a result of the
fire biomass. However, there is time lag in which the AOD has the highest value
on 26 and 30 of June between the peaks generated by hotspot source emission
(mostly on June 19th, 21st, 23rd, and 24th). So it appears that there is an increasing
level of AOD corresponding to decreasing number of fire hotspot counts on the
end of June. This may be explained as follows: firstly, the measurement of AOD
is taken from Singapore site where it is located 300–400 km from Riau. Aerosol
takes relatively short time to be transported to Singapore. Secondly, according to
280 Proceeding ASEAN COSAT 2014

Salinas [10], unlike surface flaming combustion and peat fires which are considered
to be the main source of fuel for this event, hotspots tend to be persistent and can
smolder underground long after the surface flames have subsided. Furthermore,
smoke can still be released into the atmosphere even though no direct hotspot
can be observed or attributed to it. Thirdly, even if the amount of fire hotspot
counts has truly decreased, the observed AOD peak on day 30rd, might be the
result of the cumulative effects of stagnant (aged smoke) aerosol generated earlier
(days 19th and 21st respectively) with fresh smoke contributions from day 22nd
onwards. In other tropical biomass burning regions (e.g. Amazon, Africa) burning
takes place from various sources and is so intense that fresh and aged smoke are
often found on a combined state [13].
A qualitative way of evaluating the regional trans-boundary smoke transport
patterns is to perform trajectory of smoke haze dispersion using Hysplit Model.
One of BMKG policy that has been operationalized in terms of handling forest
fire smog is doing trajectory modeling dispersion smog. Results of running the
three-dimensional trajectory modeling with Hypslit model showed that the
distribution of fire haze in Riau in June 2013 led to the East and Northeast regions
towards Singapore which is shown on Figure 3. Besides applying Hysplit Trajectory
Model, to strengthen the occurence of regional smoke haze, observation using
satellite is ex-tremely important (Figure 4). As can be seen from the Figure 4 that
AOD observation using MODIS Satellite shows that aerosol emission as a result
of smoke haze from Riau moved to Singapore and Malaysia. Furthermore, on days
23rd June, smoke haze with high level of AOD floated up to the south western
edge of the Philippines. According to Kunii et al., [5] and Heil and Goldammer
[14], the low-level, southern monsoon wind circulation which common during the
main burning season (dry season) produces northward, cross-equatorial transport
of fire emissions from Indonesia, particularly towards Singapore and Malaysia.

3.2 Properties of Aerosol and Air Quality Condition in Singapore


AOD parameter retrieves from Sun-Photometer observation is useful to study
the properties of aerosol. Furthermore, from its derivative, the source of aerosol
could be traced.
As mentioned earlier, the presence of aerosol will impact the air quality
condition. To study impact on ambient air quality in Singapore as a result of
regional smoke haze pollution, concentration of PM2.5 and visibility were also
assessed. The reduction of visibility was widely used as an indicator for ambient
air quality during the smoke-haze episode.
Innovation for Better ASEAN Community 281

1600 30 Jun 2.5


1400
26 Jun 2.0
1200

AOD (500 nm)


1000 1.5
Hotspot

800
600 1.0

400
0.5
200
0 0.0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

Hotspot AOD (500 nm) Linear (AOD (500 nm))

Figure 2. Hotspot counts (vertical bar) in Riau Province plotted together with AOD (line) taken
from Sun-Photometer measurement at Singapore site for June 2013.

Figure 3. Trajectory smoke haze dispersion by Hysplit Model. Left side is trajectory apply on 19 June, while
the right side is trajectory during whole June 2013.

Correlation among visibility, AOD, and PM 2.5 concentration is given


on Table 1. Aerosol optical depth (AOD) on lower wavelength (340 nm) has
negative and higher correlation with visibility compared than higher wavelength.
The increasing of AOD is followed by decreasing of visibility. Higher correlation
between AOD and visibility is found on 340 nm in which small particles of aerosol
is sensitively detected on lower wavelength. According to Ward [15] and Novakov
et al., [16], vegetation fires produce particle in the fine size range (PM2.5). To
support the finding, correlation between visibility and PM 2.5 are also calculated
and showing negative and high correlation in three locations. Mean concentration
of PM2.5 during June 2013 was 64 µm/m3 with highest concentration on days of
21st. Pollutant Standard Index (PSI) on this day was recorded at 239 point which
remain very unhealthy as an impact of fires occurred in Riau.
282 Proceeding ASEAN COSAT 2014

Figure 4. Observation of AOD using Aqua MODIS Satellite from 17 June to 25 June 2013.
Colours represent level of AOD.
Tabel 1. Correlation among visibility, AOD parameter, and PM2.5 concentration during
June 2013. Visibility data were measured from three different locations in Singapore. Aerosol
Optical Depth (AOD) in many different wavelength is retrieved from Sun-Photometer located
at National University of Singapore. Average PM2,5 concentration was resulted from measure-
ment in five locations all over Singapore.
AOD (nm) PM 2.5
Visibility
1640 1020 870 675 500 440 380 340 (µm/m3)

Changi 0.06 -0.06 -0.12 -0.23 -0.37 -0.42 -0.45 -0.47 -0.78
Paya lebar 0.04 -0.07 -0.13 -0.25 -0.40 -0.45 -0.48 -0.51 -0.78
Seletar 0.03 -0.09 -0.15 -0.26 -0.41 -0.45 -0.49 -0.51 -0.74

3.3 Policy Responses on Handling and Managing Fires


In order to tackle the recent hotspots and haze occurrence, coordination of various
agencies in Indonesia is needed. This section will discuss the policy responses of
Indonesian agencies particularly BMKG and many efforts which has been taken
to minimize impacts of smoke haze both at the national level and ASEAN region.
At national level, BMKG has important roles in providing services on weather,
climate, and air quality information to support sustainable development. In the
area of smoke haze, BMKG develops and integrates systems to predict fires and
Innovation for Better ASEAN Community 283

to track them once they start. Related to that, several policies implemented in
BMKG among others are (Figure 5):
1) Fire Danger Rating System (FDRS) developed by JICA, Canada, Ministry
of Forestry and the National Disaster Management Agency (BNPB). BMKG
disseminates FDRS based on real time and 7 days prediction to several agencies
i.e Ministry of Forestry, BNPB, as well as to ASEAN. This information and
then used by related agencies to take action on fire prevention.
2) Climate early warning such as drought early warning system, which is online
and automatic. It is accessible using a smartphone app.
3) Information on extreme weather such as dry spell, wet spell and tropical
cyclone.
4) Smoke haze trajectory dispersion using Hysplit Model.
5) Hotspot detection using Aqua and Terra MODIS satellite.
6) AEROSOL program collaboration with NASA to study aerosol physical
and optical properties. Three observation sites in Jambi, Pontianak, and
Palangkaraya have been established.
7) CATCOS Program (Capacity Building and Twinning for Climate Observing
Systems) for measurement of aerosols in the Global Atmospheric Monitoring
Station Kototabang Hill, West Sumatra.
8) Greenhouse Gas Monitoring Network in 15 regions.
9) Monitoring of PM 10 with Beta Attenuation Monitoring (BAM) in eight
provincial capi-tals of vulnerable forest fires (Pekanbaru, Palangkaraya
Pontianak, Jambi, Palembang, Medan, Balikpapan and Banjarmasin).
At regional level, haze pollution is one of the major and ongoing problems in
ASEAN region and becoming a political issue. All matters related to smoke haze
issue is coordinated by ASEAN through the regional and national haze action
plans and related technical assistance programs in which Indonesian government is
intensively involved. Several instruments have been developed to address the issue,
such as the ASEAN Agreement on Trans-boundary Haze Pollution (AATHP)
which emphasizes on co-operation in its approach. According to Nurhidayah [17],
arguments that emerge regarding why Indonesia has not ratified yet the agreement
because the effectiveness of a treaty or agreement required the participation and
compliance of “targeted state.” Some argue that even if Indonesia ratifies this
Agreement this would not solve haze pollution problem. This is due to complex
problem management of natural resources and environment governance in
Indonesia. In fact, to be effective the Agreement requires capacity at national,
provincial, muni-cipal and village levels to implement the Agreement.
284 Proceeding ASEAN COSAT 2014

Fire Danger Rating


System (FDRS)

Climate Early
Warning

BMKG
Policies Information on
Extreme Weather

Smoke Haze
Trajectory
Dispersion
Smoke haze &
Hotspot Detection
forest fire

AERONET
Program

CATCOS Program

Green House
gasses Monitoring

Monitoring of
PM10

Figure 5. BMKG Policies on smoke haze and forest fire

However, despite of all political issue, BMKG as a science basis institution


with its role in providing services and information on weather, climate and air
quality has obligation to monitor fires and to analyze it in order to minimize
impact. These could be done through observations, advance technology, and join
analyses in detecting, monitoring and minimizing the risk of the problem with
national and international party in particular ASEAN member countries. Such
monitoring and observation as elaborated in this paper could be an input and
support for decision maker to take further action.

IV. Conclusion
As shown in this paper that during June 2013 biomass burning that occured
in Riau Province caused the trans-boundary haze pollution over neighboring
countries. Measured parameters such as Sun-photometer AOD, in-situ PM 2.5
Innovation for Better ASEAN Community 285

particulate concentration, and visibility in Singapore were consistent with the


presence of trans-boundary biomass burning smoke. Observation through satellite
also justified the ground based on observation which showed that high value of
AOD increased from the early of June to the end of month and covered Singapore,
Malaysia, even Philippines. Good correlation also was showed between visibility
and AOD measurement in 340 nm as well as with PM 2.5 concentrations. These
conclude the source of fine aerosol come from vegetation fires.
To prevent fire such as development and integration systems and to predict
and monitor fires, several policies have been taken. Further research on fire
dynamics study and air pollution related to smoke haze is needed in order to
minimize the impact.

V. Acknowledgment
Sincere thanks are expressed to those agencies and persons who supported the
author’s work in providing data and valuable inputs, in particular to Prof. Dr.
Edvin Aldrian for the guidance and remarkable support; colleagues from BMKG
named Mizani Ahmad and Gian Gardian; AERONET Program for processing and
archiving the Sun Photometer data; and National Environment Agency Singapore.

VI. R eferences
1) L. Tacconi. (2003). CIFOR Occasional Paper No. 38, Bogor, Indonesia:
Center for International Research. Fires in Indonesia: Causes, Costs and Policy
Implications.
2) I.P. Anderson and M.R. Bowen. ( 2000). Fire Zones and the Threat to the
Wetlands of Sumatra, Indonesia. Forest Fire Prevention and Control Project.
European Union and Ministry of Forestry. [Online]. Available: http://
dephut.net/Halaman/Perlindungan%20Dan%20Konservasi/FFPCP/PDF/
Firezone_and_the_threat_to_the_wetlands_of_Sumatra.PDF
3) J. Miettinen, and S.C., Liew. (2009). Burn-scar patterns and their effect
on regional burnt-area mapping in insular South-east Asia. Int. J. Wildland
Fire, 18, 837–849.
4) Link http://satelit.bmkg.go.id/satelit/image/HOTSPOT/2013/06/).
5) O. Kunii, S. Kanagawa, I. Yajima, Y, Hisamitsu, S. Yamamura, T. Amagai,
and I.T. Ismail. (2002). The 1997 Haze Disaster in Indonesia: Its Air Quality
and Health Effects. Archives of Environment Health, 57, 16–22.
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6) R. J. Yokelson, T. Karl, P. Artaxo, D. R. Blake, T. J. Christian, D. W. T.


Griffith, A. Guenther, and W. M. Hao. (2007). The Tropical Forest and
Fire Emissions Experiment: Overview and Airborne Fire Emission Factor
Measurements. Atmospheric Chemistry Physics, 7, 5175–5196.
7) B. N. Holben, D. Tanr, A. Smirnov, T. F. Eck, I. Slutsker, N. Abuhassan,
W. W. Newcomb, J. S. Schafer, B. Chatenet, F. Lavenu, Y. J. Kaufman, J.
Vande Castle, A. Setzer, B. Markham, D. Clark, R. Frouin, R. Halthore,
A. Karneli, N. T. O’Neill, C. Pietras, R. T. Pinker, K. Voss, and G. Zibordi.
(June, 2001). Journal of Geophysical Research. [Online]. 106 (D11), 12067-
12097. Available: http://onlinelibrary.wiley.com/doi/10.1029/2001JD900014/
pdf
8) P. Artaxo, F. Gerab, M.A. Yamasoe, and J. V. Martins. (1994). Fine Mode
Aerosol Composition at Three Long-term Atmos-pheric Monitoring Sites
in the Amazon Basin. Journal of Geophysical Research: Atmospheres, 99,
22857-22868.
9) T. Budiwati, Sumaryanti, and I. Sofiati. (2001). Karakteristik Optik Aerosol
di Bandung. Kontribusi Fisika Indonesia,12 No. 4. Okt.
10) S.V. Salinas, B.B. Chew, J. Miettinen, J.R. Campbell, E.J. Welton, J.S. Reid,
L.E. Yu, and S.C. Liew. (2013). Physical and Optical Charac-teristics of
the October 2010 Haze Event over Singapore: A Photometric and Lidar
Analysis. Journal of Atmospheric Research, 122, 555–570 .
11) B.N. Chew, S.C. Liew, R. Balasubramanian, L.E. Yu, J.S. Reid. (2009). Seven
Southeast Asian Studies (7-SEAS): atmospheric supersite in Singapore, in
30th Asian Conference on Remote Sensing (ACRS 2009). Asian Association
of Remote Sensing. Beijing, China.
12) J.S Reid, et. al. (2013). Observing and under-standing the Southeast Asian
aerosol system by remote sensing: An initial review and analysis for the Seven
Southeast Asian Studies (7SEAS) program. Atmos. Res., 122, 403–468 (this
issue).
13) T.F. Eck, B.N. Holben, J.S. Reid, N.T. O’Neill, J.S. Schafer, O. Dubovik, A.
Smirnov, M.A. Yamasoe, and P. Artaxo. (2003). High aerosol optical depth
biomass burning events: a comparison of optical properties for different
source regions. Geophys. Res. Lett., 30 (20), 2035.
14) A. Heil and J.G. Goldammer. (2001). Smoke-haze pollution: a review of the
1997 episode in Southeast Asia. Regional Environmental Change, 2, 24–37.
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15) D.E. Ward. (1990). Factors influencing the emiss-ions of gases and
particulate matter from biomass burning, In: J. G. Goldammer (ed) Fire
in the tropical biota. Ecosystem processes and global challenges. Ecological
Studies, 84, 418–436.
16) T. Novakov, H. Cachier, J.S. Clark, S. Macko, and P. Masclet. (1997).
Characterisation of par-ticle products of biomass burning combust-ion. In:
J.S. Clark, H. Cachier, J.G. Goldam-mer, B. Stocks (eds) Sediment records
of biomass burning and global change. NATO ASI Series No. 1, Global
En-vironmental Change, 51, 117–144.
17) L. Nurhidayah. (2013). Legislation, regulations, and policies in Indonesia
relevant to addressing land/forest fires and transboundary haze pollution: A
critical evaluation. Asia Pacific Journal of Environmental Law, [online]. Vol.
16, pp 15-239. Available: http://search.informit.com.au/documentSumma
ry;dn=779978732417466;res=IELHSS
The Potential Impact of Carbon Monoxide
Emission to The Community Health In The
Vicinity of Baranangsiang Toll Gates
Yudith Vega Paramitadevia,*, Arief Sabdo Yuwonob, Meiske Widyartib
Diploma Program, Bogor Agricultural University
a

Jl. Kumbang No. 14, Bogor, Indonesia


b
Department of Civil and Environmental Engineering, Bogor Agricultural University
PO BOX 220, Bogor, Indonesia

Abstract
Over the past decade, emission from carbon monoxide (CO) has risen due to the increase of
vehicles per year. Bogor as a weekend tourist city has a heavy burden in terms of the volume of
motor vehicles. Object in this study is Baranangsiang Bogor toll gateway where queue of motor
vehicles often happens and allegedly produces CO pollutants.
This study was conducted to simulate the CO concentration by the method of Finite Length
Line Source (FLLS) around Baranangsiang Bogor toll gateway and recapitulate types of diseases
associated with CO impacts on communities around the toll in line with the pattern of 6%
increase of vehicle volume per year.
Based on the results of measurements and simulations conducted in four sampling points
within 20 m and 190 m from the sources of pollution on 29 August to 1 September 2013 , the
concentration of CO is still within the range of quality standards in accordance with Regulation
No. 41 Year 1999 which is 634–9,189 μg/Nm3. Dispersion of pollutants CO dominantly heading
Eastwards with the wind speed measurements 1.5–5.2 m/s and atmospheric stability class B.
Kampung Sawah RT 02 RW 07 is higher exposed with CO. Recapitulation of medical records
showed that suspected CO intoxication disease cases are more common in Kampung Sawah than
IPB Baranangsiang settlements.
Key words: Carbon Monoxide, CO Intoxication, Dispersion CO, Toll Gates Baranangsiang

I. Introduction
Carbon monoxide emitted by various vehicles to the air is one of air pollution
causes. The dispersion of pollutants in the air affects the regional air quality
that adversely impacts the public health [1][5]. Nowadays, in most countries,
air pollution caused by the vehicular exhaust emission has increased due to the
increasing amount of vehicle per year as a result of the increasing of transportation
consumer needs [13,16,20].

