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Screening of marine microalgae for biodiesel feedstock

Thi Thai Yen Doan a,*, Balasubramanian Sivaloganathan b, Jeffrey Philip Obbard a,b
a

Division of Environmental Science and Engineering, Faculty of Engineering, National University of Singapore, E1A #02-19,
1 Engineering Drive 2, Singapore 117576, Singapore
b
Tropical Marine Science Institute, National University of Singapore, S2S, 18 Kent Ridge Road, Singapore 119227, Singapore

article info

abstract

Article history:

Biodiesel production from microalgae lipids is increasingly regarded as a more sustainable

Received 9 June 2010

and feasible alternative to conventional biodiesel feedstocks derived from terrestrial bio-

Received in revised form

energy crops. A total of ninety-six strains of marine microalgae, with an elevated biomass

5 February 2011

productivity and intracellular lipid content, were isolated from the coastal waters of

Accepted 10 February 2011

Singapore using an automated flow cytometric cell-sorting technique. Cell sorting was

Available online 24 March 2011

based on the two-dimensional distribution of algal cells for red fluorescence (representing
chlorophyll auto-fluorescence) against forward-light scatter (representing cell size) and red

Keywords:

vs. green fluorescence. Twenty-one of the strains were further characterized with respect

Microalgae

to cell growth rate, biomass concentration, lipid content (total and neutral lipid) and fatty

Biodiesel

acid profile. The growth rates of Skeletonema costatum, Chaetoceros and Thalassiosira species

Flow cytometric cell sorting

were greatest among the entire strains, but in terms of absolute lipid yield Nannochloropsis

Screening

strains predominated. Nannochloropsis strains had a lipid content ranging from 39.4% to

Neutral lipid

44.9% of dry weight biomass. Transesterification of the lipids yielded 25e51% of fatty acid
methyl ester (FAME) i.e. biodiesel, where total FAME content ranged between 11 and 21% of
dry weight biomass. This study describes the microalgae screening process and demonstrates that Nannochloropsis is a promising species for biodiesel feedstock.
2011 Elsevier Ltd. All rights reserved.

1.

Introduction

Biodiesel, as derived from microalgae lipids, has become

a focal area of contemporary, global biotechnological research
as it holds the potential to provide a scalable, low-carbon,
renewable feedstock without adversely affecting the supply of
food and other crop related products [1]. The most productive
terrestrial bioenergy crops, including palm and soybean oil, do
not match the potential high productivity of microalgae [2,3].
Development of a microalgae-based biodiesel technology for
biodiesel production has the potential to dramatically
improve our common environment and move the world
beyond a petroleum-based economy. However, significant
challenges must be surmounted, including the effective and
rapid isolation of microalgae strains that have a high intrinsic
lipid content and biomass yield.

The island state of Singapore lies 180 km north of the equator

and has a year-round tropical climate with temperatures
ranging from 25
C to 32
C, and an annual precipitation rate in
excess of 2500 mm (Yearbook of Statistic Singapore, 2008).
Singapores coastal seas have a warm ambient temperature
(approximately 30
C) and contain a high biodiversity of marine
life, including microalgae. This biodiversity represents a valuable resource for the development of an indigenous biodiesel
feedstock in the form of microalgae lipid. Isolation of suitable
microalgae from the natural environment is the first critical
step in developing oil-rich strains for further exploitation in
engineered systems for the production of biodiesel feedstock.
High-throughput cell sorting, coupled with flow cytometry, is
a powerful tool to facilitate the rapid and efficient isolation of
microalgae strains for onward axenic culture. Microalgae have
different photosynthetic pigments which emit various auto-

* Corresponding author. Jalan Tiga, Blk 49, #04-58, Singapore 390049, Singapore. Tel.: 65 63007516; fax: 65 67744202.
E-mail address: doan_yen@nus.edu.sg (T.T.Y. Doan).
0961-9534/$ e see front matter 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biombioe.2011.02.021

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fluorescence. These characteristics of microalgae have been

applied in flow cytomertry to identify algae [4]. Isolation of
microalgae from natural waters using flow cytometric cell sorting has been previously reported by Reckermann [5] and Crosbie
et al. [6], where chlorophyll was used as a fluorescent probe to
distinguish between different strains of microalgae. Reckermann [5] and Sensen et al. [7] used the chlorophyll auto-fluorescence (CAF) properties of eukaryotic phytoplankton, diatoms
and picoautotrophic cells for isolation of axenic cultures,
whereas Crosbie et al. [6] used both red and orange auto-fluorescence to differentiate between algal strains. Green autofluorescence (GAF) is also considered to be a valuable taxonomic
characteristic of dinoflagellates [8], where GAF is common in
both autotrophic and heterotrophic dinoflagellate species.
Flow cytometry, in conjunction with cell sorting, also
represents an invaluable tool for screening and exploiting
microalgae strains for biodiesel feedstock development. In
this study, the GAF and CAF properties of local marine
microalgae were subjected to a cell-sorting protocol to
differentiate and isolate microalgae strains, which were then
subsequently screened for high biomass productivity and
elevated levels of intracellular lipids.

