A Stepwise NaHSO3 Addition Mode Greatly Improves H2 Photoproduction in Chlamydomonas reinhardtii

NaHSO3 addition greatly increases the yield of H2 photoproduction in a unicellular green alga Chlamydomonas reinhardtii through removing O2 and activating hydrogenase but significantly impairs the activity of PSII, an electron source for H2 photoproduction. Here, a stepwise addition mode of total 13 mM NaHSO3, an optimal concentration for H2 photoproduction of C. reinhardtii identified in a previous one step addition method, significantly improved H2 photoproduction. Such improvement was believed to be the result of increased residual PSII activity in an anaerobic background, but was at least independent of two alternative electron sinks for H2 photoproduction, cyclic electron transport around PSI and CO2 assimilation. Based on the above results, we propose that increased residual PSII activity in an anaerobic environment is an efficient strategy to enhance H2 photoproduction in C. reinhardtii, and the stepwise NaHSO3 addition mode is a case study in the strategy.


INTRODUCTION
With the increasing awareness of fossil fuel depletion and global warming, efforts have been undertaken to develop clean and sustainable energy sources (McKendry, 2002). Molecular hydrogen (H 2 ) is one of the potential future energy sources (Hansel and Lindblad, 1998;Momirlan and Veziroglu, 2002). C. reinhardtii, a unicellular green alga, has been recognized as an ideal system for sustainable H 2 photoproduction under anaerobic conditions; however, this alga cannot efficiently and continuously produce H 2 in an aerobic environment because its H 2 ase is extremely sensitive to O 2 (Ghirardi et al., 1997). To activate H 2 ase for sustainable and efficient H 2 photoproduction in C. reinhardtii, therefore, numerous strategies have been extensively developed mainly through engineering O 2 tolerance in H 2 ase (Flynn et al., 2002;Liebgott et al., 2011;Wu et al., 2011) or decreasing O 2 content around H 2 ase (Melis et al., 2000;Kruse et al., 2005;Surzycki et al., 2007;Wu et al., 2010;Xu et al., 2011;Jurado-Oller et al., 2015;Xiong et al., 2015;Shu et al., 2018).
Meanwhile, our studies demonstrate that NaHSO 3 addition strategy is capable of decreased the O 2 content around H 2 ase, thereby activating the enzyme activity and promoting H 2 photoproduction Ma et al., 2011;Wei et al., 2017). This strategy can result in an approximately 10-fold or 200-fold increase in H 2 photoproduction in the nitrogen-fixing cyanobacterium Anabaena sp. strain PCC 7120  or the unicellular green alga C. reinhardtii Wei et al., 2017), respectively. Despite these increases, this yield is still not sufficient to meet the requirements of industrial applications. Thus, extensive optimization of this NaHSO 3 addition strategy is necessary to increase H 2 photoproduction in C. reinhardtii further.
The yield of H 2 photoproduction caused by sulfur deprivation is also not sufficient to meet the requirements of industrial applications. Under sulfur deprivation conditions, therefore, many strategies have been developed to improve the yield of H 2 photoproduction in C. reinhardtii via metabolic and genetic engineering (for review, see Esquível et al., 2011;Dubini and Ghirardi, 2015). Among them, increased residual photosystem II (PSII) activity was found to play a vital role in efficient H 2 photoproduction (Zhang et al., 2002;Kosourov et al., 2005;Kim et al., 2010;Volgusheva et al., 2013;Grewe et al., 2014;Steinbeck et al., 2015;Chen et al., 2016), since the PSII activity is significantly impaired by sulfur deprivation (Melis et al., 2000). Similarly, in the NaHSO 3 addition background, the PSII activity is also significantly impaired . To test whether increased residual PSII activity in the NaHSO 3 addition background can also enhance H 2 photoproduction, we monitored the accumulated H 2 level and residual PSII activity in the stepwise mode of total 13 mM NaHSO 3 , an optimal concentration for H 2 production of C. reinhardtii identified in a previous one step addition method . We also measured the content of dissolved O 2 and activities of two alternative electron sinks for H 2 photoproduction, CET and CO 2 assimilation, in the stepwise NaHSO 3 addition mode. Our results demonstrate that the stepwise NaHSO 3 addition mode evidently enhances the yield of H 2 photoproduction in C. reinhardtii; such enhancement is mostly the result of increased residual PSII activity in an anaerobic environment, but is at least independent of two alternative electron sinks for H 2 photoproduction.

