- Open Access
Mechanistic insights into pH-dependent H2 photoproduction in bisulfite-treated Chlamydomonas cells
Biotechnology for Biofuels volume 13, Article number: 64 (2020)
Bisulfite addition is an important H2 photoproduction strategy that removes O2 and activates hydrogenase. The pH values of cell cultures can change the ratio of bisulfite to sulfite, which may affect H2 photoproduction. However, little is known regarding the pH effect of bisulfite addition on H2 photoproduction and relevant underlying mechanism.
Here, changes in H2 photoproduction with different initial extracellular pH values showed a parabolic distribution and a pH of 8.0 is an optimal value for H2 photoproduction in Chlamydomonas reinhardtii cells treated with bisulfite. Compared to the growth pH (pH 7.3), increased photoproduction of H2 at this optimal pH was primarily caused by a relatively high residual activity of photosystem II (PSII), which provides a relatively plentiful source of electrons for H2 photoproduction. Such increased H2 photoproduction was most likely a result of decreased the ratio of bisulfite to sulfite, consistent with the result that the toxicity of bisulfite on PSII was much more than that of sulfite. This possibility was corroborated by the result that treatment with a combination of 7 mM bisulfite and 6 mM sulfite further enhanced H2 photoproduction compared with 13 mM bisulfite alone.
Collectively, our findings provide novel mechanistic insights into pH-dependent H2 photoproduction in C. reinhardtii cells treated with bisulfite, and demonstrate that sulfite addition is another important strategy for H2 photoproduction, just like bisulfite addition.
Increased awareness of fossil fuel depletion and global warming has led to extensive efforts to develop clean and renewable energy sources (for reviews, see [1, 2]). Molecular hydrogen (H2) is considered to be one of the most promising future energy sources because its combustion only produces H2O as a waste product [3, 4]. Chlamydomonas reinhardtii, a unicellular green alga, has been recognized as an ideal candidate among eukaryotes for photobiological H2 production because its [Fe–Fe]-hydrogenase (H2ase) exhibits a higher specific activity than exhibited by [Ni–Fe]-H2ases in some other microorganisms [5, 6]. Under natural conditions, however, C. reinhardtii only produces H2 under anaerobic conditions because its [Fe–Fe]-H2ase is extremely sensitive to oxygen (O2) . As a consequence, numerous strategies are developed to activate [Fe–Fe]-H2ase in C. reinhardtii for efficient and sustainable H2 photoproduction (for recent reviews, see [8,9,10]), including (1) developing the O2-tolerant [Fe–Fe]-H2ase [11, 12]; and (2) decreasing the O2 content around [Fe–Fe]-H2ase [13,14,15,16,17,18,19].
Nearly one decade ago, we also developed an alternative H2 photoproduction strategy that treatment of C. reinhardtii cells with bisulfite (NaHSO3) activates H2ase by decreasing the O2 levels in those cells . Such decrease was found to be a result of efficient reaction of bisulfite with superoxide anion under sufficient light conditions . We further found that regardless of an approximately 200-fold increase in H2 photoproduction was induced by this strategy in C. reinhardtii cells , but its yield was significantly suppressed by impaired PSII , an electron source for H2 photoproduction [23,24,25]. Thus, it is logic to hypothesize that this strategy has a great potential for enhancing the yield of H2 photoproduction in C. reinhardtii cells through improving PSII activity.
Numerous studies have reported that optimal pH values are important for efficient production of H2 in cyanobacteria [26,27,28] and green algae [29, 30]. For example, in sulfur-deprived Chlamydomonas cells, a pH of 7.7 is an optimal value to lead to maximum H2 photoproduction, which is closely associated with residual PSII activity but less with starch and protein degradation . In addition, we noticed that SO2 derivatives at least contain bisulfite and sulfite (Na2SO3), and pH values can change their ratio in the cell cultures . Moreover, the toxicity of bisulfite to the growth of algal cells was much more than that of sulfite [32, 33]. Collectively, we hypothesized that pH is able to change H2 photoproduction via affecting the ratio of bisulfite to sulfite in the cell cultures. However, little is known regarding the pH effect of bisulfite addition on the yield of H2 photoproduction and relevant underlying mechanism.
To investigate the pH effect of bisulfite addition on H2 photoproduction and relevant underlying mechanism, we first examined the effects of different initial extracellular pH values on H2 photoproduction in NaHSO3-treated C. reinhardtii cells. We then assessed the degree to which H2 photoproduction increased at the optimal pH and indicated the possible action target site that was associated with this increased H2 production. Finally, we compared the residual activity of PSII and the yield of H2 photoproduction under conditions of bisulfite and sulfite both with that under conditions of bisulfite alone.
Effect of initial extracellular pH on H2 photoproduction in NaHSO3-treated C. reinhardtii cells
Changes in the rates of H2 photoproduction with different initial extracellular pH values showed a parabolic distribution (Fig. 1a). In specific, the maximum rate of H2 photoproduction was observed to occur at pH 8.0 (see red arrow in Fig. 1a), and any increase or decrease in initial extracellular pH resulted in a lower rate of H2 photoproduction (Fig. 1a). This finding indicates that H2 photoproduction is enhanced at moderate pH levels, and that a pH of 8.0 is an optimal value to result in maximum H2 photoproduction in NaHSO3-treated cells of C. reinhardtii.
