Valorization of carbon resources via in situ esterification in dark fermentation: Enabling butyl butyrate formation and boosting biohydrogen production

Biological hydrogen production via dark fermentation is a promising and sustainable approach for renewable energy generation. However, its practical application is hindered by the inefficient utilization of carbon substrates, as a large proportion is diverted into inhibitory by-products such as volatile fatty acids (VFAs). To overcome this limitation, this study developed an integrated one-pot system, combining dark fermentation with enzyme-catalyzed esterification, which converts VFAs into high-value esters while enhancing hydrogen production. Key influence factors, including lipase type, dosage, pH, and extractant type, were systematically evaluated. Under optimal conditions, cumulative hydrogen production reached 3631 ± 154 mL/L, a 58.21 % increase compared to the non-esterified group, while butyl butyrate (BB) production in extraction reached 8.66 ± 0.06 g/L. The carbon flow analysis demonstrated that approximately 7.60 % of the carbon from the consumed starch was converted into BB. Furthermore, a fed-batch fermentation resulted in simultaneous hydrogen and BB production, achieving a final BB production of 22.44 ± 0.36 g/L. Microbial community analysis indicated that the esterification strategy promoted the enrichment of Clostridium species, known for their pivotal role in hydrogen production. Under the esterification strategy, KEGG based functional genes analysis showed a notable enrichment in the relative abundances of genes associated with glycolysis and hydrogenase functions. This innovative approach not only alleviates VFAs inhibition but also provides a theoretical and technical foundation for the high-value utilization of VFAs, offering considerable potential for industrial applications.