CN108251330B - Compound microbial agent for treating soil pollution and preparation method and application thereof - Google Patents

The invention discloses a compound microbial agent, which comprises the following raw material components: pseudomonas japonicus cultures, Comamonas testosteroni cultures, Microbacterium cultures, and Acidovorax deleterii cultures; the invention prepares the compound microbial agent by mixing and fermenting a plurality of bacterial cultures, has effective degradation function on organic residues such as nitrobenzene compounds formed by pesticides or pesticides in soil, and provides a simple and easy preparation method for the mixed culture of special strains and the mixed fermentation of a plurality of strains.

Compound microbial agent for treating soil pollution and preparation method and application thereof

Technical Field

The invention relates to a microbial agent and a preparation method and application thereof, in particular to a compound microbial agent for treating soil pollution and a preparation method and application thereof.

Background

The nitrobenzene compounds are widely applied to production of the nitrobenzene compounds of pesticides, dyes, explosives, rubber and other chemical products, can enter the environment through waste water and waste gas, and also can enter the environment in large quantity due to accidents in the transportation and production processes and improper disposal of a storage tank, the nitrobenzene pollutants in the environment mainly comprise nitrobenzene, nitrochlorobenzene, nitrotoluene, nitrophenol, nitroaniline, nitrobenzoic acid and the like, most of the nitrobenzene compounds have the characteristics of stable chemical property, high toxicity and easy biological enrichment, and are listed in the United states environmental protection agency and the national priority monitoring pollutant list.

In the prior art, the restoration method of the pollution of nitrobenzene compounds mainly comprises a physical method, a chemical method and a biological method; the physical method mainly utilizes porous resin, active carbon and the like to adsorb pollutants; the chemical method mainly utilizes oxidation reaction to eliminate pollutants; the biological method mainly utilizes artificially domesticated or constructed engineering bacteria to degrade pollutants and finally mineralize the pollutants. Compared with chemical method and physical method, the biological treatment method has the advantages of low cost, difficult secondary pollution, capability of reducing the concentration of pollutants to the maximum extent and wide application range. Meanwhile, the soil containing the nitrobenzene compounds often has coexistence of various pollutants, and the various pollutants can be removed simultaneously and the degradation of the nitrobenzene compounds is accelerated by utilizing the synergistic effect among the microorganisms and the co-metabolism effect of the microorganisms on the substrate. Therefore, with the wide application and development of the biological strengthening technology and the genetic engineering technology, the biological treatment method has wide application prospect in the aspect of preventing and treating the environmental pollution of nitrobenzene compounds.

However, the complex microorganism strains in the prior art have poor treatment effect on nitrobenzene compounds, complex preparation process and easy loss of the strains, thereby causing resource waste.

Disclosure of Invention

The purpose of the invention is as follows: the first purpose of the invention is to provide a compound microbial agent.

The second purpose of the invention is to provide a preparation method of the compound microbial agent.

The third purpose of the invention is to provide the application of the compound microbial agent.

The technical scheme is as follows: in order to realize the aim, the invention provides a compound microbial agent, which comprises the following raw material components: pseudomonas japonicus cultures, Comamonas testosteroni cultures, Microbacterium cultures, and Acidovorax deleterii cultures.

Wherein the ratio of the Pseudomonas japonicus culture to the Comamonas testosteroni culture to the Microbacterium culture to the Acidovorax deleterii culture is 1 (2-3): (1.5-2.5): (3-4).

Preferably, the Pseudomonas japonicus culture is Pseudomonas japonicus ATCC 33616 or ATCC33660 culture; the comamonas testosteroni culture is ATCC 700441 or ATCC 39523 culture; the microbacterium culture is an ATCC 31001 culture; the acidovorax deleteris culture is DSM 50403 or ATCC 49664 culture.

Further, the raw material components of the compound microbial agent also comprise a bacillus subtilis culture, a nitrogen-fixing actinomycete culture, a rhodobacter sphaeroides culture and a candida culture.

Wherein the Bacillus subtilis culture is Bacillus subtilis ATCC 21556 culture; the azotobacter culture is an azotobacter ATCC 33255 culture; the rhodobacter sphaeroides culture is rhodobacter sphaeroides ATCC 49419, ATCC 17023, ATCC 33575 culture; the Candida culture is Candida CICC 33050, NYNU 14772 culture.

