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CAH1 and CAH2 as key enzymes required for high bicarbonate tolerance of a novel microalga Dunaliella salina HTBS
Enzyme and Microbial Technology 87–88 (2016) 17–23
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CAH1 and CAH2 as key enzymes required for high bicarbonate tolerance of a novel microalga Dunaliella salina HTBS Yuyong Hou a , Zhiyong Liu a , Yue Zhao a , Shulin Chen a , Yubin Zheng b , Fangjian Chen a,∗ a Tianjin Key Laboratory for Industrial Biological Systems and Bioprocessing Engineering, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, PR China b Shandong Jinjing Biotechnology Co., Ltd., Shandong 261313, PR China
a r t i c l e
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Article history: Received 14 December 2015 Received in revised form 19 February 2016 Accepted 19 February 2016 Available online 23 February 2016 Keywords: Dunaliella salina HTBS Inorganic carbon fixation Bicarbonate Carbonic anhydrase
a b s t r a c t Outdoor microalgal cultivation with high concentration bicarbonate has been considered as a strategy for reducing contamination and improving carbon supply efficiency. The mechanism responsible for algae’s strong tolerance to high bicarbonate however, remains not clear. In this study, we isolated and characterized a strain and revealed its high bicarbonate tolerant mechanism by analyzing carbonic anhydrase (CA). The strain was identified as Dunaliella salina HTBS with broad temperature adaptability (7–30 ◦ C). The strain grew well under 30% CO2 or 70 g L−1 NaHCO3 . In comparison, two periplasm CAs (CAH1 and CAH2) were detected with immunoblotting analysis in HTBS but not in a non-HCO3 − —tolerant strain. The finding was also verified by an enzyme inhibition assay in which only HTBS showed significant inhibition by extracellular CA inhibitor. Thus, we inferred that the extracellular CAH1 and CAH2 played a multifunctional role in the toleration of high bicarbonate by HTBS. © 2016 Elsevier Inc. All rights reserved.
1. Introduction The sharp rise in atmospheric CO2 directly enforces the “greenhouse effect” and causes great concerns over global warming and climate change. With the demand for CO2 capturing technologies, the biological CO2 fixation by microalgae has received increasing attention because of its sustainability [1–4]. Previous studies focused on the effects of CO2 concentrations from 0% to 15% on the microalgal growth among different species [1,5,6]. However, efficiently supplying CO2 with a high concentration is challenging as it easily escapes from the medium due to its low solubility. Supplying the inorganic carbon in the form of HCO3 − can be more convenient than dissolving gaseous CO2 thus is considered as an alternative for culturing algae. When the pH of culture medium ranges from 6.4 to 10.3, HCO3 − is predominant; otherwise, CO2 is the dominant form at lower pH levels [7]. To cope with changes in Ci species (CO2 , HCO3 − , CO3 2− ), the carbon concentrating mechanism (CCM) was evolved to regulate carbon uptake and cell acclima-
Abbreviations: HTBS, the high tolerance of bicarbonate strain; CA, carbonic anhydrase; CAH1, carbonic anhydrase I; CAH2, carbonic anhydrase II; CAH3, carbonic anhydrase III; CCM, the carbon concentrating mechanism; AZ, acetazolamide; EZ, ethoxyzolamide; SEM, scanning electron microscopic; TEM, transmission electron microscopic; DCW, dry cell weight. ∗ Corresponding author at: 32 West 7th Avenue, Tianjin Airport Economic Area, Tianjin 300308, PR China. Fax: +86 22 24828745. E-mail address: chen [email protected] (F. Chen). http://dx.doi.org/10.1016/j.enzmictec.2016.02.010 0141-0229/© 2016 Elsevier Inc. All rights reserved.
tion [8]. Carbonic anhydrase (CA, EC 4.2.1.1) as a key part of the CCM is responsible for the reversible hydration of CO2 . Two of CAs, CAH1 and CAH2 that only differ in a few amino acids [9,10] are extremely closely related active enzymes in periplasm and can be excreted into the medium in cell-wall deficient strains of Chlamydomonas reinhardtii [11]. The function of CAH1 is conversion of HCO3 − to CO2 , and CAH2 is presumed to hydrate CO2 . The activities of the two enzymes can be inhibited by acetazolamide (AZ), which results in the decreased uptake of Ci and inhibition of the cell growth [12]. Under low-CO2 conditions, CAH1 and CAH2 tend to up-regulate and down-regulate, respectively [13]. CAH3, which is found in the thylakoid lumen, can be inhibited by the internal CA inhibitor (ethoxyzolamide; EZ). The strains that are defective in CAH3 gene cannot use Ci efficiently even when its concentration is high, but carry out photosynthesis normally when the CAH3 is put back into these strains [13,14]. Although the effects of various CO2 concentrations on the expression and function of CAs have been intensively studied [7,9,10,13], the report on the high bicarbonate response mechanism in HCO3 − –tolerant microalgae is very limited. Fukuzawa et al. [9] reported that CAH1 is expressed only under low CO2 conditions at the transcript levels, no accumulation of CAH2 mRNA is observed in the dark by Northern blot analysis. Nevertheless, the work from Fujiwara et al. [10] showed the expression of CAH2 mRNA under low-CO2 condition, and CAH2 mRNA could be translated into enzymatically active CA polypeptides. In addition, Moroney et al. [7] had summarized the expression and function of 12 genes that encode
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CA isoforms in C. reinhardtii at low and elevated CO2 conditions using multiple technologies. However, it was still unclear whether there was a relationship between algal bicarbonate-tolerance and CAs. The main objective of this study was to isolate and characterize a novel high HCO3 − —tolerant microalgae and dissect the molecular mechanism of the relationship between bicarbonatetolerance and CAs. Firstly, a novel microalga HTBS that can tolerate and utilize high-bicarbonate for biomass production was isolated and identified. Then, the expression profile of the CAs was evaluated by western blot analysis. Subsequently, to further identify the location of carbonic anhydrase, two specific CA inhibitors on the growth of HTBS, AZ and EZ were applied. Finally, the effects of CO2 and NaHCO3 concentrations and temperature on the growth of HTBS were performed. These findings provide further insights into the multifunction of CAs in response to high level of HCO3 − and potential benefit to large-scale application of Ci fixation and comprehensive utilization in microalgae cultivation.
isolate. Nannochloropsis oculata (CCAP 849/1), obtained from the Culture Collection of Algae and Protozoa at the Scottish Marine Institute (Oban, Argyll, U.K.), was the used as the control strain (non-HCO3 − —tolerant strain). All seed cultures were maintained aerobically in 250-mL Erlenmeyer flasks with 100 mL of f/2 medium under continuous artificial illumination at 75 mol m−2 s−1 . The temperature and pH were maintained at 25 ◦ C and 7.5, respectively.
2. Material and methods
2.3. Molecular identification
2.1. Strains and culture condition
Firstly, genomic DNA was extracted on the basis of the Fast-Prep DNA purification method [16]. Two universal green algal primers, 18SF (forward, 5 -CCTGGTTGATCCTGCCAG-3 ) and 18SR (reverse, 5 -TTGATCCTTCTGCAGGTTCA-3 ), were then used for PCR amplification of the 18S rDNA by the method of Liu et al. [16]. NCBI BLAST (http://www.ncbi.nlm.nih.gov) was used to study the homology of the partial 18s rDNA gene sequence. The neighbor-joining method in MEGA5 was used to construct the phylogenetic tree [16].
The high HCO3 − —tolerant strain HTBS used in this study was isolated from seawater in the Bohai Gulf (Tianjin, China, 117.82◦ E, 39.01◦ N). The strain was detected in the water samples after treating with 50 g L−1 NaHCO3 . Then the NaHCO3 treated cells were spread onto f/2 agar plates and the single colony was subcultured to the f/2 medium [15]. Optical microscope was used to check the
2.2. Electron microscopy analysis The cells were harvested at day 4 during the logarithmic phase, then scanning electron microscopic (SEM) and transmission electron microscopic (TEM) studies were performed to observe the cell morphology and structure according to Liu et al. [16] with a SEM (Quanta200; FEI, Hillsboro, Oregon) and TEM (JEM-1400; JEOL, Tokyo, Japan), respectively.
Fig. 1. Microscopic and morphological appearance of strain HTBS. Scanning (A and B) and transmission (C and D) electron micrographs of the cell in f/2 medium. C, the cells cultured under the protection of N2 ; D, the cells treated by HCO3 − under the protection of N2 . ST, starch grain; CHL, chloroplast. The scale bar is indicated at the bottom of each figure.