Sample Preparation and NaHSO 3 Addition
Cells of C. reinhardtii were continuously illuminated by growth light of 40 µmol photons m −2 s −1 and were cultured in 0.5 L of TAP medium for 2 days with bubble aeration (A 750 = 0.8-1.0), after which a fixed volume of cells containing 300 µg of Chl was transferred to 60 mL serum bottles (30 mL head space and 30 mL cells) with rubber seals. After cells were statically pre-cultured under continuous illumination of 200 µmol photons m −2 s −1 for 36 h, total 13 mM of NaHSO 3 was directly or step by step added to the serum bottles, as indicated in Figures 1, 2, Supplementary Figure S1, and described in Table 1, with Lin of 5 mM (final concentration) or AA of 10 µM (final concentration) or GA of 2 mM (final concentration) or DCMU of 20 µM (final concentration) or not. Subsequently, the FIGURE 1 | A stepwise NaHSO 3 addition mode significantly increases (A) H 2 photoproduction, and (B) in vivo and (C) in vitro H 2 ase activity in C. reinhardtii. After cells were statically pre-cultured under continuous illumination of 200 µmol photons m −2 s −1 for 36 h, total 13 mM of NaHSO 3 was directly or step by step added to the serum bottles, as indicated by arrows 1-3 and described in Table 1. Values are means ± SD (n = 5).  cells were still illuminated at 200 µmol photons m −2 s −1 or were incubated in the dark to induce the production of H 2 .

Monitoring H 2 Photoproduction
At predetermined time intervals, 200 µL of gas samples were withdrawn from the bottles using a gas-tight syringe and injected into a gas chromatograph (Agilent 7890A; Agilent Technologies Inc., United States) with a thermal conductivity detector for determining the concentrations of H 2 , O 2 , and N 2 simultaneously. The column of the gas chromatograph was a molecular sieve column (type 5Å; 2 m × 1/8 mm), and argon was used as the carrier gas.

H 2 ase Activity Assay
In vivo and in vitro H 2 ase activity was monitored as described earlier Wei et al., 2013Wei et al., , 2017 with some modifications. In brief, 1 mL cell suspension samples upon exposure to 200 µmol photons m −2 s −1 were anaerobically withdrawn from the 60 mL serum bottles at designated time points (see Figures 1B,C) and then injected into 10 mL glass vials. To measure in vivo H 2 ase activity, the cell suspension samples were immediately purged with argon gas for 1 min to eliminate the inhibitory effect of O 2 on the H 2 ase activity. The cell suspension samples were then placed in a 25 • C water bath for 1 h and shaken continuously (150 rpm) whilst exposed to a constant light of 200 µmol photons m −2 s −1 . To measure in vitro H 2 ase activity, we used vials containing 1 mL of 10 mM oxidized MV prepared in O 2 -free 50 mM Tris buffer (for pH 7.1-9.0) and 0.2% (w/v) Triton X-100. The reaction was started when MV was reduced by the addition of 100 µL of 100 mM anaerobic sodium dithionite in 0.03 N NaOH. This assay was performed at 37 • C in the dark for 20 min. We determined the amount of H 2 produced in the headspace of the glass vial by gas chromatography, and the rate of H 2 production was calculated on the basis of the total Chl content in the glass vial, unless otherwise indicated.

Dissolved Oxygen Measurement
Dissolved oxygen was monitored as described earlier (Wei et al., 2017). In brief, a DO meter (Orion Star A213, Thermo Scientific, Untied States) was used to monitor the DO attenuation process after the addition of NaHSO 3 to the cell suspension cultures of C. reinhardtii. The DO meter was corrected before each measurement. The DO meter probe was placed in the middle of the cell suspension cultures and the data were recorded at several designated time points.

