Interactive Effects of Limonene and Phenol on Dark Fermentative Hydrogen Production: Metabolic and Microbial Insights

Citrus waste has been classified as inefficient for biogas production through biological processes due to the presence of limonene and phenolic compounds formed from its hydrolysis. However, the effects of limonene and phenol on hydrogen (H₂) production via dark fermentation remains unclear. To address this gap, batch experiments were conducted to evaluate the synergistic-antagonistic interactions between limonene and phenol during dark fermentation without interference from other compounds. For this reason, glucose (5 g/L) was used as the substrate, and granular anaerobic sludge was used as the inoculum. Limonene and phenol were added at known concentrations across six binary proportions (100:0, 80:20, 60:40, 40:60, 20:80, and 0:100), and interactions were assessed using the isobologram method. Results indicated an antagonistic interaction; the 40:60 limonene: phenol ratio yielded the highest H₂ production, with a total accumulated volume of 296.6 ± 7.9 mL H₂. The primary fermentation metabolites were lactic acid, acetic acid, and propionic acid. It was also observed that the concentrations of both limonene and phenol decreased after fermentation, suggesting their partial degradation by fermentative H₂-producing microorganisms. Enterobacteriaceae dominated across all treatment combinations, demonstrating their capacity to produce H₂ in the presence of limonene and phenol. These findings demonstrate that H₂ production is feasible despite the presence of the inhibitory compounds.