Extraction of organic compounds

Abstract

The present invention relates to a method of extracting at least one liquid organic product from an aqueous medium into an extraction medium, the method comprising: a) contacting the extraction medium with the aqueous medium to form a resultant mixture; and b) allowing the extraction medium and aqueous medium to settle forming almost no droplets in the resultant mixture during the extraction, wherein the extraction medium comprises at least one trialkyl-phosphine-oxide and at least one alkane.

Description

[0001] EXTRACTION OF ORGANIC COMPOUNDS

[0002] FIELD OF THE INVENTION

[0003] The invention relates to a method for extracting organic compounds from an aqueous medium using an extraction medium. In particular, there are almost no droplets in the mediums throughout the extraction process.

[0004] BACKGROUND OF THE INVENTION

[0005] There are many methods known in the art for extraction of organic compounds from aqueous solutions. For example, W02009059228 discloses a process for recovering acetic acid from a wood extract comprising: providing an aqueous wood extract containing acetic acid and dissolved hemicellulose containing uronic acid; providing a water insoluble solvent containing an extractant for the acetic acid; contacting the wood extract with the solvent and extractant in order to extract the acetic acid from the wood extract; and recovering the acetic acid from the solvent and extractant, wherein the extractant for acetic acid may comprise trioctylphosphine oxide.

[0006] US4705894 discloses a process for the recovery of a carboxylic acid selected from the group consisting of citric acid, malic acid, tartaric acid and oxalic acid from fermentation broths with an extractant being a mixture of trialkyl phosphine oxides having a total of 15 to 27 carbon atoms. A well-known process for preparing and extracting alkanoic acids involves the hydrolysis and decarboxylation of malonic esters. The malonic ester is saponified using aqueous sodium hydroxide to result in the formation of an aqueous solution of disodium salt and ethanol. The salt solution is then treated with a strong mineral acid to produce a mineral acid sodium salt and to precipitate the solid dicarboxylic acid. Simple separation procedures such as filtration or extraction, is used to then isolate the dicarboxylic acid. The sodium salt is discarded as waste. The isolated acid is further dried and heated to a temperature sufficient to cause decarboxylation to occur. This procedure is lengthy, requires numerous steps, generates waste, and is equipment intensive.

[0007] Similarly, salting-out extraction is also commonly used for extracting organic compounds such as alkanoic acids. This method however uses a system composed of ethanol and ammonium sulfate. The system parameters influencing the extraction efficiency, include tie line length, phase volume ratio, acid concentration, temperature, system pH and the like. Although the extraction efficiency of alkanoic acids was shown to increase using this method, the various parameters involved makes the method too complicated for industrial use.

[0008] WO 2021 / 018717 and WO 2019 / 158683 teach methods of extracting aliphatic alcohols and alkanoic acids respectively using an alkane and a trialkyl-phosphine-oxide. However, these methods though simple, are still inefficient in producing high yield. Accordingly, there is a need in the art for not only a simple extraction method but one that is versatile for extracting any targeted organic products from an aqueous medium and for one that results in a higher yield of the targeted organic product than currently possible with the methods known in the art.

[0009] DESCRIPTION OF THE INVENTION

[0010] The present invention solves the problems above by providing a means of extracting organic compounds / products from an aqueous solution that is not only more efficient and cheaper than the current methods available in the art but also results in a higher yield of the desired organic product. The present invention also provides a means of extracting the organic product that can be used in conjunction with a biotechnological method of producing the organic product.

[0011] According to one aspect of the present invention, there is provided a method of extracting at least one dissolved organic product from an aqueous medium into an extraction medium, the method comprising: a) contacting the extraction medium with the aqueous medium to form a resultant mixture; and b) allowing the extraction medium and aqueous medium to settle forming no visible droplets in the resultant mixture during the extraction, wherein the extraction medium comprises at least one trialkyl-phosphine-oxide and at least one alkane and the resultant mixture is shaken at a suitable velocity to produce no visible droplets.

[0012] It was found that, surprisingly, when the extraction medium is brought in contact with the aqueous medium comprising the target organic product to be extracted, without forming any visible droplets or when the extraction medium is left to settle over the aqueous medium to form almost no droplets, extraction of the organic product into the extraction medium takes place most efficiently (i.e. larger yield and less extraction medium required) compared to the extraction process with more droplets in the mixture. In the absence of droplets or in the presence of almost no droplets, no emulsion of the two solutions is formed. With less droplets formed and no emulsion produced, the extraction of the organic product from the aqueous medium to the extraction medium is more efficient and higher yield is obtained from the aqueous medium into the extraction medium compared to when the two solutions (i.e. aqueous medium with the organic product and extraction medium) comprise more droplets.