Chl Fluorescence and P700 Analysis
The yields of Chl fluorescence at a steady-state of electron transport were measured at room temperature using a Dual-PAM-100 monitoring system (Walz, Effeltrich, Germany) equipped with an ED-101US/MD unit (Schreiber et al., 1986;Ma et al., 2008;Wei et al., 2013Wei et al., , 2017. Minimal fluorescence at open PSII centers in the dark-adapted state (F o ) was excited by a weak measuring light (650 nm) at a photon flux density of 0.05-0.15 µmol photons m −2 s −1 . A saturating pulse of red light (600 ms, 10,000 µmol photons m −2 s −1 ) was applied to determine the maximal fluorescence at closed PSII centers in the dark-adapted state (F m ). F v /F m was evaluated as (F m −F o )/F m (Kitajima and Butler, 1975;Wei et al., 2013Wei et al., , 2017. The reduction of P700 + in darkness was measured with the aforementioned Dual-PAM-100 fluorometer by monitoring absorbance changes at 830 nm and using 875 nm as a reference. Cells were kept in the dark for 2 min, and 10 µM of DCMU was added to the cell suspension cultures prior to the measurement. The P700 was oxidized by far-red light with a maximum at 720 nm from a light-emitting diode lamp for 30 s, and the subsequent re-reduction of P700 + in the dark was monitored and its half-time was calculated.

Oxygen Evolution Activity
Oxygen production in intact C. reinhardtii cells by photosynthesis was determined at 25 • C by monitoring the changes in O 2 levels with a Clark-type oxygen electrode (Hansatech Instruments, King's Lynn, United Kingdom). Prior to the measurements, 10 mM of NaHCO 3 was added to the cell suspension cultures. The intensity of light used for the measurements was 1,000 µmol photons m −2 s −1 .

A Stepwise NaHSO 3 Addition Mode
Significantly Increases the Yield of H 2 Photoproduction in C. reinhardtii To test whether a stepwise NaHSO 3 addition mode can enhance the yield of H 2 photoproduction, we monitored accumulated H 2 amounts in the one step addition method and stepwise mode of total 13 mM NaHSO 3 (hereafter one step method and stepwise mode, respectively; see Table 1), an optimal concentration for H 2 photoproduction of C. reinhardtii identified in a previous one step method . Compared to the one step method, stepwise mode evidently enhanced the yield of H 2 photoproduction ( Figure 1A and Table 2). The H 2 level in stepwise mode was approximately 1.5 times greater than that in one step method and, approximately 350 times greater than that in untreated cells ( Figure 1A and Table 2). This was confirmed by the results of in vivo ( Figure 1B) and in vitro ( Figure 1C) H 2 ase activity. We therefore conclude that the stepwise mode considerably improves H 2 photoproduction in the green alga C. reinhardtii.

The Stepwise Mode Can Also Establish an Anaerobic Environment
To elucidate the mechanism underlying the increase in the H 2 yield under stepwise mode, we monitored the dissolved O 2 (DO) content in the cell suspension cultures of C. reinhardtii. The results indicated that addition of total 13 mM NaHSO 3 to the cell suspension cultures in both the one step method and stepwise mode can similarly create an anaerobic environment (Figure 2), although the stepwise mode was slightly slow to generate an anaerobic environment when compared to the one step method (insert in Figure 2). It is worthy of note that, when an initial concentration of NaHSO 3 in stepwise mode was less than or equal to 7 mM, the cell suspension cultures did not enter or maintain an anaerobic environment, which evidently suppressed the increase of H 2 photoproduction in the stepwise mode (data not shown). Based on the above results, we propose that the stepwise mode is necessary to operate in an anaerobic environment as an efficient strategy for H 2 photoproduction in C. reinhardtii.  Figure 1A) and its value in one step method was taken as 100%. 3 Time represents the time of continuous H 2 production, which was calculated from Figure 1A.

The Stepwise Mode Maintains a Relatively High Residual Activity of Electron Source for H 2 Photoproduction
To understand why the stepwise mode can increase the yield of H 2 photoproduction, we measured the activity of PSII, an electron source for H 2 photoproduction. After the addition of total 13 mM NaHSO 3 to the cell suspension cultures, the residual activity of PSII was much higher in stepwise mode than that in one step method, as evaluated by the F v /F m values (Figure 3). This was supported by the results that the stepwise mode maintained a slightly high DO content at an efficient H 2 production stage when compared to the one step method (Figure 2). This implies that the relatively high PSII activity under anaerobic conditions is an important reason for improved H 2 photoproduction in the stepwise mode.

