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Short communication

Nitrate repletion strategy for enhancing lipid production from marine microalga
Tetraselmis sp

Garam Kim, Jinsung Bae, Kisay Lee

PII: S0960-8524(16)30021-9
DOI: http://dx.doi.org/10.1016/j.biortech.2016.01.045
Reference: BITE 15956

To appear in: Bioresource Technology

Received Date: 4 November 2015

Revised Date: 13 January 2016
Accepted Date: 14 January 2016

Please cite this article as: Kim, G., Bae, J., Lee, K., Nitrate repletion strategy for enhancing lipid production from
marine microalga Tetraselmis sp, Bioresource Technology (2016), doi: http://dx.doi.org/10.1016/j.biortech.
2016.01.045

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Re-Revised manuscript of BITE-D-15-05779R2

Nitrate repletion strategy for enhancing lipid production from marine

microalga Tetraselmis sp.

Garam Kim, Jinsung Bae and Kisay Lee*

Department of Environmental Engineering and Energy, Myongji University, Yongin, 449-728,

Korea

*Corresponding author. Tel: 82-31-330-6689; Fax: 82-31-336-6336

E-mail: kisay@mju.ac.kr (K. Lee)

ABSTRACT

The cell growth rate and cellular lipid content of microalgae are affected by the nitrogen

levels during cultivation. The growth rate and lipid content of marine microalga Tetraselmis sp.

was found to increase under nitrate replete conditions, but not under deplete conditions. Thus, in

order to enhance the lipid productivity of Tetraselmis sp., a two-stage culture process utilizing

nitrate replete condition was applied. When the cells were cultivated in F/2 medium for five days

in the first stage, the obtained lipid content and productivity were 22.4% and 26.7 mg L-1 d-1,

respectively. After second stage of cultivation for a further 36 h under nitrate replete conditions

with 8.82 mM NaNO3, increased biomass concentration of 1.32 g L-1 and lipid content of 30.5%

were obtained, with an enhanced lipid productivity of 47.3 mg L-1 d-1.

Keywords: Tetraselmis; lipid productivity; two-stage process; nitrate repletion; FAME profiles

1
1. Introduction

Microalgae are regarded as an alternative feedstock for biodiesel production, owing to the

fast growth rate and high lipid productivity. Microalgal culture requires less arable land than oil

crops, does not compete with food production, and can also contribute to reducing the

greenhouse gas emissions. However, there are still several obstacles that must be overcome for

the commercial production, such as reducing costs and energy requirements, while maximizing

lipid productivity (Wijffels and Barbosa, 2010). Particularly, in order to improve the lipid

productivity of microalgae, it is important to select an appropriate strategy for inducing cellular

lipid accumulation during cultivation, but not decreasing the biomass concentration. One of the

most typical methods for increasing lipid content of microalgae is nitrogen limitation (Scott et al.,

2010). However, the nitrogen limitation is accompanied by the reduction of cell growth rate or

even a loss of biomass concentration (Huerlimann et al, 2010). Also, it is not a universal stress

factor that stimulates lipid accumulation. For some microalgae, lipid synthesis does not respond

to nitrogen limitation, and larger lipid production is observed at higher nitrogen levels (Xu et al.,

2001; Feng et al., 2011).

The present study investigated the influence of nitrate concentrations on the biomass and

lipid production of the marine green alga, Tetraselmis sp. The Tetraselmis species was reported

to grow well under a wide salinity range up to 70 practical salinity units (PSU) and a low

temperature range of 0 – 37°C, with a lipid productivity of 18.6 – 48.9 mg L-1 d-1 depending on

the culture conditions (Lee and Seong, 2015; Kim et al., 2015). In order to enhance the lipid

productivity of Tetraselmis sp., a two-stage culture process was applied; a first stage for biomass

production, followed by a second stage for stimulating lipid production through the nitrate

2
repletion. The produced cell biomass was converted to fatty acid methyl esters (FAME) biodiesel

and the composition of fatty acids at different nitrate levels were compared.

