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Gradient domestication of Haematococcus pluvialis mutant with 15% CO2 to promote biomass growth and astaxanthin yield
Accepted Manuscript Gradient domestication of Haematococcus pluvialis mutant with 15% CO2 to promote biomass growth and astaxanthin yield Jun Cheng, Ke Li, Zongbo Yang, Hongxiang Lu, Junhu Zhou, Kefa Cen PII: DOI: Reference:
S0960-8524(16)30736-2 http://dx.doi.org/10.1016/j.biortech.2016.05.095 BITE 16592
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
4 April 2016 21 May 2016 23 May 2016
Please cite this article as: Cheng, J., Li, K., Yang, Z., Lu, H., Zhou, J., Cen, K., Gradient domestication of Haematococcus pluvialis mutant with 15% CO2 to promote biomass growth and astaxanthin yield, Bioresource Technology (2016), doi: http://dx.doi.org/10.1016/j.biortech.2016.05.095
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Gradient domestication of Haematococcus pluvialis mutant with 15% CO2 to promote biomass growth and astaxanthin yield Jun Cheng*, Ke Li, Zongbo Yang, Hongxiang Lu, Junhu Zhou, Kefa Cen State Key Laboratory of Clean Energy Utilization, Zhejiang University, Hangzhou 310027, China
Abstract: In order to increase biomass yield and reduce culture cost of Haematococcus pluvialis with flue gas from coal-fired power plants, a screened mutant by nuclear irradiation was gradually domesticated with 15% CO2 to promote biomass dry weight and astaxanthin yield. The biomass yield of mutant after 10 generations of 15% CO2 domestication increased to 1.3 times as that with air. With the optimization of nitrogen and phosphorus concentration, the biomass dry weight was further increased by 62%. The astaxanthin yield induced with 15% CO2 and high light of 135 µmol photons m−2s−1 increased to 87.4 mg/L, which was 6 times higher than that induced with high light in air. Keyword: Haematococcus pluvialis mutant; 15% CO2 domestication; Astaxanthin; Biomass growth
1. Introduction Flue gas mitigation and fixation have attracted considerable attention because of greenhouse gases emission and their effects on global climates and ecosystems. Due to the large flow rates and high concentrations of CO2 in the flue gas, billions of tons of CO2 are released into the atmosphere from coal-fired power plants every year.
Among multifaceted approaches of CO2 mitigation, biosequestration of CO2 by microalgae shows high competitiveness in both CO2 capture and energy recovery. Approximately 1.83 kg of CO2 can be fixed in every 1 kg of microalgae biomass, and the efficiency of converting solar energy to chemical energy by microalgae is 10–50 times greater than that by terrestrial plants (Cheah et al., 2015). Subsequently, the lipid and carbohydrate in microalgae cells can be processed to biodiesel and bioethanol. In addition to eco-friendly biofuels, microalgaes contain various natural nutrition and pigments, e.g. astaxanthin in Haematococcus pluvialis, which is a high value-added pigment with strong antioxidant activity and applied in many nutraceuticals and in the food and feed industries. Given the slow mass transfer of CO2 in water, around 40% of the total energy consumption is consumed by delivering atmospheric air to ensure that sufficient carbon source is available for microalgae reproduction. However, the overall fossil-energy input can be significantly reduced via aeration of flue gas, and biomass and lipid can be remarkably enhanced simultaneously (Lam et al., 2012; Yoo et al., 2010；Jiang et al., 2011; Tang et al., 2011；Ji et al., 2013). But the high concentration of CO2 (above 10%) in flue gas may be toxic to some microalgaes (Ota et al., 2009; 2015; Wang et al., 2014). Several studies have focused on developing high CO2-tolerant strains for industrial cultivation with flue gas. Thermal-and CO2-tolerant mutant strains of Chlorella sp. were isolated via ultraviolet or chemical mutagenesis, and were cultured directly with flue gas generated from coke oven of steel plants (Chiu et al., 2011; Li et al., 2011; Kao et al., 2014). High CO2-tolerant strains can also
be isolated via manual selection and domestication with high CO2 (Cheng et al., 2013；Bhakta et al., 2015；Li et al., 2015). According to previous studies, the growth of H. pluvialis was enhanced by 82% when the CO2 concentration increased from air to 6%, however, cell growth was inhibited by further increment of CO2 (Lee et al., 2015; Cheng et al., 2016). The astaxanthin content was decreased by 40% when the CO2 concentration was increased to 20% (Yin et al., 2015). Further works are necessary to improve high CO2-tolerant H. pluvialis strains that can be cultivated by flue gas from coal-fired power plants with 15% CO2. γ-Ray has been recently used to screen efficient microalgae stains due to its strong penetration considerably surpassing that of UV and X-ray. γ-Rays and free radicals generating via radiolysis of water can improve the photosynthesis of microalgae cells and screen astaxanthin-hyperproducing strains of H. pluvialis simultaneously. In our previous study, the biomass and astaxanthin yields of H. pluvialis mutant irradiated with 60Co-γunder 6%CO2 before domestication were much higher than those of the wild strain (Cheng et al., 2016). In the present study, the mutant was gradually domesticated with elevated CO2 concentrations to further enhance biomass and astaxanthin yields under 15% CO2.
