Characterization of lipid and fatty acids composition of Chlorella zofingiensis in response to nitrogen starvation

Journal of Bioscience and Bioengineering VOL. xx No. xx, 1e5, 2015 www.elsevier.com/locate/jbiosc

Characterization of lipid and fatty acids composition of Chlorella zofingiensis in response to nitrogen starvation Shunni Zhu, Yajie Wang, Changhua Shang, Zhongming Wang, Jingliang Xu, and Zhenhong Yuan* Key Laboratory of Renewable Energy, Guangzhou Institute of Energy Conversion, Chinese Academy of Sciences, Guangzhou 510640, China Received 20 August 2014; accepted 18 December 2014 Available online xxx

Cellular biochemical composition of the microalga Chlorella zofingiensis was studied under favorable and nitrogen starvation conditions, with special emphasis on lipid classes and fatty acids distribution. When algal cells were grown in nitrogen-free medium (N stress), the increase in the contents of lipid and carbohydrate while a decrease in protein content was detected. Glycolipids were the major lipid fraction (50.7% of total lipids) under control condition, while neutral lipids increased to be predominant (86.7% of total lipids) under N stress condition. Triacylglycerol (TAG) content in N stressed cells was 27.3% dw, which was over three times higher than that obtained under control condition. Within neutral lipids fraction, monounsaturated fatty acids (MUFA) were the main group (40.6%) upon N stress, in which oleic acid was the most representative fatty acids (34.5%). Contrarily, glycolipids and phospholipids showed a higher percentage of polyunsaturated fatty acids (PUFA). Lipid quality assessment indicated the potential of this alga as a biodiesel feedstock when its neutral lipids were a principal lipid fraction. The results demonstrate that the neutral lipids content is key to determine the suitability of the microalga for biodiesel, and the stress cultivation is essential for lipid quality. Ó 2015, The Society for Biotechnology, Japan. All rights reserved. [Key words: Chlorella zofingiensis; Nitrogen starvation; Lipid; Fatty acids; Biodiesel]

Over-consumption of fossil fuels has led to energy crisis and environmental problems. In light of this, sustainable biofuels, especially biodiesel, have attracted much attention in recent years. Biodiesel is a mixture of fatty acid methyl esters (FAME) which is conventionally produced by transesterification of vegetable oils or animal fats (1). Currently, microalgae have been recognized as a promising feedstock for biodiesel production due to several advantages, such as high photosynthetic efficiency, rapid growth rate, and high lipid content (2,3). Chlorella has long been commercially applied for human food, animal feed, and bioactive compounds. In recent years, there were increasing reports demonstrating the potential of Chlorella for biodiesel production due to their high lipid productivity and environmental adaptation (4e6). Generally, algal cell growth and metabolism are highly influenced by environmental conditions and can be physiologically manipulated. It has been reported that nutrient limitation or starvation could significantly increase lipid accumulation of many species of Chlorella (7e9). As far as we are concerned, not only is the lipid quantity affected by stress condition in microalgae, but variations of lipid quality are occurred as well. However, much of the information about lipid and fatty acid composition corresponds to the total lipids. Few studies focused on the lipid composition as well as the distribution of fatty acids in individual lipid class in Chlorella species (1). The limited information hinders the comprehensive understanding of microalgal lipid metabolism and evaluation of the microalgal lipid suitability for biodiesel (10).

* Corresponding author. Tel.: þ86 2087057735; fax: þ86 2087057737. E-mail address: [email protected] (Z. Yuan).

