Application of rhodococcus in degradation of benzo[a]pyrene in water body

Abstract

The application relates to application of Rhodococcus in degradation of benzene[a]pyrene in a water body and belongs to the technical field of microbial engineering. A Rhodococcus activation strain is subcultured with LB liquid culture medium, the cell concentration is adjusted, and the strain is inoculated into liquid culture medium containing benzene[a]pyrene at a volume ratio of 5%, the final concentration of benzene[a]pyrene in the culture medium is 0.5 mM; and the sample is cultured in a shaking table under the conditions that the temperature is 27 DEG C, the pH is 7.0, and the rotating speed is 180 rpm for 5 days. The Rhodococcus can grow and metabolize by taking benzene[a]pyrene as the only carbon source and energy source, and the benzene[a]pyrene can be efficiently degraded. Through systematic optimization of the degradation conditions, the optimal process parameters are determined, the degradation efficiency is obviously improved, and the application has the advantages of low cost, no secondary pollution, high degradation efficiency and the like. The application provides an efficient and environment-friendly technical scheme for biological remediation of benzene[a]pyrene pollution in a water body, and has good ecological benefits and wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of microbial engineering technology, and in particular relates to a biotechnology that can degrade benzo[a]pyrene in water. Background Technology

[0002] Polycyclic aromatic hydrocarbons (PAHs) are a class of persistent organic pollutants formed by the fusion of two or more benzene rings. They are widely distributed in environmental media such as the atmosphere, water, and soil, posing a serious threat to ecosystems and human health. Benzo[a]pyrene is a typical high-molecular-weight PAH with a five-fused benzene ring structure. It is chemically stable, highly hydrophobic, and difficult to degrade naturally. Benzo[a]pyrene is widely found in water pollution sources such as industrial wastewater, urban runoff, oil spills, and atmospheric deposition. Due to its extremely strong carcinogenic, teratogenic, and mutagenic properties, it has been listed as a priority environmental pollutant for control by many countries. In aquatic environments, benzo[a]pyrene, due to its lipid solubility, easily accumulates in aquatic organisms and exhibits a biomagnification effect along the food chain. This pollutant can induce various toxic effects on aquatic organisms such as fish, crustaceans, and shellfish, including oxidative stress, tissue pathological damage, immunosuppression, and abnormal growth and reproduction. Benzo[a]pyrene in water bodies not only poses a serious threat to aquatic ecosystems, but can also accumulate in the human body through the food chain, increasing the risk of diseases such as cancer.

[0003] Currently, the main technologies for treating benzo[a]pyrene in water bodies include physical adsorption, chemical oxidation, and bioremediation. Physical methods, such as activated carbon adsorption, can quickly remove pollutants, but they suffer from high risks of secondary pollution after adsorption saturation and high treatment costs. Chemical oxidation methods, such as Fenton oxidation and ozone oxidation, can effectively degrade benzo[a]pyrene, but they easily produce toxic byproducts and can potentially damage the aquatic ecosystem. In contrast, bioremediation technology, due to its environmental friendliness, low cost, and lack of secondary pollution, has become a research hotspot for the treatment of benzo[a]pyrene pollution in water bodies.

[0004] Rhodococcus ( Rhodococcus Rhodococcus sp. P14 is a Gram-positive bacterium belonging to the genus Rhodococcus in the phylum Actinobacteria. This strain was first screened from petroleum-contaminated environments and has attracted widespread attention due to its unique metabolic capabilities and environmental adaptability. Microorganisms in the genus Rhodococcus generally possess rich enzyme systems, especially encoding various key enzymes such as aromatic ring dioxygenases, monooxygenases, and dehydrogenases, giving them a significant advantage in degrading persistent organic pollutants such as polycyclic aromatic hydrocarbons, alkanes, and chlorinated aromatic compounds. Currently, Rhodococcus (… RhodococcusRhodococcus sp.) P14 has been widely used in bioremediation research of petroleum-contaminated soils and waters. Studies have shown that this strain not only has good environmental adaptability, growing and metabolizing within a wide temperature (16–37℃) and pH (5.0–9.0) range, but also exhibits strong tolerance and degradation stability. Furthermore, Rhodococcus (… Rhodococcus sp.) P14 can synergize with a variety of indigenous microorganisms to improve overall remediation efficiency. In practical applications, this strain can serve as a candidate strain for highly efficient degradation agents, suitable for the bioremediation of polycyclic aromatic hydrocarbon (PAH) contaminated sites such as industrial wastewater, oil spill-contaminated water bodies, and urban runoff.

