Method for promoting degradation of benzo[a]pyrene by bacteria in tea residue fermentation liquor and application thereof

Abstract

The application discloses a method for promoting bacteria to degrade benzene[a]pyrene by using tea residue fermentation liquor and application thereof. The method comprises the following steps: adding the tea residue fermentation liquor into an environment containing benzene[a]pyrene, and promoting the microorganisms in the environment to degrade the benzene[a]pyrene. Under the stimulation of the tea residue fermentation liquor, the degradation effect of the bacteria in the environment on the benzene[a]pyrene is significantly improved, and the stimulation efficiency is 212.6%. The tea residue fermentation liquor is simple to make, low in production cost and convenient to obtain, and can quickly repair the polluted environment.

Description

Technical Field

[0001] This invention belongs to the field of microbial degradation of environmental organic pollutants, specifically relating to a method and application of tea residue fermentation broth to promote the degradation of benzo[a]pyrene by bacteria. Background Technology

[0002] Benzo[a]pyrene (BaP) is one of the 16 important polycyclic aromatic hydrocarbons listed by the U.S. Environmental Protection Agency (USEPA). It may induce a variety of diseases such as leukemia, kidney damage, lung cancer, stomach cancer, bladder cancer and digestive tract cancer. It also has phototoxicity and photogenotoxicity, posing a serious threat to the health of organisms.

[0003] Microorganisms, by actively degrading organic pollutants and obtaining energy from polluted environments, have become an important tool for environmental remediation. Biostimulation strategies can enhance microbial degradation activity and effectively remediate benzo[a]pyrene pollution. By studying the effects and mechanisms of biostimulation, we can better apply biostimulation technologies to improve environmental quality and promote bioremediation and degradation processes (Tripathi, S., Sharma, P. and Chandra, R. Degradation of organometallic pollutants of distillery wastewater by autochthonous bacterial community in biostimulation and bioaugmentation process[J]. BioresourceTechnology, 2021, 338: 125518.).

[0004] Therefore, providing a method for a biostimulant that can promote the degradation of benzo[a]pyrene by microorganisms is of great help in the bioremediation of polycyclic aromatic hydrocarbons. Compared with the method and application of biostimulants to promote the degradation of benzo[a]pyrene by bacteria (Patent No.: CN 116333942 A), the present invention has a more significant stimulating effect on the degradation of high concentrations of benzo[a]pyrene by dispersible pantothenic bacteria MSC14, mainly reflected in the increase in stimulating efficiency from 124.03% to 212.6%. In addition, the tea residue material used in the present invention is solid waste, which can achieve the effect of low cost and "waste treatment". Summary of the Invention

[0005] This invention aims to overcome the shortcomings and deficiencies of the prior art and proposes a method for using tea residue fermentation liquid as a biostimulant to promote the degradation of benzo[a]pyrene by bacteria.

[0006] Another object of the present invention is to provide the application of the above method in the bioremediation of benzo[a]pyrene contaminated water bodies.

[0007] To achieve the above objectives, the technical solution provided by this invention is as follows: This invention provides a method for promoting the degradation of benzo[a]pyrene by bacteria in tea residue fermentation broth, comprising the following steps: Adding tea residue fermentation liquid to an environment containing benzo[a]pyrene can promote the degradation of benzo[a]pyrene by microorganisms in the environment.

[0008] Furthermore, the method for promoting the degradation of benzo[a]pyrene by bacteria in tea residue fermentation broth also includes the following steps: Adding dispersible pantothecin to an environment containing benzo[a]pyrene Pantoea dispersa MSC14, tea residue fermentation broth promotes the degradation of benzo[a]pyrene by dispersed pantothenic bacteria MSC14.

[0009] Furthermore, dispersed pantothecin ( Pantoea dispersa The accession number of MSC14 is GDMCC No: 62680. It was deposited on August 3, 2022, at the Guangdong Provincial Microbial Culture Collection Center, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou (Patent Application Publication No.: CN 115820494 A).

[0010] Furthermore, the preparation method of tea residue fermentation liquid is as follows: Disodium hydrogen phosphate, cellulase, and Aspergillus niger spore powder are added to tea residue, stirred and mixed, then ultrapure water is added, stirred and mixed, and then placed into a sealed bag with a one-way exhaust valve for anaerobic fermentation. After fermentation, the tea residue fermentation liquid is obtained by filtration.

