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Life Science Archives (LSA)

ISSN: 2454-1354
Volume 1; Issue - 2; Year 2015; Page: 84 - 89

Research Article
BIODIESEL ENERGY PRODUCTION FROM MICROALGAE - Spirogyra sp.,
Oscillatoria foreani AND Chlorella pyrenoidosa
R. Muthulakshmi* and K. Meenatchisundaram
PG Department of Microbiology, A.V.C College (Autonomous), Mayiladuthurai, Tamil Nadu, India.
Abstract
Algae are an economical choice for biodiesel production, because of its availability and low cost.
Microalgae were a good source for biodiesel production due to its lipid content in addition to its easy growth.
Hence, in the present study, fresh water microalgae viz., Spirogyra sp., Oscillatoria foreani and Chlorella
pyrenoidosa were chosen. The total lipid content was estimated in Spirogyra sp., Oscillatoria foreani and
Chlorella pyrenoidosa. Highest lipid content was observed in Oscillatoria foreani than other two species.
The dry weight percent were found to be higher in Oscillatoria foreani. After transesterification process the
extraction of oil and biomass observed were to be more in Oscillatoria foreani compared with other two
species. The pH varies between the two algal species and it is higher in Oscillatoria foreani. The present
investigation thus revealed higher oil extraction in Oscillatoria foreani. Microalgae Oscillatoria foreani were
found to be more beneficial than Spirogyra sp. and Chlorella pyrenoidosa commercially and economically
for production of biodiesel. The study also concludes that microalgae are a best source of biodiesel.
Article History
Received : 18.05.2015
Revised : 29.03.2015
Accepted : 02.04.2015
1. Introduction

Key words: Spirogyra sp., Oscillatoria foreani,

Chlorella pyrenoidosa and Microalgae

Biodiesel is a nontoxic and biodegradable

fuel that is obtained from renewable sources.
Biodiesel can be prepared from waste cooking oil
such as palm, soyabeen, canola, rice, bran,
sunflower, coconut, cornoil, fish oil, chicken fat
and algae, which would partly decrease the
dependency on petroleum based fuels. Microalgae
are sunlight-driven cell factories that convert
carbon dioxide to potential biofuels, foods, feeds
and high-value bioactives (Metting and Pyne,
1986). In addition, these photosynthetic
microorganisms are useful in bioremediation
applications and as nitrogen fixing biofertilizers.
This article focuses on microalgae as a potential
* Corresponding author: R. Muthulakshmi
Tel.: +91-7373784146
E-mail: 80chinns@gmail.com

source of biodiesel. Microalgae can provide

several different types of renewable biofuels.
These include methane produced by anaerobic
digestion of the algal biomass (Spolaore et al.,
2006); biodiesel derived from microalgal oil
(Roessler et al., 1994).
Biodiesel is produced currently from plant
and animal oils, but not from microalgae. This is
likely to change as several companies are
attempting to commercialize microalgal biodiesel.
Biodiesel is a proven fuel. Technology for
producing and using biodiesel has been known for
more than 50 years. Bioenergy is one of the most
important components to mitigate greenhouse gas
emissions and substitute of fossil fuels. The need
of energy is increasing continuously, because of
increases in industrialization and population. The

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R. Muthulakshmi/Life Science Archives (LSA), Volume 1, Issue 2, Page 84 to 89, 2015

basic sources of this energy are petroleum, natural
gas, and coal, hydro and nuclear. The major
disadvantage of using petroleum based fuels is
atmospheric pollution created by the use of
petroleum diesel. Petroleum diesel combustion is a
major source of greenhouse gas (GHG). Apart
from these emissions, petroleum diesel is also
major source of other air contaminants including
NOx, SOx, CO, particulate matter and volatile
organic compounds. The world is entering a
period of declining non-renewable energy
resources, popularly known as Peak oil with
energy demand is increasing the worlds oil
production is expected to decline in between one
and ten decades (Crokes, 2006). As a result of this
impending energy crisis, both governments and
private industry are examining alternative source
of energy. Other non-renewable sources of energy
exist, such as coal and uranium: however these
sources are limited and will also inevitably decline
in availability. Microalgae have been suggested as
very good candidates for fuel production because
of their advantages of higher photosynthetic
efficiency, higher biomass production and faster
growth compared to other energy crops. In this
study, Biodiesel is produced from microalgae such
as Spirogyra sp., Oscillatoria foreani and
Chlorella pyrenoidosa.
2. Materials and Methods
2.1. Sample collection
The microalgae sample viz., Spirogyra sp.,
Oscillatoria foreani and Chlorella yrenoidosa and
were collected from fresh water source at
Kalyanasozhapuram Village, Nagapattinam Dt.
2.2. Microscopic observation
The Collected algal species were
confirmed based on color cultural and microscopic
observation (Desikachary, 1959).

