Optimizing Biohydrogen Production from Orange Waste in Dark Fermentation Process: Effect of Substrate Concentration and Initial pH

Recent studies have highlighted the potential of agricultural and food industry waste as substrates for biohydrogen production through dark fermentation. Among these wastes, orange waste is particularly promising due to its high water and organic matter content. It is produced in large quantities and lacks a standardized final disposal method, making it suitable for recovery. However, limonene has been reported as a limiting component in biological processes due to its inhibitory effect. The objective of this study was to determine the optimal substrate concentration and initial pH for hydrogen production from orange peels using a central composite design (22) with 13 runs. Batch experiments were conducted under mesophilic conditions (37 °C) with varying initial pH and substrate concentration, using granular anaerobic sludge as the inoculum and maintaining a substrate-to-inoculum ratio of 2.7. Additionally, metabolite production, kinetic parameters, limonene content, and microbial community composition were analyzed. The results indicated that the optimal initial pH and substrate concentration are 7.1 and 29.8 g VS/L, respectively, with a predicted cumulative hydrogen volume of 307.9 mL H2. Limonene at 402 mg/L had no effect on hydrogen production, and no inhibition was observed. Lactic acid was one of the main metabolites in runs with substrate concentrations above 30 g VS/L; however, the high lactic acid content did not correlate with decreased hydrogen production under certain conditions. The test that obtained the highest hydrogen production showed greater dominance of Citrobacter, Clostridium sensu stricto 1, Lacticaseibacillus, Levitactobacillus, and Lactiplantibacillus.