Bacillus strain GBD3 and application thereof in degrading phthalic ester plasticizer

Abstract

The invention discloses a bacillus strain GBD3 and application thereof to degradation of phthalic ester plasticizers, belongs to the field of environmental pollutant remediation, and is characterized in that the strain is classified and named as Bacillus sp. GBD3. The strain is preserved in the China General Microbiological Culture Collection Center (CGMCC) on November 03, 2023, and the preservation number is CGMCC No.28860. The invention further discloses a preparation method of the strain. The invention also discloses an application of the strain in degrading the phthalic ester plasticizer. The strain can efficiently degrade the phthalic ester plasticizer with the side chain length of C2-C5 at room temperature. The degrading bacteria screened from soil polluted by plastic agricultural films for a long time can effectively remove phthalic ester plasticizers. The degrading strain can be used for repairing the soil environment polluted by the phthalic ester plasticizer.

Description

Technical Field

[0001] The present invention belongs to the field of environmental pollutant remediation, specifically a Bacillus strain GBD3 and an application thereof, which can be used to remediate soil environments contaminated by phthalate plasticizers and has great potential in practical applications. Background Art

[0002] Phthalate plasticizers are a class of chemical substances widely used in plastic products. Their main function is to increase the flexibility, plasticity and ductility of plastics. These plasticizers have been widely used in industries, consumer products, medical devices, food packaging and building materials. Dimethyl phthalate (DMP), (2-ethylhexyl phthalate) (DEHP), dibutyl phthalate (DBP) and diethyl phthalate (DEP) are the four most widely used phthalate plasticizers in industry. Phthalate plasticizers are not bound to plastic molecules by covalent bonds but by van der Waals forces or hydrogen bonds. They are in a free state, so they are easy to migrate from polymers into the environment and accumulate over time. Phthalate plasticizers are teratogenic, mutagenic, carcinogenic and reproductively toxic. They not only harm the ecosystem and affect the growth of plants and animals, but also accumulate through the bioaccumulation of plants and animals, and are transmitted through the food chain, endangering human health. At present, this type of substance has become one of the most common important environmental organic pollutants in the world. Phthalate plasticizers have been detected in the atmosphere, soil and water. The US EPA has included them in the list of key controlled pollutants. In my country, phthalate plasticizers are also priority controlled pollutants.

[0003] Phthalate plasticizers have a relatively slow rate of photolysis and hydrolysis under natural environmental conditions, and the use of microorganisms to degrade them is considered to be the most effective method at present. However, in a complex actual environment, when free microorganisms are added to the actual environment, they may compete with the microorganisms existing in the environment or have difficulty adapting to the environment, resulting in often failure to achieve a good repair effect. Bacillus has a strong ability to adapt to the environment and can colonize and survive in a variety of environments, making it an ideal environmental bioremediation tool. At present, there are few reports on the use of Bacillus as a phthalate plasticizer degradation bacterium.

[0004] The Chinese patent number is 201510026457.4, and the invention name is "A strain of phthalate diester plasticizer-degrading bacteria and its application", which discloses a phthalate diester plasticizer-degrading bacteria. The strain classification in the patent is named Camelimonassp., which is a Gram-negative bacterium and belongs to a different category of strains from the strain of the present invention.

[0005] In addition, the inventor of this case’s earlier patent 201410677772.9, the invention name of which is “A strain for degrading di(2-ethylhexyl) phthalate and its application in tobacco cultivation”, discloses a microbial strain, the Sphingobacteriumthalpophilum zh-x-08 strain, which is also a Gram-negative bacterium and belongs to a different genus from the strain of the present invention. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a Bacillus strain GBD3 and use it to repair the soil environment polluted by phthalate plasticizers.

[0007] Another technical problem to be solved by the present invention is to provide the application of the above-mentioned strain GBD3 in degrading phthalate plasticizers.

[0008] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows: A Bacillus strain GBD3, which has been deposited in the General Microbiology Center of China Microbiological Culture Collection Administration (CGMCC), the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, the deposit date is November 03, 2023, the deposit number is CGMCC No. 28860, and the proposed classification name is Bacillus sp. GBD3. The strain was isolated by the inventor from tobacco field soil in Xuchang City, Henan Province in September 2023.

