CN117925443A

CN117925443A - Rhodococcus ruber and application thereof

Info

Publication number: CN117925443A
Application number: CN202311619852.4A
Authority: CN
Inventors: 赵琬玫; 温胜国; 孙元章; 闫明英; 王锦梅; 王文红; 刘圣鹏
Original assignee: Qingdao Weilan Saide Biotechnology Co ltd
Current assignee: Qingdao Weilan Saide Biotechnology Co ltd
Priority date: 2023-11-30
Filing date: 2023-11-30
Publication date: 2024-04-26

Abstract

The invention relates to a rhodococcus ruber (Rhodococcus ruber) KH1, which is preserved in China general microbiological culture Collection center (China Committee) for culture Collection of microorganisms, and has the following addresses: the collection number of China institute of microbiology, national academy of sciences, no. 3, west Lu 1, north Star, beijing, chao, korea, is: CGMCC No.27877, the strain has high degradation efficiency on benzene series, and the benzene degradation rate of 72 hours reaches more than 99 percent aiming at the benzene content below 50mg/L in an evaluation culture medium at 30 ℃; the strain is moderately salt-tolerant, and the number of viable bacteria is hardly affected when the strain grows under the salinity condition of less than 5 percent.

Description

Rhodococcus ruber and application thereof

Technical Field

The invention relates to screening and fermentation production of rhodococcus ruber and application thereof in environmental treatment and purification of water quality, belonging to the technical field of environmental microorganisms.

Background

Benzene series is an important component of petroleum hydrocarbon, is a common pollutant in waste water of petroleum, chemical industry and coking, and has higher content in waste water of industries such as synthetic rubber, paint, medicine, plastics, synthetic medicine, synthetic resin, synthetic fiber, pesticide, dye and the like. Benzene series have great volatility, are easily released into the environment during the production, storage and transportation of petroleum industry, cause environmental pollution and are harmful to the ecosystem and human body health.

Pyridine and nitrogen-containing heterocyclic compounds can be regarded as compounds in which one (CH) of benzene molecules is replaced by N, so that nitrogen benzene is also called colorless or yellowish liquid, and malodor exists. Pyridine and its homologs are found in bone tar, coal gas, shale oil, petroleum. Pyridine is widely used in chemical industry, pharmaceutical industry, pesticide production and other industries, and has the characteristics of teratogenicity, strong carcinogenicity, difficult biodegradation and the like.

The current benzene series and pyridine pollution restoration technology mainly comprises a physical restoration method, a chemical restoration method and a biological restoration method, wherein the biological restoration has the advantages of strong processing capacity, wide application range, simple operation, economy and applicability, no secondary pollution to the environment, and especially the biological restoration method has irreplaceable importance when mechanical devices cannot be removed and chemical medicines cannot be used.

Scientists have now found a variety of benzene-degrading bacteria including pseudomonas (pseudomonasp.), nocardia (Nocardioides sp.), micrococcus (Micrococcus sp.), etc., but these strains are still not sufficiently excellent in the effect of degrading benzene, and therefore there is a need to screen and construct strains capable of degrading a variety of benzene-based compounds and having a strong effect of degrading ability.

Since the 70 s of the 20 th century, scholars have been continually isolated with some pyridine degrading bacteria, most of which are bacteria such as pseudomonas, bacillus, achromobacter, flavobacterium, rhodococcus chaos, arthrobacter, and the like, actinomycetes and fungi such as white rot fungi, but there are few strains that can be used for practical bioremediation and have high success.

Disclosure of Invention

Aiming at the current situation that the existing benzene series and pyridine degrading strain is not remarkable in effect, the invention provides the rhodococcus ruber and the application thereof, and the strain can utilize the benzene series and pyridine in the water body as carbon sources to efficiently degrade the benzene series and pyridine in the water body, thereby achieving the effect of reducing COD in the water body.

