Complex bacteria for degrading and repairing benzo[a]pyrene and functional bacterial agent and application thereof

Abstract

The application discloses a composite bacterium for degrading and repairing benzo[a]pyrene as well as a functional bacterium agent and application thereof. The composite bacterium comprises Mycolicibacterium sp. 142 and Stenotrophomonas sp. 143, and the preservation numbers are CCTCC NO: M 20221294 and CCTCC NO: M 20221295 respectively. The composite bacterium obtained by separation and screening can effectively degrade benzo[a]pyrene and various polycyclic aromatic hydrocarbons; can quickly and effectively degrade more than 90% of benzo[a]pyrene in an inorganic salt culture medium; meanwhile, the composite bacterium agent can degrade more than 80% of high-concentration benzo[a]pyrene in soil in a short time, significantly improves the degradation effect of a single strain, and effectively solves the problem of hazards of residual benzo[a]pyrene to soil or water environment, crops and human health.

Description

Technical Field

[0001] This invention belongs to the field of environmental microbiology technology, specifically relating to a compound bacterium for degrading and repairing benzo[a]pyrene, its functional bacterial agents, and their applications. Background Technology

[0002] Benzo[a]pyrene (BaP), also known as 3,4-benzo[a]pyrene, is a pentacyclic polycyclic aromatic hydrocarbon (PAH). It is one of the most toxic and potent carcinogens among PAHs. It is not only widely present in the environment but also relatively stable, and its concentration correlates with that of other PAHs. Therefore, BaP is generally used as a representative of PAHs in the environment. It has been identified as a carcinogenic hazard requiring key monitoring and concern by environmental and health agencies in many countries and organizations. In 1995, the U.S. Environmental Protection Agency listed 16 PAHs as priority pollutants for control. China's national standards, such as the "Soil Environmental Quality Standard for Construction Land Soil Pollution Risk Control (Trial)" (GB36600-2018), include eight PAHs, including benzo[a]pyrene, 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, due to its environmental friendliness and cost-effectiveness, has become a research hotspot. Summary of the Invention

[0003] Purpose of the invention: To address the problems existing in the prior art, this invention provides a complex microbial system for the bioremediation of benzo[a]pyrene pollution that is highly efficient, low-cost, and environmentally friendly. This invention utilizes a complex microbial system composed of specifically screened strains Mycolicibacterium sp.142 and Stenotrophomonas sp.143. Both of these strains have shown highly efficient degradation capabilities for benzo[a]pyrene and can form a significant synergistic effect with each other, significantly improving the degradation efficiency.

[0004] Technical solution: In order to achieve the above objective, the present invention provides a compound microorganism for degrading and repairing benzo[a]pyrene, wherein the compound microbial agent includes Mycolicibacterium sp. and Stenotrophomonas sp.

[0005] The compound microbial agent includes Mycolicibacterium sp.142 and Stenotrophomonas sp.143, with accession numbers CCTCC NO:M 20221294 and CCTCC NO:M 20221295, respectively.

[0006] The two benzo[a]pyrene degrading strains 142 and 143 described in this invention are as follows: 142, identified as *Mycolicibacterium* sp., is deposited at the China Center for Type Culture Collection (CCTCC) on August 18, 2022, with accession number CCTCC NO:M 20221294; 143, identified as *Stenotrophomonas* sp., is also deposited at the CTCC on August 18, 2022, with accession number CCTCC NO:M 20221295.

[0007] The bacterial strains of this invention were isolated by the inventors from sludge in the Yulin oilfield area of ​​Shaanxi Province and named 142 and 143. Both strains achieved a degradation rate of over 80% for 100 mg / L benzo[a]pyrene after 10 days. Colony 142 on LB agar plates was entirely circular, with a raised surface, neat edges, moist, and yellow in color. Colony 143 on LB agar plates was entirely circular, with a raised surface, neat edges, smooth, and milky white in color. Strain 142 was short rod-shaped (0.4–0.6 μm × 1.0–1.8 μm), while strain 143 was flagellated and short rod-shaped (0.4–0.6 μm × 1.5–2.0 μm).

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

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

[0010] The present invention relates to a composite microbial functional agent of a compound bacteria that degrades and repairs benzo[a]pyrene.

