J Appl Phycol (2014) 26:1399–1413
DOI 10.1007/s10811-013-0160-y
Lipid productivity and fatty acid composition-guided selection
of Chlorella strains isolated from Malaysia
for biodiesel production
Vejeysri Vello & Siew-Moi Phang & Wan-Loy Chu &
Nazia Abdul Majid & Phaik-Eem Lim & Soh-Kheang Loh
Received: 19 July 2013 / Revised and accepted: 17 September 2013 / Published online: 18 October 2013
# Springer Science+Business Media Dordrecht 2013
Abstract The need to develop biomass-based domestic pro-
duction of high-energy liquid fuels (biodiesel) for transporta-
tion can potentially be addressed by exploring microalgae
with high lipid content. Selecting the strains with adequate
oil yield and quality is of fundamental importance for a cost-
efficient biofuel feedstock production based on microalgae.
This work evaluated 29 strains of Chlorella isolated from
Malaysia as feedstock for biodiesel based on volumetric lipid
productivity and fatty acid profiles. Phylogenetic studies
based on 18S rRNA gene revealed that majority of the strains
belong to true Chlorella followed by Parachlorella . The
strains were similarly separated into two groups based on fatty
acid composition. Of the 18 true Chlorella strains, Chlorella
UMACC187 had the highest palmitic acid (C16:0) content
(71.3±4.2 % total fatty acids, TFA) followed by UMACC84
(70.1± 0.7 %TFA), UMACC283 (63.8 ±0.7 %TFA), and
UMACC001 (60.3±4.0 %TFA). Lipid productivity of the
strains at exponential phase ranged from 34.53 to
V. Vello : S.<M. Phang : P.<E. Lim
Institute of Biological Sciences, Faculty of Science,
University of Malaya, 50603 Kuala Lumpur, Malaysia
V. Vello : S.<M. Phang (*) : P.<E. Lim
Institute of Ocean and Earth Sciences (IOES), University of Malaya,
50603 Kuala Lumpur, Malaysia
e-mail: phang@um.edu.my
W.<L. Chu
International Medical University (IMU), Jalan Jalil Perkasa 19, Bukit
Jalil, 57000 Kuala Lumpur, Malaysia
N. Abdul Majid
Genetics and Molecular Biology Unit, Institute of Biological
Sciences, Faculty of Science, University of Malaya,
50603 Kuala Lumpur, Malaysia
S.<K. Loh
Malaysian Palm Oil Board, Persiaran Institusi, Bandar Baru Bangi,
Kajang 43000, Selangor, Malaysia
230.38 mg L−1 day−1, with Chlorella UMACC050 attaining
the highest lipid productivity. This study demonstrated that
Chlorella UMACC050 is a promising candidate for biodiesel
feedstock production.
Keywords Algae . Biodiesel . Biomass productivity .
Biotechnology . Chlorella . Fatty acid . Lipid
Introduction
Biodiesel from microalgae presents a sustainable alternative to
resolve the global energy and climate change crisis.
Microalgae are considered as a promising feedstock for bio-
diesel production due to their ability to grow rapidly and to
accumulate a high quantity of lipid (20–50 % of dry weight,
dw) especially triacylglycerol (TAG) and many valuable by-
products, which are excellent feeds or fertilizers (Hu et al.
2008). The use of rapeseed, soybean, sunflower, corn, palm,
and waste cooking oil, as well as animal fat, is impractical for
large-scale biodiesel production due to a large land require-
ment besides creating food security risk in poor countries
(Chisti 2007). Microalgae, however, surpass similar oil-
producing crops as they can grow in nonarable land and are
realistic to meet the global diesel needs (Brennan and Owende
2010). Furthermore, microalgae can utilize wastewater such
as rubber, palm oil, and animal manure effluents, eliminating
the use of freshwater and further removing the toxicants,
excess nutrients, and carbon dioxide generated by chemical
and agro-industries (Phang 1990; Phang et al. 2000; Phang
and Ong 1988). Despite the worldwide growing interest in
microalgae as a clean and carbon neutral energy source, the
search for the suitable indigenous strains is in its infancy in
Malaysia. Previous efforts in screening of tropical
microalgae are limited to the use as food supplements,
aquaculture feed as well as fertilizer and bioremediation
1400
agent (Chu et al. 2009; Lim et al. 2010; Mustafa et al.
2012; Vairappan and Yen 2008).
Malaysia’s natural environment and tropical climate sup-
port a diverse range of microalgae, which are yet to be fully
explored. Amongst the microalgae, Chlorella is considered as
a robust species (Dahiya 2012). Most Chlorella strains can
grow mixotrophically with short doubling times and simple
growth requirements (Heredia Arroyo et al. 2011). Chlorella
with high biomass content is a good bioenergy source to be
coproduced with biochar, which reduces carbon input
(Phukan et al. 2011). Previous studies using Chlorella strains
cultured in a thin-layer photobioreactor have demonstrated
that lipid productivity increased up to 330 mg L−1 day−1 due
to a high biomass production rate (Pribyl et al. 2012). This
indicates that most Chlorella strains are intrinsically able to
produce high biomass despite being less oleaginous compared
to other potential microalgae species such as Nannochloropsis
(Thi et al. 2011). Therefore, screening of indigenous
Chlorella strains is potentially useful for future biodiesel
production in the tropical environment.
Production of quality feedstock from microalgae demands
not only high biomass and lipid productivity, but also a
suitable fatty acid composition for conversion to biodiesel.
The aim of this study was to investigate the biomass and lipid
productivity of 29 Chlorella strains isolated from Malaysia
from the University of Malaya Algae Culture Collection
(UMACC). The phylogenetic relationship of the 29 strains
was determined based on 18S ribosomal RNA gene se-
quences. The fatty acid (FA) distribution patterns were
assessed in a phylogenetic context for their usefulness in
species selection. This would assist in predicting the FA
content of the algae if their phylogenetic relationships corre-
late with their FA distribution patterns. Furthermore, the
growth rate, biomass, and lipid productivity as well as bio-
chemical profiles were evaluated for a cost-efficient biodiesel
production.
Materials and methods
A total of 29 Malaysian microalgal strains were obtained from
the UMACC (Phang and Chu 1999). Strains and culture
media used in this work are listed in Table 1. The mixed
cultures were originally purified by serial dilution followed
by streaking on agar plates (20 g L−1 of Bacto™) to obtain
individual colonies. The cultures were then isolated onto the
agar plates or slants and kept in a culture chamber (25±1 °C
and irradiance of 8 μmol photons m−2 s−1). The individual
colonies were inoculated into Bold’s basal medium (BBM)
and Provasoli medium buffered with 3 mM HEPES (pH 6.8–
8.2). An inoculum size of 10 % of total culture volume,
standardized at an optical density at 620 nm (OD620) of 0.4
(equivalent to 0.15 mg L−1 chlorophyll a , r 2 =0.9) from
J Appl Phycol (2014) 26:1399–1413
exponential phase cultures was used. For growth studies, the
microalgae were cultured in triplicate flasks with a working
volume of 600 mL. The flasks were incubated in a
controlled environment at 25±1 °C, illuminated with cool
white fluorescent lamps (40 μmol photons m−2 s−1) on a
12:12-h light–dark cycle and supplied with 100 mL min−1
filtered ambient air.
DNA extraction, PCR amplification, sequencing,
and phylogenetic analysis
An aliquot of cultured cells (10 mL) was harvested in the mid-
to late exponential growth phase (4–6 days) by centrifugation
(6,500×g for 3 min at 4 °C) in a sterile 1.5-mL
microcentrifuge tube. Genomic DNA was extracted using
the i-genomic Plant DNA Extraction Mini Kit (iNtRON
Biotechnology, Korea) according to the manufacturer’s in-
structions and protocols. PCR amplification of the partial
18S ribosomal RNA genes was carried out using the primers
as suggested by Wu et al. (2001). The following were the
partial 18S rRNA gene primers used:
Forward—5′-GTAGTCATATGCTTGTCTC-3′
Reverse—5′-GGCTGCTGGCACCAGACTTGC-3′
Amplification of the DNA markers was performed with a
Labnet MultiGene™ Gradient Thermalcycler (Labnet, USA)
using the i-Taq™Plus DNA Polymerase Kit (iNtRON
Biotechnology, Korea). The total volume for the PCR ampli-
fication was 50 mL consisting 5 mL of i-Taqplus buffer, 5 mL
of dNTP mixture (2.5 mM each), 0.25 mM of each primer, 1.0
unit of i-TaqTM DNA polymerase, and 50 pg to 1.0 mg DNA.
The parameters were according to Wu et al. (2001) except for
the annealing temperature which was changed to 50 °C.
Amplicons were electrophoresed through a 1.0 % agarose gel
stained with SYBR®Safe DNA gel stain (Invitrogen, USA).
DNA purification was conducted using NucleoSpin® Extract
II (Macherey-Nagel, USA) gel and PCR purification kits. The
purified PCR products were sent to AIT Biotech Pte Ltd
(Singapore) for DNA sequencing. Resulting DNA electrophe-
rograms for both molecular markers were truncated and
processed via Chromas Pro V1.5 (Technelysium Pty Ltd) and
multiple sequence alignments encoded in NEXUS format were
generated using ClustalX V2.0 (Larkin et al. 2007). The phylo-
genetic tree was inferred by neighbor-joining (NJ) analysis using
PAUP* 4.0b10 (Swofford 2003). NJ bootstrap values were
estimated using 1,000 replicates with Kimura’s two-parameter
model of substitution (K2P distance) evolution model.
Growth rate determination
Growth was monitored every day based on OD620nm and
chlorophyll a (Chla) concentration. The Chla concentration
was determined at 665, 645, and 630 nm using a UV–vis
J Appl Phycol (2014) 26:1399–1413 1401

