Gordonia polyisoprene LCF4 and application of Gordonia polyisoprene LCF4 in degradation of phthalate plasticizers

Abstract

The invention discloses a Gordonia polyisoprene LCF4 strain and application of the Gordonia polyisoprene LCF4 strain in degradation of phthalate plasticizers, and belongs to the technical field of microorganisms. The polyisoprene gordonia LCF4 is preserved in China General Microbiological Culture Collection Center (CGMCC) on December 13, 2024, the preservation number is CGMCC NO.33066, and the preservation address is No.3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The Gordonia polyisoprene LCF4 can efficiently degrade DEHP with the initial concentration of 50-2000 mg / L, the strain is inoculated to a culture medium containing the DEHP with the concentration of 2000 mg / L to be cultured for 72 h, and the degradation rate reaches 99.34%. The Gordonia polyisoprene LCF4 also can be used for effectively degrading other PAEs plasticizers (DEP, DPRP, DBP, BBP, DPP and DOP) with different side chains and DEHP intermediate products (MEHP and PCA).

Description

Technical Field

[0001] The invention relates to a polyisoprene-eating Gordonia sp. LCF4 and application thereof in degrading phthalate plasticizers, belonging to the technical field of microorganisms. Background Art

[0002] Phthalates (PAEs) are a class of commonly used plasticizers, which are widely used in food packaging materials, personal care products, children's toys and other products. Di(2-ethylhexyl) phthalate (DEHP), as a typical PAEs plasticizer, is widely used in people's daily lives, especially as a plastic additive, which can reach 20-50% by weight. Since DEHP in plastic products is easily transferred into the environment, it is widely present in the atmosphere, water, soil and organisms. DEHP is the most toxic among phthalates. It can enter the body through various pathways, seriously interfere with its endocrine, reproductive and nervous systems, and has extremely strong teratogenicity, mutagenicity and carcinogenicity. At present, DEHP has become one of the most common pollutants in the world and has been listed as a priority controlled pollutant by the US Environmental Protection Agency and the China National Environmental Monitoring Center.

[0003] The hydrolysis, photolysis and volatilization rates of DEHP in the natural environment are very slow, and biodegradation has a natural advantage in removing DEHP from the environment. However, due to the long side chain of DEHP, although many strains with DEHP degradation activity have been reported, the strains that can efficiently degrade different concentrations of DEHP at the same time and have a broad spectrum of substrates are still relatively limited. Therefore, it is of great significance to screen DEHP-efficient degrading bacteria and study their degradation characteristics. Summary of the invention

[0004] The purpose of the present invention is to provide a highly efficient DEHP-degrading bacterium, Gordonia polyisoprenosum, and application of the strain in degrading PAEs plasticizers and their intermediates.

[0005] To achieve the above object, the technical solution adopted by the present invention is as follows: The present invention provides a strain of Gordonia polyisoprenivorans LCF4, which was deposited in the General Microbiological Center of China Microbiological Culture Collection Administration on December 13, 2024, and was classified and named Gordonia polyisoprenivorans (Gordoniarpolyisoprenivorans), with a deposit number of CGMCCNO: 33066, and a deposit address of No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; The polyisoprene-eating Gordonia sp. LCF4 was isolated and purified from a landfill soil sample from Liancheng, Fujian.

[0006] The present invention also provides the use of the polyisoprene-eating Gordonia LCF4 in degrading PAEs plasticizers and their intermediates; The PAEs plasticizer is selected from di(2-ethylhexyl) phthalate (DEHP), dimethyl phthalate (DMP), diethyl phthalate (DEP), dipropyl phthalate (DPRP), dibutyl phthalate (DBP), butyl benzyl phthalate (BBP), di-n-propyl phthalate (DPP), and dioctyl phthalate (DOP); the intermediate product is selected from mono(2-ethylhexyl) phthalate (MEHP) and protocatechuic acid (PCA).

[0007] In one embodiment of the present invention, the seed liquid of the polyisoprene-eating Gordonia sp. LCF4 is inoculated into a liquid culture medium containing a PAEs plasticizer and / or its intermediates for co-cultivation; The PAEs plasticizer is selected from DEHP, DMP, DEP, DPRP, DBP, BBP, DPP, and DOP; the intermediate product is selected from MEHP and PCA; The culture conditions are: temperature 30° C., pH 8.0.

