A strain of Daqinghu golden bacillus and its application

Abstract

The present invention relates to the field of microbial technology, and in particular to a strain of Daqinghu golden bacillus and its application. The strain was deposited in the China Center for Type Culture Collection on January 17, 2022, with a deposit number of CGMCCNO.24314. Experiments have shown that the strain has a significant degradation effect on dichloromethane, and the 24h degradation rate of dichloromethane with an initial concentration of 2000 mg / L is 99.53%, indicating that the strain can effectively remove dichloromethane in the biochemical treatment system without secondary pollution, has significant economic and environmental benefits, and can be widely used in the treatment of wastewater, soil and waste gas containing dichloromethane.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a Daqinghu golden bacillus strain and application thereof. Background Art

[0002] Dichloromethane (DCM) is a widely used organic reagent, often used as an industrial solvent, and applied in the organic synthesis industry and the chemical industry, such as the manufacture of safe film films and polycarbonate. The rest is used as a coating solvent, metal degreasing agent, aerosol spray, polyurethane foaming agent, release agent, and paint stripper. With the acceleration of industrialization and urbanization, the demand for it is also increasing. It is not only a major pollutant in the atmosphere, but also an important component of urban and industrial sewage. Since it is not easily degraded and removed, its half-life in the atmosphere and water system is 70 days and 700 years respectively. More than 90% of the environment comes from the use of chemical industry and the burning of waste; since it is a volatile substance, a large amount of it will escape during its transfer and transportation, causing serious pollution to the atmospheric environment. In addition, the exhaust gas discharged after the combustion of motor vehicle fuel and the volatilization of organic pollutants in surface water bodies will also cause pollution.

[0003] Dichloromethane is a colorless, transparent liquid with an aromatic odor. Dichloromethane can produce highly toxic phosgene when it comes into contact with open flames or hot objects. It can hydrolyze to produce trace amounts of hydrogen chloride when in contact with humid air, and light can also promote hydrolysis and increase its corrosiveness to metals. Dichloromethane is slightly soluble in water and soluble in ethanol and ether. Dichloromethane enters the body through the respiratory tract, gastrointestinal tract, and skin. Once it enters the body, more than 70% of dichloromethane enters the blood circulation and is distributed throughout the body. When dichloromethane enters the body, it will first be metabolized by the cytochrome Paso-dependent mixed function oxidase (MFO). Some dichloromethane will be converted into carcinogens after being activated by the mixed function oxidase in the cell microsomes. They catalyze the keto-enol tautomerism of proteins, thereby causing irreversible changes in cell protein molecules and promoting cell carcinogenesis.

[0004] The treatment of dichloromethane at home and abroad mainly includes combustion, chemical absorption, activated carbon adsorption and biological treatment. Combustion, chemical absorption, activated carbon adsorption and other physical and chemical methods are mainly used to treat industrial waste gas with high dichloromethane content, while low concentrations (<3mg / m 3 ) Dichloromethane, the purification effect is poor, and the energy consumption is large, the cost is high, it is easy to produce secondary pollution, and it is not easy to promote. The biological treatment method is based on the fact that microorganisms have a strong adaptability to dichloromethane pollutants and can use dichloromethane as a metabolic substrate to degrade and transform dichloromethane. It has less investment than traditional processes, lower operating costs, simpler operation, and a wide range of applications.

[0005] The screening of efficient dichloromethane-degrading strains is the key to bioremediation. There are not many patents related to dichloromethane-degrading bacterial agents, and none of them indicate the degradation performance and application promotion of the strains. The screening of efficient degradation strains and the optimization of degradation performance are key factors in bioremediation, which are crucial to the promotion and application of bacterial agents and the remediation of dichloromethane pollution. Summary of the invention

[0006] In view of this, the present invention provides a Daqinghu golden bacillus strain and its application. The strain has a significant degradation effect on dichloromethane, is easy to culture and has stable propagation characteristics.

[0007] The present invention separates and purifies strain YJY22-05 from soil around a wastewater treatment device of a petrochemical company and system sludge. The strain YJY22-05 has the following morphological characteristics: it is a Gram-negative bacterium and forms round golden colonies on ordinary nutrient agar. The colonies are opaque, have irregular edges, are flat, and have a smooth surface.

