Dibutyl phthalate degrading bacterium and application thereof

Abstract

The invention discloses a dibutyl phthalate degrading bacterium and application thereof, and belongs to the technical field of biological strains, the dibutyl phthalate degrading bacterium is microbacterium paraoxidans CGY-B, and the preservation number is CGMCC No.30456. The dibutyl phthalate degrading bacterium is inoculated into an environment containing dibutyl phthalate, biodegradation of organic pollutants is achieved, and the dibutyl phthalate degrading bacterium can be used for degrading the organic pollutants. By culturing the degrading bacterium provided by the invention, an efficient, green and cheap environment restoration method can be provided for degrading dibutyl phthalate, so that the serious threat of dibutyl phthalate to human health and ecological environment is reduced.

Description

Technical Field

[0001] The invention belongs to the technical field of biological strains and relates to dibutyl phthalate degrading bacteria and application thereof. Background Art

[0002] Phthalates (PAEs) are primarily used as plasticizers in plastic manufacturing and are also widely used in many consumer products. Since they are not chemically bound to these products, they tend to escape easily into the environment during manufacturing, product use, and product disposal. As a result, traces of phthalates have been found in soil sediments, water bodies, and groundwater sources around the world. PAEs can cause carcinogenicity, endocrine disruption, and reproductive and developmental interference in humans and animals. Dibutyl phthalate (DBP) is one of the most common PAEs and has received attention as a priority pollutant by the U.S. Environmental Protection Agency and the European Parliament and Council due to its repeated toxicity. It has been associated with hepatotoxicity, mutagenicity, teratogenicity, and carcinogenicity in humans at low concentrations. Several countries, including the European Union, Argentina, Japan, Mexico, China, and the United States, have imposed limits on the concentration of DBP and other PAEs (from 60% to 0.1%) in consumable plastic products designed for children due to health concerns. However, despite these limitations, global production of PAEs, including DBP, continues to rise, posing a significant threat to human and environmental health due to their continued release into the environment.

[0003] The complex chemical structure and hydrophobicity of DBP hinder its physical degradation. Therefore, people have explored various physical, chemical and biological methods to remove these organic pollutants from the environment. Among them, microbial-mediated degradation has become an effective and environmentally friendly remediation method. Many DBP-degrading bacteria with different metabolic pathways have been isolated from contaminated soil, activated sludge, sewage, river sediments, wetlands and other soils. These include Gordonia isolated from plants; Bacillus isolated from activated sludge; Sphingococcus from gasoline-contaminated soil; Methylobacterium from plastic industrial wastewater discharge, etc. The degradation effects of different genera vary, and the application effects are also different. The bacterial-mediated biodegradation of dibutyl phthalate is greatly affected by environmental factors. High concentrations of dibutyl phthalate and pH pressure often hinder the growth of microorganisms and affect their degradation ability. Therefore, it is of great significance to study and solve the bioremediation of PAEs environmental pollution by screening strains that have strong adaptability to changes in environmental factors and have good effects on phthalate degradation.

[0004] In order to solve the above problems, the present invention is proposed. Summary of the invention

[0005] In view of the above technical problems, the present invention aims to provide a dibutyl phthalate-degrading bacterium and application thereof, wherein the degrading bacterium can effectively, quickly and efficiently degrade dibutyl phthalate in a system containing dibutyl phthalate.

[0006] To achieve the above object, the technical solution adopted by the present invention is:

[0007] A dibutyl phthalate-degrading bacterium, the degrading bacterium is Microbacterium paraoxidans CGY-B, which was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on April 26, 2024, with the deposit number CGMCC No.30456.

[0008] The phosphate-solubilizing bacteria are cultured and grown in an inorganic salt liquid medium with dibutyl phthalate as the sole carbon source; the gene sequence of the strain is shown in the bases in SEQ ID No.1;

[0009] The components of the inorganic salt liquid culture medium with dibutyl phthalate as the sole carbon source are: 1.0 g of dimethyl hydrogen phosphate heptahydrate, 0.5 g of ammonium sulfate, 1.0 g of sodium chloride, 0.4 g of magnesium sulfate heptahydrate, 0.01 g of calcium chloride, 0.01 g of ferric chloride hexahydrate, 100 μL of 10 g / L DBP methanol solution, pH=7, the inoculation amount of the degrading bacteria is 1%, and the solution volume is 20 mL.

[0010] Before inoculating into an inorganic salt liquid culture medium with dibutyl phthalate as the sole carbon source for culturing and growing, the method also includes pre-culturing the degrading bacteria, inoculating the degrading bacteria into a nutrient broth culture medium for pre-culturing for about 20 hours, wherein the ingredients of the nutrient broth culture medium are: 0.3 g beef extract powder, 1.0 g peptone, 0.5 g NaCl, pH value 7.2±0.2, the inoculation amount of the degrading bacteria is one ring, and the solution volume is 100 mL.

[0011] The invention also provides application of the degradation bacteria, and application of the strain in degrading dibutyl phthalate.

[0012] As a limitation of the application of the dibutyl phthalate degradation bacteria of the present invention, the degradation bacteria are inoculated into an environment containing the dibutyl phthalate organic pollutant which is difficult to degrade, so as to achieve the degradation of dibutyl phthalate.