[0008] The present invention also provides a bacterial agent for degrading PAEs plasticizers and intermediates thereof, wherein the bacterial agent contains the polyisoprene-eating Gordonia sp. LCF4 as an active ingredient.

[0009] The beneficial effects of the present invention are: The present invention provides a polyisoprene-eating Gordonia LCF4, which can efficiently degrade DEHP with an initial concentration of 50 to 2000 mg / L. The strain is inoculated into a culture medium containing 2000 mg / L DEHP at an inoculation amount of 1% (v / v) and cultured for 72 hours, and the degradation rate reaches 99.34%. The optimal DEHP degradation conditions of the strain are 30°C and pH 8.0. The polyisoprene-eating Gordonia LCF4 can also effectively degrade other PAEs plasticizers (DEP, DPRP, DBP, BBP, DPP and DOP) with different side chains and DEHP intermediates (MEHP and PCA). The polyisoprene-eating Gordonia LCF4 provided by the present invention provides a new solution for the efficient degradation of PAEs plasticizers and their intermediates, and also enriches the resources of DEHP-degrading strains. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 The colony morphology and bacterial morphology of Gordonia polyisoprena LCF4.

[0011] Figure 2Phylogenetic tree of 16S rDNA of Gordonia polyisoprenivora LCF4.

[0012] Figure 3 The optimal pH condition for the degradation of DEHP by Gordonia polyisoprene LCF4.

[0013] Figure 4 The optimal temperature condition for the degradation of DEHP by Gordonia polyisoprene LCF4.

[0014] Figure 5 Degradation of DEHP at different initial concentrations by Gordonia polyisoprene LCF4.

[0015] Figure 6 Degradation of different PAEs plasticizers by Gordonia polyisoprene LCF4.

[0016] Figure 7 The degradation of PAEs plasticizer intermediates PA, PCA and MEHP by polyisoprene-feeding Gordonella LCF4. DETAILED DESCRIPTION

[0017] The following examples are further elaborations of the present invention, but the protection scope of the present invention is not limited thereto.

[0018] 1 Experimental Materials and Methods 1.1 The culture medium formula described in the embodiment is as follows: MSM liquid medium (L -1 ): K 2 HPO 4 1.5g, KH 2 PO 4 0.5g, CaCl 2 ·2H 2 O 0.02g, MgSO 4 7H 2 O0.2g, (NH 4 ) 2 SO 4 1.5g, NaCl 0.5g, FeCl 3 0.001 g, sterilized at 121°C for 20 min; for solid culture medium, 1.5% (w / v) agar powder was added to the liquid culture medium.

[0019] LB liquid medium (L -1 ): 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, sterilized at 121°C for 20 min; solid culture medium was supplemented with 1.5% (w / v) agar powder on the basis of liquid culture medium.

[0020] 1.2 Screening and identification of strains (1) Screening of strains Landfill soil from Liancheng, Fujian was collected and added to MSM liquid culture containing 100, 200, 400, and 600 mg / L DEHP as the sole carbon source for gradient enrichment. After four generations of enrichment, the enrichment liquid LCF with DEHP degradation activity was selected for single bacterial isolation and screening. Take 200 μL of the diluted enrichment liquid LCF and spread it on the MSM solid culture medium with 200 mg / L DEHP as the sole carbon source, and culture it in a constant temperature incubator at 30°C for 5 days. The single bacteria grown on the solid culture medium were selected for repeated streaking purification until a single colony appeared, and the degradation activity of the purified strain on DEHP was verified by HPLC. All experiments were set up in three parallel groups.

[0021] (2) Identification of strains ① Observation of colony morphology of strains Scrape off a single colony and inoculate it onto LB solid medium for streak culture. After culturing at 30°C for 48 h, observe the colony morphology.