Our previous studies have shown that the green microalga Chlorella zofingiensis could be used as a cell factory for algal oil under nitrogen starvation (11) and utilize various wastewater for growth (12,13), thus being promising in commercial applications. Other studies have also reported the high lipid content and biomass concentration of C. zofingiensis in the past few years (4,14), although this alga was found to produce astaxanthin initially (15e16). Liu et al. (1,4) intensively compared lipid classes and fatty acids composition of C. zofingiensis under photoautotrophic and heterotrophic conditions, and suggested that heterotrophic C. zofingiensis was more feasible for biodiesel production. However, autotrophic algal cells in their studies were cultivated under normal growth condition rather than environmental stress condition, which cannot thoroughly reflect the lipid characteristics of autotrophic cells. Based on the aforementioned information, it is essential to study neutral lipids accumulation in microalgae under photoautotrophic conditions. Though the total lipids are an important indicator for selecting oleaginous species, neutral lipids and their fatty acids profiles provide a more specific signal of suitable substrate for biodiesel production. Up to now, a detailed characterization of lipid was not carried out for Chlorella grown under nitrogen starvation condition. Thus, in this study, C. zofingiensis was tested to further investigate the variations of cellular biochemical composition, especially lipid classes and the distribution of fatty acids in the lipid pool under nitrogen starvation (stress) condition in comparison to favorable growth (control) condition. The results from this study would be helpful for enhancing our understanding of lipid biosynthesis and evaluating the lipid suitability for biodiesel production.

1389-1723/$ e see front matter Ó 2015, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2014.12.018

Please cite this article in press as: Zhu, S., et al., Characterization of lipid and fatty acids composition of Chlorella zofingiensis in response to nitrogen starvation, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2014.12.018

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ZHU ET AL.

J. BIOSCI. BIOENG., MATERIALS AND METHODS

Algal strain and culture conditions C. zofingiensis was obtained from the Microalgae Culture Collection in Guangzhou Institute of Energy Conversion (Guangzhou, China) and maintained in BG11 medium. The culture conditions were performed according to our previous study (11). For the analysis of biochemical components, the cultures grown in N-free medium for 6 days (N stress condition) and in exponential phase in BG11 medium (control condition) were harvested, respectively. Each experiment was performed in triplicate. Protein measurement Freeze-dried samples were extracted with 1 M NaOH at 80 C for 20 min and then centrifuged at 4500 rpm for 10 min. The supernatant was used for protein determination by Bradford method (17). A calibration curve was prepared using BSA dissolved in distilled water. Carbohydrate measurement Carbohydrate concentration was determined by the phenol-sulfuric acid method. Briefly, freeze-dried biomass was reconstituted in water to prepare a known sample concentration for each sample. Aliquots of 2 mL sample were reacted with 5 mL of concentrated sulfuric acid (98 wt%) and 1 mL of phenol (6%, w/v). After cooled to room temperature, the absorbance of the final mixture was measured on a spectrophotometer at 490 nm. Samples were then quantified by comparison to a calibration curve made from glucose under the same conditions. Total lipids extraction Total lipids were extracted according to Bigogno et al. (18). Freeze-dried algal biomass was extracted with methanol containing 10% DMSO for 50 min under stirring. Then the mixture was centrifuged, and the supernatant was removed. The residua were re-extracted with a mixture of hexane and diethyl ether (1:1, v/v). After extraction, water was added to the combined supernatants to separate the organic phase. The mixture was shaken and then centrifuged for 5 min and the upper phase was collected. The water phase was re-extracted twice with a mixture of hexane and diethyl ether (1:1, v/v). The organic phase was combined and evaporated to dryness under a stream of N2. The lipid content was determined gravimetrically. Lipid fractionation and fatty acids profile Total lipid extracts were fractionated into neutral lipids (NL), glycolipids (GL) and phospholipids (PL) by solidphase extraction according to Liu et al. (4). A 500 mg Sep-Pak cartridge of silica gel (Waters) was initially equilibrated with 5 mL of chloroform. Subsequently, 1 mL chloroform solution containing about 50 mg of lipid was applied to it. The cartridge was eluted with 10 mL of chloroform to collect NL, 10 mL of acetone to collect GL, and 10 mL of methanol to collect PL. The different fractions obtained were evaporated and weighed. For analysis of TAG, neutral lipids were separated on a silica gel 60 TLC (Merck) using a solvent system of hexane/diethyl ether/acetic acid (70:30:1, v/v/v). After solvent evaporation, the plates were sprayed with phosphomolybdic acid solution (10% in ethanol) and heated at 90 C for 10 min. TAG was quantified using densitometry and image analysis scaled to a dilution series of TAG standard (triolein). Fatty acid methyl esters were prepared by incubating freeze-dried biomass or lipid extracts in methanol containing 2% (v/v) H2SO4 at 80 C for 2.5 h. Fatty acid analysis was performed according to our previous work (11). The analysis of algal biodiesel properties The saponification number (SN), iodine number (IN) and cetane number (CN) were estimated by empirical equations shown below (19). SN ¼ S(560  Pi)/MWi