[0011] Preferably, the tea residue is oolong tea residue.

[0012] Preferably, tea dregs, disodium hydrogen phosphate, cellulase, and Aspergillus niger spore powder are added in the following proportions: 60-70 parts, 20-30 parts, 2-5 parts, and 2-5 parts by weight.

[0013] Preferably, the mass-to-volume ratio of tea residue to ultrapure water is 10-15 g / 100 mL; Preferably, the fermentation temperature is 25-35℃; Preferably, the fermentation time is 1-2 weeks; Preferably, the filtration process involves using gauze to obtain a coarse fermentation liquid, then filtering it with filter paper and finally filtering it through a filter head to obtain a tea residue fermentation liquid.

[0014] Preferably, when the environment is liquid, the amount of tea residue fermentation liquid added is 5%-15% of the liquid volume. More preferably, the amount of tea residue fermentation liquid added is 10% of the liquid volume.

[0015] Preferably, the temperature conditions for the degradation of benzo[a]pyrene are 25-35 °C, and the pH conditions are 6.5-8.0; More preferably, the temperature conditions for the degradation of benzo[a]pyrene are 30 °C and the pH conditions are 7.0.

[0016] Preferably, the environment is a body of water.

[0017] This invention also provides the application of the above-mentioned method of promoting bacterial degradation of benzo[a]pyrene by fermentation liquid of tea residue in the bioremediation of water bodies polluted by benzo[a]pyrene.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) This invention provides a tea residue fermentation liquid as a new biostimulant. Adding tea residue fermentation liquid to an environment containing benzo[a]pyrene can promote the degradation of benzo[a]pyrene by microorganisms in the environment. The tea residue fermentation liquid is simple to prepare and is inexpensive because it uses tea residue. (2) The tea residue fermentation liquid provided by the present invention can also be added together with dispersed pantothenic bacteria MSC14 to an environment contaminated with benzo[a]pyrene. The tea residue fermentation liquid can promote the degradation of benzo[a]pyrene in the environment by dispersed pantothenic bacteria MSC14. (3) The tea residue fermentation liquid provided by the present invention can improve the degradation rate of benzo[a]pyrene by bacteria and can stimulate the dispersed pantothenic bacteria MSC14 to quickly adapt to the environment containing benzo[a]pyrene; (4) The tea residue fermentation liquid provided by this invention contains a large amount of carbon source (such as... Figure 3 As shown in the figure, it provides additional nutrients for bacterial growth; the tea residue fermentation broth effectively alleviates the toxic effects of benzo[a]pyrene through antioxidant intervention; the tea residue fermentation broth has a significant antioxidant protective effect and can effectively alleviate the oxidative damage caused by benzo[a]pyrene; thereby promoting the degradation of benzo[a]pyrene by bacteria. Attached Figure Description

[0019] Figure 1 This is a picture of fermented tea residue.

[0020] Figure 2 This is a picture of fermented tea residue liquid.

[0021] Figure 3 This is a diagram showing the composition of fermented tea residue liquid.

[0022] Figure 4 This is a comparison chart showing the effect of dispersible pantothenic acid MSC14 on the degradation of benzo[a]pyrene by various biostimulants.

[0023] Figure 5 This is a comparison of the effects of dispersed pantothenic acid MSC14 on the reduction of benzo[a]pyrene in tea residue fermentation broth under different temperature conditions.

[0024] Figure 6 This is a comparison of the effects of dispersed pantothenic acid MSC14 on the reduction of benzo[a]pyrene in tea residue fermentation broth under different pH conditions.

[0025] Figure 7 This is a comparison chart showing the effect of dispersible pantothenic acid MSC14 on the reduction of benzo[a]pyrene by tea residue fermentation broth with different addition amounts.

[0026] Figure 8 This is a scanning electron microscope (SEM) image (30.00 KX) of benzo[a]pyrene degraded by Pantotheca MSC14 before stimulation by tea residue fermentation broth.