[0013] The above technical solution of the present invention has the following advantages or beneficial effects:

[0014] The dibutyl phthalate degrading bacteria Microbacterium paraoxidans CGY-B screened by the present invention has a strong degradation ability for the difficult-to-degrade organic pollutant dibutyl phthalate. After the strain is cultivated, the degradation bacteria is inoculated into a system containing dibutyl phthalate, and the dibutyl phthalate in the system can be effectively, quickly and efficiently degraded, and has a good degradation effect. Therefore, it can be applied to environments such as soil and water bodies that are contaminated by the escape of dibutyl phthalate, reduce the concentration of dibutyl phthalate in the environment, and alleviate environmental pressure, and has good application prospects and promotion value. The degradation products of dibutyl phthalate degraded by the strain are detected, and the analysis results show that the strain CGY-B has the ability to degrade the organic dibutyl phthalate and finally completely mineralize it into water and carbon dioxide. In addition, by conducting whole genome testing on the degrading bacteria, determining their functional characteristics, predicting and mining degradation-related functional genes, and inferring their biological metabolic pathways, it is helpful to further explore the biological metabolic mechanism of DBP and has certain research significance for the bioremediation of DBP-contaminated environments.

[0015] The specific implementation of the present invention will be further described in detail below in conjunction with the embodiments of the specification and the drawings of the specification. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The colony morphology of 4 strains of dibutyl phthalate-degrading bacteria screened by the present invention, wherein: A is CGY-A, B is CGY-B, C is CGY-C, and D is CGY-D;

[0017] Figure 2 This is a graph showing the effects of the four strains screened to degrade dibutyl phthalate, where A, B, C and D represent strains CGY-A, CGY-B, CGY-C and CGY-D, respectively;

[0018] Figure 3 This is the effect of substrate concentration on the degradation rate of CGY-B;

[0019] Figure 4 This is the effect of pH on the degradation of CGY-B;

[0020] Figure 5 This is the effect of temperature on the degradation effect of bacteria CGY-B;

[0021] Figure 6 This is the colony morphology of the bacterium CGY-B;

[0022] Figure 7 is the phylogenetic tree of bacterial CGY-B;

[0023] Figure 8 This is the result of the methyl red test of bacteria CGY-B;

[0024] Fig. 9 This is the result of the indole experiment of the bacteria CGY-B;

[0025] Fig.10 This is the result of the glucose fermentation experiment of the bacteria CGY-B;

[0026] Fig.11 This is a diagram of metabolites produced by the degradation of DBP by bacteria CGY-B;

[0027] Fig.12 This is the NR annotation classification statistics of strain CGY-B;

[0028] Fig.13 This is the COG annotation classification statistics of strain CGY-B;

[0029] Fig.14 This is the GO annotation classification statistics of strain CGY-B;

[0030] Fig.15 This is the KEGG annotation classification statistics of strain CGY-B;

[0031] Fig.16 The genome circle map of strain CGY-B;

[0032] Fig.17 This is the pathway diagram for the degradation of DBP by bacteria CGY-B. DETAILED DESCRIPTION

[0033] The following embodiments are only some embodiments of the present invention, rather than all embodiments. Therefore, the detailed description in the embodiments of the present invention provided below is not intended to limit the scope of the present invention claimed for protection, but merely represents selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making creative work belong to the protection scope of the present invention.

[0034] In the present invention, unless otherwise specified, all equipment and raw materials can be purchased from the market or are commonly used in the industry. The methods in the following embodiments are all conventional methods in the art unless otherwise specified.

[0035] Materials used in the following examples:

[0036] Screening raw materials for phosphate-solubilizing bacteria: The contaminated soil used in the experiment was taken from the mulch-film-covered planting soil in the vineyard area of ​​Changli County, Hebei Province (39.70°N, 119.16°E), collected in sterile sampling bags and brought back to the laboratory and stored in a refrigerator at 4°C.

[0037] This embodiment provides a dibutyl phthalate-degrading bacterium, which is CGY-B, a Gram-positive bacterium, which has been deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration on April 26, 2024, with the deposit number CGMCC No.30456.

[0038] The dibutyl phthalate degradation strain is named Microbacterium paraoxidans CGY-B. The strain has the following morphological characteristics: short rod-shaped, purple cells after staining, and CGY-B is a Gram-positive bacterium. The strain can tolerate and degrade dibutyl phthalate in the system, and the suitable growth conditions are pH=7.0 and temperature 35°C. The phosphate-solubilizing bacteria are inoculated into an environment containing dibutyl phthalate to achieve the degradation of dibutyl phthalate.

[0039] The strain was sent to Beijing Nosai Genome Research Center Co., Ltd. for sequencing analysis, and the gene sequence was determined to be SEQ ID No. 1. The gene sequence was logged on the website of the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov) of the United States, and a nucleotide sequence Blast comparison was performed to obtain several nucleotide sequences homologous to the gene sequences of related strains. The results showed that the homology of the gene sequences of the CGY-B strain and Microbacterium paraoxydans was more than 99%, and the isolated strain was identified as Microbacterium paraoxydans.

[0040] The inorganic salt liquid culture medium of the dibutyl phthalate degrading bacteria CGY-B with the accession number of CGMCC No. 30456 provided by the present invention, which takes dibutyl phthalate as the sole carbon source, comprises the following components: 1.0 g of dimethyl hydrogen phosphate heptahydrate, 0.5 g of ammonium sulfate, 1.0 g of sodium chloride, 0.4 g of magnesium sulfate heptahydrate, 0.01 g of calcium chloride, 0.01 g of ferric chloride hexahydrate, 100 μL of 10 g / L DBP methanol solution, pH=7, the inoculum amount of the degrading bacteria is 1%, and the volume of the solution is 20 mL.

