Biotechnological
Communication
Biosci. Biotech. Res. Comm. 11(2): 318-323 (2018)
The potential of certain algal species
as source of biodiesel
Ramendra Soni, Shriya Henry and Bhavana Tandon*
Department of Molecular and Cellular Engineering, Jacob Institute of Biotechnology and Bioengineering, Sam
Higginbottom University of Agriculture, Technology & Sciences, (SHUATS), Allahabad India
ABSTRACT
Biofuels are important source of renewable chemical energy, more precisely those that are available in liquid form.
Algal biofuel offers the advantage of being a low-emission fuel. Algal biofuel offers a clean source of renewable
energy. It burns without smoke and can be easily cultured in areas which are unsuitable for agriculture due to low
production and installation costs. It releases only carbon dioxide and water on burning and carbon dioxide is one that
is xed from atmosphere during photosynthesis. In the present work, ve algae samples were collected from differ-
ent sites. According to the maximum growth of algae in BG11 media, it was selected for mass culture of potentially
growing samples. They were kept for two weeks and optimized through various con rmatory tests. The ame test and
transesteri cation test showed positive results for all ve samples. These samples were mass cultured in pond and
oil was extracted using Soxhlet extractor with ethanol as solvent. The oil was recovered and transesteri cation was
performed to convert it to biodiesel.
KEY WORDS: BIODIESEL, BIOFUEL, ALGAE, CULTURE, BG11, BOLDS BASAL, BRISTOL, SOXHLET
318
ARTICLE INFORMATION:
*Corresponding Author: btandon7@gmail.com
Received 18
th
March, 2018
Accepted after revision 16
th
June, 2018
BBRC Print ISSN: 0974-6455
Online ISSN: 2321-4007 CODEN: USA BBRCBA
Thomson Reuters ISI ESC / Clarivate Analytics USA and
Crossref Indexed Journal
NAAS Journal Score 2018: 4.31 SJIF 2017: 4.196
© A Society of Science and Nature Publication, Bhopal India
2018. All rights reserved.
Online Contents Available at: http//www.bbrc.in/
DOI: 10.21786/bbrc/11.1/19
Algae are a diverse group of eukaryotic organisms that
belong to the phylum Protista. These organisms use
light energy to convert CO
2
and H
2
O into carbohydrates
and other cellular products. During this process, oxy-
gen released. Algae are found anywhere there is water,
fresh water, salt water, and in the soil (Brown, 1969).
Macro-algae are de ned as the multicellular plants also
known as seaweeds found in salt or fresh water (Khark-
wal, 2012) whereas one of the miniscule plants known as
Micro-algae contributes in the production of around 60
percent of the earth’s oxygen. Such organisms comprise
of twenty ve to thirty thousand species representing a
range of forms and sizes that can exist from unicellular
microscopic organism (microalgae) to multicellular large
size (macroalgae) (Ugoala, 2012). Due to the fact that the
oceans cover 70% of the earth’s surface, aquatic algae
Bhavana Tandon, Shriya Henry and Ramendra Soni
are major producer of oxygen and important users of
carbon dioxide. All algae are primarily made up of pro-
teins, carbohydrates, fats and nucleic acids in varying
proportions. While the percentage can vary with the type
of algae, some types of algae are made up of up to 40%
fatty acids based on their overall mass. It is this fatty
acid that can be extracted and converted into biofuel.
Due to the high lipid content, algal strains are of great
interest in the search for sustainable sources for the pro-
duction of biodiesel. Reports proclaim that Macro-algae
is composed of more than 2400 organic and chemical
free products with a great commercial value in indus-
tries such as pharmaceutical, biomedical, neutraceutical,
etc. (Saranya, 2013).
Fuel derived from plants, animals or algal resources
can not only bring an initiative to preserve a healthful
global environment but also shall prove to be a substi-
tute in order to reduce our dependency on fossil fuels.
High lipid content, ease of cultivation, cost effective
and rapid growth rate is the factors that make microal-
gae to become a desired candidate for biofuel produc-
tion (Alam, 2015). Over the past decade there have been
plenty of advancements in algal technology for biofuel
production. Algae is considered as a traditional food or
feed and can be cultured in huge open ponds or closed
photo bioreactors placed on non-arable land. Certain
algal species have a high potential as the oil-producer
as compared to oil crops. It can be isolated from various
carbon (CO
2
) sources and further processed in a wide
spectrum of products like biodiesel, gasoline replace-
ments, green diesel, methane, bioethanol, heat, high
protein animal feed, bio-oil, etc. (Yang, 2016).
