Method for producing normal valine by fermentation

Abstract

The present invention provides a method for producing an amino acid by fermentation in a bioreactor wherein the bioreactor comprises a methanogenic microorganism in a fermentation broth, the method comprising at least the steps of: producing an amino acid from the methanogenic microorganism under conditions such that the methanogenic microorganism produces the amino acid; the bioreactor is supplied with a gaseous carbon source comprising carbon dioxide and / or carbon monoxide, a nitrogen source, and preferably a sulfur source, wherein the amino acid comprises normal valine. Preferred methanogens are, for example, archaea selected from the group consisting of Methanothermophiles, Methanothermophiles, and Methanococci. Also provided are fermentation broths comprising a methanogenic microorganism and normal valine, as well as bioreactors comprising the fermentation broths.

Description

Technical Field

[0001] The field of the invention relates to methods for producing norvaline by fermentation using microorganisms in bioreactors. Background Art

[0002] Amino acids are used in various fields: food and feed, agriculture, pharmaceuticals and even packaging and housing. The biosynthesis of protein-producing amino acids is an important branch of biotechnology (Becker and Wittmann 2012).

[0003] However, the demand for non-proteinogenic amino acids is also increasing. Norvaline (also referred to herein as Nva) is a non-canonical amino acid that is increasingly being used as a dietary supplement. In addition, it is used to induce vasodilation (see, for example, US 11,260,039 B1). It has also been suggested as a therapeutic agent for Alzheimer's disease (Polis et al. 2019).

[0004] Unrelatedly, interest also arose when valine was found to be incorporated into protein-based biopharmaceuticals produced by recombinant E. coli fermentation processes (Biermann et al. 2013), which is usually undesirable.

[0005] CN106520651A discloses a method for producing L-norvaline via enzymatic conversion.

[0006] Kisumi et al. 1976 discussed the biosynthesis of the non-proteinogenic amino acids norvaline, norleucine and homoisoleucine in the enteric bacterium Serratia marcescens.

[0007] US 2006 / 0057685 A1 relates to a fermentation method for producing norvaline using Enterobacteriaceae bacteria in which all acetohydroxyacid synthases have been inactivated.

[0008] Despite these efforts, there remains a need for further methods of producing norvaline.

[0009] Therefore, an object of the present invention is to provide improved methods for producing norvaline by fermentation with microorganisms in bioreactors. These methods should make more efficient use of (natural) resources, be more environmentally friendly, reduce greenhouse gas emissions, increase yields and / or overcome one or more disadvantages of amino acid production methods known in the art. Summary of the invention

[0010] The present invention provides a method for producing amino acids by fermentation in a bioreactor. The bioreactor contains methanogenic microorganisms in a fermentation broth. The method comprises at least the following steps: providing a gaseous carbon source including carbon dioxide and / or carbon monoxide, a nitrogen source and preferably a sulfur source to the bioreactor under conditions such that the methanogenic microorganisms produce amino acids, wherein the amino acids include norvaline (particularly L-norvaline). Typically, the method comprises the step of harvesting at least a portion of the amino acids from the bioreactor (e.g., subsequently separating the amino acids from other components of the fermentation broth by a purification method).

[0011] In one aspect, the present invention provides the use of a methanogenic microorganism (e.g., any methanogenic archaea disclosed herein, exemplified by Methanothermobacterium, Methanothermococcus, Methanococcus and Methanococcus) to produce norvaline (particularly L-norvaline).

[0012] In another aspect, the present invention provides a fermentation broth comprising a methanogenic microorganism (e.g., any methanogenic archaea disclosed herein, exemplified by Methanothermobacterium, Methanothermococcus, Methanococcus and Methanococcus) and norvaline (particularly L-norvaline), preferably wherein the norvaline is in the supernatant (i.e., the liquid fraction of the fermentation broth).

[0013] In yet another aspect, the present invention provides a bioreactor comprising the fermentation broth.

[0014] It was unexpectedly observed during the course of the present invention that methanogenic archaea are able to produce norvaline and actively excrete norvaline into the cell culture supernatant, particularly under carbon-fixing conditions (or carbon- and nitrogen-fixing conditions). Norvaline production has not been previously seen in archaea, let alone methanogenic archaea. This discovery of the present invention makes it possible to combine carbon (and N2) fixation and the concomitant norvaline production. DETAILED DESCRIPTION OF THE INVENTION

[0016] The detailed description given below relates to all aspects of the invention described above, unless explicitly excluded.

[0017] In general, the metabolic potential of archaea for amino acid production has been largely overlooked so far (Pfeifer et al. 2021). Rinke et al. 2021 published a standardized archaea classification.

[0018] It is well known that methanogenic archaea can produce methane (CH4) as the end product of their energy metabolism (e.g., Mand & Metcalf, 2019). Some of these archaea can grow autotrophically in a hydrogenotrophic manner by reducing carbon dioxide (CO2) to CH4 with molecular hydrogen (H2) and play a key role in the global carbon cycle (Lyu et al. 2018). Methanogens can be divided into different metabolomic groups based on their substrate utilization spectrum: hydrogenotrophic (H2, formate, or simple alcohols), acetate-splitting (acetate), methylotrophic (methyl-containing compounds), H2-dependent methylotrophic (methylated compounds with H2 as electron donor), and methoxytrophic (methoxylated aromatic compounds) (Mayumi et al. 2016, Kurth et al. 2020). The biology of methanogenic archaea has also been discussed in earlier publications, such as Zeikus 1977.

[0019] Taubner et al. 2019 is a non-biotechnology related exobiology research paper. Specifically, the paper deals with the membrane lipid composition and amino acid excretion patterns of Methanothermococcus okinawensis grown in the presence of inhibitors detected in the plumes of Enceladus. These findings are important for understanding the ecophysiology of methanogens on Earth and have important implications for the use of biomarkers as possible signs of extraterrestrial life in future space missions to the Solar System. Along similar lines, Taubner et al. 2018 discussed the production of biogenic methane under hypothetical Enceladus-like conditions.

[0020] Further, not related to biotechnology, studies by Kengen & Stams 1994 concern the formation of L-alanine as a reduction end product during carbohydrate fermentation by the thermophilic archaeon Pyrococcus furiosus. Sment & Konisky 1989 claim that a 1,2,4-triazole-3-alanine-resistant mutant of Methanococcus voorhizium has an opposite amino acid excretion to the wild type. In the experiments described in this document, Methanococcus voorhizium was grown in a defined medium containing 19 amino acids under a H2-CO2 atmosphere.

[0021] Also not related to biotechnology, Porat et al. 2004 discussed the biosynthetic pathway of aromatic amino acids in Methanococcus marinum.

[0022] Whitman et al. 1986 described the isolation and characterization of 22 species of mesophilic methanococci. The cultures were grown under H2-CO2 and the medium was prepared under N2-CO2. The document does not mention amino acid production at all.

[0023] Also not related to biotechnology, Fardeau et al. 1987 deals with the energetics of thermoautotrophic Methanobacterium and thermolithotrophic Methanothermococcus using ammonium chloride and nitrogen. This document does not mention amino acid production at all.

[0024] Along similar lines, Whitman et al. 1982 discuss the nutrition and carbon metabolism of Methanococcus voerei. Various nitrogen sources are discussed. It is claimed that Methanococcus voerei requires ammonia for growth in chemically defined media. Mineral requirements are also discussed in this article.

[0025] Rittmann et al. 2021 generally focus on the applications of archaea in biotechnology.

[0026] Several studies have discussed the use of methanogenic archaea in renewable energy production, i.e., the reduction of CO2 to CH4 with H2 (Pappenreiter et al. 2019, Rittmann et al. 2018, Mauerhofer et al. 2018, Abdel Azim et al. 2018, Abdel Azim et al. 2017, Rittmann 2015, Mauerhofer et al. 2021, Rittmann et al. 2012). Liu et al. 2021 focused on the effects of different amino acids and their configurations on methane production and bioconversion of intermediate metabolites during anaerobic digestion. WO 2012 / 110256 A1 discloses a method for converting carbon dioxide and hydrogen into methane by methanogenic microorganisms. WO2014 / 128300A1 relates to a method and system for producing methane using methanogenic microorganisms and applying a specific nitrogen concentration in the liquid phase.

[0027] WO 2017 / 070726 A1 relates to a method for determining the culture state of a microbial culture, for example a culture of hydrogenotrophic methanogenic microorganisms.

[0028] Hoffarth et al. 2019 focused on the effect of N2 on biological methanogenesis by Methanothermobacterium marburgii in a continuous stirred tank reactor. The article states that N2 behaves like an inert gas. The article also teaches that "N2 does not participate in catabolic reactions". The article does not mention amino acid production at all.

[0029] US 2011 / 281333 A1 relates to the production of methane from single-cell organisms (e.g., methanogens). The growth of methanogens involves the consumption of carbon dioxide to produce methane. Methods for enhancing growth are disclosed. Gaseous N2 is not considered a nitrogen source.

[0030] US 2018 / 0179559 A1 relates to a biological and chemical process that uses chemoautotrophic microorganisms to fix carbon dioxide and / or other inorganic carbon sources into organic compounds through chemical synthesis and generate additional useful products. The microorganisms can be selected from a variety of different bacterial and archaeal species.

[0031] EP 2 192 170 A1 relates to an amino acid-producing microorganism and a method for producing amino acids. A microorganism (preferably selected from γ-proteobacteria, rod-shaped bacilli or bacteria belonging to the genus Alicyclobacillus, bacillus or yeast of the genus Saccharomyces) is disclosed, which has the ability to produce L-amino acids (selected from L-lysine, L-threonine, L-tryptophan, L-phenylalanine, L-valine, L-leucine, L-isoleucine and L-serine) and has been modified so that pyruvate synthase or pyruvate:NADP + Increased activity of oxidoreductases.

[0032] WO 2016 / 179545 A1 and US 2018 / 0163240 A1 disclose compositions and methods for the biological production of methionine.

[0033] US 2019 / 0194630 A1 and US 2017 / 0130211 A1 relate to compositions and methods for the biological production of amino acids in hydrogenotrophic microorganisms. Specifically, the hydrogenotrophic microorganisms can be selected from Methanococcus and Methanosarcina.

[0034] WO 2020 / 252335 A1 relates to a method and system for producing a product by fermentation. Specifically disclosed is a method comprising: (a) adding a x and H2 (wherein x is 1 or 2), a nitrogen source and optionally a sulfur source are fed to a bioreactor containing hydrogenotrophic microorganisms under conditions such that the hydrogenotrophic microorganisms produce at least one fermentation product selected from amino acids, alcohols, aldehydes or ketones, carboxylic acids, or hydroxy or keto acids; (b) removing from the bioreactor a gas stream having a gas stream selected from sulfur-containing compounds, nitrogen-containing compounds, H2, CO x (c) removing a liquid stream comprising fermentation broth, hydrogenotrophic microorganisms and fermentation products from the bioreactor; and (d) separating the hydrogenotrophic microorganisms from the liquid stream and recycling the hydrogenotrophic microorganisms back to the bioreactor. The hydrogenotrophic microorganisms may be selected from methanogenic archaea.

[0035] According to a preferred embodiment, the method further comprises a step of harvesting at least a portion of the amino acids from the bioreactor (after which the amino acids are separated from other components of the fermentation broth, for example, by a purification method). The portion comprises norvaline but may also comprise other amino acids.

[0036] In particular for continuous cultures, the harvesting step may involve withdrawing a liquid stream from the bioreactor, the liquid stream comprising the fermentation broth, the methanogenic microorganisms and the produced amino acid, such as norvaline (particularly in the culture supernatant), separating the methanogenic microorganisms from the liquid stream (e.g. by filtration) and recycling the methanogenic microorganisms back to the bioreactor. The harvested amino acid (which may be present in the liquid fraction of the fermentation broth) is then preferably further purified, for example by methods known in the art, such as chromatography.

[0037] It was unexpectedly discovered that methanogens actively excrete (or secrete) multiple amino acids, including norvaline, into the culture supernatant. The secretion of these amino acids into the supernatant is particularly interesting because it simplifies downstream steps (e.g., product harvesting without cell lysis) while increasing productivity (because the methanogens remain viable and can be retained or returned to the bioreactor).

[0038] Therefore, according to a preferred embodiment of the present invention, the method comprises harvesting at least a portion of the amino acids (including norvaline) from the supernatant of the fermentation broth.

[0039] In the process of the present invention, it was found that a variety of methanogenic microorganisms were still alive and / or intact (because lysis was greatly reduced). Therefore, it is preferred that the protein content of the supernatant (used in the harvesting step) is less than 1000 μg / mL, preferably less than 500 μg / mL, more preferably less than 250 μg / mL, even more preferably less than 100 μg / mL, even more preferably less than 50 μg / ml or even less than 40 μg / mL, especially less than 30 μg / mL or even less than 20 μg / mL. The protein content can be measured by methods known in the art, such as the Bradford protein assay.

