RU2785987C2 - Alkane acid extraction - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: present invention relates to a method for extraction of hexanoic acid and/or its composite ester from an aqueous medium for fermentation, while hexanoic acid and/or its composite ester are or are produced by specific cells from a carbon source. The method includes following stages: (a) bringing hexanoic acid and/or its composite ester in the aqueous medium for fermentation into contact with at least one extractant for a time sufficient for extraction of hexanoic acid and/or its composite ester from the aqueous medium to the extractant, (b) separation of the extractant with extracted hexanoic acid and/or its composite ester from the aqueous medium for fermentation. Moreover, the extractant provides a mixture of at least one alkylphosphinoxide and at least one alkane, where alkane contains at least 12 carbon atoms.

EFFECT: creation of an effective extraction method.

14 cl, 12 ex

Description

The technical field to which the invention belongs

The present invention relates to a method for extracting an alkanoic acid and/or its ester from an aqueous medium. In particular, a mixture of at least one alkylphosphine oxide, preferably trioctylphosphine oxide (TOPO), and at least one alkane is used in the process.

State of the art invention

Alkanoic acids are carboxylic acids in which the oxygen atom (=O) is replaced by two hydrogen atoms in the corresponding alkane and the OH functional group is replaced by another H atom on the same carbon atom. Alkanoic acids are characterized in the art by several functions. For example, they can be used in the preparation of polymers, pharmaceuticals, solvents and food additives.

A well-known method for the preparation and extraction of alkanoic acids involves the hydrolysis and decarboxylation of malonic esters. The malonic ester is saponified with aqueous sodium hydroxide to form an aqueous solution of the disodium salt and ethanol. The brine is then treated with a strong mineral acid to give the sodium salt of the mineral acid and precipitate the dicarboxylic acid as a solid. For subsequent isolation of the dicarboxylic acid, simple separation procedures such as filtration or extraction are used. The sodium salt is discarded as waste. The recovered acid is further dried and heated to a temperature sufficient to allow the decarboxylation reaction to proceed. This procedure is lengthy, requires multiple steps, generates waste, and is labor intensive in terms of equipment.

Another method for extracting alkanoic acids such as formic, acetic, propionic, lactic, succinic and citric acids is salting out extraction. In this method, a system consisting of ethanol and ammonium sulfate is used. System parameters that affect extraction efficiency include line length, phase volume ratio, acid concentration, temperature, system pH, etc. Although extraction efficiency of alkanoic acids has been shown to improve using this method, the various parameters involved make the process too complex to handle. industrial application.

CA1167051 discloses a process for extracting or recovering certain carboxylic acids such as acetic acid and formic acid. However, the method requires the use of high temperatures and special equipment for the countercurrent heat exchange steps.

Accordingly, there is a need in the art for a cheaper and more efficient extraction process for extracting alkanoic acids, in particular industrial scale alkanoic acids. In addition, there is a need for an alkanoic acid extraction process that can be used in conjunction with a biotechnological process for producing alkanoic acids.

Description of the invention

The present invention attempts to solve the above problems by providing extraction methods for alkanoic acids and/or their ester that are more efficient and less expensive than the current methods available in the prior art. The present invention also provides methods for extracting alkanoic acids and/or their ester, which can be used in combination with a biotechnological process for the production of alkanoic acids and/or their ester.

In accordance with one aspect of the present invention, there is provided a method for extracting an alkanoic acid and/or an ester thereof from an aqueous medium, the method comprising

(a) contacting the alkanoic acid and/or its ester in an aqueous medium with at least one extractant for a period of time sufficient to extract the alkanoic acid and/or its ester from the aqueous medium into the extractant,

(b) separating the extractant with the extracted alkanoic acid and/or its ester from the aqueous medium,

where the extractant provides

- a mixture of at least one alkylphosphine oxide, preferably trioctylphosphine oxide (TOPO), and at least one alkane, where the alkane contains at least 12 carbon atoms.

In particular, the extraction process according to any aspect of the present invention provides for an increase in yield relative to the amount of extractives used. For example, less than 50% by weight of the extractant can be used to extract the same amount of alkanoic acids and/or their ester as if only pure alkanes were used. Therefore, with a small volume of extractant, a higher yield of alkanoic acids and/or their esters can be extracted. The extractant is also not harmful to microorganisms. Accordingly, an extractant according to any aspect of the present invention may be present in the biotechnological production of an alkanoic acid and/or its ester. In addition, at least in cases where the alkanoic acid is hexanoic acid, it can be easily separated from the extractant according to any aspect of the present invention by distillation. The reason is that hexanoic acid undergoes minimal distillation at a much lower boiling point than the extractant, and after separation by distillation, the extractant can be easily recycled.

The method according to any aspect of the present invention may be a method for extracting at least one isolated alkanoic acid and/or its ester from an aqueous medium. The isolated alkanoic acid and/or its ester can refer to at least one alkanoic acid and/or its ester, which can be separated from the medium in which the alkanoic acid and/or its ester was obtained. In one example, the alkanoic acid and/or its ester can be produced in an aqueous medium (eg, a fermentation medium where the alkanoic acid and/or its ester is produced by specific cells from a carbon source). Isolated alkanoic acid and/or its ester may refer to an alkanoic acid and/or its ester extracted from an aqueous medium. In particular, the extraction step involves the separation of excess water from the aqueous medium, thus leading to the formation of a mixture containing the extracted alkanoic acid and/or its ester.

The extractant may also be referred to as "extraction medium". The extraction medium can be used for extracting/isolation of the alkanoic acid and/or its ester obtained in accordance with any method of the present invention from the aqueous medium in which the alkanoic acid and/or its ester was originally obtained. At the end of the extraction step, excess water from the aqueous medium can be removed, thus obtaining an extractant containing the extracted alkanoic acid and/or its ester. The extractant may include a combination of compounds that can lead to efficient methods for extracting the alkanoic acid and/or its ester from an aqueous medium. In particular, the extractant may contain (i) at least an alkane containing at least 12 carbon atoms, and (ii) at least one alkyl phosphine oxide molecule. Using the extraction medium according to any aspect of the present invention, the alkanoic acid and/or its ester can be efficiently extracted into an extractant which is a mixture of an alkane and an alkyl phosphine oxide. This extractant, comprising a mixture of an alkyl phosphine oxide and at least one alkane, may be considered suitable for the process of any aspect of the present invention since the mixture works effectively in extracting the desired alkanoic acid and/or its ester in the presence of a fermentation medium. In particular, a mixture of an alkyl phosphine oxide and at least one alkane can be considered to work better than any method currently known in the art for the extraction of an alkanoic acid and/or its ester, since it does not require any special equipment to carry out, and with it it is relatively easy to obtain a high yield of the product.

An alkane may contain at least 12 carbon atoms. In particular, an alkane may contain on the order of 12-18 carbon atoms. In one example, the alkane may be selected from the group consisting of dodecane, tridecane, tetradecane, pentadecane, hexadecane, heptadecane, and octadecane. In an additional example, the extractant may include a mixture of alkanes.

Alkyl phosphine oxides have the general formula OPX ₃ where X is alkyl. Suitable alkyl phosphine oxides according to any aspect of the present invention include an alkyl group consisting of a linear, branched or cyclic hydrocarbon, wherein the hydrocarbon consists of from 1 to about 100 carbon atoms and from 1 to about 200 hydrogen atoms. In particular, "alkyl" as used in relation to an alkyl phosphine oxide according to any aspect of the present invention may refer to a hydrocarbon group having 1 to 20 carbon atoms, often 4 to 15 carbon atoms or 6 to 12 carbon atoms, and which may consist of straight chains, cyclic links, branched chains or mixtures thereof. The alkyl phosphine oxide may have from one to three alkyl groups at each phosphorus atom. In one example, the alkyl phosphine oxide has three alkyl groups at the P atom. In some examples, the alkyl group may contain an oxygen atom instead of one carbon atom of the C4-C15 or C6-C12 alkyl group, provided that the oxygen atom is not attached to the P atom of the alkyl phosphine oxide. Typically, the alkylphosphine oxide is selected from the group consisting of trioctylphosphine oxide, tributylphosphine oxide, hexylphosphine oxide, octylphosphine oxide, and mixtures thereof.

Even more specifically, the alkylphosphine oxide may be trioctylphosphine oxide (TOPO). Trioctylphosphine oxide (TOPO) is an organophosphorus compound of the formula OP(C ₈ H ₁₇ ) ₃ . At least one alkylphosphine oxide, preferably trioctylphosphine oxide (TOPO), may be present in the extraction medium along with at least one alkane. In particular, a mixture of at least one alkylphosphine oxide, preferably trioctylphosphine oxide (TOPO), and an alkane containing at least 12 carbon atoms, may include a weight ratio of from about 1:100 to 1:10, of at least one alkylphosphine oxide, preferably trioctylphosphine oxide (TOPO), relative to the alkane. More specifically, the weight ratio of at least one alkylphosphine oxide, preferably trioctylphosphine oxide (TOPO), and alkane in the extraction medium according to any aspect of the present invention 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 specifically, the weight ratio of at least one alkyl phosphine oxide, preferably trioctyl phosphine oxide (TOPO), and the alkane may 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 alkylphosphine oxide, preferably trioctylphosphine oxide (TOPO), and alkane may be from 1:40 to 1:15 or from 1:25 to 1:15. In one example, the weight ratio of at least one alkylphosphine oxide, preferably trioctylphosphine oxide (TOPO), and alkane may be approximately 1:15. In an example, the alkane may be hexadecane and therefore the weight ratio of at least one alkylphosphine oxide, preferably trioctylphosphine oxide (TOPO), and hexadecane may be approximately 1:15.

The term "approximately" as used in this document refers to changes within 20 percent. In particular, the term "about" as used herein refers to +/- 20%, more specifically +/-10%, even more specifically +/- 5% of a given measurement or value.

