Process for producing higher alcohols

Abstract

The invention relates to a method for producing higher alcohols. The invention also relates to a reaction mixture and a method for producing at least one higher alcohol, said method comprising a reaction mixture comprising a mixed culture of a first and a second microorganism in an aqueous medium comprising carbon monoxide gas, wherein the first microorganism is an acetogenic microorganism capable of converting a carbon source to acetic acid and / or ethanol; and-the second microorganism is selected from the group consisting of Clostridium kluyveri capable of converting acetic acid and / or ethanol to form an acid, and C. carboxidivorans; wherein the first microorganism is further capable of converting the acid to the corresponding higher alcohol, and the higher alcohol comprises at least 6 carbon atoms.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 201610054149.7, "Method for Producing Higher Alcohols", filed on January 27, 2016. TECHNICAL FIELD

[0002] The present invention relates to biotechnological methods for producing higher alcohols from carbon sources. Specifically, the mixtures and methods relate to the biotechnological production of at least one higher alcohol in the presence of carbon monoxide. BACKGROUND

[0003] Higher alcohols have several uses, including use as fuels and in the perfume and cosmetic industries. For example, hexanol is commonly used in the perfume industry.

[0004] Heptanol, for example, is used in cardiac electrophysiology experiments to block gap junctions and increase the axial resistance between muscle cells. Increasing the axial resistance decreases the conduction velocity and increases the susceptibility of the heart to re-entrant excitation and persistent arrhythmias. Also, 1-heptanol has a pleasant odor and is used in cosmetics due to its fragrance.

[0005] These higher alcohols can also be used as fuels in the future and can replace gasoline in the long term. For these and more reasons, there is a potential market for higher alcohols. Higher alcohols can also be used as industrial solvents.

[0006] Currently, higher alcohols are primarily manufactured from petroleum. These compounds are obtained by cracking gasoline or petroleum, which is harmful to the environment. Additionally, since the cost of these raw materials will be tied to the price of petroleum, as the price of petroleum is expected to increase in the future, the price of higher alcohols can also increase relative to the increasing price of petroleum.

[0007] The Alfol® alcohol process is a process for producing higher alcohols from ethylene using an organoaluminum catalyst. The reaction produces linear, long-chain primary alcohols (C2-C 28 ). The process uses an aluminum catalyst to oligomerize ethylene and allows the resulting alkyl groups to be oxidized. However, this process produces a wide range of alcohols and maintains this distribution pattern. This constant pattern limits the ability of producers to only manufacture specific ranges of alcohols that are in the highest demand or have the best economic value. Also, the gases required in the reaction must be very clean and different gas compositions are needed for successful performance of the reaction.

[0008] WO2009100434 also describes an indirect method for the production of butanol and hexanol from carbohydrates. The method comprises a homoacetogenic fermentation to produce an acetate intermediate which is then chemically converted into ethanol. The ethanol and the remainder of the acetate intermediate are then used as substrates in an acidogenic fermentation to produce butyrate and hexanoate intermediates which are then chemically converted into butanol and hexanol. However, this method uses the expensive raw material carbohydrates and has two additional method steps, ester formation and chemical hydrogenation of the ester, which makes the method not only longer but also results in a loss of useful material along the way.

[0009] Perez, J.M., 2012 discloses a method for the conversion of short-chain carboxylic acids into their corresponding alcohols using Clostridium ljungdahlii in the presence of syngas. However, short-chain carboxylic acids have to be added as substrates for the conversion into the corresponding higher alcohols. Clostridium ljungdahlii

[0010] Therefore, the currently available higher alcohol production methods have limitations in the mass transfer of the gaseous substrates into the fermentation broth, lower productivity and lower end product concentrations, resulting in higher energy costs for product purification.

[0011] Therefore, it would be desirable to find a more sustainable raw material other than pure petroleum- or corn-based sources as starting material for the production of higher alcohols by biotechnological means which also cause less damage to the environment. In particular, there is a need for a simple and efficient one-pot biotechnological production of higher alcohols from sustainable raw materials. SUMMARY

[0012] The present invention provides a reaction mixture of at least two microorganisms in the presence of carbon monoxide (CO), wherein the first microorganism can be capable of converting CO into acetic acid and / or ethanol and the second microorganism can be capable of converting acetic acid and / or ethanol into at least one acid, and the first microorganism can be subsequently capable of converting the acid into the corresponding higher alcohol, wherein all steps can be carried out in the presence of CO and the higher alcohol comprises at least 6 carbon atoms or more. In particular, CO is present as a gaseous substrate in the reaction mixture and CO is present in the gaseous substrate in a concentration of 2% and / or more.

[0013] In one aspect of the present invention, a reaction mixture is provided comprising a mixed culture of a first and a second microorganism in an aqueous medium comprising a carbon monoxide containing gas, wherein - the first microorganism is an acetogenic microorganism capable of converting a carbon source into acetic acid and / or ethanol; and - the second microorganism is selected from the group consisting of Clostridium ljungdahlii (C. ljungdahlii) and Clostridium autoethanogenum (C. autoethanogenum) capable of converting acetic acid and / or ethanol to form an acid and Clostridium kluyveri C. carboxidivorans ​​​wherein said first microorganism is further capable of converting an acid into a corresponding higher alcohol, and wherein said higher alcohol comprises at least 6 carbon atoms.

[0014] According to a further aspect of the present application, there is provided a method for producing at least one higher alcohol in an aqueous culture medium comprising a culture of a first and a second microorganism, wherein, - said first microorganism is an acetogenic microorganism capable of converting a carbon source comprising carbon monoxide into acetic acid and / or ethanol; and - said second microorganism is selected from the group consisting of Clostridium aceticum capable of converting acetic acid and / or ethanol to form an acid and C. carboxidivorans ; wherein said first microorganism is further capable of converting an acid into a corresponding higher alcohol, and wherein said higher alcohol comprises at least 6 carbon atoms.

[0015] According to a further aspect of the present application, said method for producing at least one higher alcohol in an aqueous culture medium comprises the following steps: (a) adding a first acetogenic microorganism capable of converting a carbon source comprising carbon monoxide into acetic acid and / or ethanol to an aqueous culture medium; and (b) adding a second microorganism selected from the group consisting of Clostridium aceticum capable of converting acetic acid and / or ethanol to form an acid; C. carboxidivorans ; wherein said first microorganism is further capable of converting an acid into a corresponding higher alcohol, and wherein said higher alcohol comprises at least 6 carbon atoms.

[0016] In one example, steps (a) and (b) are performed simultaneously. In another example, steps (a) and (b) are performed sequentially. The concentrations of the first and second microorganisms can be maintained continuously to keep the reaction going. In one example, the concentration of ethanol and / or acetic acid is measured prior to performing step (b) to ensure that optimal concentrations are achieved for the second microorganism. Specifically, the concentration of acetic acid and / or ethanol can be at a level that is optimal for the presence of sufficient substrate in the aqueous culture medium for the second organism to form at least one higher acid. One skilled in the art will be able to readily determine the appropriate time to include the second organism into the aqueous culture medium.

[0017] In another example, steps (a) and (b) are performed simultaneously. In this example, said second microorganism can be active (i.e. can be latent) until sufficient amounts of acetic acid and / or ethanol can be produced by the activity of the first microorganism. The presence of either microorganism in the aqueous culture medium does not disrupt or hinder the activity and / or efficiency of the other.

[0018] One advantage of the present invention can be that CO2 / CO mixtures of far more favorable raw materials can be used. These different sources include natural gas, biogas, coal, oil, plant residues, etc. Another advantage of the process can be a high carbon yield. This is made possible by the return of the formed CO2in the first stage. That is, CO2can occur in the reverse in the first stage back to acetic acid. Another advantage can be greater flexibility with respect to the fermentation conditions used, since any acetic acid-producing and any microorganism capable of the ethanol-carboxylic acid fermentation pathway can be used in combination for the actual production of higher alcohols. Another advantage of the present invention can be that, since the second microorganism can function in the presence of CO and / or produce acids from acetic acid and / or ethanol, both the first and the second microorganism can be present in a homogeneous mixture for the production of higher alcohols from carbon sources comprising CO. This feature of the second microorganism makes the production of higher alcohols from carbon sources like CO a one-step process, making the process more efficient and higher yielding. Surprisingly, because of this advantage of the second microorganism, a one-step process for making higher alcohols can be performed in a single fermenter without the need for intermediate separation steps. Using this one-step process can also have an increased concentration of the end product. This is surprising because both Baffert C., 2011 and Thauer, R.K., 1973 teach that hydrogenases are inhibited in the presence of CO. For this reason and more, WO2013 / 167663 includes a separation step between (a) the step of forming acetic acid and / or ethanol from CO and / or CO2in the presence of an acetic acid-producing organism and (b) the step of forming a hydrocarbon comprising at least one oxygen atom (e.g., hexanoic acid) in the presence of a second microorganism. The ability to produce higher alcohols, particularly higher alcohols comprising at least 6 carbon atoms, from CO in a one-pot synthesis according to any aspect of the present invention is therefore a surprising result. In any case, even if steps (a) and (b) are performed in two separate steps (i.e., two separate vessels), there can be no need for a specific extraction method for removing all traces of CO to enable the first and second microorganism to function.

[0019] As can be seen in the examples, the presence of CO allows for the production of hexanol in a process according to any aspect of the present invention, wherein the carbon source comprises at least CO.

[0020] The carbon source comprising CO can be converted into at least one acid in the presence of at least one acetic acid-producing microorganism and a second microorganism capable of the ethanol-carboxylic acid fermentation pathway. In particular, the acid can comprise 6 or more carbon atoms. More particularly, the acid formed can be selected from the group consisting of hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, etc. In particular, the carbon source comprising CO can result in the production of ethanol and / or acetic acid in the presence of at least one acetic acid-producing bacteria.

[0021] The term "acetogenic bacteria" as used herein refers to microorganisms that are capable of the Wood-Ljungdahl pathway and thus capable of converting CO, CO2 and / or hydrogen into acetate. These microorganisms include microorganisms that do not have the Wood-Ljungdahl pathway in their wild type form, but have acquired this trait due to genetic modification. These microorganisms include, but are not limited to, E. coli cells. These microorganisms can also be referred to as carboxydotrophic bacteria. Currently, 21 different genera of acetogenic bacteria are known from the state of the art (Drake et al., 2006) and these can also include some Clostridia (Drake & Kusel, 2005). These bacteria are capable of using 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 reductive pathway leading to the formation of acetate is referred to as the acetyl-CoA or Wood-Ljungdahl pathway.

[0022] Specifically, the acetogenic bacteria can be selected from Acetoanaerobium notera (ATCC 35199), Acetonema longum (DSM 6540), Acetobacterium carbinolicum (DSM 2925), Acetobacterium malicum (DSM 4132), Acetobacterium species no. 446 (Morinaga et al., 1990, J. Biotechnol., Vol. 14, p. 159-166), Acetobacterium sp. (DSM 2909), Acetobacterium sp. (DSM 2926), Acetobacterium sp. (DSM 2927), Acetobacterium sp. (DSM 2928), Acetobacterium sp. (DSM 2929), Acetobacterium sp. (DSM 2930), Acetobacterium sp. (DSM 2931), Acetobacterium sp. (DSM 2932), Acetobacterium sp. (DSM 2933), Acetobacterium sp. (DSM 2934), Acetobacterium sp. (DSM 2935), Acetobacterium sp. (DSM 2936), Acetobacterium sp. (DSM 2937), Acetobacterium sp. (DSM187-194), Acetobacterium wieringae (DSM 1911), Acetobacterium woodii (DSM 1030), Alkalibaculum bacchi (DSM 22112), Archaeoglobus fulgidus (DSM 4304), Blautia producta (DSM 2950, formerly Ruminococcus productus, formerly Peptostreptococcus productus), Butyribacterium methylotrophicum (DSM 3468), Clostridium aceticum (DSM 1496), Clostridium autoethanogenum (DSM 10061, DSM 19630 and DSM 23693), Clostridium carboxidivorans (DSM 15243), Clostridium coskatii (ATCC Accession No. PTA-10522), Clostridium drakei (ATCC BA-623), Clostridium formicoaceticum (DSM 92), Clostridium glycolicum (DSM 1288), Clostridium ljungdahlii (DSM 13528), Clostridium ljungdahlii C-01 (ATCC 55988), Clostridium ljungdahlii ERI-2 (ATCC 55380), Clostridium ljungdahlii O-52 (ATCC 55989), 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 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, p. 1607-1612), Moorella thermoacetica (DSM 521, formerly Clostridium thermoaceticum), Moorella thermoautotrophica (DSM 1974), Oxobacter pfennigii (DSM 322), 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, formerly Acetogenium kivui). More specifically, strain ATCC BAA-624 of Clostridium carboxidivorans can be used. Even more specifically, bacterial strains designated "P7" and "P11" described in U.S. 2007 / 0275447 and U.S. 2008 / 0057554, respectively, can be used. Clostridium carboxidivorans the bacterial strains designated "P7" and "P11" described in U.S. 2007 / 0275447 and U.S. 2008 / 0057554, respectively.

[0023] Another particularly suitable bacterium can be Clostridium ljungdahlii. In particular, a strain selected from Clostridium ljungdahlii PETC, Clostridium ljungdahlii ERI2, Clostridium ljungdahlii COL and Clostridium ljungdahlii O-52 can be used to convert syngas to caproic acid. These strains are described, for example, in WO 98 / 00558, WO 00 / 68407, ATCC 49587, ATCC 55988 and ATCC 55989. In another example, the selected acetogenic bacterium for the first organism can be Clostridium autoethanogenum.

