Bioresource Technology 172 (2014) 131–137

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Rapid neutral lipid accumulation of the alkali-resistant oleaginous Monoraphidium dybowskii LB50 by NaCl induction Haijian Yang a,b, Qiaoning He a,b, Junfeng Rong c, Ling Xia a,b, Chunxiang Hu a,⇑ a

Key Laboratory of Algal Biology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China University of Chinese Academy of Sciences, Beijing 100039, China c SINOPEC Research Institute of Petroleum Processing, Beijing 100083, China b

h i g h l i g h t s  0.25 g L

1

urea and 0.1 M NaHCO3 are optimal nutritions for M. dybowskii LB50. 1 NaCl.  Induction and cultivation time are shortened at optimum NaCl addition.  Induction efficiency in 140 L reactor is the same as that in 5 L flasks.  Membrane remodeling contributes to NL accumulation under salt stress.

 Neutral lipid productivity is enhanced by 20 g L

a r t i c l e

i n f o

Article history: Received 9 July 2014 Received in revised form 11 August 2014 Accepted 13 August 2014 Available online 8 September 2014 Keywords: Monoraphidium dybowskii LB50 Neutral lipid productivity NaCl induction Glycolipids Phospholipids

a b s t r a c t NaCl is an effective inducer of lipid accumulation in freshwater microalgae, but little is known on whether the enhanced lipid components are desired. To address this issue, Monoraphidium dybowskii LB50 from a freshwater habitat was selected, cultivated, and subjected to NaCl induction at different scales outdoors. Results showed that the optimal salt concentration reduced glycolipid (GL) content, as well as enhanced neutral lipid (NL) and phospholipid (PL) contents. Moreover, GL was preferentially converted to NL at 20 g L1 NaCl. Total lipid and NL contents respectively increased to 41.7% and 17.48% in 1 d. The highest NL productivity was also achieved at both the 5 L (24.13 mg L1 d1) and 140 L (13.05 mg L1 d1, 3.43 g m2 d1) scales. These results suggest that NL accumulated effectively and rapidly at different scales, indicating that this strategy has broad application prospects for the scale-up cultivation of oily algae. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Microalgae have numerous advantages as feedstock for thirdgeneration bioenergy. These advantages include photosynthetic efficiency, high lipid content, and capability to grow in extreme environments (Hu et al., 2008). Microalgae can also be cultivated as an integrated approach for wastewater treatment to optimize the energy and financial input for the production process (Sharma et al., 2012). However, to achieve the sustainable industrial production of bioenergy from microalgae, the crucial technological bottleneck of providing sufficient oleaginous feedstock must be addressed. Given that microalgae with high lipid contents are often confronted with significantly low lipid contents when grown indoors, polluted by other miscellaneous algae, or preyed ⇑ Corresponding author. Tel./fax: +86 27 68780866. E-mail address: [email protected] (C. Hu). http://dx.doi.org/10.1016/j.biortech.2014.08.066 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.

upon by protozoa (Hu et al., 2008; Moheimani, 2013a,b), largescale culture in outdoor environments is recommended. Therefore, an essential prerequisite to achieve the industrial-scale application of microalgal biofuel is the selection of robust and highly productive microalgal strains with relatively high lipid content. To accumulate large amounts of lipids and neutral lipids, microalgae are usually subjected to stress conditions, such as nutrient and phosphorus deficiency (Lacour et al., 2012), because lipid contents can be significantly enhanced by the absence of nutrients. Triacylglycerols (TAG) of Chlamydomonas reinhardtii increased in the absence of sulfur (Cakmak et al., 2012). However, high biomass cannot be obtained in this way. Therefore, the photoautotrophic two-stage process naturally becomes a very promising approach (Xia et al., 2013). However, the use of the method from nutrientrich to nitrogen-strarvation medium is difficult for outdoor largescale cultures and changes in light and temperature are limited to outdoor cultures (Rodolfi et al., 2009; Su et al., 2011). Thus, a

