Acetone production through biotechnology

Abstract

Acetone production through biotechnology The present invention is a method for producing acetone from a gaseous composition by microbial fermentation. At least one genetically modified homoacetic acid-producing bacterium is brought into direct contact with a gas composition containing at least CO, CO2, H2, and CH4. The gas composition is an off-gas from at least one steam methane reforming apparatus, and the off-gas from the steam methane reforming apparatus is brought into direct contact with the genetically modified homoacetic acid-producing bacteria. The present invention relates to a method in which the homoacetic acid-producing bacteria are genetically modified to produce acetone from the gas composition.

Description

[Technical Field]

[0001] This invention relates to a biotechnological method for producing ketones from off-gas or waste gas. Specifically, in this method, the off-gas or waste gas is fermented by directly contacting it with suitable bacteria without pretreatment of the gas for the fermentation process. The off-gas or waste gas is obtained from a steam methane reformer, and the off-gas or waste gas obtained therefrom can be directly introduced into microbial fermentation and acetone production. [Background technology]

[0002] Catalytic processes are primarily used to convert gases containing CO, CO2, and hydrogen (H2) into various fuels and chemicals. Microorganisms are also 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 high specificity, high yield, reduced energy costs, and high resistance to poisoning. In particular, the use of acetic acid-producing bacteria with various carbon sources to produce ethanol, acetic acid, and / or other alcohols is well known. The general application of genetically modified organisms in the production of CO-containing raw material chemicals is disclosed in at least European Patent No. 2678432.

[0003] The use of synthesis gas as a carbon source for gas fermentation is demonstrated in U.S. Patent No. 8,263,372. European Patent No. 3050968 also discloses the production of various alcohols using synthesis gas.

[0004] Furthermore, methods for producing acetone and other ketones from various gases using genetically modified organisms are disclosed in at least International Publication No. 2015 / 085015, European Patent No. 2181195, International Publication No. 2010 / 121849, and Nature Biotechnology 2022, 40, 335-344. In U.S. Patent No. 8,376,736, Lanzatech discloses a method for using blast furnace off-gas for acetone production. However, using blast furnace off-gas for fermentation required pre-cooling and pre-treatment of the off-gas to remove particles, long-chain hydrocarbons, and tar from the gas stream. This not only increased the time required for acetone production from off-gas, but also made process design and large-scale production more complex and costly.

[0005] Acetone is an industrial solvent used as a raw material for at least methyl methacrylate (MMA), polymethyl methacrylate (PMMA), and isobutylene, which are used in a variety of industrial applications. Acetone is also used as a raw material in the manufacture of jet fuel (Anbarasan, Nature, 491: 235-239, 2012).

[0006] However, currently available methods for producing acetone from off-gas or waste gas are inefficient and require at least one additional step of treating the off-gas before contacting the gas with bacteria for fermentation. Low productivity and low concentration of the final product result in high energy costs for product purification. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] European Patent No. 2678432 [Patent Document 2] U.S. Patent No. 8,263,372 [Patent Document 3] European Patent No. 3050968 [Patent Document 4] International Publication No. 2015 / 085015 Brochure [Patent Document 5] European Patent No. 2181195 [Patent Document 6] International Publication No. 2010 / 121849 [Patent Document 7] U.S. Patent No. 8,376,736 [Non-patent literature]

[0008] [Non-Patent Document 1] Nature Biotechnology 2022, 40, 335-344 [Non-Patent Document 2] Anbarasan, Nature, 491 : 235-239, 2012 [Overview of the Initiative] [Problems that the invention aims to solve]

[0009] Therefore, it is desirable to find other sustainable raw materials that yield equivalent or higher yields and have a lower environmental impact, as starting materials for acetone production using biotechnology. Specifically, there is a need for a simple and efficient method of producing acetone from sustainable raw materials using biotechnology. [Means for solving the problem]

[0010] The present invention attempts to solve the above problems by providing an industrial-scale production method of raw material chemical substances, particularly acetone, based on off-gas fermentation. Acetone can be used in applications in downstream manufacturing processes. Specifically, in this method, the off-gas from a steam reforming device is brought into contact with at least one bacterial cell capable of converting the off-gas into acetone. With a new combination of a steam reforming device and a gas fermentation device according to any aspect of the present invention, the steam reforming device gas (specifically, its off-gas stream) can be utilized in the production of useful chemical substances such as alcohols, acids, aldehydes, and ketones, with acetone as the main product and acetic acid and ethanol as commonly seen by-products.

[0011] An advantage of the method according to any aspect of the present invention is that it can realize a new off-gas source for the production of useful raw materials based on fermentation. Furthermore, by improving the gas mixture composition of the off-gas from a steam reforming device, particularly a steam methane reforming device, it becomes possible to directly apply the off-gas to a fermentation process, particularly an anaerobic fermentation process, without the need for prior pretreatment or washing.

[0012] Unlike many other off-gas streams, for example, at least the blast furnace off-gas stream, the off-gas stream from a steam reforming device according to any aspect of the present invention does not contain a significant oxygen concentration. This is considered to be one of the reasons why the off-gas stream from the steam reforming device can be directly used as a feed stream for an anaerobic gas fermentation process. Another advantage of the method according to any aspect of the present invention is that since the gas temperature at the outlet of the steam reforming device where the off-gas is released is about 40°C, there is no need to pre-cool the gas stream before contact with the bacteria for fermentation. Using the off-gas from the steam reforming device as a feed stream for fermentation eliminates several steps required to prepare the off-gas as a feed stream, simplifies the biotechnological production of acetone, reduces costs, and speeds up the process.

[0013] According to one aspect of the present invention, it is a method for producing acetone from a gas composition by microbial fermentation, At least one genetically modified homoacetic acid-producing bacterium is brought into direct contact with a gas composition containing at least CO, CO2, H2 and CH4, where the gas composition is offgas from at least one steam methane reformer, and the offgas from the steam methane reformer is brought into direct contact with the genetically modified homoacetic acid-producing bacterium, and there is provided a method in which the homoacetic acid-producing bacterium is genetically modified to produce acetone from the gas composition. As used herein, the term "gas composition" refers to all gas mixtures. The gas composition is a gas substrate that serves as the main carbon source in microbial fermentation according to any aspect of the present invention. In particular, the gas composition is synthesis gas (i.e., containing CO and H2). More specifically, the gas composition according to any aspect of the present invention contains at least CO, CO2, H2 and CH4. In some examples, N2, O2 and H2S are also present in the gas composition. In particular, N2, O2 and H2S are present at low concentrations compared to the main components CO, CO2, H2 and CH4 of the gas composition.

[0014] CO2 in the gas composition according to any aspect of the present invention ranges from 30 to 80% by volume, particularly 35 to 80, 40 to 80, 45 to 80, 50 to 80, 55 to 80, 60 to 80, 65 to 80, 70 to 80, 30 to 75, 35 to 75, 40 to 75, 45 to 75, 50 to 75, 55 to 75, 60 to 75, 65 to 75, 70 to 75, 30 to 70, 35 to 70, 40 to 70, 45 to 70, 50 to 70, 55 to 70, 60 to 70, 65 to 70, 30 to 65, 35 to 65, 40 to 65, 45 to 65, 50 to 65, 55 to 65, 30 to 60, 35 to 60, 40 to 60, 45 to 60, 50 to 60, 30 to 55, 30 to 55, 40 to 55, 45 to 55, 30 to 50, 35 to 50, 40 to 50, 45 to 50, 30 to 45, 35 to 45, 40 to 45, 30 to 40, or 35 to 40% by volume. More specifically, CO2 may be about 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 or 80% by volume. Even more specifically, CO2 may range from 35 to 65% by volume.

[0015] The H2 in the gas composition according to any embodiment of the present invention may be in the range of 10 to 50 by volume, particularly in the range of 15 to 50, 20 to 50, 25 to 50, 30 to 50, 35 to 50, 40 to 50, 10 to 45, 15 to 45, 20 to 45, 25 to 45, 30 to 45, 35 to 45, 40 to 45, 10 to 40, 15 to 40, 20 to 40, 25 to 40, 30 to 40, 35 to 40, 10 to 35, 15 to 35, 20 to 35, 25 to 35, 30 to 35, 10 to 30, 15 to 30, 20 to 30, 25 to 30, 10 to 25, 15 to 25, 20 to 25, 10 to 20, or 15 to 20 by volume. More specifically, H2 may be approximately 15, 20, 25, 30, 35, 40, 45, or 50 volume percent. Even more specifically, H2 may be in the range of 20 to 40 volume percent.

