Gradual development of ammonia-induced syntrophic acetate-oxidizing activities under mesophilic and thermophilic conditions quantitatively tracked using multiple isotopic approaches

Insights into microbiota adaptation to increased ammonia stress, and identification of indicator microorganisms can help to optimize the operation of anaerobic digesters. To identify microbial indicators and investigate their metabolic contribution to acetoclastic methanogenesis (AM), syntrophic acetate oxidation (SAO) or hydrogenotrophic methanogenesis (HM), 40 anaerobic batch reactors fed with acetate of 110 mmol/L were set up at NH₄⁺-N concentrations of 0.14 g/L, 5.00 g/L or 7.00 g/L, inoculated with thermophilic or mesophilic microbiota with or without pre-exposure to ammonia stress. Four stable carbon isotope probing approaches were applied in parallel, with [1,2-¹³C]-CH₃COOH, [2-¹³C]-CH₃COOH, [¹³C]NaHCO₃ or non-labeled CH₃COOH used individually. The last three approaches were used to quantify the methanogenic pathways by tracking labeled ¹³C or natural ¹³C signatures in the resulting CH₄ and CO₂, and consistently detected the dynamic transition of
dominant pathways from AM to SAO-HM under ammonia stress. Results of quantitative PCR and fluorescence in-situ hybridization illustrated the procedure, acetotrophic methanogens being outcompeted by acetate-oxidizing syntrophs. The first and last isotope-labeling approaches were designed to probe the active acetate-mineralizing microbes with DNA-SIP. Known acetate-oxidizing bacteria like Syntrophaceticus and Tepidanaerobacter, as well as novel members of Pseudomonas, Bacillus and Symbiobacteraceae were detected, with Methanoculleus as the predominant H₂/CO₂-utilizing partner. Using NanoSIMS, some bacterial cells were observed to be fixing CO₂ from [¹³C]NaHCO₃. In this study, Methanosaeta was only active with ammonia < 200 mg-N/L; the syntrophs catalyzing SAO-HM started to compete with AM-conducting Methanosarcina at intermediate concentrations of ammonia, i.e. 200–500 mg-N/L, and outcompeted the acetotrophic methanogens with ammonia > 500 mg-N/L. Under ammonia stress, diverse known and novel microbial taxa were involved in acetate mineralization, comparable with those identified in previous studies.