Clostridium and application thereof in fermentation preparation of organic acid
Through ARTP mutagenesis screening and culture condition optimization, the highly efficient fermented Clostridium sp. Clostridium P50 strain was obtained, which solved the problems of low yield and high cost in the conversion of anaerobic food gas microorganisms into organic acids, and achieved efficient production of caproic acid.
Patent Information
- Application Number
- CN202510063797.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-15
- Publication Date
- 2025-04-18
AI Technical Summary
In the prior art, anaerobic food gas microorganisms have problems with low yield and high cost during the conversion of synthesis gas into organic acids. Since these strains are strictly anaerobic bacteria, genetic modification is difficult and there is a lack of efficient conversion efficiency.
Clostridium sp. Clostridium P50 strain was obtained by ARTP mutagenesis screening, and the fermentation performance and caproic acid yield of the strain were improved by optimizing the culture conditions, including the addition of sodium molybdate and the control of the pH of the culture system.
The efficient fermentation of Clostridium sp. Clostridium P50 strain was achieved, with the cell concentration reaching OD600nm~2.0Abs, and the maximum yield of caproic acid reached 5.4g/L, which significantly increased the yield of caproic acid relative to the starting strain.
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Figure CN119842540A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of functional microorganism screening and application, and specifically relates to a clostridium and an application thereof in preparing organic acids by fermentation. Background Art
[0002] Microbial conversion of syngas to produce chemicals has been a hot topic in recent years. Organic acids are a major product of microbial conversion of syngas, including acetic acid, butyric acid, caproic acid, etc. Among them, the synthesis of medium- and long-chain organic acids, such as caproic acid, is more difficult. During the conversion process, microorganisms first synthesize small molecule organic acids such as acetic acid, and then synthesize medium- and long-chain organic acids such as butyric acid and caproic acid.
[0003] Hexanoic acid is an important chemical product that can be used as a reagent, seasoning, desiccant, and resin production. The preparation methods of hexanoic acid include direct extraction, chemical synthesis, and biosynthesis. The direct extraction method has low yield and high cost; the chemical synthesis method has high pollution and high energy consumption problems; the biosynthesis is green and environmentally friendly, but low yield and high cost are still challenges. There is a class of anaerobic gas-eating microorganisms in nature that can grow with syngas (CO, CO2, and H2) as the only carbon source and energy source, and convert it into a variety of organic acids and alcohols, including acetic acid, butyric acid, hexanoic acid and other high value-added chemicals. This bacterium can not only fix the carbon source that pollutes the environment in the air, but also produce products with industrial value, and has great research value. At present, the problem is that since this type of bacteria belongs to strict anaerobic bacteria, the understanding of its physiological mechanism is limited, and genetic modification is also extremely difficult. There is still a lack of strains with high carbon conversion efficiency. Therefore, it is necessary to use a variety of technical means, such as physical and chemical mutagenesis, to obtain high-performance strains, especially high-yield strains for target products. The screening range still needs to be expanded to provide strain resources for syngas conversion. Summary of the invention
[0004] The purpose of the present invention is to provide a clostridium and application thereof in preparing organic acids by fermentation, thereby making up for the deficiencies of the prior art.
[0005] The Clostridium sp. P50 strain provided by the present invention was deposited on November 6, 2024 at the General Microbiology Center of the China Culture Collection Administration, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No.46263.
[0006] The present invention also provides a use of the Clostridium P50 strain, which is use in fermentation to prepare organic acids.
[0007] As a specific record of the embodiment, the organic acid is hexanoic acid;
[0008] The present invention also provides a method for preparing caproic acid by fermenting synthetic gas, wherein the method uses the above-mentioned Clostridium P50 strain as a fermentation strain to catalyze the synthetic gas to prepare caproic acid;
[0009] The method, wherein the synthesis gas comprises carbon monoxide, carbon dioxide and hydrogen;
[0010] Furthermore, the volume ratio of carbon monoxide, carbon dioxide and hydrogen in the synthesis gas is 10:7:3.
[0011] The method comprises adding sodium molybdate to the culture system at a concentration of 0.8 to 1 μM, and using ammonia water to adjust the pH of the culture system to maintain it at 6.5 to 8 during the culture process to obtain a fermentation product mainly containing hexanoic acid.
