Mechanistic Insights Into PH-dependent H₂ Photoproduction In Bisulfite-treated Chlamydomonas Cells

The unicellular green alga Chlamydomonas reinhardtii has been recognized as an ideal candidate among eukaryotes for photobiological H2 production because its iron-iron hydrogenase?H2ase?exhibits a higher activity than that of nickel-iron H2 ases in some other microorganisms.Under natural conditions,however,C.reinhardtii only produces H2 under anaerobic conditions because its H2 ase is extremely sensitive to oxygen?O2?.Therefore,it is necessary to ensure an anaerobic environment to induce the activity of hydrogenase for continuous H2 photoproduction.This strategy has become one of the important methods for studying H2 photosynthesis in C.reinhardtii cells.13m M of NaHSO₃ as an optimal concentration for H2 photoproduction in C.reinhardtii can activates H2 ase by decreasing the O2 levels in those cells,and approximately 200-fold increase in H2 photoproduction is induced by this strategy.Such decrease was found to be a result of efficient reaction of bisulfite with superoxide anion under sufficient light conditions.It has been extensively reported that moderate pH values play an important role in efficient production of H2 in cyanobacteria and green algae,pH values not only affect the H2 photoproduction efficiency of microalgae,but also has certain effects on cell growth and metabolic response.However,it has not been studied whether changing the pH value of algal medium will also affect the H2 photoproduction in bisulfite-treated Chlamydomonas cells.Collectively,this thesis will test whether the H2 photoproduction rate in bisulfite-treated Chlamydomonas cells can be further optimized by changing the initial pH value of algal medium.Our results showed that changes in the rates of H2 photoproduction in bisulfite-treated Chlamydomonas cells with different initial extracellular pH values showed a parabolic distribution.Specifically,the maximum rate of H2 photoproduction was observed to occur at pH8.0.The H2 photoproduction rate ofChlamydomonas under optimal pH value?pH8.0?was mainly increased by the relatively high residual activity of photosystem II?PSII?,which provided a relatively rich source of electrons for H2 ase,compared with the growth pH value?pH7.3?.Moreover,we proved that the improvement of H2 photoproduction rate of Chlamydomonas was not related to the activity of alternative electron sinks?cyclic electron transport around photosystem I and CO2 fixation?.In addition,we noticed that SO2 derivatives at least contain bisulfite and sulfite?Na2SO3?and,pH values can change the ratio of bisulfite to sulfite in the cell cultures.Moreover,the toxicity of bisulfite to the growth of algal cells was much more than that of sulfite.As a consequence,it is logical to hypothesize that under pH8.0,increasing the H2 photoproduction rate of Chlamydomonas is likely to be a result of decreased the proportion of bisulfite to sulfite.This hypothesis was confirmed that the combination7 m M bisulfite and 6m M sulfite could further improve the efficiency of H2 photoproduction,compared with the 13 m M bisulfite alone,which is consistent with the result that the toxicity of bisulfite to PSII is much greater than that of sulfite.Based on these results,this thesis found that the H2 photoproduction in bisulfite-treated Chlamydomonas cells can be further optimized by changing the initial extracellular pH values,and the hydrogen production rate reached the maximum at pH 8.0.This is likely that the ratio of bisulfite to sulfite at optimal pH will decrease when compared to growth pH7.3,which weakens the toxicity of bisulfite to Chlamydomonas cells and improved the relative residual activity of PSII.As a consequence,abundant electron source for H2 ase finally improves the photosynthetic hydrogen production level of Chlamydomonas.Therefore,changing the initial extracellular pH values in the background of bisulfite addition is an important strategy to further optimize the photosynthetic hydrogen production of Chlamydomonas by decreasing the proportion of bisulfite to sulfite.