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Energy Procedia 105 (2017) 277 282

The 8th International Conference on Applied Energy ICAE2016

Oil Algae Extraction of Selected Microalgae Species Grown

in Monoculture and Mixed Cultures for Biodiesel Production
Yano Surya Pradanaa,d, Hanifrahmawan Sudibyoa,d, Eko Agus Suyonob,d,
Indartoc,d, Arief Budimana,d,*
a
Chemical Engineering Department, Gadjah Mada University, Jalan Grafika No. 2 Yogyakarta 55281, Indonesia
b
Biological Department, Gadjah Mada University, Jalan Grafika No. 2 Yogyakarta 55281, Indonesia
c
Mechanical Engineering Department, Gadjah Mada University, Jalan Grafika No. 2 Yogyakarta 55281, Indonesia
d
Center for Energy Studies, Gadjah Mada University, Jalan Sekip UGM K-1A Yogyakarta 55281, Indonesia

Abstract

The idea of using microalgae as a bio-source of biodiesel is being taken seriously because of the escalating price of
fossil fuel. As the tropical country, which gets the sun a year round, Indonesia has big potential for cultivating
microalgae in large scale, as biodiesel feedstock. This study examines the algal oil extraction of selected monoculture
microalgae species of Botryococcus braunii, Nannochloropsis sp., Arthrospira platensis and mixed cultures
microalgae from South Coast of Yogyakarta, Indonesia. Among all species of microalgae studied, Nannochloropsis
sp. was found to have the highest algal oil yield (0.0346 g dry algal oil/g dry microalgae) and theoretical calorific
value (187.69 kcal/kg dry microalgae).
2017 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
2016 The Authors. Published by Elsevier Ltd.
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Selection and/or
Peer-review peer-reviewofunder
under responsibility responsibility
the scientific committeeofofICAE
the 8th International Conference on Applied Energy.

Keywords: microalgae; monoculture; mixed cultures; algal oil extraction; biodiesel

1. Introduction

The world has been faced with an energy crisis associated with the lack of petroleum resources used as
primary energy in the last few decades [1]. With the increasing concerns to reduce greenhouse gas
emissions, considerable attention has been focused on the development of renewable-alternative fuels
[2,3], such as biodiesel [16]. Besides, biodiesel is also non-toxic and degradable biofuel [4]. Nowadays,
biodiesel feedstocks mostly derived from palm oil, soybean oil, jatropha oil, sunflower oil, rapeseed oil,
used cooking oil, and Palm Fatty Acid Distillate. However, the sustainability production of this mode has

* Corresponding author. Tel.: +628164262111

E-mail address: abudiman.ugm@gmail.com; abudiman@ugm.ac.id

1876-6102 2017 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Peer-review under responsibility of the scientific committee of the 8th International Conference on Applied Energy.
doi:10.1016/j.egypro.2017.03.314
278 Yano Surya Pradana et al. / Energy Procedia 105 (2017) 277 282

been concerned because of requirement of large area of plantation. Recently, algae have received a lot of
attention as a new biomass source for the production of biofuels [5]. The main characteristics which set
algae apart from other biomass sources are that algae have a high biomass yield per unit of light and area
and high oil or starch content, do not require agricultural land, fresh water is not essential and nutrients
can be supplied by wastewater and CO2 from combustion gas [6].

The cellular lipid content in microalgae reaches 75% in Botryococcus braunii, but it is associated with
low productivity of biomass [5]. The microalgae Nannochloropsis sp. and Arthrospira platensis are two
widely available microalgae strains in the commercial applications. They showed great potentials as future
industrial biodiesel producers due to their high growth rate and their high oil contents. Also, many
methods and techniques have studied such as using mixed culture of microorganism. When using a mixed
culture, two or more preselected species of microorganism are synchronously cultivated within the same
medium, where these microorganisms can mutually exploit complementary metabolic activities to
survive, grow, and reproduce [7].

Algal oil extraction is another challenge that is most easily addressed from the engineering side.
Extraction can be broadly categorized into three methods: mechanical, electrical and chemical methods.
The mechanical press generally requires drying the algae, which is energy intensive, while the use of
chemical solvents present safety and health issues [8]. In the electrical method, pulsed electric field is
extraction technology using electrical current for breaking cell wall of microalgae [9]. However, the
chemical solvent extraction is more widely used because it is easy in operation. The purpose of this study
is to extract monoculture of selected microalgae species of Botryococcus braunii, Nannochloropsis sp.,
Arthrospira platensis, and mixed cultures microalgae from South Coast of Yogyakarta, Indonesia. The
chemical solvent extraction, known as soxhlet extraction is chosen to separate algal oil from its biomass
residue.