Table 1 List of the Chlorella

strains used in this study and their Strain Medium Origin
origin
UMACC 001 BBM Pond at IPSP Farm, University of Malaya
UMACC 003 BBM Fish tank containing chicken manure, IPSP Farm, University of Malaya
UMACC 006 BBM Fish tank containing chicken manure, IPSP Farm, University of Malaya
UMACC 014 BBM IPSP Farm, University of Malaya
UMACC 017 BBM IPSP Farm, University of Malaya
UMACC 050 BBM Aerobic pond for POME treatment, Tenamaran Palm Oil Mill, Selangor
UMACC 051 BBM Aerobic pond for POME treatment, Tenamaran Palm Oil Mill, Selangor
UMACC 055 BBM Tire tracks at the Tenamaran Palm Oil Mill, Batang Berjuntai, Selangor
UMACC 084 BBM Digested POME, enriched with goat dung, IPSP Farm, University of Malaya
UMACC 087 BBM Digested POME, enriched with goat dung, IPSP Farm, University of Malaya
UMACC 094 BBM Tenamaran Palm Oil Factory, Selangor
UMACC 104 BBM Muddy water of Sementa Mangrove, Selangor
UMACC 177 BBM Plastic container, Kuantan Pahang
UMACC 187 BBM Tin, Chinese graveyard, Kuantan Pahang
UMACC 191 BBM Earthen jar, Johor Bahru
UMACC 208 BBM Concrete tank, shop houses, Kedah
UMACC 245 Prov Seawater from Terengganu
UMACC 252 Prov Sea Bass Pond at Sepang, Selangor
UMACC 253 Prov Sea Bass Pond at Sepang, Selangor
UMACC 254 Prov Sea Bass Pond at Sepang, Selangor
UMACC 255 Prov Sea Bass Pond at Sepang, Selangor
UMACC 256 Prov Sea Bass Pond at Sepang, Selangor
UMACC 257 Prov Sea Bass Pond at Sepang, Selangor
UMACC 258 Prov Sea Bass Pond at Sepang, Selangor
UMACC 261 Prov Sea Bass Pond at Sepang, Selangor
UMACC 268 BBM Raw palm oil effluent pond, Labu Palm Oil Mill, Negeri Sembilan
UMACC 283 BBM Anaerobic pond 3, Labu Palm Oil Mill, Negeri Sembilan
UMACC 322 BBM Wastewater treatment pond at oil refinery, Negeri Sembilan
BBM Bold’s basal medium, Prov UMACC 325 BBM Wastewater treatment pond at oil refinery, Negeri Sembilan
Provasoli medium

spectrophotometer (Shimadzu UV1700, Japan) after an over-
night extraction with acetone of the filtered and homogenized
cells using 0.45 μm filters (Whatman GF/C,) using the for-
mula by Strickland and Parsons (1968).
.
Chla ¼ ðA  volume of acetone in mLÞ volume of sample in mL
where A =11.6 (OD665nm)−1.31(OD645nm)−0.14 (OD630nm).
As Chla concentration was measured for all samples, specific
growth rate (μ, day−1) based on this parameter was calculated
using the following formula:

