Biotechnological production of acetone

Abstract

The invention relates to a method for producing acetone from a gas composition by microbial fermentation, said method comprising:-directly contacting at least one genetically modified homoacetogen with said gas composition, said gas composition comprising at least CO, CO2, H2 and CH4; wherein the gas composition is an exhaust gas from at least one steam methane reformer, and contacting the exhaust gas from a steam methane reformer directly with the genetically modified homoacetogenic bacterium; and the homoacetogenic bacterium is genetically modified to produce acetone from the gas composition.

Description

Technical Field

[0001] This invention relates to a biotechnological method for producing ketones from exhaust gas or waste gas. Specifically, the method involves directly contacting the exhaust gas or waste gas with bacteria suitable for fermenting the gas, without any prior steps preparing the gas for the fermentation process. The exhaust gas or waste gas originates from a steam methane reformer, and the exhaust gas or waste gas from it can be directly introduced for microbial fermentation and acetone production. Background Technology

[0002] Catalytic processes can be used to convert gases, primarily composed of CO, CO2, and hydrogen (H2), into a variety of fuels and chemicals. Microorganisms can also be used to convert these gases into useful fuels and chemicals. While these biological processes are generally slower than chemical reactions, they offer several advantages over catalytic processes, including higher specificity, higher yields, lower energy costs, and greater resistance to toxicity. The use of acetogenic bacteria to produce ethanol, acetates, and / or other alcohols from various carbon sources is particularly well-known. The general application of genetically modified organisms in the production of CO-containing feedstock chemicals is disclosed at least in EP2678432B1.

[0003] US8,263,372B2 also demonstrates the use of syngas as a carbon source for gaseous fermentation. EP3050968B1 also discloses the syngas-based production of various alcohols.

[0004] Furthermore, at least WO 2015 / 085015 A1, EP2181195 B1, WO2010 / 121849 A1, and Nature Biotechnology 2022, 40, 335–344 disclose the production of acetone and other ketones from various gases using genetically modified organisms. In US8,376,736B2, Lanzatech discloses the use of blast furnace exhaust for acetone production. However, the use of blast furnace exhaust for fermentation relies on the pre-cooling and pretreatment of the exhaust to remove particles, long-chain hydrocarbons, and tar from the gas stream. This not only increases the time required to produce acetone from the exhaust but also makes the method more complex and expensive in terms of design and large-scale production.

[0005] Acetone is an industrial solvent and precursor for at least methyl methacrylate (MMA) and polymethyl methacrylate (PMMA), as well as isobutylene, which have multiple functions in industry. Acetone is also a precursor used in the production of jet fuel (Anbarasan, Nature, 491: 235-239, 2012).

[0006] However, currently available methods for producing acetone from exhaust gas are inefficient and require at least one additional step of treating the exhaust gas before contacting the gas with bacteria for fermentation. Low productivity and low concentrations of the final product also lead to higher energy costs for product purification.

[0007] Therefore, there is a desire to find other sustainable raw materials as starting materials for the production of acetone via biotechnology, which would offer the same or higher yields with less environmental damage. In particular, there is a need for a simple and efficient biotechnology for the production of acetone from sustainable raw materials. Summary of the Invention

[0008] This invention seeks to address the aforementioned problems by providing a method for the industrial-scale production of feedstock chemicals, particularly acetone, based on exhaust gas fermentation. Acetone can be used in downstream production processes. Specifically, the method involves contacting exhaust gas from a steam reformer with at least one bacterial cell capable of converting the exhaust gas into acetone. Novel combinations of steam reformers and coupled gas fermenters according to any aspect of the invention enable the use of steam reformer gases, particularly their exhaust streams, to produce valuable chemicals, including alcohols, acids, aldehydes, and ketones, with acetone as the target major product and acetate and ethanol typically observed as byproducts.

[0009] One advantage of the method according to any aspect of the invention is the use of novel exhaust gas sources for the production of valuable fermentation-based feedstocks. Furthermore, the improved gas mixture composition of exhaust gas from steam reformers, particularly steam methane reformers, allows the exhaust gas to be used directly in fermentation processes, especially anaerobic fermentation processes, without the need for pretreatment or washing.

[0010] Compared to many other exhaust streams, such as at least blast furnace exhaust streams, steam reformer exhaust streams according to any aspect of the invention contain no significant oxygen concentration. This may be one reason why steam reformer exhaust streams can be directly used as feed streams for anaerobic gas fermentation processes. Another advantage of the method according to any aspect of the invention is that pre-cooling of the gas stream is not required before contact with the bacteria used for fermentation, since the gas temperature at the steam reformer outlet where the steam reformer exhaust is released is approximately 40°C. Therefore, using steam reformer exhaust as a feed stream for fermentation makes the biotechnological production of acetone less complex, cheaper, and faster, as it eliminates the need for several steps of preparing the exhaust as a feed stream.

[0011] According to one aspect of the present invention, a method for producing acetone from a gaseous composition via microbial fermentation is provided, the method comprising: - Directly contact at least one genetically modified homoacetic bacteria with the gas composition, the gas composition comprising at least CO, CO2, H2 and CH4; The gaseous composition is exhaust gas from at least one steam methane reformer, and the exhaust gas from the steam methane reformer is directly contacted with the genetically modified homoacetic bacteria; and the homoacetic bacteria are genetically modified to produce acetone from the gaseous composition. The term "gaseous composition" as used herein refers to any mixture of gases. The gaseous composition is a gaseous substrate that serves as the primary carbon source for microbial fermentation according to any aspect of the invention. Specifically, the gaseous composition is syngas (i.e., containing CO and H2). More specifically, the gaseous composition according to any aspect of the invention contains at least CO, CO2, H2, and CH4. In some instances, N2, O2, and H2S are also present in the gaseous composition. Specifically, N2, O2, and H2S are present in low concentrations compared to the primary components of the gaseous composition, i.e., CO, CO2, H2, and CH4.

[0012] The CO2 in the gas composition according to any aspect of the invention can be 30-80% by volume, particularly 35-80%, 40-80%, 45-80%, 50-80%, 55-80%, 60-80%, 65-80%, 70-80%, 30-75%, 35-75%, 40-75%, 45-75%, 50-75%, 55-75%, 60-75%, 65-75%, 70-75%, 30-70%, 35-70%, 40-70%, 45-70%, 50-70%. The range is 55-70, 60-70, 65-70, 30-65, 35-65, 40-65, 45-65, 50-65, 55-65, 30-60, 35-60, 40-60, 45-60, 50-60, 30-55, 30-55, 40-55, 45-55, 30-50, 35-50, 40-50, 45-50, 30-45, 35-45, 40-45, 30-40, or 35-40% by volume. More specifically, CO2 can be approximately 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80% by volume. Even more specifically, CO2 can be in the range of 35-65% by volume.

[0013] The H2 in the gas composition according to any aspect of the invention can be in the range of 10-50% by volume, particularly 15-50, 20-50, 25-50, 30-50, 35-50, 40-50, 10-45, 15-45, 20-45, 25-45, 30-45, 35-45, 40-45, 10-40, 15-40, 20-40, 25-40, 30-40, 35-40, 10-35, 15-35, 20-35, 25-35, 30-35, 10-30, 15-30, 20-30, 25-30, 10-25, 15-25, 20-25, 10-20 or 15-20% by volume. More specifically, H2 can be approximately 15, 20, 25, 30, 35, 40, 45, or 50% by volume. Even more specifically, H2 can be in the range of 20-40% by volume.

[0014] In any aspect of the invention, the CO in the gaseous composition may be in the range of 1-30% by volume, particularly 5-30, 10-30, 15-30, 20-30, 25-30, 1-25, 5-25, 10-25, 15-25, 20-25, 1-20, 5-20, 10-20, 15-20, 1-15, 5-15, 10-15, 1-10, or 5-10% by volume. More particularly, CO may be approximately 5, 10, 15, 20, 25, or 30% by volume. Even more particularly, H2 may be in the range of 5-20% by volume.

[0015] The CH4 in the gas composition according to any aspect of the invention is in the range of 0.01-30% by volume, particularly 0.01-25, 0.01-20, 0.01-15, 0.01-10, 0.01-5, 0.01-1, 0.01-0.5, 0.01-0.1, 0.01-0.05, 0.05-30, 0.05-25, 0.05-20, 0.05-15, 0.05-10, 0.05-5, 0.05-1, 0.05-0.5, 0.05-0.1, 0.1-30, 0 The range of 0.1-25, 0.1-20, 0.1-15, 0.1-10, 0.1-5, 0.1-1, 0.1-0.5, 0.5-30, 0.5-25, 0.5-20, 0.5-15, 0.5-10, 0.5-5, 0.5-1, 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 5-30, 5-25, 5-20, 5-15, 5-10, 10-30, 10-25, 10-20, 10-15, 20-30 or 20-25 volume %. More specifically, CH4 can be approximately 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 5, 10, 15, 20, 25, or 30% by volume. Even more specifically, CH4 can be in the range of 0.01-20% by volume.

