Method for producing higher alkanones, preferably 6-undecanone and its derivatives

Abstract

The present invention relates to a method for producing higher alkanones, preferably 6-undecanone, from ethanol and / or acetate, the method comprising (a) contacting ethanol and / or acetate with at least one microorganism capable of carbon chain extension to produce hexanoic acid and / or its esters from ethanol and / or acetate; (b) extracting the hexanoic acid and / or its esters from (a) using at least one extractant in an aqueous medium, wherein the extractant comprises at least one alkylphosphine oxide and at least one alkane comprising at least 12 carbon atoms; or at least one trialkylamine and at least one alkane comprising at least 12 carbon atoms; and (c) contacting the extracted hexanoic acid and / or its esters from (b) with at least one ketonization catalyst and optionally other alkanoic acids comprising 1 to 22 carbon atoms under suitable reaction conditions to chemically ketonize the hexanoic acid and optionally other alkanoic acids to higher alkanones, preferably 6-undecanone.

Description

Field of the Invention

[0001] The present invention relates to a process for producing higher alkanones, preferably 6-undecanone. In particular, the present invention relates to the production of higher alkanones, preferably 6-undecanone, using a combined biotechnological and chemical process. Background of the Invention

[0003] 6-Undecanone is commonly found in herbs and spices and is a component of Osmanthus fragrans (sweet osmanthus). 6-Undecanone is a dialkyl ketone with the formula (CH3(CH2)4)2CO. It is used as a flavor enhancer, especially in the food industry. In other industries, 6-Undecanone is used as a solvent and in paints and coatings.

[0004] Industrially, 6-undecanone is usually produced from carboxylic acids by contacting the corresponding carboxylic acids with various metal oxide catalysts at elevated temperatures. For example, a method for generating dialkyl ketones using manganese dioxide supported on alumina and other examples of catalysts that can be used to generate diethyl ketone from propionic acid are described in U.S. Patent No. 4,754,074. It is well known in the art that other catalysts such as oxides of lead, iron, zirconium, manganese, thorium and neodymium can be used when preparing ketones from carboxylic acids. Although high pressure hydrogen at high reaction temperatures of 250-450° C. is required to make the method efficient, these catalysts can be used without significant carbon loss. These conditions increase the cost of producing ketones.

[0005] Furthermore, in all these methods of practice, the dialkyl ketone produced is always formed from carboxylic acids as starting materials. For example, the production of 6-undecanone starts with caproic acid. Caproic acid is mainly obtained exclusively from plant and animal fats. Animal fats as raw materials still cannot meet customer requirements, and vegetable oils containing short-length and medium-length carboxylic acids are either difficult to obtain or are only produced in tropical areas, and often also lead to the destruction of rain forests. In addition, specific plant and animal fat raw materials have specific but limited fatty acid distributions, resulting in coupled production. Therefore, it is difficult to obtain pure caproic acid as a substrate for the production of 6-undecanone.

[0006] Therefore, there is a need in the art for more efficient means of producing 6-undecanone and other higher alkanones from other starting materials or at other starting points. In particular, there is a need in the art for a method of producing 6-undecanone from another raw material source that enables efficient and effective production.

[0007] Invention drawings

[0008] Figure 1Microbial metabolic pathways for carbon chain elongation are shown, such as (a) production of butyrate (C4) by Clostridium and Butyrivibrio (Kim BH et al., Appl Environ Microbiol. 1984; 48(4): 764–70) and (b) putative production of caproate in Megasphaera elsdenii and Clostridium kluyveri (Khan MA.. Melbourne: Victoria University; 2006). Summary of the invention

[0009] The present invention attempts to solve the above problems by providing a complete cycle for producing higher alkanones, preferably 6-undecanone, involving biotechnological and chemical means. In particular, the production of higher alkanones, preferably 6-undecanone, uses biotechnological means to produce caproic acid from simple substrates such as ethanol and / or acetate. The produced caproic acid can then be extracted and the extracted caproic acid is subjected to a chemical step that converts the caproic acid into higher alkanones, preferably 6-undecanone. The advantage of this method is that higher alkanones, preferably 6-undecanone, are produced from cheap and easily available raw materials. The raw materials (acetic acid esters and / or ethanol) are also produced using means that do not kill any animals or plants. In addition, according to any aspect of the present invention, a biotechnological step for producing caproic acid is used and then an extraction step is performed to obtain a high and pure yield of caproic acid, which can then be easily used to produce higher alkanones, preferably 6-undecanone using a chemical step.

[0010] According to one aspect of the present invention, there is provided a method for producing higher alkanones, preferably 6-undecanone, from ethanol and / or acetate, the method comprising

[0011] (a) contacting ethanol and / or acetate with at least one microorganism capable of carbon chain elongation to produce caproic acid and / or an ester thereof from ethanol and / or acetate;

[0012] (b) extracting the hexanoic acid and / or its esters from (a) using at least one extractant in an aqueous medium, wherein the extractant comprises at least one alkylphosphine oxide and at least one alkane comprising at least 12 carbon atoms; or at least one trialkylamine and at least one alkane comprising at least 12 carbon atoms; and

[0013] (c) contacting the extracted hexanoic acid and / or its ester from (b) with at least one ketonization catalyst and optionally other alkanoic acids containing 1 to 22 carbon atoms under suitable reaction conditions to chemically ketonize the hexanoic acid and optionally other alkanoic acids to higher alkanones, preferably 6-undecanone.

[0014] As used herein, the term "higher alkanones / alkanols / alkanoic acids" refers to alkanones / alkanols / alkanoic acids containing at least six carbon atoms.

[0015] The microorganism capable of carbon chain elongation to produce caproic acid in (a) may be Figure 1 Any organism that can extend the carbon chain (Jeon et al., Biotechnol Biofuels (2016) 9: 129). The carbon chain extension pathway is also disclosed in Seedorf, H., et al., 2008. The microorganism according to any aspect of the present invention may also include a microorganism whose wild-type form cannot extend the carbon chain but has acquired this property due to genetic modification. In particular, the microorganism in (a) can be selected from Clostridium carboxidivorans and Clostridium kluyveri. More particularly, the microorganism according to any aspect of the present invention can be Clostridium kluyveri.

[0016] The extraction step in (b) according to any aspect of the present invention allows an increase in yield relative to the amount of extractant used. For example, less than 50% by weight of the extractant can be used to extract the same amount of caproic acid as if only pure alkanes were used. Therefore, using a small volume of extractant, caproic acid can be extracted at a higher yield. The extractant is also harmless to microorganisms. Therefore, when caproic acid is produced biotechnologically according to any aspect of the present invention, an extractant according to any aspect of the present invention may be present. Therefore, the aqueous medium according to any aspect of the present invention, in particular after the step (b) of separating caproic acid, can be recycled back to step (a). This recycling step allows the microorganism to be recycled and reused because the extractant according to any aspect of the present invention is non-toxic to the microorganism. Another advantage of the step of recycling the aqueous medium in the method according to any aspect of the present invention is that the residue of caproic acid (which was not initially extracted from step (b) in the first cycle) can be given the opportunity to be extracted again or extracted multiple times (the same number of times the aqueous medium is recycled). In addition, caproic acid can be easily separated from the extractant according to any aspect of the present invention by distillation. This is because caproic acid distills at least at a significantly lower boiling point than the extractant and, after separation via distillation, the extractant can easily be recycled.

[0017] The method according to any aspect of the present invention may include a step of extracting separated caproic acid from an aqueous medium. Separated caproic acid may refer to caproic acid that can be separated from the medium in which the caproic acid has been produced. In one example, caproic acid can be produced in an aqueous medium (e.g., a fermentation medium in which caproic acid is produced from a carbon source by specific cells). Separated caproic acid may refer to caproic acid extracted from an aqueous medium. In particular, the extraction step allows excess water to be separated from the aqueous medium, resulting in the formation of a mixture containing extracted caproic acid.

[0018] The extractant may also be referred to as an "extraction medium" or "extracting medium". The extractant may be used to extract / separate the caproic acid produced according to any of the methods of the present invention from the aqueous medium in which the caproic acid was originally produced. At the end of the extraction step, excess water from the aqueous medium may be removed, thereby obtaining an extractant containing the extracted caproic acid. In particular, at the end of the extraction step, as the caproic acid is extracted and removed, what remains may be a fermentation medium with cells for producing caproic acid, which cells may then be recycled together with the fermentation medium for step (a). The skilled person will be able to determine whether the fermentation medium and / or cells need to be supplemented after the first cycle. In particular, the first cycle according to any aspect of the present invention involves a round of steps (a) to (c). The medium and / or cells may then be recycled starting from the second cycle. The extractant may comprise a combination of compounds that may result in an efficient method for extracting caproic acid from an aqueous medium. In particular, the extractant may include:

[0019] - at least one alkylphosphine oxide and at least one alkane comprising at least 12 carbon atoms; or

[0020] at least one trialkylamine and at least one alkane comprising at least 12 carbon atoms.

[0021] The extractant according to any aspect of the present invention can efficiently extract hexanoic acid into the extractant. It can be considered that the extractant of the mixture of alkylphosphine oxide or trialkylamine and at least one alkane is suitable in the method according to any aspect of the present invention because the mixture is efficient in extracting the desired hexanoic acid in the presence of a fermentation medium. In particular, it can be considered that the mixture of alkylphosphine oxide or trialkylamine and at least one alkane is better than any method currently known in the art for extracting hexanoic acid because it does not require any special equipment to carry out and is relatively easy to carry out with a high product yield. In addition, the extractant according to any aspect of the present invention is also not poisonous to the microorganism according to step (a).

[0022] The alkane in the extractant may contain at least 12 carbon atoms. In particular, the alkane may contain 12-18 carbon atoms. In an example, the alkane may be selected from dodecane, tridecane, tetradecane, pentadecane, hexadecane, heptadecane and octadecane. In other examples, the extractant may contain a mixture of alkanes.

[0023] Alkylphosphine oxide has the general formula OPX3, wherein X is an alkyl group. Suitable alkylphosphine oxides according to any aspect of the present invention include alkyl groups consisting of straight chain, branched or cyclic hydrocarbons, which consist of 1 to about 100 carbon atoms and 1 to about 200 hydrogen atoms. In particular, the "alkyl" used in the alkylphosphine oxide according to any aspect of the present invention may refer to a hydrocarbon group having 1 to 20 carbon atoms, typically 4 to 15 carbon atoms or 6 to 12 carbon atoms and consisting of straight chain, cyclic, branched or a mixture of these. The alkylphosphine oxide may have one to three alkyl groups on each phosphorus atom. In one example, the alkylphosphine oxide has three alkyl groups on P. In some examples, the alkyl group may include an oxygen atom to replace one carbon of a C4-C15 or C6-C12 alkyl group, provided that the oxygen atom is not connected to the P of the alkylphosphine oxide. Typically, the alkylphosphine oxide is selected from trioctylphosphine oxide, tributylphosphine oxide, hexylphosphine oxide, octylphosphine oxide and mixtures thereof. Even more particularly, the alkylphosphine oxide may be trioctylphosphine oxide (TOPO).