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promising idea is to incorporate the inducer directly into the medium. The effectiveness of this type of inducer mainly depends on the use of iron salt, zinc salt, and sodium salt (NaCl, NaHCO3, NaAC) etc. (Einicker-Lamas et al., 2002; Herrera-Valencia et al., 2011; Liu et al., 2008; Xia et al., 2013, 2014). However, the effects are not evident in numerous microalgae strains when iron salt and zinc salt are used for induction. Moreover, these salts can harm the environment when used at a large scale. Therefore, sodium salt is widely used to stimulate lipid accumulation in microalgae, with NaCl proven to be very effective. However, efficiency and induction time are directly related to production cost (Xia et al., 2014). Therefore, induction efficiency should be enhanced to shorten the culture period. Under adverse environmental or stress conditions, many microalgae form and accumulate neutral lipids (20–50% DW) in the form of TAG by altering the lipid biosynthesis pathway (Sharma et al., 2012). Although the mechanism of lipid transformation under the NaCl-induced stress is unclear, the case is sure to differ from that of nitrogen and phosphorus nutrient deficiency. Approximately 60% lipid was synthesized from de novo carbon fixation (from CO2 fixation) in cases of nitrate deprivation. The remaining lipids came from the transformation of pigment, protein, starch, and other components of lipid membranes (Burrows et al., 2012; Li et al., 2014). Under NaCl stress, the adaptability of some amino acids (proline, homoserine, etc.), carbohydrates (sucrose, trehalose, etc.) increases, which reduces the pyruvate content, which is a precursor of TAG synthesis (Bromke et al., 2013). To resist the osmotic pressure, an increase in membrane lipid content naturally reduces the TAG percentage from membrane lipid transformation (Chen et al., 2008), and the increased total lipid mainly comprise polar lipids, not TAG (Azachi et al., 2002; Chen et al., 2008). However, this condition is inconsistent with the transformation relationships among NL, PL and GL (Chen et al., 2008; Zhila et al., 2011). The effect of NaCl induction is closely related to the growth stage of microalgae. When Dunaliella tertiolecta was cultured with different concentrations of NaCl (0.5–1.0 M), high salt increased lipid content by 7%, TAG by 15.8%. Adding NaCl at mid-log phase or at the end of log phase did not cause a significant increase in lipid and TAG contents (Takagi et al., 2006). Notably, few studies have been conducted under relatively large-scale outdoor conditions, which is the key to using NaCl as an inducer. This study aims to screen the alkali-resistant oleaginous Monoraphidium dybowskii LB50 with high lipid productivity and to obtain the maximum neutral lipid productivity within a short time of NaCl induction in a 140 L bioreactor situated outdoors. Microalgal strains were first screened and then induced with different NaCl concentrations. Lipid productivity and the transformation relationships were investigated among NL, PL, and GL. Based on the above concentrations, the NL accumulation of the selected strains at different outdoor scales were induced by the optimal NaCl content. Finally, a quick and effective culture method was established.

