Influence of pH and nitrogen source on metabolism and extracellular polymeric substance production in a mixed aerobic methanotrophic community

Aerobic methanotrophs are key regulators of methane emissions and have recently gained interest for their potential in biotechnological applications. Here, enrichment cultures from activated sludge were subjected to fed-batch experiments testing two nitrogen sources (nitrate and ammonium) under three pH conditions (4.5, 6.5, and 8.5) to investigate their potential for exopolymeric substances (EPS) production. Neutral and alkaline pH resulted in a similar consumption of nitrogen independently of the nitrogen source added. However, tests conducted with acidic pH resulted in a slower nitrogen consumption rate, especially in those tests where ammonium was added, stopping its consumption after the third ammonium pulse. Community profiling revealed the dominance of the phylum Pseudomonadota with Methylocystis as the most abundant methanotroph. EPS production was strongly induced at pH 4.5, where concentrations increased substantially after 5 days and remained elevated. By contrast, neutral and alkaline conditions yielded only modest EPS levels, regardless of the nitrogen source. • Acidic pH triggers enhanced EPS production in methanotrophic consortia. • Nitrate supports higher methane oxidation than ammonium under neutral pH. • EPS mainly composed of polysaccharides indicates stress-induced protection. • Combined pH and nitrogen source strongly shape methanotrophic metabolism.