2. Materials and methods

2.1. Microalgal strain and culture conditions

Marine microalga Tetraselmis sp. KCTC 12236BP (Korean Collection for Type Cultures)

was cultivated in artificial seawater F/2 medium without sodium silicate (Guillard, 1975). The

artificial seawater had the following composition (g L-1): NaCl, 24.72; KCl, 0.67; CaCl2·2H2O,

1.36; MgCl2·6H2O, 4.66; MgSO4·7H2O, 6.29; NaHCO3, 0.18; Tris, 0.606 (adjusted to pH 8.2).

Tetraselmis cells were cultivated in aerated photobioreactors (PBRs) under the following

conditions: aeration rate of 0.2 vvm with ambient air, continuous light supply with intensity of

110 – 120 µmol m-2 s-1, temperature of 20 – 25°C. All experiments were conducted in triplicate.

2.2. Analyses

Microalgal cell growth was monitored by measuring the dry cell weight (DCW). Nitrate

concentrations were determined according to the Standard method of APHA (1995).

Carbohydrate content and protein content were determined using the phenol-sulfuric acid method

and the Lowry method, respectively. Lipids were extracted following the method described by

Bligh and Dyer (1959). The cell biomass was converted to fatty acid methyl ester (FAME)

through the acid-catalyzed transesterification proposed by the National Renewable Energy

Laboratory (Wychen and Laurens, 2013). The method is based on a whole biomass

transesterification procedure of lipids to FAME, which enables to convert all fatty acids

including free fatty acids in the biomass. Fatty acids composition was analyzed using GC-FID

(YL6500 GC, Younglin Instrument Co., Korea) equipped with an HP-INNOWAX capillary

column (Agilent 19091N-213). GC analysis for each sample was conducted in triplicate.

3
3. Results and discussion

3.1. Effect of initial nitrate concentration on the cell growth and lipid production

As shown in Fig. 1a, the higher the initial nitrate concentration, the more cell biomass

was produced. After 2 weeks of cultivation, both cultures supplemented with higher nitrate

concentrations of 1.76 and 2.65 mM reached a cell concentration of 1.5 g L-1 without significant

differences. The nitrate deplete culture had the lowest cell growth rate, however, it also showed

continuous cell growth and achieved a cell concentration of 1.2 g L-1 on the final day. The results

show that the Tetraselmis cells can survive for a relatively long time even under nitrogen deplete

conditions, although the cell growth rate decreases due to nitrogen deficiency. Many microalgae

species were reported to decrease their cell growth rate significantly under complete nitrogen

depletion. However, some microalgae species are able to sustain their growth by utilizing

intracellular nitrogen storage, such as chlorophyll, by conversion to proteins, nucleic acid, and

cell wall materials (Li et al., 2008). As shown in Fig. 1c, the higher the initial nitrate

concentration supplied, the greater the increment of cellular lipid content. At the highest nitrate

concentration of 2.65 mM, the lipid content increased from 19.0% to 26.5% within 2 days, and

reached the highest content of 27.6% on the 5th day. Conversely, the nitrate deplete culture

demonstrated the lowest lipid content during the entire cultivation process. Also, when

comparing nitrate concentration (Fig. 1b) and lipid content (Fig. 1c), it was found that lipid

content increased when nitrate was present in the medium, but the lipid content did not increase

further after the nitrate was depleted.

Owing to the higher cell growth and lipid content, lipid concentration per reactor volume

was significantly increased at higher nitrate concentrations, compared to that at lower nitrate

concentrations or N deplete conditions (Fig. 1d). The highest lipid concentration of 0.299 g L-1

4
was achieved at the nitrate level of 2.65 mM on the 5th day, however, after the 5th day, lipid

concentration did not increase further, rather showing a slight decrease due to the significant

decrement of lipid content, in spite of the continuous increase of cell concentrations. Therefore,

it is important to select an appropriate harvest time to achieve both higher biomass concentration

and lipid content, ultimately enabling the achievement of maximum lipid productivity.