40 mg K2HPO4, 75 mg MgSO4  7H2O, 20 mg Na2CO3, 36 mg CaCl2  2H2O, 6 mg ammonium ferric citrate, 6 mg ammonium citrate monohydrate, 1 mg EDTA, 2.86 lg H3BO3, 1.81 lg MnCl2  4H2O, 0.222 lg ZnSO4  7H2O, 0.39 lg Na2MoO4  2H2O, 0.079 lg CuSO4  5H2O, 0.050 lg CoCl2  6H2O in 1 L water. 2.2. Experimental design 2.2.1. Strain selection Six freshwater microalgal strains were screened indoors. These strains were cultivated in modified BG11 medium with 0.15 g L1 NaNO3 in 400 mL of the culture medium in 500 mL Erlenmeyer flasks. The algal cultures underwent continuously bubbling with filter-sterilized air (from the bottom) through the transparent glass tube. A light density of 60 lmol m2 s1 (24 h) was provided by cool white fluorescent tubes (400–750 nm), and the temperature was maintained at 25 °C by an air conditioner. Six freshwater microalgal strains were screened in 5 and 140 L bioreactors in a green house in Beijing, China (40°2200 , 116°2000 E). Gases were supplied to each bioreactor through the pipage mixed with 2% CO2 (v/v) during daytime. At night only pure air was supplied. The medium was thoroughly compounded with tap water to realize the microalgae resistance. The strains were cultivated in modified BG11 medium with 0.15 g L1 NaNO3 in 4 L of the culture medium in 5 L flasks. To keep cells in suspension, a 5 cm magnetic stirring bar (mixing at 150 rpm) was used to stir the middle of the 5 L flasks. Micractinium sp. XJ-2, M. dybowskii LB50, and M. dybowskii XJ-2 were cultivated in modified BG11 medium with 0.25 g L1 urea in the 140 L photobioreactor. The 140 L bioreactor was used to test whether the microalgae can grow well and accumulate the neutral lipid under large-scale culture conditions. This bioreactor was composed of two connected 70 L polyvinylchloride hanging bags (1800 mm in height and 220 mm in diameter) (Dalian Huixin Titanium Equipment Development Co., Ltd., China) (Xia et al., 2013). 2.2.2. Optimization of microalgal nutrition M. dybowskii LB50 was cultivated in 400 mL of culture medium in 500 mL Erlenmeyer flasks indoors. The concentrations of NaNO3 and urea were respectively set at 0.15, 0.30, 0.9, 1.50 g L1 and 0.05, 0.10, 0.25, 0.50 g L1. Under suitable concentration of NaNO3 and urea, the NaHCO3 concentrations were set to 0, 0.1, and 0.2 M. 2.2.3. Induction experiments M. dybowskii LB50 was cultivated under optimal nutrition conditions in 4 L of the culture medium in 5 L flasks outdoors. On the 12th day (the late-exponential growth phase) NaCl was added for final concentration of 0, 20, 40, and 60 g L1 in the 5 L flasks. In the 140 L bioreactor, the NaCl concentration was 20 g L1. The induction experiments were conducted from June to August 2013. The greenhouse temperature was 34.44 ± 6.10 °C during day time and 22.17 ± 3.05 °C at night. 2.3. Analytical procedures

2. Methods 2.1. Organisms The six oleaginous microalgae used in this study were provided by Prof. Xudong Xu of the Institute of Hydrobiology the Chinese Academy of Sciences. These strains were Micractinium sp. XJ-2, Westella botryoides XJ-4, M. dybowskii XJ-2, Monoraphidium sp. 1, M. dybowskii LB50, and Monoraphidium sp. LB59. M. dybowskii LB50 was isolated from an alkaline (pH 8.48–9.04) reservoir in Inner Mongolia of China. The stock cultures were maintained indoors in a sterilized BG11 medium containing 1.5 g NaNO3,

2.3.1. Biomass measurement The biomass productivity (BP, mg L1 d1) was calculated according to the following equation:

BP ¼ ðB2  B1Þ=t where B2 and B1 represent the dry weight biomass density at the time t (days) and at the start of the experiment, respectively. The algal density was determined by measuring the OD680. The relationships between the dry weight (DW, g L1) and the OD680 values of the algae can be described using the following equations:

H. Yang et al. / Bioresource Technology 172 (2014) 131–137

DW ¼ 0:287  OD  0:005 R2 ¼ 0:991 ðMicractinium sp:XJ-2Þ

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DW ¼ 0:212  OD  0:004 R2 ¼ 0:989 ðM: dybowskii LB50Þ

content and lipid productivity, the pattern of the six strains outdoors was similar to that indoors. At the 140 L scale (Table 2), M. dybowskii XJ-2 had lower biomass productivity because it was easily contaminated (miscellaneous algae or protozoa), and Micractinium sp. XJ-2 could grow normally because of its significant antipollution capability but at a lower lipid content. Only the lipid productivity of M. dybowskii LB50 was the highest (25.07 mg L1 d1), and M. dybowskii LB50 was well cultured in the 140 L photobioreactor. Therefore, M. dybowskii LB50 was studied in the subsequent trial.