[0024] The acetogenic bacterium can be used in combination with a second microorganism that can be capable of an ethanol-carboxylic acid fermentation pathway. In one example, both an acetogenic bacterium and a second microorganism that can be capable of an ethanol-carboxylic acid fermentation pathway can be used to produce higher acids from a carbon source comprising CO. The acids can be converted to corresponding higher alcohols selected from hexanol, octanol, nonanol, decanol, etc. In one example, the higher alcohols can be selected from 1-hexanol, 2-hexanol, 3-hexanol, 1-heptanol, 2-heptanol, 3-heptanol, 4-heptanol, octanol, nonanol, decanol, etc.

[0025] In one example, the ethanol and / or acetic acid can be converted to corresponding higher acids in the presence of a second microorganism that is capable of an ethanol-carboxylic acid fermentation pathway. The ethanol-carboxylic acid fermentation pathway is described in detail at least in Seedorf, H., et al., 2008. In particular, the second organism can be selected from Clostridium kluyveri, C.Carboxidivorans etc. These second microorganisms include microorganisms that do not have an ethanol-carboxylic acid fermentation pathway in their wild-type form, but have acquired this trait due to genetic modification. In particular, the second microorganism can be Clostridium kluyveri.

[0026] In another example, the second microorganism can be a wild-type organism that expresses at least one enzyme selected from E1 to E 11 , wherein E1 is an alcohol dehydrogenase (adh), E2 is an acetaldehyde dehydrogenase (ald), E3 is an acetoacetyl-CoA thiolase (thl), E4 is a 3-hydroxybutyryl-CoA dehydrogenase (hbd), E5 is a 3-hydroxybutyryl-CoA dehydratase (crt), E6 is a butyryl-CoA dehydrogenase (bcd), E7 is an electron transport flavoprotein subunit (etf), E8 is a CoA transferase (cat), E9 is an acetate kinase (ack), E 10 is a phosphotransacetylase (pta) and E 11 is a transhydrogenase. In particular, the wild-type second microorganism according to any aspect of the present invention can express at least E2, E3 and E4. Even more in particular, the wild-type second microorganism according to any aspect of the present invention can express at least E4.

[0027] In another example, the second microorganism according to any aspect of the application can be a genetically modified organism having increased expression of at least one enzyme selected from the group consisting of E1 to E 11 , wherein E1 is an alcohol dehydrogenase (adh), E2 is an acetaldehyde dehydrogenase (ald), E3 is an acetoacetyl-CoA thiolase (thl), E4 is a 3-hydroxybutyryl-CoA dehydrogenase (hbd), E5 is a 3-hydroxybutyryl-CoA dehydratase (crt), E6 is a butyryl-CoA dehydrogenase (bcd), E7 is an electron transport flavoprotein subunit (etf), E8 is a CoA transferase (cat), E9 is an acetate kinase (ack), E 10 is a phosphotransacetylase (pta) and E 11 is a transhydrogenase. In particular, the genetically modified second microorganism according to any aspect of the application can express at least enzymes E2, E3 and E4. Even more in particular, the genetically modified second microorganism according to any aspect of the application can express at least E4. Enzymes E1 to E 11 may be isolated from Clostridium kluyveri. A person skilled in the art can be able to measure the activity of each of these enzymes using methods known in the art. In particular, the activity of enzymes E1 and E2 can be measured using assays taught at least in Hillmer P., 1972, Lurz R., 1979; the activity of E2 can also be measured using assays taught in Smith L.T., 1980; the activity of enzymes E3 and E4 can be measured using assays taught at least in Sliwkowski M.X., 1984; the activity of E4 can also be measured using assays taught in Madan, V.K., 1972; the activity of E5 can also be measured using assays taught in Bartsch, R.G., 1961 ; the activity of enzymes E6 and E7 can be measured using assays taught in Li, F., 2008; the activity of E7 can also be measured using assays taught in Chowdhury, 2013; the activity of E8 can be measured using assays taught in Stadman, 1953; the activity of E9 can be measured using assays taught in Winzer, K., 1997; the activity of E 10 can be measured using assays taught in Smith L.T., 1976; and the activity of E 11 can be measured using assays taught in Wang S, 2010.

[0028] According to any aspect of the present application, the first and / or second microorganism can be a genetically modified microorganism. A genetically modified cell or microorganism can be genetically different from a wild type cell or microorganism. The genetic difference between a genetically modified microorganism according to any aspect of the present application and a wild type microorganism can be the presence of a complete gene, amino acid, nucleotide, etc. in the genetically modified microorganism that can be absent in the wild type microorganism. In one example, a genetically modified microorganism according to any aspect of the present application can comprise an enzyme that enables the microorganism to produce at least one carboxylic acid. The wild type microorganism relative to the genetically modified microorganism according to any aspect of the present application can not have or have no detectable enzymatic activity that enables the genetically modified microorganism to produce at least one carboxylic acid. The term "genetically modified microorganism" as used herein can be used interchangeably with the term "genetically modified cell". The genetic modification according to any aspect of the present application can be made on the cell of the microorganism.

[0029] The phrase "wild type" as used herein in relation to a cell or microorganism can refer to a cell having a genomic composition as found in nature in the wild. The term can apply to both whole cells and individual genes. The term "wild type" therefore does not include a cell or a gene in which the sequence of the gene has been altered at least in part by human use of recombinant methods.

[0030] The skilled person will be able to genetically modify a cell or microorganism using any method known in the art. According to any aspect of the present application, the genetically modified cell can be genetically modified so as to form at least two-fold, in particular at least 10-fold, at least 100-fold, at least 1000-fold or at least 10000-fold more carboxylic acid and / or corresponding carboxylic acid ester than a wild type cell within a defined time interval, within 2 hours, in particular within 8 hours or 24 hours. The increase in product formation can be determined, for example, by culturing a cell according to any aspect of the present application and a wild type cell separately from each other under the same conditions (same cell density, same nutrient medium, same cultivation conditions), for a specific time interval in a suitable nutrient medium, and then determining the amount of the target product (carboxylic acid) in the nutrient medium.

[0031] In another example, the acid can be produced from a carbon source comprising CO by any of the methods disclosed in Steinbusch, 2011, Zhang, 2013, Van Eerten-Jansen, M. C. A. A, 2013, Ding H. et al., 2010, Barker H. A., 1949, Stadtman E. R., 1950, Bornstein B. T., et al., 1948, and the like. Even more specifically, the acid can be produced from a carbon source comprising CO in the presence of at least Clostridium kluyveri.

[0032] Even more specifically, according to any aspect of the application, the acid is produced in the presence of at least one acetogenic microorganism and Clostridium kluyveri. In one example, the acetogenic microorganism can be Clostridium ljungdahlii or Clostridium ragsdahlei .

[0033] In the production of acid from a carbon source comprising CO, combinations of bacteria can be used. There can be more than one acetogenic bacteria combined with one or more second microorganisms. In another example, there can be more than one type of acetogenic bacteria and only one type of second microorganism. In yet another example, there can be more than one second microorganism combined with only one acetogenic bacteria.

[0034] The reaction mixture can comprise two microorganisms in a homogeneous mixture. The term "homogeneous mixture" as used herein refers to a mixture of microorganisms that are spatially uniformly distributed in the culture medium. Specifically, the mixture can comprise at least two microorganisms, an acetogenic microorganism and a second microorganism, uniformly distributed in an aqueous culture medium. In one example, there can be approximately equal numbers of acetogenic microorganisms and second microorganisms in the mixture. In another example, there can be more acetogenic microorganisms than second microorganisms in the mixture. In yet another example, there can be more second microorganisms than acetogenic microorganisms in the mixture. In all possible examples, the microorganisms are in a single homogeneous mixture, wherein they are uniformly distributed throughout the mixture. "Aqueous culture medium" as used herein can be used interchangeably with the term "reaction mixture."

[0035] The term "acetic acid" as used herein refers to both acetic acid and its salts, which is an unavoidable consequence because, as is known in the art, the microorganisms work in an aqueous environment and there is always an equilibrium between the salts and acids present.

[0036] According to any aspect of the application, the term "second microorganism" refers to a microorganism that is different from the "first microorganism."

[0037] In another example, steps (a) and (b) can be carried out in two different vessels. In one example, step (a) can be carried out in fermenter 1, wherein the first microorganism is contacted with a carbon source comprising CO to produce acetic acid and / or ethanol. The ethanol and / or acetic acid can then be contacted with a second microorganism in fermenter 2 to produce at least one acid. The acid can then be fed back into fermenter 1 to convert the acid to the desired higher alcohol. A cycle can be established, wherein acetic acid and / or ethanol produced in fermenter 1 can be periodically fed into fermenter 2, acetic acid and / or ethanol in fermenter 2 can be converted to at least one acid and the acid in fermenter 2 is fed back into fermenter 1. CO fed into fermenter 1 can be transferred to fermenter 2 along with the acetic acid and / or ethanol. No special extraction method is required, as it has surprisingly been found that the second microorganism converts acetic acid and / or ethanol to at least one acid in the presence of CO.

[0038] In another example, the medium is circulated between fermenters 1 and 2. Thus, ethanol and / or acetic acid produced in fermenter 1 can be fed into fermenter 2 and acid produced in fermenter 2 can be fed back into fermenter 1. In the process of circulating the medium, CO from fermenter 1 can be introduced into fermenter 2. Also, acid produced in fermenter 2 can subsequently be introduced again into fermenter 1. The second microorganism in fermenter 2 can be able to continue to produce acid from acetic acid and ethanol in the presence of CO circulated from fermenter 1 into fermenter 2. The alcohol accumulated in fermenters 1 and 2 can then be extracted by methods known in the art.

[0039] In a further example, there can be three vessels for carrying out the method according to any aspect of the application. A first microorganism can be present in a first fermenter, a second microorganism in a second fermenter and a third fermenter again has a first microorganism. In fermenter 1, the first microorganism is contacted with a carbon source to produce acetic acid and / or ethanol. The ethanol and / or acetic acid can then be contacted with a second microorganism in fermenter 2 to produce at least one acid. The acid is then fed into fermenter 3 to produce at least one alcohol.

[0040] In particular, CO can be provided in a continuous gas stream to the aqueous medium. The concentration of CO in the gas stream can be present in at least 2% by volume of the total amount of gas in the gas stream. In particular, CO can be present in a concentration ranging from 2 to 99% by volume, ranging from 2 to 95% by volume, ranging from 5 to 95% by volume, ranging from 10 to 90% by volume, ranging from 15 to 85% by volume, in particular ranging from 20 to 80% by volume. More in particular, the concentration of CO can be about 7%, 24% by volume. The gas phase concentration of carbon monoxide in the carbon source can at least be measured using a gas chromatograph GC 6890N from Agilent Technologies Inc. with a thermal conductivity detector.

[0041] The term "about" as used herein refers to a variation within 20%. In particular, the term "about" as used herein refers to + / - 20%, more particularly + / - 10%, even more particularly + / - 5% of a given measured value or value.

[0042] Unless otherwise stated, all percentages (%) are volume percentages.

[0043] The carbon source used according to any aspect of the present application comprises carbon dioxide and / or carbon monoxide. The skilled person will understand that there are many possible sources for providing CO and / or CO2 as a carbon source. It will be appreciated that in practice, any gas or any mixture of gases can be used as a carbon source according to any aspect of the present application which is capable of providing a sufficient amount of carbon to the microorganisms so that acetic acid and / or ethanol can be formed from the source of CO and / or CO2.

[0044] Typically, for a mixed culture according to any aspect of the present application, the carbon source comprises at least 50% by volume, at least 70% by volume, in particular at least 90% by volume of CO and / or CO2, wherein the percentage by volume - % - is relative to all carbon sources available to the first microorganism in the mixed culture.

[0045] In a mixed culture according to any aspect of the present application, a source of carbon material can be provided. Examples of carbon sources in gaseous form include off-gases such as synthesis gas, flue gas and petroleum refinery gas produced by yeast fermentation or clostridial fermentation. These off-gases are formed from the gasification or coal gasification of cellulosic material. In one example, these off-gases are not necessarily produced as a by-product of other processes, but can be specifically produced for use with a mixed culture according to any aspect of the present application.

[0046] According to any aspect of the present application, the carbon source can be synthesis gas. Synthesis gas can for example be produced as a by-product of coal gasification. Accordingly, the microorganisms of a mixed culture according to any aspect of the present application can be capable of converting a material which is a waste product into a valuable resource. In another example, synthesis gas can be a by-product of the gasification of widely available, low cost agricultural raw materials for use with a mixed culture of the present application to produce at least one higher alcohol.

[0047] There are many examples of raw materials which can be converted into synthesis gas, as almost all plants can be used for this purpose. In particular, the raw material is selected from perennial grasses such as miscanthus, corn residues, processing waste such as sawdust and the like.

[0048] Typically, the synthesis gas can be obtained in a gasification plant of dry biomass, mainly by pyrolysis, partial oxidation and steam reforming, where the main products of the synthesis gas are CO, H2and CO2. The synthesis gas can also be the product of electrolysis of CO2. The skilled person will understand the suitable conditions to carry out electrolysis of CO2to produce a synthesis gas comprising the desired amount of CO.

[0049] Typically, a portion of the synthesis gas obtained from the gasification process is first processed to optimize product yields and to avoid the formation of tars. The cracking of undesirable tars and CO in the synthesis gas can be carried out using lime and / or dolomite. These processes are described in detail in, for example, Reed, 1981.