[0016] More specifically, the O2 concentration in the gas composition may be less than 1% by volume of the total gas content in the gas composition. Specifically, oxygen may be present in concentrations ranging from 0.000005% to 2% by volume, specifically in the range of 0.00005% to 2% by volume, 0.0005% to 2% by volume, 0.05% to 2% by volume, 0.00005% to 1.5% by volume, 0.005% to 1.5% by volume, 0.05% to 1.5% by volume, 0.5% to 1.5% by volume, 0.00005% to 1% by volume, 0.005% to 1% by volume, 0.05% to 1% by volume, 0.5% to 1% by volume, 0.55% to 1% by volume, and 0.60% to 1% by volume, particularly in the ranges of 0.60% to 1.5%, 0.65% to 1%, and 0.70% to 1% by volume. In particular, acetic acid-producing microorganisms are especially suitable when the O2 ratio in the gas / gas composition is approximately 0.00005, 0.0005, 0.005, 0.05, 0.15, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, or 2% by volume relative to the gas volume in the gas composition. Those skilled in the art can measure the volume concentration of oxygen in the gas composition using any method known in the art. Specifically, the volume of oxygen can be measured using any method known in the art. In one example, the gas phase concentration of oxygen can be measured using a trace oxygen impregnation probe from PreSens Precision Sensing GmbH. Oxygen concentration can be measured by fluorescence quenching, where the degree of quenching is related to the partial pressure of oxygen in the gas phase.

[0017] The H2S concentration in the gas composition according to any aspect of the invention may be 0.00000001 volume%–0.0001 volume.

[0018] The N2 concentration in the gas composition according to any aspect of the invention may be 0.1–0.7 by volume.

[0019] As used herein, the term “approximately” means a variation within 20%. In particular, as used herein, “approximately” means + / - 20% of a given measurement or value, more specifically, + / - 10%, and even more specifically, + / - 5%.

[0020] Unless otherwise stated, all percentages (%) are volume percentages.

[0021] The gas composition according to any aspect of the invention can be exhaust gas or waste gas from any industrial process. In particular, the exhaust gas or waste gas according to any aspect of the invention can originate from at least one steam methane reformer. Exhaust gas from a steam methane reformer refers to a byproduct of the steam methane reforming process, i.e., unwanted gas produced as a result of the steam methane reforming process. Specifically, the exhaust gas from a steam methane reformer is a carbon source for an acetone production method according to any aspect of the invention.

[0022] Steam methane reforming (SMR) is a process in which methane from natural gas is heated with steam in the presence of a catalyst to produce exhaust gas or waste gas primarily containing CO, CO2, H2, and CH4. This exhaust gas or waste gas can then be used in a method according to any aspect of the invention to produce acetone. At least two reactions take place in the steam methane reformer. These are: (1) Steam-methane reforming reaction CH4 + H2O (+heat) → CO + 3H2 (2) Water-gas shift reaction CO + H2O → CO2 + H2 (+ a small amount of heat) The concentration of gases (i.e. gaseous composition) in the exhaust or waste gas from a steam methane reformer is suitable for direct use in microbial fermentation to produce acetone.

[0023] Therefore, exhaust gas or waste gas from a steam methane reformer can be directly contacted with at least one genetically modified homoacetic bacteria to produce acetone. Specifically, the bacteria are directly or immediately contacted with the exhaust gas or waste gas from the steam methane reformer without any additional steps, particularly without any purification steps to prepare the gas for fermentation. Therefore, exhaust gas or waste gas from a steam methane reformer is suitable for direct use in fermenters for acetone production. More specifically, direct contact between the exhaust gas or waste gas from the steam methane reformer and the bacteria is achieved without the need for pretreatment or washing.

[0024] As used herein, the terms “homogeneous acetogens” and “acetogens” are interchangeable and refer to microorganisms capable of executing the Wood-Ljungdahl pathway and thus converting CO, CO2, and / or hydrogen into acetate. These microorganisms include those that do not possess the Wood-Ljungdahl pathway in their wild-type form but have acquired this trait through genetic modification. Such microorganisms include, but are not limited to, *Escherichia coli* cells. These microorganisms may also be referred to as carbon monoxide-producing bacteria. Currently, 21 different genera of acetogens are known in the art (Drake et al., 2006), which may also include some *Clostridium* species (Drake & Kusel, 2005). These bacteria are able to use carbon dioxide or carbon monoxide as a carbon source and hydrogen as an energy source (Wood, 1991). In addition, alcohols, aldehydes, carboxylic acids, and many hexoses can also be used as carbon sources (Drake et al., 2004). The reducing pathway leading to acetate formation is called the acetyl-CoA or Wood-Ljungdahl pathway.

[0025] In particular, the acetogenic bacteria used according to any aspect of the invention are genetically modified bacteria that are genetically modified to produce acetone from a carbon source, particularly exhaust gas or waste gas from a steam methane reformer. The genetically modified cells are acetogenic cells that are genetically modified to increase the expression of enzymes that enable the cells to produce acetone from a carbon source, particularly exhaust gas or waste gas from a steam methane reformer, compared to wild-type cells.

[0026] As used herein, the phrase “increased heterologous expression of the enzyme” should be understood as increased intracellular activity. Essentially, increased enzyme activity can be achieved by increasing the copy number of the gene sequence encoding the enzyme, using a strong promoter, or employing a gene or allele encoding the corresponding enzyme with increased activity, and optionally by combining these measures.

[0027] Genetically modified cells used in the method according to the invention are produced, for example, by transformation, transduction, conjugation, or a combination of these methods, using a vector containing the desired gene, an allele of that gene, or a portion thereof, and a vector enabling the expression of that gene. Heterologous expression is achieved, in particular, by integrating the gene or allele into the cell chromosome or an extrachromosomal replication vector. Specifically, the increase in enzyme activity relative to wild-type cells can be 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% higher than that of wild-type cells.

[0028] Those skilled in the art can genetically modify cells using any method known in the art. Whether the nucleic acid molecule, polypeptide, and more specifically the enzyme used according to any aspect of the invention is recombinant does not necessarily affect its expression level. However, in one instance, one or more recombinant nucleic acid molecules, polypeptides, or enzymes used according to any aspect of the invention may be overexpressed. As used herein, the term "overexpression" means that the corresponding polypeptide encoded or expressed is expressed at a higher level or with higher activity than it is normally found in cells, for example, in their respective wild-type cells, under the same conditions without genetic modifications to increase expression. Those skilled in the art are familiar with many ways to induce overexpression. For example, the nucleic acid molecule to be overexpressed or the nucleic acid molecule encoding the polypeptide or enzyme to be overexpressed can be placed under the control of a strongly inducible promoter such as a lac promoter. Standard plasmids that can be used for this purpose are described in the prior art, such as vectors of the pET system, for example, pET-3a (available from Novagen). Whether a nucleic acid or polypeptide is overexpressed can be determined by quantitative PCR (in the case of nucleic acid molecules), SDS-PAGE, Western blotting, or comparative activity assays (in the case of polypeptides). Genetic modification can involve transcriptional, translational, and / or post-translational modifications that result in changes in enzyme activity and / or selectivity under selected and / or defined culture conditions. Therefore, in various embodiments of the invention, microorganisms may contain one or more gene deletions for more efficient operation. Gene deletions can be achieved by a mutant gene deletion method, and / or by starting with mutant strains that reduce or eliminate the expression of one or more of these enzymes, and / or by other methods known to those skilled in the art.

[0029] DE-A-100 31 999 provides a general survey of the possibility of increasing enzyme activity in cells, taking pyruvate carboxylase as an example. It is incorporated herein by reference, and its disclosure regarding the possibility of increasing enzyme activity in cells forms part of the disclosure of this invention.

[0030] The expression of the enzymes or genes mentioned above and subsequently all mentioned below can be detected by means of 1D and 2D protein gel separation and subsequent optical identification of the protein concentration in the gel using appropriate analytical software. If the increase in enzyme activity is entirely based on the increase in the expression of the corresponding gene, the quantification of the increase in enzyme activity can be determined in a simple manner by comparing 1D or 2D protein separations between wild-type cells and genetically modified cells. In the case of Corynebacterium, the routine method used for preparing protein gels and identifying proteins is the procedure described by Hermann et al. (Electrophoresis, 22: 1712.23 (2001)). Protein concentration can also be analyzed by Western blot hybridization using specific antibodies against the protein being tested (Sambrook et al., Molecular Cloning: a laboratory manual, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY USA, 1989) and subsequent optical analysis using appropriate concentration assay software (Lohaus and Meyer (1989) Biospektrum, 5: 32-39; Lottspeich (1999) Angewandte Chemie 111: 2630-2647). The activity of DNA-binding proteins can be measured by DNA band shift analysis (also known as gel retardation) (Wilson et al. (2001) Journal of Bacteriology, 183:2151-2155). The effects of DNA-binding proteins on the expression of other genes can be detected using various well-described reporter gene assays (Sambrook et al., Molecular Cloning: a laboratory manual, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY USA, 1989). Intracellular enzyme activity can be measured using various described methods (Donahue et al. (2000) Journal of Bacteriology 182 (19): 5624-5627; Ray et al. (2000) Journal of Bacteriology 182 (8): 2277-2284; Freedberg et al. (1973) Journal of Bacteriology 115 (3): 816-823).If the actual method for determining the activity of an enzyme is not specified in the following implementation scheme, the determination of an increase in enzyme activity and the determination of a decrease in enzyme activity are preferably performed by means of the methods described in Hermann et al., Electrophoresis, 22: 1712-23 (2001), Lohaus et al., Biospektrum 5 32-39 (1998), Lottspeich, Angewandte Chemie 111:2630-2647 (1999), and Wilson et al., Journal of Bacteriology 183: 2151-2155 (2001).