[0040] Similarly, it is very preferred that at least 50%, preferably at least 60%, more preferably at least 70%, even more preferably at least 80%, in particular at least 90% or even at least 95% of the methanogenic microorganisms remain viable and / or intact before and during harvesting. This applies in particular to methanogenic microorganisms present in a liquid stream withdrawn from the bioreactor (e.g. in a continuous culture). The methanogenic microorganisms can then be recycled back to the bioreactor.

[0041] According to another preferred embodiment, the total amino acid concentration of the fermentation broth supernatant (used in the harvesting step) is at least 1 μmol / L, preferably at least 5 μmol / L, more preferably at least 10 μmol / L, even at least 25 μmol / L, even more preferably at least 50 μmol / L, even at least 100 μmol / L, in particular at least 150 μmol / L (see Examples 1 and Figure 5 ). It is particularly preferred that the total amino acid concentration of the supernatant is at least 200 μmol / L, preferably at least 500 μmol / L, in particular at least 1000 μmol / L (or even a higher lower limit).

[0042] The amino acids produced by the methods and uses disclosed herein can be one or both of the D- or L-isomers. The amino acids can be, for example, 2-aminobutyric acid, alanine, β-alanine, arginine, aspartic acid, carnitine, citrulline, cystine, dehydroalanine, glutamic acid, glutamine, glycine, hydroxyproline, isoleucine, leucine, lysine, methionine, norleucine, norvaline, ornithine, phenylalanine, proline, pyroglutamic acid, pyrroproline, pyrrolysine, selenocysteine, selenomethionine, serine, homoserine, threonine, tryptophan, tyramine, tyrosine or valine.

[0043] In addition to norvaline, the (harvested) amino acids preferably comprise at least one, preferably at least two or even at least three, more preferably at least four or even at least five, even more preferably at least seven or even at least nine, more preferably at least 12 or even at least 15, even more preferably at least 17 or even at least 18, especially all 20 canonical amino acids (i.e. Asp, Glu, Asn, Ser, His, Gln, Gly, Thr, Arg, Ala, Tyr, Val, Met, Trp, Ile, Phe, Leu, Lys, Cys and Pro). Particularly preferably, the (harvested) amino acids comprise one or more of the following: essential amino acids (essential for human consumption), branched-chain amino acids (BCAA) and glutamic acid, preferably, wherein at least 50 mol%, preferably at least 60 mol%, especially at least 70 mol% of the (produced or harvested) amino acids are essential amino acids, branched-chain amino acids (BCAA) or glutamic acid.

[0044] According to another preferred embodiment, the production rate of norvaline per volume of fermentation broth is at least 0.001 μmol L - 1 h -1 , preferably at least 0.005 μmol L -1 h -1, more preferably at least 0.01 μmol L -1 h -1 , even more preferably at least 0.05 μmol L -1 h -1 , and more preferably at least 0.1 μmol L -1 h -1 , especially at least 0.5 μmol L -1 h -1 or even at least 1.0 μmol L -1 h -1 (or even higher).

[0045] According to another preferred embodiment, the norvaline production rate per unit biomass is at least 0.01 μmol g -1 h -1 , preferably at least 0.05 μmol g -1 h -1 , more preferably at least 0.1 μmol g -1 h -1 , even more preferably at least 0.5 μmol g -1 h -1 , and even more preferably at least 1.0 μmol g -1 h -1 , especially at least 5 μmol g -1 h -1 or even at least 10 μmol g -1 h -1 (or even higher).

[0046] In a preferred embodiment, an electron donor (or electron donor compound) suitable for methanogenic microorganisms is supplied to the bioreactor. In particular, hydrogen (i.e. molecular hydrogen or H2), acetate, methyl compounds (preferably selected from methylamine, methyl sulfide and methanol), any other alcohol (preferably a secondary alcohol, such as 2-propanol or 2-butanol), methoxylated aromatic compounds and / or formates are supplied to the bioreactor (as electron donor compounds). Hydrogen, acetate, methanol or a combination thereof (e.g. methanol and acetate) are particularly preferred.

[0047] According to a further preferred embodiment, methane (produced by methanogenic microorganisms) is harvested from the bioreactor.

[0048] According to another preferred embodiment, the total amino acid production rate per volume of fermentation broth is at least 0.01 μmol L - 1 h -1 , preferably at least 0.05 μmol L -1 h -1 , more preferably at least 0.1 μmol L-1 h -1 , even more preferably at least 0.5 μmol L -1 h -1 , and more preferably at least 1.0 μmol L -1 h -1 , especially at least 5 μmol L -1 h -1 or even at least 10 μmol L -1 h -1 (or even higher).

[0049] According to another preferred embodiment, the total amino acid production rate per biomass is at least 0.1 μmol g -1 h -1 , preferably at least 0.5 μmol g -1 h -1 , more preferably at least 1.0 μmol g -1 h -1 , even more preferably at least 5 μmol g -1 h -1 , and more preferably at least 10 μmol g -1 h -1 , especially at least 50 μmol g -1 h -1 or even at least 100 μmol g - 1 h -1 (or even higher).

[0050] The methanogenic microorganisms used may be natural (e.g., natural isolates or laboratory strains obtained therefrom) or genetically manipulated. For example, genetic manipulation in methanogenic archaea may be performed by a person skilled in the art, such as manipulations based on site-directed mutagenesis, selectable markers, transformation methods, and reporter genes, see, e.g., Sarmiento et al. 2011. CRISPR-based gene editing and other CRISPR-based genetic tools are also available to a person skilled in the art, see, e.g., Nayak & Metcalf 2017 and Dhamad & Lessner 2020.

[0051] According to a particularly preferred embodiment, the methanogenic microorganism comprises an archaea selected from any one of the orders Methanobacteriales, Methanococcales, Methanomicrobiales, Methanosarcinales, Methanopyrales, Methanocellales, Methanomassiliicoccales and Methanonatronarchaeales, preferably Methanobacteriales and Methanococcales; more preferably selected from the families Methanobacteriaceae, Methanocaldococcaceae and Methanococcaceae; in particular selected from the families Methanothermobacter, Methanothermococcus, and Methanococcus. Particularly preferred are Methanothermobactermarburgensis, Methanocaldococcus jannaschii, Methanococcus igneus and Methanocaldococcus villosus. Other suitable methanogenic archaea are, for example, Methanobacterium alcaliphilum, Methanobacterium bryantii, Methanobacterium congolense, Methanobacterium defluvii, Methanobacterium espanolae, Methanobacterium formicicum, Methanobacterium ivanovii, Methanobacterium palustre, Methanobacterium thermaggregans, Methanobacterium uliginosum, Methanobrevibacter acididurans,Methanobrevibacter arboriphilicus, Methanobrevibacter gottschalkii, Methanobrevibacter olleyae, Methanobrevibacter ruminantium, Methanobrevibacter smithii, Methanobrevibacter woesei, Methanobrevibacterwolinii, Methanocella arvoryzae, Methanocella conradii, Methanocella paludicola, Methanothermobacterthermautotrophicum, Methanothermobacter thermoflexlgs, Methanothermobacter thermophilus, Methanothermobacter wolfeii, Methanothermus sociabilis, Methanocorpusculum bavaricum, Methanocorpusculumparvum, Methanoculleus chikuoensis, Methanocullelgssubmarinus, Methanogenium frigidura, Methanogeniumliminatans, Methanogenium marinlgm, Methanomicrococcus blatticola, Methanoplanus endosymbiosus, Methanoplanus limicola), Methanoplanuspetrolearius, Methanoregula boonei, Methanosaetaconcilii,Methanosaeta harundinacea, Methanosaeta pelagica, Methanosaeta thermophila, Methanosarcina acetivorans, Methanosarcina barkeri, Methanosarcina mazei, Methanosarcinathermophila, Methanomicrobium mobile, Methanococcusaeolicus, Methanococcus maripaludis, Methanococcus vanniclii, Methanococcus voltae, Methanothermococcus thermolithotrophicus, Methanopyrus kandleri, Methanothermobacter thermautotrophicum, Methanocaldococcus fervens, Methanocaldococcus indicus, Methanocaldococcus infernus, Methanocaldococcus vulcanius.

[0052] Other species or strains of archaea suitable for use in the present invention are described in, for example: Leigh 2000; Fardeu et al. 1987; Belay et al. 1984; Murray and Zinder 1984; & Thauer, 1980; Blank et al. 1995; Bult et al. 1996; Kessler et al., 1997, Mauerhofer et al., 2021; all of which are incorporated herein by reference. Strains can be obtained, for example, from the "Deutsche Sammlung für Mikroorganismen und Zellkulturen GmbH" (DSMZ) (Braunschweig, Germany).

[0053] Methanogens can be hydrogenotrophic, aceticlastic, methylotrophic (eg, H2-dependent methylotrophic), or methoxytrophic.

[0054] The (defined) co-cultivation of methanogenic microorganisms in a bioreactor has also proven to be advantageous.Thus, the methanogenic microorganisms (in the bioreactor) preferably comprise at least two different species.

[0055] It is highly preferred to initiate the fermentation with methanogenic microorganisms in a chemically defined fermentation medium.

[0056] In another preferred embodiment, the fermentation is carried out under anaerobic conditions.

[0057] According to another preferred embodiment, the method is a continuous process, a fed-batch process, a batch process, a closed batch process, a repeated batch process, a repeated fed-batch process or a repeated closed batch process, preferably a continuous process, a fed-batch process or a repeated fed-batch process, especially a continuous process. Particularly preferably, the continuous process (cultivation) is a chemostat process (cultivation), especially with a controlled pH.

[0058] When the method is a continuous process (continuous culture), the preferred dilution rate D is 0.001h -1 ~1.5h -1 , preferably 0.01h -1 ~0.5h -1 , especially 0.0125h -1 ~0.1h -1 .

[0059] Typically, a sulfur source is also supplied to the bioreactor. The sulfur source preferably comprises cysteine ​​and / or sulfide, in particular wherein the concentration of sulfide contained in the fermentation broth is 0.001-150 mg / L, preferably 0.01-100 mg / L, in particular 1-80 mg / L, and / or wherein the sulfide feed rate or the cysteine ​​feed rate is 0.0001-0.2 mol L -1 h -1 , preferably 0.001-0.05 mol L -1 h -1 .

[0060] Biomolecular nitrogen (N2) fixation is a key process in the global nitrogen cycle and is closely related to the carbon cycle. 16 moles of ATP are produced for each mol of N2 fixed, making it one of the most expensive metabolic processes (Thamdrup 2012; Hu and Ribbe 2012). Certain archaea as well as bacteria are able to fix N2 (Fernandez et al. 2019). This biological process is called diazotrophy. The first report of nitrogen-fixing growth was in 1984 in archaea, where Methanosarcina barkeri (Murray and Zinder 1984) and Methanococcus thermophilus (Belay et al. 1984) were shown to be able to fix N2. The enzymes required for the reduction of N2 are nitrogenases, which are encoded in the nif gene cluster (Raymond et al. 2004).

[0061] Jones & Stadtman 1977 relates to the effect of selenium and tungsten on the growth of Methanococcus vannieri. The document teaches that the growth of this microorganism on formate is significantly stimulated by selenium and tungsten. Similarly, Dridi et al. 2012 relates to the growth of Methanosphaera stadtmanae enhanced by tungsten. In addition, Lobo & Zinder 1988 studied nitrogen fixation and nitrogenase activity in the archaeon Methanosarcina barkeri 227. These documents do not mention the production of amino acids at all.

[0062] Agriculture and the production of artificial nitrogen-containing fertilizers are indirect sources of greenhouse gas emissions, releasing N2O through the nitrification of ammonia (NH3). The Haber-Bosch process is the main industrial process for synthetic N2 fixation, and 1.5 tons of CO2 are released for every ton of NH3 produced. In the course of the present invention, the inventors investigated whether biological processes could be used to combine carbon and N2 fixation with concomitant amino acid production. It was unexpectedly found that adding NH4 to the fermentation medium +It is beneficial for methanogenic microorganisms to perform efficient N2 fixation. It has also been found that it is beneficial for the tungstate content in the culture medium to be as low as possible (i.e., just below 0.1 μmol / L, preferably below 0.01 μmol / L, and especially below 0.001 μmol / L), because otherwise the amino acid production under nitrogen fixation conditions will be low. Accordingly, in the course of the present invention, the inventors have found that the NH4 that is most suitable for methanogenic microorganisms to effectively generate amino acids (and actively secrete them into the cell culture supernatant) during combined carbon and N2 fixation. + and tungstate concentration range.

