Culture method and application of pit mud sourced medium and short chain fatty acid stable degradation flora

Abstract

The invention discloses a culture method and application of a pit mud-sourced medium and short chain fatty acid stable degradation flora, and belongs to the technical field of white spirit brewing. The invention firstly provides the culture method of the medium-chain and short-chain fatty acid degrading flora with the stable pit mud source, the method comprises the following steps: by taking hexanoic acid, acetic acid and formic acid as main carbon sources of an enrichment culture medium and hexanoic acid and formic acid as main carbon sources of a subculture medium, inoculating the pit mud into the enrichment culture medium for enrichment culture; primary medium-chain and short-chain fatty acid degrading flora is obtained. The medium-chain and short-chain fatty acid degrading flora obtained by adopting the culture method disclosed by the invention is combined with a diphasic anaerobic digestion process of pit mud-derived acid-producing microorganisms, so that the main organic substance lactic acid and a small amount of medium-chain and short-chain fatty acid in the bottom pot water of the liquor brewing wastewater can be finally degraded into methane, and meanwhile, the pH regulation is not required by diphasic coupling; the wastewater treatment cost is reduced to a certain extent.

Description

Technical Field

[0001] The invention relates to a method for culturing a cellar mud-derived medium- and short-chain fatty acid-stabilizing bacterial flora and application thereof, and belongs to the technical field of liquor brewing. Background Art

[0002] Baijiu is one of the six major distilled liquors in the world. According to the differences in flavor and brewing process, it can be divided into twelve flavors, mainly strong-flavor, sauce-flavor, and light-flavor. Among them, strong-flavor Baijiu is one of the most popular flavors and the Baijiu flavor with the highest production in my country. Strong-flavor Baijiu uses the fruity pineapple aroma of ethyl caproate as the main aroma, and has the flavor characteristics of "rich cellar aroma, cotton-sweet, refreshing, soft and coordinated, and clean and long aftertaste". During the fermentation process of Baijiu brewing, the microorganisms in the cellar mud can use macromolecular organic substances to produce a variety of medium and short-chain fatty acids such as acetic acid, butyric acid, and caproic acid. These fatty acids can react with ethanol that seeps into the cellar mud from the fermented grains to produce a variety of ester substances including ethyl caproate. The ester substances enter the fermented grains through volatilization and other means, and enter the Baijiu through the distillation process, providing material support for the formation of the unique aroma of Baijiu.

[0003] "Old cellars produce good wine" is a well-known practical experience gained from long-term liquor production. During the liquor production process, cellar mud is in an anaerobic environment for a long time. Bacteria (including archaea) are the main microbial groups in the old cellar mud community. Studies have shown that the microorganisms in cellar mud can be divided into hydrolytic and acidogenic fermentation bacteria, symbiotic acetogenic bacteria, and methanogenic bacteria (Ren Cong, Du Hai, Xu Yan. Research Progress on Microbiome of Traditional Fermented Foods in China [J]. Journal of Microbiology, 2017, 57(6): 885-898.). During the liquor fermentation process, hydrolytic and acidogenic fermentation bacteria will produce a large amount of medium- and short-chain fatty acids. Some of the medium- and short-chain fatty acids will enter the fermented grains through the material exchange between the cellar mud and the fermented grains and react with ethanol to form esters. Symbiotic acetogenic microorganisms can use the remaining fatty acids in the cellar mud for growth and metabolism, avoiding the acidification of the cellar mud caused by the accumulation of fatty acids, thereby maintaining the stability of the pH in the cellar mud ecosystem.

[0004] Fatty acid-degrading microorganisms are widely present in natural habitats such as soil, lakes and seawater sediments, and are also an important component of artificial habitats such as anaerobic granular sludge and anaerobic reactors. Medium- and short-chain fatty acid-degrading microorganisms can be divided into Syntrophic acetate-oxidizing bacteria (SAOB), Syntrophic propionate-oxidizing bacteria (SPOB), Syntrophic butyrate-oxidizing bacteria (SBOB) and Syntrophic valerate-oxidizing bacteria (SVOB). These acid-degrading microorganisms must metabolize with methanogenic microorganisms because the Gibbs free energy (under standard thermodynamic conditions) of their fatty acid degradation process is greater than zero. Due to the harsh culture conditions of syntrophic fatty acid microorganisms, how to study the syntrophic metabolic relationship between syntrophic acid-degrading microorganisms and methanogenic microorganisms in microbial communities and regulate them has always been a research hotspot. In order to further analyze the specific functions of acid-reducing microorganisms, there is a related culture technology that cultivates the acid-reducing microbial flora in cellar mud through the method of culture medium enrichment (CN202010466226), thereby achieving the enrichment and cultivation of short-chain fatty acid (butyric acid, acetic acid) degrading flora. However, this technology still has disadvantages such as long enrichment time and difficulty in continuous culture during the culture process. At present, there is a lack of long-term, stable culture and green preparation technology for medium-chain fatty acids derived from cellar mud.

[0005] Wastewater from the production process of liquor enterprises can be divided into non-brewing wastewater and brewing by-product wastewater. The latter is mainly composed of bottom pot water (remaining at the bottom of the retort barrel after distillation), which can reach 80% of the wastewater and is the main cause of environmental pollution caused by liquor enterprises. At present, the treatment methods for bottom pot water of liquor include aerobic biological method, anaerobic biological method and electrolysis method. The treatment process in actual engineering is mostly "physical and chemical method pretreatment + aerobic anaerobic biological method combined treatment + physical and chemical method post-treatment". In the anaerobic biological treatment process of bottom pot water of liquor, traditional single-phase anaerobic digestion is mostly used, and the acid-producing microorganisms and acid-reducing methanogenic microorganisms are located in a single reaction system to carry out the entire anaerobic digestion process. However, there are great differences between acid-producing microorganisms and methanogenic microorganisms in terms of reproduction performance, nutritional requirements and optimal environment. Therefore, in the actual operation of the single-phase anaerobic digestion reactor, it is often difficult to simultaneously meet the optimal growth, reproduction and metabolic environment conditions of acid-producing microorganisms and acid-reducing methanogenic microorganisms, and it is impossible to simultaneously exert the optimal metabolic performance of acid-producing microorganisms and acid-reducing methanogenic microorganisms. The use of two-phase anaerobic digestion technology is conducive to improving the upper limit of the influent COD load in the process of liquor brewing wastewater treatment and accelerating the wastewater treatment efficiency. However, the difficulty of two-phase anaerobic digestion of lactic acid wastewater lies in how to reduce the generation of difficult-to-degrade fatty acids such as propionic acid and the pH difference between the two phases of influent and effluent. If the treatment of liquor brewing wastewater can solve the problems of high propionic acid concentration in the acid-producing phase and the coupling of pH between the two phases, the two-phase anaerobic digestion process can be used without diluting the high-concentration wastewater, thus achieving efficient treatment of high-concentration wastewater.

[0006] In summary, enriching and screening highly efficient hexanoic acid-degrading microorganisms from cellar mud and realizing the green preparation of these microorganisms (without using or using as little artificial synthetic culture medium as possible) have important application value for the production of cellar mud and the efficient treatment of brewing by-products (bottom pot water) in the liquor brewing process. Summary of the invention

[0007] In order to clarify the specific species and functions of acid-reducing microorganisms in the research of cellar mud microorganisms and optimize the treatment process of brewing wastewater in liquor enterprises, the present invention provides an enrichment and continuous cultivation method for stable medium- and short-chain fatty acid-degrading bacterial flora derived from cellar mud, which is applied to the two-phase anaerobic digestion process of liquor brewing wastewater.

[0008] The present invention adopts the following technical solutions:

[0009] The invention provides a method for culturing a stable medium- and short-chain fatty acid-degrading bacterial flora derived from cellar mud. The method adopts caproic acid, acetic acid and formic acid as the main carbon sources of an enrichment medium, adopts caproic acid and formic acid as the main carbon sources of a passage medium, and inoculates the cellar mud into the enrichment medium for enrichment culture to obtain a primary medium- and short-chain fatty acid-degrading bacterial flora.

[0010] In one embodiment of the present invention, the method comprises: inoculating pit mud into an enrichment medium for enrichment culture, and then inoculating the obtained seed liquid into a subculture medium for step-by-step expansion culture; the expansion culture time of each level of seed liquid is 7 to 10 days.

[0011] In one embodiment of the present invention, the enrichment culture is to inoculate the cellar mud into the enrichment culture medium and culture it at 30-37°C for 15 to 30 days; preferably, the thawed cellar mud is inoculated into the above culture medium at an inoculation rate of 10% to 20% (m / v); further, the thawed cellar mud is inoculated into the above culture medium at an inoculation rate of 10% to 15% (m / v).

[0012] In one embodiment of the present invention, the enrichment medium components contain: caproic acid 5-8 g / L, acetic acid 3-6 g / L, formic acid 1-2 g / L, yeast powder 0.8-1.2 g / L, peptone 0.8-1.2 g / L, mineral element solution 45-55 mL / L, trace element solution 0.8-1.2 mL / L, B vitamin solution 4-6 mL / L, cysteine ​​hydrochloride solution 8-12 mL / L, pH 7.2-7.4;

[0013] In one embodiment of the present invention, the subculture medium components contain: caproic acid 10-20 g / L, formic acid 1-3 g / L, yeast powder 1.8-2.2 g / L, peptone 1.8-2.2 g / L, mineral element solution 80-100 mL / L, trace element solution 1.8-2.0 mL / L, B vitamin solution 8-12 mL / L, cysteine ​​hydrochloride solution 18-22 mL / L, and the pH is 7.2-7.4.

