Biotechnology and Bioprocess Engineering 2009, 14: 187-192

DOI/10.1007/s12257-008-0171-8

Evaluation of Extraction Methods for

Recovery of Fatty Acids from Botryococcus
braunii LB 572 and Synechocystis sp. PCC 6803
Hai-Linh Tran, Seong-Joo Hong, and Choul-Gyun Lee*
Institute of Industrial Biotechnology, Department of Biological Engineering, Inha University, Incheon 402-751, Korea

^Äëíê~Åí= Microalgae are a very diverse group of organisms that consist in both prokaryotic and eukaryotic forms. Some species of
microalgae can be induced to overproduce particular fatty acids through simple manipulations of the physical and chemical
properties of the culture medium. In this paper, the effect of different extraction techniques on the recovery of fatty acids from
the freeze-dried biomass from two lipid-producing microalgal strains: _çíêóçÅçÅÅìë= Äê~ìåáá LB 572 (green algae) and
póåÉÅÜçÅóëíáë sp. PCC 6803 (cyanobacteria) was examined. Five procedures were used: after conversion of the lipid mate-
rial into the corresponding fatty acid methyl esters (FAMEs) îá~ suitable derivatization reactions (extraction-transesterification)
and direct transesterification of biomass to produce FAMEs (without the initial extraction step) that used differential types of
catalysts and processing conditions. This study has shown that procedure 3, a one step practical procedure for lipid extrac-
tion and áå=ëáíì methyl ester derivation could be used successfully for the determination of the fatty acid compositions of mi-
croalgae and cyanobacteria. © KSBB

hÉóïçêÇëW=Ñ~ííó=~ÅáÇëI=íê~åëÉëíÉêáÑáÅ~íáçåI=ãáÅêç~äÖ~ÉI=Botryococcus braunii i_=RTOI=Synechocystis=ëéK=m``=SUMP=

