Clostridium autoethanogenum, isolation and purification method and use thereof

Abstract

The application provides a Clostridium autoethanogenum and a separation and purification method and application, and belongs to the field of biological fermentation. The Clostridium autoethanogenum is Clostridium autoethanogenum SGLT-Ca-0001-mut 1, the preservation number is GDMCC 63758, the preservation address is the Guangdong Microbial Culture Collection Center, and the preservation date is August 7, 2024. Through plate culture combined with first genotype identification, the mixed bacteria in the fermentation liquor can be preliminarily screened out, so that relatively pure Clostridium autoethanogenum single colonies are obtained; through enrichment culture and second gene identification, Clostridium autoethanogenum single colonies in the relatively pure Clostridium autoethanogenum single colonies can be further screened out; in addition, combined with a preset culture environment, an advantage mutant Clostridium autoethanogenum with high production efficiency and high metabolic selectivity can be directionally screened out; finally, through multiple streaking and separation, the mutant Clostridium autoethanogenum can be screened out, so that a Clostridium autoethanogenum with high strain robustness and metabolic performance can be obtained.

Description

Technical Field

[0001] This application relates to the field of bio-fermentation technology, and in particular to an ethanol-producing Clostridium, its isolation and purification method, and its application. Background Technology

[0002] The extraction and use of fossil fuels has promoted modern industrial development, but it has also led to massive carbon dioxide emissions, causing climate problems such as global warming. Therefore, the sustainable production of chemicals and fuels, and mitigating the greenhouse effect, are two major challenges facing humanity. Traditional biorefining technologies often use grains such as corn and wheat as raw materials, enabling the production of alternative petrochemical products. While these technologies are green and low-carbon, they also pose the problem of competing with humans for food and land, making large-scale sustainable development difficult in my country. One-carbon gases (CO, CO2) are widely available; industries such as iron and steel metallurgy, chemicals, power generation, and coking all produce large quantities of them. Syngas (CO, CO2, H2), containing one-carbon gases, can also be produced through the gasification of agricultural and forestry waste and municipal solid waste. Biorefining technologies using one-carbon gases as raw materials can directly reduce greenhouse gas emissions, thus giving the production process and products a negative carbon profile, and therefore have broad application prospects. This is also one of the effective ways to alleviate the two major challenges of fossil fuel depletion and the greenhouse effect.

[0003] Acetic acid-producing bacteria, such as *Clostridium autoethanogenum*, *Clostridium ljungdahlii*, *Clostridium ragsdalei*, *Clostridium carboxidivorans*, *Acetobacterium woodii*, *Moorella thermoacetica*, and *Eubacterium limosum*, can autotrophically grow and synthesize acetic acid using CO, CO2, and H2 as raw materials via the Wood-Ljungdahl pathway (WLP). Currently, the most biotechnologically feasible and valuable strains are anaerobic acetic acid-producing bacteria dependent on the WLP and aerobic hydrogen-consuming bacteria dependent on the Calvin-Benson-Bassham cycle (CBB). As a leading strain among acetic acid-producing bacteria, *Clostridium autoethanogenum* has been successfully commercialized through bio-fermentation of ethanol from tail gases of the steel and ferroalloy industries. Creating novel industrial microbial strains is key to improving biomanufacturing efficiency and reducing operating costs. For model strains such as *Escherichia coli* and *Saccharomyces cerevisiae*, gene editing tools are relatively mature, the physiological and metabolic mechanisms of chassis strains are well understood, and the relatively short doubling time facilitates rational design and improvement of strains through synthetic biology. However, for anaerobic acetic acid-producing bacteria that autotrophically grow using carbon gas as a raw material, the genetic manipulation system for these strains is not perfect, the doubling time is long, and the engineered strains obtained through modification need to be evaluated for performance through gaseous fermentation. The special nature of the fermentation process limits the throughput of strain fermentation characterization, all of which increase the difficulty of obtaining new strains through rational modification. While new engineered acetic acid-producing strains are being developed through methods such as key enzyme optimization, pathway remodeling, and global transcriptional regulation—for example, modifying the carbon monoxide dehydrogenase (CODH) to reduce oxygen sensitivity, knocking out the CODH isoenzyme CooS1 to shorten the lag phase of autotrophic growth in a CO2 / H2 environment, and knocking out the adhE1 / adhE2 genes in the direct ethanol synthesis pathway to increase ethanol yield—laboratory strains are often only tested in batches at the anaerobic flask level, failing to simulate commercial continuous fermentation processes. Furthermore, robustness evaluation is rarely conducted, hindering the commercial application of new strains. Traditional mutagenesis breeding methods, while shortening strain creation time, remain extremely difficult due to the lack of screening signals or suitable screening pressures, making it extremely challenging to select mutant strains with expected performance from a vast mutant library. Therefore, isolating mutant strains with improved fermentation performance is a pressing technical problem that needs to be solved. Summary of the Invention