[0008] The 16SrDNA nucleotide sequence of the strain is shown in SEQ ID NO: 1, which is the complete sequence of the 16SrDNA of the strain; BLAST comparison of the 16SrDNA sequence shows that the nucleotide sequence of the 16SrDNA of the strain has greater than 99% homology with the nucleotide sequences of different strains of the genus Chryseobacterium, and has 100% homology with the strain clearly marked as Chryseobacterium daecheongense.

[0009] The strain YJY22-05 was identified as Chryseobacterium daecheongense by morphology and 16s rDNA, and its deposit number is CGMCC NO.24314.

[0010] The invention also provides a microbial agent, the raw material of which includes Daqinghu golden bacillus with a preservation number of CGMCC NO.24314.

[0011] The present invention also provides a method for preparing the microbial agent, wherein the microbial agent package is a solid agent or a liquid agent.

[0012] The microbial agent is a liquid agent, and the preparation method thereof comprises: activating the Daqinghu golden bacillus with a preservation number of CGMCC NO.24314 to prepare a seed liquid, then inoculating the liquid into a fermentation medium for fermentation, and collecting the fermented bacteria.

[0013] The microbial agent is a solid agent, and its preparation method comprises: activating the Daqinghu golden bacillus with a preservation number of CGMCC NO.24314 to prepare seed liquid, then inoculating it into a fermentation medium for fermentation, collecting the fermented bacteria, and sequentially adsorbing, drying and crushing them.

[0014] The bacterial strain is activated at room temperature for 4 hours to 10 hours.

[0015] The preparation of the seed solution comprises: inoculating the activated bacterial strain into a liquid culture medium, and culturing the culture medium under the conditions of a temperature of 25 to 37° C. and a shaking speed of 50 to 170 r / min for 15 to 20 hours.

[0016] The fermentation comprises the following steps: inoculating Daqinghu golden bacillus seed liquid in a volume percentage of 1%-5% into a fermentation medium for high-density fermentation; and the conditions for the high-density fermentation are: tank pressure of 0.02-0.05MPa, temperature of 25-37°C, rotation speed of 200-300r / min, dissolved oxygen ≧20%, and time of 16-24h.

[0017] The fermentation medium comprises: 1.0%-1.5% glucose, 2.2%-3.8% soluble starch, 0.05%-0.09% magnesium sulfate, 0.1%-0.3% ammonium sulfate, 0.01%-0.03% sodium chloride, 0.5%-1.0% potassium dihydrogen phosphate, 0.005%-0.01% ferrous sulfate, 0.01%-0.05% calcium chloride, 0.05% polyether defoamer, and the balance water, with a pH value of 7.0-7.2.

[0018] In some specific embodiments, the microbial agent is a solid preparation or a liquid preparation, and the preparation method thereof comprises the following steps (1) to (3) or (1) to (4):

[0019] (1) Activation of strains: Take out the Daqinghu golden bacillus strain with the deposit number CGMCCNO.24314 stored at 4°C and activate it at room temperature for 4-10 hours. Since the strain is stored on the slant nutrient agar medium in a test tube;

[0020] (2) Liquid seed preparation: On a sterile operating table, take 10 mL of sterile water and add it to the culture storage tube. Use a pipette to repeatedly blow and beat to prepare a bacterial suspension. Then inoculate the bacterial suspension into 50-500 mL of liquid LB medium and shake and culture for 15-20 h. The culture conditions are: temperature 25-37°C, shaking speed 50-170 rpm;

[0021] (3) High-density fermentation: Inoculate the liquid seeds of Daqinghu golden bacillus at an inoculum rate of 1%-5% by volume into the corresponding fermentation medium for liquid fermentation; fermentation conditions: sterile air to maintain a tank pressure of 0.02-0.05MPa, a temperature of 25-37°C, a rotation speed of 200-300rpm, dissolved oxygen ≧20%, and a culture time of 16-24h to obtain a liquid bacterial agent;

[0022] (4) The liquid bacterial agent obtained in step (3) is adsorbed, dried, and crushed to obtain a solid bacterial agent with an effective viable bacterial count of 10 8 cfu / g or above.

[0023] The solid microbial inoculant prepared by the above method of the present invention has an effective viable count of 10 8 cfu / g or more. The solid microbial agent is activated before use, and the activation is specifically as follows: the solid agent: water is mixed evenly in a mass ratio of 1:3-10, and the activation is stirred in a stirring container for 12-20 hours. Furthermore, the water also includes 2-4% glucose, 0.03-0.06% ammonium sulfate and 0.02-0.05% potassium dihydrogen phosphate.