* Corresponding Author.Tel: +6285-717-99-555-6. E-mail: vega_paramitadevy@yahoo.com

289
290 Proceeding ASEAN COSAT 2014

Bogor, a tourist destination city on weekend, has quite heavy burden in terms
of vehicle volume. Some places with high CO concentrations in Bogor are the toll
gates, intersections and main roads [17]. The object in this study is Baranangsiang
Bogor toll gates. At the gates, heavy traffic often occurs and allegedly produces a
lot of CO gas. CO gas increasing occurs when motor vehicle running at low speed
due to braking frequency [1,15]. Based on [6] finding at Surabaya entrance toll,
it has been stated that the highest concentration of CO was 7,845 μg/Nm3 on
weekdays while on holidays was 8,708 μg/Nm3. For concentrations higher than
11,700 μg/Nm3 during 10 hours of exposure, CO may cause negative impacts
on human health such as chest pain, chronic lung disease, flu-like symptoms
and headache [4].
Based on the problems mentioned before, a more in-depth formulation
regarding CO pollutant dispersion in the vicinity of Baranangsiang Bogor toll gates
is needed. Thus, simulation of CO pollutant dispersion is required. In this study,
Finite Length Line Source (FLLS) mathematical model was used. It is a derivative
of Line Source Gaussian model with Visual Basic programming for calculating
CO concentration. Summary of medical record disease from CO intoxication
was also carried out to determine the potential impact of CO pollution.

II. Method/M aterial


A. Time and Location of CO Pollutant Measurements
Data collection and measurement of CO sample was carried out in four points in
the vicinity of Baranangsiang Bogor toll gates (managed by PT. Jasa Marga) for
a week from August 26 to September 1, 2013. Two points within ±20 m apart
perpendicular to highway axis point, located at either side of the roadside. Next
two points within ±190 m perpendicular to highway, were located in KPP IPB
Baranangsiang 4 and Kampung Sawah settlement RT 02 RW 07. CO sampling
was conducted on weekdays and weekends, four (4) times a day for a week. The
duration of each measurement was one hour.
The criteria for determining the location of ambient air quality samples refer
to National Standard, namely SNI No. 19-7119.6-2005 on The Determination
of Test Sample Location for Controlling of Ambient Atmosphere Quality. Figure
1 shows the location of pollutant CO measurement. CO sample analysis was
carried out in Laboratory of Aquaculture IPB in September–October 2013.
Innovation for Better ASEAN Community 291

B. Materials
Materials used in this research consist of primary and secondary data.
1). Primary data, such as:
a) CO pollutant concentration based on direct field measurement.
b) Meteorological data at the measurement time such as temperature, wind
direction and speed, relative humidity.
c) Coordinate of measurement points and toll axis point.

2) Secondary data, such as:


a) Numbers of vehicle passing through toll gates for three years (2010-2012),
obtained from PT Jasa Marga, Jagorawi Branch.
b) Meteorological data from Meteorology Station Class I Dramaga and
Meteorology Station Class III Citeko, i.e. temperature, relative humidity,
wind direction and speed, and radiation intensity from 2008-2012.

Figure 1 Location of Pollutant CO Measurement

C. Tools
Tools used in this study consist of data processing devices, in-field measurement
devices, and laboratory equipment for analysis. The devices are listed below:
1) Data processing devices, such as Windows-based CPU. Software used in this
process are:
a) Visual Basic 6.0 for simulation.
b) Global Mapper 11.0 for sampling points plotting.
c) Surfer 8.0 for visualizing of CO dispersion.
292 Proceeding ASEAN COSAT 2014

2) Measurement devices in field covered impinger, thermometer, anemometer,


spectrophotometer, tripod, manual counter, GPS, and portable CO meter.
3) Analysis equipment in laboratory including flasks, beakers, pipettes, and test
tubes. Chemical solution KI was needed to absorb CO and Iodine Standard
as primary solution.

D. Methodology
This study consist of five (5) phases, i.e. CO concentration measurements, CO
concentration analysis in laboratory, forming Line Source Gaussian dispersion
modeling using Finite Length Line Source (FLLS) method, model visualization
of CO pollutant dispersion pattern and recapitulation of medical records at
three public health centers near sampling points. The laboratory analysis of CO
concentration refered to SNI No. 19-4848-1996 on Pentoxide Method.
The calculation of emission load refered to [14] with the speed considered
varied from 0–60 km/hour and the duration of vehicles maintenance in India
and Indonesia was relatively similar between 10 and 15 years.
FLLS method determined the concentration of gaseous pollutant including
its dispersion by dividing the segments of line source into its smallest segments.
Having gained the smallest segments, then the distance between receptor and
lined source was calculated in order to determine the dispersion parameter of
each receptor [3,7,12]. FLLS equation according to [9] is listed below:
where :

(1)

Description :

Q : Pollutant source rate. At the finite point source, unit g/sec is used. At
the finite line source, unit g.m/sec is used.
ū : Wind speed at the-x (m/sec).
σz : Concentration dispersion parameter at the-z (m).
z : Position of Z in Cartesian coordinate (in this study, z=0).
H : Effective height ofemission source (in this study, H=0).
B : The rate of road length to dispersion parameter ().
Innovation for Better ASEAN Community 293

The calculation was performed on model using equation (1) including emis-
sion load to obtain Q, wind speed to obtain, road length and receptor location
to obtain σy and σz as well as B1 and B2. Data visualization phase using Surfer
software in the form of coordinate data, thus pollutant dispersion patterns could
be observed based on isopleth from modeling. Recommendations were given if
the resident medical records showed a positive correlation with the isopleth map.

III. R esult and Discussion


A. Traffic Density Condition In the Vicinity of Baranangsiang Bogor Toll
Gates
Baranangsiang Bogor toll gates are one of the Jagorawi toll gates managed by PT.
Jasa Marga. There are 9 toll booths, which 4 booths are for ticketing locations
(Entrance), other 4 booths are for payment points (Exit), and another one is a
stand-by booth which can be operated as entrance or exit booth. The total volume
of vehicle entering and exiting the toll gates in Bogor reaches 9 to 10 million units
per year [11]. The average number of vehicle passing by a toll gate is 270 units per
hour. On weekends, the average numbers of vehicle passing by Baranangsiang toll
gates can reach 70,000 units/day. On the other hand, on weekdays the average
is about 55,000 units. Traffic density that often occurs on holidays is caused by
family travel activity with Bogor as the main destination.
According to data from [11] during 2010-2012, the most dominant type
of vehicle passing by the gates is private vehicles. They were diesel-driven and
petrol-driven cars with two axles or Class I vehicle which cover about 97% of
total numbers of vehicle. The second most dominant vehicle found was Class II
vehicle such as diesel-driven small trucks and buses with two axles (3.05%). And
the remaining 0.17% was Class III vehicle while Class IV and V were 0.03%
and 0.02%, respectively.

B. Simulation of Modeling Results


In this study, software was developed using Visual Basic 6.0 program in order
to obtain accuracy and simplicity for calculating CO concentrations model.
Simulations for CO were carried out in July–September using Surfer 8.0 program
which is illustrated in Figure 2 (a-c). Meteorological outputs and traffic conditions
data used in the simulation are the average of five-year data in that specified
month, with the average wind speed is 1.31 m/sec, the average wind direction
is to the East at about 296°, atmospheric stability Class B, the average numbers
of vehicle is 2559 units/day.
The highest concentration of CO pollutant dispersion was 4,000 μg/Nm3 at 20
m at right roadside. According to the FLLS formulation, wind distribution factors
294 Proceeding ASEAN COSAT 2014

(a)

(b)

(c)
Figure 2 Simulation of CO in (a) July 2013; (b) August 2013 and (c)
September 2013
Innovation for Better ASEAN Community 295

were influenced the dispersion of pollutants. The smaller the angle perpendicular
to the road, relative speed to the road was small, too. Consequently, the multiplier
factor (K) decreases and the concentration of CO model become higher than CO
observed. Pollutant was dispersed to the East direction towards Kampung Sawah.

C. Medical Record Analysis of Community in The Vicinity of Baranang-


siang Toll Gates
Based on the simulation results, the dispersion of pollutant CO dominantly moved
toward the East. The residential population point exposed to higher CO was
Kampung Sawah RT 02 RW 07. The highest concentration of CO in July 2013
was 1,200 mg/Nm3. At another point, namely IPB Baranangsiang 4 Settlement,
the highest concentration of CO in the same month was 1,150 mg/ Nm3.
The number of vehicles in the Greater Jakarta increased by nearly 6% per year
[19], indicating concentration of pollutants CO increase, too. Thus, although
the concentration of the simulation results was still below the quality standard
of 24 hours i.e 10,000 mg/Nm3, the analysis of medical records of residents was
still being done.
Urban communities have HbCO levels in the blood of about 2–6% [8,10],
symptoms of CO intoxication will be felt receptors after HbCO levels in the
blood reaches 10% [2]. CO intoxication symptoms similar to the flu and diseases
associated with respiratory infections, difficulty breathing (relationship to the
lungs, hereinafter referred to exacerbation disease of chronic obstructive lung)
and chest pain (relationship to disease in the vessels of the heart/ cardio vascular)
[4,18]. This study has used the data of upper respiratory illness (URI), exacerbation
of chronic obstructive pulmonary (COPD) illness i.e emphysema, asthma and
chronic bronchitis also vascular system diseases such as ischemic cardiac disease.
Medical records obtained from three public health centers in the city of Bogor
i.e the North Bogor Health Center near the IPB Baranangsiang 4 Settlement,
the East Bogor Health Center near Kampung Sawah Housing RT 02 RW 07
and the Pulo Armyn Health Center that is located in Pulo Armyn Padjadjaran
Street. Figure 3 shows the summary results of the medical records at the three
health centers in diseases associated with CO intoxication.
Medical record refers to the data in Figure 3 was obtained from the number
of occurrences of the disease in the population who live in the catchment area of
each health center. The number of events due to the isemic cardiac diseases was
low, especially in the area of ​​IPB Baranangsiang 4 Settlement. A high number of
patients with isemic cardiac diseases were biggest in Kampung Sawah. The same
thing was found in URI and COPD.
296 Proceeding ASEAN COSAT 2014

Figure 3 Medical Report of CO Intoxication Disease Event from January 2012 to March 2013

Dispersion of pollutants CO moves toward to the East, but the conclusion


that the prevalence of respiratory disease, lung disease and cardiovascular in
Kampung Sawah is higher than in the Baranangsiang 4 Settlement should be
studied further. This is due to the HbCO analysis was not performed on receptors
in this region. According to the research results of [8], Indonesia rarely carried
out an examination of HbCO in patients. Consequently, patients were exposed
to pollutants CO has come in the acute situation when taken to the nearest
health center.

IV. Conclusion
The simulation results in July–September 2013, with the highest CO concentra-
tion of 4,000 mg/Nm3 in the right roadside. The simulations also indicate the
dispersion of pollutants CO eastward namely Kampung Sawah.
Summary medical record indicates a high number of patients with the
incidence of isemic cardiac disease, URI and COPD respectively 8, 858 and 424
events during the period from January to March 2013 in the village of Kampung
Sawah Baranangsiang.
The relationship between the high incidence of the disease in Kampung Sawah
and the dispersion of pollutants CO needs to be studied further.
Innovation for Better ASEAN Community 297

V. Acknowledgment
Authors appreciate to Program Diploma IPB for the funding support of the
research in PT Jasa Marga (Persero) Jagorawi Branch.

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The Mechanism of Dry Mid-Atmosphere in The
Western Maritime Continent during Rainy
Season in 2014

Supari a,*, A.M. Setiawan a, E. Makmur b, A. Sopaheluwakan a, Siswanto c,


W. Sulistya a
a
Center for Climate, Agro and Marine Climate - Agency for Meteorology Climatology and
Geophysics (BMKG)
b
Center for Research and Development - Agency for Meteorology Climatology and
Geophysics (BMKG)
c
Center for Aviation and Marine Meteorology - Agency for Meteorology Climatology and
Geophysics (BMKG)
Jl. Angkasa I no 2 Kemayoran, Jakarta, Indonesia

Abstract
In the late January to mid-March 2014, which is normally peak of rainy season, massive forest
fire cases were monitored over North-Western part of Maritime Continent resulting serious
environmental impact such as a huge pall of smoke, poor air quality and flight cancellation.
This paper is aimed to investigate the atmospheric setting of January-March 2014 based on site
observations and global re-analysis data which were associated to the fire cases. It was observed
that rainfall decreased up to 30 % than its long-term average. The evolution of vertical profile of
atmosphere based on sounding data from Medan (96035), Ranai (96147) and Pangkalpinang
(96237) show a dramatic drop of humidity in the mid-atmosphere (700–500 mb) starting from
the third week of January. The synoptic features of low-level atmosphere exhibit an intensified
northeasterly winds and a propagation of cold air over the South China Sea suggesting an intrusion
of dry sub-tropical air. The diagnosis of meridional wind component reveals that over the region,
the circulation was dominated by sinking air that suppressed the convection process, indicating
anomaly of Hadley cell. The negative phase of Madden-Julian Oscillation (MJO) is assumed to
play another role where the wide suppressed convection area was observed based on outgoing long-
wave radiation (OLR) data. These findings suggest that monitoring mid-atmosphere condition
is relevant for smoke trajectory forecast and fire danger early warning.
Key words: Forest fire, Maritime continent, Dry atmosphere.

I. Introduction
The forest fire case that occurred in the North-western Maritime Continent
during late January to mid-March 2014 was very unusual in term of timing
since it occurred during peak of rainy season. Although forest fire is almost yearly
hazard over the region, it usually occurs during dry season, mostly on peat lands

* Corresponding Author.Tel: +62-81315689645. E-mail: supari@bmkg.go.id

299
300 Proceeding ASEAN COSAT 2014

Figure 1. Top - Observed hotspot data from MODIS Terra-Aqua satellite during January – March
2014. Only data with at least 80 % confidence level are plotted. Gray area denotes Riau Province.
Bottom – temporal evolution of hotspot number over Riau Province, the most affected area due
to smoke-haze pollution during the event.
Innovation for Better ASEAN Community 301

and peat-swamp forest [1]. Forest fire cases in Indonesia are generally caused by
illegal human activity, including land clearing and accidental fires. Nevertheless,
the massive forest fire case will only occur when atmosphere as contributing
factor is dry enough [2]. In fact, during January–March 2014 which should be
wet months, thousands of hotspots data were monitored over almost the entire
of Sumatera and Borneo Islands as displayed in Figure 1.
Time series of hotspots number over Riau Province shows that massive hotspot
data were monitored started from mid-February till mid-March. On 11 March,
it was detected 1,149 hotspots over the province. The impact was not surprising
then, where a huge pall of smoke leading poor air quality strikes the area. Besides
causing serious health problem, the smoke also caused flight cancellation due to
minimum visibility. The economic loss is expected around USD 1 billion.
Climate over the region is mainly controlled by monsoon systems so that strong
annual rainfall variability is a key feature with wet season during October–March
and dry season during April–September. However, the semi-annual variability is
also notable characteristic over there [3] due to local response to the southward
and northward movement of the inter-tropical convergence zone (ITCZ). Dry
season is period when forest fire risk will increase and become much more risky
during ENSO year [4].
Forest fires in Indonesia have been investigated for long time both focusing
on the impact [1], [5], [6] and also focusing on the causes of fires [7]. Different
with previous studies, the analysis of climatic condition related to fire case is the
core of the present work. The aim of this study is therefore to gain insight on the
mechanisms that triggered dry condition which is favorable then for forest fire
cases, by using remote sensing data, in-situ meteorological observations and global
re-analysis data as well. The output of recent case studies, like the one presented in
this paper, is expected to be a crucial input in term of issuing warnings. It should
be noted that forest fire is truly potential treat for sustainable development due
to direct impact on environment including carbon emission [4].

II. M aterial
Data used in this study were collected from many sources. Modis/Tera-Aqua
hotspot data [8] was produced by the University of Maryland and provided by
NASA FIRMS operated by NASA/GSFC/ESDIS with funding provided by
NASA/HQ. It is available on-line (free) https://earthdata.nasa.gov/active-fire-
data#tab-content-6.
Rainfall observation and upper air sounding data were gathered from BMKG.
Relative humidity data at pressure levels were obtained from ECMWF-ERA
Interim Re-analysis [9]. Vertical velocity parameter was obtained from Japan
302 Proceeding ASEAN COSAT 2014

Re-analysis provided by Japan Met Administration (JMA) [10]. The outgoing


long-wave radiation (OLR) data were obtained from the National Oceanic
Atmospheric Agency (NOAA) Interpolated OLR [11]. OLR data characterized
the strength of longwave radiation from the earth’s surface (under clear sky
conditions) or from the top of clouds (under cloudy conditions). Lower values
of OLR indicated more enhanced convective activity under cloudy conditions.
Picture displaying OLR data is provided by Berau of Meteorology, Australia.

III. R esults and Discussion


A. Rainfall Observation
The spatial distributions of accumulated rainfall at 10-days scale indicate that
starting in the mid of January, the Northwestern Maritime Continent consistently
received very low rainfall. In the late of January, Riau, Jambi, Bangka Belitung
Province that are located in the eastern part of Sumatra got only less than 10 mm
per 10-days, very much below normal. This condition continued till the second
week of March resulting in very dry surface condition (Figure 2).
With dominant strong northerly monsoon wind, January and March should
be very wet over those regions by receiving rainfall at least 200 mm per month.
February indeed is the driest month among other wet months (DJFM), especially
for eastern part of Sumatra Island due to weakening of monsoon. However, the
normal value of monthly total is at least 100 mm. This February, therefore, was
extremely dry because monthly total rainfall was only 28 mm for Medan Station
(North Sumatera Province), 14 mm for Pekanbaru Station (Riau Province),
40.0 mm for Ranai (Riau Island province), 26.0 mm for Jambi Station (Jambi
Province) and 47.8 mm for Pangkalpinang Station (Bangka Belitung Province).