[0004] This application provides a Clostridium ethanoliferum, its isolation and purification method, and its application to solve the following technical problem: how to isolate mutant strains with improved fermentation performance.

[0005] In one aspect, this application provides an ethanol-producing Clostridium, specifically Clostridium autoethanogenum SGLT-Ca-0001-mut 1, with accession number GDMCC 63758, deposited at Guangdong Provincial Center for Microbial Culture Collection on August 7, 2024.

[0006] Optionally, the nucleotide sequence of the 16S rDNA segment of Clostridium ethanolii SGLT-Ca-0001-mut 1 includes a first nucleotide sequence and a second nucleotide sequence, the first nucleotide sequence being shown in SEQ ID NO.1 and the second nucleotide sequence being shown in SEQ ID NO.2, wherein some bases of the first nucleotide sequence and the second nucleotide sequence are complementary.

[0007] Optionally, the synthetic product of Clostridium ethanolii SGLT-Ca-0001-mut 1 includes ethanol and acetic acid, wherein the average mass concentration of ethanol is ≥25 g / L and the average mass concentration of acetic acid is ≤4 g / L.

[0008] Secondly, this application provides a method for isolating and purifying Clostridium ethanolae as described in any embodiment of the first aspect, the method comprising:

[0009] The fermentation broth from Clostridium ethanol gas fermentation was plate-cultured under a preset culture environment to obtain the first fermentation broth;

[0010] The first fermentation broth was subjected to first genotyping to screen out relatively pure Clostridium ethanolae, and the first Clostridium ethanolae single colony was obtained.

[0011] The first Clostridium ethanolica single colony was enriched and cultured, and then the enriched first Clostridium ethanolica single colony was subjected to a second genotype identification to obtain the second Clostridium ethanolica single colony.

[0012] The second Clostridium ethanolae single colony was purified by streaking multiple times to obtain Clostridium ethanolae SGLT-Ca-0001-mut1.

[0013] Optionally, the plate culture temperature is 37°C, and the plate culture time is 7 to 10 days.

[0014] Optionally, the culture medium for plate culture includes one of the following: a first PETC medium, a second PETC medium, and a third PETC medium, wherein the mass concentration of fructose and yeast extract in the first PETC medium is 0, the mass concentration of fructose in the second PETC medium is ≥5 g / L and the mass concentration of yeast extract is 0, and the mass concentration of fructose in the third PETC medium is 0 and the mass concentration of yeast extract is ≥1 g / L.

[0015] Optionally, the preset culture environment includes a high-pressure anaerobic culture atmosphere, wherein the pressure of the high-pressure anaerobic culture atmosphere is 130 kPa to 160 kPa, and the O2 concentration is 0.

[0016] Optionally, the preset culture environment further includes a carbon monoxide gas source, wherein the carbon monoxide volume V1 of the carbon monoxide gas source and the total volume V2 of the carbon monoxide gas source satisfy the relationship: V1:V2=(0.2~0.6):1.

[0017] Thirdly, this application provides a fermentation agent comprising Clostridium ethanolans SGLT-Ca-0001-mut 1 as described in any one of the embodiments of the first aspect.

[0018] Optionally, the amount of Clostridium ethanolans SGLT-Ca-0001-mut 1 added accounts for 8% to 12% of the total mass of the fermentation agent.

