Comparative effect of acid and heat inoculum pretreatment on dark fermentative biohydrogen production

This study delves into the impact of various pretreatment methods on the inoculum in dark fermentation trials, specifically exploring thermal shock at different temperatures (60, 80, and 100 °C) and durations (15, 30, and 60 min), as well as acid shock at pH 5.5. Initial acidification of the substrate/inoculum mixture facilitates H₂ generation, making acid shock an effective pretreatment option. However, it is also observed that combining thermal and acid pretreatments boosts H₂ production synergistically. The synergy between thermal and acid pretreatments results in a significant improvement, increasing the overall hydrogen production efficiency by more than 9% compared to assays involving acidification alone. This highlights the considerable potential for optimizing pretreatment strategies. Furthermore, the study sheds light on the critical role of inoculum characteristics in the process, with diverse hydrogen-generating bacteria significantly influencing outcomes. The established equivalent performance of HCl and H₂SO₄ in inoculum pretreatment demonstrates the versatility of these acids in shaping the microbial community and influencing hydrogen production. The analysis of glucose conversion data highlights a prevalence of butyric acid in all trials, irrespective of the pretreatment method, emphasizing the dominance of the butyrate pathway in hydrogen generation. Additionally, an examination of the microbial community offers valuable insights into the intricate relationships between temperature, pH, and microbial diversity. Bacteroidota established its dominance among the bacterial populations, with a relative abundance exceeding 20-25% in the raw inoculum, and this dominance further increased following the treatment. Thermal and acid pretreatments result in significant shifts in dominant microbial communities, with some non-dominant phyla like Cloacimonadota and Spirochaetota becoming more prominent. These shifts in microbial diversity underscore the sensitivity of microbial communities to environmental conditions and pretreatment methods, further highlighting the importance of understanding their dynamics in dark fermentation processes.