[0009] The 16SrDNA gene sequence table of the strain GBD3 is shown in SEQUENCE LISTING.

[0010] Said Bacillus The colonies of sp. GBD3 strain are round, light yellow, smooth and sticky. Gram staining is positive, with spores, and can decompose starch; the catalase test, glucose oxidation, VP test, and indole test are all positive, and the methyl red test is negative.

[0011] The strain GBD3 of the present invention can be used to degrade phthalate plasticizers with a side chain length of C2 to C5.

[0012] The phthalate plasticizer with a side chain length of C2 to C5 includes any one or more of diethyl phthalate, dipropyl phthalate, dibutyl phthalate and dipentyl phthalate.

[0013] When the degrading bacteria of the present invention are sprayed on soil containing phthalate plasticizers, the degradation rate of the phthalate plasticizers in the soil exceeds 95% at 30°C after 7 days.

[0014] Compared with the prior art, the present invention provides Bacillus sp. GBD3 degradation bacteria has a strong ability to degrade phthalate plasticizers in the soil, and because it is a Bacillus, the degradation bacteria agent is easy to produce and store, which can provide convenient operation for solving the phthalate plasticizer pollution in the ecological environment.

[0015] The present invention screened Bacillus sp. GBD3, which is Gram-positive, while the strains Camelimonas sp. and Sphingobacterium thalpophilum in the aforementioned prior art are Gram-negative bacteria, and their environmental adaptability is far inferior to that of the present invention. Therefore, the technical difficulty of the strain application of the present invention is lower and the application field may be wider. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Screening results of GBD3 degradation bacteria of the present invention (transparent hydrolysis circle on flat plate).

[0017] Figure 2 The substrate degradation range of the GBD3 degradation bacteria of the present invention includes: DMP: dimethyl phthalate; DEP: diethyl phthalate; DPP: dipropyl phthalate; DBP: dibutyl phthalate; DNPP: dipentyl phthalate; and DNHP: dihexyl phthalate.

[0018] Figure 3 The OD of the GBD3 degradation bacteria of the present invention when cultured with DBP as the sole carbon source 600 Growth curve.

[0019] Figure 4 Degradation of phthalate plasticizers in soil 7 days after spraying the GBD3 degradation bacteria of the present invention. DETAILED DESCRIPTION

[0020] The present invention is further described in detail below in conjunction with the embodiments (attached drawings): Example 1: A Phthalate Plasticizer Degrading Bacteria Bacillus The preparation method of the GBD3 strain comprises the following steps: Soil samples were collected from tobacco fields in Zhengzhou City, Henan Province, and placed in sterile sampling bottles. Sterile water was added and shaken to perform gradient dilution of the turbid soil solution.

[0021] The gradient diluted soil solution was coated on LB medium plates and cultured at 30°C for 24-72 hours. The LB medium formula is: tryptone 10.0 g / L, yeast powder 5.0 g / L, NaCl 10.0 g / L, agar powder 15 g / L. pH 7.0, autoclave at 121°C for 20 min. Phthalate plasticizers were added to the LB medium at a final concentration of 0.10 mM.

[0022] The bacteria producing the larger transparent hydrolysis circle in the above culture medium were cultured to obtain a strain with stronger degradation ability, named GBD3. Bacillus sp., deposited in the General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC) on November 3, 2023, with the strain deposit number CGMCC No. 28860. Its main biological characteristics are round, light yellow colonies with smooth surfaces and sticky colonies. It is Gram-positive, has spores, and can decompose starch; the catalase test, glucose oxidation, VP test, and indole test are all positive, and the methyl red test is negative.

[0023] Example 2: Substrate degradation scope of phthalate plasticizer-degrading bacteria.

