Lipid accumulation by NaCl induction at different growth stages and concentrations in photoautotrophic two-step cultivation of Monoraphidium dybowskii LB50

Accepted Manuscript Lipid accumulation by NaCl induction at different growth stages and concentrations in photoautotrophic two-step cultivation of Monoraphidium dybowskii LB50 Haijian Yang, Qiaoning He, Chunxiang Hu PII: DOI: Reference:

S0960-8524(15)00457-5 http://dx.doi.org/10.1016/j.biortech.2015.03.125 BITE 14815

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Bioresource Technology

Received Date: Revised Date: Accepted Date:

14 February 2015 24 March 2015 25 March 2015

Please cite this article as: Yang, H., He, Q., Hu, C., Lipid accumulation by NaCl induction at different growth stages and concentrations in photoautotrophic two-step cultivation of Monoraphidium dybowskii LB50, Bioresource Technology (2015), doi: http://dx.doi.org/10.1016/j.biortech.2015.03.125

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Lipid accumulation by NaCl induction at different growth stages and concentrations in photoautotrophic two-step cultivation of Monoraphidium dybowskii LB50

Haijian Yang a,b, Qiaoning Hea,b, Chunxiang Hua,*

a

Key Laboratory of Algal Biology, Institute of Hydrobiology, Chinese Academy of

Sciences, Wuhan 430072, China b

University of Chinese Academy of Sciences, Beijing 100039, China

* Corresponding author: Chunxiang Hu Key Laboratory of Algal Biology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China E-mail: [email protected] Phone/Fax: +86-27-68780866

1

Abstract NaCl induction in photoautotrophic two-step cultivation is very promising, but time node and concentration are critical to the entire production. In this study Monoraphidium dybowskii LB50 was subjected to different NaCl concentrations at different growth phases. Results showed that during the initial phase (IP), fixed carbon was used for sugar and lipid under 5 g L−1 NaCl induction, as well as for protein under 10 g L−1 NaCl induction. At late-exponential growth phase (LEGP), the highest lipid productivity was obtained at 20 g L−1 NaCl. At stationary phase (SP) the highest lipid productivity was also under 20 g L−1 NaCl but lower than that of LEGP. In summary, lipid content and quality were improved at all growth phases under NaCl induction. Therefore, cultivation scale can be sued to determine the time node and dosage of the inducer, thereby realizing the economic efficiency of the fundamental guarantee in photoautotrophic two-step cultivation.

Keyword: Monoraphidium dybowskii LB50, lipid quality, late-exponential growth phase, NaCl induction, lipid productivity

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1. Introduction Microalgae are increasingly considered as feedstock for next-generation biofuel production (Bankovic-Ilic et al., 2012). To further enhance lipid production, as well as promote economic and sustainable development of resources, photoautotrophic two-stage cultivation strategy, as a very promising approach, involves the increase of biomass density at stage I and the induction of lipid accumulation at stage II (Su et al., 2011; Xia et al., 2013). During stage II, microalgae are usually subjected to stress conditions, such as nutrient and phosphorus deficiency, light and temperature changes, and inducer additons (Fe, NaCl, NaHCO3 and NaAC) (Ho et al., 2014b; Lacour et al., 2012; Sun et al., 2014). Among numerous inducers, NaCl is relatively cheap, easily available and very effective (Venkata Mohan & Devi, 2014; Yang et al., 2014). NaCl significantly promotes the lipid accumulation of marine and freshwater microalgae (Bartley et al., 2013; Ruangsomboon, 2012). However under different growth periods and various NaCl induction concentrations, the efficiency of lipid accumulation significantly differed. When Dunaliella tertiolecta was cultured with different concentrations of NaCl (0.5–1.0 M), the high salt increased the lipid content by 7%. The additions of NaCl at the mid-log phase or at the end of log phase with an initial NaCl concentration of 1.0 M further increased lipid content (10%) (Takagi et al., 2006). To induce the lipid content of Chlorella vulgaris, an inducer (Fe) was added to the media in the exponential growth phase (Liu et al., 2008). Under induction in the late growth period, the entire lipid production was unimproved owing to the prolonged of the culture time. Other 3