[0016] The CO in the gas composition according to any aspect of the present invention may be in the range of 1 to 30 volume%, particularly in the range of 5 to 30, 10 to 30, 15 to 30, 20 to 30, 25 to 30, 1 to 25, 5 to 25, 10 to 25, 15 to 25, 20 to 25, 1 to 20, 5 to 20, 10 to 20, 15 to 20, 1 to 15, 5 to 15, 10 to 15, 1 to 10, or 5 to 10 volume%. More specifically, the CO may be about 5, 10, 15, 20, 25, or 30 volume%. Even more specifically, the H2 may be in the range of 5 to 20 volume%.

[0017] The amount of CH4 in the gas composition according to any aspect of the present invention is in the range of 0.01 to 30 volume%, particularly 0.01 to 25, 0.01 to 20, 0.01 to 15, 0.01 to 10, 0.01 to 5, 0.01 to 1, 0.01 to 0.5, 0.01 to 0.1, 0.01 to 0.05, 0.05 to 30, 0.05 to 25, 0.05 to 20, 0.05 to 15, 0.05 to 10, 0.05 to 5, 0.05 to 1, 0.05 to 0.5, 0.05 to 0.1, 0.1 to 30, 0. The range may be 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 may 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 volume percent. Even more specifically, CH4 may be in the range of 0.01 to 20 volume percent.

[0018] More specifically, the O2 concentration in the gas composition may be less than 1 volume percent of the total amount of gas in the gas composition. In particular, oxygen may be present in the following concentration ranges: 0.000005-2 vol%, 0.00005-2 vol%, 0.0005-2 vol%, 0.005-2 vol%, 0.05-2 vol%, 0.00005-1.5 vol%, 0.0005-1.5 vol%, 0.005-1.5 vol%, 0.05-1.5 vol%, 0.5-1.5 vol%, 0.00005-1 vol%, 0.0005-1 vol%, 0.005-1 vol%, 0.005-1 vol%, 0.05-1 vol%, 0.55-1 vol%, 0.60-1 vol%, and especially 0.60-1.5 vol%, 0.65-1 vol%, and 0.70-1 vol%. In particular, acetic acid-producing microorganisms are especially suitable when the proportion of O2 in the gas phase / 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, and 2 volume percent relative to the volume of gas in the gas composition. Those skilled in the art can use all methods known in the art to measure the volume concentration of oxygen in the gas composition. In particular, the volume of oxygen may be measured using all methods known in the art. As an example, the oxygen concentration in the gas phase can be measured using a micro-oxygen immersion probe from PreSens Precision Sensing GmbH. The oxygen concentration can be measured by fluorescence quenching, and the degree of quenching correlates with the partial pressure of oxygen in the gas phase.

[0019] The H2S concentration in the gas composition according to any aspect of the present invention may be 0.00000001% by volume to 0.0001% by volume.

[0020] The N2 concentration in the gas composition according to any aspect of the present invention may be 0.1 to 0.7 volume percent.

[0021] As used herein, the term "approximately" refers to a variation of no more than 20%. In particular, as used herein, the term "approximately" refers to ±20%, more specifically ±10%, and even more specifically ±5% of a given measurement or value.

[0022] All percentages (%) are volume percentages unless otherwise specified.

[0023] The gas composition according to any aspect of the present invention may be off-gas or waste gas from any industrial process. In particular, the off-gas or waste gas according to any aspect of the present invention may be off-gas or waste gas from at least one steam methane reforming plant. Off-gas from a steam methane reforming plant refers to by-products of the steam methane reforming process, i.e., unwanted gases generated as a result of the steam methane reforming process. In particular, off-gas from a steam methane reforming plant serves as a carbon source in the acetone production method according to any aspect of the present invention.

[0024] Steam methane reforming (SMR) is a method of heating methane derived from natural gas with steam in the presence of a catalyst to produce off-gas or waste gas mainly containing CO, CO2, H2, and CH4. This off-gas or waste gas may be used to produce acetone in any aspect of the present invention. At least two reactions take place in the steam methane reforming apparatus, which are as follows: (1) Steam methane reforming reaction CH4+H2O(+heat) → CO+3H2 (2) Water gas shift reaction CO + H2O → CO2 + H2 (+ a small amount of heat)

[0025] The gas concentration (i.e., gas composition) in the off-gas or waste gas from a steam methane reformer is suitable for direct use in microbial fermentation for acetone production.

[0026] Therefore, off-gas or waste gas from a steam methane reformer may be directly contacted with at least one genetically modified homoacetic acid-producing bacterium for acetone production. Specifically, this bacterium is directly or immediately contacted with the off-gas or waste gas from the steam methane reformer without any additional steps, particularly a purification step of the off-gas or waste gas for preparing the gas used for fermentation. Thus, the off-gas or waste gas from the steam methane reformer is suitable for direct use in a fermenter for acetone production. More specifically, the off-gas or waste gas from the steam methane reformer is directly contacted with the bacterium without requiring prior pretreatment or washing.

[0027] As used herein, the term “homoacetic acid-producing bacteria” is interchangeable with the term “acetic acid-producing bacteria,” and refers to microorganisms that possess the Wood-Ljungdahl pathway and can convert CO, CO2, and / or hydrogen into acetic acid. These microorganisms include those that do not possess the Wood-Ljungdahl pathway in the wild type but have acquired this characteristic as a result of genetic modification. Such microorganisms include, but are not limited to, E. coli cells. These microorganisms are also known as carboxydotrophic bacteria. Currently, 21 different genera of acetate-producing bacteria are known in the art (Drake et al., 2006), and these may include the genus Clostridia (Drake & Kusel, 2005). These bacteria can utilize carbon dioxide or carbon monoxide as a carbon source and hydrogen as an energy source (Wood, 1991). Furthermore, alcohols, aldehydes, carboxylic acids, and numerous hexoses can also be used as carbon sources (Drake et al., 2004). The reduction pathway leading to the formation of acetic acid is called the acetyl-CoA pathway or the Wood-Ljungdahl pathway.

[0028] In particular, the acetic acid-producing bacteria used according to any aspect of the present invention are genetically modified bacteria that have been genetically modified to produce acetone from a carbon source, in particular off-gas or waste gas from a steam methane reformer. The genetically modified cells are acetic acid-producing cells that, compared to wild-type cells, have been genetically modified to increase the expression of enzymes that enable acetone production from a carbon source, in particular off-gas or waste gas from a steam methane reformer.

[0029] As used herein, the phrase "enhanced heterologous expression of an enzyme" is understood to mean increased intracellular activity. Basically, increased enzyme activity can be achieved by increasing the copy number of one or more gene sequences encoding the enzyme, by using a strong promoter, or by using a gene or allele encoding the corresponding enzyme with enhanced activity, or by combining these means as necessary.

[0030] Genetically modified cells used in the method of the present invention are produced, for example, by transformation, transduction, conjugation, or a combination thereof, using a vector containing a desired gene, an allele of that gene, or a portion thereof, and a vector that enables the expression of said gene. Heterogeneous expression is achieved, in particular, by incorporating the gene or allele into the cell's chromosome or an extrachromosomal replication vector. In particular, the increase in enzyme activity compared to wild-type cells may be 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% compared to wild-type cells.

[0031] Those skilled in the art will be able to genetically modify cells using any method known in the art. Whether a nucleic acid molecule, polypeptide, or more specifically, an enzyme used according to any aspect of the present invention is recombinant or not does not necessarily affect its expression level. However, in one example, one or more recombinant nucleic acid molecules, polypeptides, or enzymes used according to any aspect of the present invention may be overexpressed. In this specification, the term “overexpression” means that each polypeptide encoded or expressed is expressed at a higher level or activity than would normally be seen in each wild-type cell under the same conditions, for example, if no genetic modification to increase expression has been performed. Those skilled in the art are familiar with various methods for inducing overexpression. For example, the nucleic acid molecule to be overexpressed, or the nucleic acid molecule encoding the polypeptide or enzyme to be overexpressed, may be under the control of a strong inducible promoter, such as the lac promoter. The state of the art describes standard plasmids that can be used for this purpose, such as the pET vector system, exemplified by pET-3a (commercially available from Novagen). Whether nucleic acids or polypeptides are overexpressed may be determined by quantitative PCR in the case of nucleic acid molecules, or by SDS-polyacrylamide electrophoresis, Western blotting, or comparative activity assay in the case of polypeptides. Genetic modifications may target transcription, translation, and / or post-translational modifications that result in changes in enzyme activity and / or selectivity under selective and / or specified culture conditions. Thus, in various examples of the present invention, microorganisms may have one or more gene deletions to function more efficiently. Gene deletions may be performed by the mutant gene deletion method and / or by starting with mutant strains in which the expression of one or more of these enzymes is reduced or absent, and / or by other methods known to those skilled in the art.

