Bioresource Technology 147 (2013) 534–538

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Orthogonal test design for optimization of lipid accumulation and lipid

property in Nannochloropsis oculata for biodiesel production
Likun Wei a, Xuxiong Huang a,b,c,⇑, Zhenzheng Huang a, Zhigang Zhou a
a
Key Laboratory of Freshwater Fishery Germplasm Resources, Ministry of Agriculture, No. 999, Hucheng Ring Road, Lingang New City, Shanghai 201306, People’s Republic of China
b
Shanghai Engineering Research Center of Aquaculture, No. 999, Hucheng Ring Road, Lingang New City, Shanghai 201306, People’s Republic of China
c
Shanghai University Knowledge Service Platform, Shanghai Ocean University Aquatic Animal Breeding Center (ZF1206), No. 999, Hucheng Ring Road, Lingang New City,
Shanghai 201306, People’s Republic of China

h i g h l i g h t s

 N–Fe–t °C orthogonal test optimize properties of N. oculata as biofuel stuffs.

 The highest total lipid content and neutral lipid proportion are 60.44% and 90.74%.
 There are significant interactions among N–Fe–t °C on lipid yield and cetane number.
 N, followed by Fe and t °C orderly, is the most influential factor in lipid yield.
1 1 1
 The optimum is combination of 0.44 mmol N L , 1.2  10 mmol Fe L and 20 °C.

a r t i c l e i n f o a b s t r a c t

Article history: In order to improve the property of Nannochloropsis oculata as biodiesel feedstock, a L9(34) orthogonal test
Received 18 June 2013 on limited nitrogen supplementation (0, 0.22 and 0.44 mmol N L 1), high iron concentration (1.2  10 2,
Received in revised form 9 August 2013 1.2  10 1 and 1.2 mmol Fe L 1) and culture temperature (10, 20 and 30 °C) was conducted to select the
Accepted 14 August 2013
most effective combinational measurement. Results showed that microalgae displayed the highest total
Available online 22 August 2013
lipid content (60.44 ± 0.68%), the highest neutral lipid proportion (90.74 ± 0.18%), the highest lipid yield
(152.70 ± 7.40 mg L 1) and the largest cetane number (CN, 64.34 ± 0.13) under different combined con-
Keywords:
ditions. There were significant interaction among nitrogen supplementation, iron concentration and cul-
Nannochloropsis oculata
Orthogonal test
ture temperature on the lipid yield and CN of N. oculata. Nitrogen supplementation, followed by iron
Biodiesel concentration and temperature orderly, was the most influential factor in lipid yield. It is therefore sug-
Nitrogen supplementation gested that the combination of 0.44 mmol N L 1, 1.2  10 1 mmol Fe L 1 and 20 °C was the best measure-
Iron concentration ment for improving the property of N. oculata as biodiesel feedstock.
Crown Copyright Ó 2013 Published by Elsevier Ltd. All rights reserved.

1. Introduction
The excessive use of fossil fuels will lead to the depletion of nat-
ural energy resources and greenhouse gas (CO2) emission increase,
thus is considered unsustainable. Biodiesel has received consider-
able attention in recent years, as it is a biodegradable, renewable
and non-toxic fuel. Biodiesel obtained from energy crops and ole-
aginous microorganisms produces favourable effects on the envi-
ronment, such as a decrease in acid rain and in the greenhouse
effect caused by combustion. Due to these factors and its biode-
gradability, the production of biodiesel is considered an advantage
to that of fossil fuels. It contributes neither net carbon dioxide nor
⇑ Corresponding author at: Key Laboratory of Freshwater Fishery Germplasm
Resources, Ministry of Agriculture, No. 999, Hucheng Ring Road, Lingang New City,
Shanghai 201306, People’s Republic of China. Tel./fax: +86 21 61900463.
E-mail address: xxhuang@shou.edu.cn (X. Huang).
sulphur to the atmosphere and emits less gaseous pollution than
petroleum diesel (Antolín et al., 2002; Vicente et al., 2004). Micro-
alga is regarded as one of the most promising feedstock for the pro-
duction of environmentally friendly biofuel. Some microalgae, such
as Nannochloropsis oculata (Huang et al., 2012), Chlorella vulgaris
(Liu et al., 2008), Scenedesmus obliquus (Abd Ei Baky et al., 2012),
Dunaliella (Takagi et al., 2006), Chaetoceros muelleri (McGinnis
et al., 1997) and Botryococcus braunii (Rao et al., 2000) have been
selected as the ideal strains for production of biofuel because of
their widespread availability and higher oil yields. However, the
commercial production of biofuel from microalgae has not begun
due to its high cost and very low or even negative profitability.
The low oil yield of microalgae, due to low biomass or low lipid
content, is the main technical limiting factor that restricts the
commercial production of biodiesel from microalgae. Environmen-
tal factors such as temperature, light, pH, salinity and nutrient
status of the culture medium, not only affect photosynthesis and