In step (a), according to any aspect of the present invention, the alkanoic acid and/or its ester in an aqueous medium may be contacted with an extractant for a time period sufficient to extract the alkanoic acid and/or its ester from the aqueous medium into the extractant. One of skill in the art may be able to determine the amount of time required to reach distribution equilibrium and the proper bubble buildup that may be needed to optimize the extraction process. In some examples, the time required may depend on the amount of alkanoic acid and/or its ester that can be extracted. In particular, the time required for extracting the alkanoic acid and/or its ester from the aqueous medium into the extractant may take only a few minutes. In examples in which the extraction is carried out as the fermentation progresses, the time for extraction is equivalent to the fermentation time.

The ratio of the extractant used and the amount of alkanoic acid and/or its ester to be extracted may vary depending on how fast the extraction is to be carried out. In one example, the amount of extractant is equal to the amount of an aqueous medium containing alkanoic acid and/or its ester. After the step of bringing the extractant into contact with an aqueous medium, the two phases (aqueous and organic) are separated using any means known in the art. In one example, the two phases can be separated using a separating funnel. The two phases can also be separated using mixer-settlers, pulse columns, etc. In one example, in which the alkanoic acid is hexanoic acid, the separation of the extractant from the hexanoic acid can be carried out using distillation, taking into account that hexanoic acid is distilled at a much lower boiling point than the extractant. The person skilled in the art will be able to select the best method for separating the extraction medium from the desired alkanoic acid and/or its ester in step (b) depending on the characteristics of the alkanoic acid and/or its ester to be extracted. In particular, step (b) according to any aspect of the present invention provides for the recovery of the alkanoic acid from step (a). Bringing the alkanoic acid into contact with an organic extractant results in the formation of two phases, the two phases (aqueous and organic) are separated using any means known in the art. In one example, the two phases can be separated using a separating funnel. The two phases can also be separated using mixer-settlers, pulse columns, temperature separation, and the like. hexanoic acid is distilled at a much lower boiling point than the extractant. One skilled in the art will be able to select the best method for separating the extractant from the desired alkanoic acid depending on the characteristics of the alkanoic acid to be recovered.

Step (b) preferably ends with an organic absorbent available again for recycling or reuse, preferably in step (0) (see below).

The alkanoic acid and/or its ester may be selected from the group consisting of alkanoic acids with 2-16 carbon atoms. In particular, the alkanoic acid may be selected from the group consisting of ethanoic acid, propionic acid, butanoic acid, pentanoic acid, hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, undecanoic acid, dodecanoic acid, tridecanoic acid, myristic acid. acids, pentadecanoic acid and palmitic acid. More specifically, the alkanoic acid may be selected from the group consisting of alkanoic acids with 4-16, 4-14, 4-12, 4-10, 5-16, 5-14, 5-12, 5-10, 6- 16, 6–14, 6–12, or 6–10 carbon atoms. Even more specifically, alkanoic acid is hexanoic acid.

The ester moiety of the alkanoic acid ester is preferably selected from the group consisting of methyl, ethyl, isopropyl, propyl, and isobutyl, and butyl.

In some examples, microorganisms capable of producing alkanoic acid and/or its ester can be cultured with any of the culture media, substrates, conditions, and methods generally known in the art for bacterial culture. This enables the production of alkanoic acid and/or its ester using a biotechnological process. Depending on the microorganism used to produce the alkanoic acid and/or its ester, suitable growth media, pH, temperature, stirring speed, inoculum level and/or aerobic, microaerobic or anaerobic conditions vary. One skilled in the art will recognize other conditions necessary to carry out the method according to any aspect of the present invention. In particular, the conditions in the container (eg fermenter) can be varied depending on the microorganisms used. Changing the conditions so that they are suitable for the optimal functioning of the microorganisms is within the knowledge of a person skilled in the art.

In one example, the method according to any aspect of the present invention can be carried out in an aqueous medium with a pH value of from 5 to 8 or from 5.5 to 7. The pressure can be from 1 to 10 bar. Microorganisms 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.

In some examples, for the growth of a microorganism and for its production of an alkanoic acid and/or its ester, the aqueous medium may contain any nutrients, ingredients and/or additives suitable for growing the microorganism or for promoting the production of an alkanoic acid and/or its ester. In particular, the aqueous medium may contain at least one of the following: carbon sources, nitrogen sources such as ammonium salt, yeast extract or peptone; minerals; salts; cofactors; buffer funds; vitamins and any other ingredients and/or extracts that may stimulate bacterial growth. The culture medium to be used must meet the requirements of the particular strains. Descriptions of culture media for various microorganisms are given in the "Manual of Methods for General Bacteriology".

Accordingly, the method for extracting alkanoic acid and/or its ester according to any aspect of the present invention can be used in conjunction with any biotechnological method for producing alkanoic acid and/or its ester. This is especially advantageous since, as a rule, during the fermentation process to obtain an alkanoic acid and/or its ester using biological methods, the alkanoic acid and/or its ester will be left to be collected in an aqueous medium, and after reaching certain concentration values in in the fermentation medium, it is the target product (alkanoic acids and/or their ester) that can inhibit the activity and productivity of the microorganism. Thus, this limits the overall yield of the fermentation process. Using this extraction method, alkanoic acids and/or their ester are extracted as they are produced, thus significantly reducing the inhibition of the final product.

The process according to any aspect of the present invention is also more efficient and cost-saving than conventional methods for removing alkanoic acids and/or their ester, in particular from a fermentation process as they are produced, since there is no primary focus on distillation and/or precipitation with recovering alkanoic acids and/or their ester. The distillation or precipitation process can lead to higher production costs, lower yields, and higher waste generation, hence reducing the overall efficiency of the process. With the method according to any aspect of the present invention attempts are made to overcome these shortcomings.

In one example, the alkanoic acid is hexanoic acid. In this example, hexanoic acid can be obtained from synthesis gas.

The synthesis gas can be converted to hexanoic acid in the presence of at least one acetogenic bacterium and/or hydrogen oxidizing bacterium. In particular, any method known from the prior art can be used. Hexanoic acid can be produced from synthesis gas using at least one kind of prokaryotes. In particular, the genus of prokaryotes may be selected from the group consisting of the genus Escherichia, such as Escherichia coli; from the genus Clostridia such as Clostridium ljungdahlii, Clostridium autoethanogenum, Clostridium carboxidivorans or Clostridium kluyveri; from the genus Corynebacteria such as Corynebacterium glutamicum; from the genus Cupriavidus such as Cupriavidus necator or Cupriavidus metallidurans; from the genus Pseudomonas such as Pseudomonas fluorescens, Pseudomonas putida or Pseudomonas oleavorans; from the genus Delftia such as Delftia acidovorans; from the genus Bacillus such as Bacillus subtillis; from the genus Lactobacillus such as Lactobacillus delbrueckii; or from the genus Lactococcus such as Lactococcus lactis.

In another example, hexanoic acid can be produced from synthesis gas using at least one eukaryotic genus. The genus of eukaryotes used in the method of the present invention may be selected from the genus Aspergillus, such as Aspergillus niger; from the genus Saccharomyces such as Saccharomyces cerevisiae; from the genus Pichia such as Pichia pastoris; from the genus Yarrowia such as Yarrowia lipolytica; from the genus Issatchenkia such as Issatchenkia orientalis; from the genus Debaryomyces such as Debaryomyces hansenii; from the genus Arxula such as Arxula adenoinivorans; or from the genus Kluyveromyces such as Kluyveromyces lactis.

More specifically, hexanoic acid can be produced from synthesis gas using any of the methods disclosed in Steinbusch, 2011, Zhang, 2013, Van Eerten-Jansen, M. C. A. A, 2013, Ding H. et al, 2010, Barker H. A., 1949, Stadtman E.R., 1950, Bornstein B.T., et al., 1948, etc. Even more specifically, hexanoic acid can be produced from synthesis gas in the presence of at least Clostridium kluyveri.

The term "acetogenic bacteria" as used herein refers to a microorganism that is capable of performing the Wood-Ljungdal pathway and thus is capable of converting CO, CO ₂ and/or hydrogen to acetate. These microorganisms include microorganisms which, in their wild-type form, do not follow the Wood-Ljungdahl pathway but have acquired this trait through genetic modification. Such microorganisms include, without limitation, E. coli cells. These microorganisms may also be known as carboxydotrophic bacteria. Currently, 21 genera of various 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). In addition, alcohols, aldehydes, carboxylic acids, as well as numerous hexoses can also be used as a carbon source (Drake et al., 2004). The reductive pathway that leads to the formation of acetate is called the 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 spp. No. 446 (Morinaga et al. , 1990, J. Biotechnol., Vol. 14, pp. 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 coskati 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), Clos tridium 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., 198 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).

More specifically, Clostridium carboxidivorans strain ATCC BAA-624 can be used. Even more specifically, a bacterial strain of Clostridium carboxidivorans labeled "P7" and "P11" which is described in, for example, U.S. 2007/0275447 and U.S. 2008/0057554.

Another particularly suitable bacterial species 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 O-52 can be used in the conversion of synthesis gas to hexanoic acid. These strains are, for example, described in WO 98/00558, WO 00/68407, ATCC 49587, ATCC 55988 and ATCC 55989.

Acetogenic bacteria can be used in conjunction with hydrogen oxidizing bacteria. In one example, both acetogenic bacteria and hydrogen oxidizing bacteria can be used to produce hexanoic acid from synthesis gas. In another example, only acetogenic bacteria can be used to metabolize syngas to produce hexanoic acid from syngas. In yet another example, only hydrogen-oxidizing bacteria can be used in this reaction.