[0041] Before inoculating into an inorganic salt liquid culture medium with dibutyl phthalate as the sole carbon source for culturing and growing, the method also includes pre-culturing the degrading bacteria, inoculating the degrading bacteria into a nutrient broth culture medium for pre-culturing for about 20 hours, wherein the ingredients of the nutrient broth culture medium are: 0.3 g beef extract powder, 1.0 g peptone, 0.5 g NaCl, pH value 7.2±0.2, the inoculation amount of the degrading bacteria is one ring, and the solution volume is 100 mL.

[0042] Example 1 Isolation and screening of degradation bacteria

[0043] (1) Selecting culture medium: Pipette 250 μL of 20 g / L DBP methanol solution into a 150 mL sterile conical flask. After the methanol is completely evaporated, add 100 mL of inorganic salt culture medium to obtain a concentration of 50 mg / L -1 Inorganic salt liquid culture medium with DBP as the only carbon source.

[0044] (2) Acclimation: Weigh 10 g of soil sample, add 90 mL of sterile water, 30 °C, 180 r / min -1 , shake for 60 minutes, and let stand for 4 hours. Take 10mL of the supernatant and inoculate it into the selection medium. Incubate it in the dark with shaking for 5 days. Use the gradient pressure acclimation method and gradually transfer the inoculation amount to 100, 200, and 400mg·L at 1% -1 The culture was continued in an inorganic salt liquid culture medium with DBP as the sole carbon source, with each 5 days as one acclimation cycle, with the culture being transferred 4 times.

[0045] (3) Isolation and purification: Use the dilution coating method to take 10 mL of acclimation solution + 90 mL of sterile water, shake for 30 seconds, and prepare 10 -1 Concentration dilution, mix well, draw 1mL10 -1 Add 9ml of sterile water to the diluent to make 10 -2 Concentration dilution, and so on, to get 10 -3 , 10 -4 , 10 -5 , 10 -6 Gradient concentration dilutions. Take 100 μL of the dilutions of different gradients and spread the mixed bacterial solution on the MSM solid medium with DBP as the only carbon source. Pick out the single bacteria with good growth and repeatedly streak them on the fresh nutrient agar plate to purify them, and obtain the strains with stable degradation ability of DBP for subsequent research.

[0046] (4) Determination of the ability of isolated bacteria to degrade DBP: The isolated degrading bacteria were inoculated into MSM medium supplemented with 100 mg / L DBP, cultured in a shaking incubator at 30°C and 150 rpm for 6 days, and samples were taken at fixed points.

[0047] like Figure 1 As shown in the figure, by using an inorganic salt liquid culture medium with gradually increasing concentrations and using DBP as the sole carbon source, a bacterial liquid with the ability to degrade DBP was domesticated, and further screening was performed using an MSM plate with DBP as the sole carbon source. Finally, four strains with DBP degradation effects were screened out, numbered CGY-A, CGY-B, CGY-C, and CGY-D. The degradation ability of these four strains was tested as follows Figure 2, and found that the four strains had a certain degradation effect on DBP. Under the condition of DBP initial concentration of 100 mg / L, the residual DBP concentration was detected at 10 points, including 0h, 6h, 12h, 24h, 36h, 48h, 72h, 96h, 120h, and 144h. It was found that strain CGY-B had the strongest degradation ability for DBP, reaching 93.25%, which was significantly higher than the other three strains. Therefore, strain CGY-B was selected as the object of this study, and subsequent related experiments were carried out.

[0048] Example 2 Study on Optimization of Strain Growth Conditions

[0049] The controlled variable method was used to study the effects of substrate concentration, pH and temperature on the growth and degradation performance of dibutyl phthalate-degrading bacteria, so as to optimize the degradation conditions of the strain and improve its ability to degrade dibutyl phthalate.

[0050] Preparation of bacterial suspension: Pick a loop of purified CGY-B bacteria from the plate and inoculate it into 100 mL of sterile nutrient broth medium, shake and culture it in a shaker at 30°C and 150 r / min for 24 h, divide the bacterial solution into two 50 mL sterile centrifuge tubes, centrifuge at 8000 rpm for 5 min, discard the supernatant, add PBS to wash twice, and resuspend in PBS to make a bacterial suspension for subsequent biodegradation experiments. Use a UV spectrophotometer to measure the absorbance (OD600) of the bacterial suspension, and adjust the OD600 value to 1.0 using phosphate buffer.

[0051] Sample preparation: In a sterile operating table, draw 200 μL of DBP mother solution with a concentration of 10 mg / L into a 50 mL conical flask, let it stand until the methanol in the mother solution evaporates completely, add 20 mL of inorganic salt liquid culture solution, and inoculate the prepared CGY-B bacterial suspension at a 1% inoculum amount to make the final DBP concentration in the culture medium 100 mg / L. The culture medium is placed in a 30°C, 150 r / min shaker in the dark, and the residual DBP concentration is tested at 10 sampling points such as 0h, 6h, 12h, 24h, 36h, 48h, 72h, 96h, 120h, and 144h.

[0052] DBP extraction method: add an equal volume of 20 mL of extracting solution n-hexane to 20 mL of 100 mg / L DBP inorganic salt culture medium, oscillate and extract at 200 r / min for 60 min, then transfer all the liquid in the culture medium to a 50 mL centrifuge tube, centrifuge at 6000 rpm for 10 min, aspirate 5 mL of the upper organic phase, use a nitrogen blower to blow the organic phase to near dryness with nitrogen, ultrasonically dissolve it in 2 mL of methanol, filter it through a 0.22 μm filter membrane, and transfer it to a brown chromatographic bottle for testing.

