Effect of Salinity Change on Biomass and Biochemical Composition of ...

Vol. 43, No. 1 February, 2012

JOURNAL OF THE WORLD AQUACULTURE SOCIETY

Effect of Salinity Change on Biomass and Biochemical Composition of Nannochloropsis oculata Na Gu Key Laboratory of Marine Bio-resource Sustainable Utilization, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, China and Graduate School of the Chinese Academy of Sciences, Beijing 100049, China

Qiang Lin1 and Gang Li Key Laboratory of Marine Bio-resource Sustainable Utilization, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, China

Geng Qin Key Laboratory of Marine Bio-resource Sustainable Utilization, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, China and Graduate School of the Chinese Academy of Sciences, Beijing 100049, China

Junda Lin Vero Beach Marine Laboratory, Florida Institute of Technology, Vero Beach, Florida 32963, USA

Liangmin Huang Key Laboratory of Marine Bio-resource Sustainable Utilization, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, China

Abstract Salinity fluctuation is an important factor affecting outdoor microalgae culture. This investigation examined the effect of salinity change (Tr-1:35–15 g/L, Tr-2:35–25 g/L, Tr-3:35–35 g/L, Tr-4:35–45 g/L, and Tr-5:35–55 g/L) on growth and the biochemical composition of Nannochloropsis oculata, a candidate for biodiesel production in indoor photo-bioreactors. Results showed that the algae increased in absorbency and dry biomass as salinity decreased. When the salinity increased, the specific growth rate (SGR) of the algae decreased significantly. The salinity stress also affected the pigments of the algae, the chlorophyll-a, and carotenoid contents of the algae which decreased with the increase of salinity from 45 to 55 g/L. The fatty acid methyl esters (FAME) content (% of dry biomass) increased with the increase of salinity (e.g., Tr-4 and Tr-5). The algae was rich in C16:0 (palmitic acid), C16:1n-7 (palmitoleic acid), and C20:5n-3 (eicosapentaenoic acid), and C16:0 content increased with decreasing salinity from 35 to 15 g/L, but C16:1n-7 content was high in all the treatments ranging from 25.25 ± 1.42% in Tr-1 to 27.05 ± 1.13% in Tr-5.

Biofuel production from photosynthetic microorganisms is considered as an effective strategy to produce renewable energy (Ma and Hanna 1999; Vicente et al. 2004; Ryan et al. 2006; Mata et al. 2010). Microalgae have 1 Corresponding

been proposed as a potential biofuel feedstock source (Huntley and Redalje 2007; Schenk et al. 2008; Pruvosst et al. 2011). Recent studies have focused on increasing the lipid content of algae through altering culture conditions, such as addition of CO2 , temperature, salinity, and nutrient concentration (Khozin-Goldberg

author.

© Copyright by the World Aquaculture Society 2012

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and Cohen 2006; Chiu et al. 2009; Converti et al. 2009; Li et al. 2010). Salinity is a primary factor influencing the growth of marine microalgae, as algae often have a negative response in morphology and physiology during the salinity fluctuation (Al-Hasan et al. 1987, 1990; Aizdaicher et al. 2010). Takagi et al. (2006) reported that high salinity inhibited the growth, lipid and triacylglyceride accumulation of Dunaliella. The hydrocarbon, carbohydrate and carotenoids contents of Botryococcus braunii are influenced by different levels of salinity (Rao et al. 2007; Zhila et al. 2011). The gross chemical and fatty acid composition of Isochrysis sp., Nannochloropsis oculata, and Nitzschia are significantly different at different salinities (Renaud and Parry 1994). The lipid content and the carotene to chlorophyll ratio of Navicula sp. increase with an increase of salinity (Al-Hasan et al. 1990). To date, one focus is to adjust the oil accumulation rate and biochemical composition of algae through salinity and nutrient stress (Yeesang and Cheirsilp 2011). Outdoor open pond raceways have been used for mass culture of microalgae because of its relatively low cost of construction and operation, large production capacity and durability (Borowitzka 1999; Khozin-Goldberg and Cohen 2006; Schenk et al. 2008; Mata et al. 2010; Krohn et al. 2011; Lin and Lin 2011). In contrast with the culture in photobioreactors, microalgae in outdoor systems are easily affected by sudden or gradual salinity fluctuations from rainfall and evaporation (Schenk et al. 2008; Mata et al. 2010). Nannochloropsis oculata, a potential candidate for the biodiesel production because of its fatty acid profiles and ability of fatty acid accumulation (Rodolfi et al. 2008; Chiu et al. 2009), was used to investigate the effects of salinity stress on growth, biochemical composition, and fatty acid profiles in indoor photo-bioreactors. Materials and Methods Algae and Culture Conditions Nannochloropsis oculata, purchased from the Key Laboratory of Aquaculture of Ocean

