Methods for fixing carbon dioxide

Abstract

A system and method for fixing carbon dioxide by fermentation are provided. More specifically, the disclosure includes the steps of producing methane by fermenting carbon dioxide with methanogenic archaea and producing a single-cell protein supplement. The disclosure further provides integration of methane-producing fermentation with additional methods to achieve improved carbon efficiency.

Description

Technical Field

[0001] A method for fixing carbon dioxide is provided. More specifically, the method includes the steps of fermenting carbon dioxide into methane and fermenting methane with methylotrophic bacteria. An additional method includes the step of processing the cell mass from the fermentation into single cell protein for use as a nutritional supplement.

Background Art

[0002] Emissions of carbon monoxide and carbon dioxide from industrial processes are two major factors in climate change and global warming. Microbial fermentation can reduce such carbon emissions by using microorganisms through their metabolic pathways to convert carbon monoxide (CO), hydrogen (H2), and / or carbon dioxide (CO2) into useful oxygenated hydrocarbon compounds such as ethanol, butanol, acetate, butyrate, 2,3 - butanediol, and other desired products. Large - scale microbial fermentation also produces large amounts of microbial biomass. Conventionally, the disposal of microbial biomass requires extremely expensive waste treatment systems, storage locations, and landfill sites. Previous findings have shown that microbial biomass can be recovered and converted into single cell protein (SCP) and other components and reused as a source of proteins, amino acids, and carbohydrates useful as nutritional supplements for animals, plants, or humans. For example, U.S. Patent No. 10,856,560 describes a method for producing whole - cell animal feed by culturing acetogens and producing microbial biomass. Therefore, there is a need for methods and systems that can effectively convert carbon dioxide into products for other uses. Furthermore, there remains a need for methods and systems for efficiently converting microbial biomass into digestion - friendly nutritional supplements and compositions of any such nutritional supplements.

Summary of the Invention

[0003] In one embodiment, a method for converting CO2 includes the steps of: fermenting a gaseous substrate containing CO2 and H2 with methane-producing archaea in a methane-producing fermentation vessel to produce a fermentation liquid broth containing methane and methane-producing archaea; and fermenting the methane with methylotropic bacteria in a methylotropic fermentation vessel to produce a fermentation liquid broth containing methylotropic bacteria and a CO2-containing vent gas. In another embodiment, a method for converting CO and CO2 includes the step of fermenting a gaseous substrate containing CO2 and H2 with methane-producing archaea in a methane-producing fermentation vessel to produce a fermentation liquid broth containing methane and methane-producing archaea. Next, the methane is fermented with methylotropic bacteria in a methylotropic fermentation vessel to produce a fermentation liquid broth containing methylotropic bacteria and a first CO2-containing vent gas. A gaseous substrate containing CO is fermented with CO-converting acetic acid-producing bacteria in a CO fermentation vessel to produce alcohol, a second CO2-containing vent gas, and a fermentation liquid broth containing acetic acid-producing bacteria. The above-mentioned features of this disclosure can be further described by reference to embodiments, some of which are illustrated in the accompanying drawings, so that the above-mentioned features of this disclosure may be understood in detail. However, it should be noted that the accompanying drawings only illustrate typical embodiments of this disclosure and should not be considered limiting in scope, for this disclosure may allow for other equally effective embodiments. [Brief explanation of the drawing]

[0004] [Figure 1A] This figure shows a method for converting CO2, including methane production and fermentation using methylotrope bacteria, in which single-cell proteins are processed together. [Figure 1B] This figure shows a method for converting CO2, including methane production and fermentation using methylotrope bacteria, in which single-cell proteins are processed separately. [Figure 2A]This figure shows a method for converting CO and CO2, including methane production, fermentation using CO-converting acetic acid-producing bacteria, and fermentation using methylotrope bacteria, in which single-cell proteins are processed together. [Figure 2B] This figure shows a method for converting CO2, including methane production, fermentation using CO-converting acetic acid-producing bacteria, and fermentation using methylotrope bacteria, in which single-cell proteins are processed separately. [Modes for carrying out the invention]

[0005] The following description should not be taken as limiting, but is provided for the purpose of illustrating general principles of exemplary embodiments. The scope of this disclosure should be determined by reference to the claims. The term “approximately” modifying any quantity refers to the variation in that quantity encountered in real-world conditions, such as a laboratory, pilot plant, or production facility. For example, when a quantity of material or measurement used in a mixture or quantity is modified by “approximately,” it includes the variation and degree of care typically used when measuring under experimental conditions in a production plant or laboratory. For example, when a quantity of a product component is modified by “approximately,” it includes variation between batches of multiple experiments in a plant or laboratory and variation inherent in the analytical method. Whether modified by “approximately” or not, quantities include quantities equivalent to those quantities. Any quantity described herein and modified by “approximately” may also be used in this disclosure as an unmodified quantity. In the context of this disclosure, the use of the terms “a,” “an,” “the,” and similar demonstrative pronouns should be interpreted as encompassing both singular and plural forms unless otherwise indicated or more clearly negated in the context.

[0006] Unless otherwise indicated, the terms “comprising,” “including,” “having,” “containing,” or “characterized by” are inclusive and do not exclude any additional elements or method steps not described herein (i.e., “including but not limited to”). Any use of examples or illustrative language used herein (e.g., “such as,” “for example,” “for instance”) is intended solely to clarify the disclosure and, unless otherwise asserted, does not limit the scope of the disclosure.

[0007] Fermentation is a metabolic process used by microorganisms to generate energy for cell growth. Certain microorganisms can sustain their growth and produce oxygen-containing hydrocarbon compounds by fermenting C1-containing gaseous substrates, such as synthesis gas, carbon monoxide (CO)-containing gaseous substrates, or carbon dioxide (CO2)-containing gaseous substrates. In such cases, the microorganisms use one or more C1 components in the C1-containing gaseous substrate as the primary carbon source for their growth. Terms such as “fermentation,” “fermentation method,” and “microbial fermentation method” are intended to encompass both the growth phase and the product biosynthesis phase of the method. During anaerobic fermentation methods, large quantities of microbial biomass are obtained, which can be purged out and processed into useful products such as nutrient supplements. Specifically, this disclosure includes a method for extracting nutrient supplements from microbial biomass by an anaerobic fermentation method. Fermentable gaseous substrates refer to C1-containing gaseous substrates that include one or more of CO, CO2, or CH2O2. Suitable gaseous substrates can include various synthesis gases (i.e., synthesis gas) and industrial off-gases.

[0008] Synthesis gas can be supplied from any known source. In one embodiment, synthesis gas can be supplied by gasification of carbonaceous material. Gasification involves partial combustion of biomass under conditions of limited oxygen supply. The resulting gas may contain CO, CO2, and H2. Several examples of suitable gasification methods and apparatus are described in U.S. Patent Applications 61 / 516,667, 61 / 516,704, and 61 / 516,646 (all filed April 6, 2011), and U.S. Patent Applications 13 / 427,144, 13 / 427,193, and 13 / 427,247 (all filed March 22, 2012), all of which are incorporated herein by reference. In another embodiment, synthesis gas can be produced from the electrolysis of water and carbon dioxide. In this embodiment, oxygen is removed from the obtained gas, and the obtained gas can be further blended with other gas sources to form a desired fermentable gaseous substrate.

[0009] Industrial off-gases may contain C1-containing waste gases from industrial processes that would normally be discharged into the atmosphere. Examples of industrial off-gases include gases produced during microbial fermentation, ferrous metal product manufacturing, non-ferrous product manufacturing, petroleum refining, coal gasification, electricity production, carbon black production, ammonia production, methanol production, coke production, and gas reforming. A C1-containing gaseous substrate may also contain H2. H2 can also be supplied to the C1-containing gaseous substrate separately to form a desired gas composition suitable for fermentation. Examples of H2 sources include gases produced during the manufacture of ferrous metal products, non-ferrous metal products, petroleum refining methods, coal gasification, biomass gasification, electricity production, carbon black production, ammonia production, methanol production, and coke production. Other hydrogen sources include, for example, H2O electrolysis and bio-generated H2. Fermentation of fermentable gaseous substrates using microorganisms is carried out in a fermentation vessel. The fermentation vessel includes a fermentation bioreactor consisting of one or more vessels and / or columns or piping, and may include batch reactors, semi-batch reactors, continuous reactors, continuous stirred tank reactors (CSTRs), bubble column reactors, external circulating loop reactors, internal circulating loop reactors, immobilized cell reactors (ICRs), trickle bed reactors (TBRs), mobile bed biofilm reactors (MBBRs), gas lift reactors, membrane reactors such as hollow fiber membrane bioreactors (HFMBRs), static mixers, gas lift fermenters, or other vessels or devices suitable for gas-liquid contact.

