Journal of Applied Microbiology ISSN 1364-5072

ORIGINAL ARTICLE

Growth optimization of algae for biodiesel production

J.L. Csavina1, B.J. Stuart2, R. Guy Riefler2 and M.L. Vis3
1 Department of Chemical and Environmental Engineering, University of Arizona, Tucson, AZ, USA
2 Department of Civil Engineering, Ohio University, Athens, OH, USA
3 Department of Environmental and Plant Biology, Ohio University, Athens, OH, USA

Keywords
algae, Amphora, biodiesel, biofuels,
bioreactor, growth, Oocystis, optimization.
Correspondence
Ben J. Stuart, Ohio University, Department of
Civil Engineering, 120 Stocker Center,
Athens, OH 45701, USA.
E-mail: stuart@ohio.edu
2011 ⁄ 0226: received 7 February 2011,
revised 12 May 2011 and accepted 25 May
2011
doi:10.1111/j.1365-2672.2011.05064.x
Abstract
Aims: Algae are favourable as a biofuel source because of the potential high oil
content and fast generation of biomass. However, one of the challenges for this
technology is achieving high oil content while maintaining exponential or high
growth of the organism. Introducing a two-stage reactor to optimize both
growth and oil content of the algae could be a solution to this hurdle. The aim
of this study was to determine the reactor design parameters of the first-stage
reactor, which would optimize growth of two algal strains, Oocystis sp. and
Amphora sp.
Methods and Results: Growth kinetics were monitored by in vivo fluorescence
and correlated to dry mass for both cultures under several environmental con-
ditions during exponential growth. Temperatures of 25 and 30C and light
intensities of 150 and 80 lmol m)2 s)1 provided the most robust growth for
Oocystis sp. and Amphora sp., respectively. Both strains showed optimized
growth at a light : dark cycle of 16 : 08. At these conditions, the doubling rate
for Oocystis sp. was 0Æ333 d)1 and for Amphora sp. was 0Æ179 d)1.
Conclusions: For both cultures, growth rate was more dependent on light :
dark cycle and temperature than light intensity. Both strains grew slower in this
work than data reported in the literature, however agitation and air ⁄ CO2
sparging were not incorporated in the system under study. The highest
doubling rate for Amphora sp. was observed near the maximum tolerable
temperature, and it is suggested to grow this strain at 30C for a consistent
high growth rate.
Significance and Impact of Study: Optimized growth conditions were
determined for two lipid producing strains identified in the Aquatic Species
Program summary report. An optimized, first-stage growth reactor operating at
these conditions would thus offer the maximum productivity for an algal bio-
mass feed stream into a lipid-optimized second-stage reactor.

produce biodiesel from high lipid content algae (Sheehan

Introduction
According to the International Energy Agency (2007), the
world relied on renewable energy for 14% of its demand,
while the US produced only 4% of total demand with
renewables in 2005. Oil is the leading source for energy
generation in the US (International Energy Agency 2007).
Biofuels are currently being evaluated to determine if they
are a viable renewable source to replace oil. In 1970, the
Aquatic Species Program grew out of a national effort to
et al. 1998). In 1996, the biofuels programme shifted its
focus to corn and soybean technologies due to the fact
that corn and soybean fuels were considered a proven
technology. However, 100% of the corn and soybean
harvest in the US would only meet 12% of the gasoline
and 6% of the diesel demand (Hill et al. 2006).
The biodiesel industry would need to produce 530
billion litres annually to satisfy the US demand for
transportation fuel. Comparing different crops used for