[0014] In one embodiment of the present invention, the seed solution is inoculated into the subculture medium at an inoculum volume of 50% (v / v);

[0015] In one embodiment of the present invention, the mineral element solution (g / L): KH2PO4 10, MgCl2·6H2O6.6, NaCl 8, NH4 Cl 8, CaCl2 1, pH 7.4; trace element solution (g / L): ZnSO4·7H2O 0.1, MnCl20.03, H3BO3 0.3, CoCl2·6H2O 0.2, CaCl2 0.01, NiCl2·6H2O 0.02, Na2MoO4 0.03, FeCl24H2O 1.5; the cysteine ​​solution contains: 12.5g cysteine ​​hydrochloride per liter;

[0016] In one embodiment of the present invention, the expansion culture is expansion culture of level 1 or more;

[0017] In one embodiment of the present invention, the inoculation amount of the cellar mud is 8-12%, and the cellar mud includes cellar bottom mud with a cellar age of not less than 5 years and a pH value of 7.2-7.4;

[0018] In one embodiment of the present invention, the cellar mud used for inoculation is selected according to the relative abundance of Syntrophomonas, Syntrophic acetic bacteria and methanogens in the cellar mud, wherein the relative abundance of Syntrophomonadaceae should be ≥1.0%, the relative abundance of Syntrophaceticus should be ≥0.1%, and the relative abundance of Methanogenus should be ≥10%;

[0019] In one embodiment of the present invention, the culturing is carried out under anaerobic conditions;

[0020] In one embodiment of the present invention, the culturing is performed at 32-37°C.

[0021] In one embodiment of the present invention, the enrichment medium components contain, per liter: 5-10 g caproic acid, 5 g acetic acid, 1.4 g formic acid, 1.0 g yeast powder, 1.0 g peptone, 50 mL mineral element solution, 1 mL trace element solution, 5 mL B vitamin solution, and 10 mL cysteine ​​hydrochloride solution.

[0022] The present invention also provides a stable medium- and short-chain fatty acid-degrading bacterial community derived from cellar mud prepared by the above method.

[0023] The present invention provides an application of a medium- and short-chain fatty acid-degrading bacterial community in pit mud in continuous caproic acid degradation. The method adopts caproic acid and formic acid as the main carbon sources of a subculture medium, and uses the subculture medium to perform continuous fed-batch fermentation on the medium- and short-chain fatty acid-degrading bacterial community to continuously degrade caproic acid.

[0024] In one embodiment of the present invention, the application is to use a subculture medium to carry out continuous fed-batch fermentation of a medium- and short-chain fatty acid-degrading bacterial community to degrade caproic acid; specifically comprising: inoculating pit mud into an enrichment medium for enrichment culture, and then inoculating the obtained seed liquid into a subculture medium for step-by-step expansion culture; the expansion culture time of each level of seed liquid is 7 to 10 days;

[0025] In one embodiment of the present invention, the enrichment medium components contain: caproic acid 5-8 g / L, acetic acid 3-6 g / L, formic acid 1-2 g / L, yeast powder 0.8-1.2 g / L, peptone 0.8-1.2 g / L, mineral element solution 45-55 mL / L, trace element solution 0.8-1.2 mL / L, B vitamin solution 4-6 mL / L, cysteine ​​hydrochloride solution 8-12 mL / L, pH 7.2-7.4;

[0026] In one embodiment of the present invention, the subculture medium components contain: caproic acid 10-20 g / L, formic acid 1-3 g / L, yeast powder 1.8-2.2 g / L, peptone 1.8-2.2 g / L, mineral element solution 80-100 mL / L, trace element solution 1.8-2.0 mL / L, B vitamin solution 8-12 mL / L, cysteine ​​hydrochloride solution 18-22 mL / L, and the pH is 7.2-7.4.

[0027] In one embodiment of the present invention, the subculture medium components contain, per liter: 5-20 g caproic acid, 2.8 g formic acid, 2.0 g yeast powder, 2.0 g peptone, 100 mL mineral element solution, 2 mL trace element solution, 10 mL B vitamin solution, and 20 mL cysteine ​​hydrochloride solution.

[0028] In one embodiment of the present invention, the mineral element solution in the above culture medium contains per liter: 10.0 g of anhydrous potassium dihydrogen phosphate, 6.6 g of magnesium chloride hexahydrate, 8.0 g of sodium chloride, 8.0 g of anhydrous ammonium chloride, and 1.0 g of anhydrous calcium chloride.

[0029] In one embodiment of the present invention, the trace element solution in the above-mentioned culture medium contains, per liter, 0.1 g zinc sulfate heptahydrate, 0.03 g manganese chloride tetrahydrate, 0.3 g boric acid, 0.2 g cobalt chloride hexahydrate, 0.01 g calcium chloride dihydrate, 0.02 g nickel chloride hexahydrate, 0.03 g sodium molybdate dihydrate, and 1.5 g ferrous chloride tetrahydrate.

[0030] In one embodiment of the present invention, the B vitamin solution in the above culture medium contains per liter: 0.02 g niacin, 0.02 g cobalamin, 0.01 g thiamine hydrochloride, 0.01 g p-aminobenzoic acid, 0.05 g pyridoxine hydrochloride, and 0.005 g calcium pantothenate.

[0031] In one embodiment of the present invention, the cysteine ​​solution in the above culture medium contains 12.5 g of cysteine ​​hydrochloride per liter.

[0032] In one implementation method of the present invention, the inoculation amount of the pit mud is 10%, the pit mud age is not less than 5 years, the inoculation amount of the subculture is 50%, the pH of the culture medium is 7.2-7.4, and the culture condition is anaerobic static culture at 37°C in an anaerobic incubator.

[0033] The present invention provides an application of a two-phase anaerobic digestion process based on the above-mentioned stable medium- and short-chain fatty acid degrading bacterial community in the treatment of bottom pot water of liquor brewing wastewater. The application can use medium- and short-chain fatty acid producing and degrading microorganisms derived from cellar mud to convert lactic acid, the main organic substance, and a small amount of medium- and short-chain fatty acids in the bottom pot water into methane.

[0034] Exemplarily, the application method may include first inoculating medium- and short-chain fatty acid producing microorganisms into the bottom pot water, and after culturing for a period of time, inoculating medium- and short-chain fatty acid degrading bacteria into the culture system, which can convert lactic acid and a small amount of medium- and short-chain fatty acids in the bottom pot water into methane.

[0035] In one embodiment of the present invention, the medium- and short-chain fatty acid producing microorganisms should be mainly composed of Bacillus lactis, and also include a variety of medium- and short-chain fatty acid producing bacteria.

[0036] In one embodiment of the present invention, the inoculation amount of the medium- and short-chain fatty acid producing microorganisms in the bottom pot water should be 5-10%, preferably 10%, and the bottom pot water should be treated until the lactic acid therein is completely degraded.

[0037] In one embodiment of the present invention, the application is specifically:

[0038] (a) Preparation of acid-producing microbial flora of pit mud:

[0039] Selecting pit mud that is over 15 years old, has a caproic acid concentration of >6 g / kg and a lactic acid concentration of <30 g / kg, and inoculating the pit mud into a composite culture medium for step-by-step expansion culture; the expansion time of each level of seed liquid is 1 to 3 days; preferably, the expansion culture is 1-4 levels;

[0040] Composite culture medium: glucose 10.0-15.0 g, sodium lactate 30-40 mL, sodium acetate 5 g, ammonium sulfate 2.0 g, dipotassium hydrogen phosphate 1.0 g, potassium dihydrogen phosphate 0.5 g, peptone 10.0 g, yeast powder 10.0 g, magnesium sulfate heptahydrate 0.1 g, ferrous sulfate heptahydrate 0.15 g, calcium chloride 0.01 g, manganese sulfate 0.01 g, cobalt chloride hexahydrate 0.002 g, zinc sulfate 0.002 g;

[0041] The sodium lactate is a sodium lactate solution with a mass fraction of 60%.

[0042] (b) Preparation of acid-producing bacteria using bottom pot water as culture medium:

[0043] The acid-producing microbial flora of the pit mud obtained in step (a) is inoculated into the bottom pot water for culturing to obtain an acid-producing system;

[0044] (c) Preparation of "caproic acid degradation-methane production" medium- and short-chain fatty acid degradation bacteria:

[0045] The stable medium- and short-chain fatty acid-degrading bacterial flora derived from the pit mud according to claim 3 is added to the acid-producing system obtained in step (b) to treat the bottom pot water.

[0046] In one embodiment of the present invention, in step (b):

[0047] The bottom pot water treatment method is: diluting with 50% deionized water, adjusting the pH to 6, and deoxygenating;

[0048] Preferably, the cellar mud acid-producing microbial flora is inoculated into the bottom pot water at an inoculation rate of 5-10%;

[0049] Preferably, the culturing is carried out under anaerobic conditions;

[0050] Preferably, the culture is carried out at 32-37°C for 7 to 10 days.

[0051] In one embodiment of the present invention, in step (a), the culture is carried out under anaerobic conditions, further, at 32-37°C, and the culture time is: 2 to 5 days. In each subculture, the pit mud or culture solution is inoculated into the composite culture medium at a ratio of 5 to 20%; preferably 10 to 20%, and more preferably 10%.

[0052] In one embodiment of the present invention, in step (c):

[0053] The stable medium- and short-chain fatty acid-degrading bacteria from the pit mud and the acid-producing system are mixed in a ratio of 1:1 to 9;

[0054] Preferably, the culturing is carried out under anaerobic conditions;

[0055] Preferably, the culture is carried out at 32-37°C for 7 to 10 days.