[0013] In particular, the extraction method according to any aspect of the present invention allows for an increase in yield relative to the amount of extractants used. For example, less than 50% by weight of extraction medium may be used to extract the same amount of organic products as if only pure alkanes were used. Therefore, with a small volume of extraction medium, a larger yield of organic products may be extracted. The extraction medium is also not harmful to microorganisms. Accordingly, the extraction medium according to any aspect of the present invention may be present when the organic product is biotechnologically produced. Further, the organic product may be easily separated from the extraction solvent and the extraction solvent may be recycled. According to any aspect of the present invention, the two liquids, the aqueous medium with the organic product (the target product) and the extraction medium are brought into contact with one another. Subsequently, the two liquids are allowed to rest or settle into two separate layers and maintained as such so that no droplets are formed or at least the minimum number of droplets formed as possible in the liquids. The phrase ‘to settle forming almost no droplets in the resultant mixture’ according to any aspect of the present invention refers to the step or process of allowing the two liquids, which have been brought in contact with each other, to rest and come to a formation of perhaps two separate layers with the minimum number of droplets formed as possible in both liquids. Besides the step of bringing the two liquids, according to any aspect of the present invention, in contact with one another, no further mixing of the liquids is done, especially not during the extraction process, so that only a minimum number of droplets are formed during the method according to any aspect of the present invention. In particular, the two liquids, the aqueous medium and extraction medium once brought into contact with one another are given time to form their separate layers and are not mixed or stirred to reduce the number of droplets formed in the two mediums. The two mediums thus have almost no droplets formed in them. Particularly, no visible droplets are seen in the two mediums. With almost no droplets present in both mediums, no emulsion is formed or present. Or at least, the two mediums appear not to form an emulsion or no emulsion appears to be formed. The reduced formation of emulsions results in a better extraction yield of the organic product. In particular, the resultant mixture is shaken at a suitable velocity, preferably not stirred, to produce no visible droplets. A skilled person would understand what a suitable velocity may be to produce no visible droplets in the resultant mixture. In traditional extraction methods, extraction is carried out using droplets where constant stirring, particularly at high velocity creates droplets, in the aqueous medium, which are then used to extract the organic compounds / products. However, in the method according to any aspect of the present invention, since minimal stirring at a low velocity or no stirring is used, no droplets are formed and the extraction is carried out using layers which is not only more efficient than the traditional method but also simpler.

[0014] In particular, the extraction medium and the aqueous medium are kept separate and are not mixed up.

[0015] More in particular, the almost no visible formation of droplets is a result of the two mediums according to any aspect of the present invention not being mixed or stirred, particularly not regularly mixed or the mediums are only minimally mixed. In one example, besides the step of bringing the two mediums in contact with one another, where they are both mixed, there may be no further step of mixing or stirring the mediums, especially not during the extraction phase. The lack of regular mixing or minimal mixing results in almost no droplets or at least no visible droplets being formed. This may then result in almost no emulsion being formed.

[0016] In particular, in the method according to any aspect of the present invention, extraction medium according to any aspect of the present invention is added to or brought into contact with the aqueous medium containing the organic product that is to be extracted. In one example, the extraction medium may be added to an aqueous medium comprising the organic product. In another example, the organic product is being produced in the aqueous medium and the extraction medium is added to the aqueous medium from the onset of production of organic product or once the concentration of organic product has reached a point where it can be extracted. In any case, the extraction medium may be present as the organic product is being produced in the aqueous medium.

[0017] In particular, the organic product present according to any aspect of the present invention may be selected from the group consisting of nitro compounds, sulfides, sulfites, alkenes, alkynes, aromatic compounds, carboxylic acids, dicarboxylic acids, hydroxycarboxylic acids, carboxylic acid esters, hydroxycarboxylic acid esters, alcohols, aldehydes, ketones, amines and amino acids. The organic product may be a substituted or unsubstituted compound. In particular, the organic product may be at least one alkanoic acid. More in particular, the organic product comprises at least hexanoic acid.

[0018] In one example, the organic product may be already present in the aqueous medium prior to the extraction step (a). In another example, the organic product is produced in the aqueous solution during step (a). That is to say, the extraction solvent is added to the aqueous medium where the organic product is being produced.

[0019] The method according to any aspect of the present invention may be a method of extracting at least one isolated dissolved organic product from an aqueous medium. An isolated organic product may refer to at least one organic product that may be separated from the medium where the organic product has been produced. In one example, the alkanoic acid and / or ester thereof may be produced in an aqueous medium (e.g. fermentation medium where the organic product is produced by specific cells from a carbon source). The isolated organic product may refer to the organic product extracted from the aqueous medium. In particular, the extracting step allows for the separation of excess water from the aqueous medium thus resulting in a formation of a mixture containing the extracted organic product.

[0020] The extracting medium may also be referred to as the ‘extraction medium’ or ‘organic solvent’. The extraction medium may be used for extracting / isolating the organic product produced according to any method of the present invention from the aqueous medium wherein the organic product was originally produced. At the end of the extracted step, excess water from the aqueous medium may be removed thus resulting in the extraction medium containing the extracted organic product thereof. The extraction medium may comprise a combination of compounds that may result in an efficient means of extracting the organic compound from the aqueous medium. In particular, the extraction medium may comprise: (i) at least alkane comprising at least 12 carbon atoms, and (ii) at least one molecule trialkyl-phosphine oxide (TAPO). The organic solvent according to any aspect of the present invention may efficiently extract the organic compound into the alkane- alkyl-phosphine oxide extracting medium. This organic solvent of a mixture of alkyl-phosphine oxides and at least one alkane may be considered suitable in the method according to any aspect of the present invention as the mixture works efficiently in extracting the desired organic product in the presence of a fermentation medium. In particular, the mixture of alkyl-phosphine oxides and at least one alkane may be considered to work better than any method currently known in the art for extraction of organic products as it does not require any special equipment to be carried out and it is relatively easy to perform with a high product yield.

