PROJECT REPORT

ON
CHARACTERIZATION OF BIOMASS & ETHANOL
PRODUCTION FROM BIOMASS BY MICROBIAL
HYDROLYSIS



UNDER THE GUIDANCE OF UNDER THE SUPERVISION OF
Er. Mohit Kamthania Dr. Maya Kumari
Lecturer, IBMER Scientist D
Mangalayatan University, Aligarh Defence Institute of Bio-Energy Research,
Haldwani, Uttarakhand



SUBMITTED BY
Hansa Saraswat
M. Sc. Biotechnology
Enrolment No-20120612

For the partial fulfillment of the requirement
For the award of
Master of Science
(Biotechnology)



INSTITUTE OF BIOMEDICAL EDUCATION & RESEARCH,
MANGALAYATAN UNIVERSITY,
BESWAN, ALIGARH
UTTAR PARDESH






Approval Sheet


This dissertation entitled Characterization of Biomass & Ethanol Production from
Biomass by Microbial Hydrolysis by Hansa Saraswat is approved for the degree of
Master of Science in Biotechnology.




Examiners


___________________________

___________________________

___________________________





Supervisor (s)
(Dr. Maya Kumari)
Scientist D, DIBER
Haldwani, Uttarakhand

(Er. Mohit Kamthania)

Faculty, Department of Biotechnology
Institute of Biomedical Education and Research
Mangalaytan University, Aligarh, Uttar Pradesh




Chairman

___________________________


Date:
Place: Beswa, Aligarh, Uttar Pradesh, India

Declaration

I declare that this written submission represents my ideas in my own words and where other
ideas or words have been included; I have adequately cited and referenced the original sources. I
also declare that I have adhered to all principles of academic honesty and integrity and have not
misrepresented or fabricated or falsified any idea/data/fact/source in my submission. I understand
that any violation of the above will be cause for disciplinary action by the Institute and can also
evoke penal action from the sources which have thus not been properly cited or from whom
proper permission has not been taken when needed.









Hansa Saraswat
Roll No.-20120612







Date:









SUPERVISORS CERTIFICATE

This is to certify that the dissertation titled Characterization of Biomass & Ethanol
Production from Biomass by Microbial Hydrolysis by Hansa Saraswat in partial fulfillment
of the requirements for the award of the Degree of Master of Science in Biotechnology is an
original work carried out by her under my supervision and guidance. It is certified that the work
has not been submitted anywhere else for the award of any other diploma or degree of this or any
other University.




Supervisor(s)

(Dr. Maya Kumari)
Scientist D, DIBER
Haldwani, Uttarakhand

(Er. Mohit Kamthania)
Faculty, Department of Biotechnology
Institute of Biomedical Education and Research
Mangalaytan University, Aligarh, Uttar Pradesh



ACKNOWLEDGEMENT
The preparation of this document would not have been possible without the support, hard work
and endless efforts of a number of individuals. I owe my gratitude to all those people who gave
me the possibility to complete this thesis.
I express my gratefulness and great sense of respect to Dr. M. Nasim, Director, DIBER,
Haldwani for giving me permission to carry out my thesis work. I sincerely thank Dr. Ankur
Agarawal, Scientist D, DIBER, Haldwani for his support during my work. I express my deep
sense of gratitude to my elegant guide Dr. Maya Kumari, Scientist D, DIBER, Haldwani. Her
deep interest in planning the experiment, inspiring guidance, constant encouragement, debonair
discussion and constructive criticism helped me to complete this thesis work.
It is my proud privilege to thank Brig. (Dr.) Surjit Singh Pabla, Vice- Chancellor, Mangalayatan
University, Aligarh for his relentless efforts and constant inspiration throughout the study. It
gives me immense pleasure in expressing my sincere gratitude to Prof. Lokesh Upadhaya, Head
of Institute of Bio-medical Education and Research, Mangalayatan University, Aligarh for their
encouragement, inspiring guidance and motivation during my work.
It is my pleasure to pen down my sincere thanks to Er. Mohit Kamthania, Faculty of Institute of
Bio-Medical Education and Research, Mangalayatan University, Aligarh for his thoughtful
guidance and co-operation during the study. I would also like to thank Dr. Kamal Kishore
Chaudhary, Co-ordinate Head of Institute of Biomedical Education and Research who inspired
me at every step during my study.
I would like to express my heartfelt gratitude to Maa and Papa who brought me to this position. I
find myself enveloped in warmth of love of my sister and brother for their immaculate affection
and motivation.
My special thanks to my friends, colleagues and juniors, for providing assistance when needed
and made difficult times easier. At last but not the least I record my sincere thanks to all the
people who directly or indirectly helped me in my endeavour.
Hansa Saraswat

INDEX

LIST OF FIGURES
LIST OF TABLES
ABSTRACT

1. INTRODUCTION
1.1 Introduction
1.2 Motivation
1.3 Objectives of study
1.4 Outline of Thesis

2. REVIEW OF LITERATURE
2.1 Lignocelluloses Biomass
2.2 Plant Cell Wall Composition
2.3 Pre-treatment of Lignocelluloses Biomass.
2.4 Microbial Hydrolysis
2.5 Fermentation for Ethanol production

3. METHODOLOGY AND CALCULATIONS
3.1 Materials
3.2 Methodology
3.3 Calculations

4. RESULT, DISCUSSION AND CONCLUSION
4.1 Result and discussion
4.2 Conclusion

REFERENCES

List of Figures

Figure
No.
Title
1.1 Structure of Plant Cell Wall
1.2 Aspergillus niger on NA Plate
1.3 Trichoderma reesei on NA Plate
1.4 Saccharomyces cerevisiae on NA Plate
1.5 Standard Curve for Reducing Sugar Estimation through DNSA Method
1.6 Standard Curve for Total Carbohydrate Estimation through Anthrone Method
1.7 Standard Curve for Ethanol Estimation through Potassium Dichromate Assay
1.8 Characterization of Biomass for its Different Components of Cell Wall
1.9 (a) Cellulose and Lignin (b) Composition in Different Biomass
1.10 Reducing Sugar Estimation for Jatropha before Microbial Incubation for Hydrolysis
1.11 Reducing Sugar Estimation for Jatropha Seed Coat after Microbial Incubation
1.12 Reducing Sugar Estimation for Jatropha Fruit Husk with Microbes Incubation
1.13 Total carbohydrate estimation for Jatropha without Microbial Incubation
1.14 Total Carbohydrate Estimation for Jatropha Seed Coat after Microbial Incubation
1.15 Total Carbohydrate Estimation for Jatropha Fruit Husk after Microbial Incubation
1.16 Ethanol estimation for Jatropha biomass from Non- hydrolyzed Fractions
1.17 Ethanol Estimation for Jatropha Seed Coat after Microbial Hydrolysis and after Fermentation
1.18 Ethanol estimation for Jatropha Fruit Husk with Microbes Incubation and after Fermentation

List of Tables

Table
No.
Title
1.1 Comparison of First and Second Generation Bio-fuel
1.2 Standard Curve for Reducing Sugar Estimation through DNSA Method
1.3 Standard Curve for Total Carbohydrate Estimation through Anthrone Method
1.4 Standard Curve for Ethanol Estimation through Potassium Dichromate Assay
1.5 Characterization of Different Biomass
1.6 Cell Wall Composition of Different Biomass
1.7 Cellulose and Lignin Composition of Different Biomass Characterized in this Study
1.8 Hydrolysis of Characterized Biomass and Reducing Sugar Estimation for through DNSA
Method
1.9 Reducing Sugar Estimation for Jatropha Seed Coat and Jatropha Fruit Husk before
Microbial Hydrolysis
1.10 Reducing Sugar Estimation for Jatropha Seed Coat after Microbial Hydrolysis
1.11 Reducing Sugar Estimation for Jatropha Fruit Husk after Microbial Hydrolysis
1.12 Total Carbohydrate Estimation for Characterized Biomass through Anthrone Method
1.13 Total Carbohydrate Estimation for Jatropha Seed Coat and Jatropha Fruit Husk before
Microbial Hydrolysis
1.14 Total Carbohydrate Estimation for Jatropha Seed Coat after Microbial Hydrolysis
1.15 Total Carbohydrate Estimation for Jatropha Fruit Husk after Microbial Hydrolysis
1.16 Ethanol Estimation for Characterized Biomass through Potassium Dichromate Assay
1.17 Ethanol Estimation for Jatropha Seed Coat and Jatropha Fruit Husk for Non- hydrolyzed
Fractions
1.18 Ethanol Estimation for Jatropha Seed Coat after Microbial Hydrolysis and after
Fermentation
1.19 Ethanol estimation for Jatropha Fruit Husk with Microbial Hydrolysis and after
Fermentation


ABSTRACT
First generation bio-fuels produced primarily from food crops such as grains, sugar beet and oil
seeds are under scanner, due to the possibility of creating undue competition for land and water
used for food and fiber production. The cumulative impacts of these concerns have increased the
interest in developing bio-fuels produced from non-food biomass. Second-generation bio-fuels
production from lignocelluloses biomass which includes agricultural residues (Corn Stover, crop
straws and bagasse), herbaceous crops (alfalfa, switch grass), short rotation woody crops,
forestry residues, waste paper and other wastes (municipal and industrial)cereal straw, bagasse,
forest residues, purpose-grown energy crops such as vegetative grasses and short rotation forests,
could avoid many of the concerns facing first-generation bio-fuels and potentially offer greater
cost reduction potential in the longer term and do not interfere with food security. Moreover, bio-
ethanol is very important in terms of energy security, environmental concern, employment
opportunities, agricultural development, foreign exchange saving, socioeconomic issues etc.
Lignocelluloses biomass consists of three main structural units: cellulose, hemicelluloses and
lignin. Cellulose is crystalline glucose polymer and hemicellulose is amorphous polymers of
xylose, arabinose, and lignin a large poly aromatic compounds. The characterization of different
biomass viz. Pine needle, Camelina, Jatropha stem, Jatropha petiole, Jatropha leaves, Jatropha
seed coat, Jatropha fruit husk, Jatropha seed cake, Wheat straw, Paddy, Sugarcane and Sugarcane
trash was carried out in the current study with the aim to explore its potential for use as feedstock
for bio-ethanol production. Camelina gave good result in the composition of cellulose and
Jatropha seed coat gave in lignin composition. The maximum amount of cellulose was seen in
Camelina (70.2 %) and maximum amount of lignin in Jatropha Seed Coat (42.9 %). Trichoderma
reesei gave good result in the production of bio-ethanol from Jatropha fruit husk and its seed
coat. The maximum amount of ethanol produced (168.2 l/ml) was on 5
th
day of incubation with
Trichoderma reesei. The result obtained here gives an indication of the usefulness of Jatropha
crop for its potential use for ethanol production through microbial hydrolysis and fermentation.
However, it needs to be validated through further experimentation and analyses.

Key Words: Lignocelluloses Biomass, Bio-ethanol, Hydrolysis, Reducing Sugar, Total
Carbohydrate, Fermentation.














