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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, Singaporeb Tropical Marine Science Institute, National University of Singapore, S2S, 18 Kent Ridge Road, Singapore 119227, Singapore

a r t i c l e i n f o

Article history:

Received 9 June 2010

Received in revised form

5 February 2011

Accepted 10 February 2011

Available online 24 March 2011

Keywords:

Microalgae

Biodiesel

Flow cytometric cell sorting

Screening

Neutral lipid

a b s t r a c t

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

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

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

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

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

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

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

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

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

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

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

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 demon-

strates that Nannochloropsis is a promising species for biodiesel feedstock.

ª 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.

Theisland stateof Singapore lies180 km northof theequator

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).

Singapore’s coastal seas have a warm ambient temperature(approximately30 C) andcontaina high biodiversityof marine

life, including microalgae. This biodiversity represents a valu-

able 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 cellsorting,coupledwithflow 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.: þ65 63007516; fax: þ65 67744202.E-mail address: [email protected] (T.T.Y. Doan).

A v a i l a b l e a t w w w . s c i e n c e d i r e c t . c o m

h t t p : / / w w w . e l s e v i e r . c o m / l o c a t e / b i o m b i o e

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

0961-9534/$ e see front matter ª 2011 Elsevier Ltd. All rights reserved.doi:10.1016/j.biombioe.2011.02.021

https://www.researchgate.net/publication/6457207_Biodiesel_From_Microalgae?el=1_x_8&enrichId=rgreq-0919a7a7-a346-444f-952b-560d6f63c5a6&enrichSource=Y292ZXJQYWdlOzIzMjQwNzI4ODtBUzoxNDQzMjEzNTc1NTM2NjRAMTQxMTQyMDMwMTQzMw==https://www.researchgate.net/publication/5629626_Biodiesel_From_Microalgae_Beats_Bioethanol?el=1_x_8&enrichId=rgreq-0919a7a7-a346-444f-952b-560d6f63c5a6&enrichSource=Y292ZXJQYWdlOzIzMjQwNzI4ODtBUzoxNDQzMjEzNTc1NTM2NjRAMTQxMTQyMDMwMTQzMw==https://www.researchgate.net/publication/5629626_Biodiesel_From_Microalgae_Beats_Bioethanol?el=1_x_8&enrichId=rgreq-0919a7a7-a346-444f-952b-560d6f63c5a6&enrichSource=Y292ZXJQYWdlOzIzMjQwNzI4ODtBUzoxNDQzMjEzNTc1NTM2NjRAMTQxMTQyMDMwMTQzMw==https://www.researchgate.net/publication/6457207_Biodiesel_From_Microalgae?el=1_x_8&enrichId=rgreq-0919a7a7-a346-444f-952b-560d6f63c5a6&enrichSource=Y292ZXJQYWdlOzIzMjQwNzI4ODtBUzoxNDQzMjEzNTc1NTM2NjRAMTQxMTQyMDMwMTQzMw==https://www.researchgate.net/publication/11057947_Botryococcus_braunii_A_Renewable_Source_of_Hydrocarbons_and_Other_Chemicals?el=1_x_8&enrichId=rgreq-0919a7a7-a346-444f-952b-560d6f63c5a6&enrichSource=Y292ZXJQYWdlOzIzMjQwNzI4ODtBUzoxNDQzMjEzNTc1NTM2NjRAMTQxMTQyMDMwMTQzMw==