2.

Instrumentations and methods

2.1.

Instrumentations

A Becton Dickinson (Mountain View, Calif.) FACSVantage SE

flow cytometer fitted with a cell-sorter and a 100 mW Argon ion
laser (Spectra Physics, Darmstadt, Germany) and a 100 mm
nozzle was used for experimentation. For single-cell sorting,
the flow cell-sorter was operated with a drop-drive attenuation
switched to a frequency of 21 kHz, a sheath pressure of
8e10 psi, a drive frequency of 15e20 kHz and an average
sample flow rate of 110e170 events s1. Prior to sorting,
instrumental tubings were sterilised by flushing with 70%
ethanol for 30 min, followed by an autoclaved phosphate
buffered saline (PBS) sheath fluid constituted from NaCl
(0.137 M), KCl (2.7 mM), KH2PO4 (1.4 mM) and Na2HPO4 (0.01 M).
A PerkineElmer LS-55 fluorescence spectrometer (PerkineElmer Corp) was used to determine Nile red (NR) fluorescence of stained microalgae. The wavelength of excitation
and emission was 480 nm and 575 nm respectively (excitation
and emission slits at 5 nm).

2.2.

Sampling sites and cultivation conditions

Seawater samples were collected at five locations in Singapores

coastal waters (see Fig. 1). Sample locations circumnavigated
Singapores coastline, so that microalgae collected represented
as diverse a taxa as possible. Samples were collected from the
water column of 50 cm depth using a 15 mm-mesh plankton net,
and then stored in cool boxes for transportation to the laboratory. Samples were then inoculated into enriched medium f/2 [9]
and Walnes medium [10] prior to placement in a photoincubator
set at 25  1
C, a light intensity of 60 mmol photon s1 m2
(as supplied by cool-white fluorescence tubes) and a 12:12 h
dark:light photoperiod for 3 days. Following incubation, microalgae were isolated via flow cytometric cell sorting.

2.3.

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Automated isolation using flow cytometric cell-sorter

The sorting protocol of microalgae cells was based upon the

differential and relative distribution of red fluorescence (representing CAF of microalgae) against forward-light scatter
(FSC), representing cell size and green fluorescence vs. red
fluorescence simultaneously. When a cell population exhibited weak CAF, the GAF was utilized for supplementary confirmation. With excitation at 488 nm using an argon laser, the
CAF of microalgae was measured through a 635  20 nm longpass filter and GAF via a 530  30 nm band-pass filter. Twodimensional profiles of FSC against red fluorescence and green
fluorescence vs. red fluorescence were electronically plotted,
where the sorting windows were positioned at optimal areas
within the distribution of an algal cell cluster. Using a twodimensional cytogram of GAF against CAF for the selection and
isolation of microalgae represents an efficient sorting technique. For example, as can be seen in Fig. 2a and b, the microalgae group labelled R4 had a weak CAF, but a high GAF. To
establish axenic cultures during the sorting process, cells were
selected on the basis of the highest relative fluorescence
according to the procedure described by Sensen et al. [7]. Fig. 2a
and b shows typical examples of flow cytometric dot plots for
an enriched microalgae culture, where regions displayed in
rectangle show the sort gates associated with each algal
cluster. Individual sorted single cells were placed into separate
culture wells of a 48-well incubation plate (Iwaki, 3830-048),
where each well contained 0.5 mL of f/2 and Walne medium.

2.4.
Control procedure for verifying a single-sorted cell
per well
Crosbies procedure was used to strictly deposit one cell into
a single culture well, where cells were sorted into odd row
numbers of a multi-well plate, leaving the even rows of the
culture plate blank [6]. After each sorting cycle, each well was
thoroughly mixed with a pipette, and half the volume was
transferred to the adjacent well on an even row. If more than one
cell was originally sorted i.e. if growth occurred in both the
collected and the transferred wells, this represented a misplacement of sorted cells and was rejected e a procedure that
improved the probability that subsequent cultures were generated from single-sorted cells. Following inoculation and colony
formation, 250 mL of medium from the well was analyzed using
an Epics Altra flow cytometer to determine the purity of isolated
strains. Expo32 version 1.2B for Altra (Beckmann Coulter) software was used for data acquisition and processing. 10 mL of either
3-mm or 10-mm fluorospheres (Beckmann Coulter) were added to
samples as an internal cell size reference standard.

2.5.

Determination of growth rate

The isolated microalgae strains were screened to identify those

which grew most rapidly and accumulated the highest biomass
and intracellular lipid. The isolated strains were scaled-up from
colonies grown in the microplates to 5 mL in culture flasks to be
used as stock solutions for further characterization. Motile
strains and flagellate microalgae were cultivated in Walne
medium, where the rest of isolated strains were grown in f/2
medium. All cultures were maintained in a photoincubator at

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Fig. 1 e Sampling locations: Kranji, Pulau Ubin, Changi, St. John Island and Second Link.

