Tetracycline in anaerobic digestion: Inhibitory effects and strategies for enhanced removal

Tetracycline is a widely used antibiotic that is frequently detected in various environmental matrices, due to its incomplete metabolism in humans and animals. The presence of tetracycline poses potential environmental risks. In anaerobic digestion systems, tetracycline can lead to the accumulation of volatile fatty acids, suppression of methane production, and inhibition of functional microorganisms. As a recalcitrant contaminant, tetracycline can be removed from anaerobic digestion systems through adsorption and biodegradation, with removal efficiency influenced by operational parameters and the presence of co-metabolizable substrates. In recent years, the addition of conductive materials, such as powdered activated carbon (PAC), has been reported as a promising strategy to enhance system performance. These materials promote electron transfer, regulate redox conditions, and facilitate the microbial activity. Therefore, a comprehensive understanding of the system-level and microbial-level responses of anaerobic digestion systems to environmental disturbances, such as tetracycline, is essential for optimizing the treatment of antibiotic-contaminated wastewater or waste. This study aims to: (i) elucidate the response of the anaerobic digestion system to tetracycline stress at both the system performance and functional gene levels; (ii) regulate the metabolism of complex carbon sources and enhance tetracycline removal by enriching microorganisms with distinct characteristics through operational parameter adjustment; and (iii) explore the application of conductive materials (PAC) for alleviating tetracycline inhibition and enhancing its removal via synergistic adsorption and biodegradation. Two operational modes, continuous-flow reactors (CFRs) and sequencing batch reactors (SBRs), were employed in this study under conditions with or without tetracycline addition, to assess the systems capacity for pollutant removal, microbial activity, and community dynamics. The results showed that tetracycline inhibited the maximum methane production rate by 24.5% in CFRs and 48.8% in SBRs. In addition, propionate accumulation was observed in tetracycline-added systems. Under steady-state conditions, over 80% of tetracycline removal was attributed to biodegradation. At the microbial level, long-term tetracycline exposure reduced the abundance of methanogens and propionate-oxidizing bacteria. Compared to SBRs, CFRs exhibited better recovery ability under shock loading, and showed advantages in propionate degradation and methane production. CFRs also facilitated the maintenance of key functional microbes. Subsequently, PAC addition further improved both system performance and tetracycline removal. PAC increased the maximum methane production rate by 15.6% in CFRs and 13.8% in SBRs, and enhanced tetracycline biodegradation by 24.4% and 19.2%, respectively. The genes encoding carbon dioxide reduction in Methanothrix and the presence of Geobacter suggested the possible involvement of direct interspecies electron transfer in methane production, particularly in PAC-added CFRs. In addition, this study further explored the system-level and gene-level responses of anaerobic digestion to tetracycline stress. At the reactor level, the effects of tetracycline dosage and sludge origin were investigated. In systems with laboratory seed sludge, increasing tetracycline concentrations (0 - 5 mg/L) inhibited ethanol production and potentially suppressed subsequent acetogenesis. When the concentration exceeded 5 mg/L, butyrate accumulated and its degradation was inhibited. Methane production was clearly impaired even with 2.5 mg/L tetracycline. In contrast, systems with farm sludge maintained stable chemical oxygen demand removal but exhibited no methane production capability. Both types of sludge exhibited tetracycline biodegradation capacity, and the biodegradation efficiency was not concentration-dependent. Metagenomic analysis further revealed mode-dependent microbial adaptation strategies. In CFRs, the microbial community was dominated by taxa resistant to tetracycline toxicity, exhibiting cohesive microbial interactions and strong functional redundancy. Meanwhile, SBRs relied on resilient microbial communities to adapt to fluctuating conditions. As for tetracycline resistance genes, ribosomal protection proteins were primarily enriched in CFRs, whereas SBRs accumulated high energy-dependent efflux pump genes. For example, the relative abundance of tetZ was actively reduced under resource-limited conditions in SBRs. With regard to energy metabolism, tetracycline stress stimulated an increase in ATPase gene abundance. While F-type ATPase genes predominated in SBRs, methanogens in CFRs played a major role in maintaining energy metabolism under antibiotic pressure. Further analysis indicated a strong coordination between genes involved in ATPase synthesis and extracellular electron transfer. This study reveals the destabilization mechanisms and adaptive pathways of anaerobic digestion systems under tetracycline stress from the perspectives of system performance, microbial community dynamics, and functional genes. A regulatory framework combining operational mode and SRT adjustment with PAC enhancement is investigated. The findings demonstrate the important role of operational parameters in stabilizing system performance and shaping functional microbes, while highlighting the synergistic effects of PAC in alleviating antibiotic inhibition and enhancing reactor performance. Gene-level analysis uncovers a dynamic trade-off between resistance burden and energy metabolism, highlighting microbial strategies for ecological adaptation under antibiotic pressure. This study establishes both theoretical and engineering foundations for enhancing the anaerobic digestion of antibiotic-contaminated wastewater and waste, and provides a conceptual framework for future investigations into microbial metabolism-energy-resistance regulation mechanisms.

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University of Galway

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University of Galway Theses (PhD Theses)