[0024] The present invention also provides the use of the Daqing Lake golden bacillus, the microbial agent or the microbial agent prepared by the preparation method in degrading dichloromethane in waste water, waste gas and soil.

[0025] The present invention has found that the strain YJY22-05 has a 24-hour degradation rate of 99.53% for dichloromethane with an initial concentration of 2000 mg / L. The bacterial agent containing the strain is applied to the biochemical treatment of dichloromethane-containing wastewater. The initial concentration of dichloromethane is 5535.20 mg / L. One ten-thousandth of the solid bacterial agent is added. After 48 hours, the dichloromethane concentration in the system is reduced to 4.26 mg / L. The results show that the strain YJY22-05 and the microbial agent prepared by the present invention have a significant degradation effect on dichloromethane.

[0026] The present invention also provides a method for degrading dichloromethane in wastewater, waste gas or soil, wherein the Daqing Lake golden bacillus, the microbial agent or the microbial agent prepared by the preparation method is added into the wastewater, waste gas or soil.

[0027] According to the effective living bacteria in the wastewater ≥ 10 5 cfu / mL; adding the Daqinghu golden bacillus or the microbial agent;

[0028] According to the effective living bacteria in the soil ≥ 10 6 cfu / g, adding the Daqinghu golden bacillus or the microbial agent,

[0029] According to the effective living bacteria in the waste gas ≥ 10 6 cfu / mL of the Daqinghu Golden Bacillus or the microbial agent is added.

[0030] In a specific embodiment of the present invention, wastewater is treated, and the treatment conditions are preferably: rotation speed 50-180rpm, temperature 25-37°C, pH 5.0-8.0, dissolved oxygen 1-8mg / L. Too high dissolved oxygen is not conducive to the division and reproduction of bacteria. During the application process, too high dissolved oxygen will also break up the activated sludge in the aerobic tank, which is not conducive to the attachment of the bacterial agent. At the same time, it will also be accompanied by floating mud and running mud, resulting in the paralysis of the entire biochemical system.

[0031] The Daqing Lake Golden Bacillus YJY22-05 provided by the present invention has a significant degradation ability for dichloromethane and can be applied to the biological treatment of dichloromethane-containing wastewater in the chemical industry. The strain has a 24-hour degradation rate of 99.53% for dichloromethane with an initial concentration of 2000 mg / L. The bacterial agent is applied to the biochemical treatment of dichloromethane-containing wastewater. The initial concentration of dichloromethane is 5535.20 mg / L, and one ten-thousandth of the solid bacterial agent is added. After 48 hours, the dichloromethane concentration in the system drops to 4.26 mg / L. The application of the strain can reduce the impact of dichloromethane on the biochemical treatment system, improve the treatment efficiency of dichloromethane-containing wastewater, and has significant economic and environmental benefits. At the same time, the strain can be applied to the treatment field of dichloromethane-contaminated wastewater and soil.

[0032] Biological Deposit Description

[0033] YJY22-05, classification name: Chryseobacterium daecheongense, was deposited on January 17, 2022 at the General Microbiology Center of China Culture Collection Administration, located at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with the deposit number CGMCCNo.24314. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is the test result of the blank system effluent in the small-scale test of the dichloromethane degradation bacterial agent in Example 3;

[0035] Figure 2 The test results of the effluent from the bacterial system in the small-scale test of the dichloromethane degradation bacterial agent in Example 3 are as follows;

[0036] Figure 3 This is the test result of the effluent from the system in the dichloromethane degradation bacterial agent application test in Example 4. DETAILED DESCRIPTION

[0037] The present invention discloses a strain of Daqinghu golden bacillus and its application. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0038] The present invention will be further described below in conjunction with embodiments:

[0039] Example 1 Screening and molecular identification of dichloromethane-degrading strains

[0040] 1) Preliminary screening of bacterial strains

[0041] The soil and system sludge around the wastewater treatment device of the petrochemical company were used as the test matrix for the isolation and purification of strains. A total of 7 strains were purified and named bn1-bn7. Inorganic salt culture medium with a dichloromethane concentration of 1000 mg / L was prepared. Single colonies were picked up in turn from the clean bench and inoculated into sterile inorganic salt culture medium. The culture was shaken at 37°C and 180 rpm for 16-20 hours, and OD600 was detected. The results are shown in Table 1. Three effective strains were obtained by preliminary screening, namely bn2, bn5, and bn6.

