Biotechnological production of caprylic acid
By combining the Clostridium krusei chain elongation reaction with the trialkyl-phosphine-oxide extraction medium, the problem of low yield in octanoic acid production was solved, achieving efficient and low-cost production of octanoic acid and its salts.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- EVONIK OPERATIONS GMBH
- Filing Date
- 2024-10-16
- Publication Date
- 2026-06-05
AI Technical Summary
Existing technologies have low production yields and high costs for octanoic acid, making it difficult to effectively produce octanoic acid and its salts through microbial chain extension methods.
The chain elongation reaction was carried out using Clostridium krychnifolia. Octanoic acid and its salts were treated separately in the adaptation and production stages using a mixed extraction medium of trialkyl-phosphine oxides and alkanes, thereby improving the yield of octanoic acid and its salts.
It significantly increased the production of octanoic acid and its salts, with a yield increase of at least 1.5 times, reduced production costs, and achieved highly efficient biotechnology production.
Abstract
Description
Technical Field
[0001] This invention relates to a biotechnological method for producing octanoic acid. Specifically, this method involves using *Clostridium kraniliforme* (Clostridium kraniliforme) from ethanol or a mixture of ethanol and acetate. Clostridium kluyveri The yield of octanoic acid produced was increased. Background Technology
[0002] Octanoic acid, also known as caprylic acid, is commonly used in several fields, including health promotion, disease control, cosmetics, and industry. For example, it can be used as an antimicrobial pesticide for surface disinfection in the food and dairy industries, and as a disinfectant in some healthcare fields and services. Octanoic acid is also widely used in the commercial production of esters and in the manufacture of fragrances and dyes.
[0003] Currently, it is a common industrial chemical produced through the oxidation of C8 aldehydes. Octanoic acid compounds are naturally found in the milk of various mammals and are minor components of coconut oil and palm kernel oil. However, environmentally friendly and bio-based alternatives are needed for the large-scale production of octanoic acid and its salts.
[0004] Octanoate is known to be produced as a byproduct in chain-elongation processes using microorganisms capable of elongating dilute ethanol and acetate into hexanoate. Clostridium krusei and Firmicutes are particularly well-known for producing hexanoate from simple carbon sources such as ethanol. In this process of producing hexanoate, n-caprylate, if produced, is a minor byproduct. n-octanoate is approximately twice as valuable as hexanoate (by weight). Because n-octanoate is far more valuable (by weight) than hexanoate, there is a need in the art to use microbial chain-elongation methods to produce more octanoate (relative to hexanoate) from ethanol.
[0005] However, in order to be feasible and superior to current methods of producing octanoic acid, not only must the yield of octanoic acid be increased, but the production must also be cost-effective. Summary of the Invention
[0006] This invention seeks to address the aforementioned problems by providing a method for producing octanoic acid and its salts from a carbon source using microorganisms capable of chain elongation. The carbon source can be ethanol or a mixture of ethanol and acetate. Specifically, the microorganisms capable of chain elongation can be *Clostridium kuristegi* or a mixed culture of microorganisms containing *Clostridium kuristegi*. In particular, the method includes the necessary step of removing some or all of the octanoic acid and / or its salts, which are produced during fermentation from ethanol or a mixture of ethanol and acetate. The step of removing the produced octanoic acid and / or its salts can be carried out during the acclimatization phase, the production phase, or both phases. Simultaneous extraction of octanoic acid and its salts during production plays a crucial role in achieving previously unreported production levels of octanoic acid and its salts, particularly during fermentation.
[0007] During the acclimatization phase, cells according to any aspect of the invention are adapted to produce caprylic acid in the fermentation medium in the presence of Clostridium kuristeum. During the production phase, the adapted cells are able to produce caprylic acid in significant yields (i.e., cells produce caprylic acid at optimal levels).
