Alcohol self-producing clostridium capable of producing ethanol by fermentation of synthesis gas

Abstract

The invention discloses an alcohol self-producing clostridium which is screened from nature and can be used for producing ethanol by fermentation of synthesis gas, belongs to clostridium and is preserved in the China Center for Type Culture Collection (CCTCC) with the preservation number of CCTCC NO: M20241614. The clostridium self-producing alcohol can be converted into ethanol through fermentation by taking a carbon gas as a raw material.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology and relates to an aerobic Clostridium, specifically a self-producing alcohol Clostridium sp. LG-6 that can produce ethanol by fermentation using syngas. Background Technology

[0002] Carbon monoxide (CO) and carbon dioxide (CO2), representing one-carbon (C1) gases, are inexpensive carbon resources with abundant sources and reserves. They can be obtained from the pyrolysis and gasification of carbon-containing minerals and biomass, and are also present in large quantities in the exhaust gases emitted by steel and coal chemical enterprises, urgently requiring effective utilization. Biocatalysis is an important option for C1 gas utilization, enabling energy conservation and emission reduction while converting C1 gas into various high-value chemicals. Compared to traditional chemical catalysis, C1 gas bioconversion has advantages such as milder reaction conditions, suitability for mixed gases with different carbon monoxide and carbon dioxide ratios, and suitability for synthesizing medium- and long-chain compounds with complex structures.

[0003] Carbon-fixing microorganisms can directly convert free carbon gas into carbon-containing organic matter through their life activities, making them a major research focus in the biological fixation and utilization of carbon gas. Currently known microorganisms capable of utilizing C1 gas can be broadly classified into three types based on their energy requirements: chemoautotrophic, photoautotrophic, and electroautotrophic microorganisms. Among these, chemoautotrophic microorganisms use hydrogen or other reducing compounds as energy donors, making them particularly suitable for the high-density fermentation modes of traditional fermentation industries, and some of their fermentation products, such as ethanol, have already been industrially produced.

[0004] Aerotrophic Clostridium species are important chemoautotrophic microorganisms capable of converting inexpensive raw materials such as H2 or CO into valuable alcohols or other chemicals. Currently known aerotrophic Clostridium species include *Clostridium ljungdahlii*, *Clostridium autoethanogenum*, *Clostridium carboxidivorans*, *Clostridium ragsdalei*, *Clostridium coskatii*, and *Acetobacterium woodii*, all of which possess significant potential for industrial applications. However, these naturally occurring chemoautotrophic Clostridium species generally suffer from slow growth, low carbon fixation rates, and weak product synthesis capabilities, limiting their application in converting industrial waste gas into chemicals and fuels. Therefore, discovering and identifying aerotrophic Clostridium species capable of more efficiently utilizing Cl gas to synthesize ethanol from specific environments is one of the main approaches to overcoming these problems. The newly isolated strains can also serve as excellent chassis microorganisms for further modification, thereby obtaining artificially synthesized strains with superior performance. Summary of the Invention

[0005] Our research group analyzed that the baijiu fermentation cellar is a micro-ecological environment with a very rich microbial community, containing a large number of anaerobic bacteria. Therefore, it is possible to isolate Clostridium aeruginosa strains with high growth rate, strong carbon fixation and ethanol synthesis capabilities from it, thereby obtaining microbial resources with potential for industrial application in gas fermentation.

[0006] Therefore, starting in 2022, we pretreated the cellar mud obtained from the winery and conducted preliminary syngas (CO-CO2-H2) fermentation on the isolated cellar mud microbiota. We found that the cellar mud microbiota could grow in syngas, but with very low biomass. Subsequently, we performed metagenomic sequencing on the cellar mud microbiota. By comparing sequences with known databases, we sorted and classified the bacterial species in the microbiota and found that some Clostridium aerogenes strains were indeed present. Therefore, these cellar mud microbiota can be used for further single-strain isolation. Furthermore, we optimized the strain screening scheme and finally screened out a Clostridium LG-6 with excellent ethanol synthesis performance.

