Extraction of aliphatic alcohols

Abstract

The present invention relates to a method for extracting aliphatic alcohols, preferably aliphatic alcohols containing 5 to 16 carbon atoms, from an aqueous culture medium, the method comprising: (a) contacting the aliphatic alcohols in the aqueous culture medium with at least one extraction medium for a time sufficient to extract the aliphatic alcohols from the aqueous culture medium into the extraction medium, (b) separating the extraction medium containing the extracted aliphatic alcohols from the aqueous culture medium, wherein the extraction medium comprises: - at least one trialkylphosphine oxide, preferably trioctylphosphine oxide, and optionally at least one alkane, and wherein the aliphatic alcohols are produced from a carbon source by contacting the carbon source with at least one microorganism capable of converting the carbon source into the aliphatic alcohols.

Description

Technical Field

[0001] The present invention relates to a method for extracting aliphatic alcohols from an aqueous culture medium. In particular, the method uses at least one trialkylphosphine oxide and, optionally, at least one alkane. Background Art

[0002] Aliphatic alcohols have a variety of functions in the art. For example, they can be used to produce polymers, drugs, solvents and food additives.

[0003] WO1985000805 discloses a process for producing alcohols comprising extracting a hydroalcoholic solution with an organic solvent system comprising an organic solvent.

[0004] US4714791 discloses a method for recovering primary normal aliphatic higher alcohols, which method comprises contacting sugar cane as an extraction raw material or a product obtained from sugar cane with a fluid in a subcritical or supercritical state.

[0005] Therefore, there is a need in the art for a cheaper and more efficient extraction method for aliphatic alcohols, especially aliphatic alcohols produced on an industrial scale. Further, there is a need for an extraction method for aliphatic alcohols that can be used in conjunction with a biotechnological method for producing aliphatic alcohols. Summary of the invention

[0006] The present invention seeks to solve the above problems by providing a means of extracting aliphatic alcohols that is more efficient and cheaper than the existing methods available in the art. The present invention also provides a means of extracting aliphatic alcohols that can be used in conjunction with a biotechnological process for producing aliphatic alcohols.

[0007] According to one aspect of the present invention, there is provided a method for extracting aliphatic alcohol from an aqueous culture medium, the method comprising:

[0008] (a) contacting the aliphatic alcohol in the aqueous medium with at least one extraction medium for a period of time sufficient to extract the aliphatic alcohol from the aqueous medium into the extraction medium,

[0009] (b) separating the extraction medium containing the extracted aliphatic alcohol from the aqueous culture medium,

[0010] The extraction medium comprises:

[0011] at least one trialkylphosphine oxide, preferably trioctylphosphine oxide (TOPO), and optionally at least one alkane, and wherein the aliphatic alcohol is produced from the carbon source by contacting the carbon source with at least one microorganism capable of converting the carbon source into an aliphatic alcohol.

[0012] Microorganisms can be

[0013] (i) a mixture of a first microorganism and a second microorganism, and

[0014] - the first microorganism is an acetogenic microorganism capable of converting a carbon source into acetate and / or ethanol; and

[0015] a second microorganism for carbon chain elongation and selected from Clostridium kluyveri and C. carboxidivorans and capable of converting acetate and / or ethanol to form acids; and

[0016] wherein the first microorganism is further capable of converting the acid into the corresponding aliphatic alcohol, or

[0017] (ii) Clostridium carboxydophorum capable of converting carbon sources into aliphatic alcohols.

[0018] Preferably, the alkane contains at least 12 carbon atoms.

[0019] In particular, the extraction method according to any aspect of the present invention allows an increase in yield relative to the amount of extractant used. For example, less than 50% by weight of the extraction medium can be used to extract the same amount of aliphatic alcohol as with pure alkanes alone. Therefore, with a small volume of extraction medium, a large yield of aliphatic alcohol can be extracted. The extraction medium is also harmless to microorganisms. Therefore, the extraction medium according to any aspect of the present invention can be present when aliphatic alcohols are produced by biotechnology. Further, the aliphatic alcohols can be easily separated from the extraction medium according to any aspect of the present invention by distillation. After separation, for example by distillation, the extraction medium can be easily recycled.

[0020] The method according to any aspect of the present invention can be a method for extracting at least one aliphatic alcohol from an aqueous medium. The aliphatic alcohol of separation can refer to at least one aliphatic alcohol that can be separated from the culture medium that has generated the aliphatic alcohol. In an example, the aliphatic alcohol can be produced in an aqueous medium (for example, a fermentation medium that produces aliphatic alcohols by a carbon source through a specific cell). The aliphatic alcohol of separation can refer to the aliphatic alcohol extracted from an aqueous medium. In particular, the extraction step allows the separation of excess water from an aqueous medium, thereby causing the formation of a mixture containing the aliphatic alcohol extracted.

[0021] The extraction medium may also be referred to as an 'extraction medium'. The extraction medium may be used to extract / separate the aliphatic alcohol produced according to any of the methods of the present invention from the aqueous medium in which the aliphatic alcohol was initially produced. At the end of the extraction step, excess water may be removed from the aqueous medium, thereby obtaining an extraction medium containing the extracted aliphatic alcohol. The extraction medium may comprise a combination of compounds that may result in an effective means of extracting the aliphatic alcohol from the aqueous medium. In particular, the extraction medium may comprise: (i) at least one alkane comprising at least 12 carbon atoms, and (ii) at least one trialkylphosphine oxide. The extraction medium according to any aspect of the present invention may effectively extract the aliphatic alcohol into the alkane-trialkylphosphine oxide extraction medium. Such an extraction medium of a mixture of trialkylphosphine oxide and at least one alkane may be considered suitable for use in the method according to any aspect of the present invention, because the mixture may effectively extract the desired aliphatic alcohol in the presence of a fermentation medium. In particular, it may be considered that a mixture of trialkylphosphine oxide and at least one alkane works better than any method currently known in the art for extracting aliphatic alcohols, because it does not require any special equipment to carry out, and it can be relatively easily carried out with high product yields.