[0056] The present invention provides an application of the above-mentioned "caproic acid degradation-methane production" medium- and short-chain fatty acid degradation bacterial flora cultivation method in the production of artificial pit mud.

[0057] Illustratively, the application method may include: using caproic acid, acetic acid, and formic acid as the main carbon sources to obtain primary medium- and short-chain fatty acid-degrading bacterial flora, and applying the flora to the production of cellar mud after step-by-step amplification and cultivation.

[0058] The present invention also provides the above method, or the use of the above-mentioned cellar mud-derived stable medium- and short-chain fatty acid-degrading bacterial flora or the bacterial flora containing Syntrophomonas in a liquor brewing system, wherein the application is to use the bacterial flora in the liquor fermentation process.

[0059] In one embodiment of the present invention, the application is to apply the bacterial community in a solid-state mash fermentation process without a cellar mud system.

[0060] The present invention also provides a method for culturing artificial cellar mud or a method for maintaining cellar mud, the method comprising: adding the above-mentioned cellar mud-derived stable medium- and short-chain fatty acid-degrading bacterial flora or the bacterial flora containing Syntrophomonas during the process of culturing artificial cellar mud or maintaining cellar mud.

[0061] Beneficial Effects

[0062] (1) The enrichment method of the present invention can be used to obtain a microbial community with a rapid hexanoic acid degradation rate, which includes a high abundance of syntrophic monads, a variety of methanogens and other functional microorganisms, which can quickly degrade the hexanoic acid in the substrate and generate methane to be discharged into the external environment.

[0063] (2) The stable medium- and short-chain fatty acid-degrading bacterial flora obtained by the cultivation method of the present invention can continuously and rapidly degrade caproic acid, and at the same time can resist the interference of a certain amount of other organic matter such as ethanol, lactic acid, and glucose, and maintain the original caproic acid degradation phenotype.

[0064] (3) The medium- and short-chain fatty acid-degrading bacterial flora obtained by the cultivation method of the present invention can not only degrade even-numbered carbon fatty acids, but also have a certain degradation ability for odd-numbered carbon fatty acids such as propionic acid.

[0065] (4) The present invention establishes a method of using caproic acid bacteria to prepare a fermentation broth containing caproic acid using bottom pot water as a culture medium, and then inoculating it with a stabilized "caproic acid degradation-methanogenesis" medium- and short-chain fatty acid degrading bacterial community, thereby achieving a green preparation of syntrophic bacteria and methanogens.

[0066] (5) The medium- and short-chain fatty acid-degrading bacterial flora obtained by the cultivation method of the present invention combined with the acid-producing microorganisms derived from the pit mud can ultimately degrade the main organic substance lactic acid and a small amount of medium- and short-chain fatty acids in the bottom pot water of liquor brewing wastewater into methane. At the same time, the two-phase coupling does not require pH adjustment, which reduces the wastewater treatment cost to a certain extent.

[0067] (6) The short-chain fatty acid-degrading bacterial community obtained by the enrichment method of the present invention has a highly efficient acid-reducing and methane-producing function, and has important application value in the production of artificial pit mud and the improvement of pit mud quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] Figure 1 This is the curve of the change in the amount of even-numbered carbon medium- and short-chain fatty acids and methane with the culture time in Example 1.

[0069] Figure 2 This is the curve of the change in the amount of even-carbon medium- and short-chain fatty acids during the continuous fed-batch culture in Example 2 as a function of the culture time.

[0070] Figure 3This is the curve of the change in the amount of caproic acid over the culture time during the culture process of the short-chain fatty acid degrading bacteria adding lactic acid, ethanol and glucose in Example 2.

[0071] Figure 4 This is the abundance change curve of the microbial genus of the short-chain fatty acid degrading flora during the continuous fed-batch culture process of Example 2 during the culture time. DETAILED DESCRIPTION

[0072] The culture medium involved in the following examples is as follows:

[0073] Enrichment medium ( / L): caproic acid 5-10 g, acetic acid 2 g, formic acid 1.4 g, yeast powder 1.0 g, peptone 1.0 g, mineral element solution 50 mL, trace element solution 1 mL, B vitamin solution 5 mL, cysteine ​​hydrochloride solution 10 mL, pH 7.3. Among them, mineral element solution (g / L): KH2PO4 10, MgCl2·6H2O 6.6, NaCl 8, NH4 Cl8, CaCl2 1, pH 7.4; trace element solution (g / L): ZnSO4·7H2O 0.1, MnCl2 0.03, H3BO3 0.3, CoCl2·6H2O 0.2, CaCl20.01, NiCl2·6H2O 0.02, Na2MoO4 0.03, FeCl2 4H2O 1.5; B vitamin solution (mg / L): niacin 20, cobalamin 20, thiamine hydrochloride 10, para-aminobenzoic acid 10, pyridoxine hydrochloride 50, calcium pantothenate 5.

[0074] Subculture medium ( / L): caproic acid 5-20 g, formic acid 2.8 g, yeast powder 2.0 g, peptone 2.0 g, mineral element solution 100 mL, trace element solution 2 mL, B vitamin solution 10 mL, cysteine ​​hydrochloride solution 20 mL. Among them, mineral element solution (g / L): KH2PO4 10, MgCl2·6H2O 6.6, NaCl 8, NH4 Cl 8, CaCl2 1, pH 7.4; trace element solution (g / L): ZnSO4·7H2O 0.1, MnCl2 0.03, H3BO3 0.3, CoCl2·6H2O 0.2, CaCl2 0.01, NiCl2·6H2O 0.02, Na2MoO4 0.03, FeCl2 4H2O 1.5; B vitamin solution (mg / L): niacin 20, cobalamin 20, thiamine hydrochloride 10, para-aminobenzoic acid 10, pyridoxine hydrochloride 50, calcium pantothenate 5.

[0075] Glucose-milk enrichment medium ( / L): 10.0 g glucose, 30 mL sodium lactate, 5 g sodium acetate, 2.0 g ammonium sulfate, 1.0 g dipotassium hydrogen phosphate, 0.5 g potassium dihydrogen phosphate, 10.0 g peptone, 10.0 g yeast powder, 0.1 g magnesium sulfate heptahydrate, 0.15 g ferrous sulfate heptahydrate, 0.01 g calcium chloride, 0.01 g manganese sulfate, 0.002 g cobalt chloride hexahydrate, 0.002 g zinc sulfate. The sodium lactate is a sodium lactate solution, and its mass fraction is 60%.

[0076] The detection methods involved in the following embodiments are as follows:

[0077] Detection method of medium and short chain fatty acids and ethanol content:

[0078] After the fermentation sample was centrifuged at 12000rpm for 5min, the supernatant was filtered using a 0.22μm filter membrane, 200μL of the filtrate was added with 50μL of the internal standard solution (12.5g / L t-pentanoic acid, pH 2.5, containing 5% concentrated hydrochloric acid) and mixed, and then subjected to gas chromatography detection. The detection instrument was an Agilent gas chromatograph GC-7890B, the chromatographic column was an Agilent Cap-Wax capillary column (50m×0.25mm×0.25μm), and the program temperature conditions were: initial temperature 60°C, maintained for 0.5min, increased to 180°C at 20°C / min, and maintained for 3min. The split ratio was 5:1, the injection port temperature was 220°C, the hydrogen flame ionization detector (FID) detection temperature was 220°C, the air flow rate was 450mL / min, the carrier gas flow rate was 2mL / min, and the hydrogen flow rate was 40mL / min.

[0079] Methane detection method:

[0080] Use an injection needle to take 500 μL of headspace gas and inject it into the instrument for detection. The detection instrument is an Agilent gas chromatograph GC-6890N, the chromatographic column is an HP-5 capillary column, the column temperature is 40°C, maintained for 5 minutes, the injector temperature is 200°C, the detector temperature is 250°C, the column flow rate is 0.9 mL / min, the split ratio is 30:1, the air flow rate is 300 mL / min, and the hydrogen flow rate is 40 mL / min.

[0081] Glucose and lactic acid content detection method:

[0082] The glucose and lactic acid contents were determined using a bioanalyzer. The glucose and lactic acid concentrations in the samples were diluted to below 1 g / L and 0.5 g / L before determination.

[0083] Calculation method of bacterial flora microbial abundance:

[0084] The DNA of the enriched samples was extracted for amplicon sequencing. After data quality control, clustering was performed according to a 99% similarity threshold to form 99% amplicon sequencing variants (ASV). The Greengenes2 database was used to annotate ASV at the kingdom, phylum, class, order, family, genus, and species levels to obtain the species composition and abundance information of the bacterial and archaeal communities in the samples.

[0085] Bacterial flora structure detection:

[0086] The bacterial community structure was detected by 16S rRNA amplicon sequencing. The V4 region of the 16SrRNA gene was selected for amplification of the bacterial community structure of the pit mud and enriched bacterial liquid. The amplification primers were 515modF (5'-GTGYCAGCMGCCGCGGTAA-3') and 806modR (5'-GGACTACNVGGGTWTCTAAT-3'). The amplified products were quality checked and the library was built. The sequencing was performed on the Illumina NovaseqPE250 platform. The original offline data was quality controlled using Trimmomatic to remove low-quality sequences. The sequence files after quality filtering were imported into Qiime2, and the DADA2 plug-in was used for denoising, splicing and clustering to obtain amplicon sequence variants. The clustering plug-in was further used to cluster 99% to obtain 99% ASV, and the 99% ASV was classified and annotated based on the Silva 138 database.

