Anaerobic gas-feeding bacteria subjected to mutagenesis screening and application of anaerobic gas-feeding bacteria in high-yield acid production of synthesis gas conversion

Abstract

The invention provides an anaerobic gas-feeding bacterium subjected to mutagenesis screening and application of the anaerobic gas-feeding bacterium in high-yield acid production through synthesis gas conversion. The anaerobic gas-feeding bacterium is a Clostridium sp. Strain P38 strain, and the preservation number of the anaerobic gas-feeding bacterium is CGMCC (China General Microbiological Culture Collection Center) No.46262. According to the invention, an acid-producing Clostridium sp. Strain P38 strain is obtained through ARTP mutagenesis screening, and through optimization of culture conditions, especially addition of sodium molybdate in a culture system and control of the pH value of the culture system, the maximum concentration of bacteria fermented by the anaerobic gas-feeding bacteria Clostridium sp. Strain P38 and the capability of fermentation acid production are improved, the maximum cell concentration of the strain can reach OD600nm-2. 0Abs, the maximum acetic acid yield is 5g / L, and the hexanoic acid content is 3.5 g / L.

Description

Technical Field

[0001] The invention belongs to the technical field of functional microorganism screening and application, and specifically relates to an anaerobic gas-eating bacterium screened by mutation and its application in high acid production by syngas conversion. Background Art

[0002] Microbial conversion of syngas to produce chemicals has been a hot topic in recent years. Not only can it help my country achieve its "dual carbon" goals, but the chemicals produced by the converted gas can also provide a cheap carbon source for synthetic biology while reducing dependence on petroleum-based products. However, the lack of excellent functional strains and the imperfection of the corresponding anaerobic gas fermentation process have hindered its further application.

[0003] Carbon-containing synthesis gas refers to a type of mixed gas with CO as the main component. This type of gas is mostly composed of CO, CO 2 and H 2 , is a kind of low-cost raw material with great development value for manufacturing petrochemical chemicals. Anaerobic gas-feeding microorganisms can convert carbon-containing components in the gas into chemicals with higher value, such as acetic acid, butyric acid, ethanol, butanol and other acid and alcohol compounds, which can be used as substitutes for petrochemical products. Depending on the product demand and downstream process requirements, the required product categories are also different.

[0004] However, the fermentation characteristics of aerobic microorganisms are complex, and there is a lack of efficient production bacteria and purposeful production and regulation methods for target fermentation products, which limits the application of anaerobic aerobic microorganisms. Therefore, if efficient conversion microorganisms can be developed, it will greatly expand the fermentation application of anaerobic aerobic bacteria and realize the industrial development of green bioprocesses. Summary of the invention

[0005] The purpose of the present invention is to provide an anaerobic aerobic bacterium screened by mutation and its application in high acid production by syngas conversion, namely, an anaerobic aerobic bacterium Clostridium sp. strain P38 obtained by screening and a method for converting syngas to produce acid.

[0006] The Clostridium sp. P38 strain provided by the present invention was deposited at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, General Microbiology Center of China Microorganism Culture Collection Administration, on November 6, 2024, with the deposit number CGMCC No.46262.

[0007] The present invention also provides a use of the Clostridium sp. P38 strain, which is the use in producing organic acids by converting syngas;

[0008] As described in the embodiment, the synthesis gas composition includes 50% CO, 35% CO2 and 15% H 2 .

[0009] The organic acid, as specifically described in the examples, is acetic acid, hexanoic acid or n-butyric acid.

[0010] Another aspect of the present invention provides a method for producing organic acids by converting synthesis gas, wherein the method uses the above-mentioned Clostridium sp. P38 strain as a fermentation bacterium.

[0011] The method comprises the following steps: sodium molybdate is added to the fermentation medium; and the addition amount is controlled at 0.75-0.85 μM; during the culture process, ammonia water is used to adjust the pH of the culture system to maintain it at 6.5-7.5.