University of China (obtained from Australian CSIRO Collection of Living Microalgae, http://www.cmar.csiro.au/microalgae), was cultured indoors in roux bottles (Pyrex, SigmaAldrich, St. Louis, MO, USA) (25.0 cm height, 11.5 cm width, and 5.5 cm depth) with 800 mL of media. Magnetic stir plates (Scholar 171, Corning Co. Ltd, Kaiserslautern, Germany) were used to continuously mix the culture media in the roux bottles. The light source was composed of the fluorescent tubes (Speethalux™ T5HO54W 6500K, China), which were placed horizontally and parallel to the front side of the roux bottles. Pure CO2 and air (2/3 by volume) were mixed by air flow meter (Dwyer Instruments, Inc., Michigan, IN, USA) before being injected into the roux bottles. Seawater used in the experiment was pumped directly from the Daya Bay (near Hong Kong) of South China Sea and treated with filtration (Waterman GF/F) and sterilization (121 C, 20 min). Experiment Design Five treatments (Tr: 35–30–25–20–15 g/L, Tr-2:35–30–25 g/L, Tr-3:35–35 g/L (control), Tr-4:35–40–45 g/L, Tr-5:35–40–45–50–55 g/L), three replicates per treatment, were used to evaluate the effects of salinity stress on growth and biochemical composition of pure culture of N. oculata. The algae was cultured in the modified F/2 media (1.5 mL Guillard’s F/2 trace metal solution and 4 mL modified Guillard’s F/2 formula [24 g urea, 6 g NaH2 PO·4 H2 O, 0.2 g MnCl·2 4H2 O, 4 g Na·2 EDTA, 0.05 g vitamin B1 , and 0.0001 g vitamin B12 per liter of the sterilized fresh seawater] in 800 mL culture media). During culture, the light was on continuously and the temperature, pH, and light intensity were 26 ± 1 C, 7.7 ± 0.2, and 9000 ± 50 lx, respectively. The initial biomass (dry weight) of algae was measured as 394 ± 27 mg/L and the initial salinity in culture media was 35 g/L. The desired salinities (15, 25, 45, and 55 g/L) at the treatments were gradually achieved at the acclimation rate of 5 ± 0.5 g/L each day. The detailed procedure of salinity regulation was

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SALINITY CHANGE ON N. OCULATA

Absorbency and Dry Biomass Approximately 20 mL of algae in each roux bottle was used to measure the absorbency and dry biomass. Absorbency of the algae (OD680 ) was determined at 680 nm by a UV–vis spectrophotometer (model Lambda 25, PerkinElmer, Milan, Italy) (Li et al. 2008). Preweighted Whatman GF/C glass microfiber filter disks (47 nm) were used to filter the algae solution, and then the filter disks with algal were dried at 105 C for 3 h and weighted again. Specific growth rate (SGR) (per d) was

