Decoding the Prolonged Suppression of Zn on Fermentative Hydrogen Generation from Sewage Sludge

The presence of zinc (Zn) in various wastes poses significant challenges to biohydrogen recovery from organic wastes. The inhibitory effect of Zn on hydrogen production from sewage sludge was investigated, and a pronounced inhibition on hydrogen production was observed across the tested Zn concentration range (200–2000 mg/L). With the increase of Zn dosage from 200 mg/L to 500 mg/L, cumulative hydrogen production (hydrogen yield) decreased from 39 mL (73.5 mL/g SCOD) to 8 mL (15.1 mL/g SCOD), corresponding to the increased inhibition rate from 23.5% to 84.3%. When the Zn dosage was further increased to 1000–2000 mg/L, hydrogen production dropped to less than 5 mL (9.42 mL/g SCOD), corresponding to the inhibition rate of over 90%. With the removal of Zn stress, hydrogen-producing capacity in all groups were recovered, and the recovery rate exhibited an increasing trend with the increase of Zn dosage. Microbial-community analysis revealed that Zn addition markedly suppressed hydrogen-producing genera such as Enterococcus and Clostridium_sensu_stricto_14. After stress removal, the system partially reconstituted its hydrogen-producing ability by enriching Zn-tolerant functional genera like Pseudomonas, Acinetobacter and Clostridium_sensu_stricto_13. Metabolic analysis revealed that Zn inhibited hydrogen production by suppressing glucose decomposing and ferredoxin-related hydrogen-producing pathways, and formate decomposition served as the main hydrogen-producing pathway in the presence of Zn stress. After stress removal, glucose decomposing and ferredoxin-related hydrogen-producing pathways replaced formate-decomposing pathway as the hydrogen-producing pathways. This study exhibited a long-term response of microorganisms to Zn inhibition, and provided a theoretical basis for understanding the metabolic mechanism of Zn inhibition on hydrogen production from actual organic waste.