[0008] In one aspect of the invention, a method for producing octanoic acid and / or its salts is provided, the method comprising: a) Contact a carbon substrate containing ethanol or a mixture of ethanol and acetate with an aqueous medium containing Clostridium krillion; b) Keep the aqueous medium in place for an appropriate period of time until Clostridium kuriformis produces large amounts of octanoic acid and / or its salts; c) Separate octanoic acid and / or its salts from an aqueous medium using an extraction medium; and d) Repeat step (c) continuously as octanoic acid and / or its salts are formed during the production stage.
[0009] The extraction medium comprises: - At least one trialkyl-phosphine oxide and at least one alkane.
[0010] The method according to any aspect of the invention is particularly advantageous because a particular combination of steps (a) to (d), especially step (d), produces an unreported amount of octanoic acid.
[0011] Octanoic acid and its salts refer to and include the undissociated species (octanoic acid) and the dissociated species (octanoate), a carboxylate. Octanoic acid and octanoate are also referred to as caprylic acid and caprylate, respectively. Octanoate can have various counter cations. Examples of suitable cations include sodium ions, potassium ions, etc. The method according to any aspect of the invention primarily produces octanoic acid and its salts. In one example, octanoic acid is produced. In another example, octanoate is produced. In yet another example, both octanoic acid and octanoate are produced. Other carboxylate can also be produced in the method according to the invention, but these carboxylate are produced at much lower concentrations and are not the target products. The method according to any aspect of the invention uses Clostridium krychnifolium, for example under environmental pressure, to reduce capital costs by ensuring a simple bioreactor design.
[0012] As used herein, the term "carbon substrate" refers to ethanol alone or a combination of ethanol and acetate in a high ethanol-acetic acid ratio. For example, a high ethanol-acetic acid ratio can be at least a 5:1 molar ratio.
[0013] As used herein, the term “acetate” refers to both acetic acid and its salts, which is unavoidable because, as is known in the art, microorganisms work in aqueous environments and there is always a balance between salts and acids.
[0014] As used herein, the term "contact" refers to the direct contact between the cells and the carbon-containing substrate in step (a) according to any aspect of the invention. For example, in step (a), the cells and the carbon-containing substrate may be in different compartments. They may then be brought together in a fermenter.
[0015] The terms "aqueous solution" or "medium" include any solution containing water, primarily water as a solvent, which can be used, at least temporarily, to maintain the metabolic activity and / or viability of cells according to any aspect of the invention, and, if necessary, to contain any additional substrate. Those skilled in the art are familiar with the preparation of various aqueous solutions, commonly referred to as media suitable for maintaining cells of the invention, such as LB medium in the case of *Escherichia coli*, ATCC1754 medium in the case of *Clostridium yangniger*, and EvoDM24 medium (pH 5.5; 0.429 g / L magnesium acetate, 0.164 g / L sodium acetate, 0.016 g / L calcium acetate, 2.454 g / L potassium acetate, 0.107 mL / L H3PO4 (8.5%), 0.7 g / L ammonium acetate, 0.35 mg / L cobalt acetate, 1.245 mg / L nickel acetate, 20 μg / L...) The formula includes d-biotin, 20 μg / L folic acid, 10 μg / L pyridoxine hydrochloride, 50 μg / L thiamine hydrochloride, 50 μg / L riboflavin, 50 μg / L niacin, 50 μg / L calcium pantothenate, 50 μg / L vitamin B12, 50 μg / L para-aminobenzoate, 50 μg / L lipoic acid, 0.702 mg / L (NH4)2Fe(SO4)2x 4H2O, 1 mL / L L-cysteine (93.5 mM), 20 mL / L ethanol, and 0.37 g / L acetic acid. It is advantageous to use a minimal medium (i.e., a rationally simple composition of medium) as an aqueous solution, containing only the minimum group of salts and nutrients necessary to maintain cells in a metabolically active and / or viable state. This avoids unnecessary contamination of the product by unwanted byproducts compared to complex media. For example, M9 medium can be used as a minimal medium.
