Heavy metal-tolerant phosphate-solubilizing Leclercia adcarboxamide YZ30 and application thereof

Abstract

The invention discloses a heavy metal-tolerant phosphate-solubilizing Leclercia adcarboxcarboxy YZ30 strain and application thereof, and belongs to the field of microorganisms, the strain is preserved in the China Center for Type Culture Collection (CCTCC), and the preservation number is CCTCC NO: M 20211025. The phosphorus solubilizing amount of the strain to insoluble inorganic phosphorus (calcium phosphate) reaches 705.82 mg / L, the phosphorus solubilizing amount to insoluble organic phosphorus (calcium phytate and calcium and magnesium phytate) reaches 698.89 mg / L and 688.48 mg / L, and the strain has high tolerance to heavy metals such as zinc, cadmium and chromium and can keep high phosphorus solubilizing amount under the maximum tolerance concentration of 1050 mg / kg (zinc), 450 mg / kg (cadmium) and 1248 mg / kg (chromium). In addition, the compound has a certain degradation effect on aniline, and the degradation rate is 32.94%. The strain has relatively strong phosphate solubilizing capacity and heavy metal tolerance, can be used for repairing and reclaiming heavy metal polluted land, and has a very wide application prospect.

Description

Technical Field

[0001] The invention belongs to the field of microorganisms, and in particular relates to a heavy metal-tolerant, phosphate-solubilizing, non-decarboxylating Luxenbergia strain YZ30 and an application thereof. Background Art

[0002] Among the macroelements required by plants, phosphorus plays an indispensable role and is closely related to protein synthesis, cell division and growth. It is also a component of many coenzymes and AMP, ADP and ATP, and participates in carbohydrate metabolism and transportation, photosynthesis, respiration and other processes. Phosphorus also promotes the growth of plant roots and improves the plant's resistance to stress and disease in the growth environment.

[0003] Plants directly absorb and utilize phosphorus in the soil in two forms: HPO4 2- and H2PO4 - These two forms of phosphorus are called "available phosphorus", accounting for about 1 / 20 of the total phosphorus in the soil. The vast majority of the other phosphorus is fixed by calcium, aluminum, iron ions and soil clay, forming ineffective phosphorus that cannot be directly absorbed and utilized by plants. The main solution to phosphorus deficiency in cultivated land has always been to apply phosphorus fertilizer, but less than 30% of the phosphorus fertilizer entering the soil is absorbed and utilized by plants. Most of the phosphorus is fixed by the soil and is difficult to absorb. The low utilization rate of phosphorus fertilizer by plants leads to a large amount of phosphorus enrichment in the soil, causing the soil quality to deteriorate, and entering surface water bodies such as rivers and lakes and groundwater systems through precipitation, runoff, and leakage, causing potential harm to the environment. Especially in recent years, with the increasing demand for high-yield crops, land resources are facing great pressure, and the demand for phosphorus fertilizers has further increased. As a non-renewable resource, phosphorus has limited reserves. Therefore, how to utilize and develop ineffective phosphorus in the soil, reduce the application of phosphorus fertilizers, and avoid land pollution is one of the problems that need to be solved urgently.

[0004] Among soil microorganisms, there is a class of microorganisms that can convert ineffective phosphorus that is difficult to absorb and utilize into a form that can be absorbed and utilized by plants. They are collectively called phosphate solubilizing microorganisms, and their efficient and environmentally friendly increase in the content of available phosphorus in the soil has been recognized by the world. In the more than 100 years since the discovery of phosphate solubilizing bacteria, researchers have come a long way from screening strains that degrade a single insoluble phosphorus source to strains that degrade multiple insoluble phosphorus sources, and then to strains that are highly resistant to heavy metals. It is particularly important to screen efficient phosphate solubilizing bacteria that tolerate heavy metals. It is not only beneficial to plant growth and enhance plant resistance, but also has a positive role in promoting the restoration of contaminated land and the reclamation of mining land.

