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Oxygen (O2) is a strong inhibitor of hydrogenase (HydA) activity and expression and altering the sulfur (S) oxidizing transitions in photosystem II (PSII) often allows algal photohydrogen production; however, this may not be practical in a wastewater environment. To counteract natural mechanisms of oxygen evolution in PSII, we utilized acetic acid and butyric acid, which are main volatile fatty acids (VFAs) found in anaerobic bacterial digestion in wastewater treatment, as oxygen regulators for photosynthetic biohydrogen production using Chlorella vulgaris. It was found that a VFA-containing synthetic wastewater promotes oxygen depletion in a photobioreactor (PBR), producing maximum hydrogen yield of 65.4 ± 0.3 μmoL H2 L−1 mM−1 acetate without artificial sulfur or chloride deprivation. Butyric acids showed no significant effect on oxygen depletion and biohydrogen production in the PBR. The measurements of both relative expression level of mRNA and specific activities of reactivate HydA revealed that repetitive algal H2 photo-evolution was possible by HydA synthesis in C. vulgaris followed by complete oxygen depletion controlled by acetic acid levels in the PBR. This emerging understanding of the role of VFAs on oxygen regulation in PSII in natural environments is expected to lead algal-driven bioenergy production technologies to the next level.

Microalgal (Micractinium reisseri YSW05) photo-heterotrophic hydrogen production was investigated with using volatile fatty acids (acetate and butyrate) from dark-fermentation effluent. Dilution ratio (effluent: deionized water) of 1:0 at pH 8.0 supported the maximum hydrogen and biomass production, and the highest acetate and butyrate consumption. Microalgal hydrogen production was higher with continuous light condition compared to alternating dark/light cycle. Hydrogen production correlated with both, biomass production and hydrogenase activity under all experimental conditions. Hydrogenase activity and apparent hydrogen production decreased significantly with increasing headspace oxygen. Interestingly substantial hydrogenase activity was observed at almost atmospheric partial pressure of oxygen (19%), indicating that the enzyme could be oxygen tolerant. The present study demonstrated the application of conventional microbial fermentation effluent for microalgae cultivation and hydrogen production, and showed the presence of oxygen tolerant hydrogenase in M. reisseri which might help to establish a novel strategy for biohydrogen technology in atmospheric condition.