[0022] The alkane may contain at least 12 carbon atoms. In particular, the alkane may contain 12-18 carbon atoms. In one example, the alkane may be selected from dodecane, tridecane, tetradecane, pentadecane, hexadecane, heptadecane and octadecane. In a further example, the extraction medium may contain a mixture of alkanes. In another example, the alkane may be a branched alkane. In particular, the branched alkane may be squalene.

[0023] Trialkylphosphine oxide has the general formula OPX3, wherein X is an alkyl group. Suitable trialkylphosphine oxides according to any aspect of the present invention include alkyl groups consisting of straight chain, branched chain or cyclic hydrocarbons, and the hydrocarbons consist of 1 to about 100 carbon atoms and 1 to about 200 hydrogen atoms. In particular, the "alkyl" used for trialkylphosphine oxide species according to any aspect of the present invention may refer to a hydrocarbon group having 1 to 20 carbon atoms, typically 4 to 15 carbon atoms or 6 to 12 carbon atoms, and the hydrocarbon group may consist of a straight chain, a ring, a branched chain or a mixture of these. X may be an alkyl group that is the same or different in a molecule. If different alkyl groups are present in a trialkylphosphine oxide molecule, the alkyl group is preferably selected from C8 and C10. Typically, trialkylphosphine oxides are selected from tributylphosphine oxide, trihexylphosphine oxide, trioctylphosphine oxide, tridecylphosphine oxide and mixtures thereof.

[0024] Even more particularly, the trialkylphosphine oxide may be trioctylphosphine oxide (TOPO). Trioctylphosphine oxide (TOPO) is a phosphine oxide having the formula OP(C8H 17)3 of an organic phosphorus compound. At least one trialkylphosphine oxide, preferably trioctylphosphine oxide (TOPO) may be present in the extraction medium together with at least one alkane. In particular, a mixture of at least one trialkylphosphine oxide, preferably trioctylphosphine oxide (TOPO) and an alkane comprising at least 12 carbon atoms may contain at least one trialkylphosphine oxide, preferably trioctylphosphine oxide (TOPO) in a weight ratio of about 1:100 to 1:10 relative to the alkane. More specifically, in the extraction medium according to any aspect of the present invention, the weight ratio of at least one trialkylphosphine oxide, preferably trioctylphosphine oxide (TOPO) to the alkane may be about 1:100, 1:90, 1:80, 1:70, 1:60, 1:50, 1:40, 1:30, 1:25, 1:20, 1:15 or 1:10. Even more particularly, the weight ratio of at least one trialkylphosphine oxide, preferably trioctylphosphine oxide (TOPO) to the alkane can be selected in the range of 1:90 to 1:10, 1:80 to 1:10, 1:70 to 1:10, 1:60 to 1:10, 1:50 to 1:10, 1:40 to 1:10, 1:30 to 1:10, or 1:20 to 1:10. The weight ratio of at least one trialkylphosphine oxide, preferably trioctylphosphine oxide (TOPO) to the alkane can be between 1:40 to 1:15 or 1:25 to 1:15. In one example, the weight ratio of at least one trialkylphosphine oxide, preferably trioctylphosphine oxide (TOPO) to the alkane can be about 1:15. In this embodiment, the alkane can be hexadecane, and therefore, the weight ratio of at least one trialkylphosphine oxide, preferably trioctylphosphine oxide (TOPO) to hexadecane can be about 1:15.

[0025] As used herein, the term "about" refers to a variation within 20%. Specifically, as used herein, the term "about" refers to + / -20%, more specifically + / -10%, even more specifically + / -5% of a given measurement or value.

[0026] In a preferred process according to the invention, the extraction medium contains a second organic component in addition to the phosphine oxide. The second organic component comprises at least 12 carbons. The second organic component is a linear or branched alkane, which may be selected from tetradecane, pentadecane, hexadecane, heptadecane, octadecane and squalene, or a mixture of alkanes such as white mineral oil (Fragoltherm-Q-32-N). Further, the second organic component may comprise an aromatic hydrocarbon, which may be selected from diisopropylbiphenyl, partially hydrogenated terphenyl, dibenzyltoluene and diisopropylnaphthalene, or a mixture of aromatic solvents such as Solvesso 200. Another possibility is to use an alcohol selected from oleyl alcohol, 2-octyldodecanol and 2-hexyldodecanol as the second organic component.

[0027] The extraction medium according to any aspect of the present invention can effectively extract the aliphatic alcohol into the extraction medium. Such an extraction medium of a mixture of at least one alkylphosphine oxide and at least one alkane containing at least two different alkyl groups per alkylphosphine oxide molecule can be considered suitable for use in the method according to any aspect of the present invention because the mixture effectively extracts the desired aliphatic alcohol in the presence of an aqueous production medium. The alkane can be a straight chain or branched chain alkane. In one example, the alkane can be a branched chain alkane, and the branched chain alkane can be squalene.

