Anaerobic gas-feeding bacterium capable of converting synthesis gas into alcohol compounds

Abstract

The invention provides an anaerobic gas-feeding bacterium capable of converting synthesis gas into an alcohol compound, the anaerobic gas-feeding bacterium is a Clostridium autoethanogenum E53 strain, and the preservation number of the anaerobic gas-feeding bacterium is CGMCC (China General Microbiological Culture Collection Center) No.46261. The invention further provides a preparation method of the anaerobic gas-feeding bacterium. According to the invention, a Clostridium autoethanogenum E53 strain with high alcohol yield is obtained through chemical mutagenesis screening, through optimization of culture conditions, especially addition of sodium bicarbonate in a culture system and control of the pH value of the culture system, the highest concentration of thalli fermented by anaerobic aerophagetes E53 and the capability of fermenting and accumulating alcohols are improved, the maximum cell concentration of the E53 strain can reach OD600nm-2.1 Abs, and the highest alcohol yield is 7.5 g / L.

Description

Technical Field

[0001] The present invention belongs to the technical field of industrial microbial screening applications, and particularly relates to an anaerobic gas-eating bacterium capable of converting syngas into alcohol compounds. Background Art

[0002] Alcohol compounds play an important role in the fields of chemical industry, medicine, biotechnology, etc., but still face many challenges in the preparation process. Alcohols can be used as solvents and reaction intermediates in the chemical industry, and are widely used in the synthesis of plastics, fibers, resins, coatings, etc. In the medical field, many drug molecules contain alcohol groups, such as vitamin E, sugars, steroids, etc. In the energy field, ethanol is an important component of biofuels and can replace traditional fossil fuels. In daily life, alcohols (such as ethanol and isopropanol) are widely used in disinfectants, cosmetics, and food additives.

[0003] Alcohol molecules contain hydroxyl groups (-OH) and can undergo various chemical reactions, such as esterification, oxidation, reduction, etc., and are important raw materials for synthesizing various organic compounds. Traditional synthesis methods are mainly chemical synthesis, which usually requires metal catalysts (such as palladium, nickel, etc.), with high costs and difficult recovery; many synthesis reactions require high temperature and high pressure, with high energy consumption and being unfavorable for environmentally friendly production; during chemical synthesis, there are many side reactions, resulting in difficult separation and purification of products and reduced efficiency. Moreover, in large-scale industrial production, relying on non-renewable resources such as petroleum, there are dual pressures of resources and the environment in the preparation of alcohols. In summary, due to their diverse applications and important chemical properties, alcohol compounds play a key role in multiple fields. However, their preparation process faces challenges in aspects such as catalysts, selectivity, greening, and large-scale production, and there is an urgent need to develop new synthesis technologies that are efficient, low-cost, and environmentally friendly.

[0004] Previous research in the prior art has found that there is a class of anaerobic gas-eating microorganisms in nature that can use syngas (CO, CO 2 and H 2 ) as the sole carbon source and energy source for growth, and convert it into various organic acids and alcohol substances, including high-value-added chemicals such as ethanol, butanol, and 2,3-butanediol. Therefore, applying syngas as a carbon source and energy source to the industrial fermentation process of microorganisms can not only form a green channel for sustainable energy and chemical development, save the operating costs of enterprises, and improve economic benefits, but also reduce carbon-containing gas emissions, accelerate the earth's carbon cycle, reduce the greenhouse effect, and create social and environmental benefits.

[0005] Currently, the main method to increase the target products of anaerobic gas-consuming microorganisms is through genetic modification. However, it is difficult to genetically modify anaerobic gas-utilizing microorganisms, lacking efficient gene editing tools and convenient genome engineering methods. This makes it complex and time-consuming to increase the target yield using genetic engineering methods, and there is an urgent need to screen and obtain efficient microorganisms for fermentation engineering. Summary of the Invention

[0006] The object of the present invention is to provide an anaerobic gas-consuming bacterium capable of converting syngas into alcohol compounds, namely Clostridium autoethanogenum E53 strain, which can catalyze syngas to convert into alcohol compounds.

[0007] The present invention first provides an anaerobic gas-consuming bacterium capable of converting syngas into alcohol compounds, which is the anaerobic gas-consuming bacterium Clostridium autoethanogenum E53 strain. It was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing on November 6, 2024, and the deposit number is CGMCC No. 46261.

[0008] The present invention also provides a use of the screened anaerobic gas-consuming bacterium E53 strain, which is used to catalyze syngas to convert into alcohol compounds;

[0009] The syngas described contains carbon monoxide, carbon dioxide and hydrogen;

[0010] As a specific record of the embodiment, the volume ratio of carbon monoxide, carbon dioxide and hydrogen is 10:7:3;

[0011] The alcohol compounds are ethanol or butanol.