2 Materials and Methods 2.1 H. pluvialis strain and growth medium The microalgae strain used in this study was H. pluvialis mutated by a 60Co-γ irradiator at 4000Gy (Cheng et al., 2016). The microalgae cells were cultured in
Bold’s Basal Medium (BBM) containing 2.94 mM NaNO3, 0.304 mM MgSO4·7H2O, 0.428 mM NaCl, 0.431 mM K2HPO4, 1.29 mM KH2PO4, 0.170 mM Ca(NO3)2·4H2O, 30.7 µM ZnSO4·7H2O, 7.28 µM MnCl2·4H2O, 4.93 µM MoO3, 6.29 µM CuSO4·5H2O, 1.68 µM Co(NO3)2·6H2O, 0.185 mM H3BO3, 0.171 mM EDTA·Na2, 0.553 mM KOH, 17.9 µM FeSO4·7H2O, 29.7 nM Vit.B1,0.102 nM Biotin, and 0.0111 nM Vit.B12 (Dominguez-Bocanegra et al., 2004).
2.2 Domestication and culture of H. pluvialis mutant with high concentrations of CO2
A H. pluvialis mutant was domesticated with gradually increased CO2 concentrations from 2% to 15% to increase its tolerance to high CO2 stress. The aeration gas in experiments was the mixture of pure CO2 and N2 rather than real flue gas. H. pluvialis mutant at the logarithmic growth phase was inoculated in 400 mL BBM medium in a 600 mL glass cylinder photobioreactor (diameter: 8.2 cm, height: 15.3 cm) covered with a silica gel stopper. Inoculums were aerated continuously with 2% CO2 at a rate of 120 mL/min for 4 days. Then, partial cells were transferred to new medium and aerated continuously with 8% CO2. These procedures were repeated under the condition of 10% and 15% CO2 gradually. The iterative cultures with 15% CO2 were performed for at least 10 generations and the H. pluvialis strain adapting to 15% CO2 was obtained. Different concentrations of nitrogen (2.94, 4.41, 5.88, 8.82, 14.70 mM NaNO3) and phosphorus ( 1.72, 2.58, 3.44, 5.16, 8.60 mM KH2PO4) were tested to determine the suitable nutrient concentrations with 15% CO2 aeration. The vegetative cultivation was performed under 25°C and 65 µmol photons m−2 s−1 of
continuous light, and the encystment induction was performed under 125 µmol photons m−2 s−1 of continuous light.
2.3 Encystment and astaxanthin induction of H. pluvialis mutant
The domesticated H. pluvialis mutant was incubated with the aeration of 15% CO2 under 25°C and 65 µmol photons m−2 s−1 of continuous light for vegetative growth. The inoculums (biomass dry weight was 1 g/L) were transferred to 500 mL volume glass tubular airlift photobioreactors (diameter: 5 cm, height: 40 cm) and aerated with 15% CO2 at a rate of 120 mL/min continuously. 135 µmol photons m−2 s−1 continuous illumination, which was suitable for astaxanthin accumulation with 15% CO2, was employed for rapid induction of astaxanthin accumulation. Ferrous sulfate at a final concentration of 450 µM was added to the inoculums for efficient inducing by facilitating Haber–Weiss reaction.
2.4 Analytical methods
2.4.1 Growth analyses Biomass yield was monitored by spectrophotometer (optical density 680 nm, A680) and by dry cell weight. For dry cell weight, the microalgae pellets collected via centrifugation were dried at 80°C for 24h and were measured gravimetrically. The biomass dry weight (BDW) shown in growth curves was calculated as: BDW (g/L) =0.512×OD680. The biomass productivity (P) and specific growth rate (µ) were calculated as follows: P = (M1－M0)/(t1－t0)
(1)
µ = (lnM1－lnM0) /(t1－t0)
(2)
where M1 was the dry weight at time t1 and M0 was the dry weight at time t0. 2.4.2. Astaxanthin determination Astaxanthin yield was measured via spectrophotometric method (Boussiba et al., 1992). The harvested cells were first treated with a solution of 5% (w/v) KOH in 30% (v/v) methanol to destroy the chlorophyll. The supernatant was discarded, and the remaining pellet was extracted with DMSO to recover the astaxanthin. The extraction procedure was repeated until the cell debris was nearly colorless. The absorbance of the combined extracts was determined at 490 nm, and the per unit volume astaxanthin concentration (c) was calculated as: c (mg/L) = 4.5×A490×Va/Vb
(3)
where Va (L) was the volume of extracts, Vb (L) was the volume of the culture sample, and A490 was the absorbance of the extracts at 490 nm. 2.4.3 Lipid extraction and estimation Lipids were extracted through direct transesterification (Johnson and Wen, 2009; Cheng et al., 2014). 0.1 g freeze-dried algal biomass was mixed with 4 mL chloroform, 4 mL methanol and 0.2 mL sulfuric acid in a digestion reactor. The digestion reactor was sealed and heated in the baking oven at 90°C for 6 h and then cooled to room temperature. The product was washed with distilled water for at least three times and dried at 60°C. The dried lipids redissolved in n-hexane were mixed with an internal standard (C19:0) and quantified with a gas chromatograph (Agilent 7890A, USA) equipped with an HP-INNOWAX column (30 m × 320 µm × 0.25 µm,
USA) and an FID detector.