[0011] The method for preparing the benzo[a]pyrene degradation compound microbial agent of the present invention involves liquid fermentation of Mycolicibacterium sp. 142 and Stenotrophomonas sp. 143 respectively; the fermented broth is then mixed to obtain the liquid compound microbial agent.

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

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

[0014] (2) Preparation of primary seed culture: Inoculate the inoculum prepared in step (1) into the liquid culture medium at an inoculation rate of 1-5% by volume, and culture to obtain primary seed culture;

[0015] (3) Preparation of fermentation broth: Inoculate the primary seed liquid prepared in step (2) into the fermenter containing fermentation medium at an inoculation rate of 1-5% by volume, and then culture to obtain the fermentation broth of each strain.

[0016] (4) Preparation of microbial agent: Mix the fermentation broth of each microorganism prepared in step (3) to obtain the compound microbial agent.

[0017] The solid culture medium for Mycolicibacterium sp. 142 consists of 3-5g beef extract, 10-15g peptone, 5-10g sodium chloride, and 15-20g agar, diluted to 1L with water. Its liquid culture medium is agar-free. The solid culture medium consists of 10-15g peptone, 10-15g sodium chloride, 5-10g yeast extract, and 20g agar, diluted to 1L with water. The solid culture medium for Stenotrophomonas sp. 143 consists of 10-15g peptone, 5-10g sodium chloride, 5-10g yeast extract, and 15-20g agar, diluted to 1L with water. Its liquid culture medium is agar-free.

[0018] In step (1), the culture is carried out until OD 600 When the OD value reaches 0.5-0.6, the inoculum is obtained; in step (2), it is cultured until the OD value reaches 0.5-0.6. 600 A value of 0.8-1.0 indicates a primary seed culture; in step (3), the primary seed culture prepared in step (2) is introduced and cultured until the viable cell count in the fermenter is greater than 5 × 10⁻⁶. 8 The fermentation broth for each strain can be obtained when the cfu / mL concentration is reached.

[0019] Furthermore, in step (3), the primary seed liquid prepared in step (2) is introduced, with an aeration rate of 2-4 m³ / h. 3 The fermentation process is carried out at a stirring speed of 150-200 rpm and a temperature of 25-35℃ until the viable cell count in the fermenter exceeds 5 × 10⁶ cells / h. 7 The fermentation broth for each strain can be obtained when the cfu / mL concentration is reached.

[0020] The application of the composite bacteria for degrading and repairing benzo[a]pyrene or the composite microbial functional agent described in this invention in the degradation and repair of benzo[a]pyrene.

[0021] The application of the composite bacteria or the composite microbial functional agent in the degradation and remediation of benzo[a]pyrene in soil or water.

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

[0023] The present invention provides a composite microbial agent that, through isolation and screening, can effectively degrade benzo[a]pyrene and various polycyclic aromatic hydrocarbons. It can rapidly degrade more than 90% of benzo[a]pyrene in inorganic salt culture media. At the same time, this composite microbial agent can degrade more than 80% of high concentrations of benzo[a]pyrene in soil in a short time, significantly improving the degradation effect of single strains and effectively solving the problem of residual benzo[a]pyrene harming soil or water environment, crops and human health.

[0024] The degradation agent of the present invention can be produced using general fermentation equipment in the fermentation industry. It has the advantages of low production cost, convenient use, and good removal effect, and is suitable for the remediation of soil contaminated with 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. Attached Figure Description

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

[0026] Figure 2 Electron micrographs of strains 142 and 143 of the present invention. Detailed Implementation

[0027] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0028] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0029] In the embodiments of the present invention:

[0030] The solid culture medium for Mycolicibacterium sp. 142 consists of 5g beef extract, 10g peptone, 10g sodium chloride, and 15g agar, diluted to 1L with water. The liquid culture medium is agar-free.

[0031] The solid culture medium for Stenotrophomonas sp. 143 consists of 10g peptone, 10g sodium chloride, 10g yeast extract, and 20g agar, diluted to 1L with water. The liquid culture medium is agar-free.

