Method for producing an amino acid in a bioreactor

Abstract

The present invention provides a method for producing an amino acid by fermentation in a bioreactor, wherein the bioreactor contains a methanogenic microorganism in a fermentation broth, and the method comprises the steps of supplying a gaseous carbon source containing carbon dioxide and / or carbon monoxide, a nitrogen source, and preferably a sulfur source to the bioreactor under conditions such that the methanogenic microorganism produces the amino acid; and recovering at least a part of the amino acid from the supernatant of the fermentation broth. Preferred methanogenic microorganisms are archaea selected from, for example, the genus Methanothermobacter, the genus Methanothermococcus, and the genus Methanococcus. Furthermore, a fermentation broth containing a methanogenic microorganism and an amino acid in the supernatant, and a bioreactor containing the same are provided.

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 with 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 is about 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 is about 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 mention 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] In some studies, the use of methanogenic archaea in renewable energy production by reducing CO2 to CH4 with H2 has been discussed (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. International Publication No. WO 2012 / 110256 discloses a method for converting carbon dioxide and hydrogen to methane by methanogenic microorganisms. International Publication No. 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, such as 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 dissimilatory 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 an L-amino acid 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] WO 2016 / 179545 and US 2018 / 0163240 disclose compositions and methods for the biological production of methionine.

[0018] US 2019 / 0194630 and US 2017 / 0130211 relate to compositions and methods for the biological production of amino acids in hydrogen -otrophic microorganisms. In particular, the hydrogen -otrophic microorganisms can be selected from the genus Methanococcus and Methanosarcina.

[0019] WO 2020 / 252335 relates to a process and system for producing products 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 hydrogen -otrophic microorganism under conditions such that the hydrogen -otrophic 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 hydrogen -otrophic microorganism, and the fermentation product; and (d) separating the hydrogen -otrophic microorganism from the liquid stream and recycling the hydrogen -otrophic microorganism to the bioreactor. The hydrogen -otrophic microorganism can be selected from methanogenic archaea.

[0020] Despite these efforts, further development of fermentation processes using methanogenic microorganisms is still needed.

[0021] Accordingly, it is an object of the present invention to provide improved methods for producing amino acids by fermentation in a bioreactor containing methanogenic 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 methanogenic microorganisms in a fermentation broth. The method includes supplying a gaseous carbon source containing 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, and recovering at least a portion of the amino acids from the supernatant of the fermentation broth.

[0023] Typically, after said recovery, a purification method is carried out to separate the amino acids from other components of the fermentation broth, for example.

[0024] In one aspect, the present invention provides the use of methanogenic microorganisms (e.g., any methanogenic archaea disclosed herein, such as Methanothermobacter, Methanothermococcus, Methanocaldococcus, and Methanococcus) for producing amino acids, wherein the amino acids are recovered from the supernatant of a fermentation broth.

[0025] In another aspect, the present invention provides a fermentation broth containing methane-producing microorganisms (e.g., any methanogenic archaea disclosed herein, such as Methanothermobacter, Methanothermococcus, Methanocaldococcus, and Methanococcus) and amino acids in the supernatant (e.g., culture supernatant).

[0026] In yet another aspect, the present invention provides a bioreactor containing this fermentation broth.

[0027] 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 emission of 1.5 tons of CO2 per ton of NH3 produced. In the process of the present invention, the inventors investigated whether biological processes could be used for carbon and / or N2 fixation and the accompanying amino acid production. Surprisingly, it was 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 for product recovery) and at the same time increases productivity (since methanogenic microorganisms are viable and can be maintained as such or resupplied to the bioreactor), which is particularly noteworthy. BRIEF DESCRIPTION OF THE DRAWINGS

[0028]

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Mode for Carrying Out the Invention

[0029] The following detailed description relates to all of the above aspects of the present invention, unless explicitly excluded.

[0030] 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, particularly a continuous process. It is particularly preferred that the continuous process (cultivation) is a chemostat process (cultivation) in which the pH is controlled.

[0031] Particularly in continuous culture, the recovery step may include removing from the bioreactor a liquid stream containing the fermentation broth, the methanogenic microorganisms, and the produced amino acids (in the supernatant of the fermentation broth), 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.

[0032] In 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 preferably less than 1000 μg / mL, more preferably less than 500 μg / mL, even more preferably less than 250 μg / mL, still more preferably less than 100 μg / mL, yet even 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.

