Sulfur-containing fermentation media

Abstract

Sulfur-containing fermentation media The present invention is an aqueous fermentation medium comprising sulfur in the form of at least one thiocarboxylate salt, said thiocarboxylate salt being represented by Formula I: [Formula 1] TIFF2024536055000008.tif38140 (wherein R is H, alkyl, COOH, COSH, and the alkyl group may also include OH, COSH, and / or COOH.) having the chemical structure The fermentation medium has a thiocarboxylate concentration of 2 to 20 mg / L.

Description

[Technical field]

[0001] The present invention relates to a fermentation medium and its use for producing organic compounds from a carbon source in the presence of hydrogen and anaerobic organisms. In particular, the fermentation medium contains sulfur in the form of a thiocarboxylate, which allows anaerobic microorganisms to produce organic products from the available carbon source. [Background technology]

[0002] In the fermentation process, anaerobic organisms convert CO2, CO, H2, and / or other carbohydrates into various organic products such as lactic acid, acetic acid, and ethanol. There are many conventional methods for maintaining cultures of microorganisms capable of producing various useful organic products during fermentation. However, these methods have many inefficiencies. Some of these microorganisms are delicate by nature and susceptible to even slight changes in the ambient conditions of the medium. This reduces their efficiency in producing useful organic products from an adequate carbon source. Besides water as a reaction medium, the microorganisms used in these fermentation processes also require certain elements and vitamins to survive, grow, and reproduce. Nutrients and micronutrients, as well as the specific supply of these nutrients, can have a significant impact on the growth and sustainability of the microorganisms.

[0003] One of the important nutrients in the fermentation medium is the sulfur source. Besides sulfur, iron, nickel, and cobalt are also essential in the fermentation medium. Usually, sulfur is in a chemically reduced state, such as sulfide. In particular, sulfur is often provided to the medium as H2S, Na2S, or even cysteine. When H2S or Na2S is used, the solubility product of the resulting Fe, Ni, or Co salts is often exceeded, and these salts eventually precipitate and have very adverse effects on pumps and valves used in fermentation. Hydrogen sulfide is also toxic, so it requires special handling, and is especially dangerous in its pure form. Even if sulfur is provided in the form of a sulfide salt, such as sodium sulfide, the hydrogen sulfide concentration in the fermenter may decrease over time due to evaporation. Hydrogen sulfide may also be highly volatile under the conditions desired for fermentation, making it difficult to use as a sulfur source. In addition, hydrogen sulfide has limited solubility in the fermentation medium. For all these and other reasons, sulfides are not the best sulfur source for efficient fermentation by cells.

[0004] Cysteine, an alternative sulfur source, is very often digested by the microorganisms themselves, so large amounts of cysteine ​​are required for each fermentation process. In particular, cysteine ​​is oxidized to dimeric cysteine. This makes the cost of cysteine ​​very high.

[0005] The reduced forms of these sulfur compounds are believed to be substantially more bioavailable as sulfur sources for use in microbial culture than the oxidized forms. The use of sulfur sources to lower the oxidation-reduction potential (ORP) of fermentation reactions reduces the effective concentration of sulfur available for microbial culture.

[0006] WO 2013 / 147621 discloses a fermentation method for producing alcohol in which sulfur is added to the fermentation medium in the form of sulfurous acid (H2SO3), SO2, Na2S2O4, Na2S, NaHS, cysteine, NH4HSO3 or (NH4)2SO3. However, for sulfur to be effectively present in the medium, CO must also be present. This limits the carbon sources that can be used as substrates for producing organic compounds. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] International Publication No. 2013 / 147621 Brochure Summary of the Invention [Problem to be solved by the invention]

[0008] Therefore, there is a need in the art to provide a sulfur source for fermentation media that is not too expensive but is also efficiently used by the microorganisms and supports the enzymatic processes occurring in the microbial culture. The added sulfur source must also be in a bioavailable form and provided in sufficient amounts to avoid inhibition of growth or production of organic products by the microorganisms. [Means for solving the problem]

[0009] Description of the invention The present invention seeks to solve the above problems by providing a fermentation medium containing sulfur in the form of at least one thiocarboxylate salt. In particular, the thiocarboxylate salt has the chemical structure of Formula I:

[0010] [ka]

[0011] wherein R is H, alkyl, aryl, COOH, COSH, and the alkyl and aryl groups may also include OH, COSH, and / or COOH. More specifically, R is H, alkyl, COOH, COSH, and the alkyl groups may also include OH, COSH, and / or COOH.

[0012] The use of thiocarboxylates according to all aspects of the invention allows sulfur to be made bioavailable for use by cells in the fermentation medium in the fermentation process to produce organic compounds without oxidizing or digesting the thiocarboxylate, thus eliminating the need to periodically replenish the thiocarboxylates in the medium.

[0013] The term "alkyl" includes saturated aliphatic groups, including straight chain alkyl groups (e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, etc.), branched chain alkyl groups (e.g., isopropyl, tert-butyl, isobutyl, etc.), cycloalkyl (alicyclic) groups (e.g., cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl), alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups. The term "alkyl" further includes alkyl groups, which may further include oxygen, nitrogen, sulfur, or phosphorus atoms replacing one or more carbons of the hydrocarbon backbone. In particular, the alkyl groups of formula I have from 1 to 6 carbon atoms. The term alkyl includes both "unsubstituted alkyl" and "substituted alkyl," the latter referring to alkyl moieties having substituents replacing hydrogen on one or more carbons of the hydrocarbon backbone. The alkyl groups may include OH, COSH, and / or COOH groups.