[0007] Therefore, the first aspect of this invention provides a self-producing alcohol-producing Clostridium autoethanogenum strain capable of producing ethanol by fermentation using syngas, belonging to the genus Clostridium sp., deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO:M 20241614. In this document, it is designated LG-6.

[0008] A second aspect of the invention provides the application of the aforementioned self-producing alcohol-producing Clostridium LG-6 in the fermentation production of ethanol.

[0009] In one embodiment, the self-producing ethanol-producing Clostridium produces ethanol through fermentation using carbon gas (C1) as a carbon source.

[0010] Preferably, the carbon gas is a synthesis gas.

[0011] For example, the carbon gas mentioned is steel plant tail gas or coal chemical tail gas rich in CO2 / CO.

[0012] In one specific embodiment, the fermentation is carried out by introducing a carbon gas into a liquid culture medium suitable for the growth and proliferation of self-producing alcohol-producing Clostridium.

[0013] The fermentation temperature can be 25-40℃, preferably 30-48℃, and more preferably 37±0.5℃.

[0014] During fermentation, the pH of the fermentation broth can be controlled at pH 5.0-6.5, preferably pH 5.5-6.0, and more preferably around pH 5.8.

[0015] This invention discovers a syngas-utilizing microorganism LG-6 from nature with promising industrial applications. It is identified as a self-producing alcohol-producing Clostridium. This strain has better growth in syngas and ethanol synthesis ability than existing standard strains of self-producing alcohol-producing Clostridium, and has good prospects for industrial applications.

[0016] The self-producing alcohol-producing Clostridium LG-6 of this invention belongs to the genus Clostridium, with the Latin name Clostridium sp. and the Chinese name Clostridium LG-6. It has been deposited at the China Center for Type Culture Collection on July 22, 2024, at the Wuhan University Collection Center, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, with the accession number CCTCCNO:M 20241614. Attached Figure Description

[0017] Figure 1 The growth of microbial communities in the pit sludge was shown. Four inoculum rates were set: 0.5%, 2%, 5%, and 10%.

[0018] Figure 2 This paper presents the enrichment culture process and results of baijiu cellar mud microorganisms under syngas conditions. A: Enrichment culture strategy of cellar mud microorganisms in syngas; B: Growth changes of cellar mud microorganisms during continuous subculturing in syngas; C: Growth history of the 21st generation of enriched microorganisms in syngas.

[0019] Figure 3The results of single-strain isolation and screening are shown. Among them, A: experimental flowchart of isolating single strains that can be used for syngas fermentation from the enriched microbial community; BH: growth curves of 7 single strains (J-1 / 2 / 3 / 6 / 11 / 12 / 16) in syngas.

[0020] Figure 4 The syngas fermentation capacity of seven single strains is shown. A: Growth curves of single strains LG-1 / 2 / 3 / 4 / 5 / 6 / 7 and the control strain in syngas fermentation; B: Bar graph comparing ethanol yield of gas fermentation broth (144h) of single strain LG-6 and the control strain.

[0021] Figure 5 These are photographs of plate colonies and microscopic images of Clostridium difficile LG-6, a self-producing alcohol-producing bacterium. In the image, A is the plate colony, and B is the optical microscope image. Detailed Implementation

[0022] In screening for fast-growing, carbon-fixing, and ethanol-synthesizing Clostridium aeruginosa from baijiu fermentation pits, we began enrichment culture of pit mud microorganisms in a medium using syngas as the carbon source in 2023. The inventors proposed adding a certain amount of fructose to the medium as an initial carbon source to ensure normal growth of the microorganisms. Therefore, Li Rui inoculated mixed pit mud microbial samples into a medium using syngas and fructose as a mixed carbon source for initial enrichment culture to increase the biomass of the microorganisms. Subsequently, the proportion of fructose in the medium was gradually reduced while maintaining a constant supply of syngas, thereby gradually increasing the microorganisms' preference for syngas until finally enriching microorganisms capable of normal growth using syngas as the sole carbon source.