[0028] In another example, an extraction medium of a mixture of at least one alkylphosphine oxide containing at least two different alkyl groups per alkylphosphine oxide molecule and at least one partially hydrogenated aromatic hydrocarbon can be considered suitable for use in the method according to any aspect of the present invention because the mixture effectively extracts the desired organic acid and / or alcohol in the presence of an aqueous production medium. In particular, a mixture of at least one alkylphosphine oxide containing at least two different alkyl groups per alkylphosphine oxide molecule and at least one partially hydrogenated aromatic hydrocarbon can be considered to work better than any method currently known in the art for extracting aliphatic alcohols because it does not require any special equipment to carry out, and it is relatively easy to carry out with a high product yield. Further, the extraction medium according to any aspect of the present invention in combination with an alkane or partially hydrogenated aromatic solvent is also not toxic to microorganisms.

[0029] In step (a) according to any aspect of the present invention, the aliphatic alcohol in the aqueous medium can be contacted with the extraction medium for a time sufficient to extract the aliphatic alcohol from the aqueous medium into the extraction medium. A skilled person can determine the amount of time required to reach distribution equilibrium and the correct bubble aggregation that may be required to optimize the extraction process. In some examples, the time required may depend on the amount of aliphatic alcohol that can be extracted. In particular, the time required to extract the aliphatic alcohol from the aqueous medium into the extraction medium may take only a few minutes. In examples where the extraction is performed while fermentation occurs, the extraction time is equivalent to the fermentation time.

[0030] The ratio of the amount of the extraction medium used to the aliphatic alcohol to be extracted can vary depending on how fast the extraction is performed. In one example, the amount of the extraction medium is equal to the amount of the aqueous medium containing the aliphatic alcohol. After the step of contacting the extraction medium with the aqueous medium, the two phases (aqueous phase and organic phase) are separated using any method known in the art. In one example, the two phases can be separated using a separatory funnel. Mixer settlers, pulse columns, etc. can also be used to separate the two phases. In one example, in the case where the aliphatic alcohol is hexanol, in view of the fact that hexanol distills at a much lower boiling point than the extraction medium, distillation can be used to separate the extraction medium from the hexanol. The technician can select the best method for separating the extraction medium from the desired aliphatic alcohol in step (b) according to the characteristics of the aliphatic alcohol to be extracted. In particular, step (b) according to any aspect of the present invention includes recovering the aliphatic alcohol from step (a).

[0031] Step (b) preferably ends with the organic absorbent being recycled or reused again, preferably in step (0) (see below).

[0032] The aliphatic alcohol is preferably selected from aliphatic alcohols containing 2 to 16, preferably 5 to 12 carbon atoms. More specifically, the aliphatic alcohol can be selected from aliphatic alcohols with 4 to 16, 4 to 14, 4 to 12, 4 to 10, 5 to 16, 5 to 14, 5 to 12, 5 to 10, 6 to 16, 6 to 14, 6 to 12 or 6 to 10 carbon atoms. In particular, the aliphatic alcohol can be selected from amyl alcohol, hexanol, heptanol and octanol. Even more particularly, the aliphatic alcohol can be selected from butanol and hexanol. In particular, the aliphatic alcohol is hexanol.

[0033] In some instances, any culture medium, substrate, condition and process for cultivating bacteria commonly known in the art can be used to cultivate microorganisms capable of producing aliphatic alcohols. This allows the use of biotechnological methods to produce aliphatic alcohols. Depending on the microorganism used for aliphatic alcohol production, suitable growth medium, pH, temperature, stirring rate, inoculum level and / or aerobic, microaerobic or anaerobic conditions are different. The technician can understand the necessary other conditions for implementing the method according to any aspect of the present invention. Especially, the conditions in the container (for example fermentor tank) can vary according to the microorganism used. The variation of the conditions for the optimal function of the microorganism is within the knowledge of the technician.

[0034] In one example, the method according to any aspect of the present invention can be carried out in an aqueous medium at a pH between 5 and 8 or between 5.5 and 7. The pressure can be between 1 bar and 10 bar. The microorganism can be cultured at a temperature ranging from about 20° C. to about 80° C. In one example, the microorganism can be cultured at 37° C.

[0035] In some examples, for the growth of microorganisms and their production of aliphatic alcohols, the aqueous culture medium may contain any nutrients, ingredients and / or supplements suitable for growing microorganisms or for promoting the production of aliphatic alcohols. In particular, the aqueous culture medium may contain at least one of the following: a carbon source, a nitrogen source such as ammonium salts, yeast extract or peptone; minerals; salts; cofactors; buffers; vitamins; and any other components and / or extracts that can promote bacterial growth. The culture medium to be used must be suitable for the requirements of the specific strain. Descriptions of culture media for various microorganisms are given in the "Manual of Methods for General Bacteriology".

[0036] Therefore, the extraction method of aliphatic alcohols according to any aspect of the present invention can be used together with any biotechnological method for producing aliphatic alcohols. This is particularly advantageous because aliphatic alcohols are usually left to accumulate in aqueous culture medium in fermentation processes using biological methods for producing aliphatic alcohols, and after reaching a certain concentration in the fermentation medium, it is precisely this target product (aliphatic alcohol) that may inhibit the activity and productivity of microorganisms. Therefore, this limits the overall yield of the fermentation process. By using this extraction method, aliphatic alcohols are extracted when they are produced, thereby greatly reducing the inhibition of the final product.