0960-8524/$ - see front matter Crown Copyright Ó 2013 Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.biortech.2013.08.079
 L. Wei et al. / Bioresource Technology 147 (2013) 534–538
productivity of algal cells, but also influence the pattern, pathway
and activity of cellular metabolism and thus dynamic cell compo-
sition (Guschina and Harwood 2009; Hu 2004). Some researches
have revealed that it is possible to manipulate cell lipid content
and lipid property by optimizing microalgae culture conditions
(temperature and light intensity) or nutrient media characteristics
(concentration of nitrogen, phosphates, and iron) (Liu et al., 2008).
In general, culture conditions for best oil accumulation are inap-
propriate to or even inconsistent with those for best cell produc-
tion of microalgae. So a two-step-manipulation method was
advised for higher lipid productivity of microalgae (Huang et al.,
2012). More specifically, the microalgae are first cultured under
favorable environmental conditions to gain higher biomass, then
transferred to environmental stress conditions to optimize lipid
characteristics for biodiesel.
Recently, the microalgae N. oculata has been investigated as a
possible source of biological material for the production of biodie-
sel (Borges et al., 2011; Chiu et al., 2009; Converti et al., 2009; Li
et al., 2011; Singh and Gu 2010). Huang et al. (2012) studied the
effect of different nitrogen supplementation (0, 0.22, 0.44, 0.88
and 1.76 mmol N L 1) in culture medium on growth, total lipid
content and fatty acid profiles of N. oculata and demonstrated that
limited nitrogen treatment (0.22 mmol N L 1) was the effective
measurement for improving the properties of N. oculata as stuff
for biodiesel. Huang et al. (2013) demonstrated that higher iron
stress (1.2 mmol L 1) induced N. oculata to accumulate more oil
in the cells, compared with other iron concentrations (1.2  10 2,
1.2  10 1 and 12 mmol L 1). Likewise, N. oculata were cultured
at 15, 20, 25, 30 and 35 °C respectively and higher temperature
(30 °C) displayed the highest total lipids (unpublished results). As
the temperature increased, neutral lipid and poly-unsaturated
fatty acids decreased while saturated fatty acids increased, accom-
panied with decreasing of monounsaturated fatty acids. But the
combined effect of nitrogen–iron–temperature on property of N.
oculata as biodiesel stuffs has not been studied. In this study, we
further investigated the combinational effect of limited nitrogen,
high iron concentration and cultured temperature on N. oculata
with a L9(34) orthogonal test from the viewpoint of biofuel produc-
tion in order to optimize the most effective measurement.
2. Methods
2.1. Culture of microalgae
N. oculata was obtained from the Culture Collection of Microal-
gae at Shanghai Ocean University, Shanghai, China. The microalgae
were grown in 60 L photobioreactor with f/2 medium at salinity
20 ppt, 25 ± 1 °C with continuous aeration. The cell pellets at the
late-exponential growth phase were then centrifuged at
8000 rpm for 15 min after a 10 days culture.
For the combinational effect trial, a L9(34) orthogonal test on
limited nitrogen supplementation (0, 0.22 and 0.44 mmol N L 1),
Table 1
The L9(34) orthogonal test used for study.
Treatments No. Nitrogen levels (mmolN
L
1 0
2 0
3 0
4 0.22
5 0.22