25  1
C, a light intensity of 60 mmol photon m2 s1 and

a 12:12 h, light/dark period with manual shaking twice per day.
The growth rate of each strain was characterized based on cell
counts every two days using an Improved Neubauer haemocytometer slide under bright-field contrast microscopy
(Olympus IMP-2, magnification 20e40) or via optical density
at 680 nm wavelength (OD680 nm) in a Hitachi U-2900

spectrophotometer. The specific growth rate of each isolate was

calculated from the slope of the linear regression of time (days)
and cell density (cells mL1), as specified by Wood et al. [11] i.e.:
m ln Nt  ln No =t  to
where Nt is cell density at time (t), and No is cell density at the
start of the exponential phase (to).

Fig. 2 e An example of a flow cytometric dot plot derived from single-cell sorting of microalgae containing four
phytoplankton clusters i.e. R1, R2, R3, R4 that represent the sort gates for each cluster: (a) a sorting window based on cell size
and chlorophyll auto-fluorescence of microalgae; and (b) a sorting window based on chlorophyll auto-fluorescence and
green auto-fluorescence of microalgae.

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2.6.

Nile red (NR) fluorescence of microalgae

The increase in intracellular neutral lipid during the growth

cycle of isolated microalgae strains was measured via fluorescence intensity of NR stained cultures [12] simultaneously to cell
growth. 10 mL of NR was added to 3 mL of algal suspensions
which was then vortexed for 1 min prior to incubation for 10 min
in darkness at room temperature. NR emits a yellow-gold fluorescence when dissolved in neutral lipid which is the best
substrate to produce biodiesel [13,14]. Fluorescence intensity of
a stained suspension of microalgae was measured at an excitation and emission wavelength of 480 nm and 575 nm respectively using an LS-55 fluorescence spectrometer (PerkineElmer
Corp.) normalized at an optical density of 0.2 at wavelength
680 nm (OD680). The microalgae suspension without NR,
considered as auto-fluorescence, was also measured and subtracted from that measured as NR fluorescence.

2.7.
Biomass dry weight and total lipid content at
stationary phase
Cells at stationary phase were harvested and lyophilized. The
total lipid content was extracted by solvent and determined
gravimetrically.

2.7.1.

Biomass dry weight (DW)

100 mL of algal suspension was centrifuged at 5000 rpm for

10 min to produce biomass pellets. Pellets were then washed
in 0.5 M of ammonium formate (twice) to remove salt. The
algal pellets were then lyophilized in a freeze drier (Christ
Alpha 2-4, Germany) to dryness to measure cell dry weight
and total lipid content.

2.7.2.

Total lipid extraction

Lipids were extracted from dry biomass pellets using a modified solvent based method derived from Folch et al. [15]. First,
lyophilized algal pellets were transferred into a chloroform/
methanol (2:1, v/v) mixture and homogenized (Heidolph
homogenizer DIAX900, Germany) for 10 min. This was followed by centrifugation at 5000 rpm at 12
C for 10 min (Biofuge Stratos, Germany). The supernatant was collected in
a separation funnel. The entire extraction process was
repeated twice. Sodium chloride solution (0.9%) was then
added at a proportion of 1:5 v/v of lipid extract. The extract
was then shaken vigorously for 1 min and allowed to undergo
phase separation for 15 min. The chloroform layer was
collected and dried initially in a rotary evaporator, and then in
an oven at 80
C to constant weight. The total lipid was then
quantified gravimetrically.

2.8.

Fatty acid determination

The fatty acid methyl esters (FAMEs) profile of the microalgae

lipids was used as a proxy for the fatty acid profile. FAMEs
were prepared via the direct methylation of fatty acids in the
presence of an HCl catalyst according to the procedure given
by Lewis et al. [16]. Dry algal biomass was reacted directly with
a mixture of methanol, chloroform and HCl (10:1:1 v/v/v) in
10 mL crimped vials at 90
C for 2 h. FAMEs were then
extracted using hexane/chloroform (4:1, v/v) and analyzed via

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gas chromatography using an ion trap (GC-QP2010, Shimadzu,

Japan). Fatty acid profiles of the microalgae strains were
deduced by comparison with a 37 FAME mix standard (i.e.
Supelco 37 FAME mix, C4-C24). Each sample was spiked with
n-heptadecanoic acid (C17:0) to a final concentration of 1 ppm
as an internal standard. The capillary column of the GC/MS
was a DB-5MS, of 30 m length, a film thickness of 0.25 mm and
an internal diameter of 0.25 mm. Helium was used as the
carrier gas. The oven temperature and injection temperature
were set at 50
C and 280
C respectively. The temperature
program of the GC was held at 50
C for 2 min and then
increased to 150
C at a rate of 10
C min1, then to 185
C for
2 min and finally to 300
C for 2 min.

