Autotrophic and mixotrophic hydrogen photoproduction in sulfur-deprived chlamydomonas cells

Abstract

In Chlamydomonas reinhardtii cells, H2 photoproduction can be induced in conditions of sulfur deprivation in the presence of acetate. The decrease in photosystem II (PSII) activity induced by sulfur deprivation leads to anoxia, respiration becoming higher than photosynthesis, thereby allowing H2 production. Two different electron transfer pathways, one PSII dependent and the other PSII independent, have been proposed to account for H2 photoproduction. In this study, we investigated the contribution of both pathways as well as the acetate requirement for H2 production in conditions of sulfur deficiency. By using 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), a PSII inhibitor, which was added at different times after the beginning of sulfur deprivation, we show that PSII-independent H2 photoproduction depends on previously accumulated starch resulting from previous photosynthetic activity. Starch accumulation was observed in response to sulfur deprivation in mixotrophic conditions (presence of acetate) but also in photoautotrophic conditions. However, no H2 production was measured in photoautotrophy if PSII was not inhibited by DCMU, due to the fact that anoxia was not reached. When DCMU was added at optimal starch accumulation, significant H2 production was measured. H2 production was enhanced in autotrophic conditions by removing O2 using N2 bubbling, thereby showing that substantial H2 production can be achieved in the absence of acetate by using the PSII-independent pathway. Based on these data, we discuss the possibilities of designing autotrophic protocols for algal H2 photoproduction.

FIG. 1.

Effects of DCMU on H₂ production, O₂, CO₂ exchange, and starch accumulation in sulfur-deprived Chlamydomonas cells. (A) Control in the absence of DCMU. (B) DCMU (20 μM final concentration) was added at t₀. Sulfur deprivation was realized by resuspending cells in sulfur-deprived TAP medium at t_0. Relative quantities of gases contained in closed flasks were measured by mass spectrometry and are expressed as the percentage of the gas phase volume. (C) Intracellular starch amounts measured at different times in the culture conditions described for panels A and B.

FIG. 2.

Effects of retarded DCMU addition on H₂ production, O₂, CO₂ exchange, and starch accumulation in sulfur-deprived Chlamydomonas cells. (A) Control in the absence of DCMU. (B) DCMU (20 μM final concentration) was added 24 h after the beginning of sulfur deprivation. (C) Intracellular starch amounts measured at different times in the culture conditions described for panels A and B. Other experimental conditions are similar to those described for Fig. 1.

FIG. 3.

Effects of acetate on H₂ production, O₂, CO₂ exchange, and starch accumulation in sulfur-deprived Chlamydomonas cells. (A) Control in the presence of acetate (TAP medium). (B) Acetate was omitted from the culture medium (minimal medium supplemented with 20 mM bicarbonate); in this experiment, N₂ bubbling was achieved 24 h after the beginning of sulfur deprivation. For both experiments, sulfur deprivation was achieved at t₀. (C) Intracellular starch amounts measured at different times in the culture conditions described for panels A and B.

FIG. 4.

Effects of DCMU addition and N₂ flushing on H₂ production, O₂, CO₂ exchange, and starch accumulation by Chlamydomonas cells in sulfur-deprived minimal medium supplemented with 20 mM bicarbonate. (A) DCMU was added at t₀, (B) DCMU was added at 24 h, and (C) DCMU was injected and N₂ bubbling was performed at 24 h. For the three experiments, sulfur deprivation was achieved at t₀. (D) Intracellular starch amounts measured at different times in the culture conditions described for panels A, B, and C.