[0010] A culture medium suitable for anaerobic microbial growth and for fermenting a fermentable gaseous substrate into one or more oxygen-containing hydrocarbon compounds is added to the fermentation vessel to support the fermentation of the gaseous substrate by acetic acid-producing bacteria. Several examples of culture medium compositions are described in U.S. Patent Applications 16 / 530,502 and 16 / 530,481 filed August 2, 2019, and U.S. Patent No. 7,285,402 filed July 23, 2001, all of which are incorporated herein by reference. The culture medium can be sterilized to remove undesirable microorganisms and inoculate the fermentation vessel with desired microorganisms. Sterilization may not always be necessary. A culture medium suitable for methanogenic fermentation is described in U.S. Patent No. 11,401,499, which is incorporated herein by reference.

[0011] methane production fermentation Methane productionIn one embodiment illustrated in Figures 1A and 1B, the method includes a methane-producing microbial fermentation vessel 105 that can be integrated with an industrial process for producing CO2. In this embodiment, the methane-producing microbial fermentation vessel 105 contains a microbial culture capable of hydrogen nutrient methane production (i.e., conversion of CO2 + H2 to methane). A separate hydrogen source 40 may be provided to the methane-producing microbial fermentation vessel 105. H2 and CO2 may be added to the methane-producing microbial fermentation vessel 105 separately, or they may be blended together and then added to the methane-producing microbial fermentation vessel 105. The method includes the step of maintaining the CO2 to H2 ratio in the fermentation vessel 105 to approximately 1:5 to approximately 1:1, in another embodiment, approximately 1:5 to approximately 1:2, in another embodiment, approximately 1:5 to approximately 1:3, in another embodiment, approximately 1:5 to approximately 1:4, in another embodiment, approximately 1:4 to approximately 1:1, in another embodiment, approximately 1:4 to approximately 1:2, in another embodiment, approximately 1:4 to approximately 1:3, in another embodiment, approximately 1:3 to approximately 1:1, in another embodiment, approximately 1:3 to approximately 1:2, in another embodiment, approximately 1:2 to approximately 1:1. A total gas delivery rate in the range of approximately 0.2 to approximately 25 gas volume, in another embodiment, approximately 2 to approximately 16, in another embodiment, approximately 1 to 22, in another embodiment, approximately 0.5 to 20 (STP, standard temperature and pressure) / volume / min is preferred. The methanogenic microbial fermentation vessel 105 can also produce a fermentation liquid broth 140 containing methanogenic archaea, which can be processed into single-cell proteins in the single-cell protein processing unit 145 to produce a nutritional supplement 147.

[0012] Suitable microbial cultures can be readily obtained from public collections of organisms or isolated from various environmental sources. Such sources include anaerobic soils and sands, swamps, wetlands, marshes, estuaries, dense algal mats, terrestrial and marine muds and sediments, deep-sea and deep wells, sewage and organic waste sites and treatment facilities, and animal intestines and feces. Many pure cultures of single species are preferred. Classified pure cultures are all members of the Archaea domain [Woese et al. Proc Natl Acad Sci USA 87:4576-4579 (1990) “Towards a natural system of organisms: Proposal for the domains Archaea, Bacteria, and Eucharya.”, incorporated herein by reference], and belong to four different classes of the Euryarchaea kingdom. Suitable organisms include four different genera within the class Methanobacteria (e.g., Methanobacterium alcaliphilum, Methanobacterium bryantii, Methanobacterium congolense, Methanobacterium defluvii, Methanobacterium espanolae, Methanobacterium formicicum, Methanobacterium ivanovii, Methanobacterium palustre, and Methanobacterium thermagregans). (thermaggregans), Methanobacterium uliginosum, Methanobrevibacter acididuransMethanobrevibacter acididurans, Methanobrevibacter arboriphilicus, Methanobrevibacter gottschalkii, Methanobrevibacter olleyae, Methanobrevibacter ruminantium, Methanobrevibacter smithii, Methanobrevibacter woesei, Methanobrevibacter wolinii, Methanothermobacter marburgensis, Methanothermobacter thermautotrophum Thermautotrophicum (also called Methanothermobacter thermoautotroiphicus), Methanothermobacter thermoflexus, Methanothermobacter thermophilus, Methanothermobacter wolfeii, Methanothermus sociabilis, and five different genera within the class Methanomicrobia (for example, Methanocorpusculum bavaricum, Methanocorpusculum parvum, Methanoculleus chikuoensis, and Methanoculleus submarinus) submarinus), Methanogenium frygidam(frigidum), Methanogenium liminatans, Methanogenium marinum, Methanosarcina acetivorans, Methanosarcina barkeri, Methanosarcina mazei, Methanosarcina thermophila, Methanomicrobium mobile), seven different genera within the class Methanococci (for example, Methanocaldococcus jannaschii, Methanococcus aeolicus, Methanococcus maripaldis) It was classified into several genera within the class Methanopyri (e.g., Methanocaldococcus maripaludis), Methanococcus vannielii, Methanococcus voltaei, Methanothermococcus thermolithotrophicus, Methanocaldococcus fervens, Methanocaldococcus indicus, Methanocaldococcus infernus, Methanocaldococcus vulcanius), and one genus within the class Methanopyri (e.g., Methanopyrus kandleri). Suitable cultures include those from public culture collections (e.g., the American Type Culture Collection, the Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH, and the Oregon)These are available from the Collection of Methanogens. Many suitable hydrogenotrophic methanogenic bacteria, isolated as pure cultures and available in public culture collections, have not yet been fully classified. Preferred pure culture organisms include Methanosarcinia barkeri, Methanococcus maripaldis, Methanothermobacter thermoautotrophicus, and Methanothermobacter marbulgensis.

[0013] Suitable cultures of mixtures of two or more microorganisms can also be readily isolated from designated environmental sources [Bryant et al. Archiv Microbiol 59:20-31 (1967) “Methanobacillus omelianskii, a symbiotic association of two species of bacteria.”, incorporated herein by reference]. The suitable mixture may be a consortium in which cells of two or more species are physically associated, or a vegetative symbiotic mixture in which two or more species cooperate metabolically without physical association. Mixed cultures may possess useful properties beyond those available from pure cultures of known hydrogenotrophic methanogenic bacteria. These properties include, for example, resistance to contaminants in the gas feed stream such as oxygen, ethanol, or other trace components, or aggregate growth that can increase the culture density and volumetric gas processing capacity of the culture.

[0014] A suitable culture of mixed organisms can also be obtained by combining cultures isolated from two or more sources. One or more species in the suitable mixed culture should be methanogenic archaea. Any non-archaeal species may be bacterial or eukaryotic. Suitable cultures can also be obtained by genetic modification of non-methane-producing organisms, in which genes essential for supporting hydrogen nutrient methane production are transferred from methane-producing microorganisms or from a combination of microorganisms that may or may not be spontaneously methane-producing. Suitable genetic modifications can be obtained by enzymatic or chemical synthesis of the necessary genes.

[0015] The methanogenic fermentation vessel 105 achieves continuous methane production using a continuous hydrogen-nutrient methane production culture operation under stable conditions. Examples of such suitable conditions are described in Schill, N., van Gulik, M., Voisard, D., & von Stockar, U. (1996) Biotcchnol & Biocng 51:645-658. “Continuous cultures limited by a gaseous substrate: development of a simple, unstructured mathematical model and experimental verification with Methanobacterium thermoautotrophicum,” which is incorporated herein by reference. The culture medium may consist of dilute mineral salts and should be adapted to the specific culture being used.

