Characterization of microalgal species isolated from fresh water bodies as a

potential source for biodiesel production

Reda A.I. Abou-Shanab
a,b
, Jae-Hoon Hwang
a
, Yunchul Cho
c
, Booki Min
d
, Byong-Hun Jeon
a,
a
Department of Environmental Engineering, Yonsei University, Wonju, Gangwon-do 220-710, South Korea
b
Department of Environmental Biotechnology, Mubarak City for Scientic Research and Technology Applications, New Borg El Arab City 21934, Alexandria, Egypt
c
Graduate School of Green Environment and Energy, Kwangwoon University, Seoul 130-701, South Korea
d
Department of Environmental Science and Engineering, Kyung Hee University, Yongin-Si, Gyeonggi-Do 446-701, South Korea
a r t i c l e i n f o
Article history:
Received 9 November 2010
Received in revised form 22 January 2011
Accepted 27 January 2011
Available online 22 February 2011
Keywords:
Microalgae
Fresh water
Biodiesel
rRNA
Fatty acids
a b s t r a c t
Due to increasing oil prices and climate change concerns, biodiesel has gained attention as an alternative
energy source. Biodiesel derived from microalgae is a potentially renewable and carbonneutral alterna-
tive to petroleum fuels. One of the most important decisions in obtaining oil from microalgae is the
choice of algal species to use. Eight microalgae from a total of 33 isolated cultures were selected based
on their morphology and ease of cultivation. Five cultures were isolated from river and identied as
strains of Scenedesmus obliquus YSR01, Nitzschia cf. pusilla YSR02, Chlorella ellipsoidea YSR03, S. obliquus
YSR04, and S. obliquus YSR05, and three were isolated from wastewater and identied as S. obliquus
YSW06, Micractinium pusillum YSW07, and Ourococcus multisporus YSW08, based on LSU rDNA (D1-D2)
and ITS sequence analyses. S. obliquus YSR01 reached a growth rate of 1.68 0.28 day
1
at 680
nm
and
a biomass concentration of 1.57 0.67 g dwt L
1
, with a high lipid content of 58 1.5%. Under similar
environmental conditions, M. pusillum reached a growth rate of 2.3 0.55 day
1
and a biomass concen-
tration of 2.28 0.16 g dwt L
1
, with a relatively low lipid content of 24 0.5% w/w. The fatty acid com-
positions of the studied species were mainly myristic, palmitic, palmitoleic, oleic, linoleic, g-linolenic, and
linolenic acids. Our results suggest that S. obliquus YSR01 can be a possible candidate species for produc-
ing oils for biodiesel, based on its high lipid and oleic acid contents.
2011 Elsevier Ltd. All rights reserved.
1. Introduction
Current energy demand is mostly fullled by conventional en-
ergy resources, such as coal, petroleum, and natural gas. Petro-
leum-based fuels have limited reserves and are concentrated in
certain regions of the world. According to many analysts, at the
present staggering rates of consumption, the world fossil oil re-
serves will be exhausted in less than 50 years. The scarcity of
known petroleum reserves makes renewable energy resources
more attractive [1]. The most feasible way to meet the growing de-
mand for energy is by utilizing alternative fuels. An alternative fuel
to petrodiesel must be technically feasible, economically competi-
tive, environmentally acceptable, and easily available [2]. One such
fuel that exhibits great potential is biofuel, in particular, biodiesel
[3]. Biofuels are generally considered to have many benets,
including sustainability, reduction of greenhouse gas emissions, re-
gional development, social structure, agriculture and security of
supply [4]. Usage of biodiesel will allow for a balance to be
achieved between agriculture, economic development and the
environment [5].
Extensive studies have been conducted on using vegetable oils
as diesel fuel. The focus has mainly been on oils, like soybean, rape-
seed, sunower and safower oils [2], which are essentially edible
in nature. Most recently, research efforts have been aimed at iden-
tifying suitable biomass species which can provide high-energy
outputs to replace conventional fossil fuels [6]. However, few at-
tempts have been made to produce biodiesel from non-edible
sources, like used frying oil, greases, tallow, lard, jatropha, and ma-
hua oils [710]. Nevertheless, the cost of biodiesel production is
still a major obstacle for large-scale commercial exploitation,
mainly due to the high feed cost of vegetable oils [2].
Microalgae have been suggested as a potential feedstock for fuel
production because of the number of advantages, including higher
photosynthetic efciency, higher biomass production, and higher
growth rates, as compared to other energy crops [11]. Microalgae
represent an exceptionally diverse but highly specialized group
of microorganisms adapted to various ecological habitats. Many
microalgae have the ability to produce substantial amounts (e.g.,
2050% dry cell weight) of triacylglycerols (TAG) as a storage lipid
under photo-oxidative stress or other adverse environmental
0306-2619/$ - see front matter 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.apenergy.2011.01.060