3.0 Tr-5 Tr-4 Tr-3 Tr-2 Tr-1

2.5

Absorbency (OD680 )

as follows. First a certain volume (depending on the variation of salinity) of algae media were taken out and countrified (within the same day, the volume of replacement was kept same among the three or five treatments); secondly the supernatant was discharged and replaced with fresh water or seawater of specific salinity, both were sterilized and free of the additional nutrients; finally the redissolved algae was placed back to the corresponding bottle. After the media replacement, 1.5 mL Guillard’s F/2 trace metal solution and 4 mL modified Guillard’s F/2 formula were added to 800 mL algae media in each roux bottle to keep the same nutrient level among the treatments. Then 150 mL of algae was used to measure the absorbency of algae, biomass, chlorophylla, and carotenoid content. Both Tr-1 and Tr-5 reached the desired salinity (15 and 55 g/L, respectively) on Day 4, and the biochemical composition (FAME and fatty acids) of the algae was measured on Day 5. Both Tr2 and Tr-4 reached the desired salinity (25 and 45 g/L, respectively) on Day 2 and the algae were sampled to measure the biochemical composition (FAME and fatty acids) on Day 3. Then the algae on Tr-2 and Tr-4 were cultured continuously under the salinity of 25 and 45 g/L, respectively, and the absorbency, biomass, chlorophyll-a, and carotenoid content for both treatments on Day 4 and Day 5 were measured to compare them with Tr-1 and Tr5. The measurement of the control (Tr-3) was conducted every day (Fig. 1).

2.0

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.5 1

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Figure 1. Effect of salinity change (Tr-1:35–15 g/L, Tr-2:35–25 g/L, Tr-3:35–35 g/L, Tr-4:35–45 g/L, Tr5:35–55 g/L) on absorbency increment during the 5-d culture of Nannochloropsis oculata. The arrows indicate new culture media addition.

calculated using the following formula SGR = ln(Wf /W0 )/t Wf and W0 were the final and initial dry biomass, respectively. t was the culture time in days (Ono and Cuello 2007). Chlorophyll-a and Carotenoid Approximately 20 mL of algae in each roux bottle were used to measure the pigments. Chlorophyll-a and carotenoid of algae were extracted by methanol (99.9%) at room temperature for 3 h in the dark. After centrifuging (800 g, 8 min), the content was determined by measurement of absorbency at 480, 652, and 665 nm with a spectrophotometer (VIS-7220/7220G, Rayleigh, Beijing, China) (Pruvost et al. 2009). Concentrations of chlorophyll-a and carotenoid were determined according to the formulas of Strickland and Parsons (1968) and Ritchie (2006): [Chl − a]μg/mL = −8.0962 × A652 + 16.5169 ×A665 [Carotenoids]μg/mL = 4 × A480 Absorbencies at 480, 652, and 665 nm were corrected from turbidity by subtracting absorbencies at 750 nm.

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FAME and Fatty Acid Profiles Five 40 mg of frozen dried algae sampled separately from Tr-2 (from 35 to 25 g/L) and Tr-4 (from 35 to 45 g/L) on Day 3 and Tr-1 (from 35 to 15 g/L), Tr-3 (from 35 to 35 g/L), and Tr-5 (from 35 to 55 g/L) on Day 5 were measured to analyze their FAME contents and fatty acid profiles. FAME and fatty acid composition were determined following the method of Lin and Lin (2011). Direct transesterification for determination of free and bound fatty acid content of the algae was conducted. Surrogate standard and transesterification reagent were prepared, and approximately vacuum-filtered 3 mg of each algae sample stored at −80 C was used. Five milliliter of transesterification reagent was added to each sample, which was heated at 100 C for 20 min with vortexing at 0 and 20 min. For rapid GC analysis, a HP 6890 series GC system with HP 6890 series auto injector and FID was used in conjunction with a Restek FAMEWAX (30 m × 0.25 mm ID × 0.25 μm) film thickness column. Duplicates of each FAME analysis were conducted and FAMEs identification was run by comparison with standards, Supelco FAME 10 mix 37 (Bellefonte, PA, USA). Instrument conditions and operation methods were the same as reported by Lin and Lin (2011).