[0033] 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 recovery. This applies particularly 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.

[0034] 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 FIG. 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).

[0035] 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 N2 fixed 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).

[0036] Jones and Stadtman, 1977 is concerned with 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 concerned with the growth of Methanosphaera stadtmanae enhanced by tungsten. Furthermore, Lobo and Zinder, 1988 is concerned with diazotrophy and nitrogenase activity in the archaeon Methanosarcina barkeri 227. These documents do not describe amino acid production at all.

[0037] When the nitrogen source contains nitrogen gas, a certain 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 10 mmol / L to 20 mmol / L. It is obvious to those skilled in the art that in continuous culture, in particular, 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).

[0038] Furthermore, the concentration of tungstate in the fermentation broth (in particular, orthotungstate, i.e., WO4 -2 concentration) is preferably less than 0.1 μmol / L, more preferably less than 0.01 μmol / L, and particularly preferably less than 0.001 μmol / L (in particular, when the fermentation broth is substantially free of tungstate). In the process of the present invention, it has been found that this enables more efficient amino acid production.

[0039] 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.

[0040] According to a further preferred embodiment, methane (produced by methanogenic microorganisms) is recovered from the bioreactor.

[0041] 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.

[0042] (The recovered) amino acids preferably include at least one, preferably at least two, more preferably at least three, still more preferably at least four, still more preferably at least five, yet more preferably at least seven, still yet more preferably at least nine, still yet more preferably at least twelve, still yet more preferably at least fifteen, still yet more preferably at least seventeen, still yet more preferably 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 that the (recovered) amino acids include 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 are essential amino acids, branched-chain amino acids (BCAAs), or glutamic acid.

[0043] According to another preferred embodiment, the total amino acid production rate per volume of the (supernatant 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, still more preferably at least 0.5 μmol / L / hour, still 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).

[0044] 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, still more preferably at least 5 μmol / g / hour, still 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).

[0045] 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.

[0046] 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, Methanocaldococcaceae, and Methanococcaceae; in particular, including 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 are, for example: Methanobacterium alcaliphilum, Methanobacterium bryantii, Methanobacterium congolense, Methanobacterium defluvii, Methanobacterium esparespanolae), Methanobacterium formicicum, 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 sociabilissociabilis), 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 vannieliivannielii), Methanococcus voltae, Methanothermococcus thermolithotrophicus, Methanopyrus kandleri, Methanothermobacter thermautotrophicum, Methanocaldococcus fervens, Methanocaldococcus indicus, Methanocaldococcus infernus, and Methanocaldococcus vulcanius.

[0047] 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).

[0048] The methanogenic microorganisms can be hydrogenotrophic, acetate-utilizing, methylotrophic (e.g., H2-dependent methylotrophic) or methoxydotrophic.

[0049] It has also been found that (defined) co-cultures of methanogenic microorganisms in the bioreactor are also advantageous. Thus, the methanogenic microorganisms (in the bioreactor) preferably contain at least two different species.

[0050] Fermentation is very preferably initiated using methanogenic microorganisms in a chemically defined fermentation medium.

[0051] In another preferred embodiment, the fermentation is carried out under anaerobic conditions.

[0052] 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, and particularly preferably from 0.0125 / hour to 0.1 / hour.

[0053] 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.

[0054] 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 / h, preferably 0.001 to 0.05 mol / L / h.

[0055] In the methods and uses disclosed herein, various 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.

[0056] 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).

[0057] 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 Escherichia coli (E. coli) fermentation process (Biermann et al., 2013).

[0058] Chinese Patent Application Publication No. 106520651 discloses a method for producing L-norvaline by enzymatic conversion.

[0059] Kisumi et al., 1976 discuss the biosynthesis of the non-proteinogenic amino acids norvaline, norleucine, and homoisoleucine in the enterobacterium Serratia marcescens.

[0060] US 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.

[0061] In 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.

[0062] Thus, in a particularly preferred embodiment, the (recovered) amino acids preferably include norvaline.

[0063] According to another preferred embodiment, the norvaline production rate 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).

[0064] According to yet another preferred embodiment, the norvaline production rate 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).

[0065] 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: supplying a gaseous carbon source containing 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 the amino acid; and recovering at least a part of the amino acid from the supernatant of the fermentation broth. A method comprising at least. Embodiment 2. The method according to Embodiment 1, wherein the methanogenic microorganisms secrete the amino acid into the supernatant of the fermentation broth.