[0016] The concentrations of various culture medium components used in the methane-producing microbial fermentation method are as follows: [Table 1]

[0017] The culture medium in the methanogenic fermentation vessel 105 should be replenished at a rate suitable for maintaining useful concentrations of essential minerals and eliminating any metabolites that can inhibit methane production. A dilution rate of less than 0.2 culture volumes / hour is preferred for obtaining high volume concentrations of active methane production capacity. In one aspect, the redox potential is maintained below -400 mV or less during methane production. In another aspect, the redox potential is maintained below -300 mV or less, in another aspect below -200 mV, and in yet another aspect below -100 mV.

[0018] In another aspect, the culture temperature is maintained near optimal for the growth of the organism used in the culture (e.g., for mesophilic organisms such as Methanosarcinia barkeri and Methanococcus maripaludis, about 35°C to about 37°C, or for thermophilic bacteria such as Methanothermobacter thermautotrophicus, about 60°C to 65°C, and for organisms such as Methanocaldococcus janaschii, Methanocaldococcus fervens, Methanocaldococcus indicus, Methanocaldococcus infernus, and Methanocaldococcus vulcanius, about 85°C to 90°C). However, it is contemplated that temperatures above or below the optimal growth temperature can be used. In another aspect, a reducing agent can be introduced into the fermentation process along with CO2 and H2. The reducing agent can preferably be hydrogen sulfide or sodium sulfide. Hydrogen itself can be used as a reductant to maintain the redox potential of the culture within the range required for optimal performance of hydrogenotrophic methane production (< -100 mV). Generally, hydrogen gas is provided at a concentration effective to convert at least a portion of the carbon dioxide in the bioreactor to methane. In another aspect, the redox potential of the culture can be maintained at < -100 mV via an electrochemical cell immersed in the medium.

[0019] In another aspect, the method includes various ways and / or features to reduce the presence of oxygen in the CO2 stream fed to the bioreactor. When using obligate anaerobic methanogenic archaea to catalyze methanogenesis, the presence of oxygen can impair the performance of the method and contaminate the product gas. Therefore, reducing the presence of oxygen in the CO2 stream helps to improve the method. In one aspect, the oxygen level is reduced by passing the H2 / CO2 mixed stream through a palladium catalyst to convert any trace oxygen to water prior to the gas inflow into the fermentation vessel. In this aspect, H2 is provided in an amount exceeding the amount required during culturing at a ratio of 2:1 with respect to the contaminating oxygen. In another aspect, oxygen is removed by pretreatment of the gas stream in the bioreactor. In this aspect, the reducing agent can be provided by the supply of a source of organic material (e.g., glucose, starch, cellulose, fermentation residues from ethanol plants, whey residues, etc.) that can serve as a substrate for oxidative fermentation. The biological catalyst of the microorganism is selected to oxidatively ferment the selected organic source and obtain CO2 from the contaminating oxygen. In this embodiment, additional H2 is supplied to enable the conversion of this additional CO2 to methane in the anaerobic fermenter.

[0020] The method results in a CO2 uptake ratio of about 0.5 to about 3 mmol of CO2 per minute per gram of cells, in another aspect about 1 to about 2 mmol of CO2 per minute per gram of cells, in another aspect about 0.5 to about 1 mmol of CO2 per minute per gram of cells, in another aspect about 1 to about 3 mmol of CO2 per minute per gram of cells, in another aspect about 0.5 to about 2 mmol of CO per minute per gram of cells. In this aspect, the method is effective in resulting in a CO2 conversion rate of 65% or more, in another aspect 70% or more, in another aspect 75% or more, in another aspect 80% or more, in another aspect 85% or more, in another aspect 90% or more, in another aspect 85% - 95%, in another aspect 90% - 99%. The method further yields cell densities of up to 100 g / L, in one embodiment 10-80 g / L, in one embodiment 15-60 g / L, in one embodiment 20-50 g / L, in one embodiment 10-30 g / L, and in another embodiment 15-45 g / L.

[0021] The method further yields a specific H2 absorption of approximately 3 to 12 mmol of H2 / min / cell per gram, in another embodiment, approximately 3 to 10 mmol of H2 / min / cell per gram, in another embodiment, approximately 3 to 8 mmol of H2 / min / cell per gram, in another embodiment, approximately 3 to 6 mmol of H2 / min / cell per gram, in another embodiment, approximately 4 to 12 mmol of H2 / min / cell per gram, in another embodiment, approximately 4 to 10 mmol of H2 / min / cell per gram, in another embodiment, approximately 4 to 8 mmol of H2 / min / cell per gram, in another embodiment, approximately 5 to 12 mmol of H2 / min / cell per gram, in another embodiment, approximately 5 to 10 mmol of H2 / min / cell per gram, and in another embodiment, approximately 5 to 8 mmol of H2 / min / cell per gram. The method further provides cell retention times of approximately 5 to 50 hours, in another embodiment, approximately 5 to 40 hours, in another embodiment, approximately 5 to 30 hours, in another embodiment, approximately 5 to 25 hours, in another embodiment, approximately 5 to 20 hours, in another embodiment, approximately 5 to 10 hours, in another embodiment, approximately 5 to 8 hours, in another embodiment, and approximately 8 to 15 hours.

[0022] The method further yields methane production of approximately 0.4 to approximately 3 mmol of methane / min / cell per gram, in another embodiment, approximately 0.4 to approximately 2 mmol of methane / min / cell per gram, in another embodiment, approximately 0.4 to approximately 1 mmol of methane / min / cell per gram, in another embodiment, approximately 1.0 to approximately 3 mmol of methane / min / cell per gram, in another embodiment, approximately 1.0 to approximately 2.5 mmol of methane / min / cell per gram, in another embodiment, approximately 1.0 to approximately 2.5 mmol of methane / min / cell per gram, and in another embodiment, approximately 1.5 to approximately 2.5 mmol of methane / min / cell per gram. In this embodiment, the method is effective in producing a methane-effluent gas having a methane concentration greater than 60%, in another embodiment greater than 65%, in another embodiment greater than 70%, in another embodiment greater than 75%, in another embodiment greater than 80%, and in yet another embodiment greater than 85%.

[0023] Methane-producing fermentation and methylotrope fermentation The CO2 conversion method illustrated in Figures 1A and 1B includes methane production and the use of methylotrope fermentation 405 to convert methane 135 into a fermented liquid broth 450 containing methylotrope bacteria, which can be processed into a single-cell protein supplement. The fermented liquid broth 140 containing methanogenic archaeal cells can also be processed into single-cell proteins. As shown in Figure 1A, methylotrope bacteria and methanogenic archaea can be processed in a single-cell protein processing unit 145 to produce a supplement 147. In an alternative embodiment shown in Figure 1B, methanogenic archaea can be processed in a first single-cell protein processing unit 146 to produce a first supplement 148, and methylotrope bacteria can be processed in a second single-cell protein processing unit 149 to produce a second supplement 150.

[0024] Methane productionIn one embodiment illustrated in Figures 1A and 1B, the method includes a methane-producing fermentation vessel 105 as described herein in the description of methane production fermentation. A supplement hydrogen stream 40 may also be supplied to the methane-producing fermentation vessel 105. CO2 produced in the methylotrope vessel 405 may be recycled back to the methane-producing fermentation vessel through line 407.