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312 Journal of Applied Microbiology 111, 312–318 ª 2011 The Society for Applied Microbiology
J.L. Csavina et al.
biodiesel and their respective oil yields, each source takes
up large areas of land leading to significant ecological
impact. The most feasible biodiesel source for the US is
microalgae (Chisti 2007; Mata et al. 2010; Scott et al.
2010). Algae grow rapidly and can double their biomass
as much as eight times in a day during exponential
growth (Chisti 2007). Algae are efficient producers of nat-
ural oils, sequester carbon dioxide thereby reducing
greenhouse gases, and do not compromise a food stock
or deplete soil nutrients. Finding high lipid producing
strains (Griffiths and Harrison 2009; Rodolfi et al. 2009;
Mutanda et al. 2011) and selecting the appropriate
culturing and processing conditions (Rodolfi et al. 2009;
Brennan and Owende 2010; Greenwell et al. 2010; Huang
et al. 2010) are critical to realize the potential and large-
scale adoption of advanced algal biofuels.
When put into stressful environments (e.g. nutrient
starvation), algae may switch carbon allocation from
reproduction to oil production (Illman et al. 2000;
Pruvost et al. 2009; Lv et al. 2010). This oil from algae
can be extracted and turned into biodiesel through a
chemical process called transesterification (Vasudevan and
Briggs 2008). For example, one strain of algae put into a
nitrogen deficient environment transitioned from 22 to
58% oil content per dry mass (Sheehan et al. 1998).
Further, Khozin-Goldberg and Cohen (2006) reported
that phosphorous starvation significantly altered the lipid
profile of Monodus subterraneus.
Some strains of algae can grow both autotrophically
and heterotrophically, if organic carbon is supplied
(Huang et al. 2010). High biomass and high lipid yield
have been demonstrated when heterotrophic algae were
placed in low light with an organic carbon (Miao and
Wu 2004; Xu et al. 2006). However, autotrophic growth
is much faster than heterotrophic in most strains, and the
need for organic carbon may make the cultivation process
more expensive and thus less economically viable on a
commercial scale.
Sheehan et al. (1998) discovered that it was difficult to
maintain laboratory algal cultures in outdoor systems due
to changing temperatures and competition from invasive
species. Another problem is that algal biomass production
may decrease in stressful conditions such as nutrient
deficiency (Pruvost et al. 2009). Therefore, the net oil pro-
ductivity may actually decrease in a stressful environment
even with increased oil content due to reduced growth.
Sheehan et al. (1998) stated, ‘With a better understanding
of these kinetics, it may be possible to provide a net
increase in total oil productivity by carefully controlling
the timing of nutrient depletion and cell harvesting’.
Based on this model, we suggest a two-stage cultivation
system for biofuel production in which growth can be
optimized in one reactor, while oil content is maximized
ª 2011 The Authors
Journal of Applied Microbiology 111, 312–318 ª 2011 The Society for Applied Microbiology
Growth of algae for biodiesel
in a second reactor. The first stage would be a continuous
reactor at conditions that optimize the algal culture
growth. The second stage would be multiple batch reactors
at conditions that optimize the oil content of the strain of
algae (Xiong et al. 2010). The batch reactors would harvest
from the first reactor at a continual rate that maintain the
first stage in exponential growth. By using bioreactors as
opposed to an open system, the environmental conditions
