Clostridium ethanolicum as well as separation and purification method and application thereof

Abstract

The invention provides clostridium ethanolicum as well as a separation and purification method and application thereof, and belongs to the field of biological fermentation. The clostridium acetobutylicum is clostridium acetobutylicum SGLT-Ca-0001-mut 1, the preservation number of the clostridium acetobutylicum SGLT-Ca-0001-mut 1 is GDMCC 63758, the preservation address of the clostridium acetobutylicum SGLT-Ca-0001-mut 1 is Guangdong Microbiological Culture Collection Center, and the preservation date of the clostridium acetobutylicum SGLT-Ca-0001-mut 1 is August 7, 2024. According to the method, plate culture is combined with first genotype identification, infectious microbes in fermentation liquor can be preliminarily screened out to preliminarily obtain relatively pure ethanol clostridium single colonies, then the ethanol clostridium single colonies in the relatively pure ethanol clostridium single colonies can be further screened out through enrichment culture and second gene identification, and in addition, the ethanol clostridium single colonies in the relatively pure ethanol clostridium single colonies can be further screened out by combining a preset culture environment. According to the method, dominant mutant clostridium ethanolicum with high production efficiency and high metabolic selectivity can be directionally screened out, and finally, the mutant clostridium ethanolicum can be screened out through multiple times of streak purification, so that clostridium ethanolicum with relatively high strain robustness and metabolic performance can be obtained.

Description

Technical Field

[0001] The present application relates to the field of biological fermentation technology, and in particular to an ethanol Clostridium and a separation and purification method and application thereof. Background Art

[0002] The exploitation and use of fossil fuels has promoted the development of modern industry, but it has also led to a large amount of carbon dioxide emissions, causing climate problems such as global warming. Therefore, the sustainable production of chemicals and fuels and the mitigation of the greenhouse effect are two major challenges currently facing mankind. Traditional biorefining technology mostly uses corn, wheat and other grains as raw materials, which can be used to replace petrochemical products and is green and low-carbon. However, there is a problem of "competing with people for food and competing with food for land", which makes it difficult to sustain large-scale development in my country. Carbon-containing gases (CO, CO2) have a wide range of sources. Industries such as steel metallurgy, chemical industry, electricity, and coking will produce a large amount of carbon-containing gases. At the same time, synthetic gas (CO, CO2, H2) containing carbon-containing gases can also be produced through the gasification of agricultural and forestry wastes and urban solid waste. Biorefining technology using carbon-containing gases as raw materials can directly reduce greenhouse gas emissions, thereby making the production process and products have negative carbon properties, so it has broad application prospects. This is also one of the effective ways to alleviate the two major challenges of fossil energy depletion and greenhouse effect.

[0003] Acetogenic bacteria, such as Clostridium autoethanogenum, Clostridium ljungdahlii, Clostridium ragsdalei, Clostridium carboxidivorans, Acetobacterium woodii, Moorella thermoacetica, Eubacterium limosum, etc., can grow autotrophically and synthesize acetic acid using CO, CO2 and H2 as raw materials through the Wood-Ljungdahl pathway (WLP). Currently, the most biotechnologically feasible and application-worthy bacteria are anaerobic acetogenic bacteria that rely on WLP and oxygen-consuming hydrogen-consuming bacteria that rely on the Calvin cycle (CBB). As a star strain among acetogenic bacteria, Clostridium autoethanogenum has successfully achieved commercial application in the production of ethanol by biofermentation of exhaust gas from the steel industry and ferroalloy industry. The creation of new industrial strains is one of the keys to improving biomanufacturing production efficiency and reducing operating costs. For model strains such as Escherichia coli and Saccharomyces cerevisiae, their gene editing tools are relatively mature, the physiological and metabolic mechanisms of chassis strains are relatively clear, and the relatively short doubling time provides convenience for rational design and improvement of strains through synthetic biology. However, for anaerobic acetogenic bacteria that grow autotrophically using one-carbon gas as raw material, the genetic manipulation system of this type of strain is not perfect, the doubling time is long, and the engineered strains obtained by transformation need to be evaluated by gas fermentation. The particularity of the fermentation process limits the flux of strain fermentation characterization, which increases the difficulty of obtaining new strains through rational transformation. Although there are also methods such as key enzyme optimization modification, pathway remodeling, and global transcriptional regulation to develop new acetogenic bacteria engineering strains, such as modifying carbon monoxide dehydrogenase CODH to reduce oxygen sensitivity, knocking out CODH isozyme CooS1 to shorten the lag period of autotrophic growth of strains in CO2 / H2 environment, and knocking out ethanol direct synthesis pathway genes adhE1 / adhE2 to increase ethanol production, the strains obtained in the laboratory are often only tested for batch fermentation at the anaerobic bottle level, which cannot simulate the continuous fermentation process of commercial production. At the same time, the robustness of the strains is rarely evaluated, which restricts the process of commercial application of new strains. Although the traditional mutagenesis breeding method shortens the time for strain creation, it is extremely difficult to screen out mutant strains that meet the expected performance from a large mutant library due to the lack of screening signals or appropriate screening pressure. Therefore, how to isolate mutant strains with improved fermentation performance is a technical problem that needs to be solved urgently. Summary of the invention