[0022] ②Molecular biological identification of strains The genomic DNA of the strain was extracted and purified by referring to the bacterial genome extraction kit TIANamp Bacteria DNA Kit 50, and the 16S rDNA fragment of the strain was amplified by PCR using universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACGGCCTACCTTGTTACGACTT-3'). The PCR amplification used a 25 μL reaction system, including: 2×Taq Master Mix 12.5 μL, 10 μmol / L upstream primer 1 μL, 10 μmol / L downstream primer 1 μL, DNA template 1 μL, ddH 2 O 9.5μL. The PCR amplification program was: 94℃ pre-denaturation for 3min; 94℃ denaturation for 30s, 50℃ annealing for 45s, 72℃ extension for 1min 40s, for a total of 35 cycles; and finally 72℃ extension for 7min. After the amplified product was detected by 1% agarose gel electrophoresis, the area with the target fragment in the gel strip was cut out and recovered using the SanPrep Column DNA Gel Extraction Kit. The target fragment was introduced into DH5α competent cells by TA cloning, and the positive cloned bacterial solution with successful TA cloning was sent to Shanghai Biotech for sequencing. The sequencing results were homologously aligned on EzBioCloud to construct a phylogenetic tree.

[0023] 1.3 Optimal growth conditions for strains to degrade DEHP (1) pH: OD600 The purified strain with a value of 0.8 was inoculated at a 1% (v / v) inoculation amount into MSM liquid culture medium with 2000 mg / L DEHP as the sole carbon source at pH 5, 6, 7, 8, 9, 10, and 11, respectively, and the MSM liquid culture medium under the same treatment conditions without inoculation of the bacterial solution was used as the control group. The culture was carried out in a constant temperature shaking incubator at 30°C and 180 rpm for 72 hours, and samples were taken. The residual amount of DEHP in the culture medium was determined by liquid chromatography and the OD of the culture medium was detected by ultraviolet spectrophotometer. 600 The degradation rate was calculated based on the values. All experiments were performed in triplicate.

[0024] (2) Temperature: set OD 600 The purified strain with a value of 0.8 was inoculated at a 1% (v / v) inoculation amount into an MSM liquid culture medium with a pH of 8 and 2000 mg / L DEHP as the sole carbon source, and the MSM liquid culture medium without inoculation under the same treatment conditions was used as a control group. The culture was cultured in a constant temperature shaking incubator at 20, 25, 30, 35, 40, 45, and 50°C and 180 rpm for 72 h, and samples were taken. The residual amount of DEHP in the culture medium was determined by liquid chromatography and the OD of the culture medium was detected by ultraviolet spectrophotometer. 600 The degradation rate was calculated based on the values. All experiments were performed in triplicate.

[0025] 1.4 Degradation of different concentrations of DEHP by strains OD 600 The purified strain culture liquid with a value of 0.8 was inoculated at an inoculum rate of 1% (v / v) into MSM liquid culture medium with a pH of 8 and 50, 100, 200, 500, 800, 1000, 1500, and 2000 mg / L DEHP as the sole carbon source, respectively, and the MSM liquid culture medium without inoculation of the bacterial liquid under the same treatment conditions was used as the control group. The culture was cultured in a constant temperature shaking incubator at 30°C and 180 rpm. Samples were taken after 12, 24, 36, 48, 60, and 72 hours of culture, and the residual amount of DEHP in the culture medium was determined by liquid chromatography and the OD of the culture medium was detected by ultraviolet spectrophotometer. 600 The degradation rate was calculated based on the values. All experiments were performed in triplicate.

[0026] 1.5 Testing of the broad-spectrum degradation of different PAEs plasticizers and their intermediates by strains OD 600The purified strain culture liquid with a value of 0.8 was inoculated at an inoculation rate of 1% (v / v) into MSM liquid culture medium with a pH of 8 and 2000 mg / L of different PAEs plasticizers (DMP, DEP, DPrP, DBP, DOP, DEHP, BBP, DCHP) as the sole carbon source, and the MSM liquid culture medium without inoculation of bacterial liquid under the same treatment conditions was used as the control group. The culture was carried out in a constant temperature shaking incubator at 30°C and 180rpm for 36 hours, and samples were taken. The residual amount of PAEs plasticizers in the culture medium was determined by liquid chromatography and the OD of the culture medium was detected by ultraviolet spectrophotometer. 600 The degradation rate was calculated based on the values. All experiments were performed in triplicate.

