CN114214254B - Rhodococcus ruber and application thereof - Google Patents

Abstract

The application relates to the fields of microorganisms and sewage treatment, in particular to rhodococcus ruber and application thereof. The rhodococcus ruber has the preservation number of CGMCC No.22759, can efficiently degrade acetonitrile when the total salt concentration is below 1%, can tolerate the initial acetonitrile concentration of up to 30g/L, has the average degradation rate of 1500mg/L for 24 hours, can be applied to the sewage treatment of acetonitrile, acrylonitrile, adiponitrile and phenylacetonitrile pollution, can be continuously cultured, has strong strain tolerance, is not easy to be impacted by external environment in the application process, has the advantages of wide application prospect and good environmental benefit, is simple to operate, is safe and environment-friendly, has strong adaptability, has no secondary pollution and the like, and can be widely applied to various high-salt sewage treatment systems containing acetonitrile and other nitrites.

Description

Rhodococcus ruber and application thereof

Technical Field

The application relates to the fields of microorganisms and sewage treatment, in particular to rhodococcus ruber and application thereof.

Background

Nitrile compounds are organic compounds with cyano groups, are important members of cyanide compounds, are widely applied to the chemical industry, are byproducts in the production processes of electroplating, metal, pharmacy and the like, and are mostly high-toxic substances. With the rapid development of modern society, the discharge of nitrile-containing waste liquid into the environment becomes an important factor for endangering ecological environment safety.

Acetonitrile is an organic chemical raw material widely used in nitrile compounds, is one of main components of nitrile compound waste water because of the irreplaceable function, and has been widely and continuously used in various industrial production, and the discharge amount of acetonitrile-containing waste water has been increased year by year with the rapid increase of acetonitrile yield and use amount in recent years. Acetonitrile can be absorbed by inhalation and skin and enter organisms, can be converted into highly toxic substances of hydrogen cyanide and acetaldehyde in the organisms, and when untreated acetonitrile-containing wastewater and waste liquid enter natural water areas, the untreated acetonitrile-containing wastewater and waste liquid can cause massive death of aquatic organisms such as fishes, so that pollution caused by the acetonitrile-containing wastewater and the waste liquid causes serious harm to human living environment.

At present, the acetonitrile wastewater treatment method mainly comprises a chemical method and a biochemical method, wherein the chemical method comprises an ozone oxidation method, a hydrogen peroxide oxidation method, an electrolysis method and the like, and the methods have the advantages of high efficiency, harsh reaction conditions, high engineering cost, low cost, simple operation and management, no secondary pollution and the like. Therefore, the separation and breeding of acetonitrile degradation strains with high tolerance and degradation capability are particularly important to be applied to the bio-enhancement technology.

Disclosure of Invention

Aiming at the problems in the prior art, the inventor firstly discovers a rhodococcus ruber in the research process, has strong acetonitrile degradation capability, strong tolerance capability and strong stress resistance, and has very remarkable environment adaptability. Can strengthen the original sewage treatment system, improve the removal efficiency of acetonitrile in sewage, and simultaneously has the capability of degrading acrylonitrile, adiponitrile and benzyl cyanide in water. The method is applied to sewage treatment, has the advantages of simple operation, safety, environmental protection, strong adaptability, no secondary pollution and the like, can be widely applied to various high-salt sewage treatment systems containing acetonitrile and other nitrites, and has wide application prospect and good environmental benefit.

Firstly, the inventor collects water samples from a sewage treatment system of a petrochemical sewage plant, enriches the water samples, and carries out gradient domestication, separation and purification on the water samples in an inorganic salt culture medium which takes acetonitrile as unique carbon, nitrogen and energy to obtain an acetonitrile degrading bacterium.

Through identification, the acetonitrile degrading bacteria are gram-positive bacteria, and the bacterial colony is orange-red, round, opaque and smooth in surface; the cell shape is spherical, and is identified as rhodococcus ruber (Rhodococcus ruber) by 16S rDNA, and is preserved in China general microbiological culture Collection center with the preservation number of CGMCC No.22759.

