Quantitative analysis of amino acid excretion by Methanothermobacter marburgensis under N₂-fixing conditions

Methanogenic archaea (methanogens) possess fascinating metabolic characteristics, such as the ability to fix molecular nitrogen (N₂). Methanogens are of biotechnological importance due to the ability to produce methane (CH₄) from molecular hydrogen (H₂) and carbon dioxide (CO₂) and to excrete proteinogenic amino acids. This study focuses on analyzing the link between biological methanogenesis and amino acid excretion under N₂-fixing conditions. Among five hydrogenotrophic, autotrophic methanogens, Methanothermobacter marburgensis was prioritized and further cultivated in closed batch cultivation mode under N₂-fixing conditions. M. marburgensis was grown on chemically defined minimal medium with different concentrations of ammonium in a H₂/CO₂/N₂ atmosphere. This enabled the quantification of ammonia uptake, N₂-fixation, amino acid excretion and the conversion of H₂/CO₂ to CH₄. To quantify N₂-fixation rates in a mass balance setting a novel method has been established. The method utilizes the pressure drop below a certain threshold pressure in closed batch cultivation mode - the threshold pressure for N₂-fixation (THp_N2fix). Using the THp_N2fix method, volumetric N₂-fixation rates of M. marburgensis as high as 0.91 mmol L^-1 h^-1 were determined. Excretion of amino acids was found with highest detected values of glutamic acid, alanine, glycine and asparagine. The highest total amino acid excretion of 7.5 µmol L^-1 h^-1 was detected with H₂/CO₂/N₂ at an ammonium concentration of 40 mmol L^-1. This study sheds light on the link between methanogenesis, biological N₂-fixation, and proteinogenic amino acid excretion. The concomitant production of amino acids and CH₄ could become of biotechnological relevance in an integrated approach coupling biomethanation and N₂-fixation in a biorefinery concept.