Clostridium butyricum produced by fermentation of synthesis gas

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to clostridium butyricum for producing short-chain fatty acid and lactic acid by utilizing synthesis gas fermentation. The clostridium butyricum is named as clostridium butyricum Gen160CO2, the clostridium butyricum is preserved in the China General Microbiological Culture Collection Center on November 18, 2024, the preservation number is CGMCC No.32696, the preservation address is Institute of Microbiology, Chinese Academy of Sciences, No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the zip code of the preservation address is 100101. The clostridium butyricum can be used for producing various short-chain fatty acids and lactic acids with industrial application values by utilizing synthesis gas through fermentation, and has relatively high economic values.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbiology, and particularly relates to a Clostridium butyricum strain that uses syngas fermentation to produce short-chain fatty acids and lactic acid. Background Art

[0002] Currently, the large consumption of fossil fuels not only gradually depletes energy but also causes a series of climate hazards such as the greenhouse effect. Through extensive research, synthesizing biofuels using industrial by-products and one-carbon gases in other industrial syngas has gradually been identified as a containment measure. Among them, using microbial fermentation of syngas has advantages over chemical methods in terms of being green and environmentally friendly, having low energy consumption, mild reaction conditions, high product selectivity, strong tolerance to impurities, and can also be used as microbial protein for consumption, offering greater flexibility and potential cost-effectiveness. With the development of microbial engineering and synthetic biology, this method has great application prospects under the background of sustainable development and carbon neutrality. However, there are still many deficiencies and challenges in this method: for example, research on using microbial fermentation of syngas only focuses on some specific microorganisms that have been reported to be able to convert CO or CO2 into products such as acetic acid, ethanol, and butanol through the Wood-Ljungdahl pathway, with significant strain limitations; the solubility and mass transfer efficiency of gas in the liquid medium are low; the microbial metabolic rate and the yield of related products are low, etc., and further research and improvement are still needed.

[0003] Short-chain fatty acids and lactic acid have various biological functions. Currently, the co-production of short-chain fatty acids and lactic acid mainly uses organic waste such as food waste for mixed-bacteria anaerobic fermentation, and short-chain fatty acids and lactic acid are produced together during the anaerobic fermentation process. This method has a complex process flow, a long treatment time cycle, and requires a large amount of manpower and material resources. At the same time, the gas generated during the anaerobic fermentation of organic waste contains a large amount of carbon dioxide, hydrogen sulfide, etc., and when discharged into the air, it emits a pungent smell and causes the greenhouse effect. The production of short-chain fatty acids and lactic acid by pure microbial fermentation has attracted increasing attention due to its mild reaction conditions, environmental friendliness, simple operation, and other advantages. Summary of the Invention

[0004] The purpose of the present invention is to provide a Clostridium butyricum strain that uses syngas fermentation to produce short-chain fatty acids and lactic acid.

[0005] To achieve the above purpose, the present invention adopts the following technical solutions: The first aspect of the present invention provides a Clostridium butyricum strain that ferments syngas to produce short-chain fatty acids and lactic acid. The Clostridium butyricum strain is named Clostridium butyricum Gen160CO2, which was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on November 18, 2024. The deposit number is CGMCC No. 32696, and the deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing 100101; Furthermore, the breeding method of the Clostridium butyricum strain is as follows: inoculate Clostridium butyricum Gen160 into a fermentation medium and carry out acclimation culture in a syngas environment. During the acclimation culture process, gradually reduce the content of organic nitrogen source in the fermentation medium through multiple transfers, and screen to obtain Clostridium butyricum Gen160CO2 that ferments syngas to produce short-chain fatty acids and lactic acid; among them, Clostridium butyricum Gen160 was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on July 5, 2012. The deposit number is CGMCC No. 6317, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing 100101.

[0006] The second aspect of the present invention provides the application of the above-mentioned Clostridium butyricum strain in the biological utilization of syngas; Furthermore, the syngas is composed of one-carbon gas and H2, and the one-carbon gas includes CO and CO2.

[0007] The third aspect of the present invention provides the application of the above-mentioned Clostridium butyricum strain in the preparation of short-chain fatty acids and lactic acid; Furthermore, the short-chain fatty acids include formic acid, acetic acid, butyric acid, and lactic acid.