The yield of H2 photoproduction at pH 8.0 is greatly enhanced in NaHSO3-treated C. reinhardtii cells
Levels of H2 increased immediately after treatment with NaHSO3 (see black arrow in Fig. 1b) and remained high, whereas the H2 level was almost unchanged and remained low in the untreated cells, regardless of cellular incubation at either pH 8.0 or pH 7.3 (growth pH; see blue arrow in Fig. 1a). This finding is in agreement with the results reported in previous studies [20, 34]. In NaHSO3-treated cells, the yield of H2 photoproduction at pH 8.0 can be further enhanced when compared to the pH 7.3 (Fig. 1b). In specific, the H2 level in NaHSO3-treated cells incubated at pH 8.0 was approximately 1.75 times greater than the level observed at pH 7.3 (Fig. 1b). We therefore conclude that the yield of H2 photoproduction at pH 8.0 is significantly increased under NaHSO3 addition conditions.
An anaerobic environment established by NaHSO3 at pH 8.0 is relatively slow but H2ase activity is more strongly stimulated
To elucidate the mechanism by which H2 photoproduction increased at pH 8.0 in NaHSO3-treated cells, we monitored dissolved O2 content alongside H2ase activity. Our results indicated that treatment with NaHSO3 rapidly creates an anaerobic environment in the cell cultures (Fig. 2a), which stimulates H2ase activity regardless of initial extracellular pH (Fig. 2b, c). Surprisingly, compared to the pH 7.3, an anaerobic environment generated by NaHSO3 addition at pH 8.0 was relatively slow (insert in Fig. 2a), but H2ase activity was more strongly increased (Fig. 2b, c). This result indicates that levels of H2 increased at pH 8.0 are independent of O2 content in the background of bisulfite addition. Future studies are required to clarify the interrelationship of the duration of H2 production at different initial extracellular pH values with O2 content in the bisulfite addition strategy.
Recently, we have demonstrated that 13 mM of NaHSO3 as an optimal concentration for H2 photoproduction in C. reinhardtii  can remove O2 efficiently in intact cells through a reaction of bisulfite with superoxide anion radicals produced at the acceptor side of PSI, especially under sufficient light conditions . A similar process has been previously reported to operate in spinach chloroplasts  and tobacco thylakoids . We named the mechanism of removal of O2 molecules as bisulfite photooxidation, since operation of this mechanism requires light irradiation and oxidation reaction of bisulfite. Here, we noticed that a photooxidation level of bisulfite at pH 8.0 was lower than that at pH 7.3 (Additional file 1: Figure S1a), consistent with the result that establishment of an anaerobic environment at pH 8.0 was slower than that at pH 7.3 (insert in Fig. 2a). This is most likely the result of less superoxide anion radicals at pH 8.0 and more superoxide anion radicals at pH 7.3 (Additional file 1: Figure S1b).
A residual activity of electron source maintained at pH 8.0 is relatively high under NaHSO3 addition conditions
To assess whether the enhanced production of H2 at pH 8.0 is driven by a relatively plentiful source of electrons (see a in Fig. 3f), we monitored the activity of PSII in NaHSO3-treated cells incubated at different pH values. The results revealed that NaHSO3 addition impaired PSII activity regardless of initial extracellular pH level [see the calculated values of maximum quantum yield of PSII (Fv/Fm); Fig. 3a]. A residual activity of PSII maintained at pH 8.0, however, is relatively high in comparison to the pH 7.3, under NaHSO3 addition conditions, consistent with the results that the amount of superoxide anion radicals at pH 8.0 was less than that at pH 7.3 (Additional file 1: Figure S1b). This finding strongly suggests that the increased production of H2 at pH 8.0 is primarily driven by the maintenance of a relatively high residual activity of PSII at this pH, which provides a relatively plentiful source of electrons for H2 photoproduction.
The activities of alternative electron sinks for H2 photoproduction at pH 8.0 are increased under NaHSO3 treatment conditions
Increased H2 production at pH 8.0 may also be driven by the activity of alternative electron sinks for H2 photoproduction (see b and c in Fig. 3f). To assess the likelihood of this possibility, we monitored the activity of cyclic electron transport around photosystem I (PSI CET) and also assessed the activity of CO2 fixation. Our results showed that treatment with NaHSO3 greatly reduced the activity of PSI CET in cells incubated at different pH values, as judged by the half-time of P700+ re-reduction in darkness (Fig. 3b). Importantly, however, the activity of PSI CET at pH 8.0 is slightly increased when compared to the pH 7.3 (Fig. 3b). We further found that treatment with NaHSO3 significantly decreased the activity of CO2 fixation in cells incubated at different pH values, as estimated by the photosynthetic production of O2 with NaHCO3 as an artificial electron acceptor (Fig. 3c). As expected, under NaHSO3 addition conditions, a residual activity of CO2 assimilation maintained at pH 8.0 was relatively higher than that at pH 7.3 (Fig. 3c). This difference was reinforced by the results of Rubisco accumulation in cells. As deduced from the accumulation levels of Rubisco large (RbcL) subunit in cells, we observed that the expression levels of Rubisco were always maintained at a relatively high level under conditions of pH 8.0 but significantly decreased under conditions of pH 7.3, especially after 12 h, with the prolonged time of NaHSO3 addition (Fig. 3d, e). Collectively, it appears plausible that at least the two alternative electron sinks for H2 photoproduction are not responsible for the increased H2 photoproduction observed at pH 8.0.
