Applied Energy 88 (2011) 36323635

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Applied Energy
journal homepage: www.elsevier.com/locate/apenergy

Microbial oil production from thermophile cyanobacteria for biodiesel production

Sevgi Ertugrul Karatay, Gnl Dnmez
Department of Biology, Faculty of Science, Ankara University, 06100 Besevler, Ankara, Turkey

a r t i c l e

i n f o

Article history:
Received 10 November 2010
Received in revised form 18 January 2011
Accepted 2 April 2011
Available online 6 May 2011
Keywords:
Cyanobacteria
Biodiesel
Microbial oil
FAME

a b s t r a c t
Compared to lipid extraction from algae, little work has been performed for cyanobacteria. In this article
it is aimed to show high lipid accumulation potential of Synechococcus sp., Cyanobacterium aponinum and
Phormidium sp. cells in BG-11 medium. Four different pH values (69) and NaNO3 (0.25, 0.5, 1.0, 1.5 g/L)
concentrations were examined at different incubation days to discover the highest lipid accumulation.
The maximum lipid content could be achieved in the medium containing 0.25 g/L NaNO3 at pH 7 for Synechococcus sp., pH 8 for C. aponinum and pH 9 for Phormidium sp. after 15 days. The maximum lipid contents and C16 and C18 methyl ester yields were measured as 42.8% and 46.9% for Synechococcus sp., 45.0%
and 67.7% for C. aponinum, 38.2% and 90.6% for Phormidium sp. The saturated compounds were 74.5%,
77.9%, 84.7% for Synechococcus sp., C. aponinum and Phormidium sp., respectively. These crude lipids could
be promising feedstock for biodiesel production.
2011 Elsevier Ltd. All rights reserved.

1. Introduction
In todays world the widespread use of fossil fuels such as
petroleum, coal and natural gas, cause several environmental consequences which triggers the researches for nding alternative
fuels. Additionally, diminishing petroleum reserves is the biggest
motivation behind the increasing number of researches [1,2].
A number of studies have shown that biodiesel fuels are dened
as fatty acid methyl esters derived from various renewable lipid resources. Because of their renewable characteristic biodiesel fuels
are notable alternatives to solve the worldwide energy problem
[3,4].
Even though vegetable oils are used as a diesel fuel source from
the beginning of early 1930s, the high cost of biodiesel (mainly that
of virgin vegetable oil) is the major obstacle for commercialization.
Biodiesel production from microbial oils are attractive since its
cheapness [5,6].
In this context, the source of oil that used in biodiesel production has a great importance on the cost of biodiesel. For instance;
expensive feedstock increases the cost of biodiesel at a rate of
40%. Therefore it is important to select appropriate raw material
for biodiesel production [7]. Although the usage of animal fats
can reduce the costs, the process requires additional cheap raw
materials [8]. In addition animal fats have higher cetane numbers
and having a tendency to oxidation because of their saturated com-

Corresponding author. Tel.: +90 312 212 6720; fax: +90 312 223 2395.
E-mail address: gdonmez@ankara.edu.tr (G. Dnmez).
0306-2619/$ - see front matter 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.apenergy.2011.04.010

pounds and crystallization unacceptably at high temperatures are

the other disadvantages of animal oils as a feedstock [9]. Therefore
microbial oils have a great potential in biodiesel production because of their structure and fatty acid composition instead of vegetable oils [10]. The microorganisms such as bacteria, yeasts, molds
and algae can accumulate lipids at more than 20% of their biomass
are dened as oleaginous species. The majority of these lipids,
which are formed from long-chain fatty acids similar to plants oils,
are comparable to conventional vegetable oils [11,12].
It is mentioned in many literature that while plants are important feedstocks for biodiesel production, photosynthetic microorganisms (algae or cyanobacteria) have many signicant
advantages [13]. These advantages are rapid growth rate, high
lipid content, smaller land usage when compared [14]. Due to
their several advantages microalgae based oils can be a promising
solution to the global renewable energy demand for transport
fuels [15].
Although these advantages of cyanobacteria, there is not enough report about the lipid accumulation properties of cyanobacteria. It is seen in the literature Chlorella sp. is the most common
studied algae species [1619].
In this work it is aimed to suggest cheaper raw material for biodiesel production by using Cyanobacterium aponinum, Synechococcus sp. and Phormidium sp. cells. Thermophilic properties of these
cyanobacteria permit the production of high volume lipids in large
and open pools which is the key step for the success in biodiesel
technology.
To our knowledge this is the unique report about the lipid accumulating properties of C. aponinum, Synechococcus sp., and Phormidium sp. cells in BG-11 medium.