[0063] When the nitrogen source comprises nitrogen gas, a specific ammonium concentration range has proven to be particularly advantageous for energy generation (under N2 fixed conditions). Thus, it is preferred that the ammonium concentration in the fermentation broth is at least 0.1 mmol / L, preferably at least 1.0 mmol / L, more preferably at least 2.0 mmol / L or even at least 4.0 mmol / L; or 0.1 mmol / L to 200 mmol / L, preferably 2 mmol / L to 100 mmol / L, more preferably 4 mmol / L to 40 mmol / L, even more preferably 5 mmol / L to 35 mmol / L, still more preferably 6 mmol / L to 30 mmol / L, especially 7 mmol / L to 25 mmol / L, or even 10 mmol / L to 20 mmol / L. It will be apparent to the skilled person that, especially in continuous cultures, the concentration in the fermentation broth may fluctuate (before reaching a steady state). However, it is preferred that the ammonium concentration is maintained at at least 0.1 mmol / L, preferably at least 1.0 mmol / L, more preferably at least 2.0 mmol / L or even at least 4.0 mmol / L or within any range as indicated above (e.g. 0.1 mmol / L to 200 mmol / L or 10 mmol / L to 20 mmol / L) for at least 5 minutes, preferably at least 10 minutes, even more preferably at least 20 minutes, even more preferably at least 1 hour, in particular at least 5 hours or even at least 10 hours (or at least 20 hours or at least 40 hours).

[0064] Alternatively, or in addition, it is preferred that the concentration of tungstate (particularly orthotungstate, i.e. WO4 -2 The concentration is below 0.1 μmol / L, preferably below 0.01 μmol / L, especially below 0.001 μmol / L (especially when the fermentation broth is substantially free of tungstate). It was found in the process of the invention that this allows for more efficient amino acid production.

[0065] Nitrogen-fixing conditions and / or carbon-fixing conditions have been shown to be favorable for the production of norvaline: Therefore, according to a preferred aspect, at least 50%, preferably at least 60%, more preferably at least 70%, even more preferably at least 80%, still more preferably at least 90% or even more preferably at least 95%, in particular at least 99% or even at least 99.9% of all nitrogen atoms of all nitrogen sources supplied to the bioreactor are supplied to the bioreactor in the form of nitrogen gas. In another preferred embodiment, at least 50%, preferably at least 60%, more preferably at least 70%, even more preferably at least 80%, still more preferably at least 90% or even more preferably at least 95%, in particular at least 99% or even at least 99.9% of all carbon atoms of all carbon sources supplied to the bioreactor are supplied to the bioreactor in the form of carbon dioxide gas and / or carbon monoxide gas.

[0066] A variety of different bioreactors can be used for methods and purposes disclosed herein. Liquid phase bioreactors (e.g., stirred tanks, packed beds, single liquid phases, two liquid phases, hollow fiber membranes) are all well known in the art. Multiphase bioreactors (e.g., bubble column bioreactors, trickle bed bioreactors (fixed bed or packed bed), fluidized bed bioreactors) can also be used. Bioreactors are usually made of (stainless) steel, plastic and / or glass in part. Typically, bioreactors include at least one inlet (which allows gas or gas mixtures to enter), and at least two outlets. An outlet allows the removal of a liquid stream, which includes one or more fermentation products (i.e., amino acids), and another outlet allows the removal of a gas stream (e.g., methane produced by methanogenic microorganisms). In a number of embodiments, the bioreactor is a chemical chemostat.

[0067] The present invention also relates to the following embodiments:

[0068] Embodiment 1. A method for producing amino acids by fermentation in a bioreactor, wherein the bioreactor comprises methanogenic microorganisms in a fermentation broth, the method comprising at least the step of supplying a gaseous carbon source comprising carbon dioxide and / or carbon monoxide, a nitrogen source and preferably a sulfur source to the bioreactor under conditions such that the methanogenic microorganisms produce amino acids, wherein the amino acids comprise norvaline, in particular L-norvaline; preferably the ammonium concentration of the fermentation broth is at least 0.1 mmol / L, preferably at least 2 mmol / L, more preferably at least 4 mmol / L, even more preferably at least 5 mmol / L, still more preferably at least 6 mmol / L, in particular at least 7 mmol / L or even at least 10 mmol / L.

[0069] Embodiment 2. The method of embodiment 1, wherein the methanogenic microorganism secretes amino acids into the supernatant of the fermentation broth.

[0070] Embodiment 3. The method of embodiment 1 or 2, wherein the ammonium concentration in the fermentation broth is maintained within 0.1 mmol / L to 200 mmol / L, preferably 2 mmol / L to 100 mmol / L, more preferably 4 to 40 mmol / L, even more preferably 5 mmol / L to 35 mmol / L, still more preferably 6 mmol / L to 30 mmol / L, especially 7 mmol / L to 25 mmol / L, or even 10 mmol / L to 20 mmol / L for at least 5 minutes, preferably at least 10 minutes, even more preferably at least 20 minutes, still more preferably at least 1 hour, especially at least 5 hours or even at least 10 hours (or at least 20 hours or at least 40 hours).

[0071] Embodiment 4. The method according to any one of embodiments 1 to 3, wherein the ammonium concentration in the fermentation broth is 0.1 mmol / L to 200 mmol / L, preferably 2 mmol / L to 100 mmol / L, more preferably 4 to 40 mmol / L, even more preferably 5 mmol / L to 35 mmol / L, still more preferably 6 mmol / L to 30 mmol / L, especially 7 mmol / L to 25 mmol / L, or even 10 mmol / L to 20 mmol / L.

[0072] Embodiment 5. The method according to any one of embodiments 1 to 4, wherein the fermentation broth is substantially free of tungstate.

[0073] Embodiment 6. The method according to any one of embodiments 1 to 5, wherein the fermentation broth is substantially free of orthotungstate.

[0074] Embodiment 7. The method according to any one of embodiments 1 to 6, wherein an electron donor compound suitable for methanogenic microorganisms is fed to the bioreactor.

[0075] Embodiment 8. A method according to embodiment 7, wherein the electron donor compound is selected from hydrogen, acetate, methyl compounds (preferably selected from methylamine, methyl sulfide and methanol), any other alcohol (preferably secondary alcohols such as 2-propanol or 2-butanol), methoxylated aromatic compounds, formates, and combinations thereof; preferably selected from hydrogen, acetate, methanol and combinations thereof.

[0076] Embodiment 9. The method of Embodiment 7, wherein the electron donor compound comprises hydrogen.

[0077] Embodiment 10. A method according to any one of embodiments 1 to 6, wherein hydrogen, acetate, methyl compounds (preferably selected from methylamine, methyl sulfide and methanol), any other alcohol (preferably a secondary alcohol, such as 2-propanol or 2-butanol), methoxylated aromatic compounds and / or formate are fed to the bioreactor; preferably, wherein hydrogen, acetate, methanol or a combination thereof are fed to the bioreactor.

[0078] Embodiment 11. The method according to embodiment 10, wherein hydrogen is fed to the bioreactor.

[0079] Embodiment 12. The method according to embodiment 10 or 11, wherein acetate and methanol are fed to the bioreactor.

[0080] Embodiment 13. The method according to any one of embodiments 1 to 12, further comprising the step of harvesting at least a portion of the amino acids from the bioreactor.

[0081] Embodiment 14. A method according to embodiment 13, wherein harvesting comprises removing a liquid stream from the bioreactor, wherein the liquid stream comprises fermentation broth, methanogenic microorganisms and produced amino acids (particularly in the supernatant), separating the methanogenic microorganisms from the liquid stream and recycling the methanogenic microorganisms back to the bioreactor; preferably wherein at least 50%, preferably at least 60%, more preferably at least 70%, even more preferably at least 80%, in particular at least 90% or even at least 95% of the methanogenic microorganisms remain viable and / or intact.

[0082] Embodiment 15. The method according to embodiment 14 further comprises the step of purifying the produced amino acids from the liquid stream, in particular from the fermentation supernatant; preferably, the total concentration of the produced amino acids in the fermentation supernatant is at least 1 μmol / L, preferably at least 5 μmol / L, more preferably at least 10 μmol / L or even at least 25 μmol / L, even more preferably at least 50 μmol / L or even at least 100 μmol / L, especially at least 150 μmol / L.

[0083] Embodiment 16. The method of any one of Embodiments 1 to 15, wherein the nitrogen source comprises nitrogen gas.

[0084] Embodiment 17. A method according to any one of embodiments 1 to 16, wherein the total protein content of the fermentation supernatant is less than 1000 μg / mL, preferably less than 500 μg / mL, more preferably less than 250 μg / mL, even more preferably less than 100 μg / mL, still more preferably less than 50 μg / ml or even less than 40 μg / mL, especially less than 30 μg / mL or even less than 20 μg / mL.

[0085] Embodiment 18. A method according to any one of embodiments 1 to 17, wherein at least 50%, preferably at least 60%, more preferably at least 70%, even more preferably at least 80%, in particular at least 90% or even at least 95% of the methanogenic microorganisms remain viable and / or intact before and / or during harvesting.

[0086] Embodiment 19. A method according to any one of embodiments 1 to 18, wherein the total amino acid concentration (especially the concentration of norvaline) in the fermentation broth supernatant is at least 1 μmol / L, preferably at least 5 μmol / L, more preferably at least 10 μmol / L or even at least 25 μmol / L, even more preferably at least 50 μmol / L or even at least 100 μmol / L, especially at least 150 μmol / L.

[0087] Embodiment 20. A method according to any one of embodiments 1 to 19, wherein the amino acids further comprise, or wherein the portion further comprises, at least one, preferably at least two or even at least three, more preferably at least four or even at least five, even more preferably at least seven or even at least nine, still more preferably at least 12 or even at least 15, even more preferably at least 17 or even at least 18, especially all 20 typical amino acids (especially their L forms).

[0088] Embodiment 21. A method according to any one of embodiments 1 to 19, wherein the amino acids further comprise, or wherein the portion further comprises, at least one, preferably at least two or even at least three, more preferably at least four or even at least five, even more preferably at least seven or even at least nine, more preferably at least 12 or even at least 15, even more preferably at least 17 or even at least 18, especially all of the following amino acids: 2-aminobutyric acid, alanine, β-alanine, arginine, aspartic acid, carnitine, citrulline, cystine, dehydroalanine, glutamic acid, glutamine, glycine, hydroxyproline, isoleucine, leucine, lysine, methionine, norleucine, norvaline, ornithine, phenylalanine, proline, pyroglutamic acid, pyrroproline, pyrrolysine, selenocysteine, selenomethionine, serine, threonine, tryptophan, tyramine, tyrosine and valine (especially their L forms).

[0089] Embodiment 22. The method according to any one of embodiments 1 to 19, wherein the amino acid further comprises, or wherein the portion further comprises, ornithine and / or norleucine, in particular L-ornithine and / or L-norleucine.

[0090] Embodiment 23. A method according to any one of embodiments 1 to 19, wherein the amino acids further comprise, or wherein the portion further comprises, one or more of the following: essential amino acids (necessary for human consumption), branched-chain amino acids (BCAAs) and glutamic acid (particularly their L-forms); preferably, at least 50 mol%, preferably at least 60 mol%, especially at least 70 mol% of the (generated or harvested) amino acids are essential amino acids, branched-chain amino acids (BCAAs) or glutamic acid (particularly their L-forms).

[0091] Embodiment 24. The method according to any one of embodiments 1 to 23, wherein the production rate of the amino acid per volume of fermentation broth is at least 0.01 μmol L -1 h -1 , preferably at least 0.05 μmol L -1 h -1 , more preferably at least 0.1 μmol L -1 h -1 , even more preferably at least 0.5 μmol L -1 h -1 , and more preferably at least 1.0 μmol L -1 h -1 , especially at least 5 μmol L -1 h -1 or even at least 10 μmol L -1 h -1 .

[0092] Embodiment 25. The method according to any one of embodiments 1 to 24, wherein the total amino acid production rate per volume of fermentation broth is at least 0.01 μmol L -1 h -1 , preferably at least 0.05 μmol L -1 h -1 , more preferably at least 0.1 μmol L -1 h -1 , even more preferably at least 0.5 μmol L -1 h -1 , and more preferably at least 1.0 μmol L -1 h -1 , especially at least 5 μmol L -1 h -1 or even at least 10 μmol L -1 h -1 .