If this possibility is true, an increase in H2 photoproduction caused by impaired the activity of either PSI CET or CO2 assimilation will be higher in cells incubated at pH 8.0 than that at pH 7.3. The results shown in Fig. 4 support our hypothesis that the increase in H2 photoproduction was slightly higher in cells incubated at pH 8.0 than that at pH 7.3 in the presence of either antimycin A (AA) that specifically inhibits the PSI CET activity  or glycolaldehyde (GA) that disrupts the Calvin–Benson cycle activity via inhibiting the phosphoribulokinase . Collectively, we may conclude that in the anaerobic background, increased residual PSII activity can significantly enhance the yield of H2 photoproduction in C. reinhardtii.
If this conclusion is true, impaired PSII activity in an anaerobic environment created by NaHSO3 addition will inevitably decrease the yield of H2 photoproduction in C. reinhardtii at a significant level, especially at pH 8.0. As expected, the H2 photoproduction rate was significantly decreased in the presence of lincomycin (Lin), which impairs the PSII activity through inhibiting the D1 protein synthesis , regardless of either optimal or growth pH (Fig. 4). Importantly, such decrease was much more in cells incubated at pH 8.0 than that at pH 7.3 (Fig. 4). These results greatly consolidate our conclusion that increased residual PSII activity in an anaerobic environment is an efficient strategy to improve H2 photoproduction in C. reinhardtii and the optimal pH is a case study in this strategy.
Treatment with a combination of bisulfite and sulfite enhances H2 photoproduction further
With the pH values increased, a ratio of bisulfite to sulfite decreased and the toxicity of bisulfite–sulfite on photosynthesis also decreased , consistent with the results that the toxicity of sulfite on photosynthesis was weaker than that of bisulfite [32, 33]. Therefore, it is logical to hypothesize that a combination of bisulfite and sulfite can improve the yield of H2 photoproduction by increasing residual PSII activity.
To test this idea, we compared the inhibitory effects of bisulfite and sulfite on PSII activity. Clearly, the toxicity of bisulfite on PSII was greater than that of sulfite as deduced from the changes in Fv/Fm values under conditions of different concentrations of bisulfite or sulfite (Fig. 5a). As a consequence, a combination of bisulfite and sulfite with a total of 13 mM alleviated their toxicity on PSII in comparison to 13 mM of bisulfite alone, regardless of cells incubated at pH 8.0 or pH 7.3 (Fig. 5b). As expected, the combination improved the yield of H2 photoproduction regardless of pH 8.0 or pH 7.3 (Fig. 5c). Furthermore, the degree of such alleviation of PSII at pH 7.3 was more evident than that at pH 8.0 (Fig. 5b). This may be because the ratio of bisulfite to sulfite changed at pH 7.3 was more than that at pH 8.0 after 13 mM of NaHSO3 was added to the cultures. Consistently, the degree of H2 photoproduction improved at pH 7.3 was greater than that at pH 8.0 (Fig. 5c). Regardless of their differences, treatment with a combination of bisulfite and sulfite can further enhance the yield of H2 photoproduction in C. reinhardtii cells.
Whether bisulfite addition functions in photosynthetic O2 evolution or photosynthetic H2 evolution depends on its concentrations: bisulfite in a low amount improves photosynthetic O2 evolution [34, 40], but in a moderate amount can significantly promote photosynthetic H2 evolution [20, 34]. It has been demonstrated that a low amount (100 μM) of bisulfite improves photosynthesis by increasing cyclic photophosphorylation and optimizing ATP/NADPH ratio required for the Calvin–Benson cycle . By contrast, a moderate amount (13 mM) of bisulfite can remove O2 efficiently through a reaction of bisulfite with superoxide anion produced at the acceptor side of PSI, especially under sufficient light conditions, and consequently activates H2ase and promotes H2 photoproduction . Consistent with other H2 photoproduction strategies [23,24,25], the source of electrons for H2 photoproduction in our bisulfite addition strategy predominantly, if not totally, comes from water photolysis via PSII  (Figs. 3, 4); unfortunately, impaired PSII by bisulfite addition greatly limits the efficient photoproduction of H2 in C. reinhardtii. Therefore, increased residual PSII activity in an anaerobic environment is an efficient strategy to improve H2 photoproduction in C. reinhardtii further.