INTRODUCTION Prokaryotic algae, cyanobacteria, resemble the chloroplasts

Fatty acid methyl esters originating from vegetable oils
and animal fats are known as biodiesel. Biodiesel has re-
ceived considerable attention in recent years, as it is a biode-
gradable, renewable, and non-toxic fuel [1]. Microalgae have
been suggested as very good candidates for fuel production
because of their advantages of higher photosynthetic effi-
ciency, higher biomass production, and faster growth com-
pared to other energy crops [1-3]. Moreover, microalgae are
eukaryotic photosynthetic microorganisms that can be used
to produce high value compounds as carbohydrates, hydro-
carbons, and natural oils. Microalgae appear to be the only
source of biodiesel that has the potential to completely dis-
place fossil diesel [4]. Microalgae with high oil productiv-
ities are desired for producing biodiesel [5]. Depending on
the species, microalgae produce many different kinds of lip-
ids, hydrocarbons, and other complex oils. Botryococcus
braunii, a green microalga that produces hydrocarbons
whose content can reach 85% of the dry biomass, has al-
ready been proposed as a future renewable source of fuel [6].
G`çêêÉëéçåÇáåÖ=~ìíÜçê
Tel: +82-32-860-7518 Fax: +82-32-875-0827
e-mail: leecg@inha.ac.kr
of eukaryotic plants with respect to lipid and fatty acid com-
position and membrane structure. The cyanobacterium, Synecho-
cystis PCC6803, is a transformable strain which may be re-
garded as a model system for elucidation of the role of the
desaturation of fatty acids [7].
Many biotechnology programs are oriented to the produc-
tion of special fatty acid compositions derived from microal-
gae, which can be used as sources for biodiesel production.
This type of research requires analyzing a large number of
samples; therefore, simple, rapid, and reliable methods for
fatty acid analysis are needed. Fatty acid identification and
quantification is most accurately performed using gas chro-
matography (GC), which requires an esterified sample of
fatty acids, usually the methyl esters (FAMEs). This process
increases the volatility of lipid components, thus providing a
better separation. Many FAME preparation methods require
prior extraction and saponification of the lipid fraction fol-
lowed by derivatization to the final FAMEs. Because these
require many manipulations, they are not suited for rapid
processing of a large number of samples. Some procedures
that allow direct transesterification of the sample, avoiding
the lipid fraction extraction, have also been developed [8-13].
Most analytical methods published to date have focused
on comparative analyses between existing derivation meth-
NUU=
ods, to define for a particular sample, which method en-
hances the amount of fatty acid extracted. In the present
study, the effect of various extraction methods on the fatty
acid composition and qualification of Botryococcus braunii
LB 572 (green algae), and Synechocystis sp. PCC 6803
(cyanobacteria) were examined and used as a model system
strain for comparison. After the weight of each processed
fatty acid component on the derivatization process is deter-
mined, it will be possible to choose existing methods to
measure fatty acid composition in microalgal strains, so as to
guarantee maximum extraction efficiency in one step with
ease of handling, high accuracy, and at minimum cost. From
that perspective, the characteristics of the transesterification
process in analyzing the composition of microalgal oil were
investigated.
MATERIALS AND METHODS
jáÅêç~äÖ~ä=mêÉé~ê~íáçå=
Botryococcus braunii LB 572 were obtained from the
University of Texas, USA. Stock cultures of B. braunii were
cultivated on liquid of BG11 medium in bubble column
photobioreactors. Column photobioreactors were made of
Pyrex glass tubes (650 mm in height and 35 mm of internal
diameter). Fluorescent lamps (FL20D, OSRAM, Korea)
were used as light source for their growth. Illuminated con-
tinuously at 100 μE m−2 s−1, the photoreactors were operated
at 25°C. Sterile-air containing 1% (v/v) CO2 by filtering us-
ing glassfiber was aerated into the column through an air
sparger at the bottom of the column at 0.5 v/v flow rate.