[0008] The fourth aspect of the present invention provides a method for preparing short-chain fatty acids and lactic acid, which is to inoculate the above-mentioned Clostridium butyricum into a deoxygenated fermentation medium and carry out shaking fermentation culture in a syngas environment to obtain a fermentation broth that may contain short-chain fatty acids and lactic acid; Furthermore, the formula of the fermentation medium is as follows: yeast powder 0.05 g / L, ammonium sulfate 2 g / L, magnesium sulfate heptahydrate 0.2 g / L, potassium dihydrogen phosphate 0.5 g / L, dipotassium hydrogen phosphate 1 g / L, calcium chloride solution 1 mL / L, Fe solution 1 mL / L, trace element solution 1 mL / L, and the rest is water; among them, the concentration of the calcium chloride solution is 20 g / L; the formula of the Fe solution is: FeSO4·7H2O 5 g / L, 37 wt% HCl 4 mL / L, and the rest is water; the formula of the trace element solution is: CoCl2·6H2O 0.2 g / L, ZnCl2 0.07 g / L, CuCl2·2H2O 0.02 g / L, MnCl2·4H2O 0.1 g / L, Na2MoO4·2H2O 0.035 g / L, H3BO3 0.06 g / L, NiCl2·6H2O 0.025 g / L, 37 wt% HCl 0.9 mL / L, and the rest is water; Furthermore, the syngas is composed of carbon monoxide gas and H2, and the carbon monoxide gas includes CO and CO2.

[0009] Furthermore, the short-chain fatty acids include formic acid, acetic acid, butyric acid, and lactic acid.

[0010] The remarkable advantages of the present invention are as follows: In the present invention, Clostridium butyricum Gen160 is inoculated into the fermentation medium and domestically cultured in a syngas environment. During the domestication culture process, the content of organic nitrogen source in the fermentation medium is gradually reduced through multiple transfers, and a strain of Clostridium butyricum Gen160CO2 that can ferment syngas to produce short-chain fatty acids and lactic acid is screened. Clostridium butyricum Gen160CO2 can use carbon monoxide gas in the syngas as a gaseous carbon source and metabolize short-chain fatty acids and lactic acid with industrial application value, having high economic value. Description of the Drawings

[0011] Figure 1 : Growth curves of Clostridium butyricum Pri.1, Clostridium butyricum 304, and Clostridium butyricum Gen160 in a syngas environment and a nitrogen environment.

[0012] Figure 2 : Growth curve of Clostridium butyricum Gen160CO2 in a syngas environment and a nitrogen environment.

[0013] Figure 3 : High-performance liquid chromatography diagram of a standard product of mixed acid substances (formic acid, acetic acid, butyric acid, lactic acid).

[0014] Figure 4 : Standard curve diagrams of various acid substances.

[0015] Figure 5: High-performance liquid chromatography (HPLC) chromatogram of Clostridium butyricum Gen160 fermentation broth.

[0016] Figure 6 : High-performance liquid chromatography (HPLC) chromatogram of Clostridium butyricum Gen160 CO2 fermentation broth.

[0017] Figure 7 : Trend of short-chain fatty acid production in Clostridium butyricum Gen160 fermentation broth over time.

[0018] Figure 8 : Trend of short-chain fatty acid production in Clostridium butyricum Gen160 CO2 fermentation broth over time. Detailed implementation mode

[0019] Now, various exemplary implementation modes of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0020] It should be understood that the terms used in the present invention are only for describing specific implementation modes and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0021] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0022] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation modes of the present invention specification, which are obvious to those skilled in the art. Other implementation modes obtained from the present invention specification are also obvious to those skilled in the art. The present invention specification and examples are only exemplary.

[0023] Regarding the use of "comprising", "including", "having", "containing", etc. in the present invention, they are all open-ended terms, meaning including but not limited to.

[0024] The Clostridium butyricum Gen160CO2 provided by the present invention (hereinafter referred to as "Clostridium butyricum Gen160CO2") is obtained by domestication and screening of Clostridium butyricum Gen160 as the starting strain in a syngas environment.

[0025] In the present invention, the syngas can be provided from any known source. In some embodiments, the syngas has carbon monoxide and hydrogen as the main components; the carbon monoxide gas can be selected from carbon monoxide and carbon dioxide. In this regard, the syngas can contain 5% mol or more of carbon monoxide gas. In another aspect, the syngas can contain 10% mol or more of carbon monoxide gas. In another aspect, the syngas can contain 20% mol or more of carbon monoxide gas. In another aspect, the syngas can contain 30% mol or more of carbon monoxide gas. In another aspect, the syngas can contain 40% mol or more of carbon monoxide gas. In another aspect, the syngas can contain 50% mol or more of carbon monoxide gas. In another aspect, the syngas can contain 60% mol or more of carbon monoxide gas. In another aspect, the syngas can contain 70% mol or more of carbon monoxide gas. In another aspect, the syngas can contain 80% mol or more of carbon monoxide gas. In another aspect, the syngas can contain 90% mol or more of carbon monoxide gas.

[0026] In the present invention, short-chain fatty acids refer to organic acids having a carbon chain of 1 to 6 carbon atoms, including formic acid, acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, caproic acid, isohexanoic acid, pyruvic acid, succinic acid and mixtures thereof.

[0027] In the present invention, the logarithmic growth phase refers to when microorganisms are cultured in a closed system (batch culture), according to the changes in the growth rate and specific growth rate of the microorganisms. After a certain stage of microbial growth, the specific growth rate of the microorganisms reaches the maximum, and at this time, it enters the logarithmic growth phase. In the logarithmic growth phase, if there are no factors inhibiting or restricting the growth of microorganisms, the microorganisms will grow at a constant maximum specific growth rate, and the number of cells will increase exponentially.