The stepwise bisulfite addition mode of our previous study  and the optimal pH of this study are two case studies to improve residual PSII activity for increasing H2 photoproduction in NaHSO3-treated cells of C. reinhardtii. It is easy to understand the reason why the stepwise bisulfite addition mode can alleviate the toxicity of bisulfite on PSII, thereby resulting in an increase in H2 photoproduction. In contrast, it is difficult to understand the reason why the optimal pH can alleviate the toxicity of bisulfite on PSII, causing an increase in H2 photoproduction. Occasionally, we noticed that treatment with the optimal pH can decrease the ratio of bisulfite to sulfite . It is logical to hypothesize that such decrease can lead to an increase in residual PSII activity, since the toxicity of sulfite on photosynthesis is lower than that of bisulfite [32, 33]. This hypothesis was confirmed by the results of this study (Fig. 5). Collectively, we propose that decreasing the ratio of bisulfite to sulfite by the optimal pH increases residual PSII activity, thereby enhancing the yield of photobiological H2 production in C. reinhardtii cells.
We also noticed that oxidation of SH groups of the enzymes that constitute the Calvin–Benson cycle, such as glyceraldehyde 3-phosphate dehydrogenase, by bisulfite and sulfite directly or indirectly [41, 42] leads to production of reactive oxygen species (ROS), which suppresses synthesis of the D1 protein during the repair of PSII after photodamage  and destabilizes the PSII architecture . Consistent with these findings, the residual Calvin–Benson cycle activity at pH 8.0 is higher and resultant ROS level is lower than that at pH 7.3 (Fig. 3c–e and Additional file 1: Figure S1b). Collectively, the toxicity of bisulfite on PSII is higher than that of sulfite possibly through suppressing more residual Calvin–Benson cycle activity and producing more ROS molecules. However, the mechanism by which bisulfite and sulfite exert their toxicity on PSII remains elusive. Future studies are required to unravel this mechanism and fully understand different effects of bisulfite and sulfite on PSII activity.
Our previous data indicated that, when an initial concentration of bisulfite in stepwise addition mode was equal to or less than 7 mM, the cell suspension cultures did not enter or maintain an anaerobic environment, which evidently suppressed the increase in H2 photoproduction in the stepwise addition mode . By comparison, a combination of 7 mM bisulfite and 6 mM sulfite can quickly establish an anaerobic environment (Additional file 1: Figure S2). It appears plausible that sulfite can also react with superoxide anion to remove O2 in cells of C. reinhardtii. This possibility was verified by the results of increased sulfate with a significant level (Additional file 1: Table S1). As a consequence, sulfite addition can also efficiently promote H2 photoproduction via removing O2 and activating H2ase (Additional file 1: Figure S3a–d and Additional file 1: Table S1) just like bisulfite addition.
Collectively, our data reported here provide novel mechanistic insights into pH-dependent H2 photoproduction in bisulfite-treated cells of C. reinhardtii. In this model, addition of bisulfite to the cell cultures incubated at pH 7.3 has a high ratio of bisulfite to sulfite, which significantly suppresses water photolysis via PSII, and leads to a low source of electrons for H2 photoproduction (Fig. 6). In contrast, a low ratio of bisulfite to sulfite created by pH 8.0 promotes water photolysis via PSII and maintains a relatively high source of electrons for H2 photoproduction (Fig. 6). Compared to the high ratio of bisulfite to sulfite created by pH 7.3, the low ratio of bisulfite to sulfite created by pH 8.0 also slightly increases the activities of two alternative sinks of electrons for H2 photoproduction, PSI CET and CO2 assimilation (Figs. 3, 6). We thus propose that the yield of H2 photoproduction was influenced by pH in C. reinhardtii cells mainly through changing the ratio of bisulfite to sulfite and subsequent the level of water photolysis via PSII, an electron source for H2 photoproduction.
Although impaired PSII has been improved by the stepwise bisulfite addition mode or the optimal pH or a combination of bisulfite and sulfite, the residual PSII activity is still at a relatively low level. This indicates that there has a great potential to enhance the yield of H2 photoproduction in the background of an anaerobic environment created by bisulfite and/or sulfite addition via increasing the residual PSII activity. Therefore, it is very important to identify these potential key targets that suppress the residual PSII activity in the background of bisulfite and/or sulfite addition using forward genetics strategy and to further enhance the yield of H2 photoproduction in algal cells in the future.
Alternatively, recent studies have demonstrated that suppression of the Calvin–Benson cycle results in a sustainable and efficient photoproduction of H2 in C. reinhardtii [25, 45]. Consistent with these findings, an evident suppression of the Calvin–Benson cycle by bisulfite at pH 7.3 causes a longer H2 photoproduction (Fig. 1b) and by GA, a Calvin–Benson cycle inhibitor, leads to a more efficient H2 photoproduction regardless of cells incubated at pH 8.0 or pH 7.3 (Fig. 4). Collectively, besides the PSII target, the Calvin–Benson cycle should be an alternative target that results in a sustainable and efficient photoproduction of H2 in bisulfite-treated algal cells.
In this study, we have demonstrated that the yield of H2 photoproduction is increased by an optimal pH in C. reinhardtii cells treated with bisulfite. Our results further revealed that this increased H2 photoproduction was mainly caused by the maintenance of a relatively high residual activity of electron source (PSII) at this optimal pH. Moreover, our results strongly suggest that the relatively high activity of electron source at this optimal pH was most likely a result of decreasing the ratio of bisulfite to sulfite in the cell cultures, since the toxicity of sulfite on PSII complex is lower than that of bisulfite. Subsequently, this suggestion is corroborated by the result that treatment with a combination of bisulfite and sulfite further enhanced the yield of H2 photoproduction in C. reinhardtii cells. During treatment with this combination of bisulfite and sulfite for H2 photoproduction, we unexpectedly found that sulfite addition can remove O2, activate H2ase and increase H2 photoproduction, just like bisulfite addition.