Rhodococcus ruber (Rhodococcus ruber) KH1, deposited in China general microbiological culture Collection center with the China Committee for culture Collection of microorganisms, at the following address: the collection number of the institute of microorganisms of China academy of sciences of China No. 3, north Star West Lu No. 1, games of Beijing is CGMCC No.27877, and the collection date is: 2023, 7 and 11 days, 16s rDNA of which is shown as SEQ ID NO. 1, and the rhodococcus ruber in the invention refers to rhodococcus ruber KH1 strain under the condition of NO special description.

The rhodococcus ruber provided by the invention has the beneficial effects that:

1) The strain has high degradation efficiency on benzene series, and the degradation rate of the strain in 72 hours reaches more than 99 percent aiming at the benzene content below 50mg/L in an evaluation culture medium at the temperature of 30 ℃; the degradation rate of the xylene in the evaluation culture medium is about 98 percent in terms of the xylene content below 50mg/L at 30 ℃;

2) Moderately salt tolerant, when grown under a salinity condition below 5%, the viable count is hardly affected, but cannot tolerate a salinity above 6%;

3) The strain can also degrade pyridine in water, and can evaluate the condition of 30 ℃ in the culture medium

Pyridine content below 50mg/L, degradation rate of 72h reaches more than 83%, and degradation capability is strong;

4) The strain has the advantages of simple culture method, high growth speed, strong environmental adaptability, high safety, no damage to the original environment and no secondary pollution.

Benzene-based compounds described in the present invention include, but are not limited to, benzene, toluene, ethylbenzene, ortho-xylene, meta-xylene, and para-xylene.

The invention also discloses a microbial agent containing the rhodococcus ruber.

Preferably, the fermentation method of rhodococcus ruber comprises the following steps:

(1) Primary seed culture: inoculating Rhodococcus ruber into enrichment medium under aseptic condition, and culturing at 25-35deg.C and 150-300rpm for 12-48 hr to obtain first seed culture solution of Rhodococcus ruber;

(2) Secondary seed culture: inoculating the first-stage seed culture solution of rhodococcus ruber into enrichment culture medium according to 1-10vol% of inoculum size under aseptic condition, and culturing at 25-35deg.C and 150-300rpm for 12-48 hr to obtain second-stage seed culture solution of rhodococcus ruber;

(3) Fermentation: and (3) after the fermentation medium in the fermentation tank is disinfected, inoculating the second-level seed culture solution of the rhodococcus ruber obtained in the step (2) into the fermentation medium according to the inoculum size of 5-10vol%, controlling the temperature to be 25-35 ℃ and the rotating speed to be 150-300rpm, fermenting under the condition that the aeration ratio is 1 (1-2), and stopping fermenting when dissolved oxygen starts to rise to obtain the fermentation solution of the rhodococcus ruber.

The aeration ratio in the present invention means the ratio of the volume of air introduced into the fermenter per minute to the total volume of the fermentation liquid.

Wherein the composition of the enrichment medium is: 5-15g/L of peptone, 3-8g/L of yeast extract or yeast powder, 5-15g/L of sodium chloride, water as solvent, and pH=6-8;

preferably, the composition of the enrichment medium is: 10g/L of tryptone, 5g/L of yeast extract or yeast powder, 10g/L of sodium chloride and water as a solvent, wherein the pH value is 7-7.5.

Further, the composition of the fermentation medium is: 10-30g/L of carbon source and 20-40g/L、PO₄ ^3-0.8-1.5g/L、K⁺0.5-1.0g/L、Mg²⁺0.05-0.2g/L、Na⁺0.1-0.3g/L、Ca²⁺0.03-0.1g/L, g/L of nitrogen source, wherein the solvent is water, and the pH value is=6-8.