2.9.

Strain screening procedure

Screening was carried out using the procedure expressed in

Fig. 3. The procedural order was: sampling, automated isolation, pre-screening and screening. Pre-screening, mainly based
on NR fluorescence of stained algal cells, and cellular neutral
lipid droplets was detected under epifluorescence microscopy.
Strains that contained cellular lipid droplets were further
screened with respect to cell division and lipid production. For
cell division, growth rate and biomass concentration of each
strain were examined. Simultaneously, lipid production of
these strains was investigated in two aspects i.e. total lipid
content and fatty acid profile. To determine lipid content,
gravimetric extraction and NR fluorescence measurements
were conducted. Fatty acid profile is a critical factor for decision
of selection of candidate strain as it ultimately determines
biodiesel quality. Selection criteria for screening were based on
biomass productivity and intracellular lipid content. Strains
which demonstrated comparatively high biomass productivity,
lipid content, and a suitable fatty acid profile were selected.

2.10.

Statistical analysis

Statistical analyses were performed using SigmaStat 3.5 software. The screening data were analyzed using either a t-test
or one-way analysis of variance (ANOVA), followed by a StudenteNewmaneKeuls (SNK) multiple comparison test.
Differences were considered significant at p < 0.05.

3.

Results and discussion

3.1.

Sampling sites

Five marine water locations were sampled around the coast of

Singapore. The average temperature of the surface water was
between 25 and 30
C, and salinity ranged from 18& (at Kranji)
to 37& (at Changi).

3.2.

Automated isolation using flow cytometric cell sorting

Isolation of suitable microalgae strains is an essential prerequisite for biodiesel feedstock production. Since microalgae are
taxonomically diverse and not all strains are able to be
cultured, the efficient isolation of microalgae is essential. The
use of flow cytometric cell sorting is a powerful tool for the

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Fig. 3 e Microalgae strain screening procedure.

selection of microalgae with the desired phenotypes for feedstock production. Flow cytometry combined with single-cell
sorting is a high-throughput technique for screening and
isolating microalgae from heterogeneous assemblages. Based
on the developed protocols for FSC (indication of cell size) vs.
red fluorescence (indication of CAF) or red fluorescence vs.
green fluorescence, a total of 96 strains of microalgae were
isolated from the five marine sample locations (see Table 1). As
seen in Fig. 2, R1 is a group of microalgae which attained weak
red and green fluorescence while group R2 had higher red and
green fluorescence. Red and green fluorescence of group R3
were highest, whereas red fluorescence of R4 was lowest
amongst the groups, but its green fluorescence was as high as

the R3 group. The result demonstrates that the size and chlorophyll content of sorted cells corresponds with sorting
criteria, i.e. sorted cells of group R1 and R2 had a size of
approximately 3 mm, and group R3 10 mm. CAF in each of the
groups differed, where group R4, which was low in red fluorescence, was successfully sorted based on its GAF. In this
study, the CAF of microalgae was used successfully as
a biomarker to distinguish and isolate microalgae in natural
seawater samples. Using the CAF vs. GAF protocol a diverse
taxa of microalgae was isolated. In agreement with previous
studies by Tang and Dobbs [8] and Lage et al. [17], the GAF
protocol aided in the taxonomic identification of dinoflagellates, diatoms, green algae, cyanobacteria, and raphidophytes.

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Table 1 e Sample locations and characteristics of local isolated microalgae strains.

Sampling
locations

Number of isolated
strains

Number of selected
strains

Number of
diatoms

Number of green
algae

Number of motile/
flagellates

Pulau Ubin island

St Johns island
Kranji coast
Second link
Changi

39
26
15
13
3

9
4
4
3
1

35 (89.7)a
23 (88.5)
9 (60)
10 (76.9)
3

4 (10.3)
3 (11.5)
3 (20)
2 (15.4)
NA

NA
NA
3 (20)
1 (0.08)
NA

Total

96

21

80 (83.3)

12 (12.5)

4 (4.2)

NA e not available.
a Numbers in parentheses are percentage of total isolates.

Isolated strains were identified using their morphological

features. The characteristics of the isolates are documented in
Table 1. Most isolates (i.e. 83% of the total) were diatom
species belonging to the genera of Skeletonema, Thalassiosira,
Chaetoceros and Cyclotella sp. Green microalgae, i.e. Nannochloropsis sp. plus some unidentified strains were less abundant than the diatoms, but many isolates from this group
exhibited the highest lipid content i.e. >40% as biomass dry
weight. Images of representative isolated strains are shown in
Fig. 4.

3.3.