6 0.22
7 0.44
8 0.44
9 0.44
535
high iron concentration (1.2  10 2, 1.2  10 1 and
1
1.2 mmol Fe L ) and temperature (10, 20 and 30 °C) was designed
according to the results of the single factor experiments (Huang
et al., 2012, 2013) (Table 1). Cell pellets were re-suspended in mod-
ified f/2 mediums containing different nitrogen and iron levels as
those showed in Table 1. The initial inoculation densities for cul-
ture were 1.0  107 cells mL 1. Then the culture was incubated in
the illuminated incubator with 3000 mL flanks at 10, 20 and
30 °C respectively under salinity 20 ppt and continuous light of
light intensity 430 lmol m 2 s 1, with continuous aeration. The
culture was incubated for another 10 days. The dry biomass and li-
pid characteristics were measured. All the experiments were car-
ried out in triplicate.
2.2. Assays on lipid and fatty acid characteristics
Cells were harvested by centrifugation at 8000 rpm for 15 min,
and pellets were freeze-dried in a freeze-drier at 46 °C. Total lipid
of the freeze-dried microalgae was extracted with chloroform–
methanol (2:1, V:V), according to the modified method of Folch
et al. (1957). In brief, 0.2 g freeze-dried microalgae were extracted
with 50 mL of chloroform–methanol for 24 h and sonicated at
70 Hz intensity with a sonicator at room temperature for twice
of 30 min during the extraction. Then the suspension was filtered
and the filtrate was washed with KCl solution twice and the lower
liquid was transferred into pre-weighed glass vial. The chloro-
form–methanol solution was evaporated to dryness at 40 °C under
vacuum. The lipid content was measured gravimetrically and cal-
culated by the equation: Y (% dw) = WL/WDA, where WL and WDA
are the weights of the extracted lipids and the dry algae biomass,
respectively.
The extracted total lipid was divided into two parts. One was
used for assays on lipid class, which was separated into polar lipid
and neutral lipid with the mixture of petroleum ether and 95%
methanol–water solution (1:1) according to the liquid–liquid
method described by Liu et al. (2009).
The other part of extracted total lipid was used for assays on
fatty acid profiles. Fatty acid methyl esters (FAMEs) were prepared
by transesterification using 0.4 M KOH–methanol. FAMEs were
analytically verified by gas chromatography–mass spectrometry.
In brief, FAMEs were detected by flame ionisation detection (FID)
after injecting the sample into an Agilent 7890A/5975C GC/MS fit-
ted with a column of HP-5MS, 30 m  0.32 mm I.D., 0.25 lm film
thickness (Catalog No. 24152). The column temperature initially
held at 50 °C for 1 min, then increased at a rate of 15 °C min 1 to
210 °C, and then held constantly for 1 min; followed by an increase
to 217 °C at 2 °C min 1, and it was then held constantly for another
1 min. Then the temperature increased to 220 °C at 0.5 °C min 1
and held for 1 min; finally, the temperature was heated up to
260 °C at 15 °C min 1 and held for 2 min, where it was held until
all FAME had been eluted. The peaks were identified by comparing
retention times with known standards (Sigma Chemical Co, St.
1 1
) Iron levels (mmolFe
L ) Temperature levels (°C)
2
1.2  10 10
1
1.2  10 20
1.2 30
2
1.2  10 20
1
1.2  10 30
1.2 10
2
1.2  10 30
1
1.2  10 10
1.2 20
536 L. Wei et al. / Bioresource Technology 147 (2013) 534–538