[0050] Mixtures of sources can be used as the carbon source.

[0051] According to any aspect of the application, a reducing agent such as hydrogen can be provided together with the carbon source. In particular, this hydrogen can be provided when C and / or CO2is provided and / or used. In one example, the hydrogen is part of the synthesis gas present according to any aspect of the application. In another example, where the hydrogen in the synthesis gas is not sufficient for the method of the application, additional hydrogen can be provided.

[0052] The skilled person will understand the other conditions required to carry out the method according to any aspect of the application. In particular, the conditions in the vessel (e.g. fermenter) can vary depending on the first and second microorganism used. Variations in conditions suitable for optimal functioning of the microorganism are within the knowledge of the skilled person.

[0053] In one example, the method according to any aspect of the application can be carried out in an aqueous medium having a pH of 5-8, 5.5-7. The pressure can be between 1 and 10 bar.

[0054] In particular, the aqueous medium can comprise a carbon source comprising CO and / or CO2. More in particular, the carbon source comprising CO and / or CO2is provided to the aqueous medium in a continuous gas flow. Even more in particular, the continuous gas flow comprises synthesis gas. In one example, the gases are part of the same flow / stream. In another example, each gas is a separate flow (stream) provided to the aqueous medium. The gases can be separated, for example, using separate nozzles, frits, membranes within the tube providing the gas into the aqueous medium, etc. opened in the aqueous medium.

[0055] Specifically, the reaction mixture according to any aspect of the present application (i.e., a mixture of a first microorganism - an acetogenic organism, a second microorganism, and a carbon source comprising carbon monoxide) can be used in any known bioreactor or fermenter to carry out any aspect of the present application.

[0056] As used herein, "higher alcohols" refer to alcohols comprising 6-10 carbon atoms and can be somewhat viscous or oily and have a heavier fruity taste. Higher alcohols can include, but are not limited to, hexanol, heptanol, octanol, nonanol, decanol, and the like. More specifically, the higher alcohols can be selected from the group consisting of 1-hexanol, 1-octanol, 1-heptanol, 3-methyl-1-pentanol, 4-methyl-1-hexanol, 5-methyl-1-heptanol, 4-methyl-1-pentanol, 5-methyl-1-hexanol, 6-methyl-1-heptanol, and combinations thereof.

[0057] According to any aspect of the present application, "corresponding higher alcohol" refers to an alcohol having the same number of carbon atoms as such an acid from which the corresponding higher alcohol is formed. For example, hexanoic acid can be converted to the corresponding alcohol - hexanol; heptanoic acid can be converted to the corresponding alcohol - heptanol; octanoic acid can be converted to the corresponding alcohol - octanol; nonanoic acid can be converted to the corresponding alcohol - nonanol; decanoic acid can be converted to the corresponding alcohol - decanol; and the like.

[0058] In one example, the method according to any aspect of the application can result in the formation of a mixture of acids and / or higher alcohols. In another example, the parameters of the method according to any aspect of the application can be adjusted to produce more of one acid and / or higher alcohol than the rest. For example, with the following parameters, a mixed culture grown in a complex medium (0.25 g / L NH4CI, 0.2 g / L MgS04x 7 H20, 0.31 g / L K2HPO4, 0.23 g / L KH2PO4, 2.5 g / L NaHCO3, 1 g / L yeast extract, 10 g / L K-acetate, 20 g / l ethanol, 0.25 g / L L-hydrochloric cysteine, 1.5 mg / L FeCI2x 4H20, 70 pg / L ZnCI2x 7 H20, 100 pg / L MnCI2x 4 H20, 6 pg / L boric acid, 190 pg / L CoCI2x 6H20, 2 pg / L CuCI2x 6 H20, 24 pg / L NiCI2x 6 H20, 36 pg / L Na2MoO4x 2 H20, 3 pg / L Na2SeOO3x 5 H20, 4 pg / L Na2WO4x 2 H20, 100 pg / L vitamin B12, 80 pg / L p-aminobenzoic acid, 20 pg / L biotin, 200 pg / L nicotinic acid, 100 pg / L calcium pantothenate, 300 pg / L pyridoxine hydrochloride, 200 pg / L thiamine hydrochloride x H20) at 37°C with a pH of about 6.5 for about 240 hours, the concentration of butanol and / or hexanol production can be controlled.

[0059] In the reaction mixture according to any aspect of the application, oxygen can be present. Accordingly, the first and second microorganism according to any aspect of the application can be aerobically growing. In particular, oxygen can be provided to the aqueous culture medium according to any aspect of the application in a continuous gas flow. More in particular, the gas flow can have an O2 concentration of less than 1% of the total amount of gas in the gas flow. In particular, oxygen can be present in a concentration ranging from 0.000005 to 2% by volume, from 0.00005 to 2% by volume, from 0.0005 to 2% by volume, from 0.005 to 2% by volume, from 0.05 to 2% by volume, from 0.00005 to 1.5% by volume, from 0.0005 to 1.5% by volume, from 0.005 to 1.5% by volume, from 0.05 to 1.5% by volume, from 0.5 to 1.5% by volume, from 0.00005 to 1% by volume, from 0.0005 to 1% by volume, from 0.005 to 1% by volume, from 0.05 to 1% by volume, from 0.5 to 1% by volume, from 0.55 to 1% by volume, from 0.60 to 1% by volume, in particular in the range from 0.60 to 1.5% by volume, from 0.65 to 1% by volume and from 0.70 to 1% by volume. In particular, the acetogenic microorganism is particularly suitable when the O2 proportion in the gas phase / flow is about 0.00005, 0.0005, 0.005, 0.05, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2% by volume relative to the volume of gas in the gas flow. The person skilled in the art will be able to measure the oxygen volume concentration in the gas flow using any method known in the art. In particular, the oxygen volume can be measured using any method known in the art. In one example, the gas phase concentration of oxygen can be measured by a micro oxygen dip probe from PreSens Precision Sensing GmbH. The oxygen concentration can be measured by fluorescence quenching, wherein the degree of quenching correlates with the oxygen partial pressure in the gas phase. Even more in particular, the first and second microorganism according to any aspect of the application can work optimally in the aqueous culture medium when oxygen is provided by a gas flow having an oxygen concentration of less than 1% by volume of the total gas (about 0.015% by volume of the total volume of gas provided in the gas flow to the reaction mixture).

[0060] The aqueous culture medium according to any aspect of the present application can comprise oxygen. The oxygen can be dissolved in the culture medium by any method known in the art. In particular, the oxygen can be present at 0.5 mg / L. In particular, the dissolved concentration of free oxygen in the aqueous culture medium can be at least 0.01 mg / L. In another example, the dissolved oxygen can be about 0.01, 0.02, 0.03, 0.04, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5 mg / L. In particular, the dissolved oxygen concentration can be 0.01-0.5 mg / L, 0.01-0.4 mg / L, 0.01-0.3 mg / L, 0.01-0.1 mg / L. In particular, the oxygen can be provided to the aqueous culture medium in a continuous gas stream. More particularly, the aqueous culture medium can comprise oxygen and a carbon source comprising CO and / or CO2. More particularly, the oxygen and the carbon source comprising CO and / or CO2 are provided to the aqueous culture medium in a continuous gas stream. Even more particularly, the continuous gas stream comprises syngas and oxygen. In one example, both gases are part of the same stream. In another example, each gas is a separate stream provided to the aqueous culture medium. The gases can be separated, for example, using separate nozzles opened into the aqueous culture medium, filter plates, membranes within the tubing providing the gases into the aqueous culture medium, etc. The oxygen can be free oxygen. According to any aspect of the present application, "reaction mixture comprising free oxygen" refers to a reaction mixture comprising elemental oxygen in the form of O2. The O2 can be dissolved oxygen in the reaction mixture. In particular, the dissolved oxygen can be at a concentration of > 5 ppm (0.000005 vol%; 5 x 10 -6 ). A person skilled in the art can be able to measure the concentration of dissolved oxygen using any method known in the art. In one example, the dissolved oxygen can be measured by an oxygen immersion probe (PSt6 from PreSens Precision Sensing GmbH, Regensburg, Germany).

[0061] In one example according to any aspect of the application, the carbon source is syngas and the carbon source can be admixed with oxygen prior to being provided to the aqueous culture medium. This admixing step can improve efficiency in the reaction and production of higher alcohols. The overall efficiency, alcohol productivity and / or overall carbon capture of the method of the application can depend on the stoichiometry of CO2, CO, H2and O2in the continuous gas stream. The continuous gas stream applied can be of composition O2, CO2and H2. In particular, in the continuous gas stream, the concentration of O2may be in the range of 0.000005 to 1% by volume, CO / CO2is about 10-50% by volume, in particular 33%, and H2will be in the range of 44% to 84% by volume, in particular 64 to 66.04%. More particularly, the gas concentrations in the continuous gas stream can be 0.15% O2by volume, 32% CO / CO2by volume and 64% H2by volume. In another example, the continuous gas stream can also contain an inert gas such as N2, up to a N2concentration of 50% by volume.

[0062] The skilled person will appreciate that it is necessary to monitor the composition and flow rate of the streams at relevant intervals. Control of the composition of the stream can be achieved by varying the ratio of the component streams to achieve a target or desired composition. The composition and flow rate of the admixed stream can be monitored by any method known in the art. In one example, the system is adapted to continuously monitor the flow rate and composition of at least two streams and combine them to produce a single admixed substrate stream in a continuous gas stream of optimal composition, and means for passing the optimised substrate stream to the mixed culture according to any aspect of the application.

[0063] It is advantageous to incorporate O2 in the reaction mixture and / or in the gas stream provided to the reaction mixture, as most off-gases comprising synthesis gas contain small or large amounts of oxygen. It is difficult and costly to remove this oxygen prior to using the synthesis gas as carbon source for the production of higher alcohols. The method according to any aspect of the present invention allows the production of at least one higher alcohol without the need to first remove any traces of oxygen from the carbon source. This allows saving time and money. In one example, the first microorganism, i.e. the acetogenic bacteria, can be present in both growth phases when O2 is present in the reaction mixture according to any aspect of the present invention. In an example, at least one acetogenic bacteria can be in the exponential growth phase and another acetogenic bacteria can be in any other growth phase of the life cycle of the acetogenic microorganism. In particular, the acetogenic bacteria in the aqueous medium according to any aspect of the present invention can comprise one acetogenic bacteria in the exponential growth phase and another acetogenic bacteria in the stationary phase. In the presence of oxygen, the acetogenic bacteria not in the exponential growth phase, the acetogenic bacteria in the stationary phase can not be able to produce acetate and / or ethanol. This phenomenon is at least demonstrated by Brioukhanov, 2006, Imlay, 2006, Lan, 2013, etc. The inventors thus surprisingly found that in the presence of the acetogenic bacteria in the exponential growth phase, the acetogenic bacteria in any growth phase can aerobically respire and produce acetate and / or ethanol in an amount that exceeds or equals the amount produced when the reaction mixture is anoxic. In one example, the acetogenic bacteria in the exponential growth phase can be able to remove free oxygen from the reaction mixture, providing a suitable environment (free of free oxygen) for the acetogenic bacteria in any growth phase to metabolize the carbon substrate comprising CO and produce acetate and / or ethanol.

[0064] In another example, the aqueous medium can already comprise the acetogenic bacteria in any growth phase, in particular in the stationary phase, in the presence of the carbon source comprising CO. In this example, oxygen can be present in the carbon source provided to the aqueous medium or in the aqueous medium itself. In the presence of oxygen, the acetogenic bacteria can be inactive and do not produce acetate and / or ethanol prior to the addition of the acetogenic bacteria in the exponential growth phase. It is in this example that the acetogenic bacteria in the exponential growth phase can be added to the aqueous medium. The inactive acetogenic bacteria already found in the aqueous medium can then be activated and can start producing acetate and / or ethanol.

[0065] In a further example, the acetogenic bacteria in any growth phase can first be mixed with the acetogenic bacteria in the exponential growth phase and then mixed with the added carbon source and / or oxygen.

[0066] According to any aspect of the present application, the microorganism in the exponential growth phase grown in the presence of oxygen can result in the microorganism to gain an adaptation to grow and metabolize in the presence of oxygen. In particular, the microorganism can be able to remove oxygen from the environment surrounding the microorganism. This newly gained adaptation allows the acetogenic bacteria in the exponential growth phase to escape the oxygen environment and thus to produce acetic acid and ethanol from the carbon source. In particular, the acetogenic bacteria with the newly gained adaptation allow the bacteria to convert the carbon source comprising CO into acetic acid and / or ethanol and to convert the newly formed acid into the corresponding higher alcohols in the presence of the alcohol. The second microorganism selected from Clostridium kluyveri and C. carboxidivorans The second microorganism can convert acetic acid and / or ethanol to form the newly formed acid. As mentioned earlier, it is advantageous for this process to be carried out in the presence of O2 (i.e. comprising O2 in the reaction mixture), because most off-gases comprising synthesis gas comprise small or large amounts of oxygen. This reaction mixture allows the production of higher alcohols from off-gases without the need for an additional expensive step of first extracting the oxygen.

[0067] The reaction mixture according to any aspect of the present application can thus comprise CO, free oxygen and the acetogenic bacteria in the exponential growth phase.