can be controlled. If theoretical yields of algae and oil con-
tent are maintained in these reactors, the increased operat-
ing costs of a closed system may be offset.
Temperature is a major factor for growth in algae cul-
tures (Andersen 2005). Cho et al. (2007) studied tempera-
tures from 15–30C for optimal growth of the green alga
Chlorella ellipsoidea and determined 25C would promote
the highest growth rate. Data from the Aquatic Species
Program identified two strains of Amphora sp. that dem-
onstrated optimal growth at temperatures of 30–35C
(Sheehan et al. 1998). In addition, temperature stability
should be maintained at ±2C, particularly for marine
strains which are less tolerant to temperature variations
(Andersen 2005).
Illumination can affect growth of algae by both length of
photoperiod and intensity (Andersen 2005). Unwanted
growth effects may occur if the culture is not maintained at
appropriate light : dark cycles, such as cyst formation with
a short photoperiod (Balzer and Hardeland 1991). While
optimal light : dark cycles have been found to vary from
12 : 12 to 16 : 08 for most cultures (Andersen 2005), some
algae may be killed by continuous light (Price et al. 1998).
Over illumination can cause photo-inhibition by photo-
oxidative stress on the algae (Leon and Galvan 1999). Some
algae prefer low light intensities (<60 lmol m)2 s)1) while
others need higher light intensities (>100 lmol m)2 s)1)
(Sheehan et al. 1998; Andersen 2005).
The current research focused on the manipulation of
environmental conditions to optimize the growth of two
algal strains, Oocystis sp. and Amphora sp. The objective
of these experiments was to determine the optimal light
intensity, temperature and light : dark cycle for both algal
strains. Temperatures of 15–40C and light : dark cycles
of 14 : 10, 16 : 08 and 18 : 06 were evaluated for optimal
growth of both strains. Light intensities from 80 to
200 lmol m)2 s)1 and 40–100 lmol m)2 s)1 were selected
based on literature recommendations for Oocystis sp. and
Amphora sp., respectively (Andersen 2005).
Materials and methods
Algal cultures
Two algal strains, the chlorophyte Oocystis sp. (OOCYS9)
and the diatom Amphora sp. (AMPHO46) were obtained
313
Growth of algae for biodiesel
from the SERI ⁄ NREL Culture Collection Catalogue at the
University of Hawaii. These strains were selected based
on reported high lipid production with Amphora sp.
yielding 63 mg l)1 day)1 triolein equivalents and Oocystis sp.
yielding 46 mg l)1 day)1 triolein equivalents (Sheehan
et al. 1998).
Artificial Sea Water (ASW) media adjusted to pH = 7Æ2
with 6 N HCl was used to cultivate the two strains of algae
in unialgal conditions (McLachlan 1964). To have the
strains in a uniform initial growth stage, inocula from a
larger stationary phase stock culture was diluted with fresh
ASW media (50 ⁄ 50) in a 1 l autoclave bottle and stirred at
300 rev min)1 for 10 days. Temperature and light inten-
sity for these cultures was maintained at 25 ± 1C and
10 ± 2 lmol m)2 s)1 with a 16 : 08 light : dark cycle.
Growth measurements
Growth kinetics were monitored by in vivo fluorescence
using a TD-700 fluorometer (Turner Designs Sunnyvale,
CA, USA). The optical kit used in the fluorometer was the
Turner Designs 10-037-R configuration which included a
daylight white lamp, a wide band-pass excitation filter
(340–500 nm), and a >665 nm long pass emission filter.
In vivo fluorescence assumes the emission of light from a
culture subjected to illumination is directly proportional
to the number of cells for a large range of culture densities
(Andersen 2005). Therefore, a linear relationship between
fluorescence and dry mass was first established for both