[0093] Embodiment 26. The method according to any one of embodiments 1 to 25, wherein the production rate of the amino acid per unit of biomass is at least 0.1 μmol g -1 h-1 , preferably at least 0.5 μmol g -1 h -1 , more preferably at least 1.0 μmol g -1 h -1 , even more preferably at least 5 μmol g -1 h -1 , and more preferably at least 10 μmol g -1 h -1 , especially at least 50ρmol g -1 h -1 or even at least 100 μmol g -1 h -1 .

[0094] Embodiment 27. The method according to any one of embodiments 1 to 26, wherein the total amino acid production rate per unit biomass is at least 0.1 μmol g -1 h -1 , preferably at least 0.5 μmol g -1 h -1 , more preferably at least 1.0 μmol g -1 h -1 , even more preferably at least 5 μmol g -1 h -1 , and more preferably at least 10 μmol g -1 h -1 , especially at least 50 μmol g -1 h -1 or even at least 100 μmol g -1 h -1 .

[0095] Embodiment 28. The method of any one of Embodiments 1 to 27, wherein methane (produced by methanogenic microorganisms) is harvested from the bioreactor.

[0096] Embodiment 29. The method according to any one of embodiments 1 to 28, wherein the methanogenic microorganism is a genetically manipulated microorganism.

[0097] Embodiment 30. The method according to any one of embodiments 1 to 28, wherein the methanogenic microorganism is a natural isolate or a laboratory strain obtained therefrom.

[0098] Embodiment 31. The method of any one of Embodiments 1 to 30, wherein the methanogenic microorganism comprises an archaea.

[0099] Embodiment 32. A method according to embodiment 31, wherein the archaea is selected from any one of Methanobacteriales, Methanococcales, Methanomicrobacteriales, Methanosarcinales, Methanopyriculares, Methanosporales, Methanomasellales and Methanoalkaliarchaeals, preferably Methanobacteriales and Methanococcales; more preferably selected from Methanobacteriaceae, Methanothermococci and Methanococcaceae; in particular selected from Methanothermophilus, Methanothermophilus, Methanothermococcus and Methanococcus.

[0100] Embodiment 33. The method of embodiment 31, wherein the archaea is selected from Methanothermosus marburgii, Methanothermosococcus jannaschii, Methanococcus pyogenes, and Methanothermosococcus vilitidis.

[0101] Embodiment 34. The method of embodiment 31, wherein the archaea belongs to the genus Methanothermophilus, in particular the species Methanothermophilus marburg.

[0102] Embodiment 35. The method of embodiment 31, wherein the archaea is selected from Methanothermophilus marburgii, Methanothermophilus jannaschii, Methanothermophilus pyrophorus, Methanothermophilus velutipes, Methanobacterium alkaliphilus, Methanobacterium brunneri, Methanobacterium congolensis, Methanobacterium desde, Methanobacterium hispanica, Methanobacterium formicum, Methanobacterium eisenii, Methanobacterium palustris, Methanobacterium thermopolymeris, Methanobacterium ulliginii, Methanobacterium acidotolerant, Methanobacterium arborophilum, Methanobacterium goerii, Methanobacterium ostreatus, Methanobacterium rumenii, Methanobacterium smithii, Methanobacterium wullingii, Methanobacterium wallinii, Methanocella arvoryzae, Methanospora connerii, Methanospora palustris, Methanobacterium thermoautotrophicum, Methanobacterium thermoflexus, Methanobacterium thermophilus, Methanobacterium wolfi, Methanobacterium agglomerate, Methanobacterium bavarianum, Methanobacterium microgranulum, Methanoculleus chikuoensis, submarine methane pocket bacteria, low-temperature methanogens, mud-swimming methanogens, marine methanogens, Methanobacterium blatti, endosymbiotic methanogens, shallow marsh methanogens, petroleum methanogens, Bune methanogens, combined methane bristles, bamboo methane bristles, pelagic methane bristles, thermophilic methane bristles, acetic methanosarcina, barberi methanosarcina, mazei methanosarcina, thermophilic methanosarcina, mobile methanobacteria, Methanococcus eicheni, marine marsh methanogens, Vanni methanococcus, Methanococcus vossen, thermolithotrophic methanothermococcus, Kansei methanothermobacterium, thermoautotrophic methanothermobacterium, boiling methanothermococcus, Indian methanothermococcus, hell methanothermococcus, volcanic methanothermococcus, and combinations thereof.

[0103] Embodiment 36. A method according to any one of embodiments 1 to 35, wherein the methanogenic microorganisms comprise at least two different species; preferably at least one of the species, in particular at least two of which are archaea, in particular selected from Methanobacteriales, Methanococcales, Methanomicrobacteriales, Methanosarcinales, Methanopyrales, Methanosporales, Methanomasellales and Methanoalkaliarchaeals, preferably Methanobacteriales and Methanococcales, more preferably selected from Methanobacteriales, Methanothermococci and Methanococcaceae; in particular selected from Methanothermophilus, Methanothermophilus, Methanothermococcus and Methanococcus.

[0104] Embodiment 37. The method of any one of Embodiments 1 to 36, wherein the methanogenic microorganism comprises a hydrogenotrophic, acetatotrophic, methylotrophic, or methoxytrophic microorganism, or a combination thereof.

[0105] Embodiment 38. The method of any one of embodiments 1 to 37, wherein the fermentation is initiated with a methanogenic microorganism in a chemically defined fermentation medium.

[0106] Embodiment 39. The method according to any one of embodiments 1 to 38, wherein the fermentation is carried out under anaerobic conditions.

[0107] Embodiment 40. The method according to any one of embodiments 1 to 39, wherein the method is a continuous process, a fed-batch process, a batch process, a closed batch process, a repeated batch process, a repeated fed-batch process or a repeated closed batch process, preferably a continuous process, a fed-batch process or a repeated fed-batch process, in particular a continuous process.

[0108] Embodiment 41. The method according to embodiment 40, wherein the method is a chemostatic process.

[0109] Embodiment 42. The method according to embodiment 40 or 41, wherein the dilution rate D is 0.001h -1 ~1.5h -1 , preferably 0.01h -1 ~0.5h -1 , especially 0.0125h -1 ~0.1h -1 .

[0110] Embodiment 43. A method according to any one of embodiments 1 to 42, wherein at least 50%, preferably at least 60%, more preferably at least 70%, even more preferably at least 80%, still more preferably at least 90% or even more preferably at least 95%, in particular at least 99% or even at least 99.9% of all nitrogen atoms of all nitrogen sources supplied to the bioreactor are supplied to the bioreactor in the form of nitrogen gas.

[0111] Embodiment 44. A method according to any one of embodiments 1 to 43, wherein at least 50%, preferably at least 60%, more preferably at least 70%, even more preferably at least 80%, still more preferably at least 90% or even more preferably at least 95%, in particular at least 99% or even at least 99.9% of all carbon atoms of all carbon sources supplied to the bioreactor are supplied to the bioreactor in the form of carbon dioxide gas and / or carbon monoxide gas.

[0112] Embodiment 45. The method of any one of embodiments 1 to 44, wherein a sulfur source is supplied to the bioreactor, preferably the sulfur source comprises cysteine ​​and / or sulfide, in particular the fermentation broth comprises sulfide at a concentration of 0.001 to 150 mg / L, preferably 0.01 to 100 mg / L, in particular 1 to 80 mg / L and / or wherein the sulfide feed rate or the cysteine ​​feed rate is 0.0001 to 0.2 mol L -1 h -1 , preferably 0.001 to 0.05 mol L -1 h -1 .

[0113] Embodiment 46. The method according to any one of embodiments 1 to 45, wherein the bioreactor is a liquid phase or multiphase bioreactor.

[0114] Embodiment 47. A method according to any one of embodiments 1 to 46, wherein the bioreactor is any one of a stirred tank, a packed bed, a single liquid phase, a two liquid phase, a hollow fiber membrane, a bubble column, a trickle bed or a fluidized bed bioreactor.

[0115] Embodiment 48. The method according to any one of embodiments 1 to 47, wherein the bioreactor is a chemostat.

[0116] Embodiment 49. A method according to any one of embodiments 1 to 48, wherein the amino acid comprises, or wherein the portion comprises L-norvaline; preferably, wherein the fermentation broth comprises ammonium at a concentration of at least 0.1 mmol / L, preferably at least 2 mmol / L, more preferably at least 4 mmol / L, even more preferably at least 5 mmol / L, still more preferably at least 6 mmol / L, especially at least 7 mmol / L or even at least 10 mmol / L.

[0117] Embodiment 50. The method according to any one of embodiments 1 to 49, wherein the production rate of norvaline (especially L-norvaline) per volume of fermentation broth is at least 0.001 μmol L -1 h -1 , preferably at least 0.005 μmol L -1 h -1 , more preferably at least 0.01 μmol L-1 h -1 , even more preferably at least 0.05 μmol L -1 h -1 , and more preferably at least 0.1 μmol L -1 h -1 , especially at least 0.5 μmol L -1 h -1 or even at least 1.0 μmol L -1 h -1 .

[0118] Embodiment 51. The method according to any one of embodiments 1 to 50, wherein the production rate of norvaline (especially L-norvaline) per unit of biomass is at least 0.01 μmol g -1 h -1 , preferably at least 0.05 μmol g -1 h -1 , more preferably at least 0.1 μmol g -1 h -1 , even more preferably at least 0.5 μmol g -1 h -1 , and even more preferably at least 1.0 μmol g -1 h -1 , especially at least 5 μmol g -1 h -1 or even at least 10 μmol g -1 h -1 .

[0119] Embodiment 52. Use of a methanogenic microorganism for producing norvaline (especially L-norvaline) and preferably other amino acids, wherein the ammonium concentration of the fermentation broth is at least 0.1 mmol / L, preferably at least 2 mmol / L, more preferably at least 4 mmol / L, even more preferably at least 5 mmol / L, still more preferably at least 6 mmol / L, in particular at least 7 mmol / L or even at least 10 mmol / L.

[0120] Embodiment 53. The use according to embodiment 52, wherein the fermentation broth is as defined in any one of embodiments 2 to 6, 17, 19 and 45.

[0121] Embodiment 54. The use of embodiment 52 or 53, wherein the methanogenic microorganism is supplied with an electron donor compound, preferably an electron donor compound as defined in any one of embodiments 7 to 9, a carbon source, a nitrogen source and preferably a sulfur source.

[0122] Embodiment 55. The use according to any one of embodiments 52 to 54, wherein the other amino acids are as defined in any one of embodiments 20 to 23 and 49.

[0123] Embodiment 56. The use of any one of embodiments 52 to 55, wherein the generation rate is as defined in any one of embodiments 24 to 27, 50 and 51.

[0124] Embodiment 57. The use according to any one of embodiments 52 to 56, wherein the methanogenic microorganism is as defined in any one of embodiments 2 and 29 to 37.

[0125] Embodiment 58. A fermentation broth comprising norvaline, especially L-norvaline (and preferably other amino acids) in the supernatant, and further comprising methanogenic microorganisms; preferably norvaline (and preferably other amino acids) are in the supernatant (of the fermentation broth); preferably the ammonium concentration of the fermentation broth is at least 0.1 mmol / L, preferably at least 2 mmol / L, more preferably at least 4 mmol / L, even more preferably at least 5 mmol / L, still more preferably at least 6 mmol / L, in particular at least 7 mmol / L or even at least 10 mmol / L.

[0126] Embodiment 59. The fermentation broth of Embodiment 58, wherein the fermentation broth is further defined as in any one of Embodiments 3 to 6, 17, 19 and 45.

[0127] Embodiment 60. The fermentation broth of embodiment 58 or 59, further comprising an electron donor compound (preferably as defined in any one of embodiments 7 to 9), a carbon source, a nitrogen source, and preferably a sulfur source.

[0128] Embodiment 61. The fermentation broth of any one of embodiments 58 to 60, wherein the other amino acids are as defined in any one of embodiments 20 to 23 and 49.

[0129] Embodiment 62. The fermentation broth of any one of Embodiments 58 to 61, wherein the methanogenic microorganism is as defined in any one of Embodiments 2 and 29 to 37.

[0130] Embodiment 63. A bioreactor comprising the fermentation broth of any one of embodiments 58 to 62. The present invention is further illustrated by the following figures and examples, but is not limited thereto. BRIEF DESCRIPTION OF THE DRAWINGS

[0131] Figure 1 :Methanothermophilic bacterium Marburg in the original culture medium filled with 7:1:1 (711) H2 / CO2 / N2 gas with different NH4 + Growth kinetics at concentrations OD 578nm. Aeration with a ratio of 4:1 (41) served as a positive control. (a) Closed batch experiment with a purge step, gas samples were collected after 40, 59 and 77 hours. (b) Fed-batch experiment without purge. Dashed lines represent overnight growth. + / - indicates the presence of carbon in the medium. 100% reference run with a 4:1 H2 / CO2 gassing (squares). Gas samples were collected after approximately 0, 13, 16, 19, 22 and 25 hours.