[0068] The person skilled in the art will understand the different growth phases of microorganisms and the methods to measure them and to identify them. In particular, most microorganisms can be found in at least four different growth phases in batch cultivation; i.e. they are: lag phase (A), log phase or exponential phase (B), stationary phase (C), and death phase (D). The log phase can be further divided into early log phase and mid to late log / exponential phase. The stationary phase can also be further distinguished into early stationary phase and stationary phase. For example, Cotter, J.L., 2009, Najafpour. G., 2006, Younesi, H., 2005 and Köpke, M., 2009 disclose acetogenic bacteria in different growth phases. In particular, the growth phase of the cells can be measured using methods at least taught in Shuler ML, 1992 and Fuchs G., 2007.

[0069] The lag phase is the period immediately following inoculation of cells into fresh medium during which the population remains temporarily constant. Although no apparent cell division occurs, the cells can grow in volume or mass, synthesize enzymes, proteins, RNA, etc., and increase in metabolic activity. The length of the lag phase can depend on a wide range of factors, including the amount of inoculum; the time necessary to recover from physical damage or shock in transfer; the time required for synthesis of essential coenzymes or splitting factors; and the time required for synthesis of new (inducible) enzymes necessary for the metabolism of substrates present in the medium.

[0070] The exponential (logarithmic) phase of growth is a mode of balanced growth in which all cells divide regularly by binary fission and grow by geometric progression. Cells divide at a constant rate, depending on the composition of the growth medium and the conditions of inoculation. The exponential growth rate of a bacterial culture is expressed as the generation time, which is also the doubling time of the bacterial population. The generation time (G) is defined as the time (t) per generation (n = number of generations). Thus, G = t / n is the formula from which the generation time calculation is derived. The exponential phase can be divided into (i) early log phase and (ii) mid to late log / exponential phase. One skilled in the art can readily identify when a microorganism, particularly an acetogenic microorganism, enters the log phase. For example, a method to calculate the growth rate of acetogenic bacteria to determine if they are in the log phase can be accomplished using methods taught at least in Henstra A.M., 2007. Specifically, a microorganism in the exponential growth phase according to any aspect of the present invention can include cells in the early log phase and the mid to late log / exponential phase.

[0071] The stationary phase is the period when exponential growth ends, as exponential growth cannot continue indefinitely in a batch culture (e.g., a closed system such as a test tube or shake flask). Population growth is limited by one of three factors: 1, depletion of available nutrients; 2. accumulation of inhibitory metabolites or end products; 3. depletion of space, in which case it is referred to as lack of "biological space". During the stationary phase, if viable cells are counted, it is not possible to determine whether some cells are on the verge of death and an equal number of cells are dividing, or whether the cell population has simply stopped growing and dividing. The stationary phase, like the lag phase, is not necessarily a quiescent phase. Bacteria that produce secondary metabolites such as antibiotics do so during the stationary phase of the growth cycle (secondary metabolites are defined as metabolites produced after the period of active growth).

[0072] The death phase follows the stationary phase. During the death phase, the number of viable cells decreases geometrically (exponentially), essentially a reversal of the growth during the log phase.

[0073] In one example, the acetogenic bacteria in the method according to any aspect of the present invention can include a combination of cells: cells in the log phase and cells in the stationary phase. The acetogenic cells in the log phase in the method according to any aspect of the present invention can include a growth rate selected from the group consisting of: 0.01 to 2 h -1 , 0.01 to 1 h -1 , 0.05 to 1 h -1 , 0.05 to 2 h -1 , 0.05 to 0.5 h -1 , and the like. In one example, the cell OD 600The OD can be selected from the range consisting of 0.001 to 2, 0.01 to 2, 0.1 to 1, 0.1 to 0.5, etc. The person skilled in the art will be able to measure the OD using any method known in the art 600 and determine the growth rate of the cells in the reaction mixture and / or to be added to the reaction mixture. For example, Koch (1994) can be used. In particular, different methods can be used to determine and monitor bacterial growth. The most common one is turbidity measurement, which relies on the optical density (OD) of bacteria in suspension and uses a spectrophotometer. The OD can be measured at 600 nm using a UV spectrometer.

[0074] In one example, the method according to any aspect of the application comprises mixing together (i) free oxygen, (ii) a population of acetogenic cells in the log phase, (iii) a population of acetogenic cells in the stationary phase, (iii) a population of Clostridium kluyveri and (iv) a carbon source comprising CO. The acetogenic cells in the log phase allow any other acetogenic cells in the aqueous medium to produce acetate and / or ethanol in the presence of oxygen. The concentration of the acetogenic cells in the log phase can be maintained in the reaction mixture. Thus, at any point in time in the reaction, the reaction mixture comprises acetogenic cells in the log phase and another growth phase, for example, acetogenic cells in the stationary phase.

[0075] The method according to any aspect of the application can further comprise the step of extracting the produced higher alcohol. The person skilled in the art will know the means to do so based on methods known in the art.

[0076] According to another aspect of the application, there is provided the use of a reaction mixture according to any aspect of the application for the production of at least one higher alcohol comprising at least 6 carbon atoms. In particular, the production of a higher alcohol from at least one carbon source comprising CO.

[0077] The present application relates in particular to the following technical solution: 1. A reaction mixture comprising a mixed culture of a first and a second microorganism in an aqueous medium comprising carbon monoxide gas, wherein - the first microorganism is an acetogenic microorganism capable of converting a carbon source into acetate and / or ethanol; and - the second microorganism is selected from Clostridium kluyveri (C. kluyveri) capable of converting acetate and / or ethanol to form an acid Clostridium kluyveri ) and C. carboxidivorans ; wherein the first microorganism is further capable of converting the acid into a corresponding higher alcohol, and the higher alcohol comprises at least 6 carbon atoms.

[0078] 2. The mixture according to solution 1, wherein the first microorganism is selected from the group consisting of Acetanaerobacterium umxidum (A. umxidum) Acetoanaerobium notera ) ( ATCC 35199Acetanaerobacterium longum (formerly Clostridium longum) (ATCC® 14865®), Acetonema longum ) ( DSM 6540 ) Acetanaerobacterium methanolicum (formerly Clostridium methanolicum) (ATCC® 13183®), Acetobacterium carbinolicum ) ( DSM 2925 ) Acetanaerobacterium malicum (formerly Clostridium malicum) (ATCC® 14868®), Acetobacterium malicum ) ( DSM 4132 ) Acetanaerobacterium sp. 446 (formerly Clostridium sp. 446) (ATCC® 33775®), Acetobacterium species no. 446 ) Acetanaerobacterium williamsii (formerly Clostridium williamsii) (ATCC® 8480®), Acetobacterium wieringae ) ( DSM 1911 ) Acetanaerobacterium woesei (formerly Clostridium woesei) (ATCC® 55329®), Acetobacterium woodii ) ( DSM 1030 ) Acetanaerobacterium xylanolyticum (formerly Clostridium xylanolyticum) (ATCC® 55380®), Alkalibaculum bacchi ( DSM 22112 ) Acetanaerobacterium fumariolicum (formerly Clostridium fumariolicum) (ATCC® 55900®), Archaeoglobus fulgidus ) ( DSM 4304 ), Blautia producta ( DSM 2950 ) Acetanaerobacterium butylicum (formerly Clostridium butylicum) (ATCC® 19360®), Butyribacterium methylotrophicum ) ( DSM 3468 ) Clostridium aceticum (ATCC® 6456®), Clostridium aceticum ) ( DSM 1496 ) Clostridium autoethanogenum (ATCC® 55900®), Clostridium autoethanogenum ) ( DSM 10061 , DSM 19630 and DSM 23693 ), Clostridium carboxidivorans ( DSM 15243 ), Clostridium coskatii ( ATCC Number PTA - 10522 ), Clostridium drakei ( ATCC BA - 623 ) Clostridium formicicum (ATCC® 27636®), Clostridium formicoaceticum ) ( DSM 92 ) Clostridium glycolicum (ATCC® 55381®), Clostridium glycolicum ) ( DSM 1288 ) Clostridium ljungdahlii (ATCC® 55905®), Clostridium ljungdahlii ) ( DSM 13528 ) Clostridium ljungdahlii C-01 (ATCC® 55872®), ATCC 55988 ) Clostridium ljungdahlii ERI-2 (ATCC® 55873®), ATCC 55380 ) Clostridium ljungdahlii O-52 (ATCC® 55874®), ATCC 55989 ) Clostridium marisruminae (ATCC® 55871®), Clostridium mayombei ) ( DSM 6539 ), Clostridium methoxybenzovorans ( DSM 12182 ), Clostridium ragsdalei ( DSM 15248 ) Clostridium scatologenes (ATCC® 19533®), Clostridium scatologenes ) ( DSM 757Clostridium species ATCC 29797, and desulfurized enterobacteria ( Desulfotomaculum kuznetsovii ) ( DSM 6115 ), heat-desulfurized enterobacteria thermosyntrophicum subspecies ( Desulfotomaculum thermobezoicum subsp . thermosyntrophicum ) ( DSM 14055 ), Eubacterium mucilaginosa ( Eubacterium limosum ) ( DSM 20543 ), Methanophora acetophenones ( Methanosarcina acetivorans C2A ( DSM 2834 ), species of the genus *Mullerella* ( Moorella sp. HUC22-1, *Acetobacter thermophilus* ( Moorella thermoacetica ) ( DSM 521 ), thermoautotrophic Murraya ( Moorella thermoautotrophica ) ( DSM 1974 ), Oxobacter pfennigii ( DSM 322 ), Sporomusa aerivorans ( DSM 13326 ), Ratella ovalis ( Sporomusa ovata ) ( DSM 2662 ), Sporomusa silvacetica ( DSM 10669 ), Globular rodentium ( Sporomusa sphaeroides ) ( DSM 2875 ), Termite Species ( Sporomusa termitida ) ( DSM 4440 ) and Kewood thermophilic bacteria ( Thermoanaerobacter kivui ) ( DSM 2030 ).

[0079] 3. A mixture according to Scheme 1 or 2, wherein the first microorganism is Clostridium yangi.

[0080] 4. A mixture according to any of the foregoing schemes, wherein the higher alcohol is a C6 to C8 alcohol.

[0081] 5. A mixture according to any of the foregoing schemes, wherein the carbon source comprises at least 2% carbon monoxide gas by volume relative to the volume of the carbon source.

[0082] 6. A mixture according to any of the foregoing schemes, wherein the carbon source comprises 2%-99% carbon monoxide gas by volume.

[0083] 7. A mixture according to any of the foregoing schemes, wherein the aqueous culture medium contains free oxygen.

[0084] 8. The mixture according to Scheme 7, wherein the mixture comprises acetotrophic bacteria in the logarithmic phase and acetotrophic bacteria in the stationary phase.

[0085] 9. The mixture according to scheme 8, wherein the first acetogenic microorganism has a growth rate of 0.01 to 2 h -1 -1 during the exponential growth phase.

[0086] 10. The mixture according to scheme 8 or 9, wherein the first acetogenic microorganism has an OD 600 of 0.01 to 2 during the exponential growth phase.

[0087] 11. A method for producing at least one higher alcohol in an aqueous culture medium, comprising a reaction mixture comprising a first and a second microorganism, wherein - the first microorganism is an acetogenic microorganism capable of converting a carbon source comprising CO into acetic acid and / or ethanol; and - the second microorganism is selected from Clostridium aceticum capable of converting acetic acid and / or ethanol to form an acid and C. carboxidivorans ; wherein the first microorganism is further capable of converting the acid into a corresponding higher alcohol comprising at least 6 carbon atoms.

[0088] 12. The method according to scheme 11, wherein the carbon source comprises 2% - 99% carbon monoxide gas by volume.

[0089] 13. The method according to scheme 11 or 12, wherein the higher alcohol is selected from the group consisting of 2-methyl-1-butanol, 3-methyl-1-butanol, 1-hexanol, 1-octanol, 1-heptanol, 3-methyl-1-pentanol, 4-methyl-1-hexanol, 5-methyl-1-heptanol, 4-methyl-1-pentanol, 5-methyl-1-hexanol, 6-methyl-1-heptanol, and combinations thereof.

[0090] 14. The method according to any one of schemes 11-13, wherein the reaction mixture is according to any one of the reaction mixtures selected from schemes 1-10.

[0091] 15. Use of a reaction mixture according to any one of schemes 1-10 for producing at least one higher alcohol comprising at least 6 carbon atoms. DETAILED DESCRIPTION EMBODIMENT

[0092] The foregoing preferred embodiments, as will be appreciated by one of ordinary skill in the art, can be subject to variations or modifications, either in design, construction or operation, without departing from the scope of the claims. Such variations, for example, are intended to be covered by the scope of the claims.

[0093] Example 1 Co-cultivation of clostridium ljungdahlii and clostridium kluyveri on a defined medium on hydrogen and carbon dioxide In this example, Y. ruckeri is cultivated autotrophically as a first organism in a defined medium in order to produce acetic acid and ethanol. After a given time, then K. europhysi is inoculated as a second organism in the same reactor for the conversion of acetic acid and ethanol into butyric acid and caproic acid. Afterwards, Y. ruckeri then converts butyric acid into butanol.