The inventor carries out further research experiments on the strain, and discovers that the strain can be applied to sewage treatment, can degrade acetonitrile, acrylonitrile, adiponitrile, benzyl cyanide and the like, has remarkable acetonitrile degradation effect, can tolerate the total salt concentration of 10000mg/L or higher, has the acetonitrile tolerance concentration of up to 30g/L, and can reach the average degradation rate of 1500mg/L or higher in a proper environment for 24 hours.

When the rhodococcus ruber is applied to sewage treatment, the rhodococcus ruber or the microbial inoculum thereof is directly added into a sewage treatment system, and the aeration treatment ensures that the dissolved oxygen is more than 2mg/L, the pH of the sewage is 5.0-7.5, and the temperature is preferably 25-35 ℃. The microbial inoculum can be a liquid microbial inoculum or a solid microbial inoculum. The sewage treatment system can be a conventional sewage biochemical treatment system such as an aeration tank, activated sludge and the like.

As a further embodiment, the addition amount of the rhodococcus ruber in the application process of the rhodococcus ruber is 10 according to the initial effective bacterial amount of the system ⁵ -10 ⁷ cfu/mL was added.

The application further provides a microbial inoculum prepared from the rhodococcus ruber, which comprises the following preparation methods:

carrying out high-density fermentation on the rhodococcus ruber to obtain a liquid microbial agent thereof; and further adsorbing, drying and crushing the fermentation product to obtain the solid microbial inoculum.

The preparation process comprises the following steps:

inoculating the rhodococcus ruber on a test tube slant culture medium, and placing the rhodococcus ruber in a constant temperature incubator, and culturing at 30-37 ℃ for about 24 hours until bacterial colonies grow on the culture medium;

secondly, the strain in the first step is transplanted into LB liquid culture medium (100 mL), shake culture is carried out for about 16 hours at 30-37 ℃ and about 160-180r/min, and primary seed liquid is obtained; inoculating the primary seed liquid into LB culture medium according to 5% (V/V), and culturing for 16h under the same condition to obtain secondary seed liquid;

thirdly, inoculating the second-level seed liquid of the rhodococcus ruber into a fermentation culture medium, performing fermentation culture, controlling the fermentation temperature to be 30-37 ℃, controlling the ventilation rate to be about 0.5-2:1, culturing for 20-30h, raising dissolved oxygen, lowering pH, and stopping fermentation to obtain the liquid microbial inoculum.

And fourthly, further adsorbing, drying and crushing the fermentation product to obtain the solid microbial inoculum. Wherein the adsorption, drying and pulverization are carried out according to the conventional technology in the field, and the detailed description is omitted.

Preferably, the liquid microbial agent has a viable count of 0.9X10 ¹⁰ ～2.5×10 ¹⁰ cfu/mL; the number of viable bacteria of the solid microbial inoculum is 1 multiplied by 10 ¹⁰ cfu/g or more.

Wherein, the fermentation medium is added in each 1L by weight: 10-30g of corn steep liquor dry powder, 10-20g of peptone, 5-8g of yeast powder, 10-20g of glucose, 20-30g of corn meal, 1-5g of sodium chloride, 0.1-0.5g of magnesium sulfate, 0.5-0.8g of potassium dihydrogen phosphate and 7.0-7.5 g of glucose.

Compared with the prior art, the application has the beneficial effects that: the rhodococcus ruber of the application, the strain comes from the sewage treatment system, the indigenous microorganism which can degrade acetonitrile is screened out from the strain, the single strain has the characteristics of simplicity and high efficiency in application, meanwhile, the strain has strong tolerance capability, can tolerate acetonitrile with high concentration, can tolerate the total salt concentration of 10000mg/L or higher, has wide application range to conditions such as temperature, pH and the like, is not easy to be impacted by external environment in the application process, has the capability of degrading acrylonitrile, adiponitrile, benzyl cyanide, pyridine, o-dichlorobenzene, trichloroethylene, methylene dichloride and trichloromethane in water, has good application value in actual production, and has wide application prospect

Preservation information

Preservation time: 2021, 6, 23;

preservation unit name: china general microbiological culture Collection center (China Committee for culture Collection);

preservation number: CGMCC No.22759;

deposit unit address: beijing, chaoyang area, north Chenxi Lu No. 1, 3;

the classification is named as rhodococcus ruber (Rhodococcus ruber).