[0005] CN 107841472 A discloses a non-decarboxylating Leclercia with phosphate solubilizing ability and its application. The strain obtained by screening in the patent has a stable activation effect on insoluble phosphorus, and can survive and function under a high heavy metal pressure environment. After the non-decarboxylating Leclercia (Leclerciaadecarboxylata) is cultured in a tricalcium phosphate liquid medium for 24 hours, the soluble phosphorus content is increased from 5mg / L to 158mg / L, and the insoluble tricalcium phosphate is continuously dissolved in the subsequent growth process. After 7 days of culture, the soluble phosphorus content reaches 218mg / L. Although the strain has the ability to solubilize phosphorus under heavy metal stress, the phosphorus solubilizing ability of the strain is not high. Summary of the invention

[0006] The technical problem to be solved by the present invention is: how to provide a strain that is tolerant to heavy metals and has excellent phosphate solubilizing ability.

[0007] The technical solution of the present invention is: a strain of non-decarboxylative Leckercia (Leckercia adecarboxylata) YZ30, which is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: M 20211025.

[0008] A microbial agent containing the above-mentioned Leckercia adecarboxylata YZ30.

[0009] The application of the non-decarboxylating Leckercia (Leckercia adecarboxylata) YZ30 or microbial agent in phosphate solubilization.

[0010] Furthermore, the dephosphorization is to dissolve poorly soluble calcium phosphate, calcium phytate or calcium magnesium phytate into soluble phosphorus.

[0011] The application of the non-decarboxylative Leckercia (Leckercia adecarboxylata) YZ30 or microbial agent in dephosphorization of heavy metal contaminated soil.

[0012] Furthermore, the dephosphorization is to dissolve poorly soluble calcium phosphate, calcium phytate or calcium magnesium phytate into soluble phosphorus.

[0013] Furthermore, the heavy metal is zinc, cadmium or chromium.

[0014] The application of the non-decarboxylating Leckercia (Leckercia adecarboxylata) YZ30 or microbial agent in degrading aniline.

[0015] Furthermore, the aniline degradation is to inoculate non-decarboxylating Leckercia adecarboxylata YZ30 or a microbial agent into the aniline-contaminated soil to degrade the aniline in the soil and thus repair the contaminated soil.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] (1) The strain of the present invention has a strong ability to solubilize insoluble inorganic phosphorus (calcium phosphate) and organic phosphorus (calcium phytate and calcium magnesium phytate), and the available phosphorus in its solution reaches 705.82 mg / L, 698.89 mg / L and 688.48 mg / L, respectively. Calcium phosphate, calcium phytate and calcium magnesium phytate are the main components of insoluble phosphorus in the soil and have great development and utilization value.

[0018] (2) Through the development of the characteristics of the strain, the strain can tolerate higher heavy metal concentrations, and its maximum tolerance is from high to low: chromium (24mM / L), zinc (16mM / L), cadmium (4mM / L). At the maximum tolerance concentration, the phosphate solubilization ability is from high to low: zinc (325.29mg / L), cadmium (301.02mg / L), chromium (231.65mg / L). Compared with the heavy metal content standards for agricultural land, the strain's tolerance to chromium, cadmium, and zinc concentrations far exceeds the regulatory standards. Therefore, the strain can be used for the reclamation and remediation of soils with excessive chromium, cadmium, and zinc heavy metals, and has high application value and prospects.

[0019] (3) The analysis of the metabolic pathway of the bacterium revealed that the bacterium has a metabolic pathway for degrading aniline. After culturing the strain with aniline as the carbon source, the aniline degradation rate was 32.94% after 72 hours of cultivation. The strain can be used as a remediation strain for aniline-contaminated land and has great development value.

[0020] Collection information:

[0021] Leckercia adecarboxylata YZ30 was deposited in the China Type Culture Collection, Wuhan University, Wuhan, China on August 13, 2021, with the deposit number CCTCC NO: M 20211025. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the phylogenetic tree of non-decarboxylating Luxenbergia YZ30;

[0023] Figure 2 is the phosphate solubilization ability of non-decarboxylating Luxenbergia sp. YZ30 to different phosphorus sources;

[0024] Figure 3The phosphate solubilizing ability of non-decarboxylating Luxenbergia sp. YZ30 to calcium phosphate under zinc stress;

[0025] Figure 4 The OD value changes of non-decarboxylating Luxenbergia YZ30 under zinc stress;

[0026] Figure 5 The phosphate solubilizing ability of non-decarboxylating Luxenbergia sp. YZ30 to calcium phosphate under cadmium stress;