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Fig. 2. Phylogenetic tree inferred from 18S rDNA sequences. The distance tree was constructed with the neighbor-joining method based on Kimura’s correction using CLUSTAL W. The horizontal lengths are proportional to the evolutionary distances. The numbers above or below the internal nodes indicate bootstrap values (1000 replicates).
2.4. Studies under varying culture conditions The effects of different CO2 concentrations and temperatures on the growth of HTBS were evaluated in glass columns (4.1 cm in diameter, 37 cm in height, a working volume of 300 mL) with f/2 and modified BG11 (seawater BG11) medium, respectively. CO2 concentrations ranged from 2% to 30% in air (v/v) were filtered and bubbled into the medium continuously at the bottom of the columns. The temperature variation studies were performed at 7 ◦ C, 15 ◦ C, 25 ◦ C, and 30 ◦ C with bubbled filtered air. Aeration flow for each column was 0.42 L min−1 , the bubble size was about 0.5 cm in diameter. NaHCO3 ranged from 0 g L−1 to 70 g L−1 with 150 mL working volume (f/2 medium) in 250-mL Erlenmeyer flasks were used to investigate the effects on HTBS, while 25 g L−1 NaHCO3 was used in the growth of N. oculata. The pH were monitored by a pH meter. NaCl was used to replace the NaHCO3 at the same concentrations to rule out the effects of Na+ on HTBS. Finally, to study the effects of CA inhibitors on HTBS and N. oculata, 150 M AZ and EZ were utilized with the same conditions. The flasks were manually shaken three times a day. All the experiments were performed with triplicates under continuous illumination at 160 ± 20 mol m−2 s−1 at 25 ◦ C except the study on the temperature effect. 2.5. Electrophoresis and Immunodetection To eliminate the influence of CO2 from the air, the algae cultured in columns were aerated with N2 . Isolation of cell fractions, protein electrophoresis and western blotting were then performed according to Sinetova et al. [17]. In particular, the membrane protein CAH3 was enriched in pellet after centrifugation at 12,000 × g for 10 min at 4 ◦ C, and the supernatant contained CAH1 and CAH2. The primary antibodies of CAH1 (sc-134853), CAH2 (sc-25596) and CAH3 (sc-99005) were obtained from Santa Cruz Biotechnology, Inc. 2.6. Determination of cell growth The cell concentration of the culture was obtained by measuring its optical density with a spectrometer at 750 nm (OD750 ). The dry
cell weight (DCW) was calculated according to Wen et al. [18]. In particular, the NaHCO3 treated cells were washed by f/2 medium with 50 mM HCl before they were used. The specific growth rate (day−1 ) was calculated as the equation: = ln (X1 /X0 )/(t1 − t0 ). Where X1 and X0 were the OD750 on days t1 and t0 , respectively. 3. Results and discussion 3.1. Isolation and identification of microalgae The isolated strain HTBS was unicellular and ellipsoidal, and the cell size was homogeneous (approximately 5 m in diameter). Two long flagella and a cell covering of salt crystals were observed with SEM (Fig. 1A and B), similar to the observations in a previous report for Dunaliella sp. [19]. Compared to the cell without bicarbonate (Fig. 1C), the more discernible starch grain and chloroplast occupied the space of the treated cells with HCO3 − (Fig. 1D). The results suggested that the more chloroplast in HCO3 − treated cells enhanced photosynthesis to uptake more Ci for growth than control groups. On the basis of 18S rDNA sequence BLAST searches in the GeneBank database, the nucleotide sequences of 15 species that included the available 18S rDNA and 5.8S rDNA gene sequences were used to construct the phylogenetic tree. The neighbor-joining tree revealed a major split between HTBS and Dunaliella sp. SAS11133 with 87% similarity (Fig. 2). HTBS fell within the evolutionary radiation occupied by the genus Dunaliella salina and was closely related to Dunaliella sp. strains SAS11133 (87%), OUC (88%), ASK-21 (90%), and 1 B2-2 (94%). Thus, the strain was named as D. salina HTBS. 3.2. Immunoblotting analysis of CAs To investigate the tolerant mechanism of high Ci at the expression level of CAs, an immunoblotting analysis was performed. The results showed that CAH1 was translated in HTBS in both 0 M and 0.2 M NaHCO3 groups. However, CAH1 was not detected in non-HCO3 − —tolerant algae N. oculata at either low or high HCO3 − concentrations (Fig. 3A). CAH1 has been shown to be strongly
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Fig. 3. Western blotting detection of ␣-CAs in HTBS and N. oculata cells. (A) Immunoblotting analyses of samples with CAH1-specific antibodies and (B) CAH2and (C) CAH3-specific antibodies. “−” and “+” indicate non-HCO3 − –treated and HCO3 − –treated groups, respectively.
expressed in a low-CO2 group and was far more abundant than that in a high-CO2 group [20]. The expression of CAH1 at a low HCO3 − level is lower than that under high-HCO3 − conditions, specifically at 24 h. This finding implied that CAH1 in the HCO3 − —treated group would be up-regulated to hydrolyze HCO3 − . CAH2 was regarded as the only enzyme that was expressed under high-CO2 conditions [10], it did not translate in N. oculata, but did translate in HTBS
under both conditions (Fig. 3B). It was presumed that CAH2 in the 0 g L−1 HCO3 − —treated group was over-expressed to address the extreme environments by fixing the CO2 to HCO3 − to prevent the leakage of CO2 from the cells. CAH3, the enzyme that played an important role in Ci absorption, converted HCO3 − to CO2 and delivered CO2 to Rubisco [21]. It was detected in both strains under both culture conditions (Fig. 3C). It seemed that the rapid growth of the HCO3 − —treated cells was related to the expression of CAs. In contrast, a high Cl− concentration could be toxic to plant cells, with critical toxicity at 15–50 mg g−1 dry weight for a Cl− —tolerant species [22]. CAH1 could not alleviate the pressure formed by Cl− when the NaHCO3 was replaced by NaCl, which finally limited the cell growth. And the other results also showed that HTBS almost did not proliferate at 25 g L−1 NaCl and died at both 50 and 70 g L−1 NaCl (Fig. 4C). It appears that the presence of CAH1 and CAH2 helps to alleviate the pressure of the high bicarbonate level and creates a suitable environment for the growth. Meanwhile, the reaction catalyzed by CAs can supply enough Ci for the cells to grow. From another point of view, the fact that CAH1 and CAH2 were required for the algae to tolerate bicarbonate could be confirmed by the fact that CAH1 and CAH2 were not translated in N. oculata (Fig. 3A and B) and that the cells in 25 g L−1 NaHCO3 tended to die (Fig. 4B). With maximum turnover numbers in excess of 106 s−1 for reversible reaction catalyzed by CAs [7], once the concentration of CO2 is sharply increased after the dehydration reactions catalyzed by CAH1, CAH2 immediately acts to reduce the
Fig. 4. Effects of different concentrations of initial NaHCO3 (A) and NaCl (C) for HTBS growth. Effects of initial NaHCO3 concentration (25 g L−1 ) on HTBS and N. oculata (B) were also studied. Finally, (D) shows the dry cell weight of HTBS in growth medium for different concentrations of NaHCO3 after culture for 6 days. Data values are means (±SE) of three replicate cultures.
Table 1 The effect of AZ and EZ for HTBS and N. oculata. HTBS
Control AZ acetazolamide EZ ethoxyzolamide
N. oculata
Cell density (× 105 cell/mL)
Inhibition (%)
Cell density (× 107 cell/mL)
Inhibition
4.25 ± 0.35 3.10 ± 0.14 1.25 ± 0.21
0 26.95 ± 2.76 70.70 ± 2.55
1.1 ± 0.03 1.00 ± 0.07 0.87 ± 0.03
0 7.25 ± 3.18 18.60 ± 0.57
(%)
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Fig. 5. Growth curves of HTBS in growth medium for different concentrations of CO2 . Data values are means (±SE) of three replicate cultures.
