The Contribution of Syntrophic Fatty-Acid Degrading Microbial Communities to Anaerobic Digester Function and Stability

Anaerobic digestion (AD), the conversion of complex organic matter to methane, occurs through a series of reactions mediated by different guilds of microorganisms. AD process imbalances, such as organic overload or high organic loading rates (OLR), can result in the accumulation of volatile fatty acids (VFA) e.g., propionate, which must be degraded to maintain stable reactor function. VFAs are metabolized by syntrophic fatty-acid degrading bacteria (SFAB) in association with methanogenic archaea (collectively, syntrophic microbial communities, SMC). Despite their indispensable role in AD, little is known about the ecology of SFAB, especially under stressed conditions. To facilitate ecological studies, four quantitative PCR assays, targeting propionate- and butyrate-degraders were developed, and applied to a variety of methanogenic environments. The highest SFAB abundance was observed in propionate enrichment cultures and anaerobic reactors. In addition, SFAB and methanogen abundance varied with reactor configuration and substrate identity. The contribution of SMC to AD function and stability was investigated in lab-scale reactors exposed to two forms of disturbance: shock overload (pulse disturbance) and increased OLR (press disturbance). SMC dynamics were linked to AD function using physicochemical and molecular techniques. The first experiment examined the effect of shock overloads on SMC structure and function. Results showed that functional resilience to the pulse disturbance in reactors was linked to the abundance of propionate-degraders and Methanosarcinaceae. Reactors with reduced numbers of these microorganisms displayed increased VFA buildup, however, there was a subsequent increase in the abundance of propionate-degraders and Methanosarcinaceae which improved the functional resilience in these reactors to the next perturbation. The second experiment examined the effect of increased OLRs on SMC structure and function. SMC decreased in abundance with increasing OLR. Prior to system collapse, a decrease in acetoclastic methanogens corresponded with an increase in syntrophic acetate oxidizers and hydrogenotrophic methanogens. In summary, this work demonstrates that an increased abundance of syntrophic fatty acid degrading microbial communities are essential in AD during stressed conditions, such as organic overload and high OLRs. These results could change how digesters are monitored and aid in the design of better anaerobic treatment processes

Quantitative Detection of Syntrophic Fatty Acid-degrading Bacterial Communities in Methanogenic Environments

In methanogenic habitats, volatile fatty acids (VFA), such as propionate and butyrate, are major intermediates in organic matter degradation. VFA are further metabolized to H2, acetate and CO2 by syntrophic fatty acid-degrading bacteria (SFAB) in association with methanogenic archaea. Despite their indispensable role in VFA degradation, little is known about SFAB abundance and their environmental distribution. To facilitate ecological studies, we developed four novel genus-specific quantitative PCR (qPCR) assays, with primer sets targeting known SFAB: Syntrophobacter, Smithella, Pelotomaculum and Syntrophomonas. Primer set specificity was confirmed using in silico and experimental (target controls, clone libraries and melt-curve analysis) approaches. These qPCR assays were applied to quantify SFAB in a variety of mesophilic methanogenic habitats, including a laboratory propionate enrichment culture, pilotand full-scale anaerobic reactors, cow rumen, horse faeces, an experimental rice paddy soil, a bog stream and swamp sediments. The highest SFAB 16S rRNA gene copy numbers were found in the propionate enrichment culture and anaerobic reactors, followed by the bog stream and swamp sediment samples. In addition, it was observed that SFAB and methanogen abundance varied with reactor configuration and substrate identity. To our knowledge, this research represents the first comprehensive study to quantify SFAB in methanogenic habitats using qPCR-based methods. These molecular tools will help investigators better understand syntrophic microbial communities in engineered and natural environments

A Microfluidic Platform For the Simultaneous Quantification of Methanogen Populations in Anaerobic Digestion Processes

Methanogens are a diverse group of archaea that play a critical role in the global carbon cycle. The lack of appropriate molecular tools to simultaneously quantify numerous methanogenic taxa, however, has largely limited our ability to study these communities in a wide variety of habitats, such as anaerobic digesters (ADs). In this study, 34 probe-based quantitative PCR (qPCR) assays were designed to target all known methanogenic genera within the archaeal phylum Euryarchaeota. These qPCR assays were adapted to a high-throughput microfluidic platform, which allowed for the simultaneous detection and absolute quantification of numerous taxa in a single run. The resulting microfluidic qPCR (MFQPCR) platform was successfully used to decipher structure–function relationships among methanogenic communities in four laboratory-scale digesters exposed to a transient organic overload. Twelve of the 34 genera targeted in the MFQPCR were detected in the ADs, similar to results obtained using high-throughput sequencing. The MFQPCR platform and conventional qPCR assays also generated similar quantitative results. The MFQPCR tool developed here will help optimize AD technologies for efficient waste treatment and enhanced biogas production and can facilitate studies that will increase our understanding of methanogenic communities in other environments