Corresponding author. Tel.: +82 33 760 2446; fax: +82 33 760 2571.
E-mail address: bhjeon@yonsei.ac.kr (B.-H. Jeon).
Applied Energy 88 (2011) 33003306
Contents lists available at ScienceDirect
Applied Energy
j our nal homepage: www. el sevi er. com/ l ocat e/ apenergy
conditions. Microalgae with high oil productivities are desired for
producing biodiesel. Depending on the species, microalgae produce
many different kinds of lipids, hydrocarbons, and other complex
oils [12,13]. Not all algal oils are adequate for making biodiesel,
but suitable oils commonly occur. In this study, the algal growth
rate, biomass production, lipid content, and productivity of micro-
algal cultures isolated from livestock wastewater and a river were
determined. Furthermore, the fatty acid composition of these iso-
lates was investigated. In addition, the identication of the micro-
algal species was conrmed by investigating the partial sequence
of the (LSU) rDNA (D1-D2) and ITS genes.
2. Materials and methods
2.1. Isolation and identication of microalgae
Water samples for microalgae isolation were collected asepti-
cally from sites that appeared to contain algal growth in a fresh
water river (YSR) and livestock wastewater treatment plant
(YSW) at Wonju, South Korea. The physicochemical characteristics
of the livestock wastewater were as follows (units in mgL
1
):
chemical oxygen demand (COD) 845, total nitrogen (TN) 1180, to-
tal phosphorus (TP) 4, chloride (Cl