CO2 level through hydration reaction. It seems that the presumed role of CAH1 and CAH2 is to maintain the CO2 /HCO3 − equilibrium for cell acclimation. The results suggested that extracellular CAH1 and CAH2 played an important role in the growth of HTBS under high bicarbonate stress. 3.3. Effect of enzyme inhibitors on HTBS and N. oculata growth As inhibitors for CA, AZ (acetazolamide) and EZ (ethoxyzolamide) were used to confirm the presence of periplasmic and intracellular CA [23]. Our results showed that the internal CA inhibitor (EZ) inhibited the growth of HTBS and N. oculata by 70.70% and 18.60%, respectively (Table 1). In addition, HTBS was significantly suppressed by AZ and the cell density decreased by 26.95%. For N. oculata, a slight difference of the growth was observed for the untreated group and the AZ group. The data reflected the presence of CAH1, CAH2, and CAH3 in HTBS and indicated that the cells of N. oculata were deficient in external CAH1 and CAH2, which was consistent with the immunoblotting results (Fig. 3A and B). As a result, without the translation of CAH1 and CAH2 in N. oculata, the HCO3 − —treated cells were unable to grow due to the lack of the ability for Ci processing (Fig. 4B). Similarly, Phaeodactylum tricornutum lacked extracellular CAs, whose photosynthesis was not negatively affected by AZ [22], and had less capacity and flexibility to take up Ci than Thalassiosira pseudonana, which contained extracellular CAs [24]. It thus suggested that CAH1 and CAH2 are required for the tolerance of bicarbonate in the novel microalga HTBS. 3.4. Effects of CO2 and NaHCO3 concentrations on HTBS As shown in Figs. 4 A and 5 , the strain could grow very well with both high bicarbonate and CO2 concentrations. The growth at 6% and 30% CO2 both reached the highest level at day 5, which
were 14.93% and 2.60% higher than that of 2% CO2 at day 5 (1.54 ± 0.03 g L−1 ), respectively. Compared to the DCW of 2% CO2 , that of 6% and 30% were lower before day 3 and day 5, respectively. It was speculated that the acidification caused by the high concentration CO2 affected the growth. With the increasing of cell biomass, the influence of acidification gradually weakened, finally these results had no significant difference. The biomass productivity reached 84 ± 2 mg L−1 day−1 with 30% CO2 . Most species grow only at low CO2 concentrations and their growth is inhibited when the CO2 level is higher than 5.0% [25]. Although, there are some strains with the capability to tolerate abnormal CO2 concentrations. Chiang et al. [26] reported that a strain Anabaena sp. CH1 isolated from central Taiwan demonstrated excellent CO2 tolerance in an environment with 15.0% CO2 . Scenedesmus obliquus and Chlorella kessleri were isolated from the waste-treatment ponds of a coal-fired thermoelectric power plant at different CO2 concentrations and grew well when the CO2 concentrations up to 18.0% (V/V) [5]. HTBS showed much higher CO2 tolerance and was regarded as a candidate for developing CO2 fixation technique for the concentrated sources like industrial flue gas (15% CO2 ). However, the low solubility of CO2 in water (only 0.81 mol/kg at 0 ◦ C and atmospheric pressure) results in the escape of a large amount of CO2 [27]. As an alternative, HCO3 − was more preferred for culturing microalgae. In this study, Four HCO3 − levels from 0 to 70 g L−1 were used to measure the effects on HTBS. The pH was increased with the microalgal growth throughout the culture period and was much higher than 6.4 (Fig. S1). According to pKa , the main form of Ci in the medium was HCO3 − [13]. HCO3 − and CO2 were taken into the cell for growth, leaving OH− in the medium, which resulted in an increase in the pH of the medium and intracellular HCO3 − accumulation. The HCO3 − was then converted to CO2 for Rubisco utilization by CAH3 [21].The growth of 25 g L−1 and 50 g L−1 NaHCO3 cultures reached the highest level at day 4, the
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Fig. 6. Growth curves of HTBS and N. oculata in f/2 medium at different temperatures. (A) 7 ◦ C, (B) 15 ◦ C, (C) 25 ◦ C, (D) 30 ◦ C. Data values are means (±SE) of three replicate cultures.
OD750 values of the strain were 0.42 ± 0.02 and 0.40 ± 0.02, which were significantly higher than those in the control group, increasing 2.23-fold and 2.08-fold, respectively (Fig. 4A). The growth rate of HTBS for the first 3 days was decreased from 1.24 day−1 of 25 g L−1 to 1.09 day−1 of 70 g L−1 . Although HTBS did exhibit a decrease in its growth rate at 70 g L−1 compared to 25 g L−1 and 50 g L−1 , the strain still grew very well, the cell density (OD750 = 0.35 ± 0.02) was 1.69fold higher than that of the control group at day 4. The highest DCW of HTBS was 0.46 g L−1 when grown in the medium with 25 g L−1 NaHCO3 , which was only 2.2% and 4.5% higher than that of cultures supplemented with 50 g L−1 and 70 g L−1 , respectively, whereas the biomass in the medium without NaHCO3 was only 0.26 g L−1 (Fig. 4D). The biomass productivity was 73 ± 12 mg L−1 day−1 with 70 g L−1 NaHCO3. Compared to HTBS, the strain N. oculata could not even grow in f/2 medium with 25 g L−1 NaHCO3 , the death of cells was observed after 24 h (Fig. 4B). Most microalgae can capture CO2 , but there are a series of technical challenges to overcome to achieve economical and efficient CO2 supplementation, such as the high cost of compression for transportation, the ease of loss into the atmosphere, and the difficulty of storage and capture. Although purified CA was reported for CO2 sequestration [28], it was a complex and costly system. As an alternative approach to address these problems, converting CO2 to HCO3 − through CO3 2− as a carbon source for algae growth was suggested [29,30]. Indeed, extensive CO2 capture through absorption in the aqueous phase is feasible with alkaline solutions [31]. However, the cells should possess a high ion strength to cope with the high level of HCO3 − in the medium. Unfortunately, a high level of HCO3 − was not suitable for culture of many algae species. Thus, the first step in the validation of the idea is to find a species that can grow in the extreme condition with high concentration of bicarbonate. In this study, the novel strain HTBS showed the potential for high HCO3 − tolerance.
To further eliminate the effects of Na+ on the bicarbonate tolerance of HTBS, we carried out sodium chloride treatment experiments in which NaHCO3 was replaced with NaCl (Fig. 4C). In the experimental design, the same molarity of NaCl and NaHCO3 will lead to the inconsistent salinities. Comparing to relevant NaHCO3 treatments, the lower salinity of NaCl groups can affect the analysis of the various treatment. Thus, the same mass concentration of NaCl and NaHCO3 were adopted to keep the salinity consistent, then the effect of Na+ was the main concern. Considering Na+ gradient test, we have designed NaHCO3 0.3–0.6 M (25–50 g L−1 ) on the sides of 0.4 M (NaCl 25 g L−1 ) at the same time. The results showed that the cells barely survived when the NaCl level reached only 25 g L−1 , their cell growth decreased even more significantly as the NaCl level increased. HTBS could not survive in an equivalent mineral salt such as sodium chloride. Although the Na+ concentrations of 50 g L−1 and 70 g L−1 NaHCO3 were much higher than those in the group treated with 25 g L−1 NaCl, the strain grew well (Fig. 4A). This finding suggested that Na+ had almost no influence on the growth of HTBS under equivalent HCO3 − and that the growth could be influenced by anion Cl− , it was consistent with previous report [22]. Compared to Cl− , HCO3 − as one of the main Ci species, could be regulated by CCM for efficient use, resulting in cell acclimation and fast growth. Our results also showed that the marine algae N. oculata was non-HCO3 − —tolerant (Fig. 4B).