[0037] The method according to any aspect of the present invention is also more effective and cost-effective than the traditional method of removing aliphatic alcohols, particularly removing aliphatic alcohols from the fermentation process when producing them, because it is not mainly dependent on distillation and / or precipitation when used to recycle aliphatic alcohols. The distillation or precipitation process can lead to higher manufacturing costs, lower yields and higher waste products, and therefore can reduce the overall efficiency of the process. The method according to any aspect of the present invention attempts to overcome these shortcomings.

[0038] In one example, the aliphatic alcohol is hexanol. In this example, the hexanol can be produced from at least one carbon source such as synthesis gas.

[0039] In the presence of at least one acetogenic bacteria and / or hydrogen oxidizing bacteria, the syngas can be converted into hexanol. In particular, any method known in the art can be used. Hexanol can be produced from the syngas by at least one prokaryotic organism. In particular, the prokaryotic organism may be selected from the genus Escherichia, such as Escherichia coli; the genus Clostridia, such as Clostridium ljungdahlii, Clostridium autoethanogenum, Clostridium carboxidivorans or Clostridium kluyveri; the genus Corynebacteria, such as Corynebacterium glutamicum; the genus Cupriavidus, such as Cupriavidus necator or Cupriavidus metallidurans; the genus Pseudomonas, such as Pseudomonas fluorescens, Pseudomonas putida. putida or Pseudomonas oleavorans; from the genus Delftia, such as Delftia acidovorans; from Bacillus, such as Bacillus subtillis; from the genus Lactobacillus, such as Lactobacillus delbrueckii; or from the genus Lactococcus, such as Lactococcus lactis.

[0040] In another example, hexanol can be produced by at least one eukaryotic organism from a carbon source such as syngas. The eukaryotic organism used in the method of the present invention can be selected from the genus Aspergillus, such as Aspergillus niger; the genus Saccharomyces, such as Saccharomyces cerevisiae; the genus Pichia, such as Pichia pastoris; the genus Yarrowia, such as Yarrowia lipolytica; the genus Issatchenkia, such as Issathenkiaorientalis; the genus Debaryomyces, such as Debaryomyces shansenii; the genus Arxula, such as Arxula adenoinivorans; or the genus Kluyveromyces, such as Kluyveromyces lactis.

[0041] As used herein, the term "acetogenic bacteria" refers to microorganisms that can perform the Wood-Ljungdahl pathway and are therefore capable of converting CO, CO2 and / or hydrogen into acetate. These microorganisms include microorganisms that do not have the Wood-Ljungdahl pathway in their wild type but have acquired this trait due to genetic modification. Such microorganisms include, but are not limited to, Escherichia coli cells. These microorganisms may also be referred to as carbon monoxide-producing bacteria. Currently, 21 different acetogenic bacteria are known in the art (Drake et al., 2006), which may also include some Clostridium (Drake & Kusel, 2005). These bacteria are capable of using carbon dioxide or carbon monoxide as a carbon source and hydrogen as an energy source (Wood, 1991). Further, alcohols, aldehydes, carboxylic acids and many hexoses may also be used as carbon sources (Drake et al., 2004). The reduction pathway that leads to the formation of acetate is referred to as acetyl-CoA or the Wood-Ljungdahl pathway. In particular, the acetogenic bacteria may be selected from: Acetoanaerobium notera (ATCC 35199), Acetonema longum (DSM 6540), Acetobacterium carbinolicum (DSM 2925), Acetobacterium malicum (DSM 4132), Acetobacterium species No. 446 (Morinaga et al., 1990, J. Biotechnol., Volume 14, Pages 187-194), Acetobacterium wieringae (DSM 1911), Acetobacterium woodii (DSM 1030), Alkalibaculum bacchi (DSM 22112), Archaeoglobus fulgidus (DSM 4304), Blautia producta (DSM 2950, ​​formerly Ruminococcus productus, formerly Peptostreptococcus productus), Butyribacterium methylotrophicum (DSM 3468), Clostridium aceticum (DSM 1496), Clostridium autoethanogenum (DSM 10061, DSM 19630 and DSM 23693), Clostridium carboxidivorans (DSM 15243), Clostridium coskatii (ATCC No. PTA-10522), Clostridium drakei (ATCC BA-623), Clostridium formicoaceticum (DSM 92), Clostridium glycolicum (DSM 1288), Clostridium ljungdahlii (DSM 13528), Clostridium ljungdahlii C-01 (ATCC 55988), Clostridium ljungdahlii ERI-2 (ATCC 55380), Clostridium ljungdahlii O-52 (ATCC 55989), Clostridium mayombei (DSM 6539), Clostridium methoxybenzovorans (DSM 12182), Clostridium ragsdalei (DSM 15248), Clostridium scatologenes (DSM 757), Clostridium species ATCC 29797 (Schmidt et al., 1986, Chem. Eng.Commun., Vol. 45, pp. 61-73), Desulfotomaculum kuznetsovii (DSM 6115), Desulfotomaculum thermobenzoicum subsp. thermosyntrophicum (DSM 14055), Eubacterium limosum (DSM 20543), Methanosarcina acetivorans C2A (DSM 2834), Moorella species HUC22-1 (Sakai et al., 2004, Biotechnol. Let., Vol. 29, pp. 1607-1612), Moorella thermoacetica (DSM 521, formerly Clostridium thermoaceticum), Moorella thermoautotrophicum (DSM 521, formerly Clostridium thermoaceticum), 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 known as Acetogenium kivui).