[0014] The term "aryl" includes groups containing 5- and 6-membered monocyclic aromatic groups that may contain 0-4 heteroatoms, such as benzene, phenyl, pyrrole, furan, thiophene, thiazole, isothiazole, imidazole, triazole, tetrazole, pyrazole, oxazole, isoxazole, pyridine, pyrazine, pyridazine, and pyrimidine. In addition, the term "aryl" includes polycyclic aryl groups, such as tricyclic aryl groups, bicyclic aryl groups, such as naphthalene, benzoxazole, benzodioxazole, benzothiazole, benzimidazole, benzothiophene, methylenedioxophenyl, quinoline, isoquinoline, naphtholidine, indole, benzofuran, purine, benzofuran, deazapurine, or indolizine. These aryl groups with heteroatoms in the ring structure are also referred to as "aryl heterocycles," "heterocycles," "heteroaryls," or "heteroaromatics." The aryl groups may contain OH, COSH, and / or COOH groups.

[0015] In particular, the thiocarboxylate may be selected from the group consisting of thioacetate, thioformate, thiobutyrate, thiolactate, thiopropionate, thiohexanoate, thiooctanoate, thiodecanoate, thiododecanoate, thiobenzoate, and thiocitrate. More particularly, the thiocarboxylate may be selected from the group consisting of thioacetate, thioformate, thiobutyrate, thiolactate, thiopropionate, thiohexanoate, thiooctanoate, thiodecanoate, thiododecanoate, thiobenzoate, and thiocitrate. Even more particularly, the thiocarboxylate may be selected from the group consisting of thioacetic acid, thiobutyric acid, and thiocitric acid. More particularly, the thiocarboxylate may be selected from the group consisting of potassium thioacetate, sodium thioacetate, calcium thioacetate, potassium thiobutyrate, sodium thiobutyrate, calcium thiobutyrate, potassium thiocitrate, sodium thiocitrate, calcium thiocitrate, and the like.

[0016] Thiocarboxylates provide high concentrations of sulfur, an important element in fermentation media, without precipitation and avoiding sulfur digestion, thus saving costs and making the fermentation process more efficient.

[0017] The sulfur concentration in the fermentation medium may be about 1-100 mg / L. In particular, the sulfur concentration in the fermentation medium may be 1-95, 1-90, 1-85, 1-80, 1-75, 1-70, 1-65, 1-60, 1-55, 1-50, 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10 mg / L. More specifically, the sulfur concentration in the fermentation medium may be about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mg / L. The sulfur in the fermentation medium is introduced using a thiocarboxylate. In particular, the thiocarboxylate concentration in the fermentation medium may be 0.02-0.3 mmol / L. More specifically, the thiocarboxylate concentration in the fermentation medium may be 0.02 to 0.25, 0.02 to 0.2, 0.02 to 0.15, 0.02 to 0.10, 0.02 to 0.05, 0.05 to 0.25, 0.05 to 0.2, 0.05 to 0.15, 0.05 to 0.10, 0.07 to 0.25, 0.07 to 0.2, 0.07 to 0.15, 0.07 to 0.10, 0.10 to 0.25, 0.10 to 0.2, 0.10 to 0.15, 0.15 to 0.25, 0.15 to 0.2, or 0.20 to 0.25.

[0018] In one example, the thiocarboxylate can be potassium thioacetate and the concentration of the thioacetate molecule in the fermentation medium can be 2-20 mg / L. In particular, the concentration of the thioacetate molecule in the fermentation medium can be 2-19, 2-18, 2-17, 2-16, 2-15, 2-14, 2-13, 2-12, 2-11, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 5-20, 5-18, 5-17, 5-16, 5-15, 5-14, 5-13, 5-12, 5-11, 5-10, 10-20, 10-19, 10-18, 10-17, 10-16, 10-15, or 15-20 mg / L. In one example, the thiocarboxylate can be potassium thioacetate and the concentration of the thioacetate molecule in the fermentation medium can be 1-14, 1-13, 1-12, 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, or 1-5 mg / L. The concentration of the thioacetate molecule in the fermentation medium can be 2-15, 2-12, or 2-10 mg / L. More specifically, the concentration of the thioacetate molecule in the fermentation medium can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mg / L.

[0019] 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%, and even more particularly ±5% of a given measurement or value.

[0020] The fermentation medium may contain other elements at specific concentrations to allow the cells to ferment efficiently. Specifically, the other elements may be selected from the group consisting of aluminum, boron, calcium, cobalt, magnesium, iron, manganese, molybdenum, potassium, nickel, selenium, tungsten, and zinc. More specifically, the other elements may be selected from the group consisting of iron, nickel, and / or cobalt. In one example, the fermentation medium may contain a thiocarboxylate according to all aspects of the invention and iron. In another example, the fermentation medium may contain a thiocarboxylate according to all aspects of the invention and nickel. In yet another example, the fermentation medium may contain a thiocarboxylate according to all aspects of the invention and cobalt. In one example, the fermentation medium may contain a thiocarboxylate according to all aspects of the invention, iron, and nickel. In a further example, the fermentation medium may contain a thiocarboxylate according to all aspects of the invention, iron, and cobalt. In one example, the fermentation medium may contain a thiocarboxylate according to all aspects of the invention, nickel, and cobalt. In another example, the fermentation medium may include a thiocarboxylate according to any aspect of the invention, iron, nickel, and cobalt.

[0021] The fermentation medium may contain between 2 mg / L and 5 mg / L of iron. More specifically, the fermentation medium may contain between 3 and 5 mg / L of iron. Even more specifically, the fermentation medium according to all aspects of the invention may contain about 3, 4.5, or 5 mg / L of iron.

[0022] In one example, the fermentation medium may further comprise cobalt. The fermentation medium may comprise 480 μg / L to 500 μg / L of cobalt. More specifically, the fermentation medium may comprise 490 to 500 μg / L of cobalt. Even more specifically, the fermentation medium according to all aspects of the invention may comprise about 495, 495.5, or 496 μg / L of cobalt.