approaches were used for the two-stage cultivation. When nitrogen consumption was over 95%, different amounts of sea salt were added to the broth to induce the lipid of Chlamydomonas sp. JSC4 (Ho et al., 2014a). However, this approach has the inconvenience for outdoor large-scale cultures. NaCl was added in an appropriate time (easily detected), and the induction efficiency should be enhanced to shorten the culture period. Thus, the time node and concentration range of the added NaCl should be determined in photoautotrophic two-step cultivation to obtain the culture technique, and provide reference for other two-step inductions. Apart from lipid productivity, the appropriate biodiesel quality (C16-18) is likewise fundamentally important to the success of the algal-based biodiesel industry, because it influences the efficiency of biodiesel conversion and quality (Nascimento et al., 2013). Although the biodiesel quality of Desmodesmus abundans can be improved by the appropriate concentration of NaCl induction (Xia et al., 2014), the long-chain fatty acids that were unsuitable for biodiesel were promoted by NaCl induction in Nitzschia Laevis (Chen et al., 2008). Moreover, the saturated fatty acids were accumulated by NaCl induction, which regulated membrane fluidity and permeability (Xu & Beardall, 1997). NaCl induction in Botryococcus braunii Kütz IPPAS H-252 and B. braunii, promoted the saturated fatty acid content (Rao et al., 2007; Zhila et al., 2011). A very high content of saturated fatty acids can reduce the cetane number (CN), which influences biodiesel quality, through which the physiological response of cell to saline stress will surely affect the synthetic lipid carbon. Thus, further studies on the influence of microalgae lipid metabolism mechanism throgh NaCl induction should be conducted. 4

This study aimed to determine the time node and concentration necessary to induce lipid accumulation, obtain the maximum lipid productivity, and accomplish a simple, feasible operation, which is crucial in photoautotrophic two-step cultivation. M. dybowskii LB50 was induced with different NaCl concentrations various growth periods. Lipid content and quality, as well as fatty acid profiling and cellular response were investigated to understand the metabolic mechanism by NaCl induction. 2. Materials and methods 2.1. Organism M. dybowskii LB50 was provided by Prof. Xudong Xu of the Institute of Hydrobiology, the Chinese Academy of Sciences. The species sample was isolated from an alkaline (pH 8.48-9.04) reservoir in Inner Mongolia of China. Stock cultures were maintained indoors in a sterilized BG11 medium (Allen & Stanier, 1968). 2.2. Experimental setup NaCl induction on the day 0 (IP) indoors: M. dybowskii LB50 was cultivated in 500 mL Erlenmeyer flasks containing 400 mL of a modified BG11 medium of 0.25 g L−1 urea with continuous illumination at 60 µmol m−2 s−1 at 25 ± 1 °C. The sample underwent continuously bubbling with filter-sterilized air (from the bottom) through a transparent glass tube. NaCl concentrations were set to 0, 5, 10 g L−1. NaCl induction on the day 0 (IP) outdoors: M. dybowskii LB50 was cultivated in the optimal nutrition in 4 L of the culture medium in 5 L flasks at a green house in Beijing, China (40°22′N, 116°20′E). Gases were supplied to each bioreactor through the pipage mixed with 2% CO2 (v/v) during daytime, and pure air only at night. The 5

medium was thoroughly compounded with tap water. To keep cells in suspension, a 5 cm magnetic stir bar (mixing at 150 rpm) was used to stir at the middle of the 5 L flasks. NaCl concentrations were set to 0, 5, 10, 20 g L−1. NaCl induction on the 12th day (LEGP): NaCl was added during LEGP for final concentrations of 0, 20, 40 and 60 g L−1 in the 5 L flasks outdoors. NaCl induction on the 15th day (SP): NaCl was added during SP for final concentrations of 0, 20, 40 and 60 g L−1 in the 5 L flasks outdoors. 2.3. Analytical procedures 2.3.1. Biomass measurement Biomass productivity (BP, mg L−1 d−1) was calculated according to the following equation: BP = (B2 –B1)/ t where B2 and B1 represent the dry weight biomass density at the time t (days) and at the start of the experiment, respectively. Algal density was determined by measuring the optical density of algal at 680 nm (OD680). Relationships between the dry weight (DW, g L−1) and the OD680 values of the algae were described using the following equation: DW = 0.2122 × OD − 0.0037