Table 2
The total lipid and lipid yield of N. oculata under different nitrogen–iron–temperature combined culture conditions.
1
No. N Fe T Total lipid contents (% dw) Neutral lipid/total lipid ratio Lipid yield (mg L )
e bcd c
1 1 1 1 51.22 ± 4.12 85.85 ± 0.25 81.23 ± 3.82
2 1 2 2 55.22 ± 0.97bcd 88.03 ± 0.54ab 133.22 ± 5.39b
3 1 3 3 57.52 ± 2.56ab 90.74 ± 0.18a 68.26 ± 7.44c
4 2 1 2 50.99 ± 2.67de 84.11 ± 2.79cde 116.81 ± 16.56b
5 2 2 3 50.29 ± 1.84e 86.94 ± 0.58abc 138.24 ± 3.29ab
6 2 3 1 60.44 ± 0.68a 83.25 ± 2.10de 131.70 ± 20.12b
7 3 1 3 45.94 ± 2.24f 87.65 ± 1.57bc 126.62 ± 6.91b
8 3 2 1 53.01 ± 0.92cde 82.33 ± 1.80e 152.70 ± 7.40a
9 3 3 2 55.86 ± 1.71bc 84.38 ± 1.54cde 152.33 ± 10.38a

Notes: Values within a column marked with different letters are significantly different (p < 0.05).

Table 3
The fatty acid profiles and predicted theoretical CN of N. oculata under different nitrogen–iron–temperature combined culture conditions.

Fatty acids Treatment 1 Treatment 2 Treatment 3 Treatment 4 Treatment 5 Treatment 6 Treatment 7 Treatment 8 Treatment 9
C14:0 2.55 ± 0.08d 3.05 ± 0.18cd 6.23 ± 0.30a 2.58 ± 0.24d 5.11 ± 0.77b 3.12 ± 0.18cd 4.60 ± 0.24b 3.50 ± 0.18c 2.89 ± 0.18d
C15:0 0.18 ± 0.07bc 0.22 ± 0.02ab 0.14 ± 0.00c 0.25 ± 0.04a 0.19 ± 0.02abc 0.19 ± 0.03abc 0.21 ± 0.03ab 0.19 ± 0.01bc 0.20 ± 0.02abc
C16:0 46.28 ± 1.07c 46.19 ± 0.81c 59.66 ± 0.91a 45.15 ± 0.93c 58.29 ± 1.31a 45.2 ± 0.43c 59.44 ± 0.76a 42.11 ± 0.90d 48.26 ± 1.19b
C16:1 36.08 ± 0.40c 32.64 ± 0.47d 23.42 ± 0.27f 33.02 ± 0.16d 25.13 ± 0.77e 37.75 ± 0.15b 24.60 ± 0.89ef 39.97 ± 0.56a 32.09 ± 1.46d
C18:0 0.50 ± 0.11ef 1.12 ± 0.04c 0.75 ± 0.10d 1.28 ± 0.07bc 1.34 ± 0.06b 0.38 ± 0.06f 1.59 ± 0.03a 0.55 ± 0.12e 0.76 ± 0.09d
C18:1 11.19 ± 0.15d 14.13 ± 0.31b 8.49 ± 0.65f 15.12 ± 0.61a 8.51 ± 0.47f 10.11 ± 0.13e 8.25 ± 0.57f 10.49 ± 0.44de 13.07 ± 0.72c
C18:2 0.34 ± 0.05a 0.20 ± 0.01cd 0.16 ± 0.02d 0.21 ± 0.03cd 0.17 ± 0.01cd 0.27 ± 0.03b 0.20 ± 0.04cd 0.22 ± 0.01bc 0.22 ± 0.03c
C20:4n6 0.23 ± 0.03d 0.32 ± 0.06b 0.38 ± 0.03a 0.32 ± 0.03b 0.30 ± 0.02bc 0.28 ± 0.02bcd 0.25 ± 0.02cd 0.26 ± 0.01bcd 0.30 ± 0.03bc
C20:5n3 2.43 ± 0.37ab 1.97 ± 0.36bc 0.67 ± 0.13d 1.94 ± 0.31c 0.81 ± 0.08d 2.59 ± 0.29a 0.71 ± 0.15d 2.59 ± 0.13a 2.11 ± 0.31abc
Others 0.22 ± 0.02c 0.31 ± 0.00a 0.25 ± 0.00b 0.30 ± 0.04ab 0.31 ± 0.02a 0.26 ± 0.03ab 0.30 ± 0.03a 0.28 ± 0.02a 0.25 ± 0.02bc
P
C16 82.36 ± 0.75b 78.82 ± 0.43d 83.08 ± 1.08ab 78.16 ± 0.79d 83.42 ± 0.67ab 82.95 ± 0.40ab 84.04 ± 0.58a 82.08 ± 0.78b 80.34 ± 0.84c
P
SFA 49.64 ± 0.95de 50.71 ± 0.59d 66.89 ± 0.63a 49.39 ± 1.08de 65.08 ± 1.03b 48.99 ± 0.53e 65.98 ± 0.53ab 46.45 ± 0.66f 52.22 ± 1.21c
P
MUFA 47.28 ± 0.54bc 46.77 ± 0.16c 31.91 ± 0.46f 48.14 ± 0.74b 33.64 ± 1.02e 47.87 ± 0.20bc 32.85 ± 0.33ef 50.46 ± 0.57a 45.15 ± 0.97d
P
PUFA 3.09 ± 0.47ab 2.52 ± 0.44bc 1.21 ± 0.18d 2.47 ± 0.36c 1.28 ± 0.06d 3.14 ± 0.34a 1.16 ± 0.21d 3.09 ± 0.11ab 2.62 ± 0.36abc
Theoretical 60.96 ± 0.23cd 61.30 ± 0.16bc 64.34 ± 0.13a 61.08 ± 0.22cd 64.05 ± 0.22a 60.79 ± 0.11d 64.27 ± 0.15a 60.29 ± 0.14e 61.56 ± 0.28b
CN

