Method for immobilizing carbon dioxide

Abstract

Systems and methods for immobilizing carbon dioxide by fermentation are provided. More specifically, the present disclosure includes fermenting carbon dioxide to methane by methanogenic archaea and producing a single cell protein nutritional supplement. The present disclosure also provides integration of methanogenic fermentation with other methods to achieve improved carbon efficiency.

Description

[0001] A method for fixing carbon dioxide is provided. More specifically, the method includes fermenting carbon dioxide into methane and fermenting the methane with methylotrophic bacteria. Another method includes processing the cell clusters from fermentation into single-cell proteins for use as a nutritional supplement. Background Technology

[0002] Carbon monoxide and carbon dioxide emissions from industrial processes are two major drivers of climate change and global warming. Microbial fermentation can reduce these emissions by utilizing microorganisms 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 desirable products through their metabolic pathways.

[0003] Large-scale microbial fermentation also produces significant amounts of microbial biomass. Traditionally, the disposal of microbial biomass requires highly expensive waste treatment systems, storage sites, and landfills. Previous findings have shown that microbial biomass can be recycled into single-cell protein (SCP) and other components that can be reused as sources of protein, amino acids, and carbohydrates, serving 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 acetic acid-producing bacteria to generate microbial biomass.

[0004] Therefore, there is a need for methods and systems capable of efficiently converting carbon dioxide into products for use in other methods. Furthermore, there remains a need for methods and systems for efficiently converting microbial biomass into digestible nutritional supplements and compositions of any such supplements. Summary of the Invention

[0005] In one aspect, methods for converting CO2 include fermenting a gaseous substrate comprising CO2 and H2 with methanogenic archaea in a methanogenic fermentation vessel to produce methane and a fermentation liquid broth containing methanogenic archaea, and fermenting methane with methyltrophic bacteria in a methyltrophic fermentation vessel to produce a fermentation liquid broth containing methyltrophic bacteria and CO2-containing exhaust gas.

[0006] On the other hand, methods for converting CO and CO2 include fermenting a gaseous substrate comprising CO2 and H2 with methanogenic archaea in a methanogenic fermentation vessel to produce methane and a fermentation broth containing methanogenic archaea. The methane is then fermented with methyltrophic bacteria in a methyltrophic fermentation vessel to produce a fermentation broth containing methyltrophic bacteria and a first CO2-containing exhaust gas. A gaseous substrate comprising CO is fermented with CO-converting acetogenic bacteria in a CO fermentation vessel to produce alcohol, a second CO2-containing exhaust gas, and a fermentation broth containing acetogenic bacteria. Attached Figure Description

[0007] To gain a more detailed understanding of the features listed above in this disclosure, reference can be made to embodiments for a more specific description of the disclosure, some of which are illustrated in the accompanying drawings. However, it should be noted that the drawings show only typical embodiments of this disclosure and should therefore not be considered as limiting its scope, as other equivalent embodiments are permissible.

[0008] Figure 1A A method for converting CO2 is shown, which includes methane production and fermentation with methyltrophic bacteria, wherein single-cell proteins are processed together.

[0009] Figure 1B A method for converting CO2 is shown, which includes methane production and fermentation with methyltrophic bacteria, wherein single-cell proteins are processed separately.

[0010] Figure 2A A method for converting CO and CO2 is shown, which includes methane production, CO conversion fermentation with acetic acid-producing bacteria, and fermentation with methyltrophic bacteria, wherein single-cell proteins are processed together.

[0011] Figure 2B A method for converting CO2 is shown, which includes methane production, CO conversion fermentation with acetic acid-producing bacteria, and fermentation with methyltrophic bacteria, wherein single-cell proteins are treated separately. Detailed Implementation

[0012] The following description should not be considered limiting, but is merely for the purpose of illustrating the general principles of exemplary embodiments. The scope of this disclosure should be determined with reference to the claims.

[0013] The term “about”, used to modify any quantity, refers to a change in quantity encountered under real-world conditions, such as in a laboratory, pilot plant, or production facility. For example, when modified by “about,” the quantity of an ingredient or measure used in a mixture or quantity includes variations and degrees of care typically used to measure under experimental conditions in a production plant or laboratory. For example, when modified by “about,” the quantity of a component of a product includes variations between batches in multiple experiments in a plant or laboratory, as well as variations inherent in the analytical method. Whether or not modified by “about,” the quantity includes equivalents of those quantities. Any quantity described herein and modified by “about” may also be used in this disclosure as a quantity not modified by “about.”

[0014] In the context of this disclosure, the terms “a”, “an”, “the” and similar references should be interpreted as encompassing both the singular and plural, unless otherwise indicated or the context clearly contradicts them.

[0015] Unless otherwise stated, the terms “comprising,” “including,” “having,” “containing,” or “characterized in” are inclusive and do not exclude any additional, unlisted elements or method steps (i.e., meaning “includes but is not limited to”). Unless otherwise claimed, any instance or exemplary language provided herein (e.g., “such as,” “for example,” “for instance”) is intended only to illustrate this disclosure and does not limit its scope.

[0016] Fermentation is a metabolic process used by microorganisms to produce energy for cell growth. Some microorganisms ferment C1-containing gaseous substrates, such as syngas, carbon monoxide (CO)-containing gaseous substrates, or carbon dioxide (CO2)-containing gaseous substrates, to sustain their growth and produce oxygenated hydrocarbons. In such cases, the microorganisms use one or more C1 components of the C1-containing gaseous substrate as their primary carbon source for growth. The terms "fermentation," "fermentation process," "microbial fermentation process," etc., are intended to encompass both the growth phase and the product biosynthesis phase of the process. In anaerobic fermentation, a large amount of microbial biomass is obtained, which can be removed and processed into useful products, such as nutritional supplements. Specifically, this disclosure includes a method for extracting nutritional supplements from microbial biomass during anaerobic fermentation.

[0017] Fermentable gaseous substrates are Cl-containing gaseous substrates containing one or more of CO, CO2, or CH2O2. Suitable gaseous substrates may include various syngas (i.e., synthesis gases) and industrial exhaust gases.

[0018] Syngas can be provided from any known source. In one aspect, syngas can be derived from the gasification of carbonaceous materials. Gasification involves the partial combustion of biomass in a limited supply of oxygen. The resulting gas may include CO, CO2, and H2. Examples of suitable gasification methods and apparatus are provided in U.S. Serial Nos. 61 / 516,667, 61 / 516,704, and 61 / 516,646 (all filed April 6, 2011) and U.S. Serial Nos. 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 aspect, syngas can be produced by the electrolysis of water and carbon dioxide. In this aspect, oxygen is removed from the resulting gas, and the resulting gas can be further blended with other gas sources to form a desired fermentable gaseous substrate.

[0019] Industrial exhaust gases can include C1-containing waste gases from industrial processes that would otherwise be released into the atmosphere. Examples of industrial exhaust gases include gases generated during microbial fermentation, ferrous metal product manufacturing, non-ferrous metal product manufacturing, petroleum refining processes, coal gasification, power generation, carbon black production, ammonia production, methanol production, coke manufacturing, and gas reforming.

[0020] C1-containing gaseous substrates may include H2. H2 may also be added alone to C1-containing gaseous substrates to form a desired gaseous composition suitable for fermentation. Examples of H2 sources include gases generated during the manufacture of ferrous and non-ferrous metal products, petroleum refining processes, coal gasification, biomass gasification, power generation, carbon black production, ammonia production, methanol production, and coke manufacturing. Other hydrogen sources may include, for example, H2 generated from H2O electrolysis and biomass production.