[0027] Figure 9 This is a scanning electron microscope (SEM) image (30.00 KX) of benzo[a]pyrene degraded by dispersed pantothenic bacteria MSC14 after stimulation by tea residue fermentation broth.

[0028] Figure 10 This is a growth curve of benzo[a]pyrene being degraded by *Panthera miltiorrhiza* MSC14 stimulated by tea residue fermentation broth.

[0029] Figure 11 This is a graph showing the change in EPS content after tea residue fermentation broth stimulates the degradation of benzo[a]pyrene by dispersed pantothenic acid MSC14.

[0030] Figure 12 This is a graph showing the changes in cell membrane permeability after tea residue fermentation broth stimulates the degradation of benzo[a]pyrene by dispersed pantothenic acid MSC14.

[0031] Figure 13 This is a graph showing the change in ROS content after tea residue fermentation broth stimulates the degradation of benzo[a]pyrene by dispersed pantothenic acid MSC14.

[0032] Figure 14 This is a graph showing the change in LPO content after tea residue fermentation broth stimulates the degradation of benzo[a]pyrene by dispersed pantothenic acid MSC14.

[0033] Figure 15 This is a graph showing the change in MDA content after tea residue fermentation broth stimulates the degradation of benzo[a]pyrene by dispersed pantothenic acid MSC14.

[0034] Figure 16 This is a graph showing the change in GSH content after tea residue fermentation broth stimulates the degradation of benzo[a]pyrene by dispersed pantothenic acid MSC14.

[0035] Figure 17 This is a graph showing the change in CAT activity after tea residue fermentation broth stimulates the degradation of benzo[a]pyrene by dispersed pantothenic acid MSC14.

[0036] Figure 18 This is a graph showing the change in SOD activity after tea residue fermentation broth stimulates the degradation of benzo[a]pyrene by dispersed pantothenic acid MSC14. Detailed Implementation

[0037] It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the reagents, methods, and equipment used in this invention are conventional in the art. Unless otherwise specified, the reagents and raw materials used in this invention are commercially available.

[0038] 1. Preparation of tea residue fermentation liquid The raw material used for the tea residue fermentation liquid is oolong tea residue from Quanzhou, Fujian Province; the auxiliary materials, cellulase and Aspergillus niger spore powder, were purchased from Shandong Hezhong Kangyuan Biotechnology Co., Ltd.; the one-way exhaust valve sealing bags used for fermentation were purchased from Jinpeinuo Color Printing Factory in Chaoan District, Chaozhou City, Guangdong Province; and the hand-operated plastic sealing machine used for sealing was purchased from Luohe Baorong Industrial Co., Ltd.

[0039] (1) Mixing tea leaves and auxiliary materials: Mix oolong tea leaves, disodium hydrogen phosphate, cellulase and Aspergillus niger spore powder in a mass ratio of 20:8:1:1. Mix the materials thoroughly, then add ultrapure water and mix well. The mass-volume ratio of tea leaves to ultrapure water is 10 g / 100 mL.

[0040] (2) Fermentation: After mixing, immediately place the mixture into a one-way degassing valve sealed bag and seal it with a hand-operated plastic sealer. Incubate at 30℃ for anaerobic fermentation for one week. During fermentation, shake the one-way degassing valve sealed bag to ensure even distribution of the material. The fermentation is complete when the material changes from a clear, tea-water-like consistency to a yellowish-brown, cloudy consistency. A photo of the fermented material is shown below. Figure 1 As shown.

[0041] (3) Fermentation broth preparation: Open the one-way exhaust valve sealed bag, pour the tea residue fermentation material into gauze for filtration to obtain coarse fermentation broth, filter it through filter paper, and then pass it through a 0.45 μm filter head and a 0.22 μm filter head in sequence for filtration and sterilization to obtain tea residue fermentation broth. Prepare and use immediately. Figure 2 The image shows the fermented tea residue liquid after filtration.