Synechocystis sp. PCC 6803 was obtained from the Pas-
teur Culture Collection of Cyanobacteria. Synechocystis sp.
PCC 6803 is unicellular, 3∼4 μm of size, and non-motile and
contains no gas vacuoles. Synechocysitis sp. PCC 6803 was
cultivated in BG-11 medium in bubble column photobiore-
actors. Column photobioreactors were made of Pyrex glass
tubes (650 mm in height and 35 mm of internal diameter).
Fluorescent lamps (FL20D, OSRAM, Korea) were used as
light source for their growth. Illuminated continuously at 40
μE m−2 s−1, the photoreactors were operated at 25°C. Sterile-
air by filtering using glassfiber was aerated into the column
through an air sparger at the bottom of the column at a flow
rate of 0.1 v/v with 100% air [14].
bñíê~Åíáçå=jÉíÜçÇë=
mêçÅÉÇìêÉ=N=E~Ç~éíÉÇ=Ñêçã=qçã=iÉïáë=Éí=~äKF=xNOz=
Freeze-dried cells were weighed in 10 mL screw-top test
tubes, to which a fresh solution of the transesterification re-
action mix (methanol/hydrochloric acid/chloroform, 10:1:1
v/v/v) was added. Cells were suspended in this solution by
vortex mixing and immediately placed at 90°C for 60 min
for transesterification. Transesterification reaction tubes were
removed from the heater and cooled to room temperature.
One milliliter of water was then added to each tube and the
fatty acids methyl esters extracted (hexane/chloroform, 4:1
v/v and 3 × 2 mL). Samples were diluted with chloroform
containing a known concentration of 19:0 (nonadecanoic
acid) as the internal injection standard and injected into the
chromatograph.
mêçÅÉÇìêÉ=O=E~Ç~éíÉÇ=Ñêçã=qçåó=oKi~êëçå=~åÇ= =
f~å=^Kdê~Ü~ãF=xNRz=
Cells were lyophilized in 2 mL screw-top vials containing
10 μg nonadecanoic acid (Sigma) as an internal standard. To
each vial, 0.5 mL 1 N HCl in methanol and 0.2 mL hexane
was added. The vials were heated at 85°C for 2 h and cooled
to room temperature. The hexane phase containing the fatty
acid methyl esters was partitioned from the aqueous phase
by the addition of 0.25 mL 0.9% KCl.
The hexane phase was transferred to Teflon-capped vials
and analyzed by gas chromatography with flame ionization
detection.
mêçÅÉÇìêÉ=P=E~Ç~éíÉÇ=Ñêçã=iÉé~ÖÉ=~åÇ=oçóI= =
ãçÇáÑáÉÇ=Äó=`çÜÉå=Éí=~äK=xVz=
Samples were dissolved in 2 mL of a freshly prepared
mixture of acetyl chloride and methanol, at a ratio of 5:100
(v/v), together with 1 mg of tricosanoic acid as an internal
standard. The reagents were placed in Teflon-capped Pyrex
tubes, and the reaction continued at 100°C for 1 h under pure
nitrogen and darkness. After cooling to 30∼40°C, 1 mL of
extracting solvent (hexane or isooctane) was added and the
FAME solvent solution was mixed in the vortex for a spe-
cific period of time (5 to 30 s). Purification of the solution
was achieved either by salting out (using 1 mL of saturated
sodium chloride solution) or washing (using 1 mL of water),
causing the formation of two immiscible phases, which were
then allowed to separate. The upper extracted solvent phase
was recovered, dried over anhydrous Na2SO4, and analyzed
using a gas chromatograph.
mêçÅÉÇìêÉ=Q=E~Ç~éíÉÇ=Ñêçã=iÉé~ÖÉ=~åÇ=oçóF=xNMz=
Samples and 5 μL 19:0 solution were placed in test tubes.
One mlliliter of freshly prepared tranesterification reagent
(methanol/acetyl chloride, 20:1 v/v) was added to each tube.
The tubes were heated at 100°C for 1 h for transmethylation,
and shaken every 10∼15 min. The mixture was cooled to
room temperature, and 1 mL each of water and hexane were
added. The tubes were then shaken and centrifuged. Two
phases were produced; the upper phase (hexane) was trans-
ferred to another tube. This operation was repeated twice, to
optimize sample lipid extraction. The hexanic phase was
dried under N2 atmosphere and FAMEs were suspended in
0.5 mL of hexane and injected into the gas chromatograph.
mêçÅÉÇìêÉ=R=E~Ç~éíÉÇ=Ñêçã=cçäÅÜI=iÉÉë=~åÇ=päç~åÉ=
pí~åäÉóF=xNSz=
The addition of 2 mL of chloroform-methanol 2:1 (v/v)
was added to each sample. The mixture was mechanically
shaken for 10 min. After centrifugation, the lower phase was
collected. Then 2 mL of chloroform-methanol 2:1 (v/v) was
added to the precipitate and the same procedure was repeated.
The lower phase was pooled and 145 mM NaCl was added

