Compound bacteria for degrading and repairing benzo [a] pyrene as well as functional bacterial agent and application of compound bacteria

Abstract

The invention discloses a compound bacterium for degrading and repairing benzo [a] pyrene as well as a functional bacterium agent and application of the compound bacterium. The compound bacterium comprises Mycolicbacteria sp.142 and Shenotrophomonas sp.143, and the preservation numbers of the Mycolicbacteria sp.142 and the Shenotrophomonas sp.143 are respectively CCTCC (China Center for Type Culture Collection) NO: M 20221294 and CCTCC NO: M 20221295. The compound bacteria obtained through separation and screening can effectively degrade benzo [a] pyrene and various polycyclic aromatic hydrocarbons; the complex microbial inoculant is capable of rapidly and effectively degrading more than 90% of benzo [a] pyrene in an inorganic salt culture medium, and meanwhile, the complex microbial inoculant can degrade more than 80% of high-concentration benzo [a] pyrene in soil within a short time, so that the degradation effect of a single strain is remarkably improved, and the problem that residual benzo [a] pyrene is harmful to soil or water environment, crops and human health is effectively solved.

Description

Technical Field

[0001] The present invention belongs to the technical field of environmental microorganisms, and in particular relates to a composite bacterium for degrading and repairing benzo[a]pyrene, a functional bacterial agent and an application thereof. Background Art

[0002] Benzo[a]pyrene (BaP), also known as 3,4-benzopyrene, is a five-ring polycyclic aromatic hydrocarbon compound and the most toxic and potent carcinogen among PAHs. It is not only widely present in the environment, but also relatively stable, and has a certain correlation with the content of other PAHs. Therefore, BaP is generally used as a representative of PAHs in the environment. It has been identified as a carcinogenic hazard that should be monitored and concerned by environmental protection and health agencies of many countries and organizations. In 1995, the U.S. Environmental Protection Agency listed 16 PAHs as priority pollutants. China's "Soil Environmental Quality Construction Land Soil Pollution Risk Control Standard (Trial)" (GB36600-2018) and other national standards include benzo[a]pyrene and other 8 PAHs in the scope of environmental pollution risk screening and control. Traditional soil remediation technologies such as physical adsorption and chemical oxidation are often costly and may cause secondary pollution. Bioremediation has become a research hotspot due to its environmental friendliness and high cost-effectiveness. Summary of the invention

[0003] Purpose of the invention: In view of the problems existing in the prior art, the present invention provides a composite bacteria for bioremediation of benzopyrene pollution with high efficiency, low cost and environmental friendliness. The present invention utilizes a composite bacteria composed of specifically screened strains Mycolicibacterium sp.142 and Stenotrophomonas sp.143. Both of these bacteria show high efficiency in degrading benzopyrene and can form significant synergistic effects with each other, thereby significantly improving the degradation efficiency.

[0004] Technical solution: In order to achieve the above-mentioned purpose, the present invention provides a composite bacteria for degrading and repairing benzo[a]pyrene, wherein the composite bacterial agent comprises Mycolicibacterium sp. and Stenotrophomonas sp.

[0005] Wherein, the composite bacterial agent includes Mycolicibacterium sp.142 and Stenotrophomonas sp.143, and their preservation numbers are CCTCC NO:M 20221294 and CCTCC NO:M 20221295, respectively.

[0006] The two benzo[a]pyrene-degrading strains 142 and 143 described in the present invention, 142 was identified as Mycolicibacterium sp., and has been deposited in the China Center for Type Culture Collection on August 18, 2022, with a deposit number of CCTCC NO: M 20221294. 143 was identified as Stenotrophomonas sp., and has been deposited in the China Center for Type Culture Collection on August 18, 2022, with a deposit number of CCTCC NO: M 20221295.

[0007] The strains of the present invention are isolated by the inventor from the sludge of the oil production area in Yulin, Shaanxi Province, and are named 142 and 143. The degradation rate of 100 mg / L benzo[a]pyrene by the two strains can reach more than 80% in 10 days. The colony morphology of colony 142 on the LB solid plate is an overall round shape, with a raised surface, neat edges, moistness, and yellowness. The colony morphology of colony 143 on the LB solid plate is an overall round shape, with a raised surface, neat edges, smoothness, and milky whiteness. Strain 142 is short rod-shaped (0.4-0.6μm×1.0-1.8μm), and strain 143 has flagella and is short rod-shaped (0.4-0.6μm×1.5-2.0μm).

[0008] The ratio of the viable counts of Mycolicibacterium sp. 142 and Stenotrophomonas sp. 143 is 0.5:1-1:0.5, or the two are mixed in equal volumes at equal OD600 concentrations.