[0021] Fermentation of fermentable gaseous substrates with microorganisms takes place in a fermentation vessel. A fermentation vessel comprises a fermentation bioreactor consisting of one or more containers and / or towers or piping arrangements, including batch reactors, semi-batch reactors, continuous reactors, continuous stirred tank reactors (CSTRs), bubble column reactors, external circulation loop reactors, internal circulation loop reactors, immobilized cell reactors (ICRs), trickle bed reactors (TBRs), moving bed biofilm reactors (MBBRs), airlift reactors, membrane reactors (e.g., hollow fiber membrane bioreactors (HFMBRs)), static mixers, airlift fermenters, or other containers or devices suitable for gas-liquid contact.

[0022] Culture media suitable for the growth of anaerobic microorganisms and for fermenting fermentable gaseous substrates into one or more oxygenated hydrocarbon compounds can be added to fermentation vessels to support the fermentation of gaseous substrates by acetic acid-producing bacteria. Examples of culture medium compositions are described in U.S. Serial Nos. 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. Culture media may be sterilized to remove undesirable microorganisms and the fermentation vessel may be inoculated with the desired microorganisms. Sterilization may not always be necessary. Suitable culture media for methanogenic fermentation are described in U.S. Patent No. 11,401,499, which is incorporated herein by reference.

[0023] methanogenic fermentation methane production :exist Figure 1A and 1B In one aspect, a method includes a methanogenic fermentation vessel 105 that can be integrated with an industrial process for producing CO2. In this aspect, the methanogenic fermentation vessel 105 contains a microbial culture capable of producing hydrogen-trophic methane (i.e., CO2 plus H2 to methane). A separate hydrogen source 40 can be provided to the methanogenic fermentation vessel 105. H2 and CO2 can be added separately to the methanogenic fermentation vessel 105 or mixed together and then added to the methanogenic fermentation vessel 105. The method involves maintaining the CO2 to H2 ratio in the fermentation vessel 105 at approximately 1:5 to approximately 1:1, or, on the other hand, approximately 1:5 to approximately 1:2, or, on the other hand, approximately 1:5 to approximately 1:3, or, on the other hand, approximately 1:5 to approximately 1:4, or, on the other hand, approximately 1:4 to approximately 1:1, or, on the other hand, approximately 1:4 to approximately 1:2, or, on the other hand, approximately 1:4 to approximately 1:3, or, on the other hand, approximately 1:3 to approximately 1:1, or, on the other hand, approximately 1:3 to approximately 1:2, and, on the other hand, approximately 1:2 to approximately 1:1. The total gas delivery rate is suitable in the range of approximately 0.2 to approximately 25 volumes of gas per minute per volume of culture, or, on the other hand, approximately 2 to approximately 16, or, on the other hand, approximately 1 to approximately 22, and, on the other hand, approximately 0.5 to approximately 20 (STP, standard temperature and pressure). The methanogenic fermentation vessel 105 can also produce a fermentation broth containing methanogenic archaea 140, which can be processed into single-cell proteins in the single-cell protein processing unit 145 to produce a nutrient supplement 147.

[0024] Suitable microbial cultures can be readily obtained from public microbial repositories or isolated from a variety of environmental sources. Such sources include anaerobic soils and sands, bogs, swamps, marshes, estuaries, dense algal mats, mud and sediments from both land and sea, deep-sea and deep-well sites, sewage and organic waste sites and treatment facilities, and animal intestines and feces. Numerous pure cultures of a single species are suitable. Taxonomically appropriate pure cultures are all members of the Archaeal 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 hereby by reference)] and belong to the four distinct classes within the phylum Euryarchaea. Instances of suitable microorganisms have been classified into four different genera within the class Methanobacteria (e.g., alkalophilic methanobacteria). Alkaliphilic Methanobacterium ), Methanobacterium brevicornu ( Bryant's Methanobacterium ), Methanobacterium congo ( Congolese Methanobacterium ), Methanobacterium sludge Defluvii Methanobacterium Methanobacterium spp. (Spanish methanogen) Spanish Methanobacterium ), Methanobacterium formate ( Formicicum Methanobacterium ), Methanobacterium ilmeni ( Methanobacterium ivanovii ), Methanobacterium swampense ( Marsh Methanobacterium ), Polymethanobacterium thermophilum ( Thermaggregans Methanobacterium ), Methanobacter wetland ( Uliginosum Methanobacterium ), acid-resistant methanogenic short bacilli ( Acididurans Methanobrevibacter ), Methanogenic short bacillus ( Arboriphilicus Methanobrevibacter ), Methanobacterium graminearum ( Methanobrevibacter gottschalkii ), Methanobacterium ostreatus ( Olley's Methanobrevibacter ), rumen methanogenic short bacilli ( Ruminantium Methanobrevibacter ), Methanobacterium spp. ( Smith's Methanobrevibacter ), Methanobacterium wartii ( Woese's Methanobrevibacter ), Methanobacterium walneri ( Methanobrevibacter wolinii Marburg methanophilic bacteria ( Marburg Methanothermobacter Thermoautotrophic methanophilic bacteria ( Thermoautotrophic Methanothermobacter (Also known as thermoautotrophic methanophilic bacillus) Thermoautotroiphicus Methanothermobacter ), Heat-curved thermophilic bacillus ( Thermoflexus Methanothermobacter ), thermophilic methanophilic thermobacter ( Methanothermobacter thermophilus), Methanophilic bacillus wolfi ( Wolfe's Methanothermobacter ), social methanotherapeutic bacteria ( Sociable Methanothermus Five different genera of the Methanomicrobia class (e.g., Methanophora bavaria) Bavaricum Methanocorpusculum ), Micrometazobium ( Parvum Methanocorpusculum ), Methanocystis bamboo ( Chikuoensis Methanoculleus ), marine methanoma ( Submarinus Methanoculleus ), cold-weather methanogens ( Frigidum Methanogenium ), mudflat methanogens ( Liminatans Methanogenium Marine methanogenic bacteria ( Marinum Methanogenium ), Methanophora acetophila ( Acetivorans Methanosarcina ), Pasteurella multocida ( Methanosarcina barkeri ), Methanococcus martensii ( Mazei Methanosarcina ), thermophilic methanococcus ( Thermophilic Methanosarcina ), methanogenic bacteria ( Mobile Methanomicrobium Seven different genera of the class Methanococci (e.g., Methanococci janniae) Methanocaldococcus jannaschii ), Methanococcus fusca ( Aeolic Methanococcus ), Methanococcus marinum ( Methanococcus maripaludis ), methanococcus vannamei ( Vanniel's Methanococcus Methanococcus warwick ( Methanococcus voltaei ), thermophilic methanococci ( Thermolithotrophic Methanothermococcus ), Methanococcus thermophilus ( Fervid Methanocaldococcus ), Indian methanococcus ( Indian Methanocaldococcus ), Hellfire Cocci ( Infernal Methanocaldococcus ), volcano methanococcus ( Methanocaldococcus vulcanius )) and a genus of the class Methanopyri (e.g., Methanopyri cannibalus) Kandler's MethanopyrusSuitable cultures are available from public culture repositories (e.g., the American Type Culture Collection, the Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH, and the Oregon Collection of Methanogens). Many suitable hydrogenotrophic methanogens isolated in pure cultures and available in public culture repositories have not been fully classified. Preferred pure cultures include *Methanococcus pasteurellii*. Barkeri Methanosarcina ), Methanococcus marinum ( Methanococcus maripaludis Thermoautotrophic methanophilic bacteria ( Thermoautotrophic Methanothermobacter ) and Marburg methanophilic bacteria ( Marburg Methanothermobacter ).

