Bioresource Technology 184 (2015) 42–46
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Isolation and characterization of microalgae for biodiesel production from seawater Liu Zhao, Yun Qi, Guanyi Chen ⇑ School of Environment Science and Engineering/State Key Lab of Engines, Tianjin University, Tianjin 300072, China Tianjin Technological Engineering Center on Biomass-derived Gas and Oil, No. 92, Weijin Rd., Nankai District, Tianjin 300072, China
h i g h l i g h t s Salinity exhibited significant positive impact on biomass and lipid production. As a light dependent strain, autotrophic cultivation was the optimally trophic type. N. gaditana Q6 was more sensitive to the variance of NH4HCO3 than NaH2PO4.
a r t i c l e
i n f o
Article history: Received 13 August 2014 Received in revised form 12 October 2014 Accepted 13 October 2014 Available online 22 October 2014 Keywords: Nannochloropsis gaditana Salinity Carbon Phosphorus Nitrogen
a b s t r a c t As green marine microalgae isolated from local seawater in Tianjin, China, Nannochloropsis gaditana Q6 was tolerant to the variation of salinity with the highest biomass and lipid concentration in natural seawater medium. Although this strain could grow mixotrophically with glycerol, the narrow gap between mixotrophic and autotrophic cultivation suggested that autotrophic cultivation was the optimal trophic type for N. gaditana Q6 growth. In addition, strain Q6 was more sensitive to the variance of NH4HCO3 concentration than NaH2PO4 concentration. Consequently, the lipid production could be maximized by the two-stage cultivation strategy, with an initial high NH4HCO3 concentration for biomass production followed by low NH4HCO3 concentration for lipid accumulation. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Microalgae have emerged as a potential sustainable biomass resource due to their neutrality towards nature and high lipid accumulation ability (Anandarajah et al., 2012; Lim et al., 2012). Generally, medium composition (such as P, N, and carbon) has direct effect on microalgae biomass and lipid production. Synthesis and accumulation of large amounts of lipid occur in microalgae cell exposed to growth constrains imposed by environmental stimuli, such as nutrient starvation (Kilham et al., 1997; Mohammady et al., 2012), and high salinity (Salama et al., 2013). However, the utilization of nutrient was species-dependent. For example, P limitation exhibited positive impact on the lipid content of Phaeodactylum tricornutum, Chaetoceros sp. and Pavlova lutheri, but negative influence on the lipid level of Nannochloris atomus and Tetraselmis sp. (Reitan et al., 1994). Consequently, the
⇑ Corresponding author. E-mail address: [email protected] (G. Chen). http://dx.doi.org/10.1016/j.biortech.2014.10.063 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.
optimal cultivation condition for particular strain was species specific. The aim of this work was to optimize the cultivation conditions (salinity, carbon, P, and N) for biomass production and lipid accumulation of a new isolated marine microalgae strain Nannochloropsis gaditana Q6. Based on the results, we hypothesis that two-stage cultivation strategy with different N concentrations can balance the rapid and efficient biomass generation with high lipid content to maximize the lipid productivity of N. gaditana Q6. 2. Methods 2.1. Isolation and cultivation Strain Q6 was isolated from seawater samples collected from Tianjin Bathing Beach, China. The microalgae were subjected to purification by serial dilution followed by plating on agar. Individual colonies were isolated and inoculated into sterilized natural seawater enriched with liquid f/2-Si medium, which consists of 880 lM NaNO3, 36 lM NaH2PO4, 76.5nM ZnSO4, 0.91 lM MnCl2,
L. Zhao et al. / Bioresource Technology 184 (2015) 42–46
26 nM Na2MoO4, 42 nM CoCl2, 39.3 nM CuSO4, 11.7 lM FeCl3, 11.7 lM Na2EDTA, 3.69 104 lM vitamin B12, 2.05 103 lM vitamin H, and 0.296 lM vitamin B1. Natural seawater was acquired from Tianjin Bathing Beach, with the salinity of 32.10‰. All cultures were grown in 250 mL flasks containing 100 mL of f/2-Si medium and incubated at 28 ± 1 °C under continuous illumination of 2500 Lux for 12 day.