[0042] More particularly, the strain of Clostridium carboxyvorans ATCC BAA-624 may be used. Even more particularly, the strains of Clostridium carboxyvorans labeled "P7" and "P11" as described in, for example, US 2007 / 0275447 and US 2008 / 0057554 may be used.

[0043] Naveira et al. in Journal of Chemical Technology & Biotechnology 92(4), April 2017, in particular, teach the production of hexanol in Clostridium carboxydophorum from syngas containing CO2.

[0044] Another particularly suitable bacterium may be Clostridium jundal. In particular, selected from Clostridium jundal PETC, Clostridium jundal ERI2, Clostridium jundal COL and Clostridium jundal O-52 can be used to convert syngas into hexanol. These strains are described, for example, in WO 98 / 00558, WO 00 / 68407, ATCC 49587, ATCC 55988 and ATCC 55989.

[0045] Acetogenic bacteria can be used in combination with hydrogen oxidizing bacteria. In one example, both acetogenic bacteria and hydrogen oxidizing bacteria can be used to produce hexanol from a carbon source such as syngas. In another example, only acetogenic bacteria can be used to metabolize syngas to produce hexanol from a carbon source such as syngas. In yet another example, only hydrogen oxidizing bacteria can be used in the reaction.

[0046] The hydrogen oxidizing bacteria may be selected from the following: Achromobacter, Acidithiobacillus, Acidovorax, Alcaligenes, Anabena, Aquifex, Arthrobacter, Azospirillum, Bacillus, Bradyrhizobium, Cupricinus, iavidus), Derxia, Helicobacter, Herbaspirillum, Hydrogenobacter, Hydrogenobaculum, Hydrogenophaga, Hydrogenophilus, Hydrogenothermus, Hydrogenovibrio, Ideon onella species O1, Kyrpidia, Metallosphaera, Methanobrevibacter, Myobacterium, Nocardia, Oligotropha, Paracoccus, Pelomonas, Polaromonas, Pseudomonas, Nocardia, Rhizobium, Rhodococcus, Rhodopseudomonas, Rhodospirillum, Streptomyces, Thiocapsa, Treponema, Variovorax, Xanthobacter and Wautersia.

[0047] In the production of hexanol from a carbon source such as syngas, a combination of bacteria may be used. There may be more than one acetogenic bacteria present in combination with one or more hydrogen oxidizing bacteria. In another example, there may be only more than one type of acetogenic bacteria. In yet another example, there may be only more than one type of hydrogen oxidizing bacteria. Hexanoic acid, also known as caproic acid, has the general formula C5H11 COOH.

[0048] In particular, the caproic acid production method may include the following steps:

[0049] - contacting a carbon source, such as syngas, with at least one bacterium capable of carrying out the Wood-Ljungdahl pathway and chain elongation to produce hexanol.

[0050] In particular, the microorganism may be

[0051] (i) a mixture of a first microorganism and a second microorganism, and

[0052] - the first microorganism is an acetogenic microorganism capable of converting a carbon source into acetate and / or ethanol; and

[0053] - a second microorganism is used for carbon chain elongation and is selected from Clostridium kluyveri and Clostridium carboxyvorans and is capable of converting acetate and / or ethanol to form acids; and

[0054] wherein the first microorganism is capable of further converting the acid into the corresponding aliphatic alcohol, or

[0055] (ii) Clostridium carboxydophorum capable of converting carbon sources into aliphatic alcohols.

[0056] As used herein, the term "contacting" refers to direct contact of the aliphatic alcohol in the culture medium with the extraction medium and / or direct contact between the microorganism and the carbon source, such as synthesis gas, in step (a). For example, the cells and the culture medium containing the carbon source can be in different compartments. In particular, the carbon source can be gaseous and added to the culture medium containing the cells according to any aspect of the present invention.

[0057] In one example, the production of hexanol from a carbon source such as syngas may involve the use of acetogenic bacteria and bacteria that can use bacteria capable of chain extension to produce hexanol. In one example, both acetogenic bacteria and bacteria capable of chain extension can be used to produce hexanol from a carbon source such as syngas. For example, Clostridium jundal can be used simultaneously with Clostridium kluyveri. In another example, only acetogenic bacteria can be used to metabolize a carbon source such as syngas to produce hexanol from a carbon source such as syngas. In this example, the acetogenic bacteria can be capable of implementing chain extension and Wood-Ljungdahl pathway. In one example, the acetogenic bacteria can be Clostridium carboxydosus that can perform both Wood-Ljungdahl pathway and chain extension.

[0058] The organism capable of chain extension can be selected from Clostridium kluyveri, Clostridium carboxydotrophicum, etc. These microorganisms include microorganisms that do not have the ability to chain extend in their wild type, but have acquired this trait due to genetic modification. In particular, the microorganism can be Clostridium kluyveri.

[0059] In one example, the bacteria used according to any aspect of the present invention are selected from Clostridium kluyveri and Clostridium carboxyvorans.

[0060] Specifically, the cell is contacted with a carbon source including monosaccharides (such as glucose, galactose, fructose, xylose, arabinose or xylulose), disaccharides (such as lactose or sucrose), oligosaccharides and polysaccharides (such as starch or cellulose), single carbon substrates and / or mixtures thereof. More specifically, the cell is contacted with a carbon source comprising CO and / or CO2 to produce an aliphatic alcohol.