Notes: Values within the same row marked with different letters are significantly different (p < 0.05).

Louis. MO, USA) and mass spectrometry. A given fatty acid content
(% in all fatty acids) was determined using the normalisation meth-
od. All measurements were performed in triplicate.
2.3. Statistics
The total lipid yield was calculated as the following equation:
P = DW⁄C, where DW (g L 1) is the dry weight of algal cells at har-
vest, C (% dw) is the lipid content of the dry algal cells at harvest.
The predicted theoretical cetane number (CN) was estimated
according to the method of Piloto-Rodríguez et al. (2013). All re-
sults were expressed as mean ± standard deviation. The data were
analysed by analysis of variance. When the analysis of variance
identified differences among groups, multiple comparisons among
means were performed using Duncan’s new multiple-range test. A
significance level of p = 0.05 was chosen.
3. Results and discussion
3.1. The total lipid contents, neutral lipid proportions and lipid yields of
N. oculata under different nitrogen–iron–temperature combined
culture conditions
The total lipid content of N. oculata was significantly affected by
nitrogen–iron–temperature combined culture conditions (Table 2).
The microalgae in treatment 6 (0.22 mmol N L 1, 1.2 mmol Fe L 1
and 10 °C) had the highest total lipid content as high as 60.44%,
which were significantly higher than those in treatment 1, 2, 4,
5, 7, 8 and 9. Microalgae in most treatments except for treatment
7 had total lipid contents more the 50%.
Under the combined effect of nitrogen–iron–temperature, N.
oculata in all the treatments displayed high neutral lipid/total lipid
ratios (Table 2). Microalgae in treatment 3 (0 mmol N L 1,
1.2 mmol Fe L 1 and 30 °C) had the highest neutral lipid/ total lipid
ratio (90.74%), then orderly followed by treatment 2, 7, 5 and 1,
which had neutral lipid/ total lipid ratio more than 85%.
After 10 days culture, the maximum total lipid yields were ap-
peared in treatment 8 (0.44 mmol N L 1, 1.2 mmol Fe L 1 and
10 °C) and treatment 9 (0.44 mmol N L 1, 1.2 mmol Fe
L 1 and
20 °C), which were 152.70 and 152.33 mg L 1 respectively, which
were significantly higher than others (Table 2).
Our previous results from single factor experiments demon-
strated that both nitrogen supplementation and iron concentration
had significant effects on the lipid contents, lipid class and fatty
acid profiles of N. oculata (Huang et al., 2012, 2013). In this study,
we also illustrated that the different combination of nitrogen sup-
plementation, iron concentration and culture temperature affected
the lipid contents and lipid class. While the highest total lipid con-
tent (60.44%) and the largest neutral lipid/ total lipid ratio (90.74%)
of N. oculata in this study were apparently higher than those of the
microalgae treated with single factor. For example, the highest to-
tal lipids and the largest neutral lipid/total lipid ratio of N. oculata
in limited nitrogen supplementation experiment were 35.85% and
58.23% (Huang et al., 2012), in high iron concentration experiment
were 37.34% and 48.37% (Huang et al., 2013). It implies that the
synergistic effects of nitrogen limitation, high iron concentration
and temperature may be more effective to induce microalgae N.
oculata to accumulate more total lipid and more neutral lipid than
the effect of single factor.
Additionally, light intensity also changes the lipid content and
lipid class of microalgae. Klok et al. (2013) demonstrated that high-
er light intensity induced microalgae to accumulate more total
 L. Wei et al. / Bioresource Technology 147 (2013) 534–538 537

