Pseudomonas putida, preparation containing pseudomonas putida and application of pseudomonas putida

Abstract

The invention relates to a Pseudomonas putida IB3, a preparation containing the Pseudomonas putida IB3 and application of the Pseudomonas putida IB3, the Pseudomonas putida IB3 is preserved in China Center for Type Culture Collection on February 27, 2023, and the preservation number of the Pseudomonas putida IB3 is CCTCC NO: M 2023201. The pseudomonas putida IB3 provided by the invention and the preparation containing the pseudomonas putida IB3, which are provided by the invention, have an excellent capability of degrading aniline and petroleum hydrocarbon, and the preparation containing the pseudomonas putida IB3 provided by the invention has an excellent capability of degrading the aniline and the petroleum hydrocarbon.

Description

Technical Field

[0001] The present invention relates to the technical field of environmental microorganisms and environmental pollution control, and in particular to a strain of Pseudomonas putida, a preparation containing the strain, and applications thereof. Background Art

[0002] Pseudomonas putida is widely present in soil, water and plants (or plant rhizosphere). It has the ability to decompose organic matter, degrade pollutants, synthesize anti-stress substances, secrete antibiotics, etc., and has broad application prospects in agricultural biological control, environmental protection and pharmaceutical development.

[0003] CN201610095155.7 discloses a bioremediation preparation for aniline-contaminated water bodies, which is composed of a composite bacterial agent and auxiliary materials. The composite bacterial agent is prepared by Pseudomonas putida, Candida, Bacillus amyloliquefaciens, Clostridium paper-soluble, Nitrosobacter, Trichoderma chrysogenum, and Nitrifying Bacteria. The preparation is added to the wastewater treatment system and has a good degradation effect on aniline-resistant compounds and is suitable for industrial wastewater treatment. However, the preparation provided by the invention requires the compounding of multiple strains and auxiliary materials, and further the compound bacterial agent and the composite auxiliary materials are compounded, and the components are relatively complex.

[0004] CN202311126460.4 discloses a multifunctional microbial strain and an organic pollution remediation agent and its application. A strain of Pseudomonas putida AM21 with the ability to efficiently repair petroleum-contaminated soil was screened from petroleum-contaminated soil. The strain can also degrade a variety of polycyclic aromatic hydrocarbons and benzene series. The agent prepared by Pseudomonas putida AM21 can effectively repair petroleum-contaminated soil and coking-contaminated soil. The remediation agent prepared by Pseudomonas putida AM21 can achieve a petroleum hydrocarbon degradation rate of more than 60% within 60 days for petroleum and coking composite contaminated soil with an oil content of 3-8%, and the degradation rate of a variety of polycyclic aromatic hydrocarbons and benzene series can reach more than 70%. However, the degradation effect of Pseudomonas putida AM21 on different pollutants provided by the invention needs to be improved. Summary of the Invention

[0005] The present invention aims to provide a strain of Pseudomonas putida and a preparation containing the strain and its application. The Pseudomonas putida and the preparation provided by the present invention have excellent aniline and petroleum hydrocarbon degradation capabilities, stable performance and outstanding effects.

[0006] The first aspect of the present invention provides a strain of Pseudomonas putida IB3, which was deposited in the China Center for Type Culture Collection on February 27, 2023, with a deposit number of CCTCC NO: M 2023201.

[0007] The main morphological characteristics of the Pseudomonas putida IB3 provided by the present invention are: after being cultured in culture medium A at 35° C. for 36 hours, the colonies are white, round, moist, opaque, and have neat edges.

[0008] The sequencing results of the 16SrDNA gene of Pseudomonas putida IB3 provided by the present invention are shown in the sequence table.

[0009] A second aspect of the present invention provides a method for culturing Pseudomonas putida IB3, which comprises using culture medium A and culturing the culture medium under aerobic conditions until the stable phase.

[0010] Furthermore, the formula of the culture medium A is: peptone 5g / L, NaCl 10g / L, MgSO4 0.5g / L, NH4Cl0.5g / L, K2HPO4 1g / L, KH2PO4 0.5g / L, FeSO40.03g / L, CaCl2 0.2g / L, aniline 0.2g / L, and diesel 1.5mL / L.

[0011] Furthermore, the culture conditions are: pH 6-9, temperature 25-40°C, dissolved oxygen 0.5-2.5 mg / L, and culture time 18-36 hours.