85

samples were inoculated at aseptic condition. The

conical flasks were kept in an alternate light and
dark regime of 2 weeks at 25 C. After incubation
the algal colonies were appeared. After 20 days
the algal mat were collected and used for further
studies.
2.3. Assay of total lipids
Total lipid content was estimated in the
Spirogyra sp. and Oscillatoria foreani by the
method of Cox and Pearson (1962). One gm of
algal sample was dissolved in 250 ml of conical
flask. Few drops of phenolphthalein were added
and titrated against 0.1 N Potassium hydroxide.
Shake constantly until a pink colour which persists
for fifteen seconds is obtained.
Calculation:
Acid value (mg KOH/g) =

./ ()
2.4. Oil extraction
Algae were ground with motor and pestle
as much as possible. The ground algae were dried
for 20 min at 80C in incubator for releasing
water. Hexane and ether solution (20 and 20 mL)
were mixed with the dried ground algae to extract
oil. Then the mixture was kept for 24 hrs for
settling (Sharif Hossain et al., 2008). The dried
ground algal sample were calculated using the
formula
% = /
100
2.5. Biomass collection
The biomass of microalgae such as
Spirogyra sp., Oscillatoria foreani and Chlorella
pyrenoidosa were collected by filtration. Biomass
percentage was calculated using the formula.
% = / 100

2.3. Mass cultivation of algal sample

The collected microalgal sample Spirogyra
sp., Oscillatoria foreani
and Chlorella
pyrenoidosa were cultivated using BG-11 medium
in the laboratory (Aneja, 2002). BG-11 medium
was prepared and 50 ml were taken at each conical
flask and autoclaved at 121 C for 30 minutes. The
medium were allowed to cool. The collected

2.6. Evaporation
The extracted oil was evaporated in
vacuum to release hexane and ether solutions
using rotary evaporator.

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86

2.7. Mixing of catalyst and methanol

3.2. Mass culturing of Algal sample

0.25 g NaOH was mixed with 24 ml

methanol and stirred properly for 20 min.

The sample such as Spirogyra sp.,

Oscillatoria foreani and Chlorella pyrenoidosa
were cultivated in sterilized BG-11 medium
separately. After two weeks greenish algal
biomass were observed in the flask, cultivated
algal species were grown on the BG-11 agar
medium for further studies.

2.8. Biodiesel production

The mixture of catalyst and methanol was
poured into the algal oil in a conical flask. The
following reaction and steps were followed
National biodiesel Board.
2.9. Transesterification
The
reaction
process
is
called
transesterification. The conical flask containing
solution was shaken for 3 h by electric shaker at
300rpm.
2.10. Settling
After shaking the solution was kept for 16
h to settle the biodiesel and sediment layers
clearly.

3.3. Estimation of total lipid

The total lipid content was estimated in
Spirogyra sp., Oscillatoria foreani and Chlorella
pyrenoidosa. In this study, maximum lipid level
was noted in Oscillatoria foreani compared than
other two species. The results were presented in
Table- 1.
3.4. Measurement of fresh weight and Dry
weight of the sample

The biodiesel was separated from

sedimentation by flask separator carefully.
Quantity sediment (glycerine, pigments, etc.) was
measured. The percentage of biodiesel were
calculated by the formula.

The fresh weight of the microalgae viz.,

Spirogyra sp., Oscillatoria foreani and Chlorella
pyrenoidosa were noted. The dry weight was
noted after drying the sample in the incubator for
20 min at 80C for releasing water content in the
sample. The percentage of dry weight was greater
in Oscillatoria foreani and Chlorella pyrenoidosa
than Spirogyra sp. species (Table - 2).