Drawings

FIG. 1 is a graph showing the effect of temperature on acetonitrile degradation rate in example 3;

FIG. 2 effect of pH on acetonitrile degradation rate in example 3;

FIG. 3 is a graph showing the comparison of acetonitrile degradation rate in example 7;

FIG. 4 is a graph showing the COD degradation rate in example 7;

FIG. 5 shows the degradation profile of acetonitrile content over time in example 8.

Detailed Description

The above-described aspects of the present application will be described in further detail with reference to the following detailed description, but it should not be construed that the scope of the above-described subject matter of the present application is limited to the following examples. All techniques realized based on the above description of the present application are within the scope of the present application, and the following examples are accomplished by conventional techniques unless otherwise specified.

In the following examples:

the inorganic salt culture medium is prepared from KH ₂ PO ₄ 1.7g/L，Na ₂ HPO ₄ 9.8g/L，(NH ₄ ) ₂ SO ₄ 1.0g/L，MgSO ₄ ·7H ₂ O 0.1g/L，MgO 10mg/L，FeSO ₄ ·7H ₂ O 0.9mg/L，CuSO ₄ ·5H ₂ O 0.5mg/L，CaCO ₃ 2mg/L，ZnSO ₄ ·7H ₂ O 1.44mg/L，H ₃ BO ₃ 0.06mg/L, ph=7.4, sterilized at 121 ℃ for 20min for later use, acetonitrile was added before use.

The enrichment medium is prepared by adding 10.0g of peptone, 5.0g of yeast powder and 10.0g of sodium chloride into 1.0L of water, adjusting the pH value to 7.0, adding 20.0g of agar into the solid medium, and sterilizing for 20min at 121 ℃.

EXAMPLE 1 acquisition of Rhodococcus ruber

The rhodococcus ruber is a strain for efficiently degrading acetonitrile, which is obtained by sampling activated sludge from a sewage treatment plant in coastal state of Shandong and performing primary screening and secondary screening. The method comprises the following specific steps:

and adding a part of acetonitrile into activated sludge of a sewage treatment plant for culture, and gradually increasing the concentration of acetonitrile to obtain the strain with the highest tolerance. The specific operation method comprises the following steps: taking 10mL of activated sludge sample, inoculating the activated sludge sample into 100mL of enrichment culture medium (initial concentration of acetonitrile is 50 mg/L), performing shaking culture at 30 ℃ and 160rpm for 2-5d of enrichment culture, sampling and detecting the acetonitrile content in the culture medium, and taking out 10mL of bacterial liquid to inoculate into fresh inorganic salt culture medium (concentration of acetonitrile is 100 mg/L) when the acetonitrile content is degraded to be lower than 5 mg/L. Repeating the steps, and gradually increasing the concentration of acetonitrile in the culture medium to 30000mg/L by taking 200, 300, 500, 1000, 2000, 5000 and 10000mg/L as gradients.

The acetonitrile domestication solution is coated on an inorganic salt culture medium flat plate which takes 10000mg/L acetonitrile as the only carbon, nitrogen and energy after being diluted in a gradient way, and is cultured for 2-3d in a 30 ℃ incubator. Single colonies with different forms which are grown on a flat plate are respectively streaked on an LB flat plate for purification for at least 3 times, the single colonies are picked up after purification and inoculated in 10mL of LB liquid medium, the single colonies are respectively inoculated in 100mL of inorganic salt liquid medium containing 2000mg/L acetonitrile with the inoculum size of 2% in the next day, the single colonies are cultured for 72 hours at 30 ℃ and 160r/min, and then the acetonitrile content is detected by adopting gas chromatography after sampling and extraction. 9 strains of the bacteria are separated by the method, wherein the bacteria with the number of A29 have the strongest acetonitrile degrading capacity within 24 hours, and can reach 1500mg/L. Through identification, the strain is a gram-positive bacterium, and is cultured in a solid state to form a red smooth circular colony with regular edges. Then, the NCBI database is used for comparing the 16S rDNA sequence of the strain, and the identification result shows that the strain belongs to rhodococcus ruber on the molecular level, and has strong acetonitrile degradation capability.

