| Summary: | ermentative H2 production was studied using raw POME as the substrate with heat-shock pre-treated POME anaerobic sludge acted as the inoculum. The effect of crucial operating variables (initial pH of medium, incubation temperature, and inoculum size) on H2 production was studied using Box−Behnken Design. A second-order polynomial regression model was generated to evaluate H2 production trend under conditions tested. The model analysis revealed the high significance of linear effects of initial pH, incubation temperature, and inoculum size (P < 0.01) towards H2 production. Similar results indicated that the interaction effect between initial pH and inoculum size, and interaction effect between incubation temperature and inoculum size were highly significant (P < 0.01). The regression model suggested that the optimum conditions were set to a pH value, incubation temperature and inoculum size of 6.4, 58.0°C and 8.0% v/v, respectively. In order to validate the optimum conditions determined by the model, heat-shock pre-treated POME anaerobic sludge was incubated with raw POME under optimum conditions. Validation experiment showed that a cumulative H2 volume of 239.0 mL was produced. Microbial community analysis of inoculum showed that mixed consortia between Clostridium sp. and other obligate anaerobic non-spore forming bacteria, mostly belonging to the Firmicutes and Bacteroidetes phyla were identified as the major H2-producers and were hugely responsible towards fermentative H2 production.
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