R2=0.9888

Cells were harvested through centrifugation and lyophilized using a vacuum freeze dryer (Alpha 1-2 LD plus, Christ, Germany). 2.3.2. Biomolecular analysis To analyze the cellular constituents (total sugars, carbohydrates, soluble sugars 6

chlorophyll, carotenoids trehaloses and proteins), 1 mL of sample cells was harvested by centrifugation during initial phase indoors. Samples were then grounded with mortar and pestle. Total sugars and soluble sugars were quantified by phenol sulfuric acid method (Dubois et al., 1956; Gorai & Neffati, 2011) with glucose (99%) as standard. Trehalose was quantified following the anthrone-sulfuric acid colorimetric method (Trevelyan & Harrison, 1952). The amounts of the chlorophyll a, chlorophyll b and carotene were measured spectrophoto metrically as described by Jeffrey and Humphrey (1975) and Jasper et al. (1965). Protein was analyzed following the method of Bradford (1976). 2.3.3. Lipid analysis Total lipid was extracted from approximately 80–100 mg the dried algae (w1) by using a Soxhlet apparatus, with chloroform–methanol (1:2, v/v) as the solvent. The total lipid was transferred into a pre-weighed beaker (w2), and blow-dried in a fume cupboard. The lipid was dried to a constant weight in an oven at 105 °C and weighted (w3). Lipid content (LC %) and productivity (LP, mg L−1 d −1) were determined using the following equations: LC (%) = (w3 − w2)/w1 × 100 LP (mg L−1 d −1) = BP × LC 2.3.4. Gas chromatography analysis Total lipid, was analyzed through gas chromatography (GC) after direct transmethylation with sulphuric acid in methanol. Fatty acid methanol esters (FAMEs) were extracted with hexane and analyzed by GC–mass spectrometry (GC–MS) (Thermo 7

Scientific ITQ 700™, USA) equipped with a flame ionization detector (FID) and a fused silica capillary column (60 m×0.25 mm×0.25 µm; Agilent Technologies, USA). The injector and detector temperatures were maintained at 270 °C and 280 °C, respectively, with an oven temperature gradient of 50–170 °C at 40 °C min−1 after a 1 min hold time at 50 °C, then with an oven temperature gradient of 170–210 °C at 18 °C min−1 after a 1 min hold. All parameters of the FAMEs were derived from the calibration curves generated from the FAME standard mix (Supelco 37 component FAME mix, Sigma–Aldrich, USA). 2.3.5. Biodiesel property analysis To analysis of the biodiesel property, the average degrees of unsaturation (ADU), iodine value (IV), CN, higher heating value (HHV), cloud point (CP), kinematic viscosity (Vis) and specific gravity (SG) were determined by empirical equations based on fatty acids (FAs) as previously described (Hoekman et al., 2012; Song et al., 2013), whereas cold filter plugging point (CFPP) and long-chain saturated factor (LCSF) were derived following the method of Nascimento et al. (2013). The Biodiesel properties are as shown in equations. ADU=∑M×Yi Vis = −0.6316×ADU+5.2065

R2=0.6704

SG = −0.0055×ADU+0.8726

R2=0.6644

CP = −13.356×ADU+19.994

R2=0.6809

CN = −6.6684×ADU+62.876

R2=0.8049

IV = 74.373×ADU+12.71

R2=0.9484 8

HHV = 1.7601×ADU+38.534

R2=0.3800

LCSF = (0.1×C16) + (0.5×C18) + (1×C20) + (1.5×C22) + (2×C24) CFPP = (3.1417×LCSF)−16.477 Where Yi is the mass fraction of each FA constituent; M is the number of carbon–carbon double bonds in each FA constituent. 2.3.6. Statistical analysis Values were expressed as means ± standard deviations. Data were analyzed by one-way ANOVA using the SPSS statistical software (version 19.0). P< 0.05 was used to denote a statistically significant difference. 3. Results and discussion 3.1. Initial phase of NaCl induction 3.1.1. Biomass and lipid content Numerous studies have been explored the effects of salinity on marine algae growth (Bartley et al., 2013; Martinez-Roldan et al., 2014). However, few studies have focused on the influence of salinity on culture media to investigate the extent and mechanism of lipid production by freshwater microalgae. Several studies suggested that lipid contents of microalgae increased under cultivation in NaCl media (Rao et al., 2007; Salama et al., 2014), although the opposite conclusion was drawn by Ruangsomboon (2012). Thus, culturing M. dybowskii LB50 under NaCl is necessary to determine the tolerance and increase lipid. As shown in Fig. 1A-B, growth was significantly reduced, and the lipid content increased with the increase of NaCl concentration indoors. The pattern of NaCl 9