Preferably, the source of the PO ₄ ^3- is dipotassium hydrogen phosphate or potassium dihydrogen phosphate, the source of the K ⁺ is one or more of dipotassium hydrogen phosphate, potassium dihydrogen phosphate, potassium sulfate, potassium chloride and potassium nitrate, the source of the Mg ²⁺ is one or more of magnesium sulfate and magnesium chloride, the source of the Na ⁺ is one or more of sodium chloride, sodium nitrate and sodium sulfate, and the source of the Mn ²⁺ is one or more of manganese sulfate monohydrate, manganese sulfate tetrahydrate, manganese nitrate and manganese chloride.

Further, the carbon source is selected from one or more of glucose, sucrose, starch, sodium citrate or molasses.

Further, the nitrogen source is selected from one or more of yeast extract powder, peptone, corn steep liquor dry powder, ammonium sulfate or potassium nitrate.

In the practical application process, the form of the final rhodococcus ruber product can be determined according to the practical use and storage requirements, when the liquid product is required to be used, the fermentation liquor is diluted to the required concentration, and can be directly used, when the solid product is required to be used, the fermentation liquor can be centrifuged to obtain bacterial mud, and then the solid bacterial powder is prepared by adopting a freeze-drying process.

The invention also claims a method for purifying sewage and wastewater by using the activation liquid or microbial agent of rhodococcus ruber, which comprises the step of applying the activation liquid of rhodococcus ruber or the microbial agent containing rhodococcus ruber to the sewage and wastewater.

Further, the benzene series concentration in the sewage is 100mg/L or less, preferably 50mg/L or less, and most preferably 30mg/L or less.

Further, the pyridine concentration in the sewage/wastewater is 50mg/L or less, preferably 30mg/L or less.

Further, the salinity of the sewage and wastewater is 5% or less, preferably 3% or less, and most preferably 2% or less.

Further, the inoculation amount of the activation solution of rhodococcus ruber or the microbial agent is more than 100ppm, preferably 100-20000ppm, most preferably 1000-10000ppm.

The invention also claims the application of the rhodococcus ruber and the microbial agent in the field of sewage and wastewater purification.

Preferably, the rhodococcus ruber and the microbial agent are used for degrading benzene compounds and pyridine in sewage; more preferably, the benzene series is one or more of benzene, toluene, ethylbenzene, ortho-xylene, meta-xylene and para-xylene.

Drawings

FIG. 1 is a photograph of a colony of Rhodococcus ruber;

FIG. 2 is a photograph of a rhodococcus ruber colony magnified by a 100-fold microscope.

Detailed Description

The principles and features of the present invention are described below in connection with examples, which are set forth only to illustrate the present invention and not to limit the scope of the invention.

The composition of each medium used in the examples is as follows:

enrichment medium: 1g of dipotassium hydrogen phosphate, 1g of monopotassium phosphate, 1g of ammonium sulfate, 0.2g of magnesium sulfate, 1g of potassium nitrate, 0.01g of calcium chloride and 1mL of microelement solution, and fixing the volume to 1L, wherein pH=7;

basal medium: 10g of peptone, 5g of yeast powder, 10g of sodium chloride, 1000ml of water and pH=7;

Evaluation of the medium: 1g of dipotassium hydrogen phosphate, 1g of monopotassium phosphate, 1g of ammonium sulfate, 0.2g of magnesium sulfate, 1g of potassium nitrate, 0.01g of calcium chloride and 1mL of microelement solution, and adjusting the concentration of benzene, dimethylbenzene and pyridine to be 1L and pH=7 according to the requirement;

trace element solution: 1.5g of ferric chloride, 0.1g of manganese sulfate, 70mg of zinc chloride, 2mg of copper chloride, 30mg of nickel chloride, 200mg of cobalt chloride, 5mg of sodium molybdate and the volume to 1L, wherein pH=7.