[0053] Detection conditions and methods: High performance liquid chromatography (Angilent-1260) was used for detection, the chromatographic column was Agilent Ecliose XDB-C18 (150×4.6mm×2.5μm), the mobile phase was methanol: water = 90:10, the injection volume was 10μL, the column temperature was 30℃, and the UV wavelength was 224nm.

[0054] (1) Effect of substrate concentration on the degradation efficiency of CGY-B

[0055] Add a certain amount of DBP mother solution to a 50mL sterile conical bottle, place it on a clean bench until the methanol evaporates, then add 20mL of sterile MSM liquid to prepare culture media with substrate concentrations of 5, 10, 20, 50, 100, and 200 mg / L, respectively, and inoculate CGY-B bacterial suspension at 1% inoculum. Place the culture medium in a shaker at 30°C and 150r / min in the dark, and take out the entire bottle of culture at a certain point for sample processing and liquid phase detection.

[0056] like Figure 3 As shown in the figure, when the initial concentration of DBP is 5-20 mg / L, the strain can achieve a degradation rate of more than 80% within 12 hours and complete the degradation of DBP within 24 hours; when the initial concentration of DBP is 50-200 mg / L, the degradation effect of the strain on DBP within 12 hours is significantly reduced, and does not exceed 60%; but with the increase of degradation time, the degradation rate of the strain gradually increases. When the initial concentration of DBP is 50 and 100 mg / L, the degradation rate of the strain in 72 hours reaches 95.31% and 86.31%, respectively; when the initial concentration of DBP is 200 mg / L, the degradation rate of the strain can reach 96.08% in 96 hours, which shows that the strain can efficiently degrade DBP below 100 mg / L, and it is difficult to degrade it within 48 hours when it exceeds 100 mg / L. This may be because high concentrations of DBP have a greater toxic effect on cells, and bacteria need a certain adaptation period to be induced to produce enough degradation enzymes to complete the degradation of DBP.

[0057] (2) Effect of pH on the degradation effect of CGY-B

[0058] Add 100 μL of DBP mother solution to a 50 mL sterilized conical flask, place it on a clean bench until the methanol evaporates, use NaOH or HCL to adjust the pH value of the MSM liquid culture medium to 4, 5, 6, 7, 8, 9, and 10, sterilize at 121 ° C for 30 minutes, and after cooling, take 20 mL of MSM with different pH values ​​into the conical flask and inoculate the CGY-B bacterial suspension at 1% inoculum. Place the culture medium in a shaker at 30 ° C and 150 r / min to avoid light and culture it. Take out the whole bottle of culture at a certain point for sample processing and liquid phase detection.

[0059] like Figure 4 As shown in the figure, according to the effect of substrate concentration on the degradation rate of CGY-B, a substrate concentration of 50 mg / L was selected for subsequent optimization experiments. The degradation efficiency of DBP by strain CGY-B reached 100% within 144 hours when the initial pH value was 7-10. When the initial pH value was 7-8 within 36 hours, the degradation rate of DBP reached 98.02% and 97.44%, respectively, and the highest was at pH 7. When the initial pH value was 4 or 5, the degradation rate dropped to 54.25% and 71.89% within 144 hours. The results showed that neutral pH was most conducive to the degradation of DBP by strain CGY-B, so the optimized degradation pH was determined to be 7. Strain CGY-B can efficiently degrade DBP in weakly acidic, neutral and alkaline environments, indicating that the strain can adapt to a wider pH range (6-10).

[0060] (3) Effect of temperature on the degradation effect of CGY-B

[0061] Add 100 μL DBP stock solution to a 50 mL sterile conical flask, place it on a clean bench until the methanol evaporates, then add 20 mL sterile MSM liquid to prepare a culture medium with a substrate concentration of 50 mg / L, and inoculate CGY-B bacterial suspension at 1% inoculum. Place it in a shaker at 15, 20, 25, 30, and 40 ° C at 150 r / min in the dark, and take out the whole bottle of culture at a certain point for sample processing and liquid phase detection.

[0062] like Figure 5 As shown in the figure, according to the results of the analysis of the effects of substrate concentration and pH on the degradation of DBP by strain CGY-B, the temperature conditions were optimized under the conditions of substrate concentration of 50 mg / L and pH value of 7. The strain CGY-B had a good degradation effect on DBP in the initial temperature range of 15-40℃, and the degradation rate could reach more than 98% within 144h. The degradation effect was best at 30℃, and the degradation rate reached 97.79% at 36h, followed by 25℃, with a degradation rate of 97.27%; when the culture temperature was 15℃ and 40℃, the 36h degradation rate decreased to 75.93% and 77.13% respectively. Although the degradation rate of the strain was relatively reduced in a short time, it did not affect the strain's completion of the degradation of DBP. It can be seen that the strain has a wide range of suitable growth temperatures and can grow normally at 15-40℃.

[0063] Example 3 Classification and identification of degradation bacteria

[0064] (1) Morphological identification

[0065] Take a certain amount of CGY-B dibutyl phthalate degrading bacteria, inoculate it on a nutrient agar solid plate under sterile conditions, put it in a 30℃ mold incubator and culture it at a constant temperature for 24 hours, select the plates with good single colonies, observe these plates, and record the performance of these colonies. Figure 6 As shown on the left, the strain CGY-B forms colonies on the agar plate with neat edges, moist and smooth surfaces, yellow and transparent, and slightly raised colonies.