Verification of single-cell sorting efficacy

In order to confirm that sorted cells were uniquely deposited

in separate incubation wells, the sorted cells were subjected to
the control procedure. The success rate of one-cell-per-well
sorting was high, ranging from 82% to 100%. Moreover,
50e75% of the sorted cells remained viable. This ratio is higher
than that previously reported by Sensen et al. [7], where only
20e30% of sorted cells grew successfully. 250 mL scaled-up
cultures of the sorted cells were further analyzed using an

Epics Altra flow cytometer to verify that isolates were in pure

culture. The cytometric dot plots confirmed that the sorted
cultures were derived from a single isolate.

3.4.
Growth rate and biomass production of isolated
microalgae
Following isolation, a total of 96 isolated strains were prescreened via NR fluorescence of cellular neutral lipid and epifluorescence microscopy. Isolates were inoculated into Guillar
f/2 or Walne medium. Almost all isolates grew robustly in f/2
medium. However, the growth of motile and flagellate microalgal isolates, e. g. strain 45 and 50, was promoted in Walne
medium and exhibited a strong neutral lipid fluorescence
when stained with Nile red. During cultivating, a number of
isolates could not survive due to challenges in culture acclimation. Isolates that grew rapidly and manifested strong
yellow-gold fluorescence from NR stained cells (i.e. reflecting
a relatively high neutral lipid content) were further screened. A
total of 21 strains of the 96 isolated microalgae were suitable for
further characterization, and results are shown in Table 2.

Fig. 4 e Photos of the representative isolated microalgal strains from Singapores coastal water. (a) Skeletonema costatum; (b)
Thalassionema nitzschoides; (c) Cyclotella sp; (d) Nannochloropsis sp; (e) Thalassiosira sp.; (f) Dunaliella sp.

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Table 2 e Genera, growth rate, biomass and lipid content

of selected microalgal strains.
Strain

1
4
5
18
19
30
31
34
35
40
41
42
43
44
45
46
47
48
49
50
51

Biomass
Lipid
concentration content
(g L1)
(% of
DW)

Genera/
species

Growth
rate
(d1)

Skeletonema
costatum
Thalassiosira sp.
Thalassionema
nitzschoides
Skeletonema sp.
Thalassiosira sp.
Thalassiosira sp.
Nannochloropsis sp.
Chaetoceros sp.
Chaetoceros socialis
Achnanthes sp.
Cyclotella sp.
Unidentified
Unidentified
Nannochloropsis sp.
Cryptomomas sp.
Nannochloropsis sp.
Nannochloropsis sp.
Nannochloropsis sp.
Nannochloropsis sp.
Heterosigma sp.
Nannochloropsis sp.

0.95

0.09  0.01

9.5  1.7c

0.61
0.58

0.08  0.03
0.05  0.03

17.8  3.8b
2.8  0.1c

0.59
0.79
0.85
0.62
0.87
0.64
0.99
0.41
0.48
0.57
0.52
0.47
0.42
0.60
0.47
0.46
0.50
0.55

0.24  0.06
0.05  0.01
0.04  0.01
0.39  0.09
0.06  0.01
0.07  0.02
0.09  0.03
0.20  0.02
0.18  0.01
0.11  0.02
0.29  0.05
0.07  0.02
0.27  0.03
0.4  0.03
0.28  0.04
0.22  0.05
0.07  0.01
0.35  0.07

2.7  0.4c
0.9  0.1c
13.6  1.2b
42.4  1.2a
16.3  4.0b
2.2  1.4c
44.5  1.7a
12.7  2.0c
11.2  1.8c
32.6  2.7b
39.5  1.9a
28.6  0.6b
41.4  1.3a
44.8  1.7a
40.3  1.3a
42.7  1.9a
39.9  2.0a
42.2  1.6a

Note: Biomass concentration and lipid content at stationary phase.

Data expressed as mean  SD (n 3). Means of total lipid content
are compared using one-way ANOVA, followed by a StudenteNewmaneKeuls (SNK) multiple comparison test. Letters
represented the order of significant differences between the
means: a > b > c.

In general, most isolated strains had a growth cycle of 8

days to stationary phase. However, some strains were found
to have a shorter growth cycle, such as the long-chain diatom
Skeletolema sp. (strain 18) and Chaetoceros socialis (strain 35).
Strains 18 and 35 bloomed at day 2, but collapsed between day
3 and 4, and had a relatively low gravimetric lipid content (i.e.
2.2e2.7% of biomass DW). These were therefore deemed
unsuitable for further culture. Other strains had an extended
growth log phase of up to 20 days, such as the group of

Nannochloropsis sp. The diatoms Skeletonema costatum, Achnanles sp. and Chaetoceros sp. had higher specific growth rates
i.e. 0.87e0.99 d1 than other strains. However, the Nannochloropsis group attained the best overall biomass yield i.e.
0.2e0.4 g L1. From the screening, Nannochloropsis and several
other strains among the best biomass producers were further
investigated for lipid accumulation. Naturally, a strain which
has high biomass productivity may manifest in a relative low
lipid productivity and vice versa [18].

3.5.

Cellular lipid accumulation

3.5.1.

