Method for producing amino acids in a bioreactor

Abstract

The present invention provides a method for producing an amino acid by fermentation in a bioreactor, wherein the bioreactor contains methanogenic microorganisms in a fermentation broth, and the method comprises at least the step of supplying a gaseous carbon source containing carbon dioxide and / or carbon monoxide, a nitrogen source containing nitrogen gas, and preferably a sulfur source to the bioreactor under conditions such that the methanogenic microorganisms produce the amino acid, wherein the fermentation broth contains ammonium at a concentration of 0.1 mmol / L to 200 mmol / L, preferably 2 mmol / L to 100 mmol / L, more preferably 4 to 40 mmol / L. Preferred methanogenic microorganisms are archaea selected from, for example, the genus Methanothermobacter, the genus Methanothermococcus, and the genus Methanococcus.

Description

Technical Field

[0001] The field of the present invention relates to a method for producing amino acids by fermentation in a bioreactor using methanogenic microorganisms.

Background Art

[0002] Amino acids are applied in various sectors: food and feed, agriculture, pharmaceuticals, and even packaging and housing. The biosynthesis of amino acids that make up proteins is an important field of biotechnology (Becker and Wittmann 2012). However, the metabolic capabilities of archaea regarding amino acid production have been largely overlooked so far (Pfeifer et al 2021). A standardized archaea classification method is disclosed in Rinke et al, 2021.

[0003] Methanogenic archaea are known for their ability to produce methane (CH4) as the final product of their energy metabolism (see, for example, Mand & Metcalf, 2019). Some of them can grow autotrophically and hydrogenotrophically by reducing carbon dioxide (CO2) to CH4 with molecular hydrogen (H2), playing an important role in the global carbon cycle (Lyu et al. 2018). According to their substrate utilization spectra, methanogens can be classified into various metabolic groups such as hydrogenotrophic (H2, formate, or simple alcohols), acetate-utilizing (acetate), methylotrophic (compounds containing a methyl group), H2-dependent methylotrophic (methylated compounds having H2 as an electron donor), and methoxydotrophic (methoxylated aromatic compounds) (Mayumi et al., 2016, Kurth et al., 2020). The biology of methanogenic archaea has also been discussed in old publications such as Zeikus, 1977.

[0004] Taubner et al., 2019 is a research paper on astrobiology unrelated to biotechnology. Specifically, this literature concerns the membrane lipid composition and amino acid excretion patterns of Methanothermococcus okinawensis grown in the presence of inhibitors detected in the Enceladian plume. These findings are important for understanding the ecophysiology of methanogens on Earth and suggest the use of biomarkers as possible signs of extraterrestrial life for future space missions in the solar system. Similarly, Taubner et al., 2018 discusses biological methane production under putative Enceladus-like conditions.

[0005] Furthermore, although unrelated to biotechnology, Kengen and Stams 1994 concerns the formation of L-alanine as the final reduction product in carbohydrate fermentation by the hyperthermophilic archaeon Pyrococcus furiosus. Sment & Konisky, 1989 claims amino acid excretion by a 1,2,4-triazole-3-alanine-resistant mutant of Methanococcus voltae, as opposed to the wild type. In the experiments described in this literature, M. voltae is grown in a defined medium containing 19 amino acids under an H2-CO2 atmosphere.

[0006] Also, although unrelated to biotechnology, Porat et al., 2004 discusses the biosynthetic pathway of aromatic amino acids in Methanococcus maripaludis.

[0007] Whitman et al., 1986 describes the isolation and characterization of 22 mesophilic methanococci. The cultures are grown under H2-CO2 and the media are prepared under N2-CO2. This literature does not describe amino acid production at all.

[0008] Also, although unrelated to biotechnology, Fardeau et al., 1987 is concerned with the energetics of the growth of Methanobacterium thermoautotrophicum and Methanococcus thermolithotrophicus on ammonium chloride and dinitrogen. This document does not mention amino acid production at all.

[0009] Similarly, Whitman et al., 1982 is concerned with the nutrition and carbon metabolism of Methanococcus voltae. Various nitrogen sources are discussed. It is stated that ammonia is required for the growth of M. voltae in defined media. The need for inorganic substances is also discussed in this document.

[0010] Rittmann et al., 2021 is generally concerned with the use of archaea in biotechnology.

[0011] Some studies have discussed the use of methanogenic archaea in renewable energy production by reducing 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 is concerned with the effects of different amino acids and their compositions on methane yield and in vivo conversion of intermediate metabolites during anaerobic digestion. WO 2012 / 110256 discloses a method for converting carbon dioxide and hydrogen to methane by methanogenic microorganisms. WO 2014 / 128300 is concerned with methods and systems for producing methane using methanogenic microorganisms and applying a specific nitrogen concentration to a liquid phase.

[0012] International Publication No. 2017 / 070726 relates to a method for determining the culture state of a microbial culture, for example, a culture containing hydrogenotrophic methanogenic microorganisms.

[0013] Hoffarth et al., 2019 relates to the effect of N2 on biological methanation in a continuously stirred tank reactor containing Methanothermobacter marburgensis. N2 is described as behaving like an inert gas. It is further taught in this document that "N2 is not involved in the dissimilation reaction". This document does not describe amino acid production at all.

[0014] U.S. Patent Application Publication No. 2011 / 281333 relates to methane production from single-celled organisms such as methanogens. Methanogen growth involves consuming carbon dioxide to produce methane. Methods for promoting growth are disclosed. Gaseous N2 is not contemplated as a nitrogen source.

[0015] U.S. Patent Application Publication No. 2018 / 0179559 relates to biological and chemical processes that utilize chemoautotrophic microorganisms for the chemical synthetic fixation of carbon dioxide and / or other inorganic carbon sources into organic compounds and for the production of further useful products. The microorganisms can be selected from many different bacterial and archaeal species.

[0016] European Patent Application Publication No. 2192170 relates to amino acid-producing microorganisms and methods for producing amino acids. Having the ability to produce L-amino acids selected from the group consisting of L-lysine, L-threonine, L-tryptophan, L-phenylalanine, L-valine, L-leucine, L-isoleucine, and L-serine, pyruvate synthase or pyruvate:NADP +Microorganisms (preferably selected from gamma-proteobacteria, coryneform bacteria, or bacteria belonging to the genus Alicyclobacillus, Bacillus, or the yeast genus Saccharomyces) modified to enhance the activity of redox enzymes are disclosed.

[0017] International Publication No. WO 2016 / 179545 and U.S. Patent Application Publication No. US 2018 / 0163240 disclose compositions and methods for the biological production of methionine.

[0018] U.S. Patent Application Publication No. US 2019 / 0194630 and U.S. Patent Application Publication No. US 2017 / 0130211 relate to compositions and methods for the biological production of amino acids in hydrogenotrophic microorganisms. In particular, the hydrogenotrophic microorganisms can be selected from the genus Methanococcus and the genus Methanosarcina.

[0019] International Publication No. WO 2020 / 252335 relates to a process and a system for producing a product by fermentation. In particular, (a) supplying a gaseous mixture containing CO x and H2 (where x is 1 or 2), a nitrogen source, and optionally a sulfur source to a bioreactor containing a hydrogenotrophic microorganism under conditions such that the hydrogenotrophic microorganism produces at least one fermentation product selected from amino acids, alcohols, aldehydes or ketones, carboxylic acids, or hydroxyl or keto acids; (b) removing from the bioreactor a gas stream having at least one compound selected from sulfur-containing compounds, nitrogen-containing compounds, H2, CO x , and hydrocarbon compounds (where x is 1 or 2); (c) removing a liquid stream from the bioreactor containing the fermentation broth, the hydrogenotrophic microorganism, and the fermentation product; and (d) separating the hydrogenotrophic microorganism from the liquid stream and recycling the hydrogenotrophic microorganism to the bioreactor. The hydrogenotrophic microorganism can be selected from methanogenic archaea.

[0020] Despite these efforts, further development of fermentation methods using methane-producing microorganisms is still needed.

[0021] Accordingly, an object of the present invention is to provide improved methods for producing amino acids by fermentation in a bioreactor containing methane-producing microorganisms. These methods should use (natural) resources more efficiently, be more environmentally friendly, lead to a reduction in greenhouse gas emissions, increase yields, and / or overcome one or more drawbacks of amino acid production methods known in the art.

Summary of the Invention

[0022] The present invention provides a method for producing amino acids by fermentation within a bioreactor. The bioreactor contains methane-producing microorganisms in a fermentation broth. The method includes at least the step of supplying to the bioreactor a gaseous carbon source containing carbon dioxide and / or carbon monoxide, a nitrogen source containing nitrogen gas (i.e., N2 gas or molecular nitrogen gas), and preferably a sulfur source, under conditions such that the methane-producing microorganisms produce amino acids. The fermentation broth contains ammonium (i.e., NH4 + ) at a concentration of 0.1 mmol / L to 200 mmol / L, preferably 2 mmol / L to 100 mmol / L, more preferably 4 to 40 mmol / L. Typically, the method includes the step of recovering at least a portion of the amino acids from the bioreactor (subsequently, for example, performing a purification method for separating the amino acids from other components of the fermentation broth).

[0023] In a further aspect, the present invention provides the use of a methanogenic microorganism for producing an amino acid from an electron donor compound, a gaseous carbon source containing carbon dioxide and / or carbon monoxide, a nitrogen source containing nitrogen gas, and preferably a sulfur source in a fermentation broth. Also, in this aspect, the fermentation broth contains ammonium at a concentration of 0.1 mmol / L to 200 mmol / L, preferably 2 mmol / L to 100 mmol / L, more preferably 4 to 40 mmol / L. The electron donor compound is preferably selected from hydrogen gas (i.e., H2 or molecular hydrogen gas), 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, formate, and mixtures thereof. Particularly preferred as the electron donor compound are hydrogen gas, methanol, and / or acetate.