[0025] Methylloaf fermentation As illustrated in Figures 1A and 1B, methane 135 may be supplied to the methylotrope fermentation vessel 405. The methylotrope fermentation vessel 405 contains methylotropes. Methylotropes are a diverse group of microorganisms that can use reduced single-carbon compounds such as methanol or methane as their carbon source for their growth. The methylotrope cells 450 can then be processed into single-cell proteins. Examples of methylotropes include Methylomonas methanica, Methylosinus trichosporium, Methylococcus capsulatus, Methylobacterium extorquens, Paracoccus denitrificans, Methylomicrobium alcaliphilum, Methylacidiphilum fumariolicum, Methylomicrobium buryatense, and Methanoperedens nitroreducens. Genetically modified organisms that can utilize methane can also be used. Examples of suitable growth conditions for methylotrope are described in U.S. Patent No. 10,934,566 and U.S. Patent Application Publication No. 20210340574, both of which are incorporated herein by reference. In methylotrope fermentation, methane and oxygen are converted into cell aggregates and CO2. The CO2-containing vent gas may then be provided as feed gas to the methane-producing microbial fermentation vessel 105. Before entering the methane-producing microbial fermentation vessel, the oxygen in the CO2-containing vent gas is removed.

[0026] CO fermentation, methane production fermentation, and methylotrope fermentation Methane production In one embodiment illustrated in Figures 2A and 2B, the method includes a methane-producing microbial fermentation vessel 105 as described herein in the description of Figures 1A and 1B.

[0027] CO fermentation Certain types of acetic acid-producing bacteria can ferment the CO-containing gaseous substrate 50 in the CO fermentation vessel 230 into useful oxygen-containing hydrocarbon compounds 255 such as ethanol and butanol, thereby producing a fermented liquid broth containing acetic acid-producing bacteria 270. In this embodiment, the preferred gaseous substrate 50 contains at least about 5 mol% CO, in one embodiment at least about 10 mol% CO, in one embodiment at least about 20 mol% CO, in one embodiment at least about 30 mol% CO, in one embodiment about 10 to about 100 mol% CO, in another embodiment about 20 to about 100 mol% CO, in another embodiment about 30 to about 90 mol% CO, in another embodiment about 40 to about 80 mol% CO, and in another embodiment about 50 to about 70 mol% CO. In this embodiment, the CO-containing gaseous substrate 50 may have about 40 mol% or less of CO2; in one embodiment, the CO-containing gaseous substrate 50 may have about 30 mol% or less of CO2; in one embodiment, the CO-containing gaseous substrate 50 may have about 20 mol% or less of CO2; in another embodiment, the CO-containing gaseous substrate 50 may have about 10 mol% or less of CO2; in yet another embodiment, the CO-containing gaseous substrate 50 may have about 1 mol% or less of CO2; and in yet another embodiment, the CO-containing gaseous substrate 50 may contain no CO2 or substantially no CO2.

[0028] Depending on the composition of the CO-containing gaseous substrate 50, the CO-containing gaseous substrate 50 may be supplied directly to the fermentation vessel 230, or it may be further modified or blended to have an appropriate H2-to-CO molar ratio. In one embodiment, the CO-containing gaseous substrate supplied to the fermentation vessel has an H2-to-CO molar ratio of about 0.1 or more, in another embodiment, about 0.2 or more, in another embodiment, about 0.25 or more, and in another embodiment, about 0.5 or more. In one embodiment, a second portion of H2 from a methane cracking device may be supplied to the CO fermentation vessel 230.

[0029] The concentrations of various culture medium components used in the CO bioconversion fermentation method are as follows: [Table 2]

[0030] Examples of acetic acid-producing bacteria useful in CO bioconversion fermentation methods include Blautia producta, Butyribacterium methylotrophicum, Caldanaerobacter subterraneous, Caldanaerobacter subterraneous pacificus, Carboxydothermus hydrogenoformans, Clostridium aceticum, Clostridium acetobutylicum, Clostridium acetobutylicum P262, and Clostridium autoethanogenum (DSM). Clostridium autoethanogenum (DSM 10061, DSMZ), Clostridium autoethanogenum (DSM 23693, DSMZ), Clostridium autoethanogenum (DSM 24138, DSMZ), Clostridium carboxidivorans, Clostridium coskatii (ATCC PTA-10522), Clostridium drakei, Clostridium ljungdahlii PETC (ATCC) 49587), Clostridium ljungdahlii ERI2 (ATCC 55380), Clostridium ljungdahlii C-01 (ATCCClostridium ljungdahlii O-52 (ATCC 55889), Clostridium magnum, Clostridium pasteurianum (DSM 525, German DSMZ), Clostridium ragsdalei P11 (ATCC BAA-622), Clostridium scatologenes, Clostridium thermoaceticum, Clostridium ultunense, Desulfotomaculum kuznetsovii, Eubacterium limosam Examples include *Lyophyllum limosum*, *Geobacter sulfurreducens*, *Methanosarcina acetivorans*, *Methanosarcina barkeri*, *Oxobacter pfennigii*, *Peptostreptococcus productus*, *Clostridium stick-landii*, and mixtures thereof.

[0031] Anaerobic bacteria are bacteria that do not require oxygen for growth. If oxygen is present above a certain threshold, anaerobic bacteria may react negatively and even die. Acetate-producing bacteria are microorganisms that can produce acetate under anaerobic respiration or fermentation by utilizing the Wood-Ljungdahl pathway as their primary mechanism for energy conservation. Other useful oxygen-containing hydrocarbon compounds, such as formic acid, propionic acid, butyric acid, heptanoic acid, decanoic acid, ethanol, butanol, 2-butanol, and 2,3-butanediol, can also be produced by acetic acid-producing bacteria. Examples of acetic acid-producing bacteria suitable for converting C1-containing gaseous substrates into useful oxygen-containing hydrocarbon compounds include strains of Clostridium lyngdaryi, including those described in International Publication No. 2000 / 68407, European Patent No. 117309, U.S. Patents No. 5,173,429, 5,593,886 and 6,368,819, International Publication No. 1998 / 00558 and 2002 / 08438, and strains of Clostridium autoethanogenum (DSM 10061 and DSM 19630, DSMZ, Germany), including those described in International Publication No. 2007 / 117157 and 2009 / 151342, U.S. Patent No. 7,704,723 and “Biofuels and Bioproducts from Biomass-Generated Synthesis Gas”, Hasan Atiyeh, presented in Examples of Clostridium bacteria include Clostridium lagsudarei (P11, ATCC BAA-622) and Alkalibaculum bacchi (CP11, ATCC BAA-1772), as well as Clostridium carboxydivorans (ATCC PTA-7827), as described in U.S. Patent Application No. 2007 / 0276447.Other suitable microorganisms include those of the genus Moorella, including the Moorella species HUC22-1, and those of the genus Carboxydothermus. Each of these references is incorporated herein by reference.

[0032] CO fermentation can be carried out under reaction conditions appropriate to the desired fermentation mode. For example, in one embodiment, CO fermentation can be set up in a mode focused on CO-to-oxygenated hydrocarbon compound (e.g., ethanol) production. In this mode, approximately 4% to 6% of the carbon derived from the CO fed into CO fermentation is converted into biomass. In another embodiment, CO fermentation can be set up in a mode focused on CO-to-microbial biomass production. In this mode, approximately 6% to 7.5% of the carbon derived from the CO fed into CO fermentation is converted into biomass. Reaction conditions to be considered include pressure, temperature, gas flow rate, liquid flow rate, medium pH, oxidation-reduction potential of the medium, stirring speed (if using a stirred-tank reactor), inoculation level, appropriate gas substrate concentration to ensure that the CO in the liquid phase is neither limiting nor inhibiting, and appropriate product concentration to avoid product inhibition. The CO fermentation method yields CO conversion rates of over 80%, over 85% in one embodiment, over 90% in another embodiment, 80% to 99% in yet another embodiment, 85% to 98% in yet another embodiment, and 90% to 97% in yet yet another embodiment. CO-converting acetic acid-producing bacteria convert CO to produce one or more alcohols and a CO2-containing vent gas. The CO2-containing vent gas is then sent to a methane-producing fermentation vessel. The CO2-containing vent gas contains 5% or less CO, in one embodiment, 3% or less CO, in one embodiment, 2% or less CO, and in another embodiment, 1% or less CO. CO can be removed from the CO2-containing vent gas before it enters the methane-producing fermentation vessel to avoid CO inhibition.