[0132] Figure 2 : NH4 in (a) closed batch and (b) fed-batch experiments + Concentration, in μM. Color indicates NH4 + 0% (orange), 5% (red), 7.5% (brown), 10% (yellow), 100% (grey) and 100% carbon or 4:1 aeration (blue).

[0133] Figure 3 : Amino acid excretion in experiments with 5%, 7.5%, 10%, 100% and 0% blocking batches. Glutamate excretion was dominant.

[0134] Figure 4 : Amino acid excretion in fed-batch 5%, 10%, 100% and 0% experiments. Alanine excretion was dominant.

[0135] Figure 5 : Total amino acids excreted (total amino acids, AA) and ammonium uptake rate (AUR) in closed batch (a) and fed batch (b) experiments. The amount of AA excreted increases with the uptake of ammonium. Example

[0136] Example 1. Amino acid production and active secretion by Methanothermobacterium marburgii under N2-fixed conditions

[0137] Our study aimed to investigate the physiological and biotechnological properties of biological nitrogen fixation linked to H2 / CO2 utilization by different methanogens.

[0138] Among the several methanogens analyzed, Methanothermosaurus marburgii was preferentially studied in closed batch and fed-batch culture modes and under different NH4 + N2 fixation, CH4 production and amino acid excretion characteristics under different concentrations.

[0139] Briefly, M. marburgii was grown in a N2 / H2 / CO2 atmosphere in a chemically defined minimal medium with varying concentrations of ammonium chloride (NH4Cl). This allowed quantification of ammonia uptake, N2 fixation, amino acid excretion, and conversion of H2 / CO2 to CH4. N2 fixation by M. marburgii was confirmed in all experiments using gas-phase H2 / N2 / CO2. In addition, active excretion of proteinogenic amino acids was found, with the highest values ​​detected for glutamate, alanine, glycine, and asparagine. In closed batches, in the presence of “100%” NH4 + At the concentration (see Table 1 below), the highest overall yield was detected after 40 hours, which was 7.5 μmol L -1 h -1 Therefore, the simultaneous production of amino acids and CH4 from CO2 in an integrated approach combining biomethanation and N2 fixation in a biorefinery concept is of biotechnological interest.

[0140] Materials and methods

[0141] strain

[0142] The following strains were selected for the experiment: Methanothermobacterium marburgii ( et al. 1980; Wasserfallen et al. 2000), Methanobacterium thermopolymeris (Blotevogel and Fischer 1985), Methanococcus maritima (Jones et al. 1983), Methanothermococcus villifolius (Bellack et al. 2011) and Methanothermococcus okinawanensis (Takai et al. 2000). These strains can be obtained, for example, from DSMZ, Braunschweig, Germany.

[0143] Culture medium

[0144] Precultures of Methanococcus villifolius and Methanothermococcus okinawanensis were grown on chemically defined medium according to Taubner & Rittmann 2016. Methanococcus marimmarum was grown on McN medium (see Mauerhofer et al. 2021). Methanobacterium thermopolymeris and Methanothermobacterium marburgii were grown on MM medium.

[0145] MM medium (see Table 1 below): NH4Cl 2.1 g / L and KH2PO4 6.8 g / L dissolved in ddH2O. 200x trace element (TE) solution (e.g., up to 5 ml per liter of MM medium) should be added: Titriplex I 9 g / L, add 800 mL of H2O, adjust pH to 6.5 with 5 mol / L NaOH solution, then add (up to target concentration): MgCl2·6H2O 8 g / L, FeCl2·4H2O 2 g / L, CoCl2·6H2O 40 mg / L, NiCl2·6H2O 240 mg / L, NaMoO4·2H2O 40 mg, then adjust pH to 7.0 and volume to 1 L with 1 mol / L NaOH and ddH2O. The medium may also contain, for example, 3.6 g / L NaHCO3 as a carbon source. As a sulfur source, 2 mL of 0.5 mol / L Na2S·9H2O can be added per liter after anaerobic and high-pressure sterilization.

[0146] Surprisingly it has been found that it is advantageous for the TE solution (and therefore the culture medium) to contain no tungstate (or at least only less than 0.1 μmol / L, preferably less than 0.01 μmol / L, in particular less than 0.001 μmol / L), since otherwise the amino acid yield under nitrogen fixing conditions would be lower.

[0147] The medium was dispensed into 117 mL serum bottles (VWR, Austria) to a total working volume of 50 mL and closed with blue rubber stoppers (pre-boiled 10 times for 30 min each, 20 mm, butyl rubber, Chemglass Life Sciences) and aluminum crimp caps (Ochs Laborbedarf, Bovenden, Germany). After autoclaving in an anaerobic glove box (Coy Laboratory Products, Grass Lake, USA), sterile L-cysteine-HCl·H2O, sterile NaHCO3 solution, and Na2S·9H2O were added according to the medium composition. To ensure anaerobic conditions, the atmosphere of the headspace was changed by evacuation and aeration with the corresponding gas mixture (H2 / CO2 or H2 / CO2 / N2) to a maximum pressure of 2 bar relative pressure (3 bar absolute pressure), and the process was repeated five times (Taubner & Rittmann 2016). For inflation, a sterile syringe filter (w / 0.2 c μm cellulose, VWR International, USA) and a sterile needle (disposable hypodermic needle, Gr 14, 0.60 X 30 mm, 23G X 11 / 4″, Braun, Germany) were used.

[0148] When preparing nitrogen-free (N-free) medium, omit NH4Cl (2.1 g L -1), replaced by a chemically equimolar amount of NaCl (2.3 g L -1 ) to ensure the correct salt concentration in the medium. To replace L-cysteine ​​monohydrate, a dilute HCl solution was used to restore the pH. + The concentrations of MM medium are shown in Table 1. To ensure that CO2 was the only carbon source, Na2CO3 was replaced by an equal molar amount of NaCl. -1 NaOH, and the medium without Na2CO3 was manually adjusted to pH 6.8.

[0149] Table 1 has different NH4 + The composition of MM medium with a concentration of *

[0150]

[0151] * Relative to the original MM medium (in g L -1 Relative to the original MM medium (containing 2.1 g L -1 NH4Cl) percentage of NH4Cl. - / + indicates the presence or absence of Na2CO3. TE: trace element solution

[0152] Chemicals

[0153] H2 (99.999%), CO2 (99.999%), N2 (99.999%), H2 / CO2 (80% / 20%), H2 / CO2 / N2 (77.74% / 11.13% / 11.13%) were used for closed batch and fed batch experiments. For gas chromatography (GC), N2 / CO2 (80% / 20%), CH4 (99.995%) and standard test gas (Messer GmbH, Wien, Austria) (0.01 Vol.-% CH4 and 0.08 Vol.-% CO2 in N2) were additionally used. Except for the standard test gas, all gases were purchased from Air Liquide GmbH, Schwechat, Austria. All other chemicals were of the highest grade available.

[0154] Closed batch experiments

[0155] Cultures were incubated in a 65°C water bath (Burgwedel, Germany) (Methanothermobacterium marburgii, Methanobacterium thermopolymeris and Methanothermococcus okinawa) or in a shaking air incubator (Burgwedel, Germany) at 37°C (Methanococcus marinum) and in a shaking air incubator (LABWIT Scientific Pty Ltd, Australia) at 80°C (Methanothermococcus vildici). For N2 fixation, all closed batch experiments were performed in a H2 / N2 / CO2 atmosphere. To select the strains of priority study, Methanothermobacterium marburgii, Methanococcus marinum, Methanobacterium thermopolymeris, Methanothermococcus vildici and Methanothermococcus okinawa were grown in triplicate (n=3) with a zero control until OD 578 About 0.7. Due to the NH4 + The highest concentration was additionally checked for NH4 + To determine whether reducing the amount of nitrogen to one-tenth of the original medium concentration would affect growth. To remove residual nitrogen compounds from the medium, pre-cultured cells were washed before inoculation. For completely nitrogen-free medium, all cultures were washed three times, while all other experiments were washed only once or not at all. Experiments with only M. marburgii were performed in quadruplicate (n=4) or octaplicate (n=8) at a relative original medium composition of 2.1 g / L -1 Different from NH4 + Concentration ("0%", "1%", "10%", "25%", "50%" and "100%", see Table 1 above). Contains one-tenth of NH4 + A preculture of 100% was used as inoculum. A batch containing 0% was used as negative control, while a batch containing 100% (gassed with 4:1 H2 / CO2) was used as positive control.

[0156] After each incubation, the serum bottle was placed at room temperature for 45 minutes to cool. The pressure was measured with a digital pressure gauge (Keller GmbH, Winterthur, Switzerland). Growth was measured by OD (λ = 578 nm, blanked with Milli-Q water) (Beckman Coulter, California, USA) spectrophotometry. 1 mL of liquid sample was centrifuged at full speed (13200 rpm) for 30 minutes. The cell pellet and supernatant of each experiment were stored in -20 ° C sterile Eppendorf tubes until further analysis.

[0157] Fed-batch experiments

[0158] All fed-batch experiments were performed in triplicate with Methanothermosus marburgii and fed with a 7:1:1 H2 / CO2 / N2 mixture. 2.2 L bioreactor system (SR1500ODLS, Eppendorf AG, Hamburg, Germany) with 1.5 L working volume of MM medium including 100 μl L -1 Defoamer (Struktol SB2023, Schill und Seilacher, Hamburg, Germany). Optimal growth conditions are pH 7 and temperature 65°C. Aeration of N2 and CO2 is controlled by an MX4 / 4 device (EppendorfAG, Hamburg, Germany). The H2 gas flow is controlled by a C100L device (Sierra Instruments, Monterey, USA). The aeration ratio is the same as for the closed batch experiment. The redox potential and pH are monitored by respective redox probes and pH probes (MettlerToledo GmbH, Vienna, Austria). Each fed-batch culture is carried out with an inoculum of a stock culture of Methanothermophilus marburgii adapted to fed-batch culture. Before inoculation, H2 / N2 / CO2 is passed into the bioreactor to ensure anaerobic conditions, and 5 mL of 0.5 mol L -1 After inoculation with 30 mL of Na2S·9H2O, 0.2 mL of feed was immediately started. -1 0.5 M Na2S·9H2O and the stirring speed was set to 1600 rpm. Gas samples were collected after approximately 0, 13, 16, 19, 22 and 25 hours. Batches with and without Na2CO3 in the culture medium under H2 / CO2 atmosphere (ratio 4:1) were used as references. For analysis of growth and amino acid excretion, liquid samples were collected and processed as described above.

[0159] Determination of Ammonium

[0160] NH4 + The assay was performed using a modification of the method described previously (Kandeler 1988). The oxidizing solution, the colorimetric reagent, and the NH4Cl stock solution were prepared freshly before the measurement. As a standard, nine different concentrations of NH4Cl were prepared, ranging from 100 μmol L -1 ~1000 μmol L -1 The samples were diluted with MilliQ to a final concentration within the standard range. Immediately before measurement, 300 μL of the colorimetric reagent and 120 μL of the oxidizing solution were added to the standards and samples and mixed immediately. After 30 minutes of protection from light, the samples were analyzed using a 96-well plate (Microtest Plate 96Well, F, Sarstedt AG&C0, Nümbrecht, Germany) and a plate photometer (Sunriseplate reader, Tecan GroupAG, Switzerland) (λ = 660 nm). Regression curve R 2Always above 0.999.

[0161] Gas chromatography

[0162] The off-gas compositions (H2, CO2, CH4 and N2) of the closed batch experiments and the gas samples collected from the fed-batch experiments were analyzed using an Agilent gas chromatograph (Agilent 7890A GC, Agilent Technologies, Santa Clara, CA, USA) equipped with a thermal conductivity detector (TDC) and a 19808 Shin Carbon ST Micropacked column (Restek GmbH, Bad Homburg, Germany).