3 Results and discussion 3.1. Domestication of H. pluvialis mutant H. pluvialis mutant isolated via the 60Co-γ irradiation showed high sensitivity to 15% CO2. The biomass productivity and biomass dry weight with the aeration of 15% CO2 were 0.05 g/L/d and 0.21 g/L, respectively, which were only 36% and 40% of those with the aeration of 2% CO2 (Fig. 1a). That might be due to the declining activity of extracellular carbonic anhydrase and the increasing energy consumption on maintaining intracellular acid-base equilibrium under the low pH condition with 15% CO2 (Tang et al., 2011; Cheng et al., 2016) (Fig. 1b). Therefore, the H. pluvialis mutant was domesticated by gradually increasing CO2 concentration (2-15%) from generation to generation to heighten its tolerance to 15% CO2. After the iterative cultivation, the biomass yield of H. pluvialis mutant obviously exceeded the original one. The biomass dry weight of the 5th generation was 2.2 times as that of the original mutant with 15% CO2. The biomass dry weight of the 10th generation increased to 0.65 g/L, which was 1.3 times as that of the original mutant with air (Fig. 1c). It might be attributed to the up-regulated expressions of many photosynthetic relevant enzymes of H. pluvialis mutant, e.g. the up-regulation of ATP synthase provided more energy in carbon fixation process and accelerated the CO2 fixation rate, the up-regulation expression of Antenna protease contributed to more electron transportation (Cheng et al., 2015), and the up-regulation expression of RuBisCO
accelerated carbohydrate synthesis. The mass transfer efficiency was considerably enhanced with 15% CO2. The CO2 fixation rate of the mutant with 15% CO2 was as two times as that with air. The CO2 transfer might be enhanced by several means as follows. First, lower pH caused by high CO2 aeration facilitated the exchange between the dissolved CO2 and the atmospheric CO2. Second, CO2 diffused into the cells other than HCO3 − active transport due to the pH difference between the neutral internal pH and the acid external pH (El-Ansari and Colman, 2015). The acid pH also activated the extracellular hydrolases and expansin secretion, which facilitated the breakage of hydrogen bonds between the cellulose fibers (Gross, 2000), so the cells with 15% CO2 had more contact points and pores for CO2 molecules (diameter: 0.33nm) diffusing into cells. Then, the CO2 was converted to HCO3− in the cytosol, and HCO3− was accumulated until the equilibrium between the intracellular and the extracellular CO2 concentration was reached (Gross, 2000). All of these scenarios were beneficial to enhance the photosynthetic efficiency under 15% CO2 condition. 3.2. Optimization of culture medium for 15% CO2 cultivation With the aeration of 15% CO2, the light-independent reactions of photosynthesis was enhanced due to the up-grading expression of RuBisCO and competitiveness of CO2 for RuBisCO against O2. Thus, more luminous energy would be necessary to promote light-dependent reactions. With the light intensity increasing from 45 to 65 µmol photons m−2 s−1, the max specific growth rate increased from 0.24 to 0.37/d. Appropriately increasing nitrogen and phosphorus concentrations were favorable for the microalgae growth. The max specific growth rate of 0.43/d with 4.41 mM nitrogen
was 20% higher than that with 2.94 mM nitrogen. The peak biomass dry weight of 0.89 g/L with 5.88 mM nitrogen was 62% higher than that with 2.94 mM nitrogen, even though the growth time was prolonged from 5 days to 9 days. However, the growth of domesticated H. pluvialis mutant was suppressed when the nitrogen and phosphorus concentrations were further increased (Fig. 2). It might be attributed to the increasing salinity caused by the increasing nutrients. High salinity might induce the degradation of the pigments in PSI, PSII and light-harvesting proteins, and lower the electron transport rate of photosynthesis. At the end of vegetative growth phase, the domesticated H. pluvialis mutant cultures were transferred to the high light of 125µmol photons m−2 s−1 to induce the astaxanthin accumulation. The peak astaxanthin yield of 42 mg/L and astaxanthin content of 15.8mg/g were obtained at the nitrogen concentration of 5.88 mM. 3.3. Astaxanthin and lipid accumulation with 15% CO2 The inoculums of the domesticated H. pluvialis mutant were incubated in tubular airlift photobioreactors with15% CO2 under continuous illumination of 135µmol photons m−2 s−1 for highly efficient astaxanthin induction. The astaxanthin accumulation rate of 8.2 mg/L/d with 15% CO2 was much higher than that with air due to the enhanced expression of most of relevant genes for astaxanthin synthesis (Wen et al., 2015) (Supplementary data). The efficient formation and deposition of astaxanthin seemed to prevent further reduction in D1 protein level of PSII, thereby enabled the cell to maintain functional and structural integrity and to recover from the damage of high light (Wang et al., 2003). Thus, 1.6 times higher biomass dry weight
was given with 15% CO2 than that of with air (Fig. 3). The astaxanthin accumulation rate reached to 12.2 mg/L/d with the addition of ferrous sulfate (450µM), and the astaxanthin yield increased from 65.7 to 87.4 mg/L compared with that induced with 15% CO2 only. The astaxanthin content was 40% higher than that with air. That might because astaxanthin production was improved by accelerating iron-catalyzed Haber–Weiss reaction, which converted less oxygen species into more reactive oxygen species (Hong et al., 2015). With increasing cultivation time, the astaxanthin content of H. pluvialis mutant with 15% CO2 increased significantly within the first 6 days, then, the astaxanthin accumulation slowed down. The expression of most of carotenogenic genes up-regulated sharply in the 24h, and then declined within 8 days depending on the different gens, e.g. ipi-2, psy and pds reached the highest levels on the 4th day, and lyc and crtO reached the highest levels on the 6th day (Wen et al., 2015). The formation of lipid droplets in H. pluvialis cell is one of the key factors driving accumulation of astaxanthin. The final steps of astaxanthin biosynthesis and the astaxanthin esterification take place in lipid droplets, and lipid droplets are the main subcellular depots for astaxanthin (Solovchenko, 2015) (Fig. 2b). There were no significant differences of lipid contents between the domesticated H. pluvialis mutant with 2 and 15% CO2. However, the lipid components changed with the elevating CO2 concentration. The proportion of saturated fatty acids C16:0 increased from 29.36 to 32.53% with the elevating CO2, while the proportion of C18:0 decreased from 3.63 to 2.64%. Fatty acids are synthesized from malonyl-ACP (acyl carrier protein), which is
formed from the generated malonyl-CoA and acetyl-CoA, by condensation reaction until palmitoyl-ACP (16:0-ACP) is formed. The elongation of the16:0-ACP to stearoyl-ACP (C18:0-ACP) proceeds according to the decarbonation reaction catalysed by β-ketoacyl-ACP synthase II (KAS II) (Equation 1), and the generated fatty acids are modified by desaturase (Sanchez and Harwood, 2002). The suppression of high CO2 to decarbonation reaction might be accounting for the reduction of C18 fatty acids.