[0028] In the present invention, Clostridium butyricum Pri.1 (hereinafter referred to as "Clostridium butyricum Pri.1") and Clostridium butyricum 304 (hereinafter referred to as "Clostridium butyricum 304") were preserved by the research group where the applicant belongs; Clostridium butyricum Gen160 (hereinafter referred to as "Clostridium butyricum Gen160") was preserved in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms on July 5, 2012, with the preservation number of CGMCC No. 6317, and the preservation address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, and the postal code of the preservation address is 100101.

[0029] Example 1: In this example, the synthesis gas flow rate was: 200 mL / min, and the synthesis gas composition was: 20% mole CO2 and 80% mole H2.

[0030] In this example, the nitrogen gas flow rate was: 200 mL / min.

[0031] In this example, the formula of the Clostridium - enriched medium was: peptone 10 g / L, yeast powder 3 g / L, NaCl 3 g / L, glucose 5 g / L, beef extract 10 g / L, soluble starch 1 g / L, sodium acetate trihydrate 5 g / L, L - cysteine hydrochloride 0.5 g / L, and the rest was water.

[0032] In this example, the formula of the fermentation medium was: yeast powder 5 g / L, ammonium sulfate 2 g / L, magnesium sulfate heptahydrate 0.2 g / L, potassium dihydrogen phosphate 0.5 g / L, dipotassium hydrogen phosphate 1 g / L, 1 mL / L of 20 g / L calcium chloride aqueous solution, 1 mL / L of Fe solution, 1 mL / L of trace element solution, and the rest was water. Among them, the formula of the Fe solution was: FeSO4·7H2O 5 g / L, 4 mL / L of 37 wt% HCl aqueous solution, and the rest was water; the formula of the trace element solution was: CoCl2·6H2O 0.2 g / L, ZnCl2 0.07 g / L, CuCl2·2H2O 0.02 g / L, MnCl2·4H2O 0.1 g / L, Na2MoO4·2H2O 0.035 g / L, H3BO3 0.06 g / L, NiCl2·6H2O 0.025 g / L, 0.9 mL / L of 37 wt% HCl aqueous solution, and the rest was water.

[0033] Preparation of resuscitated bacterial liquid: The glycerol strains of Clostridium butyricum Pri1, Clostridium butyricum 304, and Clostridium butyricum Gen160 stored at -80 °C were inoculated into the Clostridium enhanced medium sterilized at 121 °C for 15 min at an inoculation amount of 10% (v / v) respectively, and cultured at 37 °C and 200 rpm for 12 - 14 hours until OD 650 = 5 to obtain the resuscitated bacterial liquid.

[0034] Experimental group: The fermentation medium was dispensed into serum bottles with a capacity of 100 mL, with 50 mL of liquid in each bottle. The bottle mouths were sealed with rubber stoppers. A nitrogen cylinder equipped with a needle was used to fill 99.99% nitrogen into the serum bottles to remove the residual oxygen in the serum bottles. After the inflation time reached 5 min, the inflation ended. It was sterilized at 121 °C for 15 min, cooled to room temperature, and then inoculated with the resuscitated bacterial liquid at an inoculation amount of 10% (v / v). Immediately, syngas was introduced at a pressure of 0.1 Mpa for 5 min to remove the nitrogen in the serum bottles, so that Clostridium butyricum was completely in the gas environment of syngas for fermentation. Finally, the headspace pressure of the serum bottle was maintained at 0.1 MPa, sealed, and oscillated and fermented at 37 °C and 200 rpm for 10 days. Fresh syngas was replaced every day during this period. Samples were taken regularly and the absorbance (OD 650 ) of the bacterial liquid at 650 nm was measured to plot the growth curve.

[0035] Control group: The fermentation medium was dispensed into serum bottles with a capacity of 100 mL, with 50 mL of liquid in each bottle. The bottle mouths were sealed with rubber stoppers. A nitrogen cylinder equipped with a needle was used to fill 99.99% nitrogen into the serum bottles to remove the residual oxygen in the serum bottles. After the inflation time reached 5 min, the inflation ended. It was sterilized at 121 °C for 15 min, cooled to room temperature, and then inoculated with the resuscitated bacterial liquid at an inoculation amount of 10% (v / v). Immediately, nitrogen was introduced at a pressure of 0.1 Mpa for 5 min, so that Clostridium butyricum was completely in the gas environment of nitrogen for fermentation. Finally, the headspace pressure of the serum bottle was maintained at 0.1 MPa, sealed, and oscillated and fermented at 37 °C and 200 rpm for 10 days. Fresh nitrogen was replaced every day during this period. Samples were taken regularly and the absorbance (OD 650 ) of the bacterial liquid at 650 nm was measured to plot the growth curve.