[0032] German Patent Application Publication No. 10031999 outlines the possibility of increasing intracellular enzyme activity, using pyruvate carboxylase as an example. This document is inserted herein by reference, and its disclosure regarding the possibility of increasing intracellular enzyme activity constitutes part of the disclosure of the present invention.

[0033] The expression of all enzymes or genes described above and below can be detected by one-dimensional and two-dimensional protein gel separation, as well as by optical identification of protein concentrations in the gel using appropriate analytical software. If the increase in enzyme activity is based solely on the increase in the expression of the corresponding gene, the quantification of the increase in enzyme activity can be easily performed by comparing one-dimensional or two-dimensional protein separation between wild-type cells and genetically modified cells. A general method for preparing protein gels and identifying proteins in Corynebacterium is the procedure described by Hermann et al. (Electrophoresis, 22: 1712.23 (2001)). Protein concentrations can be similarly analyzed by Western blot hybridization using antibodies specific to the target protein (Sambrook et al., Molecular Cloning: a laboratory manual, 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY USA, 1989) and by optical analysis using appropriate concentration measurement 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 assay (also called 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 by various well-known reporter gene assay methods (Sambrook et al., Molecular Cloning: a laboratory manual, 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY USA, 1989).Intracellular enzyme activity can be determined according to 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). In the following embodiments, unless a practical method for measuring the activity of a particular enzyme is shown, the determination of an increase in enzyme activity and the determination of a decrease in enzyme activity are preferably carried out by the following methods: Hermann et al., Electophoresis, 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).

[0034] If the increase in enzyme activity is achieved through mutations in endogenous genes, such mutations can be induced randomly by conventional methods, such as UV irradiation or mutagenic agents, or selectively by genetic engineering techniques, such as deletion, insertion, and / or nucleotide exchange. Modified cells can be obtained from these mutations. Particularly preferred enzyme mutants are those in which feedback inhibition, product inhibition, or substrate inhibition no longer occurs, or at least to a reduced degree compared to the wild-type enzyme.

[0035] When increased enzyme activity is achieved through increased enzyme synthesis, the copy number of the corresponding gene increases, or the promoter and regulatory regions or ribosome-binding sites located upstream of the structural gene mutate. Expression cassettes integrated upstream of the structural gene act similarly. Furthermore, it is possible to increase expression at any given time using inductive promoters. In addition, "enhancers" can be added to the enzyme gene as regulatory sequences, which similarly increase gene expression by improving the interaction between RNA polymerase and DNA. Expression is also improved as a result of measures to extend the lifespan of mRNA. Furthermore, enzyme activity is similarly enhanced by suppressing the degradation of the enzyme protein. The gene or gene construct exists in plasmids with different copy numbers or is amplified by integration into chromosomes. Alternatively, overexpression of the gene can be further achieved by changing the culture medium composition and culture management.Those skilled in the art may, in particular, refer to the following as guidance: 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)), European Patent Application Publication No. 0472869, U.S. Patent No. 4,601,893, Schwarzer and Puhler (Bio / Technology 9, 84-87 (1991)), Reinscheid et al. (Applied and Environmental Microbiology 60, 126-132 (1994)), LaBarre et al. (Journal of Bacteriology 175, 1001-1007 (1993), International Publication No. 96 / 15246, Malumbres et al. (Genes 134, 15-24 (1993)), Japanese Patent Application Publication No. 10-229891, Jensen and Hammer (Biotechnology and Bioengineering 58, 191-195 (1998)), and famous textbooks on genetics and molecular biology. The above methods, as well as mutations, result in genetically modified cells.

[0036] For example, episomal plasmids are used to increase the expression of individual 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. For further preferred plasmids and vectors, see: 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 ed., Cold Spring Harbor Laboratory Press, New York.

[0037] Plasmid vectors containing the gene to be amplified are converted into the target strain by conjugation or transformation. Conjugation methods are described, for example, in Schafer 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). The resulting strain, obtained as a result of homologous recombination via the "crossover" phenomenon, contains at least two copies of the target gene. In particular, to enhance the activity of at least one enzyme E1, E2 and / or E3, the following are included: a) At least one promoter operably linked to a gene encoding any one of the enzymes E1, E2, and / or E3 in the appropriate chromosome of the cell, or b) At least one expression vector for increasing the copy number of a gene encoding any one of the enzymes E1, E2, and / or E3 in a cell, or c) A combination of (a) and (b).

[0038] According to any aspect of the present invention, cells may be genetically modified to produce at least twice, particularly at least ten times, at least 100 times, at least 1000 times, or at least 10000 times more ketone bodies than wild-type cells within a specified time interval of 2 hours, particularly within 8 hours or 24 hours. The increase in product production can be determined, for example, by culturing cells according to any aspect of the present invention and wild-type cells separately under identical conditions (same cell density, same nutrient medium, same culture conditions) in a suitable nutrient medium for a predetermined time, and then measuring the amount of the target product (lipid represented by general formula II or general formula I) in the nutrient medium.

[0039] In a similar context, "Enzyme E" as used in relation to any aspect of the present invention x The phrase "decreased activity and / or expression" should be understood to mean that the activity has decreased by at least 0.5 times, particularly at least 0.1 times, more specifically at least 0.01 times, even more specifically at least 0.001 times, and most specifically at least 0.0001 times. The phrase "decreased activity" also includes the absence of any detectable activity ("zero activity"). The activity of a particular enzyme can be reduced, for example, by selective mutation or by other means known to those skilled in the art to reduce the activity of a particular enzyme. Specifically, those skilled in the art can find instructions regarding the modification and reduction of protein expression, and the resulting decrease in enzyme activity, by inhibiting a particular gene, for example, in Dubeau et al. 2009, Singh & Rohm. 2008, Lee et al., 2009, etc. The reduction of enzyme activity in cells according to any aspect of the present invention can be achieved by modifying a gene containing one nucleic acid sequence, the modification being selected from the group consisting of insertion of foreign DNA into the gene, deletion of at least a portion of the gene, point mutation in the gene sequence, RNA interference (siRNA), antisense RNA, or regulatory sequences adjacent to the gene (e.g., promoter, terminator), or modification of the ribosome binding site (insertion, deletion, or point mutation). In particular, to reduce enzyme activity in cells, the cells may include the following: 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), antisense RNA, or in the gene encoding the enzyme and / or the regulatory sequence of the gene encoding the enzyme. d) A combination of (d), (e), and (f).

[0040] The expression of all enzymes and genes listed above and below can be measured by one-dimensional and two-dimensional protein gel separation followed by optical identification of protein concentrations in the gel using appropriate evaluation software.

[0041] Homoacetic acid-producing bacteria according to any aspect of the present invention may be genetically modified to increase the expression of at least one of the following enzymes compared to the wild type: - Thiolase (ThlA, E1) (EC2.3.1.9), - CoA transferase (CtfAB, E2) (EC2.8.3.8), and / or - Acetacetate decarboxylase (Adc, E3) (EC 4.1.1.4). and / or may be genetically modified to reduce the expression of at least the following enzymes compared to the wild type: - Secondary alcohol dehydrogenase (sAdh, E4) (EC1.1.1.1).

[0042] For example, cells may be genetically modified to increase the expression of enzymes E1, E2, and E3 compared to wild-type cells, and to decrease the expression of enzyme E4 compared to wild-type cells.

[0043] In particular, E1 may be capable of catalyzing the conversion of acetyl-CoA to acetoacetyl-CoA. E1 may be acetoacetyl-CoA thiolase, also known as acetyl coenzyme A acetyltransferase. Acetoacetyl-CoA thiolase enzymes include the gene product of atoB (Martin et al., 2003) derived from E. coli (accession number NP_416728) and thiolase derived from C. acetobutylicum. More specifically, E1 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: 2. Even more specifically, cells according to any aspect of the present invention may be genetically modified to contain the sequence of SEQ ID NO: 1.