[0042] 2. Preparation of the solutions used in the examples: LB liquid culture medium, with the following formula: 10 g / L tryptone, 10 g / L NaCl, 5 g / L yeast extract, adjusted to pH=7.0, and sterilized at 121 ℃ for 30 min; The inorganic salt culture medium containing benzo[a]pyrene was formulated as follows: 1.00 g / L NaCl, 1.00 g / L (NH4)2SO4, 0.25 g / L KH2PO4, 0.5 g / L Na2HPO4, 0.20 g / L MgSO4·7H2O, and 0.04 g / L CaCl2. The pH was adjusted to 7.0, and the medium was sterilized at 121℃ for 30 min. Then, benzo[a]pyrene stock solution that had been sterilized by filtration through a 0.22 μm filter membrane was added to make the concentration of benzo[a]pyrene in the culture medium 20 mg / L. The medium was shaken in a shaker at 30 ℃ and 150 rpm for 16 h to allow the acetone in the benzo[a]pyrene stock solution to evaporate. Phosphate-buffered saline (PBS) was prepared with the following formula: 8 g / L NaCl, 1.44 g / L Na2HPO4, and 0.24 g / L KH2PO4. The pH was adjusted to 7.4, and the solution was sterilized at 121 °C for 30 min.

[0043] 3. Detection method for benzo[a]pyrene content: A 1 g / L stock solution was prepared by dissolving benzo[a]pyrene standard in acetone, and then serially diluted to obtain samples of different concentrations. High-performance liquid chromatography (HPLC) was used for detection, and a standard curve was plotted. The sample was extracted twice with an equal volume of dichloromethane, and the volume was adjusted to 25 mL. One mL of this solution was filtered through a 0.22 μm organic filter membrane and placed in a vial. The obtained sample was then analyzed by HPLC, and its concentration was calculated based on the previously measured peak area versus concentration standard curve.

[0044] High-performance liquid chromatography (HPLC) was used for detection. The HPLC instrument used was an LC-20. The chromatographic column was a 4.6 mm × 250 mm Eclipse PAH column, the column temperature was 30 ℃, the mobile phase was a mixture of acetonitrile and ultrapure water (V:V=90:10), the flow rate was 1 mL / min, and the UV detector was set at a wavelength of 290 nm.

[0045] 4. Calculation of degradation rate: Degradation rate of nh = (initial benzo[a]pyrene concentration – residual concentration of benzo[a]pyrene after degradation of nh) / initial benzo[a]pyrene concentration.

[0046] 5. Calculation of stimulation efficiency: Stimulation efficiency = (Degradation rate of benzo[a]pyrene with added biostimulant – Degradation rate of control benzo[a]pyrene) / Degradation rate of control benzo[a]pyrene × 100%.

[0047] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the implementation of the present invention is not limited.

[0048] Example 1: Tea residue fermentation broth promotes the degradation of benzo[a]pyrene by dispersed pantothenic acid MSC14. Dispersible pantothenic acid ( Pantoea dispersa The accession number of MSC14 is GDMCC No: 62680. It was deposited on August 3, 2022, at the Guangdong Provincial Microbial Culture Collection Center, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou (Patent Application Publication No.: CN 115820494 A).

[0049] Under aseptic conditions, dispersed pantothenic MSC14 cells, stored in glycerol tubes at -40 °C, were inoculated into LB broth and cultured on a shaker at 30 °C and 150 rpm for 16 h. Then, the cells were centrifuged at 6000 rpm for 5 min, the supernatant was discarded, and the cells were collected. The cells were washed three times with sterile PBS buffer, and then centrifuged again to collect the cells. Finally, the cells were resuspended in PBS buffer and the density of the suspension was adjusted to OD using a UV-Vis spectrophotometer (Thermo Fisher, Evolution 300, USA). 600 =2.0 seed solution, ready for use.

[0050] The activated seed culture was inoculated into an inorganic salt medium containing benzo[a]pyrene at a 10% (V / V) inoculation rate. 10% (V / V) tea residue fermentation broth was added to the system. The mixture was cultured for 18 h at 30 ℃, 150 rpm and pH 7.0. Three replicates were set up, and the content of benzo[a]pyrene after culture was measured and the degradation rate was calculated.

[0051] Comparative Example 1: Commercially available biostimulants promote the degradation of benzo[a]pyrene by dispersed pantothenic acid MSC14 The activated seed culture was inoculated at a rate of 10% (V / V) in inorganic salt medium (as a blank control), inorganic salt medium supplemented with 1 g / L glucose, and inorganic salt medium supplemented with 1 g / L peptone. The culture was carried out at 30℃, 150 rpm, and pH 7.0 for 18 h. Each group was set up in triplicate. The content of benzo[a]pyrene after culture was measured and the degradation rate was calculated.