[0023] In one example, the fermentation medium may further comprise nickel. The fermentation medium may comprise between 40 μg / L and 55 μg / L of nickel. More specifically, the fermentation medium may comprise between 45 and 50 μg / L of nickel. Even more specifically, the fermentation medium according to all aspects of the invention may comprise about 49, 49.5, or 50 μg / L of nickel.

[0024] According to another aspect of the present invention, there is provided a method for producing at least one organic compound from a carbon source, the method comprising: - contacting at least one anaerobic cell with a carbon source in a fermentation medium. having The fermentation medium contains sulfur in the form of at least one thiocarboxylate, the thiocarboxylate having the chemical structure of Formula I:

[0025] [ka]

[0026] (wherein R is H, alkyl, aryl, COOH, COSH, and the alkyl and aryl groups can include OH, COSH, and / or COOH. Specifically, R is H, alkyl, COOH, COSH, and the alkyl groups can include OH, COSH, and / or COOH.)

[0027] The method maintains and / or increases the production rate of one or more organic products produced by the anaerobic cells in the fermentation medium. The fermentation efficiency of anaerobic cell cultures can be improved by using thiocarboxylates as an alternative sulfur source in the fermentation medium.

[0028] Anaerobic organisms can include carboxydotrophs, photosynthetic organisms, methanogens, and acetogens. In some examples, the anaerobic bacteria is selected from the genera Actinomyces, Bacteroides, Clostridium, Fusobacterium, Peptostreptococcus, Porphyromonas, Prevotella, Propionibacterium, or Veillonella. In one example, the anaerobic bacteria can be an acetogenic cell.

[0029] The term "acetogenic cells" or "acetogenic bacteria" as used herein refers to microorganisms that can carry out the Wood-Ljungdahl pathway and thus convert CO, CO2 and / or hydrogen into acetate. These microorganisms include those that do not have the Wood-Ljungdahl pathway in the wild type, but have acquired this trait as a result of genetic modification. Such microorganisms include, but are not limited to, Escherichia coli cells. These microorganisms may also be known as carboxydotrophic bacteria. Currently, 21 different genera of acetogenic bacteria are known in the art (Drake et al., 2006), which may include some Clostridium species (Drake & Kusel, 2005). These bacteria can use carbon dioxide or carbon monoxide as a carbon source, along with hydrogen as an energy source (Wood, 1991). In addition, alcohols, aldehydes, carboxylic acids, and many hexoses may also be used as carbon sources (Drake et al., 2004). The reductive pathway that leads to the production of acetate is called the acetyl-CoA or Wood-Ljungdahl pathway.

[0030] In particular, the acetogenic bacteria may be selected from the group consisting of Acetoanaerobium spp., Acetonema spp., Acetobacterium spp., Alkalibaculum spp., Archaeoglobus spp., Blautia spp., Butyribacterium spp., Clostridium spp., Desulfotomaculum spp., Eubacterium spp., Methanosarcina spp., Moorella spp., Oxobacter spp., Sporomusa spp., Thermoanaerobacter spp., etc. More particularly, the acetogenic bacteria may be selected from the group consisting of Acetoanaerobium notera (ATCC 35199), Acetonema longum (DSM 6540), Acetobacterium carbinolicum (DSM 2925), Acetobacterium malicum (DSM 4132), Acetobacterium sp. no. 446 (Morinaga et al., 1990, J.Biotechnol, 14 units, 187~194 units, Acetobacterium wieringae (DSM 1911), Acetobacterium woodii (DSM 1030), Alkalibaculum bacchi (DSM 22112), Archaeoglobus fulgidus (DSM). 4304), Blautia producta (DSM 2950), Ruminococcus productus, Peptostreptococcus productus, Butyribacterium methylotrophicum (DSM 3468), Clostridium aceticum (DSM). 1496) Clostridium autoethanogenum (DSM 10061; DSM 19630; DSM 23693) Clostridium carboxyvorans (DSM 15243) Clostridium coskatii (ATCC nomenclature PTA-10522), Clostridium drakei (ATCC BA-623), Clostridium formicoaceticum (DSM 92), Clostridium glycolicum (DSM 1288), Clostridium ljungdahlii (DSM 13528). Clostridium yeast C-01(ATCC 55988)、Clostridium yeast ERI-2(ATCC 55380)、Clostridium yeast O-52(ATCC 55989)、Clostridium mayombei(DSM 6539)Clostridium methoxybenzovorans(DSM). 12182) Clostridium ragsdalei (DSM 15248) Clostridium scathologenes (DSM 757) Clostridium designation ATCC 29797 (Schmidtら, 1986) Chem. Eng. Commun.45, pp. 61-73), Desulfotomaculum kuznetsovii (DSM 6115), Desulfotomaculum thermobezoicum subsp. thermosyntrophicum (DSM 14055), Eubacterium limosum (DSM 20543), Methanosarcina acetivorans C2A (DSM 2834), Moorella species HUC22-1 (Sakai et al., 2004, Biotechnol. Let., vol. 29, pp. 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).

[0031] More specifically, the acetogenic bacteria may be selected from the group consisting of Acetbacterium woodii, Alkalibaculum bacchi, Blautia producta, Clostridium aceticum, Clostridium autoethanogenum, Clostridium carboxidivorans, Clostridium drakei, Clostridium formicoaceticum, Clostridium ljungdahlii, Clostridium magnum, Butyribacterium methyotrphoicum, Clostridium scatologenes, Eubacterium limosum, Moorella thermoacetica, Sporomusa ovate, Sporomusa silvacetica, Sporomusa sphaeroides, Oxobacter pfennigii, and Thermoanaerbacter kiuvi. More specifically, the acetogenic bacterium may be selected from the group consisting of Clostridium autoethanogenum and Clostridium ljungdahlii. Even more specifically, the acetogenic bacterium may be Clostridium autoethanogenum.

