r/K Selection-based strategies for enriching acetoclastic methanogens in anaerobic digestion systems

Anaerobic digestion (AD) is a widely used biotechnology for the treatment of solid waste or wastewater and the generation of renewable energy in the form of methane. Methanogenesis is the final and often rate-limiting step in AD, directly determining overall system performance and energy recovery efficiency. Among the methanogenic pathways, acetoclastic methanogenesis, converting acetate to methane, contributes up to 60% of total methane production. Remarkably, this essential step is performed exclusively by two archaeal genera: Methanosarcina and Methanothrix. Although both utilize acetate as their substrate, they exhibit fundamentally different ecological strategies and physiological traits. Methanosarcina is an r-strategist with high growth rates and broad metabolic flexibility, while Methanothrix is a K-strategist characterized by strong substrate affinity and long-term persistence under nutrient-limited conditions. Despite their central roles in methane production, the competitive dynamics, enriching mechanisms, and syntrophic interactions of these methanogens under different operational conditions remain insufficiently understood. A deeper understanding of these mechanisms is crucial for improving AD stability, enabling targeted enrichment of functional microorganisms, and guiding the rational design of next-generation anaerobic bioreactors. This study aims to: (i) develop a novel modeling framework that integrates r/K selection theory to simulate the competitive dynamics between Methanosarcina and Methanothrix under varying operational conditions; (ii) investigate the effects of reactor configuration (continuous-flow reactors (CFRs) and sequencing batch reactors (SBRs)), solids retention time (SRT), and substrate type (acetate and ethanol) on the enrichment and energy metabolism of acetoclastic methanogens; and (iii) elucidate the mechanisms of microbial cooperation, including potential amino acid cross-feeding and interspecies electron transfer, mediated by acetoclastic methanogens in acetate- and ethanol-fed anaerobic systems. A novel AD modeling framework was developed by integrating r/K selection theory into conventional kinetic models to simulate the competitive exclusion dynamics between Methanosarcina and Methanothrix. Thermodynamic energy dissipation principles enabled the successful derivation of the kinetic parameters for Methanosarcina and Methanothrix. Sensitivity analysis identified acetate concentration and SRT as key factors influencing methanogen dominance, with low-substrate conditions and long SRTs favoring Methanothrix, and high-substrate, short-SRT environments favoring Methanosarcina. CFRs offered more stable conditions that promoted the gradual dominance of Methanothrix, especially under low acetate concentrations. Subsequent reactor-scale experiments were conducted in acetate-fed systems to explored how reactor configuration (SBR vs. CFR) and SRT influence the enrichment and energy metabolism of acetoclastic methanogens. The results showed that short SRTs (10 and 15 days) and SBRs favored the dominance of Methanosarcina, reflecting its rapid-growth, resource-responsive characteristics. In contrast, Methanothrix achieved greater relative abundance under prolonged SRTs (25 and 50 days) in CFRs, consistent with its adaptation to stable, low-substrate environments. Interestingly, SBRs supported the co-existence of both genera, possibly due to the substrate concentration gradients established during cyclic feeding. Beyond methanogens, operational parameters also impacted acetate-oxidizing bacterial communities, with genera such as Pseudomonas, Thauera, and Desulfocurvus. Furthermore, genes associated with energy conservation, such as ATPase complexes and electron transport genes (such as Ech, Vho/Vht, Fpo, Mtr), exhibited mode-dependent patterns, suggesting that methanogenesis in each system followed different metabolic metabolism. Auxotrophy of amino acid observed in dominant microbial taxa indicated potential amino acid cross-feeding interactions, which may support metabolic complementarity. In addition, operational mode significantly influenced methanogen and syntroph distribution in ethanol-fed systems. CFRs achieved full ethanol and volatile fatty acid degradation, while SBRs showed acetate and butyrate accumulation. Methanothrix displayed a higher relative abundance in CFR (6.1%) compared to SBR (1.7%), whereas Methanosarcina was more prevalent in SBR (1.2%) than in CFR (0.06%). Additionally, acetoclastic methanogenesis was not detected in SBR. Genes associated with hydrogen and electron transfer were more abundant in CFRs, supporting enhanced syntrophic cooperation and the potential occurrence of direct interspecies electron transfer (DIET) between Geobacter and Methanothrix. Overall, this research reveals how life-history strategies and environmental factors shape methanogen selection and microbial network dynamics in anaerobic digesters. It provides both theoretical insights and practical tools to guide the design and operation of AD systems toward functionally optimized and ecologically stable microbial communities.

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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)