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S.E. Karatay, G. Dnmez / Applied Energy 88 (2011) 36323635

2. Method
2.1. Cyanobacteria and cultivation conditions
Three cyanobacteria strains were used in the study, as provided
by Ankara University, Faculty of Science Laboratories from the current culture collection. Of these cyanobacteria, Synechococcus sp.
and Phormidium sp. were identied according to their morphological and physiological properties. C. aponinum was identied as in a
molecular basis using F5 AGA GTT TGA TCM TGG CTC AG, R5 TAC
GGY TAC CTT GTT ACG ACT T primers.
The cyanobacteria cells were cultured in 250 ml Erlenmeyer
asks containing 100 1 ml BG-11 fermentation medium [20].
Concentrations of nutrients in the media are; NaNO3 (17.65 mM),
K2HPO4 (0.18 mM), MgSO4  7H2O (0.30 mM), CaCl2  2H2O
(0.25 mM), Citric acid (0.029 mM), Ferric ammonium citrate
(0.030 mM), Na2-EDTA (0.003 mM), Na2CO3 (0.19 mM) and trace
elements solution (1 mL) containing H3BO3 (46 lM), Co(NO3)2  6H2O (0.17 lM), MnCl2  4H2O (9.2 lM), ZnSO4  7H2O
(0.77 lM), Na2MoO4  2H2O (1.6 lM), CuSO4  5H2O (0.32 lM).
Erlenmeyer asks were incubated under continuous illumination for 30 days in plant growth chamber. A series of batch culture
experiments were performed, in unshaken asks, illuminated by
cool white uorescent lamps emitting 12.5 wm 2 (2400 l  ) of
light intensity at 30 C [21].
2.2. Lipid accumulation assays
The total lipid of the cyanobacteria cells was estimated by
gravimetric method. The effects of initial pH on lipid accumulation
were investigated at pH values of 6, 7, 8, and 9 in the BG-11 medium for each cyanobacteria. The cyanobacteria cells were activated
in the media by transferring once before measuring cellular lipid
production and 5 ml of activated culture was inoculated into
100 ml of BG-11 media. In order to examine the effect of initial
nitrogen concentrations on lipid accumulation the cultures were
grown in BG-11 media containing 0.25, 0.5, 1.0, and 1.5 g/L NaNO3
at optimum pH values for each cyanobacteria. To see the effect of
incubation time on cellular lipid accumulation, the cyanobacteria
cells were cultivated in the media that is containing increasing
NaNO3 concentrations during 10, 15, 20, 30 days. Fatty acid proles
of microbial oils were performed by gas chromatography after cyanobacteria cells were cultured at optimum growth conditions. Each
of the experiments and measurements described below were performed in triplicate to follow the changes in the samples throughout the incubation period.
2.3. Analytical methods
The cell growth was determined by measuring the dry weight of
cyanobacteria during the incubation period. Within the incubation
process, 3 ml samples were taken daily from each ask. Samples
were centrifuged to precipitate suspended biomass at 3421  g
for 5 min. Cell growth of Synechoccoccus sp., C. aponinum and Phormidium sp. was determined by measuring dry weight of washed
biomass. The dry weight of pellets was obtained at the end of the
incubation period, by ltering the washed cultures through lter
paper, drying to a constant weight at 70 C, and weighing the biomass [22].
Absorbance measurements and centrifugation were performed
using a Shimadzu UV 2001 model spectrophotometer and Hettich
EBA12 model centrifuge, respectively. To calculate the lipid content, cells were dried to a constant weight with an oven at 70 C.
Harvested cyanobacteria cells at 4800 g for 30 min used for
extraction of lipids and biomass analysis. Extraction of lipids from

the biomass was performed to the procedure of Xu et al. [23]. Lipid

was extracted with chloroformmethanol solution.
The fatty acid methyl ester analysis was done for the lipid extracted from cyanobacterial cells. The crude lipids of the cyanobacteria that cultivated at optimum conditions solved in hexane and
transesteried to biodiesel by base catalysis with 2 N KOH dissolved in methanol. After transesterication step, 1 ll sample
was taken from the upper phase and the methylated fatty acids
were analyzed by the GC-2010 gas-chromatograph (Shimadzu, Japan). The condition of GC analysis was as follows: ame ionization
detector (FID) 240 C; column TR-CN100, 60 m  0.25 mm 
0.20 mm (Teknokroma); carrier gas N2 [24]. Fatty acid peaks were
identied against the chromotogram of a mixed fatty acid methly
ester standard (37 Comp. FAME Mix 10 mg/ml in CH2CL2; Supelco,
USA).