[0052] The results of Example 1 and Comparative Example 1 are as follows Figure 4 As shown, the degradation rate of the tea residue fermentation liquid was significantly higher than that of the blank control group. The tea residue fermentation liquid used in Example 1 was also significantly better than other biostimulants such as glucose and peptone in Comparative Example 1 in terms of improving the degradation rate. The degradation rate of benzo[a]pyrene under the stimulation of tea residue fermentation liquid was 67.2%, and the stimulation efficiency was 212.6%.

[0053] Example 2: Optimization of degradation conditions for strains stimulated by tea residue fermentation broth The experimental procedure is the same as in Example 1.

[0054] (1) Degradation temperature optimization The degradation temperature of 30 ℃ was replaced with degradation temperatures of 20 ℃, 25 ℃, 30 ℃, 35 ℃ and 40 ℃, respectively.

[0055] The results are as follows Figure 5 As shown, the degradation rate of benzo[a]pyrene by *Umbrella dispersible* under the stimulation of tea residue fermentation broth was the highest at 30 ℃, reaching 66.6%. The preferred degradation temperature for *Umbrella dispersible* MSC14 to degrade benzo[a]pyrene under the stimulation of tea residue fermentation broth was 25 ℃-35 ℃, and the optimal degradation temperature was 30 ℃.

[0056] (2) Degradation pH Optimization Based on the optimal degradation temperature, the degradation pH value of 7.0 was replaced with degradation temperatures of 6.0, 6.5, 7.0, 7.5, 8.0, and 8.5, respectively.

[0057] The results are as follows Figure 6 As shown, the highest degradation rate of benzo[a]pyrene was achieved by *Umbrella dispersans* under the stimulation of tea residue fermentation broth at a pH of 7.0, which was 67.5%. The preferred degradation pH for *Umbrella dispersans* MSC14 under the stimulation of tea residue fermentation broth for the degradation of benzo[a]pyrene was 6.5-8.0, with the optimal degradation pH being 7.0.

[0058] (3) Optimization of the amount of tea residue fermentation liquid added Based on the optimal degradation temperature and pH value, the addition of 10% (V / V) tea residue fermentation liquid was replaced with the addition of 0%, 2%, 5%, 10% and 15% (V / V) tea residue fermentation liquid, respectively.

[0059] The results are as follows Figure 7 As shown, adding tea residue fermentation liquid at a rate of 10% (V / V) yields the best effect, stimulating the degradation rate of benzo[a]pyrene by Pantothenia glutinosa to reach 66.8%. The preferred addition amount of tea residue fermentation liquid to stimulate the degradation of benzo[a]pyrene by Pantothenia glutinosa MSC14 is 5%-15%, with the optimal addition amount being 10% (V / V).

[0060] Example 3: Mechanism study of tea residue fermentation liquid promoting the degradation of benzo[a]pyrene (1) Effects of tea residue fermentation broth on bacterial morphology After culturing according to Example 1, the bacterial cells were collected by centrifugation at 8000 rpm for 5 min at 4 ℃, washed three times with PBS buffer, resuspended in 2.5% (V / V) glutaraldehyde fixative, and fixed at 4 ℃ for 12 h. The cells were then dehydrated sequentially for 5 min with gradient concentrations of 40%, 70%, 90%, and 100% (V / V) ethanol solutions, followed by replacement with a 1:1 (V / V) ethanol-tert-butanol solution for 20 min, and finally a second replacement with 100% tert-butanol solution. The dehydrated cells were freeze-dried and sputter-coated using a gold sputtering system (150 TES, EMS, USA). Cell morphology was observed under a high-resolution field emission scanning electron microscope (Merlin, Zeiss, Germany).

