Hydraulic retention time (HRT) influences the H2 generation process significantly. Reducing HRT from 18 to 12 h has improved H2 yield without affecting substrate removal efficiency [57]. Maximum H2 yield was reported between 0 and 14 h in all the experimental variations studied in batch mode during dairy, chemical and distillery wastewater treatment [22, 25, 31, 38]. Longer fermentation periods induce a metabolic shift from the acidogenic process to the methanogenic pro­cess which is unfavorable for H2 production. Shorter HRT’s have been shown to dilute out slow growing MB [86]. However, in continuous operation mode, H2 production was observed at long HRTs of 3 days (pH 6.4) without encountering problems with methanogenesis [87]. Optimal HRT mostly depends on the nature and composition of the substrate, function of biocatalyst, loading rate and fermen­tation pH employed. HRT can be considered as a readily manipulated variable for process control. Optimum HRTs from 8.0 to 14 h were reported for effective H2 production [57].