Fig. 1

H₂ production in Chlamydomonas reinhardtii. a Schematic presentation of the photosynthetic electron transport chain in green algae. Solar energy is captured by the light-harvesting complexes (LHC) of photosystem II and I (PSII and PSI). Electrons extracted from water by the oxygen-evolving complex (OEC) of PSII are transferred to the photosynthetic electron transport chain via the plastoquinone (PQ)-pool, the cytochrome b₆f complex (cyt b₆f), plastocyanin (PC), PSI and ferredoxin (Fd). From Fd, electrons can be transferred by the ferredoxin-NADP⁺ oxidoreductase (FNR) to NADP⁺ or to the hydrogenase (HydA; for clarity, oxygen-dependent alternative pathways are not shown). H⁺ accumulated in the thylakoid lumen are used for ATP production via ATP synthase. The ATP and NADPH generated during primary photosynthetic processes are consumed for CO₂ fixation in the Calvin–Benson–Bassham (CBB) cycle, which produces sugars and ultimately starch. When cultures are grown in the presence of acetate, glycerate 3-phosphate (GP) may also feed the CBB cycle. Hydrogenases are expressed under anoxic conditions; upon illumination, significant H₂ production may occur. The water-oxidation dependent pathway of H₂ production is denoted with red line. Depending on the conditions, starch degradation may also contribute to H₂ production either via the NAD dehydrogenase (NDH) complex, PSII-independent pathway, or via pyruvate-Fd-oxidoreductase (PFR). b H₂ production in CC124 Chlamydomonas cultures (50 µg chl (a + b)/ml, at 320 µmol photons/m²/s) as determined in the headspaces of sealed cultures using gas chromatography during 24 h in acetate-containing (TAP, HSA) and acetate-free media (TP, HS) following dark anaerobic incubation (4 h darkness with 3 × 10 min N₂ flushing). In the right Y axis, the percentage of H₂ in the headspaces of the cultures are shown. Time point 0 indicates the time when the cultures were transferred to the light. c Net O₂ production under the same conditions as in b. d Daily H₂ production in sulphur-containing HS medium of cultures subjected to dark anaerobic incubation and in cultures transferred to sulphur-free acetate-containing (TAP-S) or acetate-free (TP-S) media. All the alga cultures (HS, TAP-S, TP-S) were illuminated continuously and flushed every 24 h with N₂ after determining the gas concentrations in the headspaces of the sealed bottles to avoid excessive H₂ accumulation (cf. [7]) and overpressure in the headspace. e Net O₂ production under the same conditions as in d. Mean values (± SEM) are each based on 6 biological replicates. Statistical significance levels are presented relative to the Chlamydomonas culture subjected to dark anaerobic incubation in HS medium as *p < 0.05, **p < 0.01, ***p < 0.001