CHAPTER 1


1.1 INTRODUCTION
Increased concern for the security of oil supply and the negative impact of fossil fuels on the
environment, particularly greenhouse gas emissions has put pressure on society to find
renewable alternatives [Jacques; 1999]. Bio-energy from renewable resources is already today a
viable alternative to fossil fuels; however, to meet the increasing need for bio-energy several raw
materials have to be considered.
Resources for biological conversion of energy to forms useful to humanity include majorly the
plant biomass. Among forms of plant biomass, lignocelluloses biomass is particularly well suited
for energy applications because of its large scale availability, low cost and environmentally
benign production. Efficient ethanol production processes and cheap substrates are needed.
Current ethanol production processes using crops such as sugar cane and corn are well
established; however utilization of a cheaper substrate such as lignocelluloses could make bio-
ethanol more competitive with fossil fuel, the processing and utilization of this substrate is
complex and there is a requirement for efficient micro-organisms to ferment a variety of sugars
[Gunaseelan; 1997].
The amount of petrochemical like diesel and petrol is limited and the whole world is under the
shade of energy crisis and looking for the alternative fuel source. Many countries have used bio-
fuel as alternative to the petrochemicals like diesel and petrol. Ethanol is a kind of bio-fuel which
can be blended with the gasoline in fixed proportion and can be used as alternative to fuel like
diesel and petrol. Maximum of 20 % ethanol can be blended with the gasoline and can be used as
alternative fuel source with same carbonator engine [Midilli; 2006]. Bioethanol is renewable
source of energy which is generally produced by the fermentation of carbohydrate. Biomass such
as cellulose, animal fat, etc is used for the production of ethanol through fermentation. Main
source for such kind of biomass is sugarcane, corn, wheat bran, cassava, sweet potato etc. These
are used for the ethanol production but they are mainly used for the food source and if these
sources will be used for the ethanol production the whole world is going to face food crisis as the
world population is increasing rapidly. To prevent the world from fuel crisis and food crisis,
research has been focused on the production of bio-fuel from the waste biomass and the plant
source which is not for the food purpose like waste generated in sugar mill, chemical pulp
generated in paper industry and weed plant.


Bio-fuels produced from biomass such as plants or organic waste could help to reduce both the
worlds dependence on oil and CO
2
production. These bio-fuels have the potential to cut CO
2

emission because the plants they are made from use CO
2
as they grow [Osamu and Carl; 1987].
Bio-fuels and bio-products produced from plant biomass would mitigate global warming. This
may due to the CO
2
released in burning equals the CO
2
tied up by the plant during
photosynthesis and thus does not increase the net CO
2
in the atmosphere. Additionally, bio-fuel
production along with bio-products can provide new income and employment opportunities in
rural areas. 21st Century is looking for a shift to alternate industrial feedstock and green
processes to produce these chemicals from renewable biomass resources [Stevens and Verhe;
2000].First generation bio-fuels can offer some CO
2
benefits and can help to improve domestic
energy security. But concerns exist about the sourcing of feedstock, including the impact it may
have on biodiversity and land use and competition with food crops. There are concerns about
environmental impacts and carbon balances, which sets limits in the increasing production of
bio-fuels of first generation. The main disadvantage of first generation bio-fuels is the food-
versus-fuel debate; one of the reasons for rising food prices is due to the increase in the
production of these fuels [Laursen; 1993].
Second-generation bio-fuels produced from plant biomass refers largely to lignocelluloses
materials, as this makes up the majority of the cheap and abundant nonfood materials available
from plants. But, at present, the production of such fuels is not cost effective because there are a
number of technical barriers that need to be overcome before their potential can be realized
[Eisberg; 1887]. Plant biomass represents one of the most abundant and underutilized biological
resources on the planet, and is seen as a promising source of material for fuels and raw materials.
At its most basic, plant biomass can simply be burned in order to produce heat and electricity.
However, there is great potential in the use of plant biomass to produce liquid bio-fuels.
However, bio-fuel production from agricultural by-products could only satisfy a proportion of
the increasing demand for liquid fuels. This has generated great interest in making use of
dedicated biomass crops as feedstock for bio-fuel production [Gomez et.al; 1978]. The examples
of 2
nd
generation bio-fuels are cellulosic ethanol and FischerTropsch fuels. The production of
2
nd
generation bio-fuels is non-commercial at this time, although pilot and demonstration
facilities are being developed. Therefore it is anticipated that, these 2
nd
generation bio-fuels
could significantly reduce CO
2
production, do not compete with food crops and some types can

offer better engine performance. When commercialized, the cost of second generation bio-fuels
has the potential to be more comparable with standard petrol, diesel, and would be most cost
effective route to renewable, low carbon energy for road transport [ www.shell.com.].
Therefore due to many advantages and disadvantages of the 1
st
generation bio-fuels and obvious
advantages of 2
nd
generation bio-fuels as shown in Table. 1, the approaches to integral utilization
of biomass for sustainable development are more reasonable, where all parts of the plant such as
leaves, bark, fruits, and seeds can be utilized to useful products.

1
st
Generation Fuel 2
nd
Generation Bio-fuel
Technology- Economical
Feedstock- Vegetable oils, Corn sugar,
etc.
Feedstock- Non-food, Cheap and Abundant Plant
Waste Biomass (Agricultural and Forest Residue,
Grass, Aquatic Biomass and Water Hyacinth, etc.
Products- FAME or Bio-diesel, Corn
ethanol, sugar alcohol
Products- Hydro treating oil, Bio-oil, FT oil,
Lignocelluloses Ethanol, Butanol, Mixed Alcohols
Benefits- Environmental friendly,
Economic and Social Security
Advantages-
1. Not competing with food
2. Advance technology still under
development to reduce the cost of
conversion
3. Environmentally friendly
Problems-
1. Limited feedstock (Food Vs Fuel)
2. Blended partly with conventional
fuel


Table1.1- Comparison of First and Second Generation Bio-fuel





1.2 Motivation-
The amount of petrochemical like diesel and petrol is limited and the whole world is under the
shade of energy crisis and looking for the alternative fuel source. Many countries have used bio-
fuel as alternative to the petrochemicals like diesel and petrol. Ethanol is a kind of bio-fuel which
can be blended with the gasoline in fixed proportion and can be used as alternative to fuel like
diesel and petrol. Maximum of 20 % ethanol can be blended with the gasoline and can be used as
alternative fuel source with same carbonator engine. Bioethanol is renewable source of energy
which is generally produced by the fermentation of carbohydrate. Biomass such as cellulose,
animal fat, etc is used for the production of ethanol through fermentation. Main source for such
kind of biomass is sugarcane, corn, wheat bran, cassava, sweet potato etc. These are used for the
ethanol production but they are mainly used for the food source and if these sources will be used
for the ethanol production the whole world is going to face food crisis as the world population is
increasing rapidly. To prevent the world from fuel crisis and food crisis, research has been
focused on the production of bio-fuel from the waste biomass and the plant source which is not
for the food purpose like waste generated in sugar mill, chemical pulp generated in paper
industry and weed plant.

1.3 Objectives of Study-
1. Biomass characterization for bio-ethanol production
2. Hydrolysis of characterized biomass using microbes
3. Fermentation for ethanol production of characterized biomass

1.4 Outline of Thesis-
The outlines of the thesis work are structured as mentioned below:

Chapter 1:
This chapter relates to the Introduction/ foundation of the thesis work, motivation and objectives
of the study.

Chapter 2:

This chapter relates to the details about the project work carried out and contains the literature
survey pertaining to the project work.

Chapter 3:
This chapter relates to the detail about materials and methods used for biomass characterization,
hydrolysis, fermentation and calculations.

Chapter 4:
This chapter relates to the details of results obtained and discussion of the results in light of
previous findings.




























CHAPTER 2











2. REVIEW OF LITERATURE
2.1 Lignocelluloses biomass-
First generation bio-fuels produced primarily from food crops such as grains, sugar beet and oil
seeds are under scanner, due to the possibility of creating undue competition for land and water
used for food and fiber production. The cumulative impacts of these concerns have increased the
interest in developing bio-fuels produced from non-food biomass. Second-generation bio-fuels
production from lignocelluloses biomass which includes agricultural residues (Corn Stover, crop
straws and bagasse), herbaceous crops (alfalfa, switch grass), short rotation woody crops,
forestry residues, waste paper and other wastes (municipal and industrial)cereal straw, bagasse,
forest residues, purpose-grown energy crops such as vegetative grasses and short rotation forests,
could avoid many of the concerns facing first-generation bio-fuels and potentially offer greater
cost reduction potential in the longer term and do not interfere with food security [Salman ;
2011]. Moreover, bio-ethanol is very important in terms of energy security, environmental
concern, employment opportunities, agricultural development, foreign exchange saving,
socioeconomic issues etc. Lignocelluloses biomass consists of three main structural units:
cellulose, hemicelluloses and lignin. Cellulose is crystalline glucose polymer and hemicellulose
is amorphous polymers of xylose, arabinose, and lignin a large poly aromatic compounds.
The term characterization describes the measurement of the chemistry, structure, and interactions
of the biomass components [Pauly and Keegstra; 2008]. The characterization of different
biomass viz. Pine needle, Camelina, Jatropha stem, Jatropha petiole, Jatropha leaves, Jatropha
seed coat, Jatropha fruit husk, Jatropha seed cake, Wheat straw, Paddy, Sugarcane and Sugarcane
trash has been conducted by different researchers aiming to explore its potential for use as
feedstock for bio-ethanol production.
Some details of different biomass:-
Pines are conifer trees in the genus Pinus in the family Pinaceae. They are the only genus in
the sub-family Pinoideae [Barnes and Wagner; 2004].Needles, the adult leaves, which are
green (photosynthetic), bundled in clusters (fascicles) of 16, commonly 25, needles together,
each fascicle produced from a small bud on a dwarf shoot in the axil of a scale leaf. Camelina
sativa is a flowering plant in the family Brassicaceae and is usually known in English
as Camelina, gold-of-pleasure, or false flax, also occasionally wild flax, linseed dodder, German

sesame, and Siberian oilseed. It is native to Europe and to Central Asian areas. This plant is
cultivated as oil seed crop [Jones et.al; 2005]. The crop is now being researched due to its
exceptionally high levels (up to 45%) of omega-3 fatty acids, which is uncommon in vegetable
sources. Seeds contain 38 to 43% oil and 27 to 32% protein. Wheat (Triticum spp.) is a cereal
grain, originally from the Levantregion of the Near East. This grain is grown on more land area
than any other commercial food. Wheat is widely cultivated as a cash crop because it produces a
good yield per unit area, grows well in a temperate climate even with a moderately short growing
season [Francis et.al; 2009]. Sugarcane is species of tall perennial true grasses of the
genus Saccharum, tribe Andropogoneae. Sugarcane belongs to the grass family (Poaceae), an
economically important seed plant family that includes maize, wheat, rice, and sorghum and
many forage crops. The main product of sugarcane is sucrose, which accumulates in the stalk
internodes. Sucrose, extracted and purified in specialized mill factories, is used as raw material
in human food industries or is fermented to produce ethanol [Galloway; 1914]. Ethanol is
produced on a large scale by the Brazilian sugarcane industry. Jatropha curcas is a species of
spurge family, Euphorbiaceae. J. curcas is a poisonous, semi-evergreen shrub or small tree. It is
resistant to a high degree of aridity, allowing it to be grown in deserts. The seeds contain 27-
40% oil (average: 34.4%) [Achten et.al; 2008] that can be processed to produce a high-
quality bio-diesel fuel, usable in a standard diesel engine. The seeds are also a source of the
highly poisonous toxalbumin curcin or jatrophin.