[0027] Figure 6 The OD value changes of non-decarboxylating Luxenbergia YZ30 under cadmium stress;

[0028] Figure 7 The phosphate solubilization ability of non-decarboxylating Luxenbergia sp. YZ30 to calcium phosphate under chromium stress;

[0029] Figure 8 The OD value changes of non-decarboxylating Luxenbergia YZ30 under chromium stress;

[0030] Fig. 9 is the phosphorus standard curve;

[0031] Fig.10 is the aniline standard curve;

[0032] Fig.11 is the aniline concentration and the growth concentration of non-decarboxylating Luxenbergia. DETAILED DESCRIPTION

[0033] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from commercial channels unless otherwise specified.

[0034] Example 1 Isolation, Identification and Preservation of Non-decarboxylating Luxenbergia

[0035] The garlic planting soil from the Thousand Mu Garlic Demonstration Park in Yushan Town, Jinxiang County, Jining City, Shandong Province was collected as experimental materials for the screening of phosphate-solubilizing bacteria. The best growing strains were selected and cultured to the logarithmic phase, and 5 ml of bacterial solution was collected by centrifugation. The genomic DNA was amplified by PCR using the universal primers for bacterial 16S rRNA amplification (27F: 5'-AGAGTTTGA TCCTGGCTCAG-3' and 1492R: 5'-GGTTACCTTGTTACGACTT-3').

[0036] The PCR reaction system is:

[0037] 45 μl MIX, 2 μl primer 27F, 2 μl primer 1429R and 1 μl genomic DNA template.

[0038] The PCR reaction conditions were:

[0039] Pre-denaturation at 98°C for 3 min, denaturation at 98°C for 10 s, annealing at 55°C for 15 s, extension at 72°C for 30 s, 34 cycles, final extension at 72°C for 5 min, and storage at 4°C.

[0040] DNA band detection was performed using 1.2% agarose gel electrophoresis. The specific steps are as follows:

[0041] (1) Accurately weigh 1.2 g of agarose into a 250 ml Erlenmeyer flask, add 100 ml of diluted 1×TAE buffer, and heat in a microwave oven until boiling twice.

[0042] (2) Take out the gel solution and wait for it to cool to about 50°C, add 2 μl of ethidium bromide, and shake thoroughly.

[0043] (3) Pour the gel solution into the gel plate and insert a comb with a suitable pore size into the gel solution.

[0044] (4) After standing for about 20 minutes, when the gel is completely solidified, remove the comb, place it in the electrophoresis tank, and inject an appropriate amount of TAE buffer to cover the surface of the gel.

[0045] (5) Take 3 μl of 10× Loading buffer in a PCR reaction tube, mix it thoroughly with the amplified product, take 5 μl each time and add it to the spotting well, and then spot 5 μl of DNA Maker (molecular weight 100-2000).

[0046] (6) After turning on the power, perform electrophoresis at a constant voltage of 120 V for about 25 minutes.

[0047] (7) Take out the gel block and observe whether the band size is correct under ultraviolet light.

[0048] After the DNA band is correct, the gel band is cut out and purified and recovered using a DNA gel recovery kit, and the recovered DNA fragment is sent to Qingke Biotechnology for 16S rRNA sequencing. The sequence measured is shown in SEQ ID No.1.

[0049] The BLAST program of the NCBI database was used to compare the homology of the 16S rRNA gene sequences obtained by sequencing. The top 20 16S rRNA sequences with the highest homology were selected as reference sequences. After multiple sequence alignment using Muscle software, the phylogenetic tree of strain YZ30 was constructed using Maga5.1 software (see Figure 2). Figure 1As shown). The results show that the strain is more than 99% homologous to non-decarboxylative Leckercia. Therefore, it is determined that the present invention uses non-decarboxylative Leckercia (Leckerciaadecarboxylata), specifically named non-decarboxylative Leckercia Leckercia adecarboxylata YZ30, which was deposited in the China Center for Type Culture Collection on August 13, 2021, with a deposit number of CCTCC NO: M 20211025.