[0087] Functional microbial species detection:

[0088] The identification of functional microbial species in the medium- and short-chain fatty acid-degrading bacterial community was carried out using the third-generation metagenomic sequencing technology of Oxford Nanopore. Genome sequencing was performed on the Oxford Nanopore GridION X5 sequencing platform. The original offline data was removed from the machine using PoreChop (https: / / github.com / rrwick / Porechop), cut and filtered using NanoFilt, and the sequence after quality control was assembled using flye software and -meta parameters to obtain contig files. The contigs were binned using MetaBat2, MaxBin2, Semibin, Semibin2, and Metadecoder to obtain bins. The bins obtained by binning were modified and deduplicated using MetaWRAP and dRep to obtain a non-redundant draft genome (MAG), with a quality threshold of completeness ≥ 50% and contamination < 10%. The draft genome was aligned to the GTDB database using GTDB-Tk based on 120 bacterial marker genes and 122 archaeal marker genes to obtain species annotation results, and a phylogenetic tree was constructed based on the result file. The draft genome sequence was aligned to the quality-controlled sequence file using CoverM (https: / / github.com / wwood / CoverM) to obtain its abundance in each sample.

[0089] The bottom pot water involved in the following embodiments is: the bottom pot water is collected during the production process of a certain winery, and the treatment method is: the bottom pot water is diluted with 1 volume of non-sterile water, the pH is adjusted to 6.0, and nitrogen is blown for 20 minutes and then placed in an anaerobic box for use.

[0090] Example 1: Enrichment and stabilization culture of stable medium- and short-chain fatty acid-degrading bacterial flora

[0091] This embodiment provides a method for enriching and stabilizing the culture of short-chain fatty acid-degrading bacteria in pit mud, and the specific method includes the following contents:

[0092] (1) Selection of pit mud samples:

[0093] According to the following standards, cellar mud samples 1, 2, 3 and 4 with a cellar age of 5 years or more and containing a certain abundance of Syntrophomonas, Syntrophaceticus and various trophic methanogens were selected. Sample 1 came from a Luzhou-flavor liquor factory in Sichuan Province, sample 2 came from a Luzhou-flavor liquor factory in Jiangsu Province, sample 3 came from a Luzhou-flavor liquor factory in Anhui Province, and sample 4 came from a Luzhou-flavor liquor factory in Jiangsu Province.

[0094] Selection criteria: The cellar mud was selected based on the relative abundance of Syntrophomonas, Syntrophic acetic bacteria and methanogens in the cellar mud, among which the relative abundance of Syntrophomonas should be ≥1.0%, the relative abundance of Syntrophaceticus should be ≥0.1%, and the relative abundance of Methanogenus should be ≥10%.

[0095] The relative abundance of syntrophic monocytogenes, acetic acid syntrophic bacteria and methanogens in each pit mud is shown in Table 1. About 100 g of pit mud was sampled using an anaerobic bag and placed in a -20 °C refrigerator for later use, and the bacterial community structure analysis and inoculation culture were performed later.

[0096] Table 1: Relative abundance of syntrophic bacteria, acetic acid syntrophic bacteria and methanogens in samples 1, 2, 3 and 4

[0097]

[0098] (2) Culture medium preparation:

[0099] 1) For the culture medium of sample 1 and sample 2:

[0100] First, prepare the enrichment medium except B vitamin solution and cysteine ​​solution: caproic acid 5g / L, acetic acid 2g / L, formic acid 1.4g / L, yeast powder 1.0g / L, peptone 1.0g / L, mineral element solution 50mL / L, trace element solution 1mL / L, B vitamin solution 5mL, pH 7.3; mineral element solution (g / L): KH2PO4 10, MgCl2·6H2O 6.6, NaCl8, NH4Cl 8, CaCl2 1, pH 7.4; trace element solution (g / L): ZnSO4·7H2O 0.1, MnCl2 0.03, H3BO30.3, CoCl2·6H2O 0.2, CaCl2 0.01, NiCl2·6H2O 0.02, Na2MoO4 0.03, FeCl2 4H2O 1.5;

[0101] Secondly, before sterilization, adjust the pH to 7.3 with 5 mol / L NaOH solution, sterilize at 121°C for 20 min, place in an anaerobic box after sterilization, add 5 mL of B vitamin solution with a sterile 0.2 μm membrane and 10 mL of 12.5 g / L cysteine ​​hydrochloride solution after cooling, and deoxygenate for more than 48 hours for use. B vitamin solution (mg / L): 20 niacin, 20 cobalamin, 10 thiamine hydrochloride, 10 p-aminobenzoic acid, 50 pyridoxine hydrochloride, 5 calcium pantothenate. Prepare the culture medium.

[0102] 2) For the culture medium of sample 3 and sample 4:

[0103] The culture medium used for the culture is the same as the culture medium components and preparation method involved in Sample 1 and Sample 2, except that the caproic acid concentration is adjusted to 8 g / L.

[0104] (3) Bacterial flora culture:

[0105] 70 mL of the culture medium obtained in step (2) was dispensed into 100 mL saline bottles, and the selected cellar muds were respectively thawed in anaerobic boxes, and the thawed cellar muds were inoculated into the above-mentioned corresponding culture media at an inoculum amount of 10% (m / v), and anaerobically cultured at 37°C. The cellar muds of samples 1 and 4 were cultured anaerobically for 28 days, the cellar mud of sample 2 was cultured anaerobically for 24 days, and the cellar mud of sample 3 was cultured anaerobically for 16 days; medium- and short-chain fatty acid-degrading bacterial communities derived from cellar muds were respectively prepared; the proportion of methane in the headspace was regularly detected, and after the experiment, samples were taken to detect the content of medium- and short-chain fatty acids in the bacterial communities.

[0106] (4) Experimental results:

[0107] 1) Changes of medium- and short-chain fatty acids and methane during the enrichment of medium- and short-chain fatty acid-degrading bacteria Figure 1 As shown, the results show:

[0108] The medium- and short-chain fatty acid-degrading bacterial community from sample 1 was able to completely degrade the main fatty acid caproic acid (average 4.03 g / L) in the substrate and most of the acetic acid (average 1.69 g / L) through 28 days of enrichment culture, and ultimately produce methane.

[0109] The medium- and short-chain fatty acid-degrading bacterial flora from sample 2 were able to completely degrade the main fatty acid caproic acid (average 5.07 g / L) and most of the acetic acid (average 2.02 g / L) in the substrate through 24 days of enrichment culture, and ultimately produce methane.

[0110] The medium- and short-chain fatty acid-degrading bacterial flora from sample 3, after 16 days of enrichment culture, was able to completely degrade the main fatty acid caproic acid (average 7.70 g / L) in the substrate, and mostly degrade acetic acid (average 1.24 g / L), and ultimately produce methane.

[0111] The medium- and short-chain fatty acid-degrading bacterial community from sample 4, after 28 days of enrichment culture, was able to completely degrade the main fatty acid caproic acid (average 8.76 g / L) in the substrate, and mostly degrade acetic acid (average 1.11 g / L), and ultimately produce methane.

[0112] 2) In the microbial community during the enrichment of medium- and short-chain fatty acid-degrading bacteria in pit mud, the composition of the main acid-reducing functional microorganisms is shown in Tables 2 to 5.

[0113] Table 2: Changes in the community level of the main acid-reducing functional microorganisms during the enrichment of medium- and short-chain fatty acid-degrading bacteria from test sample 1

[0114]

[0115] Table 3: Changes in the community level of the main acid-reducing microorganisms during the enrichment of medium- and short-chain fatty acid-degrading bacteria from test sample 2

[0116]

[0117] Table 4: Changes in the community level of the main acid-reducing microorganisms during the enrichment of medium- and short-chain fatty acid-degrading bacteria from test sample 3

[0118]

[0119]

[0120] Table 5: Changes in the community level of the main acid-reducing microorganisms during the enrichment of medium- and short-chain fatty acid-degrading bacteria from test sample 4

[0121]

[0122] The results showed that the acid-reducing bacteria in the enriched bacterial community included Syntrophomonas and Syntrophaceticus, and the archaea were mainly Methanosarcina, Methanoculleus and Methanobacterium. Syntrophomonas degraded caproic acid into acetic acid, Syntrophaceticus oxidized acetic acid into CO2 and H2, Methanosarcina converted acetic acid into methane, and the remaining archaea were all hydrogenotrophic methanogens.

[0123] On the 28th day, the main acid-reducing microorganisms in the medium- and short-chain fatty acid-degrading bacterial community from sample 1 were Synechocystis (9.43%), Methanosarcina (12.76%), Methanosaccharomyces (15.36%), and Methanobacterium (2.34%).

[0124] On the 24th day, the main acid-reducing microorganisms of the medium- and short-chain fatty acid-degrading bacterial community from sample 2 were Synechocystis (10.71%), Methanosarcina (17.16%), and Methanosaccharomyces (13.74%).

[0125] On the 16th day, the main acid-reducing microorganisms of the medium- and short-chain fatty acid-degrading bacterial community from sample 3 were Synechocystis (14.81%), Methanosarcina (0.87%), and Methanobacterium (3.03%).

[0126] On the 28th day, the main acid-reducing microorganisms of the medium- and short-chain fatty acid-degrading bacterial community from sample 4 were Synechocystis (19.56%), Synechocystis aceticum (4.59%), Methanosarcina (5.35%), Methanosaccharomyces (1.94%), and Methanobacterium (7.85%).