 . .
μ day−1 ¼ Ln N 2 N 1 ðt 2 −t 1 Þ
where N 2 is chlorophyll content (mg mL−1) at t 2, N 1 is
chlorophyll content (mg mL−1) at t 1, and t 2 and t 1 are times
within the exponential phase. Cells were harvested between
late exponential to early stationary to determine dry weight,
lipid content, and fatty acid compositions.
Determination of biomass and biochemical composition
The samples were harvested and centrifuged at 1,200×g for
10 minutes. The supernatant was decanted, and the pellets
were freeze-dried at −51 °C, 10×10−4 atm. Freeze-dried sam-
ples were kept in the desiccator overnight to a constant weight.
Freeze-dried samples were ground using a mortar and pestle to
extract lipids. Lipids were extracted in MeOH–CHCl3–H2O
(2:1:0.8 v/v) and determined by gravimetric method (Bligh
and Dyer 1959). Proteins were extracted in 0.5 N NaOH
(80 °C, 20 min) (Teoh et al. 2004), and the concentration
was determined by the dye-binding method at 595 nm
(Bradford 1976). Carbohydrates were extracted in 2 N HCl
(80 °C, 1 h), and the concentration was determined by the
phenol–sulfuric acid reaction at 485 nm (DuBois et al. 1956).
1402
Transesterification of lipid and gas chromatography analysis
The lipids were transesterified in 1.2 % HCl in MeOH, toluene,
and water (100 °C, 1 h) (Ichihara and Fukubayashi 2010). The
extracted fatty acid methyl esters (FAME) were stored in an
inert atmosphere (N2) in a freezer at −18 °C. The composition
of FAME in the samples was analyzed using the Agilent 7820A
GC equipped with a capillary column (SLB-IL100, 30 m×
0.25 mm×0.20 μm, Supelco, USA) and a flame ionization
detector (FID), with temperatures of the injector and detector
set at 250 and 260 °C, respectively. The following thermal
program was used: 140 °C for 5 min, then increasing
8 °C min−1 up to 180 °C, following an increase of 5 °C min−1
up to 260 °C. Helium was used as carrier gas at 4.41 mL min−1.
Flow of hydrogen gas and purified air were provided at 30 and
450 mL min−1, respectively. The injections were performed in
duplicate for each extraction in a volume of 1 μL. The FAME
were identified by comparing their retention times with those of
authentic standards (Sigma-Aldrich®, USA). The quantification
of fatty acids, expressed in milligram per gram of lipids, was
performed by adding an internal standard (C7:0 Sigma®, USA).
Data analysis
Total lipid content (L c), protein content (P c), and carbohydrate
content (C c) are reported as percentage of the total biomass (in
% dry weight). The biomass productivity (P dw) is expressed
as the dry biomass produced (in g L−1 day−1), at the end of
exponential growth phase: P dw (g L−1 day−1)=biomass den-
sity (g L−1)×specific growth rate (day−1).
Volumetric productivities of lipids (P L), protein (P P), and
fatty acids (P FA) were calculated according to the equation:
P f(mg L− 1day− 1)=P dw ×C f.
where P f is productivity of lipids, protein, and fatty acids, and
C f is the final contents of lipid, protein, carbohydrate, and fatty
acids and were given as percent dry weight. All determinations
were made in triplicate and were given as average value with
standard deviation. For each detected FA, its percentage of the
total FA content of a strain was used as variable. For the inves-
tigation of the general profile of fatty acid distribution, canonical
variate analysis (CVA) was used. The percentages of dissimilarity
between group pairs were investigated by conducting SIMPER
analysis. For linear trendlines, coefficients of determination (r 2)
were determined. All statistical analyses and graphical visualiza-
tions were conducted using PAST version 2.17 software package.
Results
Strain identification and phylogenetic characterization
The microalgae strains were isolated from water samples
originated from diverse aquatic habitats especially from
J Appl Phycol (2014) 26:1399–1413
wastewater sources in Malaysia. These include samples from
waste ponds containing chicken and goat manure, as well as
treatment ponds of raw and digested palm oil mill effluent
(Table 1). Uniclonal cultures were obtained through con-
tinuous streaking onto agar plates and finally into liquid
media. Based on the microscopic observation, strains
which belonged to the genus Chlorella with morphologi-
cal characteristics of being spherical or broad oval cells,
with one pyrenoid, solitary or in mucilage-covered colo-
nies as described by Luo et al. (2010) were selected for
further analysis.
In order to specify and confirm the identity of the
Chlorella strains used in our experiments, a partial 18S
region of the ribosomal RNA gene was amplified by
PCR and sequenced. The truncated sequences comprise
537 bp of the 18S rRNA gene sequence. According to
BLAST searches, the best hits represented sequences
attributed to the class Trebouxiophyceae with level of
sequence similarity reaching up to 99 % of identical
positions. To characterize the diversity of the 29
microalgae strains and their relationship with other spe-
cies, the sequences obtained from this study were phy-
logenetically analyzed. The neighbor-joining phylogenet-
ic tree (Fig. 1) showed that all the Chlorella strains in
this study grouped within the class Trebouxiophyceae.
The phylogenetic tree of the Chlorella strains based on 18S
rRNA gene sequences was supported moderately to high with
the major clades of Trebouxiophyceae in neighbor-joining
(NJ) analyses (Fig. 1). The Chlorella strains were found to
be distributed mainly in the true Chlorella clade, which com-
prised Chlorella vulgaris Beijerinck, Chlorella lobophora
Andreyeva, Chlorella sorokiniana Shihira et Krauss, and
Chlorella variabilis Shihira et Krauss. The true Chlorella
clade contained 18 strains found in a clade with a bootstrap
value of 87 % NJ. Ten strains belonged to the Parachlorella
clade, which was supported by a high bootstrap value of 99 %
NJ. Strain UMACC191 was found to be clustered within the
Heveochlorella/Heterochlorella (Watanabea) clade. The des-
ignation of each strain into its particular group will be used
throughout the paper.
Fatty acid distribution
The FA profiles of the microalgae screened are shown in
Table 2. Palmitic acid (C16:0) followed by the α-linolenic
acid (C18:3n3), stearic acid (C18:0), and linoleic acid (C18:2)
were the predominant fatty acids in most of the algal lipid
extracts. The highest percentage of C16:0 was obtained with
the true Chlorella strains UMACC187, UMACC84,
UMACC283, and UMACC001 (71.3±4.2, 70.1±0.7, 63.8±
0.7, and 60.3±4.0 % total fatty acids (TFA), respectively).
Strains with highest C18:3 were ‘Chlorella’ sp. UMACC191,
followed by Parachlorella sp. UMACC257 and UMACC261
J Appl Phycol (2014) 26:1399–1413 1403

Fig. 1 Molecular phylogeny of the Chlorella strains based on SSU neighbor joining (using the K2P model, 1,000 replicates) are marked on the
18SrRNA sequence comparisons. The phylogenetic tree shown was inferred branching the tree. The strains in bold are sequences of authentic strains
using the neighbor-joining method, based on a data set of 537 aligned obtained from GenBank [National Center for Biotechnology Information
positions of 83 taxa using PAUP 4.0b10. Bootstrap values (>50 %) of the (NCBI)]

(26.9±1.5, 26.5±2.4, and 25.2±0.2 % TFA, respectively). On UMACC325, and UMACC177 (35.1±2.4, 31.9±3.1, and
the other hand, highest C18:0 was produced by UMACC268, 29.9±2.6 % TFA, respectively).
 1404

Table 2 Fatty acid profiles (% total fatty acid) in microalgae oils

Species Local strains Saturated fatty acids; SFA Monounsaturated fatty acids; Polyunsaturated fatty acids; PUFA
MUFA

12:0 14:0 16:0 18:0 20:0 22:0 16:1 18:1 20:1n9 16: 2 16:3 18:2 18:3n3 18:3n6 20:4n6 20:5n3 22:6n3