[0050] Example 2 Phosphate solubilization of different insoluble phosphorus sources by non-decarboxylating Luxenbergia

[0051] 1. Seed preparation

[0052] Streak strain YZ30 onto enriched solid medium. After colonies grow, select colonies with good growth and inoculate them into test tubes. Culture overnight and then inoculate 1% of the inoculum into 50 ml LB liquid medium. When OD 600 When the value reaches about 0.8, take the corresponding amount of 0.5 ml of bacterial liquid according to the amount of culture medium, centrifuge it, remove the culture medium, add 0.5 ml of sterile water to suspend it to make seed liquid, and add 0.5 ml of seed liquid to each 50 ml of insoluble phosphorus-containing liquid culture medium, and culture it in an air bath constant temperature oscillator at 30°C and 120r / min.

[0053] The enrichment culture medium is: beef extract 3g / L, peptone 10g / L, sodium chloride 5g / L, agar 20g / L; the insoluble phosphorus-containing liquid culture medium is: insoluble phosphate (calcium phosphate, calcium phytate, calcium magnesium phytate) 5g / L, glucose 10g / L, potassium chloride 0.5g / L, sodium chloride 0.3g / L, ammonium sulfate 0.5g / L, magnesium sulfate 0.3g / L, ferrous sulfate 0.03g / L, manganese sulfate 0.005g / L.

[0054] 2. Data determination

[0055] After the secondary inoculation, the culture was continued for 120 hours. The fermentation flask was taken out every 12 hours and placed on the clean bench. 250 μl of fermentation liquid was taken from each flask and placed in an EP tube. After centrifugation at 10,000 r / min for 5 minutes, 100 μl of the supernatant was taken for determination. The determination principle is: under neutral conditions, potassium persulfate (or nitric acid, perchloric acid) is used to digest the sample and all the phosphorus contained is oxidized to orthophosphate. In an acidic medium, orthophosphate reacts with ammonium molybdate to generate phosphoric acid heteropolyacid in the presence of antimony salt, which is immediately reduced by ascorbic acid to generate a blue complex.

[0056] Add 100 μl of supernatant to a 50 mL colorimetric tube, add an appropriate amount of distilled water, then add 1 to 2 drops of dinitrophenylhydrazine color developer and shake well, and add an appropriate amount of dilute sulfuric acid or sodium hydroxide solution to adjust the color to light yellow, add 5 ml of molybdenum antimony anti-color developer, shake well, add distilled water to make up the volume, place in a 30°C constant temperature box to react for 30 minutes, and measure its absorbance at a wavelength of 700 nm. Set up three parallel tests for each gradient and take the average of the results.

[0057] 3. Establishment of phosphorus standard curve and phosphorus solubilization capability

[0058] Take 8 50ml colorimetric tubes, add 0, 1, 2, 3, 4, 5, 6, 10ml of 5mg / L phosphorus standard solution respectively, then add appropriate amount of distilled water, add 1-2 drops of dinitrophenylhydrazine color developer and shake well, and adjust with sodium hydroxide solution or dilute sulfuric acid solution until the solution is just slightly yellow, accurately add 5ml of molybdenum antimony color developer, shake well, add distilled water to make up to volume, and then get standard solutions with phosphorus content of 0.0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 1.0mg / L respectively, shake well and put into 30℃ constant temperature box to react for 30min. Measure its absorbance at a wavelength of 700nm, and draw a standard curve with absorbance as the ordinate and phosphorus concentration (mg / L) as the abscissa.

[0059] According to the standard curve, the phosphorus concentration x and absorbance y satisfy the linear equation y = 0.5043x-0.0022, R 2 =0.9995, with a good linear relationship (such as Fig. 9 ), substitute the value measured in 2 above, and the value of soluble phosphorus in the actual solution is the reflection of the phosphorus solubilizing capacity (data are shown in Table 1 and Figure 2 As shown in the figure, after 36h of continuous culture, the effective phosphorus concentration in calcium phosphate medium reached a maximum value of 705.82mg / L, the effective phosphorus concentration in calcium phytate medium reached a maximum value of 698.89mg / L, and the calcium phytate magnesium medium reached a maximum value of 688.48mg / L at 48h.