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fluorescence. These characteristics of microalgae have been

applied in flow cytomertry to identify algae [4]. Isolation of

microalg ae from natural waters using flow cytometric cell sort-

ing has beenpreviouslyreported by Reckermann [5] and Crosbie

et al. [6], where chlorophyll was used as a fluorescent probe to

distinguish between different strains of microalgae. Reck-ermann [5] and Sensen et al. [7] used the chlorophyll auto-fluo-

rescence (CAF) properties of eukaryoticphytoplankton,diatoms

and picoautotrophic cells for isolation of axenic cultures,

whereas Crosbie et al. [6] used both red and orange auto-fluo-

rescence to differentiate between algal strains. Green auto-

fluorescence (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 todifferentiate 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

flowcytometerfittedwithacell-sorteranda100 mWArgonion

laser (Spectra Physics, Darmstadt, Germany) and a 100 mm

nozzle was used for experimentation. For single-cell sorting,theflow cell-sorter wasoperated witha 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 (Per-

kineElmer Corp) was used to determine Nile red (NR) fluo-

rescence 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 werecollectedat five locations in Singapore’s

coastal waters (see Fig. 1). Sample locations circumnavigated

Singapore’s coastline, so that microalgae collected represented

as diverse a taxa as possible. Samples were collected from the

water columnof 50 cm depth using a 15 mm-mesh plankton net,

and then stored in cool boxes for transportation to the labora-

tory.Samples werethen inoculated intoenrichedmedium f /2 [9]

andWalne’smedium[10] priortoplacementinaphotoincubator

set at 25 1 C, a light intensity of 60 mmol photons1 m2

(as supplied by cool-white fluorescence tubes) and a 12:12 h

dark:light photoperiod for 3 days. Following incubation, micro-

algae were isolated via flow cytometric cell sorting.

2.3. Automated isolation using flow cytometric cell-sorter

The sorting protocol of microalgae cells was based upon the

differential and relative distribution of red fluorescence (rep-

resenting CAF of microalgae) against forward-light scatter

(FSC), representing cell size and green fluorescence vs. redfluorescence simultaneously. When a cell population exhibi-

ted weak CAF, the GAF was utilized for supplementary confir-

mation. With excitation at 488 nm using an argon laser, the

CAF of microalgae was measured through a 635 20 nm long-

pass filter and GAF via a 530 30 nm band-pass filter. Two-

dimensional 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 two-

dimensional cytogramof GAFagainstCAF for theselection and

isolation of microalgae represents an efficient sorting tech-

nique. For example, as can be seen in Fig. 2a and b, the micro-

algae group labelled ‘R4’ had a weak CAF, but a high GAF. Toestablish axenic culturesduring the sorting process, cells were

selected on the basis of the highest relative fluorescence

according to theprocedure describedby 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 cellswere placed intoseparate

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

Crosbie’s 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 tothe 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 misplace-

ment of sorted cells and was rejected e a procedure that

improved the probability that subsequent cultures were gener-

ated from single-sorted cells. Following inoculation and colony

formation, 250 mL of medium from the well was analyzed using

anEpicsAltraflowcytometer todeterminethe purityof isolatedstrains. Expo32 version 1.2B for Altra (Beckmann Coulter) soft-

ware wasusedfordataacquisitionandprocessing. 10 mLofeither

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 strainswere screened to identifythose

which grew mostrapidly andaccumulated thehighest biomass

andintracellularlipid.The isolated strainswerescaled-upfrom

coloniesgrown in the microplatesto 5 mLin cultureflasks 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