[0024] Trialkylamines are organic compounds derived from ammonia (NH3) whose three hydrogen atoms are replaced by alkyl groups. Examples of trialkylamines are dimethylethylamine, methyldiethylamine, triethylamine, dimethyl-n-propylamine, dimethylisopropylamine, methyldi-n-propylamine, dimethylbutylamine, trioctylamine, etc. In particular, the trialkylamine used in the extractant according to any aspect of the present invention may be insoluble in water and may be trioctylamine.

[0025] In one example, the extractant according to any aspect of the present invention can be a combination of an alkylphosphine oxide or a trialkylamine and at least one alkane. In particular, the alkane can contain at least 12 carbon atoms. More particularly, the alkane can contain 12-18 carbon atoms. In one example, the alkane can be selected from dodecane, tridecane, tetradecane, pentadecane, hexadecane, heptadecane and octadecane. In another example, the extractant can comprise a mixture of alkanes. Even more particularly, the extractant according to any aspect of the present invention can be a combination of TOPO and tetradecane or hexadecane.

[0026] Trioctylphosphine oxide (TOPO) is a 17)3 organophosphorus compound. TOPO can be part of the extractant together with at least one alkane according to any aspect of the present invention. In particular, the weight ratio of TOPO to alkane of a mixture of TOPO and an alkane comprising at least 12 carbon atoms can be about 1:100 to 1:10. More particularly, the weight ratio of TOPO to alkane in the extractant according to any aspect of the present invention can be about 1:100, 1:90, 1:80, 1:70, 1:60, 1:50, 1:40, 1:30, 1:25, 1:20, 1:15 or 1:10. Even more particularly, the weight ratio of TOPO to alkane can be selected in the range of 1:90 to 1:10, 1:80 to 1:10, 1:70 to 1:10, 1:60 to 1:10, 1:50 to 1:10, 1:40 to 1:10, 1:30 to 1:10 or 1:20 to 1:10. The weight ratio of TOPO to the alkane may be between 1:40 and 1:15 or 1:25 and 1:15. In one example, the weight ratio of TOPO to the alkane may be about 1:15. In this example, the alkane may be hexadecane, and thus the weight ratio of TOPO to hexadecane may be about 1:15.

[0027] In another example, when the extractant comprises an alkylphosphine oxide or trialkylamine that is more soluble in the alkane in the extractant than the solubility of TOPO in the alkane containing at least 12 carbon atoms, the weight ratio of the alkylphosphine oxide (except TOPO) or trialkylamine to the alkane can be 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1 or 10:1. In one example, the extractant can be trihexylphosphine oxide and the ratio of trihexylphosphine oxide to the alkane can be 1:1. In other examples, the extractant can be a short-chain alkylphosphine oxide and the ratio of the short-chain alkylphosphine oxide to the alkane can be 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1 or 10:1. In this case, the short-chain alkylphosphine oxide refers to a phosphine oxide having a C1-C4 alkyl group. In another example, the extractant can be a trialkylamine, which is known to be more soluble in alkanes than phosphine oxide. For example, the trialkylamine may be trioctylamine (TOA) which may be present in the extractant according to any aspect of the invention in a ratio to the alkane of up to 1:1. Amines of shorter chain lengths may be used in even higher ratios. In other examples, the extractant may be a short chain trialkylamine and the ratio of the short chain trialkylamine to the alkane may be 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1 or 10:1. In this case, the short chain alkylphosphine oxide refers to a phosphine oxide having a C1-C4 alkyl group.

[0028] As used herein, the term "about" refers to a variation within 20%. In particular, as used herein, the term "about" refers to a given measurement or value + / - 20%, more specifically + / - 10%, even more specifically + / - 5%.

[0029] In step (a) according to any aspect of the present invention, ethanol and / or acetate are contacted with at least one microorganism capable of carbon chain extension to produce caproic acid and / or its esters from ethanol and / or acetate. In one example, the carbon source can be a combination of ethanol and at least one other carbon source selected from acetate and butyrate. In particular, the carbon source can be ethanol and acetate. In another example, the carbon source can be a combination of ethanol and butyric acid. In one example, the carbon substrate can be ethanol alone. In another example, the carbon substrate can be acetate alone.

[0030] The source of acetate and / or ethanol may be different according to availability. In an example, ethanol and / or acetate can be a fermentation product of synthesis gas or any carbohydrate known in the art. Especially, the carbon source for acetate and / or ethanol production can be selected from alcohol, aldehyde, glucose, sucrose, fructose, dextrose, lactose, wood sugar, pentose, polyol, hexose, ethanol and synthesis gas.

[0031] A mixed source can be used as the carbon source.

[0032] Even more particularly, the carbon source may be synthesis gas. The synthesis gas may be converted to ethanol and / or acetate in the presence of at least one acetogenic bacteria.

[0033] With regard to the source of substrates including carbon dioxide and / or carbon monoxide, the skilled person will appreciate that there are many possible sources for providing CO and / or CO2 as a carbon source. It can be seen that, since virtually any gas or any gas mixture capable of providing a sufficient amount of carbon to the microorganism can be used as a carbon source for the present invention, acetate and / or ethanol can be formed from the CO and / or CO2 source.

[0034] Typically for acetogenic cells of the invention, the carbon source comprises at least 50 wt%, at least 70 wt%, in particular at least 90 wt% CO2 and / or CO, wherein the wt% relates to all carbon sources available to the cell according to any aspect of the invention. A source of carbon material may be provided.

[0035] Examples of carbon sources in gaseous form include waste gases such as synthesis gas, flue gas and petroleum refinery gas produced by yeast fermentation or clostridial fermentation. These waste gases are formed by gasification or coal gasification of cellulose-containing materials. In an example, these waste gases may not necessarily be produced as by-products of other processes, but can be specifically produced for use with the mixed culture of the present invention.

[0036] According to any aspect of the present invention, the carbon source used in step (a) according to any aspect of the present invention for producing acetate and / or ethanol may be synthesis gas. Synthesis gas may be produced, for example, as a by-product of coal gasification. Therefore, the microorganisms according to any aspect of the present invention may be able to convert substances that are waste products into valuable resources.

[0037] In another example, syngas can be a byproduct of the gasification of widely available, low-cost agricultural raw materials for use with the mixed cultures of the present invention to produce substituted and unsubstituted organic compounds.

[0038] There are numerous examples of raw materials that can be converted into synthesis gas, since almost all forms of plants can be used for this purpose. In particular, the raw materials are selected from perennial grasses such as Miscanthus, corn residues, processing wastes such as sawdust, etc.

[0039] Typically, syngas can be obtained in a gasification unit of dry biomass, mainly by pyrolysis, partial oxidation and steam reforming, wherein the main products of syngas are CO, H2 and CO2. Syngas may also be the product of electrolysis of CO2. The skilled person will understand suitable conditions for conducting electrolysis of CO2 to produce syngas containing the desired amount of CO.

[0040] Typically, a portion of the synthesis gas obtained from the gasification process is first treated to optimize product yields and avoid the formation of tars. Cracking of unwanted tars and CO in the synthesis gas can be performed using lime and / or dolomite.

[0041] The overall efficiency, ethanol and / or acetate productivity and / or total carbon capture of the process of the present invention may depend on the stoichiometry of CO2, CO and H2 in the continuous gas stream. The applied continuous gas stream may have the components CO2 and H2. In particular, in the continuous gas stream, the concentration of CO2 may be in the range of about 10-50 wt%, in particular 3 wt% and the concentration of H2 may be in the range of 44 wt% to 84 wt%, in particular 64-66.04 wt%. In another example, the continuous gas stream may also include an inert gas such as N2, the concentration of N2 being at most 50 wt%.

[0042] Even more particularly, the carbon source comprising CO and / or CO2 is contacted with the acetogenic cells in a continuous gas stream. Even more particularly, the continuous gas stream comprises synthesis gas. These gases can be supplied, for example, using a nozzle opening into an aqueous medium, a glass frit, a membrane in a tube that feeds the gas into the aqueous medium, etc.

[0043] The skilled person will appreciate that it may be necessary to monitor the composition and flow rate of the feed stream at corresponding intervals. The control of the composition of the feed stream can be achieved by changing the ratio of each composition stream to achieve a target or desired composition. The composition and flow rate of the blended stream can be monitored by any method known in the art. In an example, the system is suitable for continuously monitoring the flow rate and composition of at least two feed streams and combining them to produce a single blended substrate stream in a continuous gas stream with an optimal composition and a device for delivering the optimized substrate stream to a fermentor.

[0044] According to any aspect of the present invention, a reducing agent such as hydrogen can be supplied with a carbon source. In particular, when supplying and / or using CO and / or CO 2 , the hydrogen can be supplied. In an example, the hydrogen is a part of the synthesis gas present according to any aspect of the present invention. In another example, in the case where the hydrogen in the synthesis gas is not enough for the method of the present invention, additional hydrogen can be supplied.

[0045] As used herein, the term "acetogenic bacteria" refers to microorganisms that can carry out the Wood-Ljungdahl pathway and thus can convert 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 property due to genetic modification. Such microorganisms include, but are not limited to, Escherichia coli (E. coli) cells. These microorganisms may also be referred to as carboxydotrophic bacteria. Currently, 21 different genera of acetogenic bacteria are known in the art (Drake et al., 2006), and these bacteria may also include some Clostridium (Drake & Kusel, 2005). These bacteria can use carbon dioxide or carbon monoxide as a carbon source and hydrogen as an energy source (Wood, 1991). In addition, alcohols, aldehydes, carboxylic acids and numerous hexoses can also be used as carbon sources (Drake et al., 2004). The reductive pathway that leads to the formation of acetate is called acetyl-CoA or the Wood-Ljungdahl pathway. In particular, the acetogenic bacteria may 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.187-194), 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 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 13326). 2030, formerly known as Acetogenium kivui). .

[0046] More particularly, the ATCC BAA-624 strain of Clostridium carboxidivorans can be used. Even more particularly, bacterial strains labeled as Clostridium carboxidivorans "P7" and "P11" as described, for example, in US 2007 / 0275447 and US 2008 / 0057554 can be used.

[0047] Another particularly suitable bacterium may be Clostridium ljungdahlii. In particular, strains selected from Clostridium ljungdahlii PETC, Clostridium ljungdahlii ERI2, Clostridium ljungdahlii COL and Clostridium ljungdahlii O-52 may be used for the conversion of syngas to hexanoic acid. These strains are described, for example, in WO 98 / 00558, WO 00 / 68407, ATCC 49587, ATCC 55988 and ATCC 55989.

[0048] In one example, the production of caproic acid is from acetic acid and / or ethanol derived from syngas and may involve the use of acetogenic bacteria in conjunction with microorganisms capable of carbon chain extension. For example, Clostridium ljungdahlii may be used simultaneously with Clostridium kluyveri. In another example, a single acetogenic cell may have the activity of both organisms. For example, the acetogenic bacteria may be C. carboxidivorans, which is capable of executing both the Wood-Ljungdahl pathway and the carbon chain extension pathway.