Table 4
The analysis of variance for four key performance indexes of biolipid.

Source SSTL SSNR SSLY SSCN df MSTL MSNR MSLY MSCN FTL FNR FLY FCN Fa
Repeat 0.0000 0.0000 432.50 0.06 2 0.0000 0.0000 216.25 0.03 0.06 0.02 2.98 0.77 F0.05(2,18) = 3.55
Treatment 0.0398 0.0151 20,927.01 65.42 8 0.0050 0.0019 2615.88 8.18 12.41** 10.88** 36.04** 224.61** F0.01(8,18) = 3.71
Nitrogen 0.0057 0.0055 12,380.01 0.24 2 0.0028 0.0027 6190.01 0.12 7.05** 15.86** 85.29** 3.23 F0.01(2,18) = 6.01
Iron 0.0270 0.0001 4531.74 0.57 2 0.0135 0.0000 2265.87 0.29 33.64** 0.23 31.22** 7.83** F0.05(2,16) = 3.63
Temperature 0.0060 0.0093 2615.30 64.21 2 0.0030 0.0047 1307.65 32.10 7.50** 26.91** 18.02** 881.83** F0.01(8,16) = 3.89
Error D 0.0012 0.0002 1399.95 0.40 2 0.0006 0.0001 699.98 0.20 1.52 0.50 9.64** 5.53* F0.01(2,16) = 6.23
Error E 0.0061 0.0029 1161.23 0.58 16 0.0004 0.0002 72.58 0.04
Total error 0.0072 0.0031 2561.18 0.99 18 0.0004 0.0002 142.29 0.05
Total variations 0.046 0.018 22,520.73 66.06 26

Note: SS, df and MS mean sum of squares, degree of freedom and mean square respectively. The subscripts TL, NR, LY and CN mean total lipid, neutral lipid/total lipid ratio,
lipid yield and theoretical CN respectively.
*
Significant difference (0.01 < p < 0.05).
**
Highly significant difference (p < 0.01).

Table 5
Estimated marginal means and pairwise comparisons of mean of different levels of three factors.