[0005] Therefore, Rhodococcus ( Rhodococcus sp.)P14 has significant research value and application potential in the field of environmental biotechnology, and is an important microbial resource for promoting the development of green and sustainable pollution control technologies. Summary of the Invention

[0006] This invention provides an application of Rhodococcus bacteria in the degradation of benzo[a]pyrene in water, in order to solve the problems of low safety and high cost in the current treatment of polycyclic aromatic hydrocarbons.

[0007] The technical solution adopted in this invention is the application of Rhodococcus bacteria in the degradation of benzo[a]pyrene in water.

[0008] The microbial method for the degradation of benzo[a]pyrene by Rhodococcus in water according to the present invention includes the following steps: (1) Rhodococcus ( Rhodococcus sp.) P14 activated strain was subcultured in LB liquid medium and the cell concentration was adjusted to OD 600nm =1.0; (2) Take the strain culture and inoculate it into a liquid culture medium containing benzo[a]pyrene at a volume ratio of 5%, wherein the final concentration of benzo[a]pyrene in the culture medium is 0.1 to 1.5 mM; (3) Place the above sample in a shaker and culture it for 1 to 7 days at a temperature of 16 to 37°C, a pH of 5.0 to 9.0 and a rotation speed of 180 rpm.

[0009] In step (1) of the present invention, the method for activating the strain is as follows: take the strain out of the -80℃ freezer, thaw it at room temperature, add LB liquid medium, and culture it overnight in a shake flask at 37℃ and 180 rpm to complete the cell activation, and then subculture it the next day.

[0010] The method for preparing the LB liquid culture medium of the present invention is as follows: 2 g peptone, 1 g yeast powder, 2 g sodium chloride, distilled water to a final volume of 200 mL, pH 7.0, sterilized at 115℃ for 30 min, and then cooled.

[0011] In step (2) of the present invention, the final concentration of benzo[a]pyrene in the culture medium is 0.5 mM.

[0012] In step (2) of the present invention, the preparation method of the benzo[a]pyrene mother liquor is as follows: dissolve the benzo[a]pyrene standard in acetone to prepare a 5 mM mother liquor, and store it in a sealed container at 4°C for later use.

[0013] The culture temperature in step (3) of this invention is 27°C.

[0014] In step (3) of this invention, the pH value of the culture medium is 7.0.

[0015] The culture time in step (3) of this invention is 5 days.

[0016] In step (3) of the present invention, the degradation efficiency of Rhodococcus P14 reaches 84.92%.

[0017] The beneficial effects of this invention are as follows: By screening multiple natural strains for their degradation ability of benzo[a]pyrene, a highly efficient degradation strain, *Rhodococcus rubrum* P14, was obtained. This strain can use benzo[a]pyrene as its sole carbon and energy source for growth and metabolism, and efficiently degrade it. Through systematic optimization of its degradation conditions, the optimal process parameters were determined, significantly improving the degradation efficiency. This invention has the advantages of simple operation, low cost, no secondary pollution, and high degradation efficiency, providing an efficient and environmentally friendly technical solution for the bioremediation of benzo[a]pyrene pollution in water bodies, with good ecological benefits and broad application prospects. Attached Figure Description

[0018] Figure 1 This is a comparison chart of the degradation efficiency of different strains of the present invention on 0.1 mM benzo[a]pyrene; Figure 2 This is a graph showing the effect of different temperatures on the degradation efficiency of benzo[a]pyrene by Rhodococcus pluvialis P14 according to the present invention; Figure 3 This is a graph showing the effect of different pH values ​​on the degradation efficiency of benzo[a]pyrene by Rhodococcus pluvialis P14. Figure 4 This is a graph showing the effect of different time points on the degradation efficiency of benzo[a]pyrene by Rhodococcus pluvialis P14 according to the present invention; Figure 5 This is a graph showing the effect of different substrate concentrations on the degradation efficiency of benzo[a]pyrene by Rhodococcus pluvialis P14. Detailed Implementation