3.2. Enhancement of lipid production through nitrate replete in 2nd stage cultivation

In order to enhance the lipid concentration per reactor volume on the final day of

cultivation, a two-stage culture process was utilized. In the first stage, Tetraselmis cells were

cultured in an original F/2 medium with 0.88 mM nitrate (control level) for five days, yielding a

cell concentration of more than 1.0 g L-1. In order to achieve the cell density of 1.0 g L-1,

excessive amounts of nitrate were unnecessary, thus the control N level was selected for the first

stage culture, and the second stage for accumulating lipids was started with different high nitrate

levels.

As shown in Fig. 2a, when the supplemented nitrate concentration was higher, more cell

biomass was produced, resulting in a maximum biomass productivity of 0.13 g L-1 d-1 at the

highest nitrate concentration of 17.6 mM. Some microalgae species were reported to show cell

growth inhibition at the high N concentration of 15 mM (Li et al., 2008), while in the present

study, the growth of Tetraselmis cells was not inhibited by high nitrate concentrations up to 17.6

mM, rather showing a slight increase in cell concentration. However, it was confirmed that

Tetraselmis cells did not fully utilize the supplied nitrogen. A high amount of nitrogen remained

in the medium indicates that the concentration supplied was extremely high and exceeded the

amount needed for growth (Fig. 2b). When high concentrations of nitrate were supplied at 5, 10,

and 20-fold the level of the control, the lipid content increased to 27.3 % within 12 h, and

5
eventually reached the highest value of 31.7 %, resulting in an increment in the lipid

concentration compared to the control culture (Fig. 2c and 2f). Compared to the fact that lipid

content increased for early 24 h in the second stage, the protein content increased after 48 h,

significantly up to 37.9 % at 72 h of the second stage culture (Fig. 2d). Interestingly, it was

clearly observed that the carbohydrate content began to decline corresponding with the increment

of lipid content (Fig. 2c and 2e). However, in a given range of high nitrate concentrations, 4.41 –

17.6 mM, the change in lipid/carbohydrate content was not so significant, showing similar

concentrations of lipid per reactor volume. This means that the nitrate concentration of 17.6 mM

may not be ideal for accumulating lipids because the oversupplied nitrogen is simply wasted.

In order to figure out the responses of Tetraselmis cells to nitrate deplete/replete

conditions, the change of biochemical composition of this microalga was investigated and shown

in Fig. 3. It clearly shows that only the carbohydrate content increased under nitrate deplete

condition, while the lipid and protein contents slightly reduced. Conversely, under nitrate replete

condition, lipid and protein contents continuously increased, while the carbohydrate content

significantly reduced. These results indicate that the carbon flux was converted from

carbohydrate to lipid and protein synthesis under nitrate replete conditions, and the nitrogen

replete condition is favorable for lipid production of Tetraselmis sp. Some microalgae species

have been found to increase their lipid content at higher N concentrations, compared to N deplete

conditions. Xu et al. (2001) found that the lipid content of a marine microalga Ellipsoidion sp.

increased with increasing nitrate concentrations. Feng et al. (2011) also reported that Chlorella

pyrenoidosa had the highest lipid content and lipid productivity under N replete conditions,

rather than in N deplete conditions. The accumulation of lipids under N repletion was attributed

to the enhanced activity of acetyl-CoA carboxylase or other key enzymes related to the

6
conversion of poly saccharides into lipids (Feng et al., 2011; Bellou et al., 2014). Although such

enzyme activity was not identified in this study, some other Tetraselmis sp. were previously

reported to demonstrate increased carbohydrate content under N deplete conditions, with reduced

lipid content (Bondioli et al., 2012). According to the review study by Griffiths and Harrison

(2009), among 24 different microalgal species including green microalgae and cyanobacteria,

seventeen species showed lipid accumulation under N deplete conditions, while seven species

showed an increase of lipid content under N replete conditions. Therefore it is thought that the

responses of algal cells to nitrogen deplete/replete conditions are species-specific as well as an

intrinsic characteristic.

The optimal N replete condition and appropriate harvest time in the second stage were

determined by comparing the lipid concentration per reactor volume and the lipid productivity

achieved for each cultivation time. As shown in Table 1, when the Tetraselmis cells were

cultivated for five days (1st stage), the achieved lipid concentration and the lipid productivity

were 0.228 g L-1 and 26.7 mg L-1 d-1, respectively. When the first stage was continued for a

further 36 h (total 6.5 days), both the lipid concentration and the lipid productivity were

decreased to 0.224 g L-1 and 19.8 mg L-1 d-1, respectively, due to the decreased lipid content.