[0012] A third aspect of the present invention provides a preparation comprising Pseudomonas putida IB3. Further preferably, the preparation further comprises at least one of an inorganic nutrient, organic nitrogen, or a preservative.

[0013] A fourth aspect of the present invention provides the use of Pseudomonas putida IB3 or a preparation containing the bacterium for degrading aniline. The strain can utilize aniline as its sole carbon and nitrogen source for growth, achieving a removal rate greater than 90% when the aniline concentration is less than 200 mg / L, preferably between 30 and 200 mg / L. The aniline can be found in industrial wastewater or in contaminated surface water or groundwater.

[0014] Furthermore, an auxiliary agent is added to the application system, the auxiliary agent is potassium phosphate, preferably at least one of KH2PO4 and K2HPO4, and the addition amount is 0.5-100 mg / L.

[0015] Furthermore, in this application system, the temperature is controlled at 25-40°C and the dissolved oxygen is controlled at 0.5-2.5 mg / L.

[0016] A fifth aspect of the present invention provides the use of Pseudomonas putida IB3 or a preparation containing the bacterium for the degradation of petroleum hydrocarbons. This strain can grow using petroleum hydrocarbons as its sole carbon source, achieving efficient removal of petroleum hydrocarbons. The petroleum hydrocarbons are a mixture of hydrocarbons typically containing paraffins, cycloalkanes, and aromatics, derived from at least one oil product, such as crude oil, gasoline, diesel, kerosene, and lubricating oil. In this application system, the removal rate is greater than 85% when the petroleum hydrocarbon concentration is less than 12,000 mg / L.

[0017] Furthermore, an auxiliary agent is added to the application system. The auxiliary agent is organic nitrogen, preferably at least one of peptone, yeast extract powder, etc., and the addition amount is 0.1-1.2 g / L.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] (1) The Pseudomonas putida IB3 strain provided by the present invention has excellent aniline degradation ability. When the aniline concentration is lower than 200 mg / L, the removal rate is greater than 90%. When the auxiliary agent potassium phosphate is added, the degradation ability is further improved.

[0020] (2) The Pseudomonas putida IB3 strain provided by the present invention has excellent petroleum hydrocarbon degradation capabilities, with a removal rate exceeding 85% when the petroleum concentration is below 12,000 mg / L. The degradation capability is further enhanced when the additive organic nitrogen is added.

[0021] (3) The present invention selects medium A to culture Pseudomonas putida IB3, which can obtain a highly efficient and stable bacterial solution in a relatively short culture time, and the prepared bacterial solution has stable performance.

[0022] (4) The Pseudomonas putida IB3 of the present invention can degrade aniline and petroleum hydrocarbons simultaneously, has simple components, does not require a complex formula, and has a stable effect.

[0023] (5) The strains and preparations of the present invention provide microbial resources for the remediation of petroleum hydrocarbon contaminated sites, the remediation of eutrophic water bodies, and the remediation of oil-contaminated oceans, and have broad application prospects.

[0024] Description of biological material deposit

[0025] The Pseudomonas putida IB3 provided by the present invention is deposited in the China Center for Type Culture Collection; deposit number: CCTCC NO: M 2023201; deposit date: February 27, 2023; deposit address: Wuhan University, Wuhan, China. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is a photo of the colony morphology of the strain of the present invention on a plate culture medium.

[0027] Figure 2 It is the phylogenetic tree of the strain of the present invention. DETAILED DESCRIPTION

[0028] The following is a further detailed description of the separation, screening, cultivation and application of the strain of the present invention in conjunction with specific examples. The examples are implemented based on the technical solution of the present invention, and provide detailed implementation methods and specific operating procedures, but the scope of protection of the present invention is not limited to the following examples.

[0029] The experimental methods in the following examples, unless otherwise specified, are conventional methods in the art. The experimental materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores. The percentages described in the present invention are all by weight.

[0030] In the present invention, aniline was measured using the "Water Quality - Determination of Aniline Compounds - N-(1-Naphthyl)ethylenediamine Azo Spectrophotometric Method" (GB / T 11889-1989). Petroleum hydrocarbons were measured using the "Water Quality - Determination of Petroleum and Animal and Vegetable Oils - Infrared Spectrophotometric Method" (HJ 637-2018).