[0049] The ethanol and / or acetate used in step (a) according to any aspect of the present invention may be a fermentation product of syngas or may be obtained by other means.The ethanol and / or acetate may then be contacted with a microorganism in step (a).

[0050] The term "contacting" as used herein refers to direct contact between a microorganism and ethanol and / or acetate. In one example, ethanol is the carbon source and the contact in step (a) involves contacting ethanol with the microorganism of step (a). The contact can be direct contact or indirect contact, and the indirect contact can include a membrane that separates the cells from the ethanol, etc. or wherein the cells and the ethanol can be maintained in two different compartments, etc. For example, in step (b), caproic acid and the extraction medium can be in different compartments.

[0051] Any culture medium, substrate, conditions and methods commonly known in the art for culturing bacteria can be used to cultivate microorganisms capable of producing caproic acid according to any aspect of the present invention. This allows the use of biotechnological methods to produce caproic acid. Depending on the microorganism used for caproic acid production, appropriate growth medium, pH, temperature, stirring rate, inoculum level and / or aerobic, microaerobic or anaerobic conditions are varied. The technician will understand the other conditions necessary for implementing the method according to any aspect of the present invention. In particular, the conditions in the container (e.g., fermentor) can vary depending on the microorganism used. Variations in conditions suitable for optimal function of the microorganism are within the knowledge of the technician.

[0052] In one example, the method according to any aspect of the present invention, in particular step (a), can be carried out in an aqueous medium with a pH between 5 and 8, 5.5 and 8, or 5.5 and 7. The pressure can be between 1 and 10 bar. The microorganism can be cultured at a temperature ranging from about 20° C. to about 80° C. In one example, the microorganism can be cultured at 37° C.

[0053] In some examples, for the growth of microorganisms and their production of caproic acid, the aqueous medium may contain any nutrients, ingredients and / or supplements suitable for the growth of microorganisms or the promotion of caproic acid production. In particular, the aqueous medium may contain at least one of the following: a carbon source, a nitrogen source such as ammonium salts, yeast extract or peptone; minerals; salts; coenzyme factors; buffers; vitamins; and any other ingredients and / or extracts that may promote bacterial growth. The culture medium used must be suitable for the requirements of the specific strain. Culture media for various microorganisms are described in the Manual of Methods for General Bacteriology.

[0054] The term "aqueous solution" or "medium" includes any solution comprising water, mainly containing water as a solvent, which can be used to keep the cell according to any aspect of the present invention in metabolic activity and / or survival state at least temporarily, and if necessary, any additional substrate is included. Those skilled in the art are familiar with the preparation of numerous aqueous solutions, which are generally referred to as culture media that can be used to preserve and / or culture cells, such as LB medium in the case of Escherichia coli (E.coli), and ATCC 1754-medium can be used in the case of Clostridium kluyveri (C.ljungdahlii). Compared with complex media, it is advantageous to use a basic medium (i.e., a medium with a reasonable simple composition of the most basic group of salts and nutrients that only contain cells to be kept in metabolic activity and / or survival state) as an aqueous solution to avoid unnecessary contamination of the product by unwanted byproducts. For example, M9 medium can be used as a basic medium. Cells are incubated for a sufficiently long time with a carbon source to produce a desired product. For example, it lasts at least 1 hour, 2 hours, 4 hours, 5 hours, 10 hours or 20 hours. The temperature selected must be such that the cells according to any aspect of the present invention remain catalytically and / or metabolically active, for example, if the cells are C. ljungdahlii cells, the temperature is 10 to 42° C., preferably 30 to 40° C., in particular 32 to 38° C. The aqueous medium according to any aspect of the present invention also includes the medium in which caproic acid is produced. It mainly refers to the medium in which the solution substantially contains water. In one example, the aqueous medium in which the cells are used to produce caproic acid is the medium that is contacted with the extractant to extract the caproic acid.

[0055] According to any aspect of the present invention, preferably the extraction is carried out in step (b) while the fermentation is carried out in step (a). This describes the in situ extraction of caproic acid and / or its esters in step (b).

[0056] In step (b) according to any aspect of the present invention, the caproic acid in the aqueous medium may be contacted with the extractant for a time sufficient to extract the caproic acid from the aqueous medium into the extractant. A skilled person may be able to determine the amount of time required to reach partition equilibrium and the correct bubble agglomeration that may be required to optimize the extraction process. In some instances, the time required may depend on the amount of caproic acid that can be extracted. In particular, the time required to extract the caproic acid from the aqueous medium into the extractant may be only a few minutes. According to any aspect of the present invention, in the case where the extraction is performed in step (b) while the fermentation is performed in step (a), the extraction time may be equal to the fermentation time.

[0057] The ratio of the amount of the extractant used to the amount of the caproic acid to be extracted can vary depending on the speed at which the extraction is to be performed. In one example, the amount of the extractant is equal to the amount of the aqueous medium containing the caproic acid. After the step of contacting the extractant with the aqueous medium, the two phases (aqueous phase and organic phase) are separated using any method known in the art. In one example, a separation funnel can be used to separate the two phases. Mixer-settlers, pulse towers, etc. can also be used to separate the two phases. In one example, in view of the fact that caproic acid distills at a significantly lower boiling point than the extractant, distillation can be used to separate the extraction medium from the caproic acid. The technician may be able to select the best method for separating the extractant from the desired caproic acid in step (b) according to the characteristics of the caproic acid. In particular, step (c) according to any aspect of the present invention involves recovering caproic acid from step (b). Contacting caproic acid with an organic extractant results in the formation of two phases, and the two phases (aqueous phase and organic phase) are separated using any method known in the art. In one example, a separation funnel can be used to separate the two phases. Mixer-settlers, pulse towers, thermal separation, etc. can also be used to separate the two phases. In one example, where the alkanoic acid is hexanoic acid, distillation can be used to separate the extraction medium from the hexanoic acid given the fact that hexanoic acid distills at a significantly lower boiling point than the extraction medium.

[0058] At the end of step (b), the organic absorbent is again available for recirculation or reuse.

[0059] Thus, the method for extracting caproic acid according to any aspect of the present invention can be used together with any biotechnological method for producing caproic acid. This is particularly advantageous because usually during a fermentation process for producing caproic acid using a biological method, caproic acid is left to be collected in an aqueous medium, and after reaching a certain concentration in the fermentation medium, it is this target product (i.e., caproic acid) that can inhibit the activity and productivity of the microorganisms. This therefore limits the overall yield of the fermentation process. Using this extraction method, caproic acid is extracted as it is produced, thereby greatly reducing the inhibitory effect of the final product.

[0060] The method according to any aspect of the present invention is also more efficient and cost-effective than traditional methods of removing caproic acid, particularly removing caproic acid from a fermentation process as it is produced, because there is no major reliance on distillation and / or precipitation to recover the caproic acid. Distillation or precipitation processes can result in higher manufacturing costs, lower yields, and higher waste products, thus reducing the overall efficiency of the process. The method according to any aspect of the present invention seeks to overcome these disadvantages.

[0061] In particular, the mixture of microorganisms and carbon sources according to any aspect of the present invention can be used in any known bioreactor or fermenter to implement any aspect of the present invention. In one example, the complete method starting from the biotechnological production of caproic acid from acetate and / or ethanol and ending with the extraction of caproic acid according to any aspect of the present invention is carried out in a single container. Therefore, there can be no separation step between the step of producing caproic acid and the step of extracting caproic acid. This saves time and cost. In particular, during the fermentation process, the microorganism can be grown in an aqueous medium and in the presence of an extractant. Therefore, the method according to any aspect of the present invention provides a "one-pot method" for producing caproic acid. In addition, since caproic acid is extracted when it is produced, no end-product inhibition occurs, ensuring that the yield of caproic acid is maintained. Further separation steps can be performed to remove caproic acid. Any separation method known in the art can be used, such as using a funnel, a tower, distillation, etc. The remaining extractant and / or cells can then be recycled.

[0062] In another example, the caproic acid extraction process can occur as a separate step and / or in another tank. After fermentation has occurred, where the desired caproic acid to be extracted has been produced, an extractant according to any aspect of the present invention can be added to the fermentation medium, or the fermentation medium can be added to the tank containing the extractant. The desired caproic acid can then be extracted by any separation method known in the art, such as using a funnel, column, distillation, etc. The remaining extractant can then be recycled. The fermentation medium with the cells can also be recycled.

[0063] Another advantage of the process is that the extractant can be recycled. Thus, once the hexanoic acid is separated from the extractant, the extractant can be recycled and reused, thereby reducing waste.

[0064] Step (c) of the process according to any aspect of the invention involves (c) contacting the extracted hexanoic acid and / or its ester from (b) with at least one ketonization catalyst and optionally other alkanoic acids comprising 1 to 22 carbon atoms under suitable reaction conditions to chemically ketonize the hexanoic acid to higher alkanones, preferably 6-undecanone.

[0065] The term "other alkanoic acids comprising 1 to 22 carbon atoms" does not encompass hexanoic acid. Preferably, the other alkanoic acids comprising 1 to 22 carbon atoms are selected from linear alkanoic acids comprising 4 to 18, preferably 5 to 12 carbon atoms.

[0066] The ketonization catalyst according to any aspect of the present invention can be any metal oxide catalyst or a mixture thereof. The ketonization reaction reacts caproic acid with the additional alkanoic acid and / or caproic acid, preferably dimerizing two caproic acid molecules into one ketone molecule and removing one water and one carbon dioxide. At least Woo, Y., Ind. Eng. Chem. Res. 2017, 56: 872-880 discloses the mechanism involved in the ketonization of caproic acid in which caproic anhydride ((CH3(CH2)4)COOCO(CH2)4CH3) may be formed. The ketonization of caproic acid in the presence of various metal oxide catalysts is also shown in Wang, SJ Phys. Chem. C 2017, 121, 18030-18046.

[0067] The ketonization catalyst used according to any aspect of the present invention may be a heterogeneous catalyst for efficiently producing a high energy density ketone, preferably a C11 ketone, from the hexanoic acid produced biologically according to step (a). In particular, the ketonization catalyst may be any metal oxide catalyst or a mixture thereof selected from the group consisting of a metal oxide catalyst or a mixture thereof selected from the group consisting of: a heteropolyacid (H3PW 12 O 40 ) catalyst, niobium oxide ((Nb2O5) catalyst, titanium oxide (TiO2) catalyst, cerium oxide (CeO2) catalyst, zinc-chromium (Zn-Cr) mixed oxide catalyst, manganese oxide (MnO x ) catalyst, lanthanum oxide (La2O3) catalyst, magnesium oxide (MgO) catalyst, iron oxide (FeO, FeO2, Fe2O3, Fe3O4, Fe4O5, Fe5O6, Fe5O7), silicon-aluminum (Si y Al z O) mixed oxide catalysts, aluminum oxide (Al2O3) catalysts and zirconium oxide (ZrO2) catalysts. MnO x The "x" in can be 1, 2 or 4. y Al z"y" and "z" in O can refer to any value, wherein the ratio of z / y is any value between 0 and 1. In one example, as described in Pham TN, ACS Catal. 2013, 3: 2456-2473, hexanoic acid is ketolyzed using a suitable heterogeneous hydrogenation metal catalyst and suitable reaction conditions. The disclosed conditions can vary according to the catalyst used to efficiently produce higher alkane ketones, preferably 6-undecanone. In another example, MnO2 and / or Al2O3 catalysts can be used to ketolyze hexanoic acid to 6-undecanone based on the contents disclosed in Gliński, M. et al., Polish J. Chem. 2004, 78: 299-302. In another example, as disclosed in US 6,265,618B1, especially in Example 3, Nb2O5 catalysts can be used to ketolyze hexanoic acid to 6-undecanone. A skilled person can determine suitable catalysts and suitable conditions for producing higher alkanones, preferably 6-undecanone, from hexanoic acid and optionally other alkanoic acids by simple trial and error based on the prior art. Orozco, LM et al. ChemSusChem, 2016, 9(17):2430-2442 and Orozco, LM et al. GreenChemistry, 2017, 19(6):1555-1569 also disclose other catalysts that can be used as ketonization catalysts according to any aspect of the present invention.