[0032] The term "fermentation" as used herein refers to a process of producing one or more organic compounds by anaerobic metabolism of acetogenic bacteria in a medium suitable for bacterial growth. A medium suitable for bacterial growth refers to a fermentation medium that contains the components necessary for anaerobic bacterial growth and alcohol production. The medium typically includes a carbon source, a nitrogen source, a phosphorus source, a sulfur source, nutrients, trace elements, salts, vitamins, and the like. Sulfur is typically an essential element in fermentation media for producing various organic compounds from a carbon source. The inventors have surprisingly found that when sulfur is present in the fermentation medium as a thiocarboxylate (salt), a smaller amount of the thiocarboxylate needs to be added to the medium compared to other sulfur sources such as sulfide and cysteine ​​that are typically used as sulfur sources in conventional fermentation media. The thiocarboxylate makes relatively more sulfur bioavailable for consumption by cells compared to conventional sulfur sources, making the fermentation process more efficient and cost-effective.

[0033] Fermentation may be carried out under suitable conditions. As used herein, the term "suitable conditions" refers to the physical and chemical parameters of the fermentation medium necessary for the growth of acetogenic bacteria and / or the production of desired organic compounds, including pH, temperature, salinity, pressure, dissolved oxygen concentration, nitrogen demand, and substrate, nutrient and trace element concentrations.

[0034] A person skilled in the art will understand the appropriate conditions required to carry out the methods according to all aspects of the invention. In particular, the conditions within the vessel (such as the fermenter) may vary depending on the acetogenic bacteria used. The modification of conditions suitable for optimal operation of the microorganism is within the knowledge of a person skilled in the art.

[0035] In one example, the methods according to all aspects of the invention may be carried out in an aqueous medium having a pH of 5 to 8, 5.5 to 7. The pressure may be from 1 to 10 bar.

[0036] Acetogenic bacteria need to convert a carbon source into at least one organic compound. In particular, the cells are contacted with a carbon source that includes monosaccharides (such as glucose, galactose, fructose, xylose, arabinose, or xylulose), disaccharides (such as lactose or sucrose), oligosaccharides, and polysaccharides (such as starch or cellulose), one-carbon substrates, and / or mixtures thereof. The carbon source used according to all aspects of the present invention may include carbon dioxide and / or carbon monoxide. Those skilled in the art will understand that there are many possible sources of CO and / or CO2 as a carbon source. In fact, it is understood that any gas or any gas mixture that can provide a sufficient amount of carbon to the microorganism can be used as a carbon source according to all aspects of the present invention, so that any organic compound can be produced from the CO and / or CO2 source.

[0037] In one example, 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, the volume percentages (%) relating to all carbon sources available to the anaerobic microorganisms according to all aspects of the invention. Examples of carbon sources in gas form include syngas, flue gas, exhaust gases such as refinery gases produced by yeast fermentation or Clostridium fermentation. These exhaust gases are produced from the gasification of cellulose-containing materials or from the gasification of coal. In one example, these exhaust gases are not necessarily produced as by-products of other processes, but may be produced specifically for use by the microorganisms according to all aspects of the invention.

[0038] According to all aspects of the present invention, the carbon source can be synthesis gas. Synthesis gas can be produced, for example, as a by-product of coal gasification. Thus, the microorganism according to all aspects of the present invention can have the ability to convert waste materials into valuable resources. In another example, synthesis gas can be a by-product of gasification of widely available low-cost agricultural feedstocks to be used by the microorganism of the present invention to produce at least one organic compound.

[0039] There are many examples of feedstocks that can be converted to synthesis gas, and almost any form of plant can be used for this purpose. In particular, the feedstocks are selected from the group consisting of perennial grasses such as miscanthus, corn residues, processing waste such as sawdust, etc.

[0040] Generally, syngas is obtained in a gasifier of dry biomass mainly through pyrolysis, partial oxidation and steam reforming, and the main products of syngas are CO, H2 and CO2. Syngas can be a product of electrolysis of CO2. Those skilled in the art will understand the appropriate conditions for carrying out electrolysis of CO2 to produce syngas containing a desired amount of CO.

[0041] Usually, a portion of the synthesis gas obtained from the gasification process is first treated to optimize product yields and avoid the formation of tars. The decomposition of the unwanted tars in the synthesis gas and the carbon dioxide can be carried out using lime and / or dolomite. These processes are described in detail, for example, in "Reed, 1981".

[0042] An advantage of the present invention may be that it allows the use of much more advantageous CO2 / CO mixtures of raw materials. These various sources include natural gas, biogas, coal, oil, plant residues, etc. The overall efficiency of the method of the present invention, the productivity of organic compounds, and / or the overall carbon recovery rate may depend on the stoichiometry of CO2, CO and H2 in the continuous gas stream. The applied continuous gas stream may be a constituent of CO2 and H2. In particular, in the continuous gas stream, the concentration range of CO2 will be about 10-50%, in particular 3% by weight, and the concentration range of H2 will be within the range of 44%-84%, in particular 64-66.04% by weight. In another example, the continuous gas stream may also contain an inert gas such as N2, with the N2 concentration being up to 50% by weight.

[0043] Generally, the carbon source comprises at least 50% by volume, at least 70% by volume, in particular at least 90% by volume of CO2, the volume percentage (%) relating to the total carbon source available to the acetogenic bacteria in the fermentation medium.

[0044] A mixture of sources can be used as the carbon source.

[0045] According to all aspects of the present invention, 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 CO2 are provided and / or used. In one example, hydrogen gas is part of the synthesis gas present according to all aspects of the present invention. In another example, additional hydrogen gas can be provided when the hydrogen gas in the synthesis gas is insufficient for the method of the present invention.