[0025] Suitable cultures of mixtures of two or more microorganisms are also readily separable 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]. Suitable mixtures can be physical associations of cells of two or more species, or they can be metabolically coordinated mixtures of two or more species without physical association. Mixed cultures may possess useful properties beyond those available from pure cultures of known hydrogenotrophic methanogens. These properties may include, for example, resistance to contaminants (e.g., oxygen, ethanol, or other trace components) in the gaseous feed stream, or aggregate growth, which can increase the culture density and volumetric gas handling capacity.

[0026] Suitable cultures of mixed microorganisms can also be obtained by combining cultures isolated from two or more sources. One or more species in a suitable mixed culture should be archaeal methanogens. Any non-archaeal species can be bacteria or eukaryotes.

[0027] Suitable cultures can also be obtained through genetic modification of non-methanogenic microorganisms, where essential genes supporting hydrogenotrophic methane production are transferred from methanogenic microorganisms or from combinations of microorganisms that may or may not produce methanogens themselves. Suitable genetic modifications can also be obtained through enzymatic or chemical synthesis of the necessary genes.

[0028] Methanogenic fermentation vessel 105 provides continuous methane production using continuous hydrogenotrophic methanogenic cultures operated under stable conditions. An example of such suitable conditions is provided in "Schill, N., van Gulik, M., Voisard, D., & von Stockar, U. (1996) Biotcchnol & Biong 51:645-658. Continuous cultures limited by a gaseous substrate: development of a simple, unstructured mathematical model and experimental verification with..." Methanobacterium thermoautotrophicum “This is incorporated herein by reference.” The culture medium may consist of diluted mineral salts and should be adapted to the specific culture being used.

[0029] The concentrations of various culture medium components used in methanogenic fermentation methods are as follows:

[0030] The culture medium in the methanogenic fermentation vessel 105 should be replenished at a rate suitable for maintaining effective concentrations of essential minerals and eliminating any metabolites that may inhibit methane production. Dilution rates below 0.2 culture volumes / hour are appropriate because they produce high volumetric concentrations of active methane-generating capacity.

[0031] In one respect, the redox potential remains below -400 mV or lower during methane formation. In another respect, the redox potential remains below -300 mV or lower, below -200 mV, and below -100 mV.

[0032] On the other hand, the temperature of the culture is maintained near the optimal value for the growth of the microorganisms used in the culture (e.g., for mesophilic organisms such as *Pamela methanogens*). Methanosarcina barkeri ) and methanococci ( Methanococcus maripaludis The optimal temperature range is approximately 35°C to approximately 37°C, or for thermophilic bacteria such as thermoautotrophic methanophilic bacteria ( ). Thermoautotrophic Methanothermobacter The optimal temperature is approximately 60°C-65°C, and is also suitable for microorganisms such as Methanococcus japonicus ( ). Jannaschii Methanocaldococcus ), Methanococcus thermophilus ( Fervid Methanocaldococcus ), Indian methanococcus ( Indian Methanocaldococcus ), Hellfire Cocci (Infernal Methanocaldococcus ) and methanococcus volcanotherae ( Methanocaldococcus vulcanius (Approximately 85°C-90°C). However, it is envisioned that temperatures higher or lower than the optimal growth temperature can be used.

[0033] On the other hand, a reducing agent can be introduced into the fermentation process along with CO2 and H2. This reducing agent can suitably be hydrogen sulfide or sodium sulfide. Hydrogen itself can be used as a reducing agent to maintain the redox potential of the culture within the range (<-100 mV) necessary for optimal performance of hydrogen-nutritive methane production. Typically, hydrogen is provided at an effective concentration that allows at least a portion of the carbon dioxide in the bioreactor to be converted to methane. Alternatively, the redox potential of the culture can be maintained at <-100 mV via an electrochemical cell immersed in the culture medium.

[0034] On the other hand, the method includes various methods and / or features for reducing the presence of oxygen in the CO2 stream supplied to the bioreactor. When using obligate anaerobic methanogenic archaea to catalyze methane formation, the presence of oxygen can be detrimental to the performance of the method and contaminate the product gas. Therefore, reducing the presence of oxygen in the CO2 stream helps improve the method. In one aspect, the oxygen level is reduced by passing a mixed H2 / CO2 stream over a palladium catalyst before the gas enters the fermentation vessel (which converts any trace oxygen into water). In this aspect, the amount of H2 provided is higher than the required 2:1 ratio in the culture relative to the amount of contaminating oxygen. On the other hand, oxygen is removed by pretreating the gas stream in the bioreactor. In this aspect, a reducing agent can be provided by providing an organic material source (e.g., glucose, starch, cellulose, fermentation residues from an ethanol plant, whey residues, etc.) that can serve as a substrate for oxidative fermentation. A microbial biocatalyst is selected to oxidatively ferment the selected organic source, producing CO2 from the contaminating oxygen. In this embodiment, additional H2 will be provided to enable the conversion of this additional CO2 into methane in the anaerobic fermenter.

[0035] The current method provides specific CO2 uptake of approximately 0.5 to approximately 3 mmol CO2 / min / g cells, on the other hand approximately 1 to approximately 2 mmol CO2 / min / g cells, on the other hand approximately 0.5 to approximately 1 mmol CO2 / min / g cells, on the other hand approximately 1 to approximately 3 mmol CO2 / min / g cells, and on the other hand approximately 0.5 to approximately 2 mmol CO2 / min / g cells. In this respect, the current method effectively provides CO2 conversion rates of 65% or greater, on the other hand 70% or greater, on the other hand 75% or greater, on the other hand 80% or greater, on the other hand 85% or greater, on the other hand 90% or greater, on the other hand 85% to 95%, and on the other hand 90% to 99%.

[0036] The method further provides cell densities of up to 100 g / L, 10 to 80 g / L in one aspect, 15 to 60 g / L in another aspect, 20 to 50 g / L in one aspect, 10 to 30 g / L in another aspect, and 15 to 45 g / L in another aspect.

[0037] The method also provides approximately 3 to approximately 12 mmol H2 / min / g cells, on the other hand approximately 3 to approximately 10 mmol H2 / min / g cells, on the other hand approximately 3 to approximately 8 mmol H2 / min / g cells, on the other hand approximately 3 to approximately 6 mmol H2 / min / g cells, on the other hand approximately 4 to approximately 12 mmol H2 / min / g cells, on the other hand approximately 4 to approximately 10 mmol H2 / min / g cells, on the other hand approximately 4 to approximately 8 mmol H2 / min / g cells, on the other hand approximately 5 to approximately 12 mmol H2 / min / g cells, on the other hand approximately 5 to approximately 10 mmol H2 / min / g cells, and on the other hand approximately 5 to approximately 8 mmol H2 / min / g cells for specific H2 uptake.

[0038] The method also provides cell retention time of about 5 to about 50 hours, on the other hand about 5 to about 40 hours, on the other hand about 5 to about 30 hours, on the other hand about 5 to about 25 hours, on the other hand about 5 to about 20 hours, on the other hand about 5 to about 10 hours, on the other hand about 5 to about 8 hours, and on the other hand about 8 to about 15 hours.

[0039] The method further provides methane production rates of approximately 0.4 to approximately 3 mmol methane / min / g cells, on another side approximately 0.4 to approximately 2 mmol methane / min / g cells, on yet another side approximately 0.4 to approximately 1 mmol methane / min / g cells, on yet another side approximately 1.0 to approximately 3 mmol methane / min / g cells, on yet another side approximately 1.0 to approximately 2.5 mmol methane / min / g cells, on yet another side approximately 1.0 to approximately 2.5 mmol methane / min / g cells, and on yet another side approximately 1.5 to approximately 2.5 mmol methane / min / g cells. In this regard, the method is effective for providing methane exhaust gases with methane concentrations greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, and greater than 85% in another aspect.

