Method for producing acid and alcohol by using synthesis gas

Abstract

The invention provides a method for producing acid and alcohol by using synthesis gas, which is characterized in that two gas-feeding anaerobic bacteria Clostridium sp.strain P38 and Clostridium autoethanobacterium E53 are combined for fermentation, culture conditions are optimized, especially sodium molybdate and sodium bicarbonate are added into a culture system and the pH value of the culture system is controlled, so that the acid and alcohol production capacity of the fermentation system is improved, the cell acetic acid yield of the mixed fermentation strain is 9.11-10.11 g / L, and the yield of the mixed fermentation strain is 9.1-10.11 g / L; the yield of the ethanol is 6.55 to 10.55 g / L.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial fermentation application, and specifically relates to a method for producing acid and alcohol by utilizing synthesis gas. Background Art

[0002] Organic acids and alcohols are important organic compounds that are widely used in food, medicine, industry and daily life. With the advancement of green chemistry, their derivatives have an increasing potential in environmentally friendly materials, bioenergy and other fields.

[0003] Ethanol Clostridium is a strict anaerobic bacterium that can use CO as the only carbon source and energy source, and can also use H2 / CO2 and some simple carbohydrates. Its metabolites are mainly acetic acid and ethanol. Microbial conversion of syngas to produce chemicals The chemicals produced by its conversion gas can also provide a cheap carbon source for synthetic biology, while reducing dependence on petroleum-based products.

[0004] Carbon-containing synthetic gas refers to a type of mixed gas with one carbon gas as the main component, generally containing CO, CO2 and H2. It is a type of cheap raw material with great development value for manufacturing petrochemical chemicals. Anaerobic gas-feeding microorganisms can convert the 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. There are some mature engineered bacteria such as Escherichia coli and yeast for converting carbon dioxide into related products, but the energy efficiency is low and cannot be applied industrially. Summary of the invention

[0005] The object of the present invention is to provide a method for producing acid and alcohol using synthesis gas, that is, using two anaerobic gas-eating bacteria Clostridium sp. strain P38 and Clostridium autoethanogenum E53 combined fermentation, a method of producing acid and alcohol using synthesis gas.

[0006] The method for producing acid and alcohol using synthesis gas provided by the present invention is to use Clostridium with a preservation number of CGMCC No.46262 ( Clostridium sp .) P38 strain and anaerobic aerobic bacteria with a deposit number of CGMCC No.46261 Clostridium autoethanogenum The E53 strain ferments syngas to produce acid and alcohol products; The acid product, as specifically described in the embodiment, is acetic acid, and the alcohol product is ethanol or n-butanol; The synthesis gas contains 10% carbon monoxide, 30% carbon dioxide and 60% hydrogen.

[0007] The method, more specifically, uses a fermentation medium composed of 1%-5% mineral element stock solution, 0.02% yeast extract (M / V), 0.3% reducing agent stock solution (reducing agent Stock (V / V)), and 0.08% trace element stock solution (V / V); Furthermore, in the method described, the fermentation temperature is 37°C.

[0008] Preferably, the initial pH during the fermentation process is adjusted to 7.5, and sodium bicarbonate is added to the system at a concentration of 3 g / L at the beginning of the fermentation.

[0009] The present invention combines two strains of gas-feeding anaerobic bacteria Clostridium sp. strain P38 and Clostridiumautoethanogenum E53 fermentation. By optimizing the culture conditions, especially the addition of sodium molybdate and sodium bicarbonate in the culture system and the control of the pH of the culture system, the ability of the fermentation system to produce acid alcohols was improved. The cell acetic acid production of the mixed fermentation strain was 9.11-10.11 g / L, and the ethanol production was 6.55-10.55 g / L. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 : Clostridium carboxidivorans p7 strain and Clostridium autoe thanogenum Results of mixed culture production of acetic acid and ethanol; Figure 2 : Concentrations of acetic acid, ethanol and butanol produced by mutant strains cultured alone and in mixed cultures; Figure 3 : Concentration diagram of acetic acid, ethanol and butanol produced by mixed culture after optimization of fermentation conditions. DETAILED DESCRIPTION

[0011] The implementation process of the present invention is described in detail below in conjunction with the embodiments and drawings.

