Abstract
Hydrogen sulfide (H2S), a product of sulfate reduction in anaerobic digestion (AD) systems, poses severe challenges by reducing methane production and destabilizing system performance. Despite extensive studies on H2S toxicity, the specific responses and adaptation mechanisms to H2S stress of key functional microorganisms in AD systems remain insufficiently elucidated. Four reactors were operated with sequencing batch reactor (SBR) and continuous flow reactor (CFR) configurations under varying COD/sulfate ratios (2 and 1) to investigate microbial response to H2S inhibition. Long-term experiments demonstrated that CFRs combined with a COD/sulfate ratio of 1 achieved superior sulfate reduction and ethanol degradation rates under H2S stress, while SBRs with a COD/ sulfate ratio of 2 facilitated methanogenic activity. Batch inhibition experiments revealed that ethanol-oxidizing bacteria (EOB) and incomplete oxidizing sulfate-reducing bacteria (IO-SRB) exhibited greater H2S tolerance in CFRs, with EOB (IC50 = 51.2-185.1 mg/L) generally outperforming IO-SRB (IC50 = 47.4-97.7 mg/L). While acetoclastic methanogens (AM) and complete oxidizing sulfate-reducing bacteria showed enhanced H2S tolerance in SBRs compared to CFRs, particularly AM in SBR with the COD/sulfate ratio of 2 (IC50 = 113.2 mg/L). Microbial adaptation analysis demonstrated that SBRs promoted Methanothrix enrichment, enhancing detoxification capacity by specifically increasing the relative abundance of genes encoding thiosulfate sulfurtransferase to mitigate H2S toxicity. Desulfomicrobium and Geobacter were significantly enriched in CFRs, and they mitigated H2S inhibition through increased cytochrome bd oxidase and cysteine synthase genes, respectively. Furthermore, thioredoxin and cysteine desulfurase protein repair genes sustained microbial metabolism under H2S stress. This study provides critical insights into microbial tolerance and adaptive strategies to H2S under different reactor configurations, offering guidance for optimizing AD processes in sulfate-rich wastewater treatment.