[0044] Those skilled in the art can identify other thioases that can perform the role of E1. In particular, those skilled in the art can use numerous known methods to evaluate whether functionally equivalent mutants have substantially the same function as the mutant nucleic acid or polypeptide. For example, the enzymatic activity of E1 can be evaluated using the methods outlined in Wiesenborn et al. 1988, Wiesenborn et al. 1989, Peterson and Bennett, 1990, Ismail et al. 1993, and de la Plaza et al. 2004.

[0045] E2 may be acetoacetate-CoA transferase (EC 2.8.3.9). Acetoacetate-CoA transferases conserve energy stored in the CoA-ester bond. These enzymes may spontaneously exhibit the desired acetoacetyl-CoA transferase activity, or they may be modified by directed evolution to more efficiently accept acetoacetyl-CoA as a substrate. In particular, such enzymes may be able to catalyze the conversion of 3-hydroxybutyryl-CoA to 3-hydroxybutyrate via the transferase mechanism. Examples of E2 include CoA transferase from E. coli (accession numbers P76459.1 or P76458.1) (Hanai et al., 2007), ctfAB from C. acetobutylicum (accession numbers NP_149326.1 or NP_149327.1) (Jojima et al., 2008), and ctfAB from Clostridium saccharoperbutylacetonicum (accession numbers AAP42564.1 or AAP42565.1) (Kosaka et al., 2007).In particular, E2 is associated with the gene products of catl, cat2, and cat3 of Clostridium kluyveri (accession numbers P38946.1, P38942.2, and EDK35586.1, respectively) (Seedorf et al., 2008; Sohling and Gottschalk, 1996), the transferase product of Trichomonas vaginalis (accession number XP_001330176) (van Grinsven et al., 2008), Trypanosoma brucei (accession number XP_828352) (Riviere et al., 2004), Fusobacterium nucleatum (Barker et al., 1982), Clostridium SB4 (Barker et al., 1978), and Clostridium acetobutylicum (Wiesenborn et al., 1989). The group may be selected from the following: FN0272 and FN0273 (accession numbers NP_603179.1 and NP_603180.1, respectively) (Kapatral et al., 2002), homologs FN1857 and FN1856 within Fusobacterium nucleatum (accession numbers NP_602657.1 and NP_602656.1) (Kreimeyer et al., 2007), the transferase product of Porphyrmonas gingivalis (accession number NP_905281.1 or NP_905290.1), and Thermoanaerobacter tengcongensis (accession number NP_622378.1 or NP_622379.1) (Kreimeyer et al., 2007). More specifically, E2 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: 4 or SEQ ID NO: 6. In particular, E2 may contain the amino acid sequences of SEQ ID NO: 4 and SEQ ID NO: 6. E2 may contain a nucleotide sequence having 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% sequence identity with SEQ ID NO: 3 or SEQ ID NO: 5.More specifically, E2 may contain nucleotide sequences having 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% sequence identity with respect to SEQ ID NO: 3 and SEQ ID NO: 5.

[0046] E2 expression may be measured using any method known in the art. In particular, an increase in E2 expression may be measured by measuring 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. Alternatively, E2 expression may be measured by measuring the amount of E2 protein expressed in the resulting culture medium. For example, E2 expression may be measured using the method disclosed in Charrier C., 2006.

[0047] E3 may be acetacetate decarboxylase (Adc; EC4.1.1.4). The acetacetate decarboxylase used according to any aspect of the present invention is selected from the proteins having polypeptide sequences in which up to 60%, preferably up to 25%, particularly preferably up to 15%, and especially up to 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% of amino acid residues are modified by deletion, insertion, substitution or combination thereof, with respect to the aforementioned reference sequences, and which still have at least 50%, preferably 65%, particularly preferably 80%, and especially more than 90% of the activity of the protein having the corresponding aforementioned reference sequence. 100% activity of a reference protein is understood to mean the increase in the activity of cells 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 relative to the amount of cells used (units [U / g CDW] per gram of cell dry weight), and this activity is related to the measurement of the activity of enzyme E3. 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. In particular, E2 may contain the amino acid sequence of SEQ ID NO: 7.

[0048] The method for measuring activity is described in Daniel et al., Appl. Environ. Microbiol. 1990, pp. 3491-3498, Vol. 56, No. 11.

[0049] E4 may be an alcohol dehydrogenase. "Alcohol dehydrogenase" may 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 and primary alcohol dehydrogenases. "Secondary alcohol dehydrogenase" is an enzyme capable of converting ketones (such as acetone) to secondary alcohols (such as isopropanol) or vice versa. "Primary alcohol dehydrogenase" is an enzyme capable of converting 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 are also called "primary-secondary alcohol dehydrogenases." The membrane-bound flavin-dependent alcohol dehydrogenase Pseudomonas putida GPO1 AlkJ type uses a flavor cofactor instead of NAD+. Another group includes iron-containing oxygen-sensitive alcohol dehydrogenases that exist in an inactive form in bacteria and yeast. Yet another group includes NAD+-dependent alcohol dehydrogenases (such as zinc-containing alcohol dehydrogenases) that have a cysteine-coordinated zinc atom at their active site, which immobilizes the alcohol substrate. For example, the term "alcohol dehydrogenase" as used herein is understood to mean an enzyme that oxidizes an aldehyde or ketone to the corresponding primary or secondary alcohol. In particular, the alcohol dehydrogenase according to any aspect of the present invention may be an NAD+-dependent alcohol dehydrogenase, i.e., an alcohol dehydrogenase that uses NAD+ as a cofactor for the oxidation of alcohol or NADH for the reduction of the corresponding aldehyde or ketone. In the most preferred embodiment, the alcohol dehydrogenase is an NAD+-dependent zinc-containing alcohol dehydrogenase.A suitable example of a suitable NAD+-dependent alcohol dehydrogenase is alcohol dehydrogenase A (database code AJ491307.1) derived from Rhodococcus ruber or its variants. Other examples include alcohol dehydrogenases derived from Ralstonia eutropha (ACB78191.1), Lactobacillus brevis (YP_795183.1), Lactobacillus kefiri (ACF95832.1), horse liver, Paracoccus pantotrophus (ACB78182.1), and Sphingobium yanoikuyae (EU427523.1), as well as their variants. For example, as used herein, the expression “NAD(P)+-dependent alcohol dehydrogenase” refers to an alcohol dehydrogenase that is NAD+ and / or NADP+-dependent.

[0050] For example, E4 may be a secondary alcohol dehydrogenase, or selected from other alcohol dehydrogenases or equivalent aldehyde reductases, and may also function as a candidate for 3-hydroxybutyraldehyde reductase. E4 may be a gene product selected from the group consisting of adhl from Geobacillus thermoglucosidasius (accession number AAR91477.1) (Jeon et al., 2008) and SADH from C. beijerinckii, or the alcohol dehydrogenase disclosed by Tani et al., 2000 (accession number BAB122273.1) may be used as E4. E4 may be selected from the group consisting of ADH2 from Saccharomyces cerevisiae (accession number NP_014032.1) (Atsumi et al., 2008), yqhD from E. coli (accession number NP_417484.1) (Sulzenbacher et al., 2004 and Perez et al., 2008), bdh I and bdh II from C. acetobutylicum (accession numbers NP_349892.1 and NP_349891.1 respectively) (Walter et al., 1992), and ADH1 from Zymomonas mobilis (accession number YP_162971.1) (Kinoshita et al., 1985).

[0051] More specifically, E4 may be a secondary alcohol dehydrogenase. More specifically, E4 may be a secondary alcohol dehydrogenase derived from C. beijerinckii. 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.

[0052] Cells according to any aspect of the present invention may be genetically modified to remove the expression of adc using standard recombinant DNA techniques known to those skilled in the art, in order to significantly reduce the expression level of adc. The gene sequences involved in adc production may be inactivated, partially or completely removed. Thus, cells according to any aspect of the present invention express adc at reduced levels, are undetectable, or express functionally inactive adc.

[0053] In one example, cells according to any aspect of the present invention may naturally express E4 and be genetically modified to reduce intracellular E4 expression to about 0% or undetectable levels compared to wild-type cells. In another example, cells according to any aspect of the present invention have an E4 expression level of about 0% or undetectable levels in their wild type. Specifically, in the case of cells according to any aspect of the present invention, the expression of enzyme E4 is undetectable.