[0163] Amino acid analysis

[0164] For amino acid analysis, the sample supernatant (obtained as described above) was diluted 1:4 with Mill-Q water. The measurements were performed on an Agilent 1260 Infinity Bioinert HPLC system, which includes a fluorescence detector, a column oven, an autosampler, and a quaternary pump. 1 mL of sample was mixed with 75 μL of borate buffer (0.4 N aqueous solution, pH = 10.2; Agilent Technologies), and then 5 μL of OPA reagent (3-mercaptopropionic acid in 0.4 mol L -1 In borate buffer, 10 mg mL -1 o-Phthalaldehyde (OPA); Agilent Technologies). After 2 min at 27°C, 100 μL of the mixture was injected into the HPLC system. The fluorescent derivative (primary dissolved free amino acid) was added at 25°C at 0.8 mL min -1 The samples were separated on a Zorbax ECLIPSE AAA column (4.6 x 150 mm, 3.5 μm particle size, Agilent Technologies) with a Zorbax ECLIPSE AAA guard column (4.6 x 150 mm, 5 μm particle size, Agilent Technologies) at a flow rate of 1.5 %. The excitation wavelength was 340 nm and the emission wavelength was 450 nm. The use of a gain factor of 9 or 10 depended on the expected concentration and was tested in advance. For peak identification and quantification, the peaks were quantified according to the concentration range of the sample (100 nmol L -1 ~15 μmol L -1) An amino acid standard primary mixture (AAS18, Sigma Aldrich) was prepared at different concentrations for each run. The AAS18 standard mixture lacked five amino acids (asparagine (Asn), glutamic acid (Glu), γ-aminobutyric acid (GABA), taurine (Tau), tryptophan (Trp); Sigma Aldrich), which were all added. A total of 20 different AAs can be measured using this method. Valine and methionine were excluded from the evaluation because they are located within the signal-to-noise "ammonium peak" and are therefore not detected in the presence of high NH4 + The method is described in detail in the previous article (Taubner et al. 2019).

[0165] GC (gas chromatography) analysis

[0166] The relative pressure (in bar) inside the serum bottle was measured with a digital manometer (Keller GmbH, Winterthur, Switzerland). The gaseous substances (n / mol) in the headspace of the serum bottle were calculated by the ideal gas law. The headspace volume was determined in earlier experiments and adjusted by the sample volume of 0.75 mL extracted after each OD measurement. All measurements were performed at room temperature (25°C).

[0167] To obtain the actual amount of N2, the pressure in the serum bottle was multiplied by 0.11392 according to the exact percentage of N2 in the gas mixture (11.392 Vol.-%) and then by the standardized gas composition obtained from the GC measurement. The value of the zero-control was used as the N2 baseline. N2 molecular absorbance (NUR / mmol L -1 h -1 ) was calculated by dividing the deviation of N2 before and after incubation (ΔN2) by the volume of liquid culture medium and the time since the last incubation (Δt):

[0168]

[0169] Quantitative / specific nitrogen uptake (qN2 / mmol h -1 g -1 ) is calculated by dividing NUR by biomass concentration (x / g L -1 ), which is calculated using an experimentally determined coefficient:

[0170]

[0171] Carbon dioxide absorption rate (CUR / mmol L -1 h -1 ), molecular hydrogen absorption rate (HUR / mmol L -1 h -1), CH4 evolution rate (MER / mmol L -1 h -1 ), carbon balance (C-balance), yield (Y (CH4 / CO2) and Y (x / CO2)) and biomass productivity (r x / c-mmol L -1 h -1 ) were calculated as described elsewhere (Taubner et al. 2016; Rittmann et al. 2012; Bernacchi et al. 2014). H2, CO2, N2, and CH4 concentrations were obtained after GC measurements.

[0172] result

[0173] Strain prioritization

[0174] The growth of Methanothermosus marburgii, Methanococcus maritima S0001, Methanobacterium thermopolymeris, Methanothermoscus velutipes and Methanothermoscus okinawanensis in a H2 / CO2 / N2 atmosphere with a defined NH4 + This allowed us to screen NH4 + Absorption, N2 fixation, amino acid excretion and conversion of H2 / CO2 to CH4. All methanogens except thermopolymeric Methanobacterium can grow in the presence of NH4 + Grow in medium to OD 578 is 0.7. Further experiments showed that under these conditions, a certain amount of NH4 + is required for growth. Due to the most favorable growth characteristics in these experiments, Methanothermophilus marburgii was selected for further experiments. + Growth experiments were performed at 2 concentrations and the results showed no growth or nitrogen-limited growth at 0% and 1% concentrations and similar growth at all other concentrations. Growth rates were higher in experiments with Na2CO3 in the medium.

[0175] NH4 from Methanothermobacterium marburgii + Absorption kinetics

[0176] Closed batch experiments with Methanothermophilus marburgii were performed with a relative concentration of 2.1 g L -1 Original medium composition 0%, 5%, 7.5%, 10% and 100% NH4 + The gas was carried out with a H2 / CO2 / N2 ratio of 7:1:1. In order to reduce NH4 +To account for the possibility of carryover, the experiment was performed in octuplicate (n=8) with one wash step and an additional zero control. Due to the biomass wash step, slower growth was observed compared to the biomass experiment without wash. After 77.17 hours, OD 578 Between 0.17 and 0.20. The 100% 4:1 positive control showed the best OD as expected 578 value, about 0.25. Gas samples were collected after about 40, 59 and 77 hours ( Figure 1 ).

[0177] Additional fed-batch experiments (n=3) were performed with NH4 + The concentrations were 0%, 1%, 5%, 10% and 100%. The reference experiment showed similar growth, OD 578 Up to 7.0 and 8.1 ( Figure 1 ). With 100% NH4 + Compared with the experiment with Na2CO3 in the medium, OD 578 2.2 times higher. This effect was also seen in closed batch experiments. + The concentration of 10% showed stagnation, and the final OD after 20 hours was 578 1.6, NH4 + The concentration of 5% showed stasis, and the OD 578 is 0.9.

[0178] The absorption rates of H2 and CO2, HUR and CUR, and MER are shown in Table 2.

[0179] Table 2: Different NH4 + Hydrogen uptake rate (HUR), carbon dioxide uptake rate (CUR), nitrogen uptake rate (NUR) and methane evolution rate (MER) and carbon balance of closed batch experiments of Methanothermophilus marburg at 240 concentrations.

[0180]

[0181] NH4 + Interaction with simultaneous absorption of N2

[0182] NH4+ uptake during N2 fixation was evident in both closed-batch and fed-batch experiments. Figure 2 (a) and Figure 2 (b) It can be seen that during the fed-batch culture, NH4 + Limits seen at 0-10% NH4 + , while in closed batch culture, NH4 + It is also worth noting that in closed batch experiments, NH4 +The concentrations did not differ much between time points, but in the fed-batch experiment, NH4 + The concentration decreases with time ( Figure 2 ). The highest consumption was achieved in the positive control. In the blocked batch experiment, the highest ammonia uptake rate (AUR) was achieved in the 100% 4:1 group, with a qN2 of 4.6 μmol h -1 g -1 243.8 μmol L -1 h -1 In the fed-batch experiment, the highest AUR of the 100% group was 577.3 μmol L -1 h -1 and qN210.8 μmol h -1 g -1 This is achieved when bicarbonate is present in the culture medium.

[0183] The highest NUR was 0.91 or 0.83 mmol L-1 after 40 h in the 7.5% and 100% blocked batch experiments, respectively. -1 h -1 , and 0.88 mmol L calculated after 59 h in the 10% experiment -1 h -1 qN2 showed the same pattern (Table 3). The earlier sampling time points showed higher NUR values, and the later time points showed lower but more balanced NUR values.

[0184] Table 3: NUR [mmol L] in 5%, 7.5%, 10% and 100% NH4Cl blocking batch experiments -1 h -1 ] and qN2[mmol h -1 g -1 ].

[0185]

[0186]

[0187] Amino Acid Excretion of Methanothermophilus Marburg

[0188] In closed batches ( Figure 3 ) and fed-batch ( Figure 4 ) were used to investigate the active excretion of amino acids by Methanothermophilus marburgii. Almost all detectable amino acids were found in all culture modes. The most highly excreted amino acids were glutamic acid (Glu), alanine (Ala), glycine (Gly), and asparagine (Asn). The concentrations of Glu, Gly, and Asn increased during the culture, but Ala was consumed after a certain time point. All culture experiments showed a significant NH4 +dependence, where amino acid excretion in the 5%, 7.5%, and 10% experiments differed from that in the 100% experiment. Figure 3 and 4 ). Looking closely at the volume values, glutamate had the highest value in the blocked batch, reaching a maximum of 4.59 μmol L after 40 h in the 5% experiment. -1 h -1 The amount of alanine was less, with a maximum of 1.36 μmol L -1 h -1 The highest value of Gly appeared in the 100% experiment after 40 h, which was 0.99 μmol L -1 h -1 Fed-batch culture resulted in Ala being the most excreted amino acid, reaching a maximum of 2.67 μmol L in the 5% experiment. -1 h -1 It is also noteworthy that the Asn yield in the 100% experiment was higher than that in the other NH4 + The concentration is 10 times higher, up to 0.79 μmolL -1 h -1 .

[0189] The total amount of excreted amino acids was examined, and closed batches showed that the amount of amino acid excretion increased over time, reaching a maximum of 14.67-18.44 μmol L -1 The fed-batch system showed a higher total AA excretion, with the highest value being 156.08 μmol L -1 ( Figure 5 ). In contrast, it is noteworthy that the productivity during the closed batch experiment [μmol L -1 h -1 ] is higher than in fed-batch experiments. Excessive NH4 + It seems to inhibit the production of amino acids, as the values ​​shown in the 100% experiment are slightly lower ( Figure 5 ). NH4 + The total absorption of NH4 was compared with the total excretion rate of amino acids, indicating that the excretion rate of amino acids increased with the increase of AUR. + During the consumption period, the concentration of AA did not increase ( Figure 5 ).

[0190] in conclusion

[0191] In the context of power-to-gas technology, biomethanation using CO2 from renewable resources combined with N2 fixation to produce amino acids by methanogens has high economic value. To the best of our knowledge, no combined CO2 / N2 fixation biological processes have been investigated.

[0192] Switching the culture mode between closed or fed-batch changed the excretion rate and concentration of amino acids, e.g., for Glu, from a maximum value of 2.74 mg L in fed-batch to -1 becomes 44.83 mg L in the closed batch -1 (Table 3).

[0193] Unexpectedly, a variety of different amino acids were actively excreted, with up to 7.5 μmol L -1 h -1 The highest total amounts were at several early time points ( Figure 5 ).

[0194] These results emphasize that methanogens are well suited for the production of amino acids in a biotechnological context, even under N2-fixing conditions.

[0195] Example 2: Amino acid production and active secretion by Methanothermophilus marburg in continuous culture A continuous culture of Methanothermophilus marburg for amino acid production was successfully established.

[0196] The experiments were carried out using Methanothermophilus marburgii in 2 L bioreactors (Eppendorf AG, Hamburg, Germany) and 15 L bioreactors (Biostat C+, Sartorius Stedim Biotech AG, Germany) ambassador.

[0197] For fermentation, the original MM medium described in Example 1 above was used.

[0198] The same medium was used as feed medium for the continuous culture mode.

[0199] To ensure anaerobic conditions in the reaction vessel, the entire system was flushed with H2 / CO2, N2 or H2 / CO2 / N2 mixed gas for 10 min before inoculation.

[0200] The cultures were maintained at 65°C with stirring speeds of 100-1200 rpm (DASGIP parallel bioreactor system, Eppendorf AG, Hamburg, Germany) and 100-1500 rpm (Biostat C+, Sartorius Stedim Biotech AG, The pH value was measured by a pH probe (Mettler Toledo GmbH, Vienna, Austria or Hamilton Bonaduz AG, Bonaduz, Switzerland) and kept constant at 7.

[0201] The redox potential (ORP) was measured by a redox probe (Mettler Toledo GmbH, Vienna, Austria). A 0.5 mol / L Na2S·9H2O solution was used as a sulfur source and was continuously fed into the bioreactor at, for example, 0.2 mL / h to 1.32 mL / h. The MM medium was supplied with a simulated peristaltic pump. The MM medium feed flow rate, the sodium Na2S·9H2O feed rate and the titration were recorded gravimetrically or adjusted by the pump speed. The bioreactor volume was kept constant by pumping away the culture suspension that exceeded the immersion tube using a peristaltic pump controlled at a fixed bioreactor weight, or by using a pipe at a fixed height as a level control system. The pumped suspension was collected in a harvest bottle and its volume was recorded gravimetrically. All solutions were converted to anaerobic solutions by flushing with N2, H2 / CO2 or H2 / CO2 / N2. To maintain anaerobic conditions, all bottles were pressurized with N2. Pure H2 / CO2 (4:1) was used as substrate for Methanothermobacterium marburgii. The CO2 gas flow was controlled by an MX4 / 4 device (Eppendorf AG, Hamburg, Germany). The H2 gas flow was controlled by a C100L device (Sierra Instruments, Monterey, USA).