induction outdoors was similar to that of induction indoors (Fig. 1C-D). Outdoors, the biomass of M. dybowskii LB50 by 5 and 10 g L−1 NaCl induction was slightly lower than that of the control group (decreased by 5.54% and 11.46%). However, the lipid content increased by 2% and 5.64%, respectively. Under the highest concentration of NaCl induction (20 g L−1), the largest increase in lipid content was 10.48%, but the biomass declined the most (25.99%). Finally, lipid productivity indicated no significant increase. The combined patterns indoors increased the lipid content on the 12th day from that on the 9th day (Table 1). These findings suggest that if biomass was unaffected, the lipid content failed to increase by increasing the NaCl concentration. Therefore, lipid productivity cannot be increased using the technology of cultivation of M. dybowskii LB50 by NaCl induction during IP. Kaewkannetra et al. (2012) reported that lipid increased with the increase of NaCl concentrations, however, the biomass was inhibited during the cultivation of Scenedesmus obliquus from natural water basins. Ruangsomboon et al. (2013) found the growth was stimulated within a PSU of 0 to 10 (10 g L−1) range; however, the growth was suppressed when PSU increased during the cultivation of Scenedesmus dimorphus KMITL from freshwater. Marine microalgae Dunaliella showed the most viable growth at 1 M (58.5 g L−1) NaCl, and the lipid content was highest at this NaCl concentration (Takagi et al., 2006). In this study, M. dybowskii LB50 was isolated from an alkaline reservoir. Thus, cells grew well at 5 g L−1 NaCl, and the lipid content increased to some extent, without increasing the lipid productivity. These results indicate that the lipid productivity of M. dybowskii LB50 was not effectively increased by NaCl induction in 10

IP. 3.1.2. Physiological response to NaCl Fig. 2 shows the physiological responses of M. dybowskii LB50 given various concentrations of NaCl. The total sugar contents of NaCl induction were higher than those of the control (Fig. 2 A), which indicated that the growth of M. dybowskii LB50 was restrained under 10 g L−1 NaCl, although the cells still synthesized more sugars than lipids to resist high osmotic stress. Total protein declined within the course of cultivation (from 331.5 mg/g to 134.7 mg/g of the control). Protein was not a significant influence under low concentration of NaCl, and the protein contents of high NaCl concentration were higher by 20% than that of the control (12d; Fig. 2B). This finding can be attributed to the cells that synthesized the soluble protein to resist the osmotic stress. Soluble sugar and trehalose were increased under 5 g L−1 and 10 g L−1 NaCl cultivation (Fig. 2C, D), which implied that the osmotic regulation substances were mainly sugar (soluble sugar, trehalose) in low concentrations of NaCl. However, cells also synthesized soluble amino acids in high NaCl concentrations to increase cellular pressure. Chlorophyll and carotenoid concentrations were reduced, when M. dybowskii LB50 cultured under NaCl (Fig. 2E, F), which suggests that either chloroplasts or thylakoid was affected. Thus, photosynthesis was affected by high concentrations of NaCl. In most cases, microorganisms use a rapid and reversible increase in the concentration of some physiological solutes to counteract osmotic fluctuations. When microalgae were subjected to NaCl stress, some salt stress proteins can be synthesized. 11

