Biomass and lipid productivities of marine microalgae

isolated from the Persian Gulf and the Qeshm Island

Nasrin Moazami
a
, Reza Ranjbar
b
, Alireza Ashori
c,
*, Mehrnoush Tangestani
a
,
Ali Sheykhi Nejad
a
a
Department of Biotechnology, Iranian Research Organization for Science and Technology (IROST), Tehran, Iran
b
Faculty of Chemical Engineering, Amirkabir University of Technology (AUT), Tehran, Iran
c
Department of Chemical Technologies, Iranian Research Organization for Science and Technology (IROST), PO Box 15815-3538,
Tehran, Iran
a r t i c l e i n f o
Article history:
Received 13 December 2010
Received in revised form
20 January 2011
Accepted 20 January 2011
Available online 12 February 2011
Keywords:
Microalgae
Biodiesel
Oil extraction
Biomass evaluation
Fatty acid
a b s t r a c t
In this work, the screening of 147 microalgal strains from the Persian Gulf and the Qeshm
Island (Iran) were done in order to choose the best ones, in terms of growth (biomass) rate
and lipid content for biodiesel production. A methodology, combining experiments in lab-
scale and pilot plant (open pond) used to produce and evaluate biomass and lipid
productivity is presented for the systematic investigation of the potential of different
microalgae species. The culture conditions, including photo ux (180 mE m
2
s
1
), photo-
period (12 h light/dark), temperature (25

C), pH (z8), air (carbon dioxide) and growth
medium, were kept constant for all experiments. Microalgae were screened in two stages
using optical density (for evaluation of biomass concentration) and Nile red and gas
chromatography (for determination of lipid content and fatty acid fractions). In general,
maximum specic growth rate and the maximum biomass productivity were obtained
after 8e12-day culture. Nannochloropsis sp. and Neochloris sp. were selected from the marine
microalgal culture collection, due to their high biomass (50 and 21.7 g L
1
, respectively) and
oil content (52% and 46%, respectively). If the purpose is to produce biodiesel only from one
species, Nannochloropsis sp. presented the most adequate fatty acid prole, namely lino-
lenic and other polyunsaturated fatty acids. However, the microalgae Chlorella sp. can also
be used if associated with other microalgal oils. In addition, selected strains could be
potent candidates for commercial production in the open pond culture.
2011 Elsevier Ltd. All rights reserved.
1. Introduction
Microalgae are unicellular photosynthetic microorganisms,
living in saline or fresh water environments that convert
sunlight, water and carbon dioxide to algal biomass [1]. In
addition, they are useful in bioremediation applications and
as nitrogen xing biofertilizers. Microalgae can provide
several different types of renewable biofuels. These include
methane produced by anaerobic digestion of the algal
biomass, biodiesel derived from microalgal oil, and photo-
biologically produced biohydrogen [2]. In recent years, usage
of microalgae as biodiesel feedstock has attracted great
attention [2e7]. The idea of using microalgae as a source of
biodiesel is not new. It was rst proposed in the 1950s and,
since the 1970s, several publicly funded research programs in
different countries (such as USA, Australia and Japan) have
* Corresponding author. Tel./fax: 98 228 2276629.
E-mail address: ashori@irost.org (A. Ashori).
Avai l abl e at www. sci encedi r ect . com
ht t p: / / www. el sevi er . com/ l ocat e/ bi ombi oe
b i o ma s s a nd b i o e ne r gy 3 5 ( 2 0 1 1 ) 1 9 3 5 e1 9 3 9
0961-9534/$ e see front matter 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biombioe.2011.01.039
investigated microalgae cultivation for producing renewable
liquid fuels [8].
Fatty acid methyl esters (FAMEs) originating from vegetable
oils are knownas biodiesel. It is a biodegradable, renewable and
non-toxic fuel [2e4]. Microalgae have been cited as one of the
best non-edible feedstock for biodiesel compared to oleaginous
crops, such as soybean, rapeseed and even oil palm (Table 1).
Thisisduetotheseveral distinct advantages that theproduction
of biodiesel using microalgae can enjoy as listed below [10,11]:
1. Higher oil productivity (at least 15e20 times higher oil yield
per hectare than conventional crops).
2. Fast reproduction (biomass doubling within 3.5 h during
exponential growth).
3. Does not necessarily require arable land and fresh water.
4. Sequestration of CO
2
and wastewater treatment (absorbs
CO
2
along with nitrates and phosphates while releasing
oxygen and water).
5. Rich in valuable co-products (Spirulina-EX and biomass for
animal feed).
6. Require much less land areas compared to conventional
crops.
7. Non-food source.
Lipid composition and productivity of microalgae depend
on growth conditions such as medium composition, irradi-
ance and temperature [12]. On the other hand, the properties
of a biodiesel fuel, including its ignition quality, combustion
heat, cold lter plugging point (CFPP), oxidative stability,
viscosity, and lubricity, are determined by the structure of its
component fatty acids [13]. Qualitatively, the proles of fatty
acids, ranging in length from 10 to 24 carbons, are often
similar between species of the same phyla or classes but differ
greatly between classes and phyla from low-yield cyanobac-
teria to oleaginous (oil-rich) strains fromeukaryotic algal taxa.
Quantitatively, the total lipid content varies between species
ranging from very low (4.5%) to very high (80%) [12].
Out of hundreds of microalgal strains reported, only very
few of them are capable of producing high contents of lipid
[3,14]. It is to be noted that most of them are marine micro-
algae [10]. Therefore, the key technical challenges include
identifying the strains with the highest growth rates and oil
contents with adequate composition, which were the main
aims of this work. The fatty acid prole was also analyzed for
the selected microalgae.
Table 1 e Comparison of some sources of biodiesel.
Feedstocks Oil yield
(kg ha
1
year
1
)
Conversion
(%)
Biodiesel yield
(kg ha
1
year
1
)
Soybean 375 95 356
Rapeseed 1000 95 950
Jatropha 2000 98 1960
Palm oil 5000 94 4700
Microalgae
a
75,000 80 60,000
a 50 wt. % oil in biomass.
Source: Lim and Teong [9].
Table 2 e Physico-chemical characteristics of the seawater of the collection sites.
Site pH D.O.
(mg L
1
)
Temp.
(