) 593, sulfate (SO

2
4
) 86, nitrate
(NO

3
) 2.6, and nitrite (NO

2
) 174. The pH of the livestock wastewa-
ter was 8.2. Ten mL of water samples were transferred to a 500 mL
conical ask containing 200 mL of sterilized Bolds Basal Medium
(BBM) [14] and then incubated on a rotary shaker at 27 C and
150 rpm under continuous illumination using white uorescent
light at intensities of 40 lmol m
2
s
1
for three weeks.
Every two days, the asks were examined for algal growth using
an optical microscope, with serial dilutions being made in BBM
from asks showing growth. Subcultures were made by inoculat-
ing 50 lL culture solution onto Petri plates containing BBM solidi-
ed with 1.5% (w/v) of bacteriological agar. Further, 50 lL aliquots
of the same dilution were placed in the wells of 96 microtiter
plates containing 200 lL BBM. These procedures were repeated
for each of the original asks. Both the Petri and microtiter plates
were incubated at 27 C under continuous illumination for two
weeks. The purity of the culture was conrmed by repeated plating
and by regular observation under a microscope. The microscopic
identication was done using botanical approaches [15] and con-
rmed using molecular markers.
2.2. DNA extraction, PCR amplication and sequencing
An aliquot of cultured cells (1 mL) was harvested in the mid- to
late-exponential phase by centrifugation (13,000g for 3 min at
4 C) in a sterile microcentrifuge tube. The genomic DNA was ex-
tracted using a Plant Genomic DNA extraction kit (SolGent,
Daejeon, South Korea), according to the manufacturers instructions
and protocols. Concentrations of DNA were measured at 260 nm
using a spectrophotometer, and DNA was amplied using a T-Gra-
dient thermocycler (Biometra GmbH, Gottingen, Germany). The
universal eukaryotic primers ITS1 (5
0
-TCCGTAGGTGAACCTGCGG-3
0
as the forward primer) and ITS4 (5
0
-TCCTCCGCTTATTGATATGC-3
0
as the reverse primer) were used to amplify the ITS14 fragments
of the ribosome with template DNA originating from the microal-
gal isolates using the PCR protocol described by Ferrer et al. [16].
For the amplication of the D1-D2 rRNA region, the primers fw1
(5
0
-AGCGGAGGAAAAGAAACTA-3
0
) and rev1 (5
0
-TACTAGAAGGTT
CG-ATTAGTC-3
0
) were used in combination with the PCR protocol
described by Sonnenberg et al. [17]. Sub-samples (10 lL) of the
reaction mixtures were analyzed using 1% horizontal agarose gel
electrophoresis to conrm the presence of product. The PCR prod-
ucts were puried using the Gel PCR Clean-Up System (Applied
Biosystems, Foster City, CA, USA). Sequencing reactions were per-
formed using a Dye Deoxy Terminator Cycle Sequencing Ready
Reaction Kit (Applied Biosystems), and sequencing fragments were
analyzed with an ABI Prism 377 DNA Sequencer.
2.3. Phylogenetic analysis
A search of GenBank with BLAST [18] was used to identify algal
species with rRNA gene sequences downloaded from the database.
Aligned sequences were checked manually and edited with Gene-
doc [19]. Sequences containing fewer than 200 nucleotides or in
excess of 1000 nucleotides were removed, and sequences not
belonging to unicellular green microalgal species were also ex-
cluded from this study. The phylogenetic tree was constructed
using the neighbor-joining (NJ) Kimuras two-parameter algo-
rithm, as implemented within the MEGA4 program package [20].
2.4. Cultivation and microalgal growth analyses
Microalgal suspension in BBM was adjusted to an absorbance of
0.05 at an OD of 680
nm
. Ten mL of each microalgal species was used
as initial algal inoculums. The microalgae were cultivated at 27 C
in 250 mL conical ask photobioreactors containing a working vol-
ume (100 mL) of BBM at 150 rpm under continuous illumination
using white uorescent light at intensities of 40 lmol m
2
s
1
for
three weeks. Algal growth was measured by daily changes in opti-
cal density at 680
nm
with a spectrophotometer (HACH

, DR/4000v,
USA), which was converted into dry cell weight. Each sample was
diluted to give an absorbance in the range of 0.11.0 if the optical
density was greater than 1.0. Microalgae dry weight per liter
(g L
1
) was measured according to the method previously reported
[21]. Microalgal cells were harvested by centrifugation and washed
twice with deionized water. Microalgal pellets were dried over-
night at 105 C for dry weight measurements [22]. Experiments
were performed in triplicate, and data are expressed as mean stan-
dard deviation (SD).
2.5. Lipid extraction and fatty acid analyses
The total lipids were extracted from the fresh microalgal bio-
mass using a slightly modied method of Bligh and Dyer [23]. In
brief, 50 mL of microalgae culture was harvested by centrifugation
at 4000 rpm, re-suspended in 1 mL distilled water, the sample was
then mixed with 1.25 mL chloroform and 2.5 mL methanol (1:2, v/
v) and subjected to sonication for 30 min at maximum power
(Power