[0060] Table 1 Phosphorus solubilization capacity

[0061]

[0062]

[0063] Example 3 Phosphate solubilization ability and growth changes of non-decarboxylating Luxenbergia under heavy metal stress

[0064] 1. Seed preparation and cultivation

[0065] Streak strain YZ30 onto enriched solid medium. After colonies grow, select colonies with good growth and inoculate them into test tubes. Culture overnight and then inoculate 1% of the inoculum into 50 ml LB liquid medium. When OD 600 When the value reaches about 0.8, take the corresponding amount of 0.5 ml of bacterial liquid according to the amount of culture medium, centrifuge it, remove the culture medium, add 0.5 ml of sterile water to suspend it to make seed liquid, and add 0.5 ml of seed liquid to each 50 ml of heavy metal calcium phosphate liquid culture medium, and culture it in an air bath constant temperature oscillator at 30°C and 120r / min.

[0066] The heavy metal calcium phosphate liquid culture medium is: calcium phosphate 5g / L, glucose 10g / L, potassium chloride 0.5g / L, sodium chloride 0.3g / L, ammonium sulfate 0.5g / L, magnesium sulfate 0.3g / L, ferrous sulfate 0.03g / L, manganese sulfate 0.005g / L, and heavy metal salts are added in sequence according to the heavy metal gradient concentration, and the concentrations are: zinc sulfate 0, 1, 2, 4, 8, 16, 24mmol / L, cadmium chloride 0, 0.5, 1, 2, 4, 8, 12mmol / L, chromium chloride 0, 4, 8, 16, 24, 32, 44mmol / L.

[0067] 2. Data determination

[0068] After the secondary inoculation, the culture was continued for 120 hours. The fermentation flasks were taken out to the clean bench every 12 hours. After standing for 20 minutes, 250 μl and 2 ml of fermentation liquid were taken from each flask and placed in EP tubes respectively. The 250 μl EP tube was centrifuged at 10000 r / min for 5 minutes, and 100 μl of the supernatant was taken for determination. The effective phosphorus of the supernatant was determined according to the determination method in Example 2.

[0069] Pour the bacterial solution in the 2ml fermentation liquid EP tube into a glass cuvette, use heavy metal calcium phosphate liquid culture medium as blank control, measure its absorbance at a wavelength of 600nm, and repeat the measurement three times, and record the average value.

[0070] 3. Phosphate solubilization ability and strain growth under stress

[0071] According to the calculation method of the standard curve in Example 2 and the bacterial solution concentration measurement data in the above 2, the data are plotted respectively, as shown in FIG. Figure 3 to Figure 8 As shown, the maximum tolerance concentration of zinc ion concentration is 16mM / L (1050mg / kg), the maximum phosphate solubilization capacity is 325.29mg / L, and the strain OD 600 The maximum value was 1.412, compared with the blank strain OD 600The maximum tolerance of cadmium ion concentration was 4mM / L (450mg / kg), the maximum phosphate solubilization capacity was 293.58mg / L, and the strain OD 600 The maximum tolerance concentration of chromium ions was 24mM / L (1248mg / kg), the maximum phosphate solubilization capacity was 231.65mg / L, and the strain OD 600 It is 1.550, which has little effect on strain growth.

[0072] Example 4 Degradation of aniline by non-decarboxylating Luxenbergia

[0073] 1. Seed preparation and cultivation

[0074] Streak the non-decarboxylated Luxenbergia on LB solid medium. After the colonies grow, select the colonies with good growth and inoculate them into test tubes. Culture them overnight and then inoculate them into 50 ml LB liquid medium at a 1% inoculum. When the OD 600 When the value reaches about 0.8, take 1.5 ml of the corresponding amount of bacterial solution according to the amount of culture medium, centrifuge, remove the culture medium, add 1.5 ml of sterile water to suspend, and make seed solution. Add 50 ml of inorganic salt culture medium with aniline concentration of 600 mg / L to a 250 ml conical flask, inoculate the bacterial suspension into the conical flask at an inoculum of 3%, and culture at 37 ° C and 180 r / min.

[0075] LB solid culture medium is: yeast powder 5g / L, peptone 10g / L, sodium chloride 10g / L, agar 20g / L; inorganic salt culture medium is: potassium dihydrogen phosphate 0.5g / L, dipotassium hydrogen phosphate 0.5g / L, sodium chloride 0.2g / L, ammonium nitrate 1g / L, magnesium sulfate 0.2g / L, calcium chloride 0.2g / L.