(Tr-1), and the algae in the control (Tr-3) had the highest absorbency on Day 5. After salinity became constant in Tr-2 and Tr-4 (from Day 3 to Day 5), the absorbency of the algae grown in 25 g/L was significantly higher than that in 45 g/L (Fig. 1). Compared with the dry biomass of the algae grown under the salinity from 35 to 15 g/L, the algae had a low increment of dry biomass in Tr-5. In contrast to the absorbency, the dry biomass of the algae at 25 and 45 g/L also increased dramatically after salinity became constant on Day 3 (Fig. 2). The control algae (Tr-3) had a significantly higher SGR than those of the treatments from Day 1 to Day 3. When the salinity changed from 25 to 15 g/L, the algae grew fast and the SGR was high during Day 3 to Day 5, and the SGR was significantly lower than that of other treatments when the salinity increased from 45 to 55 g/L (Table 1). The algae had a high increase of dry biomass with the decrease of salinity, and the dry biomass increased 226.6 ± 31.7 mg/L during the salinity decrease from 35 to 25 g/L. In contrast, when the salinity increased, especially from 45 to 55 g/L, the increment of dry biomass was the lowest (131.0 ± 1.4 mg/L; Table 1). Chlorophyll-a and Carotenoid The variations of the chlorophyll-a and carotenoid concentrations (mg/L) of the algae

Statistical Analysis 1000 Tr-5 Tr-4 Tr-3 Tr-2 Tr-1

900

Dry biomass (mg/L)

The statistical analyses were performed using the software SAS (Version 9.0). A one-way analysis of variance (ANOVA) was used to determine if there were differences in the growth and biochemical composition of the algae from the five treatments. If significant differences were detected, a least significant difference (LSD) multiple comparison was performed. We chose a significance level of 0.05.

800 700 600 500 400 300

Results Absorbency and Dry Biomass Absorbency increase of N. oculata grown under salinity stress from 35 to 55 g/L (Tr-5) was significantly lower than that of 35 to 15 g/L

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3 4 Culture time (day)

5

Figure 2. Effect of salinity change (Tr-1:35–15 g/L, Tr-2:35–25 g/L, Tr-3:35–35 g/L, Tr-4:35–45 g/L, Tr5:35–55 g/L) on dry biomass increment during the 5-d culture of Nannochloropsis oculata. The arrows indicate new culture media addition.

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The initial FAME content of the algae was 14.05 ± 0.63% of dry biomass. On Day 3, the FAME contents of the algae in Tr-2 and Tr-4 were 9.80˜± 1.07% and 11.63 ± 1.41% respectively. On Day 5, the FAME contents in Tr-1, Tr-3, and Tr-5 were 10.28 ± 1.56%, 10.98 ± 1.64%, and 13.13 ± 1.28%, respectively. The FAME content of the algae was high in the high salinity treatments (Tr-4 and Tr-5; Fig. 4).

Chlorophyll-a (mg/L)

are mean ± SD. Within the same column, significant differences are indicated by different superscripts. 1 Values

214.7 ± — 303.0 ± 8.5a — 131.0 ± 1.4c 0.0003

FAME and Fatty Acid Profiles

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Carotenoid (mg/L )

1.28 ± 0.27a — 0.72 ± 0.17a — −0.27 ± 0.05b 0.0082 — 0.92 ± 0.07b 1.14 ± 0.09a 1.19 ± 0.21a — 0.0050 6.62 ± — 5.05 ± 1.09a — −2.03 ± 0.08b 0.0028 — 6.77 ± 2.35 6.28 ± 0.36 5.41 ± 0.63 — 0.9129 — 226.6 ± 23.2a 204.0 ± 12.3a 141.5 ± 12.0b — 0.0091 0.245 ± — 0.242 ± 0.043a — 0.078 ± 0.034b 0.0328 — 0.253 ± 0.029ab 0.282 ± 0.017a 0.220 ± 0.022b — 0.0285 15 25 35 45 55 to to to to to 25 35 35 35 45 From From From From From P