[0046] The term "contacting" as used herein means direct contact between the acetogenic bacteria according to any aspect of the present invention and the carbon source. For example, the cells and the carbon source in the fermentation medium may be in different compartments. In particular, the carbon source may be in a gaseous state and may be added to the fermentation medium containing the cells according to any aspect of the present invention.

[0047] According to all aspects of the invention, the organic compound may be at least one substituted and / or unsubstituted organic compound. The substituted or unsubstituted organic compound may be selected from the group consisting of an acid, an alcohol, and / or a diol. In particular, the organic compound may be selected from the group consisting of a carboxylic acid, a dicarboxylic acid, a hydroxycarboxylic acid, a carboxylic acid ester, a hydroxycarboxylic acid ester, an alcohol, an aldehyde, a ketone, an amine, an amino acid, etc. In one example, the organic compound according to all aspects of the invention may be a carboxylic acid, a hydroxycarboxylic acid, a carboxylic acid ester, and / or an alcohol. More specifically, these organic compounds contain 1 to 36, 4 to 32, 6 to 20, or in particular 8 to 12 or 1 to 8 carbon atoms. More specifically, the alcohol is at least one C1 to C8 alcohol and the acid is at least one C1 to C8 acid. In particular, the organic compound may be selected from the group consisting of acetate, butyrate, propionate, caproate, ethanol, propanol, butanol, 2,3-butanediol, isopropanol, propylene, butadiene, isobutylene, ethylene, lactic acid, hexanoic acid, and / or acetic acid. More particularly, the organic compound according to all aspects of the present invention may be lactic acid, acetic acid, hexanoic acid, and / or ethanol.

[0048] The organic compounds produced according to all aspects of the present invention can be recovered using any separation method known in the art. For example, a portion of the organic compounds, such as ethanol, can be recovered from the fermentation medium using fractional distillation or evaporation and extractive fermentation. Extractive fermentation involves the use of water-miscible solvents that pose a low toxicity risk to the anaerobic cells used in the fermentation process to recover ethanol from the fermentation medium. Oleyl alcohol is a solvent that can be used in this type of extractive process.

[0049] In another example, alkylphosphine oxides of general formula 1 may be used to extract organic compounds produced according to any aspect of the present invention.

[0050] [ka]

[0051] (In the formula, R 1 , R 2 and R 3 is selected from alkyl groups having 6 to 12, preferably 8 to 10, more preferably 8 or 10 carbon atoms, provided that R 1 , R 2 and R 3 At least two of them are different from each other.)

[0052] For extracting organic compounds according to all aspects of the present invention, the alkylphosphine oxide may contain at least two different alkyl groups per alkylphosphine oxide molecule.

[0053] According to yet another aspect of the invention there is provided the use of a fermentation medium according to any aspect of the invention in a process for producing at least one alcohol and / or acid from a carbon source in the presence of hydrogen, the carbon source comprising carbon monoxide and / or carbon dioxide. EXAMPLES

[0054] Although the preferred embodiment has been described above, it will be understood by those skilled in the art that the design, configuration, or operation of the present invention may be changed or modified without departing from the scope of the claims. For example, these modifications are intended to be included in the scope of the claims.

[0055] Experimental Example 1 Cultivation of Clostridium autoethanogenum with potassium thioacetate. The homoacetogenic bacterium Clostridium autoethanogenum was grown on syngas in a mineral medium containing potassium thioacetate as a reduced sulfur source. All cultivation steps were carried out under anaerobic conditions in pressure-tight glass bottles that could be hermetically sealed with butyl rubber stoppers.

[0056] For the preculture of Clostridium autoethanogenum, 250 mL of EvoDM26 mineral medium (pH 6.2, 0.004 g / L magnesium acetate, 0.164 g / L sodium acetate, 0.016 g / L calcium acetate, 0.025 g / L potassium acetate, 0.107 mL / L H3PO4 (8.5%), 0.35 mg / L cobalt acetate, 1.245 mg / L nickel acetate x 4H2O, 20 μg / L d-biotin, 20 μg / L folic acid, 10 μg / L pyridoxine-HCl, 10 μg / L arginine) was added to 100 mL of EvoDM26 mineral medium (pH 6.2, 0.004 g / L magnesium acetate, 0.164 g / L sodium acetate, 0.016 g / L calcium acetate, 0.025 g / L potassium acetate, 0.107 mL / L H3PO4 (8.5%), 0.35 mg / L cobalt acetate, 1.245 mg / L nickel acetate x 4H2O, 20 μg / L d-biotin, 20 μg / L folic acid, 10 μg / L pyridoxine-HCl, 10 μg / L arginine) in a 1000 mL pressure-resistant glass bottle at 37 °C, 150 rpm, and 1 L / h ventilation in an open water bath shaker. The incubation was carried out in 50 μg / L thiamine-HCl, 50 μg / L riboflavin, 50 μg / L nicotinic acid, 50 μg / L Ca-pantothenate, 50 μg / L vitamin B12, 50 μg / L p-aminobenzoic acid, 50 μg / L lipoic acid, 2.109 mg / L (NH4)2Fe(SO4)2×6H2O, 10.69 mg / L potassium thioacetate, acetic acid, and NH3 for pH adjustment. To this, cells from a fresh culture of C. autoethanogenum were cultured at a starting OD 600nm The culture was inoculated until the pH reached 2.2 and cultivated for 200 h. Gas was released into the medium by surface aeration. The pH was kept at 6.2 by automatic addition of 4 M NH3 solution. Fresh medium was continuously fed into the reactor, and the fermentation broth was kept at 6.2 for 1.8 d. -1 The dilution rate was continuously removed from the reactor.