[0066] Embodiment 3. The method according to Embodiment 1 or 2, wherein the ammonium concentration in the fermentation culture solution 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, even more preferably at least 20 minutes, still 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 fermentation culture solution 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, 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.

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

[0069] Embodiment 6. The method according to any one of Embodiments 1 to 5, wherein the fermentation culture solution 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 combinations thereof are 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 the fermentation broth, the methane-producing microorganism, and the produced amino acid (especially in the supernatant) from the bioreactor, separating the methane-producing microorganism from the liquid stream, and recycling the methane-producing microorganism 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 methane-producing microorganism remains viable and / or intact. The method according to Embodiment 13.

[0078] Embodiment 15. The method further includes a step of purifying the produced amino acid from the liquid stream, especially from the supernatant of the fermentation broth; preferably, the total produced 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. The method according to Embodiment 14.

[0079] Embodiment 16. The method according to any one of Embodiments 1 to 15, wherein the nitrogen source contains nitrogen gas.

[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 even 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 moiety further, comprise 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 more preferably at least three, more preferably at least four or more preferably at least five, even more preferably at least seven or more preferably at least nine, still more preferably at least twelve or more preferably at least fifteen, and yet even more preferably at least seventeen or more preferably 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, pyrroproline, 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 even 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 even 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 even 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 even 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 methanogenic microorganism) is recovered from the bioreactor.

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

[0093] 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.

[0094] Embodiment 31. The method according to any one of Embodiments 1 to 30, wherein the methanogenic microorganism contains 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 archaea is selected from Methanothermobacter marburgensis, Methanocaldococcus jannaschii, Methanococcus igneus, and Methanocaldococcus villosus.

[0097] Embodiment 34. The method according to embodiment 31, wherein the archaea 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, MethanosaetaThe method according to embodiment 31, which is Clostridium acetobutylicum, 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.

[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, even more preferably at least 5 mmol / L, still 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 (particularly 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 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 (particularly 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 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, wherein the amino acid is recovered from the supernatant of the fermentation broth, and preferably, the methanogenic microorganism secretes the amino acid into the supernatant of the fermentation broth.

[0116] Embodiment 53. The use according to Embodiment 52, wherein the fermentation broth is as defined in any one of Embodiments 3 to 6, 17, 19, and 45.

[0117] Embodiment 54. The use according to Embodiment 52 or 53, wherein 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 are supplied to the methanogenic microorganism.

[0118] Embodiment 55. The 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] Embodiment 56. Use according to any one of Embodiments 52 - 55, wherein the production rate is as defined in any one of Embodiments 24 - 27, 50, and 51.

[0120] Embodiment 57. Use according to any one of Embodiments 52 - 56, wherein the methane - producing microorganism is as defined in any one of Embodiments 29 - 37.

[0121] Embodiment 58. A fermentation culture solution containing amino acids in the supernatant and methane - producing microorganisms.

[0122] Embodiment 59. The fermentation culture solution according to Embodiment 58, wherein the fermentation culture solution is further as defined in any one of Embodiments 3 - 6, 17, 19, and 45.

[0123] Embodiment 60. Further, the fermentation culture solution according to Embodiment 58 or 59, further containing an electron - donor compound (preferably an electron - donor compound as defined in any one of Embodiments 7 - 9), a carbon source, a nitrogen source, and preferably a sulfur source.

[0124] Embodiment 61. The fermentation culture solution according to any one of Embodiments 58 - 60, wherein the amino acid is as defined in any one of Embodiments 20 - 23 and 49.

[0125] Embodiment 62. The fermentation culture solution according to any one of Embodiments 58 - 61, wherein the methane - producing microorganism is as defined in any one of Embodiments 29 - 37.

[0126] Embodiment 63. A bioreactor containing the fermentation culture solution according to any one of Embodiments 58 - 62.

[0127] The present invention is further illustrated by the following drawings and examples, but is not limited thereto.

Examples

[0128] Example 1: Amino Acid Production and Active Secretion by Methanothermobacter marburgensis under N2 Fixation Conditions The aim of the research by the present inventors was to examine the physiological and biotechnological characteristics of biological N2 fixation related to H2 / CO2 utilization in different methanogens.

[0129] Among several methanogens to be analyzed, Methanothermobacter marburgensis 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.