Conversely, when the conditions were changed to nitrate-repletion and started the second stage

from the 5th day, the highest lipid concentration of 0.403 g L-1 and lipid productivity of 47.3 mg

L-1 d-1 were achieved with 8.82 mM of nitrate replete conditions within 36 h (Table 1). After 48

h in the second stage, the lipid production were not significantly enhanced (data not shown).

Therefore, when applying N replete conditions during the second stage of cultivation, 8.82 mM

of nitrate is considered to be an optimal concentration, and it is best to harvest the cell biomass

after 36 h of applying this N replete condition. Through the two-stage culture process utilizing

7
nitrate replete conditions, lipid productivity that was approximately 2.4-times higher could be

achieved, compared to single-stage cultivation for the same cultivation period of 6.5 days.

3.3. FAME profiles

The major fatty acids were C16:0, C16:4, C18:1, C18:2, and C18:3, which are comprised

of 80 – 86 % fatty acids (Table 2). It was observed that the fatty acid composition was influenced

by the nitrate levels. The two predominant fatty acids (C18:1 and C16:0) decreased at high

nitrate levels, while poly unsaturated fatty acids (PUFAs), such as C18:3 and C18:2, showed an

increase. Similar changes were observed in the study by Wu and Miao (2014), in which the

fractions of C18:3 and C18:2 increased at high nitrate concentrations at the expense of C18:1 in

C. pyrenoidosa. Xu et al. (2001) also reported that the higher the nitrate concentration, the higher

the PUFA contents of Ellipsoidion sp. These changes can affect both the oxidation stability and

the cold flow properties of biodiesel. The increase of PUFAs at high nitrate levels can decreases

the viscosity, leading to better lubricity than saturated fatty acids (Serrano et al., 2014), and

enhance cold flow performance due to the fact that the presence of unsaturated fatty acids (UFAs)

retards the crystallization process under low temperatures. Conversely, these changes adversely

affect oxidative stability, since the oxidation rates of C18:2 and C18:3 are higher compared to

that of C18:1. The problems can be solved when blended with petroleum diesel, or by utilizing

various additives, such as antioxidants. The optimal blending ratio of biodiesel/petrodiesel can be

determined experimentally and applied to improve fuel quality.

4. Conclusions

The cell growth rate and cellular lipid content of the marine microalga Tetraselmis sp.

were found to increase at higher nitrate concentrations than in nitrate deplete conditions. When

the cells were cultivated through a two-stage culture process in which lipid accumulation was

8
induced by a nitrate replete condition with nitrate concentration of 8.82 mM in the second stage,

enhanced lipid productivity of 47.3 mg L-1 d-1 was achieved. The major fatty acids produced by

Tetraselmis sp. were oleic acid (C18:1) and palmitic acid (C16:0), and the fraction of poly

unsaturated fatty acids increased with the increase of nitrate concentration.

Acknowledgements

This research was supported by a grant from the Marine Biotechnology Program (PJT200255,

Development of Marine Microalgal Biofuel Production Technology) funded by the Ministry of

Oceans and Fisheries of Korea.

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Microalgal lipids biochemistry and biotechnological perspectives. Biotechnol. Adv. 32, 1476-

1493.

Bondioli, P., Bella, L.D., Rivolta, G., Zittelli, G.C., Bassi, N., Rodolfi, L., Casini, D., Prussi, M.,

Chiaramonti, D., Tredici, M.R., 2012. Oil production by the marine microalgae

Nannochloropsis sp. F&M-M24 and Tetraselmis suecica F&M-M33. Bioresour. Technol. 114,

567-572.

Feng, D., Chen, Z., Xue, S., Zhang, W., 2011. Increased lipid production of the marine

oleaginous microalgae Isochrysis zhangjiangensis (Chrysophyta) by nitrogen supplement.

Bioresour. Technol. 102, 6710-6716.