[0036] The results are as Figure 1As shown in the figure, G160, 304, and pri1 in the figure respectively represent the growth curves of Clostridium butyricum Gen160, Clostridium butyricum 304, and Clostridium butyricum Pri.1 in a syngas environment, and G160-control, 304-control, and pri1-control respectively represent the growth curves of the control group of Clostridium butyricum Gen160, Clostridium butyricum 304, and Clostridium butyricum Pri.1. It can be seen from the figure that the growth ability of Clostridium butyricum in the syngas environment is generally higher than that of the control group; after 1 day of fermentation culture of Clostridium butyricum Gen160 in the syngas environment, the OD of the bacterial liquid 650 value has always been stable above 1; during the fermentation culture of Clostridium butyricum 304 in the syngas environment, the OD of the bacterial liquid 650 value first increases and then decreases with the extension of the fermentation culture time, which may be due to the sudden decrease in pH in the middle stage of fermentation culture, resulting in the decrease of the activity of some enzymes and the decrease of biomass; after 1 day of fermentation culture of Clostridium butyricum Pri.1 in the syngas environment, the OD of the bacterial liquid 650 value immediately decreases, and the OD of the viable bacterial liquid 650 value is stable between 0.4 and 0.5 in the later stage of fermentation culture, which may be due to the weak expression of some key enzymes in the strain and the inability to effectively utilize syngas. Therefore, Clostridium butyricum Gen160 with the strongest growth ability in the syngas environment was selected as the subsequent experimental strain.

[0037] Example 2: In this example, the syngas flow rate is: 200 mL / min, and the syngas composition is: 20% mol CO2 and 80% mol H2.

[0038] In this example, the nitrogen flow rate is: 200 mL / min.

[0039] In this example, the formula of the Clostridium enrichment medium is the same as that in Example 1.

[0040] Preparation of the resuscitated bacterial liquid: The glycerol strain of Clostridium butyricum Gen160 stored at -80°C was inoculated into the Clostridium enrichment medium sterilized at 121°C for 15 min at an inoculation amount of 10% (v / v), and cultured at 37°C and 200 rpm for 12 - 14 hours until OD 650 = 5 to obtain the resuscitated bacterial liquid, which was stored in a 4°C refrigerator for later use.

[0041] The domestication of Clostridium butyricum Gen160 was carried out according to the following steps: S1: First-cycle domestication: S1-1: Dispense the fermentation medium (the formulation of this fermentation medium is basically the same as that in Example 1, except that the content of yeast powder in the medium is reduced to 2.5 g / L) into serum bottles with a capacity of 100 mL, with 50 mL of liquid in each bottle. Seal the bottle mouths with rubber stoppers. Use a nitrogen cylinder equipped with a needle to fill the serum bottles with 99.99% nitrogen to remove the residual oxygen in the serum bottles. After the inflation time reaches 5 min, stop inflation. Sterilize at 121 °C for 15 min. After cooling to room temperature, inoculate the Clostridium butyricum Gen160 resuscitation bacterial liquid at an inoculation amount of 10% (v / v). Immediately introduce syngas at a pressure of 0.12 Mpa for 5 min to remove the nitrogen in the serum bottles, so that Clostridium butyricum is completely in the gas environment of syngas for fermentation. Finally, maintain the headspace pressure of the serum bottle at 0.1 MPa, seal it, and shake and ferment culture at 37 °C and 200 rpm for 5 days to obtain the culture solution; S1-2: Dispense the fermentation medium (the formulation of this fermentation medium is basically the same as that in Example 1, except that the content of yeast powder in the medium is reduced to 1 g / L) into serum bottles with a capacity of 100 mL, with 50 mL of liquid in each bottle. Seal the bottle mouths with rubber stoppers. Use a nitrogen cylinder equipped with a needle to fill the serum bottles with 99.99% nitrogen to remove the residual oxygen in the serum bottles. After the inflation time reaches 5 min, stop inflation. Sterilize at 121 °C for 15 min. After cooling to room temperature, inoculate the culture solution obtained in S1-1 at an inoculation amount of 10% (v / v). Immediately introduce syngas at a pressure of 0.12 Mpa for 5 min to remove the nitrogen in the serum bottles, so that Clostridium butyricum is completely in the gas environment of syngas for fermentation. Finally, maintain the headspace pressure of the serum bottle at 0.1 MPa, seal it, and shake and ferment culture at 37 °C and 200 rpm for 5 days to obtain the culture solution; S1-3: Use the culture solution obtained in S1-2 as the inoculum, repeat S1-1 to S1-2 ten times, and finally obtain the first-cycle domesticated bacterial liquid.