[0040] Methanogenic fermentation and methylotrophic fermentation like Figure 1A and 1B The methods shown for CO2 conversion include methane production and the conversion of methane 135 into a fermentation broth containing methyltrophic bacteria 450 using methyl-trophic fermentation 405, which can be processed into a single-cell protein nutritional supplement. Fermentation broth containing methanogenic archaea cells 140 can also be processed into single-cell protein. For example... Figure 1A As shown, methyltrophic bacteria and methanogenic archaea can be processed in the single-cell protein processing unit 145 to produce a nutrient supplement 147. Figure 1B In an alternative embodiment, methanogenic archaea may be treated in a first single-cell protein processing unit 146 to produce a first nutrient supplement 148, and methyltrophic bacteria may be treated in a second single-cell protein processing unit 149 to produce a second nutrient supplement 150.

[0041] Methane production :exist Figure 1A and 1B As shown in one aspect, a method includes a methanogenic fermentation vessel 105 as described herein in the description of methanogenic fermentation. A supplemental hydrogen stream 40 may also be provided to the methanogenic fermentation vessel 105. CO2 generated in the methyl nutrient vessel 405 can be recycled back to the methanogenic fermentation vessel via line 407.

[0042] Methylotrophic fermentation :like Figure 1A and 1BAs shown, methane 135 can be supplied to a methyltrophic fermentation vessel 405. The methyltrophic fermentation vessel 405 includes methyltrophic bacteria. Methyltrophic bacteria are diverse microorganisms that can use reduced one-carbon compounds (such as methanol or methane) as their carbon source for growth. The methyltrophic bacteria cells 450 can then be processed into single-cell proteins. Examples of methyltrophic bacteria include *Methanomonas methanogens* (…). Methylomonas methanica ), Campylobacter sporangiosum ( Trichosporium Methylosinus ), capsular methylcoccus ( Capsulatus Methylococcus ), Methylobacterium tumefaciens ( Extorquens Methylobacterium ), denitrifying paracocci ( Denitrifying Paracoccus ), alkaliphilic methyl microbes ( Alkaliphilic Methylomicrobium ), hot spring acidophilic methyl bacillus ( Fumariolicum Methylacidiphilum ), Buryat methyl microbes Buryatense Methylomicrobium ) and nitrate-reducing methanogenic bacteria ( Methanoperedens nitroreducens Genetically modified microorganisms capable of using methane may also be used. Examples of suitable growth conditions for methyltrophic bacteria are provided in U.S. Patent No. 10,934,566 and U.S. Publication No. US20210340574, both of which are incorporated herein by reference.

[0043] In methyltrophic fermentation, methane and oxygen are converted into cell clusters and CO2. The CO2-containing exhaust gas can then be supplied as feed gas to the methanogenic fermentation vessel 105. Before entering the methanogenic fermentation vessel, the oxygen in the CO2-containing exhaust gas is removed.

[0044] CO fermentation, methanogenic fermentation and methylotrophic fermentation Methane production :exist Figure 2A and 2B As shown in one aspect, one method includes, as described herein... Figure 1A and 1B The methanogenic fermentation vessel 105 described in the text.

[0045] CO fermentation Certain acetic acid-producing bacteria can ferment CO-containing gaseous substrate 50 in a CO fermentation vessel 230 into useful oxygenated hydrocarbon compounds 255, such as ethanol and butanol, and produce a fermentation broth containing acetic acid-producing bacteria 270.

[0046] In this respect, a suitable gaseous substrate 50 contains at least about 5 mol% CO, in one respect at least about 10 mol%, in another respect at least about 20 mol%, in another respect at least about 30 mol%, in another respect about 10 to about 100 mol%, in another respect about 20 to about 100 mol% CO, in another respect about 30 to about 90 mol% CO, in another respect about 40 to about 80 mol% CO, and in another respect about 50 to about 70 mol% CO. In this respect, the CO-containing gaseous substrate 50 may have about 40 mol% or less CO2, in one respect the CO-containing gaseous substrate 50 may have about 30 mol% or less CO2, in one respect the CO-containing gaseous substrate 50 may have about 20 mol% or less CO2, in another respect the CO-containing gaseous substrate 50 may have about 10 mol% or less CO2, in another respect the CO-containing gaseous substrate 50 may have about 1 mol% or less CO2, and in yet another respect the CO-containing gaseous substrate 50 may contain no or substantially no CO2.

[0047] 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 may be further modified or blended to include a suitable H2 to CO molar ratio. In one aspect, the CO-containing gaseous substrate supplied to the fermentation vessel has an H2 to CO molar ratio of about 0.1 or higher, in another aspect about 0.2 or higher, in yet another about 0.25 or higher, and in yet another about 0.5 or higher. In one aspect, a second portion of H2 from the methane cracker may be supplied to the CO fermentation vessel 230.

[0048] The concentrations of various culture medium components used in CO2 bioconversion fermentation methods are as follows:

[0049] Examples of useful acetic acid-producing bacteria for CO2 bioconversion fermentation methods include *Brutella proteus* (…). Blautia producta ), Methyltrophic Butyric Acid Bacillus ( Butyribacterium methylotrophicum ), underground thermophilic anaerobic bacteria ( Caldanaerobacter subterraneous ), Pacific underground thermophilic anaerobic bacteria ( Caldanaerobacter subterraneous pacificus ), Hydrogen-producing thermophilic carbon monoxide bacteria ( Carboxydothermus hydrogenoformans ), Clostridium acetic acid ( Clostridium aceticum Clostridium acetonebutanol ( Clostridium acetobutylicum Clostridium acetone-butanol P262 ( Clostridium acetobutylicum P262), Clostridium ethanolophilum ( Clostridium autoethanogenum (DSM 19630 from DSMZ, Germany), self-produced Clostridium ethanoliferum ( Clostridium autoethanogenum (DSM10061 from DSMZ, Germany), self-produced Clostridium ethanoliferum ( Clostridium autoethanogenum(DSM 23693 from DSMZ, Germany), self-produced Clostridium ethanoliferum ( Clostridium autoethanogenum (DSM 24138 from DSMZ, Germany), Clostridium carbon monoxide-eating bacteria ( Clostridium carboxidivorans Clostridium coccidioides Clostridium coskatii (ATCC PTA-10522), Clostridium deltae ( Clostridium drakei Clostridium yangii ( Clostridium ljungdahlii PETC (ATCC 49587), Clostridium yangii ( Clostridium ljungdahlii ERI2 (ATCC 55380), Clostridium yangii ( Clostridium ljungdahlii C-01 (ATCC 55988), Clostridium yangii ( Clostridium ljungdahlii O-52 (ATCC55889), Clostridium megaterium ( Clostridium magnum ), Clostridium pasteurellum ( Clostridium pasteurianum (DSM 525 from DSMZ in Germany), Clostridium difficile ( Clostridium ragsdalei P11 (ATCC BAA-622), Clostridium fecalis ( Clostridium scatologenes ), Clostridium thermophilum ( Clostridium thermoaceticum ), Clostridium urtenii ( Clostridium ultunense ), Kuhl's desulfurized enterobacteria ( Desulfotomaculum kuznetsovii ), Eubacterium mucilaginosa ( Eubacterium limosum ), sulfur-reducing bacteria ( Geobacter sulfurreducens ), Methanophora acetophila ( Methanosarcina acetivorans ), Pasteurella multocida ( Methanosarcina barkeri ), Prevotella ( Oxobacter pfennigii ), product digestive streptococci ( Peptostreptococcus productus Clostridium stearothermum ( Clostridium Stick-landii ) and its mixtures.