DW ¼ 0:197  OD þ 0:002 R2 ¼ 0:995 ðMonoraphidium sp: LB59Þ

3.2. Optimization of nutritional conditions

DW ¼ 0:269  OD  0:010 R2 ¼ 0:990 ðW: botryoides XJ-4Þ DW ¼ 0:260  OD  0:012 R2 ¼ 0:990 ðM: dybowskii XJ-2Þ DW ¼ 0:189  OD þ 0:002 R2 ¼ 0:992 ðMonoraphidium sp:1Þ

The cells were harvested by centrifugation and then lyophilized using a vacuum freeze dryer (Alpha 1-2 LD plus, Christ). 2.3.2. Lipid analysis The total lipid was extracted from approximately 80–100 mg of dried algae (w1) using a Soxhlet apparatus, with chloroform–methanol (1:2, v/v) as the solvent. The total lipid was transferred into a pre-weighted beaker (w2), and blow-dried in a fume cupboard. The lipid was dried to a constant weight in an oven at 105 °C and then weighed (w3). The total lipid content (LC, %) and the lipid productivity (LP, mg L1 d1) were determined according to the following equations:

LC ð%Þ ¼ ðw3  w2 Þ=w1  100 1

LP ðmg L1 d Þ ¼ BP  LC 2.3.3. Fractionation of Lipids Solid phase extraction was used to separate lipid extracts into three fractions: NL, GL, and PL (Bellou and Aggelis, 2012). The extracted lipids were dissolved in chloroform (1 mL), fractionated by using a column (20 mm  200 mm) of silicic acid (4 g), and activated by heating overnight at 110 °C. Successive applications of chloroform (six times the volume), acetone (four times the volume) and methanol (four times the volume) produced fractions containing NL, GL and PL, respectively. The total lipid contents were then calculated as follows: neutral lipid content (NLC, %), glycolipids content (GLC, %), and phospholipids content (PLC, %). The neutral lipid content of algal dry weight (NLB, %) and the neutral lipid productivity (NLP, mg L1 d1) were calculated according to the following equations:

NLB ð%Þ ¼ NLC  LC 1

NLP ðmg L1 d Þ ¼ NLB  BP 2.3.4. Statistical analysis The values were expressed as the mean ± standard deviation. The data were analyzed by one-way ANOVA using the SPSS statistical software (version 19.0). P < 0.05 was considered to denote a statistically significant difference. 3. Results and discussion 3.1. Strain selection As shown in Table 1, three microalgal strains had higher lipid productivity: M. dybowskii XJ-2 (32.95 mg L1 d1), Micractinium sp. XJ-2 (30.03 mg L1 d1), and M. dybowskii LB50 (31.25 mg L1 d1). To test their growth potential under outdoor conditions, the six microalgae were screened under outdoor conditions in a 5 L flask (Table 1). According to their biomass productivity, lipid

Nutritional conditions should be optimized to obtain the maximum biomass of M. dybowskii LB50. Microalgae require a large amount of nitrogen. The types and concentrations of nitrogen are important in microalgae growth and metabolism, as well as significantly affect the cost of outdoor large-scale cultivation. Therefore, NaNO3 and urea were selected to identify candidate nitrogen sources for M. dybowskii LB50 in this study. In addition, M. dybowskii LB50 was isolated from a natural alkaline environment and exhibited good growth only in such environment. Thus, M. dybowskii LB50 cultures were treated with a range of NaHCO3 concentrations from 0.0 to 0.2 M NaHCO3 to select the optimum concentration. M. dybowskii LB50 had the highest biomass yield under 0.9 g L–1 NaNO3 and 0.25 g L1 urea, and the biomass productivity under urea was higher than that of NaNO3 (Table 3). Under optimal concentrations, the highest lipid productivity were 25.18 and 25.21 mg L1 d1. However, high concentration of nitrogen sources (1.5 g L1 NaNO3 or 0.5 g L1 urea) resulted in low biomass. Therefore, the optimum nitrogen source and concentration should be chosen for oleaginous microalgae cultivations. In addition, under outdoor large-scale cultivation, using urea is significantly cheaper than using NaNO3 and helps to fight pollution by protozoa and other algae. Therefore, 0.25 g L1 of urea was used in the M. dybowskii LB50 outdoor cultures and follow-up tests. As regards alkalinity, we found that highest biomass productivity and highest lipid productivity occurred in 0.1 M NaHCO3 by adding 0, 0.1 and 0.2 M NaHCO3 to the optimal concentrations of NaNO3 (0.9 g L1) and urea (0.25 g L1) (Table 3). Therefore, regardless of the nitrogen source, the optimal concentration of NaHCO3 was 0.1 M. Mus et al. (2013) reported that Phaeodactylum tricornutum was not inhibited under suitable concentrations of NaHCO3, whereas lipid content did not increased, but merely served as a source of dissolved inorganic carbon (DIC). However, TAG increased at higher concentrations or with an inducer. The lipid content of M. dybowskii LB50 did not increase at 0.1 M NaHCO3 (Table 3). 3.3. Lipid induced 3.3.1. Lipid accumulation and transformation Although the lipid content of M. dybowskii LB50 was more than 30% in the 140 L bioreactor (Table 5), the pursuit of high lipid productivity and neutral lipid productivity is the goal in the chain of the oleaginous microalgae industry. Therefore, further improving the lipid content is necessary. Compared with other inducers, NaCl is relatively cheap and easy to obtain. Biodiesel quality is improved through NaCl induction (Xia et al., 2014). Thus, this study selected NaCl as a large-scale pilot for induction, on day 12 (the late-exponential growth phase) through the addition of different NaCl concentrations. As shown in Fig. 1, growth was significantly reduced (p < 0.05) under 40 and 60 g L1 NaCl, but the growth at 20 g L1 NaCl did not exhibit an obvious decline. As regards lipid contents, lipid