[0023] In the experiment, the enriched bacterial culture was streaked onto solid culture plates, and then a large number of single colonies were individually fermented with syngas. Seven single bacterial strains capable of utilizing syngas were successfully isolated. Subsequently, these seven strains were identified through morphological observation and molecular biological identification (including 16S rRNA gene sequencing and specific gene sequencing). The results showed that all seven identified strains were *Clostridium autoethanogenum*, with a genomic sequence similarity of 99.58%–99.86% to the reported standard strain (*Clostridium autoethanogenum* DSM10061) from the German Microbiological and Cell Culture Collection (DSMZ).

[0024] Next, the syngas fermentation capabilities of these seven strains and the standard strain of Clostridium autoethanogenum (DSM10061) from the German Culture Collection (DSMZ) were compared, focusing on key indicators such as growth rate, types and yield of metabolites. The results showed that strain LG-6 exhibited significantly higher growth and product synthesis capabilities during gas fermentation than the aforementioned standard strain DSM10061, demonstrating potential industrial application and research value.

[0025] The self-producing alcohol-producing Clostridium LG-6 screened in this invention can also be used as a chassis cell to develop genetically engineered bacteria with stronger carbon fixation and ethanol synthesis capabilities, so as to be used for the green and sustainable production of biofuels and chemicals. This has important economic and social significance because using self-producing alcohol-producing Clostridium for fermentation to produce ethanol represents a technological trend in developing a circular economy, which helps to improve the "atom economy" of carbon, reduce greenhouse gas emissions, reduce environmental pollution and dependence on fossil fuels, and achieve full utilization of natural resources.

[0026] The present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0027] Example

[0028] In embodiments of the present invention, unless otherwise specified, the experimental operating temperature generally refers to room temperature (10-30°C).

[0029] This article involves the addition amount, content and concentration of various substances. Unless otherwise specified, the percentage content mentioned refers to the weight percentage.

[0030] In this article, the terms "solution" or "liquid" generally refer to aqueous solutions, with water usually being the primary solvent, which is easily understood by those skilled in the art.

[0031] Example 1: Extraction of microbial communities from baijiu cellar mud and their syngas fermentation test

[0032] Preliminary analysis revealed that the microbial biomass in the baijiu fermentation pits was extremely high, with anaerobic bacteria, particularly Clostridium, accounting for a significant proportion. Therefore, we pretreated the baijiu pit mud, extracted the microbial community, and conducted syngas fermentation to confirm the presence of bacteria capable of utilizing syngas.

[0033] Experimental steps:

[0034] (1) Sample collection: The research subject was the baijiu cellar mud sent by the School of Bioengineering, Jiangnan University. After sampling, the cellar mud samples were placed in sterile self-sealing bags and stored in a refrigerator at 4°C.

[0035] (2) Culture medium: The culture medium used for fermentation is the fully synthetic culture medium PETC 1754.

[0036] PETC 1754 culture medium:

[0037] 30 mL / L mineral element stock solution, 1 mL / L vitamin stock solution, 2 mL / L trace element stock solution, 0.5 g / L yeast extract, 5 g / L 2-morpholine ethanesulfonic acid, 5 mL / L reducing agent stock solution, 500 μL / L resazu stock solution, dissolved in double-distilled water, and adjusted to pH 6.0 with NaOH.

[0038] Mineral element storage solution: 80 g / L sodium chloride, 100 g / L ammonium chloride, 10 g / L potassium chloride, 10 g / L potassium dihydrogen phosphate, 20 g / L magnesium sulfate heptahydrate, 4 g / L calcium chloride dihydrate, dissolved in double-distilled water.

[0039] Reducing agent stock solution: 9 g / L sodium hydroxide, 40 g / L cysteine ​​hydrochloride, 40 g / L sodium sulfide nonahydrate, dissolved in double-distilled water.