[0004] The present application provides an ethanol Clostridium and a separation and purification method and application to solve the following technical problem: how to separate a mutant strain with improved fermentation performance.

[0005] In the first aspect, the present application provides an autoethanogenum, wherein the autoethanogenum (Clostridium autoethanogenum) is autoethanogenum SGLT-Ca-0001-mut 1, with a preservation number of GDMCC 63758, a preservation address of Guangdong Provincial Microbiological Culture Collection Center, and a preservation date of August 7, 2024.

[0006] Optionally, the nucleotide sequence of the 16S rDNA segment of the ethanolic Clostridium SGLT-Ca-0001-mut 1 includes a first nucleotide sequence and a second nucleotide sequence, the first nucleotide sequence is shown in SEQ ID NO.1, the second nucleotide sequence is shown in SEQ ID NO.2, and the first nucleotide sequence and the second nucleotide sequence are partially base-complementary.

[0007] Optionally, the synthesis products of the ethanolic Clostridium SGLT-Ca-0001-mut 1 include ethanol and acetic acid, the average mass concentration of the ethanol is ≥25 g / L, and the average mass concentration of the acetic acid is ≤4 g / L.

[0008] In a second aspect, the present application provides a method for separating and purifying ethanolic Clostridium as described in any one of the embodiments of the first aspect, the method comprising:

[0009] The fermentation liquid of the gas fermentation of Clostridium ethanolum is cultured on a plate under a preset culture environment to obtain a first fermentation liquid;

[0010] Performing a first genotype identification on the first fermentation broth to screen out relatively pure ethanol Clostridium to obtain a first ethanol Clostridium single colony;

[0011] The first ethanolic Clostridium single colony is enriched and cultured, and then the enriched first ethanolic Clostridium single colony is subjected to a second genotype identification to obtain a second ethanolic Clostridium single colony;

[0012] The second ethanolic Clostridium single colony was streaked and purified multiple times to obtain ethanolic Clostridium SGLT-Ca-0001-mut1.

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

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

[0015] Optionally, the preset culture environment includes a high-pressure anaerobic culture atmosphere, 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 also includes a carbon monoxide gas source, and 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] In a third aspect, the present application provides a fermentation agent, wherein the fermentation agent comprises the ethanolic Clostridium SGLT-Ca-0001-mut 1 described in any one embodiment of the first aspect.

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

[0019] The above technical solution provided by the embodiment of the present application has the following advantages compared with the prior art:

[0020] The present application provides an ethanol Clostridium, which can be used to preliminarily screen out foreign bacteria in the fermentation broth through plate culture combined with a first genotype identification, so as to preliminarily obtain a relatively pure ethanol Clostridium single colony, and then through enrichment culture and a second gene identification, the ethanol Clostridium single colony in the relatively pure ethanol Clostridium single colony can be further screened out. In addition, combined with a preset culture environment, advantageous mutant ethanol Clostridium with high production efficiency and high metabolic selectivity can be selectively screened out, and finally, the mutant ethanol Clostridium can be screened out through multiple streaking and purification, so as to obtain ethanol Clostridium with higher strain robustness and metabolic performance. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0022] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

[0023] Figure 1 A schematic diagram of the process of separating and purifying Clostridium ethanolum provided in an embodiment of the present application;

[0024] Figure 2 A graph showing the growth of bacterial colonies cultured in a high-pressure anaerobic tank containing a CO2 gas environment provided in Example 1 of the present application;

[0025] Figure 3 16s rDNA sequencing results of single colonies on solid plates of the first PETC culture medium and the second PETC culture medium provided in Example 1 of the present application;

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

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

[0028] Figure 6 Gas continuous fermentation curves of the initial ethanolic Clostridium (A) and ethanolic Clostridium SGLT-Ca-0001-mut 1 (B) provided in Example 2 of the present application. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical solution and advantages of the embodiments of the present application clearer, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application.