[0042] S2: Second-cycle subculture domestication: S2-1: Dispense the fermentation medium (the formulation of this fermentation medium is basically the same as that of Example 1, except that the content of yeast powder in the medium is reduced to 0.5 g / L) into serum bottles with a capacity of 100 mL, with 50 mL of liquid in each bottle. Seal the bottle mouths with rubber stoppers. Use a nitrogen cylinder equipped with a needle to fill the serum bottles with 99.99% nitrogen to remove the residual oxygen in the serum bottles. After the inflation time reaches 5 minutes, end the inflation. Sterilize at 121°C for 15 minutes. After cooling to room temperature, inoculate the first-cycle acclimated bacterial liquid at an inoculation amount of 10% (v / v). Immediately introduce syngas at a pressure of 0.12 Mpa for 5 minutes to remove the nitrogen in the serum bottles, so that Clostridium butyricum is completely in the syngas gas environment for fermentation. Finally, keep the headspace pressure of the serum bottle at 0.1 MPa, seal it, and oscillate and ferment culture at 37°C and 200 rpm for 5 days to obtain the culture solution; S2-2: Dispense the fermentation medium (the formulation of this fermentation medium is basically the same as that of Example 1, except that the content of yeast powder in the medium is 0.1 g / L) into serum bottles with a capacity of 100 mL, with 50 mL of liquid in each bottle. Seal the bottle mouths with rubber stoppers. Use a nitrogen cylinder equipped with a needle to fill the serum bottles with 99.99% nitrogen to remove the residual oxygen in the serum bottles. After the inflation time reaches 5 minutes, end the inflation. Sterilize at 121°C for 15 minutes. After cooling to room temperature, inoculate the culture solution obtained in S2-1 at an inoculation amount of 10% (v / v). Immediately introduce syngas at a pressure of 0.12 Mpa for 5 minutes to remove the nitrogen in the serum bottles, so that Clostridium butyricum is completely in the syngas gas environment for fermentation. Finally, keep the headspace pressure of the serum bottle at 0.1 MPa, seal it, and oscillate and ferment culture at 37°C and 200 rpm for 5 days to obtain the culture solution; S2-3: Use the culture solution obtained in S2-2 as the inoculum, repeat S2-1 to S2-2 ten times, and finally obtain the second-cycle acclimated bacterial liquid.

[0043] S3: Third-cycle acclimation: S3-1: Aliquot the fermentation medium (the formulation of this fermentation medium is basically the same as that of Example 1, except that the content of yeast powder in the medium is reduced to 0.05 g / L) into serum bottles with a capacity of 100 mL, with 50 mL of liquid in each bottle. Seal the bottle mouth with a rubber stopper. Use a nitrogen cylinder with a needle to fill the serum bottle with 99.99% nitrogen to remove the residual oxygen in the serum bottle. After 5 minutes of inflation, end the inflation. Sterilize at 121 °C for 15 minutes. After cooling to room temperature, inoculate the second-cycle acclimated bacterial liquid at an inoculation amount of 10% (v / v). Immediately introduce syngas at a pressure of 0.12 Mpa for 5 minutes to remove the nitrogen in the serum bottle, so that Clostridium butyricum is completely in the gas environment of syngas for fermentation. Finally, keep the headspace pressure of the serum bottle at 0.1 MPa, seal it, and shake and ferment culture at 37 °C and 200 rpm until the stationary phase to obtain the culture solution; S3-2: Use the culture solution obtained in S3-1 as the inoculum and repeat S3-1 ten times to finally obtain the third-cycle acclimated bacterial liquid.

[0044] Perform plate streaking with the third-cycle acclimated bacterial liquid until a single colony is isolated to obtain Clostridium butyricum Gen160CO2. Clostridium butyricum Gen160CO2 was deposited on November 18, 2024 at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms. The taxonomic name is Clostridium butyricum, and the deposit number is CGMCC No. 32696. The deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the postal code of the deposit address is 100101.

[0045] Example 3: In this example, the syngas flow rate is: 200 mL / min, and the syngas composition is: 20% mole CO2 and 80% mole H2.

[0046] In this example, the nitrogen flow rate is: 200 mL / min.

[0047] In this example, the formulation of the Clostridium enrichment medium is the same as that of Example 1.

[0048] In this example, the formulation of the fermentation medium is basically the same as that of Example 1, except that the content of yeast powder in the medium is 0.05 g / L.

[0049] Experimental group: Clostridium butyricum Gen160CO2 was inoculated into Clostridium - enriched medium and cultured at 37°C and 200 rpm until the logarithmic growth phase to obtain the Clostridium butyricum Gen160CO2 seed solution; the fermentation medium was aliquoted into serum bottles with a capacity of 100 mL, with 50 mL of liquid in each bottle. The bottle mouths were sealed with rubber stoppers, and a nitrogen cylinder equipped with a needle was used to fill 99.99% nitrogen into the serum bottles to remove the residual oxygen in the serum bottles. After the inflation time reached 5 min, the inflation ended. It was sterilized at 121°C for 15 min, cooled to room temperature, and then inoculated with the Clostridium butyricum Gen160CO2 seed solution at an inoculation amount of 10% (v / v). Immediately, syngas was introduced at a pressure of 0.12 Mpa for 5 min to remove the nitrogen in the serum bottles, so that Clostridium butyricum was completely in the gas environment of syngas for fermentation. Finally, the headspace pressure of the serum bottle was maintained at 0.1 MPa, sealed, and cultured by shaking fermentation at 37°C and 200 rpm for 10 days, and fresh syngas was replaced every day. Samples were taken regularly and the absorbance (OD 650 ) of the bacterial solution at 650 nm was measured to plot the growth curve.