[0016] Clostridium krusei (DSM555) cells are incubated with a carbon substrate for a sufficiently long time (i.e., an appropriate time period) to produce the desired product (octanoic acid and its salt). For example, at least 1 hour, 2 hours, 4 hours, 5 hours, 10 hours, or 20 hours.
[0017] In one example, *Clostridium krillonii* cells according to any aspect of the invention, particularly step (b), are incubated with a carbon substrate for at least two phases: an acclimation phase and a production phase for producing caprylic acid and / or its salts. Caprylic acid and / or its salts are produced during both phases. For example, the carbon substrate according to any aspect of the invention is contacted with *Clostridium krillonii* cells, and during the acclimation phase (e.g., lasting at least one day), the desired amount of caprylic acid and / or its salts is produced (i.e., the optimal production rate of caprylic acid and / or its salts), wherein the *Clostridium krillonii* cells reach their optimal, efficient chain elongation level. During the production phase, the cells according to any aspect of the invention continue to produce caprylic acid and / or its salts at the optimal level.
[0018] In step (b) according to any aspect of the invention, the cells are maintained at a pH of 5-8, particularly 5-7.5, 5-7, 5-6.5, 5-6, or 5-5.5, and at a temperature of 10 to 42°C, preferably 30 to 40°C, particularly 32 to 38°C. The selected temperature and pH must allow the cells according to any aspect of the invention to maintain their catalytic and / or metabolic activity. During this step, octanoic acid and / or its salts are produced to the desired yield and continuously produced at the desired yield.
[0019] The resulting octanoic acid and / or its salts are partially or completely continuously removed or extracted from an aqueous medium (also known as a reaction mixture), wherein octanoic acid and / or its salts are produced by fermentation in the presence of Clostridium krillion and a carbon substrate, ethanol, or a mixture of ethanol and acetate. This step allows for increased yields of octanoic acid and / or its salts according to any aspect of the invention. In particular, octanoic acid and / or its salts are extracted simultaneously while they are being produced.
[0020] In one instance, the extraction medium is added to an aqueous medium containing cells and carbon substrates according to any aspect of the invention.
[0021] Specifically, in step (c), an extraction medium is used to separate octanoic acid and / or its salts from the aqueous medium. More specifically, the extraction method according to any aspect of the invention allows for increased yields 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 octanoic acid and / or its salts compared to using only pure alkanes. Thus, with a small volume of extraction medium, a greater yield of octanoic acid and / or its salts can be extracted. The extraction medium is also harmless to microorganisms. Therefore, when octanoic acid and / or its salts are produced biotechnically, an extraction medium according to any aspect of the invention can be present. Furthermore, octanoic acid and / or its salts can then be readily separated from the extraction medium using any method known in the art (e.g., distillation, funnel, column, etc.). After separating octanoic acid and / or its salts from the extraction medium, the extraction medium can be easily recycled.
[0022] The extraction medium may also be referred to as an "extraction medium". This extraction medium can be used to extract / separate octanoic acid and / or its salts produced according to any aspect of the invention from an aqueous medium in which octanoic acid and / or its salts are initially produced. At the end of the extraction step, excess water from the aqueous medium can be removed, thereby producing an extraction medium containing the extracted octanoic acid and / or its salts. The extraction medium may contain a combination of compounds that provide an effective means of extracting octanoic acid and / or its salts from an aqueous medium. In particular, the extraction medium may contain: (i) at least an alkane comprising at least 12 carbon atoms, and (ii) at least one molecule of trialkylphosphine oxide (TAPO).
[0023] Extraction media according to any aspect of the invention can effectively extract octanoic acid and / or its salts into an alkane-alkyl-phosphine oxide extraction medium. When the mixture is effectively used in the extraction of octanoic acid and / or its salts in the presence of a fermentation medium, such an extraction medium of a mixture of alkyl-phosphine oxides and at least one alkane is considered suitable for the method according to any aspect of the invention. In particular, the mixture of alkyl-phosphine oxides and at least one alkane is considered to operate better than any method currently known in the art for the extraction of octanoic acid because it requires no special equipment to implement, is relatively easy to operate, and has a high product yield.