, Sonic 420, South Korea). After sonication, the tubes were

incubated overnight at 27 C at 100 rpm. The next day, an addi-
tional portion of chloroform (1.25 mL) was added, and the extrac-
tion mixture was sonicated again for 30 min. To separate the
chloroform and aqueous methanol layers, 1.25 mL water was
added and then centrifuged at 4000 rpm for 10 min. The chloro-
form layer was gently removed from the bottom, and a second
extraction was performed by adding 2.5 mL chloroform and vor-
texing. The chloroform portions were collected and washed with
5 mL 5% NaCl solution and evaporated in an oven at 50 C to dry-
ness. Thereafter, the weight of the crude lipid obtained from each
sample was measured gravimetrically. Experiments were per-
formed in triplicate and data are expressed as mean SD.
Fatty acids were analyzed by modifying the method of Lepage
and Roy [24]. The crude lipid (10 mg) was dissolved using 2 ml
freshly prepared mixture of chloroformmethanol (2:1, v/v) and
transferred to a 10 mL Pyrex tube with a Teon-sealed screw cap.
One mL of chloroform containing nonadecanoic acid (500 g L
1
;
Sigma Chemical Co., USA) as an internal standard, and 1 mL meth-
anol and 300 lL sulfuric acid as transmethylation reagents were
added to the tube and mixed for 5 min. The tube was incubated
R.A.I. Abou-Shanab et al. / Applied Energy 88 (2011) 33003306 3301
at 100 C for 10 min and cooled to room temperature. One mL
water was added for phase separation. The tube was mixed vigor-
ously for 10 min and centrifuged at 4000 rpm for 10 min. After
phase separation, the lower phase (chloroform) was transferred
to another tube using a hypodermic disposable polypropylene syr-
inge. The organic phase was ltered using a disposable 0.22-lm
PVDF syringe lter (Millex-GV, Millipore, USA). Methyl esters of
fatty acids were analyzed with a gas chromatograph equipped with
a ame ionization detector and an HP-INNOWax capillary column
(Agilent Technologies, USA). Mix RM3, Mix RM5, GLC50, GLC70
(Supelco Co., USA), Heptadecanoic acid, and g-Linolenic acid (Sig-
ma Chemical Co.) were used as standards. Other reagents used
were of analytical grade.
3. Results and discussion
3.1. Isolation and identication of the microalgae
Microalgae are present in all existing earth ecosystems, not just
aquatic, but also terrestrial, representing a large variety of species
living in a wide range of environmental conditions [25]. It is esti-
mated that more than 50,000 species exist, but only a limited num-
ber, of around 30,000, have been studied and analyzed [26]. In this
study, a total of 33 microalgal cultures were isolated, 18 from the
river nearest to Yonsei University Campus, Wonju, South Korea,
and 15 from a livestock wastewater treatment plant located in
Wonju. Only 5 (YSR01, YSR02, YSR03, YSR04 and YSR05) and 3
(YSW06, YSW07 and YSW08) isolates of the 18 and 15 microalgal
cultures, respectively, were selected based on their purity and
growth rates. Microscopic observation of the algal isolates revealed