Hydrogen oxidizing bacteria may be selected from the group consisting of Achromobacter, Acidithiobacillus, Acidovorax, Alcaligenes, Anabena, Aquifex, Arthrobacter, Azospirillum, Bacillus, Bradyrhizobium, Cupriavidus, Derxia, Helicobacter, Herbaspirillum, Hydrogenobacter, Hydrogenobaculum, Hydrogenovibrio Hydrogenophaga, Hydrogenophilus, Hydrogenothermus, , Ideonella sp. O1, Kyrpidia, Metallosphaera, Methanobrevibacter, Myobacterium, Nocardia, Oligotropha, Paracoccus, Pelomonas, Polaromonas, Pseudomonas, Pseudonocardia, Rhizobium, Rhodococcus, Rhodopseudomonas, Rhodospirillum, Streptomyces, Thiocapsa, Treponema, Variovorax, Xanthobacter and Wautersia.

In the production of hexanoic acid from synthesis gas, a combination of bacteria can be used. More than one species of acetogenic bacteria may be present in combination with one or more species of hydrogen-oxidizing bacteria. In another example, however, more than one type of acetogenic bacteria may be present. In another example, however, more than one species of hydrogen-oxidizing bacteria may be present. Hexanoic acid, also known as caproic acid, has the general formula C ₅ H ₁₁ COOH.

In particular, the process for producing hexanoic acid may include the step of:

- bringing the synthesis gas into contact with at least one species of bacteria capable of implementing the Wood-Ljungdal pathway and the ethanol-carboxylate fermentation pathway to produce hexanoic acid.

The term "contacting" as used herein means direct contact between the alkanoic acid and/or its ester in the medium with the extraction medium in step (a) and/or direct contact between the microorganism and the synthesis gas. For example, the cell and the medium containing the carbon source may be in different compartments. In particular, the carbon source may be in a gaseous state and added to the environment containing cells according to any aspect of the present invention.

In one example, the production of hexanoic acid from synthesis gas may involve the use of acetogenic bacteria in conjunction with a bacterium capable of producing hexanoic acid using the ethanol-carboxylate fermentation pathway of hydrogen-oxidizing bacteria. In one example, both acetogenic bacteria and hydrogen oxidizing bacteria can be used to produce hexanoic acid from synthesis gas. For example, Clostridium ljungdahlii can be used simultaneously with Clostridium kluyveri. In another example, only acetogenic bacteria can be used to metabolize syngas to produce hexanoic acid from syngas. In this example, acetogenic bacteria may be capable of performing both the ethanol-carboxylate fermentation pathway and the Wood-Ljungdahl pathway. In one example, the acetogenic bacteria may be C. carboxidivorans, which may be capable of both the Wood-Ljungdahl pathway and the ethanol-carboxylate fermentation pathway.

The ethanol-carboxylate fermentation pathway is described in detail in at least Seedorf, H., et al., 2008. The organism may be selected from the group consisting of Clostridium kluyveri, C. carboxidivorans, and the like. Such microorganisms include microorganisms that in their the wild-type form does not carry out the ethanol-carboxylate fermentation pathway, but acquired this trait as a result of genetic modification. In particular, the microorganism may be Clostridium kluyveri.

In one example, the bacteria used in accordance with any aspect of the present invention are selected from the group consisting of Clostridium kluyveri and C. carboxidivorans.

In particular, cells are brought into contact with a carbon source that 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), substrates with one carbon atom and/or mixtures thereof. More specifically, the cells are brought into contact with a carbon source containing CO and/or CO ₂ to obtain an alkanoic acid and/or its ester.

With regard to the source of substrates containing carbon dioxide and/or carbon monoxide, the person skilled in the art will understand that there are many possible sources for providing CO and/or CO ₂ as a carbon source. It can be seen that any gas, or any mixture of gases, which is capable of providing sufficient amounts of carbon to the microorganisms so that acetate and/or ethanol can be formed from the source of CO and/or CO ₂ can be used as the carbon source of the present invention in practice.

Typically, for a cell of the present invention, the carbon source provides at least 50% by weight, at least 70% by weight, in particular at least 90% by weight of CO ₂ and/or CO, where the percentages by weight are % refer to all carbon sources that are available to the cell according to any aspect of the present invention. A source may be provided which is a carbon material.

Examples of carbon sources in gaseous forms include waste gases such as synthesis gas, flue gas, and gases from petroleum refining from yeast fermentation or clostridia fermentation. These waste gases are generated during the gasification of cellulose-containing materials or the gasification of coal. In one example, these waste gases are not necessarily obtained as by-products of other processes, but may be specifically obtained for use with the mixed culture of the present invention.

According to any aspect of the present invention, the carbon source may be synthesis gas, including for the production of acetate and/or ethanol used in step (0) (see below) according to any aspect of the present invention. Synthesis gas can, for example, be obtained as a by-product of coal gasification. Accordingly, a microorganism according to any aspect of the present invention may be capable of converting a waste material into a valuable resource.

In another example, synthesis gas may be a by-product of the gasification of widely available, inexpensive agricultural raw materials for use with the mixed crop of the present invention to produce substituted and unsubstituted organic compounds.

There are numerous examples of raw materials that can be converted into synthesis gas, since almost all forms of vegetation can be used for this purpose. In particular, the raw materials are selected from the group consisting of perennial grasses such as miscanthus, corn residues, processing residues such as sawdust, and the like.

In general, synthesis gas can be produced in a dry biomass gasifier mainly by pyrolysis, partial oxidation and steam reforming, where the primary products of the synthesis gas are CO, H ₂ and CO ₂ . Synthesis gas can also be a CO ₂ electrolysis product. One skilled in the art will recognize suitable conditions for carrying out electrolysis of CO ₂ to produce synthesis gas containing CO in the desired amount.

Typically, a portion of the synthesis gas produced from the gasification process is first treated to optimize yield values and prevent tar formation. Cracking of unwanted tar and CO in the synthesis gas can be done using lime and/or dolomite. These processes are described in detail, for example, in Reed, 1981.

The overall efficiency, productivity for alkanoic acid and/or its ester and/or overall carbon uptake in the process of the present invention may depend on the stoichiometry of CO ₂ , CO and H ₂ in the continuous gas stream. The continuous gas streams supplied may consist of CO ₂ and H ₂ . In particular, in a continuous gas stream, the CO ₂ concentration range may be approximately 10-50%, in particular 3% by weight, and H ₂ will be in the range from 44% to 84%, in particular from 64 to 66.04%, according to weight. In another example, the continuous gas stream may also include inert gases like N ₂ up to an N ₂ concentration of 50% by weight.

Mixtures of sources can be used as a carbon source.

According to any aspect of the present invention, a reducing agent, such as hydrogen, may be supplied along with the carbon source. In particular, this hydrogen can be supplied when C and/or CO ₂ are supplied and/or used. In one example, hydrogen gas is part of the synthesis gas present according to any aspect of the present invention. In another example, where hydrogen gas in the synthesis gas is insufficient for the process of the present invention, additional hydrogen gas may be supplied.

In one example, the alkanoic acid is hexanoic acid. More specifically, a carbon source containing CO and/or CO ₂ is contacted with the cells in a continuous gas stream. Even more specifically, the continuous gas stream contains synthesis gas. These gases can be supplied, for example, using separate nozzles that open into the aqueous medium, frits, membranes inside the gas supply pipe to the aqueous medium, and the like.

One skilled in the art will appreciate that it may be necessary to control the composition and flow rates of the jets at certain time intervals. Control of the composition of a stream can be achieved by changing the proportions of its constituent jets to achieve a target or desired composition. The composition and flow rate of the mixed stream can be controlled by any means known in the art. In one example, the system is adapted to continuously control the flow rates and compositions of at least two jets and combine them to produce a single mixed substrate jet in a continuous gas stream of optimal composition and a means to pass the optimized substrate stream to the fermenter.

The term "aqueous solution" or "medium" includes any solution containing water, mainly water, as a solvent, which can be used to maintain a cell according to any aspect of the present invention at least temporarily in a metabolically active and/or viable state, and contains if necessary, any additional substrates. The person skilled in the art is aware of methods for preparing numerous aqueous solutions, commonly referred to as media, which can be used to preserve the culture and/or cells, eg LB medium in the case of E. coli, ATCC1754 medium can be used in the case of C. ljungdahlii. It is preferable to use a minimal medium as an aqueous solution, i.e. a medium with a fairly simple composition, which contains only the minimum set of salts and nutrients necessary to maintain the cell in a metabolically active and / or viable state, in contrast to complex media, in avoiding unnecessary contamination of products with unwanted by-products. For example, the M9 environment can be used as the minimum environment. Cells are incubated with a carbon source long enough to obtain the desired product. For example, for at least 1, 2, 4, 5, 10, or 20 hours. The selected temperature should be such that the cells according to any aspect of the present invention remain catalytically competent and/or metabolically active, for example from 10 to 42°C, preferably from 30 to 40°C, in particular from 32 to 38°C in case the cell is a C. ljungdahlii cell. The aqueous medium according to any aspect of the present invention also provides for a medium in which the alkanoic acid and/or its ester is prepared. It mainly refers to a medium in which the solution contains mainly water. In one example, the aqueous medium in which the cells are used to produce the alkanoic acid and/or its ester is the one that is contacted with the extraction medium to extract the alkanoic acid and/or its ester.

In particular, the mixture of microorganism and carbon source according to any aspect of the present invention can be used in any known bioreactor or fermenter to carry out any aspect of the present invention. In one example, the complete process according to any aspect of the present invention, which begins with the preparation of the alkanoic acid and/or its ester and ends with the extraction of the alkanoic acid and/or its ester, takes place in a single container. Therefore, there may not be a separation step between the step of obtaining the alkanoic acid and/or its ester and the step of extracting the alkanoic acid and/or its ester. This saves time and costs. In particular, during the fermentation process, the microorganism can be grown in an aqueous medium and in the presence of an extraction medium. Thus, the method according to any aspect of the present invention provides a one-pot device for the production of alkanoic acids and/or their ester. In addition, since the alkanoic acid and/or its ester is extracted as it is produced, no inhibition by the final product occurs, which ensures that the yield of the alkanoic acid and/or its ester is maintained. An additional separation step may be carried out to remove the alkanoic acid and/or its ester. Any separation method known in the art can be used, such as using a funnel, column, distillation, etc. The remaining extractant and/or cells can then be recycled.