[0031] If the increase in enzyme activity is achieved through mutations in an endogenous gene, such mutations can be generated randomly by conventional methods (e.g., by UV irradiation or by mutagenic chemicals) or selectively by genetic engineering methods (e.g., deletions, insertions, and / or nucleotide exchanges). Modified cells are obtained through these mutations. Particularly preferred enzyme mutants are those that are no longer repressible for feedback, product, or substrate, or at least to a reduced extent compared to the wild-type enzyme.

[0032] If increased enzyme activity is achieved by increasing enzyme synthesis, this can be done by increasing the copy number of the corresponding gene, or by mutating the promoter and regulatory regions or ribosome binding sites upstream of the structural gene. Expression cassettes incorporated upstream of the structural gene function in the same way. Alternatively, expression can be increased at any desired time point using inducible promoters. However, "enhancers" can also be assigned as regulatory sequences to enzyme genes, which similarly increases gene expression by improving the interaction between RNA polymerase and DNA. Expression is also improved as a result of mRNA lifetime extension measures. Furthermore, enzyme activity is also increased by preventing enzyme protein degradation. The gene or gene construct can be integrated and amplified in plasmids with different copy numbers or in chromosomes. Alternatively, overexpression of the relevant gene can be achieved by altering the culture medium composition and culture management. Those skilled in the art, particularly those familiar with the publications, have noted the work of Martin et al. (Bio / Technology 5, 137-146 (1987)), Guerrero et al. (Genes 138, 35-41 (1994)), Tsuchiya and Morinaga (Bio / Technology 6, 428-430 (1988)), Eikmanns et al. (Genes 102, 93-98 (1991)), EP-A-0472 869, US 4,601,893, Schwarzer and Pühler (Bio / Technology 9, 84-87 (1991)), Reinscheid et al. (Applied and Environmental Microbiology 60, 126-132 (1994)), and LaBarre et al. (Journal of Bacteriology 175, 1001-1007). Descriptions of this can be found in (1993)), WO-A96 / 15246, Malumbres et al. (Genes 134, 15-24 (1993)), JP-A-10-229891, Jensen and Hammer (Biotechnology and Bioengineering 58, 191-195 (1998)), and known textbooks on genetics and molecular biology. Similar to mutations, the above measures also result in genetically modified cells.

[0033] Plasmids are used, for example, to increase the expression of their respective genes. Suitable plasmids or vectors are, in principle, all embodiments available to those skilled in the art for this purpose. Such plasmids and vectors can be obtained, for example, from brochures of Novagen, Promega, New England Biolabs, Clontech, or Gibco BRL. Further optimized plasmids and vectors can be found in: Glover, DM (1985) DNA cloning: a practical approach, Vol. I-III, IRL Press Ltd., Oxford; Rodriguez, RL and Denhardt, D. T (eds) (1988) Vectors: a survey of molecular cloning vectors and their uses, 179-204, Butterworth, Stoneham; Goeddel, DV (1990) Systems for heterologous gene expression, Methods Enzymol. 185, 3-7; Sambrook, J.; Fritsch, EF and Maniatis, T. (1989), Molecular cloning: a laboratory manual, 2nd edition, Cold Spring Harbor Laboratory Press, New York.

[0034] The plasmid vector containing the gene to be amplified is then transformed into the desired strain via conjugation or transformation. Conjugation methods are described, for example, in Schäfer et al., Applied and Environmental Microbiology 60: 756-759 (1994). Transformation methods are described, for example, in Thierbach et al., Applied Microbiology and Biotechnology 29: 356-362 (1988), Dunican and Shivnan, Bio / Technology 7: 1067-1070 (1989), and Tauch et al., FEMS Microbiology Letters 123: 343-347 (1994). After homologous recombination via a "cross-over" event, the resulting strain contains at least two copies of the relevant gene. In particular, to increase the activity of at least one enzyme E1, E2, and / or E3: a) At least one promoter operatively linked to a gene encoding any one of enzymes E1, E2, and / or E3 on a suitable chromosome of the cell, or b) At least one expression vector to increase the copy number of the gene encoding any one of enzymes E1, E2, and / or E3 in the cell, or c) A combination of (a) and (b).

[0035] According to any aspect of the invention, cells can be genetically modified to produce at least twice, especially at least 10, 100, 1000, or 10,000 times more ketones than wild-type cells within a defined time interval, particularly within 2 hours, especially within 8 hours or 24 hours. The increase in product formation can be determined, for example, by separately culturing cells and wild-type cells according to any aspect of the invention in a suitable nutrient medium for a specified time interval under the same conditions (same cell density, same nutrient medium, same culture conditions), and then determining the amount of the target product (lipids having general formula II or I) in the nutrient medium.

[0036] In the same context, the phrase "enzyme E" is used with respect to any aspect of the invention. x "Reduced activity and / or expression" can be understood as a reduction in activity of at least 0.5-fold, particularly at least 0.1-fold, more particularly at least 0.01-fold, even more particularly at least 0.001-fold, and most particularly at least 0.0001-fold. The phrase "reduced activity" also includes undetectable activity ("zero activity"). A reduction in the activity of an enzyme can be achieved, for example, through selective mutation or other measures known to those skilled in the art for reducing the activity of an enzyme. In particular, those skilled in the art can find descriptions, for example, at least in Dubeau et al. 2009, Singh & Röhm. 2008, Lee et al., 2009, etc., of modifying and reducing protein expression by interrupting specific genes, thereby reducing enzyme activity.

[0037] According to any aspect of the invention, the reduction of enzyme activity in cells can be achieved by modifying a gene containing one of the nucleic acid sequences, wherein said modification is selected from: insertion of exogenous DNA into the gene, deletion of at least part of the gene, point mutation in the gene sequence, RNA interference (siRNA), antisense RNA, or modified (insertion, deletion, or point mutation) regulatory sequences, such as promoters and terminators or ribosome binding sites located flanking the gene. In particular, to reduce enzyme activity in cells, the cell may contain... a) Foreign DNA in the gene encoding the enzyme; b) Deletion of at least a portion of the gene encoding the enzyme; c) At least one point mutation, RNA interference (siRNA), or antisense RNA in the gene encoding the enzyme and / or the regulatory sequence of the gene encoding the enzyme; Or a combination of (d), (e), and (f).

[0038] The expression of the enzymes and genes mentioned above and all those mentioned below can be determined by separating the proteins in the gels using 1D and 2D protein gels and then optically identifying the protein concentration in the gels using appropriate evaluation software.

[0039] Homoacetic acid-producing bacteria according to any aspect of the present invention can be genetically modified to: Compared to its wild type, the expression of at least one of the following enzymes is increased: - Thiolase (ThlA, E1) (EC2.3.1.9). - Coenzyme A transferase (CtfAB, E2) (EC 2.8.3.8) and / or - Acetoacetate decarboxylase (Adc, E3) (EC 4.1.1.4); and / or Compared to its wild type, the expression of at least the following enzymes is reduced: - Secondary alcohol dehydrogenase (sAdh, E4) (EC 1.1.1.1).

[0040] In one instance, cells can be genetically modified to increase the expression of enzymes E1, E2, and E3 relative to their wild-type cells, and genetically modified to decrease the expression of enzyme E4 relative to their wild-type cells.

[0041] Specifically, E1 may be able to catalyze the conversion of acetyl-CoA to acetoacetyl-CoA. E1 can be acetoacetyl-CoA thiolase, also known as acetyl-CoA acetyltransferase. Acetyl-CoA thiolase includes the gene product of atoB from *E. coli* (Martin et al., 2003), accession number NP_416728, derived from... Clostridium acetone-butanol (C.) acetobutylicum) The thiolytic enzyme. More particularly, E1 may comprise an amino acid sequence having 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% sequence identity with SEQ ID NO: 2. Even more particularly, cells according to any aspect of the invention may be genetically modified to comprise the sequence of SEQ ID NO: 1.