Key performance indexes Means of different levels of each factor

1 1
Nitrogen (mmolN
L ) Iron (mmolFe
L ) Temperature (°C) Std. error
2 1
0 0.22 0.44 1.2  10 1.2  10 1.2 10 20 30
Total lipid contents (% dw) 0.56a 0.55a 0.52b 0.51c 0.53b 0.58a 0.55a 0.55a 0.52b 0.006
Neutral lipid/total lipid ratio 0.88a 0.85b 0.85b 0.86a 0.86a 0.86a 0.84c 0.86b 0.89a 0.004
Lipid yield (mg L 1) 0.095c 0.131b 0.145a 0.112b 0.142a 0.117b 0.122b 0.136a 0.113b 0.004
Theoretical CN 62.20a 61.97a 62.04a 62.10a 61.88b 62.23a 60.68c 61.31b 64.22a 0.076

Note: Values of the same factor within the same row superscripted with different small letters indicate significant differences (p < 0.05).

lipid and neutral lipid in cells. The continuous light at light inten-
sity of 430 lmol m 2 s 1 in this study was different from those in
our previous studies for N. oculata, which was intermittent light
(L:D = 14:10) with light intensity of 150 lmol m 2 s 1 in nitrogen
supplementation and iron concentration experiment (Huang
et al., 2012, 2013). So the apparent increment on total lipid content
and neutral lipid/total lipid ratio in this study may partly be due to
the adopted high light intensity.
3.2. The fatty acid profiles of N. oculata under different nitrogen–iron–
temperature combined culture conditions
Under the combined effect of nitrogen–iron–temperature, all
the microalgae lipids are mainly composed of saturated fatty acids
(SFA, 46.45–66.89%) and mono-unsaturated fatty acids (MUFA,
31.91–50.46%). The significant fatty acids in N. oculata were
C16:0 and C16:1, which accounted for 42.11–59.66% and 23.42–
39.97% of the total fatty acids, respectively. All the treatments dis-
played a high level of 16-carbon fatty acids and very small
amounts of poly-unsaturated fatty acids (PUFA) (Table 3). But in
our preview studies, 20:5n-3 (EPA) was the dominant fatty acid
in N. oculata (Huang et al., 2004, 2012, 2013). Borges et al. (2011)
also demonstrated that the EPA proportion of the total fatty acid
of N. oculata was more than 20%. However, Converti et al. (2009)
reported that C16:0 was the dominant fatty acid of N. oculata that
had been treated with different temperature and nitrogen concen-
trations. In this study, we found that the fatty acid profiles of N.
oculata were significantly changed by the combined nitrogen–
iron–temperature. It also implied that the combined nitrogen–
iron–temperature was much more effective for changing the lipid
properties of N. oculata as sources for biodiesel.
Biodiesel from microalgae is in essence a set of monoalkyl es-
ters of long-chain fatty acids and at present is derived chiefly from
the acylglycerols (Meher et al., 2006). Compared to polar lipids
such as phospholipid, a neutral lipid (triacylglycerols) produces
more methyl esters of fatty acids through transesterification. The
higher total lipids and higher proportion of triacylglycerols in mic-
roalgae, the more biodiesel could be produced from microalgae.
Additionally, the degree of saturation, chain length and branching
of the fatty compounds will significantly influence the cetane num-
ber (CN) of biodiesel (Wadumesthrige et al., 2008), which has been
included as a fuel quality specification in biodiesel standards, with
a minimum of 47 prescribed for neat biodiesel or B100 in the Uni-
ted States using the American Society for Testing and Materials
(ASTM) D 6751-07a fuel standards, and a minimum of 51 in some
European countries (e.g., European standard EN 14214:2003)
(Wadumesthrige et al., 2008). In this study, all the treatments dis-
played appropriate theoretical CN values (Table 3). The highest and
lowest predicted theoretical CN were 64.32 and 60.29 which ap-
peared in treatment 3 (0 mmol N L 1, 1.2 mmol Fe L 1 and 30 °C)
and treatment 8 (0.44 mmol N L 1, 1.2  10 1 mmol Fe L 1 and
10 °C) respectively.
3.3. The variance analysis of the effects of nitrogen, iron and
temperature on lipid properties of N. oculata
Orthogonal test is an effective measurement for assaying the
comprehensive effect of more environmental factors on the organ-
