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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
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
b

article info

abstract

Article history:

In this work, the screening of 147 microalgal strains from the Persian Gulf and the Qeshm

Received 13 December 2010

Island (Iran) were done in order to choose the best ones, in terms of growth (biomass) rate

Received in revised form

and lipid content for biodiesel production. A methodology, combining experiments in lab-

20 January 2011

scale and pilot plant (open pond) used to produce and evaluate biomass and lipid

Accepted 20 January 2011

productivity is presented for the systematic investigation of the potential of different

Available online 12 February 2011

microalgae species. The culture conditions, including photo flux (180 mE m
2 s
1), photoperiod (12 h light/dark), temperature (25  C), pH (z8), air (carbon dioxide) and growth

Keywords:

medium, were kept constant for all experiments. Microalgae were screened in two stages

Microalgae

using optical density (for evaluation of biomass concentration) and Nile red and gas

Biodiesel

chromatography (for determination of lipid content and fatty acid fractions). In general,

Oil extraction

maximum specific growth rate and the maximum biomass productivity were obtained

Biomass evaluation

after 8e12-day culture. Nannochloropsis sp. and Neochloris sp. were selected from the marine

Fatty acid

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 profile, namely linolenic 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 fixing 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 photobiologically 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 first 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).
0961-9534/$ e see front matter 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biombioe.2011.01.039

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Table 1 e Comparison of some sources of biodiesel.

Feedstocks
Soybean
Rapeseed
Jatropha
Palm oil
Microalgaea

Collection of microalgal samples

from
different aquatic environments

Conversion Biodiesel yield

Oil yield
(%)
(kg ha
1 year
1)
(kg ha
1 year
1)
375
1000
2000
5000
75,000

95
95
98
94
80

356
950
1960
4700
60,000

Microalgae cultivation

a 50 wt. % oil in biomass.

Source: Lim and Teong [9].

investigated microalgae cultivation for producing renewable

liquid fuels [8].
Fatty acid methyl esters (FAMEs) originating from vegetable
oils are known as 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).
This is due to the several distinct advantages that the production
of biodiesel using microalgae can enjoy as listed below [10,11]:

Harvesting

Preliminary screening
(biomass)

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 CO2 and wastewater treatment (absorbs
CO2 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.

Secondary screening
(oil production)

Fatty acid fractions

Biodiesel production

Lipid composition and productivity of microalgae depend

on growth conditions such as medium composition, irradiance and temperature [12]. On the other hand, the properties
of a biodiesel fuel, including its ignition quality, combustion
heat, cold filter plugging point (CFPP), oxidative stability,
viscosity, and lubricity, are determined by the structure of its
component fatty acids [13]. Qualitatively, the profiles 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 cyanobacteria to oleaginous (oil-rich) strains from eukaryotic algal taxa.
Quantitatively, the total lipid content varies between species
ranging from very low (4.5%) to very high (80%) [12].

Fig. 1 e Screening of microalgae for production of biodiesel.

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 microalgae [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 profile was also analyzed for
the selected microalgae.

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
Qeshm Island

7.6
8.2

7.88
5.07

24
28

0.023
0.665

0.369
0.397

0.015
0.025

1.38
1.83

D.O.: disolved oxygen.

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1.6

Table 4 e Oil content and lipid concentration in selected

microalgal strains.

Cell concentration (OD)

1.4
1.2
1.0
0.8

PTCC
6016

PTCC
6003

PTCC
6022

PTCC
6001

52

46

38


32

Lipida
Oil content (%)

0.6
0.4
0.2
0.0
0

10

12

14

16

18

strongly red fluorescence, red fluorescence,  weakly red

fluorescence.
a Microalgae strains with high-lipid content selected by Nile red
staining.

Time ( days)

Fig. 2 e Cell concentration for the 24 highly biomass

producing microalgal strains.