In another example, the extraction process may take place as a separate step and/or in a different reactor. After a fermentation has been carried out that has already produced the desired alkanoic acid and/or its ester to be extracted, the extractant according to any aspect of the present invention may be added to the fermentation medium, or the fermentation medium may be added to the reactor containing the extractant. The desired alkanoic acid and/or its ester can then be extracted using any separation method known in the art, such as using a funnel, column, distillation, etc. The remaining extractant can then be recycled.

Another advantage of the process is that the extractant can be recycled. Therefore, once the alkanoic acid and/or its ester is separated from the extraction medium, the extraction medium can be recycled and reused, reducing waste.

According to another aspect of the present invention, the use of a mixture of at least one alkylphosphine oxide, preferably trioctylphosphine oxide (TOPO), and an alkane is contemplated for extracting an alkanoic acid from an aqueous medium, the alkane having at least 12 carbon atoms. In particular, an alkane may contain from 12 to 18 carbon atoms. More specifically, the alkane may be hexadecane. Even more specifically, the alkanoic acid and/or its ester is selected from the group consisting of alkanoic acids with 4-16 carbon atoms. In one example, the alkanoic acid may be hexanoic acid.

In a preferred process according to the present invention, ethanol and/or acetate is used as starting material.

With such a preferred method according to the present invention, the alkanoic acid and/or its ester derived from ethanol and/or acetate is extracted and includes step (0) before step (a):

(1) contacting ethanol and/or acetate with at least one species of microorganism capable of performing carbon chain extension in an aqueous medium to produce alkanoic acid and/or its ester from ethanol and/or acetate.

According to a preferred process according to the present invention, the aqueous medium after separation step (b) of the alkanoic acid and/or its ester can be recycled back to step (0). This recycling step involves the recycling and reuse of microorganisms, since the extractant according to the present invention is non-toxic to microorganisms. This aqueous medium recycling step in the process of the present invention has the additional advantage of being able to subject the residue of the alkanoic acid and/or its ester that was not extracted in the first place from steps (a) and (b) in the first cycle to extraction in for additional time or as many times as the aqueous medium is recycled.

The microorganism at (0) capable of performing carbon chain extension to produce alkanoic acid can be any organism that can be capable of carbon chain extension (see Jeon et al. Biotechnol Biofuels (2016) 9:129). The carbon chain extension pathway is also disclosed in Seedorf, H., et al., 2008. Microorganisms according to any aspect of the present invention may also include microorganisms that, in their wild-type form, are not capable of carbon chain extension, but have acquired this trait through genetic modification. . In particular, the species of microorganisms on (0) may be selected from the group consisting of Clostridium carboxidivorans, Clostridium kluyveri and C. pharus. In particular, the microorganism according to any aspect of the present invention may be Clostridium kluyveri.

In step (0), according to any aspect of the present invention, ethanol and/or acetate is contacted with at least one species of microorganism capable of carrying out carbon chain extension to form an alkanoic acid and/or its ester from ethanol and/or acetate. In one example, the carbon source may be ethanol in combination with at least one other carbon source selected from the group consisting of acetate, propionate, butyrate, isobutyrate, valerate, and hexanoate. In particular, the carbon source may be ethanol and acetate. In another example, the carbon source may 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 may be ethanol itself. In another example, the carbon substrate may be acetate itself.

The source of acetate and/or ethanol may vary depending on availability. In one example, ethanol and/or acetate can be a fermentation product of synthesis gas or any carbohydrate known in the art. In particular, the carbon source for the production of acetate and/or ethanol may be selected from the group consisting of alcohols, aldehydes, glucose, sucrose, fructose, dextrose, lactose, xylose, pentose, polyol, hexose, ethanol, and synthesis gas. Mixtures of sources can be used as a carbon source.

Even more specifically, the carbon source may be synthesis gas. The synthesis gas can be converted to ethanol and/or acetate in the presence of at least one species of acetogenic bacteria.

In one example, the preparation of an alkanoic acid and/or its ester from acetate and/or ethanol, which is obtained from synthesis gas, may involve the use of acetogenic bacteria in conjunction with a microorganism capable of carbon chain extension. For example, Clostridium ljungdahlii can be used simultaneously with Clostridium kluyveri. In another example, a single cell of acetogenic bacteria may be capable of the activity of both organisms. For example, acetogenic bacteria may be C. carboxidivorans, which may be capable of both the Wood-Ljungdahl pathway and the carbon chain extension pathway.

The ethanol and/or acetate used in step (0) according to any aspect of the present invention may be a synthesis gas fermentation product or may be produced by other means. Ethanol and/or acetate can then be brought into contact with the microorganism in step (0).

The term "bringing into contact", as used in this document, means the implementation of direct contact between the microorganism and ethanol and/or acetate. In one example, ethanol is the carbon source and contacting in step (0) involves bringing the ethanol into contact with the microorganism from step (0). The contact may be direct contact or indirect, which may involve the separation of cells from ethanol by a membrane or the like, or cases where cells and ethanol may be contained in two different compartments, etc. For example, in step (a), alkanoic acid and /or its ester and extractant may be in different compartments.

According to any aspect of the present invention where the extraction is carried out in step (a) as the fermentation progresses in step (0), the extraction time may be equivalent to the fermentation time.

Examples

The foregoing describes preferred embodiments, which, as will be appreciated by those skilled in the art, may be subject to change or modification during development, construction, or operation without departing from the scope of the claims. It is understood that these changes, for example, are covered by the scope of the claims.

Example 1

Preparation of butyric acid from acetate and ethanol with Clostridium kluyveri

For the biological conversion of ethanol and acetate to butyric acid, the bacterium Clostridium kluyveri was used. All cultivation steps were carried out under anaerobic conditions in pressure-resistant glass flasks that can be hermetically sealed with a butyl rubber stopper.

For pre-culture 100 ml DMSZ52 medium (pH = 7.0; 10 g/l K-acetate, 0.31 g/l K ₂ HPO ₄ , 0.23 g/l KH ₂ PO ₄ , 0.25 g/l l NH ₄ Cl, 0.20 g/l MgSO ₄ x 7H ₂ O, 1 g/l yeast extract, 0.50 mg/l resazurin, 10 µl/l HCl (25%, 7.7 M), 1, 5 mg/l FeCl ₂ x 4H ₂ O, 70 µg/l ZnCl ₂ x 7H ₂ O, 100 µg/l MnCl ₂ x 4H ₂ O, 6 µg/l H ₃ BO ₃ , 190 µg/l CoCl ₂ x 6H ₂ O, 2 µg/l CuCl ₂ x 6H ₂ O, 24 µg/l NiCl ₂ x 6H ₂ O, 36 µg/l Na ₂ MO ₄ x 2H ₂ O, 0.5 mg/l NaOH, 3 µg/l Na ₂ SeO ₃ x 5H ₂ O, 4 µg/l Na ₂ WO ₄ x 2H ₂ O, 100 µg/l vitamin B12, 80 µg/l p-aminobenzoic acid, 20 µg/l D(+)-biotin, 200 µg/l nicotinic acid, 100 µg/l D-Ca-pantothenate, 300 µg/l pyridoxine hydrochloride, 200 µg/l thiamine-HCl x 2H ₂ O, 20 ml/l ethanol, 2.5 g/l NaHCO ₃ , 0.25 g/l cysteine-HCl x H ₂ O, 0.25 g/l Na ₂ S x 9H ₂ O) in a 250 ml flask was inoculated with 5 ml of frozen Clostridium kluyveri cryoculture and incubated at 37°C for 144 h up to OD _{600 nm} > 0.2.

To obtain the main culture, 200 ml of fresh DMSZ52 medium in a 500 ml flask were inoculated with centrifuged cells from the preculture to an OD _{600 nm} of 0.1. This growing culture was incubated at 37°C for 27 hours until OD _{600 nm} > 0.6. The cell suspension was then centrifuged, washed with preparation buffer (pH 6.0; 8.32 g/l K-acetate, 0.5 g/l ethanol) and centrifuged again.

To obtain a production culture, 200 ml of production buffer in a 500 ml flask was inoculated with washed cells from the main culture to an OD of _{600 nm} of 0.2. The culture was closed with a butyl rubber stopper and incubated for 71 hours at 37°C and 100 rpm. in an open water bath with shaking. Samples were taken at the beginning and at the end of the culture period. They were tested for optical density, pH value and various analytes (tested by NMR).

It can be seen from the results that in the production phase, the amount of acetate decreased from 5.5 g/l to 5.0 g/l, and the amount of ethanol decreased from 0.5 g/l to 0.0 g/l. In addition, the concentration of butyric acid increased from 0.05 g/L to 0.8 g/L, and the concentration of hexanoic acid increased from 0.005 g/L to 0.1 g/L.

Example 2

Preparation of hexanoic acid from acetate and ethanol with Clostridium kluyveri

For the biological conversion of ethanol and acetate to hexanoic acid, the bacterium Clostridium kluyveri was used. All cultivation steps were carried out under anaerobic conditions in pressure-resistant glass flasks that can be hermetically sealed with a butyl rubber stopper.