[0094] A defined medium consisting of 2 g / L (NH4)2HPO4, 0.2 g / L NaCl, 0.15 g / l KCl, 1 g / l KOH, 0.5 g / L MgCl2x 6 H2O, 0.2 g / L CaCl2x 2 H2O, 15 mg / L FeCl2x 4 H2O, 0.4 g / L L-cysteine hydrochloride, 0.4 g / L Na2S x 9 H2O, 3 mg / L boric acid, 2 mg / L CoCl2x 6 H2O, 1 mg / L ZnSO4x 7 H2O, 0.3 mg / L Na2MoO4x 2 H2O, 0.3 mg / L MnSO4x H2O, 0.2 mg / L NiCl2x 6 H2O, 0.1 mg / L CuCl2x 2 H2O, 0.1 mg / L Na2SeO3, 106 µg / L biotin, 5 µg / L folic acid, 2.5 µg / L pyridoxine hydrochloride, 266 µg / L thiamine hydrochloride x H2O, 12.5 µg / L riboflavin, 12.5 µg / L nicotinic acid, 413 µg / L calcium pantothenate, 12.5 µg / L vitamin B12, 12.5 µg / L p-aminobenzoic acid, 15 µg / L lipoic acid was used for the co-cultivation of the two microorganisms.

[0095] Autotrophic cultivation was performed in 250 mL defined medium in 500 mL serum bottles, which were continuously sparged with a synthetic gas consisting of 67% H2 and 33% CO2 at a rate of 1 L / h. The gas was introduced into the liquid phase by a microbubble sparger with a pore size of 10 µm. The serum bottles were continuously shaken at 37°C in an open water bath Innova 3100 from New Brunswick Scientific at a shaking rate of 150 min -1 -1. The pH was maintained in the range of pH 5.0-6.5 by continuous addition of an oxygen-free stock solution of KOH (40 g / L).

[0096] At the beginning of the experiment, cells grown autotrophically were inoculated at an OD 600Inoculation of C. ljungdahlii. Therefore, C. ljungdahlii was grown in complex medium in 1 L serum bottles with 500 mL complex medium under continuous sparging with synthetic gas consisting of 67% H2and 33% C02at a rate of 3 L / h. Using complex medium, the medium consisted of: 1 g / L NH4CI, 0.1 g / L KCI, 0.2 g / L MgS04x 7 H20, 0.8 g / L NaCI, 0.1 g / L KH2P04, 20 mg / L CaCI2x 2 H20, 20 g / L MES, 1 g / L yeast extract, 0.4 g / L L-hydrochloric cysteine, 0.4 g / L Na2S x 9 H20, 20 mg / L nitrilotriacetic acid, 10 mg / L MnS04x H20, 8 mg / L (NH4)2Fe(S04)2x 6 H20, 2 mg / L CoCI2x 6 H20, 2 mg / L ZnS04x 7 H20, 0.2 mg / L CuCI2x 2 H20, 0.2 mg / L Na2Mo04x 2 H20, 0.2 mg / L NiCI2x 6 H20, 0.2 mg / L Na2Se04, 0.2 mg / L Na2W04x 2 H20, 20 pg / L biotin, 20 pg / L folic acid, 100 pg / L pyridoxine hydrochloride, 50 pg / L thiamine hydrochloride x H20, 50 pg / L riboflavin, 50 pg / L nicotinic acid, 50 pg / L calcium pantothenate, 1 pg / L vitamin B12, 50 pg / L p-aminobenzoic acid, 50 pg / L lipoic acid. The gas was introduced into the liquid phase by a microbubble sparger with a pore size of 10 pm. The serum bottles were continuously shaken at 37°C in an open water bath Innova 3100 from New Brunswick Scientific at a shaking rate of 150 min -1 The cells were harvested in late log phase with an OD -1 of 0.67 and a pH of 4.69 by anaerobic centrifugation (4500 min 600 , 4300 g, 20°C, 10 min). The supernatant was discarded and the pellet was resuspended in 10 mL of the above defined medium of determined composition. This cell suspension was then used to inoculate the co-cultivation experiments.

[0097] In parallel, Clostridium krusei was heterotrophically grown on acetic acid and ethanol in 200 mL of compound culture medium in a 500 mL serum bottle. A composite culture medium was used, comprising the following components: 0.25 g / L NH4Cl, 0.2 g / L MgSO4x 7H2O, 0.31 g / L K2HPO4, 0.23 g / L KH2PO4, 2.5 g / L NaHCO3, 1 g / L yeast extract, 10 g / L potassium acetate, 20 g / L ethanol, 0.25 g / L L-cysteine ​​hydrochloride, 1.5 mg / L FeCl2x 4H2O, 70 µg / L ZnCl2x 7H2O, 100 µg / L MnCl2x 4H2O, 6 µg / L boric acid, 190 µg / L CoCl2x 6H2O, 2 µg / L CuCl2x 6H2O, 24 µg / L NiCl2x 6H2O, 36 µg / L Na2MoO4x 2H2O, and 3 µg / L... Na₂SeOO₃ x 5 H₂O, 4 µg / L Na₂WO₄ x 2 H₂O, 100 µg / L Vitamin B12, 80 µg / L para-aminobenzoic acid, 20 µg / L Biotin, 200 µg / L Niacin, 100 µg / L Calcium Pantothenate, 300 µg / L Pyridoxine Hydrochloride, 200 µg / L Thiamine Hydrochloride x H₂O. Serum bottles were placed in an open water bath at 37°C for 100 min in an Innova 3100 from New Brunswick Scientific. -1 The shaking rate was continuously adjusted. The mixture was then subjected to anaerobic centrifugation (4500 min). -1 (4300 g, 20℃, 10 min) at an OD of 0.81 600 Cells were harvested at late logarithmic phase (pH 5.96). The supernatant was discarded and the precipitate was resuspended in 10 mL of the pre-defined culture medium. After running the experiment for 96 hours, the cell suspension was then used at an OD of 0.2. 600 Inoculation and co-culture experiment.

[0098] During the experiment, 5 mL of sample was taken to determine OD. 600 pH and product concentration. The latter is determined quantitatively. 1 H-NMR spectroscopy confirmed the results.

[0099] After inoculation of the Clostridium ljungdahlii, the cells started to grow and continuously produced acetate. Along with the acetate production, ethanol was produced at a lower rate compared to the acetate production. After 96 hours, Clostridium kluyveri was then inoculated into the reactor and a decrease in ethanol concentration was measured in the subsequent experiments. Then, butyrate (maximum 1163 mg / L) and caproate (maximum 136 mg / L) were measured to be produced simultaneously in the subsequent 113 hours of the experiment. In parallel with the butyrate production by Clostridium kluyveri, Clostridium ljungdahlii converted the butyrate into butanol to a maximum concentration of 20 mg / L butanol at the end of the experiment.

[0100] Example 2 Co-cultivation of clostridium ljungdahlii and clostridium kluyveri in a complex medium with CO-containing gas (25% CO) Clostridium ljungdahlii was cultivated autotrophically as a first organism in a complex medium in order to produce acetate and ethanol. After a given time, Clostridium kluyveri was then inoculated as a second organism in the same reactor for the conversion of acetate and ethanol into butyrate and caproate. Thereafter, Clostridium ljungdahlii then converted the butyrate into butanol and the caproate into caproic alcohol.

[0101] A complex medium was used for the co-cultivation of the two microorganisms, which consisted of: 1 g / L NH4CI, 0.1 g / L KCI, 0.2 g / L MgS04x 7 H20, 0.8 g / L NaCI, 0.1 g / L KH2P04, 20 mg / L CaCI2x 2 H20, 20 g / L MES, 1 g / L yeast extract, 0.4 g / L L-hydrochloric cysteine, 0.4 g / L Na2S x 9 H20, 20 mg / L nitrilotriacetic acid, 10 mg / L MnS04x H20, 8 mg / L (NH4)2Fe(S04)2x 6 H20, 2 mg / L CoCI2x 6 H20, 2 mg / L ZnS04x 7 H20, 0.2 mg / L CuCI2x 2 H20, 0.2 mg / L Na2Mo04x 2 H20, 0.2 mg / L NiCI2x 6 H20, 0.2 mg / L Na2Se04, 0.2 mg / L Na2W04x 2 H20, 20 pg / L biotin, 20 pg / L folic acid, 100 pg / L pyridoxine hydrochloride, 50 pg / L thiamine hydrochloride x H20, 50 pg / L riboflavin, 50 pg / L nicotinic acid, 50 pg / L calcium pantothenate, 1 pg / L vitamin B12, 50 pg / L p-aminobenzoic acid, 50 pg / L lipoic acid.

[0102] Autotrophic cultivation was performed in 500 mL complex medium in 1 mL serum bottles continuously sparged with a synthetic gas consisting of 5% H2, 25% C02, 25% CO and 45% N2 at a rate of ~3.6 L / h (>0.5 ppm oxygen). The gas was introduced into the liquid phase by a microbubble sparger with a 10 pm pore size. The serum bottles were continuously shaken at 37°C and a shaking rate of 120 min -1 at an open water bath Innova 3100 from New Brunswick Scientific. During this experiment, the pH was not controlled.

[0103] At the start of the experiment, C. ljungdahlii was inoculated with autotrophically grown cells at an OD 600 of 0.1. Therefore, C. ljungdahlii was grown in the above complex medium in 1 L serum bottles with 500 mL complex medium continuously sparged with a synthetic gas consisting of 67% H2 and 33% C02 at a rate of 3 L / h. The gas was introduced into the liquid phase by a microbubble sparger with a 10 pm pore size. The serum bottles were continuously shaken at 37°C and a shaking rate of 150 min -1 at an open water bath Innova 3100 from New Brunswick Scientific. Cells were harvested in late log phase with an OD -1 of 0.51 and a pH of 5.04 by anaerobic centrifugation (4500 min 600 , 4300 g, 20°C, 10 min). The supernatant was discarded and the pellet was resuspended in 10 mL of the above complex medium. This cell suspension was then used to inoculate the co-cultivation experiment.

[0104] In parallel, C. kluyveri was grown heterotrophically on acetate and ethanol in 500 mL serum bottles in 200 mL complex medium. The complex medium was used, which consisted of: 0.25 g / L NH4CI, 0.2 g / L MgS04x 7H20, 0.31 g / L K2HPO4, 0.23 g / L KH2PO4, 2.5 g / L NaHCO3, 1 g / L yeast extract, 10 g / L potassium acetate, 20 g / l ethanol, 0.25 g / L L-hydrochloric cysteine, 1.5 mg / L FeCI2x 4 H20, 70 pg / L ZnCI2x 7H20, 100 pg / L MnCI2x 4 H20, 6 pg / L boric acid, 190 pg / L CoCI2x 6 H20, 2 pg / L CuCI2x 6 H20, 24 pg / L NiCI2x 6 H20, 36 pg / L Na2MoO4x 2 H20, 3 pg / L Na2SeOO3x 5 H20, 4 pg / L Na2WO4x 2 H20, 100 pg / L vitamin B12, 80 pg / L p-aminobenzoic acid, 20 pg / L biotin, 200 pg / L nicotinic acid, 100 pg / L calcium pantothenate, 300 pg / L pyridoxine hydrochloride, 200 pg / L thiamine hydrochloride x H20. The serum bottles were continuously shaken at 37°C in an open water bath Innova 3100 from New Brunswick Scientific at a shaking rate of 100 min"1. -1 The cells were harvested in late log phase with an OD -1 of 0.54 and a pH of 6.60 by anaerobic centrifugation (4500 min 600 , 4300 g, 20°C, 10 min). The supernatant was discarded and the pellet was resuspended in 10 mL of the above complex medium. This cell suspension was then used to inoculate the co-cultivation experiments after running the experiment for 240 hours.

[0105] During the experiment, 5 mL samples were taken for determination of OD 600 , pH and product concentrations. The latter were determined by quantitative 1 H-NMR spectroscopy.

[0106] After inoculation of C. ljungdahlii, the cells started to grow and continuously produced acetate and ethanol, up to a concentration of ~3 g / L acetate and ~0.5 g / L ethanol after 71 hours. During the subsequent time course of the experiment, acetate was completely converted into ethanol, up to a concentration of 4.8 g / L after 240 hours. At a process time of 240 hours, C. kluyveri was then inoculated into the reactor. Since this organism requires acetate as a substrate in addition to ethanol, ~3 g / L acetate (in the form of sodium acetate) was anaerobically added to the reactor at the same time as the inoculation of C. kluyveri. During the subsequent time course of the experiment, production of butyrate and caproate was measured, up to a concentration of 1.6 g / L each. In parallel to the production of butyrate and caproate by C. kluyveri, C. ljungdahlii converted butyrate into butanol to a maximum concentration of 690 mg / L butanol and caproate into caproic alcohol to a maximum concentration of 1478 mg / L caproic alcohol. Table 2. Results of Example 2 (n.d. = not detected).

[0107] Example 3 Production of acetic acid and ethanol from synthesis gas without oxygen by clostridium ljungdahlii In this example, C. scindens was anaerobically cultured in complex medium with syngas (composed of H2 and CO2) in the absence of oxygen to produce acetic acid and ethanol. For cell culture of C. scindens, 2 mL of cryoculture was anaerobically cultured in 200 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 MgSO4x 7 H2O, 0.02 g / L CaCl2x 2 H2O, 20 mg / L nitrilotriacetic acid, 10 mg / L MnSO4x H2O, 8 mg / L (NH4)2Fe(SO4)2x 6 H2O, 2 mg / L CoCl2x 6 H2O, 2 mg / L ZnSO4x 7 H2O, 0.2 mg / L CuCl2x 2 H2O, 0.2 mg / L Na2MoO4x 2 H2O, 0.2 mg / L NiCl2x 6 H2O, 0.2 mg / L Na2SeO4, 0.2 mg / L Na2WO4x 2 H2O, 20 µg / L d-biotin, 20 µg / L folic acid, 100 µg / L pyridoxine hydrochloride, 50 µg / L thiamine hydrochloride x 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, about 67.5 mg / L NaOH) with about 400 mg / L L-hydrochloric acid cysteine and 400 mg / L Na2S x 9 H2O. The culture was chemotrophically autotrophic with a premixed gas mixture composed of 67% H2, 33% CO2 in a fireproof 1 L glass bottle at 37°C, 150 rpm in an open water bath shaker with 1-3 L / h fumigation for 161 hours. Gas entry into the medium was through a filter with 10 micron pore size and fixed at the center of the reactor at the sparging tube. Cells were centrifuged, washed with 10 ml of ATCC medium and centrifuged again.