Parachlorella sp. UMACC017 0.0 1.4 42.0b, c 2.1 0.0 0.0 0.4 37.7a 0.0 1.9 0.0 10.6a, b, c 3.9 0.0 0.0 0.0 0.0
UMACC245 0.3a, b 3.4 27.5 19.2b, c 0.0 1.7a, b, c 2.4a, b, c 5.9b, c 8.6b, c 0.7 26.1a 0.9 2.2 0.1c 0.4 0.2 0.4a
UMACC252 0.2c 6.1b, c 29.0 16.7b, c 0.0 0.7b, c 4.6a, b, c 1.7 0.0 0.0 11.9b, c 5.5c 19.2a, b, c 0.0 4.3b, c 0.0 0.0
UMACC253 0.3c 4.9c 27.3 19.1a, b 0.1b, c 1.0a,b, c 4.3a, b, c 2.1 0.0 0.1 16.7a, b 4.6 17.6a, b, c 0.0 1.8 0.0 0.0
UMACC254 0.2c 2.0 29.6 11.3c 0.0 0.8b, c 5.4a, b, c 4.4b, c 0.1 0.5 12.4b, c 7.8a, b, c 25.0a, b 0.1c 0.4 0.0 0.0
UMACC255 0.0 9.6a 41.6b, c 5.6 0.0 0.0 1.8c 1.6b, c 0.0 1.5 0.4 15.8a, b, c 21.7a, b, c 0.4c 0.0 0.0 0.0
UMACC256 0.3b, c 7.2a, b, c 28.1 16.9b, c 0.3b, c 1.0a, b, c 4.9a, b, c 1.0 0.0 0.0 16.4a, b 3.8 15.1a, b, c 0.0 4.8b 0.1 0.0
UMACC257 0.0 1.8 16.7 12.8c 6.1a 0.1 2.7b, c 9.2b 4.9b, c 0.0 1.5 13.8a, b, c 26.9a, b 2.2a 1.3 0.0 0.0
UMACC258 0.2c 5.3c 27.1 13.3c 0.2b, c 0.8b, c 4.5a, b, c 0.8 0.0 0.0 11.3b, c 8.8a, b, c 22.2a, b, c 0.0 5.4a 0.0 0.0
UMACC261 0.2c 2.7 28.9 13.0c 0.0 0.6c 6.7a, b 0.8 0.0 0.0 11.8b, c 5.4b, c 25.2a, b 0.0 4.7b, c 0.0 0.0
Chlorella sp. UMACC001 0.0 1.6 60.3a, b, c 6.3 0.0 0.0 0.5 0.1 0.0 3.3 1.3 17.1a, b, c 9.6c 0.0 0.0 0.0 0.0
UMACC006 0.2c 1.9 22.0 10.4 0.0 2.2a, b, c 1.8 0.0 18.3a 3.1 14.5a, b 7.7a, b, c 16.9a, b, c 0.1c 0.1 0.2 0.5a
UMACC050 0.0 1.6 38.2c 9.3 5.9a 0.0 0.5 6.8b, c 2.2b, c 7.5b, c 1.0 18.7a, b 6.0 1.5a, b 0.8 0.0 0.0
UMACC051 0.0 1.6 62.4a, b, c 6.3 0.0 0.0 0.3 0.0 0.0 2.4 1.3 16.3a, b, c 8.7 0.0 0.7 0.0 0.0
UMACC084 0.0 1.7 70.1a 4.9 0.0 0.0 0.1 0.0 0.0 2.3 0.9 14.9a, b, c 5.1 0.0 0.0 0.0 0.0
UMACC094 0.0 3.2 52.9a, b, c 10.2 0.0 0.0 0.0 0.7 0.0 5.4b, c 2.9 14.4a, b, c 10.2c 0.0 0.0 0.0 0.0
UMACC208 0.3c 3.0 26.9 13.7b, c 0.0 2.9a 2.5a, b, c 1.7b, c 16.5a 1.7 15.8a, b 0.0 14.2a, b, c 0.0 0.4 0.0 0.3a, b
UMACC268 0.8a 4.6c 34.5 35.1a 0.2b, c 2.6a, b 7.1a 0.4 0.0 0.7 6.4c 4.0 3.5 0.0 0.0 0.0 0.0
UMACC283 0.0 1.6 63.8a, b 5.2 0.0 0.0 0.0 1.4c 0.0 4.2c 1.1 15.9a, b, c 6.9 0.0 0.0 0.0 0.0
UMACC322 0.0 1.9 56.3a, b, c 8.5 0.0 0.0 1.1 1.4c 0.0 2.6 1.7 17.0a, b, c 9.0 0.0 0.4 0.0 0.0
UMACC325 0.1 2.2 29.3 31.9a 0.0 0.5 1.6 0.0 15.1a 1.9 3.3 1.2 11.0c 0.0 1.7c 0.1 0.2a, b, c
UMACC003 0.0 2.7 49.6a, b, c 5.3 0.5b, c 0.4 1.1 0.0 0.0 5.4b, c 0.8 19.6a 12.7a, b, c 0.0 1.8b, c 0.0 0.0
UMACC014 0.0 1.1 26.3 9.2 6.4a 0.0 1.1 3.7b, c 7.2a, b 14.2b 1.0 16.0a, b, c 13.1a, b, c 0.6b, c 0.1 0.0 0.0
UMACC055 0.0 1.5 58.9a, b, c 4.7 0.0 0.0 0.6 0.8 0.0 2.0 0.9 17.8a, b, c 12.3a, b, c 0.1c 0.4b, c 0.0 0.0
UMACC104 0.0 1.8 38.3c 6.4 6.9a 0.0 0.5 3.7b, c 0.1 10.4b, c 1.2 22.5a 6.5 1.4a, b 0.3 0.0 0.0
UMACC087 0.0 9.2a, b 28.9 5.2 0.0 0.0 0.5 0.0 0.0 25.5a 0.0 14.9a, b, c 15.4a, b, c 0.0 0.5 0.0 0.0
UMACC177 0.0 2.4 23.1 29.9a 1.0b 0.0 2.7a, b, c 3.2 8.8a, b 11.9b, c 0.0 3.8 12.2b, c 0.6b, c 0.4b, c 0.0 0.0
UMACC187 0.0 2.2 71.3a 2.6 0.0 0.0 0.6 0.0 0.0 1.9 0.0 15.6a, b, c 5.8 0.0 0.0 0.0 0.0
‘Chlorella’ sp.a UMACC191 0.1 3.4 29.4 11.8c 0.0 0.6c 2.3 1.8b, c 0.0 0.3 9.4b, c 9.5a, b, c 26.5a 0.0 5.0a 0.0 0.0

The data with the same lowercase letters in the same line mean there is no significant difference
a
‘Chlorella’ sp. belongs to the Watanabea clade
J Appl Phycol (2014) 26:1399–1413
J Appl Phycol (2014) 26:1399–1413 1405

The TFA contents (Table 3) varied from 5.8 ± 0.6
(UMACC087) to 44.0±3.1 mg g−1 (UMACC017) of total
dry biomass. The mean total TFA content of all the algae
was 25.4 ± 13.4 mg g −1 . The true Chlorella strains
UMACC283 and UMACC051 produced 70.6±0.9 and 70.3
±0.2 % of TFA, respectively, which were composed mainly of
saturated fatty acids (SFA). In comparison, UMACC17 pro-
duced the highest amount of monounsaturated FA (MUFA)
(38.1±1.8 % TFA) particularly C18:1. The highest percent-
ages of polyunsaturated fatty acids (PUFA) were observed for
UMACC087 and UMACC191 (56.3±3.3, and 50.6±2.4 %
TFA, respectively). For most Chlorella strains, the combina-
tion of SFA and MUFA made up 60 % of TFA. The highest
values for the sum of SFA and MUFA, however, were ob-
served for UMACC268 and UMACC017 (85.3 and 83.6 % 0f
TFA).
Analysis of FA distribution patterns in phylogenetic context
The 29 Chlorella strains were compared with each other based
on 18S rRNA gene phylogenies, which were represented by the
true Chlorella species, members of the Parachlorella clade as
well as more distantly related strains, i.e., from the Watanabea
clade. The ordination, which resulted from CVA, pointed out a
difference between members of the true Chlorella clade (left in
Fig. 2) and the strains of the Parachlorella clade (right in

Table 3 The distribution of saturated (SFA), monounsaturated (MUFA), and polyunsaturated (PUFA) fatty acids, and total fatty acid contents in the
microalgae dry biomass. Data are presented as mean ± standard deviation, n =3 (ANOVA, Tukey HSD test, p <0.05)

Species Local strains % TFA TFA (mg g−1 biomass)