Days 3–5

42.3b 0.034a

Days 3–5 Days 1–3 Days 3–5 Days 1–3 Days 1–3 Days 3–5 Days 1–3 Salinity stress (g/L)

at different treatments were similar during the 5-d culture. When salinity changed from 35 to 55 g/L (Tr-5), the chlorophyll-a and carotenoid concentrations of the algae increased significantly less than that from 35 to 15 g/L (Tr-1). After the salinity stress, the difference of chlorophyll-a and carotenoid concentrations in 25 g/L and control was not significant (Fig. 3). The increment of chlorophyll-a and carotenoid contents (mg/g of dry biomass) of the algae was also studied. The chlorophyll-a content of the algae increased with the decrease of salinity (from 35 to 15 g/L). When the salinity increased from 45 to 55 g/L, the algae dramatically decreased its chlorophyll-a content (2.03 ± 0.08 mg/g), and the carotenoid content also reduced 0.27 ± 0.05 mg/g (Table 1).

0.73a

Carotenoids increment (mg/g) Chlorophyll-a increment (mg/g) Dry biomass increment (mg/L) Specific growth rate (per d)

Table 1. Comparison of specific growth rate (SGR) and the increments of dry biomass, chlorophyll-a and carotenoid contents of Nannochloropsis oculata when the salinity was changed from 25 to 15, 35 to 25, 35 to 35, 35 to 45, and 45 to 55 g/L, respectively.1


4

Tr-5 Tr-4 Tr-3 Tr-2 Tr-1

20 15 10 5

3 2 1 1

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Culture time (day)

Figure 3. Effect of salinity change (Tr-1:35–15 g/L, Tr-2:35–25 g/L, Tr-3:35–35 g/L, Tr-4:35–45 g/L, Tr5:35–55 g/L) on chlorophyll-a and carotenoid contents of Nannochloropsis oculata during the 5-d culture. The arrows indicate new culture media addition.

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8.77% after the salinity stress). C16:0 content increased with the salinity changing from 35 to 15 g/L, but C16:1n-7 content was high while stressed by high salinity and ranged from 25.25 ± 1.42% in Tr-1 to 27.05 ± 1.13% in Tr-5. C18:1n-7, 12 contents increased with the increase of salinity, and the algae had the highest C20:5n-3 content at Tr-5 (Table 2).

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FAME content (%)

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11 Tr-5 Tr-4 Tr-3 Tr-2 Tr-1

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Discussion

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Figure 4. Effect of salinity change (Tr-1:35–15 g/L, Tr-2:35–25 g/L, Tr-3:35–35 g/L, Tr-4:35–45 g/L, Tr5:35–55 g/L) on the fatty acid methyl esters (FAME) content of Nannochloropsis oculata during the 5-d culture.

Eighteen fatty acids of FAME in the N. oculata were detected, with high C16:0 (palmitic acid), C16:1n-7 (palmitoleic acid), and C20:5n3 (eicosapentaenoic acid) contents and low C18 series (oleic acid) content (e.g. In Tr-1: the total content of C16 series was the highest at 52.57%, but the C18 series content was only

As a potential candidate for biodiesel production (Rodolfi et al. 2008; Chiu et al. 2009), N. oculata is highly productive and yields high concentrations of oil through culture in outdoor open raceways. However, the optimum salinity for the growth of the N. oculata is reported to be 22 to 25 g/L (Renaud and Parry 1994; Wilkerson 1998). The mass yielded and biochemical composition of algae is reported to decrease from sudden salinity fluctuations in open ponds (Schenk et al. 2008; Mata et al. 2010). The effects of salinity stress on growth and mass biochemical composition of microalgae has been reported for Isochrysis spp and

Table 2. Effect of salinity change (Tr-1:35-15 g/L, Tr-2:35-25 g/L, Tr-3:35-35 g/L, Tr-4:35-45 g/L, Tr-5:35-55 g/L) on fatty acid profiles (% of total FAME) of Nannochloropsis oculata after the 5-d culture. Values are mean ± SD. Percentage of total FAME Fatty acids 10:0 12:0 14:0 14:1n-5 16:0 16:1n-4 16:1n-7 18:0 18:1n-7, 12 18:1n-9 18:2n-6 18:3n-3 20:1n-9 20:1n-11 20:4n-6 20:5n-3 22:0 22:1n-9

35–15 g/L (Day 5) — — 4.92 ± 0.32 ± 26.77 ± 0.55 ± 25.25 ± 0.68 ± 1.59 ± 1.03 ± 0.53 ± 4.94 ± — — — 20.99 ± 3.42 ± 0.60 ±

0.75 0.28 1.75 0.31 1.42 0.15 0.27 0.25 0.24 0.16

4.11 0.35 0.45

FAME = fatty acid methyl esters.