[0127] 3) The types of acid-reducing microorganisms in the process of enriching medium- and short-chain fatty acid-degrading bacteria are shown in Table 6:

[0128] Table 6: Functional microbial species in the enrichment process of medium- and short-chain fatty acid-degrading bacteria from samples 1 and 2 based on nanopore sequencing

[0129]

[0130]

[0131] The results showed that the genome drafts of 6 Syntrophomonadaceae microorganisms related to hexanoate degradation and 16 methanogenic microorganisms were obtained by metagenomic sequencing technology. Syntrophomonadaceae included 5 uncultured microorganisms and 1 pure cultured Syntrophomonas methylbutyratica, accounting for about 0.4% to 20.7% of the total abundance of Syntrophomonasaceae; methanogenic microorganisms included 15 uncultured microorganisms and 1 pure cultured Methanoculleus bourgensis. Metagenomic analysis showed that except for Syntrophomonasmethylbutyratica and Methanoculleus bourgensis, all the species enriched by this technology were new species, and they could be enriched using different pit mud samples.

[0132] It should be noted that microorganisms with a certain phylogenetic relationship often have similar metabolic characteristics. For example, species of the family Syntrophomonas have the characteristics of degrading medium and short-chain fatty acids. Although the abundance of species of the family Syntrophomonas that can be enriched by different samples in the present invention is not exactly the same, the enrichment system can degrade caproic acid. This technology can achieve effective enrichment and cultivation of different species of the same family, and effectively degrade caproic acid.

[0133] Example 2: Application of stable medium- and short-chain fatty acid-degrading bacterial flora in stabilization and anti-interference test of hexanoic acid degradation

[0134] In this example, the stability of the medium- and short-chain fatty acid-degrading bacterial flora obtained by the enrichment method of the present invention in multiple batches of fermentation was investigated, and lactic acid, ethanol, and glucose were added for intervention to test the effects of three common carbon sources of brewing wastewater, lactic acid, ethanol, and glucose, on the stability of the caproic acid degradation system and the tolerance concentration ( Figures 3-4 ).

[0135] The specific methods include the following:

[0136] 1. Batch fermentation method for hexanoic acid degradation:

[0137] (1) According to the method of Example 1, sample 1 and sample 2 in Example 1 were selected as sources to prepare medium-chain fatty acid-degrading bacterial flora and short-chain fatty acid-degrading bacterial flora, respectively, specifically:

[0138] Medium preparation:

[0139] Prepare enrichment culture medium except B vitamin solution and cysteine ​​solution, specifically: caproic acid 5-8 g / L, acetic acid 5 g / L, formic acid 1.4 g / L, yeast powder 1.0 g / L, peptone 1.0 g / L, mineral element solution 50 mL / L, trace element solution 1 mL / L, pH 7.3; mineral element solution (g / L): KH2PO4 10, MgCl2·6H2O 6.6, NaCl 8, NH4 Cl 8, CaCl21, pH 7.4; trace element solution (g / L): ZnSO4·7H2O 0.1, MnCl2 0.03, H3BO3 0.3, CoCl2·6H2O0.2, CaCl2 0.01, NiCl2·6H2O 0.02, Na2MoO4 0.03, FeCl2 4H2O 1.5;

[0140] Before sterilization, adjust the pH to 7.3 with 5mol / L NaOH solution, sterilize at 121℃ for 20min, place in an anaerobic box after sterilization, add 10mL of B vitamin solution with a sterile 0.2μm membrane and 12.5g / L cysteine ​​hydrochloride solution after cooling, deoxygenate for more than 48h for use. B vitamin solution (mg / L): 20 niacin, 20 cobalamin, 10 thiamine hydrochloride, 10 p-aminobenzoic acid, 50 pyridoxine hydrochloride, 5 calcium pantothenate; prepare the culture medium.

[0141] (2) Bacterial flora culture:

[0142] 70 mL of the culture medium obtained in step (1) was dispensed into 100 mL salt water bottles, the cellar mud of sample 1 and the cellar mud of sample 2 in Example 1 were placed in an anaerobic box for thawing, the thawed cellar mud was inoculated into the above culture medium at an inoculum amount of 10% (m / v), and the culture was statically cultured at 37° C. for 28 days to prepare the short-chain fatty acid degrading bacterial flora of sample 1 and the short-chain fatty acid degrading bacterial flora of sample 2, respectively;

[0143] (3) Continuous subculture (the end of each generation is the starting point of the next generation)

[0144] 40 mL of the medium- and short-chain fatty acid-degrading bacterial flora (C0) obtained in step (2) were mixed and fermented with 40 mL of fresh subculture medium, that is, the medium- and short-chain fatty acid-degrading bacterial flora of sample 1 and the medium- and short-chain fatty acid-degrading bacterial flora of sample 2 obtained in step (2) were respectively inoculated into the subculture medium at an inoculation amount of 50% (v / v), and anaerobically fermented at 37° C. for 3 days to obtain the first-generation enriched culture fluid C1;

[0145] In order to make the nutrient concentration in the bacterial community after subculture consistent with that in the initial enrichment medium, the other components of the subculture medium except caproic acid and acetic acid are twice that of the enrichment medium. The caproic acid concentration can be set at 5-20 g / L. 12 g / L is used in this embodiment. Since some acetic acid remains in the primary medium- and short-chain fatty acid-degrading bacterial community, acetic acid is no longer added to the subculture medium.

[0146] Subculture medium ( / L): caproic acid 12g, formic acid 2.8g, yeast powder 2.0g, peptone 2.0g, mineral element solution 100mL, trace element solution 2mL, B vitamin solution 10mL, cysteine ​​hydrochloride solution 20mL; wherein, mineral element solution (g / L): KH2PO4 10, MgCl2·6H2O 6.6, NaCl 8, NH4 Cl 8, CaCl2 1, pH is 7.4; trace element solution (g / L): ZnSO4·7H2O 0.1, MnCl2 0.03, H3BO3 0.3, CoCl2·6H2O 0.2, CaCl2 0.01, NiCl2·6H2O 0.02, Na2MoO4 0.03, FeCl2 4H2O 1.5; B vitamin solution (mg / L): niacin 20, cobalamin 20, thiamine hydrochloride 10, p-aminobenzoic acid 10, pyridoxine hydrochloride 50, calcium pantothenate 5;

[0147] The first generation enriched culture fluid C1 was inoculated into the fresh subculture medium at a rate of 50% (v / v); the culture fluid was fermented anaerobically at 37° C. for 10 days to obtain the second generation enriched culture fluid C2;

[0148] The second generation enriched culture fluid C2 was inoculated into the fresh subculture medium at a rate of 50% (v / v); the culture fluid was fermented anaerobically at 37° C. for 10 days to obtain the third generation enriched culture fluid C3;

[0149] According to the above enrichment culture method, the inoculation amount and the culture temperature remain unchanged, and the fourth generation enrichment culture fluid C4 (fermentation for 7 days), the fifth generation enrichment culture fluid C5 (fermentation for 7 days), the sixth generation enrichment culture fluid C6 (fermentation for 7 days), the seventh generation enrichment culture fluid C7 (fermentation for 6 days), the eighth generation enrichment culture fluid C8 (fermentation for 6 days), the ninth generation enrichment culture fluid C9 (fermentation for 6 days), the tenth generation enrichment culture fluid C10 (fermentation for 7 days), the eleventh generation enrichment culture fluid C11 (fermentation for 10 days), the twelfth generation enrichment culture fluid C12 (fermentation for 7 days), the thirteenth generation enrichment culture fluid C13 (fermentation for 7 days), the fourteenth generation enrichment culture fluid C14 (fermentation for 7 days), the fifteenth generation enrichment culture fluid C15 (fermentation for 7 days), the sixteenth generation enrichment culture fluid C16 (fermentation for 7 days), and the seventeenth generation enrichment culture fluid C17 (fermentation for 7 days) are obtained respectively. , the 18th generation enriched culture broth C18 (fermentation for 7 days), the 19th generation enriched culture broth C19 (fermentation for 10 days), the 20th generation enriched culture broth C20 (fermentation for 10 days), the 21st generation enriched culture broth C21 (fermentation for 10 days), the 22nd generation enriched culture broth C22 (fermentation for 8 days), the 23rd generation enriched culture broth C23 (fermentation for 8 days), the 24th generation enriched culture broth C24 (fermentation for 8 days), the 25th generation enriched culture broth C25 (fermentation for 8 days), the 26th generation enriched culture broth C26 (fermentation for 8 days), the 27th generation enriched culture broth C27 (fermentation for 8 days), the 28th generation enriched culture broth C28 (fermentation for 8 days), the 29th generation enriched culture broth C29 (fermentation for 8 days), the 30th generation enriched culture broth C30 (fermentation for 8 days), and the 31st generation enriched culture broth C31 (fermentation for 8 days).

[0150] After each subculture fermentation, the contents of caproic acid, butyric acid and acetic acid in the enriched culture fluid of each bacterial colony were tested. The results were as follows: Figure 2 shown.

[0151] The results showed that at the end of fermentation, the contents of caproic acid and butyric acid in the third generation enriched culture fluid C3 to the thirty-first generation enriched culture fluid C31 obtained by culture were almost 0, and the content of acetic acid was ≤3 g / L, which proved that the enriched culture fluid obtained by the method of the present invention has the stability of succession. The contents of caproic acid, butyric acid and acetic acid at the starting point and the end point in the enriched culture fluids of C0, C1, C2, C3, C4, C10, C16, C21, C26, C30 and C31 are listed in the table, as shown in Table 7:

[0152] Table 7: Contents of caproic acid, butyric acid and acetic acid in the starting and ending points of each generation of enriched culture medium

[0153]

[0154]

[0155] 2. Fermentation method with exogenous addition of lactic acid, ethanol and glucose:

[0156] In order to explore the effects of the main organic matter in cellar mud and liquor brewing wastewater on the metabolism of enriched bacteria and microbial communities, 10 g / L lactic acid, 6 g / L ethanol and 6 g / L glucose were additionally added during the preparation of the subculture culture medium. 40 mL of medium- and short-chain fatty acid-degrading bacteria were mixed and fermented with 40 mL of the three culture media, and samples were taken regularly to detect changes in fatty acid content.

