e-ISSN: 2582-5208

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PRODUCTION OF BIODIESEL FROM MICROALGAE
(CHLORELLA PROTOTHECOIDES)
Muthu Gayathri D*1, Sathya R*2, Dharani D*3, Jayashree M*4,
Shanmugapriya K*5, Ruthra S*6
*1,2,3,4,5,6Student, Department Of Biotechnology, VSB Engineering College,
Karur, Tamil Nadu, India.
ABSTRACT
As the search for alternatives to fossil fuels continues, microalgae have emerged as a promising renewable
feedstock for biodiesel. Many species contain high lipid concentrations and require simple cultivation including
reduced freshwater and land area needs compared to traditional crops used for biofuels. Recently,
technological advancements have brought microalgae biodiesel closer to becoming economically feasible
through increased efficiency of the cultivation, harvesting, pretreatment, lipid extraction, and
transesterification subsystems. The present article makes a sweeping attempt to highlight the various methods
employed for cultivation of microalgae, techniques to harvest, extract biomass from huge algal cultures and
transesterification process, as well advantages of micro algal feedstocks for biofuel production and application
of biodiesel are also discussed.
Keywords: Biodiesel, Microalgae, Chlorella Protothecoides, Cao Nanocatalyst, Transesterification.
I. INTRODUCTION
The energy source has a significant impact on a country's economy and growth. Coal, natural gas, and natural
oil are the primary sources of energy in the manufacturing and transportation industries. According to reports,
petroleum, coal and natural gas accounted for 81 percent of world energy demand in 2016. Biofuels are fuels
made from organic biomass and lignocellulosic materials. Biodiesel, bioethanol, biogas, and biohydrogen are
some of the most regularly utilised biofuels. Biofuels are further classified into generations based on the
feedstock used. First-generation biofuels may be made from edible food crops including rapeseed, oil seeds,
soybeans, sunflowers, wheat, potato, barley, and sugarcane, among others, whose production was increased
dramatically between 2000 and 2008 to fulfil global energy demand. Second generation biofuels may be made
from non-edible crops such as pongamia, jatropha, sea mango and jojoba as well as lignocellulosic biomass
generated from saw dust, food processing industry by-products, dry wood, maize and wheat stalks and oils
extracted from food wastes. Due to high production costs and a lack of efficacious feedstock conversion
processes, second-generation biofuel yields are still low, making them unsuitable for use as a long-term
substitute for fossil fuels. Third-generation biofuels are biofuels obtained from microalgae, which have
immersed research in recent years due to their high lipid content, high growth rate, ability to live in adverse
conditions, capacity to sequester CO2, non-competition for agricultural land and appeared as the most
practicable feedstock. Currently, edible oils account for 95 percent of global biodiesel production. Fascinatingly,
the lipid (triglyceride) which are inedible, generated by microalgae may be recovered and turned to biodiesel
via the transesterification process.
Microalgae are a diverse group of photosynthetic microorganisms that can fix CO2 from atmosphere and
produce biomass more effectively and quickly than terrestrial plants. They are deliberated to be a viable
feedstock for a variety of industries including food, nutraceuticals, feed, biofuels and medicines. Microalgae
have a number of benefits for biofuel generation while compared to traditional crops including the possibility
for high carbohydrate or lipid content, high growth and productivity and the capacity to thrive in wastewater,
salty or seawater. Microalgae can also be grown in a symbiotic relationship with bacteria to boost cell growth
and biomass production. This review focus on microalgae, biosynthesis of lipid, cultivation, harvesting,
extraction of lipid and transesterification. The optimization of lipid production, other possible applications and
how to merge them with production of biodiesel are also discussed.

