Succession of the Bacterial Community and Dynamics of Hydrogen Producers in a Hydrogen-Producing Bioreactor

Biological hydrogen production is expected to be an important strategy in the development of clean and sustainable alternative energy sources (6). Anaerobic mixed cultures are frequently used as inocula in hydrogen production (3, 4, 10). Compared with pure cultures, mixed cultures have the advantage of more technical feasibility and the potential for using complex carbohydrates as substrates (14), possibly because different members of the bacterial community play complementary or mutually beneficial roles in utilizing substrates, providing growth factors, eliminating feedback inhibition, etc. (12, 17). Many sources of natural microflora, including compost (20), different sludges (4, 10, 24), and soils (21), have been used as inocula for hydrogen production after inactivation of hydrogenotrophic methanogens. However, whether pretreated natural microflora is the most efficient mixed culture for hydrogen production is still unknown. Understanding the relationships between variation in microbial composition and its hydrogen production efficiency is the first step in reconstructing more-efficient hydrogen-producing consortia. The 16S rRNA gene has been widely used as a universal molecular marker (7, 15). The Fe-hydrogenase gene (hydA), which is usually involved in proton reduction (H2 production) to dispose of excess reducing equivalents (1, 2, 22) in Clostridium spp. and sulfate reducers, has recently been used as a molecular marker to distinguish potential hydrogen-producing bacteria in mixed cultures (3, 8, 23). Therefore, in this study, the V3 regions of the 16S rRNA gene and the hydA gene were used as biomarkers to investigate the succession of the bacterial community during hydrogen production in a batch culture. Hydrogen production. Hydrogen production was conducted in a 10-liter continuously stirred tank reactor (CSTR) with a 7-liter working volume and no specific requirement to remove oxygen from the culture and headspace. Cattle dung compost was pretreated by boiling for 5 min and then used as an inoculum. The fermentation reaction mixture was kept at pH 5.8, with stirring at 100 rpm. Other fermentation conditions for batch hydrogen production from 18 g liter 1 sucrose were the same as in a previous study (27). The evolved biogas was collected by the water displacement method. The concentrations of hydrogen in the biogas were quantified as previously described (18). A total of 25.6 liters of hydrogen was evolved in the batch fermentation, corresponding to a hydrogen yield of 3.11 mol H2 mol sucrose 1 or 200 ml H2 g sucrose 1 . The reported high hydrogen yields from sucrose in dark fermentation were 3.8 mol H2 mol sucrose 1 by a Clostridium pasteurianum