[0030] Various embodiments of the present application may be presented in the form of a range; it should be understood that the description in the form of a range is only for convenience and brevity, and should not be understood as a rigid limitation on the scope of the present application; therefore, the range description should be considered to have specifically disclosed all possible sub-ranges and single numerical values ​​within the range. For example, the range description from 1 to 6 should be considered to have 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., as well as single numbers within the range, such as 1, 2, 3, 4, 5 and 6, which apply regardless of the range. In addition, whenever a numerical range is indicated herein, it is meant to include any cited number (fractional or integer) within the indicated range.

[0031] In addition, in the description of the specification of the present application, the terms "including", "comprising", etc. mean "including but not limited to". In this article, relational terms such as "first" and "second", etc. are only used 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 article, "and / or" describes the association relationship of associated objects, indicating that three relationships may exist. For example, A and / or B can represent: A exists alone, A and B exist at the same time, and B exists alone. Wherein A and B can be singular or plural. In this article, "at least one" means one or more, and "plurality" means two or more. "At least one", "the following at least one item (items)" or similar expressions refer to any combination of these items, including any combination of single items (items) or plural items (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, c can be single or plural, respectively.

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

[0033] The present application provides an autoethanogenum, wherein the autoethanogenum (Clostridium autoethanogenum) is autoethanogenum SGLT-Ca-0001-mut 1, with a preservation number of GDMCC 63758, a preservation address of Guangdong Provincial Microbiological Culture Collection Center, and a preservation date of August 7, 2024.

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

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

[0036] Adaptive evolution is the continuous subculture of microorganisms under specific conditions, and the continuous enrichment of spontaneous mutations of strains to obtain new phenotypes or physiological properties that adapt to specific conditions. Adaptive evolution can effectively change certain phenotypes or physiological characteristics of strains (such as strain specific growth rate, substrate utilization rate, tolerance, etc.) in a relatively short period of time. In the process of microbial evolution, the existence of selection pressure can ensure the survival of the fittest in the random variation of strains, so that the target trait mutant strains can be retained. In the commercial gas continuous fermentation process, factors such as fluctuations in the concentration of raw gas components, fluctuations in gas volume, and differences in the impurity content of raw gas will affect the stable operation of continuous fermentation. Long-term continuous subculture will inevitably enrich and screen mutants that adapt to specific upstream gas supply conditions. These strains will have improvements in robustness and metabolic selectivity compared to the initial strains. Screening, purifying, analyzing and evaluating these strains can, on the one hand, obtain new industrial strains with improved fermentation performance. On the other hand, through the omics analysis of mutants screened under these specific conditions, the understanding of the metabolic mechanism of the strain can be deepened.

[0037] Thus, the present invention obtains the advantageous mutant ethanol clostridium SGLT-Ca-0001-mut 1 by long-term adaptive evolution screening. In the small-scale CSTR gas continuous fermentation experiment, ethanol clostridium SGLT-Ca-0001-mut 1 is superior to the initial strain in ethanol production efficiency and metabolic selectivity, and ethanol clostridium SGLT-Ca-0001-mut 1 has stronger adaptability to the environment, which significantly improves the strain robustness and metabolic performance of ethanol clostridium. Exemplarily, the average ethanol concentration of the strain can be 25g / L, 25.5g / L, 26g / L, 26.5g / L, 27g / L, 27.5g / L, etc., and the average acetic acid concentration of the strain can be 3g / L, 3.1g / L, 3.2g / L, 3.3g / L, 3.4g / L, 3.5g / L, 3.6g / L, 3.7g / L, 3.9g / L, 4g / L, etc.

[0038] Figure 1 A schematic diagram of the process of separating and purifying Clostridium ethanolum provided in an embodiment of the present application.