palmitoyl - ACP + malonyl - ACP KASII → stearoyl - ACP + CO 2 + H 2O (1)
4. Conclusions High CO2 domestication can efficiently increase biomass and astaxanthin yields of H. pluvialis with 15% CO2 which is the CO2 content of flue gas from coal-fired power plants. The biomass yield of the domesticated mutant with 15% CO2 was 30% higher than that with air. The astaxanthin yield induced with 15% CO2 and high light was 6 times higher than that with air. The changes of H. pluvialis cell in genomics, proteomics, and metabonomics with high CO2 need further investigation.
Acknowledgements This study was supported by the National Natural Science Foundation-China (51176163, 51476141) and Zhejiang Provincial Natural Science Foundation-China (LR14E060002).
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List of Figures and Tables: Fig. 1. Biomass dry weight of H. pluvialis mutant before and after domestication with 15% CO2 (a) growth curves of mutant before domestication, (b) dynamic pH values of cultures before domestication, (c) biomass dry weight of mutant domesticated with 15% CO2. Fig. 2. Effects of nitrogen and phosphorus concentrations in growth media on (a) biomass growth during vegetative growth stage with low light of 65 µmol photons m−2 s−1 and (b) astaxanthin yield during astaxanthin accumulation stage with high light of 125 µmol photons m−2 s−1 of H. pluvialis mutant domesticated with 15% CO2. Fig. 3. Biomass dry weight and astaxanthin yield of domesticated H. pluvialis mutant induced with 15% CO2 and 135 µmol photons m−2 s−1 of high light. (a) astaxanthin yield, (b) biomass dry weight.
Biomass dry weight(g/L)
0.7
0.04% CO2 (air)
(a)
2% CO2
0.6
15% CO2
0.5 0.4 0.3 0.2 0.1 0.0
0
1
2
3
4
5
6
7
Growth time(d)
10
(b)
9
0.04% CO2 (air) 2% CO2
pH
8
15% CO2
7 6 5
Biomass dry weight (g/L)
0.8
0
(c)
1
2 3 4 Growth time(d)
5
6
Undomesticated cells 1st generation of domesticated cells under 15% CO2 5th generation 7th generation 10th generation
0.6 0.4 0.2 0.0 0.04
10 2 8 CO2 Concentration (%)
15
Fig. 1. Biomass dry weight of H. pluvialis mutant before and after domestication with 15% CO2 (a) growth curves of mutant before domestication, (b) dynamic pH values of cultures before domestication, (c) biomass dry weight of mutant domesticated with 15% CO2.
1.0
(a)
0.8
0.8
0.6
0.6
0.4
0.4
0.2
0.2
Biomass dry weight Max specific growth rate
0.0
0.0 N=2.94 P=1.72
N=4.41 P=2.58
N=5.88 P=3.44
N=8.82 P=5.16
Max specific growth rate (/d)
Biomass dry weight (g/L)
1.0
N=14.70 P=8.60
Nitrogen and phosphorus concentrations (mM)
5
(b)
40
4
30
3
20
2
10
1
Astaxanthin yield Biomass dry weight
Biomass dry weight (g/L)
Astaxanthin yield (mg/L)
50
0
0 N=2.94 P=1.72
N=4.41 P=2.58
N=5.88 P=3.44
N=8.82 P=5.16
N=14.70 P=8.60
Nitrogen and phosphorus concentrations (mM) Fig. 2. Effects of nitrogen and phosphorus concentrations in growth media on (a) biomass growth during vegetative growth stage with low light of 65 µmol photons m−2 s−1 and (b) astaxanthin yield during astaxanthin accumulation stage with high light of 125 µmol photons m−2 s−1 of H. pluvialis mutant domesticated with 15% CO2.
Astaxanthin yield (mg/L)
15% CO2
40
0.04% CO2 (air)
80
Astaxanthin content 15% CO2 and FeSO4
30
15% CO2
60
0.04% CO2 (air)
20
40 10
20 0
Biomass dry weight (g/L)
(a)
0
2
6
4 6 8 10 12 14 16 Induction time (d)
15% CO2 and FeSO4
0
Astaxanthin content in dried biomass (mg/g)
Astaxanthin yield 15% CO2 and FeSO4
100
(b)
15% CO2 0.04% CO2 (air)
4
2
0
0
2
4
6 8 10 12 14 16 Induction time (d)
Fig. 3. Biomass dry weight and astaxanthin yield of domesticated H. pluvialis mutant induced with 15% CO2 and 135 µmol photons m−2 s−1 of high light. (a) astaxanthin yield, (b) biomass dry weight.