[0033] The method for converting CO and CO2 illustrated in Figures 2A and 2B includes the step of fermenting a gaseous substrate 50 containing CO in a CO fermentation vessel 230. CO-converting acetic acid-producing bacteria in the CO fermentation vessel 230 can convert CO into one or more alcohols 255. Vent gas 481 from the CO fermentation vessel 230 contains CO2. The vent gas 481 is supplied to the methane-producing fermentation vessel 105. An additional gaseous substrate 115 containing H2 and / or CO2 may also be supplied to the methane-producing fermentation vessel 105. Methanogenic archaea in the methane-producing fermentation vessel convert CO2 into methane 135. The methane 135 may be supplied to the methylotrope fermentation vessel 405. The methylotrope fermentation vessel 405 contains methylotrope bacteria, which consume methane for microbial growth. CO-fermentation broth 270 containing CO-converting acetic acid-producing bacterial cells, methylotrope fermentation broth 450 containing methylotrope bacterial cells, and methanogenic archaeal fermentation broth 140 containing methanogenic archaeal cells can be further processed into single-cell protein supplements.

[0034] Methylloaf fermentation As illustrated in Figures 2A and 2B, and further described in Figures 1A and 1B, methane 135 may be supplied to the methylotrope fermentation vessel 405. The methylotrope fermentation vessel 405 contains methylotrope. Integrated fermentation system As will be understood by those skilled in the art, one or all of the methods described in Figures 1A, 1B, 2A, and 2B can be combined into the overall system.

[0035] Microbial biomass The fermented liquid broth from any of the fermentation vessels (Figures 1A and 1B: 140 and 450, Figures 2A and 2B: 140, 270 and 450) can be further purged from the fermentation vessel and then processed into a protein-containing nutritional supplement in one or more single-cell protein processing units. The fermented liquid broth can be processed separately or together as shown in the figures. The fermented liquid broth is separated into a cell-free permeate and a cell-containing suspension by one or more separation devices. The cell membranes and / or cell walls of the cells in the cell-containing suspension are ruptured to produce homogenates. The homogenates are then fractionated using a fractionation device into a protein-containing supernatant and a protein-containing cell debris portion.

[0036] Suitable cell separation devices include, but are not limited to, filtration devices, hollow fiber filtration devices, spiral filtration devices, ultrafiltration devices, ceramic filter devices, cross-flow filtration devices, size exclusion column filtration devices, spiral membranes, centrifugal devices, and combinations thereof. A method for producing single-cell proteins from biomass is described in U.S. Patent Application No. 16 / 416,133, filed May 17, 2019. The cell-containing suspension contains microbial cells at a higher cell concentration than the fermented liquid broth. In one embodiment, the cell concentration of the cell-containing suspension is approximately 20 g or more / L, in another embodiment, approximately 30 g or more / L, in another embodiment, approximately 40 g or more / L, in another embodiment, approximately 50 g or more / L, in another embodiment, approximately 60 g or more / L, in another embodiment, approximately 20 to approximately 300 g / L, in another embodiment, approximately 30 to approximately 250 g / L, in another embodiment, approximately 40 to approximately 200 g / L, in another embodiment, approximately 50 to approximately 150 g / L, and in yet another embodiment, approximately 100 to approximately 150 g / L.

[0037] The cells in the cell-containing suspension may be ruptured using one or more rupture devices selected from the group consisting of microfluidic devices, sonic devices, ultrasonic devices, mechanical rupture devices, French presses, freezers, heaters, heat exchangers, distillation columns, sterilization devices, UV sterilization devices, gamma ray sterilization devices, reactors, homogenizers, and combinations thereof. In one embodiment, the pH of the cell-containing suspension is adjusted to about 6 to about 12 before rupturing the cell membranes of the cell-containing suspension; in another embodiment, 7 to 12; in another embodiment, 8 to 12; in another embodiment, 7.5 to 11; in yet another embodiment, 8.5 to 11; and in yet another embodiment, 7 to 10.

[0038] In another embodiment, the cell-containing suspension is hydrolyzed by a hydrolase enzyme. In this embodiment, the cell-containing suspension and the hydrolase enzyme are incubated at a temperature of about 50 to about 70°C for about 3 to about 72 hours, in one embodiment 3 to 48 hours, in one embodiment 4 to 24 hours, in one embodiment 6 to 24 hours, in another embodiment 6 to 12 hours, and in yet another embodiment 4 to 12 hours to form a hydrolyzed lysate. The pH of the cell-containing suspension is adjusted to about 6 to about 12 pH, in another embodiment 7 to 12 pH, in another embodiment 8 to 12 pH, in another embodiment 7.5 to 11 pH, in another embodiment 8.5 to 11 pH, and in yet another embodiment 7 to 10 pH before the hydrolysis of the cell-containing suspension. The hydrolase enzyme is selected from the group consisting of subyrase, alcalase, serine protease, serine endopeptidase, and mixtures thereof. The hydrolyzed lysate is fractionated into a protein-containing supernatant and a protein-containing cell debris portion using centrifugation, ultrafiltration, and / or a combination thereof. The protein-containing supernatant has a nucleic acid content of less than approximately 5%, less than 4% in one embodiment, less than 3% in another embodiment, and less than 2% in another embodiment.

[0039] The protein-containing supernatant and protein-containing cell debris can be used as is or further processed into protein-containing nutritional supplements. A dehydration unit can be used to dry the protein-containing supernatant and produce soluble protein-containing nutritional supplements such as protein powder. Suitable dehydration units include spray drying units, drum drying units, freeze-drying units, lyophilizing units, and combinations thereof. The protein-containing supplement can be purified by further removal of other components such as water and ash. The protein-containing supplement can be used as animal feed as is, or it can be blended with other materials to produce one or more types of nutritional supplements. In one embodiment, the protein-containing supplement contains about 60 to about 99 mass percent protein; in another embodiment, about 70 to about 95 mass percent protein; in another embodiment, about 75 to about 95 mass percent protein; in another embodiment, about 80 to about 95 mass percent protein; and in another embodiment, about 85 to about 95 mass percent protein. Methods for producing protein-containing nutrient supplements from methylotropic bacteria and acetate-producing bacteria may differ from those for producing protein-containing nutrient supplements from methanogenic archaea due to the lack of cell walls in methanogenic archaea. As shown in Figures 1B and 2B, nutrient supplement 148 from methanogenic archaea can be produced in a single-cell protein processing unit 146, while nutrient supplement 150 produced from methylotropic bacteria and / or acetate-producing bacteria can be produced in a separate single-cell protein processing unit 149. In such a scenario, the operating costs and processing time in single-cell protein processing unit 146 may be significantly lower than those in single-cell protein processing unit 149. [Examples]

[0040] The following embodiments further illustrate the present disclosure and should not be construed as limiting its scope.

[0041] (Example 1) CO2 fermentation by methane-producing bacteria CO2 and H2 gases are continuously introduced into a stirred tank bioreactor containing Methanothermobacter thermautotrophicus along with a standard liquid culture medium containing trace metals and salts. The New Brunswick Bioflow 320 reactor containing the fermentation medium is started with actively growing Metanothermobacter thermautotrophus. The reactor stirring speed is set to 1200 rpm at the start of the experiment. This stirring speed is maintained throughout the experiment. The feed gas flow to the reactor is increased based on the absorption of H2 and CO2 by the culture. The bioreactor temperature is maintained at approximately 60°C throughout the experiment. Samples of the gas feed to the bioreactor, the off-gas from the bioreactor, and the fermentation broth in the bioreactor are taken at intervals. For example, feed gas, off-gas, and fermentation broth are sampled approximately daily, every 2 hours, and every 4 hours, respectively. The above samples are analyzed for the consumption or production of various gas components and the optical density (cell density) of the culture. The stationary volume of the reactor is maintained at 2000-2200 ml throughout the experiment. The gas flow to the reactor is also measured in real time using a mass flow controller that regulates the gas to the reactor. The feed gas composition for this experiment is 76% H2, 20% CO2, and 4% N2.

