Nε-acetyl- <i>β</i> -lysine or glycine betaine as compatible solutes in response to increasing ammonia in <i>Methanoculleus sp</i> strains

Methanogens rely on compatible solutes to withstand osmotic stress, yet their responses to high ammonium concentrations, common in biogas digesters, remain poorly understood. In this study, intracellular osmolyte accumulation was examined in four Methanoculleus bourgensis strains (MAB1, MAB2, MAB3, and BA1), isolated from high-ammonia biogas digesters, under progressive increase in concentrations of ammonium and sodium chloride. Their responses were compared with those of the type strain Methanoculleus bourgensis MS2T and the halophilic Methanoculleus submarinus Nankai-1T. All investigated strain grew to 12 g l-1 NH4+-N (0.3 mg l-1 NH3), and gradual adaptation increased ammonium/ammonia tolerance in some strains to 25 g l-1 NH4+-N. Whereas the reference strains accumulated glycine betaine under both ammonium and sodium chloride stress, the M. bourgensis strains from high ammonia biogas systems uniquely accumulated Nε-acetyl-β-lysine during increasing levels of ammonium chloride. This β-amino acid derivative is known as a NaCl-induced osmoprotectant in methanogens, but it´s association with high ammonium/ammonia levels in pure cultures has not previously been demonstrated. Our findings identify Nε-acetyl-β-lysine biosynthesis as a potential mechanism underpinning the exceptional ammonium/ammonia tolerance of M. bourgensis, a taxon frequently dominating methane production in high-ammonia biogas systems, while also revealing notable variation in this trait among its subspecies.