[0024] Agriculture and the production of artificial nitrogen-containing fertilizers are indirect sources of greenhouse gas emissions by releasing N2O through the nitrification of ammonia (NH3). The Haber-Bosch process is the main industrial procedure for synthetic N2 fixation and is responsible for the release of 1.5 tons of CO2 per ton of the produced NH3. In the process of the present invention, the inventors investigated whether biological processes could be used for the combination of carbon and N2 fixation and the accompanying amino acid production. Surprisingly, it was found that the addition of NH4 + to the fermentation medium is very advantageous for enabling efficient N2 fixation in methanogenic microorganisms. However, when the concentration of NH4 + in the medium becomes too high, amino acid production is inhibited. Therefore, in the process of the present invention, the inventors found the NH4 + concentration range that is most suitable for efficient amino acid production (and active secretion into the cell culture supernatant) in methanogenic microorganisms during the combination of carbon and N2 fixation.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0026] The detailed description given below relates to all of the above aspects of the present invention unless explicitly excluded.

[0027] Biological molecular nitrogen (N2) fixation is an important process in the global nitrogen cycle that is closely related to the carbon cycle. ATP per mole of fixed N2 is 16 moles, which is one of the most expensive metabolic processes (Thamdrup 2012; Hu and Ribbe 2012). Not only certain phyla of archaea but also bacteria can fix N2 (Fernandez et al. 2019). This biological process is called diazotrophy. Diazotroph growth was first reported in archaea in 1984, and it was shown that Methanosarcina barkeri (Murray and Zinder 1984) and Methanococcus thermolithotrophicus (Belay et al., 1984) can fix N2. The enzyme required for the reduction of N2 is nitrogenase encoded by the nif gene cluster (Raymond et al. 2004).

[0028] Jones and Stadtman, 1977 is about the effects of selenium and tungsten on the growth of Methanococcus vannielii. This document teaches that the growth of microorganisms on formate is significantly stimulated by selenium and tungsten. Similarly, Dridi et al., 2012 is about the tungsten-enhanced growth of Methanosphaera stadtmanae. Furthermore, Lobo and Zinder, 1988 is about diazotrophy and nitrogenase activity in the archaeon Methanosarcina barkeri 227. These documents do not describe amino acid production at all.

[0029] When the nitrogen source contains nitrogen gas, a certain specific ammonium concentration range has been found to be particularly advantageous for energy generation (under N2 fixation conditions). Therefore, the ammonium concentration in the fermentation broth is preferably 0.1 mmol / L to 200 mmol / L, more preferably 2 mmol / L to 100 mmol / L, still more preferably 4 mmol / L to 40 mmol / L, even more preferably 5 mmol / L to 35 mmol / L, yet even more preferably 6 mmol / L to 30 mmol / L, particularly 7 mmol / L to 25 mmol / L, or even more preferably 10 mmol / L to 20 mmol / L. In particular, in continuous culture, it is obvious to those skilled in the art that the concentration in the culture broth can vary (until the steady state is reached). However, the ammonium concentration preferably remains within any of the above ranges (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, still more preferably at least 20 minutes, even more preferably at least 1 hour, particularly at least 5 hours or even at least 10 hours (or at least 20 hours or at least 40 hours).

[0030] Furthermore, when the concentration of tungstate (especially orthotungstate, i.e., WO4 -2 concentration) in the fermentation broth is less than 0.1 μmol / L, preferably less than 0.01 μmol / L, particularly less than 0.001 μmol / L (especially when the fermentation broth substantially does not contain tungstate), it is preferred. In the process of the present invention, it has been found that this enables more efficient amino acid production.

[0031] In a preferred embodiment, an electron donor (or electron donor compound) suitable for methanogenic microorganisms is supplied to the bioreactor. In particular, hydrogen gas (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, a methoxylated aromatic compound, and / or formate is supplied to the bioreactor (as an electron donor compound). Hydrogen gas, acetate, methanol, or a combination thereof (e.g., methanol and acetate) is particularly preferred.

[0032] According to a more preferred embodiment, methane (produced by methanogenic microorganisms) is recovered from the bioreactor.

[0033] Typically, the method of the present invention includes recovering at least a portion of the amino acids from the bioreactor. In particular, continuous culture may include removing a liquid stream containing the fermentation broth, methanogenic microorganisms, and the produced amino acids (especially in the culture supernatant) from the bioreactor, separating the methanogenic microorganisms from the liquid stream (e.g., by filtration), and recycling the methanogenic microorganisms to the bioreactor. The recovered amino acids (which may be present in the liquid fraction of the fermentation broth) are then preferably further purified by methods known in the art, such as chromatography.

[0034] Surprisingly, it has been found that methanogenic microorganisms actively excrete (or secrete) many different amino acids into the culture supernatant. The secretion of these amino acids into the supernatant simplifies downstream processes (e.g., does not require cell lysis to recover the product) and at the same time increases productivity (since the methanogenic microorganisms remain viable and can remain in or be fed back into the bioreactor), which is particularly remarkable.

[0035] Thus, according to a preferred embodiment of the present invention, the method comprises recovering at least a portion of the amino acids from the supernatant of the fermentation broth.

[0036] During the process of the present invention, it has been found that many of the methanogenic microorganisms remain viable and / or intact (due to significantly reduced lysis). Thus, the protein content of the supernatant (used in the recovery 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, still more preferably less than 50 μg / ml, or even less than 40 μg / mL, particularly 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.

[0037] Similarly, it is highly preferred that at least 50%, preferably at least 60%, more preferably at least 70%, even more preferably at least 80%, particularly at least 90% or even at least 95% of the methanogenic microorganisms remain viable and / or intact before and during the recovery. This is particularly applicable to the methanogenic microorganisms present in the liquid stream removed from the bioreactor (e.g., in continuous culture). The methanogenic microorganisms can then be recycled to the bioreactor.

[0038] According to another preferred embodiment, the total amino acid concentration in the supernatant of the fermentation broth (used in the recovery step) 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, particularly at least 150 μmol / L (see Example 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, particularly at least 1000 μmol / L (or even higher lower limits).

[0039] The amino acids produced by the methods and uses disclosed herein can be either the D-isomer or the L-isomer or both. The amino acids can be, for example, 2-aminobutyric acid, alanine, beta-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, pyrrolidine, selenocysteine, selenomethionine, serine, homoserine, threonine, tryptophan, tyramine, tyrosine, or valine.

[0040] (The recovered) amino acids preferably include at least one, preferably at least two or even at least three, more preferably at least four or even at least five, still more preferably at least seven or even at least nine, yet more preferably at least twelve or even at least fifteen, still even more preferably at least seventeen or even at least eighteen, and particularly all of the 20 standard 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). It is particularly preferred if the (recovered) amino acids contain one or more of essential amino acids (essential for human consumption), branched-chain amino acids (BCAAs), and glutamic acid, preferably at least 50 mol%, preferably at least 60 mol%, particularly at least 70 mol% of the (produced or recovered) amino acids being essential amino acids, branched-chain amino acids (BCAAs), or glutamic acid.

[0041] According to another preferred embodiment, the total amino acid production rate per volume of the fermentation broth is at least 0.01 μmol / L / hour, preferably at least 0.05 μmol / L / hour, more preferably at least 0.1 μmol / L / hour, even more preferably at least 0.5 μmol / L / hour, still even more preferably at least 1.0 μmol / L / hour, particularly at least 5 μmol / L / hour, or even at least 10 μmol / L / hour (or higher).

[0042] According to yet another preferred embodiment, the total amino acid production rate per biomass is at least 0.1 μmol / g / hour, preferably at least 0.5 μmol / g / hour, more preferably at least 1.0 μmol / g / hour, even more preferably at least 5 μmol / g / hour, still even more preferably at least 10 μmol / g / hour, particularly at least 50 μmol / g / hour, or even at least 100 μmol / g / hour (or higher).

[0043] The methanogenic microorganisms used may be natural (e.g., natural isolates or laboratory strains derived therefrom) or genetically engineered. By way of example, genetic manipulations in methanogenic archaea, such as site-directed mutagenesis, selectable markers, transformation methods, and reporter gene-based manipulations, are available to those skilled in the art (see, e.g., Sarmiento et al., 2011). CRISPR-based gene editing and other CRISPR-based gene tools are also available to those skilled in the art; see, e.g., Nayak & Metcalf, 2017 and Dhamad & Lessner, 2020.

