A PAEs-degrading strain of Bacillus stearate AH-ZY2 and its application

Abstract

The present invention relates to a PAEs-degrading strain Pimelobacter sp. AH-ZY2 and its application. The Pimelobacter sp. AH-ZY2 is deposited in the China Center for Type Culture Collection (CCTCC), classified and named as Pimelobacter sp. AH-ZY2, address: Wuhan University, Wuhan, China, postal code 430072, deposit number CCTCC NO: M20221377, deposit date September 5, 2022. The beneficial effects of the present invention are mainly reflected in: the present invention provides a new wild-type strain capable of degrading multiple PAEs, which can effectively degrade multiple PAEs, has high efficiency in degrading both short and long side chains, and has good application prospects.

Description

(I) Technical field

[0001] The invention relates to a PAEs-degrading strain, Bacillus lipolytica AH-ZY2, and application thereof. (II) Background technology

[0002] Phthalate esters are a class of organic compounds generated by esterification reaction of phthalic anhydride and corresponding alcohols under the action of acid catalysts. They are usually colorless, transparent, oily and viscous liquids. They are insoluble in water but soluble in organic reagents such as methanol and ether. PAEs can be divided into long side chain PAEs (DEHP, DnOP, DiNP, etc.) and short side chain PAEs (DEP, DBP, etc.) according to the length of the side chain. PAEs are mainly added to polyvinyl chloride as plasticizers to make it elastic. They are widely used in materials such as plastic products, medical supplies, home appliances and toys. Statistics from the International Agency for Research on Cancer (IARC) show that the content of PAEs in finished plastics is usually 10% to 60%, while reports show that the content of PAEs mixed in plastics can reach up to 70%. The bond between PAEs as plasticizers and polymers is not a strong chemical bond, but a physical bond (hydrogen bond or van der Waals force). Therefore, slight changes in environmental factors will accelerate the separation of PAEs from polymers into the environment. PAEs are a typical environmental endocrine disruptor that has hepatotoxicity, reproductive toxicity, and "three-hazard" toxicity to humans. PAEs are stable in the environment, difficult to degrade naturally, have a long retention time, and have been detected in soil, atmosphere, and water. PAEs in the environment are mainly enriched in soil. With the extensive use of plastic products, the problem of soil contamination by PAEs has received more and more attention. Among them, DBP, BBP, DEHP, DnOP, etc. are listed as priority controlled pollutants by the US Environmental Protection Agency (EPA). In my country, DEHP and DBP are the PAEs with the highest detected content in the soil.

[0003] At present, domestic research on microbial degradation of PAEs mainly focuses on the degradation of single PAEs. In the environment, there is usually a complex pollution of multiple PAEs, and there is currently a lack of strains that can efficiently degrade complex pollution. (III) Summary of the invention

[0004] The purpose of the present invention is to provide a new wild-type strain capable of degrading multiple PAEs, the strain has a wide substrate spectrum and has good research prospects.

[0005] The technical solution adopted by the present invention is:

[0006] A PAEs-degrading bacterium, Pimelobacter sp. AH-ZY2, is deposited in the China Center for Type Culture Collection (CCTCC), classified as Pimelobacter sp. AH-ZY2, address: Wuhan University, Wuhan, China, Postal Code 430072, deposit number CCTCC NO: M 20221377, deposit date September 5, 2022.

[0007] The Pimelobacter sp. AH-ZY2 was isolated from farmland soil in Wuhu, Anhui.

[0008] The 16S rDNA sequence of the strain is shown as SEQ ID No.1.

[0009] SEQ ID No.1:

[0010]

[0011] The present invention also relates to the application of the fatty bacillus AH-ZY2 in the microbial degradation treatment of PAEs.

[0012] The present invention also relates to the application of the fatty bacillus AH-ZY2 in repairing soil PAEs pollution.

[0013] The PAEs are one of the following or a mixture of two or more thereof: dimethyl phthalate (DMP), diethyl phthalate (DEP), dipropyl phthalate (DPrP), dibutyl phthalate (DBP), butyl benzyl phthalate (BBP), di(2-ethylhexyl) phthalate (DEHP), di-n-octyl phthalate (DnOP), and diisononyl phthalate (DiNP).

[0014] Preferably, the PAEs is dibutyl phthalate.

[0015] The beneficial effects of the present invention are mainly reflected in: the present invention provides a new wild-type strain that can degrade multiple PAEs. The strain can effectively degrade multiple PAEs, has high efficiency in degrading both short-chain and long side chains, and has good application prospects. (IV) Description of the drawings

[0016] Figure 1 The identification results of Pimelobacter sp. AH-ZY2 strain; a is the colony morphology on the LB plate; b is the transmission electron microscopy picture; c is the Gram staining result.