C)
Nitrate
(mg L
1
)
Salinity
(mg L
1
)
Phosphate
(mg L
1
)
Total chlorophyll
(mg L
1
)
Persian Gulf 7.6 7.88 24 0.023 0.369 0.015 1.38
Qeshm Island 8.2 5.07 28 0.665 0.397 0.025 1.83
D.O.: disolved oxygen.
Microalgae cultivation
Preliminary screening
(biomass)
Harvesting
Secondary screening
(oil production)
Fatty acid fractions
Biodiesel production
Collection of microalgal samples
from
different aquatic environments
Fig. 1 e Screening of microalgae for production of biodiesel.
b i o ma s s a nd b i oe ne r g y 3 5 ( 2 0 1 1 ) 1 9 3 5 e1 9 3 9 1936
2. Materials and methods
2.1. Strains
One hundred and forty seven marine microalgal strains were
used in our laboratory. The samples were isolated from
mangrove forests in the northern part of Qeshm Island
(26

57
0
N, 55

25
0
E) and Persian Gulf (26

32
0
N, 53

56
0
E) (Iran).
The physico-chemical characteristics of the sites seawater
are presented in Table 2. Selected strains including three
microalgae and one diatom were identied and deposited in
Persian Type Culture Collection as strains PTCC 6016 (Nano-
chloropsis sp.), PTCC 6003 (Nanochloropsis sp.), PTCC 6022
(Chlorella sp.) and PTCC 6001 (Nitzschia sp.).
2.2. Production of microalgal biomass
Fig. 1 shows a schematic of the production and screening of
microalgal strains. After cultivation, the rst step is the selec-
tion of appropriate species with the highest growth (biomass)
rate in specic culture conditions. Experiments were per-
formed in 500 mL Erlenmeyer asks containing 750 mL of
enrichedseawater medium. Growthmediumwas basedonsea
salt and RM medium (300 mg NaNO
3
, 20 mg KH
2
PO
4
, 80 mg
K
2
HPO
4
, 58.5 mg CaCl
2
$2H
2
O, 20 mg NaCl, 10 mg MgSO
4
$7H
2
O,
1.5 mg MnSO
4
$H
2
O, 300 mg H
2
BO
3,
300 mg (NH
4
O
6
Mo
7
O
24
)$H
2
O,
260 mg Co(NO
3
)
2
$6H
2
O, 80 mg CuSO
4
$5H
2
O). The culture condi-
tions, including photo ux (180 mE m
2
s
1
), photoperiod (12 h
light:12 h dark), temperature (25