EXAMPLE 1 screening separation and purification of Rhodococcus ruber

1. Water sample enrichment

Taking 100mL of a water sample from an aerobic tank of a Shandong chemical plant, inoculating 10mL of the water sample into a triangular flask filled with 100mL of enrichment medium, adding 10mg/L of benzene into the enrichment medium, performing shaking culture at 30 ℃ for 5 days at 200r/min, measuring the content of benzene every other day, and adjusting the pH; transferring the next stage of enrichment when the benzene concentration is reduced to below 50%, namely taking 10mL of culture solution after 5 days of culture, inoculating the culture solution into 100mL of enrichment medium again, increasing the benzene concentration by 10mg/L each time, and repeatedly culturing for 5 times until the benzene concentration in the enrichment medium is 50mg/L.

2. Screening and isolation of strains

Taking 1mL of the culture solution of the last stage, carrying out gradient dilution by using sterile water until the dilution is 10 ^-4,10^-5,10^-6,10^-7 and 10 ^-8 times, coating the diluted culture solution on an LB solid culture medium flat plate, placing the LB solid culture medium flat plate in a 30 ℃ incubator, observing the morphological characteristics of the bacterial colony after the bacterial colony grows to a proper size, picking a single bacterial colony for streak purification, carrying out inclined plane preservation at 4 ℃ for three generations after purification, and separating 6 bacterial strains which are named KH 1-KH 6 respectively.

3. Double screen

The 6 strains obtained by primary screening are respectively inoculated into basic culture media, shake-cultured for 24 hours at 30 ℃,10 mL of culture solution is respectively taken and centrifuged for 10 minutes at 5000rpm, the supernatant is discarded, each strain is respectively inoculated into 100mL of evaluation culture media (benzene content is 50 mg/L), shake-cultured at 30 ℃, three groups of strains are arranged in parallel in each experimental group, sterile water is used as a blank control, and the concentration change of benzene is periodically detected, and the results are shown in Table 1.

Table 1 evaluation results of benzene by the strain obtained by preliminary screening

From the data in Table 1, it is clear that the KH1 strain has the strongest benzene-removing effect, and the benzene-degrading ability is significantly better than that of the other 5 strains.

Example 2 identification of species

1. Morphological observation

Bacterial strain KH1 was inoculated on LB solid medium plates and colony morphology was observed. The colony photograph of KH1 strain is shown in FIG. 1, and the 100-fold microscopic photograph is shown in FIG. 2.KH1 colony is orange, round, opaque, rough and dry; the cell is spherical or short rod-shaped, has no flagellum, does not move and does not produce spores.

2. Molecular biological identification

Genomic DNA of strain KH1 was extracted and 16S rDNA was amplified using this as a template.

16S rDNA primer:

F:5’-CTTACACATGCAAGTCGAA-3’

R:5’-TCCCTCGGCAGCTTCCACCT-3’

the PCR products were then extracted and DNA sequenced using a sequencer ABI 3730-XL. And (3) comparing the spliced sequence file with data in an NCBI 16S database by using a NCBIBlast program to obtain species information with the greatest sequence similarity with a species to be detected, finding that the species belongs to the rhodococcus, identifying the species as rhodococcus ruber (Rhodococcus ruber), and naming the species as rhodococcus ruber KH1.

EXAMPLE 3 salinity tolerance test of Rhodococcus ruber KH1

Under the aseptic condition, the rhodococcus ruber KH1 is inoculated into a 250mL conical flask containing 100mL of basic culture medium, and is subjected to shaking culture for 24 hours at 30 ℃ and 200rpm in a shaking table to perform strain activation, so as to obtain an activated liquid of the rhodococcus ruber, and the number of viable bacteria of the activated liquid is 90 hundred million CFU/mL through test.

Based on the basal medium (salinity: 1%), naCl 0g, 1g, 2g, 3g, 4g, 5g, 6g, 7g, 8g and 9g were additionally added to the flasks containing 100mL of basal medium, respectively, and salinity gradients of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% and 10% were set. Respectively taking 5mL of rhodococcus ruber activating solution, inoculating into culture media with different salinity gradients, placing into a shaking table at 30 ℃ for shaking culture, and periodically measuring OD ₆₀₀ value and viable count to determine the growth degree of the strain, wherein the result is shown in Table 2.

