Characterization of the transient fluorescence wave phenomenon that occurs during H2 production in Chlamydomonas reinhardtii

Abstract

The redox state of the plastoquinone (PQ) pool in sulfur-deprived, H2-producing Chlamydomonas reinhardtii cells was studied using single flash-induced variable fluorescence decay kinetics. During H2 production, the fluorescence decay kinetics exhibited an unusual post-illumination rise of variable fluorescence, giving a wave-like appearance. The wave showed the transient fluorescence minimum at ~60 ms after the flash, followed by a rise, reaching the transient fluorescence maximum at ~1 s after the flash, before decaying back to the initial fluorescence level. Similar wave-like fluorescence decay kinetics have been reported previously in anaerobically incubated cyanobacteria but not in green algae. From several different electron and proton transfer inhibitors used, polymyxin B, an inhibitor of type II NAD(P)H dehydrogenase (NDA2), had the effect of eliminating the fluorescence wave feature, indicating involvement of NDA2 in this phenomenon. This was further confirmed by the absence of the fluorescence wave in the Δnda2 mutant lacking NDA2. Additionally, Δnda2 mutants have also shown delayed and diminished H2 production (only 23% if compared with the wild type). Our results show that the fluorescence wave phenomenon in C. reinhardtii is observed under highly reducing conditions and is induced by the NDA2-mediated electron flow from the reduced stromal components to the PQ pool. Therefore, the fluorescence wave phenomenon is a sensitive probe for the complex network of redox reactions at the PQ pool level in the thylakoid membrane. It could be used in further characterization and improvement of the electron transfer pathways leading to H2 production in C. reinhardtii.

Keywords: Chlamydomonas reinhardtii; hydrogen production; plastoquinone pool; sulfur deprivation; type II NDH; variable fluorescence.

Fig. 1.

Schematic representation of the electron and proton transfer pathways leading to H₂ production in C. reinhardtii cells under S-dep conditions. Inhibitors used in this study and sites of their action are represented by blue circles with corresponding numbers: DCMU (1), DBMIB (2), antimycin A (3), gramicidin D (4), polymyxin B (5), rotenone A (6), and sodium azide (7). Photosynthetic, fermentative, and CEF pathways are indicated by black, gray, and white arrows, respectively.

Fig. 2.

Changes in the flash-induced fluorescence decay kinetics during S-dep and H₂ production in C. reinhardtii cells. (A) The amount of O₂ (blue circles) and photoproduced H₂ (red circles) in the gaseous phase during incubation of C. reinhardtii under S-dep conditions in bioreactors. The experiment time course was divided into two stages: O₂ consumption stage (I) and H₂-producing stage (II). The results represent three individual experiments, and the values are the mean values ±SD. The colored arrows (for the color code, see B and C) indicate the time points at which samples were withdrawn for the fluorescence measurements. (B) Flash-induced fluorescence traces measured during the O₂ consumption stage I after 0 h (control, black), 17 h (red), and 24 h (blue) of S-dep. (C) Flash-induced fluorescence decay traces measured during the H₂-producing stage II after 48 h (white), 72 h (green), 96 h (pink), 120 h (brown), and 144 h (gray) of S-dep. Measurements were performed after 5 min of dark adaptation. The time of the actinic flash is indicated with a black arrow.

Fig. 3.

Effect of anaerobic conditions on the flash-induced fluorescence decay kinetics in C. reinhardtii cells. Anaerobic conditions were created either by (A), S-dep (TAP-S) at 0 h (control, ●) and 48 h (○) or (B), by addition of glucose, glucose oxidase, and catalase to the cells grown in regular TAP medium as described in the Materials and methods before (●) and after 15 min of incubation (◯). Traces are normalized to the same F₀ and F_m level; non-normalized traces are shown in insets. The time of the actinic flash is indicated with a black arrow.

Fig. 4.

Effect of inhibitors of the photosynthetic linear electron flow (LEF) on the flash-induced fluorescence decay kinetics of C. reinhardtii cells during S-dep. Traces shown are (A), 0 h and (B) 48 h of S-dep with no inhibitor (●, ◯), 20 μM DCMU (▲, △), and 5 μM DBMIB (◆, ◇). Traces are normalized to the same F₀ and F_m level. The time of the actinic flash is indicated with a black arrow.