[0019] The technical solutions provided in this application have the following advantages compared with the prior art:

[0020] This application provides a Clostridium ethanoliferum strain. By plate culture combined with first genotype identification, contaminating bacteria in the fermentation broth can be initially screened out to obtain relatively pure Clostridium ethanoliferum single colonies. Then, through enrichment culture and second genotype identification, Clostridium ethanoliferum single colonies can be further screened out from the relatively pure Clostridium ethanoliferum single colonies. In addition, by combining a preset culture environment, dominant mutant Clostridium ethanoliferum with high production efficiency and high metabolic selectivity can be selectively screened. Finally, by repeated streak plating and purification, the mutant Clostridium ethanoliferum can be screened out, thereby obtaining Clostridium ethanoliferum with higher strain robustness and metabolic performance. Attached Figure Description

[0021] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with this application and, together with the description, serve to explain the principles of this application.

[0022] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 A schematic flowchart illustrating the method for isolating and purifying Clostridium ethanolans provided in this application embodiment;

[0024] Figure 2 This is a diagram showing the colony growth of bacteria cultured in a high-pressure anaerobic tank with a CO gas environment, as provided in Example 1 of this application.

[0025] Figure 3 The 16S rDNA sequencing results of single colonies on solid plates of the first and second PETC culture media provided in Example 1 of this application;

[0026] Figure 4 This is a graph showing the results of bacterial biomass determination provided in Example 1 of this application;

[0027] Figure 5 This is a diagram of the 16S rDNA sequencing results of Clostridium ethanolii SGLT-Ca-0001-mut 1 provided in Example 1 of this application;

[0028] Figure 6 The gaseous continuous fermentation curves of the initial Clostridium ethanolae (A) and Clostridium ethanolae SGLT-Ca-0001-mut 1 (B) provided in Example 2 of this application. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0030] Various embodiments of this application may exist in the form of a range; it should be understood that the description in the form of a range is merely for convenience and brevity and should not be construed as a hard limitation on the scope of this application; therefore, it should be considered that the range description has specifically disclosed all possible sub-ranges and single numerical values ​​within that range. For example, it should be considered that the range description from 1 to 6 has specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and single numbers within the range, such as 1, 2, 3, 4, 5, and 6, regardless of the range. Furthermore, whenever a numerical range is referred to herein, it means including any referenced number (fraction or integer) within the referred range.

[0031] Furthermore, in the description of this application, the terms "comprising," "including," etc., mean "including but not limited to." In this document, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. In this document, "and / or" describes the relationship between related objects, indicating that three relationships can exist; for example, A and / or B can represent: A alone, A and B simultaneously, or B alone. A and B can be singular or plural. In this document, "at least one" means one or more, and "more than" means two or more. "At least one," "at least one of the following," or similar expressions refer to any combination of these items, including any combination of single or plural items. For example, "at least one of a, b, or c" or "at least one of a, b, and c" can both mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, and c can be a single or multiple.

[0032] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this application can be purchased from the market or prepared by existing methods.

[0033] This application provides a Clostridium autoethanogenum, which is Clostridium autoethanogenum SGLT-Ca-0001-mut 1, with accession number GDMCC 63758, deposited at Guangdong Provincial Center for Microbial Culture Collection, and deposited on August 7, 2024.

[0034] In some embodiments, the nucleotide sequence of the 16S rDNA segment of Clostridium ethanolii SGLT-Ca-0001-mut 1 includes a first nucleotide sequence and a second nucleotide sequence, the first nucleotide sequence being shown in SEQ ID NO.1 and the second nucleotide sequence being shown in SEQ ID NO.2, wherein some bases of the first nucleotide sequence and the second nucleotide sequence are complementary.

[0035] In some embodiments, the synthetic product of Clostridium ethanolii SGLT-Ca-0001-mut 1 includes ethanol and acetic acid, wherein the average mass concentration of ethanol is ≥25 g / L and the average mass concentration of acetic acid is ≤4 g / L.