[0042] Table 1 Preliminary screening of dichloromethane-degrading strains

[0043]

[0044] 2) Strain rescreening and molecular identification

[0045] The three effective strains obtained in the initial screening were used as re-screening strains. Single colonies were picked up in the clean bench and inoculated into sterile LB culture medium, and cultured at 37°C and 180rpm for 16-20h. According to the inoculation amount of 5%, they were inoculated into inorganic salt culture solution containing 2000mg / L dichloromethane. At the same time, a blank group was set up, and cultured at 37°C and 180rpm for 24h. The residual dichloromethane in the system was detected. The results are shown in Table 2. Strain bn2 had the best dichloromethane degradation effect, and the 24h degradation rate reached 99.53%. Therefore, bn2 was determined to be a dichloromethane efficient degradation strain, marked as strain YJY22-05.

[0046] BLAST comparison of the 16SrDNA sequence of the YJY22-05 strain showed that the nucleotide sequence of the 16SrDNA of the strain had greater than 99% homology with the nucleotide sequences of different strains of the genus Chryseobacterium, and had 100% homology with the strain clearly marked as Chryseobacterium daecheongense.

[0047] Table 2 Dichloromethane degradation strain rescreening

[0048]

[0049] Example 2 Preparation of dichloromethane degradation bacterial agent

[0050] (1) Activation of strains: Take out the Daqinghu golden bacillus strain YJY22-05 stored at 4°C and activate it at room temperature for 4-10 hours. Since the strain is stored on a slant nutrient agar medium in a test tube;

[0051] (2) Liquid seed preparation: On a sterile operating table, take 10 mL of sterile water and add it to the culture storage tube. Use a pipette to repeatedly blow and beat to prepare a bacterial suspension. Then inoculate the bacterial suspension into 50-500 mL of liquid LB medium and shake and culture for 15-20 h. The culture conditions are: temperature 25-37°C, shaking speed 50-170 rpm;

[0052] (3) High-density fermentation: Inoculate the liquid seeds of Daqinghu golden bacillus at an inoculum rate of 1%-5% by volume into the corresponding fermentation medium for liquid fermentation; fermentation conditions: sterile air to maintain a tank pressure of 0.02-0.05MPa, a temperature of 25-37°C, a rotation speed of 200-300rpm, dissolved oxygen ≧20%, and a culture time of 16-24h to obtain a liquid bacterial agent;

[0053] (4) Medium for high-density fermentation: glucose 1.0%-1.5%, soluble starch 2.2%-3.8%, magnesium sulfate 0.05%-0.09%, ammonium sulfate 0.1%-0.3%, sodium chloride 0.01%-0.03%, potassium dihydrogen phosphate 0.5%-1.0%, ferrous sulfate 0.005%-0.01%, calcium chloride 0.01%-0.05%, polyether defoamer 0.05%, pH 7.0-7.2.

[0054] The liquid bacterial agent obtained by fermentation in the above (3) is adsorbed, dried and crushed to obtain a solid bacterial agent with an effective viable bacterial count of 10 8 cfu / mL or above.

[0055] Example 3 Application of dichloromethane degradation bacterial agent in small-scale test

[0056] The bacterial agent prepared in Example 2 was used for a small test of aerobic treatment of dichloromethane wastewater. The dichloromethane wastewater was taken from a chemical plant in Shandong. The initial concentration of dichloromethane was about 4000 mg / L. The effective volume of the aerobic device was 5 L. The aerobic sludge was also taken from the chemical plant, with the sludge accounting for 30%, and the rest being dichloromethane wastewater. The specific operation steps are as follows:

[0057] (1) Activation of solid bacterial agent: solid bacterial agent: water = 1:3-10 mass ratio mixed evenly, stirring activation in a stirring container for 12-20 hours;

[0058] (2) Dosing: After the activation of the microbial agent is completed, the microbial agent is added at a ratio of 0.02% based on the effective pool capacity of the pilot test system;

[0059] (3) Debugging and testing: After the addition of the bacterial agent is completed, the aeration parameters are debugged: dissolved oxygen 1-8 mg / L, temperature 25-30 °C, pH 5-8, and the effluent from the aeration tank is sampled every 12-24 hours to test the pollutant indicators;

[0060] The water inflow of the system is 50%, and water is added and discharged once a day. At the same time, a blank control is set up. The results are as follows Figure 1 and Figure 2 As shown, the dichloromethane content in the effluent of the blank system without the addition of bacterial agent was stable at more than 4000 mg / L, while the dichloromethane concentration in the effluent of the test system with the addition of bacterial agent dropped from 4215.36 mg / L to 8.31 mg / L after 36 hours, and was subsequently maintained below 10 mg / L.