>15% CO2-tolerant Haematococcus pluvialis mutant was obtained with domestication >The biomass yield with 15% CO2 increased by 30% than that with air >The astaxanthin yield with 15% CO2 were 6 times higher than that with air
S0960-8524(16)30736-2 http://dx.doi.org/10.1016/j.biortech.2016.05.095 BITE 16592
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Bioresource Technology
Received Date: Revised Date: Accepted Date:
4 April 2016 21 May 2016 23 May 2016
Please cite this article as: Cheng, J., Li, K., Yang, Z., Lu, H., Zhou, J., Cen, K., Gradient domestication of Haematococcus pluvialis mutant with 15% CO2 to promote biomass growth and astaxanthin yield, Bioresource Technology (2016), doi: http://dx.doi.org/10.1016/j.biortech.2016.05.095
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Gradient domestication of Haematococcus pluvialis mutant with 15% CO2 to promote biomass growth and astaxanthin yield Jun Cheng*, Ke Li, Zongbo Yang, Hongxiang Lu, Junhu Zhou, Kefa Cen State Key Laboratory of Clean Energy Utilization, Zhejiang University, Hangzhou 310027, China
Abstract: In order to increase biomass yield and reduce culture cost of Haematococcus pluvialis with flue gas from coal-fired power plants, a screened mutant by nuclear irradiation was gradually domesticated with 15% CO2 to promote biomass dry weight and astaxanthin yield. The biomass yield of mutant after 10 generations of 15% CO2 domestication increased to 1.3 times as that with air. With the optimization of nitrogen and phosphorus concentration, the biomass dry weight was further increased by 62%. The astaxanthin yield induced with 15% CO2 and high light of 135 µmol photons m−2s−1 increased to 87.4 mg/L, which was 6 times higher than that induced with high light in air. Keyword: Haematococcus pluvialis mutant; 15% CO2 domestication; Astaxanthin; Biomass growth
1. Introduction Flue gas mitigation and fixation have attracted considerable attention because of greenhouse gases emission and their effects on global climates and ecosystems. Due to the large flow rates and high concentrations of CO2 in the flue gas, billions of tons of CO2 are released into the atmosphere from coal-fired power plants every year.
Among multifaceted approaches of CO2 mitigation, biosequestration of CO2 by microalgae shows high competitiveness in both CO2 capture and energy recovery. Approximately 1.83 kg of CO2 can be fixed in every 1 kg of microalgae biomass, and the efficiency of converting solar energy to chemical energy by microalgae is 10–50 times greater than that by terrestrial plants (Cheah et al., 2015). Subsequently, the lipid and carbohydrate in microalgae cells can be processed to biodiesel and bioethanol. In addition to eco-friendly biofuels, microalgaes contain various natural nutrition and pigments, e.g. astaxanthin in Haematococcus pluvialis, which is a high value-added pigment with strong antioxidant activity and applied in many nutraceuticals and in the food and feed industries. Given the slow mass transfer of CO2 in water, around 40% of the total energy consumption is consumed by delivering atmospheric air to ensure that sufficient carbon source is available for microalgae reproduction. However, the overall fossil-energy input can be significantly reduced via aeration of flue gas, and biomass and lipid can be remarkably enhanced simultaneously (Lam et al., 2012; Yoo et al., 2010；Jiang et al., 2011; Tang et al., 2011；Ji et al., 2013). But the high concentration of CO2 (above 10%) in flue gas may be toxic to some microalgaes (Ota et al., 2009; 2015; Wang et al., 2014). Several studies have focused on developing high CO2-tolerant strains for industrial cultivation with flue gas. Thermal-and CO2-tolerant mutant strains of Chlorella sp. were isolated via ultraviolet or chemical mutagenesis, and were cultured directly with flue gas generated from coke oven of steel plants (Chiu et al., 2011; Li et al., 2011; Kao et al., 2014). High CO2-tolerant strains can also
be isolated via manual selection and domestication with high CO2 (Cheng et al., 2013；Bhakta et al., 2015；Li et al., 2015). According to previous studies, the growth of H. pluvialis was enhanced by 82% when the CO2 concentration increased from air to 6%, however, cell growth was inhibited by further increment of CO2 (Lee et al., 2015; Cheng et al., 2016). The astaxanthin content was decreased by 40% when the CO2 concentration was increased to 20% (Yin et al., 2015). Further works are necessary to improve high CO2-tolerant H. pluvialis strains that can be cultivated by flue gas from coal-fired power plants with 15% CO2. γ-Ray has been recently used to screen efficient microalgae stains due to its strong penetration considerably surpassing that of UV and X-ray. γ-Rays and free radicals generating via radiolysis of water can improve the photosynthesis of microalgae cells and screen astaxanthin-hyperproducing strains of H. pluvialis simultaneously. In our previous study, the biomass and astaxanthin yields of H. pluvialis mutant irradiated with 60Co-γunder 6%CO2 before domestication were much higher than those of the wild strain (Cheng et al., 2016). In the present study, the mutant was gradually domesticated with elevated CO2 concentrations to further enhance biomass and astaxanthin yields under 15% CO2.