[0068] It will be within the knowledge of the skilled person to determine suitable conditions for the use of different ketolysis catalysts in step (c). In particular, the ketolysis catalyst may be a zirconia aerogel catalyst and may be used for the ketolysis of caproic acid as disclosed in Woo, Y., Ind. Eng. Chem. Res. 2017, 56: 872-880. Zirconia aerogel catalysts can not only effectively produce higher alkanones, preferably 6-undecanone, but also avoid leaching of the catalyst. Lee, Y. et al. disclosed different ketolysis catalysts and their effectiveness in ketolysis of caproic acid in Applied Catalysis A: General. 2015, 506: 288-293. The skilled person can very easily use the method described by Lee, Y. et al. to determine suitable ketolysis catalysts and / or conditions for the ketolysis of caproic acid. In particular, suitable reaction conditions for step (c) include 100°C-500°C, 100°C-450°C, 100°C-400°C, 100°C-350°C, 100°C-300°C, 100°C-250°C, 100°C-200°C, 150°C-500°C, 150°C-450°C, 150°C-400°C, 150°C-350°C, 150°C-300°C. , 150°C-250°C, 150°C-200°C, 200°C-500°C, 200°C-450°C, 200°C-400°C, 200°C-350°C, 200°C-300°C, 200°C-250°C, 250°C-500°C, 250°C-450°C, 250°C-400°C, 250°C-350°C, 250°C-300°C, etc. More specifically, higher alkanones, particularly 6-undecanone, can be produced from hexanoic acid at a reaction temperature of 150°C-250°C using a zirconium oxide aerogel catalyst as a ketonization catalyst.

[0069] According to another aspect of the present invention, there is provided a method for producing a higher alkanol, preferably 6-undecanol, from ethanol and / or acetate, the method comprising: (d) contacting a higher alkanone, preferably 6-undecanone, produced according to any aspect of the present invention with at least one hydrogenation metal catalyst to catalytically hydrogenate the higher alkanone, preferably 6-undecanone, into a higher alkanol, preferably 6-undecanol. 11 H 24 O), which is a secondary alcohol) is the result of catalytic hydrogenation of a higher alkanone, preferably 6-undecanone, adding a hydrogen molecule across the carbon-oxygen double bond to ultimately provide a higher alkanol, preferably 6-undecanol, as the final product.

[0070] In particular, this aspect of the invention comprises: (a) contacting ethanol and / or acetate with at least one microorganism capable of carbon chain extension to produce hexanoic acid and / or its esters from ethanol and / or acetate; (b) extracting the hexanoic acid and / or its esters from (a) using at least one extractant in an aqueous medium, wherein the extractant comprises at least one alkylphosphine oxide and at least one alkane comprising at least 12 carbon atoms; or at least one trialkylamine and at least one alkane comprising at least 12 carbon atoms; (c) contacting the extracted hexanoic acid and / or its esters from (b) with at least one ketonization catalyst and optionally other alkanoic acids comprising 1 to 22 carbon atoms under suitable reaction conditions to chemically ketonize the hexanoic acid and optionally other alkanoic acids to higher alkanes, preferably 6-undecanone; and (d) contacting higher alkanes, preferably 6-undecanone, with at least one hydrogenation metal catalyst to catalytically hydrogenate higher alkanes, preferably 6-undecanone, to higher alkanols, preferably 6-undecanol.

[0071] The hydrogenation metal catalyst can be a homogeneous or heterogeneous catalyst. The homogeneous metal catalyst can be a metal complex known in the art. In particular, the hydrogenation metal catalyst can be a heterogeneous catalyst. Some advantages of using a solid catalyst for a heterogeneous catalytic reaction include easy separation of catalyst and product, easy recovery and recycling of the catalyst, and relatively mild operating conditions. There are also obvious economic and environmental considerations for using a heterogeneous catalyst. In particular, the hydrogenation metal catalyst can be selected from a ruthenium (Ru) catalyst, a rhenium (Re) catalyst, a nickel (Ni) catalyst, an iron (Fe) catalyst, a cobalt (Co) catalyst, a palladium (Pd) catalyst, and a platinum (Pt) catalyst. More particularly, the catalyst can be selected from a Ni catalyst, a Pd catalyst, and a Pt catalyst. In an example, the hydrogenation metal catalyst used according to any aspect of the present invention can be a nickel nanoparticle as disclosed in Alonso, F. Tetrahedron, 2008, 64: 1847-52. In another example, as described in Gorgas, N., Organometallics, 2014, 33 (23): 6905-6914, iron (II) PNP pincer complexes can be used as hydrogenation metal catalysts for hydrogenating higher alkanones to higher alcohols, preferably 6-undecanone to 6-undecanol. In another example, as disclosed in Tariq Shah M., et al., ACS Applied Materials & Interfaces, 2015: 7 (12), 6480-9, magnetite nanoparticles of ruthenium (Ru) catalysts, rhenium (Re) catalysts, nickel (Ni) catalysts, iron (Fe) catalysts, cobalt (Co) catalysts, palladium (Pd) catalysts or platinum (Pt) catalysts can be used as heterogeneous hydrogenation metal catalysts according to any aspect of the present invention. In another example, as disclosed in Chen, JX., Tetrahedron, 2000, 56: 2153-2166, copper-phosphine complex is used as a homogeneous hydrogenation metal catalyst according to any aspect of the present invention. In another example, as disclosed in Journal of Molecular Catalysis A: Chemical, 2014, 388-389: 116-122, heterogeneous Pt catalysts, particularly Pt / Al2O3 catalysts can be used to hydrogenate higher alkanones to higher alcohols, preferably 6-undecanone to 6-undecanol. ChemSusChem, 2017: 10 (11), 2527-2533 also disclose a variety of heterogeneous catalysts, such as Pt / C, Ru / C and Pd / C, which may or may not be used with an acid catalyst to hydrogenate 6-undecanone to 6-undecanol.Based on the above, a skilled person can determine a suitable hydrogenation catalyst to be used according to any aspect of the present invention to produce higher alkanols from higher alkanones, preferably 6-undecanol from 6-undecanone.

[0072] The skilled person will be able to readily determine the appropriate hydrogenation metal catalyst and modify the conditions accordingly to efficiently produce higher alkanols, preferably 6-undecanol, from the hydrogenation of 6-undecanone.

[0073] According to another aspect of the present invention, there is provided a method for producing alkanoic acid, preferably lauric acid, from ethanol and / or acetate, the method comprising

[0074] (e) contacting the higher alkanol, preferably 6-undecanol, produced according to any aspect of the present invention with CO2 and a homogeneous carboxylation catalyst capable of carboxylating the higher alkanol, preferably 6-undecanol, to alkanoic acid, preferably lauric acid.

[0075] In particular, this aspect of the invention comprises: (a) contacting ethanol and / or acetate with at least one microorganism capable of carbon chain extension to produce caproic acid and / or its esters from ethanol and / or acetate; (b) extracting the caproic acid and / or its esters from (a) using at least one extractant in an aqueous medium, wherein the extractant comprises at least one alkylphosphine oxide and at least one alkane comprising at least 12 carbon atoms; or at least one trialkylamine and at least one alkane comprising at least 12 carbon atoms; (c) reacting the extracted caproic acid and / or its esters from (b) with at least one keto group under suitable reaction conditions. (d) contacting the higher alkanone, preferably 6-undecanone, with at least one hydrogenation metal catalyst to catalytically hydrogenate the higher alkanone, preferably 6-undecanone, to higher alkanol, preferably 6-undecanol; and (e) contacting the higher alkanol, preferably 6-undecanol produced according to any aspect of the present invention with CO2 and a homogeneous carboxylation catalyst capable of carboxylating the higher alkanol, preferably 6-undecanol, to alkanoic acid, preferably lauric acid.

[0076] Alkyl acid (preferably lauric acid (C 12 H 24 O2), also known as dodecanoic acid) can be produced from higher alkanols, preferably 6-undecanol, in the presence of a carboxylation catalyst. A possible method is disclosed at least in Scheme 1.

[0077]

[0078] Scheme 1 Formation of 6-undecanone, 6-undecanol and lauric acid from hexanoic acid

[0079] Carboxylation catalyst can be selected from Rh, Ni, Ru, Co, Pt, Pd and Fe catalyst.Technicians will be able to determine the suitable conditions and ligands that may be necessary to effectively and efficiently convert the higher alkanols, preferably 6-undecyl alcohol produced according to any aspect of the present invention into alkanoic acid, preferably lauric acid in combination with carboxylation catalyst.In particular, the carboxylation catalyst according to any aspect of the present invention can be a homogeneous carboxylation catalyst.In an example, step (e) according to any aspect of the present invention includes (ei) contacting the higher alkanols, preferably 6-undecyl alcohol produced according to any aspect of the present invention with at least one nickel catalyst and carbon dioxide at atmospheric pressure to carboxylate the higher alkanols to higher alkanoic acid, preferably 6-undecyl alcohol to lauric acid.Step (ei) can include the bromide intermediate formed.Juliá-Hernández, Nature, 2017, 545: 84-88 further discloses the details of the step (ei) of the method.

[0080] In another example, step (e) according to any aspect of the present invention involves (eii) contacting the higher alkanol produced according to any aspect of the present invention, preferably 6-undecanol, with hydrogen, CO2 and a homogeneous Rh catalyst to carboxylate the higher alkanol to higher alkanoic acid, preferably 6-undecanol to lauric acid. This method of catalytic hydrocarboxylation (Scheme 2) is further described in detail in Ostapowicz, TG et al., Angew. Chem. Int, Ed., 2013, 52: 12119-23.

[0081]

[0082] Scheme 2: Catalytic hydrocarboxylation of olefins with CO2.

[0083] In particular, step (eii) can be carried out in the presence of an elevated temperature. More particularly, the elevated temperature of step (eii) can be in the range of 100 to 300° C. For example, the temperature of step (eii) can be in the range of 100-250° C., 100-200° C., 150-300° C. or 150-250° C. In addition, suitable conditions for step (eii) can include a CO2 partial pressure in the range of 0.1 to 200 bar, in particular, the CO2 partial pressure can be between 0.1-150 bar, 0.1-100 bar, 0.1-60 bar, etc. The skilled person will be able to determine suitable carboxylation conditions that do not inhibit the catalyst and produce higher alkanoic acids, preferably lauric acid, from higher alkanols in high yield.

