Method for extracting carbonic acid, fatty acids, fatty esters, and fatty alcohols from aqueous media

Abstract

The present invention provides a method for extracting at least one selected from carbonic acid, a fatty acid, a fatty acid ester, and a fatty alcohol from an aqueous medium, the method comprising: (a) contacting the carbonic acid, the fatty acid, the fatty acid ester, and / or the fatty alcohol in the aqueous medium with the extraction medium comprising at least one alkylphosphine oxide for a time sufficient to extract the carbonic acid, the fatty acid, the fatty acid ester, and / or the fatty alcohol from the aqueous medium into an extraction medium; (b) separating the extraction medium containing the extracted carbonic acid, fatty acid, fatty acid ester, and / or fatty alcohol from the aqueous medium; and the at least one alkylphosphine oxide comprises at least two different alkyl radicals per alkylphosphine oxide molecule; the aqueous medium in step (a) contains a microorganism, preferably a live microorganism that produces the carbonic acid, the fatty acid, the fatty acid ester, and / or the fatty alcohol; The present invention relates to a method characterized by:

Description

[Technical Field]

[0001] The present invention provides a method for extracting at least one selected from carbonic acid, a fatty acid, a fatty acid ester, and a fatty alcohol from an aqueous medium, the method comprising: (a) contacting carbonic acid, fatty acids, fatty acid esters, and / or fatty alcohols in an aqueous medium with an extraction medium comprising at least one alkylphosphine oxide for a time sufficient to extract alkanoic acids from the aqueous medium into an extraction medium; (b) separating the extraction medium containing the extracted carbonic acid, fatty acid, fatty acid ester, and / or fatty alcohol from the aqueous medium; and At least one alkylphosphine oxide contains at least two different alkyl radicals per alkylphosphine oxide molecule. The present invention relates to a method characterized by: [Background technology]

[0002] Patent Document 1 discloses a method for recovering acetic acid from a wood extract, which comprises preparing an aqueous wood extract containing acetic acid and dissolved hemicellulose containing uronic acid, preparing a water-insoluble solvent containing an extractant for acetic acid, contacting the wood extract with the solvent and the extractant to extract acetic acid from the wood extract, and recovering acetic acid from the solvent and the extractant, wherein the extractant for acetic acid may contain trioctylphosphine oxide.

[0003] Patent Document 2 discloses a method for recovering carboxylic acids selected from the group consisting of citric acid, malic acid, tartaric acid, and oxalic acid from a fermentation broth using an extractant that is a mixture of trialkylphosphine oxides having a total of 15 to 27 carbon atoms.

[0004] There remains a need in the art for more efficient extraction methods for fatty acids, fatty esters, and fatty alcohols, especially fatty acids, produced on an industrial scale.Furthermore, there is a need for methods for extracting fatty acids, fatty esters, and fatty alcohols, especially fatty acids, that can be used in combination with biotechnological production methods for fatty acids, fatty esters, and fatty alcohols. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2009 / 059228 [Patent Document 2] U.S. Patent No. 4,705,894 Summary of the Invention [Problem to be solved by the invention]

[0006] The present invention seeks to solve the above problems by providing a means for the extraction of fatty acids, fatty esters, and fatty alcohols, particularly fatty acids, that is more efficient than current methods available in the art. The present invention also provides a means for the extraction of fatty acids, fatty esters, and fatty alcohols, particularly fatty acids, that can be used in conjunction with biotechnological methods for the production of fatty acids, fatty esters, and fatty alcohols, particularly fatty acids.

[0007] According to one aspect of the present invention, there is provided a method for extracting at least one selected from carbonic acid, fatty acid, fatty acid ester, and fatty alcohol from an aqueous medium, as set forth in claim 1.

[0008] Another aspect of the present invention is an alkylphosphine oxide of general formula 1:

[0009] [ka] General formula 1

[0010] (In the formula, R 1 , R 2 and R 3 is selected from alkyl radicals having 6 to 12, preferably 8 to 10, more preferably 8 or 10 carbon atoms, with the proviso that R 1 , R 2 and R 3 At least two of them are different from each other.)