its colonial existence and purity (Fig. 1). The eight isolated micro-
algal cultures of YSR01, YSR02, YSR03, YSR04, YSR05, YSW06,
YSW07 and YSW08 were identied as the genera Scenedesmus,
Nitzschia, Chlorella, Scenedesmus, Scenedesmus, Scenedesmus, Micr-
actinium and Ourococcus, respectively, by morphological examina-
tion under a microscope based on cell shapes. Chinnasamy et al.
[27] found that about 27 species of green algae, 20 species of cya-
nobacteria, and 8 species of diatoms were observed in both treated
and untreated wastewaters. Among these 55 algal species, Scene-
desmus had the highest species richness, being represented by 14
species. Metzger and Largeau [13] reported that, within in each
chemical race and for the same strain, the morphology of the alga
could vary in relation to age and culture conditions. The morpho-
logical heterogeneity of the alga makes the microscopic examina-
tion difcult. Thus, in this study, the total DNA was isolated and
used for PCR amplication of the rRNA (LSU) and internal tran-
scribed spacer (ITS) as potential markers for species identication.
3.2. PCR amplication and sequence analysis
The primers used to amplify the D1-D2 LSU region successfully
amplied DNA from the YSR01, YSR02, YSR03, YSR04, YSR05,
YSW06, and YSW08 microalgal cultures. The size of the PCR prod-
ucts of LSU rDNA (D1-D2) ranged from 776 to 843-bp. While no
amplication product was detected using LSU rDNA primer pairs
with genomic DNA isolated from the YSW07 microlagal isolate, a
single band 690-bp of amplied rDNA products was obtained
from the DNA of YSW07 using ITS1ITS4 primers.
rRNA genes are good candidates for detection because they are
present in high copy numbers, and the sensitivity of their detection
S. obliquus YSR01 N. pusilla YSR02 C. ellipsoidea YSR03 S. obliquus YSR04
S. obliquus YSR05 S. obliquus YSW06 M. pusillum YSW07 O. multisporus YSW08
Fig. 1. Light microscopic (40) pictures of the tested microalgal isolates.
Table 1
The accession numbers, length base pair and the similarity between amplied sequences and the closest relative sequence for 8 strains of environmentally isolated microalgae.
Microalgal strain Accession number Length (nt
a
) Closest relative and GenBank accession number % Similarity
Scenedesmus obliquus YSR01 GU732419 811 S. obliquus AF183482 99
Nitzschia cf. pusilla YSR02 GU732414 776 N. pusilla AF417663 97
Chlorella ellipsoidea YSR03 GU732422 819 C. ellipsoidea D17810 97
Scenedesmus obliquus YSR04 HM103383 843 S. obliquus AF183482 95
Scenedesmus obliquus YSR05 GU732426 805 S. obliquus AF183482 99
Scenedesmus obliquus YSW06 GU732421 806 S. obliquus AF183482 99
Micractinium pusillum YSW07 GU732425 691 M. pusillum FM205873 99
Ourococcus multisporus YSW08 GU732424 813 O. multisporus AF277655 99
3302 R.A.I. Abou-Shanab et al. / Applied Energy 88 (2011) 33003306
may be dramatically increased by the use of nested PCR. The tran-
scriptional unit is composed of 18S, 5.8S, and 28S rRNA genes.
Intergenic transcribed spacer regions (ITS) that are not translated