[0050] Anaerobic bacteria are bacteria that do not require oxygen for growth. If oxygen levels exceed a certain threshold, anaerobic bacteria may experience adverse reactions or even die. Acetogenic bacteria are microorganisms capable of producing acetate during anaerobic respiration or fermentation using the Wodd-Ljungdahl pathway (as their primary energy conservation mechanism). Other useful oxygenated hydrocarbons, 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 oxygenated hydrocarbons include Clostridium (…). Clostridium Those belonging to the genus *Clostridium*, such as *Clostridium yangensis* (… Clostridium ljungdahliiStrains, including those described in WO 2000 / 68407, EP117309, U.S. Patents 5,173,429, 5,593,886 and 6,368,819, WO 1998 / 00558 and WO 2002 / 08438, including *Clostridium difficile* (*Clostridium ethanolophilum*). Clostridium autoethanogenum The strains (DSM10061 and DSM 19630 of the German DSMZ), including those described in WO 2007 / 117157 and WO 2009 / 151342, as well as Clostridium ranelii ( Clostridium ragsdalei (P11, ATCC BAA-622) and Pasteurella multocida ( Alkalibaculum bacchi (CP11, ATCC BAA-1772), including those described in U.S. Patent Nos. 7,704,723 and "Biofuels and Bioproducts from Biomass-Generated Synthesis Gas," by Hasan Atiyeh (presented at the Oklahoma EPSCoR Annual State Conference, April 29, 2010), and the carbon monoxide-eating Clostridium described in U.S. Patent Application No. 2007 / 0276447. Clostridium carboxidivorans (ATCCPTA-7827). Other suitable microorganisms include Mucor ( ). Moorella Those belonging to the genus *Mucor*, including *Mucor* HUC22-1 ( Moorella sp. HUC22-1) and carbon monoxide-loving bacteria (sp. HUC22-1) and carbon monoxide-loving bacteria Carboxydothermus Those belonging to the same genus. Each of these references is incorporated herein by reference.

[0051] CO2 fermentation can be carried out desirablely under appropriate reaction conditions for the desired fermentation mode. For example, in one aspect, CO2 fermentation can be configured to focus on the production of oxygenated hydrocarbons (e.g., ethanol) from CO. In this mode, approximately 4% to 6% of the carbon from the CO fed into CO2 fermentation is converted to biomass. In another aspect, CO2 fermentation can be configured to focus on the production of microbial biomass from CO. In this mode, approximately 6% to 7.5% of the carbon from the CO fed into CO2 fermentation is converted to biomass. Reaction conditions to be considered include pressure, temperature, gas flow rate, liquid flow rate, culture medium pH, culture medium redox potential, stirring rate (if a stirred tank reactor is used), inoculum level, appropriate gaseous substrate concentration to ensure that CO in the liquid phase does not become limiting or inhibitory, and appropriate product concentration to avoid product inhibition. CO2 fermentation methods also provide CO2 conversion rates of 80% or higher in one aspect, 85% or higher in another aspect, 90% or higher in another aspect, 80% to 99% in another aspect, 85% to 98% in another aspect, and 90% to 97% in yet another aspect.

[0052] Acetic acid-producing bacteria convert CO to produce one or more alcohols and CO2-containing exhaust gases. These CO2-containing exhaust gases are then fed into a methanogenic fermentation vessel. The CO2-containing exhaust gases contain 5% or less, 3% or less in one aspect, 2% or less in another aspect, and 1% or less of CO in the third aspect. CO can be removed from the CO2-containing exhaust gases before they enter the methanogenic fermentation vessel to avoid CO inhibition.

[0053] like Figure 2A and 2B The method shown for converting CO and CO2 includes fermenting a gaseous substrate 50 containing CO in a CO fermentation vessel 230. CO-converting acetotrophic bacteria in the CO fermentation vessel 230 convert CO into one or more alcohols 255. Exhaust gas 481 from the CO fermentation vessel 230 contains CO2. Exhaust gas 481 is provided to a methanogenic fermentation vessel 105. A gaseous substrate 115 containing additional H2 and / or CO2 can also be provided to the methanogenic fermentation vessel 105. Methanogenic archaea in the methanogenic fermentation vessel convert CO2 into methane 135. Methane 135 can be provided to a methyltrophic fermentation vessel 405. The methyltrophic fermentation vessel 405 includes methyltrophic bacteria that consume methane for microbial growth. The CO fermentation broth containing CO-converting acetotrophic bacteria cells 270, the methyltrophic fermentation broth containing methyltrophic bacterial cells 450, and the methanogenic fermentation broth containing methanogenic archaea cells 140 can be further processed into a single-cell protein nutritional supplement.

[0054] Methylotrophic fermentation :like Figure 2A and 2BAs shown, and in Figure 1A and 1B The description further states that methane 135 can be supplied to a methyl-trophic fermentation vessel 405. The methyl-trophic fermentation vessel 405 includes methyl-trophic bacteria.

[0055] Integrated fermentation system As will be understood by those skilled in the art, it can be Figure 1A , 1B Any or all of the processes described in 2A and 2B are combined into the whole system.

[0056] Microbial biomass Fermentation broth from any fermentation vessel ( Figure 1A and 1B 140 and 450 Figure 2A and 2B (140, 270, and 450) can be further removed from the fermentation vessel and then processed into a protein-containing nutritional supplement in one or more single-cell protein processing units. The fermentation broth can be processed individually or together, as shown in the figure. The fermentation broth is separated into cell-free permeate and a cell-containing suspension using one or more cell separators. The cell membranes and / or cell walls of the cells in the cell-containing suspension are ruptured to produce a homogenate. The homogenate is then fractionated into a protein-containing supernatant and a protein-containing cell debris fraction using a fractionator.

[0057] Suitable cell separators include, but are not limited to, filtration devices, hollow fiber filtration devices, spiral wound filtration devices, ultrafiltration devices, ceramic filtration devices, cross-flow filtration devices, size-exclusion column filtration devices, spiral wound membranes, centrifugation devices, and combinations thereof. A method for producing single-cell proteins from biomass is described in U.S. Serial No. 16 / 416,133, filed May 17, 2019.

[0058] The cell suspension contains microbial cells at a higher concentration than that of the fermentation broth. In one aspect, the cell concentration of the cell suspension is about 20 g / L or higher, in another aspect about 30 g / L or higher, in another aspect about 40 g / L or higher, in another aspect about 50 g / L or higher, in another aspect about 60 g / L or higher, in another aspect about 20 to about 300 g / L, in another aspect about 30 to about 250 g / L, in another aspect about 40 to about 200 g / L, in another aspect about 50 to about 150 g / L, and in yet another aspect about 100 to about 150 g / L.

[0059] Cells in a cell suspension can be ruptured using one or more of the following devices: microfluidics devices, sonication devices, ultrasonic devices, mechanical rupture devices, French presses, refrigerators, heaters, heat exchangers, distillation columns, pasteurization devices, UV sterilization devices, gamma-ray sterilization devices, reactors, homogenizers, and combinations thereof. In one aspect, prior to rupturing the cell membranes in the cell suspension, the pH of the cell suspension is adjusted to approximately 6 to approximately 12, in another aspect to 7 to 12, in another aspect to 8 to 12, in another aspect to 7.5 to 11, in another aspect to 8.5 to 11, and in yet another aspect to 7 to 10.