[0042] In this experiment, a cell recycling system (CRS) is attached to the reactor before starting the experiment. During the experiment, the flow rate of nutrients (growth medium) to the reactor is 2.0–5.0 ml / min. The cell purging rate is 3.0–5.0 ml / min, and the permeate is removed through the CRS at a rate of 0–2.0 ml / min.

[0043] The results can be summarized as follows. Specific CO2 absorption: 0.8-1.1 mmol CO2 / min / cell per gram Ratio H2 absorption: 3.3-3.8 mmol of H2 / min / gram of cell Average cell retention time: 11.2 hours Average cell density: 3g / L CO2 conversion rate: 90%~99% The specific methane production capacity is 0.77 mmol / min / cell / gram. The efflutent gas composition in this experiment is 13.8% H2, 7.6% CO2, 62.5% CH4, and 16.1% N2.

[0044] While the disclosure disclosed herein has been described by specific embodiments, examples, and applications thereof, other modifications and variations can be made without departing from the basic scope of the disclosure as defined in the claims.

Claims

1. CO 2 A method of converting, CO 2 and H 2 The steps include: fermenting a gaseous substrate containing methane with methane-producing archaea in a methane-producing fermentation vessel to produce a fermentation liquid broth containing methane and methane-producing archaea; Methane is fermented in a methylotrope fermentation vessel with methylotrope bacteria to produce a fermentation liquid broth containing methylotrope bacteria and CO2. 2 Steps to produce the contained vent gas and A method that includes this.

2. CO 2 The method according to claim 1, wherein at least a portion of the contained vent gas is sent to a methane-producing microbial fermentation vessel.

3. CO 2 O in the vent gas 2 At least a portion of CO 2 The method according to claim 2, wherein at least a portion of the contained vent gas is removed before it is sent to a methane-producing microbial fermentation vessel.

4. CO 2 and H 2 A gaseous substrate containing CO 2 to H 2 having a ratio of about 1:3 to 1:4, the method according to claim 1.

5. Methanogenic archaea include Methanobacterium alcaliphilum, Methanobacterium bryantii, Methanobacterium congolense, Methanobacterium defluvii, Methanobacterium espanolae, Methanobacterium formicicum, Methanobacterium ivanovii, Methanobacterium palustre, Methanobacterium thermaggregans, and Methanobacterium euriginosum. Methanobrevibacter uliginosum, Methanobrevibacter acididurans, Methanobrevibacter arboriphilicus, Methanobrevibacter gottschalkii, Methanobrevibacter olleyae, Methanobrevibacter ruminantium, Methanobrevibacter smithii, Methanobrevibacter woesei, Methanobrevibacter wolinii, Methanothermobacter Methanothermobacter marburgensis, Methanothermobacter thermautotrophumMethanothermobacter thermoflexus, Methanothermobacter thermophilus, Methanothermobacter wolfeii, Methanothermus sociabilis, Methanocorpusculum bavaricum, Methanocorpusculum parvum, Methanoculleus chikuoensis, Methanoculleus submarinus, Methanogenium frigidum, Methanogenium liminatans, Methanogenium marinum Methanosarcina marinum, Methanosarcina acetivorans, Methanosarcina barkeri, Methanosarcina mazei, Methanosarcina thermophila, Methanomicrobium mobile, Methanocaldococcus jannaschii, Methanococcus aeolicus, Methanococcus maripaludis, Methanococcus vannielii, Methanococcus voltaei, MethanothermococcusThe method according to claim 1, selected from the group consisting of *Methanocaldococcus thermolithotrophicus*, *Methanopyrus kandleri*, *Methanothermobacter thermoautotroiphicus*, *Methanocaldococcus fervens*, *Methanocaldococcus indicus*, *Methanocaldococcus infernus*, and *Methanocaldococcus vulcanius*.

6. The method according to claim 1, wherein the methanogenic archaeon is metanothermobacter thermoautotrophycus.

7. The methane-producing microbial fermentation vessel contains approximately 0.5 to 3 mmol of CO2. 2 / min / cell ratio CO2 2 The method according to claim 1, which provides absorption.

8. The methane-producing microbial fermentation vessel contains approximately 1.5 to 12 mmol of H. 2 / min / cell-to-gram ratio H 2 The method according to claim 1, which provides absorption.

9. The method according to claim 1, wherein the methane-producing microbial fermentation vessel has a cell retention time of about 5 to about 50 hours.

10. The method according to claim 1, wherein the methane-producing microbial fermentation vessel yields a methane production capacity of approximately 0.5 to approximately 2.5 mmol of methane / minute / cell per gram.

11. The methane-producing bacteria fermentation vessel contains more than 65% CO2. 2 The method according to claim 1, which provides a conversion rate.

12. The method according to claim 1, wherein the methylotrope bacterium is selected from the group consisting of Methylomicrobium alcaliphilum, Methylacidiphilum fumariolicum, Methylomicrobium buryatense, Methanoperedens nitroreducens, and combinations thereof.

13. The steps include separating a fermentation liquid broth containing methylotrope bacteria into a first cell-free permeate and a first cell-containing suspension, The steps include: rupturing the cell membranes of cells in a first cell-containing suspension to produce a first homogenate; The first homogenate is fractionated into a first protein-containing supernatant and a first protein-containing cell debris portion. The first step is to collect the protein-containing nutritional supplement. The method according to claim 1, further comprising:

14. The method according to claim 13, wherein the first cell-containing suspension has a dry cell mass concentration of about 20 g / liter to about 200 g / liter.

15. The method according to claim 13, wherein the step of rupturing the cell membrane of the first cell-containing suspension is performed using one or more rupture devices selected from the group consisting of microfluidic devices, sonic processing devices, ultrasonic devices, mechanical destruction devices, French presses, freezers, heaters, heat exchangers, distillation columns, sterilization devices, UV sterilization devices, gamma ray sterilization devices, reactors, homogenizers, and combinations thereof.

16. The method according to claim 13, wherein the pH of the first cell-containing suspension is adjusted to a pH of about 6 to about 12 before the step of rupturing the cell membranes of the first cell-containing suspension.

17. The method according to claim 13, wherein the first homogenate is a hydrolyzed lysate formed by contacting the first cell-containing suspension with a hydrolase enzyme.

18. The method according to claim 17, wherein the first cell-containing suspension and hydrolase enzyme are incubated at a temperature of about 50 to about 70°C for about 3 to about 72 hours to form a hydrolyzed lysate.

19. The method according to claim 17, wherein the hydrolase enzyme is selected from the group consisting of subyrase, alcalase, serine protease, serine endopeptidase, and mixtures thereof.

20. The method according to claim 13, wherein the first homogenate is fractionated into a first protein-containing supernatant and a first protein-containing cell debris portion using centrifugation, ultrafiltration, and / or a combination thereof.

21. The method according to claim 13, wherein the first protein-containing supernatant has a nucleic acid content of less than about 5%.

22. The method according to claim 13, wherein the first protein-containing supernatant is dehydrated to obtain a first soluble protein-containing nutritional supplement containing about 60 to about 99% dry mass of protein.

23. The steps include separating the fermentation liquid broth containing methanogenic archaea into a second cell-free permeate and a second cell-containing suspension, The steps include: rupturing the cell membranes of cells in a second cell-containing suspension to generate a second homogenate; The second homogenate is fractionated into a second protein-containing supernatant and a second protein-containing cell debris portion. The second step is to collect the protein-containing nutritional supplement. The method according to claim 1, further comprising:

24. The method according to claim 23, wherein the second cell-containing suspension has a dry cell mass concentration of about 20 g / liter to about 200 g / liter.

25. The method according to claim 23, wherein the step of rupturing the cell membrane of the second cell-containing suspension is performed using one or more rupture devices selected from the group consisting of microfluidic devices, sonic processing devices, ultrasonic devices, mechanical destruction devices, French presses, freezers, heaters, heat exchangers, distillation columns, sterilization devices, UV sterilization devices, gamma ray sterilization devices, reactors, homogenizers, and combinations thereof.

26. The method according to claim 23, wherein the pH of the second cell-containing suspension is adjusted to a pH of about 6 to about 12 before the step of rupturing the cell membranes of the second cell-containing suspension.