in order to separate the methanol and chloroform phase. Fol-
lowing centrifugation, the lower phase containing the lipids
was evaporated to dryness at room temperature under a gen-
tle stream of nitrogen. The residue was solubilized in a mix-
ture of 1 mL of methanol-benzene 3:2 (v/v) and 1 mL of
acetyl chloride-methanol 5:100 (v/v). The mixture was then
subjected to methanolysis at 100°C for 1 h, cooled to room
temperature and an internal standard was added. The speci-
mens were then processed and injected into the gas chro-
matograph.
d~ë=`Üêçã~íçÖê~éÜó=
Determination of fatty acid profiles was carried out using
gas chromatography equipped with an automatic injector and
a flame ionization detector (FID). Chromatographic condi-
tions were: carrier gas, helium; flow rate, 3 mL min−1; sam-
ple input temperature, 250°C; initial temperature, 110°C;
initial time, 1 minute; rate, 5°C/minute; and giving a total
heating time of 28 min.
`~äÅìä~íáçåë=~åÇ=s~äáÇ~íáçå=çÑ=íÜÉ=jÉíÜçÇ=
FAMEs were compared with those of authentic standards.
The peak area for each identified fatty acid was determined
by a computer programmed to calculate the amount of each
fatty acid recovered in each sample using the internal stan-
dard method. Fatty acid quantitation was done using non-
adecanoic acid (19:0) as an internal standard. The amount of
individual fatty acid was calculated using the expression: Ci
= Cp (Ai/Ap), where A is the chromatographic area units and
C is the amount of fatty acid. Subscript p represents the in-
ternal standard and i refers to any fatty acid.
RESULTS AND DISCUSSION
c~ííó=^ÅáÇë=mêçÑáäÉ=çÑ=_KÄê~ìåáá=
A gas chromatograph of fatty acid methyl esters of
B.braunii which were cultured in BG11 medium at 25°C
under continuous illumination is shown in Table 1. The dif-
ferent preparative steps on the recovery of fatty acids of
B.brauniii expressed as [mg/g DW] (Table 1A) and as [% of
total fatty acids] are shown in Table 1B. Only those peaks
are listed which could be identified beyond doubt. The pat-
tern of fatty acids in B.braunii is very typical for Chlorococ-
cales (Ahlgren et al., 1992) [17]: it is very rich in palmitic acid
(16:0), oleic acid (18:1n9c), and linoleic acid (18:3). As seen
in Table 1, oleic acid (C18:1n9c) was found to be the major
fatty acid recovered from all of the extractions. The next
dominating fatty acids were palmitic acid (C16:0) and linoleic
acid (C18:3). Anirban et al. (2002) reported that oleic acid
was the precursor for the n-alkadienes and the algal strains
used in the present study were reported to belong to race A [6].
Pooled data for all samples extracted by direct transesteri-
fication (procedures 1, 2, 3, and 4) was compared with ex-
traction-transesterification samples (procedure 5). The amount
Biotechnol. Bioprocess Eng. NUV
of total fatty acids extracted by direct transesterification
(procedures 1, 3, and 4) was significantly higher than those
extracted by procedure 5. However, the amount of fatty acids
extracted by procedure 2 was the least. This indicated that
the methylation’s solution significantly influenced the amount
of fatty acids extracted from the biomass, even though the
reaction time (2 h) was higher than in the other procedures.
Procedure 3 seemed to recover slightly greater quantities
than procedure 4 (121.345 and 109.118 mg/g dry cells
weight, respectively). As seen in the results from procedure
3 and procedure 4 in Table 1A, C16:0, C18:1n9c, C18:1n9t,
and C18:3 were the main constituents, while the amount of
C16:0, C18:1n9c, and C18:3 from procedure 3 was higher
than those from procedure 4. Therefore, we concluded that
one could take advantage using procedure 3 in order to carry
out many gas chromatographic analyses.
c~ííó=^ÅáÇë=mêçÑáäÉ=çÑ=póåÉÅÜçÅóëíáë=ëéK=m``SUMP=
A gas chromatograph of fatty acids methyl esters of Synecho-
cystis sp. PCC6803 which were cultured in BG11 medium at
25°C under continuous illumination is shown in Table 2. The
different preparative steps on the recovery of fatty acids of
Synechocystis sp. PCC6803 were expressed as [mg/g DW]
(Table 2A) and as [% of total fatty acids] (Table 2B).
The fatty acid composition of Synechocystis sp. PCC6803
which was extracted by various procedures is shown in Table
2. The major fatty acids were 16:0, 16:1, 18:0, 18:1n9c, and
18:1n9t. The proportion of C16:0 was greater than 43% of
the total fatty acids. These results are in agreement with
those for same cyanobacterium reported by Kenyon et al.
[18] and Hajime Wada et al. [7]. Pooled data for all samples
extracted by direct transesterification (procedures 1, 2, 3, and
4) was compared with the extraction-transesterification sam-
ples (procedure 5). The amount of total fatty acids extracted
by direct transesterification (procedures 1 and 4) was lower
than those extracted by procedure 5. Among them, fatty ac-
ids could not be determined by procedure 2. This indicated
that the methylation solution was not sufficient enough to
recover fatty acids from the biomass, even though the reac-
tion time (2 h) was higher than the other procedures. Proce-
dure 3 seemed to recover greater quantities (73.838 mg/g dry
cells weight) than the other procedures. As seen in Table 2A,
C16:0, C16:1, and C18:1n9t were the main constituents,
while the amount of C16:0, C16:1, and C18:1n9t from pro-
cedure 3 was higher than those from the other procedures.
Based on this result, the procedure 3 is more effective to
extract and analyze the fatty acids in B. braunii and
Synechocystis sp. PCC 6803.
CONCLUSION
Results from this study indicate that deviations from vari-
ous extraction methods significantly influenced the amount
of fatty acids recovered from Botryococcus braunii LB572
and Synechocystis sp. PCC6803. The amount of total fatty
acids extracted from Botryococcus braunii LB572 and
NVM=