[0040] (3) Syngas fermentation: The syngas used was simulated steel plant tail gas (gas composition: CO∶CO2∶H2∶N2=56%∶20%∶9%∶15%, Shanghai Haoqi Gas Co., Ltd.). Fermentation was carried out in 120mL serum bottles, with a fermentation system of 30mL. The atmospheric pressure inside the fermentation bottle was 0.2MPa. The fermentation system was cultured in a shaker at 37℃ and a shaking speed of 100rpm. All culture media and fermentation serum bottles needed to be autoclaved at 121℃ for 20min in advance. The specific operating steps are as follows:

[0041] 1) Preparation of bacterial culture: The cellar mud sample mailed by Jiangnan University was suspended in glycerol, filtered through a cell filter, and the filtered bacterial culture was stored in a -80 degree freezer.

[0042] 2) Fermentation: Syngas fermentation was carried out using filtered fermenting glycerol bacteria. First, the bacterial culture was added at different inoculum amounts to serum bottles containing 30 mL of the aforementioned PETC 1754 medium. After capping, the syngas was introduced. The aeration conditions were: aeration for 3 min 30 s, headspace pressure of 0.2 MPa atm. After aeration, the serum bottles were placed in a shaker at 37°C for incubation.

[0043] 3) Gas exchange sampling: Replace the gas in the serum bottle every 24 hours, using the same procedure as above.

[0044] 4) Growth curve determination: Samples were taken every 24 hours using a 1 mL sterile syringe, and the OD of the bacterial culture samples was measured using a visible spectrophotometer. 600 Draw the growth curve.

[0045] Experimental results:

[0046] The bacterial solution extracted from the pit mud was subjected to syngas fermentation. The results are as follows: Figure 1 As shown, after 48 hours of cultivation, the concentration of the bacterial solution increased significantly compared to 0 hours, indicating the presence of bacteria in the microbial community capable of using syngas as a carbon source for growth. Therefore, it is advisable to isolate these bacteria from the pit mud microbial community for further identification and testing.

[0047] Example 2: Enrichment and cultivation of microbial communities in baijiu cellar mud under syngas conditions

[0048] Experimental steps:

[0049] To isolate a single syngas-utilizing bacterial strain from the bacterial culture derived from the baijiu cellar mud in Example 1 above, we inoculated the bacterial culture into a medium using syngas and fructose as a mixed carbon source for fermentation. The fructose concentration in the medium was kept constant (5 g / L). When the strain achieved stable growth (stable growth rate and maximum growth rate) under 5 g / L fructose conditions, it was transferred to a 4 g / L fructose medium, and so on. Every 4–5 days, the culture was transferred to a new medium for three consecutive subcultures until the bacterial population stabilized. Then, the fructose content in the medium was gradually reduced, and continuous subculture was continued until the bacterial population could finally grow using syngas as a carbon source in a fructose-free medium. Through these experimental steps, bacteria capable of utilizing syngas were enriched from the bacterial population.

[0050] The culture medium composition is as follows: YT (yeast extract, tryptone) medium (1L): 10g yeast extract, 16g tryptone, 0.2g sodium chloride, 0.3g cysteine, 500μl resazu stock solution, 2ml trace element stock solution, 1ml vitamin stock solution, dissolved in double-distilled water, and adjusted to pH 5.8 with hydrochloric acid. For solid culture medium, 2% agar is added to this mixture.

[0051] Syngas fermentation operations are as follows:

[0052] (1) Seed culture preparation stage: Take one frozen glycerol bacteria from the -80℃ freezer and inoculate it into 5mL of YT medium for activation culture, and culture at 37℃ for about 24h.

[0053] (2) Fermentation: Take 1.5 mL of activated bacterial solution (inoculation amount of 5 v / v%) and inoculate it into 30 mL of PETC 1754 medium containing 5 g / L fructose. Place the culture medium in a 120 mL serum bottle and introduce syngas (gas composition and operation steps are the same as above).