% =/ 100

3.5. Measurement of oil extraction

2.12. Washing

Production of Biodiesel was measured by

using measuring cylinder; pH was measured and
stored for analysis.

The oil extraction of microalgae viz.,

Spirogyra sp., Oscillatoria foreani and Chlorella
pyrenoidosa
were
noted
after
the
transesterification
process.
The
obtained
percentage of oil content was seemed to be higher
in Oscillatoria foreani. This represents that the
biodiesel (Methyl ester) production was found to
be maximum in Oscillatoria foreani (Table - 3).

3. Results

3.6. Assay of total biomass

In this study the microalgae such as

Spirogyra sp., Oscillatoria foreani and Chlorella
pyrenoidosa were used for the production of
biodiesel. The investigated results were presented
in this chapter.

The biomass obtained after oil extraction

were seem to be higher in microalgae Oscillatoria
foreani when compared with other two species
(Table-4).

3.1. Microscopic observation

There is a significant difference between

pH of the two microalgae viz., Spirogyra sp.,
Oscillatoria foreani and Chlorella pyrenoidosa
were noted (Table - 5). Overall, the sediment

2.11. Separation of biodiesel

Biodiesel was washed by 5% water until it

was become clean.
2.13. Storage

The collected three micro algal species

were screened by microscopically.

3.7. Determination of pH

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R. Muthulakshmi/Life Science Archives (LSA), Volume 1, Issue 2, Page 84 to 89, 2015

(Glycerine, pigment and other element) were seen
to be higher in Oscillatoria foreani and Chlorella
pyrenoidosa than the Spirogyra sp.
4. Discussion
In the present study, microalgae viz.,
Spirogyra sp., Oscillatoria foreani and Chlorella
pyrenoidosa were analyzed for their biodiesel
production and compared with previous
theoretical and biostatistical reports. The results
revealed
microalgae
Oscillatoria
foreani
comparatively show effective production of
biodiesel than other two species. Van Gerpen
(2005) reported that the biodiesel is a primary
advantages, that it is one of the most renewable
fuels and also non-toxic and biodegradable shay
(1993) reported that today, biodiesel has come to
mean a very specific chemical modification of
natural oils. The use of vegetable oils as
alternative fuels has been around for 100 years
when the inventor of the diesel engine Rudolph
Diesel first tested Peanut oil in his compression
ignition engine. One of the biggest advantages of
biodiesel compared to many other alternative
transportation fuels is that it can be used in the
existing diesel engines without modification, and
can be blended in at any ratio with petroleum
diesel.
Barnwal and Sharma (2005) found that
biodiesel performs as well as petroleum diesel,
while reducing emissions of particulate matter,
carbon monoxide (Co), Hydrocarbons and oxides
of sulphur (Sox). Other environmental benefits of
biodiesel include the fact that it is highly
biodegrable and appear to reduce emissions of air
toxins and carcinogens (relative to petroleum
diesel). The production of biofuels from algae
does not reduce atmospheric carbon-di-oxide,
because any Co2 taken out of the atmosphere by
the algal is returned when the biofuels are burned.
They do however eliminate the introduction of
new Co2 by displacing fossil hydrocarbon fuels.
(Murdoch University, Western Australia). Many
researchers prove the Algae are a good source of
biodiesel production than the commercial food
crops.
Hall et al. (1991) reported that biomass has
been focused on as an alternative energy source.