27. The method according to claim 23, wherein the second homogenate is a hydrolyzed lysate formed by contacting the second cell-containing suspension with a hydrolase enzyme.

28. The method according to claim 27, wherein a second cell-containing suspension and a hydrolase enzyme are incubated at a temperature of about 50 to about 70°C for about 4 to about 24 hours to form a hydrolyzed lysate.

29. The method according to claim 27, wherein the hydrolase enzyme is selected from the group consisting of subyrase, alcalase, serine protease, serine endopeptidase, and mixtures thereof.

30. The method according to claim 23, wherein the second homogenate is fractionated into a second protein-containing supernatant and a second protein-containing cell debris portion by centrifugation, ultrafiltration, and / or a combination thereof.

31. The method according to claim 23, wherein the second protein-containing supernatant has a nucleic acid content of less than about 5%.

32. The method according to claim 23, wherein the second protein-containing supernatant is dehydrated to obtain a second soluble protein-containing nutritional supplement containing about 60 to about 99% dry mass of protein.

33. The steps include: mixing a fermented liquid broth containing methylotropic bacteria with a fermented liquid broth containing methanogenic archaea to produce a mixed cell-containing fermented liquid broth; The steps include separating the mixed cell-containing fermented liquid broth into a third cell-free permeate and a third cell-containing suspension, The steps include: rupturing the cell membranes of cells in a third cell-containing suspension to produce a third homogenate; The third homogenate is fractionated into a third protein-containing supernatant and a third protein-containing cell debris portion. The third step is to recover the protein-containing nutritional supplement. The method according to claim 1, further comprising:

34. The method according to claim 33, wherein the third cell-containing suspension has a dry cell mass concentration of about 20 g / liter to about 200 g / liter.

35. The method according to claim 33, wherein the step of rupturing the cell membrane of a third cell-containing suspension is performed using one or more rupture devices selected from the group consisting of microfluidic devices, sonic processing devices, ultrasonic devices, mechanical destruction devices, French presses, freezers, heaters, heat exchangers, distillation columns, sterilization devices, UV sterilization devices, gamma ray sterilization devices, reactors, homogenizers, and combinations thereof.

36. The method according to claim 33, wherein the pH of the third cell-containing suspension is adjusted to a pH of about 6 to about 12 before the step of rupturing the cell membranes of the third cell-containing suspension.

37. The method according to claim 33, wherein the third homogenate is a hydrolyzed lysate formed by contacting the third cell-containing suspension with a hydrolase enzyme.

38. The method according to claim 37, wherein a third cell-containing suspension and a hydrolase enzyme are incubated at a temperature of about 50 to about 70°C for about 3 to about 72 hours to form a hydrolyzed lysate.

39. The method according to claim 37, wherein the hydrolase enzyme is selected from the group consisting of subyrase, alcalase, serine protease, serine endopeptidase, and mixtures thereof.

40. The method according to claim 33, wherein the third homogenate is fractionated into a third protein-containing supernatant and a third protein-containing cell debris portion using centrifugation, ultrafiltration, and / or a combination thereof.

41. The method according to claim 33, wherein the third protein-containing supernatant has a nucleic acid content of less than about 5%.

42. The method according to claim 33, wherein the third protein-containing supernatant is dehydrated to obtain a third soluble protein-containing nutritional supplement containing about 60 to about 99% dry mass of protein.

43. CO and CO 2 A method of converting, CO 2 and H 2 The steps include: fermenting a gaseous substrate containing methane with methane-producing archaea in a methane-producing fermentation vessel to produce a fermentation liquid broth containing methane and methane-producing archaea; Methane is fermented in a methylotrope fermentation vessel with methylotrope bacteria to produce a fermentation liquid broth containing methylotrope bacteria and the first CO2. 2 The steps include producing the contained vent gas, A gaseous substrate containing CO is fermented in a CO fermentation vessel with CO-converting acetic acid-producing bacteria to produce alcohol and a second CO 2 The steps include producing a fermented liquid broth containing vent gas and acetic acid-producing bacteria, and A method that includes this.

44. First CO 2 The method according to claim 43, wherein at least a portion of the contained vent gas is sent to a methane-producing microbial fermentation vessel.

45. First CO 2 O in the vent gas 2 At least a portion of the first CO 2 The method according to claim 44, wherein at least a portion of the contained vent gas is removed before it is sent to a methane-producing microbial fermentation vessel.

46. Second CO 2 The method according to claim 43, wherein at least a portion of the contained vent gas is sent to a methane-producing microbial fermentation vessel.

47. Second CO 2 At least a portion of the CO in the contained vent gas is a second CO 2 The method according to claim 46, wherein at least a portion of the contained vent gas is removed before it is sent to a methane-producing microbial fermentation vessel.

48. CO 2 and H 2 A gaseous substrate containing the first CO 2 At least a portion of the contained vent gas, second CO 2 At least a portion of the contained vent gas, or the first CO 2 At least a portion of the contained vent gas and the second CO 2 The method according to claim 43, comprising a combination of at least a portion of the contained vent gases.

49. CO 2 and H 2 A gaseous substrate containing CO2 in a ratio of approximately 1:3 to 1:4 2 vs H 2 The method according to claim 43, having the ratio of .

50. Methanogenic archaea include Metanobacterium alkaliphyllum, Metanobacterium bryantyi, Metanobacterium congolense, Metanobacterium deflubyi, Metanobacterium espanolae, Metanobacterium formicicum, Metanobacterium ibanobii, Metanobacterium parstre, Metanobacterium thermagregans, Metanobacterium euriginosum, Metanobrevibacter acididurans, and Metanobrevibacter - Arboriphyllicus, Metanobrevibacter gotscharkii, Metanobrevibacter oreyae, Metanobrevibacter luminantium, Metanobrevibacter smisi, Metanobrevibacter oozei, Metanobrevibacter wolinii, Metanothermobacter marbulgensis, Metanothermobacter thermoautotrophum, Metanothermobacter thermoflexus, Metanothermobacter thermophilus, Metanothermobacter wolfei Metanotermus sochiabilis, Metanocorpus bavaricum, Metanocorpus palham, Metanocreus thikoensis, Metanocreus submarinus, Metanogenium frigidum, Metanogenium liminatans, Metanogenium marinum, Metanosarcinia acetylborans, Metanosarcinia barkerii, Metanosarcinia mazei, Metanosarcinia thermophylla, Metanomicrobium mobile, Metanocardococcus janaskii, Metanoco The method according to claim 43, selected from the group consisting of Cass aeolicus, Metanococcus maripaldis, Metanococcus van neerii, Metanococcus voltae, Metanothermococcus thermolotrophus, Metanopyrus candrelli, Metanothermobacter thermoautotrophycus, Metanocardococcus favens, Metanocardococcus indicus, Metanocardococcus infernus, and Metanocardococcus bulcanius.

51. The method according to claim 43, wherein the methanogenic archaeon is metanothermobacter thermoautotrophycus.

52. Methane-producing microbial fermentation produces approximately 1 to 3 mmol of CO2. 2 / min / cell ratio CO2 2 The method according to claim 43, which provides absorption.

53. Methanogenic microbial fermentation produces approximately 3 to 12 mmol of H 2 / min / cell-to-gram ratio H 2 The method according to claim 43, which provides absorption.

54. The method according to claim 43, wherein the methane-producing microbial fermentation has a cell retention time of about 5 to about 50 hours.

55. The method according to claim 43, wherein the fermentation by methanogenic bacteria yields a methane production capacity of approximately 1.2 to approximately 2.5 mmol of methane / minute / cell per gram.

56. The methane-producing bacteria fermentation vessel contains more than 65% CO2. 2 The method according to claim 43, which provides a conversion rate.