[0044] According to certain preferred embodiments, the methanogenic microorganism is an archaeon selected from the order Methanobacteriales, Methanococcales, Methanomicrobiales, Methanosarcinales, Methanopyrales, Methanocellales, Methanomassiliicoccales, and Methanonatronarchaeales, preferably either the order Methanobacteriales or Methanococcales; more preferably, an archaeon selected from the family Methanobacteriaceae and Methanococcaceae; in particular, it includes an archaeon selected from the genus Methanothermobacter, Methanothermococcus, and Methanococcus. Particularly preferred are Methanothermobacter marburgensis, Methanocaldococcus jannaschii, Methanococcus igneus, and Methanocaldococcus villosus. Other suitable methanogenic archaeal species include, for example: Methanobacterium alcaliphilum, Methanobacterium bryantii, Methanobacterium congolense, Methanobacterium defluvii, Methanobacterium espanolae, Methanobacterium formicicumformicicum), Methanobacterium ivanovii, Methanobacterium palustre, Methanobacterium thermaggregans, Methanobacterium uliginosum, Methanobrevibacter acididurans, Methanobrevibacter arboriphilicus, Methanobrevibacter gottschalkii, Methanobrevibacter olleyae, Methanobrevibacter ruminantium, Methanobrevibacter smithii, Methanobrevibacter woesei, Methanobrevibacter wolinii, Methanocella arvoryzae, Methanocella conradii, Methanocella paludicola, Methanothermobacter thermautotrophicum, Methanothermobacter thermoflexus, Methanothermobacter thermophilus, Methanothermobacter wolfeii, Methanothermus sociabilis, Methanocorpusculum bavaricumbavaricum), Methanocorpusculum parvum, Methanoculleus chikuoensis, Methanoculleus submarinus, Methanogenium frigidum, Methanogenium liminatans, Methanogenium marinum, Methanomicrococcus blatticola, Methanoplanus endosymbiosus, Methanoplanus limicola, Methanoplanus petrolearius, Methanoregula boonei, Methanosaeta concilii, Methanosaeta harundinacea, Methanosaeta pelagica, Methanosaeta thermophila, Methanosarcina acetivorans, Methanosarcina barkeri, Methanosarcina mazei, Methanosarcina thermophila, Methanomicrobium mobile, Methanococcus aeolicus, Methanococcus maripaludis, Methanococcus vannielii, Methanococcus voltae (Methanococcusvoltae), Methanothermococcus thermolithotrophicus, Methanopyrus kandleri, Methanothermobacter thermautotrophicum, Methanocaldococcus fervens, Methanocaldococcus indicus, Methanocaldococcus infernus, and Methanocaldococcus vulcanius.

[0045] Further archaeal species or strains suitable for the present invention are disclosed, for example, in the following research papers: Leigh 2000; Fardeu et al. 1987; Belay et al 1984; Murray and Zinder 1984; Schonheit & Thauer, 1980; Blank et al. 1995; Bult et al 1996; Kessler et al, 1997, Mauerhofer et al, 2021. All of these are hereby incorporated by reference. Strains can be obtained, for example, from "Deutsche Sammlung fur Mikroorganismen und Zellkulturen GmbH" (DSMZ) (Braunschweig, Germany).

[0046] The methanogenic microorganisms can be hydrogenotrophic, acetate-utilizing, methylotrophic (e.g., H2-dependent methylotrophic) or methoxydotrophic.

[0047] It has also been found that (defined) co-cultures of methanogenic microorganisms in a bioreactor are also advantageous. Thus, the methanogenic microorganisms (in the bioreactor) preferably comprise at least two different species.

[0048] Fermentation is very preferably initiated using methanogenic microorganisms in a chemically defined fermentation medium.

[0049] In another preferred embodiment, fermentation is carried out under anaerobic conditions.

[0050] According to another preferred embodiment, the method is a continuous process, fed-batch process, batch process, closed batch process, repeated batch process, repeated fed-batch process, or repeated closed batch process, preferably a continuous process, fed-batch process, or repeated fed-batch process, particularly a continuous process. It is particularly preferred that the continuous process (culture) is a chemostat process (culture) with particularly controlled pH.

[0051] When the method is a continuous process (continuous culture), the dilution rate D is preferably from 0.001 / hour to 1.5 / hour, more preferably from 0.01 / hour to 0.5 / hour, particularly preferably from 0.0125 / hour to 0.1 / hour.

[0052] It has been found that nitrogen fixation conditions and / or carbon fixation conditions are advantageous. Thus, according to a further preferred example, 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%, particularly 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 yet 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%, particularly 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.

[0053] Typically, a sulfur source is also supplied to the bioreactor. This source preferably contains cysteine and / or sulfide. In particular, the fermentation broth contains sulfide at a concentration of 0.001 to 150 mg / L, preferably 0.01 to 100 mg / L, particularly 1 to 80 mg / L, and / or the sulfide supply rate or cysteine supply rate is 0.0001 to 0.2 mol / L / hour, preferably 0.001 to 0.05 mol / L / hour.

[0054] In the methods and uses disclosed herein, a variety of different bioreactors can be used. Liquid-phase bioreactors (e.g., stirred tanks, packed beds, one liquid phase, two liquid phases, hollow fiber membranes) are 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 typically made, at least in part, of (stainless) steel, plastic, and / or glass. Usually, a bioreactor contains at least one inlet that allows a gas or gas mixture to enter, and at least two outlets. One outlet allows for the removal of a liquid stream containing one or more fermentation products (i.e., amino acids), and the other outlet allows for the removal of a gas stream (such as methane produced by methanogenic microorganisms). In a plurality of embodiments, the bioreactor is a chemostat.

[0055] Also, the demand for non-proteinogenic amino acids is increasing. Norvaline (also referred to herein as Nva) is a non-standard amino acid whose use as a nutritional supplement is increasing. Further, it is used to induce vasodilation (see, for example, U.S. Patent No. 11,260,039). It has also been suggested as a therapeutic agent for Alzheimer's disease (Polis et al., 2019).

[0056] Although not related to this, norvaline has also attracted interest when it has been found to be incorporated into protein-based biopharmaceuticals produced by a generally undesirable recombinant E. coli fermentation process (Biermann et al., 2013).

[0057] Chinese Patent Application Publication No. 106520651 discloses a method for producing L-norvaline by enzymatic conversion.

[0058] Kisumi et al., 1976 discussed the biosynthesis of the non-proteinogenic amino acids norvaline, norleucine, and homoisoleucine in the enterobacterium Serratia marcescens.

[0059] U.S. Patent Application Publication No. 2006 / 0057685 relates to a fermentation process for producing norvaline using bacteria of the Enterobacteriaceae family in which all acetohydroxyacid synthases have been inactivated.

[0060] During the process of the present invention, surprisingly, it has been observed that methanogenic archaea are capable of norvaline production and the active excretion of norvaline into the cell culture supernatant, particularly under carbon fixation conditions (or under carbon and nitrogen fixation conditions). Norvaline production has not been previously observed in archaea, let alone methanogenic archaea. This finding of the present invention enables combined carbon (and N2) fixation and the accompanying norvaline production.

[0061] Thus, in a particularly preferred embodiment, the (recovered) amino acids preferably include norvaline.

[0062] According to another preferred embodiment, the production rate of norvaline per volume of the fermentation broth is at least 0.001 μmol / L / hour, preferably at least 0.005 μmol / L / hour, more preferably at least 0.01 μmol / L / hour, even more preferably at least 0.05 μmol / L / hour, still even more preferably at least 0.1 μmol / L / hour, particularly at least 0.5 μmol / L / hour or even at least 1.0 μmol / L / hour (or higher).

[0063] According to yet another preferred embodiment, the production rate of norvaline per biomass is at least 0.01 μmol / g / hour, preferably at least 0.05 μmol / g / hour, more preferably at least 0.1 μmol / g / hour, even more preferably at least 0.5 μmol / g / hour, still even more preferably at least 1.0 μmol / g / hour, particularly at least 5 μmol / g / hour or even at least 10 μmol / g / hour (or higher).

[0064] The present invention further relates to the following embodiments: Embodiment 1. A method for producing an amino acid by fermentation in a bioreactor, wherein the bioreactor contains methanogenic microorganisms in a fermentation broth, and the method comprises at least the step of supplying a gaseous carbon source containing carbon dioxide and / or carbon monoxide, a nitrogen source containing nitrogen gas, and preferably a sulfur source to the bioreactor under conditions such that the methanogenic microorganisms produce the amino acid, and preferably, the fermentation broth contains ammonium at a concentration of 0.1 mmol / L to 200 mmol / L, preferably 2 mmol / L to 100 mmol / L, more preferably 4 to 40 mmol / L.

[0065] Embodiment 2. The method according to Embodiment 1, wherein the ammonium concentration in the fermentation broth is 5 mmol / L to 35 mmol / L, even more preferably 6 mmol / L to 30 mmol / L, particularly 7 mmol / L to 25 mmol / L, or even 10 mmol / L to 20 mmol / L.

[0066] Embodiment 3. The method according to Embodiment 1 or 2, wherein the ammonium concentration in the fermentation culture 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 even more preferably 6 mmol / L to 30 mmol / L, particularly 7 mmol / L to 25 mmol / L, or even 10 mmol / L to 20 mmol / L, and persists for at least 5 minutes, preferably at least 10 minutes, more preferably at least 20 minutes, even more preferably at least 1 hour, particularly at least 5 hours, or even at least 10 hours (or at least 20 hours or at least 40 hours).

[0067] Embodiment 4. The method according to any one of Embodiments 1 to 3, wherein the tungstate concentration in the fermentation culture is less than 0.1 μmol / L, preferably less than 0.01 μmol / L, particularly less than 0.001 μmol / L.

[0068] Embodiment 5. The method according to any one of Embodiments 1 to 4, wherein the fermentation culture substantially does not contain tungstate.

[0069] Embodiment 6. The method according to any one of Embodiments 1 to 5, wherein the orthotungstate concentration in the fermentation culture is less than 0.1 μmol / L, preferably less than 0.01 μmol / L, particularly less than 0.001 μmol / L, and in particular, the fermentation culture substantially does not contain orthotungstate.

[0070] Embodiment 7. The method according to any one of Embodiments 1 to 6, wherein an electron donor compound suitable for the methane-producing microorganism is supplied to the bioreactor.

[0071] Embodiment 8. The method according to Embodiment 7, wherein the electron donor compound is selected from hydrogen gas, 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, formate, and combinations thereof; preferably selected from hydrogen gas, acetate, methanol, and combinations thereof.

[0072] Embodiment 9. The method according to Embodiment 7, wherein the electron donor compound contains hydrogen gas.

[0073] Embodiment 10. Hydrogen gas, 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 supplied to the bioreactor; preferably, hydrogen gas, acetate, methanol, or a combination thereof is supplied to the bioreactor. The method according to any one of Embodiments 1 to 6.

[0074] Embodiment 11. The method according to Embodiment 10, wherein hydrogen gas is supplied to the bioreactor.