[0011] Another aspect of the present invention is the use of at least one alkylphosphine oxide for extracting at least one selected from carbonic acid, fatty acids, fatty acid esters, and fatty alcohols from an aqueous medium, The alkylphosphine oxide contains at least two different alkyl radicals per alkylphosphine oxide molecule, The aqueous medium contains live microorganisms, preferably microorganisms that produce carbon dioxide, fatty acids, fatty acid esters, and / or fatty alcohols.

[0012] The advantage of the present invention is that the extraction method according to any aspect of the present invention can increase the yield relative to the amount of extractant used. stomach The yield can be extracted from fatty acids, fatty acid esters, and fatty alcohols, especially fatty acids.

[0013] A further advantage of the present invention is that the process can be carried out at low temperatures without risk of disturbing the means by which the process is carried out.

[0014] A further advantage of the present invention is that the process can be carried out without the need for an additional extractant, such as an alkane.

[0015] Yet another advantage of the present invention is that the extractant can be easily separated from the carbonic acid, fatty acids, fatty acid esters, and fatty alcohols by distillation due to the high boiling point of the extractant.

[0016] A further advantage of the present invention is that the extractant is non-toxic to microorganisms. Yet another advantage of the present invention is that the extractant can be used over a wide pH range.

[0017] Immediately claimed is a method for extracting at least one selected from carbonic acid, a fatty acid, a fatty acid ester, and a fatty alcohol from an aqueous medium, comprising: (a) contacting carbonic acid, fatty acid, fatty acid ester, and / or fatty alcohol in an aqueous medium with an extraction medium comprising at least one alkylphosphine oxide for a time sufficient to extract the carbonic acid, fatty acid, fatty acid ester, and / or fatty alcohol from the aqueous medium into an extraction medium; (b) separating the extraction medium containing the extracted carbonic acid, fatty acid, fatty acid ester, and / or fatty alcohol from the aqueous medium; and At least one alkylphosphine oxide contains at least two different alkyl radicals per alkylphosphine oxide molecule. The method is characterized by the following.

[0018] The method of the present invention preferably includes step (c): isolating the extracted carbonic acid, fatty acid, fatty acid ester, and / or fatty alcohol from the extraction medium.

[0019] Because the extraction medium is not toxic to microorganisms, the extraction medium according to any embodiment of the present invention may be present when biotechnologically producing carbonic acid, fatty acids, fatty acid esters, and / or fatty alcohols according to any embodiment of the present invention. Therefore, the aqueous medium according to any embodiment of the present invention may be reused in step (a), particularly after step (c) of separating the extracted carbonic acid, fatty acids, fatty acid esters, and / or fatty alcohols from the extraction medium. Because the extraction medium according to any embodiment of the present invention is not toxic to microorganisms, this reuse step allows the microorganisms to be recycled and reused. The reuse step of the aqueous medium in the method according to any embodiment of the present invention has the additional advantage that residual carbonic acid, fatty acids, fatty acid esters, and / or fatty alcohols from the extraction medium that were not initially extracted from steps (a) and (b) in the first cycle may be further extracted, or may be extracted as many times as the aqueous medium is reused.

[0020] Preferably, the method according to the invention comprises the step of: , i.e., monocarboxylic acid wherein the fatty acid radical in the fatty acid ester is selected from the group consisting of a monofunctional fatty acid radical; In other words, monocarboxylic acids radicals, and the aliphatic alcohol is selected from monohydric alcohols.

[0021] Preferably, the method according to the invention is characterized in that the fatty acid is a monofunctional alkanoic acid. , i.e. saturated aliphatic carboxylic acids In the fatty acid ester, the fatty acid radical is selected from the group consisting of monofunctional alkanoic acid , i.e. saturated aliphatic carboxylic acids the aliphatic alcohol is selected from the group consisting of Alkyl alcohol, Alkyl Preferably the chains are unbranched.