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Microalgae
Microalgae are capable of producing 5,000 - 15,000 gallons of biodiesel per acre per year. Microalgae biomass
can produce a variety of biofuels including biodiesel, bio methane, bio hydrogen, bioethanol and bioelectricity.
In micro algal cells, triacylglycerol is the major type of energy storage, comprising for 60–70% of the dry cell
weight. Three fatty acid (FA) moieties are attached to the glycerol backbone of each TAG molecule. Depending
on degree of unsaturation, each FA molecule is categorised as saturated (SFA), mono saturated (MUFA), or
polyunsaturated (PUFA). As a result, the relative profusion of these various FAs in TAG determines its usability
for certain purposes such as high-value nutrient supplements, transportation fuel, industrial polymers and
emulsifiers.
Table 1. Lipid content of different species
Lipid Content Lipid Productivity
S. No Species
(% of DCW) (mg L-1 D-1 )
1. Chlorella protothecoides 43 - 46 1881.3 - 1840.0
2. Nannochloropsis sp. 35 - 48 385 - 413
3. Chlorella zofingiensis 51.1 354
4. Desmodesmus sp. 53.8 ± 6.0 263
5. Chlorella vulgaris 28.1 ± 4.3 204.9 ± 6.4
6. Scenedesmus sp. 12.6 ± 0.8 174
7. Chlorella vulgaris ESP-31 50 78
8. Ankistrodesmus falcatus 59.60 74.07
9. Chlorella sp. BUM11008 42.8 54.0 ± 0.6
Microalgae have a variety of metabolic behaviours, including (1) autotrophic, which uses light as its sole energy
source and converts it to chemical energy using CO2 by photosynthesis; (2) mixotrophic, which uses
photosynthesis as its primary energy source but requires both CO2 and organic compounds; (3) heterotrophic,
which uses only organic compounds as carbon source and energy; and (4) photoheterotrophic, which uses light
to use organic compounds as a carbon source. Changes in environmental conditions can cause algae to modify
their metabolic pathway. Algae's ability to fix CO2 is a way of removing CO2 from power plant flue gases, which
can be utilised to minimise greenhouse gas emissions. To reduce cultivation costs and use CO2 as much as
feasible for CO2 sequestration, algae utilised for biodiesel production must be chosen to grow in
a photoautotrophic mode.

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Raceway

Open Ponds

Microalgae Photo Circular

Hybrid Systems
cultivation bioreactors

Closed

Tabular Flat plate Column

Harvesting of
Microalgae

Electrolytic
centrifugati Centrifugati Screening Electrophore
on
Flocculatio Flotation Filtration Gravity
n

Oil
Extraction
Oil Press Chemical

Expeller Ultrasonic Supercritical

Fluid

Biodiesel

Transesterification Hydrogenation

Figure 1: Biodiesel production from microalgae

Biodiesel is often made by transesterifying algal oil in the presence of an acid or alkali as catalyst. Algal
biodiesel can be produced directly from algal biomass by transesterification. It can alternatively be made in a
two-step procedure in which lipids are extracted first and then transesterified, however both processes require
lipid extraction using alcohols and solvents such as petroleum ether, isopropanol and methanol. The direct
transesterification technique is a quick and low-cost technology. Biodiesel made from microalgae could be a
great alternative to the present fuel shortage, however strains with high oil content and growth rate should be
chosen in order to produce biodiesel efficiently.
Advantages of micro algal feedstocks for biofuel production
 Microalgae grow quickly and produce high yield per acre.
 The use of microalgae to produce feedstocks for biofuels will not interfere with food production.
 Waste water and saline water that cannot be used for regular agriculture or residential purposes can be
used to cultivate microalgae.
 Microalgae have enormous technical promise for reducing greenhouse gas emissions through the biological
carbon capture process.
 There is no need to remove the CO2 after it has been trapped by the microalgae. CO2 is broken down into
carbohydrates and lipids.

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 A micro algal bio-refinery could create a variety of coproducts in addition to biofuel, such as oils, protein,
and carbohydrates. For the generation of biofuels such as biodiesel, green gasoline or methane, aviation fuel,
green diesel conversion technologies can be included.
Application of biodiesel
 Railway usage
 Vehicles
 Aircraft use
 Biodiesel in generators
 Cleaning oil spills
 As a heating oil
II. METHODOLOGY
Biomass Separation
Biomass of algal species was extracted by two methods.
 Freezing
 Solvent extraction method
Freezing
The two 500 ml beakers were distributed to two separate algal crops and put within the congealer until the
solutions were frozen. The algae cultures then were thawed by putting the beakers in warm water. The abrupt
temperature change was used to break the wall and to isolate the lipid type biomass from the cell wall.
Solvent Extraction Method
The total lipids of the cell mass obtained have been collected using a slightly changed version method from
Bligh and Dyer (1959) utilizing the mixing of chloroform methanol (1:1) (1:1.5) (1:2) for 1:1 proportion. The
mixtures were placed into a conical container and shaken for 5 minutes. The mixture was then moved to the
separation funnel. The solvent was then evaporated using a rotary evaporator to extract oil from the solvent.
Oil Extraction
The centrifugation process at 3000 rpm for 30 minutes was used to remove oil from biomass.
Synthesis of Calcium Oxide Nanoparticles
In order to remove dust and impurities, the aseel egg shell washed with de-ionized water and dried for two
days in direct sunlight. The egg shell was crushed by means of the mixer grinder and sieved and then
600,700,800 and 900oC calcined for four hours.
CaCO3 CaO + CO2