Furthermore low molecular weight solutes can be synthesized to maintain membrane system integrity and protein stability. The Chl a, growth rates and net photosynthesis decreased, when the marine algae Nannochloropsis sp. was subjected to different concentrations of NaCl (13, 27, 54 and 81 g L−1 NaCl) (Martinez-Roldan et al., 2014). When green algae Botryococcus braunii were cultured under different concentrations of NaCl, carbohydrate limit increased (Rao et al., 2007). By contrary, cultivating marine alga Dunaliella salina and Dunaliella tertiolecta under different concentrations of NaCl, decreased the soluble sugar, chlorophyll a and b, and photosynthesis. Starch converted into other materials such as glycerol relative to the C flow (Tammam et al., 2011). Therefore, the physiological responses of algal cells to NaCl stress resulted in numerous substances and lipid competing for carbon, through which more substances competed with lipid as the NaCl concentration increased. 3.2. NaCl induction during late-exponential growth and stationary phases 3.2.1. Biomass and lipid contents through NaCl induction at late-exponential growth phase The 12th day (LEGP) of induced lipid accumulation through different NaCl concentrations (Fig. 3A, B), demonstrated the significant reduction of growth by 40 and 60 g L−1 NaCl induction (P< 0.05). However, such growth failed to exhibit an obvious decline after 3 d of 20 g L−1 NaCl induction. In the lipid aspect, lipid contents were significantly improved after 3 d of induction (increased by 11.15%, 11.15%, and 2.44%). The induced efficiency did not significantly change as the induction time increased (Fig. 3A). As regards lipid productivity. On the 15th day (3 d of induction), the lipid 12

productivity was the highest (37.73 mg L−1 d −1) by 20 g L−1 NaCl induction, which differed from the 60 g L−1 NaCl induction. Biomass and lipid contents were lower by 40 g L−1 NaCl induction than that by 20 g L−1 NaCl induction. On the 18th day, the time of induction increased, and the biomass and lipid contents did not significantly increase along with low lipid productivity. Thus, lipid contents and productivity can effectively increase in LEGP with suitable additions of NaCl concentrations. Increased induction time indicated no obvious contribution to the production. When Phaeodactylum tricornutum was cultured by different concentrations of NaHCO3 induction at the initial phase (0 d) or the exponential growth phase (4 d), the lipid content was the highest by 15 mM NaHCO3 induction at the exponential growth phase (Mus et al., 2013). When Dunaliella tertiolecta was cultured with different concentrations of NaCl (0.5 M to 1.0 M), the lipid content increased by 7% under high-salt concentration. Adding NaCl at the mid-log phase or at the end of log phase caused an increase of 10% in lipid (Takagi et al., 2006). Our results obtained the most feasible time node. At the appropriate stage of growth (LEGP), adding the proper NaCl concentration (20 g L−1), led to the maximum lipid content and productivity. Thus, this process not only saves time costs, but it can also obtain the highest production benefit. 3.2.2 Biomass and lipid contents through NaCl induction at the stationary phase On the 15th day (SP), the addition of different NaCl concentrations (Fig. 3C, D), indicated suppressed growth and increased inhibition with increasing NaCl concentration. The lipid contents were not higher after 3 d of 20 g L−1 NaCl induction, either with prolonged induction time or increased concentrations of NaCl. With regards 13

to lipid productivity, the efficiency was optimal after 3 d of 20 g L−1 NaCl induction (increased by 36.35%). With 10 g L−1 NaCl added at IP in combination with 20 g L−1 NaCl induction at LEGP, the lipid productivity did not significantly increase by more than only 20 g L−1 NaCl induction in LEGP (Fig. 4). Although the lipid contents increased by 20 g L−1 NaCl induction at SP, the lipid productivity (32.3 mg L−1 d−1) was lower than that (37.7 mg L−1 d−1) by 20 g L−1 NaCl induction at LEGP. The speed of increase of lipid increase was slower than the growth speed of M. dybowskii LB50 within 3 d of induction. As shown in Fig. 4, lipid productivity was divided into four parts. The low value was 20 mg L−1 d −1 to 25 mg L−1 d−1 at IP by induction with different NaCl concentrations. The lipid productivity (37.72 mg L−1 d−1) by 20 g L−1 NaCl induction at LEGP was higher than that by NaCl induction during IP and SP, as well as that by NaCl induction during IP in combination NaCl induction at LEGP. M. dybowskii LB50 was entirely different from the marine algae Chlamydomonas sp. JSC4, Nannochloropsis salina and Dunaliella (Bartley et al., 2013; Ho et al., 2014a; Takagi et al., 2006). The lipid content and productivity of Chlamydomonas sp. JSC4 were improved by 2% salinity under the initial and 1% salinity induction in the exponential growth phase (Ho et al., 2014a). However, M. dybowskii LB50 can be well cultured under outdoor conditions, which improved lipid productivity was improved in a large-scale (140 L) by 20 g L−1 NaCl induction at LEGP (Yang et al., 2014). 3.3. Fatty acid profiling and estimated biodiesel properties of M. dybowskii LB50 Fatty acid profiles for M. dybowskii LB50 under different periods by NaCl 14