[0017] Figure 2 This is the evolutionary tree of the 16S rDNA sequence of strain AH-ZY2.

[0018] Figure 3 The degradation and growth of strain AH-ZY2 on various PAEs. (V) Specific implementation methods

[0019] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0020] Embodiment 1:

[0021] Sample source: isolated from farmland soil in Baimao Town, Jiujiang District, Wuhu City, Anhui Province.

[0022] 1. Collection of soil samples: Soil samples were collected from farmland soil in Baimao Town, Jiujiang District, Wuhu City, Anhui Province (118°46′35″E, 31°1′42″N) and stored in sterile sampling tubes.

[0023] 2. Screening of PAEs-degrading bacteria: Take 5g soil sample and add 25ml water, shake and mix, and naturally settle for 30min. Take 2ml of supernatant and culture in 50ml LB. After 3 days of culture, take 2mL of upper liquid and add it to 100mL BSM medium (with 100mg / L DEP, 100mg / L DBP, 100mg / L DEHP, 100mg / L DnOP), and culture for 4 days at 30℃ and 180r / min. Then take 2mL of culture and transfer it to new 100mL BSM medium (with 100mg / L DEP, 100mg / L DBP, 100mg / L DEHP, 100mg / L DnOP) and repeat the above culture process. After several transfers, the final culture solution was diluted and spread on LB plates, and single colonies with different morphologies were picked to verify their ability to degrade PAEs: the purified strains were cultured in BSM medium supplemented with 200 mg / L of DMP, DEP, DPrP, DBP, BBP, DEHP, DnOP or DiNP to observe which PAEs the strains could specifically degrade.

[0024] 3. Identification of PAEs-degrading bacteria: The obtained strains were identified by physiological and biochemical experiments and 16S rDNA sequencing according to Bergey's Manual of Systematic Bacteriology (2012) to determine the genus of the obtained strains.

[0025] 4. Identification results of AH-ZY2: The colonies on the LB plate are white or grayish white, with a smooth and transparent surface and serrated edges. The entire colony is water-stained, and the Gram staining result is positive ( Figure 1 ).

[0026] 5. Physiological and biochemical experiments and sole carbon source utilization characteristics: The strain AH-ZY2 was identified by some physiological and biochemical experiments, and the results are shown in Table 1. In addition, its utilization characteristics of 8 sole carbon sources were also tested, and the results are shown in Table 2. In Tables 1 and 2, "+" indicates that the test result is positive, and "-" indicates that the test result is negative.

[0027] Table 1: Physiological and biochemical test results of strain AH-ZY2

[0028]

[0029]

[0030] Table 2: Sole carbon source utilization characteristics of strain AH-ZY2

[0031]

[0032] Based on the 16S rDNA sequencing results (see SEQ ID No. 1 for the 16S rDNA sequence of strain AH-ZY2), evolutionary analysis showed that it has the closest relationship with strains of the genus Pimelobacter ( Figure 2 ). Therefore, based on the physiological and biochemical and 16S rDNA identification results, the strain was named Pimelobacter sp.AH-ZY2.

[0033] Embodiment 2:

[0034] The PAEs degradation ability test and application experiment of the obtained strain are as follows:

[0035] 1. Use 200 mg / L DMP, DEP, DPrP, DBP, BBP, DEHP, DnOP, DiNP as the only carbon source to verify growth.

[0036] 2. The degradation of 200 mg / L DMP, DEP, DPrP, DBP, BBP, DEHP, DnOP and DiNP by the test strain within 7 days.

[0037] On the 7th day, the degradation and growth of strain AH-ZY2 on various PAEs were measured. Figure 3 The results showed that strain AH-ZY2 could effectively degrade the eight PAEs tested, degrading more than 50% of 200 mg / L DBP within 7 days, and had high efficiency in degrading both short-chain and long-side chains.

Claims

1. A strain of PAEs-degrading bacteria, Bacillus lipolytica ( Pimelobacter sp . )AH-ZY2, characterized by The deposit number is CCTCC NO: M 20221377.

2. Use of the fatty bacillus AH-ZY2 according to claim 1 in the microbial degradation treatment of PAEs, wherein the PAEs are one of the following or a mixture of two or more thereof: DMP, DEP, DPrP, DBP, BBP, DEHP, DnOP, DiNP.

3. Use of the fatty bacillus AH-ZY2 according to claim 1 in remediating soil PAEs contamination, wherein the PAEs are one of the following or a mixture of two or more thereof: DMP, DEP, DPrP, DBP, BBP, DEHP, DnOP, DiNP.

4. The use according to claim 2 or 3, characterized in that The PAEs are DBP.

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