[0009] The ratio of the viable counts of Mycolicibacterium sp.142 and Stenotrophomonas sp.143 is 1:1; or the two are mixed in equal volumes at a concentration of OD600 of 1.

[0010] The composite microbial functional bacterial agent of the composite bacteria for degrading and repairing benzo[a]pyrene described in the present invention.

[0011] The preparation method of the composite microbial agent for benzopyrene degradation of the present invention comprises the following steps: subjecting Mycolicibacterium sp. 142 and Stenotrophomonas sp. 143 to liquid fermentation respectively; and mixing the cultured fermentation broths to obtain a liquid composite microbial agent.

[0012] The preparation method of the composite microbial functional agent of the present invention comprises the following steps:

[0013] (1) Activation of bacterial strains: Mycolicibacterium sp.142 and Stenotrophomonas sp.143 strains were inoculated into solid culture medium, cultured, and single colonies were selected and inoculated into liquid culture medium to obtain inoculum;

[0014] (2) Preparation of primary seed solution: inoculating the inoculum prepared in step (1) into a liquid culture medium at an inoculum volume ratio of 1-5%, and culturing to obtain a primary seed solution;

[0015] (3) Preparation of fermentation broth: The first-grade seed solution prepared in step (2) is inoculated into a fermentation tank containing a fermentation medium at an inoculum ratio of 1-5% by volume, and the fermentation broth of each strain is obtained by culturing;

[0016] (4) Preparation of microbial agent: The fermentation broths of the various bacteria prepared in step (3) are mixed to obtain a composite microbial agent.

[0017] The solid culture medium of Mycolicibacterium sp.142 is 3-5g beef extract, 10-15g peptone, 5-10g sodium chloride, 15-20g agar, and water is added to make the volume to 1L, and the liquid culture medium does not contain agar; the solid culture medium: peptone 10-15g, sodium chloride 10-15g, yeast powder 5-10g, agar 20g, water is added to make the volume to 1L; the solid culture medium of Stenotrophomonas sp.143 is peptone 10-15g, sodium chloride 5-10g, yeast powder 5-10g, agar 15-20g, water is added to make the volume to 1L, and the liquid culture medium does not contain agar.

[0018] Wherein, in step (1), the culture is carried out until OD 600 When the value reaches 0.5-0.6, the inoculum is obtained; in step (2), the culture is continued until the OD 600 The value reaches 0.8-1.0, which is the first-level seed liquid; in step (3), the first-level seed liquid prepared in step (2) is added and cultured until the number of viable bacteria in the fermentation tank is greater than 5×10 8 cfu / mL, the fermentation broth of each strain was obtained.

[0019] Furthermore, in step (3), the first-level seed solution prepared in step (2) is introduced, and the ventilation volume is 2-4m 3 / h, stirring speed of 150-200rpm, 25-35℃ culture until the number of viable bacteria in the fermentation tank is greater than 5×10 7 cfu / mL, the fermentation broth of each strain was obtained.

[0020] The composite bacteria for degrading and repairing benzo[a]pyrene described in the present invention or the use of the composite microbial functional bacterial agent in degrading and repairing benzo[a]pyrene.

[0021] Among them, the composite bacteria or the composite microbial functional bacterial agent is used in the degradation and restoration of benzo[a]pyrene in soil or water.

[0022] Beneficial effects: The present invention has the following advantages:

[0023] The composite bacteria obtained by separation and screening can effectively degrade benzo[a]pyrene and various polycyclic aromatic hydrocarbons; the benzo[a]pyrene in the inorganic salt culture medium can be quickly degraded by more than 90%, and the composite bacterial agent can degrade high-concentration benzo[a]pyrene in the soil by more than 80% in a short time, significantly improving the degradation effect of a single strain, and effectively solving the problem of the harm of residual benzo[a]pyrene to the soil or water environment, crops and human health.

[0024] The degradation bacterial agent of the present invention can be produced by using general fermentation equipment in the fermentation industry, has the advantages of low production cost, convenient use, and good removal effect, and is suitable for soil remediation contaminated by polycyclic aromatic hydrocarbons. The present invention is of great significance for protecting the ecological environment, reducing the damage of organic residues to crops, and protecting human health. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 are colony photos of strains 142 and 143 of the present invention;

[0026] Figure 2 It is the electron micrograph of strains 142 and 143 of the present invention. DETAILED DESCRIPTION

[0027] The present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0028] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0029] In the embodiment of the present invention:

[0030] The solid culture medium of Mycolicibacterium sp.142 is composed of 5 g beef extract, 10 g peptone, 10 g sodium chloride, 15 g agar, and water is added to make up to 1 L. The liquid culture medium does not contain agar.