cultures under several growth conditions while maintain-
ing an exponential growth rate. Dry mass was determined
by Standard Test Method 2540D for total suspended solids
in water using an 80 ml sample size (Clesceri et al. 1998).
The fluorometer was initially calibrated with spinach
extracted chlorophyll a (Sigma-Aldrich Chemicals,
St Louis, MO, USA) and the calibration was checked
before each set of experiments with a red solid standard.
Measurements were taken directly from cultures grown in
40-ml culture tubes and reported in fluorescence units
(fsu). The algal test tubes were taken from the incubator
and placed immediately in the sample holder to eliminate
optical variations.
Experimental set-up
The growth conditions examined in these experiments
were light intensity, light : dark cycle, and temperature. A
Thermo Electron Diurnal Growth Chamber (Thermo
Electron, Marietta, OH, USA) was used to maintain
temperature ±1C and a consistent light intensity. The
light source consisted of 20 W fluorescent bulbs, and the
appropriate light : dark cycle was achieved with a timer.
The light intensity for each experiment was established by
314 Journal of Applied Microbiology 111, 312–318 ª 2011 The Society for Applied Microbiology
J.L. Csavina et al.
mapping the interior of the growth chamber using a
Li-Cor 250A light meter (Li-Cor, Lincoln, NE, USA)
facing directly towards the light source at the mid-height
of the culture.
Preliminary experiments for Amphora sp. demonstrated
photo-inhibition with light intensities above 100 lmol
m)2 s)1, while Oocystis sp. preferred higher light levels.
Further, a value of 200 lmol m)2 s)1 was the maximum
light intensity possible for this experimental set-up result-
ing in the difference in light intensity ranges for each
algal strain. Five replicate tubes were used for each exper-
iment, and the Amphora sp. and Oocystis sp. strains were
initially diluted with fresh media to 100 and 150 fsu,
respectively, and a volume of 40 ml. Fluorescence was
measured daily at approximately the same time in the
photoperiod throughout the experiment.
During the exponential growth phase, the cell concen-
tration at a given time was proportional to the initial cell
concentration (Andersen 2005). Therefore, first order
reaction kinetics can be used to describe cell growth by
the following equation:
Nt ¼ N0 ert ð1Þ
where N0 is the number of cells present in the culture at
the beginning of a time interval, Nt is the number of cells
present in the culture at the end of a time interval, r is
the exponential growth rate and t is the time interval.
Further, by dividing r by the natural log of two, dou-
blings per day can be found. The following equation
expresses the conversion of r to doublings per day (k):
k ¼ r=06931 ð2Þ
In the analysis, linear regression correlated fluorescence
(fsu) to dry weight (mg l)1). Using this relationship, all
fluorescence data was converted to dry weight to describe
the biomass present in the culture. Dry weight was then
plotted vs time, and using the log phase of the growth
curve, an exponential curve was fit to the average kinetic
data to determine the proportional rate of change,
r. Finally, r was converted into doublings per day, and all
data were compared with determine the conditions which
promoted the highest doubling rate per day. A student’s
t-test was used to prove statistical difference with 95%
confidence.
Results
FSU to Dry weight correlations
Linear relationships were established between fluorescence
and dry weight for both algal strains (Fig. 1). Twenty data
points were obtained for both correlations. Three
ª 2011 The Authors
J.L. Csavina et al. Growth of algae for biodiesel