[0075] Embodiment 12. The method according to Embodiment 10 or 11, wherein acetate and methanol are supplied to the bioreactor.

[0076] Embodiment 13. The method according to any one of Embodiments 1 to 12, further comprising a step of recovering at least a part of the amino acid from the bioreactor.

[0077] Embodiment 14. The recovery includes removing a liquid stream containing a fermentation broth, methanogenic microorganisms, and produced amino acids (especially in the supernatant) from the bioreactor, separating the methanogenic microorganisms from the liquid stream, and recycling the methanogenic microorganisms to the bioreactor; preferably, at least 50%, preferably at least 60%, more preferably at least 70%, even more preferably at least 80%, particularly at least 90% or even at least 95% of the methanogenic microorganisms remain viable and / or intact. The method according to Embodiment 13.

[0078] Embodiment 15. The method further includes a step of purifying the produced amino acids from the liquid stream, especially from the supernatant of the fermentation broth; preferably, the total concentration of the produced amino acids in the supernatant of the fermentation broth 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, particularly at least 150 μmol / L. The method according to Embodiment 14.

[0079] Embodiment 16. The method according to any one of Embodiments 1 to 15 further includes recovering at least a part of the amino acids from the supernatant of the fermentation broth.

[0080] Embodiment 17. The total protein content of the supernatant of the fermentation broth 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, particularly less than 30 μg / mL or even less than 20 μg / mL. The method according to any one of Embodiments 1 to 16.

[0081] Embodiment 18. The 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%, particularly at least 90% or even at least 95% of the methane-producing microorganisms are viable and / or intact before and / or during recovery.

[0082] Embodiment 19. The method according to any one of Embodiments 1 to 18, wherein the total amino acid concentration in the supernatant of the fermentation broth 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, particularly at least 150 μmol / L.

[0083] Embodiment 20. The method according to any one of Embodiments 1 to 19, wherein the amino acids further, or the moieties further, contain at least 1, preferably at least 2 or even at least 3, more preferably at least 4 or even at least 5, even more preferably at least 7 or even at least 9, still more preferably at least 12 or even at least 15, still even more preferably at least 17 or even at least 18, particularly all 20 standard amino acids (especially their L-forms).

[0084] Embodiment 21. The method according to any one of Embodiments 1 to 19, wherein the amino acid or the moiety comprises at least one, preferably at least two or even at least three, more preferably at least four or even at least five, still more preferably at least seven or even at least nine, yet more preferably at least twelve or even at least fifteen, and still even more preferably at least seventeen or even at least eighteen, particularly all of 2-aminobutyrate, alanine, beta-alanine, arginine, aspartic acid, carnitine, citrulline, cystine, dehydroalanine, glutamic acid, glutamine, glycine, hydroxyproline, isoleucine, leucine, lysine, methionine, norleucine, norvaline, ornithine, phenylalanine, proline, pyroglutamic acid, pyroproline, pyrrolidine, selenocysteine, selenomethionine, serine, threonine, tryptophan, triamine, tyrosine, and valine (especially their L-forms).

[0085] Embodiment 22. The method according to any one of Embodiments 1 to 19, wherein the amino acid or the moiety comprises ornithine and / or norleucine, particularly L-ornithine and / or L-norleucine.

[0086] Embodiment 23. The method according to any one of Embodiments 1 to 19, wherein the amino acid or the moiety comprises one or more of essential amino acids (essential for human consumption), branched-chain amino acids (BCAAs), and glutamic acid (especially their L-forms); preferably, at least 50 mol%, preferably at least 60 mol%, particularly at least 70 mol% of the (produced or recovered) amino acids are essential amino acids, branched-chain amino acids (BCAAs), or glutamic acid (especially their L-forms).

[0087] Embodiment 24. The production rate of the amino acid per volume of the fermentation broth is at least 0.01 μmol / L / h, preferably at least 0.05 μmol / L / h, more preferably at least 0.1 μmol / L / h, even more preferably at least 0.5 μmol / L / h, still more preferably at least 1.0 μmol / L / h, particularly at least 5 μmol / L / h, or even at least 10 μmol / L / h. The method according to any one of Embodiments 1 to 23.

[0088] Embodiment 25. The production rate of the total amino acids per volume of the fermentation broth is at least 0.01 μmol / L / h, preferably at least 0.05 μmol / L / h, more preferably at least 0.1 μmol / L / h, even more preferably at least 0.5 μmol / L / h, still more preferably at least 1.0 μmol / L / h, particularly at least 5 μmol / L / h, or even at least 10 μmol / L / h. The method according to any one of Embodiments 1 to 24.

[0089] Embodiment 26. The production rate of the amino acid per biomass is at least 0.1 μmol / g / h, preferably at least 0.5 μmol / g / h, more preferably at least 1.0 μmol / g / h, even more preferably at least 5 μmol / g / h, still more preferably at least 10 μmol / g / h, particularly at least 50 μmol / g / h or even at least 100 μmol / g / h. The method according to any one of Embodiments 1 to 25.

[0090] Embodiment 27. The production rate of the total amino acids per biomass is at least 0.1 μmol / g / h, preferably at least 0.5 μmol / g / h, more preferably at least 1.0 μmol / g / h, even more preferably at least 5 μmol / g / h, still more preferably at least 10 μmol / g / h, particularly at least 50 μmol / g / h or even at least 100 μmol / g / h. The method according to any one of Embodiments 1 to 26.

[0091] Embodiment 28. The method according to any one of Embodiments 1 to 27, wherein methane (produced by the methane-producing microorganism) is recovered from the bioreactor.

[0092] Embodiment 29. The method according to any one of Embodiments 1 to 28, wherein the methane-producing microorganism is a genetically engineered microorganism.

[0093] Embodiment 30. The method according to any one of Embodiments 1 to 28, wherein the methane-producing microorganism is a natural isolate or a laboratory strain obtained therefrom.

[0094] Embodiment 31. The method according to any one of Embodiments 1 to 30, wherein the methane-producing microorganism includes archaea.

[0095] Embodiment 32. The archaea is selected from any of Methanobacteriales, Methanococcales, Methanomicrobiales, Methanosarcinales, Methanopyrales, Methanocellales, Methanomassiliicoccales, and Methanonatronarchaeales, preferably selected from any of Methanobacteriales and Methanococcales; more preferably, selected from Methanobacteriaceae, Methanocaldococcaceae, and Methanococcaceae; particularly, selected from Methanothermobacter, Methanothermococcus, Methanocaldococcus, and Methanococcus. The method according to Embodiment 31.

[0096] Embodiment 33. The method according to embodiment 31, wherein the archaeon is selected from Methanothermobacter marburgensis, Methanocaldococcus jannaschii, Methanococcus igneus, and Methanocaldococcus villosus.

[0097] Embodiment 34. The method according to embodiment 31, wherein the archaeon is of the genus Methanothermobacter, particularly Methanothermobacter marburgensis.

[0098] Embodiment 35. The archaea are Methanothermobacter marburgensis, Methanocaldococcus jannaschii, Methanococcus igneus, Methanocaldococcus villosus, 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 oesei (MethanobrevibacterWoesei), Methanobrevibacter wolinii, Methanocella arvoryzae, Methanocella conradii, Methanocella paludicola, Methanothermobacter thermautotrophicum, Methanothermobacter thermoflexus, Methanothermobacter thermophilus, Methanothermobacter wolfeii, Methanothermus sociabilis, Methanocorpusculum bavaricum, Methanocorpusculum parvum, Methanoculleus chikuoensis, Methanoculleus submarinus, Methanogenium frigidum, Methanogenium liminatans, Methanogenium marinum, Methanomicrococcus blatticola, Methanoplanus endosymbiosus, Methanoplanus limicola, Methanoplanus petrolearius, Methanoregula boonei, Methanosaeta concilii, Methanosaeta(harundinacea), Methanosaeta pelagica, Methanosaeta thermophila, Methanosarcina acetivorans, Methanosarcina barkeri, Methanosarcina mazei, Methanosarcina thermophila, Methanomicrobium mobile, Methanococcus aeolicus, Methanococcus maripaludis, Methanococcus vannielii, Methanococcus voltae, Methanothermococcus thermolithotrophicus, Methanopyrus kandleri, Methanothermobacter thermautotrophicum, Methanocaldococcus fervens, Methanocaldococcus indicus, Methanocaldococcus infernus, Methanocaldococcus vulcanius, and combinations thereof, the method according to embodiment 31.

[0099] Embodiment 36. The methane-producing microorganism contains at least two different species; preferably, at least one, particularly at least two of these species are archaea, and in particular, it is selected from any of Methanobacteriales, Methanococcales, Methanomicrobiales, Methanosarcinales, Methanopyrales, Methanocellales, Methanomassiliicoccales, and Methanonatronarchaeales, preferably selected from either Methanobacteriales or Methanococcales; more preferably, it is selected from Methanobacteriaceae, Methanocaldococcaceae, and Methanococcaceae; in particular, it is selected from Methanothermobacter, Methanothermococcus, Methanocaldococcus, and Methanococcus, the method according to any one of Embodiments 1 to 35.

[0100] Embodiment 37. The methane-producing microorganism contains hydrogenotrophic microorganisms, acetate-utilizing microorganisms, methylotrophic microorganisms, or methoxydotrophic microorganisms, or a combination thereof, the method according to any one of Embodiments 1 to 36.

[0101] Embodiment 38. The fermentation is initiated using methane-producing microorganisms in a chemically defined fermentation medium, the method according to any one of Embodiments 1 to 37.

[0102] Embodiment 39. The fermentation is carried out under anaerobic conditions, the method according to any one of Embodiments 1 to 38.