[0061] Scanning electron microscopy results of bacteria in tea residue fermentation broth before and after stimulation are as follows: Figure 8 and Figure 9 As shown, under benzo[a]pyrene stress, bacterial cells swell, their surface wrinkles intensify, and numerous tightly packed spherical structures adhere to them. After adding tea residue fermentation broth, the bacterial density increases, the surface wrinkles decrease, smoothness improves, and bacterial adhesion is enhanced. Therefore, under the stimulation of tea residue fermentation broth, *Ureaplasma dispersans* MSC14 can rapidly adapt to an environment containing benzo[a]pyrene and efficiently degrade benzo[a]pyrene.

[0062] (2) Effect of tea residue fermentation liquid on bacterial growth Following the inoculation method described in Example 1, activated seed culture was inoculated at a 10% (V / V) inoculation rate into inorganic salt medium (as a blank control), inorganic salt medium containing benzo[a]pyrene, and inorganic salt medium containing benzo[a]pyrene supplemented with 10% (V / V) tea residue fermentation broth. The cultures were incubated at 30°C and 150 rpm. Samples were taken at 0, 2, 4, 6, 8, 10, 12, 24, 36, and 48 h. The absorbance of the bacterial culture was measured using a UV-Vis spectrophotometer at a wavelength of λ=600 nm, with PBS as a blank control. The specific steps were: sampling the relevant culture medium or sample, measuring the absorbance using a cuvette at a wavelength of 600 nm, and obtaining the OD. 600 The value indicates that the bacteria are growing better.

[0063] like Figure 10 As shown, the dispersible pantothenic acid MSC14 strain grew slowly under benzo[a]pyrene stress in inorganic salt medium with benzo[a]pyrene as the carbon source. The OD at 2 h was [data missing]. 600 The value was 0.647, approximately 3.2 times the initial bacterial count, but after 2 hours, the OD... 600The value began to decline, indicating that the living conditions were no longer suitable, the bacterial count had decreased, and a large number of Pantothenia gravis bacteria had died. After adding tea residue fermentation liquid, the bacteria multiplied rapidly, and the OD value increased significantly after 36 hours. 600 The concentration reached 1.956, approximately 6.5 times that of the inorganic salt culture medium. The tea residue fermentation broth contains a large amount of carbon source (such as...). Figure 3 As shown in the figure, this provides additional nutrients for bacterial growth.

[0064] (3) Effects of tea residue fermentation broth on bacterial EPS content and cell membrane permeability Following the inoculation method described in Example 1, activated seed culture was inoculated at an inoculation rate of 10% (V / V) in inorganic salt medium (as a blank control), inorganic salt medium containing benzo[a]pyrene, and inorganic salt medium containing benzo[a]pyrene supplemented with 10% (V / V) tea residue fermentation broth. The cultures were incubated at 30 °C and 150 rpm for 18 h. After centrifugation, the bacterial cells were collected, washed three times with PBS solution, resuspended in PBS, and OD was adjusted. 600 The concentration was 1.0, and a bacterial suspension was prepared for later use.

[0065] Determination of EPS content: EPS was extracted using a heating method. Take 5 mL of bacterial suspension and heat it in a constant temperature water bath at 70 ℃ for 2 h. After cooling to room temperature, centrifuge at 8000 rpm for 5 min, collect the supernatant, and filter it with a 0.22 μm aqueous filter membrane to remove the small amount of residual bacterial cells, thus obtaining EPS extract.

[0066] The protein content in EPS extract was determined using the Coomassie Brilliant Blue method. Coomassie Brilliant Blue Stock Solution: Dissolve 100 mg G-250 Coomassie Brilliant Blue in 50 mL of 95% (V / V) ethanol, add 100 mL of 85% (V / V) phosphoric acid, and then dilute to 200 mL with distilled water. Dilute the stock solution with pure water at a ratio of 1:4 to obtain Coomassie Brilliant Blue staining solution. Add 1 mL of EPS extract to 5 mL of dye solution and react for 5-30 min. Measure the absorbance at 595 nm using a UV-Vis spectrophotometer. A protein standard curve was plotted using standard bovine serum albumin, and its protein content was calculated.