(1)

IN ¼ S(254  D  Pi)/MWi

(2)

CN ¼ 46.3 þ 5458/SN e 0.225  IN

(3)

where Pi is the weight percentage of each FAME, MWi is the molecular mass of individual FAME, D is the number of the double bonds in each FAME.

RESULTS Cellular biochemical composition Table 1 shows the biochemical composition of C. zofingiensis under control and N stress conditions. The algal strain was grown well in N-rich

medium and showed the biomass concentration on day 4 was 2.27 g L1. However, nitrogen depletion severely inhibited cell growth with a low biomass on day 6 (0.64 g L1). The protein content showed a significant decrease under N stress (16.56% dw) compared with control (33.15% dw). In contrast, the levels of carbohydrate and total lipids under control were 31.73% dw and 26.69% dw, respectively, and increased significantly to 47.73% dw and 34.99% dw, respectively, under N stress condition. The difference of other composition of biomass except the sum of protein, carbohydrate and total lipids between two conditions may be derived from the restriction of analytical methods. For example, Bradford method was only appropriate for the measurement of soluble proteins rather than insoluble proteins. Lipid fractions GL were the main fraction of total lipids under control condition, accounting for 50.7% of total lipids (Fig. 1) with a value of 12.95% dw (Table 1). Another kind of membrane lipid (PL) were 7.5% of total lipids (Fig. 1) and 1.92% dw (Table 1), much lower than GL. NL were the second most abundant lipid class, occupying 36.4% of total lipids (Fig. 1) and 9.29% dw (Table 1) under control condition. Under N stress condition, NL content increased dramatically to 30.75% dw, while GL and PL were both reduced to less than 1% dw (Table 1). As shown in Fig. 1, NL became the major fraction that accounted for 86.7% of total lipids under N stress condition, in which TAG was the predominant component, accounting for 75% of the total lipids. Although TAG was also the most abundant component of NL under control, its cellular content was merely 7.67% dw, which was much lower than that obtained from N stressed cells (27.28% dw). Regarding lipid productivity which is an important indicator for practical application, the productivities of total lipids and NL are listed in Table 1. Total lipid productivity under control (138.60 mg L1 d1) was much higher than that under N stress (25.37 mg L1 d1), mainly attributed to the substantial biomass production. However, NL productivity under control (48.31 mg L1 d1) was less than twice that under N stress (28.88 mg L1 d1). Fatty acids profiles in total lipids and individual lipid fraction Fatty acids profiles in total lipids and major lipid fractions under control and N stress conditions are listed in Table 2. Most fatty acid species were composed of 16 and 18 carbon atoms in all lipid fractions regardless of culture conditions, but specific fatty acid species have considerable changes upon N stress. C18:0 and C18:1 species in NL increased while C16:0, C16:1, C18:2 and C18:3 species decreased under N stress condition. Especially oleic acid (C18:1) became the predominant species in NL after N stress condition, which was beneficial for producing high quality biodiesel, since high content of oleic acid can balance the oxidative and low-temperature stability (20). Fatty acids profile in total lipids has a similar pattern with that in NL in response to N stress except for C16:0 and C18:0 species. However, there were quite different patterns in GL and PL. In order to achieve clearer understanding of variations of fatty acid profiles in each lipid fraction and total lipids upon N stress, fatty acids were categorized into three groups and summarized in Fig. 2. Interestingly, the fatty acids groups of total lipids and NL followed similar trends when exposure to N stress. N starvation led

TABLE 1. Protein, carbohydrate, total lipids and lipid fractions (in percentage of dry weight biomass ¼ % dw), biomass and lipid productivity of C. zofingiensis growing under different culture conditions. Condition Control N stress

Biomass (g L1)

Protein (% dw)

Carbohydrate (% dw)

Total lipids (% dw)

