Indian Journal of Geo Marine Sciences
Vol.46 (02), February 2017, pp. 385-390
Biodiesel production from Anabaena variabilis cyanobacterium
Ali Salehzadeh1,*, Akram Sadat Naeemi2
1
2
Department of Biology, Rasht Branch, Islamic Azad University, Rasht, Iran
Department of Biology, Faculty of Science, University of Guilan, Rasht, Iran
* [E. mail: [email protected]]
Received 04 February 2014; revised 11 September 2014
Present study indicates the production of algal biodiesel from Anabaena variabilis. The research showed that the oil content in
Anabaena variabilis was 45% by weight of dry biomass. The oil was extracted from the Anabaena variabilis biomass. Gas
chromatography–mass spectrometry (GC–MS) was used to analyze the extracted oil. Several chemical constituents were defined after
GC-Mass analysis. Among them, the constituents that can be used as biodiesel were Palmitic acid (C16H32O2), 3,3-Dimethoxy-2butanone (C6H12O3), 2,2-Dimethoxybutane (C6H14O2) and 2,2-Dimethoxypropane (C5H12O2). The percentage of them were (2.3%),
(2.18%), (8.51%) and (1.54%) respectively.
[Keywords: Cyanobacterium, Biodiesel, Anabaena variabilis]
Introduction
Nowadays around 80% of global energy demand
is produced from fossil fuels. However, vast
utilization of fossil fuels has led to global climate
change, health problems, and environmental
pollution1. Some countries are, therefore, trying to
develop new sources of energy sources that are
clean sustainable. Of the potential alternate sources
of renewable energy, biofuels have received the
most consideration and are expected to play a key
role in the universal energy infrastructure in the
future. Biodiesel, one of the most frequently used
biofuels, is accepted as an ideal, recyclable energy
carrier as a possible principal energy source2.
Currently commercial biodiesel is produced from
animal fat, vegetable oils and waste frying oil 3,
whose competition with edible vegetable oil for
agricultural land is still a controversial matter4. So,
microalgae that can grow quickly and transform
solar energy to chemical energy through CO2
fixation are being considered as a hopeful oil source
for producing biodiesel4. Under suitable culture
environments, some microalgal species are capable
to gather up to 50–70% of oil/lipid per gram of dry
weight 2. The fatty acid pattern of microalgal oil is
excellent for the production of biodiesel 5. The other
advantage is that microalgae can yield up to 58,700
liter of oil per hectare, which is one to two times
higher than that of any other energy crop 6.
Yet, mass manufacture of microalgal oil
encounters several of technical obstacles that render
the current development of the algal industry.
Moreover, it is also necessary, but vastly
problematic, to establish economical technologies
that would permit effective biomass harvesting and
oil extraction. However, because microalgae
production is considered as a convincing approach
to quench global warming, it is clear that producing
oil from microalgal biomass would provide deep
benefits including fuel. The current research was
done to resolve the ability of Anabaena variabilis to
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INDIAN J. MAR. SCI., VOL. 46, NO. 02, FEBRUARY 2017
produce biodiesel by measuring the amount of oil
produced by isolated strain.
Materials and Methods
Anabaena variabilis was purchased from
National Inland Water Aquaculture Institute of Iran.
The microalgae was isolated from Anzali
international wetland and purified (Fig. 1).
Anabaena variabilis was cultured in BG11
medium 7. The pH of the medium was adjusted to
7·2 using 0·1 Ν NaOH before autoclaving. Cultures
were kept as batch cultures at 26±1°C with a light
intensity of 1500–2000 lux, and grown in a dark /
light cycle of 12/12 h for 14 days.
Microalgae biomass was harvested using a
continuous feed, fixed bowl centrifuge. The plate,
consisting of 15–20% dry matter, was directly
frozen. The paste was then dried in a vacuum oven
at 70 °C and stored at -20 °C.
The neutral lipids that were converted to FAME
(fatty acid methyl esters) using a fixed bed reactor
were extracted from dried algae powder using
hexane Soxhlet extraction. Prior to extraction, dried
algae flakes were crushed using a blender and then
pulverized in a ball grinder. When the cells had been
milled properly, the mixture was placed in the
Soxhlet extractor until the extracting hexane was
colorless. The lipid containing hexane solution was
then filtered through a 1.2 lm GFC Whatman filter.