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Table 1 Biomass productivity, lipid content and lipid productivity of microalgae under different conditions. Species

BP (mg L1 d1)

LC (%)

LP (mg L1 d1)

Indoors (250 mL)

Micractinium sp. XJ-2 Westella botryoides XJ-4 Monoraphidium dybowskii XJ-2 Monoraphidium dybowskii LB50 Monoraphidium sp. LB59 Monoraphidium sp. 1

106.59 ± 4.91 76.09 ± 3.42 92.44 ± 7.37 92.01 ± 5.22 83.71 ± 0.50 63.00 ± 5.55

30.91 ± 0.28 24.37 ± 0.91 32.49 ± 1.77 33.97 ± 1.73 28.67 ± 0.78 29.87 ± 0.19

32.95 ± 1.52 18.54 ± 0.83 30.03 ± 2.39 31.25 ± 1.75 24.00 ± 0.14 18.82 ± 1.66

Outdoors (5 L)

Micractinium sp. XJ-2 Westella botryoides XJ-4 Monoraphidium dybowskii XJ-2 Monoraphidium dybowskii LB50 Monoraphidium sp. LB59 Monoraphidium sp. 1

131.96 ± 4.94 96.58 ± 3.54 128.14 ± 6.34 112.02 ± 6.05 100.55 ± 4.06 80.48 ± 3.12

31.91 ± 1.06 23.37 ± 0.54 29.15 ± 0.87 31.57 ± 0.40 25.67 ± 1.84 29.01 ± 0.16

42.11 ± 1.56 22.57 ± 0.83 37.35 ± 1.85 35.37 ± 1.91 25.81 ± 1.04 23.35 ± 0.90

Table 2 Lipid content and lipid productivity of three microalgal strains in 140 L large-scale photobioreactors outdoors. Species

BP (mg L1 d1)

LC (%)

LP (mg L1 d1)

Micractinium sp. XJ-2 Monoraphidium dybowskii LB50 Monoraphidium dybowskii XJ-2

91.75 80.56 66.34

25.11 31.12 30.87

23.04 25.07 20.48

contents were higher under 20 g L1 and 40 g L1 NaCl induction, whereas the lipid contents were low (almost 30%) in the control group and at 60 g L1 NaCl induction. Furthermore, from the perspective of induced efficiency, the lipid contents were enhanced only 1 d after induction and did not change significantly after subsequent days of induction (Fig. 1). Lipid could be large accumulated in a short time through 20 g L1 NaCl induction, such that the culture period is shortened and higher lipid productivity is achieved. However, higher concentrations (40 and 60 g L1) are harmful to microalgae because of high osmotic stress. After total lipid content was enhanced, lipid classes, especially neutral lipid content, should be considered. As shown in Fig. 2A, the NL contents were highest under 20 and 40 g L1 NaCl induction. By contrary, PL increased instead of NL at the highest concentration of NaCl (60 g L1) induction. Along with the increase in salinity, GL rapidly decreased. At 20, 40 and 60 g L1 NaCl induction, GL reduction was equal to the amount of total PL and NL increase (Fig. 2B). This finding indicates that increase in NL can be mainly attributed to the transformation of intracellular GL under a suitable concentration of NaCl. Fig. 2C further shows the relative proportion of each component transformation. At

