Competition between Methanothrix and Methanosarcina species isolated from a butyrate-fed anaerobic digester

Methanothrix and Methanosarcina are key methanogens in anaerobic digesters that support stable methane production through direct interspecies electron transfer (DIET), but they rarely co-dominate. Methanothrix thrives under low acetate, high pH, salinity and ammonia, while Methanosarcina prefers higher acetate and is more stress-sensitive. In a butyrate-fed reactor with low acetate, Methanothrix comprised 95 % of the population, while Methanosarcina represented only 0.01 %. Methanosarcina DH-1 was isolated under high acetate, whereas Methanothrix E1 dominated under low acetate. Competition experiments showed Methanosarcina dominated at pH 6.5-7.0, while Methanothrix prevailed at pH 8.0, 5 g L^-1 NH₄Cl, and 18 g L^-1 NaCl. Genome sequencing revealed 98 % and 99 % similarity between E1 and Mx. soehngenii GP6 and DH-1 and M. vacuolata Z-761, and identified adaptive mutations in toxin-antitoxin and regulatory genes. These findings reveal mechanisms underlying competitive exclusion between methanogens that can inform strategies to optimize anaerobic digester performance.