[0012] The invention obtains an acid-producing Clostridium sp. strain P50 strain through ARTP mutagenesis screening, and improves the maximum bacterial concentration and the ability of producing caproic acid by fermentation of the anaerobic aerobic bacteria Clostridium sp. strain P50 through optimization of culture conditions, especially the addition of sodium molybdate in the culture system and the control of pH value of the culture system. The maximum cell concentration of the Clostridium sp. strain P50 strain can reach OD600nm-2.0Abs, and the maximum caproic acid yield is 5g / L. BRIEF DESCRIPTION OF THE DRAWINGS
[0013] Figure 1 : OD and high hexanoic acid producing strains screened by gas fermentation of different mutagenic strains,
[0014] Figure 2 :Photograph of the morphology of Clostridium sp.strain P50,
[0015] Figure 3 :OD and hexanoic acid production of Clostridium sp.strain P50 mutant after optimization of gas fermentation conditions. DETAILED DESCRIPTION
[0016] The present invention induces mutagenesis and screens a Clostridium sp. strain P50 mutant that can efficiently convert syngas to synthesize organic acids and has high gas conversion efficiency and product synthesis, and establishes a culture system for the bacteria to synthesize medium- and long-chain organic acids (mainly hexanoic acid) by gas fermentation, providing strong support for the further promotion and application of anaerobic gas fermentation technology.
[0017] The Clostridium sp. strain P50 provided by the present invention is obtained by subjecting the starting strain Clostridium sp. strain P38 to atmospheric pressure and room temperature plasma mutagenesis (ARTP); wherein the starting strain Clostridium sp. P38 was deposited at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, General Microbiology Center of China Microorganism Culture Collection Administration on November 6, 2024, with a deposit number of CGMCC No. 46262.
[0018] The Clostridium sp. P50 provided by the present invention was deposited on November 6, 2024 at the General Microbiology Center of the China Microorganism Culture Collection Administration, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number: CGMCC No.46263.
[0019] The implementation process of the present invention is described in detail below in conjunction with the embodiments and drawings.
[0020] Example 1: Mutagenesis screening of strains
[0021] The starting strain is Clostridium sp. strain P38, which was obtained by mutation in our laboratory based on a gas-eating acid-producing bacterium (strain number 15243) purchased from the German National Culture Collection DSMZ. The following activation medium was used: 5 g / L tryptone, 5 g / L beef extract peptone, 10 g / L yeast extract powder, 10 mg / L calcium chloride dihydrate, 20 mg / L magnesium sulfate heptahydrate, 40 mg / L dipotassium hydrogen phosphate, 40 mg / L potassium dihydrogen phosphate, 80 mg / L sodium chloride, 0.5 g / L L-cysteine hydrochloride, 1 g / L sodium bicarbonate, and 5 g / L glucose. The pH was adjusted to 7.0. After preparation, the mixture was divided into anaerobic bottles, filled with nitrogen (99.995%) for more than 5 minutes to remove the air in the bottle, and sterilized at 115°C for 30 minutes. The culture was carried out at 35°C for 24 to 48 hours, and obvious growth was observed. Take 1 mL of P38 bacterial solution (OD600≈1.1) cultured to the mid-logarithmic phase, add 154 μL of 75% glycerol to the 1 mL bacterial solution, and the final glycerol concentration of the treated bacterial solution is 10%, to obtain the bacterial suspension to be induced by mutagenesis. Set the ARTP instrument power to 100 W, the gas flow parameter to 10 SLM, and the irradiation time to 12 s for plasma mutagenesis.
[0022] Example 2: Screening method for highly active anaerobic gas-producing caproic acid bacteria
[0023] Dilute the mutagenic bacterial suspension and apply it. Dilute the resuspension by 10 2 , 10 3Two gradients. The primary screening medium is a solid screening medium containing 1-5% of a large number of element solutions (the formula of the large number of element solutions g / L: NaCl 80g / L, NH4Cl100g / L, KCl 10g / L, KH2PO4
[0024] 10g / L, MgSO4 20g / L, CaCl2 4g / L), 1-5% trace element solution (trace element solution formula g / L: nitrilotriacetic acid 10g / L, MnSO4·H2O 5g / L,
[0025] (NH4)2SO4·FeSO4·6H2O 4g / L, CoCl2·6H2O 1, ZnSO4·7H2O 1g / L, CuCl2·2H2O0.10g / L, NiCl2·6H2O 0.10g / L, Na2MoO4·2H2O0.10g / L, Na2O4Se 0.10g / L, Na2WO4·2H2O0.10g / L), 0.2-0.8% reducing agent solution (reducing agent solution formula 100ml: NaOH 0.9g, L-Cysteine·HCl4g, Na2S·9H2O 4g), the rest of nutrient water solution, 1.2% agar, resazurin indicator 5mg / L, the final pH is 6.0.