[0036] Adaptive evolution involves the continuous subculturing of microorganisms under specific conditions, leading to the accumulation of new phenotypes or physiological properties adapted to those conditions through spontaneous mutation. Adaptive evolution enables strains to effectively alter certain phenotypic or physiological characteristics (such as specific growth rate, substrate utilization rate, and tolerance) within a short period. In microbial evolution, the existence of selection pressure ensures the survival of the fittest through random mutations, preserving mutant strains with target traits. In commercial continuous gas fermentation processes, fluctuations in feed gas component concentration, gas flow rate, and impurity content can all affect the stable operation of continuous fermentation. Long-term continuous subculturing inevitably enriches and screens mutant strains adapted to specific upstream gas supply conditions. These strains exhibit improved robustness and metabolic selectivity compared to the initial strains. Screening, purifying, and analyzing these strains can yield new industrial strains with improved fermentation performance. Furthermore, omics analysis of these mutant strains obtained under specific conditions can deepen our understanding of their metabolic mechanisms.

[0037] Therefore, this invention obtains the superior mutant strain *Clostridium ethanolica* SGLT-Ca-0001-mut 1 through long-term adaptive evolutionary screening. In small-scale CSTR gas continuous fermentation experiments, *Clostridium ethanolica* SGLT-Ca-0001-mut 1 showed superior ethanol production efficiency and metabolic selectivity compared to the initial strain. Furthermore, *Clostridium ethanolica* SGLT-Ca-0001-mut 1 exhibited stronger environmental adaptability, significantly improving the strain's robustness and metabolic performance. For example, the average ethanol concentration of the strain can be 25 g / L, 25.5 g / L, 26 g / L, 26.5 g / L, 27 g / L, 27.5 g / L, etc., and the average acetic acid concentration can be 3 g / L, 3.1 g / L, 3.2 g / L, 3.3 g / L, 3.4 g / L, 3.5 g / L, 3.6 g / L, 3.7 g / L, 3.9 g / L, 4 g / L, etc.

[0038] Figure 1 This is a schematic flowchart of the method for isolating and purifying Clostridium ethanolans provided in the embodiments of this application.

[0039] Based on a general inventive concept, please refer to... Figure 1 This application provides a method for isolating and purifying Clostridium ethanolae as described in any of the above embodiments, the method comprising:

[0040] S1. The fermentation broth of Clostridium ethanol gas fermentation is plate cultured under a preset culture environment to obtain the first fermentation broth;

[0041] In some embodiments, the plate culture temperature is 37°C, and the plate culture time is 7 to 10 days.

[0042] When plate culture is limited to 37°C, *Clostridium ethanolans* can grow and multiply rapidly, forming visible colonies. A limited culture time of 7–10 days allows *Clostridium ethanolans* to grow sufficiently and form easily identifiable single colonies. Furthermore, a longer culture time helps reduce false positive or false negative results caused by excessively short culture times.

[0043] In some embodiments, the culture medium for plate culture includes one of the following: a first PETC medium, a second PETC medium, and a third PETC medium, wherein the fructose and yeast extract concentrations in the first PETC medium are both 0, the fructose concentration in the second PETC medium is ≥5 g / L and the yeast extract concentration is 0, and the fructose concentration in the third PETC medium is 0 and the yeast extract concentration is ≥1 g / L.

[0044] This invention rationally designs the chemical composition of the culture medium. In PETC medium containing no fructose and yeast extract (YE) or only fructose, Clostridium ethanolii can use CO or fructose in the environment as a carbon source and inorganic nitrogen source in the culture medium as a nitrogen source to grow, while other bacteria cannot grow under these two conditions.

[0045] In some embodiments, the preset culture environment includes a high-pressure anaerobic culture atmosphere, wherein the pressure of the high-pressure anaerobic culture atmosphere is 130 kPa to 160 kPa and the O2 concentration is 0.

[0046] In some embodiments, the preset culture environment further includes a carbon monoxide gas source, wherein the carbon monoxide volume V1 of the carbon monoxide gas source and the total volume V2 of the carbon monoxide gas source satisfy the relationship: V1:V2=(0.2~0.6):1.