[0019] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. Unless otherwise specified, the principles, methods, and equipment used in the present invention are those conventionally used in the art. Example 1

[0020] Includes the following steps: (1) Rhodococcus ( Rhodococcus sp.) P14 activated strain was subcultured in LB liquid medium and the cell concentration was adjusted to OD 600nm =1.0; (2) Take the strain culture and inoculate it into a liquid culture medium containing benzo[a]pyrene at a volume ratio of 5%, wherein the final concentration of benzo[a]pyrene in the culture medium is 0.1 mM; (3) Place the above sample in a shaker and culture it for 1 day at a temperature of 16℃, a pH of 5.0 and a rotation speed of 180 rpm. Example 2

[0021] Includes the following steps: (1) Rhodococcus ( Rhodococcus sp.) P14 activated strain was subcultured in LB liquid medium and the cell concentration was adjusted to OD 600nm =1.0; (2) Take the strain culture and inoculate it into a liquid culture medium containing benzo[a]pyrene at a volume ratio of 5%, wherein the final concentration of benzo[a]pyrene in the culture medium is 0.5 mM; (3) The above sample was placed in a shaker and cultured for 5 days at a temperature of 77℃, a pH of 7.0 and a rotation speed of 180 rpm. The degradation efficiency of Rhodococcus P14 reached 84.92%. Example 3

[0022] Includes the following steps: (1) Rhodococcus ( Rhodococcus sp.) P14 activated strain was subcultured in LB liquid medium and the cell concentration was adjusted to OD 600nm =1.0; (2) Take the strain culture and inoculate it into a liquid culture medium containing benzo[a]pyrene at a volume ratio of 5%, wherein the final concentration of benzo[a]pyrene in the culture medium is 1.5 mM; (3) Place the above sample in a shaker and culture it for 7 days at a temperature of 37℃, a pH of 9.0 and a rotation speed of 180 rpm.

[0023] The effects of the present invention will be further illustrated below through experimental examples.

[0024] Experiment 1: Degradation of benzo[a]pyrene by different strains and different culture times (a) Degradation experiment of benzo[a]pyrene by different strains after 2 days of culture (1) Rhodococcus ( Rhodococcussp.) P14, Trichomonas testis ( Comamonas testosteroni CT1, Vibrio ( Vibrio H5, Pseudomonas putida ( Pseudomonas putida PS, Pseudomonas ( Pseudomonas sp.) LY1, Acinetobacter calcium acetate ( Acinetobacter calcoaceticus LM1, Pseudomonas stearothermiae ( Pseudomonas stutzeri JP1 and Bacteroides ( Buttiauxella S19-1 was removed from the -80°C freezer, thawed at room temperature, and then added to LB broth. The cells were incubated overnight at 37°C and 180 rpm in a shake flask to complete cell activation. The cells were then subcultured the following day, and the cell concentration was adjusted to OD. 600nm =1.0; The preparation method of LB liquid culture medium is as follows: 2 g peptone, 1 g yeast powder, 2 g sodium chloride, distilled water to a final volume of 200 mL, pH 7.0, sterilize at 115℃ for 30 min, and cool; (2) Take the culture broth of each strain and inoculate it into a liquid culture medium containing benzo[a]pyrene at a volume ratio of 5%. The final concentration of benzo[a]pyrene in the culture medium is 0.1 mM. The preparation method of benzo[a]pyrene stock solution is as follows: dissolve the benzo[a]pyrene standard in acetone to prepare a 5 mM stock solution, and store it in a sealed container at 4°C for later use. (3) Place the above sample in a shaker and culture it for 2 days at a temperature of 27°C, a pH of 7.0 and a rotation speed of 180 rpm.

