Effect of COD/SO₄^2-ratio and Fe(II) under the variable hydraulic retention time (HRT) on fermentative hydrogen production

The effect of chemical oxygen demand/sulfate (COD/SO₄^2-) ratio on fermentative hydrogen production using enriched mixed microflora has been studied. The chemostat system maintained with a substrate (glucose) concentration of 15 g COD L^-1 exhibited stable H₂ production at inlet sulfate concentrations of 0-20 g L^-1 during 282 days. The tested COD/SO₄^2- ratios ranged from 150 to 0.75 (with control) at pH 5.5 with hydraulic retention time (HRT) of 24, 12 and 6 h. The hydrogen production at HRT 6 h and pH 5.5 was not influenced by decreasing the COD/SO₄^2- ratio from 150 to 15 (with control) followed by noticeable increase at COD/SO₄^2- ratios of 5 and 3, but it was slightly decreased when the COD/SO₄^2- ratio further decreased to 1.5 and 0.75. These results indicate that high sulfate concentrations (up to 20,000 mg L^-1) would not interfere with hydrogen production under the investigated experimental conditions. Maximum hydrogen production was 2.95, 4.60 and 9.40 L day^-1 with hydrogen yields of 2.0, 1.8 and 1.6 mol H₂ mol^-1 glucose at HRTs of 24, 12 and 6 h, respectively. The volatile fatty acid (VFA) fraction produced during the reaction was in the order of butyrate > acetate > ethanol > propionate in all experiments. Fluorescence In Situ Hybridization (FISH) analysis indicated the presence of Clostridium spp., Clostridium butyricum, Clostridium perfringens and Ruminococcus flavefaciens as hydrogen producing bacteria (HPB) and absence of sulfate reducing bacteria (SRB) in our study.