The Stepwise Mode Slightly Enhances the Activities of Two Alternative Electron Sinks for H 2 Photoproduction
We also measured the activities of CET and CO 2 assimilation, two alternative electron sinks for H 2 photoproduction. The rates of CET and CO 2 assimilation were slightly faster in the stepwise mode than those in the one step method, as estimated by the rate of re-reduction of P700 + (Figure 4), and photosynthetic production of O 2 with NaHCO 3 as an artificial electron acceptor (Figure 5), respectively. It appears plausible that at least the two alternative electron sinks for H 2 photoproduction do not contribute to enhance the photoproduction of H 2 in the stepwise mode. If this possibility is true, an increase in H 2 photoproduction caused by impaired the activity of either CET or CO 2 assimilation will be higher in the stepwise mode than that in the one step method. The results shown in Figure 6 support our hypothesis that the increase in H 2 photoproduction FIGURE 3 | A stepwise addition mode alleviates the inhibitory effects of NaHSO 3 on PSII activity in C. reinhardtii. The Chl concentration was adjusted to 10 µg mL −1 before measurement. PSII activity was evaluated by the F v /F m values. Values are means ± SD (n = 5).
Frontiers in Plant Science | www.frontiersin.org FIGURE 4 | A stepwise addition mode slightly alleviates the inhibitory effects of NaHSO 3 on cyclic electron transport around PSI in C. reinhardtii. The rate of cyclic electron transport around PSI was judged by half-time of P700 + dark reduction. Values are means ± SD (n = 5).
FIGURE 5 | A stepwise addition mode slightly alleviates the inhibitory effects of NaHSO 3 on CO 2 assimilation in C. reinhardtii. Activity of CO 2 assimilation was assessed by photosynthetic production of O 2 with NaHCO 3 as an artificial electron acceptor. Values are means ± SD (n = 5).
was slightly higher in the stepwise mode than that in the one step method in the presence of either AA that specifically inhibits the CET activity (Tagawa et al., 1963) or GA that disrupts the Calvin-Benson cycle activity via inhibiting the phosphoribulokinase (Rühle et al., 2008). Taking all these results together, we may conclude that in the anaerobic background, increased residual PSII activity can significantly enhance the yield of H 2 photoproduction in C. reinhardtii. If this conclusion is true, impaired PSII activity in an anaerobic environment created by NaHSO 3 addition will inevitably decrease the yield of H 2 photoproduction in C. reinhardtii at a significant level. As expected, the H 2 photoproduction rate was significantly decreased in the presence of Lin, which impairs the PSII activity via inhibiting the D1 protein synthesis (Vavilin et al., 1995), regardless of either one step method or stepwise mode (Figure 6). This consolidates our conclusion that increased FIGURE 6 | Comparison of the H 2 photoproduction in C. reinhardtii between one step method and stepwise mode with multiple inhibitors. After cells were statically pre-cultured under continuous illumination of 200 µmol photons m −2 s −1 for 36 h, NaHSO 3 was added as described in the legend for Figure 1, as well as several inhibitors, lincomycin (Lin; 5 mM), antimycin A (AA; 10 µM), and glycolaldehyde (GA; 2 mM) were also added to the serum bottles, respectively. Values are means ± SD (n = 5).
residual PSII activity in an anaerobic environment is an efficient strategy to improve H 2 photoproduction in C. reinhardtii and the stepwise NaHSO 3 addition mode is a case study in this strategy.