The genomic DNA of microalgae cells was extracted following the standard phenol–chloroform extraction protocols. Two primers, 16S1 N (forward, 50 -TCCTGCCAGTAGTCATATGC-30 ) and 16S2N (reverse, 50 -TGATCCTTCT/CGCAGGTTCAC-30 ), were used for PCR reaction (Grzebyk et al., 1998). Each PCR sample consisted of a 50 lL solution with 1 lL of DNA template (10–20 ng), 2 lL of each primer (Invitrogen, USA), 25 lL of 2 Taq Mix (Taq Mix, Japan). The PCR (Biometra, Germany) reaction was performed with a thermal program, which consisted of preheating at 94 °C for 5 min, 30 cycles of denaturation at 94 °C for 35 s, annealing at 51 °C for 35 s, and extension at 72 °C for 3 min, followed by another 10 min extension at 72 °C. PCR products were sequenced by the Shanghai Sangon Biotech (Shanghai, China). 2.3. Experimental design 2.3.1. Cultivation with different salinity, carbon, N, and P resources In order to optimize the biomass and lipid accumulation of N. gaditana Q6, the salinity of medium were 32.10‰, 25.68‰, 16.05‰ and 0‰; the carbon resources were 1.0 g/L NaHCO3, 0.01 M glucose, 0.02 M glycerol and 0.03 M NaAc; the P resources were 36 lM NaH2PO4, Na2HPO4, KH2PO4, and K2HPO4; the N resources were NaNO3, NH4HCO3 and urea with 440, 880 and 1320 lM. The other conditions were the same as in Section 2.1. 2.3.2. The response surface methodology (RSM) design In order to optimize the cultivation conditions for strain Q6, central composite design (CCD) and RSM were employed and designed in Design Expert software (version 8.0.0, Stat-Ease Inc., Minneapolis, USA). In this study, a 22 CCD with 13 runs and 5 replications of the center points were used to determine the optimal concentrations of NH4HCO3, and NaH2PO4 on the biomass and lipid production of N. gaditana Q6. The coded and corresponding actual values were given in Table S1. The corresponding central composite experimental design and their values were shown in Table S2. And all the design points were run in three replications. For statistical calculations, the relation between the coded values and actual values are described as Eq. (1): 2 2 2 X X X bi X i þ bij X i X j þ bij X 2j i¼1
Lipid concentration (CL, mg/L) was determined by the standard addition method (the excitation and emission spectra were 480 and 590 nm, respectively) of Bertozzini et al. (2011). The lipid content (CLC, %) was calculated according to the Eq. (3):
C LC ¼ C L =C B
ð3Þ
2.5. Statistical analysis
2.2. DNA analysis
Y ¼ b0 þ
43
i
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Isolation and characterization of microalgae for biodiesel production from seawater Liu Zhao, Yun Qi, Guanyi Chen ⇑ School of Environment Science and Engineering/State Key Lab of Engines, Tianjin University, Tianjin 300072, China Tianjin Technological Engineering Center on Biomass-derived Gas and Oil, No. 92, Weijin Rd., Nankai District, Tianjin 300072, China
h i g h l i g h t s Salinity exhibited significant positive impact on biomass and lipid production. As a light dependent strain, autotrophic cultivation was the optimally trophic type. N. gaditana Q6 was more sensitive to the variance of NH4HCO3 than NaH2PO4.
a r t i c l e
i n f o
Article history: Received 13 August 2014 Received in revised form 12 October 2014 Accepted 13 October 2014 Available online 22 October 2014 Keywords: Nannochloropsis gaditana Salinity Carbon Phosphorus Nitrogen
a b s t r a c t As green marine microalgae isolated from local seawater in Tianjin, China, Nannochloropsis gaditana Q6 was tolerant to the variation of salinity with the highest biomass and lipid concentration in natural seawater medium. Although this strain could grow mixotrophically with glycerol, the narrow gap between mixotrophic and autotrophic cultivation suggested that autotrophic cultivation was the optimal trophic type for N. gaditana Q6 growth. In addition, strain Q6 was more sensitive to the variance of NH4HCO3 concentration than NaH2PO4 concentration. Consequently, the lipid production could be maximized by the two-stage cultivation strategy, with an initial high NH4HCO3 concentration for biomass production followed by low NH4HCO3 concentration for lipid accumulation. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Microalgae have emerged as a potential sustainable biomass resource due to their neutrality towards nature and high lipid accumulation ability (Anandarajah et al., 2012; Lim et al., 2012). Generally, medium composition (such as P, N, and carbon) has direct effect on microalgae biomass and lipid production. Synthesis and accumulation of large amounts of lipid occur in microalgae cell exposed to growth constrains imposed by environmental stimuli, such as nutrient starvation (Kilham et al., 1997; Mohammady et al., 2012), and high salinity (Salama et al., 2013). However, the utilization of nutrient was species-dependent. For example, P limitation exhibited positive impact on the lipid content of Phaeodactylum tricornutum, Chaetoceros sp. and Pavlova lutheri, but negative influence on the lipid level of Nannochloris atomus and Tetraselmis sp. (Reitan et al., 1994). Consequently, the
⇑ Corresponding author. E-mail address: [email protected] (G. Chen). http://dx.doi.org/10.1016/j.biortech.2014.10.063 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.