[0157] Specifically:

[0158] The 22nd, 23rd, and 24th generations of medium- and short-chain fatty acid-degrading bacteria (C22, C23, and C24) of sample 1 obtained in step (2) of step 1 were inoculated at an inoculum of 50% (v / v) into a subculture medium supplemented with 10 g / L lactic acid, 6 g / L ethanol, or 6 g / L glucose, respectively, so that the final concentration of lactic acid in the subculture medium was 5 g / L, the final concentration of ethanol was 3 g / L, and the final concentration of glucose was 3 g / L;

[0159] Subculture medium ( / L): lactic acid / ethanol / glucose, caproic acid 5g, formic acid 2.8g, yeast powder 2.0g, peptone 2.0g, mineral element solution 100mL, trace element solution 2mL, B vitamin solution 10mL, cysteine ​​hydrochloride solution 20mL; wherein, mineral element solution (g / L): KH2PO4 10, MgCl2·6H2O 6.6, NaCl 8, NH4 Cl 8, CaCl21, pH is 7.4; trace element solution (g / L): ZnSO4·7H2O 0.1, MnCl2 0.03, H3BO3 0.3, CoCl2·6H2O0.2, CaCl2 0.01, NiCl2·6H2O 0.02, Na2MoO4 0.03, FeCl2 4H2O 1.5; B vitamin solution (mg / L): niacin 20, cobalamin 20, thiamine hydrochloride 10, p-aminobenzoic acid 10, pyridoxine hydrochloride 50, calcium pantothenate 5;

[0160] The results are as follows:

[0161] (1) The C24 generation bacterial colony was fermented anaerobically at 37°C for 15 days in a subculture medium supplemented with glucose, and the glucose content during the fermentation process was measured. The results are shown in Table 8.

[0162] Table 8: Changes in glucose and caproic acid concentrations during subculture with additional glucose addition

[0163]

[0164]

[0165] (2) The C22 generation bacterial colony was fermented anaerobically at 37°C for 14 days in the culture medium supplemented with lactic acid, and the lactic acid content during the fermentation process was measured. The results are shown in Table 9.

[0166] Table 9: Changes in lactic acid and caproic acid concentrations during subculture with additional lactic acid

[0167]

[0168] (3) The C23 generation bacterial colony was fermented anaerobically at 37°C for 14 days in the culture medium supplemented with ethanol, and the ethanol content during the fermentation process was measured. The results are shown in Table 10.

[0169] Table 10: Changes in ethanol and caproic acid concentrations during subculture with additional ethanol addition

[0170]

[0171] The results showed that in a culture medium containing lactic acid / glucose / lactic acid, the main organic substances in cellar mud and liquor brewing wastewater, the medium- and short-chain fatty acid-degrading bacterial community obtained by the culture method of the present invention was able to resist the influence of low concentrations of glucose (<3g / L), lactic acid (≤5g / L), and ethanol (<3g / L) to maintain basic hexanoic acid degradation ability, further demonstrating the stability of the enriched bacterial community obtained by the present invention.

[0172] 3. Bacterial flora structure detection:

[0173] The bacterial community results in the C0-C31 enriched culture fluids prepared during the batch fermentation of the medium- and short-chain fatty acid degrading bacterial community in step 1 were detected respectively, and the results are shown in Tables 11 and 12. The medium- and short-chain fatty acid degrading bacterial community obtained by this method is mainly composed of the genera of Syntrophomonas, Methanosarcina, and Methanosaccharomyces in C0-C10, and Syntrophomonas, Syntrophobacter, Methanosaccharomyces, and Methanobacterium in C11-C31. The similarity of the structures of the two medium- and short-chain fatty acid degrading bacterial communities gradually increases with the continuous feeding, indicating that the structure of the enriched bacterial community obtained by this method is stable. The relative abundances of Syntrophomonas, Syntrophaceticus, Methanosarcina, Methanoculleus, and Methanobacterium at the end points in the enriched culture media of C0, C1, C2, C3, C4, C10, C16, C21, C26, C30, and C31 are listed in the table, respectively.

[0174] Table 11: Relative abundance (%) of some functional microbial genus levels during serial passage of Sample 1.

[0175]

[0176] Table 12: Relative abundance (%) of some functional microbial genus levels during serial passage of Sample 2.

[0177]

[0178] The ASV sequences of Syntrophomonas in C0-C10 were aligned to the NCBI database, and during the fed-batch fermentation process, Syntrophomonas methylbutyratica gradually became dominant, accounting for a maximum of 13.29% of the total microbial abundance and 91.33% of the abundance of Syntrophomonas, indicating that the present invention can obtain a stable medium- and short-chain fatty acid-degrading bacterial community derived from cellar mud with Syntrophomonas methylbutyratica as the main caproic acid-degrading functional bacteria.

[0179] The above results based on artificial culture medium show that the caproic acid-degrading bacterial flora cultivation method of the present invention can achieve the semi-continuous fermentation preparation of medium- and short-chain fatty acid-degrading bacterial flora.

[0180] Example 3: Preparation of "hexanoic acid degradation-methanogenic bacteria" based on the green stabilization of medium- and short-chain fatty acid-degrading bacteria and biphasic anaerobic digestion process for boiler water treatment

[0181] In this embodiment, the bottom pot water, a byproduct of liquor brewing, is used to prepare the "hexanoic acid degradation-methanogenic bacteria group" in a green way through a two-phase anaerobic fermentation process. At the same time, the two-phase anaerobic fermentation process can also be applied to the anaerobic digestion treatment of bottom pot water.

[0182] The specific methods include the following:

[0183] (1) Cultivation of stable medium- and short-chain fatty acid-degrading bacterial flora:

[0184] The continuous cultivation of the stable medium- and short-chain fatty acid-degrading bacterial flora was carried out according to the method in Example 2. That is, according to the method in Example 2, the 30th generation enriched culture fluid C30 (sample 1) was prepared.

[0185] (2) Cultivation of acid-producing microorganisms in pit mud:

[0186] Select pit mud that is over 15 years old, has a caproic acid concentration of >6g / kg and a lactic acid concentration of <30g / kg as the seed source, and obtain the seed liquid of the acid-producing bacteria group that can produce caproic acid using glucose and lactic acid through multiple subcultures; the specific steps are:

[0187] 1) Culture medium preparation:

[0188] Prepare glucose-lactate enriched medium, specifically ( / L):

[0189] Glucose 10.0g, sodium lactate 30mL, sodium acetate 5g, ammonium sulfate 2.0g, dipotassium hydrogen phosphate 1.0g, potassium dihydrogen phosphate 0.5g, peptone 10.0g, yeast powder 10.0g, magnesium sulfate heptahydrate 0.1g, ferrous sulfate heptahydrate 0.15g, calcium chloride 0.01g, manganese sulfate 0.01g, cobalt chloride hexahydrate 0.002g, zinc sulfate 0.002g;

[0190] 2) Bacterial culture:

[0191] 70 mL of the culture medium obtained in step 1) was dispensed into 100 mL salt water bottles, and the pit mud was inoculated into the culture medium at a 10% (m / v) inoculation rate, and anaerobically cultured at 37° C. for 3 days to prepare an initial enrichment culture solution F0;

[0192] The enriched culture fluid F0 was transferred into fresh glucose-milk enriched medium at an inoculum volume of 10% (v / v), and the first generation enriched culture fluid F1 was obtained after anaerobic culture at 37°C for 2 days;

[0193] The enriched culture fluid F1 was transferred into a fresh glucose-lactide enriched medium at an inoculation rate of 10% (v / v), and the second-generation enriched culture fluid F2 was obtained after anaerobic culture at 37°C for 2 days to prepare a seed solution of acid-producing bacteria. The caproic acid production in the seed solution was detected; the results showed that the caproic acid production at the end of the F2 generation was about 11.25 g / L.

[0194] (3) Acid-producing fermentation of the bottom pot water:

[0195] The test showed that the lactic acid concentration in the bottom pot water was about 20g / L.

[0196] The acid-producing bacteria seed liquid prepared in step (2) was inoculated into the bottom pot water (50% dilution with deionized water, pH adjusted to 6, deoxygenated) at a ratio of 10%, and cultured anaerobically at 37°C for 7 days. Samples were taken every day to detect the concentrations of caproic acid and lactic acid, and the acid-producing system was obtained on the 7th day of culture. The concentrations of caproic acid and lactic acid in the acid-producing system were 2.245 g / L and 0 g / L, respectively.

[0197] (4) Fermentation of caproic acid-degrading bacteria in bottom pot water:

[0198] Experimental group: The 30th generation enrichment culture fluid C30 of the medium- and short-chain fatty acid-degrading bacterial flora obtained in step (1) and the acid-producing system obtained in step (3) were mixed at a volume ratio of 1:1 (experimental group 1), 1:3 (experimental group 2), and 1:9 (experimental group 3), respectively, and then anaerobically cultured at 37° C. for 7 days;

[0199] Control group 1: single culture of medium- and short-chain fatty acid-degrading bacteria: the 30th generation enriched culture fluid C30 obtained in step (1) was anaerobically cultured at 37°C for 7 days;

[0200] Control group 2: The enriched culture fluid C30 of the 30th generation of medium- and short-chain fatty acid-degrading bacteria obtained in step (1) was mixed with bottom pot water (50% diluted with deionized water, adjusted to pH 6, and deoxygenated) at a volume ratio of 1:1, and then anaerobically cultured at 37°C for 7 days;

[0201] Three parallel groups were set up for both the experimental group and the control group.

