Strong pH dependence of hydrogen production from glucose by Rhodobacter sphaeroides

Purple non-sulfur bacteria (PNSB) are well known for converting short-chain organic acids to H₂, however, a decrease in pH caused by metabolic acids production limited H₂ production during the photo-fermentation from glucose. Here we address why volatile fatty acids (VFA) excreted as fermentation products cannot be further degraded by R. sphaeroides that readily use them. We found that the photo-fermentation with pH controlled at 6.9 ± 0.1 resulted in a 90% increase of H₂ yield and a 107.6% increase in volume H₂ production relative to the pH-uncontrolled culture. Comparative fermentations on glucose at pH 5.8 and pH 7.1 using culture medium supplemented with 50% spent fermentation broth demonstrated that low pH alone is not the limiting factor and compounds present in the supernatants along with pH decrease were the most inhibitory to H₂ production. The impact of byproducts VFA on phototrophic H₂ production was dependent on both the pH and VFA concentrations; even 7 mM VFA addition totally inhibited H₂ production from glucose at pH 5.4. H₂ production with pH control for the Δhup strain was not discernibly different from the parent strain, which are all significantly higher than high-performance strains by metabolic engineering. These results demonstrate that pH dependent VFA inhibition can be turned into a driving force for enhanced H₂ production from glucose by pH regulation.