[0047] This invention optimizes the temperature, time, culture medium composition, and culture environment (including pressure and gas composition) of plate culture to promote the growth of Clostridium ethanolans, while inhibiting the growth of other bacteria, thus ensuring the acquisition of pure Clostridium ethanolans single colonies and significantly improving the isolation and purification efficiency of Clostridium ethanolans.

[0048] S2. The first fermentation broth is subjected to first genotyping to screen out relatively pure Clostridium ethanolae and obtain the first Clostridium ethanolae single colony.

[0049] S3. The first Clostridium ethanolica single colony is enriched and cultured, and then the enriched first Clostridium ethanolica single colony is subjected to second genotyping to obtain the second Clostridium ethanolica single colony.

[0050] S4. The second Clostridium ethanolae single colony is subjected to multiple streaking tests to obtain Clostridium ethanolae SGLT-Ca-0001-mut 1.

[0051] Based on a general inventive concept, this application provides a fermentation agent comprising Clostridium ethanolans SGLT-Ca-0001-mut 1 as described in any embodiment of the first aspect.

[0052] The starter culture of this invention uses Clostridium ethanolii SGLT-Ca-0001-mut 1 as the main active ingredient, aiming to improve the efficiency, stability, and product quality of the fermentation process through its unique metabolic capabilities and fermentation characteristics. This starter culture can be widely used in various fields such as food, beverages, bioenergy, and chemical raw materials, meeting the fermentation needs of different industries.

[0053] In some embodiments, the amount of Clostridium ethanolans SGLT-Ca-0001-mut 1 added accounts for 8% to 12% of the total mass of the fermentation agent.

[0054] To ensure optimal performance of the starter culture, this invention specifically limits the addition amount of Clostridium ethanolii SGLT-Ca-0001-mut 1 to the range of 8% to 12%, ensuring a sufficient amount of seed culture while preventing excessive entry of metabolites from the seed culture into the fermentation system. For example, the addition amount of Clostridium ethanolii SGLT-Ca-0001-mut 1 can be 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, etc.

[0055] The present application is further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the application. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to industry standards. If there is no corresponding industry standard, then common international standards, conventional conditions, or conditions recommended by the manufacturer are followed.

[0056] Example 1

[0057] Plating and streak plating are the simplest methods for microbial isolation and purification. Commercial continuous gas fermentation processes cannot guarantee the same level of sterility in materials and the fermentation environment as laboratory fermentation. Therefore, in addition to the target strain, the fermentation broth also contains other microorganisms that can survive in this environment, increasing the difficulty of isolating and screening dominant target strains from the production line fermentation broth. This invention uses the commercial gas fermentation of *Clostridium ethanolae* as an example to develop a method for isolating and purifying dominant mutant strains of *Clostridium ethanolae* from the production line fermentation broth.

[0058] The culture media used for the purification and culture of the strains were PETC and YTF, and the composition of the culture media was adjusted as needed. The composition of PETC and YTF culture media is shown in Tables 1 and 2. Since different culture environments and culture medium formulations may affect the efficiency of strain isolation and purification, the culture medium formulations used in this invention are as follows: First PETC medium (fructose and yeast extract concentrations are both 0), Second PETC medium (fructose concentration is 5 g / L and yeast extract concentration is 0), Third PETC medium (fructose concentration is 0 and yeast extract concentration is 1 g / L), and Fourth PETC medium (fructose concentration is 5 g / L and yeast extract concentration is 1 g / L). The culture environments are an anaerobic chamber (CO-free environment), an atmospheric pressure anaerobic tank (filled with 20 kPa raw material gas containing 50% CO), and a high-pressure anaerobic tank (filled with 150 kPa raw material gas containing 50% CO).

