Influence of reduced electron shuttling compounds on biological H ₂ production in the fermentative pure culture Clostridium beijerinckii

Several reports suggest that extracellular electron shuttles influence fermentative metabolism in a beneficial manner for bioremediation and biotechnology strategies. The focus of this research was to characterize the effects of reduced electron shuttling molecules on fermentative H₂ production. Reduced electron shuttles may provide reducing equivalents to generate H₂, which influences alternate cellular processes. Electron shuttling compounds cycle between reduced-oxidized states and influence fermentative physiology. Clostridium beijerinckii fermentation was altered using a physiological approach that resulted in H₂ production with the reduced extracellular electron shuttle anthrahydroquinone-2,6,-disulfonate (AH₂QDS) and biologically reduced humic substances as the primary electron donors. Cells were suspended in a buffer with an excess of the biological electron transfer molecule NAD⁺, with AH₂QDS (100-1000 μM) or biologically reduced humic substances (0.01-0.025 g/L) as the sole electron source. Increasing concentrations of AH₂QDS and reduced humics increased H₂ production, while H₂ production was suppressed by Fe(III) hydroxides, which outcompeted the cells for electrons from the reduced shuttles, suggesting that the shuttles are in fact electron donors for H₂ production. Oxidized AQDS/humics did not increase H₂ production. Organic acid production shifted toward butyric acid in the presence of reduced electron shuttles, particularly with growing cells. Growth and hydrogen production rates in growing cells were initially faster in the presence of the reduced electron shuttles; however, the final biomass yield was inversely proportional to the starting AH₂QDS concentration, which suggests that reduced shuttles may compete with anabolic cell processes for available energetic resources or that the shift to excess butyrate becomes toxic to the cells.