[0202] Methanothermophilus marburgii was cultured continuously for about thirty different rounds under anaerobic conditions. The volume of one round was 1.6L to 10.29L. The dilution rate D fluctuated between rounds, and the specific D value was 0.0125h -1 ~0.05h -1 . The gas volume per volume of liquid per minute (vvm) also fluctuates between each round, for example, 0.125 to 0.5. The stirring (rpm) also fluctuates between each round, for example, 375 to 1500. As a sulfur source, 0.5 mol / L Na2S is supplied, for example, 0.2 mL / h to 1.32 mL / h. Typically, the ammonium concentration is maintained at 15 mmol / L to 35 mmol / L.

[0203] Importantly, the volumetric amino acid production rate and the amino acid specific production rate are about 25 to about 75 μmol L -1 h -1 and about 50 to about 2000 μmol h -1 g -1(Total amount of all amino acids). Overall, the following amino acids were observed to be generated (in combination) and secreted into the culture supernatant: Asp, Glu, Asn, Ser, His, Gln, Gly, Thr, Arg, Ala, Tyr, Val, Met, norvaline (Nva), Trp, Ile, Phe, Leu, Lys. The generation rate of a single amino acid was observed to be up to about 40 μmol L - 1 h -1 (volume measurement) and up to about 900 μmol h -1 g -1 (Specific production rate per biomass). Cys and Pro were not detected due to analytical limitations, but are expected to be produced and secreted.

[0204] In summary, reliable amino acid production was observed in continuous cultures. The secretion of these amino acids into the supernatant is particularly noteworthy as it simplifies downstream steps (e.g., product harvesting without cell lysis). Also unexpected was the observation of Nva production, which has never been observed in methanogenic archaea (let alone in the Methanobacteriales order).

[0205] Example 3: Amino acid production and active secretion in other methanogenic archaea

[0206] The production of amino acids (including typical amino acids and Nva) and their active secretion are also seen in methanogenic archaea other than Methanothermobacterium marburgii, namely Methanococcus jannaschii, Methanococcus pyrophorus and Methanococcus velutipes. These methanogens were incubated in 282-basic medium under closed batch conditions similar to Example 1, but at their respective preferred temperatures (see also Mauerhofer et al. 2021). Although the production of Glu, for example, was more pronounced under these conditions, Nva production was also clearly visible in Methanococcus jannaschii, Methanococcus pyrophorus and Methanococcus velutipes (volumetric Nva production rates exceeded 1.0 μmol L -1 h -1 , the Nva specific generation rate exceeds 10 μmol g -1 h -1 The production of Nva has never been seen before in methanogenic archaea (let alone Methanococcales).

[0207] In summary, the following amino acids were observed to be produced (in combination) and actively secreted into the culture supernatant in methanogenic microorganisms: Asp, Glu, Asn, Ser, His, Gln, Gly, Thr, Arg, Ala, Tyr, Val, Met, Nva, Trp, Ile, Phe, Leu and Lys.

[0208] Example 4: Amino acid production and active secretion of Methanothermophilus marburgii in continuous culture (further experiments)

[0209] The experiments were carried out using Methanothermophilus marburgii in 2 L bioreactors (Eppendorf AG, Hamburg, Germany) and 15 L bioreactors (Biostat C+, Sartorius Stedim Biotech AG, Germany) ambassador.

[0210] For fermentation, the original MM medium described in Example 1 above was used.

[0211] The same medium was used as feed medium for the continuous culture mode.

[0212] To ensure anaerobic conditions in the reaction vessel, the entire system was flushed with H2 / CO2, N2 or H2 / CO2 / N2 mixed gas for 10 min before inoculation.

[0213] The cultures were maintained at 65°C with stirring speeds of 375-1500 rpm (DASGIP parallel bioreactor system, Eppendorf AG, Hamburg, Germany) and 375-1500 rpm (Biostat C+, Sartorius Stedim Biotech AG, The pH value was measured by a pH probe (Mettler Toledo GmbH, Vienna, Austria or Hamilton Bonaduz AG, Bonaduz, Switzerland) and kept constant at 7.

[0214] The redox potential (ORP) was measured by a redox probe (Mettler Toledo GmbH, Vienna, Austria). A 0.5 mol / L Na2S·9H2O solution was used as a sulfur source and was continuously fed into the bioreactor at, for example, 0.05 mL / h to 1.32 mL / h. The MM medium was fed with a simulated peristaltic pump. The MM medium feed flow rate, the sodium Na2S·9H2O feed rate and the titration were adjusted by the pump speed. The bioreactor volume was kept constant. The withdrawn suspension was collected in a harvest bottle. All solutions were turned into anaerobic solutions by flushing with N2, H2 / CO2 or H2 / CO2 / N2. To maintain anaerobic conditions, all bottles were pressurized with N2. Pure H2 / CO2 (4:1) was used as a substrate for Methanothermophilus marburgii. The CO2 gas flow was controlled by an MX4 / 4 device (EppendorfAG, Hamburg, Germany). The H2 gas flow was controlled by a C100L device (Sierra Instruments, Monterey, USA).

[0215] Methanothermophilus marburgii was cultured continuously for more than 100 different rounds under anaerobic conditions. The volume of one round ranged from 1.6 L to 10.29 L. The dilution rate D fluctuated between rounds, with a specific value of D of 0.00625 h -1 ~0.05h -1 , such as 0.025h -1 . The gas volume per volume of liquid per minute (vvm) also fluctuated between each round, for example, 0.125 to 0.5. The stirring (rpm) also fluctuated between each round, for example, 375 to 1500. As a sulfur source, 0.5 mol / L Na2S was supplied, for example, 0.05 mL / h to 1.32 mL / h. Typically, the ammonium concentration was maintained at 0.21 mmol / L to 41.63 mmol / L.

[0216] Importantly, the volumetric amino acid production rate and the amino acid specific production rate are about 5 to about 250 μmol L -1 h -1 and about 10 to about 150 μmol h -1 g -1 (Total amount of all amino acids). Overall, the following amino acids were observed to be generated (in combination) and secreted into the culture supernatant: Asp, Glu, Ser, His, Gly, Thr, Arg, Ala, Tyr, Val, Met, norvaline (Nva), Trp, Ile, Phe, Leu, norleucine (Nle), Lys. The generation rate of a single amino acid was observed to be as high as about 130 μmol L -1 h -1 (volume measurement) and up to about 70 μmol h-1 g -1 (Specific production rate per biomass). Cys and Pro were not detected due to analytical limitations, but are expected to be produced and secreted.

[0217] In summary, reliable amino acid production was observed in continuous cultures. The secretion of these amino acids into the supernatant is particularly noteworthy as it simplifies downstream steps (e.g., product harvesting without cell lysis). Also unexpected was the observation of production of Nva and Nle, which has never been observed in methanogenic archaea (let alone in the Methanobacteriales order).

[0218] Example 5: Amino acid production and active secretion in other methanogenic archaea

[0219] The production of amino acids (including the canonical amino acids, norvaline, ornithine and homoserine) and their active secretion into the culture supernatant were also observed in methanogenic archaea other than Methanothermococcus marburgii, namely Methanothermococcus thermoautotrophicus and a species of Methanothermococcus sp. These methanogens were incubated under closed batch conditions similar to those of Example 1, but at their respective preferred temperatures in MM medium (see Example 1; for the cultivation of Methanothermococcus, 30 g / L NaCl was added to the medium) and at various ammonium concentrations. Although the production of, for example, Ala and Glu was more pronounced under these conditions, the production of Nva was also clearly visible in each species. The production of Nva has never been seen before in methanogenic archaea (let alone Methanobacteriales or Methanococcales).

[0220] In summary, the following amino acids (combinations) were observed to be produced and actively secreted into the culture supernatant in methanogenic microorganisms: Ala, Asp, Asn, Glu, Gly, His, homoserine, Ile, Leu / Nle, Lys, Nva, ornithine, Phe, Pro, Ser, Thr, Try, Tyr, and Val. (The detection method used in this example cannot distinguish between Leu and Nle.)

[0221] Non-patent references

[0222] Abdel Azim, A., Rittmann, SK-MR, Fino, D., Bochmann, G., 2018. The physiological effect of heavy metals and volatile fatty acids on Methanococcus maripaludis S2. Biotechnol Biofuels 11, 301.

[0223] Abdel Azim,Annalisa,Christian Pruckner,Philipp Kolar,Ruth SophieTaubner,Debora Fino,Guido Saracco,Filipa L.Sousa,and SimonK.M.R.Rittmann.2017.″The Physiology of Trace Elements in Biological MethaneProduction.″Bioresource Technology 241:775-86.

[0224] Becker J and Wittmann C.Systems and synthetic metabolic engineeringfor amino acid production-the heartbeat of industrial strain development.CurrOpin Biotechnol.2012Oct;23(5):718-26.

[0225] Belay N,Sparling R and Daniels L.1984.″Dinitrogen Fixation by aThermophilic Methanogenic Bacterium.″Nature 312(5991):286-288.

[0226] Bellack,Annett,Harald Huber,Reinhard Rachel,Gerhard Wanner,andReinhard Wirth.2011.″Methanocaldococcus Villosus Sp.Nov.,a Heavily FlaellatedArchaeon That Adheres to Surfaces and Forms Cell-Cell Contacts.″

[0227] Bernacchi,Sébastien,et al.″Experimental methods for screeningparameters influencing the growth to product yield(Y(x / CH4))of a biologicalmethane production(BMP)process performed with Methanothermobactermarburgensis.″AIMS Bioengineering 1.2(2014):72-87.

[0228] Biermann,Michael,et al.″Simultaneous analysis of the non-canonicalamino acids norleucine and norvaline in biopharmaceutical-relatedfermentation processes by a new ultra-high performance liquid chromatographyapproach.″Amino Acids 44.4(2013):1225-1231.

[0229] Blank,C.E.,Peter S.Kessler,and John A.Leiqh.1995.″Genetics inMethanogens:TransposonInsertion Mutagenesis of a Methanococcus MaripaludisNifH Gene."Journal of Bacteriology 177(20):5773-77.

[0230] Blotevogel,Karl Heinz and Ulrich Fischer.1985.″Isolation andCharacterization of a New Thermophilic and Autotrophic Methane ProducingBacterium:Methanobacterium Thermoaggregans Spec.Nov.″Archives of Microbiology142(3):218-22.

[0231] Bult,Carol J.,et al.″Complete genome sequence of the methanogenicarchaeon,Methanococcus jannaschii.″Science 273.5278(1996):1058-1073.

[0232] Dhamad,Ahmed E.,and Daniel J.Lessner.″A CRISPRi-dCas9 system forarchaea and its use to examine gene function during nitrogen fixation byMethanosarcina acetivorans.″Applied and environmental microbiology 86.21(2020):e01402-20.

[0233] Dridi,Bédis,et al.″Tungsten-enhanced growth of Methanosphaerastadtmanae.″BMC Research Notes 5(2012):1-4.

[0234] Fardeau,M-L.,J-P.Peillex,and J-P.Belaich.″Enerqetics of the rowth ofMethanobacterium thermoautotrophicum and Methanococcus thermolithotrophicuson ammonium chloride and dinitrogen.″Archives of microbiology 148.2(1987):128-131.

[0235] Fernandez L,Bertilsson S,Peura S2019Non-cVanobacterial diazotrophsdominate nitrogen-fixing communities in permafrost thaw ponds,Limnol.Oceanogr.65,2020,S180-S193,Association for the Sciences of Limnologyand Oceanography.

[0236] Hoffarth,Marc Philippe,Timo Broeker,and Jan Schneider.″Effect of N2on biological methanation in a continuous stirred-tank reactor withmethanothermobacter marburgensis.″Fermentation 5.3(2019):56.

[0237] Hu Y and Ribbe MW.2012.″Nitroenase Assembly."Biochimica et BiophysicaActa 1827:1112-22.

[0238] Jones,J.B.,and Stadtman.″Methanococcus vannielii:culture and effectsof selenium and tungsten on growth.″Journal of bacteriology 130.3(1977):1404-1406.

[0239] Jones,W.Jack,M.J.B.Paynter,and R.Gupta.1983.Characterization ofMethanococcus Maripaludis Sp.Nov.,a New Methanogen Isolated from Salt MarshSediment.Vol.135.

[0240] Kandeler,Ellen.1988.″Short-Term AssaV of Soil Urease Activity UsingColorimetric Determination of Ammonium SoilCare View Project BiodiversityExploratories View Project.″Biology and Fertility of Soils 6(1):68-72.

[0241] Kengen,Servé WM,and Alfons JM Stams.″Formation of L-alanine as areduced end product in carbohydrate fermentation by thehyperthermophilicarchaeon Pvrococcus furiosus.″Archives of Microbiology 161(1994):168-175.