The extraction process comprises the steps of sampling 5mL, centrifuging at 5000rpm to remove thalli, adding equal volume of dichloromethane, standing at room temperature for 30min, and storing 1mL of organic phase in an automatic sample injection bottle for preservation and detection.

The above acetonitrile was detected by gas chromatography under the condition that the detector was FID and the column was OV-1701 (30 m.times.0.53 mm.times.1.0 μm). The sample injection amount was 1. Mu.L, and the split ratio was set to 20:1. The sample inlet temperature and the detector temperature are respectively 200 ℃ and 220 ℃ and the column temperature is 70 ℃; the carrier gas was N2 at a flow rate of 3.0mL/min.

EXAMPLE 2 preparation of microbial agent

The preparation method of the rhodococcus ruber microbial inoculum in the embodiment comprises the following steps:

firstly, inoculating purified rhodococcus ruber on a test tube slant culture medium, placing the rhodococcus ruber in a constant temperature incubator, and culturing until colonies grow out on the culture medium;

secondly, transferring the strain in the first step into an LB liquid culture medium (100 mL), performing shake culture to obtain a first-stage seed liquid, inoculating the first-stage seed liquid into the LB culture medium according to 5% (V/V), and culturing for 16 hours under the same conditions to obtain a second-stage seed liquid;

thirdly, inoculating the second-level seed liquid of the rhodococcus ruber into a fermentation culture medium, performing fermentation culture, controlling the fermentation temperature to be 30+/-1 ℃ and the ventilation rate to be about 1:1, culturing for 20-30h, raising dissolved oxygen, lowering pH, and stopping fermentation to obtain the rhodococcus ruber agent. The viable count of the microbial inoculum in this example was 1X 10 ¹⁰ cfu/mL。

The fermentation medium comprises the following components in percentage by mass: 2-3% of corn steep liquor dry powder, 0.5-1.2% of peptone, 0.5-1.2% of yeast powder, 1-2% of glucose, 2-3% of corn flour, 0.1-0.5% of sodium chloride, 0.03-0.05% of magnesium sulfate, 0.03-0.06% of potassium dihydrogen phosphate, the balance of water, pH7-7.5, and sterilizing at 121 ℃ for 20min under high pressure.

The LB culture medium is prepared from 10.0g of peptone, 5.0g of yeast powder and 10.0g of sodium chloride by adding water to 1.0L, adjusting the pH value to 7.0, and sterilizing at 121 ℃ for 20min.

Example 3 acetonitrile degradation Properties

The bacterial preparation obtained in example 2 was washed twice with physiological saline to give an effective viable count of about 1.2X10 ⁸ cfu/mL。

Inoculating 5mL to 100mL of inorganic salt culture medium, wherein acetonitrile is used as the only carbon, nitrogen and energy source, and detecting degradation performance.

Detection of maximum degradation capability:

under the conditions, the acetonitrile concentration adding amount is 1000, 2000, 3000, 5000, 8000, 10000, 30000mg/L, the shaking culture is carried out at 30 ℃ and 160r/min, every 24h sampling is carried out, the sample is centrifuged for 3-5 min at 4000-8000 r/min, the supernatant is extracted for detecting the acetonitrile content, the rhodococcus ruber can endure 30g/L of acetonitrile and maintain stable degradation capacity about 1500mg/L.d, the bacterium has strong tolerance to acetonitrile, and specific data are shown in table 1.