into rRNA are found between the 18S and 5.8S and between the
5.8S and 28S rDNA gene subunits. Although rRNA genes are highly
conserved, the ITS regions are divergent and distinctive [28]. The
LSU rRNA gene has a higher evolutionary rate, as compared to
the SSU rRNA gene [17] and should be a better tool for the discrim-
ination of closely related species using short diagnostic sequences.
Percentage similarity values obtained after pair wise alignment
of the LSU D1-D2 sequence of isolates (YSR01, YSR02, YSR03,
YSR04, YSR05, YSW06, YSW08) and ITS1ITS4 sequence of isolate
YSW07, versus EMBL database sequences are 99%, 97%, 97%, 95%,
99%, 99%, 99% and 99%, respectively. The gene sequence
YSR03
Chlorella ellipsoidea D17810.1
Leptosira terrestris Z95378.1
Chlorella vulgaris AB237642.1
Chlorella sp. OK1-ZK AB437257.1
Chlorella sp. NC64A AB236862.1
Chlorella sp. SW1-ZK AB437244.1
Chlorella sp. NTAI01 GU186909.1
Treubaria schmidlei AF183487.1
Treubaria setigera AF183488.1
YSW06
YSR01
YSR05
Scenedesmus obliquus AF183482.1
YSR04
Ourococcus multisporus AF277655.1
YSW08
Ankistrodesmus stipitatus AF183447.1
Pediastrum tetras strain Heg 1976-29 AY779890.1
Sorastrum spinulosum strain UTEX 2452 AY779899.1
Pediastrum duplex strain SB0201 VA AY534717.1
Pediastrum sp. CL0201VA AY534711.1
Pediastrum integrum strain SAG 29.81 AY534703.1
Nitzschia cf.pusilla CCMP 560 AF417663.1
Nitzschia cf. pusilla UTEX2047 AF417662.1
Nitzschia cf. pusilla ND 73 FJ214168.1
Nitzschia cf. pusila ND 53 FJ214163.1
YSR02
Nitzschia communis M1762 AF417661.1
Nitzschia alba M1354 AF417670.1
C
h
l
o
r
o
p
h
y
t
a
B
a
c
i
l
l
a
r
i
o
p
h
y
t
a
M. pusillum CCAP 248/4 FM20586.1
M. pusillum CCAP 248/5 FM205836.1
M. pusillum CCAP 248/9 FM205869.1
M. pusillum SAG 13.81 FM205866.1
M. pusillum CCAP 248/6 FM205872.1
M. pusillum CCAP 248/15 FM205873.1
YSW07
M. pusillum CCAP 248/10 FM205870.1
M. pusillum CCAP 248/12 FM205871.1
M.pusillum CCAP 248/11 FM205867.1
C
h
l
o
r
o
p
h
y
t
a
Fig. 2. Phylogenetic trees showing the relationships among LSU rDNA D1-D2 sequences of isolates YSR01, YSR02, YSR03, YSR04, YSR05, YSW06, YSW08 and the ITS1ITS4
sequence of isolate YSW07 and the most similar sequences retrieved from databases.
R.A.I. Abou-Shanab et al. / Applied Energy 88 (2011) 33003306 3303
comparison demonstrated the afliation of isolate YSR01 to Scene-
desmus obliquus (99% identity), YSR02 to Nitzschia cf. pusilla (97%
identity), YSR03 to Chlorella ellipsoidea (97% identity), YSR04 to S.
obliquus (95% identity), YSR05 to S. obliquus (99% identity),
YSW06 to S. obliquus (99% identity), YSW07 to Micractinium pusil-
lum (99% identity) and YSW08 to Ourococcus multisporus (99% iden-
tity). The DNA sequences were published in the NCBI databases
under the specic accession numbers. The lengths of the LSU rDNA
(D1-D2) and ITS1ITS4 regions of eight species of microalgae, their
specic accession numbers and the nearest identiable match
present in the GenBank nucleotide data base are shown in Table 1.
Identication of the microalgal cultures YSR01, YSR02, YSR03,