SFA MUFA PUFA

Parachlorella sp. UMACC017 45.4±1.3c 38.1±1.8a 16.4±3.1 44.0±3.1a

UMACC245 52.1±1.6 16.9±2.3b, c 31.0±7.0 25.8±2.8
UMACC252 52.7±4.8 6.4±0.7b, c 41.0±5.5b, c 11.1±1.0
UMACC253 52.7±7.0c 6.4±1.7b, c 40.8±6.4b, c 36.9±2.5b, c
UMACC254 43.9±2.3 9.9±2.1b, c 46.2±2.2a, b, c 39.8±1.4a, b, c
UMACC255 56.8±2.3b, c 3.4±2.7 39.8±0.4b, c 19.0±1.3
UMACC256 53.8±1.8 6.0±1.6b, c 40.2±3.0b, c 5.8±0.9
UMACC257 37.5±9.9 16.8±6.0b, c 45.8±6.8a, b, c 28.3±0.9
UMACC258 46.9±0.8 5.3±0.7c 47.8±1.5a, b, c 11.9±0.5
UMACC261 45.4±4.5 7.5±0.6b, c 47.1±5.1a, b, c 21.2±1.3
Chlorella sp. UMACC001 68.2±3.1a, b, c 0.6±0.2 31.2±2.9 40.6±1.4a, b, c
UMACC006 36.8±4.3 20.1±3.8a, b 43.1±2.9b, c 17.5±0.4
UMACC050 54.9±0.6c 9.6±0.4b, c 35.5±0.9c 39.5±3.2a, b, c
UMACC051 70.3±0.2a, b, c 0.3±0.3 29.4±0.5 42.9±3.9a, b
UMACC084 76.7±0.2a 0.1±0.2 23.2±0.0 37.3±2.4b, c
UMACC094 66.4±4.8a, b, c 0.7±1.2 33.0±5.9 37.5±4.1a, b, c
UMACC208 56.5±8.1b, c 14.6±3.0b, c 28.9±5.2 9.8±1.0
UMACC268 77.8±1.0a 7.5±2.0b, c 14.7±1.1 12.3±1.5
UMACC283 70.6±0.9a, b 1.4±1.2 28.0±0.3 42.2±2.3a, b
UMACC322 66.7±3.3a, b, c 2.5±0.8 30.7±3.2 9.3±1.3
UMACC325 63.9±4.1a, b, c 16.6±5.3b, c 19.4±5.6 9.7±0.5
UMACC003 58.6±1.5b, c 1.1±0.2 40.3±1.3b, c 35.6±2.7c
UMACC014 42.9±3.0 12.0±0.1b, c 45.0±3.0a, b, c 34.5±0.9c
UMACC055 65.1±2.9a, b, c 1.4±0.6 33.5±3.5 37.0±2.6b, c
UMACC104 53.4±6.8 4.4±1.8 42.2±7.4b, c 17.0±2.6
UMACC087 43.2±3.8c 0.5±0.8 56.3±3.3a 5.8±0.6
UMACC177 46.8±2.3 20.7±0.9a, b 32.5±1.5 10.9±0.9
UMACC187 76.1±3.9a 0.6±0.7 23.3±3.4 23.0±1.1
‘Chlorella’ sp.a UMACC191 45.3±5.0 4.1±3.5 50.6±2.4a, b 16.0±2.4

The data with the same lowercase letters in the same column mean there is no significant difference
The data without any lowercase letters are significantly lower (p <0.05) than those with lowercase letters
a
‘Chlorella’ sp. belongs to Watanabea clade
1406
Fig. 2). The Watanabea clade was found to be more related to
the Parachlorella clade in terms of total fatty acid pattern.
On the other hand, the two groups were found significantly
distinct from each other based on analysis of significance (NP-
MANOVA and ANOSIM; p <0.001). Following SIMPER anal-
ysis, the observed dissimilarity between the true Chlorella spp.
and the Parachlorella spp. was 42.83 %. The first canonical variate
(CV1) involved 87.41 % of all possible differences among the three
groups, thus the possible variant between this axis and FAs.
The distribution of C16:3 and C18:3n3 was mostly found
to be correlated with Parachlorella spp. followed by that of
C14:0, C16:1, and C18:1. The C16:0 followed by C18:2 and
C16:2 could be used to discriminate the true Chlorella and its
closely related species from the Parachlorella spp. The dis-
tribution of C20:4n6 was exclusively correlated with the
second canonical variate, however, with more influence on
the Watanabea clade ‘Chlorella’ sp. strain.
Growth kinetics, productivities, and biochemical composition
The 29 algal strains tested attained stationary phase after 4 to
8 days (Fig. 3). All the strains achieved exponential phase by day
2. The Parachlorella strains, UMACC256, UMACC252,
UMACC257, UMACC254, and UMACC245, showed the
highest specific growth rates, μ (0.75±0.08, 0.69±0.05, 0.68±
0.04, 0.65±0.09, and 0.64±0.05 day−1) and biomass productiv-
ities (above 0.30 g L−1 day−1), although their lipid contents were
not significantly different (p >0.05) (Table 4). Although the
5 the other indigenous strains in this study (Fig. 5). On average,
4
3 20:4n6 18:0
2
CV2 (12.59%)
16:1
1 18:2 16:3
20:0
0 18:3n3
18:1
14:0
- nearly four times the total protein (30.0 to 63.6 %) compared
16:2
-2 20:1n9
-3
-4
-5
16:0
-6
-6 - -4 -3 -2 -1 0 1 2 3
CV1 (87.41%)
Fig. 2 Comparison of fatty acid patterns of multiple strains of true Chlorella
(filled circles) with more distantly related Parachlorella sp. (filled squares);
Chlorella-like green algae of the Watanabea-clades (filled inverted trian-
gles) (canonical variate analysis; both vectors correspond to fatty acids
correlated with both canonical axes, biological replicates were used, n =3)
J Appl Phycol (2014) 26:1399–1413
biomass of these strains was low, they produced high amounts
of protein and carbohydrate (≥40 % dw and ≥20 % dw, respec-
tively). On the other hand, Parachlorella UMACC017 produced
the highest amount of lipid (49.18±3.20 % dw) with μ of 0.50±
0.01 day−1 and biomass productivity of 0.41±0.01 g L−1 day−1.
The protein content of this strain was the lowest (23.8±1.9 %
dw) with moderate amount of carbohydrate (13.4±0.1 % dw).
The strains grouped within the Parachlorella clade attained
relatively high specific growth rate and shorter exponential
phase, which lasted for 2 days compared to other groups. Upon
reaching the end of the exponential phase, the biomass produced
by those strains was also among the highest (0.31 g L−1 day−1).
Nevertheless, UMACC050, which grouped within the true
Chlorella, showed the highest P dw (0.60 g L−1 day−1) with fairly
high lipid content (38.18±2.88 % dw) compared to all other
strains. This strain produced moderate amounts of protein (35.4±
1.0 %) and carbohydrate (15.0±0.7 % dw). For the other strains,
biomass productivity varied between 0.09 and 0.52 g L−1 day−1
(Table 1). The top biomass producers in the present study did not
correspond to the top lipid producers, which is in agreement with
the fact that biomass productivity did not correlate (r 2 =0.0588;
p ≤0.05) with lipid content (in % dw) (Fig. 4a). However, there
was a correlation between μ and biomass productivity (r 2 =
0.4203; p ≤0.05) as in Fig. 4b.
The Chlorella strains isolated from the treatment pond of
palm oil mill effluent and hatchery attained the highest lipid
and fatty acid productivities. The lipid and fatty productivities
of the two strains Chlorella UMACC50 and Parachlorella
UMACC017 differed significantly (p ≤ 0.05; n =3) from all
the protein content was highest, followed by lipid content
while carbohydrate remained the lowest biochemical constit-
uent among the Chlorella strains (Fig. 6). The protein con-
tents ranged from 30.0±3.8 % dw (UMACC017) to 63.6±
3.8 % dw (UMACC258). Lipid content was at least more than
19.5±2.6 % dw (UMACC256). The carbohydrate content was
not more than 24.8±7.9 % dw (UMACC252). The majority of
species (80 %) contained only <15 % carbohydrate but had
with other species. In addition, 20 % of the strains showed
carbohydrate contents between 15 and 25 %, with nearly twice
the total protein (30.7 to 55.0 %) as compared to the majority.
As expected, the protein productivity correlated well with
biomass productivity (r 2 =0.7784; p ≤0.05) (Fig. 4c).
Discussion
4 5 6
Chlorella taxonomy and fatty acid distribution for species
selection
The proper designation of the wild strains taxonomically will
be useful for biodiesel production as it can lead to genetic
J Appl Phycol (2014) 26:1399–1413 1407