[0024] In particular, the alkyl-phosphine oxide (also known as trialkyl-phosphine oxide (TAPO)) used in the method according to any aspect of the invention is selected from the alkyl-phosphine oxides of general formula 1: General Formula 1 Where R 1 R 2 and R 3 Selected from alkyl groups, preferably straight-chain alkyl groups, containing 4 to 18, preferably 6 to 12 carbon atoms. The condition is that R 1 R 2 and R 3 At least two of them are different from each other.
[0025] More specifically, the alkyl-phosphine oxide is selected from alkylphosphine oxides of general formula 1, wherein R 1 R 2 and R 3The alkyl groups are selected from alkyl groups, preferably straight-chain alkyl groups, containing 8 to 10, particularly 8 or 10 carbon atoms. More particularly, the molar ratio of all alkyl groups containing 8 and 10 carbon atoms in all alkyl-phosphine oxides of general formula 1 contained in the extraction medium is in the range of 1.0:2.0 to 2.0:1.0, particularly 1.0:1.5 to 1.5:1.0, and even more particularly 1.0:1.2 to 1.2:1.0. Even more specifically, the alkyl-phosphine oxides in the extraction medium in any aspect of the method according to the invention are alkyl-phosphine oxides of general formula 1. General Formula 1 Where R 1 R 2 and R 3 It is selected from straight-chain alkyl groups having 6 to 12 carbon atoms, wherein one of the alkyl groups has 8 carbon atoms and the other has 10 carbon atoms. The condition is that R 1 R 2 and R 3 At least two of them are different from each other.
[0026] More specifically, in the extraction medium of any aspect of the method according to the invention, alkyl-phosphine oxides account for at least 50% by weight of the total extraction medium, particularly at least 80% by weight, even more particularly at least 90% by weight, and most particularly at least 97% by weight.
[0027] In one example, the extraction medium according to any aspect of the invention may further comprise a second organic component in addition to alkyl-phosphine oxides. The second organic component contains at least 12 carbon atoms. Specifically, the alkane may contain 12-18 carbon atoms. The second organic component is a straight-chain or branched alkane, which may be selected from tetradecane, pentadecane, hexadecane, heptadecane, octadecane, and squalene, or mixtures of alkanes such as white mineral oil (Fragoltherm-Q-32-N). Furthermore, the second organic component may comprise aromatic hydrocarbons selected from diisopropylbiphenyl, partially hydrogenated terphenyl, dibenzyltoluene, and diisopropylnaphthalene, or mixtures of aromatic solvents such as Solvsso 200. Alternatively, an alcohol may be used as the second organic component, which may be selected from oleyl alcohol, 2-octyldodecanool, and 2-hexyldodecanool.
[0028] In any aspect of the invention, the weight ratio of alkyl-phosphine oxide to alkane in the extraction medium is preferably from 1:100 to 100:1. More specifically, the weight ratio of alkyl-phosphine oxide to alkane can be selected from 1:2 to 50:1, more preferably from 1:1 to 97:3. In this example, the alkane can be hexadecane, and therefore the weight ratio of alkyl-phosphine oxide to hexadecane can be about 97:3.
[0029] The extraction medium according to any aspect of the invention can effectively extract octanoic acid. The extraction medium is a mixture of at least one alkyl-phosphine oxide and at least one alkane, each alkyl-phosphine oxide molecule containing at least two distinct alkyl groups. This extraction medium is considered suitable for the method according to any aspect of the invention because the mixture operates efficiently in extracting the desired octanoic acid. 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.