[0057] The main culture had an OD of 1.6 600nmThe required number of cells from the preculture was transferred to 250 mL of EvoDM26 medium. Chemoautotrophic cultivation was carried out in 1 L pressure bottles in an open water bath shaker at 37 °C, 150 rpm, and 1 L / h ventilation using a gas mixture of 62.5% H2, 25% CO2, and 12.5% ​​CO2 for 192 h. Gas was released into the medium by surface aeration. The pH was kept at 6.2 by automatic addition of 4 M NH3 solution. Fresh medium was continuously fed to the reactor and the fermentation broth was incubated for 1.7 d. -1 The culture was continuously removed from the reactor at a dilution rate of OD 600nm Several 5 mL samples were taken to measure the concentration, pH, and product formation. Measurement of product concentration was performed by semi-quantitative 1H-NMR method. Sodium trimethylsilylpropionate (T(M)SP) was used as an internal quantitative standard.

[0058] During the main culture in EvoDM26 medium, 2.048 g of cell dry matter and 1.84 g of acetate were produced. The sulfur concentration in the medium remained at the same level of about 3 mg / L throughout the entire culture time.

[0059] Experimental Example 2 Production of acetate and ethanol from syngas and resupply of cysteine ​​by Clostridium ljungdahlii. The homoacetogenic bacterium Clostridium ljungdahlii was grown on syngas while resupplying with cysteine ​​for the biotransformation of hydrogen and carbon dioxide into acetate and ethanol. All cultivation steps were carried out under anaerobic conditions in pressure-tight glass bottles that could be hermetically sealed with butyl rubber stoppers.

[0060] For the preculture of C. ljungdahlii, 500 mL of medium (ATCC1754 medium: pH 6.0, 20 g / L MES, 1 g / L yeast extract, 0.8 g / L NaCl, 1 g / L NH4Cl, 0.1 g / L KCl, 0.1 g / L KH2PO4, 0.2 g / L MgSO4 x 7H2O, 0.02 g / L CaCl2 x 2H2O, 20 mg / L Nitrogen, 0.02 g / L L-Cysteine ​​hydrochloride, 0.02 g / L NaCl ... Lithium triacetate, 10 mg / L MnSO4 x H2O, 8 mg / L (NH4)2Fe(SO4)2 x 6H2O, 2 mg / L CoCl2 x 6H2O, 2 mg / L ZnSO4 x 7H2O, 0.2 mg / L CuCl2 x 2H2O, 0.2 mg / L Na2MoO4 x 2H2O, 0.2 mg / L NiCl2 x 6H2O, 0.2 mg / L Na2SeO4, 0.2 mg / L Na2WO4 x 2H2O, 20 μg / L d-biotin, 20 μg / L folic acid, 100 5 mL of frozen stock was inoculated into a 100 mL flask containing 50 μg / L pyridoxine-HCl, 50 μg / L thiamine-HCl×HO, 50 μg / L riboflavin, 50 μg / L nicotinic acid, 50 μg / L calcium pantothenate, 1 μg / L vitamin B12, 50 μg / L p-aminobenzoate, 50 μg / L lipoic acid, and approximately 67.5 mg / L NaOH. For chemolithoautotrophic cultures, OD was measured in 1 L pressure-resistant glass bottles at 37°C, 100 rpm, and 3 L / h ventilation using a premix of 67% H2 and 33% CO2 in an open water bath shaker. 600nm The reaction was continued for 69 hours until the β-acetylglucosamine content exceeded 0.4. Gas was released into the medium through a 10 μm pore sparger attached to the center of the reactor. The cell suspension was then centrifuged and the cell pellet was resuspended in fresh CGF1 medium.

[0061] In the production stage, the OD of 0.2 600nmThe required number of washed cells from a preculture of C. ljungdahlii were added to 100 mL of mineral medium (CGF1 medium, pH 6.5, 1.4 g / L KOH, 2 g / L (NH4)2SO4, 1 g / L KH2PO4, 1 g / L K2HPO4, 10 mg / L FeSO4 x 7H2O, 3.8 mg / L MnSO4 x 1H2O, 246 mg / L MgSO4 x 7H2O, aerated for 30 min with a premixed gas containing H2: 67% and CO2: 33%). Initially, culture A was supplemented with an additional 400 mg / L L-cysteine ​​hydrochloride, and cultures B and C were supplemented with an additional 200 mg / L L-cysteine ​​hydrochloride, respectively. The cultivation was carried out in a 500 mL pressure-resistant glass bottle in an open water bath shaker at 37 °C and 150 rpm for 163 h. The open water bath shaker was aerated once a day with a premixed gas containing 67% H2 and 33% CO2 to an overpressure of 0.8 bar. The pH was kept above 5.0 by intermittent addition of 140 g / L KOH solution. During the cultivation, the OD 600nm Several 5 mL samples were taken to measure the concentration, pH, and product formation. After 18, 41, 65, and 89 h of cultivation, culture C was re-fed with 200 mg / L L-cysteine ​​hydrochloride. Measurement of product concentration was performed by semi-quantitative 1H-NMR method. Sodium trimethylsilylpropionate (T(M)SP) was used as an internal quantitative standard.

[0062] During the production phase, the concentrations of acetate and ethanol in culture C increased more than in cultures A and B (see Table 1). Culture C also showed more vigorous cell growth than cultures A and B. In all three cultures, all added L-cysteine ​​was completely consumed.

[0063] [Table 1]

[0064] Table 1: Cell growth and product formation of Clostridium ljungdahlii cultures in CGF1 mineral medium on syngas containing H2:67% and CO2:33% with different initial concentrations of L-cysteine ​​and partial re-feeding of L-cysteine.