[0242] Kessler,Peter S.,Jennifer McLarnan,and John A.Leigh.″Nitrogenasephylogeny and the molybdenum dependence of nitrogen fixation in Methanococcusmaripaludis.″Journal of bacteriology 179.2(1997):541-543.

[0243] Kisumi,Masahiko,Masaki Sugiura,andIchiro Chibata.″Biosynthesis ofnorvaline,norleucine,and homoisoleucine in Serratia marcescens.″The Journalof Biochemistry 80.2(1976):333-339.

[0244] Kurth JM,Op den Camp HJM,Welte CU,2020.Several ways one goal-methanogenesis from unconventional substrates.Appl Microbiol Biotechnol 104,6839-6854.

[0245] Leigh,John A.2000.″Nitrogen Fixation In Methanogens:The ArchaealPersDective.″2:125-31.

[0246] Liu,Hui,et al.″Effects of different amino acids and theirconfigurations on methane yield and biotrans formation of intermediatemetabolites during anaerobic digestion.″Journal of Environmental Management296(2021):113152.

[0247] Lobo,Anthony L.,and Stephen H.Zinder.″Diazotrophy and nitrogenaseactivitv in the archaebacterium Methanosarcina barkeri 227.″Applied andenvironmental microbiology 54.7(1988):1656-1661.

[0248] Lyu,Z,Shao N,Akinyemi T,Whitman WB,2018.Methanogenesis.CurrentBiology 28,R727-R732.

[0249] Mand,Thomas D.,and William W.Metcalf.″Energy conservation andhydrogenase function in methanogenic archaea,in particular the genusMethanosarcina.″Microbiology and Molecular Biology Reviews 83.4(2019):e00020-19.

[0250] Mauerhofer,LM.,Zwirtmayr,S.,Pappenreiter,P.et al.Hyperthermophilicmethanogenic archaea act as high-pressure CH4cell factories.Commun Biol 4,289(2021).

[0251] Mauerhofer,L.-M.,Reischl,B.,Schmider,T.,Schupp,B.,Nagy,K.,Pappenreiter,P.,Zwirtmayr,S.,Schuster,B.,Bernacchi,S.,Seifert,A.H.,Paulik,C.,Rittmann,S.K.-M.R.,2018.Physiology and methane productivitV ofMethanobacterium thermaggregans.Appl Microbiol Biotechnol 102,76437656.

[0252] Mayumi D,Hanako M,Hideyuki T,Kyosuke Y,Hideyoshi Y,Yuichiro S,YoichiK,and Susumu S.2016.″Methane Production from Coal by a Single Methanogen.″Science 354(6309):22225.

[0253] Murray PA and Zinder SH.1984.″Nitrogen Fixation by a MethanogenicArchaebacterium.″Nature 312(5991):284-86.

[0254] Nayak,Dipti D.,and William W.Metcalf.″Cas9-mediated genome editing inthe methanogenic archaeon Methanosarcina acetivorans.″Proceedings of theNational Academy of Sciences 114.11(2017):2976-2981.

[0255] Pappenreiter,P.A.,Zwirtmayr,S.,Mauerhofer,L.-M.,Rittmann,S.K.-M.R.,Paulik,C.,2019.Development of a simultaneous bioreactor system forcharacterization of gas production kinetics of methanogenic archaea at highpressure.Engineering in Life Sciences 19,537-544.

[0256] Pfei fer,Kevin,et al.″Archaea biotechnology.″Biotechnology Advances47(2021):107668.

[0257] Polis,Baruh,et al.″L-Norvaline,a new therapeutic agent againstAlzheimer′s disease.″Neural regeneration research 14.9(2019):1562.

[0258] Porat,Iris,et al.″Two biosynthetic pathways for aromatic amino acidsin the archaeon Methanococcus maripaludis.″Journal of bacteriology 186.15(2004):4940-4950,

[0259] Raymond J,Siefert JL,Staples CR,and Blankenship RE.2004.″The NaturalHistory of Nitrogen Fixation.″Molecular Biology and Evolution21(3):541-54.

[0260] Rinke,Christian,et al.″A standardized archaeal taxonomy for theGenome Taxonomy Database.″Nature Microbiology 6.7(2021):946-959.

[0261] Rittmann,S.,A.Seifert,and Christoph Herwig.″Quantitative analysis ofmedia dilution rate effects on Methanothermobacter marburgensis grown incontinuous culture on H2 and CO2.″Biomass and Bioenergy 36(2012):293-301.

[0262] Rittmann,S.K.-M.R.,2015.A Critical Assessment of MicrobiologicalBiogas to Biomethane Upgrading Systems.Adv.Biochem.Eng.Biotechnol.151,117-135.

[0263] Rittmann,S.K.-M.R.,Seifert,A.H.,Bernacchi,S.,2018.Kinetics,multivariate statistical modelling,and physiology of CO2-based biologicalmethane production.Applied Energy 216,751-760.

[0264] Rittmann,Simon K-MR,et al.″Archaea in der Biotechnologie.″BIospektrum27.1(2021):96-98.

[0265] Sarmiento,Felipe B.,John A.Leigh,and William B.Whitman.″Geneticsystems for hydrogenotrophic methanogens.″Methods inenzVmology.Vol.494.Academic Press,2011.43-73.

[0266] P,Johanna Moll,and Rudolf K.Thauer.1980.″Growth Parameters(Ks,Mmax,Ys)of Methanobacterium Thermoautotrophicum.″Archives of Microbiology127(1):5965

[0267] Peter,and Rudolf K.Thauer.″L-Alanine,a product of cell wallsynthesis in Methanobacterium thermoautotrophicum.″FEMS Microbiology Letters9.2(1980):77-80.

[0268] Sment&Konisky.″Excretion of amino acids by 1,2,4-triazole-3-alanine-resistant mutants of Methanococcus voltae.″Applied and environmentalmicrobiology 55.5(1989):1295-1297.

[0269] Takai,Ken,AkiraInoue,and Koki Horikoshi.2002.″Methanothermococcusokinawensis Sp.Nov.,a Thermophilic,Methane-Producing ArchaeonIsolated from aWestern Pacific Deep-Sea Hydrothermal Vent System.″International Journal ofSystematic and Evolutionary Microbiology 52(4):1089-95.

[0270] Taubner,Ruth Sophie and Simon K.M.R.Rittmann.2016.″Method forIndirect Quantification of CH4production via H2o Production UsingHydrogenotrophic Methanogens.″Frontiers in Microbiology 7(APR):1-11.

[0271] Taubner,R.-S.,et al,2018.Biological methane production under putativeEnceladus-like conditions.Nature Communications 9,748.

[0272] Taubner,Ruth-Sophie,et al.″Membrane lipid composition and amino acidexcretion patterns of Methanothermococcus okinawensis grown in the presenceof inhibitors detected in the Enceladian plume.″Life 9.4(2019):85.

[0273] Thamdrup B,New Pathways and Processes in the Global Nitrogen Cycle,2012,Annual Review of Ecology,Evolution,and Systematics,407-428,43,1.

[0274] Wasserfallen A,et al.2000.″Phylogenetic Analysis of 18ThermophilicMethanobacterium Isolates Supports the Proposals to Create a New Genus,Methanothermobacter Gen.Nov.,and to Reclassify Several Isolates in ThreeSpecies,Methanothermobacter Thermautotrophicus Comb.Nov.,Methano.″International Journal of Systematic and EvolutionarV Microbiology 50:4353.

[0275] Whitman,W.B.,Es Ankwanda,and R.S.Wolfe.″Nutrition and carbonmetabolism of Methanococcus voltae.″Journal of Bacteriology 149.3(1982):852-863.

[0276] Whitman,William B.,et a1.″Isolation and characterization of22mesophilic methanococci.″Systematic and applied microbiology 7.2-3(1986):235-240.

[0277] Zeikus,J.G.″The biology of methanogenic bacteria.″Bacteriologicalreviews 41.2(1977):514-541.

Claims

1. A method for producing amino acids by fermentation in a bioreactor, wherein the bioreactor comprises methanogenic microorganisms in a fermentation broth, the method comprising at least the following steps: The bioreactor is fed with a gaseous carbon source including carbon dioxide and / or carbon monoxide, a nitrogen source, and preferably a sulfur source, under conditions such that the methanogenic microorganism produces an amino acid, wherein the amino acid comprises norvaline.

2. The method according to claim 1, further comprising the step of harvesting at least a portion of the amino acids from the bioreactor.

3. The method according to claim 1 or 2, wherein the amino acids further comprise, or the portion further comprises, at least one, preferably at least two or even at least three, more preferably at least four or even at least five, even more preferably at least seven or even at least nine, more preferably at least 12 or even at least 15, even more preferably at least 17 or even at least 18, especially all 20 typical amino acids.

4. The method according to any one of claims 1 to 3, wherein the methanogenic microorganism comprises an archaea selected from any one of Methanobacteriales, Methanococcales, Methanomicrobacteriales, Methanosarcinales, Methanopyrales, Methanosporales, Methanomasellales and Methanoalkaliarchaeals, preferably selected from Methanobacteriales and Methanococcales; more preferably selected from Methanobacteriaceae, Methanothermococci and Methanococcaceae; in particular selected from Methanothermophilus, Methanothermophilus, Methanothermococcus and Methanococcus.

5. The method according to any one of claims 1 to 4, wherein the norvaline production rate per volume of fermentation broth is at least 0.001 μmol L -1 h -1 , preferably at least 0.005 μmol L -1 h -1 , more preferably at least 0.01 μmol L -1 h -1 , even more preferably at least 0.05 μmol L -1 h -1 , and more preferably at least 0.1 μmol L -1 h -1 , especially at least 0.5 μmol L -1 h -1 or even at least 1.0 μmol L -1 h -1 .

6. The method according to any one of claims 1 to 5, wherein the norvaline production rate per unit biomass is at least 0.01 μmol g -1 h -1 , preferably at least 0.05 μmol g -1 h -1 , more preferably at least 0.1 μmol g -1 h -1 , even more preferably at least 0.5 μmol g -1 h -1 , and more preferably at least 1.0 μmol g -1 h -1 , in particular at least 5 μmol g -1 h -1 or even at least 10 μmol g -1 h -1 .

7. The method according to any one of claims 1 to 6, wherein the method is a continuous process, a fed-batch process, a batch process, a closed batch process, a repeated batch process, a repeated fed-batch process or a repeated closed batch process, preferably a continuous process, a fed-batch process or a repeated fed-batch process, in particular a continuous process.

8. The method according to any one of claims 1 to 7, wherein the nitrogen source comprises nitrogen gas; preferably wherein at least 50%, preferably at least 60%, more preferably at least 70%, even more preferably at least 80%, yet more preferably at least 90% or even more preferably at least 95%, in particular at least 99% or even at least 99.9% of all nitrogen atoms of the total nitrogen source supplied to the bioreactor are supplied to the bioreactor in the form of nitrogen gas.

9. The method according to any one of claims 1 to 8, wherein at least 50%, preferably at least 60%, more preferably at least 70%, even more preferably at least 80%, still more preferably at least 90% or even more preferably at least 95%, in particular at least 99% or even at least 99.9% of all carbon atoms of the total carbon source supplied to the bioreactor are supplied to the bioreactor in the form of carbon dioxide gas and / or carbon monoxide gas.

10. The process according to any one of claims 1 to 9, wherein hydrogen, acetate, methyl compounds (preferably selected from methanol, methylamine and methyl sulfide), any other alcohol (preferably a secondary alcohol such as 2-propanol or 2-butanol), methoxylated aromatic compounds and / or formate are fed to the bioreactor; preferably hydrogen, acetate, methanol or a combination thereof are fed to the bioreactor.

11. The method according to any one of claims 1 to 10, wherein a sulfur source is supplied to the bioreactor, preferably the sulfur source comprises cysteine ​​and / or sulfide, in particular the fermentation broth comprises sulfide in a concentration of 0.001 to 150 mg / L, preferably 0.01 to 100 mg / L, in particular 1 to 80 mg / L and / or the sulfide supply rate or cysteine ​​supply rate is 0.0001 to 0.2 mol L -1 h -1 , preferably 0.001 to 0.05 mol L -1 h -1 .

12. The method according to any one of claims 1 to 11, wherein the ammonium concentration of the fermentation broth is 0.1 mmol / L to 200 mmol / L, preferably 2 mmol / L to 100 mmol / L, more preferably 4 to 40 mmol / L.

13. Use of methanogenic microorganisms for producing norvaline.

14. A fermentation broth comprising a methanogenic microorganism and norvaline, preferably the norvaline is in the supernatant.

15. A bioreactor comprising the fermentation broth of claim 14.

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