The hexane was then removed using a rotary
evaporator. The crude oil was then re-dissolved in
hexane and filtered through activated carbon to
remove pigments (Fig. 2)8.
A two-step protocol was used for the
transesterification of all extracted lipids. The first
step used an acid catalyst to methylate free fatty
acids (FFA) and to transmethylate acylglycerols.
Twenty mL methanol and 10 % H2SO4 in 1 mL
methanol were added to the lipid in hexane solution
stored in each vial. The mixture was transferred into
a flask, heated to 50 °C, and moderately agitated for
2 h. Evaporated methanol was frequently refilled. In
the second step, 25 wt% KCH3O in methanol was
added dropwise to the gently stirred reaction
mixture until a pH 13 was attained. The mixture was
then heated again to 55 °C and moderately agitated
for 2 h. Evaporated methanol was frequently
refilled. The mixture was evaporated in a 60 °C
oven to obtain dried post-methylated lipid extract.
The lipid was then re-dissolved in 20 mL hexane for
FAME analysis.
GC-MS analysis of the extract was done using an
Agilent 7890B gas chromatograph (Agilent
Technologies, USA) attached to a Agilent 5977A
mass spectrometer (Agilent Technologies, USA).
The column used was HP DB-5 capillary column
(30×0.25 mm×0.25 μm; Agilent Technologies). GC
oven initial temperature was 50°C for 2 min and
was computed to 280°C at a rate of 5°C/min, and
finally held at 280°C for 2 min. Running conditions
for GC were as follows: helium was used as carrier
gas (5 mL/min); the temperature of injector and
detector was 250°C and 280°C, respectively; the
volume injected was 2 μL in split mode (10: 1). The
mass spectra were performed at 70 eV of the mass
range of 35~400. Three replicates were done for
each sample. Elucidation on mass spectrum of GCMS was done using the database of National
Institute Standard and Technology (NIST) having
more than 62,000 patterns. The mass spectrum of
unknown components was compared with the
spectrum of the known components kept in these
libraries. The name, molecular weight and structure
of the components of the test materials were found
out 9.
Fig. 1—Anabaena variabilis isolated from Anzali wetland.
Results
The oil content in Anabaena variabilis was 45%
by weight of dry biomass. Fig. 3 presents the
GC/MS analysis of the chemicals identified in the
Anabaena variabilis extract. About 12 chemical
constituents were defined after GC-Mass analysis
(Table 1). Among them, the constituents that can be
used as biodiesel were Palmitic acid (C16H32O2), 3,
SALEHZADEH & NAEEMI: BIODIESEL PRODUCTION FROM ANABAENA VARIABILIS
387
3-Dimethoxy-2-butanone
(C6H12O3),
2,
2Dimethoxybutane
(C6H14O2) and 2, 2Dimethoxypropane (C5H12O2) (Fig. 4). The
percentage of them were (2.3%), (2.18%), (8.51%)
and (1.54%) respectively. Palmitic acid was found
to be as the major fatty acid in Anabaena variabilis
microalgae.
Fig. 2—The oils extracted from Anabaena variabilis
.
Fig. 3— GC-MS chromatogram of A. variabilis extracted
oils.
Discussion
Recently a number of investigators have
exanimated seed oils for the creation of biofuels.
Manufacturing of second generation fuels such as
bioethanol and biodiesel from biomass grown on
arable lands, especially the use of oil-seeds for
biodiesel, have raised the food prices. Third
generation biodiesel from microalgae grown on nonFig. 4— Mass spectrum and structures of oils identified by GCMS in A. variabilis
INDIAN J. MAR. SCI., VOL. 46, NO. 02, FEBRUARY 2017
388
Table 1. Chemical constituents of Anabaena variabilis, based on GC-MS analysis
No.