[0022] Preferably, the method according to the present invention is characterized in that the fatty acid, fatty acid ester and / or fatty alcohol has 4 to 18, preferably 4 to 12, more preferably 6 to 8 carbon atoms, and further preferred fatty acids, fatty acid esters and / or fatty alcohols are selected from the group consisting of butanol, pentanol, hexanol, butanoic acid, pentanoic acid, hexanoic acid, and the methyl and ethyl esters of these three acids.

[0023] The weight ratio of the extraction medium used to the amount of carbonic acid, fatty acid, fatty acid ester, and / or fatty alcohol to be extracted may vary depending on the rate at which the extraction is performed. The weight ratio preferably varies from 0.5:1 to 1:200, preferably from 1:1 to 1:100, and most preferably from 1:5 to 1:15. In one example, the amount of extraction medium is equal to the amount of aqueous medium containing carbonic acid, fatty acid, fatty acid ester, and / or fatty alcohol. After contacting the extraction medium with the aqueous medium, the two phases (aqueous and organic) are separated using any means known in the art. In one example, a separatory funnel may be used to separate the two phases. Mixer-settlers, pulsed columns, and the like may also be used to separate the two phases. In one example, the separation of the extraction medium from the carbonic acid, fatty acid, fatty acid ester, and / or fatty alcohol may be performed by distillation, especially when the carbonic acid, fatty acid, fatty acid ester, and / or fatty alcohol distill at a significantly lower boiling point than the extraction medium. Those skilled in the art will be able to select the best method for separating the extraction medium from the desired carbonic acid, fatty acid, fatty acid ester, and / or fatty alcohol depending on the properties of the carbonic acid, fatty acid, fatty acid ester, and / or fatty alcohol that they wish to extract.

[0024] Preferably, the process according to the invention is characterized in that the alkylphosphine oxide is selected from the alkylphosphine oxides of general formula 1:

[0025] [ka] General formula 1

[0026] (In the formula, R 1 , R 2 and R 3 is selected from alkyl radicals having 4 to 18, preferably 6 to 12, carbon atoms, preferably linear alkyl radicals, However, R 1 , R 2 and R 3 At least two of them are different from each other.)

[0027] Preferably, the process according to the invention is such that the alkylphosphine oxide is selected from alkylphosphine oxides of general formula 1, in which R 1 , R 2 , and R 3 are selected from alkyl radicals having 8 to 10, preferably 8 or 10, carbon atoms, preferably linear alkyl radicals, preferably characterized in that, with respect to all alkyl phosphine oxides of general formula 1 contained in the extraction medium, the molar ratio of all alkyl radicals having 8 and 10 carbon atoms is in the range of 1.0:2.0 to 2.0:1.0, preferably 1.0:1.5 to 1.5:1.0, more preferably 1.0:1.2 to 1.2:1.0.

[0028] Preferably, the method according to the invention is characterized in that in the extraction medium, the at least one alkylphosphine oxide accounts for at least 50% by weight, preferably at least 80% by weight, more preferably at least 90% by weight, most preferably at least 97% by weight of the total extraction medium.

[0029] In a preferred method according to the present invention, the extraction medium further comprises at least one alkane having at least 12 carbon atoms, preferably 12 to 18 carbon atoms, more preferably selected from the group consisting of tetradecane, pentadecane, hexadecane, heptadecane, and octadecane. In a further preferred method according to the present invention, the extraction medium may comprise a mixture of alkanes. In another example, the alkane may be a branched alkane. In particular, the branched alkane may be squalene.

[0030] In a preferred method of the present invention, the extraction medium contains a second organic component in addition to the phosphine oxide. The second organic component contains at least 12 carbon atoms. The second organic component is a linear or branched alkane that may be selected from the group consisting of tetradecane, pentadecane, hexadecane, heptadecane, octadecane, and squalene, or a mixture of alkanes such as white mineral oil (Fragoltherm-Q-32-N). Furthermore, the second organic component may contain an aromatic hydrocarbon that may be selected from the group consisting of diisopropylbiphenyl, partially hydrogenated terphenyls, dibenzyltoluene, and diisopropylnaphthalene, or a mixture of aromatic solvents such as Solvesso 200. An alcohol that may be selected from the group consisting of oleyl alcohol, 2-octyldodecanol, and 2-hexyldodecanol may also be used as the second organic component.