[0012] The method, wherein the culture medium comprises the following components in volume fractions: 1-5% of a large amount of element solution, 0.2-0.8% of a reducing agent solution, and the remainder of a nutrient solution.

[0013] The invention obtains an acid-producing Clostridium sp. strain P38 strain through ARTP mutagenesis screening, and improves the maximum bacterial concentration and the ability of fermenting and producing acids of the anaerobic aerobic bacteria Clostridium sp. strain P38 through optimization of culture conditions, especially the addition of sodium molybdate in the culture system and the control of pH of the culture system, the cell concentration of the strain can reach a maximum of OD600nm-2.0Abs, the maximum acetic acid yield is 5g / L, and the caproic acid content is 3.5g / L. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 : ARTP-induced mortality curve of Clostridiumcarboxidivorans P7,

[0015] Figure 2 :Photographs of strain screening and culture;

[0016] Figure 3 :OD and acid production of gas fermentation screening of different mutant strains,

[0017] Figure 4 :OD and acid production of Clostridium sp. strain P38 mutant after optimization of gas fermentation conditions. DETAILED DESCRIPTION

[0018] The Clostridium sp. strain P38 strain provided by the present invention is obtained by subjecting the starting strain Clostridium sp. strain P7 to atmospheric pressure room temperature plasma mutagenesis (ARTP), and was deposited on November 6, 2024 at the General Microbiological Center of the China Microbiological Culture Collection Administration, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with a deposit number of: CGMCC No.46262.

[0019] The present invention is described in detail below in conjunction with embodiments and drawings.

[0020] Example 1: Mutation of anaerobic gas-producing bacteria

[0021] 1. Preparation of original strain Clostridium sp. strain P7 bacterial suspension

[0022] The original strain Clostridium carboxidivorans P7 was purchased from the German National Culture Collection DSMZ by our laboratory. The strain number is 15243 and it is a model strain. The following activation medium is used: 5g / L tryptone, 5g / L beef extract peptone, 10g / L yeast extract powder, 10mg / L calcium chloride dihydrate, 20mg / L magnesium sulfate heptahydrate, 40mg / L dipotassium hydrogen phosphate, 40mg / L potassium dihydrogen phosphate, 80mg / L sodium chloride, 0.5g / L L-cysteine ​​hydrochloride, 1g / L sodium bicarbonate, 5g / L glucose. Adjust the pH to 7.0. After preparation, use anaerobic bottles to dispense, fill with nitrogen (99.995%) for more than 5 minutes to remove the air in the bottle, and sterilize at 115℃ for 30 minutes. Cultivate at 35℃ for 24 to 48 hours, and obvious growth can be seen. Take 1 mL of P7 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 bacterial solution to be treated is 10%, to obtain the bacterial suspension to be induced by mutation.

[0023] 2. Plasma mutagenesis operation

[0024] Place the sterile metal sheet, the prepared bacterial solution to be treated, and the centrifuge tube containing 980μL of activation culture medium in the anaerobic box in the anaerobic station; take the anaerobic box and sterile tweezers to the ARTP operation room for operation; turn on the ARTP mutagenesis system and perform UV sterilization for 10 minutes. Take the sterile metal slide out of the anaerobic box and quickly move it to the ARTP mutagenesis system operation chamber. Use sterile tweezers to place the slide in the corresponding hole position, adjust the knob under the stage so that the slide is 2mm from the air flow port, set the instrument power to 100W, the air flow parameter to 10SLM, and the irradiation time to 0s, 2s, 6s, 8s, 10s, 12s, 14s for plasma mutagenesis, and treat one slide at a time. After mutagenesis, wash the cells on the carrier with saline and draw a survival curve.

[0025] The experimental results are as follows Figure 1 In order to increase the probability of obtaining strains with high acid production activity and strong growth ability, 12s was selected as the mutagenesis irradiation time for subsequent screening according to the preliminary experimental results. At this time, the cell survival rate was 7%.