YSR04, YSR05, YSW06, YSW08 and YSL07 is also supported by
the LSU rDNA D1-D2 and ITS1ITS4 sequence-based phylogenetic
analysis, respectively. In the phylogram (Fig. 2), analysis of the
LSU rDNA sequences ties the isolates YSR01, YSR04, YSR05 and
YSW06 clearly to S. obliquus, with the closest similarity to microal-
gal strain S. obliquus AF183482. The LSU rDNA sequence of isolates
YSR02, YSR03 and YSW08 clearly conrmed the identication as
Nitzschia cf. pusilla, Chlorella ellipsoidea, and Ourococcus multisporus
with the closest similarities to the next nearest classied Nitzschia
cf. pusilla AF417663, C. ellipsoidea D17810 and O. multisporus
AF277655, respectively (Fig. 2 and Table 1). Analysis of the ITS1
ITS4 sequence ties isolates YSW07 clearly to Micractinium pusillum,
with similarity of 99% to microalagal strain M. pusillum FM205873
(Fig. 2 and Table 1).
3.3. Growth rate of microalgal strains
Under suitable conditions and sufcient nutrients, microalgae
can grow profusely. Commonly, they double their biomass within
3.5 h or 24 h during the exponential growth phase [29]. The net
growth rate differed among the examined microalgal species
(Fig. 3a and b). Algal growth is directly affected by the availability
of nutrients, light, the stability of pH, temperature, and the initial
inoculums density [30]. Under similar environmental conditions,
the average specic growth rates of 2.35 0.55, 1.68 0.28,
1.42 0.02, 1.32 0.05, 1.06 0.03, 0.99 0.02, 1.19 0.17 and
0.51 0.14 days
1
at 680
nm
were found for M. pusillum YSW07, S.
obliquus YSR01, S. obliquus YSR04, S. obliquus YSW06, S. obliquus
YSR05, C. ellipsoidea YSR03, O. multisporus YSW08 and N. pusil-
laYSR02, respectively. The growth rates of M. pusillum YSW07
and S. obliquus YSR01 reached 4.07 0.06 and 3.02 0.57, respec-
tively, compared with 0.152 0.01 and 0.168 0.004 at an OD of
680 nm after 20 days of incubation. This result indicates that the
M. pusillum YSW07 and S. obliquus YSR01 strains were suitable
for high-density cultures.
3.4. Biomass, lipid content and lipid productivity
The eight microalgal species were tested for their lipid
production by evaluating biomass productivity and lipid content in
250-mL ask laboratory cultures after 21 days of incubation under
similar conditions (Table 2). M. pusillumYSW07 showed the highest
biomass productivity at 2.28 0.16 g dwt L
1
, while O. multisporus
YSW08 showed the lowest biomass productivity at 0.95 0.11
g dwt L
1
, when compared to the other species.
The total lipid contents for the microalgae cultured in this study
ranged from 21 1.1% to 58 1.5% of the dry weight. The lipid con-
tent from S. obliquus YSR01 was 58 1.5% of the dry weight, which
was about 2.8, 2.4, and 2.1 times higher than that from S. obliquus
YSR04, M. pusillum YSW0, and S. obliquus YSR05, respectively
(Table 2). The lipid productivity of S. obliquus YSR01 was highest at
0.91 0.36 g L
1
, when compared with the other tested microalgal
0
1
2
3
4
5
0 7 14 21
Incubation days
O
p
t
i
c
a
l