2. Experimental

Monoculture-grown microalgae used in this experiment were Nannochloropsis sp., Botryococcus

Braunii, and Arthrospira platensis cultivated in Situbondo, East Java, Indonesia. Mixed cultures-grown
microalgae were isolated from South Coast of Yogyakarta, Glagah, Indonesia.

Stock of mixed cultures microalgae was rejuvenated in bolds basal medium (BBM). After 5 days
cultivation, 800L culture was collected in micro tube and added with 200 L alcohol 70%. Afterward,
the micro tube was shaken and left for 15 minutes to undergo fixation. Then, the mixture was observed
under the light microscope for identification of microalgae species composing the Glagah Strain. The
identification reference was according to Edmondson [10] and van Vuuren [11].

Samples from green microalgae were collected in the conical tube and then centrifuged at 3300 rpm for
15 minutes for dewatering. In soxhlet extraction, the ratio of 20 mL n-hexane:1 gram dry weight
microalgae (Wm.a.-dry) are used. The extraction was undergone for 3 hours at 80oC to achieve maximum
extraction efficiency. After extraction, the vacuum evaporator was used to evaporate the n-hexane. The
yield of dry oil extracted (Woil-dry) from dry microalgae calculated using Eq. 1. Lipid samples extracted
from microalgae were analyzed for their fatty acid profiles by GC-MS.

Woildry (1)
Yield x100%
Wm.a.dry
 Yano Surya Pradana et al. / Energy Procedia 105 (2017) 277 282 279

To predict which microalgae species was good for biofuel or biodiesel, the theoretical calculation of
calorific value was determined. First, the molecular weight of fatty acid mixture (Mrmixture) and the mole of
fatty acid mixture (nmixture) calculated using Eq. 2 and 3. Then, the data of specific fatty acid mole fraction
(xi) from GC-MS and heat of combustion of specific fatty acid methyl ester ('Hc,i) were used to determine
the theoretical heat of combustion of FAME mixture per dry weight of microalgae ( 'Hc,mixture) by Eq. 4.

Mrmixture x .Mr
i
i i (2)

Wm.a.dry
nmixture Yield . (3)
Mrmixture

nmixture. xi .'H c ,i .
'H c ,mixture i (4)
Wm.a. dry

3. Results and Discussion

Fig. 1a showed the microscopic scale photograph. Fig. 1b Fig. 1f shows identified species presented
in mixed cultures microalgae. From all species detected in Fig. 1, only Cylindrospermopsis raciborskii
was different from the others. As noted that Cylindrospermopsis raciborskii is a freshwater
cyanobacterium. It was predicted that mixed cultures microalgae had good resistance to the predator.
Cylindrospermopsis raciborskii can produce several toxins which affect organisms such as
cylindrospermopsin, anatoxin-a, and saxitoxin. When the system is attacked by predator, it is protected
by this bacterium species and the microalgae inside are not affected by the toxin released because they
have adapted with the existence of Cylindrospermopsis raciborskii. Qualitative interpretation of the
photograph showed that Cyclotella polymorpha and Chlamydomonas sp. were the most contributive for
the oil extract. According to the study conducted by Cavonius [12] on imaging of lipids in microalgae, it
was confirmed that the lipid or oil inside microalgae were represented by the yellow color. Both
Cyclotella polymorpha and Chlamydomonas sp. showed unclear but sufficient enough yellow color to
prove that they were the most contributive ones for the yield of oil extract.

(a) (b) (c) (d) (e) (f)

Fig. 1. The identified species [13] presented in (a) The microscopic scale photopgraph of mixed cultures microalgae
from South Coast of Yogyakarta, Indonesia are: (b). Cyclotella polymorpha (c). Cylindrospermopsis raciborskii (d).
Golenkinia radiata (e). Chlamydomonas sp. (f). Syracosphaera pirus

The results of algal oil extraction were shown in Table 1. The monoculture microalgae species of
Nannochloropsis sp. showed the highest dry algal oil yield of 0.346 g dry algal oil/g dry microalgae after
n-hexane soxhlet extraction. The yield of mixed cultures microalgae of 0.0170 g dry algal oil/g dry
microalgae was under expectation as the highest yield. As shown in Fig. 2, this microalgae strain contains
280 Yano Surya Pradana et al. / Energy Procedia 105 (2017) 277 282

Cylindrospermopsis raciborskii which produce several toxins from its metabolism. The toxins possibly
influenced the microalgae productivity of lipid.