3.5. Effects of temperature on HTBS For further assessment of the features of HTBS, we studied the effects of four different temperatures on cell growth. The algae grew well at both low (Fig. 6A and B) and high temperatures (Fig. 6C and D). The optical density (OD750 ) of the cells was 1.67 under a culture temperature of 7 ◦ C on day 12. In contrast, the growth of the control algae N. oculata was inhibited. A similar result was observed at 15 ◦ C. For high temperatures, the OD750 of HTBS could reach 2.96 and 2.03 at 25 ◦ C and 30 ◦ C, respectively. As one of the most impor-
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tant factors in microalgae culture conditions, temperature has a great effect on the uptake of nutrients, the fixation of Ci, and the growth rate. Low temperature leads to low intracellular enzyme activity and is responsible for the decreased use of Ci and other nutrients [32]. Most species cannot survive in cold weather (usually below 16 ◦ C) [33,34]. However, screening microalgal strain with the capability to grow at low temperature is meaningful for outdoor cultures, especially in the northern regions. In this study, the wide growth temperature range made HTBS as a promising candidate for commercial application. 4. Conclusions The microalgal strain HTBS isolated from a biodiverse body of seawater, the Bohai Gulf, showed tolerance to 30% CO2 , 70 g L−1 NaHCO3 and a low temperature of 7 ◦ C. The total biomass productivity reached 84 and 73 mg L−1 day−1 with 30% CO2 and 70 g L−1 NaHCO3 , respectively. CAH1 and CAH2 were required to function under the condition of a high concentration of bicarbonate as part of the mechanism for the algae to tolerate high level Ci. Acknowledgements This work was supported partially by the National Natural Science Foundation of China (Grant Nos. 31200093 and 31570047), the Hi-Tech Research and Development Program (863) of China (2014AA022003), the Major State Basic Research Development Program of China (973 Project) (Grant Nos. 2011CB200905 and 2011CB200906), and the Key Program for International S&T Cooperation Projects of China (2014DFA61040). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.enzmictec.2016. 02.010. References [1] S. Basu, A.S. Roy, K. Mohanty, et al., Enhanced CO2 sequestration by a novel microalga: Scenedesmus obliquus SA1 isolated from bio-diversity hotspot region of Assam, India, Bioresour. Technol. 143 (2013) 369–377. [2] Y. Chisti, Biodiesel from microalgae, Biotechnol. Adv. 25 (2007) 294–306. [3] A. Kumar, S. Ergas, X. Yuan, et al., Enhanced CO2 fixation and biofuel production via microalgae: recent developments and future directions, Trends Biotechnol. 28 (2010) 371–380. [4] M. Ota, Y. Kato, H. Watanabe, et al., Fatty acid production from a highly CO2 tolerant alga, Chlorocuccum littorale, in the presence of inorganic carbon and nitrate, Bioresour. Technol. 100 (2009) 5237–5242. [5] M.G. de Morais, J.A.V. Costa, Isolation and selection of microalgae from coal fired thermoelectric power plant for biofixation of carbon dioxide, Energy Convers. Manage. 48 (2007) 2169–2173. [6] E.B. Sydney, W. Sturm, J.C. de Carvalho, et al., Potential carbon dioxide fixation by industrially important microalgae, Bioresour. Technol. 101 (2010) 5892–5896. [7] J.V. Moroney, Y. Ma, W.D. Frey, et al., The carbonic anhydrase isoforms of Chlamydomonas reinhardtii: intracellular location expression, and physiological roles, Photosynth. Res. 109 (2011) 133–149. [8] A.J. Brueggeman, D.S. Gangadharaiah, M.F. Cserhati, et al., Activation of the carbon concentrating mechanism by CO2 deprivation coincides with massive transcriptional restructuring in Chlamydomonas reinhardtii, Plant Cell 24 (2012) 1860–1875. [9] H. Fukuzawa, S. Fujiwara, Y. Yamamoto, et al., cDNA cloning sequence, and expression of carbonic anhydrase in Chlamydomonas reinhardtii-regulation by environmental CO2 concentration, Proc. Natl. Acad. Sci. U. S. A. 87 (1990) 4383–4387.
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[10] S. Fujiwara, H. Fukuzawa, A. Tachiki, et al., Structure and differential expression of two genes encoding carbonic anhydrase in Chlamydomonas reinhardtii, Proc. Natl. Acad. Sci. U. S. A. 87 (1990) 9779–9783. [11] M. Rawat, J.V. Moroney, Partial characterization of a new isoenzyme of carbonic anhydrase isolated from Chlamydomonas reinhardtii, J. Biol. Chem. 266 (1991) 9719–9723. [12] S. Basu, A.S. Roy, K. Mohanty, et al., CO2 biofixation and carbonic anhydrase activity in Scenedesmus obliquus SA1 cultivated in large scale open system, Bioresour. Technol. 164 (2014) 323–330. [13] J.V. Moroney, R.A. Ynalvez, Proposed carbon dioxide concentrating mechanism in Chlamydomonas reinhardtii, Eukaryot. Cell 6 (2007) 1251–1259. [14] J. Karlsson, A.K. Clarke, Z.Y. Chen, et al., A novel ␣-type carbonic anhydrase associated with the thylakoid membrane in Chlamydomonas reinhardtii is required for growth at ambient CO2 , EMBO J. 17 (1998) 1208–1216. [15] R.R. Guillard, J.H. Ryther, Studies of marine planktonic diatoms. I. Cyclotella nana Hustedt, and Detonula confervacea (cleve) Gran, Can. J. Microbiol. 8 (1962) 229–239. [16] Z. Liu, C. Liu, Y. Hou, et al., Isolation and characterization of a marine microalga for biofuel production with astaxanthin as a co-product, Energies 6 (2013) 2759–2772. [17] M.A. Sinetova, E.V. Kupriyanova, A.G. Markelova, et al., Identification and functional role of the carbonic anhydrase Cah3 in thylakoid membranes of pyrenoid of Chlamydomonas reinhardtii, Biochim. Biophys. Acta 1817 (2012) 1248–1255. [18] Z. Wen, Z. Liu, Y. Hou, et al., Ethanol induced astaxanthin accumulation and transcriptional expression of carotenogenic genes in Haematoceccus pluvialis, Enzyme Microb. Technol. 78 (2015) 10–17. [19] A. Kacka, G. Donmez, Isolation of Dunaliella spp. from a hypersaline lake and their ability to accumulate glycerol, Bioresour. Technol. 99 (2008) 8348–8352. [20] M. Mitra, C.B. Mason, Y. Xiao, et al., The carbonic anhydrase gene families of Chlamydomonas reinhardtii, Can. J. Bot. 83 (2005) 780–795. [21] D.T. Hanson, L.A. Franklin, G. Samuelsson, et al., The Chlamydomonas reinhardtii cia3 mutant lacking a thylakoid lumen-localized carbonic anhydrase is limited by CO2 supply to Rubisco and not photosystem II function in vivo, Plant Physiol. 132 (2003) 2267–2275. [22] N.L. Teakle, S.D. Tyerman, Mechanisms of Cl− transport contributing to salt tolerance, Plant Cell Environ. 33 (2010) 566–589. [23] D. Satoh, Y. Hiraoka, B. Colman, et al., Physiological and molecular biological characterization of intracellular carbonic anhydrase from the marine diatom phaeodactylum tricornutum, Plant Physiol. 126 (2001) 1459–1470. [24] M. Samukawa, C. Shen, B.M. Hopkinson, et al., Localization of putative carbonic anhydrases in the marine diatom, Thalassiosira pseudonana, Photosynth. Res. 121 (2014) 235–249. [25] B. Zhao, Y. Su, Process effect of microalgal-carbon dioxide fixation and biomass production: a review, Renew. Sustain. Energy Rev. 31 (2014) 121–132. [26] C.L. Chiang, C.M. Lee, P.C. Chen, Utilization of the cyanobacteria Anabaena sp. CH1 in biological carbon dioxide mitigation processes, Bioresour. Technol. 102 (2011) 5400–5405. [27] Z. Duan, R. Sun, C. Zhu, et al., An improved model for the calculation of CO2 solubility in aqueous solutions containing Na+ , K+ Ca2+ , Mg2+ , Cl− , and SO4 2− , Mar. Chem. 98 (2006) 131–139. [28] A. Sharma, A. Bhattacharya, A. Shrivastava, Biomimetic CO2 sequestration using purified carbonic anhydrase from indigenous bacterial strains immobilized on biopolymeric materials, Enzyme Microb. Technol. 48 (2011) 416–426. [29] Z. Chi, J.V. O‘Fallon, S. Chen, Bicarbonate produced from carbon capture for algae culture, Trends Biotechnol. 29 (2011) 537–541. [30] Z. Chi, Y. Xie, F. Elloy, et al., Bicarbonate-based integrated carbon capture and algae production system with alkalihalophilic cyanobacterium, Bioresour. Technol. 133 (2013) 513–521. [31] M.E. Russo, G. Olivieri, C. Capasso, et al., Kinetic study of a novel thermo-stable ␣-carbonic anhydrase for biomimetic CO2 capture, Enzyme Microb. Technol. 53 (2013) 271–277. [32] S.G. Venkata, M.V. Rohit, M.P. Devi, et al., Temperature-induced stress influence on biodiesel productivity during mixotrophic microalgae cultivation with wastewater, Bioresour. Technol. 169 (2014) 789–793. [33] L. Brasanti, P. Gualtieri, Algae Anatomy, Biochemistry, and Biotechnology, CRC Press, Raton, 2006. [34] M. Onay, C. Sonmez, H.A. Oktem, et al., Thermo-resistant green microalgae for effective biodiesel production: isolation and characterization of unialgal species from geothermal flora of Central Anatolia, Bioresour. Technol. 169 (2014) 62–71.