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

[0033] Example 1

[0034] Isolation and identification of strains:

[0035] Initial benzo[a]pyrene was added to 100 ml of inorganic salt medium to prepare a 50 mg / L medium. 4 g of sludge sample was added to the prepared medium, and the shaking incubator was set to 30℃, 175 rpm, and 7 days. After the first generation, 2 ml was added to a new 50 mg / L medium for further cultivation, for a total of 4 generations. The degradation effect of the enriched solution on benzo[a]pyrene was determined by liquid chromatography to obtain a concentrated solution with effective degradation. 0.5 mL of the effective concentrated solution was then... -1 10 -2 10 -3 10 -4 10 -5 10 -6 Serial dilutions were performed, with 100 μL of each dilution enriched solution spread onto inorganic salt solid medium plates containing benzo[a]pyrene at mg / L, and incubated at 30°C for 7 days. Single colonies grown on the plates were picked and inoculated into test tubes containing liquid LB medium (10 g peptone, 5 g yeast extract, 5 g NaCl, 1000 mL water, pH 7.0-7.2), and incubated at 30°C and 180 rpm for 2 days. 1 mL of LB medium was taken, centrifuged at 5000 rpm for 5 min, and the LB medium was washed away with sterile water. The bacterial cells were resuspended in 1 mL of sterile water and inoculated into inorganic salt liquid medium containing 50 mg / L benzo[a]pyrene. The culture was incubated at 30°C and 165 rpm for 10 days. The degradation effect of benzo[a]pyrene was detected by high performance liquid chromatography.

[0036] Verification method for degradation effect by high performance liquid chromatography (HPLC): An equal volume of dichloromethane was added to the culture medium for total extraction. After vigorous shaking and standing, the layers separated. 1 mL of the lower layer was taken, and after complete evaporation of the dichloromethane, 1 mL of acetonitrile (chromatographic grade) was added to dissolve it. The solution was then filtered through a membrane (0.22 μm pore size). The content of benzo[a]pyrene in the extract was determined by UV and HPLC, respectively. HPLC conditions: mobile phase acetonitrile:water (90:10, V / V), Zorbax C218 ODS Spherex reversed-phase column (5 μm, 4.6 mm × 250 mm, Agilent, USA), column temperature 30℃, PDA detector, measurement wavelength 254 nm, injection volume 10 μL, flow rate 1.0 mL·min⁻¹. -1 External standard method: quantification based on peak area.

[0037] The bacterial culture with significant degradation effect was repeatedly plated onto drug-containing solid inorganic salt agar plates. The resulting single bacteria were then repeatedly cultured to verify the degradation effect and screen for isolates. Two single bacteria with significant degradation effects were isolated and named 142 and 143. The strains were amplified using PCR technology with primers 1492R and 27F to obtain the 16S rDNA gene sequence, which was sequenced by Platinum BioSynthetic Sequencing Co., Ltd. The 16S rDNA sequences of strains 142 and 143 are shown in SEQ ID NO. 1-2, respectively. The obtained gene sequences were compared using EzBioCloud. The gene sequence of strain 142 is most closely related to the genus *Mycolicibacterium*, while the 16S rRNA gene sequence of strain 143 is most closely related to the genus *Stenotropium*.

[0038] Colony 142 on LB agar plates is entirely round, with a raised surface, neat edges, moist, and yellow in color. Colony 143 on LB agar plates is entirely round, with a raised surface, neat edges, smooth, and milky white in color. Electron micrograph of strain 142 is shown below. Figure 2 As shown, electron micrographs of short rod-shaped (0.4–0.6 μm × 1.0–1.8 μm) and strain 143 are as follows. Figure 2 As shown, it has flagella, which are short rod-shaped (0.4–0.6 μm × 1.5–2.0 μm).

[0039] Based on gene sequence analysis and physiological and biochemical characteristics, strain 142 was preliminarily identified as belonging to the genus *Mycolicibacterium*, and has been deposited at the China Center for Type Culture Collection (CCTCC) on August 18, 2022, with accession number CCTCC NO: M20221294. Strain 143 was identified as belonging to the genus *Stenotrophomonas*, and has also been deposited at the China Center for Type Culture Collection (CCTCC) on August 18, 2022, with accession number CCTCC NO: M 20221295; they were named *Mycolicibacterium* sp. 142 and *Stenotrophomonas* sp. 143, respectively.