isms. It provides a reasonable amount of information for testing
the optimal combination from a fewer number of assays, therefore
reducing the overall cost associated with the analysis. Nitrogen
starvation is the widely applied strategy for inducing microalgae
cells to accumulate lipid. Several studies demonstrated that lipid
content in microalgae cells can be improved through nitrogen star-
vation due to different proposed mechanism (Botham and Ratledge
1979; Chen and Johns 1991; Ratledge and Wynn 2002; De Swaaf
et al., 2003; Huang et al., 2012; Garcia-Ferris et al., 1996; Ganuza
et al., 2008). According to literature reports, nitrogen limitation
may increase the intracellular content of fatty acid acyl-CoA and
activate diacylglycerol acyltransferase, which converts fatty acid
acyl-CoA to triglyceride (Sukenik and Livne, 1991; Takagi et al.,
2000). It has been reported that the lack of nitrogen can induce a
538 L. Wei et al. / Bioresource Technology 147 (2013) 534–538
significant increase in neutral lipids in green algae cells (Packer
et al., 2011). The increment of triacylglycerols in cells could be in-
creased by nitrogen starvation (Widjaja et al., 2009). Improved li-
pid contents induced by a high level of iron stress in culture
medium also has been demonstrated in Chlorella (Liu et al.,
2008) and N. oculata (Huang et al., 2013). It also was reported that
the lipid content and fatty acid profiles of N. oculata could be chan-
ged by culture temperature (Singh and Gu, 2010). Through the var-
iance analysis of the results (Table 4), we found that there were
significant interaction between nitrogen supplementation, iron
concentration and culture temperature for the lipid yield of N. ocu-
lata and CN of the biodiesel from N. oculata. With the combined
conditions, iron concentration, followed by temperature and nitro-
gen supplementation orderly, affected the total lipid in cells signif-
icantly. Temperature, followed by nitrogen supplementation,
affected the neutral lipid/total lipid ratio in cells significantly.
Nitrogen supplementation, followed by iron concentration and
temperature orderly, affected the lipid yield in cells significantly.
While temperature, followed by iron concentration, affected the
CN significantly.
With the estimated marginal means and pairwise comparisons
(Table 5), it could be concluded that the combination of
0.44 mmol N L 1, 1.2  10 1 mmol Fe L 1 and 20 °C was the most
effective for N. oculata to produce lipid while the combined treat-
ment of 0 mmol N L 1, 1.2 mmol Fe L 1 and 30 °C was the most
effective for improving CN of the microalgae. But in fact, all the
CN of N. oculata under different combined conditions in this study
accord with the USA and European biodiesel standards. So it is
therefore suggested that the combination of 0.44 mmol N L 1,
1.2  10 1 mmol Fe L 1 and 20 °C was the most effective measure-
ment for improving the property of N. oculata as biodiesel
feedstock.
4. Conclusions
N. oculata SHOU-S14 displays the highest total lipid content
(60.44 ± 0.68%), the highest neutral lipid proportion
(90.74 ± 0.18%), the highest lipid yield (152.70 ± 7.40 mg L 1) and
the largest cetane number (CN, 64.34 ± 0.13) under different com-
bined conditions. There are significant interaction among nitrogen
supplementation, iron concentration and culture temperature on
the lipid yield and CN of N. oculata. Nitrogen supplementation, fol-
lowed by iron concentration and temperature orderly, is the most
influential factor in lipid yield. Therefore the combination of
0.44 mmol N L 1, 1.2  10 1 mmol Fe L 1 and 20 °C is the best
measurement for improving the property of N. oculata as biodiesel
feedstock.
Acknowledgements
This study was financially supported by a Project of the Na-
tional High Technology Research and Development Program, China
(2009AA064401), a Project of the State Oceanic Administration of
China (SHME2011SW02) and by Shanghai Universities First-class
Disciplines Project of Fisheries.
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