2.2 PLANT CELL WALL COMPOSITION-
2.2.1 Cell Wall-
The cell wall is a tough, flexible but sometimes fairly rigid layer that surrounds some types of
cells [Alexopoulos et.al; 1996]. It is located outside the cell membrane and provides these cells
with structural support and protection, in addition to acting as a filtering mechanism. A major
function of the cell wall is to act as a pressure vessel, preventing over-expansion when water
enters the cell [Howland and John; 2000]. Cell walls are found in plants, bacteria, fungi, algae,
and some Achaea. Animals and protozoa do not have cell walls [Koivikko et.al; 2005]. The
material in the cell wall varies between species, and can also differ depending on cell type and
developmental stage.

2.2.2 Cell Wall Components-
The main ingredient in cell wall is polysaccharides (or complex carbohydrates or complex
sugars) which are built from monosaccharide (or simple sugars).Eleven different
monosaccharide are common in these polysaccharides including glucose and galactose.
Carbohydrates are good building blocks because they can produce a nearly infinite variety of
structures [Raven; 1983]. There are a variety of other components in the wall including protein,
and lignin.
Some of the wall components include as given below and the structure of cell wall is shown in
Fig. 1.1.
1. Cellulose
Cellulose is made up of as many as 25,000 individual glucose molecules. Cellobiose (glucose-
glucose disaccharide) is the basic building block. Cellulose readily forms hydrogen bonds with
itself (intra-molecular H-bonds) and with other cellulose chains (inter-molecular H-bonds). A
cellulose chain will form hydrogen bonds with about 36 other chains to yield micro-fibrils
[Hudler and George; 1998]. Micro-fibrils are 5-12 nm wide and give the wall strength - they
have a tensile strength. Some regions of the micro-fibrils are highly crystalline while others are
more "amorphous".
2. Cross linking glycans (=Hemicelluloses)
Diverse group of carbohydrates is called hemicelluloses. It is characterized when being soluble
in strong alkali. They are linear (straight), flat, with a -1, 4 backbone and relatively short side
chains. Two common types include xyloglucans and glucuronarabinoxylans. Other less common
ones include glucomannans, galactoglucomannans, and galactomannans. The main feature of
this group is that they dont aggregate with themselves- in other words, they dont form micro-
fibrils. However, they form hydrogen bonds with cellulose and hence the reason they are called
"cross-linking glycans" [Van Heijenoort; 2001]. There may be a fructose sugar at the end of the

side chains which may help keep the molecules planar by interacting with other regions of the
chain.
3.Lignin-
Polymer of phenolics, especially phenyl propanoids. Lignin is primarily a strengthening agent in
the wall. It also resists fungal/pathogen attack [Joseleau et.al; 2007].

Fig. 1.1- Structure of Plant Cell Wall
2.3 PRE-TREATMENT OF LIGNOCELLULOSES BIOMASS-
Pretreatment is an important tool for practical cellulose conversion processes. It is necessary in
order to alter the structure of cellulosic biomass, to make cellulose more accessible for
conversion of carbohydrate polymers into fermentable sugars. The goal of pretreatment is to
break the lignin seal, solubilize hemicelluloses, and disrupt the crystalline structure of cellulose
[Taherzadeh et.al; 2004]. Various pretreatment options are available now to fractionate,
solubilize, hydrolyze, and separate cellulose, hemicelluloses, and lignin components. The
biomass is treated to reduce its size and open its structure. Pretreatment usually hydrolyzes
hemicelluloses to the sugars (xylose, L-arabinose, and others) that are water soluble [Saha and
B.C.; 2004].

Pretreatment must meet the following requirements [Sun; 2002]:
1. Improve the formation of sugars or the ability to subsequently form sugars by hydrolysis,
2. avoid the degradation or loss of carbohydrate,
3. Avoid the formation of byproducts that are inhibitory to the subsequent hydrolysis and
fermentation processes, and
4. Be cost-effective.
Pre-treatment are of following types:-
1. Physical Pre-treatment
2. Chemical Pre-treatment
3. Biological Pre-treatment

Physical Pre-treatment-
Physical pretreatments do not use any chemicals. Size reduction by mechanical methods such as
grinding or milling is one of them, through which the surface area of biomass is increased, and
the degree of polymerization (DP) and crystallinity of cellulose is decreased to some extent, but
the power requirement for reducing the feedstock from millimeter size to fine particles of
micrometers is extremely high, which is unacceptable from the engineering point of view.
Radiation such as microwaves that can penetrate and heat the feedstock instantly has also been
studied [Binod et.al; 2011]. However, it is problematic to process the feedstock in large
quantities, not to mention the power requirement to generate the radiation. Therefore, more
attention regarding physical pretreatment has been focused on the hydrothermal processes of
steam explosion (SE) and liquid hot water (LHW) treatment. SE involves heating the feedstock
at elevated temperature and pressure for a short duration, followed by depressurizing the system
to disrupt the structure of LCCs. Due to lower capital investment, less impact on the
environment, and simple process design and operation, the SE process has been tested at pilot
scales worldwide. The mechanism underlying the pretreatment is assumed to be the partial
degradation of LCCs catalyzed by acetic acid released from acetylated hemicelluloses and other
organic acids such as formic and levulinic acids, making the process auto hydrolytic in nature
[Ramos; 2003].


Chemical Pre-treatment-
High temperatures applied during the hydrothermal pretreatments under SE and LHW conditions
dehydrate sugars and produce inhibitors such as furfural from xylose and hydroxyl-methyfurfural
from glucose. To address this problem, acids can be supplemented to facilitate the deconstruction
of LCCs under less severe conditions, either lower temperature or shorter reaction time. Among
various acids, sulfuric acid is most commonly used. Although the temperatures in concentrated
acid pretreatment are much lower, acid recovery presents a big challenge for the economic
viability of the process. Therefore, dilute acid with concentrations less than 2% is preferred,
which can be conveniently neutralized by lime or ammonium during the conditioning process
[Jennings and Schell; 2011]. Dilute acid pretreatments have been intensively studied over the
years with various feedstock and reactors at different scales [Lloyd and Wyman; 2005].

Biological Pre-treatment-
Compared with physical and chemical pretreatments in which expensive equipment, chemicals
and intensive energy consumption are needed, biological pretreatment by solid fermentation
employs microorganisms that degrade lignocelluloses biomass at mild conditions without special
requirements for equipment [Keller et.al; 2003]. Both bacteria and fungi have been explored,
but rot fungi associated with wood decay are the predominant species in lignocelluloses
degradation for the purpose of bio-fuel production, particularly white-rot fungi due to their
abundant ligninolytic enzymes, including lignin peroxidase, manganese peroxidase, laccases and
other enzymes, and better selectivity in lignin degradation [Dashtban et.al; 2010)]. Although
biological pretreatment is energy-saving and environmentally friendly, its disadvantages are
apparent. Firstly, the extremely low degradation rate requires times as long as weeks for a
significant change in the structure of the lignocelluloses biomass, making the process
mismatched with the subsequent hydrolysis of cellulose and fermentation of sugars. Secondly,
significant biomass is lost during the process, not only the lignin which is mineralized into low
molecular- weight fragments that might be further catabolized into the useless final product CO
2

[Steffen et.al; 2000], but also sugars released from hemicelluloses and even cellulose by the
hydrolytic enzymes (simultaneous decay with lignin degradation) as a carbon source to support
the growth of the microorganisms [Hammel; 1997]. Finally, the control of microbial growth and
metabolism under open and solid fermentation conditions with mixture species is unreliable,

which inevitably affects the subsequent processes such as cellulose hydrolysis and ethanol
fermentation.

2.4 MICROBIAL HYDROLYSIS-
2.4.1 Hydrolysis-
Hydrolysis (From Greek hydro-meaning "water", and lyses, meaning "separation") usually
means the cleavage of chemical bonds by the addition of water. Where a carbohydrate is broken
into its component sugar molecules by hydrolysis (e.g. sucrose being broken down into
glucose and fructose), this is termed saccharification. Generally, hydrolysis or saccharification
is a step in the degradation of a substance.
Hydrolysis is a chemical reaction in which water is being used to break the bonds of certain
substances. In biotechnology and living organisms, these substances are often polymers. In a
hydrolysis reaction involving an ester link, such as that found between two amino acids in a
protein, the products that result include one that receives the hydroxyl (OH) group from the
water molecule, and another that becomes a carboxylic acid with the addition of the remaining
proton (H+).
Hydrolysis reactions in living organisms are performed with the help of catalysis by a class of
enzymes known as hydrolases. The biochemical reactions that break down polymers such as
proteins (peptide bonds between amino acids), nucleotides, complex sugars and starch, and fats
are catalyzed by this class of enzymes. Within this class, lipases, amylases and proteinases
hydrolyze fats, sugars and proteins, respectively.
Cellulose-degrading bacteria and fungi play a special role in paper production and other
everyday biotechnology applications, because they have enzymes (cellulases and esterases) that
can break cellulose into polysaccharides (polymers of sugar molecules) or glucose.
2.4.2 Enzymatic Hydrolysis-
The enzymatic hydrolysis, aims at the hydrolysis of the hemicellulose and the delignification. In
the case of the hydrolysis of the hemicelluloses, in spite of the specificity of xylanases, where the
action is carried out through the synergy of the - xylosidase, endo 1,4--xylanases,

acetylxylanaesterase, -glucoronidase and Larabinofuranosidase enzymes, hurdles exist in the
form of high cost of enzyme and adequate scale up to industrial level. There are two major types
of lignolytic enzymes widely used: phenol oxidase (laccase) and peroxidases (lignin peroxidase,
LiP and manganese peroxidase, MnP) [Krause et.al; 2003]. The other enzymes also used, but
whose mode of action are not clearly known, are glyoxal oxidase, glucose oxidase, oxido-
reductase and methanol oxidase [Eriksson; 2000]. The main area of application was the paper
and pulp industry, where such enzymes are used to convert to chlorine and chorine derivative
substances. This creates another problem due to the irreversible tendency of total chlorine free
bleaching (TCF systems) and elemental chlorine free (ECF systems) [Viikari et.al; 1994].
2.4.2.1 Cellulases Enzyme System-
Natural cellulosic substrates (primarily plant cell materials) are composed of heterogeneous
intertwined polysaccharide chains with varying degrees of crystallinity, hemicelluloses and
pectin, embedded in lignin. Microorganisms produce multiple enzymes to degrade plant cell
materials, known as enzyme systems [Acebal et.al; 1986].
For microorganisms to hydrolyze and metabolize insoluble cellulose, extracellular cellulases
must be produced that are either free or cell associated. The biochemical analysis of cellulases
systems from aerobic and anaerobic bacteria and fungi has been comprehensively reviewed
during the past two decades. Components of cellulases systems were first classified based on
their mode of catalytic action and have more recently been classified based on structural
properties. Three major types of enzymatic activities are found [Aho et.al; 1990]:

1. Endoglucanases or 1,4-D-glucan-4-glucanohydrolases,
2. Exoglucanases, including 1,4-D-glucan glucanohydrolases (also known as
cellodextrinases) and 1,4-Dglucan cellobiohydrolases (cellobiohydrolases), and
3. Glucosidases or glucoside glucohydrolases





1. Endoglucanases or 1,4-D-glucan-4-glucanohydrolases-
Endoglucanases cut at random at internal amorphous sites in the cellulose polysaccharide
chain, generating oligosaccharides of various lengths and consequently new chain ends
[Barras et.al; 1994].