Neutral lipids (% dw)

Glycolipids (% dw)

Phospholipids (% dw)

Total lipid productivity (mg L1 d1)

NL producitivity (mg L1 d1)

2.27  0.03 0.64  0.01

33.15  1.77 16.56  1.23

31.73  1.40 47.73  3.63

26.69  0.90 34.99  0.95

9.29  0.67 30.75  0.50

12.95  0.28 0.68  0.17

1.92  0.39 0.73  0.04

138.60  5.69 25.37  2.01

48.31  2.59 28.88  0.26

Values are means  standard deviations of triplicates. NL, neutral lipids.

Please cite this article in press as: Zhu, S., et al., Characterization of lipid and fatty acids composition of Chlorella zofingiensis in response to nitrogen starvation, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2014.12.018

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Lipid distribution (%)

100

Control

80

N stress

60 40 20 0 NL

GL

PL

FIG. 1. Distribution of NL, GL and PL in total lipids extracted from control and N stressed cells. The horizontal line inside the NL bar marks the portion of TAG in this fraction. Values are expressed as mean  standard deviation of triplicates.

to an increase in monounsaturated fatty acids (MUFA) levels and a decrease in the proportions of polyunsaturated fatty acids (PUFA) in both total lipids and NL, whereas saturated fatty acids (SFA) levels had no significant modifications. MUFA represented the most abundant group of NL (40.6%) under N stress condition, in which oleic acid was the most representative fatty acid (34.5%). Contrarily, GL and PL showed a higher percentage of PUFA with respect to NL. As shown in Fig. 2, the proportion of PUFA within GL and PL were 37.0% and 38.7% respectively, while NL contained PUFA with a lower level of 23.9%. DISCUSSION Our previous study has reported the growth characteristics of C. zofingiensis under N-rich and N-free conditions, and found that nitrogen depletion severely inhibited the cell growth (11). As an important macronutrient, the variations of nitrogen affect not only cell growth but also cellular biochemical composition (21,22). Protein, carbohydrate and lipid are the major constituents of microalgal biomass, and our results showed that nitrogen starvation led to the increase in the contents of carbohydrate and lipid and a decrease in protein content. Similar patterns have been reported in Chlorella vulgaris and Scenedesmus acutus cells (23,24). However, different behaviors were observed in Neochloris oleoabundans (22) and Nannochloropsis sp. (25) where the carbohydrate

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content was virtually impervious to N stress. A decrease in protein content is a common response in microalgae under N depletion, as nitrogen is a necessity for protein synthesis. Several reports have suggested that N starvation increases carbon availability, causing carbon flux shifted from the pathway of protein synthesis to that of lipid and/or carbohydrate synthesis (26,27). Generally vegetative algal cells mainly synthesize membrane lipids (GL and PL), but alter lipid biosynthetic pathways to accumulate NL when subject to stress conditions (3). However, the allocation of the lipid classes in a specific microalga was not clear. In this study, GL were the main fraction of total lipids under control condition, which are characteristic of photoautotrophic microalgae in the exponential growth phase (28,29). Contrarily, NL, instead of GL, became the major fraction under N stress condition. From the perspective of biodiesel production, NL are superior to PL or GL due to their higher percentage of fatty acids and lack of phosphate and sulfur (30). The low content of PL and GL in algal oil is beneficial for downstream processing. Thus, stress cultivation is very important for producing numerous desirable lipids. On the other hand, even though total lipids content increased only 31%, NL and TAG contents both increased over twofold under N stress in comparison to control. Our results documented that, under N starvation, a degradation of membrane lipids was accompanied by the accumulation of NL (mainly TAG). Goncalves et al. (31) found that membrane lipid acyl groups remodeled into TAG during N starvation in Chlorella UTEX29. Yoon et al. (32) suggested that an acyl CoA-independent PDAT pathway may mediate membrane lipids turnover and promote TAG synthesis under stress, which could provide insight into the connection between a decrease of membrane lipids and an increase of NL. In addition to the storage form of carbon and energy, TAG might play a protective role to prevent microalgae from oxidative damage under stress condition (3,27). Lipid productivity is an important indicator for screening algal species for biodiesel production, but many concerns were focused on total lipid productivity rather than NL productivity. From our analysis above, NL productivity should be a more key characteristic for biodiesel production. In this study, even though total lipid productivity was much higher under control than under N stress, the difference of NL productivities between control and N stress was much smaller than that of total lipid productivities (Table 1). So it will be necessary to elevate biomass production under N stress condition through cultivation optimization to increase NL productivity. As can been seen in Figs. 1 and 2, fatty acids profile in total lipids was severely influenced by fatty acids profile in the principal lipid fraction. For example, under control condition, the distribution of

