Microbial ecology and the role of conductive materials in sulfate-rich anaerobic digestion systems using different reactor configurations

Anaerobic digestion (AD) is widely applied as an efficient technology for the treatment of sulfate-rich wastewater, enabling simultaneous organic removal and energy recovery. However, the existence of sulfate fundamentally alters microbial community structure and metabolic networks, intensifying competition and cooperation among key functional groups, especially between various types of sulfate-reducing bacteria (SRB) and methanogens. In addition, hydrogen sulfide (H₂S), as the terminal product of sulfate reduction, exerts severe toxicity on methanogens and other key functional microorganisms, threatening methane production and process stability. Although much attention has been given to SRB and H₂S inhibition, the ecological differentiation and metabolic roles of incomplete oxidizing SRB (IO-SRB) and complete oxidizing SRB (CO-SRB) with methanogens, as well as their adaptive responses to H₂S stress, remain poorly understood. Moreover, conductive materials (CMs) have shown great potential in enhancing anaerobic digestion of sulfate-rich wastewater by improving electron transfer. However, how the effectiveness of CMs varies under different reactor operational modes that shape microbial communities and system performance are still unclear. To comprehensively address these knowledge gaps, this study systematically investigates the distinct ecological roles, interspecies interactions, and stress adaptation mechanisms of key functional microorganisms under different reactor configurations and with the addition of CMs Four ethanol-fed bioreactors were operated under two operational modes (sequencing batch reactor, SBR; and continuous flow-reactor, CFR) and two chemical oxygen demand to sulfate (COD/sulfate) ratios (1 and 2) to systematically explore strategies for enriching IO-SRB and/or CO-SRB and their microbial interactions with other microorganisms. Compared to SBRs, CFRs could enhance sulfate removal and demonstrated higher microbial activities in sulfate and ethanol degradation. IO-SRB competed with ethanol-oxidizing bacteria (EOB) in all reactors, and IO-SRB's contribution to ethanol degradation increased from 62.9%-67.1% to 69.0%-82.1% as the COD/sulfate ratio decreased from 2 to 1. Moreover, CO-SRB competed acetoclastic methanogens (AM) exclusively in CFRs, as CO-SRB could not be efficiently enriched in SBRs. Low COD/sulfate ratios facilitated the enrichment of Desulfococcus (CO-SRB), and the CFR operational mode further strengthened its enrichment. Additionally, hydrogenotrophic SRB outperformed hydrogenotrophic methanogens in all four reactors. In general, IO-SRB and CO-SRB possessed distinct microbial interactions with methanogens, with potential syntrophic relationships between IO-SRB and AM while competitive relationships between CO-SRB and AM. In addition, this study investigated the tolerance and adaptive mechanisms of functional microorganisms to H2S. 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 EOB and 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 AM and CO-SRB 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 further compared SBRs and CFRs amended with magnetite (Fe₃O₄) or powdered activated carbon (PAC) for treating sulfate-rich wastewater. CM amendments significantly accelerated sulfate reduction, volatile fatty acids degradation, and methane production, especially with the addition of Fe₃O₄. Maximum methanogenesis rates in CFRs increased from 31.2 to 51.0 and 39.7 mg COD/(g VSS·h) with the addition of Fe₃O₄ and PAC, respectively. Methanogenesis in SBRs was severely inhibited by elevated H2S concentrations, and supplementation with 1 g/L CMs failed to alleviate this inhibition. However, CFRs favored direct ethanol-to-acetate conversion, whereas SBRs activated ethanol-to-propionate metabolic pathway mediated by Desulfobulbus. CM additions led to increased sludge conductivity and electron transport activity. Specifically, PAC strongly enhanced electron transfer in CFRs by promoting e-pili and cytochrome gene abundances, whereas Fe₃O₄ in SBRs predominantly acted as an external conductive conduit, partially substituting intrinsic microbial conductive structures. Key SRB, including Unclassified_f_Desulfovibrionaceae, Desulfomicrobium, Desulfolutivibrio, and Desulfovibrio, dominated the expression of e-pili and cytochrome genes associated with the direct interspecies electron transfer, which was promoted by CFR operation through the enrichment of SRB. Microbial co-occurrence network analysis further highlighted Desulfovibrio, Methanothrix, and Geobacter as central keystone species mediating robust syntrophic electron transfer networks. These findings provide critical insights for optimizing sulfate-rich wastewater treatment through strategic selection of reactor modes and CMs. Overall, this study provides a comprehensive understanding of how reactor configurations, COD/SO₄²⁻ ratio, and CM amendments modulate the enrichment, interaction, and resilience of key functional microorganisms in sulfate-rich anaerobic systems. By distinguishing the ecological roles of IO-SRB and CO-SRB and elucidating their interactions with methanogens under varying COD/SO₄²⁻ ratios and operational modes, this work reveals fundamental microbial mechanisms governing electron competition and H₂S stress response. The findings underscore the critical importance of tailoring reactor strategies and material interventions to drive electron flow, enhance syntrophic cooperation, and stabilize process performance. These insights contribute valuable guidance for the rational design and optimization of anaerobic treatment processes treating sulfate-rich wastewaters, advancing both theoretical microbial ecology and practical engineering applications.

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University of Galway

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CC BY-NC-ND

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University of Galway Theses (PhD Theses)