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 2535

https://www.researchgate.net/publication/5580732_Crosbie_ND_Pockl_M_Weisse_T_Rapid_establishment_of_clonal_isolates_of_freshwater_autotrophic_picoplankton_by_single-cell_and_single-colony_sorting_J_Microbiol_Meth_55_361-370?el=1_x_8&enrichId=rgreq-0919a7a7-a346-444f-952b-560d6f63c5a6&enrichSource=Y292ZXJQYWdlOzIzMjQwNzI4ODtBUzoxNDQzMjEzNTc1NTM2NjRAMTQxMTQyMDMwMTQzMw==https://www.researchgate.net/publication/249026012_The_production_of_clonal_and_axenic_cultures_of_microalgae_using_fluorescence-activated_cell_sorting_Europ_J_Phycol?el=1_x_8&enrichId=rgreq-0919a7a7-a346-444f-952b-560d6f63c5a6&enrichSource=Y292ZXJQYWdlOzIzMjQwNzI4ODtBUzoxNDQzMjEzNTc1NTM2NjRAMTQxMTQyMDMwMTQzMw==https://www.researchgate.net/publication/5580732_Crosbie_ND_Pockl_M_Weisse_T_Rapid_establishment_of_clonal_isolates_of_freshwater_autotrophic_picoplankton_by_single-cell_and_single-colony_sorting_J_Microbiol_Meth_55_361-370?el=1_x_8&enrichId=rgreq-0919a7a7-a346-444f-952b-560d6f63c5a6&enrichSource=Y292ZXJQYWdlOzIzMjQwNzI4ODtBUzoxNDQzMjEzNTc1NTM2NjRAMTQxMTQyMDMwMTQzMw==https://www.researchgate.net/publication/6527958_Green_Autofluorescence_in_Dinoflagellates_Diatoms_and_Other_Microalgae_and_Its_Implications_for_Vital_Staining_and_Morphological_Studies?el=1_x_8&enrichId=rgreq-0919a7a7-a346-444f-952b-560d6f63c5a6&enrichSource=Y292ZXJQYWdlOzIzMjQwNzI4ODtBUzoxNDQzMjEzNTc1NTM2NjRAMTQxMTQyMDMwMTQzMw==https://www.researchgate.net/publication/249026012_The_production_of_clonal_and_axenic_cultures_of_microalgae_using_fluorescence-activated_cell_sorting_Europ_J_Phycol?el=1_x_8&enrichId=rgreq-0919a7a7-a346-444f-952b-560d6f63c5a6&enrichSource=Y292ZXJQYWdlOzIzMjQwNzI4ODtBUzoxNDQzMjEzNTc1NTM2NjRAMTQxMTQyMDMwMTQzMw==https://www.researchgate.net/publication/5580732_Crosbie_ND_Pockl_M_Weisse_T_Rapid_establishment_of_clonal_isolates_of_freshwater_autotrophic_picoplankton_by_single-cell_and_single-colony_sorting_J_Microbiol_Meth_55_361-370?el=1_x_8&enrichId=rgreq-0919a7a7-a346-444f-952b-560d6f63c5a6&enrichSource=Y292ZXJQYWdlOzIzMjQwNzI4ODtBUzoxNDQzMjEzNTc1NTM2NjRAMTQxMTQyMDMwMTQzMw==https://www.researchgate.net/publication/6527958_Green_Autofluorescence_in_Dinoflagellates_Diatoms_and_Other_Microalgae_and_Its_Implications_for_Vital_Staining_and_Morphological_Studies?el=1_x_8&enrichId=rgreq-0919a7a7-a346-444f-952b-560d6f63c5a6&enrichSource=Y292ZXJQYWdlOzIzMjQwNzI4ODtBUzoxNDQzMjEzNTc1NTM2NjRAMTQxMTQyMDMwMTQzMw==https://www.researchgate.net/publication/14221488_Flow_Cytometry_and_Cell_Sorting_of_Heterogeneous_Microbial_Populations_the_Importance_of_Single-Cell_Analyses?el=1_x_8&enrichId=rgreq-0919a7a7-a346-444f-952b-560d6f63c5a6&enrichSource=Y292ZXJQYWdlOzIzMjQwNzI4ODtBUzoxNDQzMjEzNTc1NTM2NjRAMTQxMTQyMDMwMTQzMw==https://www.researchgate.net/publication/249026012_The_production_of_clonal_and_axenic_cultures_of_microalgae_using_fluorescence-activated_cell_sorting_Europ_J_Phycol?el=1_x_8&enrichId=rgreq-0919a7a7-a346-444f-952b-560d6f63c5a6&enrichSource=Y292ZXJQYWdlOzIzMjQwNzI4ODtBUzoxNDQzMjEzNTc1NTM2NjRAMTQxMTQyMDMwMTQzMw==https://www.researchgate.net/publication/249026012_The_production_of_clonal_and_axenic_cultures_of_microalgae_using_fluorescence-activated_cell_sorting_Europ_J_Phycol?el=1_x_8&enrichId=rgreq-0919a7a7-a346-444f-952b-560d6f63c5a6&enrichSource=Y292ZXJQYWdlOzIzMjQwNzI4ODtBUzoxNDQzMjEzNTc1NTM2NjRAMTQxMTQyMDMwMTQzMw==https://www.researchgate.net/publication/5580732_Crosbie_ND_Pockl_M_Weisse_T_Rapid_establishment_of_clonal_isolates_of_freshwater_autotrophic_picoplankton_by_single-cell_and_single-colony_sorting_J_Microbiol_Meth_55_361-370?el=1_x_8&enrichId=rgreq-0919a7a7-a346-444f-952b-560d6f63c5a6&enrichSource=Y292ZXJQYWdlOzIzMjQwNzI4ODtBUzoxNDQzMjEzNTc1NTM2NjRAMTQxMTQyMDMwMTQzMw==https://www.researchgate.net/publication/5580732_Crosbie_ND_Pockl_M_Weisse_T_Rapid_establishment_of_clonal_isolates_of_freshwater_autotrophic_picoplankton_by_single-cell_and_single-colony_sorting_J_Microbiol_Meth_55_361-370?el=1_x_8&enrichId=rgreq-0919a7a7-a346-444f-952b-560d6f63c5a6&enrichSource=Y292ZXJQYWdlOzIzMjQwNzI4ODtBUzoxNDQzMjEzNTc1NTM2NjRAMTQxMTQyMDMwMTQzMw==https://www.researchgate.net/publication/5580732_Crosbie_ND_Pockl_M_Weisse_T_Rapid_establishment_of_clonal_isolates_of_freshwater_autotrophic_picoplankton_by_single-cell_and_single-colony_sorting_J_Microbiol_Meth_55_361-370?el=1_x_8&enrichId=rgreq-0919a7a7-a346-444f-952b-560d6f63c5a6&enrichSource=Y292ZXJQYWdlOzIzMjQwNzI4ODtBUzoxNDQzMjEzNTc1NTM2NjRAMTQxMTQyMDMwMTQzMw==