[0054] The accession numbers used in this application refer to each sequence from the Genbank database operated by NCBI. The releases referenced herein are those available online as of March 30, 2015.

[0055] E4 expression may be measured using all methods known in the art. In particular, an increase in E4 expression may be measured by measuring 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 E4. In another example, E4 expression may be measured by measuring the amount of E4 protein expressed in the resulting culture medium. In one example, E4 expression may be measured using the method disclosed in Ismaiel, AA (1993).

[0056] In particular, the acetic acid-producing bacteria genetically modified according to any aspect of the present invention are selected from the following group: Acetoanaerobium notera (ATCC 35199), Acetonema longum (DSM 6540), Acetobacterium carbinolicum (DSM 2925), Acetobacterium malicum (DSM 4132), Acetobacterium no. 446 (Morinaga et al., 1990, J. Biotechnol., Vol. 14, p. 187-194), Acetobacterium wieringae (DSM 1911), Acetobacterium woodii (DSM 1030), Alkalibaculum bacchi (DSM 22112), Archaeoglobus fulgidus (DSM 4304), Blautia producta (DSM 2950, ​​formerly known as Ruminococcus productus) Clostridium carboxidivorans (DSM 15243), Clostridium drakei (ATCC BAA-623), Clostridium aceticum (DSM 1496), Clostridium autoethanogenum (DSM 10061) formicoaceticum (DSM 92), Clostridium glycolicum (DSM 1288), Clostridium ljungdahlii (DSM 13528), Clostridium mayombei (DSM 6539), Clostridium methoxybenzovorans (DSM 12182), Clostridium ragsdalei (DSM 15248), Clostridium scatologenes (DSM 757), Clostridium sp. ATCC 29797 (Schmidt et al., 1986, Chem. Eng.)Commun., Vol. 45, p. 61-73), Desulfotomaculum kuznetsovii (DSM 6115), Desulfotomaculum thermobezoicum subspecies thermosyntrophicum (DSM 14055), Eubacterium limosum (DSM 20543), Methanosarcina acetivorans C2A (DSM 2834), Moorella HUC22-1 (Sakai et al., 2004, Biotechnol. Let., Vol. 29, p. 1607-1612), Moorella thermoacetica (DSM 521, formerly Clostridium thermoaceticum), Moorella thermoautotrophica (DSM 1974), Oxobacter pfennigii (DSM 3222), Sporomusa aerivorans (DSM 13326) *Clostridium ovata* (DSM 2662), *Sporomusa silvacetica* (DSM 10669), *Sporomusa sphaeroides* (DSM 2875), *Sporomusa termitida* (DSM 4440), and *Thermoanaerobacter kivui* (DSM 2030, formerly *Acetogenium kivui*). More specifically, *Clostridium carboxidivorans* strain ATCC BAA-624 may be used. Even more specifically, bacterial strains of *Clostridium carboxidivorans* labeled "P7" and "P11," as described, for example, in U.S. Patent Application Publication No. 2007 / 0275447 and U.S. Patent Application Publication No. 2008 / 0057554, may be used.

[0057] Another particularly suitable bacterium is Clostridium ljungdahlii. Specifically, strains selected from the group consisting of Clostridium ljungdahlii PETC, Clostridium ljungdahlii ERI2, Clostridium ljungdahlii COL, and Clostridium ljungdahlii O-52 may be used for the conversion of synthesis gas to hexanoic acid. These strains are described, for example, in International Publication No. 98 / 00558, International Publication No. 00 / 68407, ATCC 49587, ATCC 55988, and ATCC 55989. More specifically, homoacetic acid-producing bacteria may be selected from the family Clostridium.

[0058] Homoacetic acid-producing bacteria are selected from the group consisting of Clostridium autoethanogenum (DSM 10061, DSM 19630 and DSM 23693), Clostridium ljungdahlii (DSM 13528), Clostridium carboxidivorans (DSM 15243), Acetobacterium woodii (DSM 1030), Clostridium ragsdalei (DSM 15248), Clostridium drakei (ATCC BAA-623), Moorella thermoacetica (DSM 521), Moorella thermoautotrophica (DSM 1974), Sporomusa silvacetica (DSM 10669), and Alkalibaculum bacchi (DSM 22112). In particular, homoacetic acid-producing bacteria may be selected from the group consisting of Clostridium autoethanogenum, Clostridium ljungdahlii, and Clostridium carboxidivorans. In one example, genetically modified C. autoethanogenum may be used for acetone production. In another example, genetically modified Clostridium ljungdahlii may be used for acetone production.

[0059] A method according to any aspect of the present invention is, (a) A process that receives an off-gas or exhaust gas stream from a steam methane reformer. It has. As used herein, the term “flow” refers to a flow of material that enters, passes through, and exits one or more stages of a process, for example, material supplied to a bioreactor where fermentation takes place. The composition of the flow may change as it passes through certain stages. For example, a flow entering a bioreactor may contain large amounts of CO, CO2, H2, and CH4, and low concentrations of N2, O2, and H2S. As the flow passes through the bioreactor, the CO, CO2, and H2 content in the flow may decrease.

[0060] As used herein, terms such as “fermentation process,” “fermentation reaction,” or “microbial fermentation” are intended to encompass both the growth step and the biosynthesis step of the product (particularly acetone) of the method according to any aspect of the present invention.

[0061] Further steps for acetone purification may be included. In particular, acetone may be purified directly from the crude product stream by distillation after the fermentation process.

[0062] According to another aspect of the present invention, the apparatus for producing acetone from a gaseous composition by microbial fermentation is as follows: (i) A gaseous composition source for continuously supplying off-gas or exhaust gas streams from a steam methane reformer containing at least CO, CO2, H2, and CH4, (ii) Bioreactor inlet for receiving off-gas or exhaust gas from the steam methane reformer, (iii) A bioreactor containing a culture of homoacetic acid-producing bacteria, (iv) Bioreactor outlet for discharging off-gas or waste gas after contact with homoacetic acid-producing bacteria Equipped with, The supply source is provided, which is an apparatus that supplies a gaseous composition obtained directly from a steam methane reformer.

[0063] A further aspect of the present invention provides a use of off-gas or waste gas from a steam methane reformer for the production of acetone, wherein the off-gas or waste gas comprises at least CO, CO2, H2, and CH4, and the off-gas or waste gas is brought into contact with at least one homoacetogenic bacterium.

[0064] A further aspect of the present invention provides a steam methane reformer adapted to produce acetone by microbial fermentation of off-gas or waste gas from a steam methane reformer. [Examples]

[0065] The above describes preferred embodiments, and modifications or changes can be made to the design, configuration, or operation without departing from the claims, as will be understood by those skilled in the art. For example, such modifications will be included within the claims.

[0066] Experimental Example 1 High-level acetone production by Clostridium ljungdahlii using off-gas from SMR. For the in vivo conversion of hydrogen, carbon monoxide, and carbon dioxide to acetone, Clostridium ljungdahlii (a GMO based on European Patent No. 2421960) was cultured using off-gas from a steam methane reformer as a genetically modified homoacetic acid-producing bacterium. All culture steps were carried out under anaerobic conditions in pressure-resistant glass bottles that could be airtightly sealed with butyl rubber stoppers, or in a stirred-tank type stainless steel benchtop bioreactor.

[0067] In the pre-culture, 500 mL of medium (ATCC1754- 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 MgSO4×7 H2O; 0.02 g / L CaCl2×2 H2O; 20 mg / L nitrilotriacetic acid; 10 mg / L MnSO4×H2O; 8 mg / L (NH4)2Fe(SO4)2×6 H2O; 2 mg / L CoCl2×6 H2O; 2 mg / L ZnSO4×7 H2O; 0.2 mg / L CuCl2×2 H2O; 0.2 mg / L Na2MoO4×2 H2O; 0.2 mg / L NiCl2×6 H2O; 0.2 mg / L Na2SeO4; 0.2 mg / L Na2WO4×2 H2O; 20 μg / L d-biotin; 20 μg / L folic acid; 100 μg / L pyridoxine-HCl; 50 μg / L thiamine-HCl×H2O; 50 μg / L riboflavin; 50 μg / L nicotinic acid; 50 μg / L calcium pantothenate; 1 μg / L vitamin B12; 50 μg / L p-aminobenzoic acid; 50 μg / L lipoic acid; approximately 67.5 mg / L To NaOH, 400 mg / L L-cysteine ​​hydrochloride and 400 mg / L Na2S×9 H2O were added, and 2.5 μL of frozen stock of C. ljungdahlii GMO was inoculated. Using a 1L pressure-resistant glass bottle, a mixed gas (CO2: 49%, H) from a steam methane reformer was measured under the conditions of 37°C, 150 rpm, and a ventilation rate of 1 L / hour. 2: Chemolithoautotrophic culture was performed for 66 hours in an open water bath shaker with a 30% CO:10.2%, CH4:10.2%, N2:0.7%, H2S:100ppb, O2:10ppm solution. The gas was discharged into the culture medium through a 10μm pore size sparger placed in the center of the reactor. The culture was performed without pH control.