[0108] For preculture, a number of washed cells from a growing culture of C. scindens were transferred to 200 mL of ATCC medium with about 400 mg / L L-hydrochloric acid cysteine and grown to an OD of 0.12 600The cultivation was carried out in a pressure-tight 500 ml glass bottle with a premixed gas mixture consisting of 67% H2, 33% CO2, which was in an open water bath shaker at 37°C, 150 rpm and with aeration of 3 L / h for 65 hours. The gas entry into the culture medium was through a filter with a 10 micron pore size, which was placed in the center of the reactor. The cells were centrifuged, washed with 10 ml production buffer (pH 6.2; 0.5 g / L of KOH, aerated with a premixed gas mixture of 67% H2, 33% CO2 at 1 L / hr for 1 hour) and centrifuged again.

[0109] For the production cultivation, a number of washed cells from the preculture of C. ljungdahlii were transferred into 200 mL ATCC medium with about 400 mg / L L-cysteine hydrochloride and grown to an OD 600 The cultivation was carried out in a pressure-tight 500 ml glass bottle with a premixed gas mixture consisting of 67% H2, 33% CO2, which was in an open water bath shaker at 37°C, 150 rpm and with aeration of 3 L / h for 118 hours. The gas entry into the culture medium was through a filter with a 10 micron pore size, which was placed in the center of the reactor. When the pH dropped below 5.0, 1 ml of a 140 g / l KOH solution was added. When sampling, each 5 ml sample was removed for determination of OD 600 , pH and product range. The determination of product concentrations was carried out by semi-quantitative 1 H-NMR spectroscopy. Trimethylsilylpropionic acid sodium salt (T (M) SP) was used as internal quantitative standard.

[0110] The cell density in the production culture remained constant during the cultivation period of 118 hours, which can be identified by a stable OD 600 of 0.2, corresponding to a growth rate of μ = 0 hr -1 The acetic acid concentration increased significantly from 4 mg / L to 3194 mg / L and the ethanol concentration from 17 mg / L to 108 mg / L at the same time.

[0111] Example 4 Production of acetic acid and ethanol from synthesis gas comprising CO2 and H2 with C. ljungdahlii C. ljungdahlii was cultivated in complex medium with synthesis gas and oxygen. C. ljungdahlii was first cultivated in the presence of synthesis gas (consisting of H2 and CO2) and in the absence of oxygen to produce acetic acid and ethanol. For the cultivation, the cells were grown in pressure-tight glass bottles, which can be airtight sealed with a butyl rubber plug. All steps involving C. ljungdahlii cells were carried out under anaerobic conditions.

[0112] For cell culture of C. ljungdahlii, 2 mL of a cryoculture was anaerobically cultured in 200 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 NH4CI, 0.1 g / L KCI, 0.1 g / L KH2PO4, 0.2 g / L MgSO4x 7 H2O, 0.02 g / L CaCI2x 2 H2O, 20 mg / L nitrilotriacetic acid, 10 mg / L MnSO4x H2O, 8 mg / L (NH4)2Fe(SO4)2x 6 H2O, 2 mg / L CoCI2x 6 H2O, 2 mg / L ZnSO4x 7 H2O, 0.2 mg / L CuCI2x 2 H2O, 0.2 mg / L Na2MoO4x 2 H2O, 0.2 mg / L NiCI2x 6 H2O, 0.2 mg / L Na2SeO4, 0.2 mg / L Na2WO4x 2 H2O, 20 pg / L d-biotin, 20 pg / L folinic acid, 100 pg / L pyridoxine hydrochloride, 50 pg / L thiamine hydrochloride x H2O, 50 pg / L riboflavin, 50 pg / L niacin, 50 pg / L calcium pantothenate, 1 pg / L vitamin B12, 50 pg / L p-aminobenzoic acid, 50 pg / L lipoic acid, about 67.5 mg / L NaOH) with about 400 mg / L L-cysteine hydrochloride and 400 mg / L Na2S x 9 H2O. Cultures were performed in fireproof 1 L glass bottles with chemotrophically autotrophic energy using a premixed gas mixture consisting of 67% H2, 33% CO2, at 37°C, 150 rpm in an open water bath shaker with a fumigation of 1-3 L / h for 161 hours. Gas entry into the medium was performed through a filter with a 10 micron pore size and was fixed at the reactor center at the aeration tube. Cells were centrifuged, washed with 10 ml of ATCC medium and centrifuged again.

[0113] For preculturing, a number of washed cells from a growth culture of C. ljungdahlii were transferred to 200 mL of ATCC medium with about 400 mg / L L-cysteine hydrochloride and grown to an OD of 0.12 600The cultivation was carried out in a pressure-resistant 500 ml glass bottle with a premixed gas mixture consisting of 67% H2, 33% CO2, which was in an open water bath shaker at 37°C, 150 rpm and with aeration of 3 L / h for 24 hours. Subsequently, the gas mixture was changed to a gas mixture with a composition of 66.85% H2, 33% CO2 and 0.15% O2 and the cells were further aerated with 3 L / h for 67 hours. The gas entry into the culture medium was carried out through a Begasungsfritte with a 10 micrometer pore size, which was placed in the center of the reactor at the sparger. The cells were centrifuged, washed with 10 ml ATCC medium and centrifuged again. The gas entry into the culture medium was carried out through a filter with a 10 micrometer pore size, which was placed in the center of the reactor. The cells were centrifuged, washed with 10 ml ATCC medium and centrifuged again.

[0114] For the production cultivation, a number of washed cells from a preculture of Clostridium ljungdahlii were transferred into 200 mL ATCC medium with about 400 mg / L L-cysteine hydrochloride and grown to an OD 600 The cultivation was carried out in a pressure-resistant 500 ml glass bottle with a premixed gas mixture consisting of 66.85% H2, 33% CO2 and 0.15% O2, which was in an open water bath shaker at 37°C, 150 rpm and with aeration of 3 L / h for 113 hours. The gas entry into the culture medium was carried out through a filter with a 10 micrometer pore size, which was placed in the center of the reactor. When sampling, each 5 ml sample was removed for determination of OD 600 , pH and product range. The determination of product concentrations was carried out by semi-quantitative 1 H-NMR spectroscopy. Trimethylsilylpropionic acid sodium salt (T(M) SP) was used as internal quantitative standard.

[0115] In the period of 89 hours to 113 hours no recognizable cell growth was shown. OD 600 stabilized at 0.29, which corresponds to a growth rate μ = 0 h -1 During this time course the acetic acid concentration increased slightly from 86.9 mg / L to 89.4 mg / L and the ethanol concentration decreased from 16.2 mg / L to 11.9 mg / L.

[0116] Example 5 Cultivation of Clostridium ljungdahlii in the presence of a synthesis gas comprising CO2 and oxygen in the logarithmic phase C. ljungdahlii is fed H2and C02from a feed-through gas phase and forms acetic acid and ethanol. For culturing, pressure-resistant glass bottles can be used which can be airtight sealed with butyl rubber stoppers. All culturing steps in which C. ljungdahlii cells are involved are performed under anaerobic conditions.

[0117] For cell culturing of C. ljungdahlii, 5 mL of a Cryoculture are anaerobically cultivated in 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 NH4CI, 0.1 g / L KCI, 0.1 g / L KH2P04, 0.2 g / L MgS04x 7 H20, 0.02 g / L CaCI2x 2 H20, 20 mg / L nitrilotriacetic acid, 10 mg / L MnS04x H20, 8 mg / L (NH4)2Fe(S04)2x 6 H20, 2 mg / L CoCI2x 6 H20, 2 mg / L ZnS04x 7 H20, 0.2 mg / L CuCI2x 2 H20, 0.2 mg / L Na2Mo04x 2 H20, 0.2 mg / L NiCI2x 6 H20, 0.2 mg / L Na2Se04, 0.2 mg / L Na2W04x 2 H20, 20 pg / L d-biotin, 20 pg / L folinic acid, 100 pg / L pyridoxine hydrochloride, 50 pg / L thiamine hydrochloride x H20, 50 pg / L riboflavin, 50 pg / L nicotinic acid, 50 pg / L calcium pantothenate, 1 pg / L vitamin B12, 50 pg / L p-aminobenzoic acid, 50 pg / L lipoic acid, about 67.5 mg / L NaOH) with about 400 mg / L L-cysteine hydrochloride and 400 mg / L Na2S x 9 H20. Culturing is performed chemoautotrophically with a premixed gas mixture consisting of 67% H2, 33% C02in a fireproof 1 L glass bottle, which is airtight sealed with a butyl rubber stopper, in an open water bath shaker at 37°C, 100 rpm with a fumigation of 3 L / h for 72 hours. Gas entry into the medium is performed through a filter with a pore size of 10 micrometer and is fixed at the center of the reactor at the gas inlet tube. Cells are centrifuged, washed with 10 ml of ATCC medium and centrifuged again.

[0118] For the main culture, many washed cells from the growth culture of C. ljungdahlii are transferred to 500 mL of ATCC medium with about 400 mg / L L-cysteine hydrochloride and grown to an OD 600Cultures were carried out in pressure-resistant 1 L glass bottles with a premixed gas mixture consisting of 66.85% H2, 33% C02and 0.15% 02at 37°C, 150 rpm in an open water bath shaker with aeration of 1 L / h for 45 hours. Gas entry into the culture medium was through a filter with 10 micron pore size placed in the center of the reactor. When sampling, 5 ml samples were removed for determination of OD 600 nm, pH and product range. Determination of product concentrations was carried out by semi-quantitative 1 H-NMR spectroscopy. Trimethylsilyl propionate sodium salt (T(M) SP) was used as internal quantitative standard.

[0119] During the cultivation period, significant cell growth was shown by an increase of OD 600 nm from 0.10 to 0.54, corresponding to a growth rate μ = 0.037 h -1 nm. Acetate concentration increased simultaneously from 9.6 mg / L to 3,304 mg / L and ethanol concentration from 2.2 mg / L to 399 mg / L.

[0120] Example 6 Cultivation of clostridium ljungdahlii in the exponential phase in the presence of synthesis gas (65% CO) comprising CO and oxygen Clostridium ljungdahlii was cultivated autotrophically with synthesis gas (consisting of CO, H2 and C02) in the presence of oxygen in complex medium to produce acetate and ethanol.

[0121] Using complex medium, consisting of: 1 g / L NH4CI, 0.1 g / L KCI, 0.2 g / L MgS04x 7 H20, 0.8 g / L NaCI, 0.1 g / L KH2P04, 20 mg / L CaCI2x 2 H20, 20 g / L MES, 1 g / L yeast extract, 0.4 g / L L-homocysteine hydrochloride, 0.4 g / L Na2S x 9 H20, 20 mg / L nitrilotriacetic acid, 10 mg / L MnS04x H20, 8 mg / L (NH4)2Fe(S04)2x 6 H20, 2 mg / L CoCI2x 6 H20, 2 mg / L ZnS04x 7 H20, 0.2 mg / L CuCI2x 2 H20, 0.2 mg / L Na2Mo04x 2 H20, 0.2 mg / L NiCI2x 6 H20, 0.2 mg / L Na2Se04, 0.2 mg / L Na2W04x 2 H20, 20 pg / L biotin, 20 pg / L folic acid, 100 pg / L pyridoxine hydrochloride, 50 pg / L thiamine hydrochloride x H20, 50 pg / L riboflavin, 50 pg / L nicotinic acid, 50 pg / L calcium pantothenate, 1 pg / L vitamin B12, 50 pg / L p-aminobenzoic acid, 50 pg / L lipoic acid.

[0122] Autotrophic cultivation was performed in 500 mL medium in a 1 L serum bottle continuously aerated with synthetic gas consisting of 65% CO, 4% H2 and 15% CO2 at a rate of 3.6 L / h. The gas was introduced into the liquid phase by a microbubble sparger with a 10 pm pore size. The serum bottle was continuously shaken at 37°C and a shaking rate of 120 min -1 .

[0123] The pH was not controlled.

[0124] At the start of the experiment, Clostridium ljungdahlii was inoculated with cells grown autotrophically on H2 / CO2 at an OD 600 of 0.1. Thus, Clostridium ljungdahlii was grown in complex medium in a 1 L serum bottle with 500 mL complex medium continuously aerated with synthetic gas consisting of 67% H2 and 33% CO2 at a rate of 3 L / h. The medium described above was also used for this cultivation. The gas was introduced into the liquid phase by a microbubble sparger with a 10 pm pore size. The serum bottle was continuously shaken at 37°C and a shaking rate of 150 min-1 The shaking rate was continuously varied. Cells were harvested in the log phase with an OD -1 of 0.49 and a pH of 5.03 by anaerobic centrifugation (4500 min 600 , 4300 g, 20°C, 10 min). The supernatant was discarded and the pellet was resuspended in 10 mL of the above-mentioned medium. This cell suspension was then used to inoculate the cultivation experiments. The gas phase concentration of carbon monoxide was measured as follows: The gas phase was sampled and analyzed offline by gas chromatography GC 6890N from Agilent Technologies Inc. with a thermal conductivity detector. The gas phase concentration of oxygen was measured by a micro oxygen immersion probe from PreSens Precision Sensing GmbH. The oxygen concentration was measured by fluorescence quenching, whereas the degree of quenching correlates with the oxygen partial pressure in the gas phase. The measured value of oxygen in the synthesis gas used indicates an O2 concentration of 0.1 vol%.