[0060] On the other hand, the cell-containing suspension is hydrolyzed by a hydrolytic enzyme. In this aspect, the cell-containing suspension and the hydrolytic enzyme are incubated at a temperature of about 50 to about 70°C for about 3 to about 72 hours to form a hydrolysate, for 3 to 48 hours in one aspect, 4 to 24 hours in another aspect, 6 to 24 hours in another aspect, 6 to 12 hours in another aspect, and 4 to 12 hours in yet another aspect. Prior to the hydrolysis of the cell-containing suspension, the pH of the cell-containing suspension is adjusted to about 6 to about 12, for 7 to 12 in another aspect, for 8 to 12 in another aspect, for 7.5 to 11 in another aspect, for 8.5 to 11 in another aspect, and for 7 to 10 in yet another aspect. The hydrolytic enzyme is selected from subtilase, alkaline protease, serine protease, serine endopeptidase, and mixtures thereof. The hydrolysate is fractionated into a protein-containing supernatant and a protein-containing cell debris fraction using centrifugation, ultrafiltration, and combinations thereof. The protein-containing supernatant has a nucleic acid content of less than about 5%, less than 4% in one aspect, less than 3% in another aspect, and less than 2% in yet another aspect.

[0061] Protein-containing supernatants and protein-containing cell debris fractions can be used directly or further processed into protein-containing nutritional supplements. Dehydration units can be used to dry the protein-containing supernatants and produce nutritional supplements containing soluble proteins, such as protein powder. Suitable dehydration units include spray drying units, drum dryer units, freeze-drying units, lyophilization units, and combinations thereof. Other components (e.g., moisture and ash) can be further removed to purify the protein-containing supplements. Protein-containing supplements can be used directly as animal feed or blended with other ingredients to produce one or more types of nutritional supplements. In one aspect, the protein-containing supplement contains about 60 to about 99% by weight of protein, in another about 70 to about 95% by weight of protein, in another about 75 to about 95% by weight of protein, in another about 80 to 95% by weight of protein, and in another about 85 to 95% by weight of protein.

[0062] The method for producing protein-containing nutritional supplements using methylotrophic bacteria and acetotrophic bacteria differs from the method using methanogenic archaea due to the lack of cell walls in methanogenic archaea. For example... Figure 1B and 2B As shown, nutrient supplement 148 from methanogenic archaea can be produced in single-cell protein processing unit 146, while nutrient supplement 150 produced by methyltrophic bacteria and / or acetotrophic bacteria can be produced in another single-cell protein processing unit 149. In this case, the operating cost and processing time in single-cell protein processing unit 146 can be significantly lower than those in single-cell protein processing unit 149. Example

[0063] The following examples further illustrate this disclosure and should not be construed as limiting its scope.

[0064] Example 1: Methanogenic fermentation of CO2 Gases containing CO2 and H2, along with a conventional liquid culture medium containing trace amounts of metals and salts, were continuously introduced into a culture medium containing thermoautotrophic methanophilic bacteria. Methanothermobacter thermautotrophicus A stirred tank bioreactor.

[0065] The New Brunswick Bioflow 320 reactor, containing fermentation medium, was started with actively growing thermoautotrophic methanophilic bacteria (Bacillus methanogenus). Methanothermobacter thermautotrophicusAt the start of the experiment, the reactor stirring rate was set to 1200 rpm. This stirring rate was maintained throughout the experiment. The feed gas flow to the reactor was increased based on the H2 and CO2 uptake of the culture. The temperature in the bioreactor was maintained at approximately 60°C throughout the experiment. Samples of the gas fed to the bioreactor, the exhaust gas from the bioreactor, and the fermentation broth in the bioreactor were taken at intervals, for example, approximately once per day, once every 2 hours, and once every 4 hours, respectively. The consumption or production of various gas components in the above samples, as well as the optical density (cell density) of the culture, were analyzed. Throughout the experiment, the unstirred volume of the reactor was maintained between 2000 and 2200 ml. Moreover, the gas flow to the reactor was measured in real time by regulating the gas entering the reactor using a mass flow controller. The feed gas composition for this experiment was 76% H2, 20% CO2, and 4% N2.

[0066] In this experiment, a cell recirculation system (CRS) was connected to the reactor before the experiment began. During the experiment, the flow rate of nutrients (growth medium) to the reactor was 2.0–5.0 ml / min. The cell clearance rate was 3.0–5.0 ml / min, and permeate was extracted through the CRS at a rate of 0–2.0 ml / min.

[0067] The results can be summarized as follows: Specific CO2 uptake: 0.8-1.1 mmol CO2 / min / gram of cells Specific H2 uptake: 3.3-3.8 mmol H2 / min / gram of cells Mean cell retention time: 11.2 hours Average cell density: 3g / L CO2 conversion rate: 90% to 99% The specific methane production rate is 0.77 mmol / min / gram of cells. The composition of the exhaust gas in this experiment was 13.8% H2, 7.6% CO2, 62.5% CH4 and 16.1% N2.

[0068] While the disclosure of this disclosure has been described through specific implementations, examples and applications thereof, other and further variations may be designed without departing from the basic scope of this disclosure as set forth in the following claims.

Claims

1. A method for converting CO2, the method comprising: A gaseous substrate containing CO2 and H2 is fermented with methanogenic archaea in a methanogenic fermentation vessel to produce methane and a fermentation broth containing methanogenic archaea; and The methane is fermented with methyltrophic bacteria in a methyltrophic fermentation vessel to produce a fermentation broth containing methyltrophic bacteria and CO2-containing emission gas.

2. The method according to claim 1, wherein at least a portion of the CO2-containing emission gas is fed to the methanogenic fermentation vessel.

3. The method according to claim 2, wherein at least a portion of the O2 in the CO2-containing emission gas is removed before it is sent to the methanogenic fermentation vessel.

4. The method of claim 1, wherein the gaseous substrate comprising CO2 and H2 has a CO2 to H2 ratio of about 1:3 to 1:

4.

5. The method according to claim 1, wherein the methanogenic archaea is selected from *Methanobacterium alkalophilum*, *Methanobacterium brevicornum*, *Methanobacterium congo*, *Methanobacterium sludgeii*, *Methanobacterium serratum*, *Methanobacterium formate*, *Methanobacterium ilmeni*, *Methanobacterium swampense*, *Methanobacterium thermophilum*, *Methanobacterium hygroscopicum*, *Methanobacterium spp.*, *Methanobacterium olsigmatii*, *Methanobacterium rumenense*, *Methanobacterium schlegelii*, *Methanobacterium waldii*, *Methanobacterium marburgii*, *Methanobacterium thermoautotrophum*, *Methanobacterium thermocurvatum*, and *Methanobacterium thermophilum*. Methanotherapeutic Bacillus Wolff, Methanotherapeutic Bacillus socialis, Methanotherapeutic Bacillus Bavaria, Methanotherapeutic Bacillus microsporum, Methanotherapeutic Bacillus basilica, Methanotherapeutic Bacillus serrata, Methanotherapeutic Bacillus coldus, Methanotherapeutic Bacillus serrata, Methanotherapeutic Bacillus marineis, Methanotherapeutic Bacillus acetophilus, Methanotherapeutic Bacillus pastoris, Methanotherapeutic Bacillus martensii, Methanotherapeutic Bacillus thermophilus, Methanotherapeutic Bacillus molybdenum, Methanotherapeutic Bacillus janthii, Methanotherapeutic Bacillus zebufo, Methanotherapeutic Bacillus serrata, Methanotherapeutic Bacillus venerae, Methanotherapeutic Bacillus wollenii, Methanotherapeutic Bacillus thermophilus, Methanotherapeutic Bacillus cannibalus, Thermoautotrophic Methanotherapeutic Bacillus, Methanotherapeutic Bacillus cinnamon, Methanotherapeutic Bacillus indicus, Methanotherapeutic Bacillus hellenius, and Methanotherapeutic Bacillus volcanicus.