induction in this study are listed in Table 2. The predominant FAMEs found in treatments were C16:0 and C18:1. The C16-18 FA possessed over 95% of the total FA. The contents of C16:0 decreased after NaCl induction. Monounsaturated and polyunsaturated fatty acids changed significantly with different periods of NaCl induction. At IP and LEGP by NaCl induction, the monounsaturated fatty acids increased (mainly C18:1), whereas the polyunsaturated fatty acids decreased (mainly C18:2). The monounsaturated fatty acids did not significantly change through NaCl induction at SP, but a slight decrease in the polyunsaturated fatty acids was derived. Generally, ADU was decreased under the induction of NaCl, which indicated that osmotic stress can assist the alga acclimate to the salinity stress by reducing the permeability and fluidity of the membrane lipid. These results were consistent with the FA changes observed in other algae by high NaCl concentration (Salama el et al., 2013; Xia et al., 2014). The appropriate lipid quality, apart from its yields, is also a desirable characteristic key of biodiesel. The ADU is closely related to the quality of diesel, such that if ADU is higher, CN is lower, thereby characterizing the lower oxidation stability. Moreover, according to the two most common quality standards for biodiesel, ASTM D6751 in the US and EN 14214 in Europe, the values of Vis, SG, CN and IV of different treatments satisfied the specifications (Table 2). The CN of M. dybowskii LB50 by NaCl induction was higher than that without NaCl induction. The CP values slightly increased, but CFPP failed to change significantly with a low temperature (−15 °C), which indicates that the low temperature flow property is very good and can satisfy China in addition to 15

three northeast provinces with CFPP requirements. Whenever NaCl was added to induce, the Vis of M. dybowskii LB50 was less than 5 (satisfied EN 14214). Thus, the quality of the biodiesel can be improved by NaCl induction in all growth phases. 4. Conclusions This work integrates physiological research and biochemical engineering approaches to develop a feasible and effective technology in determining the time node and dose of inducers in photoautotrophic two-stage cultivation to enhance lipid productivity and quality. The physiological responses by NaCl stress resulted in numerous substances and lipids that competed for carbon. Moreover, lipid contents and quality were improved through NaCl-inducted at different growth stages, particularly by 20 g L−1 NaCl (1.4 mg/10 7cells) induction at LEGP, lipid content and productivity can be significantly improved (increased by 10% and 30%). Therefore, this approach is a good strategy in photoautotrophic two-stage cultivation. 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 strain. We also thank Ms. Fang for her help in the analytical work

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Figure captions Fig. 1. Effect of different NaCl concentrations indoors and outdoors on growth and lipid content in M. dybowskii LB50. (A) Dry weight indoors, (B) Lipid content indoors, (C) Dry weight outdoors, (D) Lipid content and productivity outdoors. LC, lipid content; LP, lipid productivity. Fig. 2. Effect of NaCl concentration on biochemical composition of M. dybowskii LB50. Total sugar (A), Protein (B), Soluble sugar (C), Trehalose (D), ChlorophyII (E), Car (F). Fig. 3. Dry weight, lipid content (A) and lipid productivity (B) of M. dybowskii LB50 at different concentration of NaCl addition at LEGP; dry weight, lipid content (C) and lipid productivity (D) of M. dybowskii LB50 at different concentration of NaCl addition at SP. The addition times are indicated by arrows. Fig. 4. Growth and lipid content tradeoffs. Tradeoffs in growth and lipid content observed between different concentrations and different phases of NaCl addition treatments in M. dybowskii LB50. The cross-hatched is peak lipid productivity. (●, ▼, ■, ◆, represent 0, 5, 10, 20 g L−1 NaCl induction at IP; ●, ▼, ■, ◆,represent 0, 20, 40, 60 g L−1 NaCl induction 3 days at LEGP; ●, ▼, ■, ◆, represent 0, 20, 40, 60 g L−1 NaCl induction 3 days at SP; ●，■，represent control and 20 g L−1 NaCl induction at LEGP under 10 g L−1 NaCl culture).

