CAH1 and CAH2 as key enzymes required for high bicarbonate tolerance of a novel microalga Dunaliella salina HTBS

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Highlights

  • An extremely alkalihalophilic microalgae Dunaliella salina HTBS was isolated.

  • The presence of extracellular CAs (CAH1 and CAH2) in HTBS was confirmed.

  • CAH1 and CAH2 played a multifunctional role for tolerating high HCO3 concentration.

  • HTBS featured with tolerance of 70 g L−1 NaHCO3, 30% CO2 and low temperature (7 °C)

Abstract

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.

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], [2], [3], [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, CO32−), the carbon concentrating mechanism (CCM) was evolved to regulate carbon uptake and cell acclimation [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 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 bicarbonate-tolerance 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.

Section snippets

Strains and culture condition

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 isolate. Nannochloropsis oculata (CCAP 849/1), obtained from the Culture Collection of Algae and Protozoa at the

Isolation and identification of microalgae

The isolated strain HTBS was unicellular and ellipsoidal, and the cell size was homogeneous (approximately 5 μm 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

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).

References (34)

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