[0031] Example 1 Isolation and screening of strains

[0032] Strain IB3 was isolated and screened from the petroleum-contaminated soil of an oil depot using the dilution plate separation method. The sample was obtained from an oil depot of Shanghai Gaoqiao Petrochemical in November 2020.

[0033] (1) Take 10g of soil sample and add it to a 250mL Erlenmeyer flask. Then add 100mL of culture medium A. After culturing at 28℃ and 150rpm for 5 days, take 1% of the enriched solution and transfer it to fresh culture medium A. Continue culturing under the same conditions. 660 When the cell count is >0.8, transfer the cells to fresh culture medium A. Repeat the above transfer operation three times.

[0034] (2) Transfer to solid culture medium plate (liquid culture medium A + 2% agar) under sterile conditions, and use plate dilution coating method, the dilution concentration is 10 -6 -10 -16 , culture until a single colony appears on the plate, pick a single colony and streak it on the solid culture medium plate, repeat three times.

[0035] (3) Transfer the purified single colony to medium B and culture at 28°C and 150 rpm until the OD of the colony reaches 660 >0.5, a purified strain was obtained. Microscopic observation confirmed its purity. If not, the above steps were repeated until it was confirmed to be pure. After repeated cultivation, the dominant pure strain IB3 was obtained.

[0036] The formula of culture medium A is: peptone 5 g / L, NaCl 10 g / L, MgSO4 0.5 g / L, NH4Cl 0.5 g / L, K2HPO4 1 g / L, KH2PO4 0.5 g / L, FeSO4 0.03 g / L, CaCl2 0.2 g / L, aniline 0.2 g / L, and diesel 1.5 mL / L.

[0037] The formula of culture medium B is: NaCl 10g / L, MgSO4 0.5g / L, K2HPO4 1g / L, KH2PO4 0.5g / L, FeSO4 0.03g / L, CaCl2 0.2g / L, aniline 0.1g / L, and diesel 0.5ml / L.

[0038] The pure strain IB3 was identified for species identification. The 16S rDNA gene sequencing results are shown in the sequence listing. Comparison of the 16S rDNA sequence with specialized databases based on the NCBI database revealed the closest homology to Pseudomonas putida. Therefore, the strain was identified as Pseudomonas putida.

[0039] Example 2 Rapid Culture of Pseudomonas putida IB3

[0040] The strain IB3 stored at -60℃ was inoculated into LB medium (NaCl 10g / L, peptone 10g / L, yeast extract 5g / L) and cultured aerobically at 30℃ for more than 24h until the OD 660 When the concentration reaches 0.9 or above, the activated bacterial solution is obtained.

[0041] Take 5% of the activated bacterial solution and inoculate it into fresh medium A for expansion culture. Cultivate it at 30℃ under aerobic conditions for more than 18 hours. The culture conditions are: pH 7.0-8.0, dissolved oxygen 0.5-2.5mg / L. Take samples for testing, OD 660 If it can reach 0.8 or above, it is a bacterial solution that can be added for use, or made into a preparation for use.

[0042] Example 3 Application of Pseudomonas putida IB3 in Degrading Petroleum Hydrocarbons

[0043] The water body of a certain enterprise was contaminated by petroleum hydrocarbons, with the concentration of petroleum being 7350 mg / L.

[0044] (1) The bacterial solution cultured in Example 2 was added to wastewater at an inoculum rate of 8% by volume and treated at 30°C, pH 7.0-8.5, and aerobic conditions for 144 hours. Testing revealed a petroleum concentration of 1020 mg / L in the effluent.

[0045] (2) The bacterial solution cultured in Example 2 was added to the wastewater at an inoculum rate of 8% by volume, and 1 g / L of peptone was added. The wastewater was treated under the same conditions for 144 h. The petroleum concentration in the effluent was 593 mg / L after testing.

[0046] (3) The bacterial solution cultured in Example 2 was added to the wastewater at an inoculum rate of 8% by volume, and 0.5 g / L of yeast extract was added. The wastewater was treated under the same conditions for 144 h. Testing revealed a petroleum concentration of 701 mg / L in the effluent.

[0047] Example 4 Application of Pseudomonas putida IB3 in Degrading Aniline

[0048] The water body of a certain enterprise was contaminated by aniline, with the aniline concentration being 180 mg / L.