[0084] Other examples of carboxylation catalysts that can be used in step (e) according to any aspect of the present invention can be found in at least Journal of Molecular Catalysis A: Chemical, 2003, 197: 61-64; J. Am. Chem. Soc. 1985, 107: 3568-3572; J. Am. Chem. Soc. 1985, 107: 3565-3567 and Journal of Molecular Catalysts, 1987, 40: 71-82.

[0085] In one example, the hydrogenation metal catalyst of step (c) is also capable of performing the carboxylation of step (e) according to any aspect of the invention. Thus, a single catalyst can result in the direct formation of alkanoic acid from higher alkanones, preferably lauric acid from 6-undecanone. In particular, the catalyst that can be used to convert higher alkanones into alkanoic acid, preferably 6-undecanone into lauric acid (i.e. steps (d) and (e)) can be a homogeneous metal catalyst. More particularly, the homogeneous catalyst that can be used to perform steps (d) and (e) can be a homogeneous Rh catalyst. Possible examples of homogeneous Rh catalysts and suitable conditions for use are described at least in Organic Letters, 2008, 10(20):4697-4700; Organometallics, 2008, 27(21):5494-5503; European Journal of Organic Chemistry, 2002, 10:1685-1689; European Journal of Inorganic Chemistry, 2001, 1:289-296 and Angewandte Chemie, International Edition, 1998, 37(8):1100-1103. DETAILED DESCRIPTION

[0086] Example

[0087] The above describes a preferred embodiment, as will be appreciated by those skilled in the art, changes or modifications may be made to the design, construction or operation without departing from the scope of the claims. For example, these changes are intended to be included within the scope of the claims.

[0088] Example 1

[0089] Formation of hexanoic acid from acetate and ethanol using Clostridium kluyveri

[0090] For the bioconversion of ethanol and acetate into hexanoic acid, Clostridium kluyveri was used. All cultivation steps were performed under anaerobic conditions in pressure-resistant glass bottles that could be hermetically sealed with butyl rubber stoppers.

[0091] For pre-culture, 5 ml of a frozen cryogenic culture of Clostridium kluyveri was inoculated into 100 ml of DMSZ52 medium (pH = 7.0; 10 g / L potassium acetate, 0.31 g / L K2HPO4, 0.23 g / L KH2PO4, 0.25 g / l NH4Cl, 0.20 g / l MgSO4 x 7H2O, 1 g / L yeast extract, 0.50 mg / L resazurin, 10 μl / l HCl (25%, 7.7 M), 1.5 mg / L FeCl2 x 4H2O, 70 μg / L ZnCl2 x 7H2O, 100 μg / L MnCl2 x 4H2O, 6 μg / L H3BO3, 190 μg / L CoCl2 x 6H2O, 2 μg / L CuCl2 x 6H2O, 24μg / L NiCl2 x 6H2O, 36μg / LNa2MO4 x 2H2O, 0.5mg / L NaOH, 3μg / L Na2SeO3 x 5H2O, 4μg / L Na2WO4 x2H2O, 100μg / L vitamin B12, 80μg / L p-aminobenzoic acid, 20μg / LD(+) biotin, 200μg / L nicotinic acid, 100μg / L calcium D-pantothenate, 300μg / L pyridoxine hydrochloride, 200μg / L thiamine hydrochloride x 2H2O, 20ml / L ethanol, 2.5g / L NaHCO3, 0.25g / L cysteine-HCl x H2O, 0.25g / L Na2S x 9H2O) and incubated at 37°C for 144 hours until OD 600nm >0.2.

[0092] For the main culture, the centrifuged cells from the pre-culture were inoculated into 200 ml of fresh DMSZ52 medium in a 500 ml flask to an OD of 600nm The grown culture was incubated at 37°C for 27 hours until the OD 600nm >0.6. The cell suspension was then centrifuged, washed with production buffer (pH 6.0; 0.832 g / L potassium acetate, 5.0 g / l ethanol) and centrifuged again.

[0093] For production culture, washed cells from the main culture were inoculated into 200 ml of production buffer in a 500 ml flask to an OD of 600nmThe culture was covered with butyl rubber stoppers and incubated for 71 hours in an open water bath shaker at 37°C and 100 rpm. Samples were taken at the beginning and end of the incubation period. These samples were tested for optical density, pH and different analytes (by NMR testing).

[0094] The results showed that during the production stage, the amount of acetate decreased from 0.54 g / l to 0.03 g / l and the amount of ethanol decreased from 5.6 g / l to 4.9 g / l. In addition, the concentration of butyric acid increased from 0.05 g / l to 0.28 g / l and the concentration of caproic acid increased from 0.03 g / l to 0.79 g / l.

[0095] Example 2

[0096] Formation of caproic acid from butyrate and ethanol using Clostridium kluyveri

[0097] For the bioconversion of ethanol and butyrate into caproate, the bacterium Clostridium kluyveri was used. All cultivation steps were carried out under anaerobic conditions in pressure-resistant glass bottles that could be hermetically sealed with butyl rubber stoppers.

[0098] For pre-culture, 5 ml of a frozen cryogenic culture of Clostridium kluyveri was inoculated into 100 ml of DMSZ52 medium (pH = 7.0; 10 g / L potassium acetate, 0.31 g / L K2HPO4, 0.23 g / L KH2PO4, 0.25 g / l NH4Cl, 0.20 g / l MgSO4 x 7H2O, 1 g / L yeast extract, 0.50 mg / L resazurin, 10 μl / l HCl (25%, 7.7 M), 1.5 mg / L FeCl2 x 4H2O, 70 μg / L ZnCl2 x 7H2O, 100 μg / L MnCl2 x 4H2O, 6 μg / L H3BO3, 190 μg / L CoCl2 x 6H2O, 2 μg / L CuCl2 x 6H2O, 24μg / L NiCl2 x 6H2O, 36μg / LNa2MO4 x 2H2O, 0.5mg / L NaOH, 3μg / L Na2SeO3 x 5H2O, 4μg / L Na2WO4 x2H2O, 100μg / L vitamin B12, 80μg / L p-aminobenzoic acid, 20μg / LD(+) biotin, 200μg / L nicotinic acid, 100μg / L calcium D-pantothenate, 300μg / L pyridoxine hydrochloride, 200μg / L thiamine hydrochloride x 2H2O, 20ml / L ethanol, 2.5g / L NaHCO3, 0.25g / L cysteine-HCl x H2O, 0.25g / L Na2S x 9H2O) and incubated at 37°C for 144 hours until OD 600nm >0.3.

[0099] For the main culture, the centrifuged cells from the pre-culture were inoculated into 200 ml of fresh DMSZ52 medium in a 500 ml flask to an OD of 600nm The growing culture was incubated at 37°C for 25 hours until the OD 600nm >0.4. The cell suspension was then centrifuged, washed with production buffer (pH 6.16; 4.16 g / L potassium acetate, 10.0 g / l ethanol) and centrifuged again.

[0100] For multiple production cultures, inoculate washed cells from the main culture into 200 ml of production buffer in a 500 ml flask to an OD of 600nmThe culture was incubated at 37°C and 100 rpm in an open water bath shaker for 71 hours. Samples were taken at the beginning and end of the culture period. These samples were tested for optical density, pH and different analytes (by NMR).

[0101] The results showed that during the production phase of butyrate supplementation, the amount of acetate decreased from 3.1 g / l to 1.1 g / l and the amount of ethanol decreased from 10.6 g / l to 7.5 g / l. Also, the concentration of butyrate increased from 1.2 g / l to 2.2 g / l and the concentration of caproate increased from 0.04 g / l to 2.30 g / l.

[0102] In the production phase of unsupplemented culture, the amount of acetate decreased from 3.0 g / l to 1.3 g / l and the amount of ethanol decreased from 10.2 g / l to 8.2 g / l. Also, the concentration of butyric acid increased from 0.1 g / l to 1.7 g / l and the concentration of caproic acid increased from 0.01 g / l to 1.40 g / l.

[0103] Example 3

[0104] Cultivation of Clostridium kluyveri in the presence of decane and TOPO

[0105] The bacterium Clostridium kluyveri was cultured to bioconvert ethanol and acetate into caproic acid. For in situ extraction of the produced caproic acid, a mixture of decane and trioctylphosphine oxide (TOPO) was added to the culture. All cultivation steps were carried out under anaerobic conditions in pressure-resistant glass bottles, which could be hermetically sealed with butyl rubber stoppers.

[0106] For pre-culture, 10 ml of live Clostridium kluyveri culture was inoculated in 250 ml Veri01 medium (pH 7.0; 10 g / L potassium acetate, 0.31 g / L K2HPO4, 0.23 g / L KH2PO4, 0.25 g / L NH4Cl, 0.20 g / L MgSO4 x 7H2O, 10 μl / L HCl (7.7 M), 1.5 mg / L FeCl2 x 4H2O, 36 μg / L ZnCl2, 64 μg / L MnCl2 x 4H2O, 6 μg / L H3BO3, 190 μg / L CoCl2 x 6H2O, 1.2 μg / L CuCl2 x 6H2O, 24 μg / L NiCl2 x 6H2O, 36 μg / L Na2MO4 x 2H2O, 0.5mg / L NaOH, 3μg / L Na2SeO3 x 5H2O, 4μg / L Na2WO4x 2H2O, 100μg / L vitamin B12, 80μg / L p-aminobenzoic acid, 20μg / LD(+) biotin, 200μg / L nicotinic acid, 100μg / L calcium D-pantothenate, 300μg / L pyridoxine hydrochloride, 200μg / L thiamine hydrochloride x 2H2O, 20ml / L ethanol, 2.5g / L NaHCO3, 65mg / L glycine, 24mg / L histidine, 64.6mg / L isoleucine 93.8mg / L leucine, 103mg / L lysine, 60.4mg / L arginine, 21.64mg / L L-cysteine-HCl, 21 mg / L methionine, 52 mg / L proline, 56.8 mg / L serine, 59 mg / L threonine, 75.8 mg / L valine) to the starting OD 600nm is 0.1.

[0107] Cultivate in 1000 mL pressure glass bottles in an open water bath shaker at 37°C, 150 rpm, 1 L / h ventilation, 100% CO2 for 671 hours. Vent the gas to the headspace of the reactor. The pH is maintained at 6.2 by automatic addition of 100 g / L NaOH solution. Fresh medium is added at 2.0 d -1 The dilution rate is continuously fed into the reactor and passed through a pore size of 0.2 μm. A hollow fiber polyethersulfone membrane (Spectrumlabs, Rancho Dominguez, USA) continuously removed the fermentation broth from the reactor to retain the cells in the reactor.