Transesterification
Transesterification, also known as alcoholysis, is a multi-step reaction that involves reacting triglycerides with
methanol in the presence of a catalyst. By eliminating the glycerin and generating an alcohol ester,
transesterification chemically neutralises the free fatty acids in a triglyceride molecule. The triglycerides are
transformed to diglycerides in the first phase. The diglycerides are then transformed into mono glycerides.
After that, the mono glycerides are converted to esters (biofuel) and glycerol is produced as a by-product.
Oil + Methanol + catalyst → Ester + Glycerol
III. MODELING AND ANALYSIS
Reaction
Instead, the alcohol / catalyst combination was charged into a closed reaction vessel, adding the oils or fats. The
machine was completely closed to the environment from here on, to avoid alcohol loss. The reaction mix was
held at 180 min (60 °C, 65 °C, 70 °C) respectively and some systems suggest reaction at room temperature.
Normally excess alcohol was used to ensure the complete conversion of the fats or oils to their esters.

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Separation
Once the reaction was complete biodiesel is collected from the sample. It has a substantial amount of the excess
methanol that was used in the reaction.
Qualitative test for fuel
Qualitative test for fuel was done by two methods
 Ferric chloride test
 Hydroxamic acid test
Ferric Chloride Test
A sample drop was dissolved in 1ml of 95% ethanol, 1ml of 2N HCL. The color change created by adding 1drop
of 5% iron (III) chloride to the solution. If a pronounced violet, blue, red or orange color is created, the
hydroxamic acid test is not needed.
Hydroxamic Acid Test
A sample drop was combined with 1ml of 0.5N hydroxylamine hydrochloride in 95% ethanol.0.2ml of 6 M
aqueous sodium hydroxide. The mixture was heated to boil and add 2ml of 2N hydrochloric acid to it after the
solution was cooled. The solution is opaque, adding 2ml of 95 percent ethanol. The improvement in color
occurred with the addition of 1 drop of 5 percent iron (III) chloride.
IV. RESULTS AND DISCUSSION
Sample collection

Figure 2: Microscopic study of Chlorella protothecoides

Biomass separation

a b c
Figure 3: a) Before Biomass separation b) Concentrated form of Biomass c) After
biomass separation Oil Extraction
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Figure 4: Before Transesterification

FTIR Analysis of CaO nanocatalyst

Figure 5: Characterization of CaO catalyst by FTIR

SEM Analysis of CaO nanocatalyst

Figure 6: Characterization of CaO catalyst by SEM

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Transesterification

Figure 7: Separated bio diesel from the biomass with the CaO catalyst
Qualitative test for ester
Ferric Chloride Test

Figure 8: Ferric chloride test result for Chlorella protothecoides sample

Hydroxamic Acid Test

Figure 9: Hydroxamic acid test result for Chlorella protothecoides

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GC-MS analysis

Figure 10: GC-MS analysis of biodiesel produced from C.protothecoides with CaO nanocatalyst
Table 2. Fatty acid profile of biodiesel
Peak
Molecular Compound Nature
Name of the compound MW Area
RT formula
%
2-Mercaptobenzothiazole 2(3H)-
17.650 C7H5NS2 167 23.58
Benzothiazolethione
dodecyl 4-methylpent-2-yl
31.139 ester C22H40O4 368 0.24
Fumaric acid

24.34 Myristic acid n-butyl ester C18H36O2 284 2.81 n-Butyl myristate
N-cyclobutylcarbonyl-,
37.282 l-Alanine C9H15NO3 185 0.2 methyl ester

V. CONCLUSION
The present study exposed that nanocatalyst derived from aseel chicken egg shell waste was found to be
effective for the production of biodiesel from Chlorella protothecoides. High rigid and porous structure of the
synthesized CaO nanocatalyst was characterized by SEM analysis also, FTIR analysis was confirmed the active
functional group present in the catalyst. The optimum reaction conditions for biodiesel production from
Chlorella protothecoides using CaO were found to be oil to methanol molar ratio (1:7), catalyst concentration
(5% w/w), temperature (65oC) and the reaction time (70 min). The nanocatalyst stability was found and it can
be reused for four cycles. The GCMS analysis of FAME was confirmed the presence of fatty acids and fatty acid
methyl esters.
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