[0061] With regard to the substrate source comprising carbon dioxide and / or carbon monoxide, the skilled person will appreciate that there are many possible sources for providing CO and / or CO2 as a carbon source. It can be seen that in practice, any gas or any gas mixture that can provide sufficient carbon for the microorganism can be used as a carbon source for the present invention, so that acetate and / or ethanol can be formed from the CO and / or CO2 source.

[0062] Typically for the cells of the invention, the carbon source comprises at least 50 wt. %, at least 70 wt. %, in particular at least 90 wt. % CO2 and / or CO, wherein the wt. % relate to all carbon sources accessible to the cells according to any aspect of the invention. A source of carbon material may be provided.

[0063] Examples of carbon sources in gaseous form include waste gases such as synthesis gas produced by yeast fermentation or clostridial fermentation, flue gas and refinery gas. These waste gases are formed by gasification of cellulose-containing materials or gasification of coal. In one example, these waste gases are not necessarily produced as by-products of other processes, but can be produced specifically for use with the mixed culture of the present invention.

[0064] According to any aspect of the present invention, the carbon source also used for producing acetate and / or ethanol in step (a) according to any aspect of the present invention (see below) can be synthesis gas. Synthesis gas can be produced, for example, as a by-product of the gasification of coal. Therefore, the microorganism according to any aspect of the present invention is able to convert waste materials into valuable resources.

[0065] In another example, syngas can be a byproduct of the gasification of widely available, low-cost agricultural feedstocks for use with the mixed cultures of the present invention to produce substituted and unsubstituted organic compounds.

[0066] There are many examples of feedstocks that can be converted into synthesis gas, since almost all forms of plants can be used for this purpose. In particular, the feedstock is selected from perennial grasses, such as Miscanthus, corn residues, processing waste materials such as sawdust, etc.

[0067] In general, synthesis gas can be obtained in a gasification plant of dry biomass mainly by pyrolysis, partial oxidation and steam reforming, wherein the main products of synthesis gas are CO, H2 and CO2. Synthesis gas can also be the electrolysis product of CO2. The skilled person will understand the appropriate conditions for electrolysis of CO2 to produce synthesis gas containing the desired amount of CO.

[0068] Typically, a portion of the synthesis gas obtained from the gasification process is first treated to optimize product yields and avoid the formation of tars. Lime and / or dolomite can be used to crack unwanted tars and CO in the synthesis gas. These processes are described in detail in, for example, Reed, 1981.

[0069] The overall efficiency, aliphatic alcohol productivity and / or total carbon capture of the process of the present invention may depend on the stoichiometry of CO2, CO and H2 in the continuous gas stream. The applied continuous gas stream may have the components CO2 and H2. In particular, in the continuous gas stream, the concentration of CO2 may range from about 10-50% by weight, in particular 3% by weight, and H2 may be between 44% by weight and 84% by weight, in particular 64 to 66.04% by weight. In another example, the continuous gas stream may also contain an inert gas such as N2 at a concentration of up to 50% by weight.

[0070] Mixtures of sources can also be used as carbon sources.

[0071] According to any aspect of the present invention, a reducing agent, such as hydrogen, can be provided together with a carbon source. In particular, the hydrogen can be supplied when supplying and / or using C and / or CO2. In an example, the hydrogen is a part of the synthesis gas present according to any aspect of the present invention. In another example, in the case where the hydrogen in the synthesis gas is not enough for the method of the present invention, additional hydrogen can be supplied. In an example, the hydrogen can be the electrolysis product of water.

[0072] In one example, the aliphatic alcohol is hexanol. More specifically, the carbon source comprising CO and / or CO2 is contacted with the cells in a continuous gas stream. Even more particularly, the continuous gas stream comprises synthesis gas. These gases can be supplied, for example, using a nozzle opened up into an aqueous medium, a frit in a pipeline supplying gas to the aqueous medium, a membrane, etc.

[0073] The skilled person will appreciate that it may be necessary to monitor the composition and flow rate of the feed streams at relevant intervals. Control of the composition of the feed streams may be achieved by changing the proportions of the constituent streams to achieve a target or desired composition. The composition and flow rate of the mixed stream may be monitored by any means known in the art. In one example, an adaptation system is provided to continuously monitor the flow rate and composition of at least two feed streams and combine them to produce a single mixed substrate stream in a continuous gas stream of optimal composition, and a device for delivering the optimized substrate stream to a fermentor.

[0074] The term "aqueous solution" or "culture medium" includes any aqueous solution, mainly water as a solvent, which can 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, if necessary, also includes any additional substrate. Those skilled in the art are familiar with the preparation of a variety of aqueous solutions, which are generally referred to as culture mediums that can be used to maintain and / or culture cells, such as LB culture medium in the case of Escherichia coli, and ATCC1754 culture medium can be used in the case of Clostridium jundal. Compared with complex culture mediums, it is advantageous to use a basic (minimal) culture medium (that is, a culture medium that is composed of a relatively simple composition, which only contains the minimum group of salts and nutrients necessary to keep the cell in a metabolically active and / or viable state) as an aqueous solution to avoid unnecessary contamination of the product by undesirable byproducts. For example, M9 culture medium can be used as a basic culture medium. The cells are incubated with a carbon source for a sufficiently long time to produce the desired product. For example, at least 1, 2, 4, 5, 10 or 20 hours. The temperature selected must be such that the cells according to any aspect of the present invention maintain catalytic capacity and / or metabolic activity, for example, in the case where the cells are Clostridium jundal cells, 10 to 42°C, preferably 30 to 40°C, in particular 32 to 38°C. The aqueous culture medium according to any aspect of the present invention also includes a culture medium in which aliphatic alcohols are produced. It mainly refers to a culture medium in which the solution essentially contains water. In one example, the aqueous culture medium in which the cells are used to produce aliphatic alcohols is the culture medium that is contacted with the extraction medium to extract the aliphatic alcohols.