TABLE 1

Impact test of culture conditions:

acetonitrile is respectively added to 2000mg/L in 6 groups of inorganic salt culture mediums, different culture temperatures (20, 25, 28, 30, 35 and 40 ℃ respectively) are set in each group, the rotating speed is 160r/min, and the culture is carried out for 24 hours;

respectively adding acetonitrile to 2000mg/L of 10 groups of inorganic salt culture mediums, setting different pH (4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8 and 8.5) conditions for each group of culture mediums, and culturing for 24 hours at 30 ℃ at the rotating speed of 160 r/min;

after the culture solution after the culture is subjected to sampling and centrifugal extraction, acetonitrile degradation conditions in the culture solution are detected, and as shown in figures 1-2, the strain has a wide applicable range, different degradation capacities under different temperature and pH conditions, and the optimal action temperature is 25-35 ℃ and the optimal action pH is 6.5-7.5.

Example 4 degradation of other nitriles

Setting 3 groups of inorganic salt culture mediums, and adding other nitrile compounds into each group: acrylonitrile, adiponitrile and benzyl cyanide with the addition amount of 100mg/L.

Rhodococcus ruber single colonies were picked and inoculated into 10mL of LB liquid medium, and the next day was inoculated into the 3 groups of medium with an inoculum size of 2% respectively.

The culture conditions are as follows: culturing in a shaking table with the rotating speed of 160rpm at 30 ℃ for 16 hours, and measuring the degradation rate of different nitrites by using a phenol-sodium hypochlorite method.

Finally, the following steps are: the degradation rate of the rhodococcus ruber to the acrylonitrile, the adiponitrile and the benzyl cyanide is 99.9 percent, 98.6 percent and 64.3 percent respectively. The rhodococcus ruber has strong degradation capability to nitrile compounds and wide application prospect.

EXAMPLE 5 degradation of other organic contaminants

Set 5 groups of inorganic salt medium of example 1, each group being supplemented with other organic contaminants: pyridine (300 mg/L), o-dichlorobenzene (390 mg/L), trichloroethylene (150 mg/L), methylene chloride (300 mg/L), and chloroform (300 mg/L).

Rhodococcus ruber single colonies were picked and inoculated into 20mL of LB liquid medium, and the next day was inoculated into the 5 groups of medium with an inoculum size of 2% respectively.

The culture conditions are as follows: culturing in a shaker at 30deg.C and rotation speed of 160rpm for about 24 hr, extracting with n-hexane, and detecting residual amount of pollutants in gas chromatography detection system and control group with degradation rates of Rhodococcus ruber to pyridine, o-dichlorobenzene, trichloroethylene, dichloromethane and trichloromethane of 93.77%, 41.1%, 47.1%, 58.9% and 64.7%, respectively. The rhodococcus ruber can degrade pyridine, dichlorobenzene, chloralkane and the like.

EXAMPLE 6 application 1 in acetonitrile Sewage treatment

A rhodococcus ruber preparation was prepared as in example 2, the culture temperature was 30.+ -. 1 ℃ and the aeration rate was controlled to 1:1, and the viable count of the obtained microbial inoculum was 1.4X10 ¹⁰ cfu/mL。

The source of the treated water is the inlet water of an aerobic section of a petrochemical sewage treatment plant, the aeration treatment is simulated in a laboratory, the treatment capacity is 5L, the addition amount of red rhodococcus is 0.1 percent (V/V), the aeration treatment ensures that the dissolved oxygen is more than 2mg/L, the sewage pH is 6.0-8.0, the COD is about 3000mg/L, the ammonia nitrogen is about 200mg/L, the total salt is 3000mg/L, the sulfide is 50mg/L, the heavy metal chromium is about 500mg/L, the initial acetonitrile concentration in the system is artificially added to 100mg/L, the inlet water and the outlet water are once in 24 hours, the inlet water and the outlet water are 1L each time, the acetonitrile concentration in the system is gradually increased, the acetonitrile content of the water is sampled and detected, and the tracking experiment result is shown in Table 2.