[0066] A certain amount of CGY-B dibutyl phthalate-degrading bacteria was taken and inoculated into a nutrient broth medium. The strain was activated in a constant temperature shaker at 30°C and 150r / min for 20 hours, then centrifuged and collected. The strain was washed three times with PBS buffer, placed in a pre-cooled 2.5% glutaraldehyde fixative, and placed in a 4°C refrigerator overnight. The sample was washed with PBS buffer, centrifuged (8000rpm, 5min) after soaking for 10min each time, and washed repeatedly three times. The washed sample was dehydrated with an ethanol concentration gradient (50, 70, 80, 95 and 100%), and centrifuged after soaking for 15min at each stage. The sample was left to stand in a 4°C refrigerator overnight with 70% ethanol, and then soaked in 100% ethanol for 15min and centrifuged. The dehydrated sample was placed in a vacuum drying oven (50°C) and dried for 3 hours. After gold spray pretreatment, the strain morphology was observed using a field emission scanning electron microscope (Merlin). The results are as follows Figure 6 As shown on the right, the bacterial body of strain CGY-B is short rod-shaped, without flagella, and is approximately 2 μm × 8 μm in size.

[0067] (2) Molecular Biological Identification

[0068] DNA extraction: Use a sterile toothpick to pick up a small amount of purified bacteria and resuspend them in 100 μL sterile ddH 2 O, then boiled in a boiling water bath for 10 min, immediately placed in a -20 °C refrigerator for 30 min, centrifuged at 12000 rpm for 1 min, and the supernatant was used as a template.

[0069] PCR amplification: The primers used were 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACCTTGTTACGACTT-3') for amplification of a 16S rDNA fragment, and the primers were diluted to a 10 μM working solution. The reaction system is shown in Table 1, and the cycling conditions are shown in Table 2.

[0070] Table 1 PCR amplification system

[0071] name Dosage (μL) <![CDATA[ddH 2 The]]> 15 Enzyme MIX 5 Primer 27F 2 Primer 1492R 2 DNA samples 2

[0072] Table 2 PCR reaction conditions

[0073]

[0074] Product recovery and sequencing: PCR products were separated by 1% agarose electrophoresis, and the desired DNA target band was cut and purified before sequencing. The determined gene sequence is shown as SEQ ID No.1.

[0075] SEQ ID No.1

[0076]

[0077] Construction of phylogenetic tree: The above 16Sr DNA sequence determination work was completed by Beijing Nosai Genome Research Center Co., Ltd. According to the PCR sequencing results, the NCBI database was used for similarity search and homology comparison to obtain related strains with high similarity, and finally the phylogenetic tree was constructed using MEGA 11 using the Neighbor-Joining method.

[0078] The 16S rDNA gene (1232 bp) was amplified by PCR and the Genbank accession number was MK294281.1. The Blast search results showed that the sequence similarity between strain CGY-B and Microbacterium paraoxydans was 99%. The phylogenetic tree of its phylogenetic relationship with members of the genus Microbacterium is shown in Figure 1. Figure 7 ,The results showed that strain CGY-B belonged to the genus Microbacterium (1232bp) and was most closely related to Microbacterium sp.

[0079] Example 4 Physiological and biochemical characteristics of the degradation bacteria CGY-B

[0080] (1) Methyl red test

[0081] Experimental steps: Prepare buffered glucose peptone water culture medium, put it into test tubes, 10mL per tube, prepare methyl red reagent and prepare CGY-B bacterial slant culture; take 6 tubes of glucose peptone water culture medium, keep 3 tubes as control (no bacteria), and inoculate the remaining 3 tubes with strain CGY-B. Place the above 6 tubes in a 37℃ constant temperature incubator for culture. After 2 days of culture, take out and slowly add 3 drops of methyl red reagent along the wall of each tube. Carefully observe the upper layer of the culture solution. If the upper layer of the culture solution turns red, it is a positive methyl red test. If it is still yellow, it is a negative methyl red test.

[0082] Experimental results: Figure 8 , which is the result of the methyl red test. Observing the culture medium, the inoculated culture medium (third from the left) is yellow, while the uninoculated culture medium (third from the right) has not changed color, indicating that the methyl red test of the CGY-B strain is negative. The strain cannot decompose glucose to produce pyruvate. It is also possible that the strain produces organic acids in the early stage of culture, but converts the organic acids into non-acidic end products, such as ethanol pyruvate, in the later stage, so that the pH rises to about 6, so the methyl red identification result is negative.

[0083] (2) Indole experiment

[0084] Experimental steps: Prepare buffered glucose peptone water culture medium and put it into test tubes, 10mL per tube; prepare indole reagent and prepare CGY-B bacteria slant culture, take 6 tubes of glucose peptone water culture medium, keep 3 tubes as control (no bacteria), and inoculate the remaining 3 tubes with CGY-B bacteria; place the above 6 tubes in a 37℃ constant temperature incubator for culture, take them out after 2 days of culture, add 5 drops of ether to each tube of culture medium, shake it fully, and then let it stand for 3 minutes to make the ether layer float on the culture medium. Slowly add 10 drops of indole reagent along the tube wall. If a red ring appears at the interface between the ether layer and the culture medium, it is a positive indole matrix test, otherwise it is a negative indole matrix test.