For pre-culture 100 ml DMSZ52 medium (pH = 7.0; 10 g/l K-acetate, 0.31 g/l K ₂ HPO ₄ , 0.23 g/l KH ₂ PO ₄ , 0.25 g/l l NH ₄ Cl, 0.20 g/l MgSO ₄ x 7H ₂ O, 1 g/l yeast extract, 0.50 mg/l resazurin, 10 µl/l HCl (25%, 7.7 M), 1, 5 mg/l FeCl ₂ x 4H ₂ O, 70 µg/l ZnCl ₂ x 7H ₂ O, 100 µg/l MnCl ₂ x 4H ₂ O, 6 µg/l H ₃ BO ₃ , 190 µg/l CoCl ₂ x 6H ₂ O, 2 µg/l CuCl ₂ x 6H ₂ O, 24 µg/l NiCl ₂ x 6H ₂ O, 36 µg/l Na ₂ MO ₄ x 2H ₂ O, 0.5 mg/l NaOH, 3 µg/l Na ₂ SeO ₃ x 5H ₂ O, 4 µg/l Na ₂ WO ₄ x 2H ₂ O, 100 µg/l vitamin B12, 80 µg/l p-aminobenzoic acid, 20 µg/l D(+)-biotin, 200 µg/l nicotinic acid, 100 µg/l D-Ca-pantothenate, 300 µg/l pyridoxine hydrochloride, 200 µg/l thiamine-HCl x 2H ₂ O, 20 ml/l ethanol, 2.5 g/l NaHCO ₃ , 0.25 g/l cysteine-HCl x H ₂ O, 0.25 g/l Na ₂ S x 9H ₂ O) in a 250 ml flask was inoculated with 5 ml of frozen Clostridium kluyveri cryoculture and incubated at 37°C for 144 h up to OD _{600 nm} > 0.2.

To obtain the main culture, 200 ml of fresh DMSZ52 medium in a 500 ml flask were inoculated with centrifuged cells from the preculture to an OD _{600 nm} of 0.1. This growing culture was incubated at 37°C for 27 hours until OD _{600 nm} > 0.6. The cell suspension was then centrifuged, washed with preparation buffer (pH 6.0; 0.832 g/l K-acetate, 5.0 g/l ethanol) and centrifuged again.

To obtain a production culture, 200 ml of production buffer in a 500 ml flask was inoculated with washed cells from the main culture to an OD of _{600 nm} of 0.2. The culture was closed with a butyl rubber stopper and incubated for 71 hours at 37°C and 100 rpm. in an open water bath with shaking. Samples were taken at the beginning and at the end of the culture period. They were tested for optical density, pH value and various analytes (tested by NMR).

It can be seen from the results that in the production phase, the amount of acetate decreased from 0.54 g/l to 0.03 g/l, and the amount of ethanol decreased from 5.6 g/l to 4.9 g/l. In addition, the concentration of butyric acid increased from 0.05 g/L to 0.28 g/L, and the concentration of hexanoic acid increased from 0.03 g/L to 0.79 g/L.

Example 3

Preparation of hexanoic acid from butyric acid and ethanol with Clostridium kluyveri

For the biological conversion of ethanol and butyric acid to hexanoic acid, the bacterium Clostridium kluyveri was used. All cultivation steps were carried out under anaerobic conditions in pressure-resistant glass flasks that can be hermetically sealed with a butyl rubber stopper.

For pre-culture 100 ml DMSZ52 medium (pH = 7.0; 10 g/l K-acetate, 0.31 g/l K ₂ HPO ₄ , 0.23 g/l KH ₂ PO ₄ , 0.25 g/l l NH ₄ Cl, 0.20 g/l MgSO ₄ x 7H ₂ O, 1 g/l yeast extract, 0.50 mg/l resazurin, 10 µl/l HCl (25%, 7.7 M), 1, 5 mg/l FeCl ₂ x 4H ₂ O, 70 µg/l ZnCl ₂ x 7H ₂ O, 100 µg/l MnCl ₂ x 4H ₂ O, 6 µg/l H ₃ BO ₃ , 190 µg/l CoCl ₂ x 6H ₂ O, 2 µg/l CuCl ₂ x 6H ₂ O, 24 µg/l NiCl ₂ x 6H ₂ O, 36 µg/l Na ₂ MO ₄ x 2H ₂ O, 0.5 mg/l NaOH, 3 µg/l Na ₂ SeO ₃ x 5H ₂ O, 4 µg/l Na ₂ WO ₄ x 2H ₂ O, 100 µg/l vitamin B12, 80 µg/l p-aminobenzoic acid, 20 µg/l D(+)-biotin, 200 µg/l nicotinic acid, 100 µg/l D-Ca-pantothenate, 300 µg/l pyridoxine hydrochloride, 200 µg/l thiamine-HCl x 2H ₂ O, 20 ml/l ethanol, 2.5 g/l NaHCO ₃ , 0.25 g/l cysteine-HCl x H ₂ O, 0.25 g/l Na ₂ S x 9H ₂ O) in a 250 ml flask was inoculated with 5 ml of frozen Clostridium kluyveri cryoculture and incubated at 37°C for 144 h up to OD _{600 nm} > 0.3.

To obtain the main culture, 200 ml of fresh DMSZ52 medium in a 500 ml flask were inoculated with centrifuged cells from the preculture to an OD _{600 nm} of 0.1. This growing culture was incubated at 37°C for 25 hours until OD _{600 nm} > 0.4. The cell suspension was then centrifuged, washed with preparation buffer (pH 6.16; 4.16 g/l K-acetate, 10.0 g/l ethanol) and centrifuged again.

For production cultures, 200 ml of production buffer in a 500 ml flask was inoculated with washed cells from the main culture to an OD of _{600 nm} of 0.2. In the first culture, 1.0 g/L butyric acid was added to the preparation buffer at the beginning, in the second culture, no butyric acid was added to the preparation buffer. The cultures were stoppered with butyl rubber and incubated for 71 hours at 37°C and 100 rpm. in an open water bath with shaking. Samples were taken at the beginning and at the end of the culture period. They were tested for optical density, pH value and various analytes (tested by NMR).

It can be seen from the results that in the butyric acid supplemented culture phase, the amount of acetate decreased from 3.1 g/L to 1.1 g/L, and the amount of ethanol decreased from 10.6 g/L to 7.5 g/L. In addition, the concentration of butyric acid increased from 1.2 g/L to 2.2 g/L, and the concentration of hexanoic acid increased from 0.04 g/L to 2.30 g/L.

In the undersupplemented phase, acetate decreased from 3.0 g/L to 1.3 g/L and ethanol decreased from 10.2 g/L to 8.2 g/L. In addition, the concentration of butyric acid increased from 0.1 g/L to 1.7 g/L, and the concentration of hexanoic acid increased from 0.01 g/L to 1.40 g/L.

Example 4

Cultivation of Clostridium kluyveri in the presence of Dean and TOPO

The bacterium DSM555 Clostridium kluyveri (German DSMZ) was cultured for the biological conversion of ethanol and acetate to hexanoic acid. For in situ extraction of the obtained hexanoic acid for cultivation, a mixture of decane with trioctylphosphine oxide (TOPO) was added. All cultivation steps were carried out under anaerobic conditions in pressure-resistant glass flasks that can be hermetically sealed with a butyl rubber stopper.

For pre-culture 250 ml Veri01 medium (pH 7.0; 10 g/l potassium acetate, 0.31 g/l K ₂ HPO ₄ , 0.23 g/l KH ₂ PO ₄ , 0.25 g/l NH ₄ Cl, 0.20 g/l MgSO ₄ X 7H ₂ O, 10 µl/l HCl (7.7 M), 1.5 mg/l FeCl ₂ X 4H ₂ O, 36 µg/l ZnCl ₂ , 64 µg /l MnCl ₂ X 4H ₂ O, 6 µg/l H ₃ BO ₃ , 190 µg/l CoCl ₂ X 6H ₂ O, 1.2 µg/l CuCl ₂ X 6H ₂ O, 24 µg/l NiCl ₂ X 6H ₂ O, 36 µg/l Na ₂ MO ₄ X 2H ₂ O, 0.5 mg/l NaOH, 3 µg/l Na ₂ SeO ₃ X 5H ₂ O, 4 µg/l Na ₂ WO ₄ X 2H ₂ O, 100 µg/l vitamin B12, 80 µg/l p-aminobenzoic acid, 20 µg/l D(+)-biotin, 200 µg/l nicotinic acid, 100 µg/l D-Ca-pantothenate, 300 µg/l pyridoxine hydrochloride , 200 μg/l thiamine-HCl x 2H ₂ O, 20 ml/l ethanol, 2.5 g/l NaHCO ₃ , 65 mg/l glycine, 24 mg/l histidine, 64.6 mg/l isoleucine, 93, 8 mg/l leucine, 103 mg/l lysine, 60.4 mg/l arginine, 21.64 mg/l L-cysteine-HCl, 21 mg/l methionine, 52 mg/l proline, 56.8 mg/l serine, 59 mg/l threonine, 75.8 mg/l valine) were inoculated with 10 ml of a live culture of Clostridium kluyveri until starting OD _{600 nm} component 0.1.

The cultivation was carried out in a 1000 ml pressure-resistant glass flask at 37°C, 150 rpm. and ventilation intensity of 1 l/h. with 100% CO ₂ in a shaker in an open water bath for 671 h. The gas was released into the headspace of the product in the reactor. The pH was maintained at 6.2 by automatic addition of 100 g/l NaOH solution. Fresh medium was continuously fed into the reactor at a dilution rate of 2.0 d ^-1 and the fermentation broth was continuously removed from the reactor using a ^KrosFlo® hollow fiber polyethersulfone membrane with a pore size of 0.2 μm (Spectrumlabs, Rancho Dominguez, USA), with retention of cells in the reactor.

To obtain the main culture, 100 ml of fresh Veri01 medium in a 250 ml flask were inoculated with centrifuged cells from the preculture to an OD _{600 nm} of 0.1. An additional 1 ml of a mixture of 6% (w/w) TOPO in decane was added. The culture was closed with a butyl rubber stopper and incubated at 37°C and 150 rpm. in a shaker in an open water bath for 43 hours in an atmosphere of 100% CO ₂ .