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Enzyme and Microbial Technology journal homepage: www.elsevier.com/locate/emt
CAH1 and CAH2 as key enzymes required for high bicarbonate tolerance of a novel microalga Dunaliella salina HTBS Yuyong Hou a , Zhiyong Liu a , Yue Zhao a , Shulin Chen a , Yubin Zheng b , Fangjian Chen a,∗ a Tianjin Key Laboratory for Industrial Biological Systems and Bioprocessing Engineering, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, PR China b Shandong Jinjing Biotechnology Co., Ltd., Shandong 261313, PR China
a r t i c l e
i n f o
Article history: Received 14 December 2015 Received in revised form 19 February 2016 Accepted 19 February 2016 Available online 23 February 2016 Keywords: Dunaliella salina HTBS Inorganic carbon fixation Bicarbonate Carbonic anhydrase
a b s t r a c t Outdoor microalgal cultivation with high concentration bicarbonate has been considered as a strategy for reducing contamination and improving carbon supply efficiency. The mechanism responsible for algae’s strong tolerance to high bicarbonate however, remains not clear. In this study, we isolated and characterized a strain and revealed its high bicarbonate tolerant mechanism by analyzing carbonic anhydrase (CA). The strain was identified as Dunaliella salina HTBS with broad temperature adaptability (7–30 ◦ C). The strain grew well under 30% CO2 or 70 g L−1 NaHCO3 . In comparison, two periplasm CAs (CAH1 and CAH2) were detected with immunoblotting analysis in HTBS but not in a non-HCO3 − —tolerant strain. The finding was also verified by an enzyme inhibition assay in which only HTBS showed significant inhibition by extracellular CA inhibitor. Thus, we inferred that the extracellular CAH1 and CAH2 played a multifunctional role in the toleration of high bicarbonate by HTBS. © 2016 Elsevier Inc. All rights reserved.
1. Introduction The sharp rise in atmospheric CO2 directly enforces the “greenhouse effect” and causes great concerns over global warming and climate change. With the demand for CO2 capturing technologies, the biological CO2 fixation by microalgae has received increasing attention because of its sustainability [1–4]. Previous studies focused on the effects of CO2 concentrations from 0% to 15% on the microalgal growth among different species [1,5,6]. However, efficiently supplying CO2 with a high concentration is challenging as it easily escapes from the medium due to its low solubility. Supplying the inorganic carbon in the form of HCO3 − can be more convenient than dissolving gaseous CO2 thus is considered as an alternative for culturing algae. When the pH of culture medium ranges from 6.4 to 10.3, HCO3 − is predominant; otherwise, CO2 is the dominant form at lower pH levels [7]. To cope with changes in Ci species (CO2 , HCO3 − , CO3 2− ), the carbon concentrating mechanism (CCM) was evolved to regulate carbon uptake and cell acclima-
Abbreviations: HTBS, the high tolerance of bicarbonate strain; CA, carbonic anhydrase; CAH1, carbonic anhydrase I; CAH2, carbonic anhydrase II; CAH3, carbonic anhydrase III; CCM, the carbon concentrating mechanism; AZ, acetazolamide; EZ, ethoxyzolamide; SEM, scanning electron microscopic; TEM, transmission electron microscopic; DCW, dry cell weight. ∗ Corresponding author at: 32 West 7th Avenue, Tianjin Airport Economic Area, Tianjin 300308, PR China. Fax: +86 22 24828745. E-mail address: chen [email protected] (F. Chen). http://dx.doi.org/10.1016/j.enzmictec.2016.02.010 0141-0229/© 2016 Elsevier Inc. All rights reserved.
tion [8]. Carbonic anhydrase (CA, EC 4.2.1.1) as a key part of the CCM is responsible for the reversible hydration of CO2 . Two of CAs, CAH1 and CAH2 that only differ in a few amino acids [9,10] are extremely closely related active enzymes in periplasm and can be excreted into the medium in cell-wall deficient strains of Chlamydomonas reinhardtii [11]. The function of CAH1 is conversion of HCO3 − to CO2 , and CAH2 is presumed to hydrate CO2 . The activities of the two enzymes can be inhibited by acetazolamide (AZ), which results in the decreased uptake of Ci and inhibition of the cell growth [12]. Under low-CO2 conditions, CAH1 and CAH2 tend to up-regulate and down-regulate, respectively [13]. CAH3, which is found in the thylakoid lumen, can be inhibited by the internal CA inhibitor (ethoxyzolamide; EZ). The strains that are defective in CAH3 gene cannot use Ci efficiently even when its concentration is high, but carry out photosynthesis normally when the CAH3 is put back into these strains [13,14]. Although the effects of various CO2 concentrations on the expression and function of CAs have been intensively studied [7,9,10,13], the report on the high bicarbonate response mechanism in HCO3 − –tolerant microalgae is very limited. Fukuzawa et al. [9] reported that CAH1 is expressed only under low CO2 conditions at the transcript levels, no accumulation of CAH2 mRNA is observed in the dark by Northern blot analysis. Nevertheless, the work from Fujiwara et al. [10] showed the expression of CAH2 mRNA under low-CO2 condition, and CAH2 mRNA could be translated into enzymatically active CA polypeptides. In addition, Moroney et al. [7] had summarized the expression and function of 12 genes that encode
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CA isoforms in C. reinhardtii at low and elevated CO2 conditions using multiple technologies. However, it was still unclear whether there was a relationship between algal bicarbonate-tolerance and CAs. The main objective of this study was to isolate and characterize a novel high HCO3 − —tolerant microalgae and dissect the molecular mechanism of the relationship between bicarbonatetolerance and CAs. Firstly, a novel microalga HTBS that can tolerate and utilize high-bicarbonate for biomass production was isolated and identified. Then, the expression profile of the CAs was evaluated by western blot analysis. Subsequently, to further identify the location of carbonic anhydrase, two specific CA inhibitors on the growth of HTBS, AZ and EZ were applied. Finally, the effects of CO2 and NaHCO3 concentrations and temperature on the growth of HTBS were performed. These findings provide further insights into the multifunction of CAs in response to high level of HCO3 − and potential benefit to large-scale application of Ci fixation and comprehensive utilization in microalgae cultivation.
isolate. Nannochloropsis oculata (CCAP 849/1), obtained from the Culture Collection of Algae and Protozoa at the Scottish Marine Institute (Oban, Argyll, U.K.), was the used as the control strain (non-HCO3 − —tolerant strain). All seed cultures were maintained aerobically in 250-mL Erlenmeyer flasks with 100 mL of f/2 medium under continuous artificial illumination at 75 mol m−2 s−1 . The temperature and pH were maintained at 25 ◦ C and 7.5, respectively.
2. Material and methods
2.3. Molecular identification
2.1. Strains and culture condition
Firstly, genomic DNA was extracted on the basis of the Fast-Prep DNA purification method [16]. Two universal green algal primers, 18SF (forward, 5 -CCTGGTTGATCCTGCCAG-3 ) and 18SR (reverse, 5 -TTGATCCTTCTGCAGGTTCA-3 ), were then used for PCR amplification of the 18S rDNA by the method of Liu et al. [16]. NCBI BLAST (http://www.ncbi.nlm.nih.gov) was used to study the homology of the partial 18s rDNA gene sequence. The neighbor-joining method in MEGA5 was used to construct the phylogenetic tree [16].
The high HCO3 − —tolerant strain HTBS used in this study was isolated from seawater in the Bohai Gulf (Tianjin, China, 117.82◦ E, 39.01◦ N). The strain was detected in the water samples after treating with 50 g L−1 NaHCO3 . Then the NaHCO3 treated cells were spread onto f/2 agar plates and the single colony was subcultured to the f/2 medium [15]. Optical microscope was used to check the
2.2. Electron microscopy analysis The cells were harvested at day 4 during the logarithmic phase, then scanning electron microscopic (SEM) and transmission electron microscopic (TEM) studies were performed to observe the cell morphology and structure according to Liu et al. [16] with a SEM (Quanta200; FEI, Hillsboro, Oregon) and TEM (JEM-1400; JEOL, Tokyo, Japan), respectively.
Fig. 1. Microscopic and morphological appearance of strain HTBS. Scanning (A and B) and transmission (C and D) electron micrographs of the cell in f/2 medium. C, the cells cultured under the protection of N2 ; D, the cells treated by HCO3 − under the protection of N2 . ST, starch grain; CHL, chloroplast. The scale bar is indicated at the bottom of each figure.