Total lipid content (gravimetric determination)

One of the key criteria for selection of candidate microalgae

strains for biodiesel feedstock production is a high intracellular lipid content. Based on gravimetric total lipid, the
maximum yield of cellular lipid content in stationary phase,
which has been the potential source for biodiesel production,
was estimated. The results in Table 2 show that approximately half of the locally isolated marine microalgae strains
contained a total lipid content greater than 25% as biomass
DW. Among these strains, the total lipid content of strains 31,
40, 44, 46, 47, 48, 49, 50 and 51 exceeded 30%. The group of
Nannochloropsis sp. i.e. strains 31, 44, 46, 47, 48, 49, 51 had
a total lipid content of up to 45%. Next were Achnanthes sp.
(strain 40), a benthic microalga and Heterosigma sp. (strain 50),
a flagella microalga (class Raphidophycae) which contained
total lipid contents at 44.5  1.7 and 39.9  2.6%, respectively.
Among locally isolated strains, Nannochloropsis sp. displayed
the best potential since they met the balance between relatively
high biomass productivity and a high intracellular lipid content.
Previous studies have also reported Nannochloropsis sp. as
having a high biomass and lipid production potential [18e21].

3.5.2.

Neutral lipid (NR fluorescence)

Total lipids are composed of neutral lipid in the form of energy

reserve bodies, as well as glyco- and phospholipids in the
structural membranes. Neutral lipids are typically the major
constituents of algal lipid-oil in aging or stressed cultures,
mainly in the form of triacylglycerols (TAGs) [22e24]. Although
both polar and neutral lipids can be converted to biodiesel
[25,26], neutral lipid is the desirable fraction since TAGs are

Fig. 5 e NR fluorescence and cell density of Thalasiossira sp. (strain 4) over a 10-day cultivation.

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Relative fluorescence intensity (a.u.)

40
35
30
25
20
15
10
5
0

5 18 19 30 31 34 35 40 41 42 43 44 45 46 47 48 49 50 51
Strain

Fig. 6 e NR fluorescence of 21 local microalgae strains at stationary phase. Fluorescence intensity was measured at an
excitation wavelength 480 nm and emission at 575 nm. Cell density was normalized at 0.2 (OD680 nm).

easily transesterified to biodiesel. Determination of neutral

lipid using high-throughput screening technique based on NR
fluorescence has been widely used as NR staining is a rapid
method to quantify intracellular lipid in vivo [12,27]. In this
study, NR fluorescence represents as a high-throughput
screening tool to select a favourable microalgae strain from
many isolates. The screening of microalgae intracellular lipid
production was carried out in two aspects: the kinetic accumulation of neutral lipid during the microalgae growth cycle;
and the maximum level of neutral lipid in stationary phase.
Firstly, an investigation of the kinetic accumulation of
intracellular neutral lipid of selected strains during the cell
growth cycle was conducted. Fig. 5 shows the growth curve

and cellular lipid accumulation of strain 4 i.e. Thalassiosira sp.,

as a representative strain. NR fluorescence was positively
correlated to lipid accumulation (R2 0.84). Neutral lipid
content was low (intensity of 0.6e1.4 relative units) for the
first 4 days of culture, after which it increased by twelve times
to a peak value on day 10 during the stationary phase. During
the exponential phase (day 1 to day 4), cell density increased
from 1.6  104 to 10.3  104 cell mL1. Fig. 5 shows that during
early exponential phase, neutral lipid production was limited,
but then increased substantially during the stationary phase.
In addition, NR fluorescence of neutral lipids can be utilized to
determine the relationship between lipid accumulation and
the growth phase of microalgae [12,27e29]. Typically, protein

Table 3 e Fatty acid composition of several isolated microalgae strains (% of total FAME, n [ 3).
Fatty acid

Myristic acid (C14:0)

Pentadecanoic acid (C15:0)
Palmitoleic acid (C16:1)
Palmitic acid (C16:0)
Linolenic acid (C18:3)
Linoleic acid (C18:2n6)
Oleic acid (C18:1n9)
Stearic acid (C18:0)
Arachidonic acid (C20:4n6)
Eicosapentaenoic acid (C20:5n3)
Docosahexaenoic acid (C22:6n3)
Behenic acid (C22:0)
Lignoceric acid (C24:0)
Others

Strains
31

40

44

46

47

48

49

50

51

3.5  0.5
0.5  0.1
29.2  3.9
28.4  6.2
nd
2.1  1.2
5.5  1.9
2.0  0.5
4.8  1.5
18.6  4.5
nd
nd
1.4  0.5
3.9  0.8

7.0  0.3
1.5  0.1
45.0  5.5
30.4  3.5
0.5  0.1
1.0  0.2
0.94  0.2
0.6  0.2
0.6  0.1
12.3  2.5
nd
nd
0.8  0.2
2.2  0.5