Fig. 3 Semi-logarithmic growth

curves of 29 microalgal strains
phylogenetically grouped
cultivated in batch cultures at 25
°C under irradiance of 40 μmol
m−2 s−1 on a 12:12 h light-dark
cycle and supplied with 100 mL
min−1 of ambient air for 12 days.
The different stages of growth are
indicated by LA Lag phase, LO,
Logarithmic phase, LL Late
logarithmic phase, and
s Stationary phase. a, b
Parachlorella sp. c, d Chlorella
sp. e Chlorella sp. and
‘Chlorella’ sp. (Watanabea
clade). Experiments were carried
out in triplicate

mapping of the high- and low-lipid-yielding taxonomic groups.
The high- and low-yielding groups are logical candidates for
the identification of species-specific (e.g., lipid and fatty acid
profile) biofuel yield-enhancing genes existing from the natural
variations. The taxonomic grouping itself may provide clues
toward the detection of additional high-yielding strains from a
specific environment that exhibit similar genetic fingerprints as
the high-yielding strains identified here. The identification of
such genetic markers arising from the high-yielding wild strain
might be logical candidates for translating biodiesel yield-
enhancing traits between microalgae feedstock species such
as Chlamydomonas, Nannochloropsis and Chlorella.
Morphological comparisons of these strains with other
described microalgae suggested that these strains belonged
to the genus Chlorella. According to Luo et al. (2010), the
Chlorellaceae clade is characterized by a single chloroplast
1408 J Appl Phycol (2014) 26:1399–1413

Table 4 Growth kinetics and biomass productivity of microalgae strains separated into taxonomical groups based on 18S rRNA. Data are presented as
mean ± standard deviation, n =3 (ANOVA, Tukey HSD test, p <0.05)

Species Local strains Specific growth rate (μ, day−1) Biomass density (g L−1) Biomass productivity (Pdw; g L¯1 day¯1)

Parachlorella sp. UMACC017 0.50±0.01c 0.81±0.11a, b, c 0.41±0.01a, b, c

UMACC245 0.64±0.05a, b, c 0.32±0.08 0.22±0.00
UMACC252 0.69±0.05a, b, c 0.67±0.05b, c 0.46±0.00a, b, c
UMACC253 0.39±0.04 0.29±0.04 0.11±0.00
UMACC254 0.65±0.09a, b, c 0.54±0.10c 0.34±0.01b, c
UMACC255 0.31±0.08 0.80±0.14a, b, c 0.24±0.01
UMACC256 0.75±0.08a, b 0.71±0.11b, c 0.54±0.01a, b
UMACC257 0.68±0.04a, b, c 0.35±0.11 0.21±0.01
UMACC258 0.58±0.09a, b, c 0.71±0.08b, c 0.42±0.01a, b, c
UMACC261 0.51±0.08c 0.23±0.03 0.12±0.00
Chlorella sp. UMACC001 0.42±0.03 0.30±0.03 0.13±0.00
UMACC006 0.53±0.02b, c 0.52±0.11c 0.27±0.01c
UMACC050 0.52±0.05b, c 1.15±0.02a 0.60±0.01a
UMACC051 0.39±0.11 0.30±0.14 0.13±0.01
UMACC084 0.43±0.09 0.48±0.09c 0.21±0.01
UMACC094 0.30±0.06 0.68±0.05b, c 0.20±0.00
UMACC208 0.48±0.04c 0.67±0.11b, c 0.32±0.01b, c
UMACC268 0.58±0.08a, b, c 0.46±0.11c 0.27±0.01c
UMACC283 0.53±0.04b, c 0.49±0.08c 0.26±0.01
UMACC322 0.49±0.02c 1.01±0.11a, b 0.49±0.01a, b, c
UMACC325 0.51±0.10c 0.70±0.14b, c 0.36±0.01b, c
UMACC003 0.35±0.07 0.25±0.01 0.09±0.00
UMACC014 0.56±0.03b, c 0.77±0.05a, b, c 0.43±0.00a, b, c
UMACC055 0.41±0.04 0.22±0.08 0.09±0.00
UMACC104 0.53±0.05b, c 0.48±0.15c 0.26±0.01
UMACC087 0.35±0.06 0.52±0.01c 0.18±0.01
UMACC177 0.42±0.04 0.56±0.13c 0.24±0.01
UMACC187 0.46±0.06c 0.80±0.13a, b, c 0.37±0.01
a
‘Chlorella’ sp. UMACC191 0.27±0.09 0.41±0.11 0.10±0.00

The data with the same lowercase letters in the same column mean there is no significant difference
The data without any lowercase letters are significantly lower (p <0.05) than those with lowercase letters
a
‘Chlorella’ sp. belongs to Watanabea clade

with a pyrenoid. The pyrenoid is covered by a starch envelope
and traversed by thylakoid membranes. The true Chlorella is
represented by coccoid, solitary algae without any mucilage or
ornamentation like spines or bristles. Morphologically, the
true Chlorella is differentiated by Parachlorella by the pres-
ence of mucilaginous envelopes and synthesize autospore cell
wall during the late cell stage (Yamamoto et al. 2005). The
Watanabea clade, on the other hand, was described to accom-
modate ellipsoidal cells with unequal size of autospores and
distinctive parietal pyrenoid structures (Hanagata et al. 1998).
However, proper identification of these wild strains based on
morphological discrimination alone was inadequate due to
varying degree of phenotypic plasticity across the cell stage.
Therefore, the highly conserved molecular markers such as 18S
rRNA gene sequences are especially useful to identify unknown
Chlorella strains and to determine their phylogenetic relation-
ship between closely related species from Chlorellaceae.
Species and strains formerly assigned to the single genus
Chlorella were shown to be actually distributed into several
genera by rRNA gene sequence analyses. The type species of
Chlorella (C. vulgaris SAG211-11b) and Parachlorella
(Parachlorella beijerinckii SAG2046) were reported to be
distinguishable by 35 nucleotide substitutions within the l8S
rRNA gene (Krienitz et al. 2004). The Watanabea clade was
found to include intron S516 in the 18S rRNA gene, which
phylogenetically separates it from other Chlorella-like strains
(Neustupa et al. 2009).
In addition to DNA sequences, FA composition has also
been suggested as a biomarker to distinguish closely related
microalgae at least at the generic level. For example, Chlorella
J Appl Phycol (2014) 26:1399–1413 1409

a b
0.8 0.8

Biomass productivity (g L-1 day -1)