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
(26 570 N, 55 250 E) and Persian Gulf (26 320 N, 53 560 E) (Iran).
The physico-chemical characteristics of the sites seawater
are presented in Table 2. Selected strains including three
microalgae and one diatom were identified and deposited in
Persian Type Culture Collection as strains PTCC 6016 (Nanochloropsis 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 first step is the selection of appropriate species with the highest growth (biomass)
rate in specific culture conditions. Experiments were performed in 500 mL Erlenmeyer flasks containing 750 mL of
enriched seawater medium. Growth medium was based on sea
salt and RM medium (300 mg NaNO3, 20 mg KH2PO4, 80 mg
K2HPO4, 58.5 mg CaCl2$2H2O, 20 mg NaCl, 10 mg MgSO4$7H2O,
1.5 mg MnSO4$H2O, 300 mg H2BO3, 300 mg (NH4O6Mo7O24)$H2O,
260 mg Co(NO3)2$6H2O, 80 mg CuSO4$5H2O). The culture conditions, including photo flux (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 certain periods using

a wet/dry vacuum, dewatered by sieving harvested material
through 2-mm mesh nylon netting to approximately 10% 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-mm sieve and stored
in sealed plastic bags at 20e25  C. The cell density was
measured with a spectrophotometer (Unicom UVeVis Spectrometry) using 540 nm wavelength. The biomass productivity
was calculated by optical density (OD) of the cells.

2.3.

Nile red staining

At the second step, 24 microalgal stains, which had the 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 same solution (0.5 mL), the Nile red solution (0.1 mg mL
1 in
acetone) was added to cell suspensions (1:100 v/v) and incubated for 10 min. After washing once, stained microalgal cells
were observed by fluorescent microscopy.

2.4.

Oil extraction

Four microalgae which had the most lipid droplets, were used
in the third step. Oil content was extracted from algal samples
using a modified Folch and Lees method [16]. Dried algal
samples (50 g) were extracted for 20 min with 20 mL of chloroform/methanol (2:1 v/v) at 25  C after cell disruption in a 25 W
sonication bath (Branson model 2510) for 1 min. Tubes were
then incubated for 10 min at room temperature to allow the

Table 3 e Microalgae biomass productivities.

Strains no.
PTCC
PTCC
PTCC
PTCC

6016
6003
6001
6022

Average biomass
concentration (g L
1)

Maximum biomass
concentration (g L
1)

Productivities
(mg L
1 d
1)

Cell conc.
(ODa)

50.0
21.7
12.6
5.1

64.2
48.6
28.1
13.9

46.5
32.6
19.3
13.0

1.50
1.38
0.96
0.81

a Optical density at 540 nm and 8 days.

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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 figure legend, the reader is referred to the web version of this article.)

organic and aqueous layers to separate. After removing and

saving the bottom (organic) layer, the aqueous layer was reextracted 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
from Fig. 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 microalgae 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 fluorescence microscopy. Neutral lipids including hydrocarbons and triglycerides were stained in yellow, while polar
lipids were stained in red. Among 24 marine microalgal
strains, 4 strains showed obvious red fluorescence, 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.
In a previous report, palmitic, stearic, oleic, and linolenic acid
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 contained a low amount of polyunsaturated fatty acid methyl esters

Fig. 4 e (A) Indoor and (B) outdoor raceway ponds agitated by paddle wheels for production of selected microalgae.

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 ) 1 9 3 5 e1 9 3 9

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
C15:0
C16:0
C17:0
C18:0
C18:1
C18:2
C18:3

NDa
ND
23.4
ND
7.14
45.4
11.7
12.2

5.22
ND
29.4
5.2
6.6
17.5
23.6
12.6

2.8
5.7
39.9
27.8
3.4
10.5
6.2
4.0

9.0
3.5
37.4
4.6
5.3
16.9
11.6
ND

a ND: not detected.