Table 2 shows that the rhodococcus ruber has obvious effect in sewage treatment, and can degrade 1000mg/L acetonitrile stably in petrochemical sewage.

TABLE 2

EXAMPLE 7 application of Rhodococcus ruber in acetonitrile Sewage treatment 2

A rhodococcus ruber preparation was prepared as in example 2, the culture temperature was 32.+ -. 1 ℃ and the aeration rate was controlled at 1:1, and the viable count of the obtained microbial inoculum was 1.8X10 ¹⁰ cfu/mL。

The treatment foundation is petrochemical aerobic activated sludge, the source of the treated water is the aerobic section sewage of a certain agricultural chemical sewage treatment plant, the laboratory simulates the aeration treatment of a biochemical system, the treatment capacity is 5L, the experimental group is that rhodococcus ruber is added into the aerobic activated sludge, the addition amount is 0.2%, and the control group only adds the aerobic activated sludge, so that the dissolved oxygen is more than 2mg/L through the aeration treatment. The pH value of the sewage is 6.0-8.0, the COD is 2500mg/L, the initial acetonitrile concentration in the system is artificially added to 100mg/L, water is fed and discharged once in 24 hours, 1L of water is fed and discharged each time, the acetonitrile concentration in the system is increased to 1000mg/L each time of water feeding, the acetonitrile degradation rate of the discharged water is sampled and detected, the COD degradation rate index is shown in figures 3 and 4, and the detection result is shown in the drawings.

Experimental results show that the rhodococcus ruber has obvious effect when being used in the aerobic sewage treatment stage, can stably degrade acetonitrile with the content of 1000mg/L in agricultural chemical sewage, has strong degradation capability and strong pollutant impact resistance, and can be used as a biological enhancer for sewage treatment.

Example 8 application in high salt acetonitrile Sewage treatment

A microbial inoculum was prepared in the same manner as in example 2, the culture temperature was 34.+ -. 1 ℃ and the aeration rate was controlled to be 0.8:1, and the viable count of the obtained microbial inoculum was 0.9X10 ¹⁰ cfu/mL。

The source of the treated water is leather sewage, the treatment capacity is 5L, the addition amount of the microbial inoculum is 0.5% (V/V), the dissolved oxygen is above 2mg/L by aeration treatment, and the pH of the sewage is 6.0-8.0. The raw sewage total salt is 9800mg/L, COD is about 4500mg/L, ammonia nitrogen is about 280mg/L, sulfide is 50mg/L, heavy metal chromium is about 500mg/L, acetonitrile concentration is below 100mg/L, acetonitrile concentration is artificially added to 800mg/L, the acetonitrile content of the sewage after 24 hours is less than 1mg/L, degradation rate reaches more than 99.9%, and the degradation process of the sewage is shown in figure 5 by sampling, tracking and detecting at regular time.

Example 9 application in treatment of high salt organic wastewater

A rhodococcus ruber preparation was prepared as in example 2, the culture temperature was 32.+ -. 1 ℃, the aeration was controlled to 1-1.5:1, and the viable count of the obtained microbial inoculum was 2.4X10 ¹⁰ cfu/mL。

The source of the treated water is epichlorohydrin sewage, the treatment capacity is 5L, the addition amount of the microbial inoculum is 0.1% (V/V), the aeration treatment ensures that the dissolved oxygen is more than 2mg/L, the sewage pH is 6.0-8.0, the total salt is 9500mg/L, the COD is about 2500mg/L, the ammonia nitrogen is about 150mg/L, the acetonitrile concentration is less than 100mg/L, the acetonitrile concentration is artificially added to 900mg/L, the pyridine is 300mg/L, the acetonitrile content of the sewage is less than 1mg/L after 24 hours, the degradation rate is more than 99.9%, the pyridine content is less than 5mg/L, and the degradation rate is 98%.