[0125] During the experiment, 5 mL samples were taken for determination of OD 600 , pH and product concentrations. The latter were determined by quantitative 1 H-NMR spectroscopy.

[0126] After inoculation of C. ljungdahlii, the cells started to grow with a growth rate of 0.062 h -1 and continuously produced acetate until a concentration of 6.2 g / L after 94.5 h. Concomitantly with acetate production, ethanol was produced at a lower rate compared to acetate production until a concentration of 1 g / L after 94.5 h. Table 3. Results of Example 6 (n.d. = not detected).

[0127] Example 7 Growth and acetic acid production of clostridium ljungdahlii on synthesis gas with oxygen For the bioconversion of hydrogen and carbon dioxide to acetate, the homoacetogenic bacterium C. ljungdahlii was cultivated on synthesis gas with oxygen. All cultivation steps were performed under anaerobic conditions in pressure-resistant glass bottles, which can be gas-tight sealed with butyl rubber stoppers.

[0128] For pre-culture, inoculate 5 mL of frozen Clostridium yangyi culture into 500 mL of medium (ATCC1754 medium: pH 6.0, 20 g / L LMEs, 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 hypozinotriacetic 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) with an additional 400 mg / L L-cysteine ​​hydrochloride and 400 mg / L Na2S x 9 H2O. H2O, 0.2 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 Hydrochloride, 50 µg / L Thiamine Hydrochloride x H2O, 50 µg / L Riboflavin, 50 µg / L Niacin, 50 µg / L Calcium Pantothenate, 1 µg / L Vitamin B1 12 The medium contained 50 µg / L p-aminobenzoic acid, 50 µg / L lipoic acid, and approximately 67.5 mg / L NaOH. Chemoautotrophic culture was carried out in 1 L pressure-resistant glass vials at 37 °C, 100 rpm, and an aeration rate of 3 L / h in an open water bath shaker for 72 hours. The gas was then introduced into the culture medium through a 10 µm orifice atomizer fixed in the center of the reactor. The culture was conducted without pH control.

[0129] After pre-culture, the cell suspension was centrifuged (10 min, 4200 rpm), and the pellet was washed with 10 ml of culture medium and centrifuged again. For the major culture, the same amount of washed cells from the pre-culture was centrifuged at 0.1 OD. 600nmThe transfer to 200 mL medium with additional 400 mg / L L-cysteine hydrochloride was performed. The chemoautotrophic cultivation was performed in 250 mL pressure-resistant glass bottles at 37 °C, 150 rpm and a gas flow of 1 L / h with a premixed gas with 65% H2, 33% CO2, 2% O2 in an open water bath shaker for 47 h. The gas was discharged into the medium via a sparger with a pore size of 10 pm, which was fixed in the center of the reactor. The cultivation was performed without pH control. During the cultivation several 5 mL samples were taken to determine OD 600 nm, pH and product formation. The determination of product concentrations was performed by semi-quantitative 1 H-NMR spectroscopy. Trimethylsilylpropionic acid sodium salt (T(M)SP) was used as internal quantitative standard. The dissolved oxygen in the medium was also measured online by an oxygen immersion probe (PSt6 with Oxy4Trace, Presens, Germany).

[0130] During the cultivation period, cell growth was observed by OD 600 nm from 0.11 to 0.32, which correlates with a growth rate of µ = 0.022 h -1 The acetic acid concentration increased from 8 mg / L to 91 mg / L, no increase of the ethanol concentration was observed. During the cultivation period, the dissolved oxygen concentration varied between 0.06 - 0.15 mg / L.

[0131] In a similar technical setup with the same parameters (medium composition, volume, bottle, gas, gas flow, temperature, shaking frequency) but without cells in the medium, a dissolved oxygen concentration of 0.50 mg / L was measured.

[0132] Example 8 Growth and acetic acid production of clostridium ljungdahlii on synthesis gas with oxygen For the bioconversion of hydrogen and carbon dioxide to acetic acid, the homoacetogenic bacterium Yantsetta sp. was cultivated on syngas with oxygen. All cultivation steps were performed under anaerobic conditions in pressure-resistant glass bottles, which can be gas-tight sealed with a butyl rubber plug.

[0133] For pre-culture, inoculate 5 mL of frozen Clostridium yangyi culture into 500 mL of medium (ATCC1754 medium: pH 6.0, 20 g / L LMEs, 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 hypozinotriacetic 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) with an additional 400 mg / L L-cysteine ​​hydrochloride and 400 mg / L Na2S x 9 H2O. H2O, 0.2 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 Hydrochloride, 50 µg / L Thiamine Hydrochloride x H2O, 50 µg / L Riboflavin, 50 µg / L Niacin, 50 µg / L Calcium Pantothenate, 1 µg / L Vitamin B12, 50 µg / L Para-aminobenzoic Acid, 50 µg / L Alpha-lipoic Acid, approximately 67.5 mg / L NaOH). Chemoautotrophic culture was carried out in 1 L pressure-resistant glass vials at 37 °C, 100 rpm, and an aeration rate of 3 L / h in an open water bath shaker for 72 hours. The gas was then introduced into the culture medium through a sprayer with a 10 µm orifice, the sprayer being fixed in the center of the reactor. The culture was conducted without pH control.

[0134] After pre-culture, the cell suspension was centrifuged (10 min, 4200 rpm), and the pellet was washed with 10 ml of culture medium and centrifuged again. For the major culture, the same amount of washed cells from the pre-culture was centrifuged at 0.1 OD. 600nm to 200 mL medium with additional 400 mg / L L-cysteine hydrochloride. The chemoautotrophic cultivation was carried out in 250 mL pressure-resistant glass bottles at 37°C, 150 rpm and a ventilation rate of 1 L / h with a premixed gas with 66.85% H2, 33% C02, 0.15% 02 in an open water bath shaker for 47 hours. The gas was discharged into the medium via a sparger with a pore size of 10 pm, which was fixed in the center of the reactor. The cultivation was carried out without pH control. During the cultivation, several 5 mL samples were taken to determine the OD 600 nm, pH and product formation. The determination of the product concentrations was carried out by semi-quantitative 1H-NMR spectroscopy. Sodium trimethylsilylpropionate (T(M)SP) was used as internal quantitative standard. The dissolved oxygen in the medium was also measured online by an oxygen immersion probe (PSt6 with Oxy4Trace, Presens, Germany).

[0135] During the cultivation period, the OD 600 nm was increased from 0.10 to 0.45, which correlates with a growth rate of µ = 0.032 h -1 The acetic acid concentration was increased from 7 mg / L to 2347 mg / L and the ethanol concentration was increased from 2 mg / L to 319 mg / L. The dissolved oxygen concentration was 0.00 mg / L throughout the cultivation phase.

[0136] In a similar technical setup with the same parameters (medium composition, volume, bottle, gas, ventilation rate, temperature, shaking frequency) but without cells in the medium, a dissolved oxygen concentration of 0.03 mg / L was measured.

[0137] Example 9 Co-cultivation of clostridium ljungdahlii and clostridium kluyveri in a complex medium with CO-containing gas (7% CO) Clostridium kluyveri as a second organism was inoculated in the same reactor after a given time for the conversion of acetic acid and ethanol into butyric acid and caproic acid. Thereafter, Clostridium ljungdahlii then converted butyric acid into butanol and caproic acid into caproic alcohol.

[0138] A complex medium was used for the co-cultivation of the two microorganisms, consisting of: 1 g / L NH4CI, 0.1 g / L KCI, 0.2 g / L MgS04x 7 H20, 0.8 g / L NaCI, 0.1 g / L KH2P04, 20 mg / L CaCI2x 2 H20, 20 g / L MES, 1 g / L yeast extract, 0.4 g / L L-hydrochloric cysteine, 0.4 g / L Na2S x 9 H20, 20 mg / L nitrilotriacetic acid, 10 mg / L MnS04x H20, 8 mg / L (NH4)2Fe(S04)2x 6 H20, 2 mg / L CoCI2x 6 H20, 2 mg / L ZnS04x 7 H20, 0.2 mg / L CuCI2x 2 H20, 0.2 mg / L Na2Mo04x 2 H20, 0.2 mg / L NiCI2x 6 H20, 0.2 mg / L Na2Se04, 0.2 mg / L Na2W04x 2 H20, 20 pg / L biotin, 20 pg / L folic acid, 100 pg / L pyridoxine hydrochloride, 50 pg / L thiamine hydrochloride x H20, 50 pg / L riboflavin, 50 pg / L nicotinic acid, 50 pg / L calcium pantothenate, 1 pg / L vitamin B12, 50 pg / L p-aminobenzoic acid, 50 pg / L lipoic acid.

[0139] Autotrophic cultivation was performed in 500 mL complex medium in a 1 L serum bottle continuously sparged with a synthetic gas consisting of 63 % H2, 7 % CO2 and 2 % CO (≥ 0.5 ppm oxygen) at a rate of ~ 3.6 L / h. The gas was introduced into the liquid phase by a microbubble sparger with a 10 pm pore size. The serum bottle was continuously shaken at 37 °C and a shaking rate of 120 min -1 The pH was not controlled during this experiment.

[0140] At the start of the experiment, Clostridium celtum was inoculated with autotrophically grown cells at an OD 600 of 0.1. Thus, Clostridium celtum was grown in the above complex medium in a 1 L serum bottle with 500 mL complex medium continuously sparged with a synthetic gas consisting of 67 % H2 and 33 % CO2 at a rate of 3 L / h. The gas was introduced into the liquid phase by a microbubble sparger with a 10 pm pore size. The serum bottle was continuously shaken at 37 °C and a shaking rate of 150 min -1The shaking rate was continuously adjusted. The mixture was then subjected to anaerobic centrifugation (4500 min). -1 (4300 g, 20℃, 10 min) at an OD of 0.89 600 Harvest cells at the stationary phase at pH 4.52. Discard the supernatant and resuspend the precipitate in 10 mL of the above-described composite culture medium. Then use this cell suspension to inoculate for co-culture experiments.

[0141] In parallel, Clostridium krusei was heterotrophically grown on acetic acid and ethanol in 200 mL of compound culture medium in a 500 mL serum bottle. A composite culture medium was used, comprising the following components: 0.25 g / L NH4Cl, 0.2 g / L MgSO4x 7H2O, 0.31 g / L K2HPO4, 0.23 g / L KH2PO4, 2.5 g / L NaHCO3, 1 g / L yeast extract, 10 g / L potassium acetate, 20 g / L ethanol, 0.25 g / L L-cysteine ​​hydrochloride, 1.5 mg / L FeCl2x 4H2O, 70 µg / L ZnCl2x 7H2O, 100 µg / L MnCl2x 4H2O, 6 µg / L boric acid, 190 µg / L CoCl2x 6H2O, 2 µg / L CuCl2x 6H2O, 24 µg / L NiCl2x 6H2O, 36 µg / L Na2MoO4x 2H2O, and 3 µg / L... Na₂SeOO₃ x 5 H₂O, 4 µg / L Na₂WO₄ x 2 H₂O, 100 µg / L Vitamin B12, 80 µg / L para-aminobenzoic acid, 20 µg / L Biotin, 200 µg / L Niacin, 100 µg / L Calcium Pantothenate, 300 µg / L Pyridoxine Hydrochloride, 200 µg / L Thiamine Hydrochloride x H₂O. Serum bottles were placed in an open water bath at 37°C for 100 min in an Innova 3100 from New Brunswick Scientific. -1 The shaking rate was continuously adjusted. The mixture was then subjected to anaerobic centrifugation (4500 min). -1 (4300 g, 20℃, 10 min) at an OD of 0.86 600 Harvest cells during the late logarithmic phase at pH 6.01. Discard the supernatant and resuspend the precipitate in 10 mL of the above-described composite medium. After running the experiment for 49 hours, use this cell suspension to inoculate for co-culture experiments.

[0142] During the experiment, 5 mL of sample was taken to determine OD. 600 pH and product concentration. The latter is determined quantitatively. 1 H-NMR spectroscopy confirmed the results.

[0143] After inoculation of the Clostridium ljungdahlii, the cells started to grow and continuously produced acetic acid and ethanol, reaching a concentration of ~ 1.3 g / L acetic acid and ~ 0.8 g / L ethanol after 49 hours. After a process time of 49 hours, Clostridium kluyveri was then inoculated into the reactor. During the subsequent time course of the experiment, production of butyric acid and caproic acid was measured to concentrations of up to 0.6 g / L each. In parallel to the production of butyric acid and caproic acid by Clostridium kluyveri, Clostridium ljungdahlii converted butyric acid into butanol to a maximum concentration of 472 mg / L butanol and caproic acid into caproic alcohol to a maximum concentration of 630 mg / L caproic alcohol.

[0144] Example 10 Co-cultivation of clostridium autoethanogenum and clostridium kluyveri in a complex medium with CO-containing gas for the production of higher alcohols such as hexanol and octanol (10% CO) ​ In this example, Clostridium autoethanogenum was cultivated autotrophically in a complex medium as a first organism to produce acetic acid and ethanol. After a given time, Clostridium kluyveri was inoculated as a second organism in the same reactor for the conversion of acetic acid and ethanol into butyric acid and caproic acid. In a subsequent step, Clostridium acetobutylicum then converted butyric acid into butanol and caproic acid into caproic alcohol. The production of octanol was also observed.