B. Moisture Content
The upper air sounding data (not shown) reveals notable difference condition of
relative humidity related to the event. It was observed from met station of Medan
(WMO No. 96035), Ranai (WMO No. 96147) and Pangkalpinang (WMO
No. 96237) that the humidity of atmosphere decreased dramatically. Although
there was no significant feature at the surface, we found interesting feature at
mid-atmosphere level.
At the 700 mb pressure level, relative humidity was relatively high around
80 % during first and second week of January. However, it dropped significantly
up to 30 % starting from third week of January. The similar pattern was also
found at 500 mb. In Ranai, the reduction of humidity even could be recognized
at lower level, 850 mb.
Innovation for Better ASEAN Community 303

Figure 2. Observed monthly rainfall for the first 10-days of February (top), the second 10-days
(midlle) and the last 10-days (bottom).
304 Proceeding ASEAN COSAT 2014

Figure 3. Spatial distribution of relative humidity (%) at 700 mb for the first 10-days of
January (top), the second 10-days (midlle) and the last 10-days (bottom).
Innovation for Better ASEAN Community 305

Figure 4. High – Latitude plot of vertical velocity averaged over 100 – 110 E, describ-
ing configuration of Hadley circulation for the first 10-days of January (top-left), the
second 10-days (top-right), the last 10-days (bottom-left) and first 10-days of February
(bottom-right). Updraft (downdraft) fraction relates to supporting (blocking) condi-
tion for convective activity.
306 Proceeding ASEAN COSAT 2014

Spatial map of relative humidity over Southeast Asia confirms that the low
humidity actually extended southward from great Asia Continent (Figure 4).
At the 700 mb, relative humidity in the first 10-days of January (1–10 January)
was generally categorized by dry air (less than 40 %) located in the 15 N and
higher latitude. In the last 10-days (21–31 January), contour of relative humidity
of 40 % propagated southward and went up to 5 N. Over South China Sea, it
even reached equator line. At the low-level atmosphere we found an intensified
northeasterly wind particularly over South China Sea and a propagation of cold
air (not shown). This anomalous appearance triggers a hypothesis of intrusion
of dry sub-tropical air mass.
Linked to the observed rainfall data, the dry condition at mid-atmosphere
describes clearly that there was not enough moisture content in the atmosphere
to support low cloud development that was expected to generate rainfall.

C. Hadley Circulation
It is interesting to track the responsible condition for minimum convective activity
as found in the previous section. Since the relative humidity at mid-atmosphere
displays southward propagation of dry air mass, we investigate then the detail
north-south atmosphere circulation i.e. Hadley cell. It is well known that tropical
area is the region in which the up draft phase of Hadley cell is dominant. This
common state gives favorable environment for convective activity to enhance
deeply. During the event, we observed surprisingly the anomalous configuration
of Hadley cell (Figure 4).
In contrast with condition of the first 10-days of January where the up draft
phase of Hadley cell dominated region between 13 S–10 N, on the second
10-days of January, the up draft phase was only observed over 10 S–0/Equator
line. The down draft phase, on the other hand dominated strangely over wide
area, from equator line to 40 N. The major circulation over the studied area was
consequently a sinking air mass that damped convective activity.

D. Role of MJO
Such background favourable conditions related to intra-seasonal phenomena like
MJO (Madden-Julian Oscillation) may also contribute for drying atmosphere.
Despite spatially large cloud system during the active phase of MJO could increase
intense precipitation in the Maritime Continent through long-lived convection
[12], [13], the negative phase of MJO, on the other hand will bring less rainfall
and drier condition [14].
OLR data observation shows that the active phase of MJO, which is character-
ized by large area of enhanced convection, developed over the Indian Ocean during
Innovation for Better ASEAN Community 307

Figure 5. Reconstructed OLR field over tropical area from August 2013 – March 2014. Source
: BoM.

late November as shown in Figure 5. It propagated eastward slowly then to the


West Pacific Ocean by early January. Over Indonesia, negative anomalies of OLR
were observed during late January and mid-February. Afterward, strong positive
OLR anomalies followed as a signal of passive phase of MJO. This suppressed
convection was still monitored till late March and is expected being responsible
as another factor for drying atmosphere over the studied region. The interaction
between Hadley cell anomaly with this passive phase of MJO blocked convective
activity and therefore atmosphere with poor moisture dominate the region.

IV. Conclusion
The January–March forest fire over nort-western Maritime Continent is considered
as unusual event since it occurred during peak of rainy season. This anomalous
forest fire case associated with dry condition over the region which was character-
ized by very much below normal rainfall condition. The analysis state that this
atmospheric setting was triggered by strange configuration of Hadley cell. The
sinking air dominated over region wich disable convective activity. However the
mechanism causing this circulation anomaly is still not clear. The passive phase
308 Proceeding ASEAN COSAT 2014

of MJO that associates with suppressed convection might also play another role
in conditioning dryier atmosphere.

VI. R eferences
1) Harrison, M. E., Page, S. E., and S. H. Limin. (2009). The global impact of
Indonesian forest fires. Biologist, 56, 156–163.
2) South Asia Association of Regional Cooperation (SAARC) - Disaster Manage-
ment Centre (SDMC), Fire Disasters,” India. [Online]. Available: http://
saarc-sdmc.nic.in/pdf/fire.pdf
3) Aldrian E., and R. D. Susanto. (2003). Identification of three dominant
rainfall regions within indonesia and their relationship to sea surface
temperature. Int. J. Climatol, 23, 1435–1452.
4) Tacconi, L. (2003). Fires in Indonesia: Causes, Costs and Policy Implications.
CIFOR, Indonesia, CIFOR Ocassional Paper No. 28.
5) Glover, D., and T Jessup. (2006). Indonesia’s Fires and Haze: The Cost of
Catastrophe, Singapore. Institute of Southeast Asian Studies, 1­–21.
6) Heil, A. (2000). The 1997–1998 Air Pollution Episode in Southeast Asia
Generated by Vegetation Fires in Indonesia. IFFN, No. 23, 68–71.
7) Dennis, R., Huffman, A., Apllegate, G., von Gemmingen, G., and K.
Kartawinata. (2001). Large-scale fire: creator and destroyer of secondary
forests in western Indonesia. Journal of Tropical Forest Science, 13, 786–799.
8) Kaufman, Y. J., Ichoku, C., Giglio, L., Korontzi, S., Chu, D. A., Hao, W. M.,
Li, R.-R., and C. O. Justice. (2003). Fire and smoke observed from the earth
observing system MODIS instrument - products, validation, and operational
use. Int. J. Rem. Sens., 24, 1765–1781.
9) Dee, D. P., Uppala, S. M., Simmons, A. J., Berrisford, P., Poli, P. et al.
(2011). The ERA-Interim reanalysis: configuration and performance of the
data assimilation system. Q. J. R. Meteorol. Soc., 137, 553–597
10) Ebita, A., Kobayashi, S., Ota, Y., M. Moriya, Kumabe, R., Onogi, K., Harada,
Y., Yasui, S., Miyaoka, K., Takahashi, K., Kamahori, H., Kobayashi, C., Endo,
H., Soma, M., Oikawa, Y., and T. Ishimizu. (2011). The Japanese 55-year
Reanalysis “JRA-55”: an interim report. SOLA, 7, 149–152.
11) Liebmann, B., and C. A. Smith. (1996). Description of a complete (inter-
polated) outgoing longwave radiation dataset. Bull. Am. Meteorol. Soc., 77,
1275–1277.
12) Hyun Oh, J., Kim, K.Y., G. H. Lim.(2012). Impact of MJO on the diurnal
cycle of rainfall over the western Maritime Continent in the austral summer.
Clim. Dyn., 35, 1167 – 1180.
13) Wu, P., Arbain, A. A., Mori, S., Hamada, J., Hattori, M., Syamsuddin, F., and
M. D. Yamanaka. (2013). The effect of an active phase of the Madden-Julian
Oscillation on the extreme precipitation event over western Java Island in
January 2013. SOLA, 9, 79 – 83.
14) Barlow, M., and D. Salstein. (2006). Summertime influence of Madden-Julian
Oscillation on daily rainfall over Mexico and Central America. Geophys. Res.
Lett., 33, L21708.

309
Design of Automatic Measurement
Instrument for Water Discharge On Drainage
Monitoring System

Retno Tri Wahyuni*


Electronic Departement, Polytechnic of Caltex, Riau
Jl. Umban Sari No. 1, Pekanbaru, Riau, Indonesia
retnotri@pcr.ac.id

Abstract
The drainage system is one of infrastructures that is developed to prevent local flooding in urban
area. The urban drainage management system should be implemented overall. One of drainage
management component is monitoring. The monitoring system integrates meteorological data
in form of rainfall data, and also data water level, and water discharge. The water discharge is
monitored to observe whether drainage can handle the water flow. The water discharge is also a
variable that is considered in design of drainage. Design of automatic measurement instrument
of water discharge consists of propeller, magnetic sensor, water level sensor, and micro controller.
Water discharge of drainage is obtained by multiplying the velocity of water flow (v) by wet cross
section area (A). Wet cross section area is calculated using hydraulic formula depends on the type
of drainage. The magnetic sensor changes the propeller rotation to electric signal for calculating
velocity of water flow. The water level measured by an ultrasonic sensor.
Key words: Water discharge, Automatic, Drainage

I. Introduction
Flood is one of disasters that have occurred in almost all regions in Indonesia.
Based on data from Badan Nasional Penanggulangan Bencana (BNPB) since
1815 to 2014 flood has the largest percentage compared to other types of disasters
that is equal to 38% [1]. One type of flooding is puddles flood. It is caused by
drainage that can’t dispose a lot of water coused by very high rainfall. The solution
is to build adequate drainage. In general, the management of drainage system
in many urban in Indonesia is still partial, so it doesn’t resolve the problem of
puddles flooding compeletelly. The urban drainage management system should
be implemented overall, starting from the stage of survey, investigation planning,
land acquisition, construction, operation and maintenance, institutional support,
financing, community participation, evaluation and monitoring [2]. If those
components of urban drainage management doesn’t work well, the performance

* E-mail: retnotri@pcr.ac.id

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312 Proceeding ASEAN COSAT 2014

Figure 1. Block Diagram System

of drainage can’t work optimally. In fact, one of weakness in urban drainage


management is monitoring. The monitoring method is done manually, so it has
human error and poor data management system, therefore the automatic drainage
monitoring system should be implemented.
In drainage monitoring system, one of variables monitored is water discharge.
The water discharge is monitored to observe if drainage can handle the water
flow. The water discharge is also a variable considered in designs of drainage.
Some methods are implemented to measure water discharge of drainage, one of
them is to measure the velocity of water in drainage using current meter. In this
method, people measure velocity of water using current meter in some points
based on procedure of measuring. The procedures consist of laying the propeller at
a certain depth, cross-sectional area measurements, speed propeller measurement,
recording and calculating the data record.
In this research, process of water discharge measurement will be implemented
on integration system that consists of sensor, tranducer and controller so the
process done automatically. The automatic measurement instrument for drainage
can be implemented on automatic drainage monitoring system so it can help on
drainage management system.

II. Method
The design of automatic measurement instrument for water discharge of drainage
consists of sensor, tranducer, and controller. The sensor will sense the velocity of
water flow using propeller. The propeller rotation will be changed to electrical
signal by magnetic sensor as tranduser. The electric signal will be inputted to
microcontroller and processed based on algorithm calculation of water discharge
of drainage. Another input for calculating water dischare of drainage is level of
water and widht of drainage. The water level will sense using ultrasonic level
sensor and the widht of drainage will inisiate manually using keypad. Figure 1
describes block diagram of the system.
Innovation for Better ASEAN Community 313

The flow chart of system that implemented


in micro controller described in Figure 2. First
process in the algorithm is to chose the type
of drainage. There are 3 types of drainage i.e:
rectangle, trapezoidal and circle. After choos-
ing the type, input the variables needed for
calculating the wet cross sectional area that is
different for every type of drainage, ex: width of
drainage for rectangle, gradient for trapezoidal
type, diameter for circle type etc. The next
process is water level measurement by level
sensor and velocity of water flow measurement.
The variable and result of water level sensor will
be used for calculating wet cross sectional area.
Finally, the last result of velocity of water flow
measurement and result of process calculation
for wet cross sectional area will be used for
calculating water discharge of drainage.

III. R esult and Discussion


A. Metereological Aspect on Drainage
Monitoring System
Irrigation and drainage is one of the purposes in
water management. Based on technical report
“Climate and Meteorological Information
Requerements for Water Management”, the
comment for data requirement for irrigation
and drainage about spatial density must
represent demand and supply area variations,
measurement interval daily for design manage-
ment and operation, real time monitoring
needed in multipurpose systems.
In fact, the climate information present
is not widely used by water management [3].
In this research the metereological will be
integrated with hidrological data for monitoring
condition of drainage. For example, Figure 3
describes rainfall intensity from weather stations
Figure 2. Flowchart of system
of Pekanbaru. This data should be used for
reference to design drainage in Pekanbaru. The
314 Proceeding ASEAN COSAT 2014

Figure 3. Intencity Rainfall on Pekanbaru Weather Station


(Source: Pekanbaru Weather Station, BMKG)

data from rainfall sensor, water level sensor and water discharge of drainage will
be combined to describing the drainage condition.
The specific discuss in this paper only about automatic instrument for
measuring of water discharge on drainage. The complete discussion about drainage
monitoring system describe in [4].

B. Velocity of Water flow Measurement Instrument


The design of velocity of water flow measurement instrument use propeller for
sensing the velocity of water flow. The design of propeller based on propeller of
commercial current meter. That is a no new design of propeller in this research.
Figure 4 describe the design of propeller that was used.

Figure 4. Design of Velocity of Water flow Measurement Instrument (hori-


zontal position)
Innovation for Better ASEAN Community 315

The propeller coupled with part that magnetic sensor is placed there. When the
propeller rotates caused by water flow, the magnetic sensor also rotates together.
Figure 5 describes the block diagram for velocity of water flow measurement
instrument.
The magnetic sensor has different response when detecting North pole or
South magnetic pole. The type of magnetic sensor used in this design is IC
UGN3503. The IC is cheap but reliable to detect magnetic field. Figure 6 describes
output of IC when detecting magnetic sensor with varying distance . From that
figure, we can see that IC UGN 3503 can use to detect North pole or South pole.
From that chart we can consider of the placement of magnetic sensor and magnet.

Magnetic
Sensor

Propeller Gear box


System

Figure 5. Block Diagram of Velocity of Water


flow Measurement Instrument

Figure 6. Output of IC UGN3503


316 Proceeding ASEAN COSAT 2014

The voltage will be inputed to micro controller as interrupt timer. The timer
will count based on the interrupt signal. The counting result will change to velocity
of water flow with equation 1.

60 60
ω= (1)
t
ω : angular velocity of propeller/water flow (rpm)
t : time for one rotation (second)
The angular velocity is changed to linear velocity with standar international unit
with equation 2.

v = ωr (2)

v : linier velocity (m/s)


r : radius of plate (m)

C. Water Level Sensor


The water level sensor is generated based on ultrasonic principle. The transmitter
of ultrasonic sensor transmits ultrasonic signal and the water surface reflects the
signal to the receiver that produces electric signal. The duration of ultrasonic signal
propagation from transmitter to receiver is counted by timer of microcontroller.
The result of the timer will be changed to water level with equation 3.

vt vt
s =h− (3)
2
v : velocity propagation of ultrasonic signal (m/s)
t : duration of ultrasonic signal propagation (s)
s : level of water (m)
h :distance between ultrasonic sensor to the bottom of drainage channel (m)

D. Calculating Algorithm for Water Discharge of Drainage


Water discharge is defined as volume rate of water flow in some wet area. Water
discharge is obtained by multiplying the velocity of water flow (v) by wet cross
section area (A).
Q = vA (4)
Innovation for Better ASEAN Community 317

Wet cross section area is calculated using hydraulic formula. There are three
types of cross section channel drainage i.e.: trapezoidal, rectangle, and circle [5].
Figure 7 describes the type of cross section channel drainage. The height variable
(‘y’at rectangle and trapezoid type,‘d’ at circle type) obtained from measurement
result of water level sensor. The formula to calculate wet cross sectional area can
be seen in equation Table 1.
Tabel 1. Formula for Calculating Wet Cross Sectional Area
Type of Drainage Formula A (m2)
Rectangle by (4)
Trapezoidal (b+xy)y (5)
Circle 1/2(Ø–sin Ø)D2 (6)

Rectangle

Trapezoidal
a

y
x
b
Circle
B

D
d
Ø

Figure 7. Type of Cross Section Area

IV. Conclusion
1) The monitoring system integrates meteorological data in form of rainfall data,
and also water level as well as water discharge data.
2) Design of automatic measurement instrument of water discharge consist of
propeller, magnetic sensor, water level sensor and micro controller.
3) Water discharge of drainage is obtained by multiplying the velocity of water
flow (v) by wet cross section area (A).
318 Proceeding ASEAN COSAT 2014

4) Wet cross section area is calculated using hydraulic formula depends on the
type of drainage.

V. Acknowledgment
Thanks to Indonesia General Directorate of Higher Education who fund this
research through “Hibah Bersaing 2014” program.