Preferably, the proportions of the culture of Pseudomonas japonicus, Comamonas testosteroni, Microbacterium, Acidovorax deleterii, Bacillus subtilis, Actinomyces azotobacter, Rhodococcus sphaeroides and Candida are: 1 (2-3) (1.5-2.5), (3-4), (2.5-3.5), (0.5-1), (1-1.5) and (2-3).

A preparation method of a compound microbial agent comprises the following steps:

(1) carrying out mixed culture on a pseudomonas stutzeri culture, a comamonas testosteroni culture, a microbacterium culture and an acidovorax deleterii culture according to parts by weight by adopting a mixed culture medium A to obtain a mixed culture A;

(2) adding a nitrobenzene compound into the mixed culture A obtained in the step (1) to obtain a domesticated mixed culture B;

(3) respectively inoculating the domesticated mixed culture B obtained in the step (2) and a bacillus subtilis culture, a nitrogen-fixing actinomycete culture, a rhodobacter sphaeroides culture and a candida culture in the raw material components into a primary culture box according to parts by weight, carrying out aeration culture to obtain a primary bacterial liquid, and then inoculating the primary bacterial liquid into a secondary culture box to obtain a secondary bacterial liquid;

(4) and (4) uniformly mixing the secondary bacterial liquid of each strain obtained in the step (3) to prepare a bacterial suspension, and controlling the number of effective viable bacteria to be more than or equal to 1.5 multiplied by 1010/g to obtain the compound microbial agent.

In the step (1), the obtaining mode of each strain culture is as follows: inoculating each strain in the raw material components into a slant culture medium, and performing amplification culture respectively to obtain a culture of each strain;

preferably, the slant culture medium and the culture conditions are respectively: pseudomonas japonicus ATCC 33616 or ATCC33660 cultures: nutrient gravy agar medium, pH 6.8 +/-0.2, culture temperature: 28 ℃; comamonas testosteroni ATCC 700441 or ATCC 39523 cultures: nutrient Agar (NA), pH 6.8 +/-0.2, and culture temperature of 30.0 ℃; microbacterium ATCC 31001 culture: nutrient gravy agar, pH 6.8 ± 0.2, culture temperature: 37 ℃; cultures of acidovorax delemar DSM 50403 or ATCC 49664: nutrient Agar (NA), pH 6.8 +/-0.2, and culture temperature of 30.0 ℃; bacillus subtilis ATCC 21556 culture: nutrient Agar (NA) plus 1% potato starch, pH 6.8 ± 0.2 culture temperature: 37 ℃; azotobacter ATCC 33255 cultures: 130g/L of malt extract powder, 15g/L of agar, 0.1g/L of chloramphenicol, pH 6.0 +/-0.2, and culture temperature of 28.0 ℃; rhodobacter sphaeroides ATCC 49419, ATCC 17023, ATCC 33575 cultures: a vannei yeast agar culture medium, the pH value is 7.0 +/-0.2, and the culture temperature is 24.0 ℃; candida CICC 33050, NYNU 14772 cultures: YCM medium, culture temperature: at 25 ℃.

In the step (1), the mixed culture medium A comprises the following components in component concentration: 8-12 g/L tryptone, 10-20 g/L soytone and 2-5 g/L, H g of beef extract₃BO₃ 2～5g/L、NaH₂PO₄·H₂O2-4 g/L, NaCl 5-8 g/L, agar 10-20 g/L and MnCl₂0.2-0.3 g/L of 4H2O 0.2, and a pH value of 6.5-6.8.

Preferably, in the step (2), the step of acclimatization is to add a nitrobenzene compound into the culture medium of the mixed culture A in the logarithmic phase, and subculture the mixed culture A in 2-3 days, 4-6 days and 7-9 days respectively to ensure that the density of bacteria reaches 4 × 106-5 × 106/ml and the survival rate of the bacteria is more than or equal to 85%.