[0031] The solid culture medium of Stenotrophomonas sp.143 is 10 g of peptone, 10 g of sodium chloride, 10 g of yeast powder, 20 g of agar, and water is added to make up to 1 L. The liquid culture medium does not contain agar.

[0032] The fermentation medium is the same as the above liquid medium, and 2 g of ammonium sulfate and 1 g of potassium dihydrogen phosphate are added respectively.

[0033] Example 1

[0034] Isolation and identification of strains:

[0035] Add the initial benzo[a]pyrene to 100ml of inorganic salt culture medium to prepare a culture medium with a concentration of 50mg / L; take 4g of the sludge sample and add it to the prepared culture medium, set the shaking incubator conditions to 30℃, 175rpm, and 7d; after the first generation of culture, take 2ml to a new culture medium (50mg / L) and continue to culture for 4 generations; determine the degradation effect of benzo[a]pyrene in the enriched solution by liquid chromatography to obtain an enriched solution with degradation effect. Take 0.5mL of the effective enriched solution, 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 Gradient dilution, take 100 μL of each gradient dilution enrichment solution and spread it on an inorganic salt solid culture medium plate containing benzo[a]pyrene mg / L, and culture at 30℃ for 7 days. Pick a single colony grown on the plate and inoculate it in a liquid LB culture medium (10g peptone, 5g yeast extract, 5g NaCl, 1000mL water, pH 7.0-7.2) test tube, culture at 30℃, 180r / min for 2 days, take 1mL LB culture solution, centrifuge at 5000rpm for 5min, wash off the LB culture medium with sterile water, take 1mL sterile water to resuspend the bacteria, and inoculate them into inorganic salt liquid culture medium containing 50mg / L benzo[a]pyrene, culture at 30℃, 165r / min for 10d, and detect the benzo[a]pyrene degradation effect by high performance liquid chromatography.

[0036] Verification method of HPLC degradation effect: add an equal volume of dichloromethane to the culture medium for full extraction, shake vigorously and let stand to separate layers, then take 1mL of the lower layer of dichloromethane and evaporate it completely, add 1mL of acetonitrile to dissolve (chromatographic grade), and filter with a filter membrane (pore size 0.22μm). The content of benzo[a]pyrene in the extract was determined by UV and HPLC, respectively. Liquid chromatography conditions: mobile phase acetonitrile: water (90:10, V / V), Zorbax C218 ODS Spherex reverse phase column (5μm, 4.6mm×250mm, Agilent, USA), column temperature 30℃, PDA detector, measurement wavelength 254nm, injection volume 10μL, flow rate 1.0mL·min -1 . The external standard method was used for quantification based on peak area.

[0037] The bacterial solution with obvious degradation effect was repeatedly coated on the drug-containing solid inorganic salt plate culture medium, and the grown single bacteria were repeatedly cultured as described above, and the degradation effect was repeatedly verified and screened, and two single bacteria with obvious degradation effect were isolated, and the strains were named 142 and 143. The strains were amplified by PCR technology, and the primers were: 1492R and 27F. The 16SrDNA gene sequence of the strains was amplified and sequenced by a certain company of Boshang Biological Sequencing. The sequences of the 16SrDNA of the strains named 142 and 143 are shown in SEQ ID NO.1-2, respectively. The obtained strain gene sequences were compared in EzBioCloud. The gene sequence of strain 142 is most closely related to the genus Mycolicibacterium, and the 16SrRNA gene sequence of strain 143 is most closely related to the genus StenotropHomonas.

[0038] The colony morphology of colony 142 on the LB solid plate is overall round, with a raised surface, neat edges, moist, and yellow. The colony morphology of colony 143 on the LB solid plate is overall round, with a raised surface, neat edges, smooth, and milky white. The electron microscope image of strain 142 is as follows Figure 2 As shown, short rods (0.4–0.6 μm × 1.0–1.8 μm) and strain 143 electron micrographs are shown Figure 2 As shown, it has flagella, which are short rod-shaped (0.4–0.6 μm × 1.5–2.0 μm).

[0039] Through gene sequence and physiological and biochemical characteristics, strain 142 was preliminarily identified as Mycolicibacterium genus, which has been deposited in China Center for Type Culture Collection on August 18, 2022, with the deposit number CCTCC NO: M20221294. Strain 143 was identified as StenotropHomonas genus, which has been deposited in China Center for Type Culture Collection on August 18, 2022, with the deposit number CCTCC NO: M 20221295; named Mycolicibacterium sp.142 and Stenotrophomonas sp.143 respectively.