6. The method according to claim 1, wherein the methanogenic archaea is a thermoautotrophic methanophilic bacillus.

7. The method of claim 1, wherein the methanogenic fermentation vessel provides a specific CO2 uptake of about 0.5 to about 3 mmol CO2 / min / g cell.

8. The method of claim 1, wherein the methanogenic fermentation vessel provides a specific H2 uptake of about 1.5 mmol to about 12 mmol H2 / min / g cell.

9. The method of claim 1, wherein the methanogenic fermentation vessel has a cell retention time of about 5 to about 50 hours.

10. The method of claim 1, wherein the methanogenic fermentation vessel provides a methane production rate of about 0.5 to about 2.5 mmol methane / min / gram of cells.

11. The method of claim 1, wherein the methanogenic fermentation vessel provides a CO2 conversion rate of 65% or higher.

12. The method according to claim 1, wherein the methylotrophic bacteria are selected from alkaliphilic methylmicrobes, hot spring acidophilic methylbacteria, Buryat methylmicrobes, and nitrate-reducing methanogenic bacteria and combinations thereof.

13. The method according to claim 1, further comprising: The fermentation broth of methyl-containing trophic bacteria was separated into a first cell-free permeate and a first cell-containing suspension. The cell membranes of the cells in the first cell-containing suspension are ruptured to produce a first homogenate; The first homogenate is fractionated into a first protein-containing supernatant and a first protein-containing cell debris fraction. as well as The first protein-containing nutritional supplement was recycled.

14. The method of claim 13, wherein the first cell-containing suspension has a stem cell weight concentration of about 20 g / L to about 200 g / L.

15. The method of claim 13, wherein the cell membrane of the first cell-containing suspension is ruptured using one or more rupture devices selected from the group consisting of: microfluidic devices, ultrasonic devices, ultrasonic devices, mechanical rupture devices, Freund's crushers, refrigerators, heaters, heat exchangers, distillation columns, pasteurization devices, UV sterilization devices, gamma-ray sterilization devices, reactors, homogenizers, and combinations thereof.

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

17. The method of claim 13, wherein the first homogenate is a hydrolysate formed by contacting the first cell-containing suspension with a hydrolytic enzyme.

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

19. The method according to claim 17, wherein the hydrolytic enzyme is selected from subtilisin, alkaline protease, serine protease, serine endopeptidase, and mixtures thereof.

20. The method of claim 13, wherein centrifugation, ultrafiltration, and combinations thereof are used to fractionate the first homogenate into the first protein-containing supernatant and the first protein-containing cell debris fraction.

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

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

23. The method according to claim 1, further comprising: The fermentation broth containing methanogenic archaea was separated into a second cell-free permeate and a second cell-containing suspension. The cell membranes of the cells in the second cell-containing suspension are ruptured to produce a second homogenate; The second homogenate is fractionated into a second protein-containing supernatant and a second protein-containing cell debris fraction. as well as The second protein-containing nutritional supplement was recycled.

24. The method of claim 23, wherein the second cell-containing suspension has a stem cell weight concentration of about 20 g / L to about 200 g / L.

25. The method of claim 23, wherein the cell membrane rupture of the second cell-containing suspension is performed using one or more rupture devices selected from the group consisting of: microfluidic devices, ultrasonic devices, ultrasonic devices, mechanical rupture devices, Freund's crushers, refrigerators, heaters, heat exchangers, distillation columns, pasteurization devices, UV sterilization devices, gamma-ray sterilization devices, reactors, homogenizers, and combinations thereof.

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

27. The method of claim 23, wherein the second homogenate is a hydrolysate formed by contacting the second cell-containing suspension with a hydrolytic enzyme.

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

29. The method according to claim 27, wherein the hydrolytic enzyme is selected from subtilisin, alkaline protease, serine protease, serine endopeptidase, and mixtures thereof.

30. The method of claim 23, wherein centrifugation, ultrafiltration, and combinations thereof are used to fractionate the second homogenate into the second protein-containing supernatant and the second protein-containing cell debris fraction.

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

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

33. The method according to claim 1, further comprising: The fermentation broth containing methyl-trophic bacteria is mixed with the fermentation broth containing methanogenic archaea to generate a fermentation broth containing mixed cells. The fermentation broth containing mixed cells was separated into a third cell-free permeate and a third cell-containing suspension. The cell membranes of the cells in the third cell-containing suspension are ruptured to produce a third homogenate; The third homogenate is fractionated into a third protein-containing supernatant and a third protein-containing cell debris fraction. as well as The third protein-containing nutritional supplement was recycled.

34. The method of claim 33, wherein the third cell-containing suspension has a stem cell weight concentration of about 20 g / L to about 200 g / L.

35. The method of claim 33, wherein the cell membrane rupture of the third cell-containing suspension is performed using one or more rupture devices selected from: microfluidic devices, ultrasonic devices, ultrasonic devices, mechanical rupture devices, Freund's crushers, refrigerators, heaters, heat exchangers, distillation columns, pasteurization devices, UV sterilization devices, gamma-ray sterilization devices, reactors, homogenizers, and combinations thereof.

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

37. The method of claim 33, wherein the third homogenate is a hydrolysate formed by contacting the third cell-containing suspension with a hydrolytic enzyme.

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

39. The method according to claim 37, wherein the hydrolytic enzyme is selected from subtilisin, alkaline protease, serine protease, serine endopeptidase, and mixtures thereof.

40. The method of claim 33, wherein centrifugation, ultrafiltration, and combinations thereof are used to fractionate the third homogenate into the third protein-containing supernatant and the third protein-containing cell debris fraction.

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

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

43. A method for converting CO and CO2, the method comprising: A gaseous substrate containing CO2 and H2 is fermented with methanogenic archaea in a methanogenic fermentation vessel to produce methane and a fermentation broth containing methanogenic archaea; and The methane is fermented with methyltrophic bacteria in a methyltrophic fermentation vessel to produce a fermentation broth containing methyltrophic bacteria and a first CO2-containing emission gas; and A gaseous substrate containing CO is fermented in a CO fermentation vessel by CO-converting acetic acid-producing bacteria to produce alcohol, a second CO2-containing exhaust gas, and a fermentation broth containing acetic acid-producing bacteria.

44. The method of claim 43, wherein at least a portion of the first CO2-containing emission gas is fed to the methanogenic fermentation vessel.

45. The method of claim 44, wherein at least a portion of the O2 in the first CO2-containing emission gas is removed before at least a portion of the first CO2-containing emission gas is fed to the methanogenic fermentation vessel.

46. ​​The method of claim 43, wherein at least a portion of the second CO2-containing emission gas is fed to the methanogenic fermentation vessel.

47. The method of claim 46, wherein at least a portion of the CO in the second CO2-containing emission gas is removed before at least a portion of the second CO2-containing emission gas is fed to the methanogenic fermentation vessel.

48. The method of claim 43, wherein the gaseous substrate of CO2 and H2 comprises at least a portion of the first CO2-containing emission gas, at least a portion of the second CO2-containing emission gas, or a combination of at least a portion of the first CO2-containing emission gas and at least a portion of the second CO2-containing emission gas.

49. The method of claim 43, wherein the gaseous substrate comprising CO2 and H2 has a CO2 to H2 ratio of about 1:3 to 1:

4.