[0026] Example 2: Screening method for highly active anaerobic gas-producing bacteria

[0027] According to the above mutagenesis method, the sterile metal slide was taken out from the anaerobic box and quickly moved to the ARTP mutagenesis system operation chamber. The slide was placed in the corresponding hole with sterile tweezers, and the knob under the stage was adjusted so that the slide was 2mm from the air flow port, which was the experimental group; another metal slide was placed in the operation room, but not in the corresponding hole of the corresponding stage, which was set as the control group; experimental group: 20μL of bacterial suspension was added to the metal slide, the irradiation time was 12s, and the "Start Processing" button was clicked to start processing the sample. After the sample was processed After the treatment, use sterile tweezers to place the slide into an EP tube containing 980 μL of activation culture medium; control group: take 20 μL of bacterial suspension and add it to the metal slide, and use sterile tweezers to place the slide into an EP tube containing 980 μL of deoxygenated activation culture medium in the sample and corresponding experimental group; oscillate all the EP tubes of the control and treatment groups on an oscillator for 1 minute to wash the microorganisms attached to the bacterial slide into the liquid to form a new bacterial suspension; dilute the new bacterial suspension and apply it. 2 , 10 3 The primary screening medium is a solid screening medium, containing 1-5% macroelement solution, 1-5% trace element solution, and 0.2-0.8% reducing agent solution, wherein the composition of the macroelement solution (g / L): NaCl 80, NH 4 Cl 100, KCl 10, KH 2 PO 4 10, MgSO 4 20, CaCl 24. Trace element solution composition (g / L): nitrilotriacetic acid 10.00, MnSO 4 ·H 2 O 5.00, (NH 4 ) 2 SO4·FeSO 4 6H 2 O 4.00, CoCl 2 6H 2 O1.00, ZnSO 4 7H 2 O 1.00, CuCl 2 ·2H 2 O 0.10, NiCl 2 6H 2 O 0.10, Na 2 MoO 4 ·2H 2 O 0.10, Na 2 O 4 Se 0.10, Na 2 WO 4 ·2H 2 O 0.10, pH 6.0; reducing agent solution composition (100 ml): NaOH 0.9 g, L-Cysteine·HCl 4 g, Na 2 S.9H 2 O 4g. The remaining nutrient solution, 1.2% agar, 5mg / L resazurin indicator, the final pH is 6.0. The coated plate is placed in a plexiglass sealed jar for inverted culture. The culture temperature is 37°C, the gas source is 50% CO, 35% CO2, 15% H2, and the pressure is 0.08MPa. After the plate in the glass sealed jar is cultured, the larger single colonies or colonies with larger light shadow circles in the experimental group compared with the control group are picked under anaerobic conditions ( Figure 2 ), activation culture medium, activation culture, seed preservation.

[0028] The preserved strains were cultured for gas fermentation in batches, and the OD600 value, gas conversion efficiency, and acetic acid and butyric acid yields 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: add 2 ml of a large amount of element solution, 0.05 g of yeast extract to a 100 ml system, adjust the initial pH range of the system to 6.0, 5 ml of reducing agent solution, fill with 99.999% nitrogen at a rate of 2 L / min for 5 minutes, seal, and sterilize at 121 degrees for 20 minutes. After sterilization, cool to about 40 degrees, add 0.1 ml of trace element storage solution, and complete the construction of the culture system. Connect 10 ml of the culture solution of the mutagenic strain that has been cultured to the logarithmic growth phase using an activated medium, and then continuously introduce simulated synthesis gas (gas composition: 50% CO, 35% CO) at a flow rate of 2 L / min and a pressure of 0.4 bar. 2 、15%H 2 ) for 5 minutes, pressurized to 0.6 bar and sealed. Shake and culture at 37 degrees and 180 rpm, and take samples at regular intervals for fermentation product detection. Finally, it was found that the growth rate and gas conversion efficiency of Clostridium sp. strain P38 were the highest among the 10 mutant strains screened, with the highest OD reaching 1.8, the acetic acid production reaching 4 g / L, and the butyric acid production reaching 1 g / L ( Figure 2 ).