[0039] Based on a general inventive concept, see Figure 1 The present application provides a method for separating and purifying ethanolic Clostridium as described in any one of the above embodiments, the method comprising:

[0040] S1. culturing the fermentation liquid of the gas fermentation of Clostridium ethanolum on a plate under a preset culture environment to obtain a first fermentation liquid;

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

[0042] The temperature of plate culture is limited to 37°C, and ethanol Clostridium can grow and multiply rapidly to form visible colonies. The culture time is limited to 7 to 10 days, which can allow ethanol Clostridium to grow fully and form single colonies that are easy to identify. At the same time, a longer culture time can also help reduce false positive or false negative results caused by too short a culture time.

[0043] In some embodiments, the medium for plate culture includes the following: a first PETC medium, a second PETC medium and a third PETC medium, wherein the mass concentrations of fructose and yeast extract in the first PETC medium are both 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.

[0044] The chemical composition of the culture medium is rationally designed in the present invention. In a PETC culture medium without fructose and yeast extract (YE) or containing only fructose, ethanol Clostridium can utilize CO or fructose in the environment as a carbon source and use an inorganic nitrogen source in the culture medium as a nitrogen source for growth, while miscellaneous bacteria cannot grow under these two conditions.

[0045] In some embodiments, the preset culture environment includes a high-pressure anaerobic culture atmosphere, 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, and 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] The present invention optimizes the temperature, time, culture medium components and culture environment (including pressure, gas composition, etc.) of plate culture to jointly act on the growth process of ethanol Clostridium, thereby promoting the growth of ethanol Clostridium and inhibiting the growth of miscellaneous bacteria, ensuring the acquisition of pure ethanol Clostridium single colonies, and significantly improving the separation and purification efficiency of ethanol Clostridium.

[0048] S2, performing a first genotype identification on the first fermentation broth to screen out relatively pure ethanol Clostridium to obtain a first ethanol Clostridium single colony;

[0049] S3, enriching and culturing the first ethanolic Clostridium single colony, and then performing a second genotype identification on the enriched first ethanolic Clostridium single colony to obtain a second ethanolic Clostridium single colony;

[0050] S4. The second ethanolic Clostridium single colony is streaked and purified multiple times to obtain ethanolic Clostridium SGLT-Ca-0001-mut 1.

[0051] Based on a general inventive concept, the present application provides a fermentation agent, wherein the fermentation agent includes the ethanolic Clostridium SGLT-Ca-0001-mut 1 described in any one of the embodiments of the first aspect.

[0052] The fermentation agent of the present invention uses ethanolic Clostridium SGLT-Ca-0001-mut 1 as the main active ingredient, and aims to improve the efficiency, stability and product quality of the fermentation process through its unique metabolic ability and fermentation characteristics. The fermentation agent can be widely used in multiple fields such as food, beverages, bioenergy, chemical raw materials, etc. to meet the fermentation needs of different industries.

[0053] In some embodiments, the amount of Clostridium ethanolatum SGLT-Ca-0001-mut 1 added is 8% to 12% of the total mass of the starter.

[0054] In order to ensure the best effect of the fermentation agent, the present invention specifically limits the addition amount of ethanolic Clostridium SGLT-Ca-0001-mut 1 to 8% to 12%, which not only ensures a sufficient amount of seeds, but also prevents a large amount of metabolites in the seed liquid from entering the fermentation system. Exemplarily, the addition amount of ethanolic Clostridium 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 will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are intended only to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are usually measured according to industry standards. If there is no corresponding industry standard, then the conditions recommended by the manufacturer are followed.

[0056] Example 1

[0057] The coated plate separation method and the plate streak separation method are the simplest methods for separating and purifying microorganisms. The commercial gas continuous fermentation process cannot guarantee the strict sterility of materials and fermentation environment like laboratory fermentation. Therefore, in addition to the target bacteria, the fermentation broth also contains other microorganisms that can survive in the fermentation environment, which increases the difficulty of separating and screening the dominant target bacteria from the production line fermentation broth. The present invention takes the commercial gas fermentation of ethanol Clostridium as an example to develop a method for separating and purifying the dominant mutant strain of ethanol Clostridium from the production line fermentation broth.