Further, in the step (3), inoculating the strain into a primary culture box, performing aeration culture to obtain primary bacterial liquid, and then inoculating the primary bacterial liquid into a secondary culture box, wherein the specific steps are as follows: inoculating the culture medium into a 5-8L culture box according to the inoculation amount of 2-4% of the culture medium, wherein the temperature is 20-25 ℃, the pH of the solution is neutral, carrying out aeration culture for 40h, and then transferring 5-8L of primary bacterial liquid into a secondary culture box to obtain 20-30L of secondary bacterial liquid; wherein, the components of the culture medium are as follows: corn flour 10g/L, glucose 5g/L, bean cake powder 10g/L, fish meal 7g/L, CaCO g/L₃ 7g/L、(NH₄)₂SO₄ 10g/L、K₂HPO₄ 0.4g/L、 MgSO₄.7H₂O0.3 g/L and MnSO₄·H₂O 0.2g/L。

Preferably, in step (4), the bacterial suspension is prepared by the following method: sucking 3.0-5.0 ml of diluent by using a 5.0ml suction tube, adding the diluent into the culture medium of each strain culture, repeatedly blowing and sucking, and washing off the lawn; subsequently, the wash solution was transferred to another sterile test tube with a 5.0ml pipette, and mixed for several seconds with an electric mixer or shaken several times on the palm to suspend each seed culture uniformly to prepare a mixed bacterial culture suspension.

The invention also discloses the application of the compound microbial agent in treating soil pollution.

Further, the application of the compound microbial agent in soil pollution treatment is the application of the compound microbial agent in soil polluted by nitrobenzene compounds.

Has the advantages that: compared with the prior art, the composite microbial agent is prepared by mixing and fermenting a plurality of bacterial cultures, has effective degradation effect on organic residues such as nitrobenzene compounds formed by pesticides or pesticides in soil, and provides a simple and feasible preparation method for the mixed culture of special strains and the mixed fermentation of a plurality of strains.

Detailed Description

The technical solution of the present invention will be described in detail below, and the following raw materials are commercially available unless otherwise specified.

Example 1

The raw material components are as follows: the ratio of the culture of Pseudomonas japonicus ATCC 33616, the culture of Comamonas testosteroni ATCC 700441, the culture of Microbacterium ATCC 31001 and the culture of Acidovorax deleterii DSM 50403 was 1:2:1.5: 3.

Preparation and application: (1) performing mixed culture on a pseudomonas stutzeri culture, a comamonas testosteroni culture, a microbacterium culture and an acidovorax deleterii culture by using a mixed culture medium A according to parts by weight to obtain a mixed culture A, wherein the mixed culture medium A comprises the following components in component concentration: tryptone 8g/L, soytone 10g/L, beef extract 2g/L, H g₃BO₃ 2g/L、NaH₂PO₄·H₂O2g/L, NaCl 5g/L, agar 10g/L and MnCl₂.4H₂O is 0.2g/L, and the pH value is 6.5;

(2) adding nitrobenzene compounds into the mixed culture A in the step (1) to obtain a domesticated mixed culture B, wherein the domestication step is to add the nitrobenzene compounds into the culture medium of the mixed culture A, and subculture the mixed culture A at 2 days, 4 days and 7 days respectively to ensure that the density of bacteria reaches 4 multiplied by 10⁶The survival rate of the strain is 85 percent per ml.

(3) Inoculating the domesticated mixed culture B obtained in the step (2) into a primary incubator, carrying out aeration culture to obtain primary bacterial liquid, and then inoculating the primary bacterial liquid into a secondary incubator.

(4) Uniformly mixing the fermentation liquor obtained in the step (3) to prepare bacterial suspension, and controlling the effective viable count to be 1.5 multiplied by 10¹⁰And (5) obtaining the compound microbial agent.

Example 2

The raw material components are as follows: the ratio of the culture of Pseudomonas japonicus ATCC33660, the culture of Comamonas testosteroni ATCC 39523, the culture of Microbacterium ATCC 31001 and the culture of Acidovorax deleterii ATCC 49664 was 1:2.5:2: 3.5.

Preparation and application: (1) performing mixed culture on a pseudomonas stutzeri culture, a comamonas testosteroni culture, a microbacterium culture and an acidovorax deleterii culture by using a mixed culture medium A according to parts by weight to obtain a mixed culture A, wherein the mixed culture medium A comprises the following components in component concentration: tryptone 10g/L, soytone 15g/L, beef extract 3g/L, H g₃BO₃4g/L、NaH₂PO₄·H₂O3 g/L, NaCl 7g/L, agar 15g/L and MnCl₂.4H₂O is 0.25g/L, and the pH value is 6.7;

(2) adding nitrobenzene compounds into the mixed culture A in the step (1) to obtain a domesticated mixed culture B, wherein the domestication step is to add the nitrobenzene compounds into the culture medium of the mixed culture A, and subculture the mixture respectively at 3 days, 5 days and 8 days to ensure that the density of the bacteria reaches 4.5 multiplied by 10⁶The survival rate of the strain is 90 percent per ml;

(3) inoculating the domesticated mixed culture B obtained in the step (2) into a primary incubator, carrying out aeration culture to obtain primary bacterial liquid, and then inoculating the primary bacterial liquid into a secondary incubator;

(4) uniformly mixing the fermentation liquor obtained in the step (3) to prepare bacterial suspension, and controlling the effective viable count to be 2 multiplied by 10¹⁰And (5) obtaining the compound microbial agent.