C), pH (z8), air (carbon
dioxide) and nutrient concentration, were kept constant for all
strain samples. Microalgal cells have been cultured for 8e16
days. Wet algal biomass was harvested at certainperiods using
a wet/dry vacuum, dewatered by sieving harvested material
through2-mmmeshnylonnettingtoapproximately10%solids
content, then air-dried for approximately 48 h using electric
fans to approximately 90% solids content. Dried biomass was
initially ground in a Wiley Mill to pass a 3-mmsieve and stored
in sealed plastic bags at 20e25

C. The cell density was
measured with a spectrophotometer (Unicom UVeVis Spec-
trometry) using 540 nmwavelength. The biomass productivity
was calculated by optical density (OD) of the cells.
2.3. Nile red staining
At the secondstep, 24 microalgal stains, whichhadthe highest
biomass, were selected for the determination of oil content.
Nile red (9-(Diethylamino)-5H-benzo[a]phenoxazin-5-one)
staining was conducted to detect intracellular lipid droplets
[15]. Selected microalgal cells were tested for Nile red staining.
The cultured cells (0.5 mL) were collected by centrifugation for
10 min and washed with physiological saline solution (0.5 mL)
several times. After the collected cells were re-suspended in
the samesolution(0.5 mL), the Nileredsolution(0.1 mgmL
1
in
acetone) was added to cell suspensions (1:100 v/v) and incu-
bated for 10 min. After washing once, stained microalgal cells
were observed by uorescent microscopy.
2.4. Oil extraction
Four microalgae which had the most lipid droplets, were used
in the third step. Oil content was extracted fromalgal samples
using a modied Folch and Lees method [16]. Dried algal
samples (50 g) were extracted for 20 min with 20 mL of chlo-
roform/methanol (2:1v/v) at 25

Cafter cell disruptionina25W

sonication bath (Branson model 2510) for 1 min. Tubes were
then incubated for 10 min at room temperature to allow the
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
0 2 4 6 8 10 12 14 16 18
Time ( days)
D
O
(
n
o
i
t
a
r
t
n
e
c
n
o
c
l
l
e
C
)
Fig. 2 e Cell concentration for the 24 highly biomass
producing microalgal strains.
Table 3 e Microalgae biomass productivities.
Strains no. Average biomass
concentration (g L
1
)
Maximum biomass
concentration (g L
1
)
Productivities
(mg L
1
d
1
)
Cell conc.
(OD
a
)
PTCC 6016 50.0 64.2 46.5 1.50
PTCC 6003 21.7 48.6 32.6 1.38
PTCC 6001 12.6 28.1 19.3 0.96
PTCC 6022 5.1 13.9 13.0 0.81
a Optical density at 540 nm and 8 days.
Table 4 e Oil content and lipid concentration in selected
microalgal strains.
PTCC
6016
PTCC
6003
PTCC
6022
PTCC
6001
Lipid
a

Oil content (%) 52 46 38 32
strongly red uorescence, red uorescence, weakly red
uorescence.
a Microalgae strains with high-lipid content selected by Nile red
staining.
b i o ma s s a nd b i o e ne r gy 3 5 ( 2 0 1 1 ) 1 9 3 5 e1 9 3 9 1937
organic and aqueous layers to separate. After removing and
saving the bottom (organic) layer, the aqueous layer was re-
extracted by adding chloroform(6e8 mL), mixing and allowing
the layers to separate. The resulting extracts (6e10 mL each)
were stored at 4