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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 cellcounts every two days using an Improved Neubauer haemo-

cytometer 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. Thespecific growthrate of each isolatewas

calculated from the slopeof the linearregression of time (days)

and cell density (cells mL1

), asspecified by Wood etal. [11] i.e.:m ¼ ðln Nt ln NoÞ=ðt toÞ

where Nt is cell density at time (t), and No is cell density at the

start of the exponential phase (to).

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

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 fluores-

cence intensity of NR stained cultures [12] simultaneously to cell

growth. 10 mL of NR was added to 3 mL of algal suspensionswhichwasthenvortexedfor 1 minpriorto incubation for10 min

in darkness at room temperature. NR emits a yellow-gold fluo-

rescence 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 excita-

tion and emission wavelength of 480 nm and 575 nm respec-

tively 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 sub-

tracted from that measured as NR fluorescence.

2.7. Biomass dry weight and total lipid content atstationary 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 modi-

fied 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 fol-

lowed by centrifugation at 5000 rpm at 12 C for 10 min (Bio-

fuge 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 undergophase 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 etal. [16]. Dryalgal 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

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/MSwasa 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 Cmin1, 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 isola-

tion, pre-screening and screening. Pre-screening, mainly basedon 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.Fattyacid profile is a criticalfactorfor decision

of selection of candidate strain as it ultimately determines

biodiesel quality. Selection criteriafor screening were based on

biomass productivity and intracellular lipid content. Strainswhichdemonstratedcomparativelyhigh biomassproductivity,

lipid content, and a suitable fatty acid profile were selected.