d
e
n
s
i
t
y

(
O
D

6
8
0

n
m
)

S. obliquus YSR01
N. pusilla YSR02
C. ellipsoidea YSR03
S. obliquus YSR04
S. obliquus YSR05
(A)
(B)
Fig. 3. Growth curve of eight microalgal cultures, ve were isolated from a river (A)
and three from livestock wastewater (B) cultivated in a batch experiment on a
rotary shaker at 27 C and 150 rpm under continuous illumination for 21 days.
Experiments were performed in triplicate.
Table 2
Biomass productivity, lipid content and lipid productivity of the microalgal strains.
Microalgal strain Biomass productivity (g dwt L
1
) Lipid productivity (g L
1
) Lipid content (% biomass)
S. obliquus YSR01 1.57 0.67 0.91 0.36 58 1.5
N. pusilla YSR02 1.37 0.08 0.66 0.00 48 3.1
C. ellipsoidea YSR03 1.48 0.04 0.47 0.07 32 5.9
S. obliquus YSR04 1.98 0.04 0.40 0.03 21 1.1
S. obliquus YSR05 1.75 0.34 0.47 0.05 27 1.9
S. obliquus YSW06 1.80 0.13 0.49 0.06 27 5.6
M. pusillum YSW07 2.28 0.16 0.54 0.05 24 0.5
O. multisporus YSW08 0.95 0.11 0.49 0.02 52 8.3
Experiments were performed in triplicate and data are expressed as mean SD.
3304 R.A.I. Abou-Shanab et al. / Applied Energy 88 (2011) 33003306
species (Table 2). Lipid content data for different algal species are
readily available and have been consistently reported in the litera-
ture [31]. Many microalgae species can be induced to accumulate
substantial quantities of lipids [1], contributing to a high oil yield.
In previous studies [32,33], total lipid contents representing 20
50% of the dry biomass weight were found to be quite common,
and some microalgae even exceeded 90% as a response to different
culture conditions [34]. Lipid content of freshwater microalgae was
near 20%, and the best biomass producers, Chlorococcum sp.
UMACC 112 and Scenedesmus sp. DM (0.28 and 0.26 g L
1
d
1
,
respectively), were also the best lipid producers (54 mg L
1
d
1
)
[35].
3.5. Fatty acid composition
Fatty acids in the eight strains of microalgae were primarily
esteried, and the major fatty acid compositions were determined
using GC analysis (Table 3). In the eight tested microalgae, linoleic
acid (C18:2n6c), palmitic acid (C16:0), linolenic acid (C18:3n3),
oleic acid (C18:1n9c) and g-linolenic acid (C18:3n6) were com-
monly dominant, which ranged from 11% to 40%, 18% to 33%,
10% to 33%, 2% to 26% and 5% to 17%, respectively, whereas the pal-
mitoleic acid (C16:1) and eicosatetraenoic acid (C20:4) existed as
minor fatty acids. The fatty acids Nonanoic (C9:0) and Undecanoic
(C11:0) were not detected in any of the tested microalgal species.
The most commonly synthesized fatty acids have chain lengths
that range from C16 to C18, similar to those of higher plants
[36]. Specically, the major fatty acids were C16:0 and C16:1 in
the Bacillariophyceae, and C16:0 and C18:1 in the Chlorophyceae
[37].
The total amount of fatty acid of the 8 algae ranged from 0.7 to
4.7 mg g
1
dwt. The highest amount of oleic acid (1.2 mg g
1
dwt)
and palmitic acid (1.3 mg g
1
dwt) was detected in S. obliquus
YSR01 and N. pusilla YSR02, respectively. The percentage of the
oleic acid, which is known to be a main component of biodiesel,
was 26%, 25%, and 24% of total fatty acids in O. multisporus
YSW08, S. obliquus YSR05 and S. obliquus YSR01, respectively. This
proportion was closely similar to the 24.8% of oleic acid reported
by Ranga Rao et al. [38]. Oils with high oleic acid content have been
reported to have a reasonable balance of fuel, including its ignition
quality, combustion heat, cold lter plugging point (CFPP), oxida-
tive stability, viscosity, and lubricity, which are determined by
the structure of its component fatty esters [39,40]. Therefore,
among the tested microalgal species, S. obliquus YSR01 showed
the highest oleic acid content (1.2 mg g
1
dwt), making it the most
suitable for the production of good quality biodiesel.
4. Conclusions
To nd microalgae with high biomass and lipid productivity,
eight microalgal cultures were selected based on easy cultivation
and growth rate. The highest growth rates (2.35 0.55 and
1.68 0.28 day
1
) were found for M. pusillum YSW07 and S. obli-
quus YSR01, respectively. The highest total fatty acid and lipid con-
tents (4.7 mg g
1
dwt, and 58 1.5%) were found in S. obliquus
YSR01. The composition of fatty acids in the studied species was
mainly C14:0, C16:0, C18:1n9c, C18:2n6c, C18:3n6 and C18:3n3.
The results of this study indicate that the naturally isolated micro-
alga S. obliquus YSR01 is a valuable candidate for use in oil
production.
Acknowledgements
This work was supported by the 21st Frontier research project
(Sustainable Water Resources Research Center 3-4-3), the Global
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R.A.I. Abou-Shanab et al. / Applied Energy 88 (2011) 33003306 3305
Research Laboratory project (Korea Institute of Geosciences and
Mineral Resources NP2008-019), the Senior Researchers (National
Research Foundation of Korea, 2010-0026904), and the Brain Kor-
ea-21 (BK-21) program of the Ministry of Education, Republic of
Korea.
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