[0029] The screened Clostridium sp. P38 strain was deposited at the General Microbiology Center of China Microorganism Culture Collection Administration, No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing on November 6, 2024, with the deposit number CGMCC No. 46262.

[0030] Example 3: Optimization of fermentation medium to increase acid production of induced strains

[0031] On the basis of the above fermentation medium, the medium components and fermentation conditions were optimized, and the final components of the medium were determined as follows: trace elements, sodium molybdate and culture medium; the specific composition of each component is consistent with the description in Example 2. The volume of trace elements is 0.35% of the volume of the culture medium; the culture medium components: 1% of the macroelement solution, 0.5% of the reducing agent solution, and the rest of the nutrient solution. In addition, the amount of sodium molybdate added to the culture system is controlled at 0.75-0.85 μM, and ammonia water is used to adjust the pH of the culture system to 6.5-7.5 during the culture process. During the gas fermentation process, the target product is detected regularly using the gas phase. The experiment uses the internal standard method to perform qualitative and quantitative analysis of the samples. First, a standard curve needs to be made. Take 0.1g acetic acid and 0.02g hexanoic acid and use a 500mL volumetric flask to make up the volume. After mixing evenly, take out the solution for gradient dilution. Take 800 μL of the solution with different concentration gradients, add 200 μL of internal standard solution (3.5 g / L isobutanol, 2 g / L isobutyric acid and 1 M hydrochloric acid), mix well, and filter to sterilize. The ratio of the peak area of ​​different concentration gradients of acetic acid and hexanoic acid to the peak area of ​​the internal standard solution is measured by 9790 gas chromatograph. Draw the standard curve, R 2 〉0.99, at this time, the column temperature of 9790 gas chromatograph is 120℃, the detector temperature is 160℃, and the column box temperature is 160℃. For the determination of samples, 1.5ml of culture solution is taken out by syringe each time, and centrifuged at 10000r / min for 2min. After the supernatant is sterilized by filtration with a 0.22μm filter membrane, 800μL is drawn and placed in a liquid phase vial. After adding 200μL of internal standard mixed solution for acidification, it is detected by gas chromatography.

[0032] Finally, the test results showed that the use of optimized culture conditions increased the maximum bacterial concentration and the ability to produce acid by fermentation of the anaerobic aerobic bacteria Clostridium sp. strain P38. The maximum cell concentration of Clostridium sp. strain P38 could reach OD600nm~2.0Abs, the maximum acetic acid production was 5g / L, and the butyric acid content was 3.5g / L ( Figure 3 ). Compared with the original strain, the acetic acid production increased by 66% and the butyric acid production increased by 9 times ( Figure 3 ).

[0033] In summary, the anaerobic aerobic bacteria induced and screened by the present invention can efficiently convert synthesis gas into organic acids and can be used for industrial production applications.

Claims

1. A Clostridium sp. Clostridium, characterized in that The deposit number of the clostridium is CGMCC No.46262.

2. Use of the Clostridium according to claim 1 in producing organic acids by conversion of syngas.

3. The use according to claim 2, characterized in that The synthesis gas composition includes CO, CO2 and H2.

4. The use according to claim 2, characterized in that The organic acid is acetic acid, caproic acid or n-butyric acid.

5. A method for producing organic acid by converting synthesis gas, characterized in that: The method is to use the Clostridium described in claim 1 as a fermentation bacterium to produce organic acids.

6. The method according to claim 5, characterized in that The method described herein, wherein sodium molybdate is added to the fermentation medium.

7. The method according to claim 6, characterized in that The added amount of the sodium molybdate is 0.75-0.85 μM.

8. The method according to claim 5, characterized in that In the method, the pH of the culture system is adjusted to maintain at 6.5-7.5 during the culture process.

9. The method according to claim 8, characterized in that The method described is to use ammonia water to adjust the pH value of the culture system.

10. The method according to claim 5, characterized in that The culture medium of the method comprises the following components in volume fractions: 1-5% of a large amount of element solution, 0.2-0.8% of a reducing agent solution, and the remainder of a nutrient water solution.

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