2.10. Statistical analysis

Statistical analyses were performed using SigmaStat 3.5 soft-

ware. The screening data were analyzed using either a t-test

or one-way analysis of variance (ANOVA), followed by a Stu-

denteNewmaneKeuls (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 rangedfrom 18& (at Kranji)

to 37& (at Changi).

3.2. Automated isolation usingflowcytometriccellsorting

Isolation of suitable microalgae strains is an essential prereq-

uisite 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


https://www.researchgate.net/publication/223869362_Fluorometric_determination_of_the_neutral_lipid_content_of_microalgal_cells_using_Nile_Red_J_Microbiol_Meth_6333-345?el=1_x_8&enrichId=rgreq-0919a7a7-a346-444f-952b-560d6f63c5a6&enrichSource=Y292ZXJQYWdlOzIzMjQwNzI4ODtBUzoxNDQzMjEzNTc1NTM2NjRAMTQxMTQyMDMwMTQzMw==https://www.researchgate.net/publication/224094532_Practical_Flow_Cytometry?el=1_x_8&enrichId=rgreq-0919a7a7-a346-444f-952b-560d6f63c5a6&enrichSource=Y292ZXJQYWdlOzIzMjQwNzI4ODtBUzoxNDQzMjEzNTc1NTM2NjRAMTQxMTQyMDMwMTQzMw==https://www.researchgate.net/publication/10137262_A_Sample_Method_for_the_Isolation_and_Purification_of_Total_Lipids_from_Animal_Tissue_J_Biol_Chem_226_497-509?el=1_x_8&enrichId=rgreq-0919a7a7-a346-444f-952b-560d6f63c5a6&enrichSource=Y292ZXJQYWdlOzIzMjQwNzI4ODtBUzoxNDQzMjEzNTc1NTM2NjRAMTQxMTQyMDMwMTQzMw==https://www.researchgate.net/publication/223869362_Fluorometric_determination_of_the_neutral_lipid_content_of_microalgal_cells_using_Nile_Red_J_Microbiol_Meth_6333-345?el=1_x_8&enrichId=rgreq-0919a7a7-a346-444f-952b-560d6f63c5a6&enrichSource=Y292ZXJQYWdlOzIzMjQwNzI4ODtBUzoxNDQzMjEzNTc1NTM2NjRAMTQxMTQyMDMwMTQzMw==https://www.researchgate.net/publication/224094532_Practical_Flow_Cytometry?el=1_x_8&enrichId=rgreq-0919a7a7-a346-444f-952b-560d6f63c5a6&enrichSource=Y292ZXJQYWdlOzIzMjQwNzI4ODtBUzoxNDQzMjEzNTc1NTM2NjRAMTQxMTQyMDMwMTQzMw==https://www.researchgate.net/publication/248287974_Spectrofluorometric_quantification_of_neutral_and_polar_lipids_in_zooplankton_using_Nile_Red_Marine_Chem_67_289-301?el=1_x_8&enrichId=rgreq-0919a7a7-a346-444f-952b-560d6f63c5a6&enrichSource=Y292ZXJQYWdlOzIzMjQwNzI4ODtBUzoxNDQzMjEzNTc1NTM2NjRAMTQxMTQyMDMwMTQzMw==


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selection of microalgae with the desired phenotypes for feed-

stock 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 fromthe 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 chlo-

rophyll 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 fluo-

rescence, 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 dinoflagel-

lates, diatoms, green algae, cyanobacteria, and raphidophytes.