[0040] Example 2

[0041] Preparation of compound microbial agents

[0042] The liquid compound microbial inoculant was obtained by mixing the fermentation broth of Mycolicibacterium sp. 142 and Stenotrop Homonass p. 143. The specific preparation method is as follows:

[0043] (1) Activation of bacterial 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 OD200. 600 Inoculum solution is obtained when the value reaches 0.5;

[0044] (2) Preparation of primary seed culture: Inoculate the inoculum prepared in step (1) into the respective liquid culture medium at a volume ratio of 5%, and incubate at 30°C and 150 rpm until OD. 600 A value of 1.0 indicates that the seed solution is of Grade I.

[0045] (3) Preparation of liquid inoculum: Inoculate the primary seed liquid prepared in step (2) into the fermenters containing their respective fermentation media at a volume ratio of 5%, with an aeration rate of 4 m³ / s. 3 The fermentation process was carried out at a stirring speed of 200 rpm and a temperature of 30°C until the viable cell count in the fermenter reached approximately 5 × 10⁶ cells / h. 8 The fermentation broth of each strain was obtained by measuring cfu / mL. The fermentation broths of each strain were mixed so that the ratio of viable cells of Mycolicibacterium sp.142 to Stenotrop Homonas sp.143 was 1:1, thus obtaining a liquid composite microbial agent.

[0046] Example 3

[0047] The degradation effects of strains 142 and 143 and the compound bacterial agent on benzo[a]pyrene residues in inorganic salt culture medium:

[0048] Determination of the degradative properties of strains 142 and 143 against benzo[a]pyrene in inorganic salt liquid medium: Single colonies of strains 142 and 143 were picked and cultured in 50 ml LB liquid medium at 30℃ and 180 r / min for 24 h with shaking to obtain fresh bacterial suspension. 3 ml of the freshly cultured suspension was centrifuged at 5000 r / min for 5 min, the supernatant was discarded, and the suspension was resuspended in 10 ml sterile water. OD 600 The value reached 1.0, indicating the presence of bacterial suspension after centrifugation. The suspended bacterial suspensions of strains 142 and 143 were mixed at a volume ratio of 1:1 to form a compound bacterial agent.

[0049] Benzo[a]pyrene was added to an inorganic salt culture medium with a final concentration of 100 mg / L. Single or mixed bacterial strains were inoculated at a 5% (v / v) inoculum and cultured in a shaker at 30°C (150 rpm). The degradation of benzo[a]pyrene by strains 142, 143, and the compound bacterial agent was detected by high-performance liquid chromatography (HPLC) using the method described 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, while the degradation rate of the compound bacterial agent after 5 days was 90.53%. The results are shown in Table 1.

[0050] Table 1. Results of Benzo[a]pyrene degradation assays by strains 142 and 143 and the compound bacterial agent.

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

[0052] As shown in Table 1, strains 142 and 143 alone can degrade benzo[a]pyrene. However, when the two strains of this invention are used in combination, under the same total concentration of strains, the combined use of strains 142 and 143 can significantly improve the degradation effect and accelerate the degradation rate, indicating that the compound bacteria of this invention have a synergistic effect in the degradation of benzo[a]pyrene.

[0053] Example 4

[0054] Determination of the degradation effect of compound microbial agents on benzo[a]pyrene in soil:

[0055] Multiple 1000g portions of paddy field soil were weighed as test soils, air-dried, and sieved. Benzo[a]pyrene was added to each portion to make the benzo[a]pyrene concentration in the soil 10mg / kg. The compound microbial agent prepared in Example 2 was used, wherein the content of each type of bacteria was 2.5×10⁻⁶. 8 CFU / mL; the concentration of strains 142 and 143 in the inoculum was 5 × 10⁻⁶ CFU / mL. 8 CFU / mL; The single bacterial agents 142 and 143, and the compound degrading bacterial agent prepared in Example 2, were inoculated into the above-mentioned soil at an inoculum rate of 2% (ml / g) and mixed thoroughly as treatments. Soil containing the same concentration of benzo[a]pyrene without the degrading bacterial agent was set as a control group. All soils were incubated at 30℃ in the dark under constant temperature conditions, with the soil moisture content maintained at 40%. The concentration of benzo[a]pyrene in the soil was measured every week after application, and the observation was continued for 10 weeks. Each group was tested in triplicate. The residue was determined using high-performance liquid chromatography, and the average degradation rate was calculated. The results are shown in Table 2.