[0030] In another example, the extraction medium is a mixture of at least one alkyl-phosphine oxide and at least one partially hydrogenated aromatic hydrocarbon, each alkyl-phosphine oxide molecule containing at least two distinct alkyl groups. This extraction medium is considered suitable for methods according to any aspect of the invention because the mixture operates efficiently in the extraction of octanoic acid and its salts. In particular, a mixture of at least one alkyl-phosphine oxide (each alkyl-phosphine oxide molecule containing at least two distinct alkyl groups) and at least one partially hydrogenated aromatic hydrocarbon is preferred. Furthermore, extraction media according to any aspect of the invention, combined with alkanes or partially hydrogenated aromatic solvents, are also non-toxic to microorganisms.
[0031] As used herein, the term “about” refers to a change of up to 20%. In particular, as used herein, “about” means + / - 20% for a given measurement or value, more specifically, + / - 10%, and even more specifically, + / - 5%.
[0032] The source of acetate and / or ethanol may vary depending on availability. In one instance, ethanol and / or acetate may be syngas fermentation products or any carbohydrate known in the art. In particular, the carbon source for acetate and / or ethanol production may be selected from alcohols, aldehydes, glucose, sucrose, fructose, dextrose, lactose, xylose, pentose, polyols, hexoses, ethanol, and syngas.
[0033] A mixture of sources can be used as a carbon source.
[0034] More specifically, the carbon source can be syngas. Syngas can be converted into ethanol and / or acetate in the presence of at least one acetic acid-producing bacterium.
[0035] Ethanol, or an additional substrate of ethanol and acetate, can be added throughout the fermentation process to maintain the production of octanoic acid and / or its salts. In one instance, additional substrates are added periodically. In another instance, additional substrates or combinations thereof are added continuously during the fermentation or production of octanoic acid and / or its salts. It is desirable not to overfeed the cells according to any aspect of the invention (feeding exceeding the chain elongation rate, which is equal to the extraction rate).
[0036] In one example, a large amount of octanoic acid and / or its salts in step (c) refers to approximately 100 ppm. Specifically, at least about 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, or 300 ppm. Octane products can be detected by methods known in the art. For example, octane products can be detected by gravimetric methods, spectroscopic methods (e.g., nuclear magnetic resonance (NMR) spectroscopy), or mass spectrometry methods (e.g., gas chromatography or liquid chromatography / mass spectrometry).
[0037] In one instance, in addition to octanoic acid and its salts, hexanoic acid and / or its salts are produced, and the aqueous medium also contains other microorganisms such as Clostridium perfringens (C. ethanolophilus). Clostridium autoethanogenum ).
[0038] According to another aspect of the present invention, a method for increasing the yield of octanoic acid and / or its salts produced in a product composition is provided, the method comprising the following steps: i) When it forms in the product composition, simultaneously extract octanoic acid and / or its salt. Octanoic acid and its salts are formed in the presence of Clostridium krusei and a substrate containing ethanol or a mixture of ethanol and acetate.
[0039] In particular, the yield of octanoic acid and / or its salts in the product composition is increased by at least about 1.5 times, especially by 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9 or 3 times.
[0040] In particular, extraction is performed using an extraction medium comprising: - At least one trialkyl-phosphine oxide and at least one alkane.
[0041] In one example, the alkane contains 12 to 18 carbon atoms. In another example, the extraction medium comprises at least one trialkyl-phosphine oxide and at least one alkane, particularly the trialkyl-phosphine oxide selected from trialkyl-phosphine oxides of general formula 1: General Formula 1 Where R 1 R 2 and R3 Selected from alkyl groups containing 4 to 18, preferably 6 to 12, carbon atoms, wherein the condition is R 1 R 2 and R 3 At least two of them are different from each other. Example
[0042] The foregoing describes preferred embodiments, and as those skilled in the art will understand, these preferred embodiments may be subject to design, construction, or operational variations or modifications without departing from the scope of the claims. Such variations are intended, for example, to be covered by the scope of the claims.