[0031] The weight ratio of alkylphosphine oxide to alkane in the extraction medium according to any embodiment of the present invention is preferably between 1:100 and 100:1. More specifically, the weight ratio of alkylphosphine oxide to alkane may be selected within the range of 1:2 to 50:1, more preferably 1:1 to 97:3. In this example, the alkane may be hexadecane, and therefore the weight ratio of alkylphosphine oxide to hexadecane may be about 97:3. As used herein, the term "about" refers to a variation within 20%. In particular, the term "about" as used herein refers to ±20%, more specifically ±10%, and even more specifically ±5% of a given measurement or value.

[0032] The extraction medium according to any embodiment of the present invention can efficiently extract organic acids and / or alcohols (i.e., carbonic acid, fatty acids, fatty acid esters, and aliphatic alcohols) into the extraction medium. This extraction medium, a mixture of at least one alkylphosphine oxide containing at least two different alkyl radicals per alkylphosphine oxide molecule and at least one alkane, is considered suitable for the method according to any embodiment of the present invention, since the mixture functions efficiently in extracting the desired organic acids and / or alcohols in the presence of an aqueous production medium. The alkane may be a linear or branched alkane. In one example, the alkane may be a branched alkane, and the branched alkane may be squalene.

[0033] In another example, an extraction medium that is a mixture of at least one alkyl phosphine oxide containing at least two different alkyl radicals per alkyl phosphine oxide molecule and at least one partially hydrogenated aromatic hydrocarbon is believed to be suitable for the method according to any embodiment of the present invention, as the mixture functions efficiently in extracting the desired organic acid and / or alcohol in the presence of an aqueous production medium. In particular, a mixture of at least one alkylphosphine oxide containing at least two different alkyl radicals per alkylphosphine oxide molecule and at least one partially hydrogenated aromatic hydrocarbon is believed to perform better than any method currently known in the art for extracting organic acids and / or alcohols because it does not require the operation of specialized equipment, is relatively simple to carry out, and provides high product yields. Furthermore, the extraction medium according to any embodiment of the present invention in combination with an alkane or partially hydrogenated aromatic solvent is not toxic to microorganisms.

[0034] Preferably, the method according to the invention is characterized in that the aqueous medium of step (a) comprises microorganisms, preferably live microorganisms, which produce carbonic acid, fatty acids, fatty acid esters and / or fatty alcohols.

[0035] The carbonic acid, fatty acid, fatty acid ester, and / or fatty alcohol-producing microorganisms according to any embodiment of the present invention may be cultured using any culture medium, substrate, conditions, and method commonly known in the art for culturing microorganisms. This allows for the production of carbonic acid, fatty acid, fatty acid ester, and / or fatty alcohol using biotechnological methods. Depending on the microorganism used to produce carbonic acid, fatty acid, fatty acid ester, and / or fatty alcohol, the appropriate growth medium, pH, temperature, agitation rate, inoculation level, and / or aerobic, microaerobic, or anaerobic conditions will vary. Those skilled in the art will understand other conditions necessary to carry out the method according to any embodiment of the present invention. In particular, the conditions in the microbial vessel (e.g., fermenter) may vary depending on the microorganism used. Modifying the conditions to suit the optimal functionality of the microorganism is within the knowledge of those skilled in the art.

[0036] Preferably, the method according to the invention is characterized in that the pH of the aqueous medium in step a) is between 5.0 and 9.0, preferably between 5.8 and 8.0, particularly preferably between 6.5 and 7.5. "pH" in the context of the present invention is defined as the value measured on the relevant composition at 25°C after 5 minutes of stirring using a pH electrode calibrated according to ISO 4319 (1977).

[0037] The pressure may be between 1 and 10 bar. The microorganism may be cultured at a temperature ranging from about 20° C. to about 80° C. In one example, the microorganism may be cultured at 37° C.