[0130] Briefly described, M. marburgensis was grown on a chemically defined minimal medium containing different concentrations of ammonium chloride (NH4Cl) in an N2 / H2 / CO2 atmosphere. This enabled the quantification of ammonia uptake, N2 fixation, amino acid excretion, and the 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 constituting proteins was observed, and the detected values of glutamic acid, alanine, glycine, and asparagine were the highest. The highest total production rate of 7.5 μmol / L / h was detected after 40 hours under the "100%" NH4 + concentration in the closed batch (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.

[0131] 〔Materials and Methods〕 (Company name) The following strains were selected for the experiments: 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).

[0132] (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.

[0133] 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 anaerobic treatment and autoclaving.

[0134] Surprisingly, it has been found advantageous that the TE solution (and consequently the medium) does not contain tungstate (or at least contains 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.

[0135] 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 with the respective gas (H2 / CO2 or H2 / CO2 / N2) mixture to a relative pressure of 2 bar (absolute pressure 3 bar) and the procedure was repeated 5 times to vary it (Taubner & Rittmann 2016). For gas treatment, a sterile syringe filter (w / 0.2c μm cellulose, VWR International, USA) and a sterile needle (disposable hypodermic needle, Gr 14, 0.60×30 mm, 23G×1 1 / 4”, Braun, Germany) were used.

[0136] 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 equimolar NaCl. The medium without Na2CO3 was manually adjusted to pH 6.8 by titration with 10 mol / L NaOH.

[0137]

Table 1

[0138] (Chemical substance) For the closed batch experiments and fed-batch experiments, 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 gas chromatography (GC), N2 / CO2(80% / 20%), CH4(99.995%), and a standard test gas (Messer GmbH, Vienna, Austria) (containing 0.01 vol% CH4 and 0.08 vol% 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.

[0139] (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 performed 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 with a H2 / CO2 ratio of 4:1 was used as a positive control.

[0140] After each incubation time, the serum bottles were left at room temperature for 45 minutes to cool. 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 the 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.

[0141] (Fed-batch experiment) All fed-batch experiments were carried out 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 are pH 7 and temperature 65 °C. The 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). The gas treatment was carried out at the same ratio as the closed-batch experiment. The redox potential and pH values 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. Before 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 30 mL of inoculation, 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 with Na2CO3 in the medium and batches without Na2CO3, 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.

[0142] (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 chromogenic reagent and 120 μL of oxidation solution were immediately added to the standard substances and samples and mixed briefly. After 30 minutes in the dark, measurement (λ = 660 nm) was carried out 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, Männedorf, Switzerland). The regression curve R 2 was always higher than 0.999.

[0143] (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, California, USA) equipped with a thermal conductivity detector (TDC) and a 19808 Shin Carbon ST Micropacked Column (Restek GmbH, Bad Homburg, Germany).

[0144] (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 of 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 dissolved 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).

[0145] (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 a previous experiment 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).

[0146] 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 value of the zero control 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:

[0147]

Equation

[0148] The quantitative / specific nitrogen uptake (qN2 / mmol / h / g) was determined by dividing the NUR by the biomass concentration (x / g / L) calculated with an experimentally determined coefficient:

[0149]

Equation

[0150] 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 xwere 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.

[0151] 〔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 allowed for 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.

[0152] (NH4 + uptake kinetics of M. marburgensis) Next, closed batch experiments of M. marburgensis were carried out with 0%, 5%, 7.5%, 10% and 100% NH4 with respect to the original medium composition of 2.1 g / L + and gas-treated with H2 / CO2 / N2 in a ratio of 7:1:1. To reduce the possibility of NH4 + carry-in, the experiments were carried out in octuplicate (n = 8) using an additional zero control along with a single washing step. For the biomass washing step, slower growth was observed compared to the non-washed biomass experiment. After 77.17 hours, the OD 578 was between 0.17 and 0.20. As expected, the 100% 4:1 positive control showed the highest OD 578 value of around 0.25. Gas samples were taken at approximately 40 hours, 59 hours, and 77 hours (Figure 1).

[0153] Additional fed-batch experiments (n = 3) were carried out at 0%, 1%, 5%, 10%, and 100% NH4 + concentrations. In the reference run, similar growth was seen up to OD 578 7.0 and 8.1 (Figure 1). Comparing the runs at 100% NH4 + , an OD 578 2.2 times higher was obtained due to Na2CO3 in the medium. This effect was also seen in the closed batch experiments. At 10% NH4 + concentration, the final OD 578 reached 1.6 after 20 hours and showed stagnation, and at 5% it reached 0.9 after 15 hours and showed stagnation. 578

[0154] Details of the uptake rates of H2 and CO2, HUR and CUR, and MER are shown in Table 2, respectively.