[0085] Experimental results: Fig. 9 The results of the indole test are shown in the figure. Observing the culture medium, a red ring appeared at the interface between the ether layer and the culture medium in the inoculated culture medium (third from left), but not in the uninoculated culture medium (third from right), indicating that the indole test of the CGY-B strain was positive. The strain has tryptophanase, which can decompose tryptophan in the peptone water culture medium to produce indole.

[0086] (3) Glucose fermentation experiment

[0087] Experimental steps: Prepare glucose fermentation medium and E. coli slant culture, take 4 tubes of glucose fermentation medium, and mark the 4 test tubes of glucose fermentation medium with CGY-B bacteria and blank control respectively. Under sterile operation, pick a small amount of E. coli slant strains and inoculate them into the test tubes of glucose fermentation medium, and the blank control is not inoculated. Then place it in a 37℃ constant temperature incubator for 2 days. If the culture solution keeps its original color, it is a negative reaction, indicating that the bacteria cannot decompose this kind of sugar; if the culture solution turns yellow, it is a positive reaction, indicating that the bacteria can decompose this kind of sugar.

[0088] Experimental results: Fig.10 , which is the result of glucose fermentation experiment. Observing the culture medium, the inoculated culture medium (second from left) did not change color, and the uninoculated culture medium (second from right) did not change color, indicating that the glucose fermentation test of CGY-B strain was negative, and the strain could not decompose glucose and produce acid.

[0089] Example 5 Study on the biodegradation products of DBP by the degradation bacteria CGY-B

[0090] Sample preparation: In order to study the products of DBP degradation by bacteria CGY-B, a 50 mg / L MSM liquid culture medium with DBP as the sole carbon source was prepared, and a bottle of culture was taken out for treatment at 0h, 3h, 6h, 9h, 12h, 18h, and 24h.

[0091] Extraction method: The degradation samples of strain CGY-B were qualitatively analyzed by GC-MS, and the sample processing was the same as the DBP extraction method in Example 2 above.

[0092] GC-MS detection program conditions: HP-5MS (30m×0.25mm×0.25mm) fused silica capillary column was used for chromatographic separation, high purity helium (99.999%) was used as carrier gas, and the flow rate was constant at 1.0mL / min. The column oven was set to an initial temperature of 60°C for 1min, the temperature was increased to 220°C at 20°C / min, maintained at this temperature for 1min, and then increased to 280°C at 5°C / min, and maintained at this temperature for 5min. Then, the extract sample (1μL) was injected into the GC-MS instrument in non-split and non-pulse mode with an injector temperature of 250°C and a transfer line temperature of 280°C.

[0093] The culture fluid samples of different lengths were collected and processed, and the intermediate products of DBP degradation by strain CGY-B were detected by GC-MS. In addition to the parent compound dibutyl phthalate (DBP), 1-methyl-6-butyl phthalate (MBP), 1-methyl-6-propyl phthalate (MPP), 1-methyl-6-isobutyl phthalate, dimethyl phthalate (DEP), phthalic acid (PA), phthalic anhydride, and methyl 4-hydroxycyclohexanecarboxylate were also detected. According to the appearance of the key product phthalic acid (PA), it can be considered that the degradation pathway of DBP by the bacteria first shortens the side chain length through demethylation and β-oxidation, and then further generates PA through ester bond hydrolysis. However, the common intermediate PCA in the further transformation of PA was not found in the test. Based on the identification of methyl 4-hydroxycyclohexanecarboxylate as a downstream product of PCA degradation, it is speculated that protocatechuic acid (PCA) may not be captured due to its instantaneous degradation after formation. In summary, strain CGY-B can completely degrade DBP through the intermediates MBP, MPP and PA. (See details Fig.11 )

[0094] Example 6 Genome sequencing and analysis of the degradation bacteria CGY-B

[0095] (1) Sample preparation

[0096] In order to obtain the complete gene information of strain CGY-B, the bacteria were inoculated into NB medium, activated and cultured at 30°C, 150r / min in a shaking table, and the activated culture solution was centrifuged in a low-temperature centrifuge at 4°C and 8000r for 10min. The collected bacteria were washed three times with phosphate buffer (pH=7.2), and the supernatant was discarded. The precipitate was washed twice with sterile water and then quickly frozen in liquid nitrogen, and then transferred to -80°C or liquid nitrogen for long-term storage to obtain the sample of Microbacterium paraoxygensis CGY-B for inspection.

[0097] (2) Whole genome sequencing

[0098] The whole genome sequencing of the strain was commissioned to Wuhan Fraser Gene Information Co., Ltd. in Hubei Province, China, and the third-generation PacBio sequencing technology was used to complete the map sequencing of the strain CGY-B.

[0099] The total genome length of CGY-B is 3,496,921bp, including a 3,412,614bp circular chromosome and two circular plasmids (66,557bp, 17,750bp). The G+C content of the chromosome and plasmid is 71%, 66% and 69%, respectively. The coding genes of CGY-B were predicted using Glimmer (v3.02) based on the full genome length. The prediction statistics of coding genes are shown in Table 3. The total base length of the gene is 3,496,921bp, and a total of 3,357 predicted protein coding sequences (CDS) were identified. The average length of the coding genes is 970.31bp, and all genes are estimated to cover 93.15% of the entire genome. The chromosome also encodes 48 tRNAs and 6 rRNAs, including three types of 5s RNA, 16s RNA and 23s RNA.