Chlamydomonas reinhardtii cells (CC-503 strain) were cultured at 25 °C in Tris–acetate–phosphate (TAP) medium . The medium was buffered with Tris–HCl (20 mM; pH 7.3), bubbled with air under continuous illumination with cool-white fluorescence lamps (40 μE m−2 s−1), and inoculated with approximately 8.1 × 104 cells mL−1 of C. reinhardtii (inoculum size 1%).
pH, NaHSO3 and Na2SO3 treatments
Chlamydomonas reinhardtii cells were cultured in 0.5 L of TAP medium for 2 days (A750 = 0.8–1.0). Subsequently, different pH values were modulated by adding 5 mM of NaOH or HCl to the cultures incubated at corresponding buffers. Next, a fixed volume of cells containing 300 µg chlorophyll (Chl) was transferred to 60-mL serum bottles (30 mL head space and 30 mL cells) with rubber seals. After a 36-h pre-culture stage, 13 mM of NaHSO3 or a combination of 7 mM NaHSO3 and 6 mM Na2SO3 or 25 mM of Na2SO3 was added to the serum bottles. The cells were then grown under continuous illumination (200 μE m−2 s−1) to induce the production of H2.
Monitoring H2 photoproduction
At predetermined time intervals, 200 µL of gas samples were withdrawn from the bottles with a gas-tight syringe and injected into a gas chromatograph (Agilent 7890A; Agilent Technologies Inc., USA) with a thermal conductivity detector for determining the concentrations of H2, O2, and N2 simultaneously. The column was a molecular sieve column (type 5Å; 2 m × 1/8 mm). Argon was used as the carrier gas.
H2ase activity assay
In vivo and in vitro H2ase activity was monitored using a previously described method [20, 22, 29, 38] with slight modifications. In brief, 1-mL cell suspension samples were withdrawn anaerobically from the 60-mL serum bottles at designated times (see Fig. 2b, c; Additional file 1: Figure S3b, c) and then injected into 10-mL glass vials. To measure in vivo H2ase activity, the samples were immediately exposed to argon gas for 1 min to eliminate the inhibitory effect of O2 on H2ase. The samples were then placed in a 25 °C water bath for 1 h and shaken continuously (150 rpm) while exposed to constant light (200 μE m−2 s−1 intensity). To measure in vitro H2ase activity, we used vials containing 1 mL of 10 mM oxidized methyl viologen prepared in O2-free 50 mM Tris buffer (for pH 7.1–9.0) and 0.2% (w/v) Triton X-100. The reaction was started when methyl viologen 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 H2 produced in the headspace of the glass vial by gas chromatography, and the rate of H2 production was calculated based on the total Chl content in the glass vial, unless otherwise indicated.
Dissolved oxygen measurement
A dissolved oxygen (DO) meter (Orion Star A213, Thermo Scientific, USA) was used to monitor the DO attenuation process after the addition of NaHSO3 to the cultures of C. reinhardtii at different pH values. The DO meter was corrected before each measurement. The DO meter probe was placed in the middle of the cultures and the data recorded at different times.
Chl fluorescence and P700 analysis
The Chl fluorescence yields at a steady-state of electron transport were measured at room temperature with a Dual-PAM-100 monitoring system (Walz, Effeltrich, Germany) equipped with an ED-101US/MD unit [47, 48]. Minimal fluorescence at open PSII centers in the dark-adapted state (Fo) was excited by a weak measuring light (650 nm) at a photon flux density of 0.05 to 0.15 μE m−2 s−1. A saturating pulse of red light (600-ms, 10,000 μE m−2 s−1) was applied to determine the maximal fluorescence at closed PSII centers in the dark-adapted state (Fm). Maximal quantum yield of PSII (Fv/Fm) was evaluated as (Fm − Fo)/Fm [49, 50]. The redox state of P700 was measured with the aforementioned Dual-PAM-100 fluorometer. The P700 was oxidized by far-red light from a photodiode (FR-102, Walz, Effeltrich, Germany) for 30 s, and then the kinetics of re-reduction of P700+ in the dark was monitored.
The production of photosynthetic O2 in intact C. reinhardtii cells was measured at 25 °C by monitoring the evolution of O2 with a Clark-type oxygen electrode (Hansatech Instruments, Kings Lynn, UK). Oxygen production by photosynthesis was measured in the presence of 10 mM NaHCO3. The intensity of light used for the measurements of O2 evolution activity was 1000 μE m−2 s−1.
Light-induced oxygen uptake in intact cells of C. reinhardtii was determined at 25 °C by the oxygen consumption using an aforementioned Clark-type oxygen electrode as described previously [21, 35, 36]. The intact cells of C. reinhardtii were pre-illuminated by red light (> 630 nm; 1000 μE m−2 s−1) for 2 min before illumination by the same light to measure the oxygen consumption rate. The oxygen consumption was started by addition of bisulfite to the cell cultures and the photooxidation level of bisulfite was calculated as the rate of oxygen consumption in light minus that in darkness.