[0155]

Table 2

[0156] (Interaction of simultaneous uptake of NH4 + and N2) NH4 during N2 fixation +Uptake is evident in both closed-batch and fed-batch experiments. Comparing Figure 2 (Chart a) and Figure 2 (Chart b), during fed-batch culture, NH4 limitation was observed at 0 - 10%, while it can be seen 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 is noted that the NH4 concentration did not vary much between time points (Figure 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 can be seen that NH4 + was not completely consumed. + Furthermore, in contrast to the fed-batch experiment where NH4 + decreased over time, in the closed-batch experiment, it is noted that the NH4

[0157] The highest NURs were 0.91 mmo / L / h or 0.83 mmo / L / h calculated from the 7.5% and 100% closed-batch experiments after 40 hours, and 0.88 mmo / L / h calculated from the 10% after 59 hours. qN2 showed the same pattern (Table 3). Higher NURs were shown at earlier sampling time points and lower but more balanced NUR values at later time points.

[0158]

Table 3

[0159] 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. Regardless 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 clear 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 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.

[0160] Examining the total amount of excreted amino acids, in the closed-batch, amino acid excretion increased over time, being the 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. Since a slightly lower value was shown in the 100% experiment, 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).

[0161] 〔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. To the best knowledge of the inventors, there is no study that has examined the CO2 / N2 fixation composite bioprocess yet.

[0162] 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 closed-batch (Table 3).

[0163] 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).

[0164] These results emphasize that methanogenic microorganisms are well-suited for amino acid production in a biotechnology context, even under N2 fixation conditions.

[0165] 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.

[0166] 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 prior to inoculation. 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.

[0167] 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 controlled peristaltic pump with a 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).

[0168] 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 stirring (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.

[0169] 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). 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 similarly.

[0170] 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, production of Nva, which had not been previously observed in methanogenic archaea (let alone in the order Methanobacteriales), was observed.

[0171] 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).

[0172] 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.

[0173] 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.

[0174] 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.

[0175] 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 recovery 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).

[0176] 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, at a rate of 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.

[0177] 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 similarly.

[0178] 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 processing (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.

[0179] 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).

[0180] 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 comprises: supplying a gaseous carbon source containing 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 the amino acid; and recovering at least a part of the amino acid from the supernatant of the fermentation broth. A method comprising at least the above steps.

2. The method according to claim 1, wherein the protein content in the 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, particularly less than 30 μg / mL or even less than 20 μg / mL.

3. The method according to claim 1 or 2, 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 methanogenic microorganisms remain viable and / or intact before or during the recovery.

4. The method according to any one of claims 1 to 3, wherein the part includes at least 2, preferably 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 more preferably at least 17 or even at least 18, particularly all 20 standard amino acids.

5. The method according to any one of claims 1 to 4, 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.

6. The method according to any one of claims 1 to 5, wherein the methanogenic microorganism comprises an archaeon selected from any of Methanobacteriales, Methanococcales, Methanomicrobiales, Methanosarcinales, Methanopyrales, Methanocellales, Methanomassiliicoccales, and Methanonatronarchaeales; preferably, the archaeon is selected from either Methanobacteriales or Methanococcales; more preferably, the archaeon is selected from Methanobacteriaceae, Methanocaldococcaceae, and Methanococcaceae; particularly, the archaeon is selected from Methanothermobacter, Methanothermococcus, Methanocaldococcus, and Methanococcus.

7. 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; and / or 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 6.

8. The method according to any one of claims 1 to 7, 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.

9. The nitrogen source includes nitrogen gas; preferably, 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 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 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 9.

11. A sulfur source is supplied to the bioreactor, preferably the sulfur source includes cysteine and / or sulfide, particularly 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 10.

12. The bio-reactor is supplied with 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, a methoxylated aromatic compound, and / or formate; preferably, the bio-reactor is supplied with the hydrogen gas, acetate, methanol, or a combination thereof, the method according to any one of claims 1 to 11.

13. Use of a methanogenic microorganism for producing an amino acid, wherein the amino acid is recovered from the supernatant of a fermentation broth.

14. A fermentation broth comprising a methanogenic microorganism and an amino acid in the supernatant.

15. A bio-reactor comprising the fermentation broth according to claim 14.

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