Compound identified
Molecular
formula
MW
RT
(min)
Peak
area
(%)
1
Hexamethylcyclotrisiloxane
C6H18O3Si3
222.46
1.53
0.34
2
2-Propen-1-one
C9H8O
132.15
1.638
23.18
3
4-Cyanobenzophenone
C14H9NO
207.22
2.239
1.68
4
2,2-Dimethoxypropane
C5H12O2
104.14
2.284
1.54
5
Benzonitrile, m-phenethyl-
C15H13N
207.27
2.376
2.47
6
1-Methyl-2isopropylbenzene
C10H14
134.21
2.799
1.90
7
2,2-Dimethoxybutane
C6H14O2
118.17
3.217
8.51
8
3,3-Dimethoxy-2-butanone
C6H12O3
132.15
4.545
2.18
9
N,Ndimethylethanethioamide
C4H9NS
103.18
4.671
2.55
10
Octasiloxane,
1,1,3,3,5,5,7,7,9,9,11,11,13,1
3,15,15-hexadecamethyl-
22.546
1.51
11
Phenol, 2,5-bis(1,1dimethylethyl)-
C14H22O
206.32
22.895
1.43
12
Palmitic acid
C16H32O2
256.42
34.253
2.30
C16H48O7Si8 577.23
SALEHZADEH & NAEEMI: BIODIESEL PRODUCTION FROM ANABAENA VARIABILIS
arable land is the clear answer to the food-fuel
competition. The process of microalgal cultivation
could be improved for efficient yield of algal lipids
over the screening and improvement of microalgal
strains10. The oil content of Anabaena variabilis
used in the present study was 45% by weight of dry
biomass. The lipid content in dry biomass of
Anabaena variabilis is higher than other microalgae.
For example, the lipid content in dry biomass of
Chlorella emersonii 28–32%, Chlamydomonas
reinhardtii is 21%, Chlorella vulgaris 14-22%,
Crypthecodinium
cohnii
20%,
Dunaliella
primolecta 23%, Dunaliella salina 6%, Dunaliella
tertiolecta 36%, Euglena gracilis 14-20%,
Phaeodactylum tricornutum 20-30%, Chlorella
protothecoides 55% and Pleurochrysis carterae
30–50% 11, 12.
Among the chemical constituents defined, the
constituents that can used as biodiesel were Palmitic
acid,
3,3-Dimethoxy-2-butanone
,
2,2Dimethoxybutane
and 2,2-Dimethoxypropane.
Zhang et al (2013)13 showed that the 3, 3Dimethoxy-2-butanone and 2, 2-Dimethoxybutane
are the bio-oils which find in willow wood. Some
compound for example Dimethoxypropane,
Dimethoxybutane and Palmitic acid are the
compound that find in canola oil14. Palmitic acid is
finding in several cyanobacteria. From these
cyanobacteria we can point out to Calothrix fusca,
Lyngbya dendrobia, Microcystis aeruginosa,
Oscillatoria calcuttensis and Scytonema bohnerii.15
Machado et al (2012)16 used the genetically
modified Synechocystis sp. for production of some
butane derivative as biodiesel. In the present study
the 3, 3-Dimethoxy-2-butanone and 2, 2Dimethoxybutane had butane in their structure.
Sharathchandra et al (2011)17 evaluated the total
lipid and fatty acid composition in 13 species of
freshwater cyanobacteria. They showed that the
Palmitic acid was one of the major fatty acids were
found in these species. Recently, researchers
characterized the Palmitic acid in Azolla filiculoides
macroalgae18. In comparison to previous researches,
the present research showed that the Palmitic acid is
key component in microalgae, macroalgae and
plants.
Anabaena variabilis microalgae can grow on
waste water19. It is a positive note. This feature is
very important for an economic production of fuel
in comparison to fossil fuels. The major energy
sources are fossil fuels but their overconsumption
leads to catastrophic impacts such as air pollution.
389
Burning of fossil fuels lead to emissions of carbon
dioxide, nitrogen monoxide, nitrogen dioxide,
sulphur dioxide, and carbon monoxide. These gasses
have serious results for habitats and also affect
human health. Furthermore, they are non-renewable
sources of energy which derive from pre-historic
fossils and are no longer available after usage. Their
sources are restricted and they are depleting at a
faster rate. Microalgae fuels in comparison to fossil
fuels do not have aforesaid problems and above all,
they are renewable. Nevertheless, further research
and development are necessary to establish an
economical industrial scale production of Anabaena
variabilis biodiesel.
Conclusions
This research has revealed that Anabaena
variabilis biodiesel is technically achievable. It is a
renewable biodiesel that can potentially entirely
displace liquid fuels obtained from petroleum.
Acknowledgement
Authors are grateful to Dr. Jannat Sarmad,
member of scientific board, University of Guilan,
for providing facilities and encouragement to carry
out the above research work.
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