[0043] Example 1 Culturing self-producing Clostridium ethanolae and Clostridium krusei in a single-phase system to produce octanoate. Clostridium krusei (DSM555) and Clostridium difficile (DSM10061) were co-cultured to bioconvert ethanol and acetate into medium-chain fatty acids. All culturing steps were carried out under anaerobic conditions. Pre-culture was conducted in a stainless steel bubble column loop reactor, and the main culture was carried out in a pressure-resistant glass bottle that could be sealed with a butyl rubber stopper.
[0044] For pre-culture, the homotype acetic acid-producing bacterium *Clostridium autoethanogenum* and the chain-elongating bacterium *Clostridium kluyveri* were co-cultured in syngas on an inorganic medium. The culture was then carried out under anaerobic conditions in a pressure-resistant stainless steel bubble column loop reactor.
[0045] The culture was conducted as a continuous fermentation at 37°C and 2 bar superpressure, with a continuous feed of 300 L / h of a mixture of water, substrate, salt, trace elements, and vitamins. Ammonia was used as the feed to automatically maintain the pH at 5.80. The effluent stream from the fermenter was 300 L / h, of which 98.2% was permeate with cell retention and 1.8% was purge without cell retention. Gas was released into the culture medium via a sparger at a ventilation rate of ~1000 L / h in the form of a gaseous mixture of 62.5% H₂ and 37.5% CO₂.
[0046] The culture medium consists of the following components: 0.004 g / L magnesium acetate x 4H₂O, 0.164 g / L sodium acetate, 0.016 g / L calcium acetate, 0.245 g / L potassium acetate, 0.107 mL / L H₃PO₄ (8.5%), 0.35 mg / L cobalt acetate, 1.245 mg / L nickel acetate x 4H₂O, 20 µg / L d-biotin, 20 µg / L folic acid, 10 µg / L pyridoxine hydrochloride, 50 µg / L thiamine hydrochloride, 50 µg / L riboflavin, 50 µg / L niacin, 50 µg / L calcium pantothenate, and 50 µg / L vitamin B. 12 50 µg / L p-aminobenzoate, 50 µg / L lipoic acid, 2.109 mg / L (NH4)2Fe(SO4)2x6H2O, 10.69 mg / L potassium thioacetate, 6.73 g / L ethanol, and NH3 were used for pH adjustment.
[0047] Using self-produced Clostridium ethanol ( C. autoethanogenum ) and Clostridium krusei ( C.kluyveri Freshly cultured cells were pre-inoculated into the culture and had been continuously run as a stable co-culture for >30,000 hours at an optical density (OD) of ~14. 600nm ). With 2.8 days -1 The dilution rate is used to continuously supply fresh culture medium into the reactor and continuously remove fermentation broth from the reactor.
[0048] For the master culture, 180 ml of the same anaerobic inorganic medium used in the pre-culture (with a higher ethanol concentration (20 mL / L ethanol) and an additional 5 g / L ammonium acetate and 0.15 g / L NH4HCO3) was filled into 500 mL flasks and seeded with a large amount of cell broth from the pre-culture to achieve an OD of 0.15. 600nm The bottles containing a monolayer aqueous phase were then incubated for 67 h in an open water bath shaker at 37 °C, 150 rpm, and a gas atmosphere of 10% H2 and 90% N2. The gas atmosphere in the bottles was rinsed with pure N2 for 10 minutes once a day.
[0049] During the culture period, 5 mL samples were taken from the aqueous phase several times to determine OD. 600nm pH and product formation. The concentration of the product in the aqueous phase was determined by semi-quantitative ¹H-NMR spectroscopy and high-performance liquid chromatography (HPLC).
[0050] During the 67-hour culture period in the aqueous phase, the acetate concentration decreased from 4.3 g / L to 1.4 g / L, and the ethanol concentration decreased from 14.0 g / L to 9.1 g / L. During the same period, 2.1 g / L of butyrate, 3.6 g / L of hexanoate, and 0.04 g / L of octanoate were generated.
