Extraction by layers

Abstract

The invention relates to a method of extracting at least one liquid organic product from an aqueous solution, wherein the method comprises: a. adding a layer of an organic solvent to the aqueous solution comprising the organic product, and b. maintaining the layer of organic solvent separate from the aqueous solution, wherein the organic solvent comprises at least one alkane and at least one trialkyl-phosphine-oxide and the layer of organic solvent is in direct contact with the aqueous solution.

Description

[0001] EXTRACTION BY LAYERS

[0002] FIELD OF THE INVENTION

[0003] The invention relates to a method for extracting organic compounds from an aqueous medium using an extraction solvent. In particular, the extraction solvent forms a layer with the aqueous medium and this layer is maintained 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 solution, wherein the method comprises: a. adding a layer of an organic solvent to the aqueous solution comprising the organic product, and b. maintaining the layer of organic solvent separate from the aqueous solution, wherein the organic solvent comprises at least one alkane and at least one trialkyl-phosphine-oxide and the layer of organic solvent is in direct contact with the aqueous solution.

[0012] It was found that, surprisingly, when the organic solvent is maintained as a separate layer from the aqueous solution and the two liquids are not constantly mixed or combined, lesser droplets are formed between the layers and 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 solution to the organic solvent is more efficient and higher yield is obtained from the aqueous solution into the extraction solvent compared to when the two solutions (i.e. aqueous solution with the organic product and extraction solvent) are constantly stirred and combined. 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.

[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 extracting 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 extracting medium, a larger yield of organic products may be extracted. The extracting medium is also not harmful to microorganisms. Accordingly, the extracting 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.

[0014] The term ‘layer of an organic solvent’ refers to a film or sheet of extraction medium or solvent that is formed with the aqueous solution when the organic solvent is added or brought into contact with the aqueous solution. The organic solvent does not directly and / or independently mix with the aqueous solution. Both liquids form two separate layers, one on top of the other or one beside each other without combining. In one example, the organic solvent being lighter forms a layer or floats above the aqueous solution. The two liquids do not interact with one another regularly and are maintained as two separate layers without any physical boundary between the two liquids. However, the layer of organic solvent is in direct contact with the aqueous solution. The maintaining of the two separate layers reduces formation of emulsions and results in a better extraction yield of the organic product. In particular, the organic solvent and the aqueous solution according to any aspect of the present invention are not mixed, or at least not regularly mixed or minimally mixed. Even more in particular, the aqueous solution and organic solvent according to any aspect of the present invention do not form an emulsion or appear not to form an emulsion or no emulsion appears to be formed.

[0015] 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 solution containing the organic product that is to be extracted. In one example, the extraction medium may be added to an aqueous solution comprising the organic product. In another example, the organic product is being produced in the aqueous solution and the extraction medium is added to the aqueous solution 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 solution.

[0016] 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.

[0017] In one example, the organic product may be already dissolved in the aqueous solution 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 solution where the organic product is being produced.

[0018] 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.

[0019] The aqueous solution is at least one fermentation medium comprising at least one microorganism and the microorganism is in direct contact with the aqueous solution and organic solvent. In particular, the organic solvent and the aqueous solution are both not toxic to the microorganism and therefore the microorganism and / or the organic solvent may be recycled and reused in the method according to any aspect of the present invention.

[0020] The extracting medium may also be referred to as the ‘extraction medium’ or ‘organic solvent’. The organic solvent 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 organic solvent containing the extracted organic product thereof. The organic solvent 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 extracting 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. 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.

[0022] 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.

[0023] 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.

[0024] 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. 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.

[0025] 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 alkylphosphine 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.

[0026] 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.

[0027] 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.

[0028] In step (b) according to any aspect of the present invention, the organic product in the aqueous medium may contact the extracting medium for a time sufficient to extract the organic product from the aqueous medium into the organic solvent. 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 extracting 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.

[0029] The ratio of the organic solvent used to the amount of organic compound to be extracted may vary depending on how quick the extraction is to be carried out. In one example, the amount of extracting medium is equal to the amount of aqueous medium comprising the organic product. After the step of contacting the organic solvent 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 organic absorbent made available again to be recycled or reused.

[0030] In one example, the aqueous solution 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.

[0031] 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 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] 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.

[0035] 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.

[0036] 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).

[0037] 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.

[0038] 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).

[0039] 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.

[0040] Mixtures of sources can be used as a carbon source.

[0041] 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.

[0042] 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.

[0043] 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 solutions, 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. Ijungdahlii. 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. Ijungdahlii 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.

[0044] 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 extracting medium and / or the cells may then be recycled.

[0045] 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.