2. Exoglucanases, or cellodextrinases or cellobiohydrolases-
Exoglucanases act in a possessive manner on the reducing or non-reducing ends of
cellulose polysaccharide chains, liberating either glucose (glucanohydrolases) or
cellobiose (cellobiohydrolases) as major products [Boussaid et.al; 1999]. Exoglucanases
can also act on micro-crystalline cellulose, presumably peeling cellulose chains from the
micro-crystalline structure.

3. Glucosidases or glucoside glucohydrolases-
Glucosidases hydrolyze soluble cellodextrinases and cellobiose to glucose [Adney et.al;
1994].

Cellulases are distinguished from other glycoside hydrolases by their ability to hydrolyze 1, 4-
glucosidic bonds between glucosyl residues. The enzymatic breakage of the 1,4-glucosidic bonds
in cellulose proceeds through an acid hydrolysis mechanism, using a proton donor and
nucleophile or base. The hydrolysis products can either result in the inversion or retention
(double replacement mechanism) of the anomeric configuration of carbon-1 at the reducing end.
The insoluble, recalcitrant nature of cellulose represents a challenge for cellulases systems. A
general feature of most cellulases is a modular structure often including both catalytic and
carbohydrate-binding modules (CBMs) [Bagnara et.al; 1985]. The CBM effects binding to
the cellulose surface, presumably to facilitate cellulose hydrolysis by bringing the catalytic
domain in close proximity to the substrate, insoluble cellulose. The presence of CBMs is
particularly important for the initiation and processivity of exoglucanases. Revisiting the original
model of cellulose degradation proposed by Reese.et.al. a possible additional non-catalytic role
for CBMs in cellulose hydrolysis was proposed: the sloughing off of cellulose fragments from
cellulosic surfaces of, e.g., cotton fibers, thereby enhancing cellulose hydrolysis. Cellulases
systems exhibit higher collective activity than the sum of the activities of individual enzymes, a

phenomenon known as synergism. Four forms of synergism have been reported [Lemaire et.al;
1996]:

I. Endo-exo synergy between Endoglucanases and exoglucanases,
II. Exo-exo synergy between exoglucanases processing from the reducing and non-reducing
ends of cellulose chains,
III. Synergy between exoglucanases and glucosidases that remove cellobiose (and
cellodextrinases) as end products of the first two enzymes, and Intramolecular synergy
between catalytic domains and CBMs.
2.4.3 Microbial Hydrolysis-
Lignin, due to its recalcitrant structure, decreases the catalytic power and may cause inactivation
of the cellulases. Palonen; 2004 showed that the localization and the structure of lignin affect
more the enzymatic hydrolysis than the absolute lignin quantity in the lignocellulosics complex.
The study revealed, furthermore, that modifications of the lignin surface by oxidants treatments
with lactase led to a rise of the cellulose hydrolysis. There are various microorganisms present in
nature which are able to attack and degrade lignin, thus exposing the cellulose to easy action by
cellulases. These microorganisms are found in forest leaf litter/composts and include the wood
rotting fungi, actinomycetes and bacteria. They have specialized enzyme systems to attack,
depolymerize and degrade the lignocellulosics matrix to release the lignin and other polymers.
Phanerochaete chrysosporium has been the main microorganism studied for lignin degradation
by white rot fungi [Kirk and Farell; 1987]. Saritha et.al; 2012 have studied the biological
treatments with white rot fungi and Streptomyces sp. For delignifying pulp, increasing
digestibility of lignocellulosics as animal feed and for bioremediation of paper mill effluents.
Such lignocellulolytic microbes are extremely useful supplements in production of bio-ethanol as
they help in removal of lignin from lignocellulosics substrate and also aid in cellulase
production. They further studied the treatment of hardwood and softwood residues with
Streptomyces griseus isolated from leaf litter and showed that it increased the mild alkaline
dissolution of lignins and produced high levels of the cellulase complex when grown on wood
substrates. The observed loss of lignin was 10.5% and 23.5% in case of soft wood and hard
wood, respectively. The fungal breakdown of lignin is anaerobic and uses a family of

extracellular enzymes termed as lignases [Howard et.al; 2005]. Some bacterial laccases have
also been isolated from Azospirillum lipoferum, Bacillus subtilis etc. [Kunamneni et.al; 2007].
Recent researches have elucidated that microorganisms like Lentinus edodes (Songulashvilli et
al, 2005), Pleurotes spp. [Ragunathan and Swaminathan; 2004], Penicillium camemberti
cultured at 2535C for 322 days resulted in 4575% and 6580% holocellulose and lignin
degradation, respectively. Thus, biological pretreatment process for lignocellulosic substrate
using lignolytic organisms such as actinomycetes and white rot fungi can be developed for
facilitating efficient enzymatic digestibility of cellulose. Microbial hydrolysis not only offers
advantages such as low capital cost and low energy, but also added advantages of little
dependence of chemicals, and mild environmental conditions. However, the main flaw in the
process is the low hydrolysis rate obtained in most biological processes. Thus, an economic
biological hydrolysis process of lignocelluloses having improved hydrolysis leading eventually
to improved fuel yields has to be found out by researching more micro-organisms for their ability
to delignify the plant material quickly and efficiently [Saritha et.al; 2012].
2.4.3.1 Aspergillus niger-
Aspergillus niger is a haploid filamentous fungus and is a very essential microorganism in the
field of biology. In addition to producing extracellular enzymes and citric acid, A. niger is used
for waste management and bio-transformations [Adams et.al; 1997]. The fungi are most
commonly found in mesophilic environments such as decaying vegetation or soil and plants.
Aspergillus niger was isolated from the plant Welwitschia mirabilis in Namibia and Angola, a
plant estimated to be about 3000 years old [Takahashi et.al; 1991]. A. niger is easily isolated
from common thing such as dust, paint, and soil.
Aspergillus niger is usually found in common mesophilic environments such as soil, plants, and
enclosed air environments. A. niger is not only a xerophilic fungi (mold that doesnt require free
water for growth, can grow in humid environments), but is also a thermotolerant organism
(capable of growing at high temperatures). Because of this property, the filamentous fungi
exhibits a high tolerance to freezing temperatures.

The production of ochratoxin A from A. niger, is liable to cause immunotoxcitiy in animals
[Santhiya et.al; 2005]. The effects on animals include a decrease in antibody responses, a size
reduction in immune organs, and an alteration in the production of cytokine which are proteins
and peptides specifically used in signaling. Food that has been contaminated by A. niger has
toxic metabolite which had a major affect on the poultry industry. The culture plate is being
shown in Fig. 1.2.

Fig. 1.2- Aspergillus niger on NA Plate
2.4.3.2 Trichoderma reesei-
Trichoderma reesei is a mesophilic and filamentous fungus. It is an anamorph of the
fungus Hypocrea jecorina. T.reesei has the capacity to secrete large amounts
of cellulolytic enzymes (cellulases and hemicellulases).Microbial cellulases have industrial
application in the conversion of cellulose, a major component of plant biomass, into glucose.
Recent advances in the biochemistry of cellulases enzymology, the mechanism of
cellulose hydrolysis (cellulolysis), strain improvement, molecular cloning and process
engineering are bringing T. reesei cellulases closer to being a commercially viable route to
cellulose hydrolysis. The genome of this organism was released in 2008. . The culture plate is
being shown in Fig. 1.3.


Fig. 1.3- Trichoderma reesei on NA Plate

2.5 FERMENTATION FOR ETHANOL PRODUCTION-
2.5.1 Ethanol fermentation-
The chemical equation below shows the alcoholic fermentation of glucose, whose chemical
formula is C
6
H
12
O
6
.One glucose molecule is converted into two ethanol molecules and two
carbon dioxide molecules:
C
6
H
12
O
6
2 C
2
H
5
OH + 2 CO
2

C
2
H
5
OH is the chemical formula for ethanol.
Before fermentation takes place, one glucose molecule is broken down into two pyruvate
molecules. This is known as glycolysis.



2.5.2 Saccharomyces cerevisiae-
Saccharomyces cerevisiae is a eukaryotic microbe. More specifically, it is globular-shaped,
yellow-green yeast belonging to the Fungi kingdom, which includes multicellular organisms
such as mushrooms and molds. Natural strains of the yeast have been found on the surfaces of
plants, the gastrointestinal tracts and body surfaces of insects and warm-blooded animals, soils
from all regions of the world and even in aquatic environments. Most often it is found in areas
where fermentation can occur, such as the on the surface of fruit, storage cellars and on the
equipment used during the fermentation process.
Saccharomyces means "sugar fungus", which provides a clue to the function of this yeast.
Saccharomyces have the ability to ferment sugars, meaning they can convert sugar into carbon
dioxide and alcohol [Stewart and Graham; 1987].
S. cerevisiae is famously known for its role in food production. It is the critical component in the
fermentation process that converts sugar into alcohol; an ingredient shared in beer, wine and
distilled beverages [Freeman and Scott; 2005]. It is also used in the baking process as a
leavening agent; yeast releasing gas into their environment results in the spongy-like texture of
breads and cakes. Archaeologists have found evidence of a fermented beverage in a pot in China
as early as 7000BC, and molecular evidence of yeast being used in fermentation was found in a
wine jar dating back to 3150BC. Isolation of the species did not occur until 1938, when Emil
Mrak isolated it from rotten figs found in Merced, California.
S. cerevisiae is also considered to be a "model organism" by scientists [Osley et.al; 2007]. Its
big advantage is that it is both a unicellular and eukaryotic organism. Another advantage is its
fast growth rate. The culture plate is being shown in Fig. 1.4.


Fig. 1.4- Saccharomyces cerevisiae on NA Plate
2.5.2.1 Fermentation of Hexoses by Saccharomyces cerevisiae-
Wild-type S. cerevisiae ferments glucose, the dominant sugar in all plant hydrolysates, at high
rates even under anaerobic conditions. S. cerevisiae contains an elaborate system for hexose
transport [Baldoma and Aguilar; 1988]. S. cerevisiae all transport glucose via facilitated
diffusion; glucose uptake only requires a concentration gradient across the plasma membrane.
After uptake, glucose dissimilation proceeds via the Embden-Meyerhof glycolytic pathway
[Barnett et.al; 1990]. This pathway oxidizes glucose to two pyruvate, resulting in the net
formation of two ATP per glucose. In anaerobic, fermentative cultures of S. cerevisiae, the
NADH formed by glyceraldehyde-3-phosphate dehydrogenase is re-oxidized via alcoholic
fermentation. This essential redox balancing involves the combined activity of pyruvate
decarboxylase and alcohol dehydrogenase. But obviously glucose is not the only carbohydrate
present in the hydrolysates. In order to ferment such non-glucose carbohydrates with S.
cerevisiae, three key criteria have to be met:
1. Presence of a functional transporter in the plasma membrane,
2. Presence of enzyme(s) that couple metabolism of the carbohydrate to the main glycolytic
pathway and
3. Maintenance of a closed redox balance.