VI. R eferences
1) Badan Nasional Penanggulangan Bencana. Frequency of Disaster. [Online].
Available: http://dibi.bnpb.go.id. Retrieved May 2014.
2) Rauf, Syafruddin. (2012) “Pemetaan Jaringan Drainase Berbasis Quantum
GIS Open Source di Kota Makassar”, Prosiding Hasil Penelitian Fakultas
Teknik Universitas Hasanuddin, Vol 6, No 1. 2012. [online]. Available:
http://journal.unhas.ac.id/index.php/prostek/article/view/766/657
3) Dent, James E. (2012). Climate and Meteorological Information Requere-
ments for Water Management. World Meteorological Organization, Geneva,
Switzerland, Tech. Rep. WMO-1094,1-9.
4) Wahyuni, R.T., Wijaya, Y.P., dan D. Nurmalasari. (2014). Design of Wireless
Sensor Network for Drainage Monitoring System. Innovative and System
Design and Engineering, 5, no.5. 6-13.
5) Nizar, C. (2013). Pengertian Hidrolika. [Online]. Available: http://www.
ilmusipil.com/pengertian-hidrolika.
ACEHSEIS, a local seismic experiment in Bener
Meriah and Central Aceh

Muzli Muzlia,*, Muksin Umarb, c, Klaus Bauerb, Masturyonoa, Jaya


Murjayaa, Rakhindro P. Mahesworoa, Sigit Pramonoa
Meteorological, Climatological and Geophysical Agency of Indonesia (BMKG)
a

Jl. Angkasa I No. 2 Kemayoran – Jakarta Pusat 10720


b
German Research Center for Geosciences (GFZ-Potsdam)
Telegrafenberg 14473 Potsdam Germany
c
University of Syiah Kuala (UNSYIAH)
Jl. Teuku Nyak Arief, Darussalam – Banda Aceh

Abstract
Following the destructive 2013 Mw 6.2 Bener Meriah earthquake in Aceh, we conduct a temporary
seismic experiment in Bener Meriah and Central Aceh. The experiment is a collaboration work
between GFZ-Potsdam, BMKG and Unsyiah. We deploy temporarily six months 30 short period
seismic sensors. The sensors are stationed with a distance of about 5 to 7 km apart. This dense
network is expected to record the local seismic, particularly from the source of Sumatran fault and
other local faults near Central Aceh. The aim of this project is to identify the seismic structure and
faults in Aceh. Understanding the local seismic structure of the region will significantly contribute
to future disaster mitigation in this region. Moreover, this study could also be used to fill the gap
of a comprehensive investigation of the large picture of the Sumatra area of the previous studies
from Lake Toba to the southern part of the Sumatra Island.
Key words: Seismic experiment, Sumatran fault, local earthquake, tomography

I. Introduction
The tectonics setting of northern part of Sumatra particularly Aceh is controlled
by subduction in the western part of Sumatra as well as great Sumatran fault. The
pressure of Indo-Australian plate to Sumatra Island which lay on the Eurasian plate
yields the local faults along Sumatra island. Sumatran fault is the longest fault in
Indonesia. Moreover, if we look more detail to the complexity of Sumatran fault,
we will find that there are many other small faults around the great Sumatran fault.
Aceh is one of the region which has relatively large number of small faults. There
are several small faults in Aceh, for instance Seulimum fault, Aceh fault, Batee
fault and Tripa fault [1]. Therefore this region is very interesting to be investigated.
Due to the complexity of tectonics setting, Sumatra, particularly Aceh becomes
a dangerous and high risk area of the earthquakes disaster. This disaster can arise
* Corresponding Author.Tel: +62-21-4246321 ext. 8200. E-mail: muzli@bmkg.go.id

319
320 Proceeding ASEAN COSAT 2014

Figure 1. Sensors location of AcehSeis project, mainshock of the Mw 6.2 Bener Meriah earthquake
and historical seismicity in Aceh during 2009-2013.

from the shock of earthquake or tsunami following the powerful earthquake that
originated in the ocean floor. The largest earthquake in Sumatra that ever occurs
is dated December 26, 2004 earthquake with a moment magnitude of 9.0. The
earthquake has become a historical record as one of the largest magnitude of
earthquake in the world. The earthquake caused the most devastating tsunami
causing casualties and properties damages very much [2].
In addition to the above effects, Sumatra earthquake has a domino effect to
the large earthquake at subduction zones along Sumatra island. The earthquake
activity in the Sumatra region increased significantly after the mainshock of
Sumatra earthquake, both the Sumatra earthquake aftershock occurrence in
subduction zones as well as activities on the Sumatra fault. Several earthquakes
follow the mainshock of Sumatra earthquake originating in the subduction zone
are for instance the Mw 8.5, 2005 Nias earthquake [3,4], the Mw 8.4, 2007
Bengkulu earthquake [5] and the Mw 7.8, 2010 Mentawai earthquake [6,7].
Besides subduction earthquake, a series of large earthquakes with the sources in
Sumatra fault occurs after the main Sumatra earthquake occurrence.
Innovation for Better ASEAN Community 321

Figure 2. Aftershocks distribution of the Mw 6.2, 2013 Bener Meriah earthquake [9].

In Aceh, several strong earthquakes with the sources on the great Sumatra
fault or small local faults that occur after the sequence of Sumatra earthquake
period are for instance the Mw 6.0, 2013 Mane earthquake and the Mw 6.2, 2013
Bener Meriah earthquake. The overall seismicity rate in Aceh due to local fault
activities is represented on the figure of shallow earthquake epicenter distribution
in the period 2009–2013 [8] as shown in Figure 1.
Among the earthquakes above, one of the most interesting to be investigated
is Bener Meriah earthquake. The earthquake occurred on 2 July 2013 with a
magnitude of 6.2. The epicenter was on land at the geographic coordinates 4.7N
96.61E with the source depth of 10km. The devastating earthquake in Sumatra
mainland is generally sourced from the main Sumatra fault. Unlike in general,
Bener Meriah earthquake occurred at a location about 80 kilometers east of the
main Sumatra fault. This earthquake shows that other small faults have been
activated locally in Aceh outside of the main Sumatra fault. BMKG survey team
has conducted a survey to this earthquake aftershock in the period from 2 to 8
July 2013. The aftershock distribution is shown in Figure 2 [9].
Based on the BMKG report, the location of the main earthquake and the
aftershock series were not located along the main Sumatra fault but rather
occurred along the secondary fault system in the east of the Sumatra fault. This
phenomenon becomes interested to be investigated deeply. Understanding the
322 Proceeding ASEAN COSAT 2014

tectonic setting of the region will significantly contribute to future disaster


mitigation in the regions. Therefore, we propose a seismological study to better
understand the tectonic features of the region. This new study could also be
used to fill the gap of a comprehensive investigation of the large picture of the
Sumatra area of the previous studies from Lake Toba to the southern part of the
Sumatra island [e.g. 10].

II. AcehSeis
On collaboration work between German Research Center for Geosciences (GFZ-
Potsdam), Meteorological, Climatological and Geophysical Agency of Indonesia
(BMKG) and University of Syiah Kuala (Unsyiah), we conduct an experiment
of local seismic in Bener Meriah and Central Aceh, Aceh province. The network
consists of thirty short period seismic sensors distributed in the area of Bener
Meria and Central Aceh (see Figure 1). The sensors belong to and imported
from GFZ-Potsdam, Germany. The specification of the sensors uses “CUBE”
data logger, three components 100Hz seismometers. We use Mark products L-4
seismometers with the power supply from solar panels. The network is introduced
as AcehSeis network. This network is operated temporarily for six months from
August 2014 to January 2015.

A. Objectives
The objectives of AcehSeis project are to identify the local faults and seismic
structure in Aceh. Imaging the local travel time as well as attenuation tomography.
To complete the 1D and 3D database of velocity model. To identify the possibility
caldera of Danau Laut Tawar. To fill the gap of overall comprehensive investigation
of great Sumatra fault along Sumatra island from northern to southern part.

B. Methods
Several methods are poposed to be applied using data of AcehSeis project, include
1) Travel times of P and S wave arrivals will be used to derive the seismicity
distribution at high resolution [e.g. 11]. HypoDD method [12, 13] will be
applied to localize the better accuracy of hypocenters. The magnitudes of the
earthquakes will be determined and analyzed.
2) The polarity of first arrival waveforms are used to derive focal mechanisms
and to estimate the stress field in the region [e.g. 14].
3) Travel times of P and S waves are used to determine 1D and 3D Vp and Vp/
Vs velocity structures in the study area.
Innovation for Better ASEAN Community 323

4) Waveform spectral properties of P and S waves are used to derive the attenu-
ation structure of the target region [e.g. 15].
5) Long-term registrations will allow us to recover the ambient noise generated
surface waves from the data, and to apply inversion methods for the imaging
of surface wave velocities and S velocity structure [e.g. 16].

C. Time Schedule
The project was prepared during April to June 2014. The installation was done
in July 2014. Data collecting and stations services will be done monthly from
September 2014 to January 2015. Deinstallation will be done in February 2015.
The data analysis will be started from October 2014.

III. R esults
The figures below show the process of deployment of seismometers in July 2014
(see Figure 3) and data collection in September 2014 (see Figure 4).

Figure 3. Seismometer installation in Central Aceh on July 2014.


324 Proceeding ASEAN COSAT 2014

Figure 4. Station service and data collection in September 2014.

Figure 5. Waveforms of earthquake event on August 1st, 2014 with the magnitude 4.9
and depth of 10 km. The event was located at 4.37N and 96.6E.
Innovation for Better ASEAN Community 325

Several earthquakes have been recorded during the period of July to September
2014. One of relatively large magnitudes was an event on August 1st, 2014 with
the magnitude 4.9. The earthquake was located at 4.37N and 96.6E with the
depth of 10 km. Figure 5 shows the event was recorded by the whole 30 stations.
The signal to noise ratio of the records are very large, the waveforms are in a very
good quality.

IV. Conclusions and E xpected impacts


The network has been running well. Several events have been recorded during the
period of station running. At this moment we collect the data, check the quality
and localization process.
From the seismicity location and relocation as well as the focal mechanism
analysis, we could derive the detailed fault structure of the region. This is important
for the BMKG database and earthquake disaster mitigation.
The combination of the previous and the new data could be used to determine
the regional 1D and 3D model of Sumatra and the distribution of active faults.
The new regional 1D and 3D model could be assigned in the procedure of the
localization of the on shore earthquakes in Sumatra.
The seismic structure could be used to understand the volcanic activities in
the region. The seismic structure also provides the information of the sedimentary
basin as well as the possibility caldera of Danau Laut Tawar. Together with
the detailed fault structure, geological observation could be used to further
investigate the seismic hazards area and the possibility of landslides triggered by
the earthquakes. It will significantly contribute to future disaster mitigation in
the region.

V. Acknowledgement
We thanks to the local government as well as the ministry of energy and mineral
resources of Indonesia (ESDM) branch office central Aceh for supporting the
project. The project is funded by GFZ-Potsdam and supported by BMKG and
Unsyiah.

VI. R eferences
1) Wulandari, B.R. & N. Hurukawa (2013). Relocation of large earthquakes along
the Sumatran fault and their fault planes. Bulletin of the International Institute
of Seismology and Earthquake Engineering ISSN 0074-655X CODEN
IISBB2, vol. 47, pp. 25-30 [6 page(s) (article)] (1/4 p.)
326 Proceeding ASEAN COSAT 2014

2) Stein, Seth., & Emile A. Okal. (2005). Speed and size of the Sumatra earthquake.
Nature 434, 581–582.
3) Nalbant, Suleyman S., Steacy, Sandy, Sieh, Kerry, Natawidjaja, Danny &
John McCloskey. (2005). Earthquake risk on the Sunda trench, Nature 435,
756–757.
4) Walker, K. T., M. Ishii, and P. M. Shearer. (2005). Rupture details of the 28
March 2005 Sumatra Mw 8.6 earthquake imaged with teleseismic P waves,
Geophys. Res. Lett., 32, L24303, doi:10.1029/2005GL024395.
5) Borrero, Jose C., Weiss, Robert, Okal, Emile A., Hidayat, Rahman, Suranto,
Arcas, Diego, and Vasily V. Titov. (2009). The tsunami of 2007 September 12,
Bengkulu province, Sumatra, Indonesia: post-tsunami field survey and numerical
modeling, Geophys. J. Int. (2009) 178 (1): 180-194 doi:10.1111/j.1365-
246X.2008.04058.x
6) Lay, T., C. J. Ammon, H. Kanamori, Y. Yamazaki, K. F. Cheung, and A. R.
Hutko. (2011). The 25 October 2010 Mentawai tsunami earthquake (Mw 7.8)
and the tsunami hazard presented by shallow megathrust ruptures, Geophys. Res.
Lett., 38, L06302, doi:10.1029/2010GL046552
7) Muzli, M. (2013). Combined evaluation of strong motion and GPS data for
analyzing coseismic deformation caused by strong earthquakes, PhD Thesis,
(Scientific Technical Report ; 13/06), Potsdam: Deutsches GeoForschun-
gsZentrum GFZ, 142 p. DOI: http:// doi.org/ 10.2312/ GFZ.b103-13067
8) Sari, N. (2013). Hypocenter relocation of earthquakes in Aceh using
HypoDD, unpublished results. STMKG.
9) Tim Survey BMKG. (2013). Laporan gempabumi Bener Meriah 2013,
unpublished reports, BMKG.
10) Masturyono, R. McCaffrey, Wark, D. A., Roecker, S. W., Fauzi, Ibrahim,
G., and Sukhyar. (2001). Distribution of magma beneath the Toba caldera
complex, north Sumatra, Indonesia, constrained by three-dimensional P wave
velocities, seismicity, and gravity data, Geochem. Geophys. Geosyst., 2, 1014,
doi:10.1029/2000GC000096
11) Muksin, U., Bauer, K., and C. Haberland. (2013a). Seismic Vp and Vp/Vs
structure of the geothermal area around Tarutung (North Sumatra, Indonesia)
derived from local earthquake tomography. Journal of Volcanology and Geo.
Research 260, 27–42.
12) Waldhauser, F. and W.L. Ellsworth. (2000). A double-difference earthquake
location algorithm: Method and application to the northern Hayward fault, Bull.
Seism. Soc. Am., 90, 1353–1368.
Innovation for Better ASEAN Community 327

13) Waldhauser, F. (2001). HypoDD: A computer program to compute double-


difference earthquake locations, USGS Open File Rep., 01–113
14) Muksin, U., Haberland, C., Bauer, K., M., Weber. 2013b. Three-dimensional
upper crustal structure of the geothermal system in Tarutung (North Sumatra,
Indonesia) revealed by seismic attenuation tomography. Geophysical Journal
International 195, 2037–2049.
15) Muksin, U., Haberland, C., Nukman, M., Bauer, K., M., Weber. 2014.
Detailed fault structure of the Tarutung pull-apart basin in Sumatra-Indonesia
derived from local earthquake data. Journal of Asian Earth Sciences (submitted).
16) Stankiewicz, J., Ryberg, T., Haberland, C., Fauzi, D. Natawidjaja. (2010).
Lake Toba volcano magma chamber imaged by ambient seismic noise tomography.
Geophys. Res. Lett. 37, L17306. ­
Selection of Global GMPEs models
for Seismic Hazards Assessments in Indonesia
(Case Study Sumatra-Java area)
Ariska Rudyantoa,*, Phil Cummins b,c, Hadi Ghasemic, I Nyoman Sukantaa
Badan Meteorologi Klimatologi dan Geofisika,
a

Jl. Angkasa I, No.2 Kemayoran Jakarta Pusat, 10720 Indonesia


b
Research School of Earth Sciences-The Australian National University
Bld 142 Mills Road Acton ACT, 0200 Australia
c
Geoscience Australia
Cnr Jerrabomberra Ave and Hindmarsh Drive, Symonston ACT, 2609 Australia

Abstract
This study focuses on investigation published by Ground Motion Prediction Equations (GMPE)
which is appropriate to be used in Indonesian earthquake hazard assessment, especially for study
area in Sumatra-Java region. The relevant GMPEs compared in this study are based on the
resemblance of geologic and tectonic conditions of the regions where the GMPEs were developed
to the study area. Twelve GMPEs have been considered in this study, consisting of nine GMPEs
derived for subduction-zone event types (intraslab and interface regimes) and three GMPEs
derived for the crustal regime. The analysis of GMPEs in this study was done using the graphical
analysis of residuals between the observed ground motion value and the corresponding values
predicted by each GMPE. The visual analysis of the statistical graphs presented in this study
indicates four GMPEs (Youngs (1997), Zhao (2006), Kanno (2006) and Lin-Lee (2008) that
match with the recorded data well, while the others have poor ft with the data. In this study, we
also rank the GMPEs using the quantitative method proposed by Scherbaum et.al (2004). The
Scherbaum e.al (2004) scheme shows that comparison of PGA/PSA with threshold value 0.0005
m/s2 gives a better output than using all data. In this study, we found that among all models,
only the Youngs (1997) and Zhao (2006) models provide predictions that are consistent with
the data from BMKG’s network.
Key words: Ground Motion Prediction Equations (GMPE), Seismic hazard assesment, Strong-ground
motion.

I. Introduction
Indonesia is one of the most seismically active country in the world. The
Indonesian tectonic system is highly complex, resulted from the interaction of
three major plates: the Eurasia plate in the north-northwest, the Indian-Australian
plate in the south and the Pacific plate in the east. This tectonic activity results in
rugged topography, frequent earthquakes (sometimes also followed by tsunamis)

* Corresponding Author.Tel: +062-8563322617. E-mail: ariska.rudyanto@bmkg.go.id, rudyantoariska@gmail.com

329
330 Proceeding ASEAN COSAT 2014

and volcanic eruptions that affect not only in Indonesia but also the surrounding
of Southeast Asian region as well. In addition, the tectonic setting in Indonesia
includes dozens of active faults spread throughout the country.
As a part of efforts to improve seismic hazard assesment, the Indonesian
government suported the development of the latest Indonesian seismic hazard
map in 2010 [1]. This map followed the International Building Code 2009 (IBC
2009) [2] recommendation in presenting spectral hazard maps. This work utilised
many new findings from various research fields related to the seismic activity and
the tectonic setting of Indonesia.
Some of the most important inputs to this map are appropriate attenuation
models for earthquake ground motion, known as Ground Motion Prediction
Equations (GMPEs). GMPEs are represented as functions of the earthquake
source, propagation path and local site conditions. However, due to the lack of
strong ground motion records for Indonesia, there is no current Indonesia-specific
GMPE available for seismic hazard studies and for this reason, they were forced
to rely on GMPEs developed for other tectonically active regions [3].
Since 2006, the Indonesian Agency for Meteorology, Climatology and
Geophysics (BMKG) has operated a seismographic network of approximately
158 broadband seismographs (InaTEWS, 2010) [4]. Subsequently, BMKG
supplemented this seismograph network with acceleropgraphs, in order to monitor
strong ground motion caused by earthquakes. This strong-motion network has
grown steadily since its establishment in 2007. Many earthquakes recorded by
this strong-motion network since its establishment provide a potentially useful
source of unique data that could be used to constrain GMPEs used in Indonesian
seismic hazard assessments and impact analysis [5].