[0202] The headspace methane content was tested and samples were taken every day, and the samples were stored at -20°C to measure the metabolic phenotype and bacterial community structure.

[0203] The results show:

[0204] 1) The fatty acid content of different groups at different fermentation times is shown in Table 13:

[0205] Table 13: Changes in fatty acids, lactic acid, ethanol and methane during two-phase anaerobic digestion of bottom boiler water.

[0206]

[0207]

[0208] The results of single culture acid-reducing bacteria (control group 1) show that when the medium and short-chain fatty acid degrading bacteria are directly inoculated into the bottom pot water for fermentation, propionic acid will be produced, and the odd-carbon short-chain fatty acids represented by propionic acid are difficult to be further degraded by the acid-reducing bacteria. After the bottom pot water is inoculated with caproic acid-producing microorganisms, 2.245g / L of caproic acid can be produced. After completing the caproic acid fermentation, the acid-reducing bacteria ("caproic acid degradation-methane production" bacteria) are inoculated. When the inoculation amount of the acid-reducing bacteria: the acid-producing bacteria (acid-reducing: acid-producing) is maintained between 1:1 and 1:9, the effective degradation of caproic acid can be achieved, and the ratio of 1:9 is excellent. At this time, the amount of acid-reducing bacteria that needs to be inoculated is relatively low. The double-step fermentation method in the present embodiment is also called a two-phase anaerobic digestion process, which can avoid the pH difference problem in the coupling process of the acid-producing phase and the methane-producing phase, and can directly enter the methane-producing phase to produce methane after the acid-producing phase reacts.

[0209] 2) The bacterial community structure of different groups at different fermentation times is shown in Tables 14 to 19.

[0210] Table 14: Changes in abundance of core functional microbial genera of the community during fermentation of bottom pot water by acid-producing bacteria (acid-producing system in step (3)) over culture time (%)

[0211]

[0212] Table 15: Changes in abundance of core functional microbial genera in the fermentation process of acid-reducing bacteria with culture time (%)

[0213]

[0214] Table 16: Changes in abundance of core functional microbial genera in fatty acid degradation community over culture time (%)

[0215]

[0216]

[0217] Table 17: Changes in abundance of core functional microbial genera in the community over culture time during the 1:1 fermentation of fatty acid degrading bacteria and acid production system (%)

[0218]

[0219] Table 18: Changes in abundance of core functional microbial genera in the community during the fermentation of fatty acid-degrading bacteria and acid-producing system 1:3 with culture time (%)

[0220]

[0221] Table 19: Changes in abundance of core functional microbial genera in the fatty acid degrading bacterial community and the acid production system during the 1:9 fermentation process with culture time (%)

[0222]

[0223] The results showed that the dominant genera after the medium- and short-chain fatty acid-degrading bacteria were mixed with the acid-producing system were Enterobacter and Caproicibacterium in the acid-producing system and Syntrophomonas, Syntrophaceticus and Methanobacterium in the acid-reducing bacteria. The bacterial community results of the three experimental groups showed the same trend during the co-culture process. As the fermentation process progressed, the relative abundance of Syntrophomonas, Syntrophaceticus and Methanobacterium decreased, while the relative abundance of Syntrophomonas, Petrimonas and Proteiniphilum gradually increased, and the total biomass gradually increased with the fermentation process; the dominant microbial genera at the end of the fermentation were Enterobacter, Syntrophaceticus, Proteiniphilum, Syntrophomonas, Petromonas and Methanobacterium. Microbial community analysis showed that the two-phase fermentation process of first fermenting for caproic acid production and then fermenting for caproic acid degradation can use a high proportion of bottom pot water for the green preparation of "caproic acid degradation-methane production" medium- and short-chain fatty acid degrading bacteria.

[0224] Example 4: Preparation of artificial pit mud based on medium- and short-chain fatty acid degrading bacteria

[0225] This embodiment adopts the above-prepared medium- and short-chain fatty acid-degrading bacterial flora cultivation method to prepare artificial pit mud, and the specific method includes the following contents:

[0226] (1) Preparation of seed solution:

[0227] According to Example 2, 500 mL of medium- and short-chain fatty acid-degrading bacterial culture (any one of Sample 1 / Sample 2, and any one of C0-C30 enriched culture fluids) was prepared, inoculated into the subculture medium (Step 1 of Example 2) at a ratio of 50%, and fermented at 37° C. in an anaerobic box, and the generated methane gas was discharged regularly. On the 8th day, the caproic acid and butyric acid were completely consumed, and the acetic acid was at a low level (≤3 g / L).

[0228] (2) Second level amplification:

[0229] The bacterial liquid prepared in step (1) above was pumped into a 3 L fermentation tank containing 1 L of subculture medium (step 1 of Example 2) using nitrogen gas, and fermented at 37° C. with stirring for 8 days, during which the caproic acid and butyric acid were completely consumed, and the acetic acid was at a low level.

[0230] (3) Third level amplification:

[0231] 2 L of the secondary amplified bacterial liquid obtained in step (2) was pumped into a 30 L fermentation tank containing 20 L of subculture medium (bottom pot water (diluted with 50% deionized water, adjusted to pH 6, and deoxygenated with nitrogen) using high-purity nitrogen, and fermented at 37° C. for 14 days. After 9 days, caproic acid and butyric acid were completely consumed, and acetic acid was degraded to a lower level after 14 days.

[0232] (4) Fourth level amplification:

[0233] 20 L of the tertiary amplified bacterial liquid obtained in step (3) was pumped into a 200 L fermentation tank containing 120 L of subculture medium (bottom pot water (diluted with 50% deionized water, adjusted to pH 6, and deoxygenated with nitrogen) using high-purity nitrogen, and fermented at 37° C. for 14 days. After 10 days, caproic acid and butyric acid were completely consumed, and acetic acid was degraded to a lower level after 14 days.

[0234] (5) Artificial pit mud production:

[0235] 500 kg of artificial cellar mud is prepared, the main components of which are: 250 kg of yellow mud, 100 kg of cellar skin mud, 25 kg of old cellar mud, 25 kg of medium-temperature Daqu powder, 5 kg of yeast powder, 5 kg of corn steep liquor, 50 kg of medium- and short-chain fatty acid degrading bacterial flora liquid obtained in step (4), water is added to the mixture to make the water volume 35-40%, the mixture is evenly mixed and then left to stand in a fermentation tank, the room temperature is controlled at 30 DEG C for fermentation, and the fermentation time is 50 days for cellar construction.

[0236] Example 5: Effect of different concentrations of nitrogen sources

[0237] Specific implementation method The same as Example 2, the difference is that the carbon source in the culture medium is adjusted, specifically: pit mud sample 1 is selected as the source to prepare medium- and short-chain fatty acid degrading bacteria, and compared with the original culture medium, the nitrogen source in the culture medium is adjusted to be: 5g / L yeast powder, 5g / L peptone; or 10g / L yeast powder, 10g / L peptone, the initial caproic acid concentration is about 8g / L, and the primary enrichment culture solution (C0) is prepared according to the method of Example 2. The changes in the concentrations of caproic acid, butyric acid, propionic acid, and acetic acid in the culture solution during the enrichment culture were detected, and the results are shown in Tables 20 and 21.

[0238] Table 20: Changes in the concentrations of caproic acid, butyric acid, propionic acid, and acetic acid during the cultivation of primary syntrophic caproic acid-degrading bacterial flora under medium nitrogen source concentration conditions

[0239] time Nitrogen source ratio (yeast powder + peptone) Hexanoic acid(g / L) Butyric acid (g / L) Propionic acid(g / L) Acetic acid (g / L) Day 0 5g / L+5g / L 7.825 0 0 0 4 days 5g / L+5g / L 11.559 1.293 0.615 6.567 8 days 5g / L+5g / L 11.894 1.379 0.775 7.117 12 days 5g / L+5g / L 11.595 1.394 0.827 5.362 16 days 5g / L+5g / L 12.255 1.268 0.763 4.81 22 days 5g / L+5g / L 11.662 0.947 0.689 4.079 26 days 5g / L+5g / L 11.579 0.936 0.707 4.069 30 days 5g / L+5g / L 11.544 0.881 0.706 4.074

[0240] Table 21: Changes in the concentrations of caproic acid, butyric acid, propionic acid and acetic acid during the cultivation of primary syntrophic caproic acid-degrading bacteria under high nitrogen source concentration conditions

[0241] time Nitrogen source ratio (yeast powder + peptone) Hexanoic acid(g / L) Butyric acid (g / L) Propionic acid(g / L) Acetic acid (g / L) Day 0 10g / L+10g / L 9.18 0.048 0.007 0.314 4 days 10g / L+10g / L 8.879 2.582 0.695 9.313 8 days 10g / L+10g / L 9.716 2.971 0.859 9.492 12 days 10g / L+10g / L 10.047 2.941 0.871 6.743 16 days 10g / L+10g / L 10.563 3.142 0.944 7.135 22 days 10g / L+10g / L 10.158 2.342 0.876 6.931 26 days 10g / L+10g / L 9.679 2.345 0.886 6.877 30 days 10g / L+10g / L 9.05 2.428 0.907 6.239

[0242] The results showed that compared with the medium of 1g / L yeast powder + 1g / L peptone nitrogen source, caproic acid showed an upward trend under medium and high nitrogen source concentrations, and only showed a weak trend of caproic acid degradation after 22-26 days, indicating that the caproic acid bacteria in the pit mud grew under high nitrogen source conditions, indicating that the nitrogen source concentration is extremely critical for caproic acid degradation, and both medium and high nitrogen source concentrations are not conducive to the enrichment and stabilization of acid-reducing bacteria. Therefore, a lower single nitrogen source concentration (2g / L) is a necessary condition for enriching caproic acid-degrading bacteria.