In conclusion, the rhodococcus ruber disclosed by the application is added into sewage impacted by acetonitrile, acrylonitrile, adiponitrile, benzyl cyanide, pyridine, o-dichlorobenzene, trichloroethylene, methylene dichloride and trichloromethane pollutants, so that different degrees of detoxification effects are realized on the sewage, the rhodococcus ruber has a good effect on acetonitrile, adiponitrile, benzyl cyanide and pyridine, particularly has a very remarkable degradation effect on acetonitrile, and has a wide application prospect.

Sequence listing

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Claims (10)

1. A strain of rhodococcus ruber is characterized in thatRhodococcus ruber) The strain is preserved in China general microbiological culture Collection center (CGMCC) with a preservation number of CGMCC No.22759.

2. The use of rhodococcus ruber as claimed in claim 1, for the treatment of sewage, for the degradation of acetonitrile or acrylonitrile or adiponitrile or phenylacetonitrile or pyridine.

3. The use of rhodococcus ruber as claimed in claim 2, for the treatment of sewage and for the degradation of acetonitrile.

4. The method for using the rhodococcus ruber according to claim 1, wherein when the rhodococcus ruber is used for sewage treatment, the rhodococcus ruber or the microbial inoculum thereof is directly added into a sewage treatment system for aeration treatment.

5. The method for using rhodococcus ruber according to claim 4, wherein the aeration treatment makes the dissolved oxygen above 2mg/L, the pH of the sewage is 5.0-7.5, and the temperature is 25-35 ℃.

6. The method of claim 4, wherein the microbial agent is a liquid microbial agent or a solid microbial agent.

7. The method of using rhodococcus ruber as defined in claim 4, wherein the addition amount of rhodococcus ruber in the application process of rhodococcus ruber is 10 according to the initial effective bacterial amount of the system ⁵ -10 ⁷ cfu/mL was added.

8. The rhodococcus ruber microbial inoculum is characterized by comprising the following preparation methods: carrying out high-density fermentation on the rhodococcus ruber to obtain a liquid microbial agent thereof; or further adsorbing, drying and pulverizing the fermentation product to obtain a solid microbial agent;

the rhodococcus ruber is preparedRhodococcus ruber) The strain is preserved in China general microbiological culture Collection center (CGMCC) with a preservation number of CGMCC No.22759.

9. The rhodococcus ruber microbial inoculum according to claim 8 is characterized in that the preparation process of the specific liquid microbial inoculum is as follows:

firstly, inoculating the rhodococcus ruber on a test tube slant culture medium, and placing the rhodococcus ruber in a constant temperature incubator to culture for 24 hours at 30-37 ℃ until bacterial colonies grow on the culture medium;

secondly, transferring the strain in the first step into an LB liquid culture medium, and performing shake culture for 16 hours at the temperature of 30-37 ℃ and the speed of 160-180r/min to obtain primary seed liquid; inoculating the primary seed liquid into LB culture medium according to 5% V/V, and culturing for 16h under the same condition to obtain secondary seed liquid;

thirdly, inoculating the second-level seed liquid of the rhodococcus ruber into a fermentation culture medium, performing fermentation culture, controlling the fermentation temperature to be 30-37 ℃, controlling the ventilation rate to be 0.5-2:1, culturing for 20-30h, raising dissolved oxygen, lowering pH, and stopping fermentation to obtain a liquid microbial inoculum;

wherein, the fermentation medium is added in each 1L by weight: 10-30g of corn steep liquor dry powder, 10-20g of peptone, 5-8g of yeast powder, 10-20g of glucose, 20-30g of corn meal, 1-5g of sodium chloride, 0.1-0.5g of magnesium sulfate, 0.5-0.8g of potassium dihydrogen phosphate and 7.0-7.5 g of glucose.

10. The microbial inoculum of rhodococcus ruber as set forth in claim 8, wherein the viable count of the liquid microbial inoculum is 0.9x10 ¹⁰ ~2.5×10 ¹⁰ cfu/mL; the number of viable bacteria of the solid microbial inoculum is 1 multiplied by 10 ¹⁰ cfu/g or more.