Fig. 5.

Effect of inhibitors of the photosynthetic cyclic electron flow (CEF) and proton gradient (A), and mitochondrial electron transport (B) on the flash-induced fluorescence decay kinetics of C. reinhardtii cells during S-dep. Traces shown are after 48 h of S-dep with no inhibitor (◯), 4 μM antimycin A (△), 10 μM gramicidin D (◇), 20 μM rotenone A (☐), and 1 mM sodium azide, 5 (*). Traces are normalized to the same F₀ and F_m level; non-normalized traces are shown in insets. The time of the actinic flash is indicated with a black arrow.

Fig. 6.

Effect of inhibition of type-II NDH by polymyxin B on the flash-induced fluorescence decay kinetics of C. reinhardtii cells. (A), Cells grown in regular TAP medium with no inhibitor (●) or with 400 μM polymyxin B (◆). (B) Cells after 48 h of S-dep (TAP-S) with no inhibitor (◯) or with 400 μM polymyxin B (◇). Traces are normalized to the same F₀ and F_m level; non-normalized traces are shown in insets. The time of the actinic flash is indicated with a black arrow.

Fig. 7.

H₂ production in the presence of the different inhibitors used in this study (Table 1). Chlamydomonas reinhardtii cells in the H₂-producing stage at 48 h of S-dep were incubated with different inhibitors, and H₂ production was measured after 22 h of incubation: control (no inhibitor), 20 μM DCMU, 5 μM DBMIB, 4 μM antimycin A, 10 μM gramicidin D, 20 μM rotenone A, 400 μM polymyxin B, and 1 mM sodium azide. Control (no inhibitor) was set to 100%. The presented results were obtained in three individual experiments, and values represented are the mean values ±SD.

Fig. 8.

Effect of gramicidin D, a proton gradient inhibitor, on the flash-induced variable fluorescence decay kinetics of C. reinhardtii cells under different anaerobic conditions. (A) S-dep cells incubated under aerobic conditions for 0 h (black), 17 h (red), and 42 h (white). (B) S-dep cells incubated under aerobic conditions for 42 h with no addition (◯), or further incubated for 15 min after addition of glucose, glucose oxidase, and catalase in the absence (◇) or presence of gramicidin D (△). Traces are normalized to the same F₀ and F_m level; non-normalized traces are shown in insets. The time of the actinic flash is indicated with a black arrow.

Fig. 9.

S-dep and H₂ production in the Δnda2 mutant and WT-CC4533 strains of C. reinhardtii. (A) Genomic PCR analysis with the primers NDA2MC F and NDA2MC R for nda2, N, and with the primers oMJ282 F and oMJ284 R for the control gene, C; M, 1 kb DNA ladder; ∆nda2, PCR product with ∆nda2 DNA as a template; WT, PCR product with wild-type genomic DNA as a template. Changes in the amount of O₂ (blue circles) and photoproduced H₂ (red circles) in the gaseous phase during incubation of the C. reinhardtii WT-CC4533 cells (B) and the Δnda2 mutant cells (C). In addition to the O₂ consumption stage I and the H₂-producing stage II, the O₂-producing stage was observed in the Δnda2 mutant. The results represent three individual experiments, and values represented are the mean values ±SD. The colored arrows (for the color code, see Fig. 10A and B, respectively) indicate the time points where samples were withdrawn for the fluorescence measurements.

Fig. 10.

Flash-induced fluorescence decay traces measured during S-dep of C. reinhardtii cells in the WT-CC4533 cells (A) after 0 h (black) and 24 h (white) (see Fig. 9B) and in the Δnda2 mutant cells (B) after 0 h (black), 65 hrs (red), 96 h (blue), and 113 h (white) (see Fig. 9C). (C) Comparison of the normalized fluorescence decay kinetics from the H₂-producing WT-CC4533 cells (24 h of S-dep, ◯) and the Δnda2 mutant cells (113 h of S-dep, ☐). The time of the actinic flash is indicated with a black arrow.

Fig. 11.

Proposed scheme (B) to explain different phases (A) during the transient fluorescence wave phenomenon at the H₂-producing stage in C. reinhardtii (see Conclusions for explanation).