Mannose and fructose are two isomers of glucose that occur in all plant-derived biomass
hydrolysates and that can be fermented by all wild-type S. cerevisiae strains. The general
observation that yeast capable of fermenting glucose can also ferment fructose and mannose is
known as the Kluyver rule. After phosphorylation by hexokinase, mannose-6-phosphate is
isomerizes to fructose-6-phosphate by phosphomannose isomerase, encoded by the PMI40 gene.
Hexokinase is also responsible for phosphorylation of fructose to fructose-6-phosphate, which is
subsequently metabolized through glycolysis. As mannose and glucose compete for the same
hexose transporters, kinetics of mixed-substrate utilization is determined by their relative and
absolute concentrations in hydrolysates. The galactose permease Gal2p subsequently converted
into glucose-6-phosphate via the Leloir pathway [Blank et.al; 2005]. This pathway, which links
galactose catabolism to the main glycolytic pathway, consists of three reactions. After
phosphorylation of galactose by galactokinase (Gal1p), galactose-1-phosphate
uridylyltransferase (Gal7p) converts UDP-glucose and galactose-1-phosphate to UDP-galactose
and glucose-1-phosphate. UDP galactose is reconverted into UDP-glucose by the uridine-
diphosphoglucose 4-epimerase (Gal10p). Finally, glucose-1-phosphate is converted to glucose-6-
phosphate by phosphoglucomutase, major and minor isoforms of which are encoded by PGM2
(also called GAL5) and PGM1, respectively. In wild-type S. cerevisiae strains, growth rates on
galactose are generally lower than those on glucose.
2.5.3 Zymomonas mobilis-
Zymomonas mobilis is a bacterium belonging to the genus Zymomonas. It is notable for
its bioethanol-producing capabilities, which surpass yeast in some aspects. It was originally
isolated from alcoholic beverages. Z. mobilis degrades sugars to pyruvate using the Entner-
Doudoroff pathway. The pyruvate is then fermented to produce ethanol and carbon dioxide as
the only products (analogous to yeast).
The advantages of Z. mobilis over S. cerevisiae with respect to producing bio-ethanol:
Higher sugar uptake and ethanol yield (up to 2.5 times higher),
Lower biomass production,
Higher ethanol tolerance up to 16% (v/v),

Does not require controlled addition of oxygen during the fermentation,
Amenability to genetic manipulations.
However, in spite of these attractive advantages, several factors prevent the commercial usage
of Z. mobilis in cellulosic ethanol production. The foremost hurdle is that its substrate range is
limited to glucose, fructose and sucrose [Zhang et.al; 1995].


























CHAPTER 3













3. MATERIALS AND METHODOLOGY-
3.1 Materials-
1. Biomass- Different biomass has been used for the characterization. Listing below different
biomass used for characterization:-
1. Pine Needle
2. Paddy
3. Sugarcane
4. Jatropha Stem
5. Jatropha Leaves
6. Jatropha Petiole
7. Camelina
8. Sugarcane trash
9. Wheat straw
10. Jatropha seed cake
11. Jatropha fruit husk
12. Jatropha seed coat

2. Microbes- Hydrolysis was carried on different biomass with the incubation of different
microbes. Each biomass is treated with separate microbe for hydrolysis. Microbes used were
Aspergillus niger and Trichoderma reesei. The incubation was done on Jatropha Seed Coat and
Jatropha Fruit Husk. The fermenting microbe used was Saccharomyces cerevisiae.

3. Chemicals- Chemicals of Hi-Media and Sigma-Aldrich were used for making different
solutions.

4. Glassware- Glassware of Borosil was used for conducting experiment.

3.2 Methodology-
1. Characterization of Biomass- The biomass is characterized into holo-cellulose, cellulose,
hemi-cellulose and lignin.


a. Holo Cellulose Determination-
To 2 g of the extractive free sample, 180 ml distilled water; 8.6 g sodium chloride, 6.0ml
ethanoic acid and 6.6 g sodium chloride were added. The mixture was then digested in a 250 ml
conical flask under reflux at 70C for 3 hours. It was then allowed to cool, filtered and the
residue washed with five 20 ml portions of 100 ml distilled water, the residue was then dried at
105C for 24 hours to attain constant weight [Dubois et.al; 1956].

2 g of the extractive free sample

Add 180 ml distilled water
8.6 g sodium chloride
6.0ml ethanoic acid
6.6 g sodium chloride

Mixture digested in a 250 ml conical flask under reflux at 70C for 3 hours

Cool, filtered and the residue washed with five 20 ml portions of 100 ml distilled water

Residue dried at 105C for 24 hours to attain constant weight


b. Cellulose Determination-
To 2 g of the extractive free sample was taken and put into a 250 ml beaker, 100 ml of 17.5%
NaOH solution was added and stirred at 25C for 30 minutes. The content of the beaker was then
filtered, washed with 25 ml of 9.5% NaOH solution and 20 ml portions of 100 ml distilled water.
The residue was again washed with distilled water and 40 ml of 10% acetic acid and further with
1 L distilled water. The residue was then dried at 105C for 24 hours to constant weight
[Association of Official Analytical Chemists; 1990].






2 g of the extractive free sample

Add 100 ml of 17.5% NaOH solution and stirred at 25C for 30 minutes

Content filtered & washed 25 ml of 9.5% NaOH solution
20 ml portions of 100 ml distilled water

Residue washed Distilled water
40 ml of 10% acetic acid
1 L distilled water

Residue dried at 105C for 24 hours to constant weight

c. Hemi-cellulose Determination-
On the basis of solubility in 17.5% NaOH solution, holo-cellulose is sub divided into:-
1. Insoluble cellulose
2. Soluble hemi-cellulose

Hemi-cellulose -Difference between the weight of holo-cellulose and weight of cellulose present
[Raghuramulu et.al; 1983].

d. Lignin Determination-
To 1 g of the extractives free sawdust sample, 14 ml of cold 72% sulphuric acid was added and
stirred. The mixture was left to stand for 2 hours. After the 2 hours, the mixture was then washed
in a 1 L conical flask and diluted to 3% sulfuric acid. The mixture was then boiled for 4 hours
under reflux. The insoluble material was al- lowed to settle and filtered. The residue was washed
and dried in an oven at 105C after 2 hours this then cooled and weighed as the lignin content
[Templeton and Ehrman; 1995].





1 g of the extractives free sawdust sample

Add 14 ml of cold 72% sulphuric acid and stirred

Left mixture to stand for 2 hours

After the 2 hours Mixture was then washed in a 1 L conical flask
Diluted to 3% sulfuric acid
Boil mixture for 4 hours under reflux

Insoluble material settled and filtered

Residue was washed and dried in an oven at 105C

After 2 hours cooled and weighed as the lignin content
2. Hydrolysis-
To 1 gm powdered biomass 100 ml Mandels Media was added. 1ml sample was collected in
two appendrof each to estimate total carbohydrate and reducing sugar. Three replicates of each
sample were made and each microbe was incubated in each sample.1 ml sample was taken in
each day incubation.
1 gm sample + 100 ml Mandels Media
Mandels Media-
Urea-0.3 gm
(NH
4
)
2
SO
4
-1.4 gm
KH
2
PO
4
-2.0 gm
CaCl
2
.2H
2
O-0.4 gm
MgSO
4
.7H
2
O-0.15 gm
Bactological Peptone-1.0 gm
Yeast Extract-0.25 gm
FeSO
4
.7H
2
O-0.015 gm

MnSO
4
.H
2
O-0.16 gm
ZnSO
4
.7H
2
O-0.14 gm
Co.Cl
2
-0.2 gm
Distill Water-1000 ml
pH- 5.5 to 6.0
a. Total Carbohydrate Estimation-
To 1 ml sample 4 ml Anthrone Reagent was added. Mix and boil in water bath 10 mins. Cool for
10 mins. Keep at room temperature for 20 mins. Optical density was calculated at 620 nm.
1 ml sample + 4 ml Anthrone Reagent

Mix and Boil for 10 mins

Cool for 10 mins

Kept at Room Temperature for 20 mins

OD at 620 nm
[Anthrone Reagent Preparation- 0.2 gm Anthrone in 100 ml 92% conc. H
2
SO
4
]
[92% conc. H
2
SO
4
Preparation- 93.6 ml conc.H
2
SO
4
+ 6.4 ml Distill water]
b. Reducing Sugar Estimation-
To 2 ml sample 1 ml distill water was added along with 3 ml DNSA (Di-nitro salicylic acid).
Place in water bath for 15 mins. Add 1ml Rochelle salt. OD was recorded at 510 nm.
2 ml sample + 1 ml distill water

Add 3 ml DNSA

Put in water bath for 15 mins

Add 1 ml Rochelle salt

OD at 510 nm

[DNSA Preparation - 1 gm DNSA dissolved in 100 ml of 1% NaOH + Add 0.05 gm Sodium
Sulphite + 0.2 gm Crystalline Phenol]
[Rochelle Salt Preparation- 40 gm Sodium Potassium Tartarte + 100 ml Distill water

3. Fermentation-
Ethanol Estimation-
The replicates were incubated with Saccharomyces cerevisiae. The observations were made for
next 6 days.
Potassium Dichromate Assay-
To 1ml sample 3ml chromic acid was added. Place in water bath at 90C for 10 mins. Add 1ml
Rochelle salt. Optical density of sample in each day observation was calculated at 600 nm.
1 ml sample + 3 ml Chromic Acid

Put in water bath at 90

C for 10 mins

Add 1 ml Rochelle salt

OD at 600 nm
[Chromic Acid Preparation- 1 gm Potassium Dichromate + 100 ml 5M H
2
SO
4
]
[5 M H
2
SO
4
- 27 ml H
2
SO
4
in 100 ml distill water]

4. Standard Curve Preparation-
The stock is prepared by making solution of 50 mg glucose in 50 ml distill water.
Stock Solution- 50 mg/ 50 ml solution

a. Reducing Sugar Estimation-
The reducing sugar estimation is done by DNSA method. The observations are made by
determining the optical density. The control OD obtained is 0.004. The observation made for the
estimation of reducing sugar for standard curve preparation is shown in Table 1.2. Fig. 1.5 shows
graphical analysis of reducing sugar estimated for standard curve preparation. Control OD-
0.004


Table 1.2- Standard Curve for Reducing Sugar Estimation through DNSA Method

Fig. 1.5- Standard Curve for Reducing Sugar Estimation through DNSA Method
Slope- 0.002900571
Factor- 344.7596533
b. Total Carbohydrate Estimation-
The total carbohydrate estimation is done by Anthrone method. The observations are made by
determining the optical density. The control OD obtained is 0.003. The 20 l of the sample is
being taken from original sample and further dilution is being done. The original sample is made
by different concentration of glucose dissolved in distill water making the solution of 1 ml. The
observation made for the estimation of reducing sugar for standard curve preparation is shown in
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
0 50 100 150 200 250 300 350
O
D

a
t

5
1
0

n
m

Concentration
S.No. Sample(l) Distill Water
(ml)
DNSA (ml) Rochelle
Salt (ml)
OD at 510 nm
1. 50 950 3 1 0.156
2. 100 900 3 1 0.310
3. 150 850 3 1 0.442
4. 200 800 3 1 0.589
5. 250 750 3 1 0.738
6. 300 700 3 1 0.885