q~ÄäÉ=NK Concentration of fatty acids (FA) per biomass related to different preparative steps of _K=Äê~ìåáá (A), relative amounts of FA [%
of total FA] (B).
^
Fatty acids _çíêóçÅçÅÅìë=Äê~ìåáá=
(mg/g DWa) Procedure 1 Procedure 2 Procedure 3 Procedure 4 Procedure 5
C6:0 0.212 ± 0.299 1.549 ± 0.018 2.158 ± 0.740 1.545 ± 1.107 n. d.b
C8:0 0.34 ± 0.088 n. d. n. d. n. d. 0.373 ± 0.205
C14:0 0.154 ± 0.004 n. d. 2.546 ± 0.096 2.637 ± 0.171 0.539 ± 0.076
C14:1 1.281 ± 0.578 n. d. 0.479 ± 0.041 0.481 ± 0.014 n. d.
C16:0 9.811 ± 0.704 0.876 ± 0.04 14.796 ± 1.277 13.202 ± 0.311 10.061 ± 1.022
C16:1 0.389 ± 0.110 n. d. 0.617 ± 0.188 0.619 ± 0.134 0.191 ± 0.135
C18:0 0.196 ± 0.012 n. d. 0.397 ± 0.018 0.359 ± 0.035 5.218 ± 0.489
C18:1n9c 22.919 ± 1.263 1.746 ± 0.061 68.375 ± 3.878 61.501 ± 0.447 12.865 ± 0.449
C18:1n9t 3.83 ± 0.283 0.214 ± 0.003 5.728 ± 0.395 5.183 ± 0.044 1.291 ± 0.023
C18:3 15.788 ± 1.113 0.791 ± 0.041 23.069 ± 1.657 20.774 ± 0.295 5.093 ± 0.309
C20:1 0.282 ± 0.025 n. d. 1.134 ± 0.043 1.077 ± 0.037 0.178 ± 0.0287
C22:0 n. d. n. d. n. d. n. d. n. d.
C22:1 n. d. n. d. 0.660 ± 0.229 0.382 ± 0.017 n. d.
C24:0 n. d. n. d. 0.576 ± 0.042 0.589 ± 0.091 n. d.
C26:0 n. d. n. d. 0.810 ± 0.055 0.769 ± 0.013 n. d.
Total FA 55.158 ± 4.481 5.176 ± 0.163 121.345 ± 8.658 109.118 ± 2.715 35.809 ± 2.737
Values are given as means, with the standard deviation of three replicates.
a
dry cell weight, bnot detected.