[0038] In some examples, the aqueous medium for microbial growth and production of carbonic acid, fatty acids, fatty acid esters, and / or fatty alcohols may contain any nutrients, components, and / or supplements suitable for promoting microbial growth or production of carbonic acid, fatty acids, fatty acid esters, and / or fatty alcohols. In particular, the aqueous medium may contain at least one of the following: a carbon source, a nitrogen source such as an ammonium salt, yeast extract, or peptone; minerals; salts; cofactors; buffers; vitamins; and other components and / or extracts that may promote microbial growth. The medium used should be suitable for the requirements of the particular strain. Descriptions of media for various microorganisms can be found in the "Manual of Methods for General Bacteriology."

[0039] The terms "an aqueous solution" or "medium" include any solution consisting of water (primarily water as a solvent that can be used to maintain a microorganism according to any embodiment of the present invention, at least temporarily, in a metabolically active and / or viable state), and optionally containing additional substrates. Those skilled in the art are familiar with the preparation of numerous aqueous solutions, commonly referred to as media, that can be used to maintain and / or cultivate cells; for example, LB medium for Escherichia coli and ATCC 1754 medium for Clostridium ljungdahlii can be used. To avoid unwanted contamination of the product with undesired by-products, it is advantageous to use a minimal medium as the aqueous solution, i.e., a medium with a fairly simple composition that, in contrast to complex media, contains only a minimal set of salts and nutrients essential for keeping cells metabolically active and / or viable. For example, M9 medium may be used as a minimal medium. The cells are cultured with a carbon source for a time sufficient to produce the desired product. For example, at least 1 hour, 2 hours, 4 hours, 5 hours, 10 hours, or 20 hours. The selected temperature should be such that the cells according to any embodiment of the present invention maintain catalytic activity and / or metabolic activity, for example, 10 to 42°C, preferably 30 to 40°C, and particularly 32 to 38°C. The aqueous medium according to any embodiment of the present invention also includes a medium in which carbonic acid, fatty acids, fatty acid esters, and / or fatty alcohols are produced. This primarily refers to a medium in which the solution is substantially composed of water. In one example, the aqueous medium in which cells are used to produce carbonic acid, fatty acids, fatty acid esters, and / or fatty alcohols is the medium itself that is contacted with the extraction medium to extract the carbonic acid, fatty acids, fatty acid esters, and / or fatty alcohols.

[0040] A preferred method according to the present invention is characterized in that the aqueous medium contains a total of 0.00001 to 0.00100% by weight, preferably 0.00005 to 0.00050% by weight, and more preferably 0.00008 to 0.00012% by weight of carbonate, where % by weight is a proportion relative to the total weight of the aqueous medium.

[0041] In a preferred method according to the present invention, when the fatty acid, fatty acid ester, and / or fatty alcohol has two carbon atoms, they are contained in an aqueous medium in a total amount of 0.0001% by weight to 0.0100% by weight, preferably 0.0005% by weight to 0.0050% by weight, more preferably 0.0008% by weight to 0.0012% by weight. % The weight percentage is the ratio to the total aqueous medium.

[0042] A preferred method according to the present invention is characterized in that, when the fatty acid, fatty acid ester, and / or fatty alcohol has 3 or 4 carbon atoms, they are contained in the aqueous medium in a total amount of 0.01% by weight to 1.00% by weight, preferably 0.05% by weight to 0.50% by weight, and more preferably 0.08% by weight to 0.12% by weight, where % by weight is a proportion relative to the total aqueous medium.

[0043] A preferred method according to the present invention is characterized in that when the fatty acid, fatty acid ester, and / or fatty alcohol has 5 or 6 carbon atoms, they are contained in the aqueous medium in an amount of 0.1% by weight to 10.00% by weight in total, preferably 0.5% by weight to 5.0% by weight, and more preferably 0.8% by weight to 1.2% by weight, where % by weight is a proportion relative to the total aqueous medium.

[0044] Another aspect of the present invention is an alkylphosphine oxide of general formula 1:

[0045] [ka] General formula 1

[0046] (In the formula, R 1, R 2 , and R 3 is selected from alkyl radicals having 6 to 12, preferably 8 to 10, more preferably 8 or 10 carbon atoms; However, R 1 , R 2 , and R 3 at least two of the following are different from each other, Preferably, the molar ratio of all alkyl radicals having 8 or 10 carbon atoms is in the range of 1.0:2.0 to 2.0:1.0, preferably 1.0:1.5 to 1.5:1.0, more preferably 1.0:1.2 to 1.2:1.0.

