Inhibition of norfloxacin on fermentative hydrogen production: Performance evaluation and metagenomic analysis

Toxic chemicals can inhibit the dark fermentation process for renewable biohydrogen production. Norfloxacin (NOR), a typical antibiotic, is present in various feedstocks of dark fermentation, which has excellent antibacterial properties. It is hypothesized that NOR could cause adverse effects on fermentative hydrogen production, while current understanding of this subject is scare. This study investigated the potential inhibitory effect of NOR on dark hydrogen fermentation, and explored the underlying mechanisms from the perspectives of bacterial community structure and functional gene abundance. The results showed that NOR significantly reduced the hydrogen-producing capacity upon exposure to concentrations exceeding 10 mg/L. The hydrogen yield decreased from 1.56 to 0.27 H₂/mol-glucose at NOR concentrations of 50 mg/L. NOR also prolonged the hydrogen-producing lag period. Bacterial community analysis revealed that NOR reduced the abundance of high-yielding H₂-generating bacteria, such as Clostridium sp., while increasing the abundance of H₂-consuming bacteria (e.g. Enterococcus sp.). Metagenomic analysis further indicated that crucial genes involved in glycolysis (e.g. HK, tal-pgi, pfk and PK) and hydrogen formation (e.g. por and E1.12.7.2) were remarkably downregulated under NOR exposure, essentially causing the inhibition of hydrogen production. This study contributes to a better understanding of how antibiotics inhibit dark hydrogen fermentation and provides a theoretical basis for reducing potential inhibitory effects.