DISCUSSION
Whether NaHSO 3 addition promotes photosynthesis or H 2 photoproduction depends on its concentrations: NaHSO 3 in a low amount improves photosynthesis (Wang et al., 2003) but in a moderate amount can enhance H 2 photoproduction Ma et al., 2011). Wang et al. (2003) demonstrate that a low amount (100 µM) of NaHSO 3 increases cyclic photophosphorylation and consequently improves photosynthesis via optimizing the ATP/NADPH ratio. By contrast, Wei et al. (2017) demonstrate that a moderate amount (13 mM) of NaHSO 3 can remove O 2 efficiently through the reaction of bisulfite with superoxide anion produced at the acceptor side of PSI, especially under sufficient light conditions, consequently activates H 2 ase and promotes H 2 photoproduction. The results of this study indicate that a moderate amount of NaHSO 3 under a stepwise addition mode can quickly establish an anaerobic environment (Figure 2) and significantly improves H 2 photoproduction in a unicellular green alga C. reinhardtii (Table 1 and Figure 1). Such improvement is at least independent of two alternative electron sinks for H 2 photoproduction, CET (Figures 4, 6), and CO 2 assimilation (Figures 5, 6) and, most likely the result of maintained a relatively high electron source for H 2 photoproduction, PSII activity (Figures 3, 6).
Under a photon flux density of 200 µmol photons m −2 s −1 , the Mehler reaction is usually considered to also be an important alternative electron sink for H 2 photoproduction. However, we found that the Mehler reaction is almost absent in NaHSO 3 addition strategy, regardless of either one step method or stepwise mode (data not shown), possibly because addition of NaHSO 3 to the cell suspension cultures quickly results in entering of cells to an anaerobic environment (less than 800 s) (Figure 2). Therefore, the improvement of H 2 photoproduction in C. reinhardtii by a moderate amount of NaHSO 3 under a stepwise addition mode is also independent of a third alternative electron sink for H 2 photoproduction, Mehler reaction, and consolidating the above mentioned possibility that such improvement is the result of increased residual PSII activity, an electron source for H 2 photoproduction.
Based on different sources of electrons to H 2 ase, three pathways for H 2 production have been identified in C. reinhardtii. Their sources of electrons to H 2 ase come from water photolysis via PSII (Melis et al., 2000;Kosourov et al., 2003), NADPH through type II NAD(P)H dehydrogenase (Baltz et al., 2014) and the fermentative degradation of endogenous compounds (Gfeller and Gibbs, 1985), respectively. We observed that production of H 2 under photon flux densities of 200 µmol photons m −2 s −1 by NaHSO 3 addition was almost completely suppressed in cells incubated in the dark (Supplementary Figures S1A,B) or treated with DCMU (Supplementary Figures S1A,C). A quick establishment of anaerobic environment by NaHSO 3 addition (Figure 2) suppresses the acetate uptake (Jurado-Oller et al., 2015) and impairs the mitochondrial respiratory electron transport chain function. Taking all these results together, we propose that in the NaHSO 3 addition strategy, the source of electrons for H 2 production predominantly, if not totally, comes from water photolysis via PSII, regardless of either the one step method or the stepwise mode.
The results of this study further indicate that the stepwise mode increased the maximum accumulated H 2 levels, produced a higher maximum velocity of H 2 photoproduction, and prolonged the time length of H 2 photoproduction when compared to the one step method ( Table 2). We thus propose that the stepwise mode developed in this study is an efficient and sustained strategy for improving H 2 photoproduction in the green alga C. reinhardtii.
Although a moderate amount of NaHSO 3 can remove efficiently O 2 , the activity of PSII, an electron source for H 2 photoproduction, is also significantly impaired Ma et al., 2011;Wei et al., 2017). The results of this study observe that in the anaerobic background, a stepwise mode maintains a relatively high PSII activity (Figure 3) and consequently promotes H 2 photoproduction (Figure 1). The cause and effect of PSII activity and H 2 photoproduction is also present in the sulfur-deprived strategy (Zhang et al., 2002;Kosourov et al., 2005;Kim et al., 2010;Volgusheva et al., 2013;Grewe et al., 2014;Steinbeck et al., 2015;Chen et al., 2016) but the reasons why H 2 photoproduction is terminated in sulfur deprivation and NaHSO 3 addition strategies are distinctly different. It is known that H 2 photoproduction is terminated in the sulfur deprivation strategy because of cell death (Nguyen et al., 2008) and in the NaHSO 3 addition strategy because of conversion of too much bisulfite to sulfate (Wei et al., 2017). It is worthy of note that the relationship between H 2 production and biomass accumulation in sulfur deprivation and NaHSO 3 addition strategies is also distinctly different. Regardless of either one step method or stepwise mode, the simultaneous production of H 2 and biomass is present in NaHSO 3 addition strategy, as observed in mixotrophic nutrient-replete cultures under low light conditions (Jurado-Oller et al., 2015), but is absent in sulfur deprivation strategy. Therefore, it appears reasonable that improved PSII activity in the NaHSO 3 background is considered to be a better strategy to meet future application requirements in comparison with that in the sulfur-deprived background.

AUTHOR CONTRIBUTIONS
WM designed and supervised the experiments. XL, BF, and ZR performed the experiments and analyzed the data. LW and WM analyzed and interpreted the data and wrote the article.