[0021] In particular, the alkyl-phosphine oxide also known as a tri-alkyl-phosphine oxide (TAPO) used in the method according to any aspect of the present invention is selected from an alkyl-phosphine oxide of general formula 1 general formula 1 with R1, R2and R3selected from alkyl radicals, preferably linear alkyl radicals, containing 4 to 18, preferably 6 to 12, carbon atoms, with the proviso, that at least two of R1, R2and R3differ from each other.

[0022] More in particular, the alkyl-phosphine oxide is selected from an alkyl-phosphine oxide of general formula 1 with R1, R2and R3selected from alkyl radicals, preferably linear alkyl radicals, containing 8 to 10, particularly 8 or 10, carbon atoms, more particularly with the proviso, that referring to all alkyl-phosphine oxide of general formula 1 contained in the extracting medium, the molar ratio of all alkyl radicals containing 8 and 10 carbon atoms is in the range of from 1.0:2.0 to 2.0:1.0, particularly from 1.0:1.5 to 1 .5:1 .0, even more particularly from 1 .0:1 .2 to 1 .2:1 .0. Even more in particular, the alkyl-phosphine oxide in the extracting medium in the method according to any aspect of the present invention is an alkyl- phosphine oxide of general formula 1 general formula 1 with R1, R2and R3selected from linear alkyl radicals with 6 to 12, carbon atoms and wherein one alkyl radical has 8 carbon atoms and the other 10 carbon atoms, with the proviso, that at least two of R1, R2and R3differ from each other.

[0023] Even more in particular, in the extracting medium in the method according to any aspect of the present invention the alkyl-phosphine oxide accounts for at least 50 wt.-%, particularly at least 80 wt.-%, even more particularly at least 90 wt.-%, the most particularly at least 97 wt.-%, of the total extracting medium.

[0024] In one example, the extracting medium according to any aspect of the present invention may further comprise other than the alkyl-phosphine oxide, a second organic component. The second organic component contains at least 12 carbons. In particular, the alkane may comprise at 12- 18 carbon atoms.The second organic component is an alkane linear or branched that may be selected from the group consisting, tetradecane, pentadecane, hexadecane, heptadecane, octadecane and squalene or mixtures of alkanes such as white mineral oil (Fragoltherm-Q-32-N). Furthermore the second organic component may comprise of an aromatic hydrocarbon that may be selected from the group consisting, diisopropylbiphenyl, partly hydrogenated terphenyl, dibenzyltoluol and diisopropylnaphthalene or a mix of aromatic solvents such as Solvesso 200. Another possibility is to use an alcohol that may be selected from the group consisting, oleyl alcohol, 2-octyldodecanol and 2-hexyldodecanol as the second organic component.

[0025] The weight ratio of the alkyl-phosphine oxide to alkane in the extracting medium according to any aspect of the present invention preferably is between 1 :100 to 100:1. Even more in particular, the weight ratio of the alkyl-phosphine oxide to alkane may be selected within the range of from 1 :2 to 50:1 , more preferably from 1 :1 to 97:3. In the example, the alkane may be hexadecane and therefore the weight ratio of the alkyl-phosphine oxide to hexadecane may be about 97:3.

[0026] The extracting medium according to any aspect of the present invention may efficiently extract the organic product into the extracting medium. This extracting medium of 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 alkane may be considered suitable in the method according to any aspect of the present invention as the mixture works efficiently in extracting the desired organic product in the presence of the aqueous medium or aqueous production medium. The alkane may be a straight or a branched alkane. In one example, the alkane may be a branched alkane and the branched alkane may be squalene.

[0027] In another example, the extracting medium of 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 may be considered suitable in the method according to any aspect of the present invention as the mixture works efficiently in extracting the desired organic product in the presence of the aqueous production medium. In particular, the 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 may be considered to work better than any method currently known in the art for extraction organic product as it does not require any special equipment to be carried out and it is relatively easy to perform with a high product yield. Further, the extracting medium according to any aspect of the present invention in combination with alkane or partially hydrogenated aromatic solvent is also not toxic for microorganisms.

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

[0029] The term “contacting”, as used herein, means bringing about direct contact between the aqueous medium and the extraction medium according to any aspect of the present invention. For example, the aqueous medium and the extraction medium may be in different compartments and brought together in a single compartment. In step (b) according to any aspect of the present invention, the extraction medium and aqueous medium are allowed to settle forming almost no droplets in the resultant mixture during the extraction. In this step, the aqueous medium and the extraction medium are left to stand for a time sufficient to form no droplets and to extract the organic product from the aqueous medium into the organic medium. A skilled person may be capable of determining the amount of time needed to reach distribution equilibrium and the right bubble agglomeration that may be needed to optimize the extraction process. In some examples the time needed may be dependent on the amount of organic product that may be extracted. In particular, the time needed to extract the organic product from the aqueous medium into the extraction medium may only take a few minutes. In examples where the extraction is carried out as fermentation takes place, the time for extraction is equivalent to the time of fermentation.