[0076] 2. Determination of aniline concentration and strain OD value

[0077] After the secondary inoculation, culture was continued for 72 hours. The fermentation flask was taken out on the clean bench every 12 hours. After standing for 20 minutes, 800 μl and 2 ml of the fermentation liquid were taken into EP tubes respectively. The 800 μl EP tube was centrifuged at 10000 r / min for 5 minutes, and 500 μl of the supernatant was diluted 4 times to obtain the test liquid for determination. The determination principle is: the λmax of the E2 band of aniline in aqueous solution is 230 nm, and the aniline in water is determined by ultraviolet spectrophotometer.

[0078] Place the test liquid in a quartz cuvette, measure the absorbance at 230nm, and measure three times in succession to take the average value; take another 2ml of fermentation liquid and pour it into a glass cuvette, use aniline inorganic salt liquid culture medium as a blank control, measure its absorbance at a wavelength of 600nm, repeat three times and take the average value for record.

[0079] 3. Aniline standard curve and aniline degradation rate

[0080] Add 0.1g aniline to a volumetric flask, add distilled water to 1000mL, and prepare a stock solution with a concentration of 100mg / L. Use a pipette to transfer 1, 2, 4, 6, 8, 10 and 15ml of the stock solution to a colorimetric tube, dilute the stock solution to 50mL with distilled water, and obtain aniline standard solutions with concentrations of 2, 4, 8, 12, 16, 20 and 30mg / L, respectively. Shake well, let stand for 10min, and immediately measure the absorbance at a wavelength of 230nm with distilled water as a reference. Draw a standard curve for aniline based on the measured OD value.

[0081] With aniline concentration as the horizontal axis and absorbance (λ=230nm) as the vertical axis, draw a standard curve for aniline. Fig.10 It can be seen that at a wavelength of 230 nm, when the aniline concentration ranges from 0 to 30 mg / L, the aniline concentration x and absorbance y satisfy the linear equation y = 0.1761x-0.0005, R 2 =0.9997, which has a good linear relationship. The aniline content of the culture solution can be calculated through the linear relationship between aniline concentration and absorbance, and a graph can be drawn according to the aniline concentration and the strain growth concentration, such as Fig.11 As shown in the figure, in the inorganic salt medium with an initial aniline concentration of 600 mg / L, the strain basically stagnated in the initial 24 hours, during which the non-decarboxylating Leukemia spp. YZ30 adapted to the new environment. After that, the strain entered the logarithmic growth phase and grew rapidly. After 72 hours of continuous culture, the aniline concentration dropped from 600 mg / L to 402.36 mg / L, and the aniline degradation rate was 32.94%. During the entire growth and metabolic process, the growth of the strain was consistent with the change trend of the aniline concentration, proving that the strain YZ30 can use aniline for metabolism and growth.

Claims

1. A strain of Leckercia adecarboxylata YZ30, deposited in the China Center for Type Culture Collection, with the deposit number being CCTCC NO: M 20211025.

2. A microbial agent comprising the non-decarboxylative Leckeria decarboxylata YZ30 according to claim 1.

3. Use of the non-decarboxylative Leckercia (Leckercia adecarboxylata) YZ30 described in claim 1 or the microbial agent described in claim 2 in phosphate solubilization.

4. The use according to claim 3, characterized in that: The dephosphorization is to dissolve the poorly soluble calcium phosphate, calcium phytate or calcium magnesium phytate into soluble phosphorus.

5. Use of the non-decarboxylative Leckercia (Leckercia adecarboxylata) YZ30 described in claim 1 or the microbial agent described in claim 2 in dephosphorizing heavy metal contaminated soil.

6. The use according to claim 5, characterized in that: The dephosphorization is to dissolve the poorly soluble calcium phosphate, calcium phytate or calcium magnesium phytate into soluble phosphorus.

7. The use according to claim 5, characterized in that: The heavy metal is zinc, cadmium or chromium.

8. Use of the non-decarboxylative Leckercia (Leckercia adecarboxylata) YZ30 described in claim 1 or the microbial agent described in claim 2 in degrading aniline.

9. The use according to claim 8, characterized in that: The aniline degradation is to inoculate the non-decarboxylative Leckercia (Leckercia adecarboxylata) YZ30 described in claim 1 or the microbial agent described in claim 2 into the aniline-contaminated soil to degrade the aniline in the soil and thus repair the contaminated soil.

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