50. The method according to claim 43, wherein the methanogenic archaea is selected from *Methanobacterium alkalophilum*, *Methanobacterium brevicornum*, *Methanobacterium congo*, *Methanobacterium sludgeii*, *Methanobacterium mysae*, *Methanobacterium formate*, *Methanobacterium ilmeni*, *Methanobacterium swampii*, *Methanobacterium thermophilum*, *Methanobacterium hygrophilum*, *Bacillus acidophilus*, *Bacillus tree-like*, *Bacillus griseus*, *Bacillus ostreatus*, *Bacillus rumenaeus*, *Bacillus stearothermia*, *Bacillus wollenii*, *Bacillus walinii*, *Methanophilus marburgii*, *Methanophilus thermoautotrophic*, *Methanophilus thermocurvatus*, and *Methanophilus thermophilus*. The following are listed: *Methanophilus wolfii*, *Methanophilus socialis*, *Methanophora bivalves*, *Methanophora septemlobus ...

51. The method of claim 43, wherein the methanogenic archaea is a thermoautotrophic methanophilic bacillus.

52. The method of claim 43, wherein the methanogenic fermentation provides a specific CO2 uptake of about 1 to about 3 mmol CO2 / min / g cell.

53. The method of claim 43, wherein the methanogenic fermentation provides a specific H2 uptake of about 3 to about 12 mmol H2 / min / g cell.

54. The method of claim 43, wherein the methanogenic fermentation has a cell retention time of about 5 to about 50 hours.

55. The method of claim 43, wherein the methanogenic fermentation provides a methane production rate of about 1.2 to about 2.5 mmol methane / min / gram of cells.

56. The method of claim 43, wherein the methanogenic fermentation vessel provides a CO2 conversion rate of 65% or higher.

57. The method according to claim 43, wherein the CO-converting acetic acid-producing bacteria is selected from *Clostridium acetic acid*, *Clostridium acetobutane*, *Clostridium acetobutane* P262, *Clostridium ethanolophilum* (DSM 19630 from DSMZ, Germany), *Clostridium ethanolophilum* (DSM10061 from DSMZ, Germany), *Clostridium ethanolophilum* (DSM 23693 from DSMZ, Germany), *Clostridium ethanolophilum* (DSM24138 from DSMZ, Germany), *Clostridium carbon monoxide-eating*, *Clostridium coeruleus* (ATCC PTA-10522), *Clostridium deltae*, *Clostridium yolkeris* PETC (ATCC49587), *Clostridium yolkeris* ERI2 (ATCC 55380), *Clostridium yolkeris* C-01 (ATCC 55988), *Clostridium yolkeris* O-52 (ATCC55889), *Clostridium megaterium*, *Clostridium pasteurellium* (DSM 525 from DSMZ, Germany), and *Clostridium laurethrum* P11 (ATCC). Clostridium paedae, Clostridium tumefaciens, Clostridium urtenensis, Clostridium stearothermii, and mixtures thereof.

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

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

60. The method according to claim 43, wherein the methylotrophic bacteria are selected from alkaliphilic methylmicrobes, hot spring acidophilic methylbacteria, Buryat methylmicrobes, and nitrate-reducing methanogenic bacteria and combinations thereof.

61. The method of claim 43, further comprising: The fermentation broth containing methanogenic archaea was separated into a first cell-free permeate and a first cell-containing suspension. The cell membranes of the cells in the first cell-containing suspension are ruptured to produce a first homogenate; The first homogenate is fractionated into a first protein-containing supernatant and a first protein-containing cell debris fraction. as well as The first protein-containing nutritional supplement was recycled.

62. The method of claim 61, wherein the second cell-containing suspension has a stem cell weight concentration of about 20 g / L to about 200 g / L.

63. The method of claim 61, wherein the cell membrane of the first cell-containing suspension is ruptured using one or more rupture devices selected from the group consisting of: microfluidic devices, ultrasonic devices, ultrasonic devices, mechanical rupture devices, Freund's crushers, refrigerators, heaters, heat exchangers, distillation columns, pasteurization devices, UV sterilization devices, gamma-ray sterilization devices, reactors, homogenizers, and combinations thereof.

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

65. The method of claim 61, wherein the first homogenate is a hydrolysate formed by contacting the first cell-containing suspension with a hydrolytic enzyme.

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

67. The method according to claim 65, wherein the hydrolytic enzyme is selected from subtilisin, alkaline protease, serine protease, serine endopeptidase, and mixtures thereof.

68. The method of claim 61, wherein centrifugation, ultrafiltration, or a combination thereof is used to fractionate the first homogenate into the first protein-containing supernatant and the first protein-containing cell debris fraction.

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

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

71. The method of claim 43, further comprising: The fermentation broth containing methyltrophic bacteria is mixed with the fermentation broth containing acetic acid-producing bacteria to generate a first fermentation broth containing mixed cells. The first fermentation broth containing mixed cells was separated into a second cell-free permeate and a second cell-containing suspension. The cell membranes of the cells in the second cell-containing suspension are ruptured to produce a second homogenate; The second homogenate is fractionated into a second protein-containing supernatant and a second protein-containing cell debris fraction. as well as The second protein-containing nutritional supplement was recycled.

72. The method of claim 71, wherein the second cell-containing suspension has a stem cell weight concentration of about 20 g / L to about 200 g / L.

73. The method of claim 71, wherein the cell membrane rupture of the second cell-containing suspension is performed using one or more rupture devices selected from the group consisting of: microfluidic devices, ultrasonic treatment devices, ultrasonic devices, mechanical rupture devices, Freund's crushers, refrigerators, heaters, heat exchangers, distillation columns, pasteurization devices, UV sterilization devices, gamma-ray sterilization devices, reactors, homogenizers, and combinations thereof.

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

75. The method of claim 71, wherein the second homogenate is a hydrolysate formed by contacting the second cell-containing suspension with a hydrolytic enzyme.

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

77. The method according to claim 75, wherein the hydrolytic enzyme is selected from subtilisin, alkaline protease, serine protease, serine endopeptidase, and mixtures thereof.

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

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

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

81. The method of claim 43, further comprising: The fermentation broth containing methyl-trophic bacteria and the fermentation broth containing methanogenic archaea are mixed with the fermentation broth containing acetic acid-producing bacteria to generate a second fermentation broth containing mixed cells. The second fermentation broth containing mixed cells was separated into a third cell-free permeate and a third cell-containing suspension. The cell membranes of the cells in the third cell-containing suspension are ruptured to produce a third homogenate; The third homogenate is fractionated into a third protein-containing supernatant and a third protein-containing cell debris fraction. as well as The third protein-containing nutritional supplement was recycled.

82. The method of claim 81, wherein the third cell-containing suspension has a stem cell weight concentration of about 20 g / L to about 200 g / L.

83. The method of claim 81, wherein the cell membrane rupture of the third cell-containing suspension is performed using one or more rupture devices selected from the following: microfluidic device, ultrasonic treatment device, ultrasonic device, mechanical rupture device, Freund's crusher, refrigerator, heater, heat exchanger, distillation column, pasteurization device, UV sterilization device, gamma ray sterilization device, reactor, homogenizer, and combinations thereof.

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

85. The method of claim 81, wherein the third homogenate is a hydrolysate formed by contacting the third cell-containing suspension with a hydrolytic enzyme.

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

87. The method according to claim 85, wherein the hydrolytic enzyme is selected from subtilisin, alkaline protease, serine protease, serine endopeptidase, and mixtures thereof.

88. The method of claim 81, wherein centrifugation, ultrafiltration, and combinations thereof are used to fractionate the third homogenate into the third protein-containing supernatant and the third protein-containing cell debris fraction.

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

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

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