[0054] Experimental results:

[0055] We enriched and cultured the microbial community in the pit mud based on the following experimental protocol (see...). Figure 2 (See section A). We first fixed the fructose concentration in the culture medium (5 g / L) and introduced the aforementioned syngas into the fermentation serum flask for bacterial culture. When the bacterial population could grow stably under these culture conditions, the fructose concentration in the culture medium was reduced to 4 g / L, and syngas was introduced for bacterial culture. This process was repeated until the fructose concentration in the culture medium was reduced to 0 g / L (see section A). Figure 2 (B). After twenty-one subcultures, we finally enriched a bacterial community that grows using syngas as a carbon source without relying on fructose, with a maximum biomass (OD). 600 It can reach around 1.0. Figure 2 (C)

[0056] Subsequently, we conducted further gaseous fermentation tests on the enriched bacterial community, using nitrogen as a control, and observed and recorded the growth of the community. The experimental results showed that the community could indeed grow under syngas conditions, but hardly grew under nitrogen conditions (see [link to experimental data]). Figure 2 The presence of CO in the syngas further confirms that the microbial community uses CO and CO2 from syngas as carbon sources.

[0057] Example 3: Single-strain isolation and screening

[0058] Experimental steps:

[0059] (1) Stranding isolation of single bacteria: The liquid bacterial population enriched in the 21st generation in Example 2 above was isolated by streaking on the surface of a solid agar plate. The plates were incubated at 37°C for 48 hours, and a large number of single colonies grew on the solid agar plate.

[0060] (2) Preparation of fermentation seed culture and gas fermentation: Single bacteria from the plate were inoculated into 5 mL of YT medium and cultured at 37°C for about 24 h. When the bacterial cell growth was concentrated, the seed culture was transferred to the above-mentioned PETC 1754 medium at an inoculation rate of 5 v / v% for gas fermentation. The specific operation was the same as the syngas fermentation described above.

[0061] Experimental results:

[0062] We isolated single strains using multiple streak plating methods (see...). Figure 3 Twenty single strains (named J-series) were isolated from strain A, and their gas utilization capacity was determined for each. The results showed that seven single strains exhibited good growth in syngas fermentation: strains J-1, J-2, J-3, J-6, J-11, J-12, and J-16 (see [reference needed]). Figure 3 (BH). Therefore, these 7 strains were used for further identification and performance testing.

[0063] Example 4: Identification of 7 single strains

[0064] Experimental steps:

[0065] (1) Take the above 7 single strains (J-1, J-2, J-3, J-6, J-11, J-12 and J-16) frozen in a -80℃ freezer, inoculate them into 5 mL of YT medium for activation culture, and culture at 37℃ for about 24 h.

[0066] (2) Sequencing analysis and sequence alignment

[0067] Method 1: 16S rDNA gene sequencing identification. Using activated bacterial culture as a template, the 16S rRNA gene fragment of the strain was amplified by PCR using universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTTACCTTGTTACGACTT-3'). The amplified products were detected by agarose gel electrophoresis and sent to Shanghai Platinum Semiconductor Co., Ltd. for sequencing. The sequencing results were analyzed by BLAST comparison in the NCBI database.

[0068] Method 2: Specific gene sequencing identification. A review of the literature (CANTOS-PARRAE, RAMIó-PUJOL S, COLPRIMJ, et al. Specific detection of "Clostridium autoethanogenum", Clostridium ljungdahlii and Clostridium carboxidivorans in complex bioreactor samples[J]. FEMS Microbiol Lett, 2018, 365(18)) revealed that the formic acid / nitrite transporter (fnt) sequences, namely the gene (CLJU_c37490, NC 014328.1) in Clostridium ljungdahlii and the gene (CLAU_1560, NC 022592.1) in Clostridium autoethanogenum, exhibit certain sequence differences, which can be used to distinguish between Clostridium ljungdahlii and Clostridium autoethanogenum. Therefore, using bacterial culture as a template, we designed primers CL-F (5'-AAGCAGTACTTGGAATACTCGC-3') and CL-R (5'-CCTCCAACTATATTACCTAAAGTTACAGGT-3') targeting the aforementioned fnt gene for PCR amplification. The PCR products were sent to Shanghai Platinum Semiconductor Co., Ltd. for sequencing. The sequencing results were analyzed using BLAST in the NCBI database.