C prior to removing aliquots for oil analysis.
A fatty acid composition analysis was performed using gas
chromatography (GC) method.
3. Results and discussion
3.1. Biomass productivity
The tested microalgae strains revealed biomass maximum
concentration within 10 days (Fig. 2). Out of 147 strains, only
24 of them had the fastest growth rate during 8e12 days,
a period that is economical for large-scale production.
Maximum dry biomass values were observed at the 8th and
10th days, which were 50 and 21.7 g L
1
, respectively. This
data is in agreement with literature [17,18]. As can be seen
fromFig. 2, four strains including PTCC 6016, PTCC 6003, PTCC
6022 and PTCC 6001 had the highest optical density of the
cells, which ranged between 0.81 and 1.50. The biomass
productivity of each selected microalgal species is shown in
Table 3. Based on the daily biomass, the highest productivity
was for PTCC 6016 at 46.5 mg L
1
d
1
. Moreover, average
concentration and productivities were varied for all micro-
algae tested ranging from 3.4 to 46.5 mg L
1
d
1
.
3.2. Selection of highly lipid-producing microalgal
strains
Nile red staining of microalgal cells cultured for 8e12 days
were performed for the detection of intracellular lipid droplets
by uorescence microscopy. Neutral lipids including hydro-
carbons and triglycerides were stained in yellow, while polar
lipids were stained in red. Among 24 marine microalgal
strains, 4 strains showed obvious red uorescence, i.e. neutral
lipid accumulation (Table 4). In particular, strains PTCC 6016,
PTCC 6003, PTCC 6022 and PTCC 6001 contained remarkable
lipid droplets. Fig. 3 shows microscopic images of strain PTCC
6016 (A) and PTCC 6003 (B). The microscopic images indicated
that the cell size of PTCC 6003 was bigger than other tested
strains. Furthermore, the extracellular lipid droplets were
ejected from the cells by the pressure on the cover glass. All
the selected strains, which were re-cultured in the large-scale
indoor and outdoor open ponds agitated by paddle wheels,
accumulated neutral lipids in less than two weeks (Fig. 4). The
above-mentioned strains were further analyzed in the
following experiments.
Inapreviousreport, palmitic, stearic, oleic, andlinolenicacid
were recognized as the most common fatty acids contained in
biodiesel [19]. As indicated in Table 5, for the four tested
microalgae, palmitic acid (C16:0) and oleic acid (C18:1) were
commonly dominant. Linoleic acid (C18:2) was highest in PTCC
6003 at 23.6% (based on dry weight), while other samples con-
tainedalowamount of polyunsaturatedfattyacidmethyl esters
Fig. 3 e Nile red staining of microalgae cells, PTCC 6016 (A) and PTCC 6003 (B). (For interpretation of the references to colour
in this gure legend, the reader is referred to the web version of this article.)
Fig. 4 e (A) Indoor and (B) outdoor raceway ponds agitated by paddle wheels for production of selected microalgae.
b i o ma s s a nd b i oe ne r g y 3 5 ( 2 0 1 1 ) 1 9 3 5 e1 9 3 9 1938
(C18:2 and C18:3), which is a signicant difference compared to
edible oils such as soybean and sunower. Among the tested
microalgal species, PTCC 6016 showed the highest oleic acid
(45.4%) content, making it the most suitable for the production
of goodqualitybiodiesel. Rashidet al. [20] reportedthat oils with
a high oleic acid content have a reasonable balance of fuel
properties. As such, a higher oleic acid content increases the
oxidativestabilityfor longer storageanddecreases thecoldlter
plugging point for use in cold regions [13].
4. Conclusion
Microalgae, the largest primary biomass, have been attracting
attention as a source of high-lipid material to produce biofuel.
A methodology combining experiments in lab-scale and pilot
plant used to predict biomass and lipid productivity is used for
the systematic investigation of the potential of different
microalgae species for biodiesel production. Maximum specic
growth rate, the maximum biomass production and the maxi-
mum biomass productivity were obtained after 8e12-day
culture. Out of 147 microalgae that were evaluated in this work,
strains PTCC 6016 and 6003 proved to be suitable as raw mate-
rials for commercial production, due to their high biomass
(50 and 21.7 g L
1
, respectively) and oil content (52% and 46%,
respectively). They are marine microalgae, which do not
compete with food crops while increasing the environmental
cultivation possibilities. However, strains PTCC 6022 and 6001
can also be used if associated with other microalgae oils. Pal-
mitic acid (C16:0) is the predominate fatty acid in the four
selected microalgal strains, while oleic acid (C18:1) was highest
inPTCC6016at 45.4%. Inconclusion, amicroalga, PTCC6016was
identied as high biomass and lipid producers. Furthermore, all
selected strains could be potent candidates for commercial
production of biofuel in the open pond (large-scale) culture.
Acknowledgments
The Ministry of Industries and Mines of Iran is gratefully
acknowledged for providing nancial support to carry out this
research work.
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Table 5 e Main fatty acids present in selected microalgal
strains in dry weight %.
Fatty
acid
PTCC
6016
PTCC
6003
PTCC
6022
PTCC
6001
C14:0 ND
a
5.22 2.8 9.0
C15:0 ND ND 5.7 3.5
C16:0 23.4 29.4 39.9 37.4
C17:0 ND 5.2 27.8 4.6
C18:0 7.14 6.6 3.4 5.3
C18:1 45.4 17.5 10.5 16.9
C18:2 11.7 23.6 6.2 11.6
C18:3 12.2 12.6 4.0 ND
a ND: not detected.
b i o ma s s a nd b i o e ne r gy 3 5 ( 2 0 1 1 ) 1 9 3 5 e1 9 3 9 1939