TABLE 2 OD ₆₀₀ values and viable count of Rhodococcus ruber cultivated under different salinity conditions

As can be seen from the data in Table 2, rhodococcus ruber KH1 can grow normally at a salinity of 1-5%, 1% is the most suitable salinity for growth, and the strain growth is significantly better than that at a salinity of more than 5% below 5%.

Example 4 determination of degradation efficiency of Rhodococcus ruber KH1 at different benzene concentrations

Under the aseptic condition, the rhodococcus ruber KH1 is inoculated into a 250mL conical flask containing 100mL of basic culture medium, and the strain is activated by shaking culture for 24 hours in a shaking table at 30 ℃ and 200rpm, so as to obtain an activation solution of the rhodococcus ruber. The number of viable bacteria of the activating solution is 93 hundred million CFU/mL.

Evaluation media with benzene content of 200mg/L, 100mg/L, 50mg/L, 40mg/L, 30mg/L, 20mg/L and 10mg/L were prepared respectively. Rhodococcus erythropolis activating solutions were inoculated in an inoculum size of 1vol% to 100mL of sterilized evaluation medium, and then placed in shaking tables at 30℃and 200rpm for shaking culture, 3 replicates per group, and sterile water was added to the evaluation medium as a blank. The experimental group arrangement is shown in Table 3, and the benzene content in the culture medium was measured every 24 hours later, and the results are shown in Table 4.

The benzene content detection method utilizes a gas chromatograph-mass spectrometer to detect.

Table 3 evaluation of benzene content in the Medium for each experimental group

Table 4 each experimental group and control group evaluate the change in benzene content in the medium

As can be seen from the data in Table 4, rhodococcus erythropolis KH1 showed a good benzene degrading effect at benzene concentrations of 100mg/L or less. When the benzene concentration is below 50mg/L, the degradation rate of 72 hours is above 99 percent; when the benzene concentration is 100mg/L, the degradation rate of 72 hours can reach 87 percent. When the benzene concentration is 200mg/L, the degradation efficiency of rhodococcus ruber KH1 is reduced, and the benzene degradation rate is about 27% in 72 hours.

The results show that rhodococcus ruber KH1 can tolerate benzene with concentration below 100mg/L, has higher degradation efficiency, but has poor tolerance to benzene with concentration of 200mg/L and above.

Example 5 determination of degradation efficiency of Rhodococcus ruber KH1 at different xylene concentrations

Under the aseptic condition, the rhodococcus ruber KH1 is inoculated into a 250mL conical flask containing 100mL of basic culture medium, and the strain is activated by shaking culture for 24 hours in a shaking table at 30 ℃ and 200rpm, so as to obtain an activation solution of the rhodococcus ruber. The number of viable bacteria of the activating solution is 90 hundred million CFU/mL.

Evaluation media with xylene concentration of 100mg/L, 50mg/L, 40mg/L, 30mg/L, 20mg/L and 10mg/L were prepared respectively. Rhodococcus ruber activating solutions were inoculated into 100mL of sterilized evaluation medium at 1vol% of the inoculum size, and then placed in shaking tables at 30℃and 200rpm for shaking culture, 3 replicates each, sterile water was added to the evaluation medium as a blank control, the experimental group was arranged as shown in Table 5, and the xylene content in the medium was measured every 24 hours, and the results are shown in Table 6.

The detection method of the xylene content utilizes a gas chromatograph-mass spectrometer for detection.