[0047] Yet another aspect of the present invention is the use of at least one alkylphosphine oxide comprising at least two different alkyl radicals per alkylphosphine oxide molecule for extracting at least one selected from carbonic acid, fatty acids, fatty acid esters, and fatty alcohols from an aqueous medium. The use according to the invention preferably uses alkylphosphine oxides in the same preferred manner as are used in the process according to the invention, and of course the use according to the invention is preferably used for the same preferred fatty acids, fatty acid esters and fatty alcohols as mentioned above.

[0048] The above describes preferred embodiments, and as will be understood by those skilled in the art, changes or modifications in design, structure, or operation may be made without departing from the scope of the claims. These modifications are, for example, intended to be included within the scope of the claims. [Example]

[0049] Toxicity testing Experimental Example 1 General Exam Description To test the toxicity of various trialkylphosphines (TAPO), batch fermentations were mixed with these trialkylphosphines as additives, and the reactivity of the fermenters was monitored. These results were compared with the corresponding positive controls, which were batch fermentations under standard conditions without the additive. Bacterial reactivity was monitored by the formation of carboxylic acids (C4 + C6). The concentrations of butyric acid and hexanoic acid were analyzed by HPLC and 1H-NMR. If the ratio between the working test and the positive control was close to 100%, the corresponding additive was considered non-toxic. If the ratio between the working test and the positive control was close to 0%, the corresponding additive was considered non-toxic. Each test was replicated at least once. The results shown are average values.

[0050] General description of standard fermentation EvoDM24 medium (250 mL) in a 1000 mL pressure-resistant glass bottle (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 NH4 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-HCl, 50 μg / L riboflavin). Clostridium kluyveri was precultured in a 2.0-mL flask containing 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)2 × 4H2O, 1 mL / L L-cysteine ​​(93.5 mM), 20 mL / L ethanol, and 0.37 g / L acetic acid, at 37°C, 150 rpm, and a 1 L / h aeration rate of 1 L / h with a mixture of 25% CO2 and 75% N2. The gas was vented into the headspace above the reactor. The pH was maintained at 5.5 by automatic addition of 2.5 M NH3 solution. Fresh medium was added for 2.0 days. -1The fermentation broth was continuously fed to the reactor at a dilution rate of 0.05% and removed from the reactor by a 0.2 μm pore size KrosFlo® hollow fiber polyethersulfone membrane (Spectrumlabs, Rancho Dominguez, CA, USA), keeping the cells in the reactor at an OD of approximately 1.5. 600nm held. For the main culture, 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 / L MgSO4 × 7H2O, 10 μL / L HCl (7.7 M), 1.5 mg / L FeCl2 × 4H2O, 36 μg / L ZnCl2, 10 μL / L HCl (7.7 M) was used in a 250 mL bottle. 64 μg / L MnCl2×4H2O, 6 μg / L H3BO3, 190 μg / L CoCl2×6H2O, 1.2 μg / L CuCl2×6H2O, 24 μg / L NiCl2×6H2O, 36 μg / L Na2MO4×2H2O, 0.5 mg / L NaOH, 3 μg / L Na2SeO3×5H2O, 4 μg / L Na2WO4×2H2O, 100 μg / L Bi Vitamin B12, 80 μg / L p-aminobenzoic acid, 20 μg / L LD(+)biotin, 200 μg / L nicotinic acid, 100 μg / L D-Ca-pantothenate, 300 μg / L pyridoxine hydrochloride, 200 μg / L thiamine-HCl × 2H2O, 20 mL / L ethanol, 2.5 g / L NaHCO3, 65 mg / L glycine, 24 mg / L histidine leucine, 64.6 mg / L isoleucine, 93.8 mg / L leucine, 103 mg / L lysine, 60.4 mg / L arginine, 21.64 mg / 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, 2.5 mL / L HCl; 25%), OD 600nm Centrifuged cells from the preculture were inoculated until the r.t. was 0.1. The culture was capped with a butyl rubber stopper and incubated in an open water bath shaker at 37°C and 150 rpm in a 100% CO2 atmosphere for 47 hours. The incubation period was approximately 140 hours. During culture, OD 600nmSeveral 5 mL samples were taken to measure pH, pH, and product formation. Product concentration was determined by semi-quantitative 1H-NMR. Sodium trimethylsilylpropionate (T(M)SP) was used as an internal quantification standard. When added, the corresponding trialkylphosphine was added briefly after inoculation of the main culture at a ratio of 1 mL trialkylphosphine / 100 mL broth volume.