[0059] Table 1 Components of PETC culture medium

[0060] <![CDATA[NH4Cl]]> 1g / L KCl 0.1g / L NaCl 0.8g / L <![CDATA[KH2PO4]]> 0.2g / L <![CDATA[MgSO4·7H2O]]> 0.2g / L <![CDATA[CaCl2·2H2O]]> 0.02g / L <![CDATA[FeSO4·7H2O]]> 0.05g / L <![CDATA[NaHCO3]]> 1g / L yeast extract 1g / L fructose 5g / L L-cysteine ​​HCL 0.75g / L Trace element (TSE) solution 10mL / L Wolfe's vitamin solution 10mL / L 2g / L of resazurin 0.5 mL / L

[0061] Table 2 Components of YTF Culture Medium

[0062] NaCl 0.2g / L Yeast extract 10g / L Tryptone 16g / L fructose 10g / L L-cysteine ​​HCL 0.75g / L Trace element (TSE) solution 10mL / L Wolfe's vitamin solution 10mL / L 2g / L of resazurin 0.5 mL / L

[0063] The specific components of TSE are shown in Table 3, as shown in Tables 1 and 2, while the specific components of Wolfe's vitamin solution are shown in Table 4.

[0064] Table 3. Composition of Trace Element Solution

[0065] nitrilotriacetic acid 2g / L <![CDATA[MnSO4·H2O]]> 1g / L <![CDATA[Fe(SO4)2(NH4)2·6H2O]]> 0.8g / L <![CDATA[CoCl2·6H2O]]> 0.2g / L <![CDATA[ZnSO4·7H2O]]> 0.2 mg / L <![CDATA[CuCl2·2H2O]]> 0.02g / L <![CDATA[NaMoO4·2H2O]]> 0.02g / L <![CDATA[Na2SeO3]]> 0.02g / L <![CDATA[NiCl2·6H2O]]> 0.02g / L <![CDATA[Na2WO4·2H2O]]> 0.02g / L

[0066] Table 4. Composition of Wolfe's Vitamin Solution

[0067] Biotin (Vitamin H) 2mg / L folic acid (vitamin B) 2mg / L pyridoxine hydrochloride (vitamin B6) 10mg / L Thiamine HCl (Vitamin B1) 5mg / L Riboflavin (Vitamin B2) 5mg / L Nicotinic acid (vitamin B3) 5mg / L Calcium pantothenate (vitamin B5) 5mg / L Vitamin B12 0.1 mg / L p-aminobenzoic acid (vitamin Bx) 5mg / L thioctic acid 5mg / L

[0068] The fermentation broth from the production line was spread onto plates containing the four different culture medium formulations described above in an anaerobic chamber. Each culture medium plate was coated with 100 μL of undiluted fermentation broth and 10 μL of... -6 The fermentation broth was diluted several times, and the prepared plates were incubated at 37°C under three different culture conditions to observe the growth of the strains (the pressure drop in the high-pressure anaerobic tank was obvious).

[0069] The results showed that no colonies grew on plates cultured in the anaerobic chamber that contained no fructose or yeast extract, or only fructose. However, large, flat colonies and smaller, round colonies grew on plates containing yeast extract or both yeast extract and fructose that were coated with undiluted fermentation broth. After coating with undiluted broth, a single layer of bacteria should have grown on the plate. Therefore, it was speculated that the single colonies grown by this culture method were either mixed bacteria or a mixture of Clostridium ethanolans and mixed bacteria.

[0070] Similarly, the blackening of solid culture medium plates cultured in atmospheric pressure anaerobic tanks may be due to oxygen intake during the culture process. The single colonies growing on the plates vary in color and morphology, making the selection process laborious and unsuitable for the isolation and purification of Clostridium ethanolae.

[0071] The colony composition of bacteria cultured in a high-pressure anaerobic reactor with a CO2 gas environment is as follows: Figure 2 As shown, after the undiluted fermentation broth was spread onto solid plates of the first and second PETC media, a layer of bacteria grew. After dilution, uniform single colonies grew. When the undiluted fermentation broth was spread onto the third and fourth PETC media, single colonies appeared on the surface of the bacterial layer. The bacterial layer on the more nutrient-rich fourth PETC medium was thinner, and the single colonies on the surface were larger. Diluted plates produced two types of colonies of varying sizes. Therefore, it is preliminarily speculated that in PETC media without fructose and yeast extract (YE) or containing only fructose, *Clostridium ethanolans* can utilize CO2 or fructose from the environment as a carbon source and inorganic nitrogen from the medium as a nitrogen source for growth, while other bacteria cannot grow under these conditions. However, in solid media containing only YE, other bacteria showed signs of growth, and the bacterial layer formed by *Clostridium ethanolans* became thinner. When the medium contained both fructose and YE, the *Clostridium ethanolans* bacterial layer was even thinner, but the single colonies on the surface could grow larger. This indicates that other bacteria have a certain dependence on organic nitrogen sources, and that they have a significant competitive advantage under nutrient-rich conditions. The colony growth under different culture environments and culture media is shown in Table 5.

