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

Abstract

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.

Keywords: Anaerobes; Archaea; Biofuel; Bioproduct; Biorefinery; Biotechnology; Diazotrophy; Methanogens; Microbiology.

Fig. 1

Time-series experiment showing the headspace pressure results of M. marburgensis (a) and M. maripaludis (b) in the prioritization phase. The ∆p of M. marburgensis reached up to 2 bar and outcompeted M. maripaludis. For all experiments n = 3.

Fig. 2

Growth kinetics of M. marburgensis during the THp_N2fix methodology experiment without Na₂CO₃ in the medium and at different NH₄⁺ concentrations in relation to original media composition (n = 4). Due to undercutting the THp_N2fix in all four replicates of 5% NH₄⁺ experiments at two subsequent time points, growth could not be monitored further since the method of destructive sampling has been employed for verifying THp_N2fix independently twice through GC measurements.

Fig. 3

NUR / mmol L⁻¹ h⁻¹ of M. marburgensis with different NH₄⁺ concentrations calculated by the THp_N2fix methodology. Negative values for NUR / mmol L⁻¹ h⁻¹ on the y-axis indicate N₂-fixation. Sample size varies due to the time point of undercutting THp_N2fix. The sample number (n) is indicated in brackets on the x-axis. In addition, the sampling time points (in h) and the values of the applied NH₄⁺ concentration (in %) are also indicated on the x-axis.

Fig. 4

Results of NURs during the amino acid excretion and N₂-fixation time series experiment. The experiment was performed using destructive sampling procedure. M. marburgensis biomass was washed before inoculation. NUR / mmol L^-1h^-1 of M. marburgensis during growth on H₂/CO₂/N₂ are shown as individual box plots indicating NH₄⁺ concentration and the time point. The first number on the x-axis labelling indicates the percentages of NH₄⁺ in the medium. The first number on the x-axis labelling indicated the sampling time point. The legend on the right-hand side of the graph indicates the respective group in colours (red: 5% NH₄⁺, green: 7.5% NH₄⁺, blue: 10% NH₄⁺, magenta: 100% NH₄⁺ as well as the time point of sampling (40 h, 59 h, 77 h). All results are n = 8. Thus, 93 out of 96 individual NUR results show N₂-fixation whereas only 3 NUR results from 3 different set-ups do not show N₂-fixation.

Fig. 5

Results of individual amino acid concentrations during the amino acid excretion and N₂-fixation time series experiment. M. marburgensis biomass was washed before inoculation. Amino acid concentration / µmol L⁻¹ of M. marburgensis during N₂-fixation on H₂/CO₂/N₂ shown as individual bar charts with standard deviation for each amino acid. The legend on the right-hand side of the graphs indicates the quantified amino acids. On the left-hand y-axis, the amino acid (AA) concentration / µmol L⁻¹ is shown. NH₄⁺ concentrations of the respective time series are indicated on the right-hand y-axis from top to bottom: 0%, 5%, 7.5%, 10%, 100% and 100%_41. 100%_41 denotes amino acid excretion during incubation with H₂/CO₂ (4:1). The sampling time is shown on the x-axis as headers from left to right. After biomass washing negligible amino acid concentration is measurable at time point 0 h. All results are n = 8.