[0050] Control group: Clostridium butyricum Gen160CO2 was inoculated into Clostridium - enriched medium and cultured at 37°C and 200 rpm until the logarithmic growth phase to obtain the Clostridium butyricum Gen160CO2 seed solution; the fermentation medium was aliquoted into serum bottles with a capacity of 100 mL, with 50 mL of liquid in each bottle. The bottle mouths were sealed with rubber stoppers, and a nitrogen cylinder equipped with a needle was used to fill 99.99% nitrogen into the serum bottles to remove the residual oxygen in the serum bottles. After the inflation time reached 5 min, the inflation ended. It was sterilized at 121°C for 15 min, cooled to room temperature, and then inoculated with the Clostridium butyricum Gen160CO2 seed solution at an inoculation amount of 10% (v / v). Immediately, nitrogen was introduced at a pressure of 0.12 Mpa for 5 min, so that Clostridium butyricum was completely in the gas environment of nitrogen for fermentation. Finally, the headspace pressure of the serum bottle was maintained at 0.1 MPa, sealed, and cultured by shaking fermentation at 37°C and 200 rpm for 10 days, and fresh nitrogen was replaced every day. Samples were taken regularly and the absorbance (OD 650 ) of the bacterial solution at 650 nm was measured to plot the growth curve.

[0051] The results are as Figure 2 shown. During the entire fermentation and culture period, the growth ability (OD 650 value) of Clostridium butyricum Gen160CO2 using syngas as the gaseous carbon source was overall increased by more than 33% compared with the control group. It shows that Clostridium butyricum Gen160CO2 has the ability to convert syngas into cell growth.

[0052] Example 4: In this example, the flow rate of the synthesis gas is: 200 mL / min, and the composition of the synthesis gas is: 20% mol CO2 and 80% mol H2.

[0053] In this example, the flow rate of nitrogen gas is: 200 mL / min.

[0054] In this example, the formula of the Clostridium - enhanced medium is the same as that in Example 1.

[0055] In this example, the formula of the fermentation medium is basically the same as that in Example 1, except that the content of yeast powder in the medium is 0.05 g / L.

[0056] The ability of Clostridium butyricum Gen160CO2 to ferment synthesis gas to produce short - chain fatty acids and lactic acid: Inoculate Clostridium butyricum Gen160CO2 into the Clostridium - enhanced medium, and culture it at 37 °C and 200 rpm until the logarithmic growth phase to obtain the Clostridium butyricum Gen160CO2 seed solution; Dispense the fermentation medium into serum bottles with a capacity of 100 mL, with 50 mL of liquid in each bottle. Seal the bottle mouth with a rubber stopper, and use a nitrogen gas cylinder connected with a needle to fill 99.99% nitrogen gas into the serum bottle to remove the residual oxygen in the serum bottle. After 5 minutes of inflation, stop inflation. Sterilize at 121 °C for 15 minutes, cool to room temperature, and then inoculate the Clostridium butyricum Gen160CO2 seed solution at an inoculation amount of 10% (v / v). Immediately introduce synthesis gas at a pressure of 0.12 Mpa for 5 minutes to remove nitrogen gas in the serum bottle, so that Clostridium butyricum is completely in the gas environment of synthesis gas for fermentation. Finally, keep the headspace pressure of the serum bottle at 0.1 MPa, seal it, and culture it by shaking fermentation at 37 °C and 200 rpm for 10 days. During this period, replace the fresh synthesis gas every day. Regularly take samples and determine the contents of short - chain fatty acids (formic acid, acetic acid, butyric acid) and lactic acid in the fermentation broth by high - performance liquid chromatography.

[0057] The ability of Clostridium butyricum Gen160 to ferment syngas to produce short-chain fatty acids and lactic acid: Inoculate Clostridium butyricum Gen160 into Clostridium enhanced medium and culture it at 37 °C and 200 rpm until the logarithmic growth phase to obtain the Clostridium butyricum Gen160 seed solution; Dispense the fermentation medium into serum bottles with a capacity of 100 mL, with 50 mL of liquid in each bottle, seal the bottle mouth with a rubber stopper, use a nitrogen cylinder connected with a needle to fill 99.99% nitrogen into the serum bottle to remove the residual oxygen in the serum bottle, end the inflation after 5 minutes of inflation, sterilize at 121 °C for 15 minutes, cool to room temperature, and inoculate the Clostridium butyricum Gen160 seed solution at an inoculation amount of 10% (v / v). Immediately introduce syngas at a pressure of 0.12 Mpa for 5 minutes to remove the nitrogen in the serum bottle, so that Clostridium butyricum is completely in the gas environment of syngas for fermentation. Finally, keep the headspace pressure of the serum bottle at 0.1 MPa, seal it, and shake and ferment at 37 °C and 200 rpm for 10 days, and replace the fresh syngas every day during this period. Regularly take samples and determine the contents of short-chain fatty acids (formic acid, acetic acid, butyric acid) and lactic acid in the fermentation broth by high-performance liquid chromatography sampling.