[0072] Table 5. Colony growth under different culture environments and media.

[0073]

[0074] In Table 5, 1 indicates that under this specific culture environment and culture medium conditions, the bacterial layer is a uniform single colony of Clostridium ethanolae or has few mixed bacterial colonies, and 0 indicates that under this culture environment and culture medium conditions, no colonies grow or the bacterial layer contains a large number of mixed bacterial colonies.

[0075] Using bacterial colonies grown on solid plates of the first, second, and third PETC media after spreading the stock solution, as templates, the key genes for ethanol synthesis, AOR1 and 16S rDNA, were amplified for Clostridium ethanolans identification. It was found that the bacterial colonies grown on the first and second PETC media successfully amplified AOR1, but only the latter amplified 16S rDNA. Sequencing results showed that the bacterial cells in the colonies were Clostridium ethanolans (seq3.PETC-F-1492R_R_C03, seq4. Clostridium ethanolans 16S rDNA sequence). However, the bacterial colonies grown on the third PETC media failed to amplify AOR1 and 16S rDNA genes. Based on the above analysis, speculation, and verification, this invention selected single colonies grown on solid plates of the first and second PETC media in a CO gas-containing environment using a high-pressure anaerobic digester for subsequent purification verification experiments.

[0076] >seq3.PETC-F-1492R_R_C03

[0077] CAGTGTACCCCACCTTCGGCCGCGTCCTCCTAAGTTAGACTACGGACTTCGGGTGTTGCCAGCTCTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGACATTCTGATTCGCGATTACTAGCAACTCCAACTTCATGTAGGCGAGTTTCAGCCTGCAATCCGAACTGGGGGCAGTTTTTGAGGTTTGCTCCACCTTGCGGTCTTGCTTCTCTCTGTACTGCCCATTGTAGCACGTGTGTTGCCCTGGACATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGCGTTAACCGCGGCAGTCTTGCTAGAGTGCTCAACTAAATGTTAGCAACTAACAACAGGGGTTGCGCTCGTTGCAGGACTTAACCTAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTATCCCTGCCCCGAAGGGCTTCTCTTATCTCTAAGATATTCAGGGTATGTCAAGTCCAGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCGCTGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTTAATCTTGCGATCGTACTTCCGAGACGGAGTACTTATTGTGTTTACTGCGGCACAGAAGGGGTCGATACCTCCTACACCTAGTACTCATCGTTTACGGCGTGGACTACCAGGGGTATCTAATCCTGTTTGCTACCCACGCTTTT

[0078] seq4. Clostridium autoethanogenum 16s rDNA sequence[[ID=​

[0080] Single colonies grown on solid plates of the first and second PETC media were streaked onto solid plates with the same medium formulation for enrichment. After incubation in a high-pressure anaerobic CO2 environment, PCR amplification and 16S rDNA sequencing were performed for verification. Figure 3 The selected single colonies were found to be all *Clostridium ethanolans*. The enriched single colonies were streaked and purified on new plates. After further culturing in a high-pressure anaerobic digester under CO2 conditions, the resulting single colonies were streaked and purified a second time, and then cultured again in a high-pressure anaerobic digester under CO2 conditions. For ease of operation, the single colonies purified a second time under CO2 conditions were streaked on YTF plates and then cultured in an anaerobic chamber. The growth rate of the strains in the first round of culture was very slow, but the growth of the single colonies increased rapidly after inoculation with YTF liquid medium. After 48 hours of anaerobic incubation at 37°C and 220 rpm, the biomass of the single colonies on YTF plates was measured. The results are as follows: Figure 4 As shown, the fastest-growing Clostridium ethanoliferous production line was purified and labeled as SGLT-Ca-0001-mut 1 (abbreviated as Ca_mut1) and preserved as a glycerol bacterium. This strain was verified as Clostridium ethanoliferous by PCR amplification of its 16S rDNA and sequencing (seq1.Ca_mut1-1492R, seq2.Ca_mut1-27F). Figure 5 ).