[0103] Embodiment 40. The method according to any one of Embodiments 1 to 39, which is a continuous process, a fed-batch process, a batch process, a closed batch process, an iterative batch process, an iterative fed-batch process, or an iterative closed batch process, preferably a continuous process, a fed-batch process, or an iterative fed-batch process, particularly a continuous process.

[0104] Embodiment 41. The method according to Embodiment 40, which is a chemostat process.

[0105] Embodiment 42. The method according to Embodiment 40 or 41, wherein the dilution rate D is from 0.001 / hour to 1.5 / hour, preferably from 0.01 / hour to 0.5 / hour, particularly from 0.0125 / hour to 0.1 / hour.

[0106] Embodiment 43. The 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%, particularly 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.

[0107] Embodiment 44. The 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%, particularly 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.

[0108] Embodiment 45. A sulfur source is supplied to the bioreactor. Preferably, the sulfur source contains cysteine and / or sulfide. In particular, the fermentation broth contains sulfide at a concentration of 0.001 to 150 mg / L, preferably 0.01 to 100 mg / L, particularly 1 to 80 mg / L, and / or the sulfide supply rate or cysteine supply rate is 0.0001 to 0.2 mol / L / h, preferably 0.001 to 0.05 mol / L / h. The method according to any one of Embodiments 1 to 44.

[0109] Embodiment 46. The bioreactor is a liquid-phase or multiphase bioreactor. The method according to any one of Embodiments 1 to 45.

[0110] Embodiment 47. The bioreactor is any one of a stirred tank, a packed bed, one liquid phase, two liquid phases, a hollow fiber membrane, a bubble column, a trickle bed, or a fluidized bed bioreactor. The method according to any one of Embodiments 1 to 46.

[0111] Embodiment 48. The bioreactor is a chemostat. The method according to any one of Embodiments 1 to 47.

[0112] Embodiment 49. The amino acid or the moiety contains norvaline, particularly L-norvaline. Preferably, the fermentation broth contains ammonium at a concentration of at least 0.1 mmol / L, preferably at least 2 mmol / L, more preferably at least 4 mmol / L, still more preferably at least 5 mmol / L, even more preferably at least 6 mmol / L, particularly at least 7 mmol / L, or even at least 10 mmol / L. The method according to any one of Embodiments 1 to 48.

[0113] 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 the fermentation broth is at least 0.001 μmol / L / hour, preferably at least 0.005 μmol / L / hour, more preferably at least 0.01 μmol / L / hour, even more preferably at least 0.05 μmol / L / hour, still even more preferably at least 0.1 μmol / L / hour, particularly at least 0.5 μmol / L / hour, or even at least 1.0 μmol / L / hour.

[0114] Embodiment 51. The method according to any one of Embodiments 1 to 50, wherein the production rate of norvaline (especially L-norvaline) per biomass is at least 0.01 μmol / g / hour, preferably at least 0.05 μmol / g / hour, more preferably at least 0.1 μmol / g / hour, even more preferably at least 0.5 μmol / g / hour, still even more preferably at least 1.0 μmol / g / hour, particularly at least 5 μmol / g / hour, or even at least 10 μmol / g / hour.

[0115] Embodiment 52. Use of a methanogenic microorganism for producing an amino acid from an electron donor compound, a gaseous carbon source containing carbon dioxide and / or carbon monoxide, a nitrogen source containing nitrogen gas, and preferably a sulfur source in a fermentation broth, wherein the fermentation broth contains ammonium at a concentration of 0.1 mmol / L to 200 mmol / L, preferably 2 mmol / L to 100 mmol / L, more preferably 4 to 40 mmol / L.

[0116] 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.

[0117] Embodiment 54. The use according to Embodiment 52 or 53, wherein the electron donor compound is as defined in any one of Embodiments 7 to 9.

[0118] Use according to any one of Embodiments 52 to 54, wherein the amino acid is as defined in any one of Embodiments 20 to 23 and 49.

[0119] Use according to any one of Embodiments 52 to 55, wherein the production rate is as defined in any one of Embodiments 24 to 27, 50, and 51.

[0120] Use according to any one of Embodiments 52 to 56, wherein the methane-producing microorganism is as defined in any one of Embodiments 29 to 37.

[0121] The present invention is further illustrated by the following drawings and examples, but is not limited thereto.

Examples

[0122] Example 1: Amino Acid Production and Active Secretion by Methanothermobacter marburgensis under N2 Fixation Conditions The purpose of the research by the present inventors was to investigate the physiological and biotechnological characteristics of biological N2 fixation related to H2 / CO2 utilization in different methanogens.

[0123] Among several methanogens analyzed, Methanothermobacter marburgenis was prioritized, and the N2 fixation, CH4 production, and amino acid excretion characteristics were investigated in closed batch and fed batch culture modes and at different NH4 + concentrations.

[0124] Briefly described, Methanothermobacter marburgensis was grown on a chemically defined minimal medium containing different concentrations of ammonium chloride (NH4Cl) in an N2 / H2 / CO2 atmosphere. This enabled quantification of ammonia uptake, N2 fixation, amino acid excretion, and conversion of H2 / CO2 to CH4. N2 fixation by M. marburgensis was confirmed in all experiments using H2 / N2 / CO2 in the gas phase. Furthermore, active excretion of amino acids that make up proteins was observed, and the detected values of glutamic acid, alanine, glycine, and asparagine were the highest. At "100%" NH4 + concentration in a closed batch, the highest total production rate of 7.5 μmol / L / h was detected after 40 hours (see Table 1 below). Therefore, the simultaneous production of amino acids and CH4 from CO2 was found to be biotechnologically relevant in an integrated approach combining biomethanation and N2 fixation in the concept of biorefinery.

[0125] [Materials and Methods] [Company Name] The following strains were selected for the experiment: Methanothermobacter marburgensis (Schonheit et al. 1980; Wasserfallen et al. 2000), Methanobacterium thermaggregans (Blotevogel and Fischer 1985), Methanococcus maripaludis (Jones et al. 1983), Methanocaldococcus villosus (Bellack et al. 2011), and Methanothermococcus okinawensis (Takai et al. 2000). These strains may be obtained, for example, from DSMZ (Braunschweig, Germany).

[0126] [Culture medium] Pre-cultures of M. villosus and M. okinawensis were grown on a chemically defined medium according to Taubner and Rittmann, 2016. M. maripaludis was grown in McN medium (see Mauerhofer et al. 2021). Methanobacterium thermaggregans and Methanothermobacter marburgensis were cultured on MM medium.

[0127] MM medium (see also Table 1 below): NH4Cl 2.1 g / L and KH2PO4 6.8 g / L (in ddH2O). 200× trace element (TE) solution added (e.g., at a concentration of 5 mL per liter of MM medium): Titriplex I 9 g / L, add 800 mL of H2O, adjust the pH to 6.5 with 5 mol / L NaOH solution, then (to the target concentration), add 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 the pH to 7.0 and the volume to 1 L with 1 mol / L NaOH and ddH2O. The medium may further contain, for example, 3.6 g / L of NaHCO3 as a carbon source. As a sulfur source, for example, 2 mL of 0.5 mol / L Na2S·9H2O per liter may be added after anaerobicization and autoclaving.

[0128] Surprisingly, it has been found to be advantageous that the TE solution (and consequently the medium) does not contain tungsten (or, at least, is less than 0.1 μmol / L, preferably less than 0.01 μmol / L, particularly less than 0.001 μmol / L). This is because otherwise the amino acid yield under nitrogen fixation conditions is lower.

[0129] The medium was dispensed into 117 mL serum bottles (VWR, Austria) to a total working volume of 50 mL and sealed with a blue rubber stopper (pre-boiled 10 times for 30 minutes, 20 mm, butyl rubber, Chemglass Life Sciences) and an aluminum crimp cap (Ochs Laborbedarf, Boveninden, Germany). According to the medium composition, sterilized L-cysteine-HCl·H2O, sterilized NaHCO3 solution, and Na2S·9H2O were added after autoclaving in an anaerobic glove box (Coy Laboratory Products, Grass Lake, USA). To ensure anaerobic conditions, the atmosphere in the headspace was evacuated and gas-treated to a relative pressure of 2 bar (absolute pressure 3 bar) using each gas (H2 / CO2 or H2 / CO2 / N2) mixture, and the procedure was varied by repeating it 5 times (Taubner & Rittmann 2016). For gas treatment, a sterilized syringe filter (w / 0.2c μm cellulose, VWR International, USA) and a sterilized needle (disposable hypodermic needle, Gr 14, 0.60×30 mm, 23G×1 1 / 4”, Braun, Germany) were used.

[0130] NH4Cl (2.1 g / L) was omitted to prepare a nitrogen-free (N-free) medium, which was replaced with a chemically equal molar amount of NaCl (2.3 g / L) to ensure the correct salt concentration in the medium. To replace L-cysteine monohydrate, a diluted HCl solution was used to restore the pH value. As shown in Table 1, MM media with various NH4 + concentrations were prepared. To ensure that CO2 was the sole carbon source, Na2CO3 was replaced with an equimolar amount of NaCl. The medium without Na2CO3 was manually adjusted to pH 6.8 by titration with 10 mol / L NaOH.

[0131]

Table 1

[0132] (Chemical substance) For the closed batch experiments and fed-batch experiments, H2 (99.999%), CO2 (99.999%), N2 (99.999%), H2 / CO2 (80% / 20%), and H2 / CO2 / N2 (77.74% / 11.13% / 11.13%) were used. For gas chromatography (GC), N2 / CO2 (80% / 20%), CH4 (99.995%), and a standard test gas (Messer GmbH, Vienna, Austria) (containing 0.01% by volume of CH4 and 0.08% by volume of CO2 in N2) were further used. All gases except the standard test gas were purchased from Air Liquide (Air Liquide GmbH, Schwechat, Austria). All other chemicals were of the highest available grade.