Biomass productivity (g L-1 day-1) 0.7 0.7

0.6 0.6

0.5 0.5

0.4 0.4

0.3 0.3

0.2 0.2

0.1 0.1

0.0 0.0
0 20 40 60 0.0 0.2 0.4 0.6 0.8 1.0
Lipid content (% dw) Specific growth rate (µ day -1)
c
0.8
Biomass productivity (g L-1 day -1)

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0.0
0 100 200 300 400 500
Protein productivity (mg L-1 day -1)
Fig. 4 Correlation of biomass productivity with lipid content (r 2 =0.0588), biomass productivity with specific growth rate (r 2 =0.4203), and biomass
productivity with protein productivity (r 2 =0.7784) in 29 microalgae Chlorella strains. Biological triplicates were used, n =3

minutissima which is rich in EPA was reclassified as with respect to taxonomical difference. The CVA ordination
Nannochloropsis sp. based on its fatty acid profile (abundant plot of FA distribution patterns revealed a tendency among the
in EPA) and pigmentation (Gladu et al. 1995). Using fatty acid strains to be distributed into two clusters, i.e., one cluster of
markers as a guide, the intrinsic characteristics pertaining to strains of the true Chlorella was clearly separated from another
biodiesel such as FA distribution can be assessed in wild strains cluster containing mainly strains of the Parachlorella based on

Fig. 5 Lipid productivity (filled 250.0 50.0

circles) and productivity of fatty 45.0 Fatty acid productivity (mg L-1 day -1)
Lipid productivity (mg L-1 day -1)

acids (open circles) of 29

screened Chlorella strains 200.0 40.0
35.0
150.0 30.0
25.0
100.0 20.0
15.0
50.0 10.0
5.0
0.0 0.0
UMACC256
UMACC017
UMACC245
UMACC252
UMACC253
UMACC254
UMACC255

UMACC257
UMACC258
UMACC261
UMACC001
UMACC006
UMACC050
UMACC051
UMACC084
UMACC094
UMACC208
UMACC268
UMACC283
UMACC322
UMACC325
UMACC003
UMACC014
UMACC055
UMACC104
UMACC087
UMACC177
UMACC187
UMACC191
1410 J Appl Phycol (2014) 26:1399–1413