Griffiths, M.J., Harrison, S.T.L., 2009. Lipid productivity as a key characteristic for choosing

algal species for biodiesel production. J. Appl. Phycol. 21, 493-507.

9
Guillard, R.R.L., 1975. Culture of phytoplankton for feeding marine invertebrates in: Smith,

W.L., Chanley, M.H. (Eds.), Culture of Marine Invertebrate Animals. Plenum Press, New

York, pp. 26-60.

Huerlimann, R., Nys, R., Heimann, K., 2010. Growth, lipid content, productivity, and fatty acid

composition of tropical microalgae for scale-up production. Biotechnol. Bioeng. 107 (2), 245-

257.

Kim, G., Lee, C.H., Lee, K., 2015. Enhancement of lipid production in marine microalga

Tetraselmis sp. through salinity variation. Korean J. Chem. Eng. 33 (1), 230-237.

Lee, C.G., Seong, D.H., 2015. A novel Tetraselmis sp. and method for preparing biodiesel with

this strain. Korean Patent 10-1509562.

Li, Y., Horsman, M., Wang, B., Wu, N., Lan, C.Q., 2008. Effects of nitrogen sources on cell

growth and lipid accumulation of green alga Neochloris oleoabundans. Appl. Microbiol.

Biotechnol. 81, 629-636.

Scott, S.A., Davey, M.P., Dennis, J.S., Horst, I., Howe, C.J., Lea-Smith, D.J., Smith, A.G., 2010.

Biodiesel from algae: challenges and prospects. Curr. Opin. Biotechnol. 21, 277-286.

Serrano, M., Oliveros, R., Sanchez, M., Moraschini, A., Martinez, M., Aracil, J., 2014. Influence

of blending vegetable oil methyl esters on biodiesel fuel properties: Oxidative stability and

cold flow properties. Energy. 65, 109-115.

Wijffels, R.H., Barbosa, M.J., 2010. An outlook on microalgal biofuels. Science. 329, 796-799.

Wu, H., Miao, X., 2014. Biodiesel quality and biochemical changes of microalgae Chlorella

pyrenoidsa and Scenedesmus obliquus in response to nitrate levels. Bioresour. Technol. 170,

421-427.

10
Wychen, S.V., Laurens, L.M.L., 2013. Determination of total lipids as fatty acid methyl esters

(FAME) by in situ transesterification. Laboratory analytical procedure, NREL, U.S.A.

Xu, N., Zhang, X., Fan, X., Han, L., Zeng, C., 2001. Effects of nitrogen source and concentration

on growth rate and fatty acid composition of Ellipsoidion sp. (Eustigmatophyta). J. Appl.

Phycol. 13, 463-469.

11
Figure Captions

Figure 1. The change of (a) cell concentration, (b) remaining nitrate concentration in medium, (c)

cellular lipid content, and (d) lipid concentration per reactor volume at different initial nitrate

levels in the first stage of cultivation.

Figure 2. The change of (a) cell concentration, (b) remaining nitrate concentration in medium, (c)

cellular lipid content, (d) protein content, (e) carbohydrate content, and (f) lipid concentration per

reactor volume at different high nitrate levels in the second stage of cultivation.

Figure 3. The change of biochemical composition at (a) nitrate deplete and (b) nitrate replete

conditions in the second stage of cultivation. (N-; nitrate deplete; 0 mM, N+; nitrate replete; 8.82

mM)

12
Table 1. Comparison of cell concentration, biomass productivity, cellular lipid content, lipid
concentration per reactor volume and lipid productivity in the single-stage and two-stage
cultivation with different nitrate concentrations.
1st stage 1st stage
Initial 1st stage (5 d) + 2nd stage (1.5 d)
(for 5 days) (for 6.5 days)
N concentration (mM) 0 0.88 0 0.88 0.88 4.41 8.82 17.6
-1
Cell concentration (g L ) 0.5 0.87 1.02 0.89 1.05 1.27 1.30 1.32 1.34