[0145] A complex medium was used for the co-cultivation of the two microorganisms, consisting of: 1 g / L NH4CI, 0.1 g / L KCI, 0.2 g / L MgS04x 7 H20, 0.8 g / L NaCI, 0.1 g / L KH2P04, 20 mg / L CaCI2x 2 H20, 20 g / L MES, 1 g / L yeast extract, 0.4 g / L L-hydrochloric cysteine, 20 mg / L nitrilotriacetic acid, 10 mg / L MnS04x H20, 8 mg / L (NH4)2Fe(S04)2x 6 H20, 2 mg / L CoCI2x 6 H20, 2 mg / L ZnS04x 7 H20, 0.2 mg / L CuCI2x 2 H20, 0.2 mg / L Na2Mo04x 2 H20, 0.2 mg / L NiCI2x 6 H20, 0.2 mg / L Na2Se04, 0.2 mg / L Na2W04x 2 H20, 20 pg / L biotin, 20 pg / L folic acid, 100 pg / L pyridoxine hydrochloride, 50 pg / L thiamine hydrochloride x H20, 50 pg / L riboflavin, 50 pg / L nicotinic acid, 50 pg / L calcium pantothenate, 1 pg / L vitamin B12, 50 pg / L p-aminobenzoic acid, 50 pg / L lipoic acid.

[0146] Autotrophic culture was performed in 1L serum bottles in 500mL of composite medium, with continuous aeration of syngas (60% H₂, 30% CO₂, and 10% CO) at a rate of 1.0L / h. The gas was introduced into the liquid phase through a microbubble disperser with 10µm pore size. The serum bottles were incubated in an open water bath (Innova 3100) from New Brunswick Scientific at 37°C for 150 min. -1 The shaking rate was maintained during continuous shaking. The pH was adjusted once during this experiment by adding anaerobic KOH.

[0147] At the beginning of the experiment, autotrophic cells were used with an OD of 0.1. 600 Clostridium ethanoliferum was inoculated. Therefore, Clostridium ethanoliferum was grown in a 1L serum bottle containing 500mL of composite medium under continuous aeration of syngas consisting of 67% H2 and 33% CO2 at a rate of 1 L / h. The gas was introduced into the liquid phase through a microbubble disperser with 10 µm pore size. The serum bottle was incubated in an open water bath Innova 3100 from New Brunswick Scientific at 37°C for 150 min. -1 The shaking rate was continuously adjusted. The mixture was then subjected to anaerobic centrifugation (4500 min). -1 (4300 g, 20℃, 10 min) at an OD of 0.62 600 Cells were harvested at late logarithmic growth phase (pH 5.15). The supernatant was discarded, and the precipitate was resuspended in 10 mL of the above-described composite medium. This cell suspension was then used to seed co-culture experiments. Later in the experiment, cells were harvested from pre-cultures grown under the same conditions as described above at an OD of 0.2. 600 The ethanol-producing Clostridium was inoculated again into the experimental setup. The mixture was then subjected to anaerobic centrifugation (4500 min). -1 (4300 g, 20℃, 10 min) at an OD of 0.55 600 Harvest cells during the late logarithmic phase at pH 5.12. Discard the supernatant and resuspend the precipitate in 10 mL of culture medium taken from the anaerobic culture run. After 74 hours of running, use this cell suspension to transfer the cells to the co-culture experiment again.

[0148] In parallel, Clostridium krusei was heterotrophically grown on acetic acid and ethanol in 200 mL of compound culture medium in a 500 mL serum bottle. A composite culture medium was used, comprising the following components: 0.25 g / L NH4Cl, 0.2 g / L MgSO4x 7H2O, 0.31 g / L K2HPO4, 0.23 g / L KH2PO4, 2.5 g / L NaHCO3, 1 g / L yeast extract, 10 g / L potassium acetate, 20 g / L ethanol, 0.25 g / L L-cysteine ​​hydrochloride, 1.5 mg / L FeCl2x 4H2O, 70 µg / L ZnCl2x 7H2O, 100 µg / L MnCl2x 4H2O, 6 µg / L boric acid, 190 µg / L CoCl2x 6H2O, 2 µg / L CuCl2x 6H2O, 24 µg / L NiCl2x 6H2O, 36 µg / L Na2MoO4x 2H2O, and 3 µg / L... Na₂SeOO₃ x 5 H₂O, 4 µg / L Na₂WO₄ x 2 H₂O, 100 µg / L Vitamin B12, 80 µg / L para-aminobenzoic acid, 20 µg / L Biotin, 200 µg / L Niacin, 100 µg / L Calcium Pantothenate, 300 µg / L Pyridoxine Hydrochloride, 200 µg / L Thiamine Hydrochloride x H₂O. Serum bottles were placed in an open water bath at 37°C for 100 min in an Innova 3100 from New Brunswick Scientific. -1 The shaking rate was continuously adjusted. The mixture was then subjected to anaerobic centrifugation (4500 min). -1 (4300 g, 20℃, 10 min) at an OD of 0.86 600 Cells were harvested at late logarithmic phase 6.01. The supernatant was discarded and the pellet was resuspended in 10 mL of culture medium taken from the anaerobic culture medium used in the run experiment. After 23 hours of run experiment, this cell suspension was then used to transfer cells to the co-culture experiment. Later in the experiment, cells were harvested from the pre-culture grown under the same conditions as described above at an OD of 0.15. 600 Clostridium krusei was inoculated into the experimental setup again. The mixture was then subjected to anaerobic centrifugation (4500 min). -1 (4300 g, 20℃, 10 min) at an OD of 0.38 600 Harvest cells at the logarithmic phase (pH 6.67). Discard the supernatant and resuspend the precipitate in 10 mL of culture medium taken from the anaerobic culture run. After 74 hours of the run, use this cell suspension to transfer the cells back to the co-culture experiment.

[0149] During the experiment, 5 mL samples were taken for determination of OD 600 , pH and product concentrations. The latter was determined by quantification 1 of H-NMR spectroscopy.

[0150] After inoculation of the ethanol-producing Clostridia, the cells started to grow and produced acetic acid to a concentration of ~ 2.2 g / L to a concentration of ~ 0.5 g / L after 23 hours. At this time point, Clostridium kluyveri was inoculated into the running experiment, followed by the production of butyric acid and caproic acid. Then butyric acid and caproic acid were reduced to the corresponding alcohols by the ethanol-producing Clostridia. After 69 hours of running the experiment, the pH was increased from pH 4.74 to pH 6.01 by the addition of anaerobic KOH. Afterwards, 50 mg / L L-cysteine hydrochloride was added to the medium and cells of both the ethanol-producing Clostridia and Clostridium kluyveri were inoculated into the running experiment. After 140 hours of cultivation, 650 mg / L butanol, 220 mg / L capryl alcohol and 4.5 mg / L octanol were produced.

[0151] References .

Claims

1. A reaction mixture comprising a mixed culture of a first and a second microorganism in an aqueous culture medium containing a carbon monoxide gas, wherein - the first microorganism is an acetogenic microorganism capable of converting a carbon source into acetic acid and / or ethanol; and - The second microorganism is selected from Clostridium krusei, which is capable of converting acetic acid and / or ethanol to form acid. Clostridium kluyveri )and C. carboxidivorans ; wherein the first microorganism is further capable of converting the acid into a corresponding higher alcohol, and the higher alcohol comprises at least 6 carbon atoms.

2. Mixture according to claim 1, wherein the first microorganism is selected from the group consisting of Acetanaerobacterium umxidum (ATCC 43769) ( Acetoanaerobium notera ) ( ATCC 35199 ), Acetanaerobutylicum longum (ATCC 43184) ( Acetonema longum ) ( DSM 6540 ), Acetobacterium woodii (ATCC 26042) ( Acetobacterium carbinolicum ) ( DSM 2925 ), Acetobacterium malicum (ATCC 43769) ( Acetobacterium malicum ) ( DSM 4132 ), Acetobacterium sp. 446 (ATCC 43769) ( Acetobacterium species no. 446 ), Acetobacterium wieringae (ATCC 43769) ( Acetobacterium wieringae ) ( DSM 1911 ), Acetobacterium woodyi (ATCC 43769) ( Acetobacterium woodii ) ( DSM 1030 ), Alkalibaculum bacchi ( DSM 22112 ), Archaeoglobus fulgidus (ATCC 43524) ( Archaeoglobus fulgidus ) ( DSM 4304 ), Blautia producta ( DSM 2950 ), Butyribacterium methylotrophicum (ATCC 43210) ( Butyribacterium methylotrophicum ) ( DSM 3468 ), Clostridium aceticum (ATCC 26863) ( Clostridium aceticum ) ( DSM 1496 ), Clostridium autoethanogenum (ATCC 55988) ( Clostridium autoethanogenum ) ( DSM 10061 , DSM 19630 and DSM 23693 ), Clostridium carboxidivorans ( DSM 15243 ), Clostridium coskatii ( ATCC No. PTA - 10522 ), Clostridium drakei ( ATCC BA - 623 ), Clostridium formicicum (ATCC 19358) ( Clostridium formicoaceticum ) ( DSM 92 ), Clostridium glycolicum (ATCC 43172) ( Clostridium glycolicum ) ( DSM 1288 ), Clostridium ljungdahlii (ATCC 55987) ( Clostridium ljungdahlii ) ( DSM 13528 ), Clostridium ljungdahlii C-01 (ATCC 55987) ( ATCC 55988 ), Clostridium ljungdahlii ERI-2 (ATCC 55987) ( ATCC 55380 ), Clostridium ljungdahlii O-52 (ATCC 55987) ( ATCC 55989 ), Clostridium marismigre (ATCC 43206) ( Clostridium mayombei ) ( DSM 6539 ), Clostridium methoxybenzovorans ( DSM 12182 ), Clostridium ragsdalei ( DSM 15248 ​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​Clostridium scatologenes (Clostridium scatologenes), Clostridium scatologenes DSM 757 Clostridium sp. ATCC 29797, Desulfomonile kistiae (Desulfomonile kistiae), Desulfotomaculum kuznetsovii DSM 6115 Desulfotomaculum thermosulfurigenes subsp. thermopilei thermosyntrophicum Desulfotomaculum thermobezoicum subsp thermosyntrophicum DSM 14055 Eubacterium limosum DSM 20543 Methanosarcina acetivorans (Methanosarcina acetivorans) C2A, Methanosarcina acetivorans DSM 2834 Moorella sp. Moorella sp. HUC22-1, Moorella thermoacetica (Moorella thermoacetica), Moorella thermoacetica DSM 521 Moorella thermoautotrophica Moorella thermautotrophica (Moorella thermautotrophica), DSM 1974 Oxobacter pfennigii DSM 322 Sporomusa aerivorans DSM 13326 Sporomusa ovata DSM 2662 Sporomusa silvacetica DSM 10669 Sporomusa sphaeroides DSM 2875 Sporomusa termitida DSM 4440 Thermoanaerobacter kivui DSM 2030 Clostridium scatologenes (Clostridium scatologenes), Clostridium scatologenes DSM 757 Clostridium sp. ATCC 29797, Desulfomonile kistiae (Desulfomonile kistiae), Desulfotomaculum kuznetsovii DSM 6115 Desulfotomaculum thermosulfurigenes subsp. thermopilei thermosyntrophicum Desulfotomaculum thermobezoicum subsp thermosyntrophicum DSM 14055 Eubacterium limosum DSM 20543 Methanosarcina acetivorans (Methanosarcina acetivorans) C2A, Methanosarcina acetivorans DSM 2834 Moorella sp. Moorella sp. HUC22-1, Moorella thermoacetica (Moorella thermoacetica), Moorella thermoacetica DSM 521 Moorella thermoautotrophica Moorella thermautotrophica (Moorella thermautotrophica), DSM 1974 Oxobacter pfennigii DSM 322 Sporomusa aerivorans DSM 13326 Sporomusa ovata DSM 2662 Sporomusa silvacetica DSM 10669 Sporomusa sphaeroides DSM 2875 Sporomusa termitida DSM 4440 Thermoanaerobacter kivui DSM 2030 Clostridium scatologenes (Clostridium scatologenes), Clostridium scatologenes DSM 757 Clostridium sp. ATCC 29797, Desulfomonile kistiae (Desulfomonile kistiae), Desulfotomaculum kuznetsovii DSM 6115 Desulfotom 3. The mixture according to claim 1 or 2, wherein the first microorganism is Clostridium ljungdahlii.

4. The mixture according to any one of the preceding claims, wherein the higher alcohol is a C6 to C8 alcohol.

5. The mixture according to any one of the preceding claims, wherein the carbon source comprises at least 2% carbon monoxide gas relative to the volume of the carbon source by volume.

6. The mixture according to any one of the preceding claims, wherein the carbon source comprises 2% to 99% carbon monoxide gas by volume.

7. The mixture according to any one of the preceding claims, wherein the aqueous culture medium comprises free oxygen.

8. The mixture according to claim 7, wherein the mixture comprises both log phase acetogenic bacteria and stationary phase acetogenic bacteria.

9. The mixture according to claim 8, wherein said first acetogenic microorganism has a growth rate of 0.01 to 2 h -1 -1 during the exponential growth phase.

10. The mixture according to claim 8 or 9, wherein the first acetogenic microorganism of the exponential growth phase has an OD 600 .

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