[0051] [Table 1]

[0052] As can be seen from the above product yields, trialkylphosphines with two different alkyl chains are less harmful to cells than pure C8-substituted trialkylphosphines.

[0053] Experimental Example 2 To test the toxicity of various aromatic hydrocarbons, batch fermentations were mixed with those aromatic hydrocarbons as additives, and the reactivity of the fermenters was monitored. These results were compared with the corresponding positive controls, which were batch fermentations under standard conditions without additives. The reactivity of the bacteria was monitored by the formation of carboxylic acids (C4 + C6). The concentrations of butyric acid and hexanoic acid were analyzed by HPLC. If the ratio between the working test and the positive control was close to 100%, the corresponding additive was considered non-toxic. If the ratio between the working test and the positive control was close to 0%, the corresponding additive was considered toxic. For the main culture, 90 mL of VeriOl medium as described in Example 1 was cultured at OD 600nm The cells from the preculture were inoculated until the OD reached 0.06. The culture was capped with a butyl rubber stopper and incubated in an open water bath shaker under a N2 / H2 atmosphere at 37°C and 150 rpm for 140 hours. During the incubation, the OD 600nm Several 5 mL samples were taken to measure temperature, pH, and product formation. Product concentrations were determined by HPLC analysis. When added, the corresponding aromatic hydrocarbon was added at a ratio of 1 mL aromatic hydrocarbon per 100 mL broth volume before inoculating the main culture.

[0054] [Table 2]

[0055] As can be seen from the above product yields, the partially aromatic solvents have little effect on the productivity of the cells.

[0056] Extraction experiment Experimental Example 3 General description of organic acid extraction A mixture of hexanoic acid (10 g / kg), butyric acid (2.5 g / kg), acetic acid (0.25 g / kg), and ethanol (12 g / kg) in distilled water was neutralized by adding aqueous ammonia until the pH reached 5.8. This aqueous solution was placed in a separatory funnel and vigorously mixed with an organic mixture of trialkylphosphine in alkane or pure trialkylphosphine. The weight ratio of aqueous to organic phase was 9:1. Due to extraction of the acid into the organic phase, the pH of the aqueous phase increased significantly at TAPO concentrations above 50%. Accordingly, the pH of these samples was corrected to 5.8 by adding a sufficient amount of aqueous acetic acid. After vigorously mixing, the phases were separated and analyzed by HPLC or 1 The compound concentrations in each phase were measured by H-NMR analysis. The distribution of the compounds was expressed as the partition constant Kd, which is the ratio of the concentration in the organic phase divided by the concentration in the aqueous phase.

[0057] [Table 3]

[0058] As can be seen from the table above, trialkylphosphines with two different alkyl chains show much higher Kds than pure C8-substituted trialkylphosphines.

[0059] Experimental Example 4 General description of hexanol extraction A solution of hexanol (4 g / Kg) in distilled water was prepared. Ammonium acetate buffer (0.6 g / Kg ammonium acetate adjusted to pH 5.8 by adding acetic acid) was added to the solution to bring the pH close to 5.8 during extraction. The aqueous solution was placed in a separatory funnel and vigorously mixed with an organic mixture of trialkylphosphine in alkane or pure trialkylphosphine. The weight ratio of aqueous to organic phase was 9:1. After vigorously mixing, the phases were separated and analyzed by HPLC or 1 The hexanol concentration in each phase was measured by separate H-NMR analysis. The distribution of hexanol was expressed as the partition constant Kd, which is the ratio of the concentration in the organic phase divided by the concentration in the aqueous phase.