[0024] The phthalate plasticizer degradation bacteria obtained above were cultured in LB medium at 30°C and 250 rpm for 24 hours, centrifuged at 12000 rpm for 5 minutes under sterile conditions, washed twice with sterile 1.0 mM sodium phosphate buffer, and then inoculated with 5% of the inoculum into an inorganic salt liquid medium containing phthalate plasticizers of different side chain lengths at a final concentration of 0.10 mM, and cultured at 30°C and 250 rpm for 72 hours. The formula of the inorganic salt liquid medium is: NaCl 1.0 g / L, (NH4)2S041.0g / L, K2HPO41.5g / L, KH2PO41.5 g / L, MgSO4•7H2O 0.01 g / L, pH 7.0, and autoclaved at 121°C for 20 minutes. Add 2 times the volume of methanol, mix thoroughly, centrifuge, and take the supernatant to detect the degradation effect by HPLC / MS. The detection conditions are as follows: drying gas temperature: 250°C; drying gas flow rate (nitrogen): 8.0 L / min; nebulizer gas pressure (nitrogen): 35 psi; capillary voltage: 4000 V; sheath gas temperature: 300°C; sheath gas flow rate: 10 L / min; nozzle voltage: 400 V. The HPLC column is an Eclipse XDB-Ci column with a particle size of 4.6 mm × 250 mm and 5.0 μm (Agilent). The mobile phase is 90% methanol and 10% water at a flow rate of 1 mL / min. Figure 2As shown in the figure, the obtained degradation bacteria can degrade and transform phthalate plasticizers of different chain lengths, including C2-C5: diethyl phthalate (DEP), dipropyl phthalate (DPP), dibutyl phthalate (DBP), and dipentyl phthalate (DNPP), and dipropyl phthalate (DBP) has the strongest degradation ability, which can be completely degraded within 72 hours, followed by dibutyl phthalate (DPP), which is degraded by about 95% within 72 hours, diethyl phthalate (DEP) is degraded by about 80% within 72 hours, and dipentyl phthalate (DNPP) is degraded by about 65% within 72 hours. However, there is basically no degradation activity for the short-chain ester dimethyl phthalate (DMP) and the long-chain ester dihexyl phthalate (DNHP).

[0025] Example 3: OD of Phthalate Plasticizer Degrading Bacteria When Cultivated with DBP as the Sole Carbon Source 600 Growth curve ( Figure 3 Show).

[0026] The phthalate plasticizer-degrading bacteria obtained above were cultured in LB medium at 30°C and 250 rpm for 24 h, centrifuged at 12,000 rpm for 5 min under sterile conditions, washed twice with sterile 1.0 mM sodium phosphate buffer, and then inoculated with 0.5% of the inoculum into an inorganic salt liquid medium containing a final concentration of 0.40 mM dibutyl phthalate (DBP), shaken at 30°C and 250 rpm, and the culture fluid OD was measured regularly. 600 . Bacillus sp. GBD3 strain can grow with DBP as the only carbon source.

[0027] Example 4: Degradation of Phthalate Plasticizers in Soil by Phthalate Plasticizer Degrading Bacteria The phthalate plasticizer-degrading bacteria obtained above were cultured in LB medium at 30°C and 250 rpm for 24 h, centrifuged at 12,000 rpm for 5 min under sterile conditions, washed twice with sterile 1.0 mM sodium phosphate buffer, and then sprayed at a 1% inoculum into the garden soil to which phthalate plasticizers were added at a final concentration of 0.10 mM. The phthalate plasticizers here include diethyl phthalate (DEP), dipropyl phthalate (DPP), dibutyl phthalate (DBP) and dipentyl phthalate (DNPP). After culturing at room temperature for 7 days, soil samples were collected, 2 volumes of methanol were added, mixed thoroughly, and the supernatant was taken after centrifugation to detect the degradation effect of phthalate plasticizers in the soil using HPLC / MS. The removal effect of phthalate plasticizers is as follows. Figure 4 As shown in the figure, the degradation rates of the four phthalate plasticizers in the soil by the degrading bacteria were all above 95%.