TABLE 2. Fatty acids profiles (percentage of total fatty acids) in total lipids, neutral lipids, glycolipids and phospholipids under control and N stress conditions. Fatty acids

C10:0 C12:0 C14:0 C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 C20:1 C20:2 C20:4 C22:0 C22:1 C24:0 C24:1

Total lipids

Neutral lipids

Glycolipids

Phospholipids

Control

N stress

Control

N stress

Control

N stress

Control

N stress

tr tr 1.05  0.31 23.80  2.72 6.80  0.06 8.11  0.72 20.30  0.35 25.34  0.66 13.53  0.79 tr tr nd nd 0.64  0.19 tr 1.16  0.03

tr tr 0.21  0.29 27.52  0.28 3.35  0.18 6.97  0.72 31.96  1.08 18.08  0.15 11.06  0.28 tr tr nd nd 0.24  0.33 tr 0.59  0.41

0.89  0.08 0.97  0.20 0.94  0.08 26.72  0.25 4.68  0.36 4.97  0.28 26.49  0.17 23.58  0.13 9.73  0.09 0.31  0.28 nd nd 0.44  0.25 0.28  0.03 nd nd

tr tr 0.86  0.19 25.90  0.36 2.96  0.48 6.70  1.92 34.46  2.06 15.22  1.58 8.65  0.35 tr 0.27  0.09 tr 1.33  0.17 1.23  0.39 nd 1.86  0.68

nd tr 1.15  0.11 22.56  1.36 6.39  0.29 8.25  0.51 19.69  0.93 24.25  1.67 13.64  0.27 tr tr tr 1.23  0.87 1.18  0.64 nd 2.47  1.23

nd nd tr 25.67  1.13 5.52  1.77 14.49  0.21 12.13  1.18 20.16  2.30 16.82  1.11 0.97  0.71 nd nd nd nd tr 3.51  1.20

nd nd tr 35.85  1.31 7.31  1.45 10.66  1.10 12.98  0.17 19.77  0.75 12.45  0.46 0.27  0.18 tr nd nd nd nd nd

nd nd nd 33.48  0.96 2.81  1.20 3.90  1.00 21.33  2.41 21.80  2.06 12.15  0.40 tr tr nd nd tr 1.83  0.35 tr

Values are means  standard deviations of triplicates. tr, trace; nd, no detected.

Please cite this article in press as: Zhu, S., et al., Characterization of lipid and fatty acids composition of Chlorella zofingiensis in response to nitrogen starvation, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2014.12.018

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J. BIOSCI. BIOENG.,

50

% wt

50 Control

N stress

TL

NL

40

40

30

30

20

20

10

10 0

0 SFA 50

% wt

MUFA

SFA

PUFA

% wt GL

50

PUFA

% wt PL

40

40

30

30

20

20

10

10

0

MUFA

0 SFA

MUFA

PUFA

SFA

MUFA

PUFA

FIG. 2. Fatty acids groups (% wt of TFA) of C. zofingiensis cultured under control (closed bars) and N stress (shaded bars) conditions (means  SD, n ¼ 3). SFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids; TL, total lipids; NL, neutral lipids; GL, glycolipids; PL, phospholipids. Significant variations (p <0.05) between control and N stress are marked with asterisks.