57. CO-converting acetic acid-producing bacteria include Clostridium aceticum, Clostridium acetobutylicum, Clostridium acetobutylicum P262, Clostridium autoethanogenum (DSM 19630, German DSMZ), Clostridium autoethanogenum (DSM 10061, German DSMZ), Clostridium autoethanogenum (DSM 23693, German DSMZ), Clostridium autoethanogenum (DSM 24138, German DSMZ), and Clostridium carboxydivorans. carboxidivorans), Clostridium coskatii (ATCC PTA-10522), Clostridium drakei, Clostridium ljungdahlii PETC (ATCC 49587), Clostridium ljungdahlii ERI2 (ATCC 55380), Clostridium ljungdahlii C-01 (ATCC 55988), Clostridium ljungdahlii O-52 (ATCC 55889), Clostridium magnum, Clostridium pasteurianum (DSM) 525, German DSMZ), Clostridium ragsdalei P11 (ATCC BAA-622), Clostridium scatogenesThe method according to claim 43, selected from the group consisting of Clostridium scatologenes, Clostridium thermoaceticum, Clostridium ultunense, Clostridium stick-landii, and mixtures thereof.

58. The method according to claim 43, wherein the alcohol is ethanol.

59. The method according to claim 43, wherein the CO fermentation vessel yields a CO conversion rate of 80% or more.

60. The method according to claim 43, wherein the methylotrope is selected from the group consisting of Methylomicrobium alkaliphyllum, Methylacidiphyllum fumariolycum, Methylomicrobium briatens, Methanopelledens nitroredusens, and combinations thereof.

61. The steps include separating a fermentation liquid broth containing methanogenic archaea into a first cell-free permeate and a first cell-containing suspension, The steps include: rupturing the cell membranes of cells in a first cell-containing suspension to produce a first homogenate; The first homogenate is fractionated into a first protein-containing supernatant and a first protein-containing cell debris portion. The first step is to collect the protein-containing nutritional supplement. The method according to claim 43, further comprising:

62. The method according to claim 61, wherein the second cell-containing suspension has a dry cell mass concentration of about 20 g / liter to about 200 g / liter.

63. The method according to claim 61, wherein the step of rupturing the cell membrane of a first cell-containing suspension is performed using one or more rupture devices selected from the group consisting of microfluidic devices, sonic processing devices, ultrasonic devices, mechanical destruction devices, French presses, freezers, heaters, heat exchangers, distillation columns, sterilization devices, UV sterilization devices, gamma ray sterilization devices, reactors, homogenizers, and combinations thereof.

64. The method according to claim 61, wherein the pH of the first cell-containing suspension is adjusted to a pH of about 6 to about 12 before the step of rupturing the cell membranes of the first cell-containing suspension.

65. The method according to claim 61, wherein the first homogenate is a hydrolyzed lysate formed by contacting the first cell-containing suspension with a hydrolase enzyme.

66. The method according to claim 65, wherein the first cell-containing suspension and hydrolase enzyme are incubated at a temperature of about 50 to about 70°C for about 4 to about 24 hours to form a hydrolyzed lysate.

67. The method according to claim 65, wherein the hydrolase enzyme is selected from the group consisting of subyrase, alcalase, serine protease, serine endopeptidase, and mixtures thereof.

68. The method according to claim 61, wherein the first homogenate is fractionated into a first protein-containing supernatant and a first protein-containing cell debris portion by centrifugation, ultrafiltration, and / or a combination thereof.

69. The method according to claim 61, wherein the first protein-containing supernatant has a nucleic acid content of less than about 5%.

70. The method according to claim 61, wherein the first protein-containing supernatant is dehydrated to obtain a first soluble protein-containing nutritional supplement containing about 60 to about 99% dry mass of protein.

71. The process involves mixing a fermented liquid broth containing methylotrope bacteria with a fermented liquid broth containing acetic acid-producing bacteria to produce a first mixed cell-containing fermented liquid broth, The first step is to separate the mixed cell-containing fermented liquid broth into a second cell-free permeate and a second cell-containing suspension, The steps include: rupturing the cell membranes of cells in a second cell-containing suspension to generate a second homogenate; The second homogenate is fractionated into a second protein-containing supernatant and a second protein-containing cell debris portion. The second step is to collect the protein-containing nutritional supplement. The method according to claim 43, further comprising:

72. The method according to claim 71, wherein the second cell-containing suspension has a dry cell mass concentration of about 20 g / liter to about 200 g / liter.

73. The method according to claim 71, wherein the step of rupturing the cell membrane of a second cell-containing suspension is performed using one or more rupture devices selected from the group consisting of microfluidic devices, sonic processing devices, ultrasonic devices, mechanical destruction devices, French presses, freezers, heaters, heat exchangers, distillation columns, sterilization devices, UV sterilization devices, gamma ray sterilization devices, reactors, homogenizers, and combinations thereof.

74. The method according to claim 71, wherein the pH of the second cell-containing suspension is adjusted to a pH of about 6 to about 12 before the step of rupturing the cell membranes of the second cell-containing suspension.

75. The method according to claim 71, wherein the second homogenate is a hydrolyzed lysate formed by contacting the second cell-containing suspension with a hydrolase enzyme.

76. The method according to claim 75, wherein a second cell-containing suspension and a hydrolase enzyme are incubated at a temperature of about 50 to about 70°C for about 3 to about 72 hours to form a hydrolyzed lysate.

77. The method according to claim 75, wherein the hydrolase enzyme is selected from the group consisting of subyrase, alcalase, serine protease, serine endopeptidase, and mixtures thereof.

78. The method according to claim 71, wherein the second homogenate is fractionated into a second protein-containing supernatant and a second protein-containing cell debris portion by centrifugation, ultrafiltration, and / or a combination thereof.

79. The method according to claim 71, wherein the second protein-containing supernatant has a nucleic acid content of less than about 5%.

80. The method according to claim 71, wherein the second protein-containing supernatant is dehydrated to obtain a second soluble protein-containing nutritional supplement containing about 60 to about 99% dry mass of protein.

81. The process involves mixing a fermented liquid broth containing methylotropic bacteria and a fermented liquid broth containing methanogenic archaea with a fermented liquid broth containing acetic acid-producing bacteria to produce a second mixed cell-containing fermented liquid broth, The second step is to separate the mixed cell-containing fermented liquid broth into a third cell-free permeate and a third cell-containing suspension, The steps include: rupturing the cell membranes of cells in a third cell-containing suspension to produce a third homogenate; The third homogenate is fractionated into a third protein-containing supernatant and a third protein-containing cell debris portion. The third step is to recover the protein-containing nutritional supplement. The method according to claim 43, further comprising:

82. The method according to claim 81, wherein the third cell-containing suspension has a dry cell mass concentration of about 20 g / liter to about 200 g / liter.

83. The method according to claim 81, wherein the step of rupturing the cell membrane of a third cell-containing suspension is performed using one or more rupture devices selected from the group consisting of microfluidic devices, sonic processing devices, ultrasonic devices, mechanical destruction devices, French presses, freezers, heaters, heat exchangers, distillation columns, sterilization devices, UV sterilization devices, gamma ray sterilization devices, reactors, homogenizers, and combinations thereof.

84. The method according to claim 81, wherein the pH of the third cell-containing suspension is adjusted to a pH of about 6 to about 12 before the step of rupturing the cell membranes of the third cell-containing suspension.

85. The method according to claim 81, wherein the third homogenate is a hydrolyzed lysate formed by contacting the third cell-containing suspension with a hydrolase enzyme.

86. The method according to claim 85, wherein a third cell-containing suspension and a hydrolase enzyme are incubated at a temperature of about 50 to about 70°C for about 3 to about 72 hours to form a hydrolyzed lysate.

87. The method according to claim 85, wherein the hydrolase enzyme is selected from the group consisting of subyrase, alcalase, serine protease, serine endopeptidase, and mixtures thereof.

88. The method according to claim 81, wherein the third homogenate is fractionated into a third protein-containing supernatant and a third protein-containing cell debris portion by centrifugation, ultrafiltration, and / or a combination thereof.

89. The method according to claim 81, wherein the third protein-containing supernatant has a nucleic acid content of less than about 5%.

90. The method according to claim 81, wherein the third protein-containing supernatant is dehydrated to obtain a third soluble protein-containing nutritional supplement containing about 60 to about 99% dry mass of protein.

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