Crude protein extract and immunoblotting analysis
To obtain crude protein extracts of C. reinhardtii, cells were harvested by centrifugation at 5000g for 2 min at 4 °C. The cell pellet was resuspended in 50 mM Tris–HCl, pH 8.3 plus 1% Triton X-100 and the suspension was shaken for 30 min in the dark . Subsequently, the homogenate was centrifuged at 5000g for 5 min at 4 °C to remove unbroken cells and debris. A 200-µL aliquot of the supernatant was mixed with 100 µL of protein lysis buffer [2 M urea; 0.5 M Tris–HCl, pH 8.0; 20% glycerol; 7.5% SDS; 2% (v/v) mercaptoethanol; 0.05% (w/v) bromphenol blue] and heated for 5 min at 95 °C. After centrifugation at 12,000g for 10 min at 4 °C, the crude protein extracts were loaded onto the gels. SDS–PAGE was conducted as described before  using a 10% (w/v) separating gel. Immunoblotting was performed with an enhanced chemiluminescence (ECL) assay kit (Amersham Pharmacia Biotech) according to the manufacturer’s protocol. Antibody against Rubisco large subunit (RbcL) of C. reinhardtii was kindly provided by Dr. Lili Xu (College of Life Sciences, Shanghai Normal University).
Superoxide anion assay
Superoxide anion was measured according to the instruction of superoxide anion kit (Nanjing Jiancheng Bioengineering Institute, China). The superoxide anion radicals in the assay kit were generated by the xanthine/xanthine oxidase reaction system to form a colored compound with a peak absorbance at 550 nm.
Sulfate concentration was measured using a previously described method [21, 53] with some modifications. In brief, 1-mL cell suspension samples treated by Na2SO3 or not were withdrawn from the 60-ml serum bottles at 0 or 12 h (Additional file 1: Figure S3b) and were supersonically disrupted with a power of about 100 W by six repetitions of a 20-s pulse followed by 3-min incubation on ice. The homogenate was centrifuged at 12,000g for 5 min at 4 °C to remove unbroken cells and debris. Prior to measurement, the supernatant was filtered through a 0.22-µm filter. Subsequently, SO42− in the supernatant was determined using an ICS-5000 ion chromatograph (Dionex) equipped with an IonPac AG19 guard column (4 mm × 50 mm) and an IonPac AS19 separation column (4 mm × 250 mm), connected with a conductivity detector (Dionex).
Availability of data and materials
All data generated or analyzed during this study are included in this published article and Additional file 1.
Coomassie brilliant blue
- C. reinhardtii :
- Fv/Fm :
Maximum quantum yield of photosystem II
- PSI CET:
Cyclic electron transport around photosystem I
Rubisco large subunit
Kotay SM, Das D. Biohydrogen as a renewable energy resource—prospects and potentials. Int J Hydrogen Energy. 2008;33:258–63.
Bičáková O, Straka P. Production of hydrogen from renewable resources and its effectiveness. Int J Hydrogen Energy. 2012;37:11563–78.
Hansel A, Lindblad P. Towards optimization of cyanobacteria as biotechnologically relevant producers of molecular hydrogen, a clean and renewable energy source. Appl Microbiol Biotechnol. 1998;50:153–60.
Momirlan M, Veziroglu TN. Current status of hydrogen energy. Renewable Sustainable Energy Rev. 2002;6:141–79.
Adams MW, Mortenson LE, Chen JS. Hydrogenase. Biochim Biophys Acta. 1980;594:105–76.
Frey M. Hydrogenases: hydrogen-activating enzymes. ChemBioChem. 2002;3:153–60.
Ghirardi ML, Togasaki RK, Seibert M. Oxygen sensitivity of algal H2 production. Appl Microbiol Biotechnol. 1997;63:141–51.
Allakhverdiev SI, Thavasi V, Kreslavski VD, Zharmukhamedov SK, Klimov VV, Ramakrishna S, Los DA, Mimuro M, Nishihara H, Carpentier R. Photosynthetic hydrogen production. J Photochem Photobiol C. 2010;11:101–13.
Esquível MG, Amaro HM, Pinto TS, Fevereiro PS, Malcata FX. Efficient H2 production via Chlamydomonas reinhardtii. Trends Biotechnol. 2011;29:595–600.
Oh YK, Raj SM, Jung GY, Park S. Current status of the metabolic engineering of microorganisms for biohydrogen production. Bioresour Technol. 2011;102:8357–67.
Flynn T, Ghirardi ML, Seibert M. Accumulation of O2-tolerant phenotypes in H2-producing strains of Chlamydomonas reinhardtii by sequential applications of chemical mutagenesis and selection. Int J Hydrogen Energy. 2002;27:1421–30.
Kruse O, Rupprecht J, Bader KP, Thomas-Hall S, Schenk PM, Finazzi G, Hankamer B. Improved photobiological H2 production in engineered green algal cells. J Biol Chem. 2005;280:34170–7.
Melis A, Zhang L, Forestier M, Ghirardi ML, Seibert M. Sustained photobiological hydrogen gas production upon reversible inactivation of oxygen evolution in the green alga Chlamydomonas reinhardtii. Plant Physiol. 2000;122:127–36.