During cultivation, several samples of 5 ml were taken to determine the OD _{600 nm} , the pH value and the degree of product formation. Determination of product concentrations was performed by semiquantitative analysis using 1H-NMR spectroscopy. Sodium trimethylsilyl propionate (T(M)SP) was used as an internal quantitative standard.

During the main cultivation, the concentration of butyrate increased from 0.14 g/l to 2.12 g/l, and the concentration of hexanoate increased from 0.22 g/l to 0.91 g/l, while the concentration of ethanol decreased from 15.04 to 11.98 g/l, and the concentration of acetate decreased from 6.01 to 4.23 g/l.

OD _{600 nm} decreased during this time from 0.111 to 0.076.

Example 5

Cultivation of Clostridium kluyveri in the presence of tetradecane and TOPO

The bacterium Clostridium kluyveri was cultivated for the biological conversion of ethanol and acetate to hexanoic acid. For in situ extraction of the obtained hexanoic acid for cultivation, a mixture of tetradecane with trioctylphosphine oxide (TOPO) was added. All cultivation steps were carried out under anaerobic conditions in pressure-resistant glass flasks that can be hermetically sealed with a butyl rubber stopper.

Clostridium kluyveri was pre-cultured in a 1000 ml pressure-resistant glass flask in 250 ml EvoDM24 medium (pH 5.5; 0.429 g/l Mg-acetate, 0.164 g/l Na-acetate, 0.016 g/l Ca-acetate, 2.454 g/l K-acetate, 0.107 ml/l H ₃ PO ₄ (8.5%), 0.7 g/l NH ₄ acetate, 0.35 mg/l Co-acetate, 1.245 mg/l Ni-acetate, 20 μg/l d-biotin, 20 μg/l folic acid, 10 μg/l pyridoxine-HCl, 50 μg/l thiamine-HCl, 50 μg/l riboflavin, 50 μg/l nicotinic acid, 50 μg/l Ca- pantothenate, 50 µg/l vitamin B12, 50 µg/l p-aminobenzoate, 50 µg/l lipoic acid, 0.702 mg/l (NH4) ₂ Fe(SO ₄ ) ₂ x 4H ₂ O, 1 ml/l KS-acetate (93.5 mM), 20 ml/l ethanol, 0.37 g/l acetic acid) at 37°C, 150 rpm. and a ventilation rate of 1 l/h with a mixture containing 25% CO ₂ and 75% N ₂ in a shaker in an open water bath. The gas was released into the headspace above the product in the reactor. The pH was maintained at 5.5 by automatic addition of 2.5 M NH ₃ solution. Fresh medium was continuously fed into the reactor at a dilution rate of 2.0 d ^-1 and the fermentation broth was continuously removed from the reactor using a ^KrosFlo® hollow fiber polyethersulfone membrane with a pore size of 0.2 μm (Spectrumlabs, Rancho Dominguez, USA), with retention of cells in the reactor and maintenance of OD _{600 nm} component ~1.5.

For the main culture 100 ml Veri01 medium (pH 6.5; 10 g/l potassium acetate, 0.31 g/l K ₂ HPO ₄ , 0.23 g/l KH ₂ PO ₄ , 0.25 g/l NH ₄ Cl, 0.20 g/l MgSO ₄ X 7H ₂ O, 10 µl/l HCl (7.7 M), 1.5 mg/l FeCl ₂ X 4H ₂ O, 36 µg/l ZnCl ₂ , 64 µg /l MnCl ₂ X 4H ₂ O, 6 µg/l H ₃ BO ₃ , 190 µg/l CoCl ₂ X 6H ₂ O, 1.2 µg/l CuCl ₂ X 6H ₂ O, 24 µg/l NiCl ₂ X 6H ₂ O, 36 µg/l Na ₂ MO ₄ X 2H ₂ O, 0.5 mg/l NaOH, 3 µg/l Na ₂ SeO ₃ X 5H ₂ O, 4 µg/l Na ₂ WO ₄ X 2H ₂ O, 100 µg/l vitamin B12, 80 µg/l p-aminobenzoic acid, 20 µg/l D(+)-biotin, 200 µg/l nicotinic acid, 100 µg/l D-Ca-pantothenate, 300 µg/l pyridoxine hydrochloride , 200 μg/l thiamine-HCl x 2H ₂ O, 20 ml/l ethanol, 2.5 g/l NaHCO ₃ , 65 mg/l glycine, 24 mg/l histidine, 64.6 mg/l isoleucine, 93, 8 mg/l leucine, 103 mg/l lysine, 60.4 mg/l arginine, 21.64 mg/l L-cysteine-HCl, 21 mg/l methionine, 52 mg/l proline, 56.8 mg/l serine, 59 mg/l threonine, 75.8 mg/l valine, 2.5 ml/l HCl 25%) in a 250 ml flask were inoculated subjected to mi centrifugation of cells from pre-culture to OD _{600 nm} component 0.1. An additional 1 ml of a mixture of 6% (w/w) TOPO in tetradecane was added. The culture was closed with a butyl rubber stopper and incubated at 37°C and 150 rpm. in a shaker in an open water bath for 47 hours in an atmosphere of 100% CO ₂ .

During cultivation, several samples of 5 ml were taken to determine the OD _{600 nm} , the pH value and the degree of product formation. Determination of product concentrations was performed by semiquantitative analysis using 1H-NMR spectroscopy. Sodium trimethylsilyl propionate (T(M)SP) was used as an internal quantitative standard.

During the main cultivation, the concentration of butyrate increased from 0.05 g/l to 3.78 g/l, and the concentration of hexanoate increased from 0.09 g/l to 4.93 g/l, while the concentration of ethanol decreased from 15.52 to 9.36 g/l, and the concentration of acetate decreased from 6.36 to 2.49 g/l.

OD _{600 nm} increased during this time from 0.095 to 0.685.

Example 6

Cultivation of Clostridium kluyveri in the presence of hexadecane and TOPO

The bacterium Clostridium kluyveri was cultivated for the biological conversion of ethanol and acetate to hexanoic acid. For in situ extraction of the obtained hexanoic acid for cultivation, a mixture of hexadecane with trioctylphosphine oxide (TOPO) was added. All cultivation steps were carried out under anaerobic conditions in pressure-resistant glass flasks that can be hermetically sealed with a butyl rubber stopper.

For pre-culture 250 ml Veri01 medium (pH 7.0; 10 g/l potassium acetate, 0.31 g/l K ₂ HPO ₄ , 0.23 g/l KH ₂ PO ₄ , 0.25 g/l NH ₄ Cl, 0.20 g/l MgSO ₄ X 7H ₂ O, 10 µl/l HCl (7.7 M), 1.5 mg/l FeCl ₂ X 4H ₂ O, 36 µg/l ZnCl ₂ , 64 µg /l MnCl ₂ X 4H ₂ O, 6 µg/l H ₃ BO ₃ , 190 µg/l CoCl ₂ X 6H ₂ O, 1.2 µg/l CuCl ₂ X 6H ₂ O, 24 µg/l NiCl ₂ X 6H ₂ O, 36 µg/l Na ₂ MO ₄ X 2H ₂ O, 0.5 mg/l NaOH, 3 µg/l Na ₂ SeO ₃ X 5H ₂ O, 4 µg/l Na ₂ WO ₄ X 2H ₂ O, 100 µg/l vitamin B12, 80 µg/l p-aminobenzoic acid, 20 µg/l D(+)-biotin, 200 µg/l nicotinic acid, 100 µg/l D-Ca-pantothenate, 300 µg/l pyridoxine hydrochloride , 200 μg/l thiamine-HCl x 2H ₂ O, 20 ml/l ethanol, 2.5 g/l NaHCO ₃ , 65 mg/l glycine, 24 mg/l histidine, 64.6 mg/l isoleucine, 93, 8 mg/l leucine, 103 mg/l lysine, 60.4 mg/l arginine, 21.64 mg/l L-cysteine-HCl, 21 mg/l methionine, 52 mg/l proline, 56.8 mg/l serine, 59 mg/l threonine, 75.8 mg/l valine) were inoculated with 10 ml of a live culture of Clostridium kluyveri until starting OD _{600 nm} component 0.1.

The cultivation was carried out in a 1000 ml pressure-resistant glass flask at 37°C, 150 rpm. and ventilation intensity of 1 l/h. with 100% CO ₂ in a shaker in an open water bath for 671 h. The gas was released into the headspace of the product in the reactor. The pH was maintained at 6.2 by automatic addition of 100 g/l NaOH solution. Fresh medium was continuously fed into the reactor at a dilution rate of 2.0 d ^-1 and the fermentation broth was continuously removed from the reactor using a ^KrosFlo® hollow fiber polyethersulfone membrane with a pore size of 0.2 μm (Spectrumlabs, Rancho Dominguez, USA), with retention of cells in the reactor.

To obtain the main culture, 100 ml of fresh Veri01 medium in a 250 ml flask were inoculated with centrifuged cells from the preculture to an OD _{600 nm} of 0.1. An additional 1 ml of a mixture of 6% (w/w) TOPO in hexadecane was added. The culture was closed with a butyl rubber stopper and incubated at 37°C and 150 rpm. in a shaker in an open water bath for 43 hours in an atmosphere of 100% CO ₂ .

During cultivation, several samples of 5 ml were taken to determine the OD _{600 nm} , the pH value and the degree of product formation. Determination of product concentrations was performed by semiquantitative analysis using 1H-NMR spectroscopy. Sodium trimethylsilyl propionate (T(M)SP) was used as an internal quantitative standard.

During the main cultivation, the concentration of butyrate increased from 0.14 g/l to 2.86 g/l, and the concentration of hexanoate increased from 0.20 g/l to 2.37 g/l, while the concentration of ethanol decreased from 14.59 to 10.24 g/l, and the concentration of acetate decreased from 5.87 to 3.32 g/l.

