Long-term effects of operating temperature and sulphate addition on the methanogenic community structure of anaerobic hybrid reactors

The diversity, population dynamics, and activity profiles of methanogens in anaerobic granular sludges from two anaerobic hybrid reactors treating a molasses wastewater both mesophilically (37°C) and thermophilically (55°C) during a 1081 day trial were determined. The influent to one of the reactors was supplemented with sulphate, after an acclimation period of 112 days, to determine the effect of competition with sulphate-reducing bacteria on the methanogenic community structure. Sludge samples were removed from the reactors at intervals throughout the operational period and examined by amplified ribosomal DNA (rDNA) restriction analysis (ARDRA) and partial sequencing of 16S rRNA genes. In total, 18 operational taxonomic units (OTUs) were identified, 12 of which were sequenced. The methanogenic communities in both reactors changed during the operational period. The seed sludge and the reactor biomass sampled during mesophilic operation, both in the presence and absence of sulphate, was characterised by a predominance of Methanosaeta spp. Following temperature elevation, the dominant methanogenic sequences detected in the non-sulphate supplemented reactor were closely related to Methanocorpusculum parvum. By contrast, the dominant OTUs detected in the sulphate-supplemented reactor upon temperature increase were related to the hydrogen-utilising methanogen, Methanobacterium thermoautotrophicum. The observed methanogenic community structure in the reactors correlated with the operational performance of the reactors during the trial and with physiological measurements of the reactor biomass. Both reactors achieved chemical oxygen demand (COD) removal efficiencies of over 90% during mesophilic operation, with or without sulphate supplementation. During thermophilic operation, the presence of sulphate resulted in decreased reactor performance (effluent acetate concentrations of >3000mg/l and biogas methane content of <25%). It was demonstrated that methanogenic conversion of acetate at 55°C was extremely sensitive to inhibition by sulphide (50% inhibition at 8-17mg/l unionised sulphide at pH 7.6-8.0), while the conversion of H₂/CO ₂ methanogenically was favoured. The combination of experiments carried out demonstrated the presence of specific methanogenic populations during periods of successful operational performance.