[0058] The culture medium used for strain separation and culture is PETC and YTF, and the culture medium components are adjusted as needed. The components of PETC culture medium and YTF culture medium are shown in Table 1 and Table 2. Since different culture environments and culture medium formulas may affect the efficiency of strain separation and purification, the culture medium formulas used in the present invention are respectively a first PETC culture medium (the mass concentration of fructose and yeast extract powder are both 0), a second PETC culture medium (the mass concentration of fructose is 5 g / L and the mass concentration of yeast extract powder is 0), a third PETC culture medium (the mass concentration of fructose is 0 and the mass concentration of yeast extract powder is 1 g / L) and a fourth PETC culture medium (the mass concentration of fructose is 5 g / L and the mass concentration of yeast extract powder is 1 g / L), and the culture environments are respectively an anaerobic operation box (no CO environment), a normal pressure anaerobic tank (filled with 20 kPa containing 50% CO raw gas), and a high pressure anaerobic tank (filled with 150 kPa containing 50% CO raw gas).

[0059] Table 1 Components of PETC culture medium

[0060] PETC medium components Component content <![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 resazurin 0.5mL / L

[0061] Table 2 Components of YTF medium

[0062] YTF medium components Component content 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 resazurin 0.5mL / L

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

[0064] Table 3 Components of trace element solution

[0065] TSE solution components Component content 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]]> 0.2mg / 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] Wolfe's vitamin solution ingredients Component content 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 0.1mg / L p-aminobenzoic acid (Vitamin Bx) 5mg / L Thioctic acid 5mg / L

[0068] The production line fermentation liquid was coated on the above four different formulations of culture medium plates in the anaerobic operation box. Each culture medium plate was coated with 100ul of undiluted fermentation liquid and 10 -6 The diluted fermentation liquid and the coated plates were cultured at 37°C in three different culture environments to observe the growth of the strain (the high-pressure anaerobic tank had an obvious pressure drop).

[0069] The results showed that no colonies grew on the plates cultured in the anaerobic operation box that did not contain fructose and yeast extract powder or only contained fructose, while large flat colonies and smaller round colonies grew on the plates containing yeast extract powder or yeast extract powder and fructose that were coated with undiluted fermentation liquid. A layer of bacteria should grow on the plate after the undiluted liquid is coated. Therefore, it is speculated that the single colonies grown by this culture method are miscellaneous bacteria or a combination of ethanol Clostridium and miscellaneous bacteria.

[0070] Similarly, the solid culture medium plate cultured in a normal pressure anaerobic tank showed a blackening phenomenon, which may be caused by the inflow of oxygen during the culture process. The single colonies grown on the plate had different colors and shapes, and the selection workload was large, which was not suitable for the separation and purification of ethanol Clostridium.

[0071] The bacterial colonies cultured in a high-pressure anaerobic tank containing CO2 gas are as follows Figure 2 As shown, after the fermentation liquid was coated on the solid plates of the first PETC medium and the second PETC medium, a layer of bacteria grew out, and after the diluted fermentation liquid was coated, a uniform single colony grew out; after the fermentation liquid was coated on the third PETC medium and the fourth PETC medium, there were single colonies on the surface of the bacterial layer. The fourth PETC medium with richer nutrition had a thinner bacterial layer and larger single colonies on the surface. The diluted coated plate grew two types of colonies of different sizes. Therefore, it is preliminarily speculated that in the PETC medium without fructose and YE or containing only fructose, ethanol Clostridium can use CO or fructose in the environment as a carbon source and grow with the inorganic nitrogen source in the culture medium as a nitrogen source, while the miscellaneous bacteria cannot grow under these two conditions; while in the solid culture medium containing only YE, the miscellaneous bacteria showed signs of growth, and the bacterial layer formed by the growth of ethanol Clostridium became thinner. When the culture medium contained both fructose and YE, the ethanol Clostridium bacterial layer was thinner, and the single colony on the surface of the bacterial layer could grow. This shows that the miscellaneous bacteria have a certain dependence on organic nitrogen sources, and the growth competitive advantage of miscellaneous bacteria is obvious under nutrient-rich conditions. The growth of bacterial colonies under different culture environments and culture media are shown in Table 5.

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

[0073]

[0074] In Table 5, 1 means that under this specific culture environment and culture medium conditions, the bacterial layer is a uniform single colony of ethanol Clostridium or has fewer miscellaneous bacterial colonies, and 0 means that under this culture environment and culture medium conditions, no colonies grow or the bacterial layer contains more miscellaneous bacterial colonies.