In the future of transport sector various biofuels,
including bio-ethanol, -methanol, -diesel- and –hydro-
gen appear to be appealing options. Therefore, it is
essential to look for novel feedstock sources which ar
suitable for biofuel production and does not withdraw
the supply of edible feedstock. Algae can be an alterna-
tive to the conventional crop. Algae or cyanobacteria
based third generation technology contains an elevated
oil mass fraction grown in ponds (Suganya, 2016). It is
a favourable source of third generation renewable fuels.
However, the procedure of biofuel production from the
growth of microalgae till the last step is still not contem-
plated economically viable (Laamanen, 2016). Microal-
gae have various autotrophic capabilities which add an
element of exibility as compared to conventional fuels.
They are also supported by fundamental nutrients which
capture solar energy to x the amount of carbon dioxide
and split water (Hallenbeck, 2016). The production of
biodiesel using algae demands the conversion of algal
biomass through transester cation (Kandiyoti, 2017).
It is a process in which lipids are simultaneously sepa-
rated and trans esteri ed from microalgae cell walls and
trans esteri cation is generally used for heterotrophic
microalgae (Veillette, 2017). Further research is needed
to discover the most energy ef cient, cost effective and
high yield extraction process to amplify the economic
viability of global algae biofuels sector. This will address
all the crucial technical trials which affect the produc-
tion processes involved. Hence, these aforementioned
research when incorporated with government emission
policies will fast-track the date when algal biofuel pro-
duction can be a commercial enterprise. In the eld of
science and research to succeed fossil fuels, algae feed-
stock has appeared as a suitable candidate not only for
its renewable and sustainable characteristics but also
for its economic reliability based on the prospective to
match up with the global demand for transportation
fuels (Adeniyi, 2018).
In the present study, fresh water algae were collected
from 5 below mentioned sites and denoted as R1, R2, R3,
R4 and R5. (Orchid, SHIATS R1, Agriculture Department,
SHIATS: R2, Forestry Department, SHIATS:R3, Arail
Ghat, Allahabad:R4 and Saraswati Ghat, Allahabad: R5).
Algae culture was carried out in tissue culture bot-
tles with 3 different growth media and one was in dis-
tilled water as control. 2 gm of algae was inoculated in
each media and further in open land area in polybags,
these media were following-Bolds Basal Medium (BBM)
(Nichols and Bold, 1965), Bristol Medium, (Bold, 1949)
and BG11 medium (Stanier, 1971). Oil extraction from
algae using Soxhlet apparatus was performed as per the
method of Franz von Soxhlet, (1879). The algae was
dried by exposure to hot air oven at 60
0
C for 1 hour.
After complete drying, the algae were blended to get
powder. A 25 gm sample of the dried algae was placed
in the thimble, which was loaded in the main chamber of
Soxhlet extractor. The Soxhlet extractor was placed onto
a ask containing extraction solvent i.e. 95% ethanol.
The solvent was heated to re ux, so it forms vapors,
which travels up a distillation arm, and oods into the
chamber housing the thimble of algae powder. Some of
the desired compound will then dissolve in the warm
ethanol. When the Soxhlet chamber was almost full, the
chamber was automatically emptied by the siphon side
arm, with ethanol running back to the distillation ask.
This cycle was repeated for three to four times. During
each cycle, a portion of the oil is dissolved in ethanol.
Con rmatory Tests for Biodiesel:-Transesteri cation
is the most commonly used method for Biodiesel oil pro-
duction from algae. Harvested biomass from these algal
species was dried, ground and oil was extracted by Sox-
hlet extractor using ethanol as a solvent. The extracted
oil was trans esteri ed to biodiesel using sodium meth-
oxide as a catalyst.1ml of extracted algal oil was taken
with 3ml of methanol and 0.075 g of NaOH and it was
shaken vigorously and then kept in a shaker for over-
BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS THE POTENTIAL OF CERTAIN ALGAL SPECIES AS SOURCE OF BIODIESEL 319
Bhavana Tandon, Shriya Henry and Ramendra Soni
FIGURE 1. Different sites from where algal samples were collected
FIGURE 2. Steps involved in biodiesel production from algal biomass
FIGURE 3. Algal growth of R3
FIGURE 4. Growth of algal sample in rst week of
inoculation
320 THE POTENTIAL OF CERTAIN ALGAL SPECIES AS SOURCE OF BIODIESEL BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS
Bhavana Tandon, Shriya Henry and Ramendra Soni
Table 2. List of the species
present in the algal samples
Samples Present species
R-1 Microcystis
R-2 Vaucheria
Microcystis
R-3 Microcystis
R-4 Vaucheria
Microcystis
Nostoc
R-5 Microcystis
FIGURE 5. The remaining extract after Ethanol
evaporation is the desired algal lipid
FIGURE 5. Transester cation
FIGURE 6. Flame Test
night. Two layers were observed the next day in which
the lower layer was glycerol and the upper layer was
biodiesel. The upper layer i.e. biodiesel was extracted for
further experiments.
