CN115261248A

CN115261248A - Efficient degrading strain of long side chain PAEs (polycyclic aromatic hydrocarbons) and application thereof

Info

Publication number: CN115261248A
Application number: CN202210149789.1A
Authority: CN
Inventors: 钟卫鸿; 胡彤; 杨宸; 刘腾飞; 侯正雨
Original assignee: Zhejiang University of Technology ZJUT
Current assignee: Zhejiang University of Technology ZJUT
Priority date: 2022-02-18
Filing date: 2022-02-18
Publication date: 2022-11-01
Anticipated expiration: 2042-02-18
Also published as: CN115261248B

Abstract

The invention discloses a long side chain PAEs high-efficiency degradation strain Gondonia sp.GZ-YC7 which is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of CCTCC M2022045. The invention can be used for degrading long side chain PAEs and repairing DEHP polluted soil, and has better effect.

Description

Efficient degrading strain of long side chain PAEs (polycyclic aromatic hydrocarbons) and application thereof

Technical Field

The invention belongs to the field of bioengineering, and provides a strain Gordonia sp.GZ-YC7 which has high-efficiency degradation capability of various PAEs and strong degradation capability to high-concentration PAEs and is obtained from refuse landfill soil through microorganism screening.

Technical Field

PAEs (phthalic acid esters) are organic compounds generated by esterification of phthalic anhydride and corresponding alcohols under the action of an acid catalyst, are usually colorless transparent oily viscous liquids, are insoluble in water, and are soluble in organic reagents such as methanol and diethyl ether. PAEs can be largely classified into long side chain PAEs according to side chain length: DEHP (di (2-ethylhexyl) phthalate), dnOP (di-n-octyl phthalate), diNP (diisononyl phthalate), etc., and short side chain PAEs: DEP (diethyl phthalate), DBP (di-n-butyl phthalate), and the like. PAEs are added to polyvinyl chloride as a plasticizer to impart elasticity thereto, and are widely used as materials for plastic products, medical supplies, home appliances, toys, and the like. The international agency for research on cancer (IARC) has shown that the content of PAEs in the finished plastic is usually 10% to 60%, while the content of mixed PAEs in the plastic is reported to be up to 70%. The bonding between PAEs as a plasticizer and polymer is not a strong chemical bond but a physical bond (hydrogen bonds or van der waals forces), so that small changes in environmental factors accelerate the release of PAEs from the polymer into the environment. PAEs are a typical environmental endocrine disrupting substance and have hepatorenal toxicity, reproductive toxicity and "triple-cause" toxicity to humans. The PAEs have stable properties in the environment, are difficult to naturally degrade, have long retention time and are detected in soil, atmosphere and water, wherein the PAEs in the environment are mainly enriched in the soil. With the use of a large amount of plastic products, the problem of soil pollution caused by PAEs is more and more emphasized by people, wherein DBP, BBP (butyl benzyl phthalate), DEHP, dnP and the like are prioritized to control pollutants by the United states Environmental Protection Agency (EPA), and DEHP and DBP in China are PAEs with the highest detection content in soil.

At present, domestic research on the degradation of PAEs by microorganisms mainly focuses on the degradation of PAEs with single or short side chains, but is generally the combined pollution of a plurality of PAEs in the environment, and PAEs with long side chains are also more difficult to degrade.

Disclosure of Invention

The invention aims to provide a bacterial strain with high-efficiency degradation capability and tolerance capability on various PAEs (polycyclic aromatic hydrocarbons), particularly long-side-chain PAEs (polycyclic aromatic hydrocarbons), and the bacterial strain is applied to actual environmental remediation.

Gondonia sp.GZ-YC7 is isolated from Guizhou refuse landfill soil by the applicant, and is preserved in China Center for Type Culture Collection (CCTCC) at 1 month and 10 days in 2022 with the preservation number of CCTCC M2022045.

1. The method for screening and identifying the GZ-YC7 strain comprises the following steps:

1.1 soil sample collection: a soil sample was taken from a Laibashan landfill (39 DEG 52 'N48' 105 DEG 30 '5' E) of Liudianshui city, guizhou province and stored with an aseptic sampling tube.

1.2 screening PAEs degrading bacteria: adding 25g of soil sample into 20mL of sterile water, fully shaking and uniformly mixing, then adding 2mL of upper layer liquid into 100mL of BSM culture medium (added with 200mg/L DEHP), and culturing for 4 days at 30 ℃ and 180 r/min. Then 2mL of the culture was transferred to a new 100mL BSM medium (with 200mg/L DEHP added) and the above procedure was repeated. After transferring for several times, the final culture solution is diluted and spread on an LB plate, and single colonies with different forms are picked to verify the degradation capability of PAEs: the purified strains were cultured in BSM medium supplemented with 200mg/L of DEP, DPrP (dipropyl phthalate), DBP, BBP, DEHP, dnOP, diNP, respectively, and the strains with the most variety of PAEs were selected.