[0030] The ratio of the extraction medium used to the amount of organic product to be extracted may vary depending on how quick the extraction is to be carried out. In one example, the amount of extraction medium is equal to the amount of aqueous medium comprising the organic product. After the step of contacting the extraction medium with the aqueous medium (i.e. step (a)), the two phases (aqueous and organic) are separated using any means known in the art. In one example, the two phases may be separated using a separation funnel or simple decantation. In one example, the separation of the organic solvent from the organic product may be carried out using distillation in view of the fact that some organic products distill at a significantly lower boiling point than the extracting medium. A skilled person may be able to select the best method of separating the extraction medium from the desired organic product depending on the characteristics of the organic product desired to be extracted. Step (b) preferably ends with the extraction medium made available again to be recycled or reused.

[0031] In one example, the aqueous medium is at least one fermentation medium comprising at least one microorganism. The microorganisms capable of producing the organic product may be cultivated with any culture media, substrates, conditions, and processes generally known in the art for culturing bacteria. This allows for the organic product to be produced using a biotechnological method. Depending on the microorganism that is used for the organic product production, appropriate growth medium, pH, temperature, agitation rate, inoculum level, and / or aerobic, microaerobic, or anaerobic conditions are varied. A skilled person would understand the other conditions necessary to carry out the method according to any aspect of the present invention. In particular, the conditions in the container (e.g. fermenter) may be varied depending on the microorganisms used. The varying of the conditions to be suitable for the optimal functioning of the microorganisms is within the knowledge of a skilled person. In one example, the method according to any aspect of the present invention may be carried out in an aqueous medium with a pH between 5 and 8, or 5.5 and 7. The pressure may be between 1 and 10 bar. The microorganisms 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. In some examples, for the growth of the microorganism and for its production of the organic product, the aqueous medium may comprise any nutrients, ingredients, and / or supplements suitable for growing the microorganism or for promoting the production of the organic product. In particular, the aqueous medium may comprise at least one of the following: carbon sources, nitrogen sources, such as an ammonium salt, yeast extract, or peptone; minerals; salts; cofactors; buffering agents; vitamins; and any other components and / or extracts that may promote the growth of the bacteria. The culture medium to be used must be suitable for the requirements of the particular strains.

[0032] Accordingly, the method of extraction of the organic product according to any aspect of the present invention may be used together with any biotechnological method of producing the organic product. This is especially advantageous as usually during the fermentation process to produce organic product using biological methods, the organic product would be left to collect in the aqueous medium and after reaching certain concentrations in the fermentation medium, the very target product (organic product) may inhibit the activity and productivity of the microorganism. This thus limits the overall yield of the fermentation process. With the use of this extraction method, the alkanoic acids and / or ester thereof are extracted as they are produced thus reducing end-product inhibition drastically.

[0033] The method according to any aspect of the present invention is also more efficient and cost effective than the traditional methods of removing organic products, particularly from a fermentation method as they are produced, as there is no primary reliance on distillation and / or a precipitation for recovering of the organic products. Distillation or precipitation process may lead to higher manufacturing costs, lower yield, and higher waste products therefore reducing the overall efficiency of the process. The method according to any aspect of the present invention attempts to overcome these shortcomings.

[0034] The number of droplets in the resultant mixture is almost not visible. In particular, the number of droplets is about 0.1 %, 0.05, 0.15, 0.2, 0.25, or 0.3 of the total volume of medium. Even more in particular, the number of droplets is about 0.1% of the total volume of medium. Any method known in the art may be used to measure the number of droplets in the medium. More in particular, the method used to measure the number of droplets may be the naked eye or a microscope and manual counting.

[0035] In another example, the microorganism according to any aspect of the present invention is at least one acetogenic bacteria or at least one microorganism for carbon chain elongation or a combination thereof.

[0036] In particular, the cells are brought into contact with a carbon source which includes monosaccharides (such as glucose, galactose, fructose, xylose, arabinose, or xylulose), disaccharides (such as lactose or sucrose), oligosaccharides, and polysaccharides (such as starch or cellulose), one-carbon substrates and / or mixtures thereof. More in particular, the cells are brought into contact with a carbon source comprising CO and / or CO2 to produce an organic product. With respect to the source of substrates comprising carbon dioxide and / or carbon monoxide, a skilled person would understand that many possible sources for the provision of CO and / or CO2 as a carbon source exist. It can be seen that in practice, as the carbon source of the present invention any gas or any gas mixture can be used which is able to supply the microorganisms with sufficient amounts of carbon, so that acetate and / or ethanol, may be formed from the source of CO and / or CO2. In particular, the microorganism may use synthesis gas, CO2, CO, combinations thereof or any other carbon source as a substrate for the organic product production.