[0025] (II) Degradation experiment of benzo[a]pyrene by different strains after 4 days of culture (1) Rhodococcus ( Rhodococcus sp.) P14, Trichomonas testis ( Comamonas testosteroni CT1, Vibrio ( Vibrio H5, Pseudomonas putida ( Pseudomonas putida PS, Pseudomonas ( Pseudomonas sp.) LY1, Acinetobacter calcium acetate ( Acinetobacter calcoaceticus LM1, Pseudomonas stearothermiae ( Pseudomonas stutzeri JP1 and Bacteroides ( Buttiauxella S19-1 was removed from the -80°C freezer, thawed at room temperature, and then added to LB broth. The cells were incubated overnight at 37°C and 180 rpm in a shake flask to complete cell activation. The cells were then subcultured the following day, and the cell concentration was adjusted to OD. 600nm =1.0; The preparation method of LB liquid culture medium is as follows: 2 g peptone, 1 g yeast powder, 2 g sodium chloride, distilled water to a final volume of 200 mL, pH 7.0, sterilize at 115℃ for 30 min, and cool; (2) Take the culture broth of each strain and inoculate it into a liquid culture medium containing benzo[a]pyrene at a volume ratio of 5%. The final concentration of benzo[a]pyrene in the culture medium is 0.1 mM. The preparation method of benzo[a]pyrene stock solution is as follows: dissolve the benzo[a]pyrene standard in acetone to prepare a 5 mM stock solution, and store it in a sealed container at 4°C for later use. (3) Place the above sample in a shaker and culture it for 4 days at a temperature of 27°C, a pH of 7.0 and a rotation speed of 180 rpm.

[0026] (III) Degradation experiment of benzo[a]pyrene by different strains after 6 days of culture (1) Rhodococcus ( Rhodococcus sp.) P14, Trichomonas testis ( Comamonas testosteroni CT1, Vibrio ( Vibrio H5, Pseudomonas putida ( Pseudomonas putida PS, Pseudomonas ( Pseudomonas sp.) LY1, Acinetobacter calcium acetate ( Acinetobacter calcoaceticus LM1, Pseudomonas stearothermiae ( Pseudomonas stutzeri JP1 and Bacteroides ( Buttiauxella S19-1 was removed from the -80°C freezer, thawed at room temperature, and then added to LB broth. The cells were incubated overnight at 37°C and 180 rpm in a shake flask to complete cell activation. The cells were then subcultured the following day, and the cell concentration was adjusted to OD. 600nm =1.0; The preparation method of LB liquid culture medium is as follows: 2 g peptone, 1 g yeast powder, 2 g sodium chloride, distilled water to a final volume of 200 mL, pH 7.0, sterilize at 115℃ for 30 min, and cool; (2) Take the culture broth of each strain and inoculate it into a liquid culture medium containing benzo[a]pyrene at a volume ratio of 5%. The final concentration of benzo[a]pyrene in the culture medium is 0.1 mM. The preparation method of benzo[a]pyrene stock solution is as follows: dissolve the benzo[a]pyrene standard in acetone to prepare a 5 mM stock solution, and store it in a sealed container at 4°C for later use. (3) Place the above sample in a shaker and culture it for 6 days at a temperature of 27°C, a pH of 7.0 and a rotation speed of 180 rpm.

[0027] The results are attached. Figure 1 As shown, Rhodococcus P14 exhibited the strongest degradation ability for benzo[a]pyrene after 6 days of degradation, with a degradation rate of 78.21%.

[0028] Experiment Example 2: Degradation of benzo[a]pyrene by strain P14 at different temperatures The activated strain of Rhodococcus P14 was cultured in LB liquid medium, and the bacterial concentration was adjusted to OD. 600nm=1.0, and inoculated into a liquid culture medium containing benzo[a]pyrene at a volume ratio of 5%, wherein the final concentration of benzo[a]pyrene in the culture medium is 0.1 mM. The above sample was placed in a shaker and cultured for 3 days at temperatures of 16℃, 20℃, 27℃ and 37℃, pH 7.0 and rotation speed of 180 rpm.