[0061] Example 4 Application of dichloromethane degradation bacterial agent in chemical wastewater treatment

[0062] In mid-January 2022, a chemical plant in Shandong Province had an additive leakage accident, which caused dichloromethane to enter the wastewater biochemical pretreatment device in the plant area along with the wastewater. The dichloromethane detection concentration in the aeration tank reached about 5000 mg / L, causing a serious impact on the biochemical system, resulting in the effluent COD and ammonia nitrogen indicators not meeting the standards. Therefore, dichloromethane degradation bacteria (Example 2 bacteria) was urgently added, and the specific use method is as follows:

[0063] (1) Activation of solid bacterial agent: solid bacterial agent: water = 1:10 mass ratio mixed evenly, stirred in a stirring container for 15 hours; (water contains 2-4% glucose, 0.03-0.06% ammonium sulfate, 0.02-0.05% potassium dihydrogen phosphate)

[0064] (2) Dosing: After the activation of the bacterial agent is completed, the effective pool capacity of the aeration tank of the sewage treatment system is calculated, and the dichloromethane degradation bacterial agent is added to the aeration tank;

[0065] (3) Debugging and testing: After the addition of the bacterial agent is completed, the aeration tank parameters are debugged: dissolved oxygen 8 mg / L, temperature 37°C, pH 7.0, and the effluent from the aeration tank is sampled every 12 hours to test the pollutant indicators;

[0066] Tracking test for 60 hours, the results are as follows Figure 3 The dichloromethane concentration in the aerobic effluent showed a downward trend. After 48 hours of tracking, the dichloromethane concentration in the effluent dropped from the initial 5535.20 mg / L to 4.26 mg / L, and stabilized below 5 mg / L in the later period.

[0067] In summary, the strain provided by the present invention has an obvious degradation effect on dichloromethane.

[0068] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Daqinghu golden bacillus with the deposit number of CGMCC NO.24314 ( Chryseobacterium daecheongense ).

2. A microbial agent, characterized in that: The raw material includes Daqinghu golden bacillus with a preservation number of CGMCC NO.24314.

3. The method for preparing the microbial agent according to claim 2, characterized in that: include: The Daqinghu golden bacillus with the collection number of CGMCC NO.24314 was activated to prepare seed liquid, which was then inoculated into a fermentation medium for fermentation, and the fermented bacteria were collected.

4. The preparation method according to claim 3, characterized in that: The method also includes the steps of sequentially adsorbing, drying and crushing the fermented bacteria.

5. The preparation method according to claim 3, characterized in that: The activation is performed at room temperature for 4h-10h; The preparation of the seed solution comprises: inoculating the activated bacterial strain into a liquid culture medium, and culturing the culture medium under the conditions of a temperature of 25 to 37° C. and a shaking speed of 50 to 170 r / min for 15 to 20 hours.

6. The preparation method according to claim 3, characterized in that: The fermentation comprises the following steps: inoculating Daqinghu golden bacillus seed liquid in a volume percentage of 1%-5% into a fermentation medium for high-density fermentation; and the conditions for the high-density fermentation are: tank pressure of 0.02-0.05MPa, temperature of 25-37°C, rotation speed of 200-300r / min, dissolved oxygen ≧20%, and time of 16-24h.

7. Use of the Daqinghu golden bacillus according to claim 1, the microbial agent according to claim 2, or the microbial agent prepared by the preparation method according to any one of claims 3 to 6 in degrading dichloromethane in wastewater, waste gas and soil.

8. A method for degrading dichloromethane in wastewater, waste gas or soil, characterized in that: The Daqinghu golden bacillus described in claim 1, the microbial agent described in claim 2, or the microbial agent prepared by the preparation method described in any one of claims 3 to 6 is added into wastewater, waste gas or soil.

9. The method according to claim 8, characterized in that: According to the effective living bacteria in the wastewater ≥ 10 5 cfu / mL; adding the Daqinghu golden bacillus or the microbial agent; According to the effective living bacteria in the soil ≥ 10 6 cfu / g, adding the Daqinghu golden bacillus or the microbial agent, According to the effective living bacteria in the waste gas ≥ 10 6 cfu / mL of the Daqinghu Golden Bacillus or the microbial agent is added.

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