Fig. 1. Influence of different concentrations of NaCl induction on biomass productivity and lipid content of M. dybowskii LB50 under outdoor conditions in 5 L flasks. The top graph represents DW, and bottom graph represents LC at different concentrations of NaCl induction on day 12.

20 g L1 NaCl induction, the reduced GL transformed in NL (Fig. 2B), such that the NL increased by 7% (% DW). For the increase in NL, approximately 18% came from GL, and another 82% came from the de novo synthesis of pyruvate. At 40 g L1 NaCl induction, a part of the increase in NL comprised 32% from GL transformation, and the remaining 68% was from de novo synthesis. This finding may be attributed to the fact that cell growth was inhibited at 40 g L1 NaCl induction (Fig. 1), and CO2 fixation was reduced, such that the de novo synthesis of TAG was reduced. Meanwhile, when cells were subjected to strong osmotic stress, PL increased with

Table 3 Medium optimization for M. dybowskii LB50 growth in 500 mL flasks indoors. Cultures

Concentration 1

BP (mg L1 d1)

LC (%)

LP (mg L1 d1)

Nitrate

0.15 g L 0.30 g L1 0.90 g L1 1.50 g L1

39.08 ± 0.90 56.70 ± 4.60 69.79 ± 1.40 60.77 ± 0.50

38.46 ± 3.23 34.36 ± 2.59 36.08 ± 1.79 23.08 ± 0.22

15.03 ± 0.35 19.49 ± 1.58 25.18 ± 0.51 14.02 ± 0.12

Urea

0.05 g L1 0.10 g L1 0.25 g L1 0.50 g L1

43.29 ± 4.60 32.11 ± 6.26 76.86 ± 7.78 42.72 ± 11.08

42.69 ± 2.36 36.73 ± 0.47 32.80 ± 3.79 30.65 ± 1.13

18.48 ± 1.96 11.79 ± 2.30 25.21 ± 2.55 13.09 ± 3.40

Nitrate (0.9 g L1)+NaHCO3

0.0 M 0.1 M 0.2 M

98.41 ± 1.33 117.94 ± 0.30 97.38 ± 2.94

30.57 ± 0.90 32.55 ± 1.14 33.42 ± 1.78

30.08 ± 0.41 38.39 ± 0.10 32.54 ± 0.98

Urea (0.25 g L1)+NaHCO3

0.0 M 0.1 M 0.2 M

62.39 ± 10.06 118.83 ± 2.09 99.55 ± 4.26

31.36 ± 1.18 33.75 ± 1.96 31.32 ± 2.11

19.57 ± 3.16 40.11 ± 0.71 31.18 ± 1.33

Data are represented as mean ± standard.

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Fig. 3. Variations in irradiance and air temperature of cultivated the M. dybowskii LB50 in 140 L photobioreactor. The data were obtained from a detection system that monitored the irradiance and temperature every 10 min.

Fig. 2. Contents of individual lipid classes [% total lipid (A)] of M. dybowskii LB50 at different inductions in 5 L flasks outdoors. The increment and decrement [% total lipid (B), % DW (C)] of lipid classes relative to the control after 1 d of the induction. Data are presented mean ± SD of two independent experiments. Mean values in a column sharing common alphabets are statistically not significant at P < 0.05 by one-way ANOVA. [a,b (NL) and c (PL)].