3. Results and discussion

The value of dry weight biomass was used in a calculation of lipid content which corresponds to the weight of wet biomass. The
lipid content of the cells was described as mg lipid amount/dry cell
weight in% [23]. The lipid accumulation properties of the three cyanobacteria strains were investigated in the BG-11 medium with pH
6, 7, 8, 9 at the end of twenty days of the incubation time. Table 1
shows the lipid contents of the cyanobacteria cells as%, lipid
weight/dry cell weight at the end of incubation time. The
Table depicts the effect of medial pH value on lipid accumulation
of the cyanobacteria cells at the end of twenty days incubation
time. The experiments were performed at pH 6, 7, 8, and 9. Synechococcus sp. showed 19.2%, 19.5% and 18.6% lipid content at pH
6, 8 and 9, respectively. At pH 7 the cyanobacteria showed its maximum lipid accumulation as 20.0%. In terms of optimum pH value
for the lipid accumulation of C. aponinum, the highest lipid content
was obtained in the medium with pH 8 as 34.0% where as lamentous cyanobacteria (Phormidium sp.) cells showed their best lipid
accumulation at pH 9 with 30.0% yield. It is seen from the results
that both of unicellular and lamentous cyanobacteria cells did
not effected from the changes in pH values signicantly when producing lipid components. Therefore microbial cell growth was also
considered for cyanobacteria strains when choosing the optimum
pH value for lipid accumulation.
Although there are many papers about eukaryotic microalgaes,
there are not any literature that are using cyanobacterial lipids as a
feedstock for biodiesel production to our knowledge. In the papers
about microalgal oils, researchers did not study the effect of different pH ranges but they studied different pH values.
In the previous papers about the effect of initial pH on the
growth and lipid accumulation rates of the microalgae, it was
shown that lipid production capacity affected by the pH values of
the culture medium, where the optimum pH values were found
to be between 4.9 and 8.3 in microalgae cell cultures [2527]. Widjaja et al. studied the lipid production capacity of Chlorella vulgaris
cells in the media containing NaNO3. The researchers found that

Table 1
pH effect on the lipid content of different cyanobacteria cells (1.5 g/L NaNO3; 2400
lux; 30 C 1; 20 days).
Cyanobacteria

Synechococcus sp.
C.aponinum
Phormidium sp.

pH
6

19.2 1.7
32.7 2.7
29.9 2.5

20.0 2.3
30.3 2.4
29.7 2.8

19.5 2.1
34.0 3.3
29.6 3.2

18.6 1.3
32.8 3.2
30.0 2.2

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S.E. Karatay, G. Dnmez / Applied Energy 88 (2011) 36323635

Table 2
The effect of different initial NaNO3 concentrations and incubation time on the lipid
accumulation of Synechococcus sp., C. aponinum, Phormidium sp. cells (2400 lux;
30 C 1; pH 7 for Synechococcus sp.; pH 8 for C. aponinum; pH 9 for Phormidium sp.).
Cyanobacteria

NaNO3 (g/L)

Incubation time (day)

15

20

30

Synechococcus sp.

0.25
0.5
1.0
1.5

42.8 3.1
32.4 3.0
31.8 2.9
30.8 2.9

44.4 3.7
27.4 2.6
31.4 2.5
22.6 1.8

27.6 2.7
42.8 3.3
27.0 2.2
20.2 1.6

C. aponinum

0.25
0.5
1.0
1.5

45.0 4.0
30.2 2.6
35.4 3.0
37.2 3.7

21.6 1.4
31.2 2.2
24.4 2.5
36.4 3.1

24.0 1.8
29.0 1.7
32.4 2.9
29.6 2.8

Phormidium sp.