[0069] Experimental results:

[0070] (1) Identification of the 16S rRNA gene sequence of the strain

[0071] Amplification was performed using primers 27F / 1492R (partial 16S rRNA sequence). Target fragments of approximately 1,500 bp were amplified from bacterial cultures of *C. autoethanogenum* (J-1 / 2 / 3 / 6 / 11 / 12 / 16). The fragments were recovered and sequenced, and the resulting gene sequences were compared with those in the NCBI database using BLAST. The results showed that the nucleotide similarity of the 16S rRNA gene sequences of the seven *C. autoethanogenum* strains was between 99.58% and 99.86% with that of strain *C. ljungdahlii* DSM 13528 (see Table 1). Therefore, the 16S rRNA gene sequences of the seven single bacteria in the pit mud were highly similar to those of strains C. ljungdahlii DSM 13528 and C. autoethanogenum DSM 10061, making it impossible to distinguish which strain they belonged to.

[0072] Table 1. Comparison of nucleotide similarity of 16S rRNA genes between single bacteria (J-1 / 2 / 3 / 6 / 11 / 12 / 16) in pit mud and strains C. ljungdahlii DSM 13528 and C. autoethanogenum DSM 10061. Note: +: positive.

[0073]

[0074] (2) Identification of specific gene sequences

[0075] Therefore, we turn to analyze the aforementioned literature ( We identified the seven strains using the formic acid / nitrite transporter (fnt) sequence reported by Baneras et al. (2018). Using CL-F / CL-R primers (fnt partial sequence), we amplified DNA fragments of approximately 670 bp in all seven strains. The target fragments were recovered and sequenced. Sequencing results showed that the amplified gene sequences of the seven isolated *Clostridium autoethanogenum* DSM10061 showed 100% similarity to the gene (CLAU_1560) and 95.08%–95.43% similarity to the gene (CLJU_c37490) of *C. ljungdahlii* DSM 13528 (Table 4-2). Therefore, these seven strains belong to *C. autoethanogenum*. We named them *Clostridium* sp. LG-1 / 2 / 3 / 4 / 5 / 6 / 7.

[0076] Table 2. Comparison of nucleotide similarity of the fnt gene in single bacteria (J-1 / 2 / 3 / 6 / 11 / 12 / 16) from pit mud with strains C. ljungdahlii DSM 13528 and C. autoethanogenum DSM 10061. Note: +: positive.

[0077]

[0078] Example 5: Detection of the syngas fermentation capacity of 7 single strains

[0079] We used the standard strain Clostridium autoethanogenum DSM10061, purchased from the German Culture Collection (DSMZ), as a control to compare its syngas fermentation capacity with the seven C. autoethanogenum strains (LG-1 / 2 / 3 / 4 / 5 / 6 / 7) isolated above. Biomass was measured by sampling at regular intervals (every 24 hours), and growth curves were plotted for each strain. Simultaneously, gas chromatography was used to determine the yields of ethanol and acetic acid in the fermentation broth.

[0080] Experimental steps:

[0081] (1) Take the above 7 single bacteria (LG-1 / 2 / 3 / 4 / 5 / 6 / 7) frozen in a -80℃ freezer and the standard strain Clostridium autoethanogenum DSM10061 purchased from the German Culture Collection Center (DSMZ) and inoculate them into 5 mL of YTF medium for activation culture. Culture at 37℃ for about 24 h.

[0082] The YTF (yeast extract, tryptone, fructose) liquid medium (1L) formulation is as follows: 10g yeast extract, 16g tryptone, 10g fructose, 0.2g sodium chloride, 0.3g cysteine, 500μl resazu stock solution, 2ml trace element stock solution, 1ml vitamin stock solution, dissolved in double-distilled water, and adjusted to pH 5.8 with hydrochloric acid. The YTF solid medium is prepared by adding 2% agar to the liquid medium.

[0083] (2) Take a small amount of the above bacterial solution and streak it on a solid plate culture medium using the three-line method. Incubate the streaked solid plate culture medium at 37°C for 2-3 days.

