The synergy of light irradiation, TiO2, and Fe3O4 boosts biohydrogen production from the dark fermentation at room temperature.

Biohydrogen production via dark fermentation (DF), which has energy-saving and environmental protection advantages, has been widely regarded as a viable option for sustainable global energy development. However, DF faces limitations due to the variability of metabolites and metabolic conditions, particularly concerning the cost-intensive temperature control, which typically ranges from 35 ℃ to 70 ℃. This study introduced a stimulation strategy through the synergy of light irradiation, N-TiO2, and N-Fe3O4 on the metabolic pathway of DF at room temperature in batch tests and an up-flow packed bed reactor (UPBR). The hydrogen yield in the batch test was 1.56 mol-H2/mol-glucose, comparable to mesophilic or thermophilic DF. The highest hydrogen yield of 0.56 mol-H2/mol-glucose was observed in the continuous UPBR. The intermediates were predominantly acetate and butyrate, with acetate increasing by 56.5 % compared to the dark stage, indicating a regulated hydrogen pathway. The microbial community and correlation networks in the UPBR demonstrated that the synergistic effects resulted in significant changes in microbial diversity and structure, with key functional microbes shifting from Clostridium sensu stricto_12 (58.91 %) to Caproiciproducens (60.14 %). The PICRUSt2 predictions suggested that the synergistic effects promote the enrichment of hydrogen production-related metabolic pathways and key enzymes within the DF system. Finally, a synergistic mechanism was speculated whereby light irradiation stimulated ATPase-related processes through N-TiO2, and N-Fe3O4 may interact with ferredoxin and hydrogenase during the conversion from pyruvate to acetyl-CoA, as supported by the spectra of intermediates and gas production. This study aims to provide an efficient strategy for DF biohydrogen production at room temperature.