Effects of pH and Carbon Sources on Biohydrogen Production by Co-Culture of Clostridium butyricum and Rhodobacter sphaeroides

To improve the hydrogen yield from biological fermentation of organic wastewater, a co-culture system of dark- and photo-fermentation bacteria was investigated. In a pureculture system of the dark-fermentation bacterium Clostridium butyricum, a pH of 6.25 was found to be optimal, resulting in a hydrogen production rate of 18.7 ml-H₂/l/h. On the other hand, the photosynthetic bacterium Rhodobacter sphaeroides could produce the most hydrogen at 1.81 mol-H₂/mol-glucose at pH 7.0. The maximum specific growth rate of R. sphaeroides was determined to be 2.93 h⁻¹ when acetic acid was used as the carbon source, a result that was significantly higher than that obtained using either glucose or a mixture of volatile fatty acids (VFAs). Acetic acid best supported R. sphaeroides cell growth but not hydrogen production. In the co-culture system with glucose, hydrogen could be steadily produced without any lag phase. There were distinguishable inflection points in a plot of accumulated hydrogen over time, resulting from the dynamic production or consumption of VFAs by the interaction between the dark- and photofermentation bacteria. Lastly, the hydrogen production rate of a repeated fed-batch run was 15.9 ml-H₂/l/h, which was achievable in a sustainable manner.