[0037] The term "acetogenic bacteria" as used herein refers to a microorganism which is able to perform the Wood-Ljungdahl pathway and thus is able to convert CO, CO2 and / or hydrogen to acetate. These microorganisms include microorganisms which in their wild-type form do not have a WoodLjungdahl pathway, but have acquired this trait as a result of genetic modification. Such microorganisms include but are not limited to E. coli cells. These microorganisms may be also known as carboxydotrophic bacteria. Currently, 21 different genera of the acetogenic bacteria are known in the art (Drake et al., 2006), and these may also include some Clostridia (Drake & Kusel, 2005). These bacteria are able to use carbon dioxide or carbon monoxide as a carbon source with hydrogen as an energy source (Wood, 1991). Further, alcohols, aldehydes, carboxylic acids as well as numerous hexoses may also be used as a carbon source (Drake et al., 2004). The reductive pathway that leads to the formation of acetate is referred to as acetyl-CoA or Wood-Ljungdahl pathway. In particular, the acetogenic bacteria may be selected from the group consisting of Acetoanaerobium notera (ATCC 35199), Acetonema longum (DSM 6540), Acetobacterium carbinolicum (DSM 2925), Acetobacterium malicum (DSM 4132), Acetobacterium 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 drake! (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 0-52 (ATCC 55989), Clostridium mayombei (DSM 6539), Clostridium methoxybenzovorans (DSM 12182), Clostridium ragsdalei (DSM 15248), Clostridium scatologenes (DSM 757), Clostridium species ATCC 29797 (Schmidt et al., 1986, Chem. Eng. Commun., Vol. 45, p. 61-73), Desulfotomaculum kuznetsovii (DSM 6115), Desulfotomaculum thermobezoicum subsp. thermosyntrophicum (DSM 14055), Eubacterium limosum (DSM 20543), Methanosarcina acetivorans C2A (DSM 2834), Moorella sp. HUC22-1 (Sakai et al., 2004, Biotechnol. Let., Vol. 29, p. 1607-1612), Moorella thermoacetica (DSM 521 , formerly Clostridium thermoaceticum), Moorella thermoautotrophica (DSM 1974), Oxobacter pfennigii (DSM 322), Sporomusa aerivorans (DSM 13326), Sporomusa ovata (DSM 2662), Sporomusa silvacetica (DSM 10669), Sporomusa sphaeroides (DSM 2875), Sporomusa termitida (DSM 4440) and Thermoanaerobacter kivui (DSM 2030, formerly Acetogenium kivui).

[0038] More in particular, the strain ATCC BAA-624 of Clostridium carboxidivorans may be used. Even more in particular, the bacterial strain labelled "P7" and "P11" of Clostridium carboxidivorans as described for example in U.S. 2007 / 0275447 and U.S. 2008 / 0057554 may be used. Another particularly suitable bacterium may be Clostridium ljungdahlii. In particular, strains selected from the group consisting of Clostridium ljungdahlii PETC, Clostridium ljungdahlii ERI2, Clostridium ljungdahlii COL and Clostridium ljungdahlii 0-52 may be used in the conversion of synthesis gas to the organic product. These strains for example are described in WO 98 / 00558, WO 00 / 68407, ATCC 49587, ATCC 55988 and ATCC 55989.

[0039] The chain elongating bacteria may be selected from the group consisting of Clostridium kluyveri (DSM 555), C.carboxidivorans (DSM 15243), Megasphaera cerevisiae (ATCC 43254; DSM 20462), Pseudoram ibacter alactolyticus (ATCC 23263; DSM 3980;), Eubacterium pyruvativorans (ATCC BAA- 574), Acinetobacter sp. (DSM 586 and DSM 1845), Eubacterium limosum (ATCC 8486; DSM 20543), Megasphaera elsdenii (ATCC 25940; DSM 20460), Sporanaerobacter acetigenes (DSM 13106), Desulfosporosinus meridie (ATCC BAA-275; DSM 13257), Rhodobacteraceae DSM 15280, and Acetanaerobacterium sp. (DSM 102114) In particular the chain elongating bacteria may be selected from the group consisting of Clostridium kluyveri (DSM 555), and C.carboxidivorans (DSM 15243).

[0040] The overall efficiency, alkanoic acid and / or ester thereof productivity and / or overall carbon capture of the method of the present invention may be dependent on the stoichiometry of the CO2, CO, and H2 in the continuous gas flow. The continuous gas flows applied may be of composition CO2 and H2. In particular, in the continuous gas flow, concentration range of CO2 may be about 10-50 %, in particular 33 % by volume and H2 would be within 44 % to 84 %, in particular, 64 to 66.04 % by volume. In another example, the continuous gas flow can also comprise inert gases like N2, up to a N2 concentration of 50 % by volume.

[0041] Mixtures of sources can be used as a carbon source.

[0042] According to any aspect of the present invention, a reducing agent, for example hydrogen may be supplied together with the carbon source. In particular, this hydrogen may be supplied when the C and / or CO2 is supplied and / or used. In one example, the hydrogen gas is part of the synthesis gas present according to any aspect of the present invention. In another example, where the hydrogen gas in the synthesis gas is insufficient for the method of the present invention, additional hydrogen gas may be supplied. In one example, for the production of the organic product, a carbon source comprising CO and / or CO2 contacts the cells in a continuous gas flow. Even more in particular, the continuous gas flow comprises synthesis gas. These gases may be supplied for example using nozzles that open up into the aqueous medium, frits, membranes within the pipe supplying the gas into the aqueous medium and the like.

[0043] A skilled person would understand that it may be necessary to monitor the composition and flow rates of the streams at relevant intervals. Control of the composition of the stream can be achieved by varying the proportions of the constituent streams to achieve a target or desirable composition. The composition and flow rate of the blended stream can be monitored by any means known in the art. In one example, the system is adapted to continuously monitor the flow rates and compositions of at least two streams and combine them to produce a single blended substrate stream in a continuous gas flow of optimal composition, and means for passing the optimised substrate stream to the fermenter.