[0027] OD 600 The purified strain culture liquid with a value of 0.8 was inoculated at an inoculation rate of 1% (v / v) into MSM liquid culture medium with a pH of 8 and different PAEs plasticizer intermediates (1000 mg / L MEHP, 1000 mg / L PA, 1000 mg / L PCA) as the sole carbon source, and the MSM liquid culture medium without inoculation of the bacterial liquid under the same treatment conditions was used as the control group. The culture was carried out in a constant temperature shaking incubator at 30°C and 180 rpm. Samples were taken after 12, 24, 36, 48, 60, and 72 hours of culture, and the residual amount of PAEs plasticizer intermediates in the culture medium was determined by liquid chromatography and the OD of the culture medium was detected by ultraviolet spectrophotometer. 600 The degradation rate was calculated based on the values. All experiments were performed in triplicate.

[0028] 2 Results and analysis 2.1 Identification of strain LCF4 (1) Morphology of strain LCF4 The strain LCF4 is a highly efficient DEHP-degrading bacterium isolated from the bacterial community LCF. The colony morphology of the strain LCF4 streaked on the LB plate is as follows: Figure 1 As shown in a, the colony morphology is light yellow and opaque spherical, with a smooth and moist surface, slightly convex, and neat edges. The morphological characteristics of strain LCF4 under SEM magnified 10,000 times are shown in Figure 1 As shown in b, its bacterial morphology is a long rod-shaped structure and it is a bacillus.

[0029] (2) Molecular biological identification of strain LCF4 The 16S rDNA sequence of strain LCF4 is accessed in Genbank as PP053495 (SEQ ID NO.1). Homology comparison was performed on EzBioCloud. The results showed that strain LCF4 was similar to Gordonia polyisoprenivorans NBRC 16320. TThe phylogenetic tree analysis results are as follows: Figure 2 As shown, strain LCF4 and Gordonia polyisoprenivorans NBRC 16320 T In the same branch, the bootstrap value is 100. Therefore, strain LCF4 is a microorganism of the genus Gordonia. Strain LCF4 was deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration on December 13, 2024, and was classified and named Gordonia polyisoprenivorans, with a deposit number of CGMCC NO.33066, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0030] 2.2 Optimal growth conditions for strain LCF4 to degrade DEHP (1) pH: The degradation of DEHP by strain LCF4 under different pH conditions Figure 3 When the pH is between 5 and 8, the degradation rate of DEHP and the OD value of the culture medium increase with the increase of pH. 600 When the pH is between 8 and 9, as the pH increases, the DEHP degradation rate is basically not affected by the pH change and remains at around 99%. When the pH is between 9 and 11, as the pH increases, the DEHP degradation rate and the culture medium OD 600 The values ​​decreased rapidly, especially when the pH was 11, the degradation rate dropped rapidly from 70.31% at pH 10 to 9.06%. 600 The value was only 0.199. Therefore, the optimal pH for strain LCF4 to degrade DEHP was 8, under which the strain had the highest DEHP degradation rate and the largest biomass.

[0031] (2) Temperature: The degradation of DEHP by strain LCF4 under different temperature conditions is as follows Figure 4 When the temperature is between 20 and 30°C, the degradation rate of DEHP and the OD value of the culture medium increase with the increase of temperature. 600 When the temperature is between 30 and 40°C, the degradation rate of DEHP does not decrease significantly with the increase of temperature, and the degradation rate is above 99%. 600 When the temperature is 45℃, the degradation rate of DEHP and the OD value of the culture medium 600 The value began to decrease slowly. When the temperature rose to 50℃, the degradation rate of DEHP dropped sharply from 84.97% to 5.43%. 600The value dropped sharply from 1.564 to 0.126. Therefore, the optimal temperature for strain LCF4 to degrade DEHP is 30℃, under which the strain has the highest DEHP degradation rate and the largest biomass.

[0032] 2.3 Degradation of DEHP at different initial concentrations by strain LCF4 The degradation of DEHP by strain LCF4 at different initial concentrations is shown in Figure 5 As shown. The degradation rate of strain LCF4 was very low in the first 12 hours. After 12 hours of inoculation, the degradation rate of DEHP began to increase rapidly. At 36 hours, the degradation rate of DEHP in the concentration range of 50-1000 mg / L was above 90%. At 48 hours, the DEHP in the concentration range of 50-1500 mg / L was almost completely degraded. At 72 hours, the degradation rate of strain LCF4 for 2000 mg / L DEHP was as high as 99.34%. Therefore, strain LCF4 can efficiently degrade DEHP with an initial concentration of 50-2000 mg / L.