[0133] (Closed batch experiment) The cultures were incubated in a water bath at 65 °C (Büch-Vedder, Germany) for (M. marburgensis, M. thermaggregans, and M. okinawensis), or in a shaking air incubator at 37 °C (M. maripaludis) (Büch-Vedder, Germany) and 80 °C (M. villosus) (LABWIT Scientific Pty Ltd, Australia). For the purpose of N2 fixation, all closed batch experiments were carried out in an H2 / N2 / CO2 atmosphere. To select strains for prioritization, M. marburgensis, M. maripaludis, M. thermaggregans, M. villosus, and M. okinawensis were grown in triplicate (n = 3) with one zero control until the OD 578 reached approximately 0.7. M. marburgensis had one of the highest NH4 + concentrations in the medium, so NH4 +The amount was further examined for its effect on growth by reducing it to 1 / 10 of the original medium concentration. To remove residual nitrogen compounds from the medium, the cells before culturing were washed before inoculation. For the medium without complete N, all cultures were washed three times, and for all other experiments, they were either washed once or not washed at all. Only for the experiments with M. marburgensis, different NH4 + concentrations (“0%”, “1%”, “10%”, “25%”, “50%” and “100%”, see Table 1 above) were carried out in quadruplicate (n = 4) or octuplicate (n = 8). The preculture containing 1 / 10 NH4 + was used as the inoculum. The 0% batch was used as a negative control, and the one treated with 100% gas at a ratio of H2 / CO2 of 4:1 was used as a positive control.

[0134] After each incubation time, the serum bottles were left to cool at room temperature for 45 minutes. The pressure was measured with a digital manometer (Keller GmbH, Winterthur, Switzerland). Growth was measured spectrophotometrically by OD (λ = 578 nm, with Milli-Q water as blank) (Beckman Coulter, California, USA). 1 mL of the liquid sample was taken and centrifuged at the maximum speed (13200 rpm) for 30 minutes. The cell pellets and supernatants of each experiment were stored in sterile Eppendorf tubes until further analysis at -20 °C.

[0135] (Fed-batch experiment) All fed-batch experiments were performed in triplicate using M. marburgensis in a DASGIP® 2.2 L bioreactor system (SR1500ODLS, Eppendorf AG, Hamburg, Germany) with a working volume of 1.5 L of MM medium containing 100 μl / L of antifoam agent (Struktol SB2023, Schill und Seilacher, Hamburg, Germany), gas-treated with H2 / CO2 / N2 in a ratio of 7:1:1. The best growth conditions were pH 7 and temperature 65 °C. Gas treatment of N2 and CO2 was controlled via an MX4 / 4 unit (Eppendorf AG, Hamburg, Germany). The H2 gas flow rate was controlled via a C100L unit (Sierra Instruments, Monterey, USA). Gas treatment was carried out at the same ratio as in the closed-batch experiments. The redox potential and pH value were monitored by individual redox and pH probes (Mettler Toledo GmbH, Vienna, Austria). All fed-batch cultures were inoculated with a stock culture of M. marburgensis adapted to fed-batch culture. Prior to inoculation, the bioreactor was gas-treated with H2 / N2 / CO2 to ensure anaerobic conditions and 5 mL of 0.5 mol / L Na2S·9H2O was added. Immediately after inoculation of 30 mL, the supply of 0.5 M Na2S·9H2O at 0.2 mL / h was started and the stirring speed was set to 1600 rpm. Gaseous samples were taken approximately 0 h, 13 h, 16 h, 19 h, 22 h, and 25 h later. Batches containing Na2CO3 and batches without Na2CO3 in the medium, carried out under an H2 / CO2 atmosphere (ratio 4:1), were used as references. Liquid samples were taken and processed as described above to analyze growth and amino acid excretion.

[0136] (Determination of ammonium) NH4 +The determination was carried out using a procedure modified according to the method reported previously (Kandeler 1988). The oxidation solution, chromogenic reagent, and NH4Cl stock solution were freshly prepared before measurement. As standard substances, nine different concentrations in the range of 100 μmol / L to 1000 μmol / L of NH4Cl were prepared. The samples were diluted with MilliQ to the final concentration between the standard ranges. Before measurement, 300 μL of the chromogenic reagent and 120 μL of the oxidation solution were immediately added to the standard substances and samples and mixed briefly. After 30 minutes in the dark, measurements (λ = 660 nm) were performed using a 96-well plate (Microtest Plate 96 Well, F, Sarstedt AG & C0, Nuembrecht, Germany) with a plate photometer (Sunrise plate reader, Tecan Group AG, Mannedorf, Switzerland). The regression curve R 2 was always higher than 0.999.

[0137] (Gas chromatography) The exhaust gas composition (H2, CO2, CH4, and N2) of gas samples collected from closed-batch experiments and fed-batch experiments was 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).

[0138] (Amino acid analysis) For amino acid analysis, the supernatant of the sample (obtained as described above) was diluted with Mill-Q water at a ratio of 1:4. Measurements were carried out using an Agilent 1260 Infinity Bioinert HPLC system including a fluorescence detector, a column oven, an autosampler, and a quaternary pump. 1 mL of the sample was mixed with 75 μL of boric acid buffer (0.4 N in water, pH = 10.2; Agilent Technologies), followed by 5 μL of OPA reagent (3-mercaptopropionic acid in 0.4 mol / L boric acid buffer, and 10 mg / mL o-phthalaldehyde (OPA); Agilent Technologies). 100 μL of the mixture was injected into the HPLC system after 2 minutes at 27 °C. The fluorescent derivatives (primary soluble free amino acids) were separated on a Zorbax ECLIPSE AAA column (4.6 × 150 mm, particle size 3.5 μm, Agilent Technologies) equipped with a Zorbax ECLIPSE AAA guard cartridge (4.6 × 150 mm, particle size 5 μm, Agilent Technologies) at a flow rate of 0.8 mL / min at 25 °C. The excitation wavelength was 340 nm and the emission was 450 nm. A gain factor of 9 or 10 was used according to the expected concentration, which was pre-tested in advance. For peak identification and quantification, different concentrations of a primary amino acid standard mixture (AAS18, Sigma Aldrich) were prepared for each run according to the concentration range of the sample (100 nmol / L to 15 μmol / L). Since the AAS18 standard mixture lacked five amino acids (asparagine (Asn), glutamic acid (Glu), gamma-aminobutyric acid (GABA), taurine (Tau), tryptophan (Trp); Sigma Aldrich), these were added. In this way, a total of 20 different AAs could be measured. Valine and methionine were located within the signal noise "ammonium peak" and were excluded from the evaluation because measurements were difficult in experiments using high NH4 + concentrations. The details of this method are as previously published (Taubner et al. 2019).

[0139] (GC analysis) The relative pressure in the headspace of 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 according to the ideal gas law. The headspace volume was determined in previous experiments and adjusted after each OD measurement with an extraction sample volume of 0.75 mL. All measurements were carried out at room temperature (25 °C).

[0140] To obtain the actual amount of N2, the pressure in the serum bottle was multiplied by 0.11392 based on the exact percentage of N2 (11.392% by volume) in the gas mixture, and then multiplied by the normalized gas composition obtained from the GC measurement. The zero control value was taken as the N2 baseline. The nitrogen uptake rate (NUR / mmol / L / h) was calculated by dividing the deviation of N2 (ΔN2) before and after incubation by the volume of the liquid medium and the time (Δt) from the last incubation:

[0141]

Equation

[0142] The quantitative / specific nitrogen uptake (qN2 / mmol / h / g) was determined by dividing the NUR by the biomass concentration (x / g / L) calculated with experimentally determined coefficients:

[0143]

Equation

[0144] Carbon dioxide uptake rate (CUR / mmol / L / h), molecular hydrogen uptake rate (HUR / mmol / L / h), CH4 generation rate (MER / mmol / L / h), carbon balance (C balance), yield (Y (CH4 / CO2) and Y (x / CO2) ), and biomass productivity (r xwas calculated as described elsewhere (Taubner et al. 2016; Rittmann et al. 2012, Bernacchi et al. 2014). Concentrations of H2, CO2, N2, and CH4 after GC measurement were obtained.

[0145] 〔Results〕 (Ranking of companies) Growth of M. marburgensis, M. maripaludis S0001, M. thermaggregans, M. villosus, and M. okinawensis was analyzed in an H2 / CO2 / N2 atmosphere in defined medium containing NH4 + and no N. This enabled parallel screening of NH4 + uptake, N2 fixation, amino acid excretion, and conversion of H2 / CO2 to CH4. All methanogens except M. thermaggregans were able to grow to an OD + of 0.7 in medium containing NH4 578 . Further experiments showed that a certain amount of NH4 + was required for growth under these conditions. For the most favorable growth characteristics in these experiments, M. marburgensis was selected for further experiments. Growth at specific NH4 + concentrations with or without bicarbonate (Na2CO3) had already been carried out, showing no growth or nitrogen-limited growth at 0% and 1%, and similar growth at all other concentrations. Higher growth rates were shown in experiments with Na2CO3 in the medium.

[0146] (NH4 + uptake kinetics of M. marburgensis) A closed batch experiment with M. marburgensis was then performed with 0%, 5%, 7.5%, 10% and 100% NH4 for the original medium composition of 2.1 g / L. + The experiment was carried out using NH4 + Experiments were performed in eight replicates (n=8) with one washing step as well as an additional zero control to reduce the possibility of carryover. Due to the biomass washing step, slower growth was observed compared to the non-washed biomass experiment. After 77.17 h, OD 578 The OD ranged from 0.17 to 0.20. As expected, the 100% 4:1 positive control had the highest OD around 0.25. 578 Gas samples were taken after approximately 40, 59, and 77 hours (Figure 1).