0.25
0.5
1.0
1.5

38.2 3.9
33.6 3.0
37.0 3.3
35.8 3.2

24.4 2.2
27.6 2.8
24.0 2.4
33.2 2.7

34.2 3.4
26.4 2.5
24.2 2.4
28.8 2.4

the microalgae cells were grown in all the pH values between 58

ranges [28].
NaNO3 is the nitrogen source that commonly preferred for cellular lipid accumulation of microalgae cells in the literature
[26,28]. The effect of increasing NaNO3 concentrations and incubation time on cellular lipid content was summarized in Table 2. In
our study the lipid content of the cyanobacteria cells was decreased when initial NaNO3 concentrations were increased up to
1.5 g/L. The highest lipid contents were obtained in the media containing 0.25 g/L NaNO3 for each of three cyanobacteria cells. The lipid contents of Synechococcus sp. cells were close to each other
after incubation period for 15 and 20 days such as 42.8% and
44.4%, respectively. It is obviously seen in the table that the highest
lipid content was 45.0% for C. aponinum cells at the end of 15 days
in the media containing the lowest nitrogen amount. Phormidium
sp. cells did not affected from the increasing nitrogen concentrations signicantly during the incubation time for 15 days. Their lipid contents were between 33.6% and 38.2% in the media with
different initial nitrogen concentrations. However the cells showed
their maximum lipid accumulation in the media containing 0.25 g/
L NaNO3 as 38.2%.
It is known that microorganisms need to be grown in a medium
with a limiting amount of nitrogen in order to achieve high lipid
accumulation [29]. Damiani et al. found Haematococcus pluvialis
cells could reach their lipid content to 32.99% in high light intensity with nitrogen-deprivation medium whereas the total lipid
content of control cells was 15.61% [26]. In another study the
researchers found Neochloris oleoabundans microalgae could increase its lipid content from 23% to 37% in the presence of nitrogen
limitation [30]. It is reported in the literature that Chlorella sp. cells
which is commonly studied microalgae for lipid production also increase its lipid content in the media with urea limitation [31]. In
our trials it is found that nitrogen depletion in the medium causes
an increase in cellular lipid accumulation.
To investigate the effect of time on lipid accumulation, lipid
extractions of the cyanobacteria cells were performed at the 10,
15, 20, and 30 days of the incubation time. There was no sufcient
biomass for lipid extraction at 10th day of the incubation time.
However it is seen in Table 2 that, C. aponinum and Phormidium
sp. cells showed their highest lipid content at 15th day of the incubation period for all of the tested NaNO3 concentrations. On the
other hand the lipid contents of Synechococcus sp. cells were close
to each other at 15th and 20th days for the lowest NaNO3 concentration. The dry weight of the cyanobacteria cells were 0.61 g/L for
Synechococcus sp., 0.66 g/L for C. aponinum, and 0.55 g/L for Phormidium sp. at the and of the 15th incubation time in the media with
0.25 g/L NaNO3. Widjaja et al., report that C. vulgaris cells increase

Table 3
Distribution of fatty acid methyl ester yields (%) of the lipids accumulated in
Synechococcus sp., C. aponinum, Phormidium sp. grown on BG-11 medium.
Fatty acid methyl ester yields
(%)

Synechococcus
sp.

C.
aponinum

Phormidium
sp.

Caprylic acid (C10:0)

Myristic acid (C14:0)
Palmitic acid (C16:0)
Palmitoleic acid (C16:1)
Cis-10-heptadecanoic acid
(C17:1)
Stearic acid (C18:0)
Oleic acid (C18:1n9c)
Linoleic acid (C18:2n6c)
Arachidonic acid (C20:4n6)

23.8
23.8
13.7
20.0
2.9

18.3
8.3
21.9
6.6
5.6

4.1
33.0
3.8
5.3

13.2

2.6

29.4
5.0
4.8

47.6
6.2

their lipid content in the medium supplemented with limited

NaNO3. In the study the effect of incubation time was compromised since higher lipid content was obtained when the growth
was very slow due to nitrogen starvation.
Both of algae and cyanobacteria can accumulate high levels of
lipids. Although algae accumulate lipids as storage materials when
they are under stress, cyanobacteria accumulate lipids in thylakoid
membranes. It means cyanobacteria accumulate lipids with high
levels of photosynthesis and a rapid growth rate. In addition to this
cyanobacteria are appropriate for genetic manipulations much
more than eukaryotic algae [13].
Saturated compounds were 74.5%, 77.9%, and 84.7% for the lipids extracted from Synechococcus sp., C. aponinum, and Phormidium
sp. cells, respectively. The current state shows, biodiesel that is
produced from these crude lipids will have a high cetane number
and a high oxidative stability. These are important advantages
for storing biodiesel for a long period. In addition, high methyl ester yields with 16 and 18 carbon atoms were achieved by transesterication of the cellular crude lipids (Table 3). The results
indicated that each of the crude lipid of cyanobacteria cells that
are fermented in BG-11 medium can be used as a raw material
for biodiesel production.
4. Conclusions
Even though there are not any reports on the lipid accumulation
properties of the cyanobacterial cells for biodiesel production, high
lipid accumulation properties were found for the Synechococcus sp.,
C. aponinum, and Phormidium sp. in the present study. The highest
lipid contents were found as 42.8% for Synechococcus sp., as 45.0%
for C. aponinum, as 38.2% for Phormidium sp. at optimum conditions. The highest C16 and C18 methyl ester yields and saturated
compounds were obtained from Phormidium sp. cells as 90.6%
and 84.7%, respectively. The results obtained from the study shows
that the crude lipids obtained from cyanobacteria could be promising feedstock for biodiesel production.
Acknowledgments
Financial support was provided by the Research Foundation of
Ankara University Project No: 09B4240007 and the Scientic and
_
_
Technological Research Council of Turkey (TBITAK-B
IDEB).
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