membrane lipid remodeling, which increased the transformation of GL into NL. At 60 g L1 NaCl induction, with the highest osmotic stress, GL was transformed into PL (74%) by DAG. These findings

suggest that membrane remodeling contributed to the observed NL accumulation under salt stress. Increasing findings have shown that high salt concentration induced high lipid accumulation in microalgae. However, the mechanism is unclear. Starch synthesis decreased when microalgae were cultured in high NaCl concentration (Yao et al., 2013). Many studies suggested that starch synthesis decreased with increased lipid synthesis (Fernandes et al., 2013; Siaut et al., 2011). However, whether starch was degraded and partially converted into lipid at high NaCl concentrations remains unclear. However, higher NaCl concentration (60 g L1) did not cause a greater increase in lipid content. Possibly because of high osmotic stress, cells performed essential metabolic activities, such as respiration enhancement (Martinez-Roldan et al., 2014), to respond to high osmotic pressure at higher NaCl concentration (60 g L1). However, these assumptions are physiological speculations and are inconsistent with the lipid class transformation observed in other algae. Zhila et al. (2011) reported that Botryococcus braunii Kütz IPPAS H-252 could increase lipid and TAG along with increasing salt stress. Chen et al. (2008) found that TAG of Nitzschia laevis decreased but the PL increased in heterotrophic cultures at high NaCl concentrations. In this study, the change in NL was similar to that of B. braunii Kütz IPPASH-252 in the range of 20 and 40 g L1 NaCl induction. However, at 60 g L1 NaCl induction, the change in PL was similar to that in N. laevis. PL is the main component of membrane lipids, and PL accumulation could stimulate membrane biosynthesis (Elkahoui et al., 2004). Microalgae synthesized more PL by reducing membrane fluidity and permeability to help algae adapt to salt stress at higher salt concentrations. However, at 20 and 40 g L1, NaCl induction resulted in the accumulation of more TAG possibly because NL can increase membrane hardness to adapt to the corresponding salt stress (Lu et al., 2012).This finding implies that under salt stress, membrane lipid

Table 4 Biomass and lipid productivity of M. dybowskii LB50 different inductions under outdoor conditions in 5 L flasks in the first 13 d. NaCl (g L1) 0 20 40 60

BP (mg L1 d1) a

140.26 ± 8.70 137.98 ± 1.80a 118.83 ± 6.00b 120.11 ± 1.80b

LP (mg L1 d1)

LC (%) c

32.45 ± 1.80 41.73 ± 0.75d 39.97 ± 1.17d 35.74 ± 1.99c

e

45.51 ± 2.82 57.50 ± 0.75f 47.50 ± 2.40e 42.92 ± 0.64e

NLB (% DW) g

10.23 ± 0.73 17.48 ± 0.64h 17.70 ± 0.01h 11.85 ± 0.45g

NLP (mg L1 d1) 14.35 ± 1.03i 24.13 ± 0.88j 21.03 ± 0.02k 14.23 ± 0.54i

Data represents mean ± SD of two independent experiments, mean values in a column sharing common alphabets are statistically not significant at P < 0.05 by one way ANOVA [a,b (BP); c,d (LC); e,f (LP); g,h (NLC); i,j,k (NLP)].

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Table 5 Comparisons of biomass and lipid productivity of different sizes in some microalgae outdoors. Strains

LC (%)

VBP (mg L1 d1)

ABP (g m2 d1)

VLP (mg L1 d1)

ALP (g m2 d1)

Culture

Bioreactor

References

Tetraselmis suecica Chlorella sp. S. obtusus XJ-15 S. rubescens N. salina C. zofingiensi C. sp. FC2 IITG M. dybowskii LB50

32 23 42.1 13.8 16.33 41.3 35.12 32.78 38.57

51.54 60 – – – 64.4 44 89.2 81.43

– – – 4.02 24.5 – – 23.79 21.71

14.8 13.7 23.2 – – 26.6 10.7 29.16 32.48

– – – 0.56 4 – – 7.8 8.59

CO2

120 L

(Moheimani, 2013a)

Two-step Normal Normal Air CO2

140 L RCS RCS 60 L FPPs 300 L OP 140 L

(Xia et al., 2013) (Lin and Lin, 2011) (Boussiba et al., 1987) (Feng et al., 2012) (Muthuraj et al., 2014) this study