Biomass productivity (g L-1 d-1) 0.074 0.104 0.078 0.110 0.118 0.123 0.126 0.129

Cellular lipid content (wt %) 19.0 21.3 22.4 19.9 21.3 26.7 30.2 30.5 29.6

Lipid concentration (g L-1) 0.095 0.185 0.228 0.177 0.224 0.339 0.393 0.403 0.397

Lipid productivity (mg L-1 d-1) 18.1 26.7 12.6 19.8 37.6 45.8 47.3 46.4

Table 2. The change of fatty acids composition (wt %) at different nitrate levels

Lipid Common Initial N-deplete NaNO3 NaNO3 NaNO3 NaNO3

No. Name (0 h) (0 mM) 0.08 mM 4.41 mM 8.82 mM 17.6 mM

C10:0 Capric acid 0.27a ± 0.16 0.20 ± 0.19 0.46 ± 0.27 0.49 ± 0.11 0.45 ± 0.07 0.49 ± 0.11
C14:0 Myristic acid 0.66 ± 0.08 1.60 ± 0.80 1.58 ± 0.76 1.24 ± 0.94 1.29 ± 1.03 1.24 ± 0.94
C16:0 Palmitic acid 27.89 ± 0.23 27.20 ± 0.46 26.09 ± 0.26 25.01 ± 0.40 24.49 ± 0.33 25.01 ± 0.40
Palmitoleic
C16:1 2.39 ± 0.45 2.21 ± 0.12 2.17 ± 0.11 1.68 ± 0.04 1.70 ± 0.07 1.68 ± 0.04
acid
C17:0 Margaric acid 2.71 ± 0.16 2.08 ± 0.10 2.41 ± 0.02 4.00 ± 0.05 4.36 ± 0.08 4.00 ± 0.05
C16:4 HDTA 8.48 ± 0.32 9.98 ± 0.19 10.47 ± 0.11 10.86 ± 0.11 10.98 ± 0.18 10.86 ± 0.11
C18:0 Stearic acid 0.58 ± 0.10 0.57 ± 0.16 0.52 ± 0.08 0.40 ± 0.11 0.43 ± 0.05 0.04 ± 0.11
C18:1 Oleic acid 28.17 ± 0.36 24.34 ± 0.19 22.60 ± 0.17 18.40 ± 0.45 18.00 ± 0.22 18.40 ± 0.45
C18:2 Linoleic acid 6.92 ± 0.04 6.79 ± 0.05 7.48 ± 0.19 11.02 ± 0.09 11.39 ± 0.20 11.02 ± 0.09
α-Linolenic
C18:3 14.54 ± 0.21 17.01 ± 0.22 17.69 ± 0.14 18.53 ± 0.29 18.54 ± 0.21 18.53 ± 0.29
acid
Stearidonic
C18:4 2.02 ± 0.06 1.97 ± 0.05 2.35 ± 0.10 2.50 ± 0.12 2.40 ± 0.05 2.50 ± 0.12
acid
C22:0 Behenic acid 5.39 ± 0.52 6.05 ± 0.11 6.20 ± 0.27 5.86 ± 0.45 5.98 ± 0.59 5.86 ± 0.45
SFA 37.49 37.70 37.25 37.01 37.00 37.01
UFA 62.51 62.30 62.75 62.99 63.00 62.99
MUFA 30.56 26.55 24.77 20.08 19.70 20.08
PUFA 31.95 35.75 37.99 42.91 43.30 42.91
Total 100.00 100.00 100.00 100.00 100.00 100.00
a
Data expressed as mean ± SD (n = 3)

13
 1.6 3.0
(a) (b) NaNO3 0 mM (N-deplete)
1.4 NaNO3 0.88 mM (1-fold)
2.5
NaNO3 1.77 mM (2-fold)
1.2 NaNO3 2.64 mM (3-fold)

NO3-N (mmol L-1)

2.0
DCW (g L-1)

1.0

0.8 1.5

0.6
1.0

0.4 NaNO3 0 mM (N-deplete)

NaNO3 0.88 mM (control)
0.5
0.2 NaNO3 1.76 mM (2-fold)
NaNO3 2.65 mM (3-fold)
0.0 0.0
0 2 4 6 8 10 12 14 0 2 4 6 8 10 12