II. Strong Motion Data


Strong-motion records used in this paper come from events with greater magni-
tude than or equal 5 and epicentral distance less than or equal 500 km. In this
study, we are interested in Sumatra and Java islands region inside the area of 2o
North–11o South and 90o East–116o East. In the development of a strong-motion
database, it is important to know what type of earthquake generated the ground
motion at a particular site. The classification of the earthquake plays a vital role
to understand the characteristics of ground motion attenuation because ground
motion from different types of earthquake will attenuate differently at similar
distance due to e.g., their different stress drop and path effects.
Innovation for Better ASEAN Community 331

Figure 1. Cross-section of the subduction zone in Java (up) and Sumatra (below)
segment and distribution of earthquakes used in this study.
332 Proceeding ASEAN COSAT 2014

A. Earthquake Classification
Commonly, for seismology and engineering purposes, earthquakes are classified
into 3 different categories: interface, intraslab and shallow crustal earthquakes.
The scheme to classify the events is not only based on the focal depth and focal
mechanism, but it also should consider the geometry of the slab. Along the
Indo-Australia subduction zone in the Sumatra and the Java segments, events are
classified as intraslab events if they have depths more than 40 km, while crustal
events are defined as those that is located in the distance more than 200 km from
trench and with depth less than 40 km. For events less than 200 km from the
trench and with depth less than 40 km, they will inspect carefully to determine
whether they are intraslab or megathrust/interface events using focal mechanism
information. For example if the focal mechanism is thrust, they are classified as
interface; if not, they are classified as unknown [6].
Based on this approach, it was found among 249 earthquakes considered in
this study 63 earthquakes categorizes are interface/megathrust, 161 earthquakes
are intraslab, 4 earthquakes are crustal and 21 earthquakes are unknown types. The
database used in this study consists of 3,090 records from about 249 earthquakes
(Figure 1).

B. Data Distribution
According to the magnitude-depth distribution, the data are primarily dominated
by shallow earthquakes with depth less than 60 km (more than half of the
records) and only a few records were for event with depths more than 150 km.
Furthermore, the data mostly came from small earthquakes with magnitude less
than or equal to magnitude 6 and only a small amount of data were recorded for
events of magnitude greater than or equal 7. The strong motion records in this
study were dominated by the earthquake from the subduction zone (i.e. intraslab
and interface regimes). Only a few strong-motion records comprise the crustal
regime. Moreover, intraslab event records comprise almost a half of the data.

III. Data Processing


In addition to selection of data based on geographic region (i.e., Sumatra-Java)
distance to event (less than 500 km), pre-processing was applied to remove data
with non-standard errors from the database. Following selection of data and
pre-processing, the processing stages used in this study involve: mean removal,
baseline adjustment and instrumental correction, band pass filtering, integration
of the acceleration to obtain velocity and displacement and calculation of response
spectra of acceleration, velocity and displacement waveforms. The processing
Innovation for Better ASEAN Community 333

approaches used in this study followed the scheme proposed by Boore et al.
(2002) [7] and Boore and Bommer (2005) [8].
An outline of this procedure was as follows (Boore, et al., 2005) [8]: (1)
Compute mean of the pre-event portion of the record (stopping a second or
so short of the estimated firrst arrival) and subtract that mean from the whole
record (the zeroth order correction), (2) Integrate to velocity, (3) Fit a quadratic
to velocity, starting at the time of the first arrival and constraine to be 0.0 at
the start time, (4) Remove the derivative of the quadratic from the zeroth order
corrected acceleration, and (5) filtering. In most cases, the long and short-period
noise in the accelerograms are removed by filtering. An acausal Butterworth
filter of fourth order was applied in this study for baseline adjustment and noise
reduction in the accelerograms.
In this study, the desired ground motion parameters were computed for each
set of acceleration, velocity and displacement records as peak ground acceleration
(PGA, in m/s2), peak ground velocity (PGV, in m/s) and peak ground displace-
ment (PGD, in m). The spectra were calculated using 5% damping and for about
100 frequencies, spanning from 0.25 hz up to 25 hz. The calculation of these
spectra followec the scheme by Nigam and Jennings (1968) [9].

IV. Goodness-of-fit of Selected GMPEs


A. Selected GMPEs
GMPEs are commonly used to describe the efects of source, propagation medium
and local site condition on ground motion. Since the dataset is dominated by
strong ground motion records from the subduction zones (intraslab or interface
earthquake type), the GMPEs considered in this study are also dominated by
those derived for subduction type earthquakes.
Nine GMPEs considered in this study are derived for subduction-zone event
types (intraslab and interface regimes) using worldwide or regional strong-motion
records. These models are Youngs et,al. (1997) [10], Atkinson and Boore (2003)
[11], Garcia et. al. (2005) [12], Zhao et. al. (2006) [13], Kanno et. al. (2006)
[14], Lin and Lee (2008) [15], Hong et. al. (2009) [16], Arroyo et al. (2010)
[17] and Gupta et al. (2010) [18].

B. Analysis of Residual and Statistical Measurement


The analysis of ground motion residuals plays an important role in the selection
of appropriate GMPEs. If the error term in a GMPE is consistent with the
observed data, then the model is considered valid. A graphical residual analysis
is a useful tool for validating the model even though there are many others that
can be used (NIST/SEMATEC, 2012) [19]. The graphical analysis of residuals
334 Proceeding ASEAN COSAT 2014

is worthwhile because it is efective in discerning patterns in the residuals, but it


is not recommended to use this technique as a quantitative analysis for selection
of GMPEs.
The normalised residuals used in this study are defined as the diferences
between the observed ground motion value (either both peak ground accelerations

(log(obs.value)- log (predict.value) (1)


R=
(standard deviation of predict.value)

or peak spectral accelerations) and the corresponding values predicted by each


GMPE, and divided by the corresponding standard deviation of the GMPE.
In this study, we focus on observing the patterns of residual variance. For that
purpose, we employ histogram plot as a graphical residual analysis. The histogram
of the normal (Gaussian) distribution can be used to assess whether the variance
of the data is normally distributed or not. The assumption that the measurement
variables follow or at least approximate log normal distributions has been tested
empirically (NIST/SEMATEC, 2012) [19].
Since the graphical residual analysis cannot provide a quantitative measure-
ment of misfit, it is necessary to apply other statistical tests for the model selection.
In this study, we employ the likelihood-based measurement known as the LH
test. This test is a recent technique developed solely to investigate and evaluate
GMPEs (Scherbaum et al.l, 2004) [20]. The LH test is a data-driven test that
calculates likelihood parameters and not only quantifes the model fit, but also
the shape of the underlying distribution of model residuals.
The LH value used in this approach is as follows:

where Erfc is the error functions, and Res is stands for the normalised residuals.
The Erfc is define by:

V. Conclusion
Since the strong ground motion dataset used in this study is dominated by the
data associated with the subduction-zone regime (intraslab and interface) and
only a small amount comes from crustal environment, it is not adequate to apply
such statistical tests to data from crustal earthquakes.
Innovation for Better ASEAN Community 335

Figure 2. Distribution of normalised residuals and associated likelihood for interface (left) and
intraslab (right) regime (from upo to down respectively) of A. Youngs (1997); B. Zhao (2006);and
C. Lin-Lee (2008) at PGA for all dataset

Based on plots comparing normal (Gaussian) distributions with histogram


normalised residuals, only three GMPEs give a good matching (Figure 2). Those
three GMPEs are Youngs (1997), Zhao (2006) and Lin and Lee (2008). These
results are similar both for PGA and PSA at 1 second period, and also the same
for both intraslab and interface earthquake regimes. These analysis express the
suitability of the use of those three GMPEs in the area of study.
336 Proceeding ASEAN COSAT 2014

Table 1. Summarised mean, median and standard deviation of different GMPEs for PGA and
their ranking using Indonesian strong ground motion dataset.

The analysis of capability of selected GMPEs using all dataset based on


Scherbaum et.al (2004) scheme shows that all selected GMPEs are unacceptable
(Table 1 and Table 2). The larger variability on residuals might be because of poor
quality of the data, especially from early years, and the lack of knowledge on site
information of the network.
Explanation for poor at between recorded data and associated predictions by
GMPEs are presented by many researchers. Boomer et.al. (2007) [21] demonstrate
that small earthquakes will produce ground motion with larger variability than
moderate and large earthquakes. Douglas et.al (2006) [39] explain that the data
from different magnitude and distance ranges between observations and those
used in development of the selected GMPEs can be responsible for the low quality
of GMPE predictions.
Accordingly, we try to investigate the capability of selected GMPEs by control-
ling some aspect of the recorded strong ground motion data. Here, we restrict the
PGA or PSA value of the data, and use only the data that have the values larger
Innovation for Better ASEAN Community 337

Table 2. Summarised mean, median and standard deviation of different GMPEs for PSA T= 1
s and their ranking.

than or equal to 0.0005 m/s2 in our analysis. In general as seen in Table 10, the
performance of Youngs (1997), Zhao (2006), Kanno (2006) and Lin-Lee (2008)
GMPEs on predicting PGA of the recorded strong ground motion data with this
threshold value are better than others (Table 3 and Table 4). The Zhao (2006)
GMPE gives the best performance in PGA prediction in this analysis, both for
interface and Intraslab regimes, and is classifed as C (the lowest capability class).
Conversely, Youngs (1997), Kanno (2006) and Lin-Lee (2008) are not good
enough to be classified as C class, even though their parameters are close to the
standard values of the C class. The quality of prediction of most target models
for PSA at 1 second period show the same pattern as for the PGA prediction,
with the Zhao (2006) and Youngs (1997) GMPE has better performance, while
the others have results consistent with PGA.
338 Proceeding ASEAN COSAT 2014

Table 3. Summarised mean, median and standard deviation of different


GMPEs for PGA and their ranking with PGA more than 0.0005 m/s2.

Table 4. Summarised mean, median and standard deviation of different


GMPEs for PSA T= 1 s and their ranking with PSA more than 0.0005 m/s2.
Innovation for Better ASEAN Community 339

VI. R eferences
1) Tim Revisi Peta Gempa Indonesia. (2010). Ringkasan Hasil Studi Tim Revisi
Peta Gempa Indonesia 2010. Bandung 1 Juli 2010. Laporan Studi.
2) International Code Council, Inc. (2009). Internasional Building Code.
3) Sengara, I W. (2010). Analisis hazard gempa probalistik pulau jawa untuk
masukan dalam peta Zonasi gempa Indonesia. BMKG Scientific Journal Club.
Jakarta. (in Bahasa Indonesia).
4) Inatews (2010). Indonesia Country Report. ICG/IOTWS meeting. Banda Aceh.
5) Rudyanto, A. (2013). Development of Strong-motion Database for The
Sumatra-Java Region. Thesis. ANU-Canberra.
6) Boore, D. M. and J. J. Bommer. (2005). Processing of strong-motion ac-
celerograms: Needs, options and consequences, Soil Dynamics and Earthquake
Engineering 25, 93.
7) Nigam, N.C., and P. C. Jennings (1968). Digital calculation of response spectra
from strong-motion earthquake records, Report, Earthquake Engineering Research
Laboratory, California Institute of Technology. Pasadena, California.
8) Douglas, J. (2011). Ground-motion prediction equations 1964 - 2010, PEER
Research Reports,
9) Villaverde, R. (2009). Fundamental Concepts of Earthquake Engineering,
246-260. CRC Press..
10) Youngs, R. R., Silva, W. J., et.al. (1997). Strong ground motion attenuation
relationships for subduction zone earthquakes. Seismological Research Letters
1997(1), 58–73.
11) Atkinson and David M. Boore. (2003). Empirical Ground-Motion Relations
for Subduction Zone Earthquakes and Their Application to Cascadia and
Other Regions. Bulletin of the Seismological Society of America, Vol. 93, No. 4,
1703–1729.
12) Garca D, Singh SK, Herriz M, Ordaz M, and JF., Pacheco. (2005). Inslab
earthquakes of central Mexico: Peak ground-motion parameters and response
spectra. Bull Seismol Soc Am 95(6):2272–22
13) Zhao JX, Irikura K, Zhang J, Fukushima Y, Somerville PG, Saiki T, et.al.
(2004). Site classifcation for strong motion stations in Japan using H/ V
response spectral ratio. 13th World conference of earthquake engineering. Paper
no. 278 Vancouver, BC, Canada.
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14) Kanno, T., Narita, A., Morikawa, N., Fujiwara, H., and Y., Fukushima.
(2006). A new attenuation relation for strong ground motion in Japan based
on recorded data. BSSA, 96(3), 879–897.
15) Lin P-S, and C-T., Lee. (2008). Ground-motion attenuation relationships
for subduction-zone earthquakes in northeastern Taiwan. Bull Seismol Soc
Am 98(1):220–240.
16) Hong, H. P., Pozos-Estrada, A., and R. Gomez. (2009). Orientation efect
on ground motion measurements for Mexican subduction earthquakes.
Earthquake Engineering and Engineering Vibration, 8(1), 1–16.
17) Arroyo, D., GarciLa, D., Ordaz, M., Mora, M. A., S. K., Singh. (2010).
Strong ground-motion relations for Mexican interplate earthquakes. Journal
of Seismology, 14(4), 769–785.
18) Gupta, I. D. (2010). Response spectral attenuation relations for in-slab
earthquakes in IndoBurmese subduction zone. Soil Dynamics and Earthquake
Engineering, 30(5), 368-377.
19) NIST/SEMATECH. (2012), e-Handbook of Statistical Methods, http://
www.itl.nist.gov/div898/handbook/.
20) Scherbaum F, Cotton F, P., Smit. (2004). On the use of response spectral-
reference data for the selection andranking of ground-motion models for
seismic-hazard analysis in regions of moderate seismicity: The case of rock
motion. Bull Seismol Soc Am 94(6), 2164–2185.
21) Bommer, J. J., P. Staford, J. E. Alarcn, and S. Akkar. (2007). The inuence of
magnitude range on empirical ground-motion prediction, Bull. Seismol. Soc.
Am. 97, 2152-2170.
SCMIT
LED-based Spectrometer for Advanced
Chemistry Laboratory Experiments
Mary Angelie Alagaoa ,*, Dwight Angelo Bruzonb, Imee Su Martinezb,
Giovanni Tapanga
National Institute of Physics
a

University of the Philippines-Diliman, Quezon City, Philippines


b
Institute of Chemistry
University of the Philippines-Diliman, Quezon City, Philippines

Abstract
An LED-based spectrometer that can scan in the visible range is demonstrated in a classroom
environment using the Arduino platform. The performance of the device was tested and evaluated
using solutions with absorbance in the visible region. The results obtained using the fabricated
instrument were compared to theoretical values obtained using a commercial available UV-VIS
Spectrometer. The development of a low-cost LED-based spectrometer, which is an initiative
of the Versatile Instrumentation System for Science Education and Research (VISSER), serves
as a tool to improve education, especially in laboratory class experiments, since commercial
spectrometers are expensive.
Keywords: Spectrometer, Light emitting diodes, Chemical sensors

I. Introduction
Spectrometers have always kept its timeless appeal in physical and analytical
chemistry for its applications in quantitative and qualitative analysis of various
chemical solutions. Its applications include, but are not limited to, kinetic studies,
wherein it is used to determine the rate of a reaction by analyzing the absorbance
over time of a reactant or product in the visible or ultraviolet region, and analytics,
wherein it is used to determine the concentration of an analyst through either
fitting from a calibration curve using a set of standards or using the standard
addition method. However, due to the optical components that make up a typical
spectrometer, commercially available ones are expensive.
The growing demand for portable sensing devices to monitor health,
environment and security has led to the use of light emitting diodes (LEDs) as
alternative to conventional light sources [1]. Compared to other light sources,
such as fluorescent lamps and incandescent bulbs, an LED offers convenience
by having a relatively longer lifespan, smaller size, lower cost and better optically

* Corresponding Author.Tel: +63 (02) 9818737. E-mail: malagao@nip.upd.edu.ph.

343
344 Proceeding ASEAN COSAT 2014

Figure 1. Typical Schematic Diagram of a Spectrometer

stability (as light source), which makes it more useful in battery-powered or


energy-saving devices.
It was in 1970 that the use of LEDs for photometers has been conceptualized
by Barnes, et al [2]. The LEDs were coupled with a phototransistor, which serves
as the detector. Different configurations have been designed to maximize the use
of LEDs in spectroscopy. Hauser, et al., used a blue LED as the light source to
test for Cr, Mn, Zn, Fe and Cl. Results were compared to those obtained from
conventional molecular absorption spectroscopy [3]. An array of LEDs coupled
with a fiber optic cable was also used to cover a wide range of wavelengths for
colorimetric methods [4].
While it is a known fact that LEDs emit light, a study by Forrest Mims in
1992 showed that an LED could also become a detector when reverse-biased.
The range of wavelength of light emitted by an LED is shifted from the range
that can detect [5]. This discovery has led to the use of LEDs in sun photometers
to measure perceptible water and atmospheric turbidity in a wavelength range
of 555 nm to 940 nm. These sun photometers were developed for the Global
Learning and Observation to Benefit for the Environment (GLOBE) program
that compare the performance of the LED-based sun photometers to conventional
filter-based instruments [6].
Multiple LED detectors of various colors were used to determine pH and
analyze heavy metals using a tungsten halogen lamp as a light source [7]. In 2013,
a paper by Shim and Eom suggested using a pair of LEDs to measure absorbance.
Two red LEDs were placed across each other to measure the absorbance of
Bromothymol blue (BTB), a common pH indicator [7].
The emitter-detector capability of light emitting diodes can be used to develop
a spectrometer for various spectrometry experiments, such as determining the
concentration of analyses in a solution. Using only LEDs, a spectrometer can be
constructed, owing to the pressing demand for alternative instrumentation that
are economical and highly convenient.
In the Philippines, Versatile Instrument System for Science Education and
Research (VISSER) develops microcontroller-based instruments and sensors for
Innovation for Better ASEAN Community 345

education and research applications. Its goal is to be able to develop reliable


instruments at a low cost. Among the instruments developed is a low-cost
spectrometer using LEDs for chemical analysis.
This study investigates the use of LEDs functioning both as emitters and
detectors in spectrometry. For signal processing, an open source software, such
as Arduino is used. The instrument can scan in the visible range or wavelengths,
making it a useful tool in performing spectrometry experiments in a laboratory
class setting where a hands-on experience on using the instrument is important
and the availability of the commercial instrument to the students is limited.