[0058] Figure 3 It is the high-performance liquid chromatography diagram of the standard product of mixed acid substances (formic acid, acetic acid, butyric acid, lactic acid). The peak emergence time of formic acid is 16.199 min, the peak emergence time of acetic acid is 17.671 min, the peak emergence time of butyric acid is 25.468 min, and the peak emergence time of lactic acid is 14.968 min. Figure 4 It is the standard curve diagram of each acid substance. Figure 5 It is the high-performance liquid chromatography diagram of the fermentation broth of Clostridium butyricum Gen160. Figure 6 It is the high-performance liquid chromatography diagram of the CO2 fermentation broth of Clostridium butyricum Gen160. Figure 7 It is the change trend of the short-chain fatty acid production in the fermentation broth of Clostridium butyricum Gen160 with time. Figure 8 It is the change trend of the short-chain fatty acid production in the CO2 fermentation broth of Clostridium butyricum Gen160 with time.

[0059] The formic acid production in the fermentation broth of Clostridium butyricum Gen160 showed an increasing trend from day 0 to day 1, reaching the highest value of 0.15 g / L on day 1. Thereafter, it gradually decreased as the fermentation culture proceeded and was consumed completely by day 7. The acetic acid production showed an increasing trend from day 0 to day 2, and then the growth rate slowed down and tended to be stable, reaching the highest value of 0.23 g / L on day 10. The butyric acid production generally showed an increasing trend as the fermentation culture proceeded and reached the highest value of 1.32 g / L on day 10. Lactic acid was produced after day 3, showed an increasing trend in production from day 3 to day 4, reaching the highest value of 0.12 g / L on day 4. Thereafter, it gradually decreased as the fermentation culture proceeded and was consumed completely by day 5. From day 1 to day 3 and from day 5 to day 6, the fermentation broth of Clostridium butyricum Gen160 contained formic acid, acetic acid and butyric acid, but no lactic acid. On day 4, the fermentation broth of Clostridium butyricum Gen160 contained formic acid, acetic acid, butyric acid and lactic acid. From day 7 to day 10, the fermentation broth of Clostridium butyricum Gen160 contained acetic acid and butyric acid, but no formic acid and lactic acid.

[0060] Formic acid in the fermentation broth of Clostridium butyricum Gen160 CO2 was produced at the initial stage of fermentation. As the fermentation culture proceeded, the production generally showed an increasing trend and reached the highest value of 0.23 g / L on day 9. Acetic acid was produced after day 2. As the fermentation culture proceeded, the production generally showed an increasing trend and reached the highest value of 0.16 g / L on day 10. Butyric acid was produced after day 1. As the fermentation culture proceeded, the production generally showed an increasing trend and reached the highest value of 0.25 g / L on day 9. Lactic acid was produced after day 1, showed an increasing trend in production from day 1 to day 4, reaching the highest value of 0.16 g / L on day 4. Thereafter, it gradually decreased as the fermentation culture proceeded and was consumed completely by day 7. On day 1, the fermentation broth of Clostridium butyricum Gen160 CO2 contained formic acid, but no acetic acid, butyric acid and lactic acid. On day 2, the fermentation broth of Clostridium butyricum Gen160 CO2 contained formic acid, butyric acid and lactic acid, but no acetic acid. From day 3 to day 6, the fermentation broth of Clostridium butyricum Gen160 CO2 contained formic acid, acetic acid, butyric acid and lactic acid. From day 7 to day 10, the fermentation broth of Clostridium butyricum Gen160 CO2 contained formic acid, acetic acid and butyric acid, but no lactic acid.

[0061] The ability differences of Clostridium butyricum Gen160CO2 and Clostridium butyricum Gen160 in fermenting syngas to produce short-chain fatty acids (formic acid, acetic acid, butyric acid) and lactic acid are relatively large. During the entire fermentation cycle, the highest value of formic acid production in the fermentation broth of Clostridium butyricum Gen160CO2 increased by 53% compared with the highest value of formic acid production in the fermentation broth of Clostridium butyricum Gen160, and the highest value of lactic acid production in the fermentation broth of Clostridium butyricum Gen160CO2 increased by 33% compared with the highest value of lactic acid production in the fermentation broth of Clostridium butyricum Gen160. At the end of the entire fermentation cycle, the cumulative production of formic acid in the fermentation broth of Clostridium butyricum Gen160CO2 was 0.2 g / L, the cumulative production of acetic acid was 0.16 g / L, the cumulative production of butyric acid was 0.24 g / L, and the cumulative production of lactic acid was 0, while the cumulative production of formic acid in the fermentation broth of Clostridium butyricum Gen160 was 0, the cumulative production of acetic acid was 0.23 g / L, the cumulative production of butyric acid was 1.32 g / L, and the cumulative production of lactic acid was 0.