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Fig. 2. Phylogenetic tree inferred from 18S rDNA sequences. The distance tree was constructed with the neighbor-joining method based on Kimura’s correction using CLUSTAL W. The horizontal lengths are proportional to the evolutionary distances. The numbers above or below the internal nodes indicate bootstrap values (1000 replicates).
2.4. Studies under varying culture conditions The effects of different CO2 concentrations and temperatures on the growth of HTBS were evaluated in glass columns (4.1 cm in diameter, 37 cm in height, a working volume of 300 mL) with f/2 and modified BG11 (seawater BG11) medium, respectively. CO2 concentrations ranged from 2% to 30% in air (v/v) were filtered and bubbled into the medium continuously at the bottom of the columns. The temperature variation studies were performed at 7 ◦ C, 15 ◦ C, 25 ◦ C, and 30 ◦ C with bubbled filtered air. Aeration flow for each column was 0.42 L min−1 , the bubble size was about 0.5 cm in diameter. NaHCO3 ranged from 0 g L−1 to 70 g L−1 with 150 mL working volume (f/2 medium) in 250-mL Erlenmeyer flasks were used to investigate the effects on HTBS, while 25 g L−1 NaHCO3 was used in the growth of N. oculata. The pH were monitored by a pH meter. NaCl was used to replace the NaHCO3 at the same concentrations to rule out the effects of Na+ on HTBS. Finally, to study the effects of CA inhibitors on HTBS and N. oculata, 150 M AZ and EZ were utilized with the same conditions. The flasks were manually shaken three times a day. All the experiments were performed with triplicates under continuous illumination at 160 ± 20 mol m−2 s−1 at 25 ◦ C except the study on the temperature effect. 2.5. Electrophoresis and Immunodetection To eliminate the influence of CO2 from the air, the algae cultured in columns were aerated with N2 . Isolation of cell fractions, protein electrophoresis and western blotting were then performed according to Sinetova et al. [17]. In particular, the membrane protein CAH3 was enriched in pellet after centrifugation at 12,000 × g for 10 min at 4 ◦ C, and the supernatant contained CAH1 and CAH2. The primary antibodies of CAH1 (sc-134853), CAH2 (sc-25596) and CAH3 (sc-99005) were obtained from Santa Cruz Biotechnology, Inc. 2.6. Determination of cell growth The cell concentration of the culture was obtained by measuring its optical density with a spectrometer at 750 nm (OD750 ). The dry
cell weight (DCW) was calculated according to Wen et al. [18]. In particular, the NaHCO3 treated cells were washed by f/2 medium with 50 mM HCl before they were used. The specific growth rate (day−1 ) was calculated as the equation: = ln (X1 /X0 )/(t1 − t0 ). Where X1 and X0 were the OD750 on days t1 and t0 , respectively. 3. Results and discussion 3.1. Isolation and identification of microalgae The isolated strain HTBS was unicellular and ellipsoidal, and the cell size was homogeneous (approximately 5 m in diameter). Two long flagella and a cell covering of salt crystals were observed with SEM (Fig. 1A and B), similar to the observations in a previous report for Dunaliella sp. [19]. Compared to the cell without bicarbonate (Fig. 1C), the more discernible starch grain and chloroplast occupied the space of the treated cells with HCO3 − (Fig. 1D). The results suggested that the more chloroplast in HCO3 − treated cells enhanced photosynthesis to uptake more Ci for growth than control groups. On the basis of 18S rDNA sequence BLAST searches in the GeneBank database, the nucleotide sequences of 15 species that included the available 18S rDNA and 5.8S rDNA gene sequences were used to construct the phylogenetic tree. The neighbor-joining tree revealed a major split between HTBS and Dunaliella sp. SAS11133 with 87% similarity (Fig. 2). HTBS fell within the evolutionary radiation occupied by the genus Dunaliella salina and was closely related to Dunaliella sp. strains SAS11133 (87%), OUC (88%), ASK-21 (90%), and 1 B2-2 (94%). Thus, the strain was named as D. salina HTBS. 3.2. Immunoblotting analysis of CAs To investigate the tolerant mechanism of high Ci at the expression level of CAs, an immunoblotting analysis was performed. The results showed that CAH1 was translated in HTBS in both 0 M and 0.2 M NaHCO3 groups. However, CAH1 was not detected in non-HCO3 − —tolerant algae N. oculata at either low or high HCO3 − concentrations (Fig. 3A). CAH1 has been shown to be strongly
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Fig. 3. Western blotting detection of ␣-CAs in HTBS and N. oculata cells. (A) Immunoblotting analyses of samples with CAH1-specific antibodies and (B) CAH2and (C) CAH3-specific antibodies. “−” and “+” indicate non-HCO3 − –treated and HCO3 − –treated groups, respectively.
expressed in a low-CO2 group and was far more abundant than that in a high-CO2 group [20]. The expression of CAH1 at a low HCO3 − level is lower than that under high-HCO3 − conditions, specifically at 24 h. This finding implied that CAH1 in the HCO3 − —treated group would be up-regulated to hydrolyze HCO3 − . CAH2 was regarded as the only enzyme that was expressed under high-CO2 conditions [10], it did not translate in N. oculata, but did translate in HTBS
under both conditions (Fig. 3B). It was presumed that CAH2 in the 0 g L−1 HCO3 − —treated group was over-expressed to address the extreme environments by fixing the CO2 to HCO3 − to prevent the leakage of CO2 from the cells. CAH3, the enzyme that played an important role in Ci absorption, converted HCO3 − to CO2 and delivered CO2 to Rubisco [21]. It was detected in both strains under both culture conditions (Fig. 3C). It seemed that the rapid growth of the HCO3 − —treated cells was related to the expression of CAs. In contrast, a high Cl− concentration could be toxic to plant cells, with critical toxicity at 15–50 mg g−1 dry weight for a Cl− —tolerant species [22]. CAH1 could not alleviate the pressure formed by Cl− when the NaHCO3 was replaced by NaCl, which finally limited the cell growth. And the other results also showed that HTBS almost did not proliferate at 25 g L−1 NaCl and died at both 50 and 70 g L−1 NaCl (Fig. 4C). It appears that the presence of CAH1 and CAH2 helps to alleviate the pressure of the high bicarbonate level and creates a suitable environment for the growth. Meanwhile, the reaction catalyzed by CAs can supply enough Ci for the cells to grow. From another point of view, the fact that CAH1 and CAH2 were required for the algae to tolerate bicarbonate could be confirmed by the fact that CAH1 and CAH2 were not translated in N. oculata (Fig. 3A and B) and that the cells in 25 g L−1 NaHCO3 tended to die (Fig. 4B). With maximum turnover numbers in excess of 106 s−1 for reversible reaction catalyzed by CAs [7], once the concentration of CO2 is sharply increased after the dehydration reactions catalyzed by CAH1, CAH2 immediately acts to reduce the
Fig. 4. Effects of different concentrations of initial NaHCO3 (A) and NaCl (C) for HTBS growth. Effects of initial NaHCO3 concentration (25 g L−1 ) on HTBS and N. oculata (B) were also studied. Finally, (D) shows the dry cell weight of HTBS in growth medium for different concentrations of NaHCO3 after culture for 6 days. Data values are means (±SE) of three replicate cultures.
Table 1 The effect of AZ and EZ for HTBS and N. oculata. HTBS
Control AZ acetazolamide EZ ethoxyzolamide
N. oculata
Cell density (× 105 cell/mL)
Inhibition (%)
Cell density (× 107 cell/mL)
Inhibition
4.25 ± 0.35 3.10 ± 0.14 1.25 ± 0.21
0 26.95 ± 2.76 70.70 ± 2.55
1.1 ± 0.03 1.00 ± 0.07 0.87 ± 0.03
0 7.25 ± 3.18 18.60 ± 0.57
(%)
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Fig. 5. Growth curves of HTBS in growth medium for different concentrations of CO2 . Data values are means (±SE) of three replicate cultures.