Ideally, there were two distinct layers: an amber
(ranging from very light to very dark depending on the
oil used) biodiesel layer on top and a darker glycerol
layer on the bottom (usually contaminated with catalyst,
alcohol, or dust particulates). Sometimes, there will be a
third or fourth layer between the glycerol and the bio-
diesel. These layers are soap from too much catalyst or
water and often appear milky or yellowish. The property
of a biodiesel is to be highly ammable when in con-
tact of re. So for the ame test of biodiesel we lit the
matchstick in front of the upper layer of solution which
was taken out by the process of transesteri cation.
Growth rate measurement: The tissue culture bottles
were kept in open area where proper sun-light and aera-
tion was available for the suitable growth of algal sam-
ple as shown in gure 3 and 4.
In all these above 4 growth medium BBM, Bristol,
BG11 including one control (distilled water) BG11 media
showed maximum algal growth, so it was taken for the
mass culture of algal sample in open ground.
Identi cation: This process was carried out at depart-
ment of biological sciences, SHIATS, Allahabad, using a
compound microscope for analysis of temporary slides
of Algae samples collected from different sites. Different
Algae species were identi ed by observing their unique
reproductive structures. The table 2 shows the results of
identi cation of algae samples showing the type of spe-
cies which constitute every sample.
After the transesteri cation process, a separate layer
of biodiesel was obtained from the extracted algal oil.
The upper layer contained biodiesel which was carefully
extracted with pipette in a separate ask, and the lower
layer was glycerol so it was discarded.
As Biodiesel has the highly in ammable property, it
caught re rapidly when a matchstick was passed over
it.In the current study, 5 algae samples were used to
extract oil and it was converted to biodiesel. The study
revealed that algae are fast growing and effective organ-
ism for biodiesel production as these can be grown in
BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS THE POTENTIAL OF CERTAIN ALGAL SPECIES AS SOURCE OF BIODIESEL 321
Bhavana Tandon, Shriya Henry and Ramendra Soni
normal water as well as in arti cial media. Oil extracted
from harvested biomass of these algae was trans esteri-
ed to biodiesel using sodium methoxide as a catalyst.
It was found that properties of biodiesel so it can be
blended with fossil fuels or can be used individually as
an alternative of fossil fuels.
REFERENCES
Ad eniyi, Oladapo, Azimov, Ulugbek and Burluka,
Alexey (2018) Algae biofuel: Current status and future
applications.Renewable and Sustainable Energy Reviews, 90.
pp. 316-335. ISSN 1364-0321
Alam F, Mobin S, Chowdhury H. (2015) Third generation bio-
fuel from algae. Procedia Eng 2015; 105:763–8
Becker, E.W. (1994) Microalgae: biotechnology and microbiol-
ogy. New York: Cambridge University Press; 181-182. Biotech-
nology: 235-240.
Brown, A., Knights, B. and Conway, E. (1969) Hydrocarbon
content and its relationship to physiological state in the green
algae Botryococcus braunii. Phytochem 8:543-547.
Chisty, Y. (2007) Biodiesel from microalgae. Biotechnology
Advance 25:294-306.
Chisty, Y.(2007) Chlorella Isolated from Soil in the Algerian
Sahara, International Journal Biotechnology Hydrogen Energy
4941-4946.
Douche, J, Syraka, F. and Livansky, (2005) Utilization of fuel
gas for cultivation of micro algae in an outdoor open thin-
layer photobio reactor. J. Appl Phycol 17:403.
Douskova, I., Kastanek, F., Maleterova, Y., Kastanek, P., Dou-
cha, J., and Zachleder, V. (2010) utilization of distillery stil-
lage for energy generation and concurrent production of
valuable microalgal biomass in the sequence: biogas cogen-
eration-microalgae-products. Energy convers Manage 51:606-
611.
Dukes, J.S. (2003) Burning buried sunshine: human consump-
tion of ancient solar energy. Clim Change 61:31-41.
Fargione, J., Hill, J., Tilman, D., Polasky, S. and Hawthrone,
P. (2008) Land clearing and the biofuel carbon debt. Science
319:1235-1238.
Folch, J., Lees,M., Stanley, G.H. (1957) A simple method for the
isolation and puri cation of total lipids from animals tissues,
Journal of Biological Chemistry:497-509.
Grima, M.E, Belarbi, E., Fernandez, F.A., Medina, A.R., and
Chisti, Y. (2003) Recovery of algal biomass and metabolites:
process options and economics. Biotechnol Adv 20:491-
515.