Example 3

The raw material components are as follows: the ratio of the culture of Pseudomonas japonicus ATCC33660, the culture of Comamonas testosteroni ATCC 39523, the culture of Microbacterium ATCC 31001 and the culture of Acidovorax deleterii ATCC 49664 was 1:3:2.5: 4.

Preparation and application: (1) performing mixed culture on a pseudomonas stutzeri culture, a comamonas testosteroni culture, a microbacterium culture and an acidovorax deleterii culture by using a mixed culture medium A according to parts by weight to obtain a mixed culture A, wherein the mixed culture medium A comprises the following components in component concentration: tryptone 12g/L, soytone 20g/L, beef extract 5g/L, H g₃BO₃ 5g/L、NaH₂PO₄·H₂O4g/L, NaCl 8g/L, agar 20g/L and MnCl₂.4H₂O is 0.3g/L, and the pH value is 6.8;

(2) adding nitrobenzene compounds into the mixed culture A in the step (1) to obtain a domesticated mixed culture B, wherein the domestication step is to add the nitrobenzene compounds into the culture medium of the mixed culture A, and subculture the mixture respectively at 3 days, 6 days and 9 days to ensure that the density of the bacteria reaches 5 multiplied by 10⁶The survival rate of the strain is 90 percent per ml;

(3) inoculating the domesticated mixed culture B obtained in the step (2) into a primary incubator, carrying out aeration culture to obtain primary bacterial liquid, and then inoculating the primary bacterial liquid into a secondary incubator;

(4) uniformly mixing the fermentation liquor obtained in the step (3) to prepare bacterial suspension, and controlling the effective viable count to be 2.0 multiplied by 10¹⁰And (5) obtaining the compound microbial agent.

Example 4

The raw material components are as follows: the proportions of the culture of Pseudomonas japonicus ATCC33660, the culture of Comamonas testosteroni ATCC 39523, the culture of Microbacterium ATCC 31001, the culture of Acidovorax deleterii ATCC 49664, the culture of Bacillus subtilis ATCC 21556, the culture of Actinomyces azotobacter ATCC 33255, the culture of Rhodococcus rhodochrous ATCC 49419 and the culture of Candida CICC 33050 are: 1:2:1.5:3:2.5:0.5:1:2.

Preparation and application: (1) performing mixed culture on a pseudomonas stutzeri culture, a comamonas testosteroni culture, a microbacterium culture and an acidovorax deleterii culture by using a mixed culture medium A according to parts by weight to obtain a mixed culture A, wherein the mixed culture medium A comprises the following components in component concentration: tryptone 8g/L, soytone 10g/L, beef extract 2g/L, H g₃BO₃ 2g/L、NaH₂PO₄·H₂O2g/L, NaCl 5g/L, agar 10g/L and MnCl₂.4H₂O is 0.2g/L, and the pH value is 6.5;

(2) adding nitrobenzene compounds into the mixed culture A in the step (1) to obtain a domesticated mixed culture B, wherein the domestication step is to add the nitrobenzene compounds into the culture medium of the mixed culture A, and subculture the mixed culture A at 2 days, 4 days and 7 days respectively to ensure that the density of bacteria reaches 4 multiplied by 10⁶The survival rate of the strain is 85 percent per ml;

(3) respectively inoculating the domesticated mixed culture B obtained in the step (2) and a bacillus subtilis culture, a nitrogen-fixing actinomycete culture, a rhodobacter sphaeroides culture and a candida culture in the raw material components into a primary culture box according to parts by weight, carrying out aeration culture to obtain a primary bacterial liquid, and then inoculating the primary bacterial liquid into a secondary culture box to obtain a secondary bacterial liquid;

(4) uniformly mixing the fermentation liquor obtained in the step (3) to prepare bacterial suspension, and controlling the effective viable count to be 1.5 multiplied by 10¹⁰And (5) obtaining the compound microbial agent.