(C18:2 and C18:3), which is a significant difference compared to

edible oils such as soybean and sunflower. Among the tested
microalgal species, PTCC 6016 showed the highest oleic acid
(45.4%) content, making it the most suitable for the production
of good quality biodiesel. Rashid et al. [20] reported that oils with
a high oleic acid content have a reasonable balance of fuel
properties. As such, a higher oleic acid content increases the
oxidative stability for longer storage and decreases the cold filter
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 specific
growth rate, the maximum biomass production and the maximum 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 materials 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. Palmitic acid (C16:0) is the predominate fatty acid in the four
selected microalgal strains, while oleic acid (C18:1) was highest
in PTCC 6016 at 45.4%. In conclusion, a microalga, PTCC 6016 was
identified 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 financial support to carry out this
research work.

1939

references

[1] Demirbas A. Use of algae as biofuel sources. Energy Convers

Manage 2010;51:2738e49.
[2] Chisti Y. Biodiesel from microalgae. Biotechnol Adv 2007;25:
294e306.
[3] Sharma YC, Singh B, Upadhyay SN. Advancements in
development and characterization of biodiesel: a review.
Fuel 2008;87:2355e73.
[4] Gouveia L, Oliveira AC. Microalgae as a raw material for
biofuels production. J Ind Microbiol Biotechnol 2009;36:
269e74.
[5] Ahmad AL, Yasin NHM, Derek CJC, Lim JK. Microalgae as
a sustainable energy source for biodiesel production:
a review. Renew Sust Energy Rev 2011;15:584e93.
[6] Halim R, Gladman B, Danquah MK, Webley PA. Oil extraction
from microalgae for biodiesel production. Bioresour Technol
2011;102:178e85.
[7] Eriksen N. The technology of microalgal culturing.
Biotechnol Lett 2008;30:1525e36.
[8] Rodolfi L, Zittelli GC, Bassi N, Padovani G, Biondi N,
Bonini G, et al. Microalgae for oil: strain selection,
induction of lipid synthesis and outdoor mass cultivation
in a low-cost photobioreactor. Biotechnol Bioeng 2009;102:
100e12.
[9] Lim S, Teong LK. Recent trends, opportunities and challenges
of biodiesel in Malaysia: an overview. Renew Sust Energy Rev
2010;14:938e54.
[10] Miao X, Wu Q. Biodiesel production from heterotrophic
microalgal oil. Bioresour Technol 2006;97:841e6.
[11] Widjaja A, Chien C-C, Ju Y-H. Study of increasing lipid
production from fresh water microalgae Chlorella vulgaris.
J Taiwan Inst Chem Eng 2009;40:13e20.
[12] Huerlimann R, de Nys R, Heimann K. Growth, lipid content,
productivity, and fatty acid composition of tropical
microalgae for scale-up production. Biotechnol Bioeng 2010;
107:245e7.
[13] Lee J-Y, Yoo C, Jun S-Y, Ahn C-Y, Oh H-M. Comparison of
several methods for 2 effective lipid extraction from
microalgae. Bioresour Technol 2010;101:S75e7.
[14] Solovchenco AE, Khozin-Goldberg I, Didi-Cohen S, Cohen Z,
Merzlyak MN. Effects of light intensity and nitrogen
starvation on growth, total fatty acids and arachidonic acid
in the green microalga Parietochloris incise. J Appl Phycol 2008;
20:245e51.
[15] Greenspan P, Mayer EP, Fowler SD. Nile red: a selective
fluorescent stain for intracellular lipid droplets. J Cell Biol
1985;100:965e73.
[16] Folch J, Lees M. A simple method for isolation and
purification of total lipids from animal tissues. J Biol Chem
1957;226:497e509.
[17] Mulbry W, Kondrad S, Buyer J, Luthria DL. Optimization of an
oil extraction process for algae from the treatment of
manure effluent. J Am Oil Chem Soc 2009;86:909e15.
[18] Leung DYC, Wu X, Leung MKH. A review on biodiesel
production using catalyzed transesterification. Appl Energy
2010;87:1083e95.
[19] Knothe G. Designer biodiesel: optimizing fatty ester
composition to improve fuel properties. Energy Fuels 2008;
22:1358e64.
[20] Rashid U, Anwar F, Moser BR, Knothe G. Moringa oleifera oil:
a possible source of biodiesel. Bioresour Technol 2008;99:
8175e9.