[0068] In the main culture, OD 600nmThe number of cells required to achieve a value of 0.1 were transferred from the pre-culture to 500 mL of fresh medium. For main culture, LM33 mineral medium (pH = 5.8, 0.5 g / L MgCl2, 0.21 g / L NaCl, 0.135 g / L CaCl2× 2H2O, 2.65 g / L NaH2PO4× 2H2O, 0.5 g / L KCl, 2.5 g / L NH4Cl, 15 mg / L nitrilotriacetic acid, 30 mg / L MgSO4× 7 H2O, 5 mg / L MnSO4× H2O, 1 mg / L FeSO4× 7 H2O, 8 mg / L Fe(SO4)2(NH4)2× 6 H2O, 2 mg / L CoCl2× 6 H2O, 2 mg / L ZnSO4× 7 H2O, 200 μg / L CuCl2× 2 H2O, 200μg / L KAl(SO4)2 × 12 H2O, 3 mg / L H3BO3, 300 μg / L Na2MoO4 × 2 H2O, 200 μg / L Na2SeO3, 200 μg / L NiCl2 × 6 H2O, 200 μg / L Na2WO4 × 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 Nicotinic Acid, 500 μg / L Calcium Pantothenate, 500 μg / L Vitamin B12, 500 μg / L p-Aminobenzoic Acid, 500 μg / L Lipoic Acid, 10 mg / L FeCl3, The mixture was aerated for 30 minutes in the off-gas mixture of a steam methane reformer, and then 500 mg / L of L-cysteine ​​hydrochloride was added before use.

[0069] Using a 1L pressure-resistant glass bottle, a mixed gas (CO2: 49%, H) from a steam methane reformer was measured at 37°C, 150 rpm, and a ventilation rate of 1 L / hour. 2:Chemolithoautotrophic culture was performed for 164 hours in an open water bath shaker using a 30% CO:10.2%, CH4:10.2%, N2:0.7%, H2S:100ppb, O2:10ppm mixture. Gas was discharged into the culture medium through a 10μm pore size sparger located in the center of the reactor. pH was maintained at 5.0 by automatically adding 100g / L NaOH solution using a Titrino pH control system (Methrom, Switzerland). During culture, OD 600nm Several 5 mL samples were taken to determine pH and product formation. Product concentration was measured by semi-quantitative 1H-NMR. Sodium trimethylsilylpropionate (T(M)SP) was used as an internal standard.

[0070] During the main culture in LM33 medium, the ethanol concentration was increased from 0 g / L to over 2 g / L, the acetic acid concentration from 0 g / L to over 0.5 g / L, and the acetone concentration from 0 g / L to over 2 g / L. 600nm The value reached a maximum of >2 after 100 hours of culture.

[0071] Experimental Example 2 Acetone production using off-gas from SMR with Acetobacterium woodii For the in vivo conversion of hydrogen, carbon monoxide, and carbon dioxide to acetone, Acetobacterium woodii (a GMO based on European Patent No. 2421960) was cultured using off-gas from a steam methane reformer as a genetically modified homoacetic acid-producing bacterium. All culture steps were carried out under anaerobic conditions in pressure-resistant glass bottles that could be airtightly sealed with butyl rubber stoppers, or in a stirred-tank type stainless steel benchtop bioreactor.

[0072] In pre-culture, 500 mL of medium (DSMZ135- medium: pH = 8.2; 1.0 g / L NH4Cl; 0.33 g / L KH2PO4; 0.45 g / L K2HPO4; 0.1 g / L MgSO4 × 7 H2O; 2.0 g / L yeast extract; 500 mg / L L-cysteine-HCl × H2O; 500 mg / L Na2S × 9 H2O; 10 g / L NaHCO3; 30 mg / L nitrilotriacetic acid; 60 mg / L MgSO4 × 7 H2O; 10 mg / L MnSO4 × H2O; 20 mg / L NaCl; 2 mg / L FeSO4 × 7 H2O; 3.6 mg / L CoSO4 × 7 H2O; 2 g / L CaCl2 × 2 H2O; 3.6 mg / L ZnSO4 × 7 H2O; 0.2 mg / L CuSO4 × 7 H2O; 0.4 mg / L KAl(SO4)2 × 12 H2O; 0.2 mg / L H3BO3; 0.2 mg / L Na2MoO4 × 2 H2O; 0.6 mg / L NiCl2 × 6 H2O; 6 μg / L Na2SeO3 × 5 H2O; 40 μg / L d-biotin; 40 μg / L folic acid; 200 μg / L pyridoxine-HCl; 100 μg / L thiamine-HCl × H2O; 100 μg / L riboflavin; 100 μg / L nicotinic acid; 100 μg / L calcium pantothenate; 2 μg / L vitamin B12; 100 μg / L p-aminobenzoic acid (100 μg / L lipoic acid) was inoculated with 2.5 μL of frozen stock of A. woodii GMO. In a 1 L pressure-resistant glass bottle, the mixture from a steam methane reformer (CO2: 49%, H) was collected under the conditions of 37°C, 150 rpm, and a ventilation rate of 1 L / hour. 2: Chemolithoautotrophic culture was performed for 66 hours in an open water bath shaker with a 30% CO:10.2%, CH4:10.2%, N2:0.7%, H2S:100ppb, O2:10ppm solution. The gas was discharged into the culture medium through a 10μm pore size sparger placed in the center of the reactor. The culture was performed without pH control.

[0073] In the main culture, OD 600nmThe necessary number of cells to set it to 0.1 was transferred from the preculture to 500 mL of fresh medium. In the main culture, DSMZ135 medium (aerated for 30 minutes with the off-gas mixture of a steam methane reformer) was used. Using a 1 L pressure-resistant glass bottle, at 37 °C, 150 rpm, and an aeration rate of 1 L / hour, a mixed gas from the steam methane reformer (CO2: 49%, H 2: 30%, CO: 10.2%, CH4: 10.2%, N2: 0.7%, H2S: 100 ppb, O2: 10 ppm) was chemolithoautotrophically cultured for 164 hours in an open water bath shaker. The gas was discharged into the medium through a sparger with a pore size of 10 μm installed at the center of the reactor. Regarding pH, it was maintained at 7.5 by automatically adding a 100 g / L NaOH solution with a Titrino pH control system (Methrom, Switzerland). During the culture, 5 mL samples were taken several times to determine OD 600nm , pH, and product formation. The measurement of the product concentration was performed by semi-quantitative 1H-NMR method. Sodium trimethylsilylpropionate (T(M)SP) was used as an internal standard.

[0074] During the main culture in DSMZ135 medium, the acetic acid concentration increased from 0 g / L to over 4 g / L, the acetone concentration increased from 0 g / L to over 0.2 g / L, and the isopropanol concentration increased from 0 g / L to over 4 g / L. OD 600nm reached a maximum value >2 after 100 hours of culture.

[0075] As can be seen from this experiment, compared with Experimental Example 1, A. woodii can produce acetone from the SMR off-gas, but the production amount was less than that in Experimental Example 1.

[0076] Experimental Example 3 Low production of acetone by Clostridium ljungdahlii using syngas For the in vivo conversion of hydrogen and carbon dioxide to acetone, Clostridium ljungdahlii (a GMO based on European Patent No. 2421960) was cultured using a synthesis gas mixture containing hydrogen, carbon dioxide, and methane as a genetically modified homoacetic acid-producing bacterium. All culture steps were carried out under anaerobic conditions in pressure-resistant glass bottles that could be airtightly sealed with butyl rubber stoppers, or in a stirred-tank type stainless steel benchtop bioreactor.