Example 5

The raw material components are as follows: the proportions of the culture of Pseudomonas japonicus ATCC 33616, the culture of Comamonas testosteroni ATCC 700441, the culture of Microbacterium ATCC 31001, the culture of Acidovorax deleterii DSM 50403, the culture of Bacillus subtilis ATCC 21556, the culture of Actinomyces azotobacter ATCC 33255, the culture of Rhodococcus rhodochrous ATCC 17023 and the culture of Candida NYNU 14772 are: 1:2.5:2:3.5:3:0.75:1.25:2.5.

Preparation and application:(1) performing mixed culture on a pseudomonas stutzeri culture, a comamonas testosteroni culture, a microbacterium culture and an acidovorax deleterii culture by using a mixed culture medium A according to parts by weight to obtain a mixed culture A, wherein the mixed culture medium A comprises the following components in component concentration: tryptone 10g/L, soytone 15g/L, beef extract 4g/L, H g₃BO₃ 3g/L、NaH₂PO₄·H₂O3 g/L, NaCl 7g/L, agar 15g/L and MnCl₂.4H₂O is 0.25g/L, and the pH value is 6.7;

(2) adding nitrobenzene compounds into the mixed culture A in the step (1) to obtain a domesticated mixed culture B, wherein the domestication step is to add the nitrobenzene compounds into the culture medium of the mixed culture A, and subculture the mixed culture A at 2 days, 5 days and 8 days respectively to ensure that the density of bacteria reaches 4.5 multiplied by 10⁶The survival rate of the strain is 90 percent per ml;

(3) respectively inoculating the domesticated mixed culture B obtained in the step (2) and a bacillus subtilis culture, a nitrogen-fixing actinomycete culture, a rhodobacter sphaeroides culture and a candida culture in the raw material components into a primary culture box according to parts by weight, carrying out aeration culture to obtain a primary bacterial liquid, and then inoculating the primary bacterial liquid into a secondary culture box to obtain a secondary bacterial liquid;

(4) uniformly mixing the fermentation liquor obtained in the step (3) to prepare bacterial suspension, and controlling the effective viable count to be 2.0 multiplied by 10¹⁰And (5) obtaining the compound microbial agent.

Example 6

The raw material components are as follows: the proportions of the culture of Pseudomonas japonicus ATCC 33616, the culture of Comamonas testosteroni ATCC 700441, the culture of Microbacterium ATCC 31001, the culture of Acidovorax deleterii DSM 50403, the culture of Bacillus subtilis ATCC 21556, the culture of Actinomyces azotobacter ATCC 33255, the culture of Rhodococcus rhodochrous ATCC 33575 and the culture of Candida NYNU 14772 are: 1:3:2.5:4:3.5:1:1.5:3.

Preparation and application: (1) mixed culture is carried out on the culture of the pseudomonas, the culture of the comamonas testosteroni, the culture of the microbacterium and the culture of the acidovorax deleterii according to the parts by weight by adopting a mixed culture medium A to obtain mixed cultureSubstance A, mixed medium A contains the following components in component concentration: tryptone 12g/L, soytone 20g/L, beef extract 5g/L, H g₃BO₃ 5g/L、NaH₂PO₄·H₂O4g/L, NaCl 8g/L, agar 20g/L and MnCl₂.4H₂O is 0.3g/L, and the pH value is 6.8;

(2) adding nitrobenzene compounds into the mixed culture A in the step (1) to obtain a domesticated mixed culture B, wherein the domestication step is to add the nitrobenzene compounds into the culture medium of the mixed culture A, and subculture the mixture respectively at 3 days, 6 days and 9 days to ensure that the density of the bacteria reaches 5 multiplied by 10⁶The strain/ml and the bacterium survival rate is 93 percent;

(3) respectively inoculating the domesticated mixed culture B obtained in the step (2) and a bacillus subtilis culture, a nitrogen-fixing actinomycete culture, a rhodobacter sphaeroides culture and a candida culture in the raw material components into a primary culture box according to parts by weight, carrying out aeration culture to obtain a primary bacterial liquid, and then inoculating the primary bacterial liquid into a secondary culture box to obtain a secondary bacterial liquid;

(4) uniformly mixing the fermentation liquor obtained in the step (3) to prepare bacterial suspension, and controlling the effective viable count to be 2.5 multiplied by 10¹⁰And (5) obtaining the compound microbial agent.

Comparative example 1

The raw material components are as follows: the ratio of the bacillus subtilis ATCC 21556 culture, the azotobacter actinomyces ATCC 33255 culture, the rhodobacter sphaeroides ATCC 33575 culture and the candida candidas NYNU 14772 culture is 3.5:1:1.5: 3.