[0075] In particular, the mixture of microorganisms and carbon sources according to any aspect of the present invention can be used in any known bioreactor or fermenter to implement any aspect of the present invention. In one example, the complete method starting with the production of aliphatic alcohols and ending with the extraction of aliphatic alcohols according to any aspect of the present invention is carried out in a single container. Therefore, there can be no separation step between the step of producing aliphatic alcohols and the step of extracting aliphatic alcohols. This saves time and cost. In particular, during the fermentation process, microorganisms can grow in aqueous culture medium and in the presence of extraction medium. Therefore, the method according to any aspect of the present invention provides a one-pot method for producing aliphatic alcohols. In addition, since aliphatic alcohols are extracted during their production, final product inhibition does not occur, thereby ensuring that the yield of aliphatic alcohols is maintained. Further separation steps can be performed to remove aliphatic alcohols. Any separation method known in the art can be used, such as using funnels, columns, distillation, etc. for separation. The remaining extraction medium and / or cells can then be recycled.

[0076] In another example, the extraction process can be performed as a separate step and / or in another tank. After fermentation has occurred (wherein the desired aliphatic alcohol to be extracted has been produced), an extraction medium according to any aspect of the present invention can be added to the fermentation medium, or the fermentation medium can be added to the tank containing the extraction medium. The desired aliphatic alcohol can then be extracted by any separation method known in the art, such as using a funnel, column, distillation, etc. The remaining extraction medium can then be recycled.

[0077] Another advantage of this method is that the extraction medium can be recycled. Thus, once the aliphatic alcohol is separated from the extraction medium, the extraction medium can be recycled and reused, thereby reducing waste.

[0078] According to another aspect of the present invention, there is provided the use of at least one trialkylphosphine oxide, preferably trioctylphosphine oxide and optionally at least one alkane for extracting an aliphatic alcohol from an aqueous medium, wherein the alkane preferably comprises at least 12 carbon atoms. In particular, the alkane may comprise 12 to 18 carbon atoms. More particularly, the alkane may be hexadecane. Even more particularly, the aliphatic alcohol is selected from aliphatic alcohols having 5 to 16 carbon atoms. In one example, the aliphatic alcohol may be hexanol.

[0079] In a preferred process according to the invention, ethanol and / or acetate are used as starting materials.

[0080] The preferred method according to the invention for extracting aliphatic alcohols produced from ethanol and / or acetate comprises, prior to step (a), step (0):

[0081] (0) contacting ethanol and / or acetate with at least one microorganism capable of carbon chain elongation in an aqueous medium to produce aliphatic alcohols from ethanol and / or acetate.

[0082] According to a preferred method of the invention, the aqueous culture medium after step (b) of separating the aliphatic alcohol can be recycled back to step (0). This recycling step allows the microorganisms to be recycled and reused, since the extraction medium according to the invention is not toxic to the microorganisms. This step of recycling the aqueous culture medium in the method according to the invention has the further advantage of enabling the residues of the aliphatic alcohol that were not initially extracted from steps (a) and (b) in the first cycle to have the opportunity to be extracted once more, or to be extracted as many times as the aqueous culture medium is recycled.

[0083] The microorganism capable of carbon chain extension to produce aliphatic alcohols in step (0) can be any organism capable of carbon chain extension (compare Jeon et al. Biotechnol Biofuels (2016) 9: 129). The pathway of carbon chain extension is also disclosed in Seedorf, H. et al., 2008. The microorganism according to any aspect of the present invention may also include microorganisms that are unable to perform carbon chain extension in their wild-type form but have acquired this trait due to genetic modification. In particular, the microorganism in (0) can be selected from Clostridium monoxide-eating, Clostridium kluyveri and Clostridium parus (C. pharus). In particular, the microorganism according to any aspect of the present invention can be Clostridium kluyveri.

[0084] In step (0) according to any aspect of the present invention, ethanol and / or acetate are contacted with at least one microorganism capable of carbon chain extension to produce aliphatic alcohols from ethanol and / or acetate. In one example, the carbon source can be ethanol in combination with at least one other carbon source selected from acetate, propionate, butyrate, isobutyrate, valerate and caproate. In particular, the carbon source can be ethanol and acetate. In another example, the carbon source can be a combination of propionic acid and ethanol, acetate and ethanol, isobutyric acid and ethanol, or butyric acid and ethanol. In one example, the carbon substrate can be ethanol alone. In another example, the carbon substrate can be acetate alone.

[0085] The source of acetate and / or ethanol can be different because of availability. In an example, ethanol and / or acetate can be the fermentation product of synthesis gas or any carbohydrate known in the art. Especially, the carbon source that is used for acetate and / or ethanol production can be selected from alcohol, aldehyde, glucose, sucrose, fructose, dextrose, lactose, wood sugar, pentose, polyol, hexose, ethanol and synthesis gas. The mixture in the source can be used as the carbon source.

[0086] Even more particularly, the carbon source may be synthesis gas. In the presence of at least one acetogenic bacteria, the synthesis gas may be converted into ethanol and / or acetate.