[0100] Table 3 Coding gene prediction statistics

[0101]

[0102] (3) Whole genome annotation

[0103] This sample was assembled using flye-2.9 software, and Glimmer (v3.02) was used to predict the coding genes of microbial DNA. tRNAscan-SE (2.0.9) and RNAmmer (v1.2) were used to predict the tRNA and rRNA of microbial genes, respectively. The basic database annotations include NR, COG / KOG, GO, SwissProt, KEGG, and InterPro, and the protein sequences of the predicted genes were annotated to the corresponding databases.

[0104] 1) NR Annotation

[0105] The comparison results of NR database are as follows Fig.12 As shown in the figure, according to the number of genes annotated by species statistics, the top 10 species and their proportions were marked. It was found that Microbacterium paraoxydans species was the most annotated, accounting for 68.74%, which was consistent with the previous sequencing results. The second-ranked species was Microbacterium sp.SSW1-51, accounting for 10.59%.

[0106] 2) COG Annotation

[0107] A total of 2778 functional genes of strain CGY-B were assigned to the COG database, accounting for 82.75% of the total number of genes. Fig.13 It can be seen that more proteins are involved in nucleotide transport and metabolism (G) and transcription (K), accounting for 13.10% and 12.23% of the predicted functional genes, respectively, followed by 333 genes annotated as amino acid transport and metabolism (E), accounting for 11.99%. A total of 278 genes were classified as general function prediction only (R), accounting for 10.01%; relatively few proteins are involved in inorganic ion transport and metabolism (P); translation, ribosomal structure and biogenesis (K); coenzyme transport and metabolism (H), accounting for 7.02%, 6.69% and 6.34% respectively.

[0108] 3) GO annotation

[0109] The statistical results of this classification are as follows Fig.14As shown in the figure, 2282 genes annotated by GO are involved in 47 secondary classification functional proteins. Among them, biological processes are divided into 27 categories, with cellular metabolism accounting for the highest proportion, involving 1363 genes, followed by metabolic process (1205), molecular localization (359), and response to stimulus (276). The genes identified in cellular components involve three categories, namely protein complex cellular organism (cellular anatomical entity) (1304), protein-containing complex (144), and virion component (4). Genes related to molecular functions are divided into 17 categories, mainly catalytic activity (1344), binding (1263), and transporter activity (317). In addition, there are genes showing ATP-dependent activity (186) and transcription regulator activity (125).

[0110] 4) KEGG annotation

[0111] The strain CGY-B has a total of 3002 genes annotated to participate in 294 metabolic pathways in the KEGG database. Fig.15 It can be seen that the annotation results are divided into 6 categories: Cellular Processes, Environmental Information Processing, Genetic Information Processing, Human Diseases, Metabolism, and Organic Systems. Among them, the number of genes involved in carbohydrate metabolism and amino acid metabolism is relatively large, 317 and 253 respectively. In addition, there are 18 xenobiotic degradation metabolic pathways involving 92 related annotated genes, of which 27 genes are predicted to be involved in benzoate degradation, 14 genes are involved in drug metabolism-other enzymes pathway, and 7 genes are involved in aminobenzoate degradation pathway.

[0112] (4) Bacterial genome circle map

[0113] The genome circle map can comprehensively display the characteristics of the genome, such as the distribution of genes on the forward and antisense strands, the COG functional classification of genes, GC content, genomic islands, homologous genes, etc. Various information (here refers to GC content, GC deviation, tRNA / rRNA, COG annotation, base modification and restriction modification system related enzymes) are drawn in a genome circle map, and the drawing software used is: circos-0.69-9.

[0114] The average gene length of strain CGY-B was 970.31 bp, and the coding sequence accounted for 93.15% of the entire genome sequence. Fig.16 The genome circle map of the bacterium CGY-B is displayed. From the outer circle to the inner circle are the genomic position coordinates of the target strain, restriction modification enzymes, base modification sites located on the positive chain (the height of the column indicates the coverage of the site), base modification sites located on the negative chain (the height of the column indicates the coverage of the site), coding genes located on the positive chain (the color corresponds to the COG classification), coding genes located on the negative chain (the color corresponds to the COG classification), rRNA and tRNA distribution, genome GC-skew value (the specific algorithm is GC / G+C, the inward purple part indicates that the G content in this area is lower than the C content, and the outward pink part is the opposite), genome GC content (the outward green part indicates that the GC content in this area is higher than the average GC content of the whole genome, the inward blue part is the opposite, and the higher the peak value, the greater the difference with the average GC content).

[0115] The total genome length of strain CGY-B is 3,496,921bp, with a GC content of 71%, and contains 3557 CDSs. Among them, 3305 CDSs were annotated by NR; 2778 CDSs were annotated by COG into 20 functional categories; GO annotated 2282 CDSs, of which 1363, 1205 and 359 were related to cell metabolism, metabolic processes and molecular localization, respectively; KEGG annotated 3002 genes involved in 294 metabolic pathways.

[0116] In addition, strain CGY-B also involves metabolic pathways of many different types of compounds and pathways related to the synthesis of drugs such as antibiotics. A total of 92 genes are involved in 18 heterologous substance metabolic pathways, with abundant heterologous substance degradation genes. The genes that may be involved in the degradation process are counted as shown in Table 4. Strain CGY-B involves metabolic pathways of many different types of compounds and pathways related to the synthesis of drugs such as antibiotics, such as benzoate, chlorocyclohexane and chlorobenzene, fluorobenzoate, xylene, toluene, chloroalkanes and chloroolefins, atrazine drugs, etc. Therefore, it can be inferred that there are a large number of unknown functional genes in strain CGY-B that can be further analyzed and mined.