[0033] 2.4 Degradation of different PAEs plasticizers and their intermediates by strain LCF4 The degradation of different PAEs plasticizers with an initial concentration of 2000 mg / L by strain LCF4 is shown in Figure 6 As shown. At 36h, the degradation rates of strain LCF4 for PAEs plasticizers DMP, DEP, and DPRP with shorter side chains were 34.25%, 35.79%, and 24.16%, respectively, and the degradation rates for PAEs plasticizers DBP, BBP, DPP, DEHP, and DOP with longer side chains were 95.22%, 94.96%, 91.84%, 67.09%, and 60.01%, respectively, while the degradation rate for DCHP was extremely low. Therefore, strain LCF4 can effectively degrade the other 8 PAEs plasticizers except DCHP, and has a good substrate broad spectrum.

[0034] The degradation of PAEs plasticizer intermediates MEHP, PA, and PCA by strain LCF4 is shown in Figure 7 As shown. In the first 24 hours, the degradation rate and growth rate of strain LCF4 for MEHP were significantly lower than those of PCA; at 12h and 24h, the degradation rates of MEHP were 4.77% and 14.95%, respectively, while the degradation rates for PCA were 30.33% and 39.60%, respectively; after 24h, the degradation rate of MEHP began to accelerate significantly, reaching 82.44% at 60h and 99.75% at 72h, at which time MEHP was almost completely degraded. In the process of MEHP degradation by strain LCF4, the bacterial concentration OD 600 The OD at 72h has been on an upward trend. 600The value was 1.368. The PCA degradation rate of the strain was higher than that of MEHP before 60h of degradation. At 60h, the PCA degradation rate reached 94.77%. At 72h, the PCA degradation rate was almost unchanged. This may be due to the low PCA concentration in the later period, which is not conducive to the absorption and utilization of the strain. The growth rate of the strain was relatively fast when the strain degraded PCA. At 36h, the bacterial concentration OD 600 The maximum value reached 1.769, and the bacterial concentration decreased with the extension of degradation time. The strain LCF4 could not grow under the condition of PA concentration of 1000 mg / L, and the PA concentration did not decrease with the extension of culture time, indicating that the strain LCF4 had no degradation activity for PA.

[0035] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A polyisoprenophagous Gordonia strain LCF4, characterized in that: The polyisoprene-eating Gordonia LCF4 was deposited in the General Microbiological Center of China Microbiological Culture Collection Administration on December 13, 2024, and was classified and named as polyisoprene-eating Gordonia ( Gordonia polyisoprenivorans ), the deposit number is CGMCC NO.33066, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

2. Use of the polyisoprene-eating Gordonia sp. LCF4 as claimed in claim 1 in degrading PAEs plasticizers and their intermediates.

3. The use according to claim 2, characterized in that: The PAEs plasticizer is selected from DEHP, DMP, DEP, DPRP, DBP, BBP, DPP, and DOP; and the intermediate product is selected from MEHP and PCA.

4. A method for degrading PAEs plasticizers, characterized in that: The polyisoprene-eating Gordonia LCF4 of claim 1 is co-cultured with a PAEs plasticizer.

5. The method according to claim 4, characterized in that: The PAEs plasticizer is selected from DEHP, DMP, DEP, DPRP, DBP, DOP, DPP, and BBP.

6. The method according to claim 4, characterized in that: The culture conditions are: temperature 30° C., pH 8.

0.

7. A method for degrading PAEs plasticizer intermediates, characterized in that: The polyisoprene-eating Gordonia LCF4 of claim 1 is co-cultured with a PAEs plasticizer intermediate.

8. The method according to claim 7, characterized in that: The PAEs plasticizer intermediate products are selected from MEHP and PCA respectively.

9. The method according to claim 7, characterized in that: The culture conditions are: temperature 30° C., pH 8.

0.

10. A bacterial agent for degrading PAEs plasticizers and their intermediates, characterized in that: The bacterial agent contains the polyisoprenophagous Gordonella LCF4 described in claim 1 as an active ingredient.

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