[0084] (3) Pick a single bacterium from the plate and inoculate it into 5 mL of YTF medium. Incubate at 37°C for about 24 hours until the OD of the bacterial culture reaches the target value. 600 Greater than 1.0.

[0085] (4) Take 500 μl of primary seed solution and measure OD. 600According to OD 600 The number of secondary seeds transferred is calculated to be approximately 500 / OD. 600 μL, incubate at 37℃ for 12-16h.

[0086] (5) Take 0.5 mL of secondary seed solution to measure OD. 600 When OD 600 Fermentation can proceed when the pH is between 0.85 and 1.2. First, pipette 1.5 mL (5 v / v%) of the secondary seed culture into a serum bottle. Then, measure 30 mL of PETC 1754 culture medium into the serum bottle using a graduated cylinder. After sealing the bottle, introduce synthesis gas (gas composition and operating procedures are the same as above). Re-aerate every 24 hours. After fermentation, take a sample to determine the amount of product synthesized.

[0087] (6) The ethanol content in the fermentation broth was determined using gas chromatography. The gas chromatograph and detection method were as follows: Agilent 7890 gas chromatograph, with a flame ionization detector (FID), an Alltech ECTM-WAX capillary column, and the column oven temperature was gradually increased from 85℃ to 200℃; the column pressure was 10psi to 30psi; the injector temperature was 250℃; the detector temperature was 300℃; the carrier gas was nitrogen at a flow rate of 25mL / min; the hydrogen flow rate was 30mL / min; and the air flow rate was 400mL / min. The internal standards were isobutanol, isobutyric acid, and hydrochloric acid (for acidification). The ethanol content was calculated using the internal standard method.

[0088] Experimental results:

[0089] Biomass (OD) of 7 single strains of pit mud during gas fermentation 600 Both were significantly higher than the control strain Clostridium autoethanogenum DSM10061 (see [link to relevant documentation]). Figure 4 (A). Subsequently, the ethanol yield of these 7 single strains at the fermentation endpoint (144 h) was detected by gas chromatography, and it was found that the ethanol yield of single strain LG-6 was significantly higher than that of the control strain (A). Figure 4 (B). Therefore, we finally screened out a strain of Clostridium autoethanogenum LG-6 with significantly superior traits, showing potential industrial application and research value.

[0090] Strain LG-6 belongs to the genus *Clostridium*, and its colony morphology is as follows: Single colonies of LG-6 are pale yellow, raised, and opaque, relatively uniform in size, and nearly circular in shape, with a moist and viscous surface. Under an optical microscope, the strain appears as single or paired rod-shaped cells, without other extracellular structures. During gas fermentation, the fermentation broth is yellow. In the later stages of gas fermentation, some degree of flocculation occurs in the fermentation broth.

[0091] The plate colony morphology and optical microscope morphological characteristics of strain LG-6 are as follows: Figure 5 As shown in Figures A and B. Given the potential for industrial applications of this strain, it has been deposited with the China Center for Type Culture Collection.

Claims

1. A self-producing alcohol-producing Clostridium autoethanogenum strain belonging to the genus Clostridium sp., deposited at the China Center for Type Culture Collection (CCTCCNO:M 20241614).

2. The application of the self-producing alcohol-producing Clostridium as described in claim 1 in the fermentation production of ethanol.

3. The application as described in claim 2, characterized in that, The self-producing ethanol-producing Clostridium uses carbon gas as a carbon source to produce ethanol through fermentation.

4. The application as described in claim 3, characterized in that, The carbon gas mentioned is syngas.

5. The application as described in claim 3, characterized in that, The carbon gas in question is either steel plant tail gas or coal chemical tail gas.

6. The application as described in claim 3, characterized in that, The fermentation process involves introducing a carbon gas into a liquid culture medium suitable for the growth and proliferation of self-producing alcohol-producing Clostridium.

7. The application as described in claim 6, characterized in that, The fermentation temperature is 25-40℃.

8. The application as described in claim 6, characterized in that, The pH of the fermentation broth is controlled at 5.0-6.5.

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