[0147] 0%, 1%, 5%, 10%, and 100% NH4 + Additional fed-batch experiments (n=3) were performed at concentrations of 0.01%. In the reference run, OD 578 Similar growth was observed up to 7.0 and 8.1 (Fig. 1). + When comparing runs with those with Na2CO3 in the medium, OD was 2.2-fold higher. 578 This effect was also observed in closed batch experiments. + At this concentration, the final OD was 20 hours later. 578 The OD reached 1.6 and plateaued after 15 hours at 5%. 578 reaches 0.9, indicating stagnation.

[0148] A detailed description of the uptake rates of H2 and CO2, HUR, and CUR, respectively, and MER is given in Table 2.

[0149] [Table 2]

[0150] (NH4 + and N2 simultaneous uptake interactions) NH4 during N2 fixation +Uptake is evident in both closed-batch and fed-batch experiments. Comparing Fig. 2 (Chart a) and Fig. 2 (Chart b), NH4 limitation was observed at 0 - 10% during fed-batch culture, while it was found that NH4 was not completely consumed in the closed-batch culture. Furthermore, in contrast to the fed-batch experiment where NH4 decreased over time, in the closed-batch experiment, it was noted that the NH4 concentration did not vary much between time points (Fig. 2). The highest consumption was achieved in the positive control experiment. In the case of the closed-batch experiment, the highest ammonia uptake rate (AUR) of 243.8 μmol / L / h was achieved at a qN2 of 4.6 μmol / h / g in the 100% 4:1 run, and in the fed-batch experiment, the highest AUR of 577.3 μmol / L / h and a qN2 of 10.8 μmol / h / g were obtained using bicarbonate in the medium. + with NH4 + limitation observed, while in the closed-batch culture + it was found that NH4 + was not completely consumed. Moreover, in contrast to the fed-batch experiment where NH4 + decreased over time, in the closed-batch experiment, it was noted that the NH4

[0151] concentration did not vary much between time points (Fig. 2). The highest consumption was achieved in the positive control experiment. In the case of the closed-batch experiment, the highest ammonia uptake rate (AUR) of 243.8 μmol / L / h was achieved at a qN2 of 4.6 μmol / h / g in the 100% 4:1 run, and in the fed-batch experiment, the highest AUR of 577.3 μmol / L / h and a qN2 of 10.8 μmol / h / g were obtained using bicarbonate in the medium.

[0152]

Table 3

[0153] Amino acid excretion by M. marburgensis Active amino acid excretion by M. marburgenis was investigated in closed-batch (Figure 3) and fed-batch (Figure 4) experiments. Irrespective of the culture mode, almost all detectable amino acids were found. The most excreted amino acids were glutamic acid (Glu), alanine (Ala), glycine (Gly), and asparagine (Asn). The concentrations of Glu, Gly, and Asn continuously increased during the culture, while Ala was consumed after a certain point. All culture experiments showed a distinct NH4 + dependency in the change of amino acid excretion at 5%, 7.5%, and 10% from 100% (Figures 3 and 4). Looking at the volume values in more detail, in the closed-batch, a maximum value of 4.59 μmol / L / h of glutamic acid was obtained at 5% after 40 hours, and a smaller amount of alanine with a maximum of 1.36 μmol / L / h was obtained. The highest value of Gly was achieved at 100% after 40 hours and was 0.99 μmol / L / h. In the fed-batch culture, the most excreted amino acid was Ala, which was the highest at 5% with a maximum of 2.67 μmol / L / h. It is also worth noting that at 100%, Asn produced a maximum of 0.79 μmol / L / h, which was 10 times higher compared to other NH4 + concentrations.

[0154] Examining the total amount of excreted amino acids, in the closed-batch, amino acid excretion increased over time, being highest at a late point and ranging from 14.67 to 18.44 μmol / L. In the fed-batch, the total amount of AA excretion was generally higher, with a maximum value of 156.08 μmol / L (Figure 5). Conversely, it should be noted that during the closed-batch experiment, a higher production rate [μmol / L / h] was observed compared to the fed-batch experiment. In the experiment at 100%, a slightly lower value was shown, so the presence of excess NH4 + in the medium seems to function as an inhibitor of amino acid production (Figure 5). The comparison between the total uptake of NH4 + and the total amino acid excretion rate showed that the amino acid excretion rate increased with the increase in AUR. Also, during the depletion of NH4 + the concentration of AA did not increase (Figure 5).

[0155] 〔Conclusion〕 In the context of "power to gas" technology, biological methanation using CO2 derived from renewable resources, and N2 fixation, amino acid production by methanogens is of high economic interest. As far as the inventors know, there is no research that has examined the CO2 / N2 fixation composite bioprocess yet.

[0156] Switching between closed batch or fed-batch culture modes can change the amino acid excretion rate and concentration. For example, in the case of Glu, it changes from a maximum fed-batch value of 2.74 mg / L to 44.83 mg / L in the closed batch (Table 3).

[0157] Surprisingly, it was found that various amino acids were actively excreted, with a total amount of up to 7.5 μmol / L / h at the initial time point (Figure 5).

[0158] These results emphasize that methanogenic microorganisms are well-suited for amino acid production in a biotechnology context even under N2 fixation conditions.

[0159] Example 2: Amino Acid Production and Active Secretion by Methanothermobacter marburgensis in Continuous Culture Success was achieved in establishing a continuous culture of M. marburgensis (M. marburgensis) for amino acid production.

[0160] Experiments were conducted using M. marburgensis (M. marburgensis) in a 2 L bioreactor (Eppendorf AG, Hamburg, Germany) and a 15 L bioreactor (Biostat C+, Sartorius Stedim Biotech AG, Göttingen, Germany). For fermentation, the original MM medium described in Example 1 above was used. The same medium was used as the feed medium for the continuous culture mode. To ensure anaerobic conditions in the reaction vessel, the entire system was flushed with a mixture of H2 / CO2, N2, or H2 / CO2 / N2 for 10 minutes before inoculation. The cultivation was carried out at a stirring speed of 65 °C, 100 - 1200 rpm (DASGIP parallel bioreactor system, Eppendorf AG, Hamburg, Germany) and 100 - 1500 rpm (Biostat C+, Sartorius Stedim Biotech AG, Göttingen, Germany). The pH was measured by a pH probe (Mettler Toledo GmbH, Vienna, Austria or Hamilton Bonaduz AG, Bonaduz, Switzerland) and kept constant at a value of 7.

[0161] The oxidation-reduction potential (ORP) was measured by a redox probe (Mettler Toledo GmbH, Vienna, Austria). A 0.5 mol / L Na2S·9H2O solution was used as the sulfur source and continuously supplied to the bioreactor, for example, at a rate of 0.2 mL / h to 1.32 mL / h. The MM medium was supplied using an analog peristaltic pump. The MM medium supply flow rate, the sodium Na2S·9H2O supply rate, and the titration were recorded by gravimetry or adjusted by the pump speed. The volume of the bioreactor was kept constant by withdrawing the culture suspension through an immersion pipe using a peristaltic pump controlled by the fixed bioreactor weight, or by using a pipe of a fixed height as a level control system. The withdrawn suspension was collected in a recovery bottle and its volume was recorded by gravimetry. All solutions were made anaerobic 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 the substrate for M. marburgensis. The CO2 gas flow rate was controlled via an MX4 / 4 unit (Eppendorf AG, Hamburg, Germany). The H2 gas flow rate was controlled via a C100L unit (Sierra Instruments, Monterey, USA).

[0162] Approximately 30 different runs of continuous culture of M. marburgensis were carried out under anaerobic conditions. The volume of the runs ranged from 1.6 L to 10.29 L. The dilution rate D was varied between runs, especially at D values of 0.0125 / h to 0.05 / h. The volume of gas per volume of liquid per minute (vvm) was also varied between runs, for example from 0.125 to 0.5. The agitation (rpm) was also varied between runs, for example from 375 to 1500. As a sulfur source, 0.5 mol / L of Na2S was supplied, for example, from 0.2 mL / h to 1.32 mL / h. Typically, the ammonium concentration was maintained at 15 mmol / L to 35 mmol / L.

[0163] Importantly, the volumetric amino acid production rate and the specific amino acid production rate were in the ranges of approximately 25 to approximately 75 μmol / L / h and approximately 50 to approximately 2000 μmol / h / g, respectively (total over all amino acids). The production and secretion of the following amino acids (combinations) into the culture supernatant were typically observed: Asp, Glu, Asn, Ser, His, Gln, Gly, Thr, Arg, Ala, Tyr, Val, Met, norvaline (Nva), Trp, Ile, Phe, Leu, Lys. The individual amino acid production rates were observed to be up to approximately 40 μmol / L / h (volumetric) and up to approximately 900 μmol / h / g (per biomass). Cys and Pro were not detected due to analytical constraints but are expected to be produced and secreted as well.

[0164] In conclusion, reliable production of amino acids was observed in continuous culture. The secretion of these amino acids into the supernatant is particularly notable as it simplifies downstream processes (e.g., does not require cell lysis to recover the product). Also, surprisingly, the production of Nva, which had not been previously observed in methanogenic archaea (let alone in the order Methanobacteriales), was observed.

[0165] Example 3: Amino acid production and active secretion in additional methanogenic archaea Amino acid production, including standard amino acids and Nva, and their active secretion were observed in methanogenic archaea other than M. marburgensis, namely Methanocaldococcus jannaschii, Methanococcus igneus, and Methanocaldococcus villosus. These methanogenic microorganisms were incubated under closed batch conditions at their respective preferred temperatures in 282 medium (see also Mauerhofer et al., 2021), which is similar to the conditions disclosed in Example 1. For example, Glu production was more prominent under these conditions, but Nva production was also clearly observed for each of Methanocaldococcus jannaschii, Methanococcus igneus, and Methanocaldococcus villosus (the volumetric Nva production rate exceeded 1.0 μmol / L / h, and the specific Nva production rate exceeded 10 μmol / g / h). Nva production has not been previously observed in methanogenic archaea (let alone in the order Methanococcales).