[0108] For the main culture, the centrifuged cells from the pre-culture were inoculated into 100 ml of fresh Veri01 medium in a 250 ml flask to an OD of600nm The pH was 0.1. An additional 1 ml of a 6% (w / w) TOPO mixture in decane was added. The culture was capped with a butyl rubber stopper and incubated in an open waterbath shaker at 37°C and 150 rpm in a 100% CO2 atmosphere for 43 hours.

[0109] During the incubation period, several 5 mL samples were collected to determine the OD 600nm , pH and product formation. The product concentration was determined by semi-quantitative 1H-NMR spectroscopy. Sodium trimethylsilylpropionate (T(M)SP) was used as a quantitative internal standard.

[0110] During the main culture period, the concentration of butyrate increased from 0.14 g / L to 2.12 g / L and the concentration of hexanoate increased from 0.22 g / L to 0.91 g / L, while the concentration of ethanol decreased from 15.04 g / L to 11.98 g / L, and the concentration of acetate decreased from 6.01 g / L to 4.23 g / L.

[0111] During this period, OD 600nm Reduced from 0.111 to 0.076.

[0112] Example 4

[0113] Cultivation of Clostridium kluyveri in the presence of tetradecane and TOPO

[0114] Clostridium kluyveri bacteria were cultured to bioconvert ethanol and acetate into caproic acid. In order to extract the produced caproic acid in situ, a mixture of tetradecane and trioctylphosphine oxide (TOPO) was added to the culture. All cultivation steps were carried out under anaerobic conditions in pressure-resistant glass bottles that could be hermetically sealed with butyl rubber stoppers.

[0115] The preculture of Clostridium kluyveri was carried out in an open water bath shaker at 37°C, 150 rpm, aeration rate of 1 L / h, a mixture of 25% CO2 + 75% N2 in a 1000 mL pressure glass bottle in 250 ml EvoDM24 medium (pH 5.5; 0.429 g / L magnesium acetate, 0.164 g / L sodium acetate, 0.016 g / L calcium acetate, 2.454 g / L potassium acetate, 0.107 mL / L H3PO4 (8.5%), 0.7 g / L ammonium acetate, 0.35 mg / L cobalt acetate, 1.245 mg / L nickel acetate, 20 μg / L d-biotin, 20 μg / L folic acid, 10 μg / L pyridoxine hydrochloride, 50 μg / L thiamine hydrochloride, 50 μg / L riboflavin, 50 μg / L nicotinic acid, 50 μg / L calcium pantothenate, 50 μg / L vitamin B12, 50 μg / L p-aminobenzoate, 50 μg / L lipoic acid, 0.702 mg / L (NH4)2Fe(SO4)2x 4H2O, 1 ml / L KS-acetate (93,5 mM), 20 mL / L ethanol, 0.37 g / L acetic acid). The gas was vented to the headspace of the reactor. The pH was maintained at 5.5 by the automatic addition of 2.5 M NH3 solution. Fresh culture medium was added at 2.0 d -1 The dilution rate is continuously fed into the reactor and passed through a pore size of 0.2 μm. A hollow fiber polyethersulfone membrane (Spectrumlabs, Rancho Dominguez, USA) was used to continuously remove the fermentation broth from the reactor to retain the cells in the reactor and maintain the OD 600nm It is about 1.5.

[0116] For the main culture, centrifuged cells from the preculture were inoculated into 100 ml of Veri01 medium (pH 6.5; 10 g / L potassium acetate, 0.31 g / L K2HPO4, 0.23 g / L KH2PO4, 0.25 g / L NH4Cl, 0.20 g / LMgSO4 x 7H2O, 10 μl / L HCl (7.7 M), 1.5 mg / L FeCl2 x 4H2O, 36 μg / L ZnCl2, 64 μg / L MnCl2 x4H2O, 6 μg / L H3BO3, 190 μg / L CoCl2 x 6H2O, 1.2 μg / L CuCl2 x 6H2O, 24 μg / L NiCl2 x 6H2O, 36 μg / L Na2MO4 x 2H2O, 0.5 mg / L NaOH, 3μg / L Na2SeO3 x 5H2O, 4μg / L Na2WO4 x2H2O, 100μg / L vitamin B12, 80μg / L p-aminobenzoic acid, 20μg / L (+) biotin, 200μg / L nicotinic acid, 100μg / L calcium D-pantothenate, 300μg / L pyridoxine hydrochloride, 200μg / L thiamine hydrochloride x 2H2O, 20ml / L ethanol, 2.5g / L NaHCO3, 65mg / L glycine, 24mg / L histidine, 64.6mg / L isoleucine, 93.8mg / L leucine, 103mg / L lysine, 60.4mg / L arginine, 21.64mg / L L-cysteine-HCl, 21mg / L methionine, 52mg / L proline, 56.8mg / L serine, 59mg / L threonine, 75.8mg / L valine, 2.5mg / L HCL 25%) to OD 600nm The pH was 0.1. An additional 1 ml of a 6% (w / w) TOPO mixture in tetradecane was added. The culture was capped with a butyl rubber stopper and incubated in an open waterbath shaker at 37°C and 150 rpm in a 100% CO2 atmosphere for 47 hours.

[0117] During the incubation period, several 5 mL samples were collected to determine the OD 600nm , pH and product formation. The product concentration was determined by semi-quantitative 1H-NMR spectroscopy. Sodium trimethylsilylpropionate (T(M)SP) was used as a quantitative internal standard.

[0118] During the main culture period, the concentration of butyrate increased from 0.05 g / L to 3.78 g / L and the concentration of hexanoate increased from 0.09 g / L to 4.93 g / L, while the concentration of ethanol decreased from 15.52 g / L to 9.36 g / L, and the concentration of acetate decreased from 6.36 g / L to 2.49 g / L.

[0119] During this period, OD 600nm Increased from 0.095 to 0.685.

[0120] Example 5

[0121] Cultivation of Clostridium kluyveri in the presence of hexadecane and TOPO

[0122] Clostridium kluyveri bacteria were cultured to bioconvert ethanol and acetate into caproic acid. In order to extract the produced caproic acid in situ, a mixture of hexadecane and trioctylphosphine oxide (TOPO) was added to the culture. All cultivation steps were carried out under anaerobic conditions in pressure-resistant glass bottles that could be hermetically sealed with butyl rubber stoppers.

[0123] For pre-culture, 10 ml of live Clostridium kluyveri culture was inoculated in 250 ml Veri01 medium (pH 7.0; 10 g / L potassium acetate, 0.31 g / L K2HPO4, 0.23 g / L KH2PO4, 0.25 g / L NH4Cl, 0.20 g / L MgSO4 x 7H2O, 10 μl / L HCl (7.7 M), 1.5 mg / L FeCl2 x 4H2O, 36 μg / L ZnCl2, 64 μg / L MnCl2 x 4H2O, 6 μg / L H3BO3, 190 μg / L CoCl2 x 6H2O, 1.2 μg / L CuCl2 x 6H2O, 24 μg / L NiCl2 x 6H2O, 36 μg / L Na2MO4 x 2H2O, 0.5mg / L NaOH, 3μg / L Na2SeO3 x 5H2O, 4μg / L Na2WO4x 2H2O, 100μg / L vitamin B12, 80μg / L p-aminobenzoic acid, 20μg / LD(+) biotin, 200μg / L nicotinic acid, 100μg / L calcium D-pantothenate, 300μg / L pyridoxine hydrochloride, 200μg / L thiamine hydrochloride x 2H2O, 20ml / L ethanol, 2.5g / L NaHCO3, 65mg / L glycine, 24mg / L histidine, 64.6mg / L isoleucine 93.8mg / L leucine, 103mg / L lysine, 60.4mg / L arginine, 21.64mg / L L-cysteine-HCl, 21 mg / L methionine, 52 mg / L proline, 56.8 mg / L serine, 59 mg / L threonine, 75.8 mg / L valine) to the starting OD 600nm is 0.1.

[0124] Cultivate in 1000 mL pressure glass bottles in an open water bath shaker at 37°C, 150 rpm, 1 L / h ventilation, 100% CO2 for 671 hours. Vent the gas to the headspace of the reactor. The pH is maintained at 6.2 by automatic addition of 100 g / L NaOH solution. Fresh medium is added at 2.0 d -1 The dilution rate is continuously fed into the reactor and passed through a pore size of 0.2 μm. A hollow fiber polyethersulfone membrane (Spectrumlabs, Rancho Dominguez, USA) continuously removed the fermentation broth from the reactor to retain the cells in the reactor.

[0125] For the main culture, the centrifuged cells from the pre-culture were inoculated into 100 ml of fresh Veri01 medium in a 250 ml flask to an OD of 600nm The pH was 0.1. An additional 1 ml of a 6% (w / w) TOPO mixture in hexadecane was added. The culture was capped with a butyl rubber stopper and incubated in an open waterbath shaker at 37°C and 150 rpm in a 100% CO2 atmosphere for 43 hours.

[0126] During the incubation period, several 5 mL samples were collected to determine the OD 600nm , pH and product formation. The product concentration was determined by semi-quantitative 1H-NMR spectroscopy. Sodium trimethylsilylpropionate (T(M)SP) was used as a quantitative internal standard.

[0127] During the main culture period, the concentration of butyrate increased from 0.14 g / L to 2.86 g / L and the concentration of hexanoate increased from 0.20 g / L to 2.37 g / L, while the concentration of ethanol decreased from 14.59 g / L to 10.24 g / L, and the concentration of acetate decreased from 5.87 g / L to 3.32 g / L.

[0128] During this period, OD 600nm Increased from 0.091 to 0.256.

[0129] Example 6

[0130] Cultivation of Clostridium kluyveri in the presence of heptadecane and TOPO

[0131] Clostridium kluyveri bacteria were cultured to bioconvert ethanol and acetate into caproic acid. In order to extract the produced caproic acid in situ, a mixture of heptadecane and trioctylphosphine oxide (TOPO) was added to the culture. All cultivation steps were carried out under anaerobic conditions in pressure-resistant glass bottles that could be hermetically sealed with butyl rubber stoppers.

[0132] For pre-culture, 10 ml of live Clostridium kluyveri culture was inoculated in 250 ml Veri01 medium (pH 7.0; 10 g / L potassium acetate, 0.31 g / L K2HPO4, 0.23 g / L KH2PO4, 0.25 g / L NH4Cl, 0.20 g / L MgSO4 x 7H2O, 10 μl / L HCl (7.7 M), 1.5 mg / L FeCl2 x 4H2O, 36 μg / L ZnCl2, 64 μg / L MnCl2 x 4H2O, 6 μg / L H3BO3, 190 μg / L CoCl2 x 6H2O, 1.2 μg / L CuCl2 x 6H2O, 24 μg / L NiCl2 x 6H2O, 36 μg / L Na2MO4 x 2H2O, 0.5mg / L NaOH, 3μg / L Na2SeO3 x 5H2O, 4μg / L Na2WO4x 2H2O, 100μg / L vitamin B12, 80μg / L p-aminobenzoic acid, 20μg / LD(+) biotin, 200μg / L nicotinic acid, 100μg / L calcium D-pantothenate, 300μg / L pyridoxine hydrochloride, 200μg / L thiamine hydrochloride x 2H2O, 20ml / L ethanol, 2.5g / L NaHCO3, 65mg / L glycine, 24mg / L histidine, 64.6mg / L isoleucine 93.8mg / L leucine, 103mg / L lysine, 60.4mg / L arginine, 21.64mg / L L-cysteine-HCl, 21 mg / L methionine, 52 mg / L proline, 56.8 mg / L serine, 59 mg / L threonine, 75.8 mg / L valine) to the starting OD 600nm is 0.1.

