Clostridium strains and their applications

Abstract

The invention discloses a Clostridium strain and its application. The strain is a Clostridium strain Clostridium sp.LY-ap1, and its deposit number is GDMCC No: 61746. The strain provided by the invention can degrade lignite into small molecular organic matter, and provides a new way for later methanogens to effectively utilize organic matter to produce methane.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a Clostridium strain and an application thereof. Background Art

[0002] Inner Mongolia Autonomous Region is a major coal-producing province, and its proven coal reserves rank second in the country. The coal types in this region are mainly lignite with the lowest degree of coalification. Its high moisture content, high ash content, and low calorific value have greatly hindered its comprehensive utilization. However, the above characteristics of lignite are suitable for microbial degradation and utilization to produce coalbed methane. This process is pollutant-free and environmentally friendly. However, most of the research on this topic at home and abroad is still in the experimental stage, and the microbial flora used are mostly thermophilic bacteria, mostly endogenous methanogenic flora taken from coal seams. The experimental process requires an anaerobic environment with high temperature and high pressure. The environmental conditions required for lignite degradation are harsh, and the operation process is somewhat dangerous; therefore, it is found that the microbial flora that can degrade lignite under simple and controllable conditions, as well as the effective strains in the flora, play an important role in the lignite degradation process. Screening out effective strains, testing their performance, and clarifying the metabolic pathways of the strains are of great significance for the control of lignite degradation. Summary of the invention

[0003] The purpose of the present invention is to degrade lignite into small molecular organic matter, and to provide a new strain of Clostridium and a method for obtaining the same.

[0004] The invention provides a Clostridium sp.LY-ap1 strain of the genus Clostridium, whose deposit number is GDMCC No:61746.

[0005] The colonies of this strain on the culture medium are round, moist, smooth, milky white and opaque, with irregular edges, uniform texture and flat shape; the cells are Gram-positive rods; physiological and biochemical characteristics: this strain grows well in the range of 37-39°C and pH 7.0, is motile and is an obligate anaerobe.

[0006] The strain is deposited in the Guangdong Microbiological Culture Collection Center, located on the 5th floor of the Experimental Building, No. 100, Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province. The deposit name is Clostridium sp.LY-ap1, the taxonomic name is Clostridium sp., the deposit number is GDMCC NO.61746, and the deposit date is June 28, 2021.

[0007] The method for obtaining the above new strain comprises the following steps:

[0008] (1) Sampling: Using the stable methanogenic bacteria that can degrade lignite in the laboratory as the original bacterial liquid, the Clostridium species therein were isolated;

[0009] (2) Preliminary screening: Prepare with deionized water. Add 0.4 g K2HPO4, 1.0 g NH4Cl, 2.0 g MgCl2, 0.5 g cysteine, 0.4 g KH2PO4, 0.2 g Na2S, 2.0 g yeast extract powder, 0.2 g NaHCO3, 2.0 g sodium acetate, 2.0 g NaCl, 0.2 g KCl, 10 mL vitamin solution, and 10.0 mL trace element solution to 1 L deionized water. Na2S, NaHCO3, and cysteine ​​in the culture medium formula act as reducing agents to remove dissolved oxygen in the culture medium. At the same time, boil for 5 minutes to completely remove oxygen from the culture medium. Each 5 ml of culture medium B was dispensed into a Hungate roller tube, sealed and capped, and then sterilized at 121°C for 30 min after vacuuming. After UV sterilization on a clean bench for 30 min, a beaker was prepared and filled with hot water above 50°C and the culture medium in the roller tube was completely immersed in the hot water. The bacterial solution in step 1 was inoculated into the Hungate roller tube at a 5% inoculation rate according to the bacterial solution inoculation, and the tube was quickly rolled to ensure that the culture medium was fully, smoothly and evenly spread on the inner wall of the tube roller, and then placed in a 37°C shaker to observe the growth of the colony.