2 Materials and Methods 2.1 H. pluvialis strain and growth medium The microalgae strain used in this study was H. pluvialis mutated by a 60Co-γ irradiator at 4000Gy (Cheng et al., 2016). The microalgae cells were cultured in
Bold’s Basal Medium (BBM) containing 2.94 mM NaNO3, 0.304 mM MgSO4·7H2O, 0.428 mM NaCl, 0.431 mM K2HPO4, 1.29 mM KH2PO4, 0.170 mM Ca(NO3)2·4H2O, 30.7 µM ZnSO4·7H2O, 7.28 µM MnCl2·4H2O, 4.93 µM MoO3, 6.29 µM CuSO4·5H2O, 1.68 µM Co(NO3)2·6H2O, 0.185 mM H3BO3, 0.171 mM EDTA·Na2, 0.553 mM KOH, 17.9 µM FeSO4·7H2O, 29.7 nM Vit.B1,0.102 nM Biotin, and 0.0111 nM Vit.B12 (Dominguez-Bocanegra et al., 2004).
2.2 Domestication and culture of H. pluvialis mutant with high concentrations of CO2
A H. pluvialis mutant was domesticated with gradually increased CO2 concentrations from 2% to 15% to increase its tolerance to high CO2 stress. The aeration gas in experiments was the mixture of pure CO2 and N2 rather than real flue gas. H. pluvialis mutant at the logarithmic growth phase was inoculated in 400 mL BBM medium in a 600 mL glass cylinder photobioreactor (diameter: 8.2 cm, height: 15.3 cm) covered with a silica gel stopper. Inoculums were aerated continuously with 2% CO2 at a rate of 120 mL/min for 4 days. Then, partial cells were transferred to new medium and aerated continuously with 8% CO2. These procedures were repeated under the condition of 10% and 15% CO2 gradually. The iterative cultures with 15% CO2 were performed for at least 10 generations and the H. pluvialis strain adapting to 15% CO2 was obtained. Different concentrations of nitrogen (2.94, 4.41, 5.88, 8.82, 14.70 mM NaNO3) and phosphorus ( 1.72, 2.58, 3.44, 5.16, 8.60 mM KH2PO4) were tested to determine the suitable nutrient concentrations with 15% CO2 aeration. The vegetative cultivation was performed under 25°C and 65 µmol photons m−2 s−1 of
continuous light, and the encystment induction was performed under 125 µmol photons m−2 s−1 of continuous light.
2.3 Encystment and astaxanthin induction of H. pluvialis mutant
The domesticated H. pluvialis mutant was incubated with the aeration of 15% CO2 under 25°C and 65 µmol photons m−2 s−1 of continuous light for vegetative growth. The inoculums (biomass dry weight was 1 g/L) were transferred to 500 mL volume glass tubular airlift photobioreactors (diameter: 5 cm, height: 40 cm) and aerated with 15% CO2 at a rate of 120 mL/min continuously. 135 µmol photons m−2 s−1 continuous illumination, which was suitable for astaxanthin accumulation with 15% CO2, was employed for rapid induction of astaxanthin accumulation. Ferrous sulfate at a final concentration of 450 µM was added to the inoculums for efficient inducing by facilitating Haber–Weiss reaction.
2.4 Analytical methods
2.4.1 Growth analyses Biomass yield was monitored by spectrophotometer (optical density 680 nm, A680) and by dry cell weight. For dry cell weight, the microalgae pellets collected via centrifugation were dried at 80°C for 24h and were measured gravimetrically. The biomass dry weight (BDW) shown in growth curves was calculated as: BDW (g/L) =0.512×OD680. The biomass productivity (P) and specific growth rate (µ) were calculated as follows: P = (M1－M0)/(t1－t0)
(1)
µ = (lnM1－lnM0) /(t1－t0)
(2)
where M1 was the dry weight at time t1 and M0 was the dry weight at time t0. 2.4.2. Astaxanthin determination Astaxanthin yield was measured via spectrophotometric method (Boussiba et al., 1992). The harvested cells were first treated with a solution of 5% (w/v) KOH in 30% (v/v) methanol to destroy the chlorophyll. The supernatant was discarded, and the remaining pellet was extracted with DMSO to recover the astaxanthin. The extraction procedure was repeated until the cell debris was nearly colorless. The absorbance of the combined extracts was determined at 490 nm, and the per unit volume astaxanthin concentration (c) was calculated as: c (mg/L) = 4.5×A490×Va/Vb
(3)
where Va (L) was the volume of extracts, Vb (L) was the volume of the culture sample, and A490 was the absorbance of the extracts at 490 nm. 2.4.3 Lipid extraction and estimation Lipids were extracted through direct transesterification (Johnson and Wen, 2009; Cheng et al., 2014). 0.1 g freeze-dried algal biomass was mixed with 4 mL chloroform, 4 mL methanol and 0.2 mL sulfuric acid in a digestion reactor. The digestion reactor was sealed and heated in the baking oven at 90°C for 6 h and then cooled to room temperature. The product was washed with distilled water for at least three times and dried at 60°C. The dried lipids redissolved in n-hexane were mixed with an internal standard (C19:0) and quantified with a gas chromatograph (Agilent 7890A, USA) equipped with an HP-INNOWAX column (30 m × 320 µm × 0.25 µm,
USA) and an FID detector.