[0075] The bacterial layer grown after the first PETC culture medium, the second PETC culture medium, and the third PETC culture medium solid flat plate coated with the stock solution was used as a template to amplify the key genes AOR1 and 16s rDNA of ethanol synthesis for identification of ethanol Clostridium. It was found that the bacterial layer grown by the first PETC culture medium and the second PETC culture medium was used as a template to amplify AOR1 successfully, but only the latter amplified 16s rDNA. The sequencing results showed that the bacterial layer bacteria were ethanol Clostridium (seq3.PETC-F-1492R_R_C03, seq4. ethanol Clostridium 16s rDNA sequence); while the bacterial layer grown by the third PETC culture medium was used as a template to fail to amplify AOR1 and 16s rDNA genes. Based on the above analysis, speculation and verification, the present invention selects single colonies grown by culturing the first PETC culture medium and the second PETC culture medium solid flat plate in a CO2-containing gas environment using a high-pressure anaerobic tank for subsequent purification verification experiments.

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

[0077] CAGTGTACCCCACCTTCGGCCGCGTCCTCCTAAGTTAGACTACGGACTTCGGGTGTTGCCAGCTCTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGACATTCTGATTCGCGATTACTAGCAACTCCAACTTCATGTAGGCGAGTTTCAGCCTGCAATCCGAACTGGGGGCAGTTTTTGAGGTTTGCTCCACCTTGCGGTCTTGCTTCTCTCTGTACTGCCCATTGTAGCACGTGTGTTGCCCTGGACATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGCGTTAACCGCGGCAGTCTTGCTAGAGTGCTCAACTAAATGTTAGCAACTAACAACAGGGGTTGCGCTCGTTGCAGGACTTAACCTAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTATCCCTGCCCCGAAGGGCTTCTCTTATCTCTAAGATATTCAGGGTATGTCAAGTCCAGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCGCTGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTTAATCTTGCGATCGTACTTCCGAGACGGAGTACTTATTGTGTTTACTGCGGCACAGAAGGGGTCGATACCTCCTACACCTAGTACTCATCGTTTACGGCGTGGACTACCAGGGGTATCTAATCCTGTTTGCTACCCACGCTTTT

[0078] seq4. 16S rDNA sequence of Clostridium autoethanogenum

[0079]

[0080] The single colonies grown on the solid plates of the first PETC medium and the second PETC medium were streaked and enriched on solid plates with the same medium formula, and cultured in a high-pressure anaerobic tank CO environment and then amplified by PCR and 16s rDNA sequencing was verified ( Figure 3 ), and found that the single colonies picked were all alcohol Clostridium. The single colonies after enrichment culture were streaked on a new plate for purification, and the single colonies grown after continued cultivation in the high-pressure anaerobic tank CO environment were streaked for purification for the second time, and continued to be cultivated in the high-pressure anaerobic tank CO environment. For ease of operation, the single colonies purified in the CO environment were streaked on the YTF plate for purification and then cultured in the anaerobic operation box. The growth rate of the strain in the first round of cultivation was very slow, but the single colonies grown grew faster after inoculation with YTF liquid culture medium. After the single colonies on the YTF plate were inoculated with YTF liquid culture medium at 37℃ and 220rpm for 48h, the bacterial biomass was measured. The results are as follows Figure 4 As shown, the fastest growing pure strain of ethanolic Clostridium production line was recorded as SGLT-Ca-0001-mut 1 (abbreviated as Ca_mut1) and preserved as glycerol bacteria. The strain was verified by PCR amplification and 16srDNA sequencing to be ethanolic Clostridium (seq1.Ca_mut1-1492R, seq2.Ca_mut1-27F, Figure 5 ).