[0040] Example 2

[0041] Preparation of composite microbial agents

[0042] The liquid composite microbial agent is obtained by mixing the fermentation liquid of Mycolicibacterium sp.142 and the fermentation liquid of StenotropHomonas sp.143. The specific preparation method is as follows:

[0043] (1) Activation of strains: The two strains were inoculated into their respective solid culture media and cultured at 30°C for 2 days. Single colonies were picked and inoculated into their respective liquid culture media and cultured at 30°C and 150 rpm until OD 600 When the value reaches 0.5, the inoculum is obtained;

[0044] (2) Preparation of primary seed solution: The inoculum prepared in step (1) was inoculated into each liquid culture medium at a volume ratio of 5%, and cultured at 30°C and 150 rpm until the OD 600 When the value reaches 1.0, it is the first-grade seed solution;

[0045] (3) Preparation of liquid bacterial agent: The first-grade seed solution prepared in step (2) was inoculated into the fermentation tank containing the respective fermentation medium at an inoculum rate of 5% by volume, and the ventilation volume was 4 m 3 / h, stirring speed 200rpm, 30℃ culture until the number of viable bacteria in the fermenter is about 5×10 8 cfu / mL to obtain the fermentation broth of each strain, and mix the fermentation broths of each strain so that the ratio of the viable counts of Mycolicibacterium sp.142 and Stenotrop Homonas sp.143 is 1:1 to obtain a liquid composite microbial agent.

[0046] Example 3

[0047] Degradation effect of strains 142, 143 and composite bacterial agents on benzo[a]pyrene residues in inorganic salt culture medium:

[0048] Determination of the benzo[a]pyrene degradation characteristics of strains 142 and 143 in inorganic salt liquid medium: single colonies of 142 and 143 were selected and placed in 50 ml LB liquid medium, and cultured at 30°C and 180 r / min for 24 h to obtain fresh bacterial culture. 3 ml of the cultured fresh bacterial culture was centrifuged at 5000 r / min for 5 min, the supernatant was discarded, and 10 ml of sterile water was added to resuspend the culture, and the OD 600 The value reaches 1.0, which is the bacterial solution after centrifugation suspension. The suspended bacterial solutions of strains 142 and 143 were mixed at a volume ratio of 1:1 to form a composite bacterial agent.

[0049] Benzo[a]pyrene was added to the inorganic salt medium at a final concentration of 100 mg / L, and a single strain or a mixed strain of bacterial liquid was inoculated at a volume ratio of 5%, and the mixture was cultured in a 30°C constant temperature shaker (150 rpm). The degradation of benzo[a]pyrene by strains 142, 143 and the composite bacterial agent was detected by high performance liquid chromatography using the verification method of the degradation effect in Example 1, and the degradation rate was calculated. The degradation rates of benzo[a]pyrene by strains 142 and 143 within 10 days were 80.57% and 83.46%, respectively, and the 5-day degradation rate of the composite bacterial agent was 90.53%. The test results are shown in Table 1:

[0050] Table 1 Determination results of benzopyrene degradation by strains 142, 143 and composite bacterial agents

[0051] strains Degradation rate 142 80.57％(10d) 143 83.46％(10d) Compound bacteria 90.53％(5d)

[0052] From the results in Table 1, it can be seen that the single use of strains 142 and 143 can degrade benzopyrene, and when the two specific strains of the present invention are used in combination, under the same total concentration system of strains, the combined use of strains 142 and 143 can significantly improve the degradation effect and accelerate the degradation rate, indicating that the composite bacteria of the present invention produces a synergistic effect in the degradation of benzopyrene.

[0053] Example 4

[0054] Determination of the degradation effect of composite bacterial agent on benzo[a]pyrene in soil:

[0055] Weigh several portions of 1000 g paddy field soil as test soil, air-dry and sieve, add benzo[a]pyrene to each portion to make the concentration of benzo[a]pyrene in the soil 10 mg / kg, and use the composite microbial agent prepared in Example 2, wherein the content of each bacterium is 2.5×10 8 cfu / mL; the content of single strains of strains 142 and 143 in the inoculum was 5×10 8 cfu / mL; the single bacterial agents 142 and 143 and the composite degradation bacterial agent prepared in Example 2 were inoculated into the above soil and mixed at an inoculation rate of 2% (ml / g) as treatment, and the soil containing the same concentration of benzo[a]pyrene without the corresponding degradation bacterial agent was set as the control group, and was placed in a 30°C incubator under dark conditions for constant temperature culture, during which the water holding capacity of the soil was maintained at 40%, and the benzo[a]pyrene concentration in the soil was measured every week after application, and observed continuously for 10 weeks. Each group was subjected to three parallel experiments, and the residual amount was determined by high performance liquid chromatography, and the average degradation rate was calculated for multiple times. The results are shown in Table 2.