100 exponential stage occurred at day four in all trials, while

90 Amphora sp. indicated a one-day lag phase and an expo-
nential stage that ended at day five. Amphora sp. lag
80
Dry weight (mg l–1)

phase and end of exponential stage varied by temperature,

70
60
50
40
30
20
0 100 200 300 400 500
Fluorescence (fsu)
Figure 1 Oocystis sp. (n) and Amphora sp. (¤) fluorescence to dry
weight correlation. y = 0Æ0841 x + 14Æ4 with R2 = 0Æ965, and
y = 0Æ126 x + 24Æ5 with R2 = 0Æ959, respectively.
and two data points were taken out of the data set for
Oocystis sp. and Amphora sp., respectively due to nonlin-
earity at high cell densities. Nonlinearity in the relation-
ship between fluorescence and dry weight occurs because
of changes in fluorescence associated with self-shading
(Andersen 2005). Therefore, the linear relationship is
valid from 180 to 560 fsu for Oocystis sp., and from 95 to
490 fsu for Amphora sp. Both correlations yielded an R2
value above 0Æ95. These relationships were used to convert
fluorescence data obtained during growth experiments to
dry weight.
Exponential growth curves
Exponential curves were developed for each growth
condition, and each data point on the curves represents
the average of five replicates (Fig. 2). Oocystis sp. consis-
tently demonstrated no lag phase, and the end of the
70
60
Dry weight (mg l–1)
but remained consistent at different light intensities and
light : dark cycles at the same temperature.
Growth condition dependence
Doublings per day were used to compare growth condi-
tions for Oocystis sp. and Amphora sp. seen in Figs 3 and
600
4, respectively. Conditions tested shown on the x-axis in
Figs 3 and 4 are light intensity in lmol m)2 s)1, light:
dark cycle in h : h, and temperature in C (from top to
bottom). Student’s t-test for unequal sample sizes and
unequal variance was used to determine statistical differ-
ence between experimental results. Letters shown in Figs 3
and 4 designate groups that were found to be not statisti-
cally different (alpha = 0Æ05). In determining optimal
growth conditions, it was important to consider the indi-
vidual growth parameter at various conditions, such as a
specific light intensity at various temperatures, in case the
parameter combination limited the full scale design.
From Fig. 3, Oocystis sp. optimal conditions occur at
25C, 16 : 08 h : h, and 150 lmol m)2 s)1. Though no
statistical difference was seen between light conditions of
150 and 200 lmol m)2 s)1, averages at 150 lmol m)2 s)1
were consistently higher at the optimal temperature of
25C. The light : dark cycle and temperature were both
statistically different when comparing light intensity
replicates. Experiments at the optimal conditions were
conducted twice for verification of repeatability. The aver-
age doublings per day for the duplicate experiments were
0Æ48 ± 0Æ01 and 0Æ47 ± 0Æ02, showing good repeatability
and no statistical difference.
Amphora sp. growth (shown in Fig. 4) resulted in optimal
conditions at 30C, 16 : 08 h : h, and 80 lmol m)2 s)1. No
statistical difference was found between these conditions
and at 35C, 18 : 06 h : h, and 80 lmol m)2 s)1. However,
replicates of the 35C, 18 : 06 h : h, and 80 lmol m)2 s)1

50
experiment showed high variability (0Æ224 ± 0Æ02 and
40 0Æ16 ± 0Æ04 doublings per day) indicating that the organism
30 was at the threshold of heat tolerance. At 40C, the culture
died. Repeated experiments were performed at 30C,
20
16 : 08 h : h, and 80 lmol m)2 s)1 and resulted in
10 0Æ18 ± 0Æ01 and 0Æ17 ± 0Æ01 average doublings per day.
0
These experiments showed good repeatability and no statis-
0 1 2 3 4 5 6 tical difference.
Time (day)

Figure 2 Exponential growth curve for Oocystis sp. (n) (y = 22Æ7 Discussion
e0Æ266 x with R2 = 0Æ994; Conditions: 25C, 18 : 06 light : dark,
100 lmol m)2 s)1) and Amphora sp. (¤) (y = 32Æ3 e0Æ124 x with It is important to determine a correlation between fluo-
R2 = 0Æ999; Conditions: 30C, 16 : 08 light : dark, 80 lmol m)2 s)1). rescence and dry mass for each algal strain, because no

ª 2011 The Authors

Journal of Applied Microbiology 111, 312–318 ª 2011 The Society for Applied Microbiology 315
Growth of algae for biodiesel J.L. Csavina et al.

0·6

k
gh

k
0·5

bg

bh
cde

bd
bd
cd
cfj
Doublings per day

aef
af
0·4

cf

aj

af
a

ai

ai
0·3

i
0·2
Figure 3 Oocystis sp. doublings per day
versus experimental conditions light intensity
0·1
(lmol m)2 s)1), light : dark cycle (h : h) and
temperature (C). Error bars show standard
0·0
deviation of measurement. Letters designate
200
150
100
150
100

200
150
100
200
150
100
200
150
100
150
100

200
150
100
200
150
100
80

80
groups that were found to be not statistically
18 : 06 18 : 06 18 : 06 16 : 08 14 : 10 18 : 06 18 : 06 18 : 06 different (alpha = 0Æ05). A zero doublings per
day indicates culture died in experimental
15 20 25 30 35 40 conditions.