[0049] (1) The bacterial solution cultured in Example 2 was added to wastewater at a volume inoculum rate of 2%, and treated at 30°C, pH 7.5-8.0, and aerobic conditions for 96 hours. The aniline concentration in the effluent was 16.4 mg / L.

[0050] (2) The bacterial solution cultured in Example 2 was added to the wastewater at a volume inoculum rate of 2%, and 0.5 mg / L of KH2PO4 was added. The wastewater was treated under the same conditions for 96 h. The aniline concentration in the effluent was 4.5 mg / L after testing.

[0051] (3) The bacterial solution cultured in Example 3 was added to the wastewater at a volume inoculum rate of 2%, and 0.5 mg / L of K2HPO4 was added. The wastewater was treated under the same conditions for 96 h. The aniline concentration in the effluent was 4.2 mg / L.

[0052] Example 6

[0053] A certain enterprise's water body was contaminated with aniline and petroleum hydrocarbons, with aniline at 100 mg / L and petroleum hydrocarbons at 3200 mg / L. The bacterial culture from Example 3 was added to the wastewater at a 6% inoculum volume. The wastewater was treated at 30°C, pH 7.0-8.5, and aerobic conditions for 144 hours. Testing revealed petroleum hydrocarbon concentrations of 418 mg / L and aniline of 8.8 mg / L in the effluent.

Claims

1. A strain of Pseudomonas putida, which is Pseudomonas putida ( Pseudomonas putida )IB3, the bacteria was deposited in the China Center for Type Culture Collection on February 27, 2023, with the deposit number CCTCC NO: M 2023201.

2. The Pseudomonas putida according to claim 1 ( Pseudomonas putida ) IB3, characterized by: Its morphological characteristics are: The colonies are white, round, moist, opaque and have neat edges.

3. The Pseudomonas putida according to claim 1 ( Pseudomonas putida ) IB3, characterized by: Pseudomonas putida ( Pseudomonas putida ) The 16S rDNA gene sequencing results of IB3 are shown in the sequence listing.

4. A Pseudomonas putida ( Pseudomonas putida ) A method for culturing IB3, characterized in that: Culture medium A was used and cultured under aerobic conditions until the stationary phase; the formula of the culture medium A was as follows: 5 g / L peptone, 10 g / L NaCl, 0.5 g / L MgSO4, 0.5 g / L NH4Cl, 1 g / L K2HPO4, 0.5 g / L KH2PO4, 0.03 g / L FeSO4, 0.2 g / L CaCl2, 0.2 g / L aniline, and 1.5 mL / L diesel.

5. The culture method according to claim 4, wherein: The culture conditions are as follows: pH 6-9, temperature 25-40° C., dissolved oxygen 0.5-2.5 mg / L, and culture time 18-36 h.

6. A method comprising the Pseudomonas putida according to any one of claims 1 to 3 ( Pseudomonas putida ) Preparation of IB3.

7. The preparation according to claim 6, characterized in that: The formulation further includes at least one of an inorganic nutrient, organic nitrogen, or a preservative.

8. A Pseudomonas putida according to claim 1-3 ( Pseudomonas putida ) Use of IB3 or the preparation according to claim 6 or 7 in degrading aniline.

9. The use according to claim 8, characterized in that: The strain can use aniline as the sole carbon and nitrogen source for growth. When the aniline concentration is 30-200 mg / L, the removal rate is greater than 90%.

10. The use according to claim 8, characterized in that: An auxiliary agent is added to the application system. The auxiliary agent is potassium phosphate, preferably at least one of KH2PO4 and K2HPO4, and the addition amount is 0.2-100 mg / L.

11. A Pseudomonas putida according to claim 1-3 ( Pseudomonas putida ) Use of IB3 or the preparation described in claim 6 or 7 in the degradation of petroleum hydrocarbons.

12. The use according to claim 11, characterized in that: The strain can grow using petroleum hydrocarbons as the sole carbon source. When the concentration of petroleum hydrocarbons is not higher than 12,000 mg / L, the removal rate is greater than 85%.

13. The use according to claim 11, characterized in that: An auxiliary agent is added to the application system, wherein the auxiliary agent is at least one of peptone and yeast extract powder, and the addition amount is 0.1-1.2 g / L.

14. The use according to any one of claims 8 to 13, characterized in that: In the application system, the temperature is controlled at 25-40°C and the dissolved oxygen is 0.5-2.5 mg / L.

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