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Fig. 1

Fig. 2

Fig. 3

Fig. 4

Table captions

Table 1 Dry weight, lipid content and lipid productivity of M. dybowskii LB50 at different concentrations of NaCl culture. [LC, lipid content (%); LP, lipid productivity (mg L−1 d−1)]. Table 2 Fatty acid profile (%) of biodiesel and estimated properties of biodiesel from the M. dybowskii LB50 in maximum lipid productivity of different induction periods.

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Table 1 Dry weight, lipid content and lipid productivity of M. dybowskii LB50 at different concentrations of NaCl cultivation. [BP, (mg L-1 d -1); LC, lipid content (%); LP, lipid productivity (mg L−1 d−1)] time

NaCl (g L-1)

BP (mg L-1 d-1)

LC (%)

LP (mg L-1 d-1)

9d

0

141.78±3.69a

30.97±0.81 d

43.90±1.44g

5

136.04±7.21a

33.97±0.95 e

46.21±1.29g

10

118.28±4.74b

35.69±1.15 e

42.21±1.36g

0

124.02±5.59b

33.91±0.92 e

42.06±1.14g

5

121.25±2.87b

36.03±0.46 f

43.68±0.56g

10

99.56±2.14c

35.93±0.96 f

35.77±0.95h

12d

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, c (BP), d, e, f (LC), g, h (LP)].

Table 2 Fatty acid profile (%) of biodiesel and estimated properties of biodiesel from the M. dybowskii LB50 in maximum lipid productivity of different induction periods IP (0d)

LEGP

(12d)

SP （15d）

Control

10 g/L

Control

20 g/L

Control

20g/L

C16:0

26.55

30.31

21.46

41.87

27.34

33.88

C16:1

2.31

0.06

2.80

4.26

2.68

3.48

C18:0

4.46

1.65

1.11

1.65

1.14

0.89

C18:1

40.13

46.74

42.88

12.18

31.34

30.06

24

C18:2

9.88

5.09

16.25

23.60

12.90

10.97

C18:3

11.73

11.38

8.78

12.40

17.58

16.18

C18:4

3.56

3.14

4.83

0.00

5.72

3.92

C16-C18

98.62

98.37

98.11

95.96

98.70

99.38

SFA

31.01

31.96

22.57

43.52

28.48

34.77

MUFA

42.44

46.80

45.68

16.44

34.02

33.54

PUFA

25.17

19.61

29.86

36.00

36.20

31.07

ADU

1.12

1.04

1.24

1.01

1.35

1.20

CN

55.43

55.96

54.62

56.15

53.84

54.89

CP (℃)

5.08

6.15

3.45

6.53

1.90

4.01

CFPP (℃)

-15.29

-16.48

-15.24

-15.55

-15.30

-15.39

IV (g I2 100 g-1)

95.73

89.82

104.82

87.70

113.44

101.73

SG (Kg L-1)

0.87

0.87

0.87

0.87

0.87

0.87

HHV (MJ/Kg)

40.50

40.36

40.71

40.31

40.92

40.64

Vis (mm2s-1)

4.50

4.55

4.42

4.57

4.35

4.45

SFA = saturated fatty acids; MUFA = monounsaturated fatty acids; PUFA = polyunsaturated fatty acids; Vis = Kinematic viscosity 40℃; SG = Specific gravity; CP = Cloud point; CN = Cetane number; IV = Iodine value; HHV = Higher heating value； ADU = Average degree of unsaturation; CFPP = Cold filter plugging point.

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Highlights ▶Lipid production in LEGP is the highest with 20g/L NaCl induction. ▶With NaCl induction, lipid content and quality were improved in all growth phases. ▶Sugar or protein competed for carbon with lipid under different NaCl concentrations. ▶ADU decreased, reducing the membrane fluidity when NaCl induction was applied. ▶The improved lipid quality was mainly for increasing CN and a slight decline of CFPP.

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