[0067] The polysaccharide content in EPS extract was determined using the phenol-sulfuric acid method. Take 2 mL of the extract, add 1 mL of 6% (V / V) phenol solution and 5 mL of concentrated sulfuric acid in sequence, mix thoroughly, and react in the dark for 10 min; after the dark reaction is completed, heat in a boiling water bath for 15 min. After the reaction solution cooled, the absorbance was measured at 490 nm using a UV-Vis spectrophotometer. A glucose standard curve was plotted using anhydrous glucose, and the polysaccharide content was calculated.

[0068] Measurement of cell membrane permeability: Bacterial cell membrane permeability was characterized using the membrane permeability of 4,6-diamidinyl-2-phenylindole (DAPI) and its strong binding to DNA: Take 2 mL of bacterial suspension, add 1 mL of DAPI-PBS solution (DAPI concentration of 4 μg / mL), mix well, and incubate in the dark at 37 ℃ for 10 min. Using a multi-functional microplate reader (Cytation 5, BioTek, USA), under the conditions of Ex / Em=340±1.5 nm / 488±1.5 nm, the fluorescence value A1 after the dark reaction and the fluorescence value A2 after the bacterial suspension was thawed and frozen 5 times were measured.

[0069] The calculation formula is as follows: Cell membrane permeability = A1 / A2 × 100%.

[0070] The results are as follows Figure 11 and Figure 12 As shown, under benzo[a]pyrene stress, EPS content typically decreases significantly due to oxidative stress-induced inhibition of anabolism or enhancement of catabolism. However, under the stimulation of tea residue fermentation broth, the protein content in EPS almost returned to the original level before benzo[a]pyrene stress, and the polysaccharide content was significantly increased compared to the control group under benzo[a]pyrene stress alone. Simultaneously, tea residue fermentation broth significantly inhibited the abnormal increase in cell membrane permeability induced by benzo[a]pyrene, maintaining the integrity of the cell membrane phospholipid bilayer. These results indicate that tea residue fermentation broth effectively alleviated the toxic effects of benzo[a]pyrene through antioxidant intervention, restoring microbial metabolic homeostasis to baseline.

[0071] (4) Effects of tea residue fermentation broth on bacterial oxidative stress levels Following the inoculation method described in Example 1, activated seed culture was inoculated into inorganic salt medium (as a blank control), inorganic salt medium containing benzo[a]pyrene, and inorganic salt medium containing benzo[a]pyrene with 10% (V / V) tea residue fermentation broth at an inoculation rate of 10% (V / V). The culture was carried out at 30 °C and 150 rpm for 18 h. After centrifugation, the bacterial cells were collected and washed three times with PBS solution. The cells were then resuspended in PBS to prepare a bacterial suspension. The contents of ROS, LPO, GSH, and MDA, as well as the activities of CAT and SOD, were determined according to the kit instructions.

[0072] The ROS content assay kit was purchased from Beijing Solarbio Science & Technology Co., Ltd., and the LPO, GSH and MDA content assay kits, as well as the CAT and SOD activity assay kits, were purchased from Nanjing Jiancheng Bioengineering Research Institute Co., Ltd.

[0073] like Figure 13-18 As shown, the contents of ROS, LPO, GSH, and MDA, as well as the activities of CAT and SOD, increased significantly under benzo[a]pyrene stress, but returned to near-original levels under the stimulation of tea residue fermentation broth. Under benzo[a]pyrene stress, the large amount of reactive oxygen species (ROS) produced by the bacteria attacked lipids in the cell membrane, leading to an increase in lipid peroxides (LPO) and the production of malondialdehyde (MDA) as a byproduct, which exacerbated intracellular oxidative damage. Faced with oxidative stress, cells activate antioxidant defense mechanisms. Glutathione (GSH), as a direct antioxidant, can neutralize ROS and protect cells from damage; catalase (CAT) decomposes hydrogen peroxide, and superoxide dismutase (SOD) decomposes superoxide anions. These two enzymes work synergistically to reduce ROS accumulation, which is an adaptive response of cells to benzo[a]pyrene-induced oxidative stress.

[0074] The addition of tea residue fermentation broth reduced cell ROS, inhibiting the production of LPO and MDA, restoring them to near-original levels. This indicates that tea residue fermentation broth has a significant antioxidant protective effect and can effectively alleviate oxidative damage caused by benzo[a]pyrene. With reduced ROS levels, cellular oxidative stress decreased, reducing the need for antioxidant defense; therefore, GSH, CAT, and SOD levels decreased from high levels under benzo[a]pyrene stress alone.