[0028] Sequence Listing <110> China National Tobacco Corporation Zhengzhou Tobacco Research Institute <120> A Bacillus strain GBD3 and its application in degrading phthalate plasticizers <160> 1 <211> 1442 <212> DNA <213> Strain GBD3 <400> 1 TGGGCGGCGT GCTATACATG CAAGTCGAGC GAACCACTTC GGTGGTTAGC GGCGGACGGG 60 TGAGTAACAC GTGGGCAACC TGCCTGTAAG ACTGGGATAA CTTCGGGAAA CCGGAGCTAA 120 TACCGGATAA TCCTTTTCCT CACATGAGGA AAAGCTGAAA GTCGGTTTCG GCTGACACTT 180 ACAGATGGGC CCGCGGCGCA TTAGCTAGTT GGTGAGGTAA CGGCTCACCA AGGCGACGAT 240 GCGTAGCCGA CCTGAGAGGG TGATCGGCCA CACTGGGACT GAGACACGGC CCAGACTCCT 300 ACGGGAGGCA GCAGTAGGGA ATCTTCCACA ATGGACGAAA GTCTGATGGA GCAACGCCGC 360 GTGAGCGATG AAGGCCTTCG GGTCGTAAAG CTCTGTTGTT AGGGAAGAAC AAGTATCGGA 420 GTAACTGCCG GTACCTTGAC GGTACCTAAC CAGAAAGCCA CGGCTAACTA CGTGCCAGCA 480 GCCGCGGTAA TACGTAGGTG GCAAGCGTTG TCCGGAATTA TTGGGCGTAA AGCGCGCGCA 540 GGCGGTCCTT TAAGTCTGAT GTGAAAGCCC ACGGCTCAAC CGTGGAGGGT CATTGGAAAC 600 TGGGGGACTT GAGTGCAGAA GAGGAAAGCG GAATTCCACG TGTAGCGGTG AAATGCGTAG 660 AGATGTGGAG GAACACCAGT GGCGAAGGCG GCTTTCTGGT CTGTAACTGA CGCTGAGGCG 720 CGAAAGCGTG GGGAGCAAAC AGGATTAGAT ACCCTGGTAG TCCACGCCGT AAACGATGAG 780 TGCTAAGTGT TAGGGGGTTT CCGCCCCTTA GTGCTGCAGC TAACGCATTA AGCACTCCGC 840 CTGGGGAGTA CGGCCGCAAG GCTGAAACTC AAAGGAATTG ACGGGGGCCC GCACAAGCGG 900 TGGAGCATGT GGTTTAATTC GAAGCAACGC GAAGAACCTT ACCAGGTCTT GACATCCTCT 960 GACACTCCTA GAGATAGGAC TTTCCCCTTC GGGGGACAGA GTGACAGGTG GTGCATGGTT1020 GTCGTCAGCT CGTGTCGTGA GATGTTGGGT TAAGTCCCGC AACGAGCGCA ACCCTTGATC1080 TTAGTTGCCA GCATTCAGTT GGGCACTCTA AGGTGACTGC CGGTGACAAA CCGGAGGAAG1140 GTGGGGATGA CGTCAAATCA TCATGCCCCT TATGACCTGG GCTACACACG TGCTACAATG1200 GATGGTACAA AGGGCTGCAA GACCGCGAGG TTTAGCCAAT CCCATAAAAC CATTCTCAGT1260 TCGGATTGTA GGCTGCAACT CGCCTACATG AAGCCGGAAT CGCTAGTAAT CGCGGATCAG1320 CATGCCGCGG TGAATACGTT CCCGGGCCTT GTACACACCG CCCGTCACAC CACGAGAGTT1380 TGTAACACCC GAAGTCGGTG GGGTAACCGT AAGGAGCCAG CCGCCTAAGG TCACCAGAAA1440 TG 1442

Claims

1. A Bacillus strain GBD3, characterized in that: Its classification is named Bacillus sp . GBD3, this strain has been deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, the deposit date is November 03, 2023, and the deposit number is CGMCC No. 28860.

2. The Bacillus strain GBD3 according to claim 1, characterized in that: The 16SrDNA gene sequence table of the strain GBD3 is shown in SEQUENCE LISTING.

3. The Bacillus strain GBD3 according to claim 1 or 2, characterized in that: The colonies of the GBD3 strain are round, light yellow, smooth, sticky, Gram-positive, spore-forming, and capable of decomposing starch; the catalase test, glucose oxidation test, VP test, and indole test are all positive, and the methyl red test is negative.

4. Use of the strain GBD3 according to claim 1 in degrading phthalate plasticizers with a side chain length of C2 to C5.

5. The use according to claim 4, characterized in that: The phthalate plasticizer with a side chain length of C2 to C5 includes a mixture of any one or more of diethyl phthalate, dipropyl phthalate, dibutyl phthalate and dipentyl phthalate.

6. The use according to claim 4, characterized in that: The strain GBD3 is suitable for degrading phthalate plasticizers in the soil at 30°, and the degradation rate of phthalate plasticizers in the soil exceeds 95% after 7 days.

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