[0044] The term “an aqueous solution” or “medium” comprises any solution comprising water, mainly water as solvent that may be used to keep the cell according to any aspect of the present invention, at least temporarily, in a metabolically active and / or viable state and comprises, if such is necessary, any additional substrates. The person skilled in the art is familiar with the preparation of numerous aqueous mediums, usually referred to as media that may be used to keep and / or culture the cells, for example LB medium in the case of E.coli, ATCC1754-Medium may be used in the case of C.ljungdahlii. It is advantageous to use as an aqueous solution a minimal medium, i.e. a medium of reasonably simple composition that comprises only the minimal set of salts and nutrients indispensable for keeping the cell in a metabolically active and / or viable state, by contrast to complex mediums, to avoid dispensable contamination of the products with unwanted side products. For example, M9 medium may be used as a minimal medium. The cells are incubated with the carbon source sufficiently long enough to produce the desired product. For example for at least 1 , 2, 4, 5, 10 or 20 hours. The temperature chosen must be such that the cells according to any aspect of the present invention remains catalytically competent and / or metabolically active, for example 10 to 42 °C, preferably 30 to 40 °C, in particular, 32 to 38 °C in case the cell is a C.ljungdahlii cell. The aqueous medium according to any aspect of the present invention also includes the medium in which the organic product is produced. It mainly refers to a medium where the solution comprises substantially water. In one example, the aqueous medium in which the cells are used to produce the organic product is the very medium which contacts the extraction medium for extraction of the organic product.

[0045] In particular, the mixture of the microorganism and the carbon source according to any aspect of the present invention may be employed in any known bioreactor or fermenter to carry out any aspect of the present invention. In one example, the complete method according to any aspect of the present invention that begins with the production of the organic product and ends with the extraction of the organic product takes place in a single container. There may therefore be no separation step between the step of producing the organic product and the step of extracting the organic product. This saves time and costs. In particular, during the fermentation process, the microorganism may be grown in the aqueous medium and in the presence of the extraction medium. The method according to any aspect of the present invention thus provides for a one pot means of producing the organic product. Also, since the organic product is being extracted as it is produced, no end-product inhibition takes place, ensuring that the yield of the organic product is maintained. A further step of separation may be carried out to remove the organic product. Any separation method known in the art such as using a funnel, column, distillation and the like may be used. The remaining extraction medium and / or the cells may then be recycled.

[0046] In another example, the extraction process may take place as a separate step and / or in another pot. After fermentation has taken place, where the desired organic product to be extracted has already been produced, the extracting medium according to any aspect of the present invention may be added to the fermentation medium or the fermentation medium may be added to a pot comprising the extracting medium. The desired the organic product may then be extracted by any separation method known in the art such as using a funnel, column, distillation and the like. The remaining extracting medium may then be recycled. Another advantage of the method is that the extracting medium may be recycled. Therefore, once the organic product is separated from extraction medium, the extraction medium can be recycled and reused, reducing waste.

[0047] According to a further aspect of the present invention, there is provided a use of an extraction medium comprising at least one trialkyl-phosphine-oxide and at least one alkane for extracting at least one liquid organic product from an aqueous medium into an extraction medium, wherein the extraction medium forms no visible droplets when in contact with the aqueous medium. In particular, the liquid organic product is hexanoic acid.

[0048] Unless stated otherwise, all percentages (%) given are percentages by mass.

[0049] The examples adduced hereinafter describe the present invention by way of example, without any intention that the invention, the scope of application of which is apparent from the entirety of the description and the claims, be restricted to the embodiments specified in the examples.

[0050] EXAMPLES

[0051] Example 1

[0052] Droplet formation during shaking of Clostridium aoutoethanogenum (DSM 10061) and Clostridium kluyveri (DSM 555) fermentation broth with extracting agent

[0053] The homoacetogenic bacterium Clostridium autoethanogenum was cultivated together with the chain elongating bacterium Clostridium kluyveri in a coculture on synthesis gas in a mineral medium with potassium thioacetate as reduced sulfur source. The cultivation was carried out under anaerobic conditions in a pressure-resistant stainless steel bubble column loop reactor.

[0054] The cultivation was run at 37°C and an overpressure of 2 bar as a continuous fermentation with continuous feeding of 300 L / h of a mixture of water, substrates, salts, trace elements and vitamins. The pH was automatically hold at 5.80 with ammonia feeding. The outlet stream of 300 L / h out of the fermenter was for 98,2% as permeate with cell retention and for 1 ,8% as purge without cell retention. The gas was discharged into the medium through a sparger with a ventilation rate of - 1000 L / h as a gas mixture of 62,5% H2 and 37,5% CO2.

[0055] The media feed consisted of 0.004 g / L Mg-acetate x 4 H2O, 0.164 g / L Na-acetate, 0.016 g / L Ca-acetate, 0.245 g / l K-acetate, 0.107 mL / L H3PO4 (8.5%), 0.35 mg / L Co-acetate, 1 .245 mg / L Ni-acetate x 4 H2O, 20 pg / L d-biotin, 20 pg / L folic acid, 10 pg / L pyridoxine-HCI, 50 pg / L thiamine-HCI, 50 pg / L Riboflavin, 50 pg / L nicotinic acid, 50 pg / L Ca-pantothenate, 50 pg / L Vitamin B12, 50 pg / L p-aminobenzoate, 50 pg / L lipoic acid, 2.109 mg / L (NH4)2Fe(SO4)2 x 6 H2O, 10.69 mg / L potassium thioacetate, 6,73 g / L ethanol and NH3 for pH regulation.

