Elucidating Metabolic Shifts in Thermophilic Biohydrogen Production: Effects of Inoculum Source, Pre-treatment and Substrate Hydrolysis

Abstract

Biohydrogen production through dark fermentation (DF) is a promising process that attracts increasing attention in the context of energy transition. However, a deeper understanding is necessary to enable its upscale to industrial applications. This study investigated DF using three inocula sources - anaerobic UASB sludge (PS), activated sludge (AS), and cow manure (CM) – all subjected to acid (APT) and thermal pretreatments (TPT). To assess microbial dynamics, three substrates were tested: a simple sugar (glucose); sugarcane vinasse with filter cake (in natura), and sugarcane vinasse with filter cake enzymatically hydrolysed. The process was evaluated from the metabolic and microbiological perspectives, based on electron balance, volatile fatty acids (VFA) profiles and 16S rRNA sequencing. With glucose, thermally pretreated inocula showed stronger acidogenic activity, and electron balance calculations revealed distinct metabolic pathways for each microbial community, even with similar hydrogen yields (HY). Using vinasse and filter cake - both in natura and pretreated - AS achieved higher HY, followed by PS and then CM. Hydrolysis improved HY until 262%, correlating with increased butyrate concentration in the digestates and reduced homoacetogenesis. Acid pretreatment was able to inhibit homoacetogenesis in PS inoculum. Microbial analysis highlighted the crucial role of diversity in AS community and revealed that Thermoanaerobacterium strongly depend on the presence of hydrolytic or lactic acid bacteria for efficient H₂ production from sugarcane byproducts.