[0081] Example 2

[0082] The pure strain Ca_mut1 and the initial ethanol Clostridium glycerol bacteria were inoculated into YTF medium as primary seeds and cultured at 37°C and 220rpm. It was found that the bacterial solution of Ca_mut1 became turbid 17 hours after inoculation, while the initial ethanol Clostridium had no signs of growth. After 48 hours of culture, the primary seeds were inoculated into 300mL fresh YTF medium at a 3% inoculation rate as secondary seeds and cultured at 37°C and 220rpm. After 24 hours of culture, the biomass of Ca_mut1 reached 0.5g / L, and the biomass of the initial ethanol Clostridium reached 0.45g / L. All the secondary seed liquid was inoculated into a 3L CSTR containing 1.7L PETC medium (without fructose and yeast extract powder) for gas continuous fermentation. The metabolic curve of 20 days of fermentation is as follows: Figure 6As shown in the figure, the control parameters of the entire fermentation process, including stirring speed, gas volume, and dilution rate, remained completely consistent for the two strains. In the metabolic enhancement stage, Ca_mut1 increased ethanol faster than the initial ethanol Clostridium, indicating that the purified strain had better ethanol metabolic selectivity; after the metabolism reached steady state, the average ethanol concentration of the initial ethanol Clostridium was 23.4 g / L, and the average acetic acid concentration was 6.1 g / L; the average ethanol concentration of the Ca_mut1 strain was 25.4 g / L, and the acetic acid concentration was 3.5 g / L. Ca_mut1 entered metabolic steady state two days earlier than the initial ethanol Clostridium, and the ethanol concentration of the purified strain Ca_mut1 was 8.5% higher than that of the initial ethanol Clostridium, which will produce considerable economic benefits in the commercial production process.

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

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

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

[0086] The above description is only a specific implementation of the present application, so that those skilled in the art can understand or implement the present application. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to the embodiments shown herein, but will conform to the widest range consistent with the principles and novel features applied for herein.

Claims

1. An ethanolic Clostridium, characterized in that The Clostridium autoethanogenum is Clostridium autoethanogenum SGLT-Ca-0001-mut 1, with a preservation number of GDMCC 63758, a preservation address of Guangdong Provincial Microbiological Culture Collection Center, and a preservation date of August 7, 2024.

2. The ethanolic Clostridium according to claim 1, characterized in that The nucleotide sequence of the 16S rDNA segment of the ethanolic Clostridium SGLT-Ca-0001-mut 1 includes a first nucleotide sequence and a second nucleotide sequence, the first nucleotide sequence is shown in SEQ ID NO.1, the second nucleotide sequence is shown in SEQ ID NO.2, and the first nucleotide sequence and the second nucleotide sequence are partially complementary base pairing.

3. The ethanolic Clostridium according to claim 1, characterized in that The synthetic products of the ethanolic Clostridium SGLT-Ca-0001-mut 1 include ethanol and acetic acid, the average mass concentration of the ethanol is ≥25 g / L, and the average mass concentration of the acetic acid is ≤4 g / L.

4. A method for separating and purifying ethanolic Clostridium according to any one of claims 1 to 3, characterized in that: The method comprises: The fermentation liquid of the gas fermentation of Clostridium ethanolum is cultured on a plate under a preset culture environment to obtain a first fermentation liquid; Performing a first genotype identification on the first fermentation broth to screen out relatively pure ethanol Clostridium to obtain a first ethanol Clostridium single colony; The first ethanolic Clostridium single colony is enriched and cultured, and then the enriched first ethanolic Clostridium single colony is subjected to a second genotype identification to obtain a second ethanolic Clostridium single colony; The second ethanolic Clostridium single colony was streaked and purified multiple times to obtain ethanolic Clostridium SGLT-Ca-0001-mut1.

5. The method according to claim 4, characterized in that The temperature of the plate culture is 37° C., and the time of the plate culture is 7 to 10 days.

6. The method according to claim 5, characterized in that The medium for the plate culture includes the following: a first PETC medium, a second PETC medium and a third PETC medium, wherein the mass concentrations of fructose and yeast extract in the first PETC medium are both 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.

7. The method according to claim 4, characterized in that The preset culture environment includes a high-pressure anaerobic culture atmosphere, the pressure of the high-pressure anaerobic culture atmosphere is 130 kPa to 160 kPa, and the O2 concentration is 0.

8. The method according to claim 7, characterized in that The preset culture environment also includes a carbon monoxide gas source, and 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.

9. A leavening agent, characterized in that The fermentation agent comprises the ethanolic Clostridium SGLT-Ca-0001-mut 1 according to any one of claims 1 to 3.

10. The leavening agent according to claim 9, characterized in that The added amount of the ethanolic Clostridium SGLT-Ca-0001-mut1 accounts for 8% to 12% of the total mass of the starter.

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