[0077] In the pre-culture, 500 mL of medium (ATCC1754- 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 MgSO4×7 H2O; 0.02 g / L CaCl2×2 H2O; 20 mg / L nitrilotriacetic acid; 10 mg / L MnSO4×H2O; 8 mg / L (NH4)2Fe(SO4)2×6 H2O; 2 mg / L CoCl2×6 H2O; 2 mg / L ZnSO4×7 H2O; 0.2 mg / L CuCl2×2 H2O; 0.2 mg / L Na2MoO4×2 H2O; 0.2 mg / L NiCl2×6 H2O; 0.2 mg / L Na2SeO4; 0.2 mg / L Na2WO4×2 H2O; 20 μg / L d-biotin; 20 μg / L folic acid; 100 μg / L pyridoxine-HCl; 50 μg / L thiamine-HCl×H2O; 50 μg / L riboflavin; 50 μg / L nicotinic acid; 50 μg / L calcium pantothenate; 1 μg / L vitamin B12; 50 μg / L p-aminobenzoic acid; 50 μg / L lipoic acid; approximately 67.5 mg / L To NaOH, 400 mg / L L-cysteine ​​hydrochloride and 400 mg / L Na2S×9 H2O were added, and 2.5 μL of frozen stock of C. ljungdahlii GMO was inoculated. Using a 1L pressure-resistant glass bottle, a mixed gas (CO2: 49%, H) from a steam methane reformer was measured under the conditions of 37°C, 150 rpm, and a ventilation rate of 1 L / hour. 2:Chemolithoautotrophic culture was performed for 66 hours in an open water bath shaker with a 30% CO:10.2%, CH4:10.2%, N2:0.7%, H2S:100ppb, O2:10ppm solution. The gas was discharged into the culture medium through a 10μm pore size sparger placed in the center of the reactor. The culture was performed without pH control.

[0078] In the main culture, OD 600nm The number of cells required to achieve a value of 0.1 were transferred from the pre-culture to 500 mL of fresh medium. For main culture, LM33 mineral medium (pH = 5.8, 0.5 g / L MgCl2, 0.21 g / L NaCl, 0.135 g / L CaCl2× 2H2O, 2.65 g / L NaH2PO4× 2H2O, 0.5 g / L KCl, 2.5 g / L NH4Cl, 15 mg / L nitrilotriacetic acid, 30 mg / L MgSO4× 7 H2O, 5 mg / L MnSO4× H2O, 1 mg / L FeSO4× 7 H2O, 8 mg / L Fe(SO4)2(NH4)2× 6 H2O, 2 mg / L CoCl2× 6 H2O, 2 mg / L ZnSO4× 7 H2O, 200 μg / L CuCl2× 2 H2O, 200μg / L KAl(SO4)2 × 12 H2O, 3 mg / L H3BO3, 300 μg / L Na2MoO4 × 2 H2O, 200 μg / L Na2SeO3, 200 μg / L NiCl2 × 6 H2O, 200 μg / L Na2WO4 × 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 Nicotinic acid, 500 μg / L Calcium pantothenate, 500 μg / L Vitamin B12, 500 μg / L p-Aminobenzoic acid, 500 μg / L Lipoic acid, 10 mg / L The culture solution was prepared by adding 500 mg / L of L-cysteine ​​hydrochloride to FeCl3 (aeration for 30 minutes with the H2 / CO2 / CH4 mixture used for the main culture).

[0079] Using a 1L pressure-resistant glass bottle, a gas mixture containing hydrogen, carbon dioxide, and methane (CO2:30%, H) was tested at 37°C, 150 rpm, and a ventilation rate of 1 L / hour. 2: A 60% (CH4:10%) culture was chemolithoautotrophically incubated in an open water bath shaker for 164 hours. Gas was discharged into the culture medium through a 10 μm pore size sparger located in the center of the reactor. pH was maintained at 5.0 by automatically adding 100 g / L of NaOH solution using a Titrino pH control system (Methrom, Switzerland). During incubation, OD 600nm Several 5 mL samples were taken to determine pH and product formation. Product concentration was measured by semi-quantitative 1H-NMR. Sodium trimethylsilylpropionate (T(M)SP) was used as an internal standard.

[0080] During the main culture in LM33 medium, the ethanol concentration was increased from 0 g / L to 1 g / L, the acetic acid concentration from 0 g / L to 0.5 g / L, and the acetone concentration from 0 g / L to over 1 g / L. 600nm The value reached a maximum of approximately 1 after 100 hours of incubation.

[0081] In this experimental example, when C. ljungdahlii was grown using a mixture of synthesis gas and a different carbon source that did not contain CO, the growth rate was lower compared to Experimental Example 1, and the amount of acetone produced was also reduced.

[0082] Experimental Example 4 Low production of acetone using Acetobacterium woodii with synthesis gas For the in vivo conversion of hydrogen, carbon monoxide, and carbon dioxide to acetone, Acetobacterium woodii (a GMO based on European Patent No. 2421960) was cultured using a synthesis gas mixture containing hydrogen, carbon dioxide, carbon monoxide, and methane as a genetically modified homoacetic acid-producing bacterium. All culture steps were carried out under anaerobic conditions in pressure-resistant glass bottles that could be airtightly sealed with butyl rubber stoppers, or in a stirred-tank type stainless steel benchtop bioreactor.

[0083] In pre-culture, 500 mL of medium (DSMZ135- medium: pH = 8.2; 1.0 g / L NH4Cl; 0.33 g / L KH2PO4; 0.45 g / L K2HPO4; 0.1 g / L MgSO4 × 7 H2O; 2.0 g / L yeast extract; 500 mg / L L-cysteine-HCl × H2O; 500 mg / L Na2S × 9 H2O; 10 g / L NaHCO3; 30 mg / L nitrilotriacetic acid; 60 mg / L MgSO4 × 7 H2O; 10 mg / L MnSO4 × H2O; 20 mg / L NaCl; 2 mg / L FeSO4 × 7 H2O; 3.6 mg / L CoSO4 × 7 H2O; 2 g / L CaCl2 × 2 H2O; 3.6 mg / L ZnSO4 × 7 H2O; 0.2 mg / L CuSO4 × 7 H2O; 0.4 mg / L KAl(SO4)2 × 12 H2O; 0.2 mg / L H3BO3; 0.2 mg / L Na2MoO4 × 2 H2O; 0.6 mg / L NiCl2 × 6 H2O; 6 μg / L Na2SeO3 × 5 H2O; 40 μg / L d-biotin; 40 μg / L folic acid; 200 μg / L pyridoxine-HCl; 100 μg / L thiamine-HCl × H2O; 100 μg / L riboflavin; 100 μg / L nicotinic acid; 100 μg / L calcium pantothenate; 2 μg / L vitamin B12; 100 μg / L p-aminobenzoic acid (100 μg / L lipoic acid) was inoculated with 2.5 μL of frozen stock of A. woodii GMO. In a 1 L pressure-resistant glass bottle, the mixture from a steam methane reformer (CO2: 49%, H) was collected under the conditions of 37°C, 150 rpm, and a ventilation rate of 1 L / hour. 2: Chemolithoautotrophic culture was performed for 66 hours in an open water bath shaker with a 30% CO:10.2%, CH4:10.2%, N2:0.7%, H2S:100ppb, O2:10ppm solution. The gas was discharged into the culture medium through a 10μm pore size sparger placed in the center of the reactor. The culture was performed without pH control.

[0084] In the main culture, OD600nm The number of cells required to achieve a pH of 0.1 were transferred from the pre-culture to 500 mL of fresh medium. For the main culture, DSMZ135 medium (aerated for 30 minutes with the H2 / CO2 / CO / CH4 mixture for main culture) was used. Using a 1 L pressure-resistant glass bottle, a gas mixture containing hydrogen, carbon dioxide, carbon monoxide, and methane (CO2: 22%, CO: 25%, H2: 43%, CH4: 10%) was chemolithoautotrophically cultured for 164 hours in an open water bath shaker at 30°C, 150 rpm, and a ventilation rate of 1 L / hour. The gas was discharged into the medium through a 10 μm pore size sparger located in the center of the reactor. pH was maintained at 7.5 by automatically adding 100 g / L of NaOH solution using a Titrino pH control system (Methrom, Switzerland). During culture, OD 600nm Several 5 mL samples were taken to determine pH and product formation. Product concentration was measured by semi-quantitative 1H-NMR. Sodium trimethylsilylpropionate (T(M)SP) was used as an internal standard.