[0042] Technicians may be able to identify other thiolases that can function as E1. In particular, technicians may be able to use many known methods to assess whether functionally equivalent variants have substantially the same function as the nucleic acid or polypeptide to which they are variants. In one instance, the methods outlined in Wiesenborn et al., 1988, Wiesenborn et al., 1989, Peterson and Bennet, 1990, Ismail et al., 1993, and de la Plaza et al., 2004 can be used to assess the enzymatic activity of E1.

[0043] E2 can be an acetoacetate-CoA transferase (EC 2.8.3.9). Acetoacetate-CoA transferases conserve energy stored in the coenzyme A-ester bond. These enzymes naturally exhibit the desired acetoacetate-CoA transferase activity, or they can be engineered through directed evolution to accept acetoacetate-CoA as a substrate with increased efficiency. In particular, such enzymes may also be able to catalyze the conversion of 3-hydroxybutyryl-CoA to 3-hydroxybutyrate via a transferase mechanism. Examples of E2 can include coenzyme A transferases from *E. coli* with accession numbers P76459.1 or P76458.1 (Hanai et al., 2007) and those from *E. coli* with accession numbers NP_149326.1 or NP_149327.1. Clostridium acetobutylicum The CTF-AB (Jojima et al., 2008), accession number AAP42564.1 or AAP42565.1, are from Clostridium saccharoperbutylacetonicum CTFAB (Kosaka et al., 2007), etc. In particular, E2 can also be selected from accessions P38946.1, P38942.2, and EDK35586.1. Clostridium kluyveri of catl , cat2 and cat3 The gene product (Seedorf et al., 2008; Sohling and Gottschalk, 1996), accession number XP_001330176 Trichomonas vaginalis The transferase product (van Grinsven et al., 2008), accession number XP_828352 Trypanosoma brucei The transferase product (Riviere et al., 2004). Fusobacterium nucleatum The transferase product (Barker et al., 1982). Clostridium SB4 The transferase product (Barker et al., 1978). Clostridium acetonebutanolThe transferase products (Wiesenborn et al., 1989), accession numbers NP_603179.1 and NP_603180.1, respectively, are FN0272 and FN0273 (Kapatral et al., 2002). Fusobacterium nucleatum Homologous to these, such as FN1857 and FN1856 with accession numbers NP_602657.1 and NP_602656.1 (Kreimeyer et al., 2007), and those with accession numbers NP_905281.1 or NP_905290.1. Porphyrmonas gingivalis And login numbers NP_622378.1 or NP_622379.1 Thermoanaerobacter tengcongensis The transferase product (Kreimeyer et al., 2007). More specifically, E2 may comprise an amino acid sequence having 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% sequence identity with SEQ ID NO: 4 or 6. More specifically, E2 may comprise amino acid sequences SEQ ID NO: 4 and 6. E2 may comprise a nucleotide sequence having 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% sequence identity with SEQ ID NO: 3 or 5. More specifically, E2 may comprise a nucleotide sequence having 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% sequence identity with SEQ ID NO: 3 and 5.

[0044] E2 expression can be measured using many methods known in the art. In particular, an increase in E2 expression can be measured by determining the amount of the final product obtained in the presence of the enzyme and comparing the result with the amount of the final product obtained in the absence of the enzyme E2. In another example, E2 expression can be determined by determining the amount of E2 protein expressed in the resulting culture medium. In one example, E2 expression can be measured using the method disclosed in Charrier C., 2006.

[0045] E3 can be acetoacetate decarboxylase (Adc; EC 4.1.1.4). The acetoacetate decarboxylase used according to any aspect of the invention is selected from NP_149328.1, YP_001310906.1, and CAQ57986.1, and a protein having at least 50%, preferably 65%, particularly preferably 80%, and especially greater than 90% of a polypeptide sequence having at least 60%, preferably 65%, particularly preferably 80%, and especially greater than 90% of amino acid residues modified relative to the aforementioned reference sequence by deletion, insertion, substitution, or a combination thereof, and still possessing the activity of a protein containing the corresponding aforementioned reference sequence. 100% activity of the reference protein is understood to mean an increase in the activity of the cell used as a biocatalyst compared to the activity of a biocatalyst without the reference protein, i.e., the amount of substance converted per unit time based on the amount of cells used (units [U / g CDW] per gram of dry cell weight), wherein this activity and the activity related to the determination of enzyme E3 activity are considered. More specifically, E3 may contain an amino acid sequence having 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% sequence identity with SEQ ID NO: 7. More specifically, E2 may contain the amino acid sequence SEQ ID NO: 7.

[0046] A method for determining activity is described in Daniel et al., Appl. Environ. Microbiol. 1990, pp. 3491-3498, Vol. 56, No. 11.

[0047] E4 can be an alcohol dehydrogenase. "Alcohol dehydrogenase" can include alcohol dehydrogenases capable of catalyzing the conversion of ketones (such as acetone) to secondary alcohols (such as isopropanol), or vice versa. Such alcohol dehydrogenases include secondary alcohol dehydrogenases and primary alcohol dehydrogenases. A "secondary alcohol dehydrogenase" is a dehydrogenase that can convert ketones (such as acetone) to secondary alcohols (such as isopropanol), or vice versa. A "primary alcohol dehydrogenase" is a dehydrogenase that can convert aldehydes to primary alcohols, or vice versa; however, many primary alcohol dehydrogenases can also catalyze the conversion of ketones to secondary alcohols, or vice versa. These alcohol dehydrogenases can also be called "primary-secondary alcohol dehydrogenases." There are membrane-bound flavin-dependent alcohol dehydrogenases of the type *Pseudomonas putida* GPO1 AlkJ, which use flavor cofactors instead of NAD+. Another group includes iron-containing oxygen-sensitive alcohol dehydrogenases present in bacteria and in an inactive form in yeast. Another group includes NAD+-dependent alcohol dehydrogenases, including zinc-containing alcohol dehydrogenases, wherein the active site has a cysteine ​​residue coordinated with a zinc atom that fixes the alcohol substrate. In one instance, under the term "alcohol dehydrogenase" as used herein, it is understood to refer to an enzyme that oxidizes an aldehyde or ketone to the corresponding primary or secondary alcohol. Specifically, the alcohol dehydrogenase according to any aspect of the invention can be an NAD+-dependent alcohol dehydrogenase, i.e., an alcohol dehydrogenase that uses NAD+ as a cofactor for alcohol oxidation or uses NADH to reduce the corresponding aldehyde or ketone. In the most preferred embodiment, the alcohol dehydrogenase is an NAD+-dependent zinc-containing alcohol dehydrogenase. Examples of suitable NAD+-dependent alcohol dehydrogenases may include those from… Rhodococcus Alcohol dehydrogenase A (database code AJ491307.1) or its variants. Other examples include those from horse liver. Ralstonia eutropha (ACB78191.1) Lactobacillus brevis ( Lactobacillus brevis (YP_795183.1) Lactobacillus kefir ( Lactobacillus kefiri (ACF95832.1) Paracoccus pantrophic ( Paracoccus pantotrophus (ACB78182.1) and Yano Sphingosine ( Sphingobium yanoikuyae (EU427523.1) alcohol dehydrogenases and their respective variants. In one instance, as used herein, the expression "NAD(P)+ dependent alcohol dehydrogenase" refers to an alcohol dehydrogenase that is dependent on NAD+ and / or NADP+.

[0048] In one instance, E4 can be a secondary alcohol dehydrogenase or selected from other alcohol dehydrogenases or equivalent aldehyde reductases, and can also serve as a candidate for 3-hydroxybutyral reductase. T. E4 can be selected from [accession number AAR91477.1]. thermoglucosidase Bacillus subtilis ( Geobacillus thermoglucosidasius ADH1 (Jeon et al., 2008), from Clostridium beijerinckii The product of the SADH gene, the alcohol dehydrogenase disclosed in Tani et al., 2000 with accession number BAB122273.1, can be used as E4. E4 can also be selected from ADH2 from *Saccharomyces cerevisiae* with accession number NP_014032.1 (Atsumi et al., 2008), yqhD from *Escherichia coli* with accession number NP_417484.1 (Sulzenbacher et al., 2004 and Perez et al., 2008), and from [other organisms] with accession numbers NP_349892.1 and NP_349891.1 respectively. Clostridium acetobutylicum of bdh I and bdh II (Walter et al., 1992) and the oracle YP_162971.1 from Zymomonas mobilis ADH1 (Kinoshita et al., 1985).

[0049] More specifically, E4 can be a secondary alcohol dehydrogenase. Even more specifically, E4 can be derived from... Clostridium beyerridis (C.) beijerinckii) The secondary alcohol dehydrogenase. E4 may contain an amino acid sequence having 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% sequence identity with SEQ ID NO: 8. E4 may contain a nucleotide sequence having 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% sequence identity with SEQ ID NO: 7.

[0050] To express significantly reduced ADC levels, cells according to any aspect of the invention can be genetically modified to eliminate ADC expression, which can be achieved using standard recombinant DNA techniques known to those skilled in the art. The gene sequences responsible for producing ADC can be inactivated or partially or completely eliminated. Therefore, cells according to any aspect of the invention express reduced or undetectable levels of ADC or express functionally inactive ADC.