1.3 identification of PAEs degrading bacteria: the obtained strain is subjected to physiological and biochemical experimental identification and 16S rDNA sequence sequencing comparison by referring to Bergey' S Manual of systematic bacteriology (2012), and the genus of the obtained strain is determined.

2. PAEs degradability of Gordonia sp.GZ-YC7 and application thereof:

2.1 has better degradation effect on DEP, DPrP, DBP, BBP, DEHP, dnOP and DiNP with the concentration of 500mg/L within 2 days;

2.2 the mixed pollution of four PAEs with initial concentrations of 200mg/L, namely DEP, DBP, DEHP and DnOP has better degradation capability;

2.3 for high concentrations of 4g/L five PAEs: DBP, BBP, DEHP, dnP and DiNP all have better degradation capability;

2.4 has the capability of restoring the soil polluted by 500mg/kg DEHP in the soil.

Drawings

FIG. 1 is colony morphology (a) of Gordonia sp.GZ-YC7 on LB plates; 60,000x transmission electron microscopy micrograph (b); as a result of gram staining, E.coli was stained in red, and GZ-YC7 (c) was stained in purple.

FIG. 2 is a 16S rDNA sequence evolutionary tree.

FIG. 3 shows the degradation and growth of various PAEs by GZ-YC7.

FIG. 4 shows the combined degradation and growth of four PAEs by GZ-YC7.

FIG. 5 shows the degradation of high concentrations of PAEs by GZ-YC7.

Fig. 6 is the degradation of DEHP contamination in soil by GZ-YC7, where NSS: non-sterilized soil without adding bacteria, NSS7: non-sterilized soil added bacteria, SS: sterile soil is not added with bacteria, and SS7: and (5) adding bacteria into the sterilized soil.

Detailed Description

1. Sample source: the culture medium is isolated from Guizhou refuse landfill soil, and is preserved in China Center for Type Culture Collection (CCTCC) at 1 month and 10 days in 2022, with the preservation number of CCTCC M2022045.

2. And GZ-YC7 identification result: see fig. 1. Colonies on the LB plate are orange, have regular shapes and clear boundaries, and can utilize glucose, fructose, xylose, mannose and starch; gram stain, catalase and hydrogen sulfide tests were positive and negative for urease, V-P, methyl red and casein hydrolysis tests.

See fig. 2. Evolution analysis is carried out according to the sequencing comparison result of the 16S rDNA, and the strain has a recent relationship with the Gordonia, so that the strain is identified as Gordonia sp.GZ-YC7 by combining the physiological and biochemical results and the identification result of the 16S rDNA.

3. The GZ-YC7 is used for testing the degradation capability of various PAEs: see fig. 3. The strain GZ-YC7 can efficiently degrade PAEs in 7 to be tested, 500mg/L BBP and DEHP can be completely degraded within 48h, and the degradation rates of DnOP and DiNP with long side chains are 91.22% and 97.57%.

3. The strain GZ-YC7 can be used for mixed degradation of four PAEs including DEP, DBP, DEHP and DnOP: see fig. 4. GZ-YC7 can completely degrade DBP and DEHP. The degradation of DEP and DnP also reaches 77.56% and 79.97%, so that GZ-YC7 has a remarkable degradation effect on the pollution of the composite PAEs and is more beneficial to the application in environmental remediation.

4. Degradation of high-concentration PAEs by the strain GZ-YC 7: see fig. 5. In 5 days of degradation, 4g/L DEHP can be degraded by 70.71%, and the degradation efficiency in DnOP and DiNP which are long-side chain PAEs also reaches 47.82% and 54.54% respectively. The GZ-YC7 has strong degradation capability on PAEs and has higher PAEs tolerance and application potential.

5. Restoration experiment of DEHP contaminated soil: see fig. 6. In the soil to which the strain GZ-YC7 had not been added, the concentration of DEHP did not change significantly within 5 days, regardless of whether the soil was subjected to sterilization treatment or not (NSS and SS groups). After the addition of GZ-YC7, DEHP in the soil was significantly degraded, and the soil contaminated with DEHP at an initial concentration of 500mg/kg was degraded by 45.22% and 47.33% within 5 days (NSS 7 and SS7 groups), respectively. Therefore, whether the soil is subjected to sterilization treatment in the test or not has no obvious influence on the capacity of GZ-YC7 for degrading DEHP in the soil, and the result shows that the DEHP can be effectively degraded in a complex soil environment only by the GZ-YC7. The high-concentration resistance and high-efficiency degradation capability of the previously mentioned GZ-YC7 to DEHP are shown. The strain GZ-YC7 has great potential for environmental remediation.

Claims

1. The strain Gondonia sp.GZ-YC7, CCTCC M2022045 is efficiently degraded by the long side chain PAEs.

2. The strain of claim 1, which is used for degrading long side chain PAEs.

3. The strain of claim 1, applied to the remediation of DEHP contaminated soil.