Hallenbeck PC, Grogger M, Mraz M, Veverka D. (2016) Solar
biofuels production with microalgae. Appl Energy 2016;
179:136–45 Vol-5 ISSN - 0974-2441
Harsh Kharkwal, DD Joshi, Preeti Panthari, Manish Kant Pant,
Amit C Kharkwal (2012) Algae as future drugs. Academic Sci-
ences Asian journal of pharmaceutical and clinical research
Johnson, M.B., and Wen, Z. (2009) Production of biodiesel fuel
from microalgae Schichytrium limacinum by direct transes-
teri cation of algal biomass. Energy fuels 23:5179-5183.
Kim, S., and Dale, B., (2005) Global potential bioethanol pro-
duction from wasted crops and crop residues. Biomass bioen-
ergy 26:361-375.
Liu, J., Huang, J., Sun, Z., Zhong, Y., Jiang, Y., and Chen F.
(2011) Differential lipid and fatty acid pro les of photoauto-
trophic and heterotrophic Chlorella zo ngiensis: assessment of
algal oils for biodiesel production. Bioresour Technol 120:106-
110.
Laamanen CA, Ross GM, Scott JA. (2016) Flotation harvest-
ing of microalgae. Renew Sustain Energy Rev 2016; 58:75–
86
Muf er,K.and Ulber,R. (2005) Downstream processing in
marine biotechnology.Adv biochem Eng biotechnology.
Olaizola, M. (2003) Phototrophic: ef cient alternatives to
land based crops for biofuels. Current opinion in eng: 459-
466.
Pinto, M. M., Raposo M.F., Bowen,J., Young,A.J., and Min
orais, R. (2001) Evaluation of different cell disruption pro-
cesses on encysted cells of Haematococcus pluvial: effect on
astaxanth recovery and implication for bio-availability. J Appl
Phycol 13:18-24
Rebolloso, F., Navaro, P., Garcia, C., Ramos, M. and Guil, G.
(2001) Optimisation of Biodiesel Production by Sun ower oil
transesteri cation’. J.Agric. Food. Chem: 2966-2972.
Sheehan, J., Cambreco, Duf eld, J., Graboski, M., and Shapouri,
H. (1998). An overview of biodiesel and petroleum diesel life
cycles. US Department of agriculture and Energy Report;
1-35.
Stephenson, A. L., Kazamia, E Dennis, J. S. Hawe, C. J. Scott,
S. A. and smith (2010) Lifecycle assessment potential algal
biodiesel production in the United Kingdom: a comparison of
raceways and airlift tubular bioreactor. Energy ues 24:4062-
4077.
Saranya C, Girija K, (2013) Estimation of major pigment con-
tent in seaweeds collected from Pondicherry coast, The Experi-
ment, 9(1), 2013, 522-525.
Suganya T, Varman M, Masjuki HH, Renganathan S. (2016)
Macroalgae and microalgae as a potential source for commer-
cial applications along with biofuels production: a biore nery
approach. Renew Sustain Energy Rev 2016; 55:909–41.
Ugoala, Emeka, Ndukwe G I, Mustapha K B and Ayo R I (2012)
Constraints to large scale algae biomass production and utili-
zation. J. Algal Biomass Utln. 2012, 3 (2): 14- 32
Veillette M, Giroir-Fendler A, Faucheux N, Heitz M. (2018) Bio-
diesel from microalgae lipids: from inorganic carbon to energy
production. Biofuels 2018; 9:175–202
Wijffels, R.H. (2008) Advances in biochemical engineering/
biotechnology. Trend. Biotechnol: 26-30.
Williams, P. R. D., Inman, D., Aden, A. Anheath, G. A. (2009)
Environmental and sustainability factor associated with
322 THE POTENTIAL OF CERTAIN ALGAL SPECIES AS SOURCE OF BIODIESEL BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS
Bhavana Tandon, Shriya Henry and Ramendra Soni
next generation befoul in U.S. Environment Science Tech-
nol.43:4763-4775.
Yang C. Hua, Q. and Shimizu, K. (2000) Energetics and carbon
metabolism during growth of microalgal cells under photo-
autotrophic, mixotrophic and cyclic light-autotrophic/dark-
heterotrophic conditions. Biochem Eng J 6:87-102.
Yuanjun Li (2011). Inexpensive culturing of freshwater algae in
a simulated warm environment using chicken manure medium.
Chalmers University of Technology (Master’s thesis).
Yang C, Li R, Cui C, Liu S, Qiu Q, Ding Y, et al. (2016) Cata-
lytic hydroprocessing of microalgae-derived biofuels: a review.
Green Chem 2016; 18:3684–99.
BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS THE POTENTIAL OF CERTAIN ALGAL SPECIES AS SOURCE OF BIODIESEL 323