35–25 g/L (Day 3) 0.71 ± — 4.51 ± 0.49 ± 25.85 ± 0.54 ± 26.07 ± 0.72 ± 0.99 ± 1.16 ± 0.59 ± 5.17 ± 0.60 ± — 0.15 ± 22.71 ± 2.57 ± 0.41 ±

0.12 0.43 0.37 2.04 0.21 2.63 0.13 0.45 0.10 0.13 0.30 0.54 0.09 2.15 0.14 0.17

35–35 g/L (control) 0.49 ± — 5.42 ± 0.43 ± 24.81 ± 0.62 ± 26.05 ± 0.66 ± 2.14 ± 0.90 ± 0.70 ± 4.10 ± — 0.13 ± — 22.94 ± 2.18 ± 0.31 ±

0.27 0.81 0.08 3.28 0.43 0.91 0.08 0.13 0.13 0.31 0.13 0.21 1.41 0.18 0.36

35–45 g/L (Day 3) 0.52 ± 0.29 ± 5.13 ± 0.65 ± 25.26 ± 0.67 ± 26.16 ± 0.47 ± 2.39 ± 1.23 ± 0.67 ± 4.20 ± 0.41 ± — — 22.21 ± 2.34 ± —

0.14 0.08 1.32 0.23 3.14 0.16 0.40 0.21 0.26 0.11 0.15 0.09 0.46

1.38 0.31

35–55 g/L (Day 5) 0.89 ± — 5.32 ± 1.26 ± 19.30 ± 0.69 ± 27.05 ± 0.56 ± 4.30 ± 1.73 ± 0.72 ± 3.78 ± — — — 23.46 ± 2.07 ± 0.40 ±

0.11 0.55 0.52 5.44 0.09 1.13 0.15 1.44 0.17 0.21 0.18

2.36 0.12 0.29


other species (Ben-Amotz et al. 1985; Jeffrey et al. 1990). Lower dry biomass and SGR were found when the algae was stressed by high salinities, similar to the responses of Trichodesmium sp., Cyanobacterium synechococcus sp., Dunaliella salina, and Arthrospira (Spirulina) platensis to high salinity (Fu and Bell 2003; Rosales et al. 2005; Takagi et al. 2006; Ravelonandro et al. 2011). In this study, the algae preferred the low salinity media and had slow growth during the increase of salinity (Tr-5), which may be because that the microalgae would expend energy while attempting to maintain the turgor pressure, and this resulted in a decrease in productivity or reduction in growth (Kirst 1989). Chlorophyll-a and cartenoid contents were the highest during the salinity change from 35 to 25 g/L, and this was in accordance with the reports that low salinity could increase both chlorophyll-a and carotenoid production in Dunaliella tertiolecta (Cifuentes et al. 2001; Fazeli et al. 2006) and Tetraselmi chuii (Ghezelbash et al. 2008). Generally, the chlorop hyll-a and cartenoid are essential for microalgae to maintain regular growth through the photosynthesis (Warr et al. 1985). When the pigment contents, especially the chlorophyll-a, were low under high salinity, the lower photosynthetic efficiency resulted in a significantly lower biomass production in some microalgae (Warr et al. 1985). In this study, the green color of the algae in the media lightened when the salinity increased from 45 to 55 g/L, and likely contributed to significant decreases in chlorophyll-a and cartenoid contents and also the slow growth rate. Vonshak et al. (1988) also found that the photosynthetic efficiency and PSII activity of A. (Spirulina) platensis decreased with the increase of salinity. But chlorophyll-a content in Trichodesmium sp. and carotenoid content in B. braunii increased with the increase of salinity (Fu and Bell 2003; Rao et al. 2007). Positive effects of nutrient limitation on lipid accumulation and productivity have been widely studied in many microalgae species (Takagi et al. 2000; Rodolfi et al. 2008; Hsieh