[0010] (3) Rescreening: Select the roller tube with uniform colony density for strain separation and purification, and inoculate the selected single colony into the culture medium for cultivation;

[0011] (4) When the bacterial solution becomes turbid, repeat steps (2) and (3) until the colonies in the rolling tube have the same morphology;

[0012] (5) Identification: 16S rDNA identification was performed on strains with consistent colony morphology.

[0013] The present invention also provides the use of the above-mentioned strain or its culture solution or fermentation solution or a bacterial agent containing the same in degrading lignite.

[0014] The present invention also provides the use of the above-mentioned strain or its culture solution or fermentation solution or a bacterial agent containing the same in the preparation of a degraded lignite product.

[0015] The present invention also provides a product for degrading lignite, comprising the above-mentioned strain or its culture solution or fermentation solution or a bacterial agent containing the same.

[0016] The bacterial agent may also include a carrier. The carrier may be a solid carrier or a liquid carrier. The solid carrier is a mineral material or a biological material; the mineral material may be at least one of peat, clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica and diatomaceous earth; the biological material may be at least one of various crop straws, pine husks, straw, peanut shells, corn flour, soybean flour, starch, peat and animal feces; the liquid carrier may be water; in the bacterial agent, Pseudomonas rugosa S58 or / and the metabolites of Pseudomonas rugosa S58 may exist in the form of cultured living cells, fermentation broth of living cells, filtrate of cell culture or a mixture of cells and filtrate. The dosage form of the bacterial agent may be a variety of dosage forms, such as liquid, emulsion, suspension, powder, granule, wettable powder or water-dispersible granule.

[0017] As required, a surfactant (such as Tween 20, Tween 80, etc.), a binder, a stabilizer (such as an antioxidant), a pH adjuster, etc. may also be added to the bacterial agent.

[0018] The present invention also provides a method for degrading lignite, comprising adding the above-mentioned Clostridium sp. LY-ap1 to a culture medium containing lignite.

[0019] Optionally, according to the above-mentioned method for degrading lignite, in the culture medium containing lignite, the content of yeast extract powder is 2.00 g / L, the content of dipotassium hydrogen phosphate is 0.40-0.42 g / L, the content of magnesium chloride is 2.0-2.50 g / L, the content of sodium chloride is 1.98-2.00 g / L, the content of potassium chloride is 0.20 g / L, the content of L-cysteine ​​is 0.50 g / L, the content of NH4Cl is 1.00 g, the content of sodium bicarbonate is 0.20-0.26 g / L, the content of sodium sulfide is 0.20 g / L and the content of lignite is 2.00 g / L.

[0020] Optionally, according to the above-mentioned method for degrading lignite, the culture is anaerobic culture.

[0021] Optionally, according to the above-mentioned method for degrading lignite, the culture temperature is 37°C.

[0022] Optionally, according to the above-mentioned method for degrading lignite, the pH of the culture medium containing lignite is 6.5-7.5.

[0023] The strain provided by the invention can degrade lignite into small molecular organic matter, and provides a new way for later methanogens to effectively utilize organic matter to produce methane.

[0024] Collection Instructions

[0025] Bacteria name: Clostridium sp.LY-ap1

[0026] Latin name: Clostridium sp.

[0027] Strain ID: LY-ap1

[0028] Depository: Guangdong Microbial Culture Collection Center

[0029] Depository Abbreviation: GDMCC

[0030] Address: 5th Floor, Building 59, No. 100, Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province

[0031] Date of deposit: June 28, 2021

[0032] Registration number of the depository center: GDMCC No: 61746 DETAILED DESCRIPTION

[0033] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0034] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0035] The formula of trace element solution is: aminoacetic acid 1.5g / L, CaCl2 0.1g / L, MgSO4·7H2O 3.0g / L, H3BO30.05g / L, FeSO4 0.1g / L, NaCl 1.0g / L, CoCl2 0.1g / L, MnSO4 0.5g / L, ZnSO40.1g / L, NaMoO40.05g / L, AlK(SO4)2 0.01g / L, NiCl2 0.1g / L, CuSO4 0.01g / L.