[0085] During the main culture in DSMZ135 medium, the acetic acid concentration was increased from 0 g / L to 2 g / L, the acetone concentration from 0 g / L to 0.1 g / L, and the isopropanol concentration from 0 g / L to 0.2 g / L. 600nm The value reached a maximum of approximately 1 after 100 hours of incubation.

[0086] In this experimental example, when using a synthesis gas mixture with a high CO content, cell proliferation and acetone production were reduced in A. woodii compared to experimental example 2.

Claims

1. This is a method for producing acetone from a gaseous composition by microbial fermentation. At least one genetically modified homoacetic acid-producing bacterium, at least CO, CO ２ H ２ and CH ４ By bringing it into direct contact with a gas composition containing, The gas composition is an off-gas from at least one steam methane reforming apparatus, and the off-gas from the steam methane reforming apparatus is brought into direct contact with the genetically modified homoacetic acid-producing bacteria. A method wherein the homoacetic acid-producing bacteria are genetically modified to produce acetone from the gas composition.

2. The aforementioned homoacetic acid-producing bacteria produce the following enzyme: - Thiolase (ThlA) (E.C. 2.3.1.9), - CoA transferase (CtfAB) (EC 2.8.3.8) and / or - Acetacetate decarboxylase (Adc) (EC 4.1.1.4) Genetically modified to increase the expression of at least one of the following compared to its wild type, and / or at least - Secondary alcohol dehydrogenase (sAdh) (EC 1.1.1.1) The method according to claim 1, wherein the gene is modified to reduce the expression of compared to its wild type.

3. The method according to claim 1 or 2, wherein the off-gas from the steam methane reformer is brought into direct contact with the genetically modified homoacetic acid-producing bacteria without undergoing additional pretreatment and / or washing steps.

4. The homoacetic acid-producing bacteria to be genetically modified are: Acetoanaerobium notera (ATCC 35199), Acetonema longum (DSM 6540), Acetobacterium carbinolicum (DSM 2925), Acetobacterium malicum (DSM 4132), Acetobacterium genus no. 446, Acetobacterium wieringae (DSM 1911), Acetobacterium woodii (DSM 1030), Alkalibaculum bacchi (DSM 22112), Archaeoglobus fulgidus (DSM 4304), Blautia producta (DSM 2950), Butyribacterium methylotrophicum (DSM 3468), 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 ljungdahlii (DSM 13528), Clostridium mayombei (DSM 6539), Clostridium methoxybenzovorans (DSM 12182 [SR3]), Clostridium ragsdalei (DSM 15248), Clostridium scatologenes (DSM 757), Clostridium ATCC 29797, Desulfotomaculum kuznetsovii (DSM 6115), Desulfotomaculum thermobezoicum subspecies, thermosyntrophicum (DSM 14055), Eubacterium limosum (DSM 20543), Methanosarcina acetivorans C2A (DSM 2834), Moorella spp. HUC22-1, Moorella thermoacetica (DSM 521), Moorella thermoautotrophica (DSM 1974), Oxobacter pfennigii (DSM 3222), Sporomusa aerivorans (DSM 13326), Sporomusa ovata (DSM 2662), Sporomusa silvacetica (DSM 10669), Sporomusa sphaeroides (DSM 2875), Sporomusa termitida (DSM 4440), and Thermoanaerobacter kivui (DSM A method according to any one of claims 1 to 3, selected from the group consisting of 2030).

5. The method according to any one of claims 1 to 4, wherein the homoacetic acid-producing bacteria is selected from the group consisting of Clostridium autoethanogenum (DSM 10061), Clostridium ljungdahlii (DSM 13528), Clostridium carboxidivorans (DSM 15243), Acetobacterium woodii (DSM 1030), Clostridium ragsdalei (DSM 15248), Clostridium drakei (ATCC BAA-623), Moorella thermoacetica (DSM 521), Moorella thermoautotrophica (DSM 1974), Sporomusa silvacetica (DSM 10669), and Alkalibaculum bacchi (DSM 22112).

6. The gas composition further contains N ２ , O ２ , and H ２ A method according to any one of claims 1 to 5, comprising S.

7. The CO ２ is in the range of 35 to 65% by volume, the H ２ is in the range of 20 to 40% by volume, the CO is in the range of 5 to 20% by volume, and / or the CH ４ is in the range of 0.01 to 20% by volume, the method according to any one of claims 1 to 6.

8. The aforementioned O ２ The concentration is in the range of 0.000005 to 1 volume%, and / or the H ２ The method according to claim 6 or claim 7, wherein the S concentration is in the range of 0.00000001 to 0.0001 volume percent.

9. The method according to any one of claims 1 to 8, wherein the homoacetic acid-producing bacterium is a genetically modified Clostridium autoethanogenum or Clostridium ljungdahlii.

10. (a) The method according to any one of claims 1 to 9, comprising the step of receiving the off-gas flow from the steam methane reforming apparatus.

11. This is an apparatus for producing acetone from a gaseous composition by microbial fermentation. (i) at least CO, CO ２ H ２ and CH ４ A gaseous composition supply source for continuously supplying an off-gas flow from a steam methane reformer containing the following: (ii) Bioreactor inlet for receiving the off-gas from the steam methane reformer, (iii) Bioreactor containing culture of genetically modified homoacetic acid-producing bacteria, (iv) Bioreactor outlet for discharging the off-gas after contact with the homoacetic acid-producing bacteria Equipped with, The supply source provides the gaseous composition obtained directly from the steam methane reforming apparatus, The apparatus wherein the homoacetic acid-producing bacteria are genetically modified to produce acetone from the gaseous composition.

12. This involves using off-gas from a steam methane reformer to produce acetone. The off-gas is at least CO, CO ２ H ２ and CH ４ It includes and is brought into direct contact with at least one genetically modified homoacetic acid-producing bacterium, The aforementioned homoacetic acid-producing bacteria have been genetically modified to produce acetone from the gaseous composition.

13. The genetically modified homoacetic acid-producing bacterium is Acetoanaerobium notera (ATCC 35199), Acetonema longum (DSM 6540), Acetobacterium carbinolicum (DSM 2925), Acetobacterium malicum (DSM 4132), Acetobacterium sp. no. 446, Acetobacterium wieringae (DSM 1911), Acetobacterium woodii (DSM 1030), Alkalibaculum bacchi (DSM 22112), Archaeoglobus fulgidus (DSM 4304), Blautia producta (DSM 2950), Butyribacterium methylotrophicum (DSM 3468), Clostridium aceticum (DSM 1496), Clostridium autoethanogenum (DSM 10061), Clostridium carboxidivorans (DSM 15243), Clostridium drakei (ATCC BAA-623), Clostridium formicoaceticum (DSM 92), Clostridium glycolicum (DSM 1288), Clostridium ljungdahlii (DSM 13528), Clostridium mayombei (DSM 6539), Clostridium methoxybenzovorans (DSM 12182 [SR3]), Clostridium ragsdalei (DSM 15248), Clostridium scatologenes (DSM 757), Clostridium sp. ATCC 29797, Desulfotomaculum kuznetsovii (DSM 6115), Desulfotomaculum thermobezoicum subsp. thermosyntrophicum (DSM 14055), Eubacterium limosum (DSM 20543), Methanosarcina acetivorans C2A (DSMThe use according to claim 12, selected from the group consisting of 2834), Moorella HUC22-1, Moorella thermoacetica (DSM 521), Moorella thermoautotrophica (DSM 1974), Oxobacter pfennigii (DSM 3222), Sporomusa aerivorans (DSM 13326), Sporomusa ovata (DSM 2662), Sporomusa silvacetica (DSM 10669), Sporomusa sphaeroides (DSM 2875), Sporomusa termitida (DSM 4440), and Thermoanaerobacter kivui (DSM 2030).

14. The aforementioned off-gas or waste gas is further N ２ , O ２ , and H ２ Use according to claim 12 or claim 13, including S.

15. The use according to any one of claims 12 to 14, wherein the off-gas from the steam methane reformer is in direct contact with the genetically modified homoacetic acid-producing bacteria without undergoing additional pretreatment and / or washing steps.

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