[0026] The coated plate was placed in an organic glass sealed jar for inverted culture at a temperature of 37°C, a volume ratio of syngas of 50% CO, 35% CO2, and 15% H2, and a pressure of 0.08 MPa. After the plate in the glass sealed jar was cultured, the larger single colony or colony with a larger light shadow circle in the mutagenesis compared to the starting strain was picked under an anaerobic station, and the activated culture medium was activated and cultured to preserve the seed.
[0027] The preserved strains were cultured by gas fermentation in batches, and the OD600 value, gas conversion efficiency, and hexanoic acid yield were detected. The gas fermentation performance of the strains was compared to screen out high-yield strains. The formula of the gas fermentation medium is as follows: 2 ml of a large amount of element solution, 0.05 g of yeast extract, the initial pH range of the system was adjusted to 6.0, 5 ml of reducing agent solution, 99.999% nitrogen was filled at a rate of 2 L / min for 5 min and then sealed, and sterilized at 121 ° C for 20 min. After sterilization, it was cooled to about 40 ° C, and then 0.1 ml of trace element storage solution was added to complete the construction of the culture system. 10 ml of the culture solution of the mutagenic strain cultured to the logarithmic growth phase using the activated medium was connected, and then the simulated synthesis gas (gas composition: 50% CO, 35% CO2, 15% H2) was continuously introduced at a flow rate of 2 L / min and a pressure of 0.4 bar to balance for 5 min, and pressurized to 0.6 bar and sealed. The culture was shaken at 37°C and 180 rpm, and samples were taken at regular intervals for fermentation product detection. Finally, it was found that the growth rate and hexanoic acid production of Clostridium sp. strain P50 were the highest among the 8 mutant strains screened, with the highest OD reaching 2 and the hexanoic acid production reaching 3.5 g / L ( Figure 1 ). The plate culture morphology of Clostridium sp.strain P50 is shown in Figure 2 .
[0028] Example 3: Verification of hexanoic acid production of the induced strain in 4 L fermentation medium
[0029] P50 (mutagenized strain) and P38 strain (starting strain) were activated in activation medium, and the caproic acid production of P50 was verified in a 4L gaslift fermentation device. The fermentation medium formula is as follows: 5% macroelement solution, 5% trace element solution, 0.2% reducing agent solution, and the remaining nutrient solution. In addition, the addition amount of sodium molybdate is controlled at 0.8-1μM. Simulated synthesis gas (gas composition: 50% CO, 35% CO2, 15% H2) is continuously introduced during the fermentation process, and ammonia water is used to adjust the pH of the culture system to maintain at 6.5-8 during the fermentation process. The fermentation was carried out for 4 days, and the maximum cell concentration of Clostridium sp. strain P50 strain could reach OD600nm~2.0Abs, and the maximum caproic acid content was 5.4g / L ( Figure 3 ). Compared with the starting strain, the hexanoic acid production increased by 5.75 times.
Claims
1. A Clostridium sp. P50 strain, characterized in that: The deposit number of the Clostridium sp. P50 strain is CGMCC No.46263.
2. Use of the Clostridium sp. P50 strain according to claim 1 in the fermentation preparation of organic acids.
3. The use according to claim 2, characterized in that The organic acid is caproic acid.
4. A method for preparing caproic acid by fermenting synthesis gas, characterized in that: The method is to use the Clostridium sp. P50 strain described in claim 1 as a fermentation strain to catalyze the synthesis of gas to produce caproic acid.
5. The method according to claim 4, characterized in that The method described herein, wherein the synthesis gas contains carbon monoxide, carbon dioxide and hydrogen.
6. The method according to claim 5, characterized in that The volume ratio of carbon monoxide, carbon dioxide and hydrogen in the synthesis gas is 10:7:
3.
7. The method according to claim 4, characterized in that The method is to add sodium molybdate into the culture system, and the pH of the culture system is maintained at 6.5-8 during the culture process.
8. The method according to claim 7, characterized in that The added concentration of the sodium molybdate is 0.8-1 μM.