[0056] The culture was previously inoculated with cells from fresh cultures of C.autoethanogenum and C.kluyveri and was already running since > 32.000 h full continuously as stable coculture at an optical density (ODeoonm) of - 14.8. Fresh medium was continuously feeded into the reactor and fermentation broth continuously removed from the reactor with a dilution rate of 2.8 d-1During cultivation several 5 mL samples were taken to determinate ODeoonm, pH und product formation. The determination of the product concentrations was performed by semiquantitative 1 H-NMR spectroscopy. As an internal quantification standard sodium trimethylsilylpropionate (T(M)SP) was used.

[0057] The steady state concentration of the educts and products in the reactor were about 1.6 g / L ethanol, 1.6 g / L acetate, 1 .3 g / L butyrate and 3.5 g / L hexanoate. The fermentation broth was filtered through a cell retention ceramic membrane to get a clear, cell free supernatant. This supernatant was used for further extraction experiments.

[0058] 200 ml supernatant was filled into a 500 ml glass bottle and overlayed with 100 ml extracting agent (20% trialkylphosphineoxid (TAPO) in Fragoltherm Q-32-N). The bottle with the two layer phases was then put in a tempered (37°C) shaker. Beginning from 0 rpm the shaking frequency was increased continuously in 5 rpm steps. From 0 rpm till 130 rpm the organic phase just rotated over the aqueous phase as one layer, no aqueous droplets in the organic phase were seen. From 130 rpm on first aqueous droplets in the organic phase were seen (-5 droplets / mL). The shaking velocity was increased again and more droplets were observed. At 150 rpm 10 droplets / mL were seen, at 160 rpm 20 droplets / mL. At 170 rpm a complete emulsion was formed very fast during 30 seconds. Droplets could not be counted anymore, the organic phase was completely turbid and an interphase between the aqueous and organic phase started to form. Also first organic droplets in the aqueous phase were observed.

[0059] Example 2:

[0060] Cultivation of Clostridium autoethanogenum and Clostridium kluyveri in a two-phase system with low shaking velocity

[0061] The bacterium Clostridium kluyveri was cultivated in a co culture with Clostridium autoethanogenum for the biotransformation of ethanol and acetate to hexanoic acid. For the inSitu extraction of the produced hexanoic acid the aqueous phase was overlayed with a mixture of Fragoltherm Q-32-N with trialkylphosphineoxide (TAPO). All cultivation steps were carried out under anaerobic conditions, the preculture in a stainless steel bubble column loop reactor and the main culture in a pressure-resistant glass bottle that can be closed airtight with a butyl rubber stopper. For the pre-cultivation the homoacetogenic bacterium Clostridium autoethanogenum was cultivated together with the chain elongating bacterium Clostridium kluyveri in a coculture on synthesis gas in a mineral medium. The cultivation was carried out under anaerobic conditions in a pressure-resistant stainless steel bubble column loop reactor.

[0062] The cultivation was run at 37°C and an overpressure of 2 bar as a continuous fermentation with continuous feeding of 300 L / h of a mixture of water, substrates, salts, trace elements and vitamins. The pH was automatically hold at 5.80 with ammonia feeding. The outlet stream of 300 L / h out of the fermenter was for 98,2% as permeate with cell retention and for 1 ,8% as purge without cell retention. The gas was discharged into the medium through a sparger with a ventilation rate of - 1000 L / h as a gas mixture of 62,5% H2 and 37,5% CO2.

[0063] The media feed consisted of 0.004 g / L Mg-acetate x 4 H2O, 0.164 g / L Na-acetate, 0.016 g / L Ca-acetate, 0.245 g / l K-acetate, 0.107 mL / L H3PO4 (8.5%), 0.35 mg / L Co-acetate, 1 .245 mg / L Ni-acetate x 4 H2O, 20 pg / L d-biotin, 20 pg / L folic acid, 10 pg / L pyridoxine-HCI, 50 pg / L thiamine-HCI, 50 pg / L Riboflavin, 50 pg / L nicotinic acid, 50 pg / L Ca-pantothenate, 50 pg / L Vitamin B12, 50 pg / L p-aminobenzoate, 50 pg / L lipoic acid, 2.109 mg / L (NH4)2Fe(SO4)2 x 6 H2O, 10.69 mg / L potassium thioacetate, 6,73 g / L ethanol and NH3 for pH regulation.

[0064] The culture was previously inoculated with cells from fresh cultures of C. autoethanogenum and C. kluyveri and was already running since > 30.000 h full continuously as stable coculture at an optical density (ODeoonm) of ~ 14. Fresh medium was continuously feeded into the reactor and fermentation broth continuously removed from the reactor with a dilution rate of 2.8 d1.

[0065] For the main culture 200 ml of the same mineral medium as used in the preculture with a higher ethanol concentration (20 mL / L ethanol) and additional 5 g / L NH4acetate and 0.15 g / L NH4HCO3 were filled in a 500 mL bottle and inoculated with as many cell broth from the preculture to reach an ODeoonm of 0.15. The aqueous phase was then overlayed with 100 ml extracting agent (20% trialkylphosphineoxid (TAPO) in Fragoltherm Q-32-N). The bottle with the two-layer phases was then cultivated at 37°C, 50 rpm and with a gas atmosphere of 10 % H2 and 90 % N2 in an open water bath shaker for 116 h.