[0075] In summary, the present invention has the following beneficial effects: (1) This invention provides a tea residue fermentation liquid as a new biostimulant. Adding tea residue fermentation liquid to an environment containing benzo[a]pyrene can promote the degradation of benzo[a]pyrene by microorganisms in the environment. The tea residue fermentation liquid is simple to prepare and is inexpensive because it uses tea residue. (2) The tea residue fermentation liquid improved by the present invention can also be added together with dispersed pantothenic acid MSC14 to the environment contaminated with benzo[a]pyrene. The tea residue fermentation liquid can promote the degradation of benzo[a]pyrene in the environment by dispersed pantothenic acid MSC14. (3) The tea residue fermentation liquid provided by the present invention can improve the degradation rate of benzo[a]pyrene by bacteria and can stimulate the dispersed pantothenic bacteria MSC14 to quickly adapt to the environment containing benzo[a]pyrene; (4) The tea residue fermentation liquid provided by this invention contains a large amount of carbon source (such as... Figure 3As shown in the figure, it provides additional nutrients for bacterial growth; the tea residue fermentation broth effectively alleviates the toxic effects of benzo[a]pyrene through antioxidant intervention; the tea residue fermentation broth has a significant antioxidant protective effect and can effectively alleviate the oxidative damage caused by benzo[a]pyrene; thereby promoting the degradation of benzo[a]pyrene by bacteria.

[0076] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for promoting the degradation of benzo[a]pyrene by bacteria in tea leaf residue fermentation liquor, characterized in that, Includes the following steps: Adding tea residue fermentation liquid to an environment containing benzo[a]pyrene promotes the microbial degradation of benzo[a]pyrene in the environment; also includes the following steps: Adding pantoea dispersa to an environment containing benz[a]pyrene Pantoea dispersa MSC14, tea dregs fermentation liquor promotes MSC14 to degrade benz[a]pyrene; the preservation number of pantoea dispersa MSC14 is: GDMCC No: 62680; The preparation method of the tea residue fermentation liquid is as follows: Disodium hydrogen phosphate, cellulase, and Aspergillus niger spore powder are added to tea residue, stirred and mixed, then ultrapure water is added, stirred and mixed, and then placed into a sealed bag with a one-way exhaust valve for anaerobic fermentation. After fermentation, the tea residue fermentation liquid is obtained by filtration.

2. The method for promoting the degradation of benzo[a]pyrene by bacteria in tea residue fermentation liquid according to claim 1, characterized in that: The tea residue mentioned is oolong tea residue.

3. The method for promoting the degradation of benzo[a]pyrene by bacteria in tea residue fermentation liquid according to claim 1, characterized in that: The mass fractions of tea residue, disodium hydrogen phosphate, cellulase, and Aspergillus niger spore powder added are 60-70 parts, 20-30 parts, 2-5 parts, and 2-5 parts, respectively.

4. The method for promoting the degradation of benzo[a]pyrene by bacteria in tea residue fermentation liquid according to claim 1, characterized in that: The mass-to-volume ratio of tea residue to ultrapure water is 10-15 g / 100 mL; Fermentation temperature is 25-35℃; Fermentation time is 1-2 weeks; The filtration process involves using gauze to obtain a coarse fermentation liquid, then filtering it with filter paper and finally filtering it through a filter head to obtain a tea residue fermentation liquid.

5. The method for promoting the degradation of benzo[a]pyrene by bacteria in tea residue fermentation liquid according to claim 1, characterized in that: When the environment is liquid, the amount of tea residue fermentation liquid added is 5%-15% of the liquid volume. The temperature conditions for the degradation of benzo[a]pyrene are 25-35 ℃ and the pH conditions are 6.5-8.

0.

6. The method for promoting the degradation of benzo[a]pyrene by bacteria in tea residue fermentation liquid according to claim 1, characterized in that: The environment is a body of water.

7. The method for promoting the degradation of benzo[a]pyrene by fermentation liquid of tea residue as described in any one of claims 1-6 is used in the bioremediation of water bodies polluted by benzo[a]pyrene.

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