Fig. 3 e Microalgae strain screening procedure.


https://www.researchgate.net/publication/6527958_Green_Autofluorescence_in_Dinoflagellates_Diatoms_and_Other_Microalgae_and_Its_Implications_for_Vital_Staining_and_Morphological_Studies?el=1_x_8&enrichId=rgreq-0919a7a7-a346-444f-952b-560d6f63c5a6&enrichSource=Y292ZXJQYWdlOzIzMjQwNzI4ODtBUzoxNDQzMjEzNTc1NTM2NjRAMTQxMTQyMDMwMTQzMw==https://www.researchgate.net/publication/11910563_Flow_cytometric_analysis_of_chronic_and_acute_toxicity_of_copperII_on_the_marine_dinoflagellate_Amphidinium_carterae?el=1_x_8&enrichId=rgreq-0919a7a7-a346-444f-952b-560d6f63c5a6&enrichSource=Y292ZXJQYWdlOzIzMjQwNzI4ODtBUzoxNDQzMjEzNTc1NTM2NjRAMTQxMTQyMDMwMTQzMw==https://www.researchgate.net/publication/6527958_Green_Autofluorescence_in_Dinoflagellates_Diatoms_and_Other_Microalgae_and_Its_Implications_for_Vital_Staining_and_Morphological_Studies?el=1_x_8&enrichId=rgreq-0919a7a7-a346-444f-952b-560d6f63c5a6&enrichSource=Y292ZXJQYWdlOzIzMjQwNzI4ODtBUzoxNDQzMjEzNTc1NTM2NjRAMTQxMTQyMDMwMTQzMw==https://www.researchgate.net/publication/11910563_Flow_cytometric_analysis_of_chronic_and_acute_toxicity_of_copperII_on_the_marine_dinoflagellate_Amphidinium_carterae?el=1_x_8&enrichId=rgreq-0919a7a7-a346-444f-952b-560d6f63c5a6&enrichSource=Y292ZXJQYWdlOzIzMjQwNzI4ODtBUzoxNDQzMjEzNTc1NTM2NjRAMTQxMTQyMDMwMTQzMw==


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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. Nanno-chloropsis sp. plus some unidentified strains were less abun-

dant 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 weresubjected to

the control procedure. The success rate of one-cell-per-well

sorting was high, ranging from 82% to 100%. Moreover,

50e

75% of thesorted cells remained viable. This ratio is higherthan 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 isolatedmicroalgae

Following isolation, a total of 96 isolated strains were pre-

screened via NR fluorescence of cellular neutral lipid and epi-

fluorescence 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 micro-

algal 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 accli-

mation. Isolates that grew rapidly and manifested strong

yellow-gold fluorescence from NR stained cells (i.e. reflecting a relativelyhigh neutrallipid content)were further screened. A

totalof21strainsofthe96isolatedmicroalgaeweresuitablefor

further characterization, and results are shown in Table 2.

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

Sampling locations

Number of isolatedstrains

Number of selectedstrains

Number of diatoms

Number of greenalgae

Number of motile/flagellates

Pulau Ubin island 39 9 35 (89.7)a 4 (10.3) NA

St John’s island 26 4 23 (88.5) 3 (11.5) NAKranji coast 15 4 9 (60) 3 (20) 3 (20)

Second link 13 3 10 (76.9) 2 (15.4) 1 (0.08)

Changi 3 1 3 NA 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.

Fig. 4 e Photos of the representative isolated microalgal strains from Singapore’s coastal water. (a) Skeletonema costatum; (b)

Thalassionema nitzschoides; (c) Cyclotella sp; (d) Nannochloropsis sp; (e) Thalassiosira sp.; (f) Dunaliella sp.



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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, Ach-

nanles sp. and Chaetoceros sp. had higher specific growth rates

i.e. 0.87e0.99 d1 than other strains. However, the Nanno-

chloropsis 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 furtherinvestigated 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 intracel-

lular 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 approxi-

mately 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

thebest potential since they metthe balancebetween relatively

high biomassproductivity anda 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 lipidsare composed of neutral lipid in theform 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

Table 2 e Genera, growth rate, biomass and lipid content of selected microalgal strains.