_
_çíêóçÅçÅÅìë=Äê~ìåáá=
% weight of total fatty acids
Procedure 1 Procedure 2 Procedure 3 Procedure 4 Procedure 5
C6:0 0.384 25.917 1.779 1.416 −
C8:0 0.615 − − − 1.042
C14:0 0.28 − 2.098 2.417 1.506
C14:1 2.32 − 0.394 0.441 −
C16:0 17.778 14.667 12.193 12.099 28.099
C16:1 0.704 − 0.508 0.568 0.533
C18:0 0.355 − 0.327 0.329 14.571
C18:1n9c 41.518 29.225 56.348 56.362 35.926
C18:1n9t 6.939 3.579 4.720 4.75 3.605
C18:3 28.60 13.231 19.011 19.038 14.222
C20:1 0.511 − 0.935 0.987 0.497
C22:0 − − 0.544 0.350 1.042
C22:1 − − 0.477 0.54 −
C24:0 − − 0.668 0.705 −
C26:0 − − 2.098 2.417 −

Synechocystis sp. PCC6803 by various extraction procedures vast majority of saturated and unsaturated fatty acids. Under
was significantly affected by the sequence in which solvents identical conditions, B. braunii gave the highest yields for fatty
were added to the samples. Our data also indicated that a acid content and for most of the individual fatty acids. This re-
reaction time of 60 min at 100°C was sufficient to extract the sult may be considered interesting in future studies.
 Biotechnol. Bioprocess Eng. NVN

q~ÄäÉ=OK= Concentration of fatty acids (FA) per biomass related to different preparative steps of póåÉÅÜçÅóëíáë sp. PCC6803 (A), relative
amounts of FA [% of total FA] (B).
^=
Fatty acids póåÉÅÜçÅóëíáë=sp. PCC6803
(mg/g DWa) Procedure 1 Procedure 2 Procedure 3 Procedure 4 Procedure 5
b
C6:0 n. d. n. d. 3.025 ± 1.048 2.782 ± 0.743 n. d.
C8:0 0.156 ± 0.111 n. d. 0.413 ± 0.164 0.877 ± 0.504 0.762 ± 0.463
C10:0 1.334 ± 0.799 n. d. 0.873 ± 0.877 0.289 ± 0.005 n. d.
C14:0 0.671 ± 0.012 n. d. 1.178 ± 0.089 n. d. n. d.
C14:1 n. d. n. d. 0.384 ± 0.272 n. d. n. d.
C16:0 36.789 ± 0.1 n. d. 43.741 ± 0.322 35.237 ± 0.981 29.605 ± 0.359
C16:1 4.862 ± 0.277 n. d. 5.822 ± 0.057 4.639 ± 0.899 2.689 ± 0.054
C18:0 2.457 ± 0.568 n. d. 1.901 ± 0.044 1.366 ± 0.633 16.11 ± 0.06
C18:1n9c 3.327 ± 0.071 n. d. 3.849 ± 0.037 2.943 ± 0.559 12.039 ± 0.019
C18:1n9t 10.597 ± 0.351 n. d. 12.321 ± 0.167 9.661 ± 1.737 6.173 ± 0.068
C20:0 0.280 ± 0.021 n. d. 0.33 ± 0.035 n. d. n. d.
Total FA 60.473 ± 3.209 n. d. 73.838 ± 3.113 57.795 ± 6.063 67.378 ± 1.023
Values are given as means, with the standard deviation of three replicates.
a
dry cell weight, bnot detected.

_=
póåÉÅÜçÅóëíáë sp. PCC6803
% weight of total fatty acids
Procedure 1 Procedure 2 Procedure 3 Procedure 4 Procedure 5
C6:0 − − 4.0970 4.8134 −
C8:0 0.2582 − 0.5596 1.517 1.131
C10:0 2.2052 − 1.1827 0.5008 −
C14:0 1.1099 − 1.5957 − −
C14:1 − − 0.5196 − −
C16:0 60.8359 − 59.2400 60.9695 43.938
C16:1 8.0397 − 7.8855 8.0268 3.991
C18:0 4.0624 − 2.5749 2.3643 23.91
C18:1n9c 5.5019 − 5.2122 5.09159 17.868
C18:1n9t 17.5235 − 16.6860 16.7167 9.162
C20:0 0.4633 − 0.4467 − −

For the two strains of microalgae and cyanobacteria exam-
ined, greater recovery of fatty acids was achieved with pro-
cedure 3 than by using the other procedures. The transesteri-
fication reaction mix used in procedure 3 contained about
5% (v/v) acetyl chloride and was highly basic which could
be expected to cause severe disruption to cell integrity. This
study has shown that Procedure 3, a one step practical pro-
cedure (adapted from Lepage and Roy, modified by Cohen
et al.) [9] for lipid extraction and in situ methyl ester deriva-
tion, could be used successfully for the determination of the
fatty acid compositions of microalgae and cyanobacteria.
^ÅâåçïäÉÇÖÉãÉåí= = This work was supported by Korea
Energy Management Corporation (KEMCO) Grant (2007-
N-BI02-P-02-0-000), for which authors are thankful.
Received August 19, 2008; accepted January 22, 2009
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