[0029] Subsequently, benzo[a]pyrene was extracted to prepare dry samples: 2 mL of chloroform was added to each sample, and the sample was placed on a horizontal shaker and shaken for 30 min, with vigorous shaking every 10 min to ensure that the benzo[a]pyrene in the sample was fully dissolved in the chloroform. After centrifugation at 5000 rpm for 10 min, 1.5 mL of the lower organic phase was carefully aspirated and added to a new tube, and centrifuged again (13,000 rpm for 10 min). 1 mL of the lower phase was aspirated and transferred to a new tube for vacuum drying.

[0030] The specific conditions for sample detection using high-performance liquid chromatography (HPLC) are as follows: the detector used is a UV (2988 Water Detector), and the detection wavelength (λ) is set to 254 nm; a C18 column (4.6 mm × 250 mm, particle size 5 μm) is used; the mobile phase is a mixture of acetonitrile and water with a volume ratio of 75:25 (v / v); the flow rate is set to 1 mL·min. -1 Use 50 µL of mobile phase to dissolve the sample; set the injection volume to 5 µL; maintain the column temperature at 30℃. Repeat the detection process three times for each experimental control. Calculate the degradation efficiency using the degradation rate formula (η = (1 - A1 / A2) × 100%), where η is the degradation rate of benzo[a]pyrene, A1 is the integral area of ​​the remaining BaP peak after degradation, and A2 is the integral area of ​​the remaining BaP peak before degradation.

[0031] Experimental results show that: (see attached) Figure 2 At a temperature of 27℃, P14 exhibits a higher degradation efficiency, with benzo[a]pyrene reaching a degradation rate of 70.51%. The LB liquid culture medium consists of: 2 g peptone, 1 g yeast extract, 2 g sodium chloride, distilled water to a final volume of 200 mL, pH 7.0, sterilized at 115°C for 30 min, and then cooled.

[0032] Experiment 3: Degradation of benzo[a]pyrene by strain P14 at different pH values The activated strain of Rhodococcus P14 was cultured in LB liquid medium, and the bacterial concentration was adjusted to OD. 600nm=1.0, inoculated at a volume ratio of 5% into a liquid culture medium containing benzo[a]pyrene, wherein the final concentration of benzo[a]pyrene in the culture medium was 0.1 mM. The above sample was placed in a shaker and cultured for 3 days at a temperature of 27°C, pH values ​​of 5.0, 6.0, 7.0, 8.0 and 9.0, and a rotation speed of 180 rpm. The experimental results are as follows: Figure 3 At pH 7.0, P14 exhibits higher degradation efficiency, with a degradation rate of 71.84% for benzo[a]pyrene. The LB liquid culture medium consists of: 2 g peptone, 1 g yeast extract, 2 g sodium chloride, distilled water to a final volume of 200 mL, pH 7.0, sterilized at 115°C for 30 min, and then cooled.

[0033] Experiment 4: Degradation of benzo[a]pyrene by strain P14 at different time points The activated strain of Rhodococcus P14 was cultured in LB liquid medium, and the bacterial concentration was adjusted to OD. 600nm =1.0, inoculated at a volume ratio of 5% into a liquid culture medium containing benzo[a]pyrene, wherein the final concentration of benzo[a]pyrene in the culture medium was 0.1 mM. The above sample was placed in a shaker and cultured for 1 day, 3 days, 5 days and 7 days under the conditions of 27°C, pH 7.0 and 180 rpm. The experimental results are as follows: Figure 4 Based on time cost, the optimal degradation time was determined to be 5 days, at which time the degradation rate of benzo[a]pyrene was 67.57%. The LB liquid culture medium consists of: 2 g peptone, 1 g yeast extract, 2 g sodium chloride, distilled water to a final volume of 200 mL, pH 7.0, sterilized at 115°C for 30 min, and then cooled.