[0133] Cultivate in 1000 mL pressure glass bottles in an open water bath shaker at 37°C, 150 rpm, 1 L / h ventilation, 100% CO2 for 671 hours. Vent the gas to the headspace of the reactor. The pH is maintained at 6.2 by automatic addition of 100 g / L NaOH solution. Fresh medium is added at 2.0 d -1 The dilution rate is continuously fed into the reactor and passed through a pore size of 0.2 μm. A hollow fiber polyethersulfone membrane (Spectrumlabs, Rancho Dominguez, USA) continuously removed the fermentation broth from the reactor to retain the cells in the reactor.

[0134] For the main culture, the centrifuged cells from the pre-culture were inoculated into 100 ml of fresh Veri01 medium in a 250 ml flask to an OD of 600nmThe pH was 0.1. An additional 1 ml of a 6% (w / w) TOPO mixture in heptadecane was added. The culture was capped with a butyl rubber stopper and incubated in an open waterbath shaker at 37°C and 150 rpm in a 100% CO2 atmosphere for 43 hours.

[0135] During the incubation period, several 5 mL samples were collected to determine the OD 600nm , pH and product formation. The product concentration was determined by semi-quantitative 1H-NMR spectroscopy. Sodium trimethylsilylpropionate (T(M)SP) was used as a quantitative internal standard.

[0136] During the main culture period, the concentration of butyrate increased from 0.15 g / L to 2.82 g / L and the concentration of hexanoate increased from 0.19 g / L to 2.85 g / L, while the concentration of ethanol decreased from 14.34 g / L to 9.58 g / L, and the concentration of acetate decreased from 5.88 g / L to 3.20 g / L.

[0137] During this period, OD 600nm Increased from 0.083 to 0.363.

[0138] Example 7

[0139] Cultivation of Clostridium kluyveri in the presence of dodecane and TOPO

[0140] Clostridium kluyveri bacteria were cultured to bioconvert ethanol and acetate into caproic acid. In order to extract the produced caproic acid in situ, a mixture of dodecane and trioctylphosphine oxide (TOPO) was added to the culture. All cultivation steps were carried out under anaerobic conditions in pressure-resistant glass bottles that could be hermetically sealed with butyl rubber stoppers.

[0141] For pre-culture, 10 ml of live Clostridium kluyveri culture was inoculated in 250 ml Veri01 medium (pH 7.0; 10 g / L potassium acetate, 0.31 g / L K2HPO4, 0.23 g / L KH2PO4, 0.25 g / L NH4Cl, 0.20 g / L MgSO4 x 7H2O, 10 μl / L HCl (7.7 M), 1.5 mg / L FeCl2 x 4H2O, 36 μg / L ZnCl2, 64 μg / L MnCl2 x 4H2O, 6 μg / L H3BO3, 190 μg / L CoCl2 x 6H2O, 1.2 μg / L CuCl2 x 6H2O, 24 μg / L NiCl2 x 6H2O, 36 μg / L Na2MO4 x 2H2O, 0.5mg / L NaOH, 3μg / L Na2SeO3 x 5H2O, 4μg / L Na2WO4x 2H2O, 100μg / L vitamin B12, 80μg / L p-aminobenzoic acid, 20μg / LD(+) biotin, 200μg / L nicotinic acid, 100μg / L calcium D-pantothenate, 300μg / L pyridoxine hydrochloride, 200μg / L thiamine hydrochloride x 2H2O, 20ml / L ethanol, 2.5g / L NaHCO3, 65mg / L glycine, 24mg / L histidine, 64.6mg / L isoleucine 93.8mg / L leucine, 103mg / L lysine, 60.4mg / L arginine, 21.64mg / L L-cysteine-HCl, 21 mg / L methionine, 52 mg / L proline, 56.8 mg / L serine, 59 mg / L threonine, 75.8 mg / L valine) to the starting OD 600nm is 0.1.

[0142] Cultivate in 1000 mL pressure glass bottles in an open water bath shaker at 37°C, 150 rpm, 1 L / h ventilation, 100% CO2 for 671 hours. Vent the gas to the headspace of the reactor. The pH is maintained at 6.2 by automatic addition of 100 g / L NaOH solution. Fresh medium is added at 2.0 d -1 The dilution rate is continuously fed into the reactor and passed through a pore size of 0.2 μm. A hollow fiber polyethersulfone membrane (Spectrumlabs, Rancho Dominguez, USA) continuously removed the fermentation broth from the reactor to retain the cells in the reactor.

[0143] For the main culture, the centrifuged cells from the pre-culture were inoculated into 100 ml of fresh Veri01 medium in a 250 ml flask to an OD of 600nmThe pH was 0.1. An additional 1 ml of a 6% (w / w) TOPO mixture in dodecane was added. The culture was capped with a butyl rubber stopper and incubated in an open waterbath shaker at 37°C and 150 rpm in a 100% CO2 atmosphere for 43 hours.

[0144] During the incubation period, several 5 mL samples were collected to determine the OD 600nm , pH and product formation. The product concentration was determined by semi-quantitative 1H-NMR spectroscopy. Sodium trimethylsilylpropionate (T(M)SP) was used as a quantitative internal standard.

[0145] During the main culture period, the concentration of butyrate increased from 0.14 g / L to 2.62 g / L and the concentration of hexanoate increased from 0.22 g / L to 2.05 g / L, while the concentration of ethanol decreased from 14.62 g / L to 10.64 g / L, and the concentration of acetate decreased from 5.92 g / L to 3.54 g / L.

[0146] During this period, OD 600nm Increased from 0.091 to 0.259.

[0147] Example 8

[0148] Determination of the partition coefficient of caproic acid between water and a mixture of hexadecane and TOPO

[0149] During all stages of the experiment, samples were taken from both phases to measure pH and caproic acid concentration by high performance liquid chromatography (HPLC). A mixture of 100 g of an aqueous solution of caproic acid (5 g / kg) and 33 g of 6% trioctylphosphine oxide (TOPO) in hexadecane was filled into a separatory funnel and mixed at 37 ° C for 1 minute. The funnel was then placed in a tripod ring and the emulsion was allowed to stand for spontaneous separation. The pH of the aqueous phase was measured. Then 1 M NaOH solution was added to the funnel and mixed. The separation and sampling steps were repeated until the pH in the aqueous phase reached 6.2. Samples were taken from both phases at this time for subsequent analysis. The aqueous phase can be directly analyzed by HPLC. In order to analyze the organic phase, the diluted caproic acid was first re-extracted into water (pH was 12.0 by adding 1 M NaOH) and then analyzed by HPLC. The partition coefficient K of caproic acid in the system of water and 6% TOPO / hexadecane was calculated by the concentration of caproic acid in the two phases. D

[0150]

[0151] Partition coefficient K of hexanoic acid in water and 6% TOPO / hexadecane at pH 6.2 D is 4.7.

[0152] Example 9

[0153] Determination of the partition coefficient of hexanoic acid between water and a mixture of heptadecane and TOPO

[0154] During all stages of the experiment, samples were taken from the two phases to measure pH and caproic acid concentration by high performance liquid chromatography (HPLC). A mixture of 100 g of an aqueous solution of caproic acid (5 g / kg) and 33 g of 6% trioctylphosphine oxide (TOPO) in heptadecane was filled into a separatory funnel and mixed for 1 minute at 37 ° C. The funnel was then placed in a tripod ring and the emulsion was allowed to stand for spontaneous separation. The pH of the aqueous phase was measured. 1 M NaOH solution was added to the funnel and mixed. Separation and sampling steps were repeated until the pH in the aqueous phase reached 6.2. Samples were taken from the two phases at this time for subsequent analysis. The aqueous phase can be directly analyzed by HPLC. In order to analyze the organic phase, the diluted caproic acid was first re-extracted into water (pH was 12.0 by adding 1 M NaOH) and then analyzed by HPLC. The partition coefficient K of caproic acid in the system of water and 6% TOPO / heptadecane was calculated by the concentration of caproic acid in the two phases. D .

[0155]

[0156] Partition coefficient K of hexanoic acid in water and 6% TOPO / heptadecane system at pH 6.2 D is 5.0.

[0157] Example 10

[0158] Determination of the partition coefficient of hexanoic acid between water and a mixture of tetradecane and TOPO

[0159] During all stages of the experiment, samples were taken from both phases to measure pH and caproic acid concentration by high performance liquid chromatography (HPLC). A mixture of 130 g of caproic acid (5 g / kg) plus an aqueous solution of acetic acid (0.5 g / kg) and 15 g of 6% trioctylphosphine oxide (TOPO) in tetradecane was filled into a separatory funnel and mixed at 37 ° C for 1 minute. The funnel was then placed in a tripod ring and the emulsion was allowed to stand for spontaneous separation. The pH of the aqueous phase was measured. 1 M NaOH solution was added to the funnel and mixed. The separation and sampling steps were repeated until the pH in the aqueous phase reached 6.2. Samples were taken from both phases at this time for subsequent analysis. The aqueous phase can be directly analyzed by HPLC. In order to analyze the organic phase, the diluted caproic acid was first re-extracted into water (pH was 12.0 by adding 1 M NaOH) and then analyzed by HPLC. The partition coefficient K of caproic acid in the system of water and 6% TOPO / tetradecane was calculated by the concentration of caproic acid in the two phases. D .

[0160]

[0161] Partition coefficient K of hexanoic acid in water and 6% TOPO / tetradecane system at pH 6.9 D is 1.3.

[0162] Embodiment 11

[0163] In situ extraction of caproic acid by culturing Clostridium kluyveri

[0164] Clostridium kluyveri bacteria were cultured to bioconvert ethanol and acetate into caproic acid. In order to extract the produced caproic acid in situ, a mixture of tetradecane and trioctylphosphine oxide (TOPO) was continuously passed through the culture. All cultivation steps were carried out under anaerobic conditions in pressure-resistant glass bottles, which could be hermetically sealed with butyl rubber stoppers.