[0060] [Table 4]

[0061] [Table 5]

[0062] As can be seen from the table above, trialkylphosphines with two different alkyl chains can be applied over a wide pH range.

[0063] Experimental Example 5 General description of organic acid extraction A mixture of hexanoic acid (5 g / kg), butyric acid (2.84 g / kg), acetic acid (1.34 g / kg), and ethanol (2.77 g / kg) in distilled water was neutralized with aqueous ammonia until the pH reached 5.8. This aqueous solution was placed in a separatory funnel and vigorously mixed with an organic mixture of trialkylphosphine in a Fragoltherm 660 column. The weight ratio of the aqueous phase to the organic phase was 9:1. Extraction of the acid into the organic phase significantly increased the pH of the aqueous phase. The pH of these samples was accordingly corrected to 5.8 by adding sufficient hexanoic acid. After vigorously mixing, the phases were separated and analyzed individually by HPLC to determine the compound concentrations in each phase. The distribution of the compounds was expressed as the partition constant, Kd, ​​which is the ratio of the concentration in the organic phase divided by the concentration in the aqueous phase.

[0064] [Table 6]

[0065] As can be seen from the table above, the Kd values ​​are not affected when using partially aromatic solvents.

Claims

1. A method for extracting at least one selected from carbonic acid, a fatty acid, a fatty acid ester, and an aliphatic alcohol from an aqueous medium, comprising: (a) removing the aqueous medium containing live microorganisms that produce the carbonic acid, the fatty acid, the fatty acid ester, and / or the fatty alcohol, and contacting the carbonic acid, the fatty acid, the fatty acid ester, and / or the fatty alcohol in the aqueous medium containing the live microorganisms with the extraction medium containing at least one trialkylphosphine oxide for a period of time sufficient to extract the carbonic acid, the fatty acid, the fatty acid ester, and / or the fatty alcohol produced by the live microorganisms from the aqueous medium into an extraction medium; (b) separating the extraction medium containing the extracted carbonic acid, fatty acid, fatty acid ester, and / or fatty alcohol from the aqueous medium; and The pH of the aqueous medium in the step (a) is 5.0 to 9.0; The microorganism is cultured at a temperature in the range of 20 to 80°C, the fatty acid, the fatty acid ester, and the fatty alcohol are selected from the group consisting of butanol, pentanol, hexanol, butanoic acid, pentanoic acid, hexanoic acid, and the methyl and ethyl esters of these three acids; In the extraction medium, the total amount of the at least one trialkylphosphine oxide accounts for at least 50% by weight of the total extraction medium; The trialkylphosphine oxide is represented by the general formula 1: 【Chemical 1】 wherein R 1 , R 2 , and R 3 are selected from alkyl radicals having 8 to 10 carbon atoms, with the proviso that at least two of R 1 , R 2 , and R 3 are different from one another. be selected from the group consisting of trialkylphosphine oxides A method characterized by:

2. the fatty acid is selected from monocarboxylic acids; in the fatty acid ester, the fatty acid radical is selected from monocarboxylic acid radicals; and the aliphatic alcohol is selected from monohydric alcohols; The method of claim 1, characterized in that

3. the fatty acid is selected from saturated aliphatic carboxylic acids; in the fatty acid ester, the fatty acid radical is selected from saturated aliphatic carboxylic acid radicals; and the aliphatic alcohol is selected from monohydric alkyl alcohols; 3. The method according to claim 1 or claim 2, characterized in that:

4. The extraction medium further comprises at least one alkane having at least 12 carbon atoms. The method according to any one of claims 1 to 3, characterized in that

5. 5. The method according to claim 1, wherein the extraction medium further comprises at least one aromatic solvent.

6. 6. The method of claim 5, wherein the aromatic solvent is selected from the group consisting of partially hydrogenated terphenyls and dibenzyltoluenes.

7. The method according to any one of claims 1 to 6, wherein the pH of the aqueous medium in step (a) is 5.8 to 8.0.

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