[0034] Experiment 5: Degradation of benzo[a]pyrene by strain P14 with different substrate concentrations The activated strain of Rhodococcus P14 was cultured in LB liquid medium, and the bacterial concentration was adjusted to OD. 600nm =1.0, inoculated at a volume ratio of 5% into a liquid culture medium containing benzo[a]pyrene, wherein the final concentrations of benzo[a]pyrene in the culture medium were 0.1 mM, 0.5 mM, 1.0 mM, and 1.5 mM, respectively. The above samples were placed in a shaker and cultured for 5 days at a temperature of 27°C, a pH of 7.0, and a rotation speed of 180 rpm. The experimental results are as follows: Figure 5 At a substrate concentration of 0.5 mM, P14 exhibited high degradation efficiency, with a degradation rate of 84.92% for benzo[a]pyrene. The LB liquid culture medium consists of: 2 g peptone, 1 g yeast extract, 2 g sodium chloride, distilled water to a final volume of 200 mL, pH 7.0, sterilized at 115°C for 30 min, and then cooled.

[0035] The above results demonstrate that the microbial method of this invention can efficiently degrade benzo[a]pyrene in water, and Rhodococcus p14 has extremely high application value in the bioremediation of benzo[a]pyrene-contaminated water. Furthermore, this invention has the advantages of low cost, simple operation, high degradation efficiency, good environmental compatibility, reduced use of chemical reagents, and avoidance of secondary pollution, showing promising application prospects in the bioremediation of benzo[a]pyrene-contaminated water. Therefore, it can serve as a reference for the commercial production of benzo[a]pyrene-degrading bacterial agents.

Claims

1. Application of Rhodococcus in the degradation of benzo[a]pyrene in water.

2. The application of Rhodococcus in the degradation of benzo[a]pyrene in water according to claim 1, characterized in that, A microbial method for the degradation of benzo[a]pyrene by Rhodococcus in water includes the following steps: (1) Rhodococcus ( Rhodococcus sp.) P14 activated strain was subcultured in LB liquid medium and the cell concentration was adjusted to OD 600nm =1.0; (2) Take the strain culture and inoculate it into a liquid culture medium containing benzo[a]pyrene at a volume ratio of 5%, wherein the final concentration of benzo[a]pyrene in the culture medium is 0.1 to 1.5 mM; (3) Place the above sample in a shaker and culture it for 1 to 7 days at a temperature of 16 to 37°C, a pH of 5.0 to 9.0 and a rotation speed of 180 rpm.

3. The application of Rhodococcus in the degradation of benzo[a]pyrene in water according to claim 2, characterized in that: In step (1), the method for activating the strain is as follows: take the strain out of the -80℃ freezer, thaw it at room temperature, add LB liquid medium, and culture it overnight in a shake flask at 37℃ and 180 rpm to complete the cell activation. Then, subculture it the next day.

4. The application of Rhodococcus in the degradation of benzo[a]pyrene in water according to claim 3, characterized in that: The method for preparing the LB liquid culture medium is as follows: 2 g peptone, 1 g yeast extract, 2 g sodium chloride, distilled water to a final volume of 200 mL, pH 7.0, sterilized at 115℃ for 30 min, and then cooled.

5. The application of Rhodococcus in the degradation of benzo[a]pyrene in water according to claim 2, characterized in that: In step (2), the final concentration of benzo[a]pyrene in the culture medium is 0.5 mM.

6. The application of Rhodococcus in the degradation of benzo[a]pyrene in water according to claim 5, characterized in that: In step (2), the preparation method of the benzo[a]pyrene mother liquor is as follows: dissolve the benzo[a]pyrene standard in acetone to prepare a 5 mM mother liquor, and store it in a sealed container at 4°C for later use.

7. The application of Rhodococcus in the degradation of benzo[a]pyrene in water according to claim 2, characterized in that: The culture temperature in step (3) is 27°C.

8. The application of Rhodococcus bacillus in degrading benzo[a]pyrene in water according to claim 7, characterized in that: In step (3), the pH value of the culture medium is 7.

0.

9. The application of Rhodococcus in the degradation of benzo[a]pyrene in water according to claim 8, characterized in that: The culture time in step (3) is 5 days.

10. The application of Rhodococcus in the degradation of benzo[a]pyrene in water according to claim 9, characterized in that: In step (3), the degradation efficiency of Rhodococcus P14 reached 84.92%.

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