[0117] Table 4 Coding gene prediction statistics

[0118]

[0119]

[0120] Example 7 Analysis of biodegradation pathways of CGY-B for degradation of DBP

[0121] According to the functional annotation information of the whole genome of Microbacterium paraoxydans sp. CGY-B, key genes related to PAEs degradation were retrieved. Generally speaking, the biodegradation process of PAEs first undergoes the hydrolysis of the ester group of the benzene ring to generate the intermediate product PA. In this process, enzymes responsible for the hydrolysis of ester bonds, such as deesterification enzymes, β-oxidases, and demethylases that encode the hydrolysis of the alkyl ester bonds of PAEs are indispensable. Based on the genome annotation, there are 34 esterase / hydrolase genes in the CGY-B genome, including 4 genes encoding esterase / carboxylesterase, 2 genes encoding lipase, and 28 genes encoding hydrolases or α / β hydrolases (see Table 5). The gene encoding carboxylesterase catalyzes the hydrolysis of DMP and MPP to generate PA, which is consistent with the degradation products generated in this study (see Fig.11 ). These coding gene annotation results indicate that CGY-B has the potential ability and molecular versatility to degrade PAEs pollutants. Based on the analysis of DBP degradation products by strain CGY-B, the degradation pathway from PA to TCA was inferred. PA is converted to BA through decarboxylation, and BA is further converted to PCA through the action of hydroxylase. Para-hydroxybenzoate monooxygenase pobA, which can convert BA into 4-hydroxybenzoic acid, was annotated in the CGY-B genome. Then PCA is further degraded to oxaloacetate through the 4,5-ring cleavage pathway encoded by the lig gene cluster and enters the TCA cycle. At the same time, according to the KEGG annotation results, the catechol ortho-cleavage pathway completed by the catechol cleavage metabolism genes xylE, xylH and mhpD (see Table 6) was also found, indicating that the intermediate BA may also be degraded through the form of ring cleavage.

[0122] Table 5 Genes related to esterase or hydrolase in strain CGY-B

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[0124]

[0125] Table 6 Genes that may be involved in PA degradation in strain CGY-B

[0126]

[0127]

[0128]

[0129] Through KEGG annotation, the complete degradation pathway of PA, a key metabolite of PAEs in CGY-B, was inferred, such as Fig.17 As shown. PA is first converted to BA under the action of decarboxylase, and BA is further converted to PCA by the hydroxylase action of p-hydroxybenzoate monooxygenase pobA, or directly converted to PCA by oxygenase. The ring-opening degradation of PCA is carried out by PCA 4,5 dioxygenase [EC:1.13.11.8] (a kind of exodiol ring cleavage dioxygenase, encoded by ligA and ligB), and the benzene ring is cleaved at the 4,5 position to generate 4-carboxy-2-hydroxymucosuccinic semialdehyde, which is then dehydrogenated under the catalysis of 2-hydroxy-4-carboxysuccinic semialdehyde hemiacetal dehydrogenase [EC:1.1.1.312] to produce 2-hydroxy-hydropyran-4,6-dicarboxylic acid. The product is catalyzed by 2-pyrone-4,6-dicarboxylate lactonase [EC:3.1.1.5 7] to generate 2-pyrone-4.6-dicarboxylic acid and undergo esterification and dehydration to generate 4-glyphosate, which is then catalyzed by ligJ-encoded 4-oxaloacetate hydratase [EC:4.2.1.83] to undergo hydration to generate 2-carboxy-4-hydroxy-2-oxopropionic acid, which is then catalyzed by ligK-encoded 4-hydroxy-4-methyl-2-oxoglutarate aldolase [EC:4.1.3.17] to undergo cleavage and decomposition into oxaloacetate and pyruvate, which enter the TCA cycle and are completely degraded. In addition, centrosomal BA can also be degraded into catechol, which can be further converted into 4-oxocyclohexanone acid by the catechol degradation gene cluster encoded by xyl, which is then converted into 4-hydroxy-2-oxopentanoic acid by the action of mhpD-encoded 2-keto-4-pentenoate hydratase [EC:4.2.1.80] and then degraded, reflecting the coexistence of multiple degradation pathways of PAEs ( Figure 4-8 The intermediate PCA was not found in the metabolite detection, which may be due to the obvious difference between the Pca cluster located on CGY-B and all other homologous gene clusters. CGY-B has a PcaADFJ gene cluster with a different genetic structure, indicating a selective advantage in the face of complex external environmental changes, which may help to avoid the accumulation of the toxic intermediate protocatechuic acid.

[0130] In summary, the phthalate-degrading bacteria provided by the present invention can achieve efficient degradation of the organic pollutant dibutyl phthalate, reduce the concentration of dibutyl phthalate in the environment, alleviate environmental pressure, and have good application prospects and promotion value.

Claims

1. A dibutyl phthalate-degrading bacterium, characterized in that: The degrading bacteria is Microbacterium paraoxidans CGY-B, which was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on April 26, 2024, with the deposit number CGMCC No.30456.

2. An application of the degrading bacteria according to claim 1, characterized in that: Application of the strain in the degradation of dibutyl phthalate.

3. The use of the degradation bacteria according to claim 2, characterized in that: The degradation bacteria are inoculated into an environment containing the organic pollutant dibutyl phthalate to achieve degradation of the dibutyl phthalate.

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