[0012] The present invention also provides a method for converting syngas into alcohol compounds. In the method, the fermentation medium includes 2%-4% of the mineral element stock solution, 0.05% of yeast extract, 0.5% of the reducing agent stock solution, and 0.1% of the trace element stock solution.

[0013] The composition of the mineral element stock solution is as follows: 8% sodium chloride, 10% ammonium chloride, 1% potassium chloride, 1% potassium dihydrogen phosphate, 2% magnesium sulfate and 0.4% calcium chloride;

[0014] The composition of the reducing agent stock solution is as follows: 0.9% sodium hydroxide, 4% L-cysteine hydrochloride and 4% sodium sulfide nonahydrate.

[0015] The composition of the trace element stock solution is as follows: 1% nitrilotriacetic acid, 0.5% magnesium sulfate, 0.4% ammonium ferrous sulfate, 0.1% cobalt chloride, 0.1% zinc sulfate, 0.01% nickel chloride, 0.01% sodium selenate, and 0.01% sodium tungstate.

[0016] Furthermore, the initial pH range of the method of the present invention is adjusted to 4.7 - 7.5, and the concentration of sodium bicarbonate is 1 - 2 g / L to obtain a fermentation product mainly composed of alcohols.

[0017] The present invention obtained a high - alcohol - producing Clostridium autoethanogenum E53 strain through chemical mutagenesis screening. By optimizing the culture conditions, especially the addition of sodium bicarbonate and the control of the pH of the culture system in the culture system, the maximum cell concentration of anaerobic gas - consuming bacteria E53 fermentation and the ability to accumulate alcohols during fermentation were improved. The cell concentration of E53 strain can reach up to OD600nm ~ 2.1 Abs at most, and the highest alcohol yield is 7.5 g / L. Description of the Drawings

[0018] Figure 1 : Lethality curve of EMS - mutagenized Clostridium autoethanogenum;

[0019] Figure 2 : OD and acid - alcohol yield diagrams of different mutagenized strains in gas fermentation screening;

[0020] Figure 3 : Plate culture morphology diagram of Clostridium autoethanogenum E53;

[0021] Figure 4 : OD and alcohol yield diagrams after optimization of gas fermentation conditions of E53 mutagenized strain;

[0022] Figure 5 : OD and alcohol yield diagrams after optimization of gas fermentation conditions of E53 mutagenized strain. Detailed Embodiments

[0023] The present invention screened and obtained a (Clostridium autoethanogenum) E53 mutant strain, and used the screened strain to catalyze the synthesis of alcohols by gas fermentation. The starting strain for mutant screening was purchased from the German National Collection of Microorganisms (DSMZ), and the strain number was: 10061. The mutagenized strain (Clostridium autoethanogenum) E53 was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, located at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing on November 6, 2024, and the deposit number was: CGMCC No. 46261.

[0024] The present invention also provides a method for highly producing alcohols by using the screened Clostridium autoethanogenum E53.

[0025] The culture system of the anaerobic acidogenic bacteria using syngas provided by the present invention comprises trace elements, sodium bicarbonate and a culture medium; the culture medium includes 2%-4% of a mineral solution stock (V / V), 0.05% of yeast extract (M / V), 0.5% of a reducing agent stock (V / V), and 0.1% of a trace element stock solution (V / V). The composition of the Mineral solution stock (M / V) is: 8% sodium chloride, 10% ammonium chloride, 1% potassium chloride, 1% potassium dihydrogen phosphate, 2% magnesium sulfate and 0.4% calcium chloride; the composition of the Reducing Agent Stock (M / V) is 0.9% sodium hydroxide, 4% L-cysteine hydrochloride and 4% sodium sulfide nonahydrate. The composition of the trace element stock solution (M / V) is 1% nitrilotriacetic acid, 0.5% magnesium sulfate, 0.4% ammonium ferrous sulfate, 0.1% cobalt chloride, 0.1% zinc sulfate, 0.01% nickel chloride, 0.01% sodium selenate and 0.01% sodium tungstate. The culture conditions of the anaerobic acidogenic bacteria using syngas provided by the present invention are as follows: adjusting the initial pH range of the system to 4.7-7.5, and the concentration of sodium bicarbonate to 1-2 g / L, to obtain a fermentation product mainly composed of alcohols.

[0026] The following describes the implementation process of the present invention in detail with reference to examples.