CO2 level through hydration reaction. It seems that the presumed role of CAH1 and CAH2 is to maintain the CO2 /HCO3 − equilibrium for cell acclimation. The results suggested that extracellular CAH1 and CAH2 played an important role in the growth of HTBS under high bicarbonate stress. 3.3. Effect of enzyme inhibitors on HTBS and N. oculata growth As inhibitors for CA, AZ (acetazolamide) and EZ (ethoxyzolamide) were used to confirm the presence of periplasmic and intracellular CA [23]. Our results showed that the internal CA inhibitor (EZ) inhibited the growth of HTBS and N. oculata by 70.70% and 18.60%, respectively (Table 1). In addition, HTBS was significantly suppressed by AZ and the cell density decreased by 26.95%. For N. oculata, a slight difference of the growth was observed for the untreated group and the AZ group. The data reflected the presence of CAH1, CAH2, and CAH3 in HTBS and indicated that the cells of N. oculata were deficient in external CAH1 and CAH2, which was consistent with the immunoblotting results (Fig. 3A and B). As a result, without the translation of CAH1 and CAH2 in N. oculata, the HCO3 − —treated cells were unable to grow due to the lack of the ability for Ci processing (Fig. 4B). Similarly, Phaeodactylum tricornutum lacked extracellular CAs, whose photosynthesis was not negatively affected by AZ [22], and had less capacity and flexibility to take up Ci than Thalassiosira pseudonana, which contained extracellular CAs [24]. It thus suggested that CAH1 and CAH2 are required for the tolerance of bicarbonate in the novel microalga HTBS. 3.4. Effects of CO2 and NaHCO3 concentrations on HTBS As shown in Figs. 4 A and 5 , the strain could grow very well with both high bicarbonate and CO2 concentrations. The growth at 6% and 30% CO2 both reached the highest level at day 5, which
were 14.93% and 2.60% higher than that of 2% CO2 at day 5 (1.54 ± 0.03 g L−1 ), respectively. Compared to the DCW of 2% CO2 , that of 6% and 30% were lower before day 3 and day 5, respectively. It was speculated that the acidification caused by the high concentration CO2 affected the growth. With the increasing of cell biomass, the influence of acidification gradually weakened, finally these results had no significant difference. The biomass productivity reached 84 ± 2 mg L−1 day−1 with 30% CO2 . Most species grow only at low CO2 concentrations and their growth is inhibited when the CO2 level is higher than 5.0% [25]. Although, there are some strains with the capability to tolerate abnormal CO2 concentrations. Chiang et al. [26] reported that a strain Anabaena sp. CH1 isolated from central Taiwan demonstrated excellent CO2 tolerance in an environment with 15.0% CO2 . Scenedesmus obliquus and Chlorella kessleri were isolated from the waste-treatment ponds of a coal-fired thermoelectric power plant at different CO2 concentrations and grew well when the CO2 concentrations up to 18.0% (V/V) [5]. HTBS showed much higher CO2 tolerance and was regarded as a candidate for developing CO2 fixation technique for the concentrated sources like industrial flue gas (15% CO2 ). However, the low solubility of CO2 in water (only 0.81 mol/kg at 0 ◦ C and atmospheric pressure) results in the escape of a large amount of CO2 [27]. As an alternative, HCO3 − was more preferred for culturing microalgae. In this study, Four HCO3 − levels from 0 to 70 g L−1 were used to measure the effects on HTBS. The pH was increased with the microalgal growth throughout the culture period and was much higher than 6.4 (Fig. S1). According to pKa , the main form of Ci in the medium was HCO3 − [13]. HCO3 − and CO2 were taken into the cell for growth, leaving OH− in the medium, which resulted in an increase in the pH of the medium and intracellular HCO3 − accumulation. The HCO3 − was then converted to CO2 for Rubisco utilization by CAH3 [21].The growth of 25 g L−1 and 50 g L−1 NaHCO3 cultures reached the highest level at day 4, the
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Fig. 6. Growth curves of HTBS and N. oculata in f/2 medium at different temperatures. (A) 7 ◦ C, (B) 15 ◦ C, (C) 25 ◦ C, (D) 30 ◦ C. Data values are means (±SE) of three replicate cultures.
OD750 values of the strain were 0.42 ± 0.02 and 0.40 ± 0.02, which were significantly higher than those in the control group, increasing 2.23-fold and 2.08-fold, respectively (Fig. 4A). The growth rate of HTBS for the first 3 days was decreased from 1.24 day−1 of 25 g L−1 to 1.09 day−1 of 70 g L−1 . Although HTBS did exhibit a decrease in its growth rate at 70 g L−1 compared to 25 g L−1 and 50 g L−1 , the strain still grew very well, the cell density (OD750 = 0.35 ± 0.02) was 1.69fold higher than that of the control group at day 4. The highest DCW of HTBS was 0.46 g L−1 when grown in the medium with 25 g L−1 NaHCO3 , which was only 2.2% and 4.5% higher than that of cultures supplemented with 50 g L−1 and 70 g L−1 , respectively, whereas the biomass in the medium without NaHCO3 was only 0.26 g L−1 (Fig. 4D). The biomass productivity was 73 ± 12 mg L−1 day−1 with 70 g L−1 NaHCO3. Compared to HTBS, the strain N. oculata could not even grow in f/2 medium with 25 g L−1 NaHCO3 , the death of cells was observed after 24 h (Fig. 4B). Most microalgae can capture CO2 , but there are a series of technical challenges to overcome to achieve economical and efficient CO2 supplementation, such as the high cost of compression for transportation, the ease of loss into the atmosphere, and the difficulty of storage and capture. Although purified CA was reported for CO2 sequestration [28], it was a complex and costly system. As an alternative approach to address these problems, converting CO2 to HCO3 − through CO3 2− as a carbon source for algae growth was suggested [29,30]. Indeed, extensive CO2 capture through absorption in the aqueous phase is feasible with alkaline solutions [31]. However, the cells should possess a high ion strength to cope with the high level of HCO3 − in the medium. Unfortunately, a high level of HCO3 − was not suitable for culture of many algae species. Thus, the first step in the validation of the idea is to find a species that can grow in the extreme condition with high concentration of bicarbonate. In this study, the novel strain HTBS showed the potential for high HCO3 − tolerance.
To further eliminate the effects of Na+ on the bicarbonate tolerance of HTBS, we carried out sodium chloride treatment experiments in which NaHCO3 was replaced with NaCl (Fig. 4C). In the experimental design, the same molarity of NaCl and NaHCO3 will lead to the inconsistent salinities. Comparing to relevant NaHCO3 treatments, the lower salinity of NaCl groups can affect the analysis of the various treatment. Thus, the same mass concentration of NaCl and NaHCO3 were adopted to keep the salinity consistent, then the effect of Na+ was the main concern. Considering Na+ gradient test, we have designed NaHCO3 0.3–0.6 M (25–50 g L−1 ) on the sides of 0.4 M (NaCl 25 g L−1 ) at the same time. The results showed that the cells barely survived when the NaCl level reached only 25 g L−1 , their cell growth decreased even more significantly as the NaCl level increased. HTBS could not survive in an equivalent mineral salt such as sodium chloride. Although the Na+ concentrations of 50 g L−1 and 70 g L−1 NaHCO3 were much higher than those in the group treated with 25 g L−1 NaCl, the strain grew well (Fig. 4A). This finding suggested that Na+ had almost no influence on the growth of HTBS under equivalent HCO3 − and that the growth could be influenced by anion Cl− , it was consistent with previous report [22]. Compared to Cl− , HCO3 − as one of the main Ci species, could be regulated by CCM for efficient use, resulting in cell acclimation and fast growth. Our results also showed that the marine algae N. oculata was non-HCO3 − —tolerant (Fig. 4B).