[0081] Example 2

[0082] The purified strain Ca_mut1 and the initial Clostridium ethanoliferum were inoculated into YTF medium as primary seed culture and cultured at 37℃ and 220 rpm. It was found that the Ca_mut1 culture became turbid 17 h after inoculation, while the initial Clostridium ethanoliferum showed no signs of growth. After 48 h of primary seed culture, a 3% inoculation rate was added to 300 mL of fresh YTF medium as secondary seed culture and cultured at 37℃ and 220 rpm. After 24 h of secondary seed culture, the biomass of Ca_mut1 reached 0.5 g / L, and the biomass of the initial Clostridium ethanoliferum reached 0.45 g / L. The entire secondary seed culture was then inoculated into a 3 L CSTR containing 1.7 L of PETC medium (without fructose and yeast extract) for continuous gas fermentation. The metabolic curves after 20 days of fermentation are shown below. Figure 6As shown, the control parameters for the entire fermentation process, including stirring speed, gas flow rate, and dilution rate, remained completely consistent between the two strains. During the metabolic uptake phase, Ca_mut1 showed faster ethanol growth compared to the initial Clostridium ethanoliferum, indicating that the purified strain had better ethanol metabolic selectivity. After reaching steady state, the average ethanol concentration of the initial Clostridium ethanoliferum was 23.4 g / L, and the average acetic acid concentration was 6.1 g / L; while the average ethanol concentration of the Ca_mut1 strain was 25.4 g / L, and the average acetic acid concentration was 3.5 g / L. Ca_mut1 reached metabolic steady state two days earlier than the initial Clostridium ethanoliferum, and the purified strain Ca_mut1 had an ethanol concentration 8.5% higher than the initial Clostridium ethanoliferum, which would generate considerable economic benefits in commercial production.

[0083] Furthermore, one or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0084] In this embodiment of the invention, the dominant mutant strain of Clostridium ethanolae obtained through long-term adaptive evolution screening on a commercial production line is used directly for production, and the strain has stronger robustness and adaptability to the environment.

[0085] In this embodiment of the invention, through laboratory-scale CSTR gas continuous fermentation, the purified strains in the production line are superior to the initial strains in terms of ethanol production efficiency and metabolic selectivity. This will translate into actual economic benefits in large-scale commercial production, and will be amplified as the production scale expands.

[0086] The above description is merely a specific embodiment of this application, enabling those skilled in the art to understand or implement this application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of this application. Therefore, this application is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features claimed herein.

Claims

1. A type of Clostridium ethanolans ( Clostridium autoethanogenum SGLT-Ca-0001-mut 1, characterized in that, The Clostridium ethanoliferum has the accession number GDMCC NO: 63758, is deposited at the Guangdong Provincial Microbial Culture Collection Center, and was deposited on August 7, 2024. The nucleotide sequence of the 16S rDNA segment of the Clostridium ethanoliferum includes a first nucleotide sequence and a second nucleotide sequence, the first nucleotide sequence being shown in SEQ ID NO.1 and the second nucleotide sequence being shown in SEQ ID NO.

2. After the Clostridium ethanoliferum SGLT-Ca-0001-mut 1 reaches steady state in gaseous continuous fermentation, the average mass concentration of ethanol in the synthesized product is ≥25 g / L and the average mass concentration of acetic acid is ≤4 g / L.

2. A fermenting agent, characterized in that, The fermentation agent includes Clostridium ethanolans SGLT-Ca-0001-mut 1 as described in claim 1.

3. The fermenting agent according to claim 2, characterized in that, The amount of Clostridium ethanolans SGLT-Ca-0001-mut 1 added accounts for 8% to 12% of the total mass of the fermentation agent.

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