[0166] In summary, the production and active secretion of the following amino acids (combinations) into the culture supernatant were observed for methanogenic microorganisms: Asp, Glu, Asn, Ser, His, Gln, Gly, Thr, Arg, Ala, Tyr, Val, Met, Nva, Trp, Ile, Phe, Leu, and Lys.

[0167] Example 4: Amino acid production and active secretion by Methanothermobacter marburgensis in continuous culture (further experiments) Experiments were conducted using M. marburgensis in a 2.2 L bioreactor (Eppendorf AG, Hamburg, Germany) and a 15 L bioreactor (Biostat C+, Sartorius Stedim Biotech AG, Göttingen, Germany). For fermentation, the original MM medium described in Example 1 above was used. The same medium was used as the feed medium for the continuous culture mode. To ensure anaerobic conditions in the reaction vessel, the entire system was flushed with a mixture of H2 / CO2, N2 or H2 / CO2 / N2 for 10 minutes before inoculation.

[0168] The culture was carried out at a stirring speed of 65 °C, 375 - 1500 rpm (DASGIP parallel bioreactor system, Eppendorf AG, Hamburg, Germany) and 375 - 1500 rpm (Biostat C+, Sartorius Stedim Biotech AG, Göttingen, Germany). The pH was measured by a pH probe (Mettler Toledo GmbH, Vienna, Austria or Hamilton Bonaduz AG, Bonaduz, Switzerland) and kept constant at a value of 7.

[0169] The oxidation-reduction potential (ORP) was measured by a redox probe (Mettler Toledo GmbH, Vienna, Austria). A 0.5 mol / L Na2S·9H2O solution was used as the sulfur source and continuously supplied to the bioreactor, for example, at a rate of 0.05 mL / h to 1.32 mL / h. The MM medium was supplied using an analog peristaltic pump. The supply flow rate of the MM medium, the supply rate of sodium Na2S·9H2O, and the titration were adjusted by the pump speed. The volume of the bioreactor was kept constant. The withdrawn suspension was collected in a collection bottle. All solutions were made anaerobic 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 the substrate for M. marburgensis. The CO2 gas flow rate was controlled via an MX4 / 4 unit (Eppendorf AG, Hamburg, Germany). The H2 gas flow rate was controlled via a C100L unit (Sierra Instruments, Monterey, USA).

[0170] Over 100 different runs of continuous culture of M. marburgensis were carried out under anaerobic conditions. The volume of the runs ranged from 1.6 L to 10.29 L. The dilution rate D was varied between runs, especially in the range of 0.00625 / h to 0.05 / h, for example, at a D value of 0.025 / h. The gas volume per liquid volume per minute (vvm) was also varied between runs, for example, from 0.125 to 0.5. The stirring (rpm) was also varied between runs, for example, from 375 to 1500. As the sulfur source, 0.5 mol / L Na2S was supplied, for example, from 0.05 mL / h to 1.32 mL / h. Typically, the ammonium concentration was maintained between 0.21 mmol / L and 41.63 mmol / L.

[0171] Importantly, the volumetric amino acid production rate and the specific amino acid production rate were in the ranges of about 5 to about 250 μmol / L / h and about 10 to about 150 μmol / h / g, respectively (total over all amino acids). Production and secretion into the culture supernatant of the following amino acids (combinations) were typically observed: Asp, Glu, Ser, His, Gly, Thr, Arg, Ala, Tyr, Val, Met, norvaline (Nva), Trp, Ile, Phe, Leu, norleucine (Nle), Lys. The individual amino acid production rates were observed to be up to about 130 μmol / L / h (volumetric) and up to about 70 μmol / h / g (per biomass). Cys and Pro were not detected due to analytical constraints but are expected to be produced and secreted as well.

[0172] In conclusion, reliable production of amino acids was observed in continuous culture. Secretion of these amino acids into the supernatant is particularly notable as it simplifies downstream processes (e.g., does not require cell lysis to recover the product). Also, surprisingly, production of Nva and Nle, which had not been previously observed in methanogenic archaea (let alone in the order Methanobacteriales), was observed.

[0173] Example 5: Amino Acid Production and Active Secretion in Additional Methanogenic Archaea The production of amino acids, including standard amino acids, norvaline, ornithine, and homoserine, and their active secretion into the culture supernatant were observed in methanogenic archaea other than M. marburgensis, namely Methanothermobacter thermautotrophicus and Methanothermococcus sp. These methanogenic microorganisms were incubated under closed-batch conditions similar to those disclosed in Example 1 but at their respective preferred temperatures and various ammonium concentrations in MM medium (see Example 1; for the cultivation of Methanothermococcus, 30 g / L of NaCl was added to the medium). For example, the production of Ala and Glu was more prominent under these conditions, but the production of Nva was also clearly observed respectively. The production of Nva has not been previously observed in methanogenic archaea (let alone in Methanobacteriales or Methanococcales).

[0174] In summary, the production and active secretion into the culture supernatant of the following amino acids (or combinations thereof) were observed for 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 could not distinguish between Leu and Nle.)

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Claims

1. A method for producing an amino acid by fermentation in a bioreactor, comprising: the bioreactor contains methanogenic microorganisms in the fermentation broth; the method includes at least the step of supplying a gaseous carbon source containing carbon dioxide and / or carbon monoxide, a nitrogen source containing nitrogen gas, and preferably a sulfur source to the bioreactor under conditions such that the methanogenic microorganisms produce the amino acid; the fermentation broth contains ammonium at a concentration of 0.1 mmol / L to 200 mmol / L, preferably 2 mmol / L to 100 mmol / L, more preferably 4 to 40 mmol / L.

2. Hydrogen gas, acetate, a methyl compound (preferably selected from methylamine, methyl sulfide, and methanol), any other alcohol, preferably a secondary alcohol such as 2-propanol or 2-butanol, a methoxylated aromatic compound, and / or formate is supplied to the bioreactor; preferably, hydrogen gas, acetate, methanol, or a combination thereof is supplied to the bioreactor. The method according to claim 1.

3. The method according to any one of claims 1 to 2, further comprising the step of recovering at least a part of the amino acid from the bioreactor.

4. The amino acid further, or the part further, contains at least one, preferably at least two or further at least three, more preferably at least four or further at least five, even more preferably at least seven or further at least nine, still more preferably at least twelve or further at least fifteen, still even more preferably at least seventeen or further at least eighteen, particularly all of the 20 standard amino acids. The method according to any one of claims 1 to 3.

5. The method according to any one of claims 1 to 4, wherein the methanogenic microorganism contains archaea selected from any one of Methanobacteriales, Methanococcales, Methanomicrobiales, Methanosarcinales, Methanopyrales, Methanocellales, Methanomassiliicoccales, and Methanonatronarchaeales; preferably contains archaea selected from any one of Methanobacteriales and Methanococcales; more preferably contains archaea selected from Methanobacteriaceae, Methanocaldococcaceae, and Methanococcaceae; particularly contains archaea selected from Methanothermobacter, Methanothermococcus, Methanocaldococcus, and Methanococcus.

6. The method according to any one of claims 1 to 5, wherein the methanogenic microorganism contains at least two different species.

7. The method according to any one of claims 1 to 6, wherein the production rate of total amino acids per volume of the fermentation broth is at least 0.01 μmol / L / h, preferably at least 0.05 μmol / L / h, more preferably at least 0.1 μmol / L / h, even more preferably at least 0.5 μmol / L / h, still more preferably at least 1.0 μmol / L / h, particularly at least 5 μmol / L / h, or even at least 10 μmol / L / h.

8. The production rate of total amino acids per biomass is at least 0.1 μmol / g / h, preferably at least 0.5 μmol / g / h, more preferably at least 1.0 μmol / g / h, even more preferably at least 5 μmol / g / h, still more preferably at least 10 μmol / g / h, particularly at least 50 μmol / g / h or even at least 100 μmol / g / h, the method according to any one of claims 1 to 7.

9. 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, particularly a continuous process, the method according to any one of claims 1 to 8.

10. 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%, particularly 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, the method according to any one of claims 1 to 9.

11. 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%, particularly 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, the method according to any one of claims 1 to 10.

12. A sulfur source is supplied to the bioreactor, preferably, the sulfur source contains cysteine and / or sulfide. In particular, the fermentation broth contains sulfide at a concentration of 0.001 to 150 mg / L, preferably 0.01 to 100 mg / L, particularly 1 to 80 mg / L, and / or the sulfide supply rate or cysteine supply rate is 0.0001 to 0.2 mol / L / h, preferably 0.001 to 0.05 mol / L / h. The method according to any one of claims 1 to 11.

13. The method according to any one of claims 1 to 12, wherein methane is recovered from the bioreactor.

14. The method according to any one of claims 1 to 13, wherein the fermentation is initiated using the methanogenic microorganism in a chemically defined fermentation medium.

15. Use of a methanogenic microorganism for producing an amino acid from an electron donor compound, a gaseous carbon source containing carbon dioxide and / or carbon monoxide, a nitrogen source containing nitrogen gas, and preferably a sulfur source in a fermentation broth, wherein the fermentation broth contains ammonium at a concentration of 0.1 mmol / L to 200 mmol / L, preferably 2 mmol / L to 100 mmol / L, more preferably 4 to 40 mmol / L; preferably, the electron donor compound is selected from hydrogen gas, 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, a methoxylated aromatic compound, formate, and combinations thereof.

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