Table 5 evaluation of xylene content in the Medium for each experimental group

Table 6 each of the experimental and control groups evaluated the change in xylene content of the medium

As is clear from the data in Table 6, red coccus KH1 showed a good effect of degrading xylene at a concentration of 50mg/L or less, and the degradation rate of red coccus KH1 was about 98% at 72 hours, but only about 12% at a concentration of 100 mg/L. The result shows that the rhodococcus ruber KH1 can tolerate less than 50mg/L of xylene, has higher degradation efficiency, but can not tolerate more than 100mg/L of xylene.

Example 6 determination of degradation efficiency of Rhodococcus ruber KH1 at different pyridine concentrations

Evaluation media with pyridine concentration of 100mg/L, 50mg/L, 40mg/L, 30mg/L, 20mg/L and 10mg/L were prepared respectively. The activated liquid was inoculated into 100mL of sterilized evaluation medium at 1vol% and then placed in shaking tables at 30℃and 200rpm, 3 replicates were set per group, sterile water was added to the evaluation medium as a blank control, the experimental group was set up as in Table 7, and the pyridine content in the medium was measured every 24 hours, and the results are shown in Table 8.

The pyridine content was detected using a gas chromatograph-mass spectrometer.

Table 7 evaluation of pyridine content in the Medium for each experimental group

TABLE 8 evaluation of the variation of pyridine content in the culture Medium for each of the experimental group and the control group

As can be seen from the data in Table 8, the rhodococcus ruber KH1 has a certain degradation effect on pyridine, and the degradation rate of the rhodococcus ruber KH1 is up to about 99% at 72h and is reduced with the increase of the pyridine concentration; when the pyridine concentration is 100mg/L, the pyridine degradation rate in 72 hours is only about 6 percent.

The results show that rhodococcus erythropolis KH1 can tolerate pyridine with the concentration below 50mg/L, has high degradation efficiency, but can not tolerate pyridine with the concentration above 100 mg/L.

Example 7 treatment efficiency of Rhodococcus ruber KH1 on raw water of chemical plant

Rhodococcus erythropolis KH1 is inoculated into a 250mL conical flask containing 100mL of basic culture medium under the aseptic condition, and is subjected to shaking culture for 24 hours at 30 ℃ and 200rpm in a shaking table to obtain an activated liquid, and the activated liquid has the viable count of 95 hundred million CFU/mL through test.

The evaluation object is raw water of a certain dyeing chemical factory in Taian city of Shandong province, the COD content before treatment is 2937mg/L, the benzene content is 661.28 mug/L, the pyridine content is 128.20 mug/L, and the pH=7.8.

100ML of the chemical raw water is respectively placed in two identical 250mL serum bottles, an experimental group is inoculated with strain activation liquid according to 1% of the volume of a water body, a control group is inoculated with sterile water with the same volume, and the sterile water is placed in a shaking table at the temperature of 30 ℃ and at 200rpm for shaking treatment, and the pH value is natural. Samples were taken every 24 hours during the reaction and the results are shown in tables 9, 10 and 11.

Wherein the COD detection method is implemented according to the technical requirement of the rapid tester of HJ 924-2017COD photometry and the detection method. Benzene and pyridine contents were detected using a gas chromatograph-mass spectrometer.

TABLE 9 variation of COD content in raw water of chemical plant with time

TABLE 10 variation of benzene content in raw water of chemical plant with time

TABLE 11 change of pyridine content in raw water of chemical plant with time

As shown by the data in tables 9-11, the COD degradation rate is 32%, the benzene degradation rate is 92% and the pyridine degradation rate is 71% within five days after the microbial inoculum is added, which indicates that the rhodococcus ruber has high-efficiency benzene and pyridine degradation capability and a certain COD degradation capability.