[0062] Formic acid, acetic acid, butyric acid and lactic acid have various biological functions. For example, formic acid has bactericidal effects, can destroy bacterial cell walls, inhibit the activity of bacterial enzymes, and affect the replication of pathogen DNA; it can also reduce the pH value of the gastrointestinal tract, thereby inhibiting the reproduction of harmful microorganisms and promoting the proliferation of beneficial bacteria. Acetic acid helps to maintain an appropriate acidic pH value in the intestine, enabling beneficial intestinal microorganisms to survive and thrive, but preventing the invasion and stay of those harmful bacteria and opportunistic pathogens; it can also bind to certain receptors in the intestine to help control appetite and regulate fat storage. Butyric acid can provide energy for colon cells, promote the growth and development of intestinal villi, improve the digestion and absorption ability of animals, and repair damaged intestinal mucosal cells; it can also reduce the expression of pro-inflammatory cytokines and control the expression of pro-inflammatory genes by inhibiting nuclear factor κB. Lactic acid is an essential organic acid for the human body, which can promote the growth of Bifidobacterium; lactic acid also has an appetizing effect, promoting gastrointestinal secretion and helping digestion. Clostridium butyricum Gen160CO2 provided by the present invention has the ability to ferment syngas as a gaseous carbon source to produce a variety of short-chain fatty acids (formic acid, acetic acid, butyric acid) and lactic acid, and has high industrial application value and economic value.

[0063] The above are only the preferred embodiments of the present invention, and all equivalent changes and modifications made according to the scope of the patent application of the present invention shall fall within the scope covered by the present invention.

Claims

1. A Clostridium butyricum strain for fermenting syngas to produce short-chain fatty acids and lactic acid, characterized in that: The named Clostridium butyricum is Clostridium butyricum ( Clostridium butyricum ), Gen160CO2, which was deposited at the General Microbiology Center of the China Committee for Culture Collection of Microorganisms on November 18, 2024, with the deposit number CGMCC No. 32696, and the deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the postal code of the deposit address is 100101.

2. The Clostridium butyricum according to claim 1, characterized in that: The breeding method of the Clostridium butyricum is as follows: inoculate Clostridium butyricum Gen160 into a fermentation medium and carry out acclimation culture in a syngas environment. During the acclimation culture process, gradually reduce the content of organic nitrogen source in the fermentation medium through multiple transfers, and screen to obtain Clostridium butyricum Gen160CO2 that can ferment syngas to produce short-chain fatty acids and lactic acid. Among them, Clostridium butyricum Gen160 was deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on July 5, 2012, with the deposit number CGMCC No. 6317, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the postal code of the deposit address is 100101.

3. The application of the Clostridium butyricum according to claim 1 in the biological utilization of syngas.

4. The application according to claim 3, characterized in that: The syngas is composed of single-carbon gas and H2, and the single-carbon gas includes CO and CO2.

5. The application of the Clostridium butyricum according to claim 1 in the preparation of short-chain fatty acids and lactic acid.

6. The application according to claim 4, wherein: The short-chain fatty acids include formic acid, acetic acid, and butyric acid.

7. A method for preparing short-chain fatty acids and lactic acid, characterized in that: Inoculate the Clostridium butyricum according to claim 1 into the deoxygenated fermentation medium and carry out shake flask fermentation culture in a syngas environment to obtain a fermentation broth containing short-chain fatty acids and lactic acid.

8. The method according to claim 7, wherein: The formula of the fermentation medium is: 0.05 g / L of yeast powder, 2 g / L of ammonium sulfate, 0.2 g / L of magnesium sulfate heptahydrate, 0.5 g / L of potassium dihydrogen phosphate, 1 g / L of dipotassium hydrogen phosphate, 1 mL / L of calcium chloride solution, 1 mL / L of Fe solution, 1 mL / L of trace element solution, and the rest is water; among them, the concentration of the calcium chloride solution is 20 g / L; the formula of the Fe solution is: 5 g / L of FeSO4•7H2O, 4 mL / L of 37 wt% HCl, and the rest is water; the formula of the trace element solution is: 0.2 g / L of CoCl2•6H2O, 0.07 g / L of ZnCl2, 0.02 g / L of CuCl2•2H2O, 0.1 g / L of MnCl2•4H2O, 0.035 g / L of Na2MoO4•2H2O, 0.06 g / L of H3BO3, 0.025 g / L of NiCl2•6H2O, 0.9 mL / L of 37 wt% HCl, and the rest is water.

9. The method according to claim 7, wherein: The syngas is composed of single-carbon gas and H2, and the single-carbon gas includes CO and CO2.

10. The method according to claim 7, characterized in that: The short-chain fatty acids include formic acid, acetic acid, and butyric acid.

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