[0243] Example 6: Effects of different passage doses

[0244] The specific implementation method is the same as Example 2, except that the inoculation amount during the subculture process is adjusted, specifically: Sample 4 is selected as the source to prepare the medium- and short-chain fatty acid degrading bacteria, and the inoculation amount of the subculture is 10% (v / v), and the culture medium is selected as the enriched culture medium. According to the method of Example 2, the second generation enriched culture solution is prepared; the changes in the caproic acid concentration in the final enriched culture solution are respectively detected, as shown in Table 22.

[0245] Table 22: Changes in hexanoic acid concentration during subculture with 10% inoculum

[0246]

[0247] The results showed that compared with the 50% subculture medium, the degradation of caproic acid was slow when the culture medium was used for subculture at a ratio of 10%. Therefore, the technical solution of the present invention can also be realized with an inoculation amount of 10% to 50%. However, the use of the subculture medium at an inoculation amount of 50% is a better subculture strategy for obtaining a stable medium- and short-chain fatty acid-degrading bacterial community.

[0248] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for culturing a stable medium- and short-chain fatty acid-degrading bacterial community derived from pit mud or a method for enriching a bacterial community containing Syntrophomonas, characterized in that: The method comprises: inoculating pit mud into an enrichment medium for enrichment culture, and then inoculating the obtained seed liquid into a subculture medium for step-by-step expansion culture; the expansion culture time of each level of seed liquid is 3 to 10 days, preferably 7 to 10 days; The enrichment medium contains: 5-8 g / L caproic acid, 3-6 g / L acetic acid, 1-2 g / L formic acid, 0.8-1.2 g / L yeast powder, 0.8-1.2 g / L peptone, 45-55 mL / L mineral element solution, 0.8-1.2 mL / L trace element solution, 4-6 mL / L B vitamin solution, 8-12 mL / L cysteine ​​hydrochloride solution, and a pH of 7.2-7.4; The subculture medium contains: 10-20 g / L caproic acid, 1-3 g / L formic acid, 1.8-2.2 g / L yeast powder, 1.8-2.2 g / L peptone, 80-100 mL / L mineral element solution, 1.8-2.0 mL / L trace element solution, 8-12 mL / L B vitamin solution, 18-22 mL / L cysteine ​​hydrochloride solution, and the pH is 7.2-7.

4.

2. The method according to claim 1, characterized in that The seed solution is inoculated into the subculture medium at an inoculation rate of 10% to 50% (v / v); Preferably, the mineral element solution (g / L): KH2PO4 10, MgCl2·6H2O 6.6, NaCl 8, NH4Cl 8, CaCl2 1, pH 7.4; trace element solution (g / L): ZnSO4·7H2O 0.1, MnCl2 0.03, H3BO3 0.3, CoCl2·6H2O0.2, CaCl2 0.01, NiCl2·6H2O 0.02, Na2MoO4 0.03, FeCl2 4H2O 1.5; the cysteine ​​solution contains: 12.5 g cysteine ​​hydrochloride per liter; Preferably, the step-by-step expansion culture is expansion culture of more than one level; Preferably, the inoculation amount of the cellar mud is 8-12%, and the cellar mud includes cellar bottom mud with a cellar age of not less than 5 years and a pH value of 7.2-7.4; the cellar mud used for inoculation: the cellar mud is selected according to the relative abundance of Syntrophomonas, Syntrophic acetic bacteria and methanogens in the cellar mud, wherein the relative abundance of Syntrophomonas should be ≥1.0%, the relative abundance of Syntrophaceticus should be ≥0.1%, and the relative abundance of Methanogenus should be ≥10%; Preferably, the subculture is carried out under anaerobic conditions; Preferably, the subculture is carried out at 32-37°C; Preferably, the enrichment culture is to inoculate the cellar mud into the enrichment medium and culture it statically at 30-37° C. for 15 to 30 days; preferably, the cellar mud is inoculated into the enrichment medium at an inoculation rate of 10% to 20% (m / v).

3. The stable medium- and short-chain fatty acid-degrading bacterial flora derived from pit mud or the bacterial flora containing Syntrophomonas prepared by the method of claim 1 or 2.

4. Use of the stable medium- and short-chain fatty acid-degrading bacterial flora derived from pit mud or the bacterial flora containing Syntrophomonas as claimed in claim 3 in the continuous degradation of caproic acid, characterized in that: The application is to use the subculture medium to carry out continuous feeding fermentation of the medium- and short-chain fatty acid-degrading bacteria to degrade caproic acid; specifically comprising: inoculating the pit mud into the enrichment medium for enrichment culture, and then inoculating the obtained seed liquid into the subculture medium for step-by-step expansion culture; the expansion culture time of each level of seed liquid is 7 to 10 days; The enrichment medium contains: 5-8 g / L caproic acid, 3-6 g / L acetic acid, 1-2 g / L formic acid, 0.8-1.2 g / L yeast powder, 0.8-1.2 g / L peptone, 45-55 mL / L mineral element solution, 0.8-1.2 mL / L trace element solution, 4-6 mL / L B vitamin solution, 8-12 mL / L cysteine ​​hydrochloride solution, and a pH of 7.2-7.4; The subculture medium contains: 10-20 g / L caproic acid, 1-3 g / L formic acid, 1.8-2.2 g / L yeast powder, 1.8-2.2 g / L peptone, 80-100 mL / L mineral element solution, 1.8-2.0 mL / L trace element solution, 8-12 mL / L B vitamin solution, 18-22 mL / L cysteine ​​hydrochloride solution, and the pH is 7.2-7.

4.

5. The use of the stable medium- and short-chain fatty acid-degrading bacterial flora derived from pit mud or the bacterial flora containing Syntrophomonas as claimed in claim 3 in the treatment of bottom pot water of liquor brewing wastewater, characterized in that: The applications specifically include: (a) Preparation of acid-producing microbial flora of pit mud: Selecting pit mud that is over 15 years old, has a caproic acid concentration of >6 g / kg and a lactic acid concentration of <30 g / kg, and inoculating the pit mud into a composite culture medium for step-by-step expansion culture; the expansion time of each level of seed liquid is 1 to 3 days; preferably, the expansion culture is 2-4 levels; Composite culture medium / L: glucose 10.0-12.0 g, sodium lactate 25-35 mL, sodium acetate 4-6 g, ammonium sulfate 1.9-2.1 g, dipotassium hydrogen phosphate 0.9-1.1 g, potassium dihydrogen phosphate 0.4-0.6 g, peptone 8.0-12.0 g, yeast powder 8.0-12.0 g, magnesium sulfate heptahydrate 0.1-0.15 g, ferrous sulfate heptahydrate 0.12-0.15 g, calcium chloride 0.008-0.012 g, manganese sulfate 0.008-0.012 g, cobalt chloride hexahydrate 0.0018-0.0022 g, zinc sulfate 0.0018-0.0022 g; (b) Acidogenic fermentation of the bottom pot water: The acid-producing microbial flora of the pit mud obtained in step (a) is inoculated into the bottom pot water for culturing to obtain an acid-producing system; (c) Fermentation of hexanoic acid-degrading bacteria in bottom water: The stable medium- and short-chain fatty acid-degrading bacterial flora derived from pit mud or the bacterial flora containing Syntrophomonas as described in claim 3 is added to the acid-producing system obtained in step (b) to treat the bottom pot water.

6. The use according to claim 5, characterized in that: In step (b): the bottom pot water is treated by diluting it with 50% water, adjusting the pH to 6, and then deoxygenating it; Preferably, in step (b), the cellar mud acid-producing microbial flora is inoculated into the bottom pot water at an inoculation rate of 5-10%; Preferably, in step (b), the culture is carried out under anaerobic conditions, further, at 32-37° C., and the culture time is: 7 to 10 days; Preferably, in step (a), the culturing is carried out under anaerobic conditions, and further, at 32-37° C., and the culturing time is 2 to 5 days.

7. The use according to claim 5 or 6, characterized in that: In step (c): The stable medium- and short-chain fatty acid-degrading bacterial flora or the bacterial flora containing Syntrophomonas from the pit mud and the acid-producing system are mixed in a ratio of 1:1 to 9; Preferably, the treatment is carried out under anaerobic conditions; Preferably, the treatment is carried out at 32-37°C for 7 to 10 days.

8. The method of claim 1 or 2, or the stable medium- and short-chain fatty acid-degrading bacterial flora or bacterial flora containing Syntrophomonas from pit mud of claim 3, or the use of any one of claims 4 to 7 in a liquor brewing system, characterized in that: The application is to apply the bacterial community in the process of liquor fermentation.

9. The use according to claim 8, characterized in that: The application is to apply the bacterial community in a solid-state mash fermentation process without a cellar mud system.

10. A method for cultivating artificial cellar mud or a method for maintaining cellar mud, the method comprising: During the cultivation of artificial pit mud or the maintenance of pit mud, the pit mud-derived stable medium- and short-chain fatty acid-degrading bacterial flora or the bacterial flora containing Syntrophomonas as claimed in claim 3 is added.

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