Dark fermentation: isolation and characterization of hydrogen-producing strains from sludges

To improve bacterial hydrogen production, ten hydrogen-producing strains belonging to Clostridium spp. were isolated from various sludges under low vacuum. Hydrogenogenesis by dark fermentation in batch cultures of these strains was optimal at about 35 degrees C and an initial pH of 6.5, which for all strains gradually dropped to ca. pH 4 during the fermentation. Clostridium roseum H5 and C. diolis RT2 had the highest hydrogen yields per total substrate (120 ml H-2/g initial COD). Substrate consumption alone by C. beijerinckii UAM and C. diolis RT2 reached 573 and 475 ml H-2/g consumed COD, respectively. Butyric acid fermentation was predominant, with butyrate and acetate as the major by-products and propionate, ethanol, and lactate as secondary metabolites. The acetate:butyrate ratios and fermentation pathways varied depending on the strains and environmental conditions. Hydrogenogenesis was studied in greater detail in C. saccharobutylicum Hi. In butyric acid fermentation by this representative strain, acetoacetate was detected as an intermediate metabolite. Hydrogenogenesis was also analyzed in an enrichment culture, which behaved similarly to the axenic cultures.