Preparation and application: other specific steps and implementation conditions are the same as those of example 6.

Comparative example 2

The raw material components are as follows: the ratio of the comamonas testosteroni ATCC 700441 culture, the microbacterium testosteroni ATCC 31001 culture, the acidovorax deleteriae DSM 50403 culture, the bacillus subtilis ATCC 21556 culture, the actinomyces azotobacter ATCC 33255 culture, the rhodobacter sphaeroides ATCC 33575 culture and the candida candidus NYNU 14772 culture was 3:2.5:4:3.5: 1.5: 3. (lack of Pseudomonas culture in Japan)

Preparation and application: other specific steps and implementation conditions are the same as those of example 6.

Comparative example 3

The raw material components are as follows: the proportions of the culture of Pseudomonas japonicus ATCC 33616, the culture of Microbacterium ATCC 31001, the culture of Acidovorax delemar DSM 50403, the culture of Bacillus subtilis ATCC 21556, the culture of Actinomyces azotobacter ATCC 33255, the culture of Rhodococcus rhodochrous ATCC 33575 and the culture of Candida NYNU 14772 are as follows: 1: 2.5:4:3.5:1:1.5:3. (Co. comamonas Leontolor cultures lacking testosterone)

Preparation and application: other specific steps and implementation conditions are the same as those of example 6.

Comparative example 4

The raw material components are as follows: the proportions of the culture of Pseudomonas japonicus ATCC 33616, the culture of Comamonas testosteroni ATCC 700441, the culture of Acidovorax deleterii DSM 50403, the culture of Bacillus subtilis ATCC 21556, the culture of Actinomyces azotobacter ATCC 33255, the culture of Rhodococcus rhodochrous ATCC 33575 and the culture of Candida NYNU 14772 are: 1:3:4:3.5:1:1.5:3. (lack of Microbacterium culture)

Preparation and application: other specific steps and implementation conditions are the same as those of example 6.

Comparative example 5

The raw material components are as follows: the proportions of the culture of Pseudomonas japonicus ATCC 33616, the culture of Comamonas testosteroni ATCC 700441, the culture of Microbacterium ATCC 31001, the culture of Bacillus subtilis ATCC 21556, the culture of Actinomyces azotobacter ATCC 33255, the culture of Rhodococcus sphaeroides ATCC 33575 and the culture of Candida NYNU 14772 are: 1:3:2.5:3.5:1:1.5:3. (lack of culture of acidovorax delemar.)

Preparation and application: other specific steps and implementation conditions are the same as those of example 6.

The complex microbial agents of examples 1 to 6 and comparative examples 1 to 5 were used to treat the pollutants in the soil contaminated by the nitrobenzene compounds, and the results of the detection of the nitrobenzene compounds in the soil are shown in table 1:

TABLE 1 influence of composite microbial agents of different raw material components on the degradation rate of nitrobenzene compounds in contaminated soil

As can be seen from Table 1, comparative example 1 shows that only Bacillus subtilis culture, azotobacter culture, rhodobacter sphaeroides culture and Candida culture have a small effect of about 9.54% on the nitrobenzene compounds in contaminated soil; comparative examples 2 to 5 show that when the complex microbial agent applied to soil lacks a culture of Pseudomonas japonicus, a culture of Comamonas testosteroni, a culture of Microbacterium or a culture of Acidovorax delemar, respectively, the degradation rate of the contaminant nitrobenzene compound does not exceed 40.57%; examples 1-3 show that when only a bacillus subtilis culture, a nitrogen-fixing actinomycete culture, a rhodobacter sphaeroides culture and a candida culture jointly act on polluted soil, the nitrobenzene compound can be well degraded, and the degradation effect is more than 72.44%; in particular, in examples 4 to 6, when the culture of Pseudomonas, Comamonas testosteroni, Microbacterium, Acidovorax deleterii, Bacillus subtilis, Actinomyces azotobacter, Rhodococcus sphaeroides and Candida mycoides act on the contaminated soil together, the degradation rate of the nitrobenzene compound is excellent and is at least 80%; among them, example 6 has the best effect of degrading nitrobenzene compounds, and the highest degradation rate of nitrobenzene compounds can reach 88.51%. Therefore, the raw material components in the compound microbial agent are absent and can play a non-negligible role in treating the nitrobenzene compound of the soil pollutant.