[0051] In one example, cells according to any aspect of the invention can naturally express E4 and can be genetically modified to reduce E4 expression in the cells to approximately 0% or undetectable levels relative to wild-type cells. In another example, cells according to any aspect of the invention have approximately 0% or undetectable levels of E4 expression in their wild-type form. In particular, cells according to any aspect of the invention have undetectable expression of the enzyme E4.

[0052] Any login number used in this application refers to the corresponding sequence from the Genbank database operated by NCBI, and the version mentioned is the one available online in March 2015.

[0053] E4 expression can be measured using any method known in the art. Specifically, an increase in E4 expression can be measured by determining the amount of the final product obtained in the presence of the enzyme and comparing the result with the amount of the final product obtained in the absence of enzyme E4. In another example, E4 expression can be determined by measuring the amount of E4 protein expressed in the resulting culture medium. In one example, E4 expression can be measured using the method disclosed in Ismaiel, AA (1993).

[0054] In particular, the acetic acid-producing bacteria genetically modified according to any aspect of the present invention are selected from... Moist anaerobic acetic acid bacteria (Acetoanaerobium notera) (ATCC 35199), Acetonema longum (DSM 6540), Acetonema longum (Acetonema longum) ... Acetobacterium carbinolicum (DSM 2925), Acetobacter malate (Acetobacterium malicum) (DSM 4132), Acetobacter species no. 446 (Morinaga et al., 1990, J.). Biotechnol., Vol. 14, pp. 187-194), Acetobacterium wieringae (DSM) 1911), Acetobacterium woodii (DSM 1030), Alkalibaculum bacchi (DSM 22112), Archaeococcus fulgidus (DSM 4304), Blautia producta (DSM 4304) 2950, ​​formerly known as Ruminococcus productus, formerly known as Peptostreptococcus *Bacillus productus*, *Butyribacterium methylotrophicum* (DSM 3468), and *Clostridium acetate* Clostridium aceticum (DSM 1496) and Clostridium autoethanogenum. (DSM 10061), Clostridium carboxidivorans (DSM 15243), Clostridium drakei (ATCC BAA-623), Clostridium formicoaceticum (DSM 92), Clostridium glycolicum (DSM 1288), Clostridium yunnanense (DSM 92) *Clostridium jungdahlii* (DSM 13528) and *Clostridium mayombei* (DSM 6539) Clostridium methoxybenzovorans (DSM 12182), Clostridium ragsdalei (DSM 15248), feces Clostridium scatologenes (DSM 757), Clostridium species ATCC 29797 (Schmidt et al., 1986, Chem. Eng. Commun., Vol. 45, pp. 61-73), Desulfotomaculum *Kuznetsovii* (DSM 6115), *Desulfotomaculum* subspecies *thermobezoicum* thermobezoicum subsp. thermosyntrophicum (DSM 14055), Eubacterium limosum (DSM 20543), Methanosarcina acetivorans C2A (DSM 2834), Moorella sp. HUC22-1 (Sakai et al., 2004, Biotechnol. Let., Vol. 29, No. 1607- Page 1612), Moorella thermoacetica (DSM 521, formerly known as Clostridium) thermoaceticum, Moorella thermoautotrophica (DSM 1974) Oxobacter pfennigii (DSM 3222), Sporoma aerivorans (DSM 13326), and Ovoobacter pfennigii (DSM 3222). (Sporomusa ovata) (DSM 2662), Sporomusa silvacetica (DSM 10669), *Sporomus* var. *spheroides* (Sporomusa sphaeroides) (DSM 2875), *Sporomusa termitida* (DSM 4440) and Thermoanaerobacter kivui (DSM 2030, formerly known as Acetogenium kivui) kivui)) More specifically, it can be used Clostridium carboxidivorans The strain ATCC BAA-624. More specifically, strains such as those described in, for example, US 2007 / 0275447 and US 2008 / 0057554 can be used. Clostridium carboxidivorans Bacterial strains labeled "P7" and "P11".

[0055] Another particularly suitable bacterium could be Clostridium ljungdahlii Specifically, selected from Clostridium yongdar PETC Clostridium yongdar ERI2, Clostridium yongdar COL and Clostridium yongdar Strains of O-52 can be used to convert syngas into hexanoic acid. These strains are described, for example, in WO 98 / 00558, WO 00 / 68407, ATCC 49587, ATCC55988, and ATCC 55989. More specifically, homoacetogenic bacteria can be selected from the Clostridium family (…). Clostridium family).

[0056] homoacetic acid-producing bacteria selected Clostridium autoethanogenum (DSM 10061, DSM) 19630 and DSM 23693) , Clostridium ljungdahlii (DSM 13528) , carbon monoxide shuttle Clostridium carboxidivorans (DSM 15243) , Acetobacterium glutamicum woodii) (DSM 1030), Clostridium ragsdal ei (DSM) 15248） , Clostridium drakei (ATCC) BAA-623) , Moorella thermoacetica (DSM 521), thermoautotrophic Moorella (Moorella thermoautotrophica) (DSM 1974), Sporomusa silvacetica (DSM 10669) and Alkalibaculum bacchi (DSM 22112) In particular, homoacetic bacteria can be selected from... Clostridium ethanoliferum (Clostridium autoethanogenum), Clostridium ljungdahlii and Carbon monoxide Clostridium carboxidivorans In one example, for acetone production, genetically modified ethanol-producing Clostridium autoethanogenum can be used. In another example, for acetone production, genetically modified... Clostridium ljungdahlii .

[0057] The method according to any aspect of the invention includes the steps of (a) Receive the feed stream from the exhaust gas or waste gas from the steam methane reformer.

[0058] As used herein, the term "feed" refers to a stream of material that enters, passes through, and exits one or more stages of a process, such as material fed into a bioreactor for fermentation. The composition of a feed stream can vary as it passes through a particular stage. For example, when a feed stream enters a bioreactor, it may contain high concentrations of CO, CO2, H2, and CH4, as well as low concentrations of N2, O2, and H2S. As the feed stream passes through the bioreactor, the CO, CO2, and H2 content of the feed stream may decrease.

[0059] As used herein, the terms “fermentation process” or “fermentation reaction” or “microbial fermentation” are intended to encompass the growth phase and product biosynthesis (particularly acetone) phase of the method according to any aspect of the invention.

[0060] Further acetone purification steps may be required. In particular, acetone can be purified directly from the crude product stream by distillation after the fermentation process.

[0061] According to another aspect of the present invention, an apparatus is provided for producing acetone from a gaseous composition via microbial fermentation, the apparatus comprising: (i) The source of the gas composition, which is used to continuously supply exhaust or waste gas stream from a steam methane reformer containing at least CO, CO2, H2 and CH4. (ii) An inlet on the bioreactor for receiving exhaust or waste gas from the steam methane reformer. (iii) A bioreactor containing a culture of homoacetic bacteria, (iii) An outlet on the bioreactor, used to discharge the exhaust gas or waste gas after contact with the homologous acetic acid-producing bacteria. The source provided is the gas composition directly from the steam methane reformer.

[0062] According to a further aspect of the invention, there is a use for exhaust gas or waste gas from a steam methane reformer for the production of acetone, wherein the exhaust gas or waste gas contains at least CO, CO2, H2 and CH4, and the exhaust gas or waste gas is contacted with at least one homoacetic acid-producing bacterium.

[0063] According to a further aspect of the invention, a steam methane reformer is provided, which is adapted to produce acetone by microbial fermentation of exhaust gas or waste gas from said steam methane reformer. Example

[0064] The preferred embodiments described above may be varied or modified in design, construction, or operation without departing from the scope of the claims, as will be understood by those skilled in the art. For example, these variations are intended to be covered by the scope of the claims.

[0065] Example 1 Acetone is produced in large quantities using exhaust gas from SMR via Clostridium ljungdahlii. To bioconvert hydrogen, carbon monoxide, and carbon dioxide into acetone, genetically modified homoacetic bacteria were cultured on exhaust gas from a steam methane reformer. Clostridium ljungdahlii (GMO from EP2421960B1). All culture steps were carried out under anaerobic conditions in pressure-resistant glass vials that could be hermetically sealed with butyl rubber stoppers or in stirred stainless steel benchtop bioreactors.