[0056] Table 2. Degradation effect of degrading microbial agents 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 degrading bacteria 142, 143, and the composite bacterial agent reached 61.0%, 68.7%, and 98.1%, respectively, after 10 weeks using the degrading bacterial agent prepared in Example 2. Under the same total bacterial load system, the composite bacterial agent achieved a degradation rate of 80.3% in week 6 and was basically completely degraded in week 8, significantly exceeding the 45.2% and 49.2% of the single bacterial agents 142 and 143, respectively. The results indicate that the composite degrading bacterial agent can effectively degrade benzo[a]pyrene in the soil and produces a synergistic effect.

Claims

1. A complex bacteria for degrading and repairing benzo[a]pyrene, characterized in that, The compound microbial agent is Mycobacterium 142 ( Mycolicibacterium sp. 142) and oligotrophic monoclonal bacteria 143 ( Stenotrophomonas sp.143), whose accession numbers are CCTCC NO:M 20221294 and CCTCC NO:M 20221295 respectively; the Mycolicibacterium sp.142 and Stenotrophomonas The ratio of viable bacteria in sp. 143 was 0.5:1 to 1:0.

5.

2. The complex bacteria degrading and remediating benz[a]pyrene according to claim 1, characterized in that, The Mycolicibacterium sp. 142 and Stenotrophomonas sp. 143 ratio of viable cell counts was 1:

1.

3. A complex microbial functional microbial inoculum containing the complex bacteria degrading and repairing benzo[a]pyrene according to claim 1.

4. A preparation method of the complex microbial functional microbial inoculum according to claim 3, comprising the following steps: (1) Activation of strains: Mycolicibacterium sp. 142 and Stenotrophomonas sp. 143 strains were inoculated into solid culture medium, cultured, and single colonies were inoculated into liquid culture medium to obtain inoculum. (2) Preparation of primary seed liquid: inoculate the inoculum prepared in step (1) into the liquid culture medium according to the inoculation amount of 1-5% by volume, and culture to obtain the primary seed liquid; (3) Preparation of fermentation liquid: inoculate the primary seed liquid prepared in step (2) into the fermentation tank containing the fermentation culture medium according to the inoculation amount of 1-5% by volume, and culture to obtain the fermentation liquid of each strain; (4) Preparation of microbial inoculum: mix the fermentation liquid of each strain prepared in step (3) to obtain the complex microbial functional microbial inoculum.

5. The preparation method according to claim 4, characterized in that, The Mycolicibacterium The solid medium of Bacillus sp. 142 is beef extract 3-5 g, peptone 10-15 g, sodium chloride 5-10 g, agar 15-20 g, and water to 1 L, and the liquid medium is without agar; the solid medium of Bacillus sp. 143 is peptone 10-15 g, sodium chloride 5-10 g, yeast powder 5-10 g, agar 15-20 g, and water to 1 L, and the liquid medium is without agar. Stenotrophomonas The solid medium of Bacillus sp. 142 is beef extract 3-5 g, peptone 10-15 g, sodium chloride 5-10 g, agar 15-20 g, and water to 1 L, and the liquid medium is without agar; the solid medium of Bacillus sp. 143 is peptone 10-15 g, sodium chloride 5-10 g, yeast powder 5-10 g, agar 15-20 g, and water to 1 L, and the liquid medium is without agar.

6. The preparation method according to claim 4, characterized in that, The culture in step (1) is cultured to OD 600 0.5-0.6, and the inoculum is obtained; the culture in step (2) is cultured to OD 600 0.8-1.0, and the first-stage seed liquid is obtained; the first-stage seed liquid prepared in step (2) is inoculated in step (3), and the culture is cultured to more than 5×10 8 cfu / mL of viable bacteria in the fermenter, and the fermentation liquid of each strain is obtained.

7. The use of the complex bacteria degrading and repairing benzo[a]pyrene according to claim 1 or the complex microbial functional microbial inoculum according to claim 3 in degrading and repairing benzo[a]pyrene.

8. Use according to claim 7, characterized in that, The use of the complex bacteria or the complex microbial functional microbial inoculum in degrading and repairing benzo[a]pyrene in soil or water.

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