NaCl

VBP, volumetric biomass productivity; ABP, areal biomass productivity; RCS, raceway cultivation system; FPPs, flat plate photobioreactors; OP, open ponds.

remodeling and turnover demonstrate adaptation to high osmotic stress. Microalgae could accumulate lipids at a suitable salt concentration, but the increases in total lipid is lower than that under N, P, S absence conditions (Pal et al., 2011; Yeesang and Cheirsilp, 2011). This is important because under osmotic stress, membrane lipid and other soluble substances increase, and carbon can be synthesized to achieve NL reduction. The reduction mainly refers to the amount of transformation between intracellular components, whereas algae biomass (CO2 fixation) and growth were unaffected. Therefore, higher lipid productivity can more effectively be achieved through NaCl induction than through nutritional deficiency. Thus, NaCl induction has more broad application prospects.

3.3.2. Neutral lipid productivity of induction As shown in Table 4, under the induction of 20 g L1 NaCl, lipid productivity increased to 57.50 mg L1 d1, which was 1.26 times higher than 45.51 mg L1 d1 without NaCl induction. This value was almost twice that of the lipid productivity (29.2 mg L1 d1) of Monoraphidium sp. SB2 (Wu et al., 2013). The neutral lipid content of dry weight also increased from 10% to 17.48%, and neutral lipid productivity increased by 10 mg L1 d1 within one day. In such a short time, lipid productivity was improved, and neutral lipid was significantly enhanced under outdoor large-scale induction. To detect NaCl-induced effects in a large-scale bioreactor, M. dybowskii LB50 was inoculated in a 140 L bioreactor during clear summer days (Fig. 3). In the 140 L bioreactor, biomass was slightly reduced after 20 g L1 NaCl induction, but the lipid content increased to 38.57%, such that volumetric lipid productivity (VLP) and areal lipid productivity (ALP) increased by 11%. In particular, neutral lipid also has good prospects in the large bioreactor (to 15.80% of dry weight), the areal productivity of neutral lipid reached 13.05 mg L1 d1 or 3.43 g m2 d1, an increase of 54% (Table 5). According to the analysis of lipid classes, the neutral lipid (% total lipid) increased from 28.57% to 40.97%. The glycolipid content decreased from 26.25% to 12.90%. The phospholipid content did not significantly change. Induced neutral lipid was higher than the control, similar to that observed in the 5 L flask outdoors. Moreover, induction efficiency is the same as that in the 5 L flasks after scale-up. The maximum advantage of this strains is that volumetric lipid productivity and areal lipid productivity are much higher than reported, reaching to 32.48 mg L1 d1 and 8.59 g m2 d1, respectively. These values are twice those of Tetraselmis suecica and Chlorella sp. and 30% more than those of Scenedesmus obtusus XJ15 (Table 5). The lipid contents of Desmodesmus abundans T12 and S. obtusus XJ-15 reached 39% and 47.7% after 9 d through NaCl induction (Xia et al., 2013, 2014). However, the induction time of M. dybowskii LB50 in this study was short (1 d). This strain could obtain the highest lipid content and neutral lipid productivity, thus

significantly shortening the culture cycle and enhancing the lipid productivity and the neutral lipid productivity. Consequently, this strain of microalgae and its culture mode have promising prospects for commercial-scale application.

4. Conclusions In this study M. dybowskii LB50 was selected from six microalgal strains for its obvious advantages in terms of resistance and high lipid productivity under optimum nutrition. The suitable NaCl concentration could further promote the conversion of GL and lipid into NL. Finally lipid content (38.57%) and neutral lipid productivity (3.43 g m2 d1) had been well improved by 20 g L1 NaCl induction within a short time (1 d) at the 140 L scale outdoors. Therefore, M. dybowskii LB50 is a promising feedstock for biodiesel production, and NaCl induction is a good strategy for increasing neutral lipid productivity.

Acknowledgements This work was funded by National 863 program (2013AA065804) and the Program of Sinopec, international partner program of innovation team (Chinese Academy of Sciences), Platform construction of oleaginous microalgae (Institute of Hydrobiology, CAS of China). We are indebted to Prof. Xu (Institute of Hydrobiology, CAS of China) for providing us the microalgal strains.

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