Strain Genera/species

Growthrate(d1)

Biomassconcentration

(g L1)

Lipidcontent

(% of DW)

1 Skeletonema

costatum

0.95 0.09 0.01 9.5 1.7c

4 Thalassiosira sp. 0.61 0.08 0.03 17.8 3.8b

5 Thalassionema

nitzschoides

0.58 0.05 0.03 2.8 0.1c

18 Skeletonema sp. 0.59 0.24 0.06 2.7 0.4c

19 Thalassiosira sp. 0.79 0.05 0.01 0.9 0.1c

30 Thalassiosira sp. 0.85 0.04 0.01 13.6 1.2b

31 Nannochloropsis sp. 0.62 0.39 0.09 42.4 1.2a

34 Chaetoceros sp. 0.87 0.06 0.01 16.3 4.0b

35 Chaetoceros socialis 0.64 0.07 0.02 2.2 1.4c

40 Achnanthes sp. 0.99 0.09 0.03 44.5 1.7a

41 Cyclotella sp. 0.41 0.20 0.02 12.7 2.0c

42 Unidentified 0.48 0.18 0.01 11.2 1.8c

43 Unidentified 0.57 0.11 0.02 32.6 2.7b


45 Cryptomomas sp. 0.47 0.07 0.02 28.6 0.6b





50 Heterosigma sp. 0.50 0.07 0.01 39.9 2.0a


Note: Biomass concentration and lipid content at stationary phase.

Data expressed as mean SD (n ¼ 3). Means of total lipid content

are compared using one-way ANOVA, followed by a Stu-

denteNewmaneKeuls (SNK) multiple comparison test. Letters

represented the order of significant differences between themeans: a > b > c.

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



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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 accu-mulation 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 ¼ 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

0

5

10

15

20

25

30

35

40

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

Strain

R e l a t i v e f l u o r e s c e n c e i n t e n s i t y ( a . u . )

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 ).

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

Fatty acid Strains

31 40 44 46 47 48 49 50 51

Myristic acid (C14:0) 3.5 0.5 7.0 0.3 3.5 0.1 3.7 0.2 3.4 0.2 3.4 0.3 3.8 0.6 9.0 02.3 4.4 0.0

Pentadecanoic acid (C15:0) 0.5 0.1 1.5 0.1 0.5 0.0 0.6 0.1 0.5 0.1 0.6 0.1 nd 0.7 0.3 nd

Palmitoleic acid (C16:1) 29.2 3.9 45.0 5.5 29.8 6.5 29.4 6.5 31.0 6.1 30.7 4.9 32.8 3.2 20.5 4.3 30.0 0.3

Palmitic acid (C16:0) 28.4 6.2 30.4 3.5 29.6 2.4 29.9 5.8 29.5 4.5 29.2 4.5 31.4 1.8 33.4 2.6 40.8 0.1

Linolenic acid (C18:3) nd 0.5 0.1 nd nd nd nd 0.6 0.1 3.6 0.3 nd

Linoleic acid (C18:2n6) 2.1 1.2 1.0 0.2 0.6 0.3 1.2 0.1 1.4 0.5 2.1 0.8 2.1 0.9 5.0 0.9 0.7 0.1

Oleic acid (C18:1n9) 5.5 1.9 0.94 0.2 5.9 1.4 6.4 1.5 6.3 1.7 6.6 1.2 6.4 0.9 10.6 2.3 12.0 0.0

Stearic acid (C18:0) 2.0 0.5 0.6 0.2 3.2 0.9 2.4 0.9 1.9 0.1 1.8 0.1 2.3 0.5 0.9 0.3 1.8 0.2

Arachidonic acid (C20:4n6) 4.8 1.5 0.6 0.1 4.1 2.3 6.2 0.4 5.3 0.0 5.4 0.1 6.4 0.9 1.8 0.3 2.5 0.1

Eicosapentaenoic acid (C20:5n3) 18.6 4.5 12.3 2.5 18.2 4.5 18.5 4.5 17.9 5.5 17.6 5.2 12.4 4.3 13.0 4.3 7.5 0.1

Docosahexaenoic acid (C22:6n3) nd nd nd nd nd nd nd 2.1 0.7 nd

Behenic acid (C22:0) nd nd nd nd nd nd nd 1.5 0.1 nd

Lignoceric acid (C24:0) 1.4 0.5 0.8 0.2 1.9 0.5 1.6 0.5 1.2 0.4 0.5 0.1 1.3 0.3 0.6 0.1 0.45 0.1

Others 3.9 0.8 2.2 0.5 3.4 1.5 1.4 0.4 2.1 0.8 2.9 0.9 1.5 0.1 4.1 0.9 0.3 0.0

nd¼ 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 ¼ 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.