optimal cultivation condition for particular strain was species specific. The aim of this work was to optimize the cultivation conditions (salinity, carbon, P, and N) for biomass production and lipid accumulation of a new isolated marine microalgae strain Nannochloropsis gaditana Q6. Based on the results, we hypothesis that two-stage cultivation strategy with different N concentrations can balance the rapid and efficient biomass generation with high lipid content to maximize the lipid productivity of N. gaditana Q6. 2. Methods 2.1. Isolation and cultivation Strain Q6 was isolated from seawater samples collected from Tianjin Bathing Beach, China. The microalgae were subjected to purification by serial dilution followed by plating on agar. Individual colonies were isolated and inoculated into sterilized natural seawater enriched with liquid f/2-Si medium, which consists of 880 lM NaNO3, 36 lM NaH2PO4, 76.5nM ZnSO4, 0.91 lM MnCl2,
L. Zhao et al. / Bioresource Technology 184 (2015) 42–46
26 nM Na2MoO4, 42 nM CoCl2, 39.3 nM CuSO4, 11.7 lM FeCl3, 11.7 lM Na2EDTA, 3.69 104 lM vitamin B12, 2.05 103 lM vitamin H, and 0.296 lM vitamin B1. Natural seawater was acquired from Tianjin Bathing Beach, with the salinity of 32.10‰. All cultures were grown in 250 mL flasks containing 100 mL of f/2-Si medium and incubated at 28 ± 1 °C under continuous illumination of 2500 Lux for 12 day.
The genomic DNA of microalgae cells was extracted following the standard phenol–chloroform extraction protocols. Two primers, 16S1 N (forward, 50 -TCCTGCCAGTAGTCATATGC-30 ) and 16S2N (reverse, 50 -TGATCCTTCT/CGCAGGTTCAC-30 ), were used for PCR reaction (Grzebyk et al., 1998). Each PCR sample consisted of a 50 lL solution with 1 lL of DNA template (10–20 ng), 2 lL of each primer (Invitrogen, USA), 25 lL of 2 Taq Mix (Taq Mix, Japan). The PCR (Biometra, Germany) reaction was performed with a thermal program, which consisted of preheating at 94 °C for 5 min, 30 cycles of denaturation at 94 °C for 35 s, annealing at 51 °C for 35 s, and extension at 72 °C for 3 min, followed by another 10 min extension at 72 °C. PCR products were sequenced by the Shanghai Sangon Biotech (Shanghai, China). 2.3. Experimental design 2.3.1. Cultivation with different salinity, carbon, N, and P resources In order to optimize the biomass and lipid accumulation of N. gaditana Q6, the salinity of medium were 32.10‰, 25.68‰, 16.05‰ and 0‰; the carbon resources were 1.0 g/L NaHCO3, 0.01 M glucose, 0.02 M glycerol and 0.03 M NaAc; the P resources were 36 lM NaH2PO4, Na2HPO4, KH2PO4, and K2HPO4; the N resources were NaNO3, NH4HCO3 and urea with 440, 880 and 1320 lM. The other conditions were the same as in Section 2.1. 2.3.2. The response surface methodology (RSM) design In order to optimize the cultivation conditions for strain Q6, central composite design (CCD) and RSM were employed and designed in Design Expert software (version 8.0.0, Stat-Ease Inc., Minneapolis, USA). In this study, a 22 CCD with 13 runs and 5 replications of the center points were used to determine the optimal concentrations of NH4HCO3, and NaH2PO4 on the biomass and lipid production of N. gaditana Q6. The coded and corresponding actual values were given in Table S1. The corresponding central composite experimental design and their values were shown in Table S2. And all the design points were run in three replications. For statistical calculations, the relation between the coded values and actual values are described as Eq. (1): 2 2 2 X X X bi X i þ bij X i X j þ bij X 2j i¼1
Lipid concentration (CL, mg/L) was determined by the standard addition method (the excitation and emission spectra were 480 and 590 nm, respectively) of Bertozzini et al. (2011). The lipid content (CLC, %) was calculated according to the Eq. (3):
C LC ¼ C L =C B
ð3Þ
2.5. Statistical analysis
2.2. DNA analysis
Y ¼ b0 þ
43
i