3.5  0.1
0.5  0.0
29.8  6.5
29.6  2.4
nd
0.6  0.3
5.9  1.4
3.2  0.9
4.1  2.3
18.2  4.5
nd
nd
1.9  0.5
3.4  1.5

3.7  0.2
0.6  0.1
29.4  6.5
29.9  5.8
nd
1.2  0.1
6.4  1.5
2.4  0.9
6.2  0.4
18.5  4.5
nd
nd
1.6  0.5
1.4  0.4

3.4  0.2
0.5  0.1
31.0  6.1
29.5  4.5
nd
1.4  0.5
6.3  1.7
1.9  0.1
5.3  0.0
17.9  5.5
nd
nd
1.2  0.4
2.1  0.8

3.4  0.3
0.6  0.1
30.7  4.9
29.2  4.5
nd
2.1  0.8
6.6  1.2
1.8  0.1
5.4  0.1
17.6  5.2
nd
nd
0.5  0.1
2.9  0.9

3.8  0.6
nd
32.8  3.2
31.4  1.8
0.6  0.1
2.1  0.9
6.4  0.9
2.3  0.5
6.4  0.9
12.4  4.3
nd
nd
1.3  0.3
1.5  0.1

9.0  02.3
0.7  0.3
20.5  4.3
33.4  2.6
3.6  0.3
5.0  0.9
10.6  2.3
0.9  0.3
1.8  0.3
13.0  4.3
2.1  0.7
1.5  0.1
0.6  0.1
4.1  0.9

4.4  0.0
nd
30.0  0.3
40.8  0.1
nd
0.7  0.1
12.0  0.0
1.8  0.2
2.5  0.1
7.5  0.1
nd
nd
0.45  0.1
0.3  0.0

nd non-detectable level. Others: fatty acid constituents as identified using a 37 FAME standard, but where concentration was below 0.5% of
total FAME. Data expressed as mean  SD (n 3). Fatty acids are compared using one-way ANOVA, followed by a StudenteNewmaneKeuls
(SNK) multiple comparison test. Letters represented the order of significant differences between the means: a > b > c.

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b i o m a s s a n d b i o e n e r g y 3 5 ( 2 0 1 1 ) 2 5 3 4 e2 5 4 4

Table 4 e Composition (% of total FAME) and yield of FAME (% of DW) of microalgae strains.
Properties

SFA (% of total FAME)

MFA (% of total FAME)
PUFA (% of total FAME)
Total FAME content (% DW)
Total FAME content (% total lipid)

Strains
31

40

44

46

47

48

49

50

51

35.7  1.8
34.8  2.9
27.0  6.8
19.8  2.7a
46.7  2.5

40.2  5.4
45.9  3.4
14.3  3.7
15.8  2.7b
35.5  2.9

35.5  4.9
35.6  1.8
25.9  1.7
15.5  1.2b
39.2  3.1

38.1  5.6
35.7  4.9
25.2  2.7
11.2  2.7b
25.0  2.3

36.5  1.2
37.3  3.9
24.6  1.6
16.6  1.4a
37.1  4.1

35.3  1.3
37.3  3.7
25.2  1.9
14.7  3.1b
36.7  2.6

39.0  0.8
39.1  5.3
21.1  1.6
14.1  2.2b
33.5  2.8

45.4  2.5
31.0  5.7
23.7  3.7
19.6  1.7a
48.8  3.3

47.5  0.7
41.8  1.7
10.7  0.9
21.3  1.6a
49.8  2.5

SFA Saturated fatty acids (14:0, 15:0, 16:0, 18:0, 22:0, 24:0), MUFA monounsaturated fatty acids (16:1, 18:1n9), PUFA polyunsaturated fatty
acids (18:2n6, 18:3n3, 20:4n6, 20:5n3, 22:6n3). Data expressed as mean  SD (n 3). Means of total FAME content are compared using one-way
ANOVA, followed by a StudenteNewmaneKeuls (SNK) multiple comparison test. Letters represented the order of significant differences
between the means: a > b > c.

synthesis is predominant over neutral lipid accumulation in

the exponential phase of cell growth [30], where neutral lipid
accumulation is elevated in the stationary phase as prevailing
nutrients become limited and cell division is reduced. Based
on the NR fluorescence measurement as an indicator for
intracellular neutral lipid content, the harvesting time of
microalgae can be optimized for maximum yield.
Secondly, to isolate candidate strain(s) that accumulate
high neutral lipid, NR was used to stain selected strains in
stationary phase. The NR fluorescence of neutral lipid content
of selected strains is shown in Fig. 6. Most diatoms responded
rapidly to NR staining, where emission of NR fluorescence was
representative of total intracellular neutral lipid [12,31]. Achnanthes sp. (strain 40) had the highest NR fluorescence intensity which corresponded to total lipid, at a relative intensity of
32 au. and 44.5%, respectively. However, Nannochloropsis sp.
was not efficiently stained using the conventional NR fluorescence method. Thus, the level of total lipid and neutral
lipid did not correspond, where the fluorescence of stained
cells was weak (see Fig. 6) but gravimetric total lipid content in
stationary phase was high (see Table 2). Inefficient NR staining
of Nannochloropsis strain has been previously reported by
Sheehan et al. [32], and is likely due to cell wall properties of
Nannochloropsis sp. which resist dye absorption. In order to
address this problem, a modified Nile red staining procedure
for Nannochloropsis is required. In this study, therefore only
gravimetric lipid content was used to select the most favourable Nannochloropsis strains.