OD _{600 nm} increased during this time from 0.091 to 0.256.

Example 7

Cultivation of Clostridium kluyveri in the presence of heptadecane and TOPO

The bacterium Clostridium kluyveri was cultivated for the biological conversion of ethanol and acetate to hexanoic acid. For in situ extraction of the obtained hexanoic acid for cultivation, a mixture of heptadecane with trioctylphosphine oxide (TOPO) was added. All cultivation steps were carried out under anaerobic conditions in pressure-resistant glass flasks that can be hermetically sealed with a butyl rubber stopper.

For pre-culture 250 ml Veri01 medium (pH 7.0; 10 g/l potassium acetate, 0.31 g/l K ₂ HPO ₄ , 0.23 g/l KH ₂ PO ₄ , 0.25 g/l NH ₄ Cl, 0.20 g/l MgSO ₄ X 7H ₂ O, 10 µl/l HCl (7.7 M), 1.5 mg/l FeCl ₂ X 4H ₂ O, 36 µg/l ZnCl ₂ , 64 µg /l MnCl ₂ X 4H ₂ O, 6 µg/l H ₃ BO ₃ , 190 µg/l CoCl ₂ X 6H ₂ O, 1.2 µg/l CuCl ₂ X 6H ₂ O, 24 µg/l NiCl ₂ X 6H ₂ O, 36 µg/l Na ₂ MO ₄ X 2H ₂ O, 0.5 mg/l NaOH, 3 µg/l Na ₂ SeO ₃ X 5H ₂ O, 4 µg/l Na ₂ WO ₄ X 2H ₂ O, 100 µg/l vitamin B12, 80 µg/l p-aminobenzoic acid, 20 µg/l D(+)-biotin, 200 µg/l nicotinic acid, 100 µg/l D-Ca-pantothenate, 300 µg/l pyridoxine hydrochloride , 200 μg/l thiamine-HCl x 2H ₂ O, 20 ml/l ethanol, 2.5 g/l NaHCO ₃ , 65 mg/l glycine, 24 mg/l histidine, 64.6 mg/l isoleucine, 93, 8 mg/l leucine, 103 mg/l lysine, 60.4 mg/l arginine, 21.64 mg/l L-cysteine-HCl, 21 mg/l methionine, 52 mg/l proline, 56.8 mg/l serine, 59 mg/l threonine, 75.8 mg/l valine) were inoculated with 10 ml of a live culture of Clostridium kluyveri until starting OD _{600 nm} component 0.1.

The cultivation was carried out in a 1000 ml pressure-resistant glass flask at 37°C, 150 rpm. and ventilation intensity of 1 l/h. with 100% CO ₂ in a shaker in an open water bath for 671 h. The gas was released into the headspace of the product in the reactor. The pH was maintained at 6.2 by automatic addition of 100 g/l NaOH solution. Fresh medium was continuously fed into the reactor at a dilution rate of 2.0 d ^-1 and the fermentation broth was continuously removed from the reactor using a ^KrosFlo® hollow fiber polyethersulfone membrane with a pore size of 0.2 μm (Spectrumlabs, Rancho Dominguez, USA), with retention of cells in the reactor.

To obtain the main culture, 100 ml of fresh Veri01 medium in a 250 ml flask were inoculated with centrifuged cells from the preculture to an OD _{600 nm} of 0.1. An additional 1 ml of a mixture of 6% (w/w) TOPO in heptadecane was added. The culture was closed with a butyl rubber stopper and incubated at 37°C and 150 rpm. in a shaker in an open water bath for 43 hours in an atmosphere of 100% CO ₂ .

During cultivation, several samples of 5 ml were taken to determine the OD _{600 nm} , the pH value and the degree of product formation. Determination of product concentrations was performed by semiquantitative analysis using 1H-NMR spectroscopy. Sodium trimethylsilyl propionate (T(M)SP) was used as an internal quantitative standard.

During the main cultivation, the concentration of butyrate increased from 0.15 g/l to 2.82 g/l, and the concentration of hexanoate increased from 0.19 g/l to 2.85 g/l, while the concentration of ethanol decreased from 14.34 to 9.58 g/l, and the concentration of acetate decreased from 5.88 to 3.20 g/l.

OD _{600 nm} increased during this time from 0.083 to 0.363.

Example 8

Cultivation of Clostridium kluyveri in the presence of dodecane and TOPO

The bacterium Clostridium kluyveri was cultivated for the biological conversion of ethanol and acetate to hexanoic acid. For in situ extraction of the obtained hexanoic acid for cultivation, a mixture of dodecane with trioctylphosphine oxide (TOPO) was added. All cultivation steps were carried out under anaerobic conditions in pressure-resistant glass flasks that can be hermetically sealed with a butyl rubber stopper.

For pre-culture 250 ml Veri01 medium (pH 7.0; 10 g/l potassium acetate, 0.31 g/l K ₂ HPO ₄ , 0.23 g/l KH ₂ PO ₄ , 0.25 g/l NH ₄ Cl, 0.20 g/l MgSO ₄ X 7H ₂ O, 10 µl/l HCl (7.7 M), 1.5 mg/l FeCl ₂ X 4H ₂ O, 36 µg/l ZnCl ₂ , 64 µg /l MnCl ₂ X 4H ₂ O, 6 µg/l H ₃ BO ₃ , 190 µg/l CoCl ₂ X 6H ₂ O, 1.2 µg/l CuCl ₂ X 6H ₂ O, 24 µg/l NiCl ₂ X 6H ₂ O, 36 µg/l Na ₂ MO ₄ X 2H ₂ O, 0.5 mg/l NaOH, 3 µg/l Na ₂ SeO ₃ X 5H ₂ O, 4 µg/l Na ₂ WO ₄ X 2H ₂ O, 100 µg/l vitamin B12, 80 µg/l p-aminobenzoic acid, 20 µg/l D(+)-biotin, 200 µg/l nicotinic acid, 100 µg/l D-Ca-pantothenate, 300 µg/l pyridoxine hydrochloride , 200 μg/l thiamine-HCl x 2H ₂ O, 20 ml/l ethanol, 2.5 g/l NaHCO ₃ , 65 mg/l glycine, 24 mg/l histidine, 64.6 mg/l isoleucine, 93, 8 mg/l leucine, 103 mg/l lysine, 60.4 mg/l arginine, 21.64 mg/l L-cysteine-HCl, 21 mg/l methionine, 52 mg/l proline, 56.8 mg/l serine, 59 mg/l threonine, 75.8 mg/l valine) were inoculated with 10 ml of a live culture of Clostridium kluyveri until starting OD _{600 nm} component 0.1.

The cultivation was carried out in a 1000 ml pressure-resistant glass flask at 37°C, 150 rpm. and ventilation intensity of 1 l/h. with 100% CO ₂ in a shaker in an open water bath for 671 h. The gas was released into the headspace of the product in the reactor. The pH was maintained at 6.2 by automatic addition of 100 g/l NaOH solution. Fresh medium was continuously fed into the reactor at a dilution rate of 2.0 d ^-1 and the fermentation broth was continuously removed from the reactor using a ^KrosFlo® hollow fiber polyethersulfone membrane with a pore size of 0.2 μm (Spectrumlabs, Rancho Dominguez, USA), with retention of cells in the reactor.

To obtain the main culture, 100 ml of fresh Veri01 medium in a 250 ml flask were inoculated with centrifuged cells from the preculture to an OD _{600 nm} of 0.1. An additional 1 ml of a mixture of 6% (w/w) TOPO in dodecane was added. The culture was closed with a butyl rubber stopper and incubated at 37°C and 150 rpm. in a shaker in an open water bath for 43 hours in an atmosphere of 100% CO ₂ .

During cultivation, several samples of 5 ml were taken to determine the OD _{600 nm} , the pH value and the degree of product formation. Determination of product concentrations was performed by semiquantitative analysis using 1H-NMR spectroscopy. Sodium trimethylsilyl propionate (T(M)SP) was used as an internal quantitative standard.

During the main cultivation, the concentration of butyrate increased from 0.14 g/l to 2.62 g/l, and the concentration of hexanoate increased from 0.22 g/l to 2.05 g/l, while the concentration of ethanol decreased from 14.62 to 10.64 g/l, and the concentration of acetate decreased from 5.92 to 3.54 g/l.

OD _{600 nm} increased during this time from 0.091 to 0.259.

Example 9

Determination of the partition coefficient of hexanoic acid between water and a mixture of hexadecane and TOPO

During all stages of the experiment, samples were taken from both phases to determine the pH value and the concentration of hexanoic acid using high performance liquid chromatography (HPLC). With 100 g of an aqueous solution of 5 g/kg of hexanoic acid and 33 g of a mixture of 6% trioctylphosphine oxide (TOPO) in hexadecane, a separating funnel was filled and mixed for 1 minute at 37°C. Then the funnel was placed in a ring on a tripod and the emulsion was left to settle with spontaneous separation. The pH value of the aqueous phase was measured. Then 1 M NaOH solution was added to the funnel and mixed. The separation and sampling step was repeated until a pH of 6.2 was reached in the aqueous phase. Samples were taken from both phases for subsequent analysis at this point in time. The aqueous phase can be analyzed directly with HPLC. For organic phase analysis, diluted hexanoic acid was first re-extracted into water (pH 12.0 by adding 1 M NaOH) and then analyzed by HPLC. The partition coefficient K _D of hexanoic acid in water and 6% TOPO in hexadecane was calculated from the concentrations of hexanoic acid in both phases.

The K _D for hexanoic acid in water and 6% TOPO in hexadecane at pH 6.2 was 4.7.