[0056] Table 2 Degradation effect of degradation bacteria on benzo[a]pyrene residues in soil

[0057]

[0058] As shown in Table 2, when the concentration of benzo[a]pyrene in the soil was 10 mg / kg, the degradation rates of the degradation bacteria 142, 143 and the composite bacteria reached 61.0%, 68.7% and 98.1% after 10 weeks using the degradation bacteria prepared in Example 2. Under the same total bacterial amount system, the composite bacteria reached a degradation rate of 80.3% in the 6th week, and the degradation was basically complete in the 8th week, significantly surpassing the 45.2% and 49.2% of the single bacteria 142 and 143. The results show that the composite degradation bacteria can effectively degrade benzo[a]pyrene in the soil and produce a synergistic effect.

Claims

1. A composite bacteria for degrading and repairing benzo[a]pyrene, characterized in that: The composite bacterial agent comprises Mycolicibacterium sp. and Stenotrophomonas sp.

2. The composite bacteria for degrading and repairing benzo[a]pyrene according to claim 1, characterized in that: The composite bacterial agent comprises Mycolicibacterium sp.142 and Stenotrophomonas sp.143, and their preservation numbers are CCTCC NO:M 20221294 and CCTCC NO:M 20221295 respectively.

3. The composite bacteria for degrading and repairing benzo[a]pyrene according to claim 2, characterized in that: The ratio of the viable bacteria counts of Mycolicibacterium sp.142 and Stenotrophomonas sp.143 is 0.5:1-1:0.5; or the two are mixed in equal volumes at equal OD600 concentrations.

4. The composite bacteria for degrading and repairing benzo[a]pyrene according to claim 2, characterized in that: The preferred ratio of the number of live bacteria of Mycolicibacterium sp.142 and Stenotrophomonas sp.143 is 1:1; or the two are mixed in equal volumes at an OD600 of 1.

0.

5. A composite microbial functional bacterial agent containing the composite bacteria for degrading and repairing benzo[a]pyrene as claimed in claim 1.

6. A method for preparing the composite microbial functional agent according to claim 5, comprising the following steps: (1) Activation of bacterial strains: Mycolicibacterium sp.142 and Stenotrophomonas sp.143 strains were inoculated into solid culture medium, cultured, and single colonies were selected and inoculated into liquid culture medium to obtain inoculum; (2) Preparation of primary seed solution: inoculating the inoculum prepared in step (1) into a liquid culture medium at an inoculum volume ratio of 1-5%, and culturing to obtain a primary seed solution; (3) Preparation of fermentation broth: The first-grade seed solution prepared in step (2) is inoculated into a fermentation tank containing a fermentation medium at an inoculum rate of 1-5% by volume, and the fermentation broth of each strain is obtained by culturing; (4) Preparation of solid bacterial agent: The fermentation broths of the bacteria prepared in step (3) are mixed to obtain a composite microbial agent.

7. The preparation method according to claim 6, characterized in that: The solid culture medium of Mycolicibacterium sp.142 is 3-5g of beef extract, 10-15g of peptone, 5-10g of sodium chloride, and 15-20g of agar, and water is added to make the volume to 1L, and the liquid culture medium does not contain agar; the solid culture medium of Stenotrophomonas sp.143 is 10-15g of peptone, 5-10g of sodium chloride, 5-10g of yeast powder, and 15-20g of agar, and water is added to make the volume to 1L, and the liquid culture medium does not contain agar.

8. The preparation method according to claim 6, characterized in that: In step (1), the culture is continued until OD 600 When the value reaches 0.5-0.6, the inoculum is obtained; in step (2), the culture is continued until the OD 600 The value reaches 0.8-1.0, which is the first-level seed liquid; in step (3), the first-level seed liquid prepared in step (2) is added and cultured until the number of viable bacteria in the fermentation tank is greater than 5×10 8 cfu / mL, the fermentation broth of each strain was obtained.

9. Use of the composite bacteria for degrading and repairing benzo[a]pyrene as claimed in claim 1 or the composite microbial functional bacterial agent as claimed in claim 5 in degrading and repairing benzo[a]pyrene.

10. The use according to claim 9, characterized in that: The composite bacteria or the composite microbial functional bacterial agent is used in degrading and repairing benzo[a]pyrene in soil or water.

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