Table 1. Yield of oil extracted from selected microalgae species

No. Species Yield, g dry algal oil/g dry microalgae

1. Botryococcus braunii 0.0187
2. Nannohloropsis sp. 0.0346
3. Arthrospira platensis 0.0121
4. Mixed cultures microalgae 0.0170

Fig. 2 shows GC-MS Chromatogram of algae oil extracted from mono and mixed cultures. Fatty acid
composition of algal oil extracted from various microalgae species presented in Table 2. From this figure
and table, we may see that Nannochloropsis sp. had the fewest number of fatty acid components (palmitic,
elaidic, and stearic acid) composing the algal oil, as shown in Fig. 2.b and Table 2. Algal oil extracted
from Botryococcus braunii and Nannochloropsis sp. were dominated by palmitic and elaidic acid.

Algal oil of mixed cultures microalgae showed more various fatty acid components (Fig. 2.d and Table
2). This algal oil had similarity with algal oil of Botryococcus braunii and Nannochloropsis sp., in which
the elaidic acid was contributed as the major fatty acid components composing the algal oil. As the
microalgae with the lowest algal oil yield, Arthrospira platensis had oleic acid and palmitic acid as the
major ones composing the algal oil (Fig. 2.c and Table 2).

(a) (b)

(c) (d)
Fig. 2. The GC-MS Chromatogram of algal oil extracted of : (a) Botryococcus braunii; () Nannochloropsis sp.; (a)
Arthrospira platensis; (d) mixed cultures microalgae

The fatty acid components detected in algal oil were classified into three categories: saturated fatty acid
(SFA), mono unsaturated fatty acid (MUFA), and poly unsaturated fatty acid (PUFA). Both mixed
cultures microalgae and Arthrospira platensis were suitable for food sector because of the high percentage
of PUFA content and the low percentage of SFA. However, the presence of both bacteria and its toxins
released in mixed cultures microalgae should be a concern in food sector. On the other hand,
Nannochloropsis sp. did not only present as the highest yield in producing algal oil, but also positioned as
the most prospective microalgae species for biofuel. It has the highest percentage of saturated fatty acid,
 Yano Surya Pradana et al. / Energy Procedia 105 (2017) 277 282 281

as shown in Fig. 3. Saturated fatty acid is required in the biodiesel production which is mainly supplied by
crude palm oil or any other crop-based oils. Theoretical calorific value was then investigated in Fig. 4.

Table 2. Fatty acid composition of algal oil extracted from monoculture and mixed cultures

Percentage, %w/w of algal oil

Component Type of Fatty Acid Botryococcus Arthrospira Mixed
Nannochloropsis sp.
braunii platensis cultures
Palmitic acid SFA 43.06 47.44 35.31 -
Linolelaidic acid PUFA 8.60 - - 10.0
Elaidic acid MUFA 43.06 46.49 - 47.6
Stearic acid SFA 5.29 6.07 5.04 29.3
Linoleic Acid PUFA - - 12.74 9.9
Oleic Acid MUFA - - 46.91 1.2
Isostearic Acid SFA - - - 2.1

Fig. 3. Distribution of SFA, MUFA, and PUFA in algal oil from selected microalgae species

Fig. 4. Theoretical calorific value calculated based on converted fatty acid into FAME

Fig. 4 shows theoretical calorific value calculated based on converted fatty acid into FAME. The
calculation (using Eqs. 2, 3 and 4) was supported by the data of the heat of combustion of FAME gained
by Freedman [14], Keffler [15], and Gerasimov [16]. The calculation gave similar interpretation with the
interpretation based on the distribution of SFA, MUFA, and PUFA. From Fig. 4 we may see that
Nannochloropsis sp. has the highest calorific value of saturated fatty acid. The calorific value of algal oil
of Botryococcus braunii was the closest to Nannochloropsis sp. The close calorific value of algal oil of
Botryococcus braunii and Nannochloropsis sp. was occurred because they had similar type of fatty acid
profile with the different percentage of the SFA.
282 Yano Surya Pradana et al. / Energy Procedia 105 (2017) 277 282

4. Conclusions

Nannochloropsis sp. was found to have the highest algal oil yield and theoretical calorific value
(187.69 kcal/kg dry microalgae). The major fatty acid component in algal oil of Nannochloropsis sp.,
Botryococcus braunii and mixed cultures microalgae from South Coast of Yogyakarta, Indonesia was
elaidic acid, different from Arthrospira platensis which had oleic acid and palmitic acid as the major ones.

Acknowledgements

The authors are grateful for the financial support from Directorate General of Higher Education, Indonesia
through Research Grant of Penelitian Unggulan Perguruan Tinggi 2015.

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Biography
Yano Surya Pradana is officially the faculty member of Chemical Engineering Department,
Universitas Gadjah Mada. He is also the young researcher at Center for Energy Studies,
Universitas Gadjah Mada focusing on bio-energy, waste to energy and process system
engineering.