Example 10

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

During all stages of the experiment, samples were taken from both phases to determine the pH value and the concentration of hexanoic acid using high performance liquid chromatography (HPLC). With 100 g of an aqueous solution of 5 g/kg of hexanoic acid and 33 g of a mixture of 6% trioctylphosphine oxide (TOPO) in heptadecane, a separating funnel was filled and mixed for 1 minute at 37°C. Then the funnel was placed in a ring on a tripod and the emulsion was left to settle with spontaneous separation. The pH value of the aqueous phase was measured. Added 1 M NaOH solution to the funnel and mixed. The separation and sampling step was repeated until a pH of 6.2 was reached in the aqueous phase. Samples were taken from both phases for subsequent analysis at this point in time. The aqueous phase can be analyzed directly with HPLC. For organic phase analysis, diluted hexanoic acid was first re-extracted into water (pH 12.0 by adding 1 M NaOH) and then analyzed by HPLC. The partition coefficient K _D of hexanoic acid in water and 6% TOPO in heptadecane was calculated from the concentrations of hexanoic acid in both phases.

The K _D for hexanoic acid in water and 6% TOPO in heptadecane at pH 6.2 was 5.0.

Example 11

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

During all stages of the experiment, samples were taken from both phases to determine the pH value and the concentration of hexanoic acid using high performance liquid chromatography (HPLC). A separating funnel was filled with 130 g of an aqueous solution of 5 g/kg of hexanoic acid plus 0.5 g/kg of acetic acid and 15 g of a mixture of 6% trioctylphosphine oxide (TOPO) in tetradecane and mixed for 1 minute at 37°C. Then the funnel was placed in a ring on a tripod and the emulsion was left to settle with spontaneous separation. The pH value of the aqueous phase was measured. Added 1 M NaOH solution to the funnel and mixed. The separation and sampling step was repeated until a pH of 6.2 was reached in the aqueous phase. Samples were taken from both phases for subsequent analysis at this point in time. The aqueous phase can be analyzed directly with HPLC. For organic phase analysis, diluted hexanoic acid was first re-extracted into water (pH 12.0 by adding 1 M NaOH) and then analyzed by HPLC. The partition coefficient K _D of hexanoic acid in water and 6% TOPO in tetradecane was calculated from the concentrations of hexanoic acid in both phases.

The K _D for hexanoic acid in water and 6% TOPO in tetradecane at pH 6.9 was 1.3.

Example 12

Cultivation of Clostridium kluyveri with in situ extraction of hexanoic acid

The bacterium Clostridium kluyveri was cultivated for the biological conversion of ethanol and acetate to hexanoic acid. For in situ extraction of the obtained hexanoic acid, a mixture of tetradecane with trioctylphosphine oxide (TOPO) was continuously passed through during cultivation. All cultivation steps were carried out under anaerobic conditions in pressure-resistant glass flasks that can be hermetically sealed with a butyl rubber stopper.

Pre-cultivation of Clostridium kluyveri was carried out in a pressure-resistant 1000 ml glass flask in 250 ml of EvoDM45 medium (pH 5.5; 0.004 g/l Mg-acetate, 0.164 g/l Na-acetate, 0.016 g/l Ca-acetate, 0 .25 g/l K-acetate, 0.107 ml/l H ₃ PO ₄ (8.5%), 2.92 g/l NH ₄ acetate, 0.35 mg/l Co-acetate, 1.245 mg/l Ni- acetate, 20 µg/l d-biotin, 20 µg/l folic acid, 10 µg/l pyridoxine-HCl, 50 µg/l thiamine-HCl, 50 µg/l riboflavin, 50 µg/l nicotinic acid, 50 µg/l Ca-pantothenate, 50 µg/l vitamin B12, 50 µg/l p-aminobenzoate, 50 µg/l lipoic acid, 0.702 mg/l (NH4) ₂ Fe(SO ₄ ) ₂ x 4 H ₂ O, 1 ml/l KS-acetate (93.5 mM), 20 ml/l ethanol, 0.37 g/l acetic acid) at 37°C, 150 rpm. and a ventilation rate of 1 l/h with a mixture containing 25% CO ₂ and 75% N ₂ in a shaker in an open water bath. The gas was released into the headspace above the product in the reactor. The pH was maintained at 5.5 by automatic addition of 2.5 M NH ₃ solution. Fresh medium was continuously fed into the reactor at a dilution rate of 2.0 d ^-1 and the fermentation broth was continuously removed from the reactor using a ^KrosFlo® hollow fiber polyethersulfone membrane with a pore size of 0.2 μm (Spectrumlabs, Rancho Dominguez, USA), with retention of cells in the reactor and maintenance of OD _{600 nm} component ~1.5.

For base culture 150 ml EvoDM39 medium (pH 5.8; 0.429 g/l Mg-acetate, 0.164 g/l Na-acetate, 0.016 g/l Ca-acetate, 2.454 g/l K-acetate, 0.107 ml/l H ₃ PO ₄ (8.5%), 1.01 ml/l acetic acid, 0.35 mg/l Co-acetate, 1.245 mg/l Ni-acetate, 20 µg/l d-biotin, 20 µg/l folic acid, 10 µg/l pyridoxine-HCl, 50 µg/l thiamine-HCl, 50 µg/l riboflavin, 50 µg/l nicotinic acid, 50 µg/l Ca-pantothenate, 50 µg/l vitamin B12, 50 µg/l l p-aminobenzoate, 50 µg/l lipoic acid, 0.702 mg/l (NH4) ₂ Fe(SO ₄ ) ₂ x 4H ₂ O, 1 ml/l KS-acetate (93.5 mM), 20 ml/l ethanol , 8.8 ml NH ₃ solution (2.5 mol/l), 27.75 ml/l acetic acid (144 g/l))

in a 1000 ml flask, 100 ml of cell broth from the pre-culture was inoculated to an OD of _{600 nm} of 0.71.

Cultivation was carried out at 37°C, 150 rpm. and a ventilation rate of 1 l/h with a mixture of 25% CO ₂ and 75% N ₂ in a shaker in an open water bath for 65 hours. The gas was released into the headspace in the reactor. The pH was maintained at 5.8 by automatic addition of 2.5 M NH ₃ solution. Fresh medium was continuously fed into the reactor at a dilution rate of 0.5 d ^-1 and the fermentation broth was continuously removed from the reactor by maintaining an OD _{600 nm} of ~0.5. An additional 120 g of a mixture of 6% (w/w) TOPO in tetradecane was added to the fermentation broth. Then this organic mixture was continuously fed into the reactor and the organic phase was also continuously removed from the reactor with a dilution ratio of 1 d ^-1 .

During cultivation, several 5 ml samples were taken from both the aqueous and organic phases to determine the OD _{600 nm} , the pH value and the degree of product formation. Determination of product concentrations was performed by semiquantitative analysis using 1H-NMR spectroscopy. Sodium trimethylsilyl propionate (T(M)SP) was used as an internal quantitative standard.

During the main cultivation in the aqueous phase, a steady state concentration of 8.18 g/l ethanol, 3.20 g/l acetate, 1.81 g/l butyrate and 0.81 g/l hexanoate was reached. The OD _{600 nm} value remained constant at 0.5. Steady-state concentrations of 0.43 g/kg ethanol, 0.08 g/kg acetate, 1.13 g/kg butyrate and 8.09 g/kg hexanoate were reached in the organic phase. After the experiment, the cells remained viable despite being transferred to additional culture steps.

The partition coefficient K _D of the substrates and products in the aqueous medium system and 6% TOPO in tetradecane was calculated from the concentrations in both phases.

K _D at equilibrium was 0.05 for ethanol, 0.03 for acetic acid, 0.62 for butyric acid and 9.99 for hexanoic acid.

Claims (20)

1. A method for extracting hexanoic acid and/or its ester from an aqueous fermentation medium, wherein the hexanoic acid and/or its ester is or is produced by specific cells from a carbon source, the method comprising (a) contacting hexanoic acid and/or its ester in the aqueous fermentation medium with at least one extractant for a period of time sufficient to extract the hexanoic acid and/or its ester from the aqueous medium into the extractant, (b) separating the extractant with extracted hexanoic acid and/or its ester from the aqueous fermentation medium, where the extractant provides - a mixture of at least one alkylphosphine oxide and at least one alkane, where the alkane contains at least 12 carbon atoms. 2. The method according to claim 1, where the alkane contains from 12 to 18 carbon atoms. 3. The method of claim 2 wherein the alkane is hexadecane. 4. Process according to any of the preceding claims, wherein hexanoic acid is produced from synthesis gas, wherein the process for producing hexanoic acid comprises - bringing the synthesis gas into contact with at least one species of bacteria capable of implementing the Wood-Ljungdahl and ethanol-carboxylate fermentation pathways to produce hexanoic acid. 5. The method according to claim 4, where the bacteria are selected from the group, consisting of Clostridium kluyveri and C. carboxidivorans. 6. The method according to any one of the preceding claims, wherein the weight ratio of at least one alkyl phosphine oxide and alkane is from 1:100 to 1:10. 7. The method according to any one of the preceding claims, wherein at least one alkylphosphine oxide is trioctylphosphine oxide (TOPO). 8. A method according to any one of the preceding claims, wherein the pH of the aqueous medium is maintained between 5.5 and 7. 9. The method according to any one of the preceding claims, wherein the extractant is recycled. 10. The method according to any one of paragraphs. 1-9, where the extractant is present in the fermentation medium when hexanoic acid and/or its ester is produced by specific cells from a carbon source. 11. The use of a mixture of at least one alkyl phosphine oxide and an alkane for extracting hexanoic acid and/or its ester from an aqueous fermentation medium, where the hexanoic acid and/or its ester is or is produced by specific cells from a carbon source, where the alkane contains at least 12 carbon atoms. 12. Use according to claim 11, where the alkane contains from 12 to 18 carbon atoms. 13. Use according to claim 11 or 12, where the alkane is hexadecane. 14. Use according to any one of the preceding claims, wherein at least one alkylphosphine oxide is trioctylphosphine oxide (TOPO).