[0165] The preculture of Clostridium kluyveri was carried out in an open water bath shaker at 37°C, 150 rpm, aeration rate of 1 L / h, a mixture of 25% CO2 + 75% N2 in a 1000 mL pressure glass bottle in 250 ml of EvoDM45 medium (pH 5.5; 0.004 g / L magnesium acetate, 0.164 g / L sodium acetate, 0.016 g / L calcium acetate, 0.25 g / L potassium acetate, 0.107 mL / L H3PO4 (8.5%), 2.92 g / L ammonium acetate, 0.35 mg / L cobalt acetate, 1.245 mg / L nickel acetate, 20 μg / L d-biotin, 20 μg / L folic acid, 10 μg / L pyridoxine-HCl, 50 μg / L thiamine hydrochloride, 50 μg / L riboflavin, 50 μg / L nicotinic acid, 50 μg / L calcium pantothenate, 50 μg / L vitamin B12, 50 μg / L p-aminobenzoate, 50 μg / L lipoic acid, 0.702 mg / L (NH4)2Fe(SO4)2x 4H2O, 1 ml / L KS-acetate (93,5 mM), 20 mL / L ethanol, 0.37 g / L acetic acid). The gas was vented to the headspace of the reactor. The pH was maintained at 5.5 by automatic addition of 2.5 M NH3 solution. -1 The dilution rate is continuously fed into the reactor and passed through a pore size of 0.2 μm. A hollow fiber polyethersulfone membrane (Spectrumlabs, Rancho Dominguez, USA) was used to continuously remove the fermentation broth from the reactor to retain the cells in the reactor and maintain the OD 600nm It is about 1.5.

[0166] For the main culture, 100 ml of cell broth from the pre-culture was inoculated into 150 ml of EvoDM39 medium (pH 5.8; 0.429 g / L magnesium acetate, 0.164 g / L sodium acetate, 0.016 g / L calcium acetate, 2.454 g / L potassium acetate, 0.107 mL / L H3PO4 (8.5%), 1.01 mL / L acetic acid, 0.35 mg / L cobalt acetate, 1.245 mg / L nickel acetate, 20 μg / L d-biotin, 20 μg / L folic acid, 10 μg / L pyridoxine hydrochloride, 50 μg / L thiamine hydrochloride, 50 μg / L riboflavin, 50 μg / L nicotinic acid, 50 μg / L calcium pantothenate, 50 μg / L vitamin B12, 50 μg / L p-aminobenzoate, 50 μg / L lipoic acid, 0.702 mg / L (NH4)2Fe(SO4)2x 4H2O, 1 ml / L KS-acetate (93.5 mM), 20 mL / L ethanol, 8.8 mL NH3 solution (2.5 mol / L), 27.75 ml / L acetic acid (144 g / L)) to OD 600nm It is 0.71.

[0167] The culture was carried out in an open water bath shaker at 37°C, 150 rpm, 1 L / h ventilation rate, and a mixture of 25% CO2 and 75% N2 for 65 hours. The gas was vented to the headspace of the reactor. The pH was maintained at 5.8 by automatic addition of 2.5 M NH3 solution. Fresh medium was added at 0.5 d -1 The dilution rate was continuously fed into the reactor and the OD 600nm The fermentation broth was continuously removed from the reactor at a rate of about 0.5. An additional 120 g of a 6% (w / w) mixture of TOPO in tetradecane was added to the fermentation broth. The organic mixture was then continuously fed to the reactor and also at a rate of 1 d -1 The organic phase is continuously removed from the reactor at a dilution rate of 1.1 %.

[0168] During the incubation period, several 5 mL samples were taken from both the aqueous and organic phases to determine the OD 600nm , pH and product formation. The product concentration was determined by semi-quantitative 1H-NMR spectroscopy. Sodium trimethylsilylpropionate (T(M)SP) was used as a quantitative internal standard.

[0169] During the main culture period, steady-state concentrations of 8.18 g / L ethanol, 3.20 g / L acetate, 1.81 g / L butyrate, and 0.81 g / L hexanoate were achieved in the aqueous phase. 600nmremained stable at 0.5. Steady-state concentrations of 0.43 g / kg ethanol, 0.08 g / kg acetate, 1.13 g / kg butyrate and 8.09 g / kg hexanoate were achieved in the organic phase. After the experiment, the cells remained viable when transferred for further cultivation.

[0170] The distribution coefficients K of the substrate and product in the system of aqueous medium and 6% TOPO / tetradecane were calculated from the concentrations in the two phases. D

[0171]

[0172] The K of ethanol at steady state D is 0.05, the K of acetic acid D is 0.03, the K of butyric acid D is 0.62, and the K of hexanoic acid D It is 9.99.

[0173] Example 12

[0174] Ketonization of hexanoic acid

[0175] Ketonization is carried out in a heated continuous fluidized bed reactor. First, magnesium oxide (50% by weight, 14.00 g) on ​​silica is loaded into the reactor and heated at 330 ° C for 1 hour under an argon stream (54 mL / min). The temperature is raised to 360 ° C. Then a mixture of hexadecanoic acid in tetradecane (v / v: 3 / 1) is continuously fed into the reactor at a rate of 3.3 mL / h. The gaseous effluent stream is collected by two cold traps, which are cooled with a mixture of water and dry ice and isopropanol. The collected fractions are weighed and analyzed for their composition by gas chromatography (GC). A total of 370.65 g of hexanoic acid is fed into the reactor, which is equal to a maximum theoretical yield of 271.70 g of 6-undecanone and 28.75 g of water and 70.21 g of carbon dioxide as by-products. The amount of 6-undecanone obtained is 267.67 g, and the amount of water is 28.32 g. This corresponds to a mass recovery of 99% at complete conversion. The high productivity and selectivity were confirmed by conventional GC measurements, as only traces of hexanoic acid and no by-products were detected.

[0176] Example 13

[0177] Cross-ketolation of hexanoic acid and palmitic acid.

[0178] The technical procedure of cross-ketolysis is the same as that of hexanoic acid alone ketolysis (Example 12), except for the composition of the substrate feed. The feed consists of hexanoic acid (116.16 g, 1.00 mol) and palmitic acid (256.43 g, 1.00 mol) as substrates and tetradecane (124.20 g) as an internal standard. The substrate feed is added at a rate of 3.3 mL / h and reacted at a temperature of 360° C. The presence of two alkanoic acids results in a product mixture of the following three ketones: 6-undecanone, 6-heneicosaneone and 16-triacontaneone. At the time of complete conversion, the amount obtained is 42.58 g 6-undecanone, 155.29 g 6-heneicosaneone and 112.71 g 16-triacontaneone.

[0179] Embodiment 14

[0180] Cross-ketonization of hexanoic acid and acetic acid.

[0181] The technical procedure of cross ketolysis is the same as that of hexanoic acid alone ketolysis (Example 12), except for the composition of the substrate feed. The feed consists of hexanoic acid (232.32g, 2.00mol) and acetic acid (120.10g, 2.00mol) as substrates and tetradecane (117.47g) as internal standard. The substrate feed is added at a rate of 3.3mL / h and reacted at a temperature of 360°C. The presence of two alkanoic acids results in a product mixture of the following three ketones: 2-acetone, 2-heptanone and 6-undecanone. When fully converted, the amount obtained is 29.04g 2-acetone, 114.19g 2-heptanone and 85.15g 6-undecanone.

Claims

1. A method for producing higher alkanones from ethanol and acetate, the method comprising (a) contacting ethanol and acetate with at least one microorganism capable of carbon chain extension to produce caproic acid and / or its esters from ethanol and acetate, wherein the microorganism is capable of carbon chain extension to produce caproic acid and / or its esters from ethanol and acetate; (b) extracting the hexanoic acid and / or its esters from (a) in situ in an aqueous medium using at least one extractant, wherein the extractant comprises at least one alkylphosphine oxide and at least one alkane comprising at least 12 carbon atoms, the alkylphosphine oxide comprising trioctylphosphine oxide; and (c) contacting the extracted hexanoic acid and / or its esters from (b) with at least one ketonization catalyst and optionally other alkanoic acids containing 1 to 22 carbon atoms under suitable reaction conditions to chemically ketonize the hexanoic acid and optionally other alkanoic acids to higher alkanones, The suitable reaction conditions of step (c) include a reaction temperature of 150°C to 350°C, and the microorganism in step (a) is selected from Clostridium carboxidivorans and Clostridium kluyveri.

2. The method according to claim 1, wherein the higher alkanone is 6-undecanone.

3. The process according to claim 1, wherein the ketonization catalyst of (c) is a metal oxide catalyst or a mixture thereof.

4. The method according to claim 3, wherein the metal oxide catalyst or a mixture thereof is selected from heteropolyacids (H3PW 12 O 40 ) catalyst, titanium oxide (TiO2) catalyst, cerium oxide (CeO2) catalyst, zinc-chromium (Zn-Cr) mixed oxide catalyst, manganese oxide (MnO2) catalyst, lanthanum oxide (La2O3) catalyst, magnesium oxide (MgO) catalyst, iron oxide, silicon-aluminum (Si-Al) mixed oxide catalyst and zirconium oxide (ZrO2) catalyst.

5. The method according to any one of claims 1 to 3, wherein the ketonization catalyst is a zirconium oxide aerogel catalyst.

6. The method according to claim 1, wherein the alkane is selected from the group consisting of pentadecane, hexadecane, heptadecane, octadecane and tetradecane.

7. The method of claim 1 or 2, wherein the alkylphosphine oxide is trioctylphosphine oxide (TOPO) and the alkane is tetradecane.

8. The method of claim 7, wherein the weight ratio of TOPO to tetradecane is between 1:100 and 1:

10.

9. The method according to claim 1 or 2, wherein the pH of the aqueous medium in (b) is maintained between 5.5 and 8.

10. A method for producing higher alkanols from ethanol and acetate, the method comprising: (d) contacting the higher alkanone produced according to any one of claims 1 to 9 with at least one hydrogenation metal catalyst to catalytically hydrogenate the higher alkanone to higher alkanol. The method according to claim 10 , wherein the higher alkanol is 6-undecanol, and the higher alkanone is 6-undecanone.

12. The method according to claim 10, wherein the hydrogenation metal catalyst is selected from the group consisting of a ruthenium (Ru) catalyst, a rhenium (Re) catalyst, a nickel (Ni) catalyst, iron (Fe), cobalt (Co) and a platinum (Pt) catalyst.

13. A method for producing alkanoic acid from ethanol and acetate, the method comprising (e) contacting the higher alkanol produced according to any one of claims 10 to 12 with CO2 and a homogeneous carboxylation catalyst capable of carboxylating the higher alkanol to alkanoic acid.

14. The method according to claim 13, wherein the alkanoic acid is lauric acid, and the higher alkanol is 6-undecanol.

15. The method of claim 13, wherein step (e) comprises (ei) contacting the higher alkanol produced according to any one of claims 10 to 12 with at least one nickel catalyst and carbon dioxide at atmospheric pressure to carboxylate the higher alkanol to a higher alkanoic acid; or (eii) contacting the higher alkanol produced according to any one of claims 10 to 12 with hydrogen, CO2 and a homogeneous Rh catalyst to carboxylate the higher alkanol to a higher alkanoic acid.

16. The method according to claim 15, wherein the higher alkanol is 6-undecanol.

17. The method of claim 15, wherein step (e) comprises (ei) contacting 6-undecanol produced according to claim 11 with at least one nickel catalyst and carbon dioxide at atmospheric pressure to carboxylate the 6-undecanol to lauric acid; or (eii) contacting 6-undecanol produced according to claim 11 with hydrogen, CO2 and a homogeneous Rh catalyst to carboxylate the 6-undecanol to lauric acid.

18. The process according to any one of claims 1, 10, 13, wherein the extractant is recycled.

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