Table 1.3. Fig. 1.6 shows graphical analysis of reducing sugar estimated for standard curve
preparation. Control OD- 0.003
S.No. Sample from Original
sample (l)
Distill Water (ml) OD at 620 nm
1. 20 980 0.132
2. 20 980 0.233
3. 20 980 0.342
4. 20 980 0.457
5. 20 980 0.575
6. 20 980 0.695
Table 1.3- Standard Curve for Total Carbohydrate Estimation through Anthrone Method

Fig. 1.6- Standard Curve for Total Carbohydrate Estimation through Anthrone Method
Slope- 0.002260571
Factor- 442.3660263


0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0 50 100 150 200 250 300 350
O
D

a
t

6
2
0

n
m

Concentration

c. Ethanol Estimation-
The ethanol estimation is done by Potassium dichromate assay. The observations are made by
determining the optical density. The control OD obtained is 0.001. The observation made for the
estimation of reducing sugar for standard curve preparation is shown in Table 1.3. Fig. 1.6 shows
graphical analysis of reducing sugar estimated for standard curve preparation.
Control OD- 0.001.
Table 1.4- Standard Curve for Ethanol Estimation through Potassium Dichromate Assay

Fig. 1.7- Standard Curve for Ethanol Estimation through Potassium Dichromate Assay


0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0 50 100 150 200 250 300 350
O
D

a
t

6
0
0

n
m

Concentration
S.No. Sample (l) Chromic Acid (ml) Rochelle Salt (ml) OD at 600 nm
1. 50 3 1 0.107
2. 100 3 1 0.218
3. 150 3 1 0.315
4. 200 3 1 0.405
5. 250 3 1 0.524
6. 300 3 1 0.625

3.3 Analysis-
1. Characterization of Biomass-
The characterization of biomass is done. The sample collected in dry form; so the amount of
sample is calculated in grams. The value in gram is converted in percentile to get the exact
composition of different biomass. The conversion of gram into percentile is being done for the
values of holo-cellulose, hemi-cellulose, cellulose and insoluble lignin. The conversion is done
by using the formula as given below:-
Conversion of composition of biomass in gram into percentile = Amount of the dry biomass*
100/ (Total biomass used * Total Volume)
The value of soluble lignin is obtained in gram/litre. The soluble lignin composition is obtained
by using formula:-
Acid Soluble Lignin (g/l) = A
205
*Dilution Factor/ Absorpitivity Co-efficient * Cell Path Length
{Where Absorpitivity co-efficient = 110 g/L-cm and Cell Path Length = 1 cm}
The value in g/l in converted into percentile by the multiplication with 100 of the obtained
composition.

2. Hydrolysis-
The value of reducing sugar and total carbohydrate is determined in the process of hydrolysis.
The concentration of unknown sample is calculated by using formula as below:-

Concentration of unknown sample = OD * Standard Factor
{Where Standard Factor = 1/ Slope}

3. Fermentation-
The value of ethanol is determined in the process of fermentation. The concentration of unknown
sample is calculated by using formula as below:-
Concentration of unknown sample = OD * Standard Factor
{Where Standard Factor = 1/ Slope}











CHAPTER 4










4.RESULT AND DISCUSSION-
1. Characterization of Biomass-
The characterization of different biomass was done to find the different composition of plant cell
wall viz. holo-cellulose, celulose, hemi-cellulose and lignin for its use in the production of
bioethanol through microbial hydrolysis. Lignin content is important to know as this component
is non degradable and also limits the access of enzymes for cellulose and hemicellulose
degradation. The different biomass characterized was pine needle, paddy, sugarcane, jatropha
stem, jatropha petiole, jatropha leaves, camelina, sugarcane trash, wheat straw, jatropha seed coat
and jatropha seed cake. The contents of holo-cellulose was observed maximum in jatropha fruit
husk, cellulose was maximum in camelina, hemi-cellulose was maximum in paddy and lignin
observed to be greater in jatropha seed coat than other biomass characterized [Table 1.5].
S.No. Sample Holo-cellulose
(%)
Cellulose
(%)
Hemi-cellulose
(%)
Lignin
(%)
1. Pine Needle 59.2 50.5 8.6 25.7
2. Paddy 74.8 59.8 25.6 13.6
3. Sugarcane 75.8 61.7 14 8.9
4. Jatropha Stem 50.5 47 3.4 9.2
5. Jatropha Leaves 60.7 58.1 2.6 19.8
6. Jatropha Petiole 54.4 49 5.3 11
7. Camelina 77.5 70.2 6.4 14.9
8. Sugarcane Trash 71.8 67.3 4.5 24.2
9. Wheat Straw 79 66.9 12.1 14.9
10. Jatropha Seed Coat 74.4 67.3 7.15 42.9
11. Jatropha Fruit Husk 84.5 66.8 17.7 16.1
12. Jatropha Seed Cake 70.3 56.4 13.9 19.2
Table 1.5- Characterization of Different Biomass
Analysis was done of composition of plant cell wall in different biomass. The biomass was
grouped in three categories:-
1. Grasses: Wheat straw, Paddy leaves, Sugarcane leaves and Sugarcane trash. The maximum
holo-cellulose content was observed in wheat straw (71.8), cellulose in sugarcane trash (67.3),

hemi-cellulose was maximum in paddy (25.6) and lignin was found to be higher in sugarcane
trash (24.2) than other biomass characterized [Fig 1.8.1].

Cell Wall
Components
Wheat Straw Paddy Sugarcane Sugarcane Trash
Holo-cellulose 79 74.8 75.8 71.8
Cellulose 66.9 59.8 61.7 67.3
Hemi-cellulose 12.1 25.6 14 4.5
Lignin 14.9 13.6 8.9 24.2
Table1.6.1: Cell Wall Composition of Different Grasses

Fig 1.8.1- Characterization of Different Biomass (Grasses) for its Different Components of
Cell Wall

2. Shrub-Tree: Jatropha. Different plants parts of Jatropha viz. Jatropha leaves, Jatropha petiole,
Jatropha stem, Jatropha seed coat, Jatropha fruit husk and Jatropha seed cake were characterized
for its cell wall components. Holo-cellulose, cellulose and lignin content was observed to be
maximum in Jatropha seed coat (74.4, 67.3 and 7.15 respectively) than other biomass
characterized [Fig 1.8.2]. The study shows that Jatropha seed coat has maximum lignin
component in the plant cell wall composition than other Jatropha plant parts.


0
10
20
30
40
50
60
70
80
Wheat
Straw
Paddy Sugarcane Sugarcane
Trash
C
o
m
p
o
s
i
t
i
o
n

o
f

B
i
o
m
a
s
s

Different Biomass
Holocellulose
Cellulose
Hemicellulose
Lignin

Cell wall
components
Jatropha
Leaves
Jatropha
Petiole
Jatropha
Stem
Jatropha
Seed Coat
Jatropha
Fruit Husk
Jatropha
Seed Cake
Holo-cellulose 60.7 54.4 50.5 74.4 84.5 70.3
Cellulose 58.1 49 47 67.3 66.8 56.4
Hemi-
cellulose
2.6 5.3 3.4 7.15 17.7 13.9
Lignin 19.8 11 9.2 42.9 16.1 19.2
Table 1.6.2: Cell Wall Composition of Different Jatropha Plant Parts


Fig 1.8.2- Characterization of Different Plant Parts of Jatropha

3. Tree-Shrub: Tree: Pine needle, Shrub: Camelina. The maximum content of holo-cellulose
was observed in Jatropha fruit husk (84.5), cellulose in Camelina (70.2), hemi-cellulose in
Jatropha fruit husk (17.7) and lignin in pine needle (25.7) than other biomass characterized [Fig
1.8.3]. The results show that Jatropha fruit husk cell wall has maximum of cellulose component.

Cell wall components Pine Needle Camelina
Holo-cellulose 59.2 77.5
Cellulose 50.5 70.2
0
10
20
30
40
50
60
70
80
90
Jatropha
Leaves
Jatropha
Petiole
Jatropha
Stem
Jatropha
Seed Coat
Jatropha
Fruit Husk
Jatropha
Seed Cake
C
o
m
p
o
s
i
t
i
o
n

o
f

B
i
o
m
a
s
s

Plant Parts of Jatropha
Holo-cellulose
Cellulose
Hemi-cellulose
Lignin

Hemi-cellulose 8.6 6.4
Lignin 25.7 14.9
Table 1.6.3: Cell wall composition of Pine Needle and Camelina


Fig 1.8.3- Characterization of Pine Needle and Camelina
It is important to know cellulose and lignin components for microbial hydrolysis and production
of ethanol. The result shows that the content of cellulose was highest in Jatropha Petiole and
lignin has highest in Pine Needle. The Jatropha Petiole has greater value of cellulose than all
other biomass and it has an increment of 1.43 fold than wheat straw [Table 1.7].
Treatment Cellulose % Lignin %
PN 50.57 23.92
C 59.87 16.85
PA 61.77 9.08
SC 47.07 11.87
SC TR 58.17 18.67
WS 49 6.47
JP 70.27 19.63
JL 67.3 22.97
JS 66.93 15.43
0
10
20
30
40
50
60
70
80
C
o
m
p
o
s
i
t
i
o
n

o
f

B
i
o
m
a
s
s

Plant Parts of Pine Needle and Camelina
Pine Needle
Camelina

JSD & JSC 61.88 22.08
J F 66.8 11.37
SE 2.49515 1.51
LSD 7.26725 4.41
Table 1.7- Cellulose and Lignin Composition of Different Biomass Characterized.

Cellulose and Lignin composition of different biomass is indicated in Table 1.7 and Fig 1.9 a &
b. Least Significant Difference (LSD) test indicates significant differences at 5% level [PN- Pine
Needle, C- Camelina, PA- Paddy, SC- Sugarcane, SC TR- Sugarcane Trash, WS- Wheat Straw,
JP- Jatropha Petiole, JL- Jatropha Leaves, JS- Jatropha Stem, JSD & JSC- Jatropha Seed Coat &
Jatropha Seed Cake , JF- Jatropha Fruit Husk].
The result shows that grasses are low in lignin content compared to shrubs and trees. since lignin
imparts recalcitrance for hydrolysis, hence, it can be concluded that grasses are more amicable to
microbial degradation compared to shrubs and tree species.
(a)
0
10
20
30
40
50
60
70
80
PN C PA SC SC TR WS JP JL JS J SD J F
C
e
l
l
u
l
o
s
e

(
%
)

Different Biomass

(b)
Fig. 1.9- (a) Cellulose and Lignin (b) Composition in Different Biomass
2. Hydrolysis-
a. Reducing Sugar Estimation-
Hydrolysis is the process which leads to break down of sucrose to glucose. In this project
Jatropha seed coat and Jatropha fruit husk were subjected to microbial hydrolysis using microbes
Aspergillus Niger and Trichoderma Reesei, studied by the lab earlier for its cellulose and laccase
activities. The reducing sugar content was estimated before incubation and after incubation of
microbes for 10 days at 2 days interval. The result shows that reducing sugar content in Jatropha
fruit husk was maximum (237.1mg/ml) before incubation compared to Jatropha seed coat. The
reducing sugar estimated was maximum in Jatropha seed coat (267.8mg/ml) with the incubation
of A. niger after 2 days, and in Jatropha fruit husk (245.4mg/ml) with the incubation of T. reesei
after 4 days. After 6 days reducing sugar content showed higher value in Jatropha fruit husk
(241.6mg/ml) and with the incubation of T. reesei for 8 days showed the maximum value in
Jatropha seed coat (225.4mg/ml) with the incubation of T. reesei and the 10 day result showed
the maximum reducing sugar content in Jatropha seed coat (218.2mg/ml) with the incubation of
T. reesei [Table 1.8].