UMACC191 Diacylglycerol (DAG), which is the substrate for

UMACC187 monogalactosyldiacylglycerol (MGDG), the main component
UMACC177 of galactolipids, exclusively contains C16:0 acids.
UMACC087 Galactolipids are especially essential as building blocks for
UMACC104 thylakoids, which aid in photosynthesis and growth
UMACC055
(Nakamura and Ohta 2010). The higher biomass productivity
UMACC014
possessed by the Chlorella as compared to the Parachlorella
UMACC003
can be explained by the presence of a higher reserve of C16:0
UMACC325
for C16:3 production. Thus, it can be concluded that the true
UMACC322
Chlorella have more active prokaryotic pathway as compared
UMACC283
UMACC268
to the Parachlorella. The eukaryotic pathway, which involves
UMACC208 C18:1, is incorporated into MGDG and further desaturated to
UMACC094 C18:3 in the cytoplasm and chloroplast, also present in green
UMACC084 algae. However, it is less active as it is only favorable for
UMACC051 evolution into land plants (Nakamura and Ohta 2010).
UMACC050 Given the tendency of the true Chlorella to produce a
UMACC006 higher amount of saturated fatty acids coupled with higher
UMACC001 biomass productivity, this group of strains is therefore more
UMACC261 suitable for production of quality biodiesel. Moreover, true
UMACC258 Chlorella contains a relatively low level of unsaturated lipids;
UMACC257 the ratio of total unsaturated to saturated FAs was between
UMACC256 0.28 (UMACC268) and 1.72 (UMACC006). In comparison,
UMACC255 higher ratios were found in other strains of Chlorella: 2.11 in
UMACC254 C. vulgaris CCALA 256 (Pribyl et al. 2012), 2.23–3.17 in C.
UMACC253 minutissima UTEX 2241 (Tang et al. 2011), and 2.54 in C.
UMACC252 vulgaris CCTCC M 209256 (Zheng et al. 2011), which were
UMACC245
all cultivated under photoautotrophic condition. According to
UMACC017
Knothe et al. (2003), a good-quality biodiesel has a high
0.0 20.0 40.0 60.0 80.0 100.0 cetane number, which is associated with saturated fatty acids
Content (% dw) such as C16:0 and C18:0 while, a low cetane number was
Lipid (Lc) Protein (Pc) Carbohydrate (Cc) observed with more highly unsaturated fatty acids such as
Fig. 6 Biochemical composition of 29 Chlorella strains as percent dry
C16:3 and C18:3. As opposed to fuels composed mainly of
weight (dw). Data are presented as mean ± standard deviation, n =3 polyunsaturated FAME, the high proportion of saturated FA in
the Chlorella strains may provide increased energy yield,
superior oxidative stability, and higher cetane numbers that
C16:0 followed by C18:2 and C16:2. Linoleic acid (C18:2) cause less problems in fuel polymerization during combustion
was found to clearly separate Trebouxiophyceae from other (Canakci and Sanli 2008). The biodiesel also has to be low in
major algal classes, though the use of this fatty acid as a oxidative potential while retaining good cold-flow characteris-
chemotaxonomic marker is yet to be elucidated (Lang et al. tics. This can be achieved by mixing fatty acids C16:1, C18:1,
2011). In general, the true Chlorella strains produced higher and C14:0 in the ratio 5:4:1 (Schenk et al. 2008). However, this
content of C16:0 and C18:2 compared to Parachlorella, which “ideal mix” is not practical as both C16:1 and C14:0 are not
had lower content of C14:0, C16:1, C18:0, and C18:1. In the common in most microalgae oils. Cha et al. (2011) has suggested
Parachlorella , C16:3 and C18:3 were predominant at the blending two (binary) or even more different microalgae oils,
expense of C16:0. In general, the true Chlorella had more i.e., ternary and so forth, to attain an optimum fatty acid profile
saturated fatty acids while Parachlorella had higher polyun- for biodiesel production. On the other hand, the oils rich in
saturated fatty acids. PUFA have very good cold-flow properties, although they are
Green algal species are postulated to use a prokaryotic susceptible to oxidation (Monyem and Van Gerpen 2001). The
pathway for the synthesis of chloroplast lipids, being similar Parachlorella strains also contain a significant amount of PUFA,
to a group of higher plants designated as C16:3 plants in terms which are mainly linoleic acid (LA, C18:2) and α-linolenic acid
of the cooperation of prokaryotic and eukaryotic pathways to (ALA, C18:3n3). These fatty acids are highly valued for human
synthesize chloroplast lipids (Sato et al. 2003). In the prokary- nutrition and food additives (Petkov and Garcia 2007). The ALA
otic pathway, which is entirely chloroplastic, C16 fatty acids of Parachlorella can be extracted before the rest of the oil can be
are desaturated up to C16:3 for galactolipid synthesis. converted to biodiesel in order to comply with the biodiesel
J Appl Phycol (2014) 26:1399–1413
standard on the PUFA ratio suggested by the International
Biodiesel Standard for Vehicles (EN14214) (European
Committee for Standardization CEN 2013). This makes the
biodiesel less susceptible to oxidation in storage.
Lipid and fatty acid productivities as selective criteria
The large-scale cultivation of microalgae for biofuel produc-
tion is dependent on growth rate and oil content (in % dw)
(Griffiths and Harrison 2009). Both lipid and biomass pro-
duction are energy-demanding process and may be mutually
exclusive. The lower growth rate and/or small cell size con-
tributes to lower lipid productivity, even when the lipid con-
tent is high (Rodolfi et al. 2009). Generally, cell proliferation
affects specific growth rate, which has to complement high
lipid contents to result in high lipid productivity. The accumu-
lation of lipids is commonly observed during the onset of the
stationary phase when most of the growth nutrients and light
availability become limited. During this period, cell division
slows down due to a decrease in chlorophyll a content when
exogenous nitrogen is exhausted. As a result, the primary
carbon source, acetate, was no longer converted to cell build-
ing blocks by the glyoxylate cycle and gluconeogenesis but
funneled directly into fatty acid biosynthesis to produce triac-
ylglycerols or hydrocarbon lipids (Miller et al. 2010).
The lipid productivity should be considered as the most
appropriate factor to facilitate species selection for biodiesel
(Huerlimann et al. 2010). Lipid productivity is correlated well
with biomass productivity, which substantiates its usefulness
as a suitable indicator for biodiesel production. Therefore,
high-lipid-producing strains should be primarily tested for
lipid yield per unit volume or area. In general, species from
the class Trebouxiophyceae, especially from the family
Chlorellaceae, have been cited in the literature as promising
for biofuel production, mainly because they are fast growing
and produce substantial amounts of lipid. The specific growth
rate of Chlorella UMACC was high, 0.31±0.08 to 0.75±
0.08 day−1 within 6 to 8 days of cultivation (Table 4, Fig. 3).
The biomass yield of Chlorella UMACC50 was up to 1.15±
0.02 g L−1 after 6 days of cultivation. The results on biomass
productivity, which ranged from 0.1 to 0.6 g L−1 day−1 in the
present study, are in agreement with previous findings by
Rodolfi et al. (2009) (0.25 to 0.31 g L−1 day−1) and Hempel
et al. (2012) (0.20 to 0.49 g L−1 day−1) for batch cultures under
controlled conditions. Higher values using autotrophic culti-
vation (up to 12 g L−1) were reported in C. vulgaris supplied
with 2 % of CO2 and under constant light intensity of
500 μmol photons m−2 s−1 (Pribyl et al. 2012), which explains
the large difference in production capacity. The lipid content
of Chlorella was reported to be between 23 and 60 % of DW
depending on the strain used, which is similar to findings in
this study (Pribyl et al. 2012; Feng et al. 2011; Zhou et al.
2013; Tang et al. 2011).
1411
The lipid productivity of 55 microalgae species grown in the
laboratory was reported to range from 97 to 160 mg L−1 day−1
(Griffiths and Harrison 2009). Strains grouped within Chlorella
and Parachlorella showed a similar range of lipid productivity
(140.8 and 136.2 mg L−1 day−1, respectively) with one strain,
Chlorella UMACC50, with the highest lipid productivity of
230.38±3.64 mg L−1 day−1 showing it to be a promising
candidate for biofuel production. Recently, other photoautotro-
phic lipid productions under laboratory conditions were report-
ed for C. minutissima UTEX 221, with a lipid productivity of
0.155 g L−1 day−1 (Tang et al. 2011), C. vulgaris FACHB1068
with lipid productivity of 0.147 g L−1 day−1 (Feng et al. 2011),
and C. vulgaris SAG 211/11B with lipid productivity of
0.079 g L−1 day−1 (Talebi et al. 2013). All these values were,
however, markedly lower than described for Chlorella
UMACC50. Chlorella and Parachlorella have been shown to
have high practicable suitability for biodiesel production using
photobioreactors. Pribyl et al. (2012) reported that Chlorella sp.
achieved higher lipid productivity (576 mg L−1 day−1) com-
pared to Parachlorella sp. grown outdoors using
photobioreactors. The true Chlorella strains were reported to
be most resistant to heat and high light intensity (Morita et al.
2000). These innate characteristics enable these strains to
achieve high photosynthetic productivity leading to higher bio-
mass production. The intrinsic ability of the Chlorella strains to
produce high biomass and fatty acids suggests that it is best
utilized to overproduce oils under nutrient depletion after a rapid
production of biomass in a two-stage cultivation system
(Praveenkumar et al. 2012). A high coefficient of r 2 =0.7037
was also estimated for correlation between lipid and fatty acid
productivity (data not shown). This estimation is important
when the direct transesterification process is applied in large-
scale biodiesel production. Direct transesterification is rapid and
low cost because the biodiesel is produced directly from cells
without the need for oil extraction (D’oca et al. 2011).
Protein contents are the major organic constituents in
Chlorella. According to Renaud et al. (1994), the amount of
protein in Chlorophyceae ranges from 20 to 67 % dw, similar
to the findings of the present study. Microalgae with high
protein production and biomass productivity will be favorable
for coproduction of food and feed supplements. According to
Lammens et al. (2012), residual algal biomass after lipid
extraction can be used to enhance the nutritional value of
conventional food and livestock feed preparations.
According to Becker (1994) and Safi et al. (2013), protein
from Chlorella is highly valued because it constitutes many
essential amino acids which are an added advantage in bio-
diesel production. Carbohydrate residues resulting from the
extraction process can be fermented for bioethanol and biogas
production (Doan et al. 2012; Harun et al. 2010). Biogas from
anaerobic digestion of algal biomass can be integrated into the
biodiesel production system to reduce the overall cost of
production per unit of biodiesel (Harun et al. 2011).
1412
In conclusion, this study characterized 29 microalgae strains
taxonomically and compared them according to their biomass
and lipid productivities. The strains were separated into three
distinct taxonomical groups based on the 18S rRNA genes
namely true Chlorella, Parachlorella and Chlorella-like green
algae of the Watanabea-clades. Major taxonomic groups were
also separated into two clusters based on fatty acid distribution.
Overall, the true Chlorella group tends to produce saturated
fatty acids with carbon chain lengths of C16 and C18 com-
pared to the Parachlorella group, which produced more
PUFA. The top biomass producers did not correspond to the
top lipid producers. The total lipid per dry biomass varied
between 20.9±4.0 % dw (UMACC322) and 49.2±3.2 % dw
(UMACC017). Lipid productivity was from 34.53±6.90
to 230.38±3.64 mg L−1 day−1. The highest lipid pro-
ductivities were observed for Chlorella UMACC050
(230.38 ± 3.64 mg L − 1 day − 1 ) and Parachlorella
UMACC017 (198.98±3.4 mg L−1 day−1), which differed
significantly (p ≤ 0.05) from all the other indigenous strains
tested in this study. The most promising strain is Chlorella
UMACC050. The significant finding of this study is that fatty
acid composition is correlated to taxonomic grouping.
Therefore, correct identification of a strain is crucial for
selecting good biodiesel feedstocks.
Acknowledgments The first author is grateful to the University of
Malaya Bright Scheme Program for the financial support given. Special
thanks to the collaborator from the National University of Singapore
(NUS), Prof. Dr. Chew Fook Tim, for technical support and advice. The
project was funded by the Postgraduate Research Fund (Ref: PS301/
2010B and PV032/2011B) and Malaysian Palm Oil Board (MPOB,
Ref: 55-02-03-1054).
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