Multiple isotope-tracking approaches quantitatively recorded the gradual development of diverse acetate-oxidizing syntrophs from microbiota dominated by acetotrophic methanogens

Background: Deep insights into the adaptation procedure of the microbiota to increased ammonia stress, and identification of indicator microorganisms can help to optimize operational strategies of anaerobic digesters. To identify such microbial indicators and establish links between their appearance with acetoclastic methanogensis (AM), syntrophic acetate oxidation (SAO) or hydrogenotrophic methanogenesis (HM), 40 anaerobic reactors fed with acetate of 110 mmol/L were launched at NH4+-N concentration 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.Results: Four stable carbon isotope probing strategies were applied in parallel, with [1, 2-13C]-CH3COOH, [2-13C]-CH3COOH, [13C]NaHCO3 or non-labelled CH3COOH utilized individually. The last three settings were used to quantify the methanogenic pathways by tracking labelled 13C or natural 13C signature in the produced CH4 and CO2, which consistently detected the dynamic transition of dominant pathways from AM to SAO-HM under ammonia stress. Quantitative PCR and fluorescence in-situ hybridization recorded the procedure acetotrophic methanogens being outcompeted by acetate-oxidizing syntrophs. The first and last strategies were designed to probe the active acetate-mineralizing microbes with DNA-SIP, which detected known syntrophs like Syntrophaceticus and Methanoculleus, as well as novel members of Pseudomona, Bacillus and Symbiobacteraceae. Nevertheless, some bacterial cells were observed to be fixing CO2 with NanoSIMS.Conclusions: Therefore, multiple isotope tracking methods are efficient in quantifying the competition between AM- and SAO-HM catalyzing microbial consortia. Under ammonia stress, diverse known and novel microbial taxa were involved, which are actually comparable between individual studies. The diversity of acetate-oxidizing syntrophs still need to be explored. The AM-conducting Methanosarcina demonstrated advantage of faster growth rate than acetate-oxidizing syntrophs under medium-level ammonia stress for recovering methanogenesis