[0087] In one example, aliphatic alcohols are produced from acetate and / or ethanol, which are produced from syngas, and may involve the use of acetogenic bacteria in combination with microorganisms capable of extending carbon chains. For example, Clostridium jungdahl may be used simultaneously with Clostridium kluyveri. In another example, a single acetogenic cell may be capable of having the activity of two organisms. For example, the acetogenic bacteria may be Clostridium carboxydophorum, which may be capable of both the Wood-Ljungdahl pathway and the carbon chain elongation pathway.

[0088] According to any aspect of the present invention, the ethanol and / or acetate used in step (a) can be a fermentation product of a carbon source such as syngas, or can be obtained in other ways. Then ethanol and / or acetate can be contacted with the microorganism in step (a).

[0089] The term "contacting" as used herein refers to contacting the microorganism with ethanol and / or acetate directly. In one example, ethanol is the carbon source and the contacting in step (a) includes contacting ethanol with the microorganism of step (a). The contacting can be direct contact or indirect contact, and the indirect contact can include a membrane separating the cell from the ethanol, or wherein the cell and the ethanol can be maintained in two different compartments, etc. For example, in step (a), the aliphatic alcohol and the extraction medium can be in different compartments.

[0090] According to any aspect of the present invention, in the case where extraction is performed in step (a) while fermentation is performed in step (0), the extraction time may be equal to the fermentation time.

[0091] The foregoing describes preferred embodiments, and as those skilled in the art will appreciate, these preferred embodiments may be changed or modified in design, construction or operation without departing from the scope of the claims. For example, these changes are intended to be covered by the scope of the claims. DETAILED DESCRIPTION

[0092] Example

[0093] Example 1

[0094] General description of extraction hexanol:

[0095] A solution of hexanol (4 g / Kg) in distilled water was prepared. Ammonium acetate buffer (0.6 g / Kg of ammonium acetate, adjusted to pH 5.8 by adding acetic acid) was added to the solution to keep the pH close to 5.8 during extraction. The aqueous solution was placed in a separatory funnel and vigorously mixed with an organic mixture of trialkylphosphine (TAPO) in alkane or pure trialkylphosphine. The mass ratio of aqueous phase to organic phase was 9:1. After vigorous mixing, the phases were separated and analyzed by HPLC or 1 H-NMR analysis was performed separately to determine the concentration of hexanol in each phase. The distribution of hexanol is represented by the distribution constant Kd, where Kd is the ratio of the concentration in the organic phase divided by the concentration in the aqueous phase.

[0096] Results of ethanol extraction using buffer in aqueous phase:

[0097]

[0098] *The carbon number indicates the length of the alkyl chain in TAPO. If two carbon numbers are shown, the corresponding trialkylphosphine exhibits two chain lengths in statistical distribution.

[0099] **Kd is the ratio of the concentration in the organic phase divided by the concentration in the aqueous phase.

[0100] Results of ethanol extraction without buffer in the aqueous phase:

[0101]

[0102] *The carbon number indicates the length of the alkyl chain. If two carbon numbers are shown, the corresponding trialkylphosphine exhibits two chain lengths in the statistical distribution.

[0103] **Kd is the ratio of the concentration in the organic phase divided by the concentration in the aqueous phase.

[0104] As shown in the table above, trialkylphosphines can be used over a wide pH range.

Claims

1. A method for extracting an aliphatic alcohol containing 5 to 16 carbon atoms from an aqueous medium, the method comprising: (a) contacting the aliphatic alcohol in the aqueous culture medium with at least one extraction medium for a period of time sufficient to extract the aliphatic alcohol from the aqueous culture medium into the extraction medium, (b) separating the extraction medium containing the extracted aliphatic alcohol from the aqueous culture medium, The extraction medium comprises: - at least one trialkylphosphine oxide, wherein the alkyl group of the trialkylphosphine oxide is a hydrocarbon group having 4 to 15 carbon atoms, and wherein the aqueous culture medium is a fermentation medium, wherein the aliphatic alcohol is produced from a carbon source comprising carbon dioxide and / or carbon monoxide by contacting the carbon source with at least one microorganism capable of converting the carbon source into the aliphatic alcohol, and wherein the fermentation medium is recycled.

2. The method according to claim 1, wherein the microorganism is: (i) a mixture of a first microorganism and a second microorganism, and - the first microorganism is an acetogenic microorganism capable of converting the carbon source into acetate and / or ethanol; and - the second microorganism is used for carbon chain elongation and is selected from Clostridium kluyveri and C. Carboxidivorans and is capable of converting the acetate and / or ethanol to form acids; and wherein the first microorganism is further capable of converting the acid into the corresponding aliphatic alcohol, or (ii) Clostridium carboxydophorum capable of converting the carbon source into aliphatic alcohols.

3. The process of claim 1 or 2, wherein the extraction medium further comprises at least one alkane, wherein the alkane comprises 12 to 18 carbon atoms.

4. The method of claim 3, wherein the alkane is hexadecane. The method according to claim 3 , wherein the alkane is a branched alkane.

6. The method according to claim 1 or 2, wherein the aliphatic alcohol is hexanol.

7. The process according to claim 3, wherein the weight ratio of the at least one trialkylphosphine oxide to the alkane is from 1:100 to 1:

10.

8. The method of claim 1 or 2, wherein the pH of the aqueous culture medium is maintained between 5.5 and 7.

9. The method according to claim 1 or 2, wherein the trialkylphosphine oxide is trioctylphosphine oxide.

10. The process of claim 1 or 2, wherein the extraction medium is recycled.

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