3 Results and discussion 3.1. Domestication of H. pluvialis mutant H. pluvialis mutant isolated via the 60Co-γ irradiation showed high sensitivity to 15% CO2. The biomass productivity and biomass dry weight with the aeration of 15% CO2 were 0.05 g/L/d and 0.21 g/L, respectively, which were only 36% and 40% of those with the aeration of 2% CO2 (Fig. 1a). That might be due to the declining activity of extracellular carbonic anhydrase and the increasing energy consumption on maintaining intracellular acid-base equilibrium under the low pH condition with 15% CO2 (Tang et al., 2011; Cheng et al., 2016) (Fig. 1b). Therefore, the H. pluvialis mutant was domesticated by gradually increasing CO2 concentration (2-15%) from generation to generation to heighten its tolerance to 15% CO2. After the iterative cultivation, the biomass yield of H. pluvialis mutant obviously exceeded the original one. The biomass dry weight of the 5th generation was 2.2 times as that of the original mutant with 15% CO2. The biomass dry weight of the 10th generation increased to 0.65 g/L, which was 1.3 times as that of the original mutant with air (Fig. 1c). It might be attributed to the up-regulated expressions of many photosynthetic relevant enzymes of H. pluvialis mutant, e.g. the up-regulation of ATP synthase provided more energy in carbon fixation process and accelerated the CO2 fixation rate, the up-regulation expression of Antenna protease contributed to more electron transportation (Cheng et al., 2015), and the up-regulation expression of RuBisCO
accelerated carbohydrate synthesis. The mass transfer efficiency was considerably enhanced with 15% CO2. The CO2 fixation rate of the mutant with 15% CO2 was as two times as that with air. The CO2 transfer might be enhanced by several means as follows. First, lower pH caused by high CO2 aeration facilitated the exchange between the dissolved CO2 and the atmospheric CO2. Second, CO2 diffused into the cells other than HCO3 − active transport due to the pH difference between the neutral internal pH and the acid external pH (El-Ansari and Colman, 2015). The acid pH also activated the extracellular hydrolases and expansin secretion, which facilitated the breakage of hydrogen bonds between the cellulose fibers (Gross, 2000), so the cells with 15% CO2 had more contact points and pores for CO2 molecules (diameter: 0.33nm) diffusing into cells. Then, the CO2 was converted to HCO3− in the cytosol, and HCO3− was accumulated until the equilibrium between the intracellular and the extracellular CO2 concentration was reached (Gross, 2000). All of these scenarios were beneficial to enhance the photosynthetic efficiency under 15% CO2 condition. 3.2. Optimization of culture medium for 15% CO2 cultivation With the aeration of 15% CO2, the light-independent reactions of photosynthesis was enhanced due to the up-grading expression of RuBisCO and competitiveness of CO2 for RuBisCO against O2. Thus, more luminous energy would be necessary to promote light-dependent reactions. With the light intensity increasing from 45 to 65 µmol photons m−2 s−1, the max specific growth rate increased from 0.24 to 0.37/d. Appropriately increasing nitrogen and phosphorus concentrations were favorable for the microalgae growth. The max specific growth rate of 0.43/d with 4.41 mM nitrogen
was 20% higher than that with 2.94 mM nitrogen. The peak biomass dry weight of 0.89 g/L with 5.88 mM nitrogen was 62% higher than that with 2.94 mM nitrogen, even though the growth time was prolonged from 5 days to 9 days. However, the growth of domesticated H. pluvialis mutant was suppressed when the nitrogen and phosphorus concentrations were further increased (Fig. 2). It might be attributed to the increasing salinity caused by the increasing nutrients. High salinity might induce the degradation of the pigments in PSI, PSII and light-harvesting proteins, and lower the electron transport rate of photosynthesis. At the end of vegetative growth phase, the domesticated H. pluvialis mutant cultures were transferred to the high light of 125µmol photons m−2 s−1 to induce the astaxanthin accumulation. The peak astaxanthin yield of 42 mg/L and astaxanthin content of 15.8mg/g were obtained at the nitrogen concentration of 5.88 mM. 3.3. Astaxanthin and lipid accumulation with 15% CO2 The inoculums of the domesticated H. pluvialis mutant were incubated in tubular airlift photobioreactors with15% CO2 under continuous illumination of 135µmol photons m−2 s−1 for highly efficient astaxanthin induction. The astaxanthin accumulation rate of 8.2 mg/L/d with 15% CO2 was much higher than that with air due to the enhanced expression of most of relevant genes for astaxanthin synthesis (Wen et al., 2015) (Supplementary data). The efficient formation and deposition of astaxanthin seemed to prevent further reduction in D1 protein level of PSII, thereby enabled the cell to maintain functional and structural integrity and to recover from the damage of high light (Wang et al., 2003). Thus, 1.6 times higher biomass dry weight
was given with 15% CO2 than that of with air (Fig. 3). The astaxanthin accumulation rate reached to 12.2 mg/L/d with the addition of ferrous sulfate (450µM), and the astaxanthin yield increased from 65.7 to 87.4 mg/L compared with that induced with 15% CO2 only. The astaxanthin content was 40% higher than that with air. That might because astaxanthin production was improved by accelerating iron-catalyzed Haber–Weiss reaction, which converted less oxygen species into more reactive oxygen species (Hong et al., 2015). With increasing cultivation time, the astaxanthin content of H. pluvialis mutant with 15% CO2 increased significantly within the first 6 days, then, the astaxanthin accumulation slowed down. The expression of most of carotenogenic genes up-regulated sharply in the 24h, and then declined within 8 days depending on the different gens, e.g. ipi-2, psy and pds reached the highest levels on the 4th day, and lyc and crtO reached the highest levels on the 6th day (Wen et al., 2015). The formation of lipid droplets in H. pluvialis cell is one of the key factors driving accumulation of astaxanthin. The final steps of astaxanthin biosynthesis and the astaxanthin esterification take place in lipid droplets, and lipid droplets are the main subcellular depots for astaxanthin (Solovchenko, 2015) (Fig. 2b). There were no significant differences of lipid contents between the domesticated H. pluvialis mutant with 2 and 15% CO2. However, the lipid components changed with the elevating CO2 concentration. The proportion of saturated fatty acids C16:0 increased from 29.36 to 32.53% with the elevating CO2, while the proportion of C18:0 decreased from 3.63 to 2.64%. Fatty acids are synthesized from malonyl-ACP (acyl carrier protein), which is
formed from the generated malonyl-CoA and acetyl-CoA, by condensation reaction until palmitoyl-ACP (16:0-ACP) is formed. The elongation of the16:0-ACP to stearoyl-ACP (C18:0-ACP) proceeds according to the decarbonation reaction catalysed by β-ketoacyl-ACP synthase II (KAS II) (Equation 1), and the generated fatty acids are modified by desaturase (Sanchez and Harwood, 2002). The suppression of high CO2 to decarbonation reaction might be accounting for the reduction of C18 fatty acids.