Cultivation time (d) Cultivation time (d)

40 0.35
(c) (d)
35 0.30

30
Lipid (g L-1) 0.25
Lipid (wt %)

25
0.20
20
0.15
15

0.10
10 NaNO3 0 mM (N-deplete) NaNO3 0 mM (N-deficient)
NaNO3 0.88 mM (1-fold) NaNO3 0.88 mM (1-fold)
5 NaNO3 1.76 mM (2-fold) 0.05 NaNO3 1.77 mM (2-fold)
NaNO3 2.65 mM (3-fold) NaNO3 2.65 mM (3-fold)
0 0.00
0 2 4 6 8 10 12 0 2 4 6 8 10 12

Cultivation time (d) Cultivation time (d)

Figure 1. The change of (a) cell concentration, (b) remaining nitrate concentration in medium, (c)

cellular lipid content, and (d) lipid concentration per reactor volume at different initial nitrate

levels in the first stage of cultivation.

14
 1.5 20
(b) NaNO3 0.88 mM (1-fold)
(a)
NaNO3 4.41 mM (5-fold)
NaNO3 8.82 mM (10-fold)
1.4
NaNO3 17.6 mM (20-fold)
15

NO3-N (mmol L-1)

DCW (g L -1)

1.3

10

1.2

NaNO3 0.88 mM (1-fold) 5

1.1
NaNO3 4.41 mM (5-fold)
NaNO3 8.82 mM (10-fold)
NaNO3 17.6 mM (20-fold)
1.0 0
0 12 24 36 48 60 72 0 12 24 36 48 60 72

Cultivation time (h) Cultivation time (h)

50
40
(c) (d)

40
30 Protein (wt %)
Lipid (wt %)

30

20

20

10 NaNO3 0.88 mM (1-fold) NaNO3 75 mg/L (1-fold)

NaNO3 4.41 mM (5-fold) 10
NaNO3 375 mg/L (5-fold)
NaNO3 8.82 mM (10-fold) NaNO3 750 mg/L (10-fold)
NaNO3 17.6 mM (20-fold) NaNO3 1500 mg/L (20-fold)
0 0
0 12 24 36 48 60 72 0 12 24 36 48 60 72

Cultivation time (h) Cultivation time (h)

40 0.5
(e) (f)

0.4
Carbohydrate (wt %)

30
Lipid (g L-1)

0.3

20

0.2

10 NaNO3 0.88 mM (1-fold) NaNO3 0.88 mM (1-fold)

NaNO3 4.41 mM (5-fold) 0.1 NaNO3 4.41 mM (5-fold)
NaNO3 8.82 mM (10-fold) NaNO3 8.82 mM (10-fold)
NaNO3 17.6 mM (20-fold) NaNO3 17.6 mM (20-fold)
0 0.0
0 12 24 36 48 60 72 0 12 24 36 48 60 72

Cultivation time (h) Cultivation time (h)

Figure 2. The change of (a) cell concentration, (b) remaining nitrate concentration in medium, (c)

cellular lipid content, (d) protein content, (e) carbohydrate content, and (f) lipid concentration per

reactor volume at different high nitrate levels in the second stage of cultivation.

15
 60
Carbohydrate (N-) (a)
Lipid (N-)
50 Protein (N-)

40
(wt %)

30

20

10

0
0 24 48 72

Cultivation time (h)

60
Carbohydrate (N+) (b)
Lipid (N+)
50 Protein (N+)

40
(wt %)

30

20

10

0
0 24 48 72

Cultivation time (h)

Figure 3. The change of biochemical composition at (a) nitrate deplete and (b) nitrate replete

conditions in the second stage of cultivation. (N-; nitrate deplete; 0 mM, N+; nitrate replete; 8.82

mM)

16
HIGHLIGHTS

A two-stage process was applied to enhance the lipid productivity of Tetraselmis sp.

Nitrate repletion enhanced both the cell growth and lipid content of Tetraselmis sp.

Nitrate depletion resulted in the lowest lipid productivity from Tetraselmis sp.

The fraction of polyunsaturated fatty acids (PUFAs) increased under N repletion.

17