Metabolomics of photobiological hydrogen production induced by CCCP in Chlamydomonas reinhardtii

• Addition of a proton uncoupler in Chlamydomonas reinhardtii yields 13-fold H2 increase for 12 h.
• This treatment reduces free amino acids and unsaturated free fatty acids.
• Conversely, saturated free fatty acids levels are increased.
• CCCP might become a useful treatment for production of biohydrogen in reactors.

The green alga Chlamydomonas reinhardtii can produce hydrogen gas (H2) in the presence of the proton uncoupler carbonyl cyanide m-chlorophenyl hydrazone (CCCP). The addition of 15 μM CCCP to the algal cultures led to 13-fold increase in H2 photoproduction compared to the control cultures without CCCP treatment. CCCP completely inhibited the photochemical activity of photosystem (PS) II under illumination. In order to better understand metabolic conditions necessary for sustained H2 production, we have used gas chromatography coupled to time-of-flight mass spectrometry (GC-TOF) for metabolomics analysis that is independent of nutritional stress, specifically, sulfur deprivation, which had been used previously to induce H2 photoproduction. Even 10 min after addition of CCCP, metabolites from many metabolic modules were found drastically decreased, including levels of free amino acids, unsaturated free fatty acids and nucleotides. During prolonged CCCP exposure H2 production was found to be stable for at least 12 h with a continued increase in levels of free fatty acids. These results indicate that CCCP might become a useful treatment for production of biohydrogen in reactors. The increase in fatty acid production might then be a useful addition for production of carbon-derived biofuels.