[0036] The formula of vitamin solution is: biotin 2mg / L, nicotinic acid 5mg / L, folic acid 2mg / L, thiamine 5mg / L, pyridoxine hydrochloride 10mg / L, cobalamin 0.1mg / L, riboflavin 5mg / L, para-aminobenzoic acid 5mg / L, pantothenic acid 5mg / L, and lipoic acid 5mg / L.

[0037] Example 1. Acquisition and identification of Clostridium strain LY-ap1

[0038] 1. Obtaining Clostridium strain LY-ap1

[0039] The Clostridium strain LY-ap1 was obtained from the methanogenic anaerobic bacteria in the sludge of the anaerobic digester of Chunhua Wastewater Treatment Plant in Hohhot, Inner Mongolia Autonomous Region in our laboratory.

[0040] The specific acquisition method is as follows:

[0041] (1) Sampling: Using the stable methanogenic bacteria that can degrade lignite in the laboratory as the original bacterial liquid (i.e., the bacterial flora obtained by culturing and domesticating the sample collected above), the Clostridium species therein are isolated;

[0042] (2) Preliminary screening: Prepare the culture medium with deionized water. Add 0.4 g K2HPO4, 1.0 g NH4Cl, 2.0 g MgCl2, 0.5 g cysteine, 0.4 g KH2PO4, 0.2 g Na2S, 2.0 g yeast extract powder, 0.2 g NaHCO3, 2.0 g sodium acetate, 2.0 g NaCl, 0.2 g KCl, 10 mL vitamin solution, and 10.0 mL trace element solution to 1 L deionized water. Na2S, NaHCO3, and cysteine ​​in the culture medium formula act as reducing agents to remove dissolved oxygen in the culture medium. At the same time, boil the culture medium for 5 minutes to completely remove oxygen from the culture medium. Each 5 ml of culture medium is dispensed into a Hungate roller tube, sealed and capped, and after vacuuming, sterilized at 121°C for 30 min; after ultraviolet sterilization on a clean bench for 30 min, prepare a beaker and fill it with hot water above 50°C and completely immerse the culture medium in the roller tube in the hot water; inoculate the original bacterial liquid in step (1) into the Hungate roller tube at an inoculum volume of 5%, roll the tube quickly to ensure that the culture medium is fully, smoothly and evenly spread on the inner wall of the tube roller, place it in a 37°C shaker, and observe the growth of the colony;

[0043] (3) Rescreening: Select the roller tube with uniform colony density for strain separation and purification, and inoculate the selected single colony into the culture medium for cultivation;

[0044] (4) After the bacterial solution becomes turbid, repeat the test contents of steps (2) and (3) until the colonies in the rolling tube have the same morphology, and obtain the strain LY-ap1.

[0045] 2. Identification of strain LY-ap1

[0046] Morphological identification

[0047] The colonies of strain LY-ap1 on the culture medium are round, moist, smooth, milky white and opaque, with irregular edges, uniform texture and flat; the cells are Gram-positive rods. Physiological and biochemical characteristics: The strain grows well at 37-39℃ and pH 7.0, is motile and is an obligate anaerobe.

[0048] 16S rDNA sequence homology analysis

[0049] Primers were selected for amplification of the bacterial 16S rRNA gene, 27F (5'-AGAGTTTGATCCTGGCTCAG-3') (SEQ ID No. 1 in the sequence table) and 1492R (5'-GGTTACCTTGTTACGACTT-3') (SEQ ID No. 2 in the sequence table); the PCR reaction system was 40 μL: Premix Taq 20 μL (purchased from TaKaRa Engineering Co., Ltd.), 20 μM primers 27F and 1492R 2 μL each, 2 μL bacterial solution, supplemented with 14 μL of sterile distilled water, and PCR reaction was performed. The reaction conditions were 94°C 2min, (94°C 30s, 55°C 30s and 72°C 60s), 72°C 5min for 30 cycles. The amplified product was sequenced to obtain the 16S rDNA sequence SEQ ID No. 3 in the sequence list. The obtained 16S rDNA sequence was compared with the NCBI gene library, and it was found that the genus with a genome coverage rate of 100% was Clostridium. Then, an evolutionary tree was constructed and the species with high similarity were given. According to the evolutionary tree, the species with the highest similarity was Clostridium bifermentans.