II. Method/M aterial


A. Test solutions
Three test solutions were used: a solution of pH 4, containing 1% KHP and 0.04%
CH2O, a solution of pH 7, containing 0.68% KH2PO4 and 0.08% NaOH, and
a solution of pH 10, containing 0.38% Na2B4O7 · 10 H2O and 0.05% NaOH,
all obtained from LabChem.
To determine the response of the instrument in varying concentrations of an
analyte, five (5) solutions of copper (II) sulphate (CuSO4) were used. Required
amounts of solid copper (II) sulphate were dissolved in 50 ml distilled water to
make standard solutions with concentrations ranging from 0.02 mol/L to 0.1
mol/L.

B. Instrument
Ten (10) different colors of off-the-shelf available LEDs were used both as the
emitter and the detector in determining the spectra of solutions. LEDs were
chosen based on the color they emit as to have a representative emitter and
detector in the visible range.
The constructed spectrometer was tested on different solutions previously
prepared. The LED emitter-detector pair were placed one across the other with
a standard quartz cuvette in-between. The relative intensity detected by the LED
detector was read using an Arduino. The schematic diagram and an image of the
prototype are shown in Figure 2.
A quartz cuvette was 4/5 filled with the test solutions and was inserted in
the fabricated device. Three trials/readings were done for each color and for each
solution. Each reading taken was the average of five consecutive outputs of the
instrument.
The spectral plot obtained using the LED-based spectrometer was compared
to results obtained using a Vernier spectrometer in order to determine the
performance of the fabricated instrument.
346 Proceeding ASEAN COSAT 2014

(a)

(b)
Figure 2. LED-based Spectrometer (a) Schematic Diagram
(b) Image of the Prototype

III. R esults and Discussion


The spectral emission and detection of the LEDs used were obtained and shown
in Figures 3a–3e, respectively.
It can be noted that the relative intensity of its emission is higher than its
detection and the range of the wavelength the LED detects is not the same as
the range it emits.

(a) Blue LED


Innovation for Better ASEAN Community 347

(b) Green LED

(c) Yellow LED

(d) Orange LED


348 Proceeding ASEAN COSAT 2014

(e) Red LED


Figure 3a-3e. Spectral emission and detection of the LEDs used in the instrument

The spectrum of a solution, Q, is a superposition of the basis spectra of the


different LEDs as shown in Equation 1.
Q = ΣaiSi(λ) (1)
where ai is the voltage reading for LED detector, i, and Si(λ) is the basis spectra
or the detection spectra of the LED detectors.
In this prototype, the highest voltage that the Arduino can detect is 3 volts,
giving us a resolution of 2.9 mV per unit. Each reading obtained per LED, given
by I, for a solution is divided over its reading on a blank solution, Io in order to
compute for the transmittance as shown in Equation 2.
%T = I/Io x 100% (2)
sing Equations 1 and 2, the transmittance plots of the test solutions were
U
obtained. Figure 4 shows the superimposed spectral plots of the solutions
obtained using the commercial spectrometer and the LED-based spectrometer.
The spectra of the different buffer solutions were compared in the wavelength
range where the transmittance peaks are expected. Results obtained using the
LED-based spectrometer are comparable to the results from the commercial
spectrometer for the blue and red buffer solutions.
The LED-based spectrometer was also tested on varying concentrations of
copper sulfate solutions. Figures 5 and 6 show the transmittance spectra for
Innovation for Better ASEAN Community 349

(a)

(b)

(c)
Figure 4. Spectra of the (a) blue, (b) green and (c) red buffer solutions
350 Proceeding ASEAN COSAT 2014

the copper (II) sulphate solutions using the LED-based spectrometer and the
commercial spectrometer, respectively.
On Figure 7, it can be noted that the transmittance decreases as the concentra-
tion increases from 0.02 M of Cu2+ to 0.1 M Cu2+. However, for the solutions
with 0.08 M of Cu2+ and 0.1 M of Cu2+, the LED-based spectrometer was not
able to distinguish the varying concentration.

Figure 6. Spectral plot of the copper sulphate solutions using the LED-based spectrometer

Figure 7. Spectral plot of the copper sulphate solutions using the commercial spectrometer
Innovation for Better ASEAN Community 351

Figure 8. Optimal Resolution of the LED detectors

On the other hand, Figure 8 shows that the transmittance at increasing


concentrations increases in the range 400nm to 650nm while decreases beyond
the visible range.
It can also be noted that the LED-based spectrometer failed to produce the
expected shape of the transmittance spectra of the solutions. This can be due to
the resolution of the LEDs used. At 430 nm, by fitting a Gaussian function with
varying standard deviation, thereby the spectral bandwidth, it was observed that
a blue LED detector could only reconstruct a spectrum in the blue range if it has
a spectral width of at least 30 nm. Shown in Figure 8 is the optimal resolution
of the LED-based spectrometer for each wavelength. This shows the spectral
bandwidth at which the developed LED-based spectrometer can reconstruct at
a particular wavelength.

IV. Conclusion
A spectrometer was constructed using different LEDs functioning both as the
emitters and detectors to measure transmittance of various solutions. Solutions
of varying absorbance in the visible region and concentration were tested using
the fabricated spectrometer and results were compared to values obtained from
a commercial spectrometer.
The LED-based spectrometer was able to show that at increasing concentra-
tions, the transmittance decreases. However, due to the current resolution of
the LED-based spectrometer, it was not able to distinguish all concentrations
and produce the expected transmittance spectra of the solutions. The device can
352 Proceeding ASEAN COSAT 2014

further be improved by adding more LEDs at varying wavelength in order to


cover the whole spectrum.

V. Acknowledgment
This research is under the Versatile Instrumentation System for Science Education
and Research (VISSER), a project funded by the Philippines Council for Industry,
Energy and Emerging Technology Research and Development (PCIEERD) and
the Office of the Vice Chancellor for Academic Affairs– Emerging Interdisciplinary
Research Grant (OVPAA-EIDR).

VI. R eferences
1) O’Toole, M. and D. Diamond. (2008). Absorbance Based Light Emitting
Diode Optical Sensors and Sensing Devices. Sensors, 8, 2453-2479.
2) Flaschka, H., McKeithan, C., and R. Barnes. (1973). Light Emitting Diodes
and phototransistors in photometric modules. Analytical Letters, . 6, 585–594.
3) Schawarz, M.A. and P.C. Hauser. (2001). Recent developments in detection
methods for microfabriacated analytical devices. Lab on a Chip, 1. 1–6.
4) Hauser, P.C., Rupasinghe, T.W.T and N.E. Cates. (1995). Amulti-wavelength
photometerbased on light-emitting diodes. Talanta, 42. 605–612.
5) Acharya. Y.B. (2005). Spectral and emission characteristics of LED and its
application to LED-based sun-photometry. Optics and Laser Technology,. 37
547–550.
6) Mims, F. (1992). Sun photometer with light emitting diodes as spectrally
selective detectors. Optical Society of Americal.
7) Berry, R.J. Harris, J.E., and R.R. Williams. (1997). Light-Emitting Diodes
as Sensors for Colorimetric Analyses. Applied Spectroscopy,. 51, 1521–1524.
8) Shin, Dong-Yong, and In-Young Eom. (2013). A Pair of Light Emitting
Diodes for Absorbance Measurement. Bulletin Korean Chemical Society,. 34,
No. 10.
Introduction investigation: Executive
Information System for University

Spits Warnarsa*, Sasmokoa, b, Nancy Susianna b


a
Surya University
Jl. Boulevard Gading Serpong Blok O/1 Summarecon Serpong, Tangerang, Indonesia
b
STKIP Surya
Jl. Scientia Boulevard Blok U/7 Gading Serpong, Tangerang, Indonesia

Abstract
University as higher education implementer has responsibility to accelerate their national human
capital in order to compete in highly competitive economic.The higher the national human capital,
the more prepared they are to compete in highly skilled labour. University as a knowledge keeper
should be equipped with information technology in order to establish well prepared university,
particularly to support high level management. High level management university, such as rector,
vice rector, dean and head of study program should be armed with information technology tool
which can help them when making their decisions become better and accurate. EIS (Executive
Information System) as highly DSS (Decision Support System) which is designed for high
level management is the appropriate information technology tool in order to sharp high level
management’s decision making. The EIS will be built up by 24 Indonesian national standards of
higher education, where each of standards will be generated as Key Performance Indicator (KPI)
to evaluate and justify the university performance from 3 perspectives such as education, research
and community services. The average KPI score of national standard of higher education for each
study program will show the quality assurance of each study program or faculty in university.
The study program or faculty performance should be assessed based on KPI national standard of
higher education and the higher KPI score total, the better quality of the study program. When
one of standards has poor quality then it will automatically alert the high level management.
EIS will be developed with web based programming and equipped with technology such as data
warehouse and data mining. Data warehouse should be used to extract data from university
data transaction into fact constellation schema, in order to increase performance including for
multidimensional purposing. Meanwhile, data mining will equipped with mining unexpected
patterns particularly from data warehouse. At the end, the better and accurate decision making
which is produced by high level management university will make it easy for the decision maker
to create well established higher education for the society and having high accreditation score
from Indonesian government. As a reward, the university will have a good reputation among the
competitors, invite best students, lecturers, researchers and funding as well.
Key words: Executive information system, Data warehouse for higher education, Quality assurance
of Indonesian higher education, Indonesian national standard of higher education, Indonesian higher
education accreditation.

* Corresponding Author.Tel: 62181318992388. E-mail: spits.warnarsr@surya.ac.id

353
354 Proceeding ASEAN COSAT 2014

I. Introduction
Recently, the Ministry of Education of the Republic of Indonesia has launched
12 years compulsory education, where in 2020 Indonesian citizens will have
minimum high school education [1]. The big concern of Indonesian government
upon the acceleration of their human capital shows that well higher educated
human capital as the most priority to build a nation, particularly to face ASEAN
Economic Community (AEC) in 2015. Obviously, concerning Indonesian
government in accelerating national human capital to prepare the nation to be
ready in highly competitive economic region. Nonetheless, the declaration of 12
years compulsory education is not enough in order to face element free flow of
skilled labour as one of five core elements of ASEAN single market. Indonesian
human capital should be prepared to accelerate more than 12 years compulsory
education and university as higher education has huge responsibility to develop
and maintain their national human capital.
Information technology as tools should be needed by organization in order to
run their organization activity, particularly in business or university organization.
Using information technology to support daily activity organization should
give value added benefit for the organization in order to compete with their
competitor. Those organizations that accelerate their organization activity will
be one step ahead rather than their competitor. University as high education
organization should be equipped with information technology tools in order to
run their university activities and nowadays there is no university still not using
information technology tools to support their daily university activity.
High level management as the university decision maker should be supported
with information technology. High level management such as rector, vice rector,
dean and head of study program should be equipped with high level management
application software which can help them to make sharp and better decisions.
This high level management application software should be connected and inline
between high level management university positions. It means that the data or
information input or output by head of study program should be connected and
inline with the data and information which are accessed by rector, vice rector or
dean of faculty.
Executive Information System (EIS) is proposed as high level management ap-
plication software to provide high level management with information technology
support in order to make sharp and better decisions. This EIS should be supported
with datawarehouse technology where star or snowflake or fact constellation
schema as the answer to make fast response creating reporting. In the future,
data mining technique can be implemented for mining education patterns, such
as frequent pattern, similar pattern, association rules and so on. At the end, this
Innovation for Better ASEAN Community 355

EIS will help high level management to provide excellent university services for
public. Indeed, EIS is a tool which provides relevant interesting information senior
manager and different with traditional information system, tailor to executive’s
information needs and access broad range of internal and external data, easy to
use, used directly without assistance and in graphical form [16].

II. R esearch Method


This research is categorized as applied research where the outcome of this research
should be implemented in the implementation of real software application. The
research should be done in 4 steps, they are
1) Literature survey.
The literature survey should be done in three sub activities, they are:
a) Information technology literature survey.
Do literature survey for current information technology application which
is used by university high level management to make their decisions.
b) Indonesian rules literature survey
Read regulation product such as law, presiden regulation, government
regulation, minister regulation which are issued for Indonesian higher
education accreditation both of internal or external quality assurance.
c) Quality assurance survey.
Do literature survey for national and international quality assurance.
2) Mapping national standard of higher education framework
Based on Indonesian rules literature survey, 24 national standards of higher
education should be implemented as Key Performance Indicator (KPI) to
evaluate and justify the university performance from 3 perspectives such as
education, research and community services.
3) Design prototyping application of EIS
Designing EIS application which is should be done in 6 steps, as follows:
a) Collect current university database Online Transcational Processing
(OLTP) and model the database design with Unified Modeling Languages
(UML) class diagram.
b) Generate reporting based on KPI from 24 national standard of higher
education, including survey result for qualitative KPI.
c) Model database design for star or snowflake or fact constellation schema
data warehouse with Unified Modeling Languages (UML) class diagram.
d) Design Extraction Transformation and Loading (ETL) algorithm as a
process to load data from university database OLTP into data warehouse.
356 Proceeding ASEAN COSAT 2014

e) Design EIS application business process with Unified Modeling Language


(UML) use case.
f ) Design user interface to entry qualitative KPI and EIS application.
4) Implementation prototyping
The EIS application would be implemented with web based programming
like Personal Home Pages (PHP) as open source server programming, whilst
database would be implemented with MySQL database as open source
database.

III. Quality assurance of Indonesian Higher Education


Universities in Indonesia both private and public should assure the quality of their
education process, in transparent and accountable ways, in order to protect the
community interest as regulated by the law [8]. The Ministry of Education and
Culture of the Republic of Indonesia has legislated the regulation [7,9] which
regulate quality assurance in higher education should consist of both of Internal
and external (article 3 clause 1) [7].
Quality assurance of Indonesian higher education both of internal and external
should be implemented by Indonesian higher education institution and has
purposes to gain and maintain the quality of learning, research and community
services [9]. The output of internal quality assurance will be used by Indonesian
National Accreditation Agency of Higher Education Institution (BAN-PT: Badan
Akreditasi Nasional Perguruan Tinggi, http://ban-pt.kemdiknas.go.id) to establish
accreditation level of study program faculty (article 3 clause 2 sub clause f ) [6],
(article 3 clause 4) [7]. Moreover, planning, executing, managing and developing
both internal and external quality assurance should refer to national standard of
higher education [7].
BAN-PT as mentioned in article 1 clause 1 [2] is an independent evaluation
board which has a duty to establish program eligibility and/or education unit on
higher eduation with reference to national standard’s education. It is nonstructural,
nonprofit and independent body which responsible to minister of education, as
mentioned in article 2 clause 2 [2].
However, regulation of ministry of national education of the Republic of
Indonesia, number 73 year 2009 [3,4] as an old regulation is not suitable with
regulation of ministry of national education of the Republic of Indonesia, number
49 and 50 year 2014 [6,7]. In old regulation, BAN-PT has seven instruments
standard accreditation (section 3, sub section A [3] or section 2) [4] such as:
1) Standard of visions, missions, goals and objectives, including strategies for
achieving.
2) Standard of governance, leadership, management and quality assurance.
Innovation for Better ASEAN Community 357

3) Standard of students and graduates.


4) Standard of human resources.
5) Standard of curriculum, learning process and academic atmosphere.
6) Standard of funding, infrastructure including information system.
7) Standard of research, community service and collaboration.
As mentioned before, both internal and external quality assurance should
refer to 24 national standards of higher education [7] which consist of 8 national
standards of education, 8 national standards or research and 8 national standards
of community services [6]. The 8 National Standards of education are:
1) Standard of graduate competence.
2) Standard of learning contents.
3) Standard of learning process.
4) Standard of learning assessment.
5) Standard of lecturer and educational personnel.
6) Standard of learning tool and infrastructure.
7) Standard of learning management.
8) Standard of learning funding.
The 8 National Standards of research are:
1) Standard of research product.
2) Standard of research content.
3) Standard of research process.
4) Standard of research assessment.
5) Standard of researcher.
6) Standard of research tool and infrastructure.
7) Standard of research management.
8) Standard of research funding and budgeting.
The 8 National Standards of community services are :
1) Standard of community services product.
2) Standard of community services content.
3) Standard of community services process.
4) Standard of community services assessment.
5) Standard of community services implementor.
6) Standard of community services tool and infrastructure.
7) Standard of community services management.
8) Standard of community services funding and budgeting.
358 Proceeding ASEAN COSAT 2014

However, Indonesia with BAN-PT as Indonesian higher education quality


assurance accreditation has been recognized by Southeast Asian Ministers of
Education Organization-Regional Centre for Higher Education and Development
(SEAMEO RIHED) which can be reached at www.rihed.seameo.org. SEAMEO
RIHED is the Regional Centre for Higher Education and Development working
for the 11 Member Countries in Southeast Asia under the umbrella of the
Southeast Asian Ministers of Education Organisation (SEAMEO). SEAMEO
RIHED promotes ASEAN Quality Assurance Network (AQAN) as a common
regional framework for Southeast Asian nations for quality assurance in higher
education [5]. AQAN by ASEAN University Network, www.aunsec.org (AUN)
promotes the harmonization of the quality assurance for ASEAN higher education
where all universities in ASEAN country can have different quality assurance
system and approach.
Indonesia by BAN-PT as quality assurance body or agency has the same model
of independent body or agency with countrie such as Cambodia, Malaysia and
Thailand, whilst countries such as Brunei, Laos, and Vietnam have centralised
government body or agency within ministry responsible for higher education.
Meanwhile, Singapore and Philippines have mixed system and Myanmar alone
does not have an established quality assurance body or agency where all quality
functions are delegated to individual higher education which has responsibility
to report to Minister of Education. BAN-PT and The Commission on Higher
Education (CHED) as Indonesian and Philippines higher education quality
assurance agencies respectively, have the same common purpose of quality by
providing information on accreditation status.