Surzycki R, Cournac L, Peltier G, Rochaix JD. Potential for hydrogen production with inducible chloroplast gene expression in Chlamydomonas. Proc Natl Acad Sci USA. 2007;104:17548–53.
Wu S, Huang R, Xu L, Yan G, Wang Q. Improved hydrogen production with expression of hemH and lba genes in chloroplast of Chlamydomonas reinhardtii. J Biotechnol. 2010;146:120–5.
Xu FQ, Ma W, Zhu XG. Introducing pyruvate oxidase into the chloroplast of Chlamydomonas reinhardtii increases oxygen consumption and promotes hydrogen production. Int J Hydrogen Energy. 2011;36:10648–54.
Jurado-Oller JL, Dubini A, Galván A, Fernández E, González-Ballester D. Low oxygen levels contribute to improve photohydrogen production in mixotrophic non-stressed Chlamydomonas cultures. Biotechnol Biofuels. 2015;8:149.
Xiong W, Zhao X, Zhu G, Shao C, Li Y, Ma W, Xu X, Tang R. Silicification-induced cell aggregation for the sustainable production of H2 under aerobic conditions. Angew Chem Int Ed. 2015;54:11961–5.
Shu L, Xiong W, Shao C, Huang T, Duan P, Liu K, Xu X, Ma W, Tang R. Improvement in the photobiological hydrogen production of aggregated Chlorella by dimethyl sulfoxide. ChemBioChem. 2018;19:669–73.
Ma W, Chen M, Wang L, Wei L, Wang Q. Treatment with NaHSO3 greatly enhances photobiological H2 production in the green alga Chlamydomonas reinhardtii. Bioresour Technol. 2011;102:8635–8.
Wei L, Yi J, Wang L, Huang T, Gao F, Wang Q, Ma W. Light intensity is important for hydrogen production in NaHSO3-treated Chlamydomonas reinhardtii. Plant Cell Physiol. 2017;58:451–7.
Wei L, Li X, Fan B, Ran Z, Ma W. A stepwise NaHSO3 addition mode greatly improves H2 photoproduction in Chlamydomonas reinhardtii. Front Plant Sci. 2018;9:1532.
Liran O, Semyatich R, Milrad Y, Eilenberg H, Weiner I, Yacoby I. Microoxic niches within the thylakoid stroma of air-grown Chlamydomonas reinhardtii protect [FeFe]-hydrogenase and support hydrogen production under fully aerobic environment. Plant Physiol. 2016;172:264–71.
Milrad Y, Schweitzer S, Feldman Y, Yacoby I. Green algal hydrogenase activity is outcompeted by carbon fixation before inactivation by oxygen takes place. Plant Physiol. 2018;177:918–26.
Nagy V, Podmaniczki A, Vidal-Meireles A, Tengölics R, Kovács L, Rákhely G, Scoma A, Tóth SZ. Water-splitting-based, sustainable and efficient H2 production in green algae as achieved by substrate limitation of the Calvin–Benson–Bassham cycle. Biotechnol Biofuels. 2018;11:69.
Jeffries TW, Timourian H, Ward RL. Hydrogen production by Anabaena cylindrica: effects of varying ammonium and ferric ions, pH, and light. Appl Environ Microbiol. 1978;35:704–10.
Antal TK, Lindblad P. Production of H2 by sulphur-deprived cells of the unicellular cyanobacteria Gloeocapsa alpicola and Synechocystis sp. PCC 6803 during dark incubation with methane or at various extracellular pH. J Appl Microbiol. 2005;98:114–20.
Burrows EH, Wong WK, Fern X, Chaplen FW, Ely RL. Optimization of pH and nitrogen for enhanced hydrogen production by Synechocystis sp. PCC 6803 via statistical and machine learning methods. Biotechnol Prog. 2009;25:1009–17.
Kosourov S, Seibert M, Ghirardi ML. Effects of extracellular pH on the metabolic pathways in sulfur-deprived, H2-producing Chlamydomonas reinhardtii cultures. Plant Cell Physiol. 2003;44:146–55.
Rashid N, Lee K, Han JI, Gross M. Hydrogen production by immobilized Chlorella vulgaris: optimizing pH, carbon source and light. Bioprocess Biosyst Eng. 2013;36:867–72.
Ough CS, Were L. Sulfur dioxide and sulfites. In: Davidson PM, Sofos JN, Branen AL, editors. Antimicrobials in Food. 3rd ed. Boca Raton: CRC Press; 2005. p. 143–67.
Yang S, Wang J, Cong W, Cai Z, Ouyang F. Effects of bisulfite and sulfite on the microalga Botryococcus braunii. Enzyme Microb Technol. 2004;35:46–50.
Jiang Y, Zhang W, Wang J, Chen Y, Shen S, Liu T. Utilization of simulated flue gas for cultivation of Scenedesmus dimorphus. Bioresour Technol. 2013;128:359–64.
Wang L, Chen M, Wei L, Gao F, Lv Z, Wang Q, Ma W. Treatment with moderate concentrations of NaHSO3 enhances photobiological H2 production in the cyanobacterium Anabaena sp. strain PCC 7120. Int J Hydrogen Energy. 2010;35:12777–83.