[0066] For pre-culture, add 500 ml of medium (ATCC1754-) containing an additional 400 mg / L L-cysteine ​​hydrochloride and 400 mg / L Na₂S x 9 H₂O. Medium: pH = 6.0; 20 g / L MES; 1 g / L yeast extract, 0.8 g / L NaCl; 1 g / L NH₄Cl; 0.1 g / L KCl; 0.1 g / L KH₂PO₄; 0.2 g / L MgSO₄ x 7 H₂O; 0.02 g / L CaCl₂ x 2 H₂O; 20 mg / L nitric acid; 10 mg / L MnSO₄ x H₂O; 8 mg / L (NH₄)₂Fe(SO₄)₂ x 6 H₂O; 2 mg / L CoCl₂ x 6 H₂O; 2 mg / L ZnSO₄ x 7 H₂O; 0.2 mg / L CuCl₂ x 2 H₂O; 0.2 mg / L Na₂S x 9 H₂O. mg / L Na₂MoO₄ x 2 H₂O; 0.2 mg / L NiCl₂ x 6 H₂O; 0.2 mg / L Na₂SeO₄; 0.2 mg / L Na₂WO₄ x 2 H₂O; 20 µg / L d-Biotin; 20 µg / L Folic Acid; 100 µg / L Pyridoxine-HCl; 50 µg / L Thiamine-HCl x H₂O; 50 µg / L Riboflavin; 50 µg / L Niacin; 50 µg / L Calcium Pantothenate; 1 µg / L Vitamin B 12 ; 50 µg / L para-aminobenzoate; 50 µg / L lipoic acid; approximately 67.5 mg / L NaOH) inoculated into 2.5 µL of [unclear - possibly a solution or solution]. Clostridium ljungdahlii Cryogenic stock solution of GMO. A chemoautotrophic mixture (49% CO2, 30% H2, 10.2% CO, 10.2% CH4, 0.7% N2, 100 ppb H2S, 10 ppm O2) was vented from a steam methane reformer in 1 L pressure-resistant glass vials at 37 °C, 150 rpm, and 1 L / h in an open water bath shaker for 66 h. Gas was released into the culture medium via a 10 µm bubbler installed in the center of the reactor. The culture was conducted without pH control.

[0067] For the master culture, 0.1% OD 600nmThe required number of cells from the preculture were transferred from the preculture to a fresh 500 mL culture medium. For the master culture, LM33 mineral medium with an additional 500 mg / L L-cysteine ​​hydrochloride was used (pH = 5.8, 0.5 g / L MgCl2, 0.21 g / L NaCl, 0.135 g / L CaCl2X 2H2O, 2.65 g / L NaH2PO4X 2H2O, 0.5 g / L KCl, 2.5 g / L NH4Cl, 15 mg / L hyponitrotriacetic acid, 30 mg / L MgSO4x 7H2O, 5 mg / L MnSO4x H2O, 1 mg / L FeSO4x 7H2O, 8 mg / L Fe(SO4)2(NH4)2x 6H2O, 2 mg / L CoCl2x 6H2O, 2 mg / L ZnSO4x 7H2O, 200 µg / L CuCl2x 2H2O, 200 µg / L KAl(SO4)2x 12H2O, 3 mg / L H3BO3, 300 µg / L Na2MoO4x 2 H2O, 200 µg / L Na2SeO3, 200 µg / L NiCl2x 6 H2O, 200 µg / L Na2WO4x 6 H2O, 200 µg / L d-Biotin, 200 µg / L Folic Acid, 100 µg / L Pyridoxine-HCl, 500 µg / L Thiamine-HCl; 500 µg / L Riboflavin; 500 µg / L Niacin; 500 µg / L Calcium Pantothenate; 500 µg / L Vitamin B1 12 ; 500 µg / L p-aminobenzoate; 500 µg / L lipoic acid, 10 mg / L FeCl3, purged with the exhaust mixture from a steam methane reformer for 30 minutes).

[0068] A chemoautotrophic mixture (49% CO2, 30% H2, 10.2% CO, 10.2% CH4, 0.7% N2, 100 ppb H2S, 10 ppm O2) was vented from a steam methane reformer in a 1 L pressure-resistant glass bottle at 37 °C, 150 rpm, and 1 L / h aeration rate in an open water bath shaker for 164 h. Gas was vented into the culture medium via a 10 µm bubbler installed in the center of the reactor. A 100 g / L NaOH solution was automatically added using a Titrino pH control system (Methrom, Switzerland) to maintain the pH at 5.0. Several 5 mL samples were taken during the culture period to determine OD. 600nmpH and product formation. Product concentration was determined by semi-quantitative 1H-NMR spectroscopy. Sodium trimethylsilylpropionate (T(M)SP) was used as an internal quantitative standard.

[0069] During the primary culture in LM33 medium, the concentrations of ethanol, acetate, and acetone were increased from 0 g / L to >2 g / L, from 0 g / L to >0.5 g / L, and from 0 g / L to >2 g / L. OD 600nm It reaches a maximum value of >2 after 100 hours of cultivation.

[0070] Example 2 Acetone production from exhaust gas from SMR using *Acetobacterium woodii*. To bioconvert hydrogen, carbon monoxide, and carbon dioxide into acetone, genetically modified homoacetic bacteria were cultured on exhaust gas from a steam methane reformer. Acetobacterium woodii (GMO from EP2421960B1). All culture steps were carried out under anaerobic conditions in pressure-resistant glass vials that could be hermetically sealed with butyl rubber stoppers or in stirred stainless steel benchtop bioreactors.

[0071] For pre-culture, add 500 ml of DSMZ135 medium (medium: pH = 8.2; 1.0 g / L NH4Cl; 0.33 g / L KH2PO4; 0.45 g / L K2HPO4; 0.1 g / L MgSO4 x 7 H2O; 2.0 g / L yeast extract; 500 mg / L L-cysteine-HCl x H2O; 500 mg / L Na2S x 9 H2O; 10 g / L NaHCO3; 30 mg / L nitric acid; 60 mg / L MgSO4 x 7 H2O; 10 mg / L MnSO4 x H2O; 20 mg / L NaCl; 2 mg / L FeSO4 x 7 H2O; 3.6 mg / L CoSO4 x 7 H2O; 2 g / L CaCl2 x 2 H2O; 3.6 mg / L ZnSO4 x 7 H2O). H2O; 0.2 mg / L CuSO4 x 7 H2O; 0.4 mg / L KAl(SO4)2 x 12 H2O; 0.2 mg / L H3BO3; 0.2 mg / L Na2MoO4 x 2 H2O; 0.6 mg / L NiCl2 x 6 H2O; 6 µg / L Na2SeO3 x 5 H2O; 40 µg / L d-Biotin; 40 µg / L Folic Acid; 200 µg / L Pyridoxine-HCl; 100 µg / L Thiamine-HCl x H2O; 100 µg / L Riboflavin; 100 µg / L Niacin; 100 µg / L Calcium Pantothenate; 2 µg / L Vitamin B112 ; 100 µg / L p-aminobenzoate; 100 µg / L lipoic acid) inoculated into 2.5 µL Acetobacter woodii (A. woodii) Cryogenic stock solution of GMO. A mixture of exhaust gas from a steam methane reformer (49% CO2, 30% H2, 10.2% CO, 10.2% CH4, 0.7% N2, 100 ppb H2S, 10 ppm O2) was chemoautotrophically cultured for 66 hours in an open water bath shaker at 30°C, 150 rpm, and 1 L / h. Gas was released into the culture medium via a 10 µm bubbler installed at the center of the reactor. The culture was conducted without pH control.

[0072] For the master culture, 0.1% OD 600nm The required number of cells from the preculture were transferred from the preculture to fresh 500 mL of medium. For the master culture, DSMZ135 medium was also used (aerated with a steam-formed methane venting mixture for 30 min). Chemoautotrophic culture was performed for 164 h in 1 L pressure-resistant glass vials at 30 °C, 150 rpm, and 1 L / h using a steam-formed methane venting mixture (49% CO2, 30% H2, 10.2% CO, 10.2% CH4, 0.7% N2, 100 ppb H2S, 10 ppm O2) in an open water bath shaker. Gas was vented into the medium via a 10 µm bubbler installed in the center of the reactor. 100 g / L NaOH solution was automatically added using a Titrino pH control system (Methrom, Switzerland) to maintain the pH at 7.5. Several 5 mL samples were taken during the culture period to determine OD. 600nm pH and product formation. Product concentration was determined by semi-quantitative 1H-NMR spectroscopy. Sodium trimethylsilylpropionate (T(M)SP) was used as an internal quantitative standard.

[0073] During the primary culture in DSMZ135 medium, the concentrations of acetate, acetone, and isopropanol were increased from 0 g / L to >4 g / L, acetone from 0 g / L to >0.2 g / L, and isopropanol from 0 g / L to >0.4 g / L. OD 600nm It reaches a maximum value of >2 after 100 hours of cultivation.

[0074] As can be seen here, compared to Example 1, Acetobacter woodii (A. woodii) Acetone can be produced from SMR exhaust gas. However, less acetone is produced compared to Example 1.

[0075] Example 3 Acetone produced in low quantities from syngas by Clostridium ljungdahlii To bioconvert hydrogen and carbon dioxide into acetone, genetically modified homoacetic bacteria were cultured on a syngas mixture containing hydrogen, carbon dioxide, and methane. Clostridium ljungdahlii (GMO from EP2421960B1). All culture steps were carried out under anaerobic conditions in pressure-resistant glass vials that could be hermetically sealed with butyl rubber stoppers or in stirred stainless steel benchtop bioreactors.

