Response surface methodology was employed to establish a thermophilic hydrogen production process from co-substrate of food waste (FW) and waste activated sludge (WAS), resulting in a maximum hydrogen production of 2602.68 ± 54.88 mL/L, which was 5.4 and 21.9 times of FW and WAS, respectively. The co-substrate facilitated the butyrate pathway of hydrogen production and decreased lactate accumulation, and significantly increased the activity of butyric kinase and hydrogenase (p < 0.05). Meanwhile, it could promote microbial growth by creating a more suitable redox environment. Microbial community analysis showed that Thermoanaerobacterium (hydrogen production genus) was specifically enriched and dominant in co-substrate (82.3%). Further functional prediction analysis showed that the co-substrate effectively promoted carbohydrate metabolism. Furthermore, pretreatment improved sludge biodegradability. This study establishes a feasible hydrogen production process, profoundly revealed the mechanism of enhanced anaerobic fermentation from the perspective of microbial growth and metabolism, which lays solid foundation on the hydrogen production from waste biomass.
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