[0012] Example 1: Conventional strain combination ( Clostridium carboxidivorans p7 and Clostridium autoethanogenum )Acid and alcohol production assay Use conventional aerobic bacteria Clostridium carboxidivorans p7 and Clostridium autoethan ogenumMixed culture was carried out and the acid and alcohol content was detected. The culture medium used included 1%-5% mineral solution stock (V / V), 0.02% yeast extract (M / V), 0.3% reducing agent stock (V / V), and 0.08% trace element stock (V / V); the composition of mineral solution stock (M / V) was 5% sodium chloride, 8% ammonium chloride, 0.8% potassium chloride, 0.7% potassium dihydrogen phosphate, 1.5% magnesium sulfate and 0.3% calcium chloride; the composition of reducing agent stock (M / V) was 1% sodium hydroxide, 2% L-cysteine ​​hydrochloride and 3% sodium sulfide nonahydrate. The composition of the trace element storage solution (M / V) is 1.2% nitrilotriacetic acid, 0.4% magnesium sulfate, 0.2% ammonium ferrous sulfate, 0.1% cobalt chloride, 0.1% zinc sulfate, 0.02% nickel chloride, and 0.01% sodium tungstate.

[0013] Constant temperature shaking culture conditions: add 2ml of mineral solution stock, 0.02g of yeast extract to 100ml system, adjust the initial pH of the system to 6.5, 5ml reducing agent stock, fill with 99.999% nitrogen at a rate of 2L / min for 5min, seal, and sterilize at 121℃ for 20min. After sterilization, cool to about 40℃, add 0.1ml of trace element storage solution, and complete the construction of the culture system. Clostridium carboxidivorans p7 strain and Clostridium autoethanogenum 10 ml of culture solution was then continuously introduced with simulated synthesis gas (gas composition: 10% CO, 30% CO2, 10% H2) at a flow rate of 2 L / min and a pressure of 0.4 bar for 5 min, and then 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.

[0014] The acetic acid, ethanol and butanol in the product were detected by the following method: 9790 gas chromatograph was used for determination, and the gas chromatograph column temperature was set to 120°C, the detector temperature was set to 160°C, and the column box temperature was set to 160°C. For the determination of the sample, 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 sterilized by filtration with a 0.22μm filter membrane, 800μL was drawn and placed in a liquid phase vial. 200μL of internal standard mixture (3.5 g / L isobutanol, 2 g / L isobutyric acid and 1M hydrochloric acid) was added and detected by gas chromatography. The standard curves of acetic acid and ethanol standards were prepared according to the above method for the calculation of product concentration.

[0015] The fermentation products were measured on the 0th, 1st, 2nd, 3rd, 4th and 5th days respectively. The results showed that after 5 days of fermentation, the ethanol concentration in the system was 3.12 g / L, butanol was not detected, and acetic acid was 6.85 g / L ( Figure 1 ).

[0016] Example 2: Mutagenic strain combination ( Clostridium carboxidivorans P38 and Clostridium autoethanogenum E53) Acid and alcohol production assay The microorganisms used in this example are anaerobic gas-eating microorganisms, Clostridium sp. strain P38, which is a physically induced mutant strain of Clostridium carboxidivorans p7. Clostridium autoethanogenum E53 is a chemically induced mutant strain of Clostridium autoethanogenum.

[0017] The Clostridium used in the present invention ( Clostridium sp .) P38 strain, which is obtained by Clostridium sp.strain P7 was obtained by atmospheric pressure room temperature plasma mutagenesis (ARTP), and has high acid production activity and growth ability. It was deposited at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China General Microbiological Culture Collection Center on November 6, 2024, with the deposit number CGMCC No.46262.