IV. K ey Performance Indicator


The 24 national standards of higher education should be generated as KPI. Each
standard will have many KPI which are used to control the quality of standard.
For example, standard of graduate competence will have KPI such as :
1) Number of students who graduate on time.
2) Employer opinion regarding alumni quality.
3) Time waiting for first job.
Each of KPI will be quantified to 5 points rating scale with quantitative
number range such as 0,1,2,3 and 4 to qualitative range score such as Very Poor,
Poor, Fair, Good and Excellent respectively. Each of KPI must have quantitative or
qualitative data such as number of students or employer good opinion regarding of
alumni quality, respectively. The score of KPI quality range refer to old regulations
of BAN-PT which can be read at copy of attachment 7 of Regulation of Minister
of National education of the Republic of Indonesia, Number 73 Year 2009 [10].
Innovation for Better ASEAN Community 359

Table 1. KPI of standard of graduate competence for HCI study program


KPI of standard of graduate competence Score
HCI Study Program
1. Number of students who graduate on time 3
2. Employer Opinion regarding of alumni quality 2
3. Tine Waiting time for first job 2
KPI Score Total 2.33

Score: Very Poor(0), Poor(1), Fair(2), Good(3), Excellent(4)

Table 1 shows the example of KPI quantification of graduate competence


standard which are categorized as fair KPI when it has KPI score total 2.33. KPI
score total is KPI average from all KPI score lists (3+2+2=7/3=2.33) and KPI
score total will be rated as well to Very Poor, Poor, Fair, Good and Excellent.
Finally, based on the 24 national standards of higher education, there will
be 24 tables as shown in Table 1 that each of table will show the quality for each
national standard of higher education. Moreover, the average of all 24 tables KPI
score total will show the quality of study program based on national standard of
higher education. The higher KPI score total, the better study program quality.
Controlling of each study program quality will be based on the KPI national
standard of higher education and when one standard has poor quality then it will
automatically alert high level management. High level management, such as rector,
vice rector, dean, vice dean, head of study program and etc will be convenient
to use the university executive information system and easy to control the study
program or faculty. Meanwhile, other university stakeholder such as government,
society, community, student’s parent, should have benefit from this university
executive information system where the quality of university can be accessed in
transparent and accountable way in order to protect the community interests as
regulated in law [8].

V. EIS Database architecture


Figure 1 shows EIS database architecture with 3 data marts and 1 data warehouse.
Data mart and data warehouse are recognized as Online Analytical Processing
(OLAP) database [11]. Figure 1 shows that data warehouse technology is designed
as centralized data warehouse architecture. The data warehouse in figure 1 is
representation of national standard of higher education, whilst the 3 of data
marts are representation of national standards such as education, research and
community services.
The data marts or data warehouse will have star or snowflake or fact constel-
lation scheme, represent unnormalized database which will be used to increase
360 Proceeding ASEAN COSAT 2014

the performance to read the database records with SQL select statement. Indeed,
data warehouse technology is used in order to increase the performance of SQL
select statement since there is decreasing of number managed tables, records and
data byte [11].
The data warehouse will be loaded with the data from the 3 of data marts
with ETL (Extraction Transformation and Loading) process. Meanwhile, the 3
data marts will be loaded from OLTP/TPS (Online Transactional Processing/
Transactional Processing System) database with ETL process as well. The ETL
process should be done on extraction scheduling mode with constructive merge
loading which differ old and new records [12]. The ETL algorithm will be run
automatically at the end of office hour, parallel with OLTP daily backup database
[12].
The data mart of national standard of education will be loaded with data
from OLTP database, such as student administration, academic administration,
human resources, general affair and finance. Meanwhile, the data mart of national
standard of research should be loaded with data from OLTP database such as
human resources, general affair, finance and research. Finally, the data mart of
national standard of community services should be loaded with data from OLTP
database such as human resources, general affair, financial and community services.
Transformation of chosen OLTP database into each data mart should be
done based on standard content in each of data mart which represent between

Figure 1. EIS database architecture


Innovation for Better ASEAN Community 361

national standard of education, research or community services. For example,


data marts national standard of research and community services have the same
chosen OLTP databases such as human resources, general affair and finance. Both
of standards of research and community services have standard of researcher and
community service implementer respectively.
It is possible to design without data warehouse and using only 3 data marts
to input the data warehouse before the 3 data marts. The number of data mart or
data warehouse, the chosen between centralized or uncentralized data warehouse
architecture should consider based on university needs, cost and performance.
Hardware architecture implementation for EIS database architecture, such as
the number of server should be designed as user’s requirement needs, cost and
performance. The OLTP database in figure 1 is an example where it is possible
to have different database architecture which depends on each university.
The 3 data marts and data warehouse can be accessed by novice or expert user,
where the application will provide reporting, graphic reporting, multidimensional
as the power of data warehouse. Moreover the intelligence of data warehouse could
be increased by adding data mining technique which can find pattern such as
classification, discriminant, association, frequent, similar and so on with chosen
of data mining algorithm such as Attribute Oriented Induction (AOI) [15],
Emerging pattern (EP), Attribute Oriented Induction High Emerging Pattern
(AOI-HEP) [13,14], Neural Network, Bayesian Network, Decision tree and so on.

VI. Conclusion
24 national standards of higher education as a KPI framework to build EIS
application for university will fulfill the Indonesian government regulation in
order to assure the higher education quality.
Study program quality assurance can be controlled based on this KPI national
standard of higher education framework and when one of the standard has poor
quality assurance then it will automatically alert the high level management and
surely increase the quality by fulfilling the KPI requirement. Moreover, university
high level management will be convenient and easy to use the EIS in order to
assure the quality of their study program or faculty.
University stakeholder such as government, society, community, student’s
parent should have benefit from the university EIS where the quality of higher
education can be accessed in transparent and accountable ways in order to protect
the community interest.
This research should need more exploration where KPI framework from 24
national standard of higher education should be generated more accurately where
362 Proceeding ASEAN COSAT 2014

each national standard will have more than 1 KPI. Furthermore, EIS application
infrastructure should be defined in order to show the EIS business process.

VI. R eferences
1) Damarjati., D. (2012). Mendikbud: 2013, Wajib belajar 12 Tahun &
Kurikulum baru diterapkan. [Online] accessed on 9 July 2014, from : news.
detik.com/read/2012/12/04/150638/2109092/10/mendikbud-2013-wajib-
belajar-12-tahun--kurikulum-baru-diterapkan .
2) Sudibyo., B. (2005). Agency of National Accreditation of Higher Education
institution. Regulation of Minister of National education of the Republic of
Indonesia, Number 28.
3) Sudibyo., B. (2009). Accreditation instruments of Bachelor study program.
Copy of attachment 1 of Regulation of Minister of National education of the
Republic of Indonesia, Number 73.
4) Sudibyo. B. (2009). Accreditation instruments of Bachelor study program.
Copy of attachment 2 of Regulation of Minister of National education of the
Republic of Indonesia, Number 73. SEAMEO RIHED. (2012). A study on
Quality Assurance Models in Southeast Asian Countries: Towards a Southeast
Asian Quality Assurance Framework [Online] accessed on 9 July 2014, from:
www.rihed.seameo.org/wp-content/uploads/2013/FrequentlyRequested/
SEAMEO_RIHED_QA_in_SEA_report_2012.pdf .
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Minister of education and culture of the Republic of Indonesia, Number 49.
6) Nuh., M. (2014). Quality Assurance of Higher education. Regulation of
Minister of education and culture of the Republic of Indonesia, Number 50.
7) Yudhoyono., S.B. (2012). Higher Education. Law of the Republic of Indonesia,
Number 12.
8) Yudhoyono. ,S.B. (2014). Organization of higher education and management
of higher education institution. Regulation of Government of the Republic of
Indonesia, Number 4
9) Sudibyo., B. (2009). Accreditation instruments of Bachelor study program.
Copy of attachment 7 of Regulation of Minister of National education of the
Republic of Indonesia, Number 73
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tion and Innovative Technology) journal, 7(4).
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11) Warnars., S. (2009). Desain ETL dengan contoh kasus perguruan tinggi.
Jurnal Informatika, 10(2), 86-93.
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13) Warnars., S. (2014). Mining Frequent Pattern with Attribute Oriented
Induction High level Emerging Pattern (AOI-HEP). IEEE the 2nd International
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14) Warnars., S. (2010). Star schema design for concept hierarchy in Attribute
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from: dssresources.com/papers/features/kelly11072002.html
Design of Implementation Delay Tolerant At
Wireless Mesh Networks using IBR-DTN and
Batman-adv
Herman Yuliandokoa,b, Sritrusta Sukaridhotob, M. Udin Harun Al Rasyidb
a
State Polytechnic of Banyuwangi
Jl. Raya Jember Km.13 Labanasem-Kabat, Banyuwangi, Indonesia
b
Postgraduate of Information Engineering and Computer, Electronics Engineering,
Polytechnic Institute of Surabaya
Jl. Raya ITS Sukolilo 60111, Surabaya, Indonesia

Abstract
Wireless technology is one of the fast growing technology. Research on wireless technology has
been widely applied, and one of them is a mesh network technology research. Mesh networking
technology is considered as one solution to the problem of network with complex equipment.
By applying a mesh network, each node has the same level with other nodes without the need
of an access point. Delay-tolerant networking (DTN) is an approach to computer network
architecture that seeks to address the technical issues in heterogeneous networks that may lack
continuous network connectivity. In our research, we use propose a design of implementation
of Batman-adv software for mesh network in addition, we also will combine and implementing
delay tolerant technology IBR-DTN to support inter-node communication. It is appropriate to
mention that previous research IBR-DTN is an efficient technology to be applied compared to
other technologies. In the end, we also include the analysis and design implementation of mesh
technology will be carried out the Batman-adv and IBR-DTN running together.
Key words: Wireless mesh, Batman-adv, IBR-DTN, Combine.

I. Introduction
Kevin Fall in “A Delay Tolerant Network Architecture for Challenged Internet”
mentioned that Delay tolerance network is a technology that is very useful for
network communication has a great challenge (obstacle) [1]. DTN can solve
intermittent connectivity problems and long or variable delay because DTN is
a technology that allows to send data packets in a network that has a difficult
environment or not continuously available connection. In this technology each
node has a network storage device so that when there is network connection
problem, the data will still be kept in storage and will be resent again when the
network has been formed again. Although DTN has any advantages, DTN also
needs long time and more energy consumption. Currently, researchers study
focuses on the architecture, algorithms, routing and network design on Delay
Tolerant [1][2][3]. While research on the Delay Tolerant Network manet is still
rare.

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Wireless mesh is currently used in many application. It is due to dynamic


mobility user, disaster area and low cost network manufacture. Research study
in rooting protocol that performed evaluation of wireless mesh have been carried
out [4]. However, research DTN for mesh network application or ad-hoc mode
is still rarely done, and majority research DTN on mesh network is still on
simulation. For an example, Laurent and Simin research on Batman simulation
with Store-and-Forward mechanism. In this research they use NS3 simulator [5].
DTN has grown so rapidly in line with DTN applications in several fields.
In the fields of transportation, DTN is used in data transmission on vehicular
network in intelligent transport system [7]. Due to the environmental conditions
of transport that has variables, they cause unstable communications networks
as well as user data to mobile users. DTN applications in the field of health
support are also very important to support communication in long distance and
long delay. Research on Rural Telemedicine Networks Using Store-and-Forward
Voice-over- IP [6]. DTN is used to support VoIP for medical field by connecting
doctor and patients in the remote areas.
In this paper, we will make research design of implementation of DTN by
using wireless mesh network. Overview on BATMAN and DTN will explain on
the first, then we review related work on performance evaluation of BATMAN
and IBR-DTN. We present setup and results of our experiment. Finally, we
summarize our contribution and describe further work.

II. Method/M aterial


Wireless mesh networks (WMN) are self-organized wireless networks in which
component parts (nodes) can all connect to each other via multiple hops. In
WMN, nodes are comprised of mesh routers and mesh clients. Each node operates
not only as a host but also as a router, forwarding packets on behalf of other
nodes that may not be within direct wireless transmission range of their destina-
tions[8]. Other than the routing capability for gateway/repeater functions as in
a conventional wireless router, a wireless mesh router contains additional routing
functions to support mesh networking. To further improve the flexibility of mesh
networking, a mesh router is usually equipped with multiple wireless interfaces
built on either the same or different wireless access technologies. Compared with a
conventional wireless router, a wireless mesh router can achieve the same coverage
with much lower transmission power through multi-hop communications. In
detail, WMN should have the following properties [9]:
1) Multi-hop wireless network
2) Dynamically self-organized, self configured and self-healing
3) Resilience to device failures
Innovation for Better ASEAN Community 367

4) Highly scalable

A. Routing Protocols
Wireless mesh network is an unstructured network and have a high mobility. It
means the WMN (Wireless Mesh Network) have to make sure the communication
can be done. Routing protocols are responsible for discovering, establishing
and maintaining such routes. Routing protocols for WMN are mostly based on
protocols designed for mobile ad-hoc networks. These can be classified in the
following categories [10].
1) Proactive protocols construct the routing table periodically.
2) Reactive protocols construct the routing table on-demand.
3) Hybrid protocols are a mixed design of the two approaches mentioned above.
These protocols typically use a proactive approach to keep routes to nodes
in the vicinity of the source, but for nodes beyond that area, the protocol
behaves like a reactive one.

B. BATMAN-ADV
BATMAN is a proactive routing protocol for WMN. It uses a distance-vector
approach and a routing metric which incorporates the reliability of the radio links.
Despite being developed and publicly available since 2006, BATMAN, especially
its newer batman-adv variant, has received sparse attention in the scientific
community. Optimized Link State Routing (OLSR) and BATMAN are two
popular protocol. Better Approach to Mobile Ad-Hoc Networking (BATMAN)
[11] is a new routing protocol for multi-hop ad-hoc mesh networks. BATMAN
uses a simple and robust algorithm for establishing multi-hop routes in mobile
ad-hoc networks. This protocol for WMN, based on distance vector routing
and a proactive protocol in that each node maintains a routing table containing
potential next hops to all other nodes forming the WMN.
Batman algorithm protocol can be described, that each node transmits
broadcast messages (originator messages or OGM) to inform the neighboring
nodes about its existence. These neighbors re-broadcast the OGM according
to specific rules to inform their neighbors about the existence of the original
initiator of this message and so on and so forth. Thus, the network is flooded
with originator messages [2]. An OGM is used in BATMAN to distribute node
specific information in the mesh network. This OGM is encapsulated in a raw
Ethernet frame and broadcasted in a fixed interval (1 second) on all used hardware
interfaces. The frame includes all information a receiving BATMAN node needs
to build up its routing table.
368 Proceeding ASEAN COSAT 2014

In the beginning, BATMAN is like other routing protocol which running in


layer 3 (batman). In 2007, Layer 2 routing (BATMAN Advanced or batman-adv)
was introduced, moving the daemon into the Linux kernel for better performance.
The development of batman was mostly stopped later on, focusing the efforts on
batman-adv. The BATMAN wiki [11] lists some characteristics of this design:
1) network-layer agnostic - you can run whatever you wish on top of batman-adv:
IPv4, IPv6, DHCP, IPX .
2) nodes can participate in a mesh without having an IP .
3) easy integration of non-mesh (mobile) clients.
4) roaming of non-mesh clients optimizing the data flow through the mesh (e.g.
interface alternating, multicast, forward error correction, etc.).
5) running protocols relying on broadcast/multicast over the mesh and non-mesh
clients (Windows neighborhood, mDNS, streaming, etc.).

1) Batman-adv Setup
In this research, we used Ubuntu 12.04 for operation system in each node and
we used wireless too connect each node on mesh network designed. Before setup
Batman-adv in our tools, it was important to update our tools repository for
implemented Batman in Ubuntu.
Batman-adv also has some tool and C program that why it was needed to
make sure that there was C compiler program in our nodes. This compiler will
be used to compile our program when running Batman-adv.
The software of Batman-adv could be downloaded from www.open-mesh.org.
There were some release series of Batman-adv and make sure that our Ubuntu
kernel was compatible with Batman-adv version. After downloaded, we extracted
and compiled them.
To make sure our Batman-adv could run smoothly, we had to prepare some
tools, for examples qt4-dev-tools, xconfig, libncurses5-dev, menuconfig and
oldconfig.
After we installed and prepared Batman-adv tools, then we needed to set our
interface. In this research, we used wlan0 (wireless) interface to connect nodes
each other. It was important to make sure that nodes firewall is disable.
Wireless mesh was one of ad-hoc mode wireless network. It was also needed
to create essid and channel. The essid and channel should be same for all nodes.
iwconfig wlan0 mode ad-hoc essid [essid] channel [channel]
Innovation for Better ASEAN Community 369

C. Activated Batman-adv and batctl


Although we already setup Batman-adv in our nodes, it was also needed to activate
our Batman-adv to operate routing protocol. Batman-adv had one advantages
compared to the another routing protocol, Nodes which use Batman-adv for
their routing protocol could connect each other by using layer 2 or layer 3. It
means nodes in Batman-adv could ping another by using IP address or MAC
address. That was why in the Batman-adv also has batctl software to support
communication process.
Wireless mesh by using Batman-adv routing protocol was ready to be used
if nodes interface status was active. To know that our interface status was active
by accessing iface_status file on Batman-adv folder.

D. DTN Store-and-Forward
DTN overcame the problems associated with intermittent connectivity, long or
variable delay, asymmetric data rates, and high error rates by using store-and- for-
ward message switching. This was an old method, used by pony-express and postal
systems since ancient times. Whole messages (entire blocks of application-program
user data) or pieces (fragments) of such messages were moved (forwarded) from
a storage place on one node (switch intersection) to a storage place on another
node, along a path that eventually reached the destination.
There were several tools or software for DTN implementation. Delay
Tolerant Network Research Group (DTNRG) to produce DTN2 to implement
basic functional of DTN. Another Linux implementation was ION. Symbian
smart-phone usually use DASM for DTN implementation and DTNLite was
designed for sensor networks running tiny OS, but did not allow for using
common applications and programming languages. IBR-DTN was to develop
a powerful and efficient implementation that runs on embedded devices as
well as on standard Linux systems. Michael Doering in IBR-DTN: An Efficient
Implementation for Embedded Systems explains that the implementation of IBR-
DTN is more efficient. Not only the slim application itself, but also the lower

Figure 1.: Store and Forward


370 Proceeding ASEAN COSAT 2014

consumption of memory makes IBR-DTN suitable to run on embedded devices


with limited resources [12].