fatty acids groups in total lipids was similar with that in GL, i.e., PUFA were the most abundant fatty acids, while MUFA were the lowest abundant fatty acids. After N starvation, NL became the principal lipid fraction, therefore total lipids showed a higher MUFA level and a lower PUFA level. PUFA are the main fatty acids in the composition of chloroplast membrane structures and their role in membrane structural integrity is critical in green algae (33). Thus, the decrease in PUFA level might be associated with strong effect of N starvation on the algal photosynthesis system. In the present study, within NL fraction, MUFA were the predominant group (40.6%) upon N stress, in which oleic acid was the most representative fatty acid (34.5%). This feature of NL indicates a good performance of this oil for biodiesel production. Apart from lipid productivity, another important characterstic for biodiesel production is the lipid suitability for biodiesel in light of the carbon chain length and degree of saturation produced by a microalgal species. In this alga, the major fatty acids is intermediate chain length with 16 and 18 carbons, and the maximum degree of unsaturation is three double bonds. As a rule, SFA are oxidative stable, while unsaturated fatty acids have low-temperature stability. In this study, SFA level in the NL fraction did not increase while PUFA showed a significant decrease under N stress condition with respect to control. Moreover, it is noteworthy that the MUFA represented the most abundant group of NL under N stress condition due to high percentage of oleic acid (Fig. 2 and Table 2). Levine et al. (34) suggested that oleic acid is considered ideal for biodiesel because it has better cold flow properties without losses to oxidative degradation. As FAME are relatively consistent with fatty acids present in the feedstock, this relation could be used to estimate the properties of

biodiesel produced from the feedstock (19,35). Cetane number (CN) is a prime indicator of biodiesel quality and used to justify the biodiesel’s ignition quality. It increases with increasing saturation and increasing chain length in the fatty acids (20). In this study, CN of total lipids under control condition could not meet European biodiesel standard (Table 3), probably due to the high PUFA content. Basically, NL had higher CN than total lipids, while N stress showed a higher CN than control condition (Table 3). Iodine number (IN) is a measurement of the oil unsaturation. The calculated IN of total lipids and NL under both conditions were all well below the allowed standards (120 gI2/100 g). In particular, NL under N stress exhibited the lowest IN (83.68 gI2/100 g). Regarding PUFA, the European EN 14214 standard limits linolenic acid’s methyl ester for vehicle use to 12% (m/m) and the methyl esters with four and more double bonds to a maximum of 1% (m/m) (36). It can be seen from Table 2, NL meets these specifications under both conditions, while total lipids meets these specifications only under N stress condition. From the evaluation above, NL generally presented good biodiesel properties whatever the conditions, while total lipids

TABLE 3. The biodiesel properties in total lipids and neutral lipid fraction under control and N stress conditions. Biodiesel property

SN CN IN

Total lipids

Neutral lipids

Control

N stress

Control

N stress

196.36 50.7 104.01

194.81 53.82 91.12

197.04 52.94 93.59

192.31 55.85 83.68

EN 14214 standard

e 51 120

SN, saponification number; IN, iodine number; CN, cetane number.

Please cite this article in press as: Zhu, S., et al., Characterization of lipid and fatty acids composition of Chlorella zofingiensis in response to nitrogen starvation, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2014.12.018

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became suitable for biodiesel when NL were prevailing under environmental stress conditions. In other words, the suitability of the microalga for biodiesel depends on its NL content. Therefore, environmental stress conditions are very essential for lipid quality from the perspective of biodiesel. Although N starvation could significantly elevate the microalgal NL content, its major drawback is low biomass production (Table 1). Thus, it will be necessary to further optimize culture conditions for this alga in order to obtain desirable lipid quantity and quality for biodiesel production. Systematic investigation on the effects of other factors, for example, increasing inoculum size, high light, limitation of other nutrients, cultivation mode, etc. on the quality and quantity of lipids in C. zofingiensis should be further made.

ACKNOWLEDGMENTS This research was financially supported by National Natural Science Foundation of China (No. 31100189), the National Basic Research Program of China (973 Program) (2011CB200905), the National Key Technology R&D Program of China (2011BAD14B03), National High-tech R&D Program of China (863 program) (2013AA065803), and the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry.

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Please cite this article in press as: Zhu, S., et al., Characterization of lipid and fatty acids composition of Chlorella zofingiensis in response to nitrogen starvation, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2014.12.018