Optimal operational conditions for biohydrogen production from sugar refinery wastewater in an ASBR

•Organic loading rate had the greatest impact on H2 production rate.•Hydraulic retention time had less significant influence on H2 content and yield.•pH was a critical factor affecting the bacterial species that contributed to H2 production.•Clostridium spp. were dominant H2-producing microbes even without seed sludge pretreatment.•Ethanol-to-acetic acid ratio was highly proportional to H2 content for pH 5.0 and 5.5.

The performance of biohydrogen production in an anaerobic sequencing batch reactor (ASBR) was evaluated with respect to variations in the key operational parameters – pH, hydraulic retention time HRT, and organic loading rate OLR using sugar refinery wastewater as substrate. Analysis of variance (ANOVA) indicated HRT had less significant influence on hydrogen content and yield in comparison to pH and OLR, whereas OLR has much impact on hydrogen production rate. Taxonomic analysis results showed that diverse bacterial species contributed to hydrogen production and the dominant species in the bioreactor were governed by all operational parameters. Even without pretreatment of the seed sludge, a high proportion of Clostridium spp. over the other bacterial species was observed at pH 5.5, and this is compatible with the high hydrogen productivity. Consequently, pH 5.5, HRT 10 h, and OLR 15 kg/m3 d were delineated as the optimal operational conditions for an ASBR fed with sugar refinery wastewater.