Example 8 fermentation production of Rhodococcus ruber KH1

The fermentation method of rhodococcus ruber comprises the following steps:

1) Picking slant seeds of rhodococcus ruber by an inoculating loop, inoculating the slant seeds into 100mL of basic culture medium, and culturing at 30 ℃ and 200rpm for 18 hours to obtain primary seed liquid;

2) Inoculating the cultured primary seed liquid into 1L of basic culture medium according to 10vol% of inoculation amount, and culturing at 30 ℃ and 200rpm for 18 hours to obtain secondary seed liquid;

3) Under the aseptic condition, inoculating the secondary seed liquid into a fermentation medium according to an inoculum size of 5-10vol%, wherein the formula of the fermentation medium is as follows: 15g/L of glucose, 10g/L of molasses, 20g/L of peptone, 10g/L of corn steep liquor dry powder, 1g/L of monopotassium phosphate, 1g/L of dipotassium phosphate, 0.5g/L of magnesium sulfate, 0.5g/L of sodium chloride and 0.1g/L of calcium chloride, fermenting under the conditions of controlling the temperature to be 25-35 ℃ and the rotating speed to be 150-300rpm and the ventilation ratio to be 1 (1-2), stopping fermenting when dissolved oxygen starts to rise, and obtaining fermentation liquor of rhodococcus ruber, wherein the viable count of the fermentation liquor is 210 hundred million CFU/mL through test.

The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the invention are intended to be included within the scope of the invention.

Claims

1. Rhodococcus ruber (Rhodococcus ruber) is characterized by being preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.27877.

2. A microbial agent comprising the rhodococcus ruber of claim 1 as an active ingredient.

3. The fermentation method of rhodococcus ruber of claim 1, which comprises the steps of: (1) first-stage seed culture: inoculating Rhodococcus ruber into enrichment medium under aseptic condition, and culturing at 25-35deg.C and 150-300rpm for 12-48 hr to obtain first seed culture solution of Rhodococcus ruber;

(3) Fermentation: and (3) after the fermentation medium in the fermentation tank is disinfected, inoculating the second-stage seed culture solution of the rhodococcus ruber obtained in the step (2) into the fermentation medium according to the inoculum size of 5-10vol%, controlling the temperature to be 25-35 ℃ and the rotating speed to be 150-300rpm, fermenting under the condition that the aeration ratio is 1 (1-2), and stopping fermenting when dissolved oxygen starts to rise to obtain the fermentation solution of the rhodococcus ruber.

4. A fermentation process according to claim 3, wherein the enrichment medium has a composition of: 5-15g/L of peptone, 3-8g/L of yeast extract or yeast powder, 5-15g/L of sodium chloride, water as solvent, and pH=6-8;

the composition of the fermentation medium is as follows: 10-30g/L of carbon source and 20-40g/L、PO₄ ^3-0.8-1.5g/L、K⁺0.5-1.0g/L、Mg²⁺0.05-0.2g/L、Na⁺0.1-0.3g/L、Mn²⁺0.03-0.1g/L, g/L of nitrogen source, wherein the solvent is water, and the pH value is=6-8.

5. The fermentation process of claim 4, wherein the carbon source is selected from one or more of glucose, sucrose, starch, sodium citrate, or molasses;

The nitrogen source is selected from one or more of yeast extract powder, peptone, corn steep liquor dry powder, ammonium sulfate or potassium nitrate.

6. A method for purifying waste water, comprising the step of applying the activation solution of rhodococcus ruber of claim 1 or the microbial agent of claim 2 to the waste water.

7. The method according to claim 6, wherein the benzene-based compound concentration in the sewage water is 100mg/L or less, preferably 50mg/L or less, most preferably 30mg/L or less.

8. A method according to claim 6 or 7, characterized in that the salinity of the sewage water is below 5%, preferably below 3%, most preferably below 2%.

9. The method according to claim 6, wherein the pyridine concentration in the sewage water is 50mg/L or less, preferably 30mg/L or less.

10. Use _; of the rhodococcus ruber of claim 1 and the microbial agent of claim 2 for degrading benzene-based compounds and pyridine in waste water is preferred in the field of waste water purification; more preferably, the benzene series is one or more of benzene, toluene, ethylbenzene, ortho-xylene, meta-xylene and para-xylene.