[0051] Example 2 Culturing self-producing ethanol-producing Clostridium and Clostridium krusei in a two-phase system to produce octanoate. Clostridium krusei (DSM555) and Clostridium difficile (DSM10061) were co-cultured to bioconvert ethanol and acetate into medium-chain fatty acids. For in-situ extraction of the generated acids, the aqueous phase was covered with a mixture of Fragoltherm Q-32-N and trialkylphosphine oxide (TAPO). All culturing steps were carried out under anaerobic conditions, with pre-culture in a stainless steel bubble column loop reactor and main culture in a pressure-resistant glass bottle that could be sealed with a butyl rubber stopper.
[0052] For pre-culture, the homologous acetic acid-producing bacterium *Clostridium perfringens* and the chain-elongating bacterium *Clostridium krusei* were co-cultured in an inorganic medium with syngas. The culture was then carried out under anaerobic conditions in a pressure-resistant stainless steel bubble column loop reactor.
[0053] The culture was conducted as a continuous fermentation at 37°C and 2 bar superpressure, with a continuous feed of 300 L / h of a mixture of water, substrate, salt, trace elements, and vitamins. Ammonia was used as the feed to automatically maintain the pH at 5.80. The effluent stream from the fermenter was 300 L / h, of which 98.2% was permeate with cell retention and 1.8% was purge without cell retention. Gas was released into the culture medium via a sparger at a flow rate of ~1000 L / h in the form of a gaseous mixture of 62.5% H₂ and 37.5% CO₂.
[0054] The culture medium consists of the following components: 0.004 g / L magnesium acetate x 4H₂O, 0.164 g / L sodium acetate, 0.016 g / L calcium acetate, 0.245 g / L potassium acetate, 0.107 mL / L H₃PO₄ (8.5%), 0.35 mg / L cobalt acetate, 1.245 mg / L nickel acetate x 4H₂O, 20 µg / L d-biotin, 20 µg / L folic acid, 10 µg / L pyridoxine hydrochloride, 50 µg / L thiamine hydrochloride, 50 µg / L riboflavin, 50 µg / L niacin, 50 µg / L calcium pantothenate, and 50 µg / L vitamin B. 1250 µg / L p-aminobenzoate, 50 µg / L lipoic acid, 2.109 mg / L (NH4)2Fe(SO4)2x6H2O, 10.69 mg / L potassium thioacetate, 6.73 g / L ethanol, and NH3 were used for pH adjustment.
[0055] Using self-produced Clostridium ethanol ( C. autoethanogenum ) and Clostridium krusei ( C.kluyveri Freshly cultured cells were pre-inoculated into the culture and had been continuously run as a stable co-culture for >30,000 hours at an optical density (OD) of ~14. 600nm ). With 2.8 days -1 The dilution rate is used to continuously supply fresh culture medium into the reactor and continuously remove fermentation broth from the reactor.
[0056] For the master culture, 180 ml of the same anaerobic inorganic medium used in the pre-culture (with a higher ethanol concentration (20 mL / L ethanol) and an additional 5 g / L ammonium acetate and 0.15 g / L NH4HCO3) was filled into 500 mL flasks and seeded with a large amount of cell broth from the pre-culture to achieve an OD of 0.15. 600nm The aqueous phase was then covered with 20 ml of anaerobic extractant (20% trialkylphosphine oxide (TAPO) in Fragoltherm Q-32-N). The flasks containing the bilayer phases were then incubated for 67 h in an open water bath shaker at 37 °C, 150 rpm, and a gas atmosphere of 10% H₂ and 90% N₂. The gas atmosphere in the flasks was rinsed with pure N₂ for 10 minutes once daily.
[0057] During the culture period, 5 mL samples were taken from both the aqueous and organic phases several times to determine OD. 600nm pH and product formation. The concentration of the product in the aqueous phase was determined by semi-quantitative ¹H-NMR spectroscopy and high-performance liquid chromatography (HPLC), and in the organic phase by gas chromatography (GC).