10/12


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. Basedon 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,whereemission of NR fluorescencewas

representative of total intracellular neutral lipid [12,31]. Ach-

nanthes sp. (strain 40) had the highest NR fluorescence inten-

sity 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 fluo-rescence 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 favour-

able Nannochloropsis strains.

3.6. Fatty acid profile of isolated strains

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].

Thecarbonchainlengthofsaturatedandunsaturatedfattyacids

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,oilsdominatedbythesefattyacidsarepronetosolidify

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.

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 characterizedwith 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 thetotalFAME. Threestrains 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 fromfurther evaluation. SFA andMUFA in

Nannochloropsis strains31, 47 and51 were predominant at>70%

of the total lipid content (see Table 4) which is favourable forhigh 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-valuefattyacidsforhumannutritionandfoodadditives.In

order to comply with biodiesel standard on the PUFA ratio, AA

and EPA of Nannochloropsis canbe extracted before the restof oil

can be converted to biodiesel. This makes Nannochloropsis-

derived biodiesel less susceptible to oxidation in storage and

takes full advantage of Nannochloropsis.

4. Conclusions

High-throughput isolation, using flow cytometric cell sor-

ting based on red and green fluorescence, and forward-light

scatter was used to isolate a diverse range of microalgae taxa

from Singapore’s 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

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

Properties Strains

31 40 44 46 47 48 49 50 51

SFA (% of total FAME) 35.7 1.8 40.2 5.4 35.5 4.9 38.1 5.6 36.5 1.2 35.3 1.3 39.0 0.8 45.4 2.5 47.5 0.7MFA (% of total FAME) 34.8 2.9 45.9 3.4 35.6 1.8 35.7 4.9 37.3 3.9 37.3 3.7 39.1 5.3 31.0 5.7 41.8 1.7

PUFA (% of total FAME) 27.0 6.8 14.3 3.7 25.9 1.7 25.2 2.7 24.6 1.6 25.2 1.9 21.1 1.6 23.7 3.7 10.7 0.9

Total FAME content (% DW) 19.8 2.7a 15.8 2.7b 15.5 1.2b 11.2 2.7b 16.6 1.4a 14.7 3.1b 14.1 2.2b 19.6 1.7a 21.3 1.6a

Total FAME content (% total lipid) 46.7 2.5 35.5 2.9 39.2 3.1 25.0 2.3 37.1 4.1 36.7 2.6 33.5 2.8 48.8 3.3 49.8 2.5

SFA¼ Saturated fatty acids (14:0, 15:0, 16:0, 18:0, 22:0, 24:0), MUFA ¼monounsaturated fatty acids (16:1, 18:1n9), PUFA¼ polyunsaturated fatty

acids (18:2n6, 18:3n3, 20:4n6, 20:5n3, 22:6n3). Data expressed as mean SD (n ¼ 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.



11/12


number for biodiesel feedstock. The screening procedure

provided useful ways to select microalgae, and is recom-

mended as an essential process for biodiesel feedstock

production. In the screening process, NR fluorescence was

used as a high-throughput screening method for determina-

tion of microalgae cellular lipid content. Besides lipid content,biomass production is also a critical factor to select a favour-

able microalgae strain. Nannochloropsis strains are undergoing

on-going investigation to further enhance biomass produc-

tivity 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 theuse of the flowcytometer, Dang TheCuong and

Sarah Siti Binte Aziz for microalgae sampling and

identification.

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