palmitoyl - ACP + malonyl - ACP KASII → stearoyl - ACP + CO 2 + H 2O (1)
4. Conclusions High CO2 domestication can efficiently increase biomass and astaxanthin yields of H. pluvialis with 15% CO2 which is the CO2 content of flue gas from coal-fired power plants. The biomass yield of the domesticated mutant with 15% CO2 was 30% higher than that with air. The astaxanthin yield induced with 15% CO2 and high light was 6 times higher than that with air. The changes of H. pluvialis cell in genomics, proteomics, and metabonomics with high CO2 need further investigation.
Acknowledgements This study was supported by the National Natural Science Foundation-China (51176163, 51476141) and Zhejiang Provincial Natural Science Foundation-China (LR14E060002).
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List of Figures and Tables: Fig. 1. Biomass dry weight of H. pluvialis mutant before and after domestication with 15% CO2 (a) growth curves of mutant before domestication, (b) dynamic pH values of cultures before domestication, (c) biomass dry weight of mutant domesticated with 15% CO2. Fig. 2. Effects of nitrogen and phosphorus concentrations in growth media on (a) biomass growth during vegetative growth stage with low light of 65 µmol photons m−2 s−1 and (b) astaxanthin yield during astaxanthin accumulation stage with high light of 125 µmol photons m−2 s−1 of H. pluvialis mutant domesticated with 15% CO2. Fig. 3. Biomass dry weight and astaxanthin yield of domesticated H. pluvialis mutant induced with 15% CO2 and 135 µmol photons m−2 s−1 of high light. (a) astaxanthin yield, (b) biomass dry weight.
Biomass dry weight(g/L)
0.7
0.04% CO2 (air)
(a)
2% CO2
0.6
15% CO2
0.5 0.4 0.3 0.2 0.1 0.0
0
1
2
3
4
5
6
7
Growth time(d)
10
(b)
9
0.04% CO2 (air) 2% CO2
pH
8
15% CO2
7 6 5
Biomass dry weight (g/L)
0.8
0
(c)
1
2 3 4 Growth time(d)
5
6
Undomesticated cells 1st generation of domesticated cells under 15% CO2 5th generation 7th generation 10th generation
0.6 0.4 0.2 0.0 0.04
10 2 8 CO2 Concentration (%)
15
Fig. 1. Biomass dry weight of H. pluvialis mutant before and after domestication with 15% CO2 (a) growth curves of mutant before domestication, (b) dynamic pH values of cultures before domestication, (c) biomass dry weight of mutant domesticated with 15% CO2.
1.0
(a)
0.8
0.8
0.6
0.6
0.4
0.4
0.2
0.2
Biomass dry weight Max specific growth rate
0.0
0.0 N=2.94 P=1.72
N=4.41 P=2.58
N=5.88 P=3.44
N=8.82 P=5.16
Max specific growth rate (/d)
Biomass dry weight (g/L)
1.0
N=14.70 P=8.60
Nitrogen and phosphorus concentrations (mM)
5
(b)
40
4
30
3
20
2
10
1
Astaxanthin yield Biomass dry weight
Biomass dry weight (g/L)
Astaxanthin yield (mg/L)
50
0
0 N=2.94 P=1.72
N=4.41 P=2.58
N=5.88 P=3.44
N=8.82 P=5.16
N=14.70 P=8.60
Nitrogen and phosphorus concentrations (mM) Fig. 2. Effects of nitrogen and phosphorus concentrations in growth media on (a) biomass growth during vegetative growth stage with low light of 65 µmol photons m−2 s−1 and (b) astaxanthin yield during astaxanthin accumulation stage with high light of 125 µmol photons m−2 s−1 of H. pluvialis mutant domesticated with 15% CO2.
Astaxanthin yield (mg/L)
15% CO2
40
0.04% CO2 (air)
80
Astaxanthin content 15% CO2 and FeSO4
30
15% CO2
60
0.04% CO2 (air)
20
40 10
20 0
Biomass dry weight (g/L)
(a)
0
2
6
4 6 8 10 12 14 16 Induction time (d)
15% CO2 and FeSO4
0
Astaxanthin content in dried biomass (mg/g)
Astaxanthin yield 15% CO2 and FeSO4
100
(b)
15% CO2 0.04% CO2 (air)
4
2
0
0
2
4
6 8 10 12 14 16 Induction time (d)
Fig. 3. Biomass dry weight and astaxanthin yield of domesticated H. pluvialis mutant induced with 15% CO2 and 135 µmol photons m−2 s−1 of high light. (a) astaxanthin yield, (b) biomass dry weight.
>15% CO2-tolerant Haematococcus pluvialis mutant was obtained with domestication >The biomass yield with 15% CO2 increased by 30% than that with air >The astaxanthin yield with 15% CO2 were 6 times higher than that with air