0
5
10
15
20
25
30
PN C PA SC SC TR WS JP JL JS JSD &
JSC
JFH
L
i
g
n
i
n

(
%
)

Different Biomass

S.N. Sample Microbe Reducing Sugar Concentration (mg/ml) (mean values of 3
replicates)
Days to
incubation
0 day 2 days 4 days 6 days 8 days 10 days
1 Jatropha
seed coat
Aspergillus
niger

196.9 264.23 227.27 219.8 213.03 191.53
Trichoderma
reesei
199.67 258.53 236.7 228.87 221.5 215.2

2 Jatropha
Fruit Husk
Aspergillus
niger
229.13 249.33 243.23 227.03 206.8 180.17
Trichoderma
reesei
230.23 249.77 234.07 227.6 208.9 178.17

Table 1.8- Hydrolysis of Biomass and Reducing Sugar Estimation through DNSA Method
The reducing sugar estimation for Jatropha seed coat and Jatropha fruit husk was carried out
before incubation. The reducing sugar content in Jatropha fruit husk was greater than Jatropha
seed coat by 1.15 fold [Table 1.9 and Fig. 1.10].
Treatment Reducing Sugar (mg/ml)
JSC 198.283
JFH 229.683
SE 2.716
LSD 8.557
Table 1.9- Reducing Sugar Estimation for Jatropha Seed Coat and Jatropha Fruit Husk before
microbial hydrolysis is estimated. Least Significant Difference (LSD) test indicated significant
differences at 5% level [JSC- Jatropha Seed Coat and JFH- Jatropha Fruit Husk].


Fig. 1.10- Reducing Sugar Estimation for Jatropha before Microbial Incubation for Hydrolysis

The reducing sugar estimation for Jatropha seed coat was done before and after incubation with
A. niger and T. reesei. The reducing sugar estimated for Jatropha seed coat with incubation of A.
niger was greater (1.02 fold) than Jatropha seed coat incubated with T. reesei after 2 days.
Reducing sugar content in Jatropha seed coat incubated with T. reesei was greater than Jatropha
seed coat incubated with A. niger by 1.04 fold after 4 days. Reducing sugar content in Jatropha
seed coat incubated with T. reesei was greater than Jatropha seed coat incubated with A. niger by
1.04 fold after 6 days. Reducing sugar content in Jatropha seed coat incubated with T. reesei was
greater than Jatropha seed coat incubated with A. niger by 1.03 fold after 8 days. Reducing sugar
content in Jatropha seed coat incubated with T. reesei was greater than Jatropha seed coat
incubated with A. niger by 1.12 fold after 10 days [Table 1.10 and Fig. 1.11].
Treatment Reducing sugar content after incubation(mg/ml)
Days to incubation 2 4 6 8 10
Js An 264.233 227.267 219.8 213.033 191.533
Js Tr 258.533 236.7 228.867 221.5 215.2
SE 2.213 1.916 3.619 3.751 3.051
LSD 8.673 7.51 14.188 14.703 11.957
Table 1.10- Reducing Sugar Estimation for Jatropha Seed Coat after Microbial Hydrolysis. Least
Significant Difference (LSD) test indicates significant differences at 5% level. [Js An- Jatropha
Seed Coat with A. niger incubation & Js Tr- Jatropha Seed Coat T. reesei incubation].
170
180
190
200
210
220
230
240
JSC JFH
R
e
d
u
c
i
n
g

S
i
g
a
r
(
m
g
/
m
l
)

Different Biomass


Fig. 1.11- Reducing Sugar Estimation for Jatropha Seed Coat after Microbial Incubation

The reducing sugar estimation for Jatropha Fruit Husk was done after incubation. The incubation
was done with A. niger and T. reesei. The least significant difference is not observed. The
reducing sugar estimated for Jatropha Fruit Husk with incubation of T. reesei is greater than
Jatropha Fruit Husk with incubation of A. niger by 1.0 fold after 2 days, Jatropha Fruit Husk with
incubation of T. reesei is greater than Jatropha Fruit Husk with incubation of A. niger by 1.03
fold after 4 days, Jatropha Fruit Husk with incubation of T. reesei is greater than Jatropha Fruit
Husk with incubation of A. niger by 1.0 fold after 6 days, Jatropha Fruit Husk with incubation of
T. reesei is greater than Jatropha Seed Coat with incubation of A. niger by 1.01 fold after 8 days
and Jatropha Fruit Husk with incubation of T. reesei is greater than Jatropha Fruit Husk with
incubation of A. niger by 1.01 fold after 10 days [Table 1.11 and Fig. 1.12].
Treatment Reducing sugar content after incubation(mg/ml)
Days to incubation 2 4 6 8 10
Jf An 249.333 243.233 227.033 206.8 180.167
Jf Tr 249.767 234.067 227.6 208.9 178.167
SE 1.567 1.531 5.243 1.7602 10.936
LSD 6.144 5.998 20.551 6.899 42.866
Table 1.11- Reducing Sugar Estimation for Jatropha Fruit Husk after Microbial Hydrolysis is
estimated. Least Significant Difference (LSD) test indicates significant differences at 5% level.
[Jf An- Jatropha Fruit Husk with A. niger incubation & Jf Tr- Jatropha Fruit Husk T. reesei
incubation].
0
50
100
150
200
250
300
2 4 6 8 10
R
e
d
u
c
i
n
g

S
u
g
a
r

(
m
g
/
m
l
)

Days of Incubation
Js An
Js Tr


Fig. 1.12- Reducing Sugar Estimation for Jatropha Fruit Husk with Microbes Incubation
Reducing sugar content increased after microbial incubation. After 2 days of microbial
hydrolysis, the reducing sugar content declined with increasing days which may be beacause of
the consumption of sugar produced by the microbes for their growth.
b. Total Carbohydrate Estimation-
The hydrolysis leads to break down of sucrose to glucose. In this project hydrolysis is performed
by using microbes viz. Aspergillus Niger and Trichoderma Reesei, i.e., microbial hydrolysis.
Jatropha seed coat and Jatropha Fruit Husk were undergone microbial hydrolysis in this project.
The observations were made in different days before and after incubation of microbes. The result
shows that Jatropha Fruit Husk has maximum of total carbohydrate contents (412.6mg/ml)
before incubation. The total carbohydrate estimated was maximum in Jatropha Fruit Husk
(600.9mg/ml) with the incubation of T. reesei after 2 days, contents was greater in Jatropha Fruit
Husk (556.7mg/ml) with the incubation of T. reesei after 4 days, after 6 days contents shows
higher value in Jatropha Seed Coat (496.5mg/ml) with the incubation of A. niger, 8 day
observation shows the maximum value in Jatropha Seed Coat (441.2mg/ml) with the incubation
of T. reesei and the 10 day result shows the maximum contents value in Jatropha Fruit Husk
(381.0mg/ml) with the incubation of A. niger [Table 1.12].



0
50
100
150
200
250
300
2 4 6 8 10
R
e
d
u
c
i
n
g

S
u
g
a
r

(
m
g
/
m
l
)

Days of Incubation
Jf An
Jf Tr

S.N Sample Microbe Total Carbohydrate Concentration (mg/ml) (mean values)
Days to
incubation
0 day 2 days 4 days 6 days 8 days 10 days
1 Jatropha
seed coat
Aspergillus
niger
282.17 318.43 466.83 476.23 383.87 317.9
Trichoderma
reesei
244.73 329.37 449.9 462.6 414.67 340.6

2 Jatropha
Fruit Husk
Aspergillus
niger
389.23 490.53 533.53 420.8 344.37 330.23
Trichoderma
reesei
363.33 541.73 510.8 424.4 426.73 221.13
Table 1.12- Total Carbohydrate Estimation for Characterized Biomass through Anthrone
Method.LSD (Least Significant Difference) test and SE (Standard Error) was estimated.
The reducing sugar estimation for Jatropha Seed Coat and Jatropha Fruit Husk was done before
incubation. The reducing sugar estimated for Jatropha Fruit Husk is greater than Jatropha Seed
Coat by 1.4 fold [Table 1.13 and 1.13].
Treatment Total carbohydrate (mg/ml)
JSC 263.45
JFH 376.283
SE 14.1113
LSD 44.4652
Table 1.13- Total Carbohydrate Estimation for Jatropha Seed Coat and Jatropha Fruit Husk
before Microbial Hydrolysis. [JSC- Jatropha Seed Coat and JFH- Jatropha Fruit Husk].

Fig. 1.13- Total carbohydrate estimation for Jatropha without Microbial Incubation
0
100
200
300
400
500
J s c J F H
T
o
t
a
l

C
a
r
b
o
h
y
d
r
a
t
e

(
m
g
/
m
l
)

Different Biomass

The total carbohydrate estimation for Jatropha seed coat was done after incubation with A. niger
and T. reesei. The total carbohydrate estimated for Jatropha seed coat with incubation of T.
reesei was greater than Jatropha seed coat incubated with A. niger by 1.03 fold after 2 days.
Total carbohydrate in Jatropha seed coat incubated with A. niger was greater than Jatropha seed
coat incubated with T. reesei by 1.03 fold after 4 days. Total carbohydrate in Jatropha seed coat
incubated with A. niger was greater than Jatropha seed coat incubated with T. reesei by 1.02 fold
after 6 days. Total carbohydrate in Jatropha seed coat incubated with T. reesei was greater than
Jatropha seed coat incubated with A. niger by 1.08 fold after 8 days. Total carbohydratein
Jatropha seed coat incubated with T. reesei was greater than Jatropha seed coat incubated with A.
niger by 1.07 fold after 10 days [Table 1.14 and Fig. 1.14].

Treatment Total Carbohydrate estimated after incubation (mg/ml)
Days to incubation 2 days 4 days 6 days 8 days 10 days
Js An 318.433 466.833 476.233 383.867 317.9
Js Tr 329.367 449.9 462.6 414.667 340.6
SE 14.538 8.605 8.618 13.289 14.991
LSD 56.985 33.729 33.781 52.091 58.763
Table 1.14- Total Carbohydrate Estimation for Jatropha Seed Coat after Microbial Hydrolysis.
[Js An- Jatropha Seed Coat with A. niger incubation & Js Tr- Jatropha Seed Coat T. reesei
incubation].