[0050] Based on the above morphological, physiological and biochemical characteristics analysis and 16s rDNA sequence homology analysis results, strain LY-ap1 was identified as Clostridium sp.

[0051] The registration number of Clostridium strain LY-ap1 in Guangdong Microbiological Culture Collection Center is GDMCC No: 61746, and the preservation name is Clostridium sp.LY-ap1.

[0052] Example 2: Degradation of lignite by Clostridium strain LY-ap1

[0053] 5 mL of the bacterial solution of the Clostridium strain LY-ap1 obtained in Example 1 was inoculated into 50 mL of lignite culture medium, cultured at 37° C. and pH 7.0 under anaerobic conditions for 15 days, and the degradation products were detected.

[0054] Lignite culture medium composition:

[0055] Yeast extract powder 2.00 g / L, dipotassium hydrogen phosphate 0.42 g / L, potassium dihydrogen phosphate 0.42 g / L, magnesium chloride 2.50 g / L, sodium chloride 1.98 g / L, potassium chloride 0.20 g / L, L-cysteine ​​0.50 g / L, NH4Cl 1.00 g, sodium bicarbonate 0.26 g / L, sodium sulfide 0.20 g / L, lignite 2.00 g / L, dilute to 1 L with deionized water, add 10.00 mL of vitamin solution and 10.00 mL of trace element solution, stir thoroughly and adjust pH to 7.00 ± 0.10 with NaOH pellets.

[0056] Degradation product detection method: The products after 5 days (5d) and 15 days (15d) of the above-mentioned anaerobic culture are first filtered to obtain product liquid, then the product liquid is subjected to organic matter extraction, and finally the extracted organic matter is analyzed by liquid chromatography-mass spectrometry to obtain qualitative and quantitative analysis of the products obtained by the bacteria degrading lignite.

[0057] The test results are shown in Table 1. The content of organic matter in samples taken at different stages of the lignite degradation process by LY-ap1 changed significantly and developed towards an unstable structure, indicating that the Clostridium strain LY-ap1 can degrade lignite into small molecular organic matter, providing a new way for later methanogens to effectively utilize organic matter to produce methane.

[0058] Table 1 Organic matter components of lignite at different stages of microbial degradation

[0059]

[0060] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. Clostridium sp. LY-ap1, deposit number is GDMCC No: 61746.

2. Use of the strain according to claim 1 or its culture solution or a bacterial agent containing the same in degrading lignite.

3. Use of the strain according to claim 1 or its culture solution or a bacterial agent containing the strain in the preparation of a degraded lignite product.

4. A product for degrading lignite, characterized in that: The invention comprises the strain according to claim 1 or its culture solution or a bacterial agent containing the same.

5. A method for degrading lignite, characterized in that: The method comprises culturing the Clostridium strain LY-ap1 according to claim 1 in a culture medium containing lignite.

6. The method for degrading lignite according to claim 5, characterized in that: In the culture medium containing lignite, the content of yeast extract powder is 2.00 g / L, the content of dipotassium hydrogen phosphate is 0.40-0.42 g / L, the content of magnesium chloride is 2.0-2.50 g / L, the content of sodium chloride is 1.98-2.00 g / L, the content of potassium chloride is 0.20 g / L, the content of L-cysteine ​​is 0.50 g / L, the content of NH4Cl is 1.00 g / L, the content of sodium bicarbonate is 0.20-0.26 g / L, the content of sodium sulfide is 0.20 g / L and the content of lignite is 2.00 g / L.

7. The method for degrading lignite according to claim 5, characterized in that: The culture is anaerobic culture.

8. The method for degrading lignite according to claim 5, characterized in that: The culture temperature is 37°C.

9. The method for degrading lignite according to claim 5, characterized in that: The pH of the culture medium containing lignite is 6.5-7.5.

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