[0018] Anaerobic bacteria Clostridium autoethanogenumThe E53 strain is a high-alcohol-producing mutant strain that has the ability to ferment and accumulate alcohol. The maximum cell concentration of the E53 strain can reach OD600nm~2.1Abs, and the maximum alcohol production is 7.5 g / L. It was deposited at the General Microbiology Center of the China Microbiological Culture Collection Administration at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing on November 6, 2024, with the deposit number CGMCC No.46261.

[0019] The two mutants were cultured separately and mixed in the fermentation medium described in Example 1. The specific operation process and parameters were referred to Example 1. The production of acetic acid, butyric acid and butanol in the separate culture and mixed culture systems was measured. The results showed that the mixed culture group had a higher product yield than the separate culture group. After 5 days of culture in the mixed group, the production of acetic acid in the system reached 8.10 g / L, ethanol 4.50 g / L, and some 1.5 g / L butanol ( Figure 2 ).

[0020] Example 3: Optimizing fermentation conditions The gas metabolism process of anaerobic aerobic bacteria is very complex, involving the production and recycling of a variety of fermentation products, as well as the balance between the production and consumption of energy ATP. The regulation of the fermentation system conditions has a great influence on the final fermentation results. The applicant found that pH and sodium bicarbonate can significantly affect the production of organic acids and alcohols in the fermentation products. Higher pH and sodium bicarbonate concentrations are conducive to the accumulation of organic acids and the production of ATP. Conversely, lower pH and sodium bicarbonate concentrations are conducive to the accumulation of alcohols, but the total yield is not high due to the lack of ATP accumulation in the acid production stage. Therefore, how to balance the bacterial fermentation process and maximize the yield of the target product is very critical.

[0021] Based on Example 2, this example adjusted the initial pH of the fermentation process to 7.5, and added sodium bicarbonate to the system at a concentration of 3 g / L at the beginning of the fermentation. The system was shaken in a constant temperature shaker, and the pH of the system was adjusted to 4.7 after 72 hours. Samples were taken out at regular intervals to measure the concentrations of acetic acid, ethanol, and butanol in the fermentation broth. After 5 days of fermentation, the ethanol concentration in the system was 10.05 g / L, butanol 2.0 g / L, and acetic acid 10.11 g / L. Under this control condition, the yields of organic acids and acid substances were improved ( Figure 3 ).

[0022] In summary, the present invention combines two strains of gas-feeding anaerobic bacteria Clostridium strain P38 and Clostridium autoethanogenum E53 fermentation, and the ability of the fermentation system to produce acid alcohols was improved by optimizing the culture conditions.

Claims

1. A method for producing acid and alcohol using synthesis gas, characterized in that: The method uses Clostridium with a preservation number of CGMCC No. 46262 and anaerobic aerobic bacteria with a preservation number of CGMCC No. 46261 to ferment synthetic gas to prepare acid and alcohol products.

2. The method according to claim 1, characterized in that The acid product is acetic acid.

3. The method according to claim 1, characterized in that The alcohol product is ethanol or n-butanol.

4. The method according to claim 1, characterized in that The synthesis gas contains 10% carbon monoxide, 30% carbon dioxide and 60% hydrogen.

5. The method according to claim 1, characterized in that The fermentation medium used in the method comprises 1%-5% mineral element stock solution, 0.02% yeast extract, 0.3% reducing agent stock solution and 0.08% trace element stock solution.

6. The method according to claim 5, characterized in that In the method, the initial pH during the fermentation process is 7.

5.

7. The method according to claim 5, characterized in that In the described method, sodium bicarbonate is also added to the fermentation medium.

8. The method according to claim 7, characterized in that The added concentration of the sodium bicarbonate is 3g / L.

9. The method according to claim 1, characterized in that The method described, the fermentation temperature is 37 ℃.

Images