[0058] During a 67-hour culture period in the aqueous phase, the acetate concentration decreased from 4.2 g / L to 0.3 g / L, and the ethanol concentration decreased from 13.1 g / L to 5.0 g / L. During the same period, 1.1 g / L of butyrate, 5.7 g / L of hexanoate, and 0.01 g / L of octanoate were generated. In the organic phase, the concentrations increased from 0 g / L to 0.75 g / L for ethanol, from 0 g / L to 0.50 g / L for butyrate, from 0 g / L to 25.7 g / L for hexanoate, and from 0 g / L to 1.08 g / L for octanoate. Acetic acid was not observed.
Claims
1. A method for producing octanoic acid and / or its salts, said method comprising: a) Contact a carbon substrate containing ethanol or a mixture of ethanol and acetate with an aqueous medium containing Clostridium krillion; b) Keep the aqueous medium in place for an appropriate period of time until Clostridium kuriformis produces large amounts of octanoic acid and / or its salts; c) Separate octanoic acid and / or its salts from an aqueous medium using an extraction medium; and d) Repeat step (c) continuously during the formation of octanoic acid and / or its salts. The extraction medium comprises: - At least one trialkyl-phosphine oxide and at least one alkane.
2. The method according to claim 1, wherein the alkane comprises 12 to 18 carbon atoms.
3. The method according to claim 1 or 2, wherein the trialkyl-phosphine oxide is selected from trialkyl-phosphine oxides of general formula 1: General Formula 1 Where R 1 R 2 and R 3 Selected from alkyl groups containing 4 to 18, preferably 6 to 12 carbon atoms. The condition is that R 1 R 2 and R 3 At least two of them are different from each other.
4. The method according to claim 3, wherein the trialkyl-phosphine oxide is selected from trialkyl-phosphine oxides of general formula 1, wherein R 1 R 2 and R 3 Selected from alkyl groups containing 8 to 10 carbon atoms.
5. The method according to claim 4, wherein the molar ratio of all alkyl groups containing 8 and 10 carbon atoms in the trialkyl-phosphine oxide is in the range of 1.0:2.0 to 2.0:1.
0.
6. The method according to any one of the preceding claims, wherein ethanol alone or ethanol and acetate are further added as a substrate.
7. The method according to any one of the preceding claims, wherein the amount of octanoic acid in step (c) refers to approximately 100 ppm.
8. The method according to any one of the preceding claims, wherein in addition to octanoic acid and its salts, hexanoic acid is also produced, and the aqueous medium further comprises other microorganisms, such as Clostridium difficile, which produces ethanol.
9. A method for increasing the yield of octanoic acid and / or its salts produced in a product composition, the method comprising the following steps: i) When octanoic acid and / or its salts are formed in the product composition, octanoic acid and / or its salts are simultaneously extracted. Octanoic acid and its salts are formed in the presence of Clostridium krusei and a substrate containing ethanol or a mixture of ethanol and acetate. Extraction is performed using an extraction medium comprising: - At least one trialkyl-phosphine oxide and at least one alkane.
10. The method of claim 9, wherein the yield of octanoic acid and / or its salt in the product composition is increased by at least about 1.5 times.
11. The method according to claim 9 or 10, wherein the alkane comprises 12 to 18 carbon atoms.
12. The method according to any one of claims 9 to 11, wherein the trialkyl-phosphine oxide is selected from trialkyl-phosphine oxides of general formula 1: General Formula 1 Where R 1 R 2 and R 3 Selected from alkyl groups containing 4 to 18, preferably 6 to 12 carbon atoms. The condition is that R 1 R 2 and R 3 At least two of them are different from each other.
13. The method of claim 12, wherein the trialkylphosphine oxide is selected from trialkylphosphine oxides of general formula 1, wherein R 1 R 2 and R 3 Selected from alkyl groups containing 8 to 10 carbon atoms.