[0076] For pre-culture, add 500 ml of medium (ATCC1754-) containing an additional 400 mg / L L-cysteine ​​hydrochloride and 400 mg / L Na₂S x 9 H₂O. Medium: pH = 6.0; 20 g / L MES; 1 g / L yeast extract, 0.8 g / L NaCl; 1 g / L NH₄Cl; 0.1 g / L KCl; 0.1 g / L KH₂PO₄; 0.2 g / L MgSO₄ x 7 H₂O; 0.02 g / L CaCl₂ x 2 H₂O; 20 mg / L nitric acid; 10 mg / L MnSO₄ x H₂O; 8 mg / L (NH₄)₂Fe(SO₄)₂ x 6 H₂O; 2 mg / L CoCl₂ x 6 H₂O; 2 mg / L ZnSO₄ x 7 H₂O; 0.2 mg / L CuCl₂ x 2 H₂O; 0.2 mg / L Na₂S x 9 H₂O. mg / L Na₂MoO₄ x 2 H₂O; 0.2 mg / L NiCl₂ x 6 H₂O; 0.2 mg / L Na₂SeO₄; 0.2 mg / L Na₂WO₄ x 2 H₂O; 20 µg / L d-Biotin; 20 µg / L Folic Acid; 100 µg / L Pyridoxine-HCl; 50 µg / L Thiamine-HCl x H₂O; 50 µg / L Riboflavin; 50 µg / L Niacin; 50 µg / L Calcium Pantothenate; 1 µg / L Vitamin B 12 ; 50 µg / L para-aminobenzoate; 50 µg / L lipoic acid; approximately 67.5 mg / L NaOH) inoculated into 2.5 µL of [unclear - possibly a solution or solution]. Clostridium ljungdahlii Cryogenic stock solution of GMO. A chemoautotrophic mixture (49% CO2, 30% H2, 10.2% CO, 10.2% CH4, 0.7% N2, 100 ppb H2S, 10 ppm O2) was vented from a steam methane reformer in 1 L pressure-resistant glass vials at 37 °C, 150 rpm, and 1 L / h in an open water bath shaker for 66 h. Gas was released into the culture medium via a 10 µm bubbler installed in the center of the reactor. The culture was conducted without pH control.

[0077] For the master culture, 0.1% OD 600nm The required number of cells from the preculture were transferred from the preculture to a fresh 500 mL culture medium. For the master culture, LM33 mineral medium with an additional 500 mg / L L-cysteine ​​hydrochloride was used (pH = 5.8, 0.5 g / L MgCl2, 0.21 g / L NaCl, 0.135 g / L CaCl2X 2H2O, 2.65 g / L NaH2PO4X 2H2O, 0.5 g / L KCl, 2.5 g / L NH4Cl, 15 mg / L hyponitrotriacetic acid, 30 mg / L MgSO4x 7H2O, 5 mg / L MnSO4x H2O, 1 mg / L FeSO4x 7H2O, 8 mg / L Fe(SO4)2(NH4)2x 6H2O, 2 mg / L CoCl2x 6H2O, 2 mg / L ZnSO4x 7H2O, 200 µg / L CuCl2x 2H2O, 200 µg / L KAl(SO4)2x 12H2O, 3 mg / L H3BO3, 300 µg / L Na2MoO4x 2 H2O, 200 µg / L Na2SeO3, 200 µg / L NiCl2x 6 H2O, 200 µg / L Na2WO4x 6 H2O, 200 µg / L d-Biotin, 200 µg / L Folic Acid, 100 µg / L Pyridoxine-HCl, 500 µg / L Thiamine-HCl; 500 µg / L Riboflavin; 500 µg / L Niacin; 500 µg / L Calcium Pantothenate; 500 µg / L Vitamin B1 12 ; 500 µg / L p-aminobenzoate; 500 µg / L lipoic acid, 10 mg / L FeCl3, aerated with H2 / CO2 / CH4 mixture for 30 minutes for the main culture).

[0078] Chemoautotrophic culture was performed for 164 hours in an open water bath shaker using a gas mixture (30% CO2, 60% H2, 10% CH4) containing hydrogen, carbon dioxide, and methane in a 1 L pressure-resistant glass bottle at 37 °C, 150 rpm, and a ventilation rate of 1 L / h. Gas was vented into the culture medium via a 10 µm bubbler installed at the center of the reactor. The pH was maintained at 5.0 by automatically adding 100 g / L NaOH solution using a Titrino pH control system (Methrom, Switzerland). Several 5 mL samples were taken during the culture for OD measurement. 600nmpH and product formation. Product concentration was determined by semi-quantitative 1H-NMR spectroscopy. Sodium trimethylsilylpropionate (T(M)SP) was used as an internal quantitative standard.

[0079] During the main culture in LM33 medium, the concentrations of ethanol, acetate, and acetone were increased from 0 g / L to ~1 g / L, from 0 g / L to ~0.5 g / L, and from 0 g / L to ~1 g / L. OD 600nm It reaches a maximum value of ~1 after 100 hours of cultivation.

[0080] In this embodiment, compared to Example 1, when using another carbon source – a CO-free syngas mixture, Clostridium ljungdahlii It not only has lower production rates, but also produces less acetone.

[0081] Example 4 Acetone produced in low quantities using syngas by *Acetobacterium woodii* To bioconvert hydrogen, carbon monoxide, and carbon dioxide into acetone, genetically modified homoacetic bacteria were cultured on a syngas mixture containing hydrogen, carbon dioxide, carbon monoxide, and methane. Acetobacter wuerii (Acetobacterium woodii) (GMO from EP2421960B1). All culture steps were carried out under anaerobic conditions in pressure-resistant glass vials that could be hermetically sealed with butyl rubber stoppers or in stirred stainless steel benchtop bioreactors.

[0082] For pre-culture, add 500 ml of DSMZ135 medium (medium: pH = 8.2; 1.0 g / L NH4Cl; 0.33 g / L KH2PO4; 0.45 g / L K2HPO4; 0.1 g / L MgSO4 x 7 H2O; 2.0 g / L yeast extract; 500 mg / L L-cysteine-HCl x H2O; 500 mg / L Na2S x 9 H2O; 10 g / L NaHCO3; 30 mg / L nitric acid; 60 mg / L MgSO4 x 7 H2O; 10 mg / L MnSO4 x H2O; 20 mg / L NaCl; 2 mg / L FeSO4 x 7 H2O; 3.6 mg / L CoSO4 x 7 H2O; 2 g / L CaCl2 x 2 H2O; 3.6 mg / L ZnSO4 x 7 H2O). H2O; 0.2 mg / L CuSO4 x 7 H2O; 0.4 mg / L KAl(SO4)2 x 12 H2O; 0.2 mg / L H3BO3; 0.2 mg / L Na2MoO4 x 2 H2O; 0.6 mg / L NiCl2 x 6 H2O; 6 µg / L Na2SeO3 x 5 H2O; 40 µg / L d-Biotin; 40 µg / L Folic Acid; 200 µg / L Pyridoxine-HCl; 100 µg / L Thiamine-HCl x H2O; 100 µg / L Riboflavin; 100 µg / L Niacin; 100 µg / L Calcium Pantothenate; 2 µg / L Vitamin B1 12 ; 100 µg / L p-aminobenzoate; 100 µg / L lipoic acid) inoculated into 2.5 µL Acetobacter woodii (A. woodii) Cryogenic stock solution of GMO. A mixture of exhaust gas from a steam methane reformer (49% CO2, 30% H2, 10.2% CO, 10.2% CH4, 0.7% N2, 100 ppb H2S, 10 ppm O2) was chemoautotrophically cultured for 66 hours in an open water bath shaker at 30°C, 150 rpm, and 1 L / h. Gas was released into the culture medium via a 10 µm bubbler installed at the center of the reactor. The culture was conducted without pH control.

[0083] For the master culture, 0.1% OD 600nmThe required number of cells from the preculture were transferred from the preculture to fresh 500 mL of medium. For the master culture, DSMZ135 medium (aerated with a mixture of H2 / CO2 / CO / CH4 for 30 min) was also used. Chemoautotrophic culture was performed for 164 h in 1 L pressure-resistant glass vials at 30 °C, 150 rpm, and 1 L / h using a gas mixture containing hydrogen, carbon dioxide, carbon monoxide, and methane (22% CO2, 25% CO, 43% H2, 10% CH4). Gas was vented into the medium via a 10 µm bubbler installed in the center of the reactor. 100 g / L NaOH solution was automatically added using a Titrino pH control system (Methrom, Switzerland) to maintain the pH at 7.5. Several 5 mL samples were taken during the culture period to determine OD. 600nm pH and product formation. Product concentration was determined by semi-quantitative 1H-NMR spectroscopy. Sodium trimethylsilylpropionate (T(M)SP) was used as an internal quantitative standard.