1) IBR-DTN Setup
There were many kinds of IBR-DTN Setup and in this paper we used repository
to setup IBR-DTN tools. We need to download Release key and make sure the
distribution as Ubuntu version which used. It can be downloaded on http://
download.opensuse.org/ after that download and install IBR-DTN tools.

E. Related Works
An extensive amount of work has been done in the field of wireless mesh network
routing protocol and delay tolerant networks. In the following, we present related
work with a focus on Batman routing protocol and IBR-DTN.
Some research on wireless mesh focus on routing protocol performance only.
Laurent et al. [5] used NS3 to make analysis and demonstrate Batman with
additional mechanism store-and-forward. They have implemented SF-Batman
(Store-and-Forward Batman) in a packet level simulation and demonstrated
its performance in a scenario that consists two region of connectivity: a well
connected mesh network and a set of sparser sub networks.
A Practical Evaluation of BATMAN Advance of the Routing Performance of
Wireless Mesh Networks in the realistic office environment [4]. It was done to
evaluate parameters which influence Batman routing performance. It was also to
demonstrate failure modes of the studied protocols.
DTN2 and IBR-DTN are two famous delay tolerant tools and Michael Doer-
ing [12], research on efficiency IBR-DTN compare to DTN2 when implemented
on embedded system. Their investigation shows that IBR-DTN is an efficient
system for embedded implementation.

D. Combine Batman-adv and IBR-DTN


In this research, we wanted to make the combination of IBR-DTN and
Batman-adv. This combination to make Batman-adv could run together with
IBR-DTN. Before that, we had to make sure that batman interface (bat0) could
detect another node.
This combination would be very useful when our node operate on challenge
network area. By using IBR-DTN the data would be kept in storage if node cannot
connect to another node and would forward the data when the node could get
a network connection to another node.
Innovation for Better ASEAN Community 371

F. Implementation
1) Evaluation
The goal of our evaluation was to assess the ability of Batman-adv on wireless
mesh network in real condition and to know the IBR-DTN performance on
Batman-adv protocol. Routing protocol performance could be seen from the
indication of packet loss, delay, throughput and another aspect. These conditions
were influenced by multiple parameters, and we would discuss and consider the
location of communication. The time at which communication was taken also
can influence the performance of routing protocol.
2) Experiment Design
To get valid result in our research we made design of experiment as actual condi-
tion. We built a test bed in the storey building which put node on different room.

Table 1. Experiment Design


Hardware Nodes Asus core i3, Lenovo corei5, Compact corei3
Ubuntu 12.04 i386
Operating System
Bat0 IP and Mac
- node 1 192.168.10.20 / 6c:71:d9:7a:37:99
- node 2 192.168.10.30 / 48:d2:24:ca:d9:62
- node 3 192.168.10.50 / 00:08:ca:b1:58:b4
- node 4 192.168.10.40 / d0:df:9a:23:b4:31
Scenario 1 One node in the 3rd floor, one node in the 2nd floor, 2 nodes
in the 1st floor, and all node in fix position
Scenario 2 One node in the 3rd floor, one node in the 2nd floor, 2 nodes
in the 1st floor and both of node mobile
Scenario 3 One node in the 3rd floor, one node in the 2nd floor, 2 nodes
in the outside building (outside range), and all moving to the
building/wifi range

160

140

120
500 byte
100 750 byte
1000 byte
80 2000 byte
5000 byte
60
10000 byte
40 50000 byte

20

0
Max Delay (ms) Min Delay (ms) Rountrip Avg delay (ms)

Figure 2. Nodes Positioning


372 Proceeding ASEAN COSAT 2014

Figure 3. Delay Transmit Data From Node 1 to Node 4

III. R esult and Discussion


We make 3 scenarios to check performance of Batman-adv and IBR-DTN on
wireless mesh communication.
Scenario 1, in this trial all nodes in fix position and separate in 3 locations.
After activated Batman-adv then try to send data packet from node 4 to node 1.
Based on standard TIPHON version [13], the performance of delay can be
classified as below table.
Refer to Table 2 condition showing that performance Batman is very good.
Scenario 2, in this scenario we want to know routing characteristic of
Batman-adv, and make test route from node 4 to node 1. Before checking route
we check potential next hope information from another node by using “batctl o”.
Nodes send OGM to inform potential next hop to neighbor node. After that
we check route transfer data as below route.
Table 2. Delay Category
Category Delay (ms) Indexs
Very good <150 4
Good 150 ~ 300 3
Fair 300 ~ 450 2
Poor >450 1
Innovation for Better ASEAN Community 373

Figure 4.: Potential Next Hope Position

Figure 5.: Trace Route

Scenario 3, in this experiment node 3 and 4 are mobile until go outside of


wireless coverage area node 2 and 1, this condition node 4 can only connect to
the node 3.
On “btctl o” neighbor checking we will see only node 3 which detected by
node 4. After that node 4 try to send data to node 1 although node 4 cannot detect
node 1, and node 4, 3 and 2 already setup IBR-DTN to handle connection less
374 Proceeding ASEAN COSAT 2014

Figure 6. Nodes 3 & 4 moving more than 50 meter from building

Figure 7. Transfer data process

condition. When sending data process is occuring we are also walking toward to
the wireless mesh coverage are of node 1 and 2. This condition also influences
the data sending process from node 4 to node 1.
If we see the graphic, it shows the process of transfer data from node 4 to the
node 1. Refer to above graphic that we sent three time data from node 4 to node
1 (500 byte, 1,000 byte and 1,500 byte). In the first experiment with sending
500 byte data, the data cannot be received in the node 1 because there is no
Innovation for Better ASEAN Community 375

connection between node 3 and node 2, but the data is kept on storage node 3
by using IBR-DTN mechanism. The data forward by node 3 to node 2 after there
is connection again, and it is happened on the 38th sequence. This IBR-DTN
mechanism is also happen second data sending process (1,000 byte) and third
data sending process (1,500 byte). On second data sending process, IBR-DTN
forward mechanism is happened on the 21th sequence and the 39th for third data.

IV. Conclusion
In the following we present the result of our measurement. We expect that
batman-adv is showing very good performance in relation to delay result. It can
see from maximum delay measurement only 41 ms. Refer to standard TIPHON,
that delay with 41ms measurement result is in the very good range. However, we
see that deviation of delay ratio need to be a concern. Beside that a the quantity
data transfer also influence to the delay ratio and sending data stability. It is needed
to improve research with also consider to environment condition.
The number of nodes needs to be added to get more valid data in trace route
testing. Although Batman-adv has tools and library to inform user easily about
next hop priority.
Applications of Batman-adv and combining with IBR-DTN have been work-
ing. It can see from data transfer result. But in the installations process we also
have take attention to the compatibility Batman-adv version and Ubuntu kernel.
Beside that some of Ubuntu version cannot install IBR-DTN. We also want to
continue this research with implementation this result in the embedded system.
Because it will take more advantages for communication in disaster condition.

V. Acknowledgment
This work supported by IR2C Lab project and DIKTI Funding Scholarship
(BPPDN). It will be the basis for further research application of delay-tolerant
and batman-adv.
VI. R eferences
1) Fall, Kevin. (2003). A Delay Tolerant Network Architecture for Challenged
Internets. SIGCOMM ’03, New York, NY, USA: ACM 2003, 27-34. Available
at http://doi.acm.org/10.1145/863955.863960
2) Bulut, Eyuphan. (2011). Opportunistic Routing Algorithm in Delay Tolerant
NetworksPPORTUNISTIC ROUTING ALGORITHMS IN DELAY TOLER-
ANT NETWORKS. Rensselaer Polytechnic Institute Troy, New York.
376 Proceeding ASEAN COSAT 2014

3) Zhao, Wenrui. (2006). Routing and Network Design in Delay Tolerant Networks.
Georgia Institute of Technology.
4) Seither, Daniel, Konig, Andre, and Matthias Hollick. (2011). Routing
Performance of Wireless Mesh Networks: A Practical Evaluation of BATMAN
Advanced. TechnischeUniversit,Darmstadt, Bonn.
5) Laurent, Delosieres. (2012). Simin Nadjm-Tehrani, BATMAN Store-and-
Forward: the Best of Two Worlds. Linkoping University SE-581 83, Linkoping,
Sweden.
6) Warthman, Forest. (2003). Delay-Tolerant Networks (DTNs): A Tutorial v1.1.
[Online]. Available http://www.dtnrg.org/ docs/tutorials/warthman-1.1.pdf.
7) André, Rein. (2012). TrustMANET Development and Evaluation of a Trusted
Mobile Ad-hoc Network. FachbereichMathematik, Naturwissenschaften und
Informatik der TechnischenHochschuleMittelhessen
8) Babu, Karisma, Cortes-Pena, Luis Miguel, Shah, Prateek, and Shivaranjani
Sankara Krishnan. (2007). Wireless Mesh Network Implementation. School of
Electrical Engineering Georgia Institute of Technology Atlanta, GA.
9) Ricardo, Pinto. WMM - Wireless Mesh Monitoring. [Online]. Available: http://
www.gsd.inesc-id.pt/~ler/reports/ricardopinto-midterm.pdf. Retrieved June
2014.
10) Abolhasan, M., Wysocki, T., and E. Dutkiewicz. (2014). A review of routing
protocols for mobile ad hoc networks. Ad Hoc Networks 2(1),1–22
11) Lindner, M., Eckelmann, S., Wunderlich, S., et al.The B.A.T.M.A.N. Project.
[Online]. Available: http://www.open-mesh.org/. Retrieved May 2014.
12) Doering, Michael Lahde, Sven, Morgenroth, Johannes and Lars Wolf. (2008).
IBR- DTN: An Efficient Implementation for Embedded Systems. Institute of
Operating Systems and Computer Networks, TechnischeUniversitätBraun-
schweig
13) Tiphon. (1999). Telecommunications and Internet Protocol Harmonization
Over Networks (TIPHON) General aspects of Quality of Service (QoS). DTR/
TIPHON-05006 (cb0010cs.PDF).
Development of a programmable
multipurpose forced convection type dryer

Emeline C. Guevarra*
Cavite State University
Indang, Cavite, Philippines

Abstract
Drying is considered as one of the effective methods of food preservation to prolong the storage
life of foods. This study is focused on the development of a programmable multipurpose forced
convection type dryer which is designed to dry agricultural crops. The hardware of the system is
composed of a drying chamber, heater, blower, buzzer, PIC16F877 microcontroller, liquid crystal
display, temperature sensor, and keypad. PBASIC is the language used in the development of
the software which interfaces the microcontroller with the dryer. Testing of the system was done
with banana, cassava, and malunggay leaves which are loaded to the dryer by batch. Results
showed that the microcontroller is able to control and monitor the temperature inside the drying
chamber. The heater automatically stopped once the desired temperature is reached while the
blower continuously blows the air inside the chamber. Blower and heater automatically shut off
when drying time is reached.
Key words: Artificial drying, Drying, Food dryer, Microcontroller, Temperature sensor.

I. Introduction
Drying is considered as the oldest method of preserving foods and also maintains
the best quality of the product by lowering the moisture content so that molds
and spoilage organisms cannot grow. The method can be traced back since ancient
times where Egyptian tribes preserved fish, meat, vegetables and fruits by drying
and salting. The common practice is to use a safe place to spread the food where
dry air can pass over while others drape foods on branches or on wide shallow
baskets on the roof.
Nowadays, there are several methods of drying, such as convective or direct
drying, indirect drying, dielectric drying, super critical drying, freeze drying and
natural drying. Among these methods, the most common is natural drying where
unheated forced air is used. This method of drying is slow and weather dependent
and may last from days to several weeks. This leads to the development of dryers
which can reduce the moisture content level of foods in a short period of time.

* Tel: (0915)949-1578. E-mail: emi_guevarra@yahoo.com

377
378 Proceeding ASEAN COSAT 2014

Through the development of a programmable multipurpose forced convection


type dryer, the temperature and relative humidity inside the drying chamber
is controlled. The dryer monitors and maintains the needed temperature of
agricultural products inside the drying chamber which leads to the shortened time
of drying. Small scale farmers or backyard farmers of the agricultural sector might
benefit in using the dryer since excessive production losses during harvest may
be prevented. Harvested agricultural products can be dried anytime regardless of
varying weather conditions producing better quality of agricultural products since
the growth of molds and microorganisms which cause food spoilage are reduced.

II. method/material
This study employed prototyping technique. Prototyping is a technique that proves
the effectiveness of the design, the computations, and the concepts applied, by
making an actual machine. It attempts to develop the existing machine into more
substantial purposes. Prototype model allows the identification of materials used
in the development of the system. The model also allows the simulation of the
design in terms of its functionality.
A functional or working prototype was constructed to simulate the functional-
ity of the system. Software is developed to interface the microcontroller with the
drying chamber.
Data gathering is considered to identify the different requirements needed
in the development of the system. This involves gathering data from reference
materials, such as books, researches both published and unpublished, and
literatures from the internet. Observation of existing dryers is also considered.
Figure 1 shows the system block diagram, which enables easy identification
of the different components to be used in the development of the system. This
identifies the specific component which will transmit and receive signals from
different components of the system. The hardware components used in the devel-
opment of a programmable multipurpose forced convection type dryer include
the drying chamber, fan or blower, heater, microcontroller circuit, temperature
sensor, Liquid Crystal Display (LCD) and keypad.
Heated air needed for the drying process is supplied by the heating element
within the dryer. The heat from the heating element is transferred through the
forced convection process with the help of the blower. Internal temperature is
regulated and monitored by the microcontroller through the use of a temperature
sensor.
The main function of the program is to monitor the temperature readings
of sensors and to control the proper operation of the blower and heater. Based
on the readings from the sensors, the proper operation of heater and blower is
Innovation for Better ASEAN Community 379

Figure 1. System Block Diagram

considered to maintain the necessary temperature requirements for the selected


crop subjected for drying. The program is also responsible in stopping the drying
process automatically.
The machine is tested if it functioned as planned. The microcontroller is
also tested if it controls the overall operation of the dryer. Proper monitoring of
temperature inside the chamber is considered to achieve the desired output. The
system automatically shut off once the drying process has ended.
Banana, cassava and malunggay leaves are used as samples in the drying
process. Crops are cut into smaller pieces and scattered on the drying trays by
batch system, only one type of crop is dried at a certain period of time. Malunggay
leaves are removed from the stalk before it is scattered on the drying trays. Three
trials of test are conducted to get the initial moisture content of each sample and
the proper time setting for the drying process. The initial moisture content of
each crop is determined using the gravimetric method. Weights are monitored
and the initial moisture content is computed using equation (1).
MC = [(Wi - Wf ) / Wi][100%] (1)
380 Proceeding ASEAN COSAT 2014

Drying time is defined as the residence time of the sample inside the drying
chamber during the drying process. Time is determined through monitoring the
initial and final weights of the sample followed by the computation of the initial
and final moisture contents after every hour. Constant final weight record for
three consecutive readings determines the safe moisture content level at which
the crop is safe for storage.
The prototype is evaluated by experts in the field of crop processing and
machine designers. The evaluation is focused on the microprocessor’s capability
to control the temperature inside the drying chamber. For checking, preset
temperature values and read out values during the drying process is considered.
Readings are displayed on the LCD.

III. R esults and Discussions


The programmable multipurpose forced convection type dryer shown in figure 2
was made up of different components which make it capable of drying different
crops based on the samples temperature requirements at specific time.

A. Construction of the Machine


The project was composed of a drying chamber, microcontroller, relays, buzzer,
blower, heater, liquid crystal display (LCD), temperature sensor, keypad and
power supply. The input and output ports of the microcontroller were configured
to control the operation of the whole system using the software while the

Figure 2. The Programmable Multipurpose Forced Convection Type Dryer


Innovation for Better ASEAN Community 381

temperature, time of drying and selection of crops were set using the keypad.
The temperature inside the drying chamber was monitored through the use of a
temperature sensor and relays were used to control the operation of the blower
and heater. Finned tube provided the needed heat while the blower blew the hot
air inside the drying chamber. Likewise, the buzzer was used to indicate that the
time for drying had ended while the LCD displayed the menu for the operation
of the dryer. The power supply was responsible for providing the necessary voltage
needed by the system.
The microcontroller switched on the blower and heater as soon as the crop to
be dried and its corresponding weight had been selected or the drying temperature
and drying time had been defined. The dryer automatically shut off once the set
time had elapsed.
The drying chamber was constructed using stainless steel, fiber glass, heat
resistant gasket, hinges, angular bars, screen and metal footings. The main
components of the chamber were the blower and the heater. The blower was a
4-pole motor which operates at 208V, 60 watts and 60 Hz. The main component
of the unit includes PIC16F877, where the software for the system was stored.
The microcontroller had five ports: Port A, B, C, D and E. Port A had 6 terminals,
Port B, C and D had 8 terminals each while Port E had 3 terminals. Ports A and B
were used as input terminals while Ports C and D were used as output terminals.
Pin 2 of Port A was used as input line for the signal coming from the
temperature sensor while pins 33, 34, 35, 36, 37, 38 and 39 of Port B were used
as input lines for the signal coming from keypad. Additionally, pins 15, 16, 17,
18, 23 and 24 of Port C were used as output lines for the signal coming from
the microcontroller to the LCD. Moreover, pins 19, 20 and 21 of Port D were
connected to the relay and pins 13 and 14 were both connected to 4-MHz crystal
oscillator, which was connected to two 27-pF capacitors for the oscillation of clock
in and out timing. The crystal oscillator was used for attuning the speed of the
timer in the microcontroller. Furthermore, pins 12 and 31 of the microcontroller
were connected to the ground and pins 32 and 11 were connected to the power
source.
The transformer, bridge rectifier diode, capacitors and the 7805 integrated
chip were the main components that comprised the power suppl