[0046] The volume of the organic phase at any point in time relative to the aqueous phase in the method according to any aspect of the present invention may always be lower than about 50%. That is to say, there is always a lesser volume of the organic phase compared to the aqueous phase such that a layer between the two phases is formed.

[0047] According to a further aspect of the present invention, there is provided a use of layer extraction to extract at least one dissolved organic product from an aqueous solution into an organic solvent, wherein phase separation between the aqueous solution and organic solvent is maintained throughout the extraction and the organic solvent comprises at least one alkane and at least one trialkyl-phosphine-oxide and the organic solvent and the aqueous solution form two layers that are in direct contact with one another. In particular, aqueous solution is at least one fermentation medium comprising at least one microorganism. More in particular, the microorganism is at least one acetogenic bacteria or at least one microorganism for carbon chain elongation or a combination thereof.

[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 (comparative)

[0052] Cultivation of Clostridium autoethanogenum and Clostridium kluyveri in a two-phase system with high shaking velocity

[0053] The bacterium Clostridium kluyveri (DSM 555) was cultivated in a co culture with Clostridium autoethanogenum (DSM 10061) for the biotransformation of ethanol and acetate to hexanoic acid. For the in Situ 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.

[0054] 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.

[0055] 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.

[0056] 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.

[0057] 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 d-1

[0058] 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, 250 rpm and with a gas atmosphere of 10 % H2 and 90 % N2 in an open water bath shaker for 116 h.

[0059] 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.

[0060] After 116 h of cultivation the two-layer system in the bottle with a lower aqueous phase and an upper organic phase was still visible, but both phases contained a stable emulsion with aqueous droplets in the organic phase and organic droplets in the aqueous phase. Both phases appeared very turbid with a third phase between them. This interphase consisted of aqueous parts, organic parts and a lot of protein and cell debris. The interphase was about 10% of all the liquid content of the bottle.

[0061] In the aqueous phase the concentration of acetate decreased from 4.3 g / L to 2.2 g / L and the concentration of ethanol decreased from 14.1 g / L to 8.9 g / L during this time. During the same time 0.4 g / L butyrate and 4.14 g / L hexanoate were built. In the organic phase the concentrations raised from 0 g / L to 1 .2 g / L for ethanol and 6.4 g / L for hexanoic acid. Butyric acid and acetic acid were not found.

[0062] Some of the hexanoic acid could be back extracted with an alkaline aqueous solution from the organic phase, but -10% of the products and the organic phase were lost in the very stable interphase and could not be reconditioned. So only a part of the organic solution could be used again for the next extraction. Also the organic phase could not be cleared completely and was in worse condition for future use.

[0063] Example 2

[0064] Cultivation of Clostridium autoethanogenum and Clostridium kluyveri in a two-phase system with low shaking velocity 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.

[0065] 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.

[0066] 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.

[0067] 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.

[0068] 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 d-1

[0069] 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.

[0070] 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.

[0071] 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 dissolved organic product from an aqueous solution, wherein the method comprises: a. adding a layer of an organic solvent to the aqueous solution comprising the organic product, and b. maintaining the layer of organic solvent separate from the aqueous solution, wherein the organic solvent comprises at least one alkane and at least one trialky I- phosphine-oxide and the layer of organic solvent is in direct contact with the aqueous solution.

2. The method according to claim 1 , wherein the aqueous solution is at least one fermentation medium comprising at least one microorganism and the microorganism is in direct contact with the aqueous solution and organic solvent.

3. The method according to claim 2, wherein the microorganism is at least one acetogenic bacteria or at least one microorganism for carbon chain elongation or a combination thereof.

4. The method according to claim 3, 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.

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

6. The method according to any one of the preceding claims, wherein the alkane is a branched alkane.

7. 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.

8. The method according to claim 7, 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.

9. The method according to claim 8, 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.

10. The method according to any one of the preceding claims, wherein the organic solvent and the aqueous solution are not mixed and / or do not form an emulsion.11 . The method according to any one of the preceding claims, wherein the organic product comprises at least hexanoic acid.

12. The method according to any one of the preceding claims, wherein there is no physical barrier between the aqueous solution and organic solvent.

13. Use of layer extraction to extract at least one dissolved organic product from an aqueous solution into an organic solvent, wherein phase separation between the aqueous solution and organic solvent is maintained throughout the extraction and the organic solvent comprises at least one alkane and at least one trialkyl-phosphine-oxide and the organic solvent and the aqueous solution form two layers that are in direct contact with one another.

14. Use according to claim 13, wherein the aqueous solution is at least one fermentation medium comprising at least one microorganism and the microorganism is in direct contact with the aqueous solution and organic solvent.

15. Use according to claim 14, wherein the microorganism is at least one acetogenic bacteria or at least one microorganism for carbon chain elongation or a combination thereof.

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