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and Wu 2009; Lin and Lin 2011; Yeesang and Cheirsilp 2011). In response to environmental stress, many microalgae species can synthesize large amounts of storage lipids such as triacylglycerols (Harwood and Jones 1989). In this study, the initial FAME content of the algae was high, which should be due to the low nutrient concentration in the culture media of pre-culture. Available nutrients in the experimental media led to the high growth and low FAME contents among all the treatments in this study. This was similar to other marine microalgae, such as Chlorella vulgaris, Neochloris oleoabundans, and Scenedesmus rubescens-like microalgae (Li et al. 2008; Converti et al. 2009; Lin and Lin 2011). Al-Hasan et al. (1990) and Takagi et al. (2006) reported that the lipid content of Navicula sp. and D. tertiolecta increased with the increase of salinity, but decreased with the increasing salinity in D. salina cells (AlHasan et al. 1987), and even the lipid content of the B. braunii CHN did not change during the salinity fluctuation (Li and Qin 2005). In this study, the FAME content of the algae was relatively high when stressed by high salinity, but this did not clearly show a positive correlation between the FAME content and salinity for the algae. However, the SGR of algae was the lowest in Tr-5 and highest in Tr-2, which provided an opposite correlation between the FAME contents and SGR of the algae in this study. Many microalgae, such as Ellipsoidion sp., Chlorella sp., and S. rubescens-like microalgae, also have been reported that their high biomass productivities often led to a relatively low lipid contents (Xu et al. 2001; Hsieh and Wu 2009; Lin and Lin 2011). Salinity fluctuation always leads to the variation of the fatty acid profiles in algae, which can influence the functions of algal cell membranes and metabolic processes (Guschina and Harwood 2006), and the degree of unsaturation of membrane fatty acids is also an important parameter in adaption of algae to environmental conditions (Zhila et al. 2011). In this study, C16:1, C18:1, and C18:2 in N. oculata increased in an elevated salinity concentration during the stress, and this might be

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due to the response to the high salinity. BenAmotz et al. (1985) also found that the proportions of polyunsaturated fatty acids (C18–C22) increased under elevated NaCl concentrations in the medium. Moreover, the unsaturated fatty acids, such as n-3/n-6 series polyunsaturated fatty acids, varied significantly during the salinity fluctuation, and linoleic acids (C18:2) and eicosapentaenoic acid (C20:5) contents increased with the increase of salinity. This is consistent with the report that the B. braunii changed its fatty acid profiles in response to the elevated salinity to keep the membrane fluid and prevent its destruction (Zhila et al. 2011). Nannochloropsis oculata has been widely used as the feedstock in aquaculture for its high eicosapentaenoic acid content (Richmond et al. 2001; Rodolfi et al. 2003), and it has also been studied as a potential microalgae for the biodiesel recently (Rodolfi et al. 2008). Generally, as for mass productivity of microalgae biofuels, C16 and C18 series of fatty acids are often used to evaluate the oil/biodiesel productivity from algae (Xu et al. 2004; KhozinGoldberg and Cohen 2006; Converti et al. 2009). In this study, the total proportions of C16 series fatty acids was approximately 50% of the total fatty acids, and relatively high compared with other microalgae (Xu et al. 2004; KhozinGoldberg and Cohen 2006). In contrast, C18 series fatty acids accounted for a low proportion of the FAME and never varied much under salinity stress; this is not a desired result for biodiesel productivity. Although the presence of low and high salinity stress inhibited the growth of N. oculata during the 5-d experiment, causing some variations in pigments, FAME and fatty acid profiles, and the evaluation of oil accumulation and productivity of algae might be balanced before confirming this strain of N. oculata a candidate for biofuels. Acknowledgment We are grateful to the staff of Shenzhen Huici Biotech., Inc. for technical assistance. This study was funded by the National Natural Science Foundation of China (No. 30901109),

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