Asada K, Kiso K. Initiation of aerobic oxidation of sulfite by illuminated spinach chloroplasts. Eur J Biochem. 1973;33:253–7.
Wu Y, Zheng F, Ma W, Han Z, Gu Q, Shen Y, Mi H. Regulation of NAD(P)H dehydrogenase-dependent cyclic electron transport around photosystem I by NaHSO3 at low concentrations in tobacco chloroplasts. Plant Cell Physiol. 2011;52:1734–43.
Tagawa K, Tsujimoto HY, Arnon DI. Role of chloroplast ferredoxin in the energy conversion process of photosynthesis. Proc Natl Acad Sci USA. 1963;49:567–72.
Rühle T, Hemschemeier A, Melis A, Happe T. A novel screening protocol for the isolation of hydrogen producing Chlamydomonas reinhardtii strains. BMC Plant Biol. 2008;8:107.
Vavilin DV, Tyystjärvi E, Aro EM. In search of a reversible stage of photoinhibition in a higher plant: no changes in the amount of functional photosystem II accompany relaxation of variable fluorescence after exposure of lincomycin-treated Cucurbita pepo leaves to high light. Photosynth Res. 1995;45:239–47.
Wang H, Mi H, Ye J, Deng Y, Shen Y. Low concentrations of NaHSO3 increase cyclic photophosphorylation and photosynthesis in cyanobacterium Synechocystis PCC6803. Photosynth Res. 2003;75:151–9.
Tanaka K, Otsubo T, Kondo N. Participation of hydrogen peroxide in the inactivation of Calvin-cycle SH enzymes in SO2-fumigated spinach leaves. Plant Cell Physiol. 1982;23:1009–18.
Würfel M, Häberlein I, Follmann H. Inactivation of thioredoxin by sulfite ions. FEBS Lett. 1990;268:146–8.
Takahashi S, Murata N. Interruption of the Calvin cycle inhibits the repair of photosystem II from photodamage. Biochim Biophys Acta. 2005;1708:352–61.
Kobayashi S, Tsuzuki M, Sato N. Sulfite-stress induced functional and structural changes in the complexes of photosystems I and II in a cyanobacterium, Synechococcus elongatus PCC 7942. Plant Cell Physiol. 2015;56:1521–32.
Kosourov S, Jokel M, Aro EM, Allahverdiyeva Y. A new approach for sustained and efficient H2 photoproduction by Chlamydomonas reinhardtii. Energy Environ Sci. 2018;11:1431–6.
Harris EH. The Chlamydomonas sourcebook: a comprehensive guide to biology and laboratory use. New York: Academic Press; 1989.
Schreiber U, Schliwa U, Bilger W. Continuous recording of photochemical and non-photochemical chlorophyll fluorescence quenching with a new type of modulation fluorometer. Photosynth Res. 1986;10:51–62.
Ma W, Wei L, Wang Q. The response of electron transport mediated by active NADPH dehydrogenase complexes to heat stress in the cyanobacterium Synechocystis 6803. Sci China C Life Sci. 2008;51:1082–7.
Kitajima M, Butler WL. Quenching of chlorophyll fluorescence and primary photochemistry in chloroplasts by dibromothylmoquinone. Biochim Biophys Acta. 1975;376:105–15.
Wei L, Li X, Yi J, Yang Z, Wang Q, Ma W. A simple approach for the efficient production of hydrogen from Taihu Lake Microcystis spp. blooms. Bioresour Technol. 2013;139:136–40.
Hemschemeier A, Fouchard S, Cournac L, Peltier G, Happe T. Hydrogen production by Chlamydomonas reinhardtii: an elaborate interplay of electron sources and sinks. Planta. 2008;227:397–407.
Laemmli UK, Favre M. Maturation of the head of bacteriophage T4. J Mol Biol. 1973;80:575–99.
Huang J, Wen Y, Ding N, Xu Y, Zhou Q. Effect of sulfate on anaerobic reduction of nitrobenzene with acetate or propionate as an electron donor. Water Res. 2012;46:4361–70.
We thank Tingting Huang (Shanghai Normal University) for sulfate measurement.
This work was supported by the Shanghai Science and Technology Committee (Grant Nos. 17070502900 and 18DZ2260500) and National Natural Science Foundation of China (Grant Nos. 31570235 and 31770259).
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Treatment with optimal pH decreases the photooxidation (a) and superoxide anion (b) levels. Figure S2. Comparison of decreased dissolved oxygen (DO) levels caused by addition of 7 mM NaHSO3 alone and a combination of 7 mM NaHSO3 and 6 mM Na2SO3 to the serum bottles. Figure S3. Treatment with Na2SO3 significantly increases the yield of H2 photoproduction in C. reinhardtii. Table S1. Concentrations of SO42− in Na2SO3-treated cultures of C. reinhardtii for different times.
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Wei, L., Fan, B., Yi, J. et al. Mechanistic insights into pH-dependent H2 photoproduction in bisulfite-treated Chlamydomonas cells. Biotechnol Biofuels 13, 64 (2020). https://doi.org/10.1186/s13068-020-01704-0
- H2 photoproduction
- Chlamydomonas reinhardtii