[0065] Experimental Example 3 Ethanol production by Clostridium autoethanogenum on boron-containing synthesis gas. The homoacetogenic bacterium Clostridium autoethanogenum was grown on syngas to bioconvert hydrogen, carbon monoxide, and carbon dioxide into ethanol. All cultivation steps were carried out under anaerobic conditions in pressure-tight glass bottles that could be hermetically sealed with butyl rubber stoppers.

[0066] For preculture, 400 mL of medium (EvoDM01-medium: pH 5.8, 0.407 g / L MgCl2×6H2O, 0.117 g / L NaCl, 0.294 g / L CaCl2×2H2O, 1.864 g / L KCl, 0.375 mL / L H3PO4, 19.8 mg / L FeCl2×4H2O, 0.5 g / L cysteine-HCl, 3.92 g / L NH4-acetate, 0.396 mg / L MnCl2×4H2O, 0.476 mg / L CoCl2×6H2O, 0.682 mg / L ZnCl2, 0.124 mg / L H3BO3, Cells from a fresh culture of C. autoethanogenum were cultured in a 100 mL culture of 0.484 mg / L Na2MoO4 x 2H2O, 0.346 mg / L Na2SeO3 x 5H2O, 1.189 mg / L NiCl2 x 6H2O, 0.660 mg / L Na2WO4 x 2H2O, 20 μg / L d-biotin, 20 μg / L folic acid, 10 μg / L pyridoxine-HCl, 50 μg / L thiamine-HCl x H2O, 50 μg / L riboflavin, 50 μg / L nicotinic acid, 50 μg / L calcium pantothenate, 50 μg / L vitamin B12, 50 μg / L p-aminobenzoic acid, and 50 μg / L lipoic acid at a starting OD of 1. 600nmThe culture was inoculated until the pH reached 0.1. Chemoautotrophic culture was carried out in a 0.5 L pressure bottle at 37°C, 150 rpm, and 2.3 L / h ventilation using a premixed gas mixture containing 60% H2, 20% CO2, and 20% CO in an open water bath shaker for 476 h. Gas was released into the medium through an aeration membrane attached to the center of the reactor. The pH was kept at 5.5 by automatic addition of 2.5 M NH3 solution. Fresh medium was continuously fed to the reactor, and the fermentation broth was diluted with water for 1.0 d. -1 The dilution rate was continuously removed from the reactor.

[0067] After preculture, the cell suspension was centrifuged (10 min, 4200 rpm) and the pellet was resuspended in fresh main culture medium. The main culture was cultured at an OD of 1.0. 600nm The required number of cells from the preculture was transferred to 400 mL of medium. EvoDM01 mineral medium was also used for the main culture. Chemoautotrophic cultures were carried out in 0.5 L pressure bottles at 37 °C, 150 rpm, and 2.3 L / h ventilation in an open water bath shaker using a premixed gas mixture containing 60% H2, 20% CO2, and 20% CO2 for 45 h. Gas was released into the medium through an aeration membrane attached to the center of the reactor. The pH was kept at 5.5 by automatic addition of 2.5 M NH3 solution. Fresh medium was continuously fed to the reactor, and the fermentation broth was kept at 1.0 d for 1 h. -1 The culture was continuously removed from the reactor at a dilution rate of OD 600nm Several 5 mL samples were taken to measure the concentration, pH, and product formation. Measurement of product concentration was performed by semi-quantitative 1H-NMR method. Sodium trimethylsilylpropionate (T(M)SP) was used as an internal quantitative standard. During the main culture in EvoDM01 medium, 3.22 g of ethanol and 1.11 g of acetate were produced.

[0068] Experimental Example 4 Cultivation of Clostridium autoethanogenum at low potassium thioacetate concentrations. The homoacetogenic bacterium Clostridium autoethanogenum was co-cultured with the chain-elongating bacterium Clostridium kluyveri on syngas in a mineral medium containing potassium thioacetate as a reduced sulfur source. Cultivations were carried out under anaerobic conditions in a pressure-resistant stainless steel bubble-column loop reactor.

[0069] The cultivation was carried out as continuous fermentation at 37°C and 2 bar overpressure with a mixture of water, substrate, salts, trace elements and vitamins continuously fed at 300 L / h. The pH was automatically kept at 5.80 by feeding ammonia. Of the 300 L / h outlet flow leaving the fermenter, 98.2% was cell-retaining permeate and 1.8% was cell-free purge. Gas was released into the medium through a sparger with an air exchange rate of approximately 1000 L / h as a gas mixture of H2:62.5% and CO2:37.5%.

[0070] The medium feed composition was as follows: 0.004 g / L magnesium acetate x 4H2O, 0.164 g / L sodium acetate, 0.016 g / L calcium acetate, 0.245 g / L potassium acetate, 0.107 mL / L H3PO4 (8.5%), 0.35 mg / L cobalt acetate, 1.245 mg / L nickel acetate x 4H2O, 20 μg / L d-biotin, 20 μg / L folic acid, 10 μg / L pyridoxine. The buffer consisted of 50 μg / L thiamine-HCl, 50 μg / L riboflavin, 50 μg / L nicotinic acid, 50 μg / L calcium pantothenate, 50 μg / L vitamin B12, 50 μg / L p-aminobenzoic acid, 50 μg / L lipoic acid, 2.109 mg / L (NH4)2Fe(SO4)2 × 6H2O, 10.69 mg / L potassium thioacetate, 6.73 g / L ethanol, and NH3 for pH adjustment.