[0027] Example 1: Mutagenesis screening of anaerobic syngas-utilizing bacteria capable of converting syngas into alcohol compounds

[0028] 1. Prepare a suspension of the starting strain Clostridium autoethanogenum

[0029] The starting strain is Clostridium gasigenes (strain number 10061) purchased by our laboratory from the German National Collection of Microorganisms DSMZ. The following activation medium is used: fructose 10 g / L, beef extract peptone 16 g / L, yeast extract 10 g / L, calcium chloride dihydrate 0.04 g / L, magnesium sulfate heptahydrate 0.2 g / L, ammonium chloride 1 g / L, potassium dihydrogen phosphate 0.1 g / L, sodium chloride 1 g / L, L-cysteine hydrochloride 0.75 g / L, potassium chloride 0.1 g / L. Adjust the pH to ~7.0. After preparation, aliquot into anaerobic bottles, fill with nitrogen (99.995%) for more than 5 minutes to remove the air in the bottles, and sterilize at 115°C for 30 minutes. The inoculation ratio is 10%, and culture at 37°C for 4 - 5 days, and obvious growth can be seen. Take 1 mL of the Clostridium autoethanogenum bacterial solution cultured to the mid-logarithmic phase (OD600≈0.6 - 0.8) to obtain the bacterial suspension to be mutagenized.

[0030] 2. Chemical mutagenesis screening with EMS (ethyl methane sulfonate)

[0031] Add EMS to the activated medium so that its contents are 0, 0.2%, 0.4%, 0.6%, 0.8%, 1.0%, 1.2%, 1.4%, 1.6%, 1.8%, 2% respectively, and culture at 37°C for 6 h. Dilute the above culture solution and spread it on the activated medium containing agar (activated medium × 3). Calculate the lethality rate according to the control group. Finally, at the EMS addition concentration of 1.4%, the lethality rate is 94.5%. The lethality rate curve is shown in Figure 1 .

[0032] Select the plate with the mutagenesis concentration of 1.4% EMS, and transfer the bacteria to the plate with fermentation medium. The formula of the fermentation medium is as follows: 2% - 4% mineral element stock solution (mineral solution stock (V / V)), 0.05% yeast extract (M / V), 0.5% reducing agent stock solution (reducing agent Stock (V / V)), 0.1% trace element stock solution (V / V). The composition of Mineral solution stock (M / V) is: 8% sodium chloride, 10% ammonium chloride, 1% potassium chloride, 1% potassium dihydrogen phosphate, 2% magnesium sulfate and 0.4% calcium chloride; the composition of Reducing Agent Stock (M / V) is 0.9% sodium hydroxide, 4% L-cysteine hydrochloride and 4% sodium sulfide nonahydrate. The composition of the trace element stock solution (M / V) is 1% nitrilotriacetic acid, 0.5% magnesium sulfate, 0.4% ammonium ferrous sulfate, 0.1% cobalt chloride, 0.1% zinc sulfate, 0.01% nickel chloride, 0.01% sodium selenate and 0.01% sodium tungstate.

[0033] The coated plates were placed in an acrylic sealed jar for inverted cultivation at a temperature of 30 °C, with a gas source of 50% CO, 35% CO 2 , 15% H 2 , and a pressure of 0.08 MPa. After the plates in the glass sealed jar were cultivated, under anaerobic conditions, compared with the control group, the larger single colonies in the experimental group were picked, activated in the activation medium, and preserved.

[0034] The preserved strains were batch-cultivated by gas fermentation, and the OD600 value, gas conversion efficiency, and acid and alcohol yields were detected to compare the gas fermentation performance of the strains, so as to screen out high-yield strains. The preparation method of the gas fermentation medium is as follows: Add 2 ml of mineral solution stock, 0.05 g of yeast extract to a 100 ml system, adjust the initial pH range of the system to 6.0, add 5 ml of reducing agent Stock, fill with 99.999% nitrogen at a speed of 2 L / min for 5 min and then seal, sterilize at 121 °C for 20 min. After sterilization, cool to about 40 °C, and then add 0.1 ml of trace element stock solution to complete the construction of the culture system. Add 10 ml of the mutagenized strain culture solution cultivated to the logarithmic growth phase using the activation medium, and then continuously introduce simulated syngas (gas composition: 50% CO, 35% CO 2 , 15% H 2 ) at a flow rate of 2 L / min and a pressure of 0.4 bar for 5 min to balance, and then pressurize to 0.6 bar and seal. Cultivate with shaking at 37 °C and 180 rpm, and sample at regular intervals for detection of fermentation products. Finally, it was determined that the growth rate and gas conversion efficiency of Clostridium autoethanogenum E53 strain were the highest among the 12 mutagenized strains screened, with its OD reaching up to 1.8, and the alcohol yield could reach 5.1 g / L ( Figure 2 ). The plate culture morphology diagram of Clostridium autoethanogenum E53 is shown in Figure 3 .

