Effects of nitrate concentration on biohydrogen production and substrate utilization in dark-fermentation

Batch experiments were conducted to investigate the effect of the nitrate (NO₃ ⁻) concentration on the hydrogen production and substrate utilization in a dark fermentation process. The nitrate concentration was varied from 0 to 5,000 mg L⁻¹ in experiments with a mixed culture of anaerobic sludge. Anaerobic sludge was treated with 2 M HCl at pH 2 for the enhanced biohydrogen production. With added NO₃ ⁻ concentration of 0 g/L, the maximum hydrogen production was 2.2 mol-H₂ mol- glucose⁻¹. But, an increase in NO₃ ⁻ concentration, the hydrogen production decreased to 0.3 from 2.2 mol-H₂ mol- glucose⁻¹ at 4,000 mg NO ₃ ⁻  L⁻¹. But, in all conditions, more than 90 % of initially added glucose (10 g L⁻¹) was removed and the microbial community structures were observed similarly in every condition and main germinated species was detected as Clostridium perfringens by DNA cloning. As a result, it was confirmed that the high nitrate concentration at the initial stage, glucose was utilized for nitrate removal reaction than hydrogen production.