87

Since it is a renewable resource and it fixes Co2 in

the atmosphere through photosynthesis. If biomass
is grown in a sustained way, its combustion has no
impact on the Co2 fixed by photosynthesis. Shay
(1993) reported that among biomass, algae (Macro
and
microalgae)
usually
have
higher
photosynthetic efficiency than other biomass.
Shay (1993) suggested that algae were one of the
best sources of biodiesel. In fact algae are the
highest yielding feed stock for biodiesel. It can
produce up to 250 times the amount of oil per acre
as soyabeans.
Ladygina and Dehyukhina (2006) stated
that the national renewable energy laboratory
(NREL) has categorized approximately 300 sp of
microalgae that could be potential fuel sources. In
this study we selected microalgae viz., Spirogyra
sp., Oscillatoria foreani
and Chlorella
pyrenoidosa for the production of biodiesel.
Spolaore et al. (2006) evaluated that microalgae
can provide several different types of renewable
biofuels. Many researchers reported the biodiesel
was derived from microalgal oil. Sheehan et al.
(1998) reported that the average lipid content of
the green algae, blue green algae ranges the
production of biodiesel. Canakci and van Gerpen
(2001) reported that Chlorella vulgaris known for
shorter chain fatty acids which are ideally suited
for biodiesel production. In my present research
work there is an increase in lipid content in
Oscillatoria foreani and than the blue green algae
Spirogyra sp. and finally this leads to the
increased production of biodiesel in the
microalgae Oscillatoria foreani.
Chisti (2007) suggested that microalgae
are capable of producing 30 times the amount of
oil per unit area of land, compared to terrestrial oil
seed crops. Metting (1996) oil content of
microalgae are usually between 20-50% (Dry
weight) while some strains can reach as high as
80%. Chisti (2007) found that oil level of 20-50%
is common in microalgae. Miao and Wu (2006)
reported that Algal biodiesel production was
typically performed by the extraction of algal oil
followed by transesterification. Hu et al. (2008)
reported that algae produced more oil in stressed
or unfavourable condition in comparison to
optimal growth condition. There are other ways to

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R. Muthulakshmi/Life Science Archives (LSA), Volume 1, Issue 2, Page 84 to 89, 2015

make biodiesel, including direct use of blending,
microemulsions and pyrolysis (Ma and Hanna,
1999), but transesterification is the most common
method since the biodiesel product can be used
directly or in blends (Miao and Wu, 2006). In my
work the obtained biodiesel from algal extract
were also undergone the transesterification
process. Fukuda et al. (2001) suggested that
transesterification was catalysed by acids, alkalis
and
lipase
enzymes.
Alkali
catalysed
transesterification in about 4000 times faster than
the acid catalyzed reaction. Transesterification is a
typical process for the commercial production of
biodiesel. The use of organic solvents can increase
this extraction level to 99% but there is an
increased cost in processing to achieve this
(Metzger
and
Largeau,
2005).
Using
transesterification allows for a single step process
that extracts and the algal oils and reacts then with
methanol result in biodiesel.
Belarbi et al. (2000) suggested that
production of methyl esters, or biodiesel, from
micro algal oil has been demonstrated although
the product was intended for pharmaceutical use.
Oil productivity, that is the mass of oil produced
per unit volume of the micro algal broth per day,
depends on the algal growth rate and the oil
content of the biomass. Microalgae with high oil
productivities are desired for producing biodiesel.
Yusuf (2007) explained about biofuel from
microalgae. He mentioned the biodiesel derived
from the oil crop is a potential renewable and
carbon neutral alternative to petroleum fuels.

88

Table 2: Measurement of Fresh weight and

Dry weight of the test organisms
S.
No.

Test
organisms

Spirogyra sp.

Oscillatoria
foreani
Chlorella
vulgaris

Fresh
Dry
Dry
weight weight weight
(g)
(g)
(%)
20
12.5
37.5
0.95
0.14
20
9.5
52.5
1.13
0.58
20
10.5
47.5
1.12
0.64

Table 3: Measurement of oil extraction

S.
No
1
2
3

Test
Organisms
Spirogyra sp.
Oscillatoria
foreani
Chlorella
vulgaris

Oil
extraction (g)
2.8 0.41
4.3 0.20

% of oil
extraction
10.7
17.9

3.6 0.30

16.4

Table 4: Assay of Total Biomass

S.
No.
1
2
3

Test
Organisms

Total Biomass
% of
(After
Biomass
Extraction)
Spirogyra sp. 3.5 0.14
71.42
Oscillatoria
3.8 0.16
73.07
foreani
Chlorella
3.7 0.12
70.06
vulgaris

Table 1: Total lipid content of Test organisms

S.No
1

Test organisms
Spirogyra sp.

Total lipid
content (%)
14

Oscillatoria foreani

29

Chlorella vulgaris

18

Table 5: Determination of pH from Algal

Biodiesel
S.
Test Organisms
No
1 Spirogyra sp.
2 Oscillatoria foreani
3 Chlorella vulgaris

pH Value
Biodiesel
6 0.30
7 0.12
7 0.21

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