Fig. 1.14- Total Carbohydrate Estimation for Jatropha Seed Coat after Microbial Incubation
0
50
100
150
200
250
300
2 4 6 8 10
R
e
d
u
c
i
n
g

S
u
g
a
r

(
m
g
/
m
l
)

Days of Incubation
Js An
Js Tr

The total carbohydrate estimation for Jatropha fruit husk was done after incubation with A. niger
and T. reesei. The total carbohydrate estimated for Jatropha fruit husk incubated with T. reesei
was greater than Jatropha fruit husk with incubation of A. niger by 1.1 fold after 2 days, Jatropha
fruit husk with incubation of A. niger is greater than Jatropha fruit husk with incubation of T.
reesei by 1.04 fold after 4 days, Jatropha fruit husk with incubation of T. reesei is greater than
Jatropha fruit husk with incubation of A. niger by 1.0 fold after 6 days, Jatropha fruit husk with
incubation of T. reesei is greater than Jatropha seed coat with incubation of A. niger by 1.23 fold
after 8 days and Jatropha fruit husk with incubation of A. niger is greater than Jatropha fruit husk
with incubation of T. reesei by 1.49 fold after 10 days [Table 1.15 and Fig. 1.15].

Treatment Total Carbohydrate Estimated after incubation (mg/ml)
Days to incubation 2 days 4 days 6 days 8 days 10 days
Jf An 490.533 533.533 420.8 344.367 330.233
Jf Tr 541.733 510.8 424.4 426.733 221.133
SE 29.678 16.388 12.724 9.5016 20.247
LSD 116.332 64.239 49.873 37.244 79.365
Table 1.15- Total Carbohydrate Estimation for Jatropha Fruit Husk after Microbial Hydrolysis.
[Jf An- Jatropha Fruit Husk with A. niger incubation & Jf Tr- Jatropha Fruit Husk T. reesei
incubation].

Fig. 1.15- Total Carbohydrate Estimation for Jatropha Fruit Husk after Microbial Incubation
0
100
200
300
400
500
600
2 4 6 8 10
T
o
t
a
l

C
a
r
b
o
h
y
d
r
a
t
e

(
m
g
/
m
l
)

Days of Incubation
Jf An
Jf Tr

3. Fermentation-
Microbial fermentation of released sugars after hydrolysis of biomass leads to formation of bio-
ethanol. In this project hydrolysis of Jatropha seed coat and Jatropha fruit husk was performed by
using microbes viz. Aspergillus Niger and Trichoderma Reesei. After hydrolysis the hydrolysates
was treated with yeast viz. Saccharomyces cerevisiae for fermentation. The fermented fractions
were estimated for ethanol produced by potassium dichromate assay starting from day one till six
days. The result shows that the ethanol produced was maximum in Jatropha fruit husk
(61.2l/ml) with the incubation of A. niger after 1 day, contents was greater in Jatropha seed coat
(89.9l/ml) with the incubation of T. reesei after 2 days, after 3 days contents shows higher value
in Jatropha fruit husk (157.5l/ml) with the incubation of T. reesei, 4
th
day observation shows the
maximum value in Jatropha fruit husk (172.1l/ml) with the incubation of T. reesei, 5
th
day
result shows the maximum value in Jatropha fruit husk (168.2l/ml) with the incubation of T.
reesei and the 6
th
day result shows the highest value of ethanol in Jatropha fruit husk
(166.3l/ml) with the incubation of T. reesei [Table 1.16].

S.N. Sample Microbe
(S. cerevisiaefor
fermentation)
Ethanol Concentration (l/ml) (mean values)
Days to
incubation
1 day 2 days 3 days 4 days 5 days 6 days
1 Jatropha
Seed Coat
Aspergillus niger 56.067 85.067 98.367 98.367 97.867 95.6
Trichoderma reesei 54.567 82.133 102.433 100 104.2 103.7

2 Jatropha
Fruit Husk
Aspergillus niger 55.567 81.333 124.133 130.433 127.533 95.333
Trichoderma reesei 56.033 76.967 130.133 123.3 132.867 134.833

Table 1.16-Ethanol Estimation for Characterized Biomass through Potassium Dichromate Assay
The ethanol estimation is being done for Jatropha. LSD (Least Significant Difference) test and
SE (Standard Error) was estimated. The ethanol estimated for Jatropha fruit husk was greater
than Jatropha seed coat by 1.18 fold [Table 1.17 and Fig. 1.16].



Treatment Ethanol from Non- hydrolyzed Fractions (l/ml)
J S C 21.2667
J F H 25.25
SE 4.15702
LSD 13.0989

Table 1.17- Ethanol estimation for Jatropha seed coat and Jatropha fruit husk for non-
hydrolyzed fractions. [JSC- Jatropha Seed Coat and JFH- Jatropha Fruit Husk].

Fig. 1.16- Ethanol estimation for Jatropha biomass from Non- hydrolyzed Fractions
The ethanol estimation for Jatropha Seed Coat was done after incubation. The incubation was
done with A. niger and T. reesei. The ethanol estimated for Jatropha Seed Coat with incubation
of T. reesei was greater than Jatropha Seed Coat after S. cerevisiae incubation with incubation of
A. niger by 1.02 fold after 1 day, Jatropha Seed Coat with incubation of A. niger was greater than
Jatropha Seed Coat with incubation of T. reesei by 1.03 fold after 2 days, Jatropha Seed Coat
with incubation of T. reesei is greater than Jatropha Seed Coat with incubation of A. niger by
1.04 fold after 3 days, Jatropha Seed Coat with incubation of T. reesei was greater than Jatropha
Seed Coat with incubation of A. niger by 1.01 fold after 4 days, Jatropha Seed Coat with
incubation of T. reesei was greater than Jatropha Seed Coat with incubation of A. niger by 1.06
fold after 5 days and the result of 6 day shows that Jatropha Seed Coat with incubation of T.
reesei has greater value of ethanol than Jatropha Seed Coat with incubation of A. niger by 1.08
fold [Table 1.18 and Fig. 1.17].
0
5
10
15
20
25
30
35
J s c J F H
E
t
h
a
n
o
l

(

l
/
m
l
)

Different Biomass

Treatment Ethanol produced after fermentation (l/ml)
Days to incubation 1 days 2 days 3 days 4 days 5 days 6 days
Js An 56.067 85.067 98.367 98.367 97.867 95.6
Js Tr 54.567 82.133 104.2 103.7 102.433 100.1
SE 2.051 2.9282 3.109 3.382 3.474 3.831
LSD 8.039 11.478 12.189 13.257 13.616 15.018
Table 1.18- Ethanol Estimation for Jatropha Seed Coat after Microbial Hydrolysis and after
Fermentation. [Js An- Jatropha Seed Coat with A. niger incubation & Js Tr- Jatropha Seed Coat
T. reesei incubation].

Fig. 1.17- Ethanol Estimation for Jatropha Seed Coat after Microbial Hydrolysis and after
Fermentation.
The ethanol estimation for Jatropha Fruit Husk was done after hydrolysis with A. niger and T.
reesei and fermentation by incubation with T. reesei. Ethanol produced was greater (1.0 fold) in
seed coat than fruit husk after S. cerevisiae incubation after incubation of A. niger after 1 day,
Jatropha fruit husk with incubation of A. niger was greater (1.05 fold) than Jatropha fruit husk
incubated with T. reesei after 2 days, Jatropha fruit husk incubated with T. reesei was greater
(1.04 fold) than Jatropha fruit husk incubated with A. niger after 3 days, Jatropha fruit husk
incubated with A. niger was greater (1.05 fold) than Jatropha fruit husk incubated with T. reesei
after 4 days, Jatropha fruit husk incubated with T. reesei was greater (1.04 fold) than Jatropha
fruit husk incubated with A. niger after 5 days and the result of 6
th
day shows that Jatropha fruit
husk incubated with T. reesei gave greater (1.41 fold) value of ethanol than Jatropha fruit husk
incubated with A. niger by [Table 1.19 and Fig. 1.18].
0
20
40
60
80
100
120
1 2 3 4 5 6
E
t
h
a
n
o
l

(

l
/
m
l
)

Days of Incubation
js an
js tr


Treatment Ethanol produced after fermentation (l/ml)
Days to
incubation
1 days 2 days 3 days 4 days 5 days 6 days
Jf An 55.5667 81.3333 124.133 130.433 127.533 95.3333
Jf Tr 56.0333 76.9667 132.867 134.833 130.133 123.3
SE 3.41288 3.40628 11.3611 18.8634 19.0454 19.0264
LSD 13.3778 13.3519 44.533 73.9404 74.6537 74.5793
Table 1.19- Ethanol estimation for Jatropha fruit husk with Microbial Hydrolysis and after
Fermentation. [Jf An- Jatropha Fruit Husk with A. niger incubation & Jf Tr- Jatropha Fruit Husk
T. reesei incubation].

Fig. 1.18- Ethanol estimation for Jatropha Fruit Husk with Microbes Incubation and after
Fermentation

The ethanol production delined after 4 days of incubation with yeast. This may be due to ethanol
toxicity which leads to the killing of yeast and hence decline in rate of fermentation.
0
20
40
60
80
100
120
140
160
180
1 2 3 4 5 6
E
t
h
a
n
o
l

(

l
/
m
l
)

Days of Incubation
Jf An
Jf Tr

4.2 SUMMARY AND CONCLUSION-
Lignocellulosic biomass is the most abundant feed stock available for biofuel production.
Biofuel generation from 1
st
generation feedstocks had limitations inviting food vs fuel debate
which is being overcome by 2
nd
generation feedstocks which include lignocellulosic biomass.
The present study was conducted with the aim to characterize different biomass from different
categories of plants like, shrubs, semi-tress and trees to find their cell wall composition for their
suitability for microbial hydrolysis and fermentation for ethanol production.
The characterization of different biomass viz. pine needle, Camelina, Jatropha Stem, Jatropha
Petiole, Jatropha Leaves, Jatropha Seed Coat, Jatropha Fruit Husk, Jatropha Seed Cake, Wheat
Straw, Paddy, Sugarcane and Sugarcane Trash was carried out in the current study with the aim
to explore its potential for use as feedstock for bio-ethanol production. Camelina gave good
result in the composition of cellulose and Jatropha Seed Coat gave in lignin composition. The
maximum amount of cellulose was seen in Camelina (70.2 %) and maximum amount of lignin in
Jatropha Seed Coat (42.9 %). Lignin content was high in pine needles which is the reason for its
slow degradation even in nature. Grasses are comparatively low in lignin content than trees and
even shrubs which makes it more amicable for microbial hydrolysis, since lignin acts as a barrier
for enzymes to act on other cell wall components.
Two different microbes Aspergillus niger and Trichoderma reesei evaluated by this lab earlier
for its cellulase and lacase activity were used for hydrolysis of two different biomass namely
Jatropha Seed Coat and Jatropha Fruit Husk since these biomass are available in abundant with
the lab. Trichoderma reesei gave good result in the production of bio-ethanol from Jatropha fruit
husk and its seed coat. The maximum amount of ethanol produced (168.2 l/ml) was on 4
th
day
of incubation with Trichoderma reesei. The ethanol production delined after 4 days of incubation
with yeast. This may be due to ethanol toxicity which leads to the killing of yeast and hence
decline in rate of fermentation.
The result obtained here gives an indication of the usefulness of Jatropha crop for its potential
use for ethanol production through microbial hydrolysis and fermentation. However, it needs to
be validated through further experimentation and analyses.

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