[0084] During the primary culture in DSMZ135 medium, the concentrations of acetate, acetone, and isopropanol were increased from 0 g / L to ~2 g / L, acetone from 0 g / L to ~0.1 g / L, and isopropanol from 0 g / L to ~0.2 g / L. OD 600nm It reaches a maximum value of ~1 after 100 hours of cultivation.

[0085] In this embodiment, compared to Example 2, a syngas mixture with a high CO content is used. Wood's acetic acid A. woodii This results in lower cell growth and lower acetone production.

Claims

1. A method for producing acetone from a gaseous composition via microbial fermentation, the method comprising: - Directly contact at least one genetically modified homoacetic bacteria with the gas composition, the gas composition comprising at least CO, CO2, H2 and CH4; The gas composition is exhaust gas from at least one steam methane reformer, and the exhaust gas from the steam methane reformer is directly contacted with the genetically modified isoacetic bacteria; and The homoacetic acid-producing bacteria are genetically modified to produce acetone from the gaseous composition.

2. The method according to claim 1, wherein the homoacetic bacteria are genetically modified to: Compared to its wild type, the expression of at least one of the following enzymes is increased: - Thiolase (ThlA) (EC2.3.1.9). - Coenzyme A transferase (CtfAB) (EC 2.8.3.8) and / or - Acetoacetate decarboxylase (Adc) (EC 4.1.1.4); and / or Compared to its wild type, the expression of at least the following enzymes is reduced: - Secondary alcohol dehydrogenase (sAdh) (EC 1.1.1.1).

3. The method according to claim 1 or 2, wherein the exhaust gas from the steam methane reformer is directly contacted with the genetically modified homoacetic bacteria without any additional steps of pretreatment and / or washing of the gas.

4. The method according to any one of the preceding claims, wherein the homoacetic bacterium to be genetically modified is selected from... tide Acetoanaerobium notera (ATCC 35199), Acetonema longum (DSM) 6540), Acetobacterium carbinolicum (DSM 2925), and Acetobacter malate Acetobacterium malicum (DSM 4132), Acetobacter species no. 446, Acetobacter wilfordii Acetobacterium wieringae (DSM 1911), Acetobacterium woodii (DSM 1911) 1030), Alkalibaculum bacchi (DSM 22112), Archaeoglobus fulgidus (DSM 4304), Blautia producta (DSM 2950), Butyribacterium methylbutyrate methylotrophicum (DSM 3468), Clostridium aceticum (DSM 1496), ethanol-producing Clostridium Clostridium autoethanogenum (DSM 10061), Clostridium carbon monoxide-eating bacteria *Clostridium carboxidivorans* (DSM 15243), *Clostridium drakei* (ATCC BAA-623), *Clostridium formicum* (acetic acid clostridium) (Clostridium formicoaceticum) (DSM 92), Clostridium glycolicum (DSM 1288), Clostridium ljungdahlii (DSM 13528), Clostridium majugbeum mayombei) (DSM 6539), Clostridium methoxybenzovorans (DSM 12182 [SR3]), Clostridium ragsdalei (DSM 15248), Clostridium scatologenes (DSM 757), Clostridium species ATCC 29797, Desulfotomaculum kuznetsovii (DSM 6115), heat Desulfotomaculum thermobezoicum subsp. Thermosyntrophicum (DSM 14055), Eubacterium limosum (DSM 20543), vinegar Methanosarcina acetivorans C2A (DSM 2834), Moorella sp. HUC22-1, Moorella thermoacetica (DSM 521), Moorella thermoautotrophicis thermoautotrophica) (DSM 1974), Oxobacter pfennigii (DSM 3222), Sporomusa aerivorans (DSM 13326), Sporomusa ovata (DSM 2662), Sporomusa Silvacetica (DSM 10669), Sporoma sphaeroides (DSM 2875), and Sporoma sphaeroides (termite sphaeroides) (Sporomusa termitida) (DSM 4440) and Thermoanaerobacter kivui (DSM 4440) 2030） .

5. The method according to any one of the preceding claims, wherein the homoacetic bacteria is selected from... Clostridium ethanoliferum (Clostridium autoethanogenum) (DSM 10061) , Clostridium ljungdahlii (DSM 13528) , Clostridium carboxidivorans (DSM 15243) , Wood's Acetic Acid Rod Acetobacterium woodii (DSM 1030), Clostridium ragsdal ei (DSM) 15248） , Clostridium drakei (ATCC BAA-623) , Moorella thermoacetica (DSM) 521), Moorella thermoautotrophica (DSM 1974), Sporoma silvacetica (DSM 10669) and Alkalibaculum bacchi (DSM 22112) .

6. The method according to any one of the preceding claims, wherein the gaseous composition further comprises N2, O2 and H2S.

7. The method according to any one of the preceding claims, wherein CO2 is in the range of 35–65 vol%, H2 is in the range of 20–40 vol%, CO is in the range of 5–20 vol%, and / or CH4 is in the range of 0.01–20 vol%.

8. The method according to claim 6 or 7, wherein the O2 concentration is between 0.000005 vol% and 1 vol%, and / or the H2S concentration is between 0.00000001 vol% and 0.0001 vol%.

9. The method according to any one of the preceding claims, wherein the homoacetic bacterium is genetically modified. Ethanol production Clostridium autoethanogenum or Clostridium ljungdahlii .

10. The method according to any one of the preceding claims, comprising the steps of (a) Receive the feed stream from the exhaust gas of the steam methane reformer.

11. An apparatus for producing acetone from a gaseous composition via microbial fermentation, the apparatus comprising: (i) The source of the gas composition, which is used to continuously supply an exhaust stream from a steam methane reformer containing at least CO, CO2, H2 and CH4. (ii) An inlet on the bioreactor for receiving exhaust gas from the steam methane reformer. (iii) A bioreactor containing a culture of genetically modified homoacetic bacteria. (iii) An outlet on the bioreactor for discharging the exhaust gas after contact with the homologous acetic acid-producing bacteria. The source provided is the gas composition directly from the steam methane reformer; and The homoacetic acid-producing bacteria are genetically modified to produce acetone from the gaseous composition.

12. Use of exhaust gas from a steam methane reformer for the production of acetone, wherein the exhaust gas contains at least CO, CO2, H2 and CH4, and the exhaust gas is directly contacted with at least one genetically modified homoacetic bacterium, wherein the homoacetic bacterium is genetically modified to produce acetone from the gas composition.

13. The use according to claim 12, wherein the genetically modified homoacetic bacterium is selected from... Moist anaerobic acetic acid bacteria (Acetoanaerobium notera) (ATCC 35199), Acetonema longum (DSM 6540), Acetonema longum (Acetonema longum) ... Acetobacterium carbinolicum (DSM 2925), Acetobacter malate Acetobacterium malicum (DSM 4132), Acetobacter species no. 446, Acetobacter wilfordii Acetobacterium wieringae (DSM 1911), Acetobacterium woodii (DSM 1911) 1030), Alkalibaculum bacchi (DSM 22112), Archaeoglobus fulgidus (DSM 4304), Blautia producta (DSM 2950), Butyribacterium methylbutyrate methylotrophicum (DSM 3468), Clostridium aceticum (DSM 1496), ethanol-producing Clostridium Clostridium autoethanogenum (DSM 10061), Clostridium carbon monoxide-eating bacteria *Clostridium carboxidivorans* (DSM 15243), *Clostridium drakei* (ATCC BAA-623), *Clostridium formicum* (acetic acid clostridium) (Clostridium formicoaceticum) (DSM 92), Clostridium glycolicum (DSM 1288), Clostridium ljungdahlii (DSM 13528), Clostridium majugbeum mayombei) (DSM 6539), Clostridium methoxybenzovorans (DSM 12182 [SR3]), Clostridium ragsdalei (DSM 15248), Clostridium scatologenes (DSM 757), Clostridium species ATCC 29797, Desulfotomaculum kuznetsovii (DSM 6115), heat Desulfotomaculum thermobezoicum subsp. Thermosyntrophicum (DSM 14055), Eubacterium limosum (DSM 20543), vinegar Methanosarcina acetivorans C2A (DSM 2834), Moorella sp. HUC22-1, Moorella thermoacetica (DSM 521), Moorella thermoautotrophicis thermoautotrophica) (DSM 1974), Oxobacter pfennigii (DSM 3222), Sporomusa aerivorans (DSM 13326), Sporomusa ovata (DSM 2662), Sporomusa Silvacetica (DSM 10669), Sporoma sphaeroides (DSM 2875), and Sporoma sphaeroides (termite sphaeroides) (Sporomusa termitida) (DSM 4440) and Thermoanaerobacter kivui (DSM 4440) 2030 ).

14. The use according to claim 12 or 13, wherein the exhaust gas or waste gas further comprises N2, O2 and H2S.

15. The use according to any one of claims 12 to 14, wherein the exhaust gas from the steam methane reformer is directly contacted with the genetically modified homoacetic bacteria without any additional steps of pretreatment and / or washing of the gas.