1H-NMR metabolomic profiling of Methanobacterium and Methanosarcina reveals unusual intracellular trimethylamine and acetamide production

Objectives Ony a limited number of metabolomic datasets are available for pure cultures of methanogenic archaea, and none have reported 1 H-NMR-based intracellular profiles for Methanobacterium and Methanosarcina . We aimed to characterize intracellular metabolite profiles in selected strains and to identify metabolites that challenge current metabolic models. Materials and methods Nine lyophilized biomass samples representing three Methanobacterium species and three Methanosarcina species (including M. acetivorans grown on methanol, acetate and trimethylamine) were analyzed as single biological samples. Intracellular metabolites were extracted with methanol/water and analyzed by 600 MHz 1 H-NMR. Spectra were processed using COW/icoshift alignment, PQN normalization and PCA. Results Seventeen metabolites were identified across all samples, including amino acids, adenine nucleotides and small organic acids. PCA separated three metabolomic clusters corresponding to Methanobacterium , Methanosarcina (excluding M. acetivorans ) and M. acetivorans grown on different substrates. Succinate and trimethylamine were detected only in Methanobacterium strains, whereas betaine and acetamide were restricted to M. acetivorans biomass. NAD(H) and pyruvate were not detected. Conclusions These exploratory data reveal unusual intracellular metabolites (trimethylamine, acetamide) not accounted for in current methanogenic pathways, suggesting missing reactions in existing metabolic models. The observed genus- and strain-specific metabolite fingerprints may support NMR-based identification of methanogens and motivate targeted studies on archaeal contributions to trimethylamine metabolism.