[0071] The cultures were pre-inoculated with cells from fresh cultures of C. autoethanogenum and C. kluyveri and were already at an optical density (OD ) of approximately 9.0. 600nm ) and was continuously operated at full capacity for more than 10,000 hours as a stable co-culture. Fresh medium was continuously fed into the reactor and the fermentation broth was maintained for 2.8 days. -1 The culture was continuously removed from the reactor at a dilution rate of OD 600nm Several 5 mL samples were taken to measure the concentration, pH, and product formation. Measurement of product concentration was performed by semi-quantitative 1H-NMR method. Sodium trimethylsilylpropionate (T(M)SP) was used as an internal quantitative standard.

[0072] The steady-state concentrations of educts and products in the reactor were approximately 3.27 g / L ethanol, 0.90 g / L acetate, 0.91 g / L butyrate, and 3.61 g / L hexanoate. 50 hours after reducing the potassium thioacetate concentration in the medium feed to 10% (1.69 mg / L), the steady-state concentrations had decreased to 0.50 g / L acetate, 0.53 g / L butyrate, and 2.41 g / L hexanoate. The ethanol concentration had risen to 4.37 g / L. During this period, the OD 600nm decreased from 9.0 to 7.60, and CO2 turnover decreased from 70% to 50%.

[0073] Experimental Example 5 Cultivation of Clostridium autoethanogenum at high potassium thioacetate concentrations. The homoacetogenic bacterium Clostridium autoethanogenum was co-cultured with the chain-elongating bacterium Clostridium kluyveri on syngas in a mineral medium containing potassium thioacetate as a reduced sulfur source. Cultivations were carried out under anaerobic conditions in a pressure-resistant stainless steel bubble-column loop reactor.

[0074] The cultivation was carried out as continuous fermentation at 37°C and 2 bar overpressure with a mixture of water, substrate, salts, trace elements and vitamins continuously fed at 300 L / h. The pH was automatically kept at 5.80 by feeding ammonia. Of the 300 L / h outlet flow leaving the fermenter, 98.2% was cell-retaining permeate and 1.8% was cell-free purge. Gas was released into the medium through a sparger with an air exchange rate of approximately 1000 L / h as a gas mixture of H2:62.5% and CO2:37.5%.

[0075] The culture was pre-inoculated with cells from fresh cultures of C. autoethanogenum and C. kluyveri and was already at an optical density (OD ) of approximately 11.8. 600nm ) and was continuously operated at full capacity for more than 13,000 hours as a stable co-culture. Fresh medium was continuously fed into the reactor and the fermentation broth was maintained for 2.8 days. -1 The culture was continuously removed from the reactor at a dilution rate of OD 600nm Several 5 mL samples were taken to measure the concentration, pH, and product formation. Measurement of product concentration was performed by semi-quantitative 1H-NMR method. Sodium trimethylsilylpropionate (T(M)SP) was used as an internal quantitative standard.

[0076] The steady-state concentrations of educts and products in the reactor were approximately 2.67 g / L ethanol, 1.10 g / L acetate, 1.08 g / L butyrate, and 3.75 g / L hexanoate. 50, 100, and 150 hours after increasing the potassium thioacetate concentration in the medium feed to 300% (32.07 mg / L), the steady-state concentrations of educts and products remained at the same levels. Also, during this period, the OD 600nm remained at 11.80 and the CO2 turnover rate remained at 70%.

Claims

1. 1. An aqueous fermentation medium containing sulfur in the form of at least one thiocarboxylate salt, said thiocarboxylate salt having Formula I: 【Chemical 1】 wherein R is H, alkyl, COOH, COSH, and the alkyl group may also contain OH, COSH, and / or COOH. and having the chemical structure The aqueous fermentation medium, wherein the concentration of the thiocarboxylate in the fermentation medium is 2 to 20 mg / L.

2. 2. The fermentation medium of claim 1, wherein the thiocarboxylate is selected from the group consisting of thioacetate, thioformate, thiobutyrate, thiolactate, thiopropionate, thiohexanoate, thiooctanoate, thiodecanoate, thiododecanoate, and thiocitrate.

3. 2. The fermentation medium of claim 1, wherein the thiocarboxylate is selected from the group consisting of thioacetate, thiobutyrate, and thiocitrate.

4. 2. The fermentation medium of claim 1, further comprising an iron salt, a nickel salt and / or a cobalt salt.

5. 1. A method for producing at least one organic compound from a carbon source, comprising contacting at least one anaerobic bacterium with the carbon source in a fermentation medium; The fermentation medium contains sulfur in the form of at least one thiocarboxylate salt, the thiocarboxylate salt having Formula I: 【Chemistry 2】 wherein R is H, alkyl, COOH, COSH, and the alkyl group may also contain OH, COSH, and / or COOH. and having the chemical structure The method, wherein the concentration of the thiocarboxylate in the fermentation medium is 2 to 20 mg / L.

6. 6. The method of claim 5, wherein the anaerobic bacteria is an acetogenic cell.

7. 6. The method of claim 5, wherein the thiocarboxylate is selected from the group consisting of thioacetate, thioformate, thiobutyrate, thiolactate, thiopropionate, thiohexanoate, thiooctanoate, thiodecanoate, thiododecanoate, and thiocitrate.

8. 6. The method of claim 5, wherein the thiocarboxylate is selected from the group consisting of thioacetate, thiobutyrate, and thiocitrate.

9. The method of claim 5 , wherein the medium further comprises iron salts, nickel salts and / or cobalt salts.

10. 6. The method of claim 5, wherein the carbon source is in the presence of hydrogen, and the carbon source comprises carbon monoxide and / or carbon dioxide.

11. The method of claim 5 , wherein the organic compound is at least one alcohol and / or acid.

12. The alcohol has at least one C １ ~C ８ alcohol, and the acid is at least one C １ ~C ８ 12. The method of claim 11, wherein the acid is

13. 10. Use of the fermentation medium of claim 1 in a process for producing at least one alcohol and / or acid from a carbon source comprising carbon monoxide and / or carbon dioxide in the presence of hydrogen.

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