[0035] Example 2: Optimization of the fermentation medium to improve the alcohol yield of mutagenized strains

[0036] In the above fermentation medium, Clostridium autoethanogenum E53, the parental strain Clostridium autoethanogenum, and the related strain Clostridium carboxidivorans P7 were used for fermentation culture, and samples were taken daily to detect the yields of alcohols (ethanol, butanol). During the gas fermentation process, the target products were regularly detected by gas phase. The internal standard method was used for qualitative and quantitative analysis of the samples in the experiment. First, a standard curve needed to be made. 0.1 g of ethanol and 0.02 g of n-butanol were taken and made up to the mark with a 500 mL volumetric flask. After mixing evenly, the solution was taken out for gradient dilution. 800 μL of solutions with different concentration gradients were taken, 200 μL of internal standard solution (3.5 g / L isobutanol, 2 g / L isobutyric acid, and 1 M hydrochloric acid) was added, mixed evenly, and filtered to remove bacteria. The ratios of the peak areas of ethanol and n-butanol at different concentration gradients to the peak area of the internal standard solution were determined by a 9790 gas chromatograph. The standard curve was drawn, and R 2 〉0.99. At this time, the column temperature of the 9790 gas chromatograph was 150 °C, the detector temperature was 180 °C, and the column oven temperature was 180 °C. For the determination of samples, 1.5 ml of the culture solution was taken out with a syringe each time and centrifuged at 10000 r / min for 2 min. After the supernatant was filtered to remove bacteria with a 0.22 μm filter membrane, 800 μL was taken and placed in a liquid phase vial. After adding 200 μL of the internal standard and mixing, it was detected by gas chromatography. Each sample had three replicates. After 4 days of culture, it was shown that the accumulations of ethanol and butanol yields of Clostridium autoethanogenum E53 were the highest, reaching 5.15 g / L, which was significantly higher than the alcohol yields of the other two strains ( Figure 4 ).

[0037] Based on the above medium composition, the medium components and fermentation conditions were further optimized. The concentration of sodium bicarbonate in the culture system was controlled at 1 - 2 g / L, the initial pH range of the system was adjusted to 4.7 - 7.5, which improved the maximum cell concentration of the anaerobic gas-fermenting bacterium Clostridium autoethanogenum E53 during fermentation and its ability to ferment and produce ethanol and butanol. The maximum cell concentration of the Clostridium autoethanogenum E53 strain could reach OD600nm~2.0 Abs, and the highest alcohol content was 7.5 g / L ( Figure 5 ).

[0038] In summary, through chemical mutagenesis screening, the present invention obtained a high-alcohol-producing E53 strain, which can efficiently catalyze syngas to generate alcohol substances.

Claims

1. An anaerobic gas-eating bacterium capable of converting synthesis gas into alcohol compounds, characterized in that: The preservation number of the anaerobic aerobic bacteria is CGMCC No.46261.

2. Use of the anaerobic aerobic bacteria according to claim 1 in catalyzing the conversion of synthesis gas into alcohol compounds.

3. A method for converting synthesis gas into alcohol compounds, characterized in that: The method is to use the anaerobic aerobic bacteria described in claim 1 to catalyze the synthesis gas to convert it into alcohol compounds.

4. The method according to claim 3, characterized in that The synthesis gas contains carbon monoxide, carbon dioxide and hydrogen.

5. The method according to claim 4, characterized in that The volume ratio of carbon monoxide, carbon dioxide and hydrogen is 10:7:

3.

6. The method according to claim 3, characterized in that The alcohol compound is ethanol or butanol.

7. The method according to claim 3, characterized in that The fermentation culture system in the method comprises 2%-4% mineral element storage solution, 0.05% yeast extract, 0.5% reducing agent storage solution and 0.1% trace element storage solution.

8. The method according to claim 7, characterized in that The mineral element storage solution is composed of: 8% sodium chloride, 10% ammonium chloride, 1% potassium chloride, 1% potassium dihydrogen phosphate, 2% magnesium sulfate and 0.4% calcium chloride; The reducing agent stock solution is composed of: 0.9% sodium hydroxide, 4% L-cysteine ​​hydrochloride and 4% sodium sulfide nonahydrate; The composition of the trace element storage solution is as follows: 1% nitrilotriacetic acid, 0.5% magnesium sulfate, 0.4% ammonium ferrous sulfate, 0.1% cobalt chloride, 0.1% zinc sulfate, 0.01% nickel chloride, 0.01% sodium selenate and 0.01% sodium tungstate.

9. The method according to claim 7, characterized in that The method described herein adjusts the initial pH of the fermentation culture system to 4.7-7.5 and the sodium bicarbonate concentration to 1-2 g / L.

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