3.6.

On the basis of cell growth rate and lipid content (see data in
Table 2 and Fig. 5), nine strains of microalgae which had a total
lipid content significantly greater (SNK test, p < 0.05) than
others, ranging from 39.5 to 44.8%, were further characterized
with fatty acid profile using FAME profile as a proxy. Results in
Tables 3 and 4 show that the majority of fatty acids present in
isolated microalgae were palmitic (C16:0), palmitoleic (C16:1)
and eicosapentaenoic (C20:5n3) acids which comprised 63e80%
of the total FAME. Three strains of Nannochloropsis (i.e. strains 31,
47, 51) and Heterosigma (strain 50) had a fatty acid content
significantly higher (SNK test, p < 0.05) than the rest (see Table
3). Although strain 50 accumulated high total lipid and FAME
content (39.9% and 19.6% of biomass DW, respectively), it had
a relatively low biomass concentration i.e. 0.07 g L1, and was
therefore eliminated from further evaluation. SFA and MUFA in
Nannochloropsis strains 31, 47 and 51 were predominant at >70%
of the total lipid content (see Table 4) which is favourable for
high cetane number [20,33,37]. However, Nannochloropsis had
PUFAs in range of 10.7e27% which exceeded the requirements
in the International Biodiesel Standard for Vehicles (EN14214).
The PUFA of Nannochloropsis were mainly arachidonic acid (AA,
C20:4n6) and eicosapentaenoic acid (EPA, C20:5n3) which are
high-value fatty acids for human nutrition and food additives. In
order to comply with biodiesel standard on the PUFA ratio, AA
and EPA of Nannochloropsis can be extracted before the rest of oil
can be converted to biodiesel. This makes Nannochloropsisderived biodiesel less susceptible to oxidation in storage and
takes full advantage of Nannochloropsis.

Fatty acid profile of isolated strains

4.
In addition to screening microalgae for elevated biomass
productivity and intrinsic cellular lipid content, the fatty acid
profile of microalgae is also an important characteristic as it
ultimately affects the quality of the biodiesel product [20,33,34].
The carbon chain length of saturated and unsaturated fatty acids
affects biodiesel properties such as cetane number, oxidative
stability and cold-flow properties [35,36]. Generally, high
proportion of saturated fatty acids (SFA) and monounsaturated
fatty acids (MUFA) are preferred for increased energy yield,
superior oxidative stability, and higher cetane numbers.
However, oils dominated by these fatty acids are prone to solidify
at low temperature. While oils rich in polyunsaturated fatty
acids (PUFAs) have very good cold-flow properties, they are, on
the other hand, more susceptible to oxidation.

Conclusions

High-throughput isolation, using flow cytometric cell sorting based on red and green fluorescence, and forward-light
scatter was used to isolate a diverse range of microalgae taxa
from Singapores coastal marine waters. Of 96 strains isolated,
a total of 21 strains were characterized for intrinsic growth
rate, biomass production and intracellular lipid content. The
three best strains with respect to lipid accumulation and
biomass generation were all Nannochloropsis sp. (strains 31, 47
and 51), with a rapid and robust growth (growth rate > 0.55 d1), lipid contents ranging from 42.5 to 45% as biomass
DW, and a corresponding FAME yield ranging between 16 and
22% of DW. Furthermore, these strains have a predominance
of SFA and MUFA e corresponding to a favourably high cetane

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b i o m a s s a n d b i o e n e r g y 3 5 ( 2 0 1 1 ) 2 5 3 4 e2 5 4 4

number for biodiesel feedstock. The screening procedure

provided useful ways to select microalgae, and is recommended as an essential process for biodiesel feedstock
production. In the screening process, NR fluorescence was
used as a high-throughput screening method for determination of microalgae cellular lipid content. Besides lipid content,
biomass production is also a critical factor to select a favourable microalgae strain. Nannochloropsis strains are undergoing
on-going investigation to further enhance biomass productivity and lipid yield to improve their viability as a biodiesel
feedstock.

Acknowledgements
Funding of this research project was provided by Agency for
Science, Technology and Research of Singapore (A* STAR). We
also thank Toh Kok Tee (NUMI) and Yeo Yin Sheng Wilson of
the Tropical Marine Science Institute (TMSI) for technical
support on the use of the flow cytometer, Dang The Cuong and
Sarah Siti Binte Aziz for microalgae sampling and
identification.

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