[0066] During cultivation several 5 mL samples from both, the aqueous and the organic phase, were taken to determinate ODeoonm, pH und product formation. The determination of the product concentrations was performed by semiquantitative 1 H-NMR spectroscopy. As an internal quantification standard sodium trimethylsilylpropionate (T(M)SP) was used.

[0067] After 116 h of cultivation it was still a two-layer system in the bottle with a lower aqueous phase and an upper organic phase which were not mixed. In the aqueous phase the concentration of acetate decreased from 4.28 g / L to 0.20 g / L and the concentration of ethanol decreased from 14.12 g / L to 4.98 g / L during this time. During the same time 0.8 g / L butyrate and 6.14 g / L hexanoate were built. In the organic phase the concentrations raised from 0 g / L to 1 .12 g / L for ethanol and 8.98 g / L for hexanoic acid. Butyric acid and acetic acid were not found. All of the organic and aqueous phase stayed clear and no emulsion was formed. All of the hexanoic acid could be back extracted with an alkaline aqueous solution from the organic phase so that the organic solution could be used again for the next extraction. No product or extracting agent was lost.

Claims

CLAIMS1 . A method of extracting at least one liquid organic product from an aqueous medium into an extraction medium, the method comprising: a) contacting the extraction medium with the aqueous medium to form a resultant mixture; and b) allowing the extraction medium and aqueous medium to settle forming no visible droplets in the resultant mixture during the extraction, wherein the extraction medium comprises at least one trialkyl-phosphine-oxide and at least one alkane and the resultant mixture is shaken at a suitable velocity to produce no visible droplets.

2. The method according to claim 1 , wherein the resultant mixture is not stirred to produce no visible droplets.

3. The method according to either claim 1 or 2, wherein the number of droplets in the resultant mixture is about 0.1%.

4. The method according any one of the preceding claims, wherein the aqueous medium is at least one fermentation medium comprising at least one microorganism.

5. The method according to claim 4, wherein the microorganism is at least one acetogenic bacteria or at least one microorganism for carbon chain elongation or a combination thereof.

6. The method according to claim 5, wherein the acetogenic bacteria is selected from the group consisting of A cetoanaerobium notera, Acetonema longum, Acetobacterium carbinolicum, Acetobacterium malicum, Acetobacterium species no. 446, Acetobacterium wieringae, Acetobacterium woodii, Alkalibaculum bacchi, Archaeoglobus fulgidus, Blautia producta, Butyribacterium methylotrophicum, Clostridium aceticum, Clostridium autoethanogenum, Clostridium carboxidivorans, Clostridium coskatii, Clostridium drakei, Clostridium formicoaceticum, Clostridium glycolicum, Clostridium ljungdahlii, Clostridium ljungdahlii C-01, Clostridium ljungdahlii ERI-2, Clostridium ljungdahlii 0-52, Clostridium mayombei, Clostridium methoxybenzovorans, Clostridium ragsdalei, Clostridium scatologenes, Clostridium species ATCC 29797, Desulfotomaculum kuznetsovii, Desulfotomaculum thermobezoicum subsp. thermosyntrophicum, Eubacterium limosum, Methanosarcina acetivorans C2A, Moorella sp. HUC22-1, Moorella thermoacetica, Moorella thermoautotrophica, Oxobacter pfennigii, Sporomusa aerivorans, Sporomusa ovata, Sporomusa silvacetica, Sporomusa sphaeroides, Sporomusa termitida, Thermoanaerobacter kivui and a mixture thereof; and the microorganism for carbon chain elongation is Clostridium kluyveri, C. Carboxidivorans or a mixture thereof.

7. The method according to any one of the preceding claims, wherein the alkane comprises 12 to 18 carbon atoms.

8. The method according to any one of the preceding claims, wherein the trialkyl-phosphine oxide is selected from a trialkyl-phosphine oxide of general formula 1general formula 1 with R1, R2and R3selected from alkyl radicals containing 4 to 18, preferably 6 to 12, carbon atoms, with the proviso, that at least two of R1, R2and R3differ from each other.

9. The method according to claim 8, wherein the trialkyl-phosphine oxide is selected from a trialkyl-phosphine oxide of general formula 1 with R1, R2and R3selected from alkyl radicals containing 8 to 10 carbon atoms.

10. The method according to claim 9, wherein the molar ratio of all alkyl radicals containing 8 and 10 carbon atoms in the trialkyl-phosphine oxide is in the range of from 1 .0:2.0 to 2.0:1 .0.11 . The method according to any one of the preceding claims, wherein the extraction medium and the aqueous medium are kept separate and are not mixed up.

12. The method according to any one of the preceding claims, wherein the organic product comprises at least hexanoic acid.

13. The method according to any one of the preceding claims, wherein the aqueous medium and extraction medium do not form an emulsion.

14. Use of an extraction medium comprising at least one trialkyl-phosphine-oxide and at least one alkane for extracting at least one liquid organic product from an aqueous medium into an extraction medium, wherein the extraction medium forms no visible droplets when in contact with the aqueous medium.

15. Use according to claim 14, wherein the liquid organic product is hexanoic acid.

Images