3.5. Effects of temperature on HTBS For further assessment of the features of HTBS, we studied the effects of four different temperatures on cell growth. The algae grew well at both low (Fig. 6A and B) and high temperatures (Fig. 6C and D). The optical density (OD750 ) of the cells was 1.67 under a culture temperature of 7 ◦ C on day 12. In contrast, the growth of the control algae N. oculata was inhibited. A similar result was observed at 15 ◦ C. For high temperatures, the OD750 of HTBS could reach 2.96 and 2.03 at 25 ◦ C and 30 ◦ C, respectively. As one of the most impor-
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tant factors in microalgae culture conditions, temperature has a great effect on the uptake of nutrients, the fixation of Ci, and the growth rate. Low temperature leads to low intracellular enzyme activity and is responsible for the decreased use of Ci and other nutrients [32]. Most species cannot survive in cold weather (usually below 16 ◦ C) [33,34]. However, screening microalgal strain with the capability to grow at low temperature is meaningful for outdoor cultures, especially in the northern regions. In this study, the wide growth temperature range made HTBS as a promising candidate for commercial application. 4. Conclusions The microalgal strain HTBS isolated from a biodiverse body of seawater, the Bohai Gulf, showed tolerance to 30% CO2 , 70 g L−1 NaHCO3 and a low temperature of 7 ◦ C. The total biomass productivity reached 84 and 73 mg L−1 day−1 with 30% CO2 and 70 g L−1 NaHCO3 , respectively. CAH1 and CAH2 were required to function under the condition of a high concentration of bicarbonate as part of the mechanism for the algae to tolerate high level Ci. Acknowledgements This work was supported partially by the National Natural Science Foundation of China (Grant Nos. 31200093 and 31570047), the Hi-Tech Research and Development Program (863) of China (2014AA022003), the Major State Basic Research Development Program of China (973 Project) (Grant Nos. 2011CB200905 and 2011CB200906), and the Key Program for International S&T Cooperation Projects of China (2014DFA61040). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.enzmictec.2016. 02.010. References [1] S. Basu, A.S. Roy, K. Mohanty, et al., Enhanced CO2 sequestration by a novel microalga: Scenedesmus obliquus SA1 isolated from bio-diversity hotspot region of Assam, India, Bioresour. Technol. 143 (2013) 369–377. [2] Y. Chisti, Biodiesel from microalgae, Biotechnol. Adv. 25 (2007) 294–306. [3] A. Kumar, S. Ergas, X. Yuan, et al., Enhanced CO2 fixation and biofuel production via microalgae: recent developments and future directions, Trends Biotechnol. 28 (2010) 371–380. [4] M. Ota, Y. Kato, H. Watanabe, et al., Fatty acid production from a highly CO2 tolerant alga, Chlorocuccum littorale, in the presence of inorganic carbon and nitrate, Bioresour. Technol. 100 (2009) 5237–5242. [5] M.G. de Morais, J.A.V. Costa, Isolation and selection of microalgae from coal fired thermoelectric power plant for biofixation of carbon dioxide, Energy Convers. Manage. 48 (2007) 2169–2173. [6] E.B. Sydney, W. Sturm, J.C. de Carvalho, et al., Potential carbon dioxide fixation by industrially important microalgae, Bioresour. Technol. 101 (2010) 5892–5896. [7] J.V. Moroney, Y. Ma, W.D. Frey, et al., The carbonic anhydrase isoforms of Chlamydomonas reinhardtii: intracellular location expression, and physiological roles, Photosynth. Res. 109 (2011) 133–149. [8] A.J. Brueggeman, D.S. Gangadharaiah, M.F. Cserhati, et al., Activation of the carbon concentrating mechanism by CO2 deprivation coincides with massive transcriptional restructuring in Chlamydomonas reinhardtii, Plant Cell 24 (2012) 1860–1875. [9] H. Fukuzawa, S. Fujiwara, Y. Yamamoto, et al., cDNA cloning sequence, and expression of carbonic anhydrase in Chlamydomonas reinhardtii-regulation by environmental CO2 concentration, Proc. Natl. Acad. Sci. U. S. A. 87 (1990) 4383–4387.
23
[10] S. Fujiwara, H. Fukuzawa, A. Tachiki, et al., Structure and differential expression of two genes encoding carbonic anhydrase in Chlamydomonas reinhardtii, Proc. Natl. Acad. Sci. U. S. A. 87 (1990) 9779–9783. [11] M. Rawat, J.V. Moroney, Partial characterization of a new isoenzyme of carbonic anhydrase isolated from Chlamydomonas reinhardtii, J. Biol. Chem. 266 (1991) 9719–9723. [12] S. Basu, A.S. Roy, K. Mohanty, et al., CO2 biofixation and carbonic anhydrase activity in Scenedesmus obliquus SA1 cultivated in large scale open system, Bioresour. Technol. 164 (2014) 323–330. [13] J.V. Moroney, R.A. Ynalvez, Proposed carbon dioxide concentrating mechanism in Chlamydomonas reinhardtii, Eukaryot. Cell 6 (2007) 1251–1259. [14] J. Karlsson, A.K. Clarke, Z.Y. Chen, et al., A novel ␣-type carbonic anhydrase associated with the thylakoid membrane in Chlamydomonas reinhardtii is required for growth at ambient CO2 , EMBO J. 17 (1998) 1208–1216. [15] R.R. Guillard, J.H. Ryther, Studies of marine planktonic diatoms. I. Cyclotella nana Hustedt, and Detonula confervacea (cleve) Gran, Can. J. Microbiol. 8 (1962) 229–239. [16] Z. Liu, C. Liu, Y. Hou, et al., Isolation and characterization of a marine microalga for biofuel production with astaxanthin as a co-product, Energies 6 (2013) 2759–2772. [17] M.A. Sinetova, E.V. Kupriyanova, A.G. Markelova, et al., Identification and functional role of the carbonic anhydrase Cah3 in thylakoid membranes of pyrenoid of Chlamydomonas reinhardtii, Biochim. Biophys. Acta 1817 (2012) 1248–1255. [18] Z. Wen, Z. Liu, Y. Hou, et al., Ethanol induced astaxanthin accumulation and transcriptional expression of carotenogenic genes in Haematoceccus pluvialis, Enzyme Microb. Technol. 78 (2015) 10–17. [19] A. Kacka, G. Donmez, Isolation of Dunaliella spp. from a hypersaline lake and their ability to accumulate glycerol, Bioresour. Technol. 99 (2008) 8348–8352. [20] M. Mitra, C.B. Mason, Y. Xiao, et al., The carbonic anhydrase gene families of Chlamydomonas reinhardtii, Can. J. Bot. 83 (2005) 780–795. [21] D.T. Hanson, L.A. Franklin, G. Samuelsson, et al., The Chlamydomonas reinhardtii cia3 mutant lacking a thylakoid lumen-localized carbonic anhydrase is limited by CO2 supply to Rubisco and not photosystem II function in vivo, Plant Physiol. 132 (2003) 2267–2275. [22] N.L. Teakle, S.D. Tyerman, Mechanisms of Cl− transport contributing to salt tolerance, Plant Cell Environ. 33 (2010) 566–589. [23] D. Satoh, Y. Hiraoka, B. Colman, et al., Physiological and molecular biological characterization of intracellular carbonic anhydrase from the marine diatom phaeodactylum tricornutum, Plant Physiol. 126 (2001) 1459–1470. [24] M. Samukawa, C. Shen, B.M. Hopkinson, et al., Localization of putative carbonic anhydrases in the marine diatom, Thalassiosira pseudonana, Photosynth. Res. 121 (2014) 235–249. [25] B. Zhao, Y. Su, Process effect of microalgal-carbon dioxide fixation and biomass production: a review, Renew. Sustain. Energy Rev. 31 (2014) 121–132. [26] C.L. Chiang, C.M. Lee, P.C. Chen, Utilization of the cyanobacteria Anabaena sp. CH1 in biological carbon dioxide mitigation processes, Bioresour. Technol. 102 (2011) 5400–5405. [27] Z. Duan, R. Sun, C. Zhu, et al., An improved model for the calculation of CO2 solubility in aqueous solutions containing Na+ , K+ Ca2+ , Mg2+ , Cl− , and SO4 2− , Mar. Chem. 98 (2006) 131–139. [28] A. Sharma, A. Bhattacharya, A. Shrivastava, Biomimetic CO2 sequestration using purified carbonic anhydrase from indigenous bacterial strains immobilized on biopolymeric materials, Enzyme Microb. Technol. 48 (2011) 416–426. [29] Z. Chi, J.V. O‘Fallon, S. Chen, Bicarbonate produced from carbon capture for algae culture, Trends Biotechnol. 29 (2011) 537–541. [30] Z. Chi, Y. Xie, F. Elloy, et al., Bicarbonate-based integrated carbon capture and algae production system with alkalihalophilic cyanobacterium, Bioresour. Technol. 133 (2013) 513–521. [31] M.E. Russo, G. Olivieri, C. Capasso, et al., Kinetic study of a novel thermo-stable ␣-carbonic anhydrase for biomimetic CO2 capture, Enzyme Microb. Technol. 53 (2013) 271–277. [32] S.G. Venkata, M.V. Rohit, M.P. Devi, et al., Temperature-induced stress influence on biodiesel productivity during mixotrophic microalgae cultivation with wastewater, Bioresour. Technol. 169 (2014) 789–793. [33] L. Brasanti, P. Gualtieri, Algae Anatomy, Biochemistry, and Biotechnology, CRC Press, Raton, 2006. [34] M. Onay, C. Sonmez, H.A. Oktem, et al., Thermo-resistant green microalgae for effective biodiesel production: isolation and characterization of unialgal species from geothermal flora of Central Anatolia, Bioresour. Technol. 169 (2014) 62–71.