0·25
bkl

0·20
l
k
Doublings per day

k
b

0·15
b

cfgi

bj
b

cfgi
g
cf

cfi
cf
f

c
dhi

0·10
dem
ahmn

d
dn

Figure 4 Amphora sp. doublings per day

ae

eh

0·05 versus experimental conditions light intensity

a
a

(lmol m)2 s)1), light : dark cycle (h : h) and

a

temperature (C). Error bars show standard

0·00 deviation of measurement. Letters designate
100

100

100
80
60
40
80
60
40
80
60
40
80
60
40
80
60
40
80
60
40

80
60

80
60

80
60
80
60
40
40

groups that were found to be not statistically

18 : 06 18 : 06 18 : 06 16 : 08 14 : 10 18 : 06 16 : 08 18 : 06 16 : 08 18 : 06 different (alpha = 0Æ05). A zero doublings per
day indicates culture died in experimental
15 20 25 30 35 40 conditions.

two strains will have the same relationship, and with and temperature were held constant, significant differ-
some strains, a linear relationship may not exist (Andersen ences were observed as light : dark cycle was changed,
2005). However, numerous studies have demonstrated with 16 : 08 h : h being optimal for both.
this in vivo fluorescence growth monitoring method to be Temperature dependence was also observed for Oocystis
applicable for many microalgal groups under a wide sp. Looking at experiments with a light intensity of
range of conditions (Brand 1985; Maldonado and Price 150 lmol m)2 s)1 and a light : dark cycle of 18 : 06 h : h,
2001). Andersen (2005) specifically found a strain of temperatures of 15, 20, 25, 30, 35 and 40C were tested.
diatom, various picoplankton and dinoflagellates were Most of these differed significantly over the range tested
able to be monitored by this method. Figure 1 clearly with growth rates decreasing as expected above and below
supports the need for separate correlation curves for the the optimum. The Amphora sp. culture showed more
strains used in this study. erratic behaviour in response to temperature variations.
Many of the experiment sets that kept light : dark cycle Light intensity over the ranges tested had the least impact
and temperature the same but varied the light intensity on growth rate, with very few examples of significantly
produced growth rates that were statistically indistin- different results when light : dark cycle and temperature
guishable or very close. In every case, when light intensity were held constant, and light intensity was varied.

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316 Journal of Applied Microbiology 111, 312–318 ª 2011 The Society for Applied Microbiology
J.L. Csavina et al.
Growth conditions for Oocystis sp. were shown to be
optimal at a temperature of 25C, a light intensity of
150 lmol m)2 s)1, and a 16 : 08 light : dark cycle. Expo-
nential growth typically lasted for 4 days without changing
the medium or parameters. For Amphora sp., consistent
optimized growth was established at a temperature of
30C, a light intensity of 80 lmol m)2 s)1, and a 16 : 08
light : dark cycle. Although higher doubling rates were
observed at 35C, the results were not repeatable, were
not true for other light intensities and appeared close
to the maximum tolerable temperature for Amphora sp.
Therefore, it was concluded that Amphora sp. would
be optimally grown at 30C for a consistently high
growth rate. For both cultures, growth rate was more
dependent on light : dark cycle and temperature, than
light intensity.
When comparing the doubling rates from these experi-
ments to the literature, both strains grew slowly (Brand
1985; Illman et al. 2000; Andersen 2005; Chisti 2007; Lv
et al. 2010). Possible reasons for the slower doubling rates
include a lack of continuous agitation and the absence of
carbon dioxide enhanced air bubbling through the culture
as in previous studies (Lv et al. 2010). As our experiment
was designed to identify optimal conditions based on
temperature, light : dark cycle, and light intensity, neither
agitation nor carbon enhancement through air ⁄ CO2
sparging was used. It is anticipated that growth rates for
Oocystis sp. and Amphora sp. could be substantially
increased over the results obtained through continuous
agitation and enhanced carbon dioxide addition (Andersen
2005).
Acknowledgements
Funding for this research was provided by an Ohio Uni-
versity 1804 Grant and the Ohio Coal Research Center.
Undergraduate student assistance in maintaining the algal
cultures and guidance from Coal Center staff is greatly
appreciated.
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