Method and application of biostimulants to promote bacterial degradation of benzo[a]pyrene

Abstract

The present invention discloses a method and application of four biostimulants to promote bacterial degradation of benzo[a]pyrene. The method comprises the following steps: adding a biostimulant to an environment containing benzo[a]pyrene to promote bacterial degradation of benzo[a]pyrene; the biostimulant comprises at least one of sodium gluconate, sodium citrate, a fermentation broth of peanut meal and wheat bran, and a fermentation broth of soybean meal and corn stalk meal. The biostimulant enhances the benzo[a]pyrene degradation efficiency of the bacteria in the environment. The biostimulant is inexpensive to produce, readily available, and can quickly repair polluted environments.

Description

Technical Field

[0001] The present invention belongs to the field of microbial and environmental organic pollutant restoration and treatment, and particularly relates to a method and application of using a biostimulant to promote bacterial degradation of benzo[a]pyrene. Background Art

[0002] Benzo[a]pyrene is one of the most resistant PAHs to degradation due to its high ring number. As a result, it remains in the environment longer and has a correspondingly higher toxicity. Benzo[a]pyrene ranks 8th out of 275 chemicals on the Substance Priority List (ATSDR) of environmentally hazardous substances.

[0003] PAHs can be degraded through biological, physical, and chemical methods, and biodegradation is considered an economical and safe method. Biostimulation and bioaugmentation are the main methods for improving bioremediation efficiency. Bioaugmentation involves adding microorganisms to contaminated sites, while biostimulation involves adding nutrients (organic biostimulants such as N, P, and K), surfactants, water, and oxygen to enhance the activity of functional microorganisms. The addition of biostimulants primarily promotes rapid microbial growth, shortens remediation time, and induces the growth of genes and enzymes involved in degradation. The core of biostimulants is to use inexpensive and common substances to stimulate microbial degradation of target pollutants, thereby achieving a green, efficient, and pollution-free process. The core of biostimulants is to use inexpensive and common substances to stimulate microbial degradation of target pollutants, thereby achieving a green, efficient, and pollution-free process. However, the production cost of some biostimulants is too high, and the cost of use in large-scale contaminated sites is also high. Therefore, there is a need to find economical biostimulants to reduce the cost of remediating contaminated sites. Summary of the Invention

[0004] The primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide a method for promoting bacterial degradation of benzo[a]pyrene by using at least one of sodium gluconate, sodium citrate, peanut cake powder and wheat bran fermentation liquid, and soybean cake powder and corn straw powder fermentation liquid as a biostimulant.

[0005] Another object of the present invention is to provide application of the above method in environmental remediation.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] A method for promoting bacterial degradation of benzo[a]pyrene by using a biostimulant comprises the following steps:

[0008] Adding biostimulants to the environment where benzo[a]pyrene exists to promote the degradation of benzo[a]pyrene by bacteria in the environment;

[0009] The biostimulant comprises at least one of sodium gluconate, sodium citrate, fermentation liquid of peanut cake powder and wheat bran, and fermentation liquid of soybean cake powder and corn stalk powder.

[0010] The preparation method of the fermentation liquid of described peanut cake powder and wheat bran is as follows:

[0011] EM fermentation bacteria liquid is added to peanut cake powder and wheat bran, and the mixture is dried after fermentation; the dried fermented product is mixed with water and shaken in a culture bottle, and the supernatant is collected after centrifugation to obtain the fermentation liquid of peanut cake powder and wheat bran.

[0012] The preparation method of the fermentation liquid of the soybean cake powder and corn straw powder is as follows:

[0013] EM fermentation bacteria liquid is added to soybean cake powder and corn straw powder, and the mixture is dried after fermentation; the dried fermented product is mixed with water and shaken in a culture bottle, and the supernatant is collected after centrifugation to obtain fermentation liquid of soybean cake powder and corn straw powder.

[0014] Preferably, the EM fermentation bacteria liquid is prepared by the following preparation method:

[0015] The EM fermentation bacteria, brown sugar and water are mixed, left to stand for activation, and then diluted to obtain the EM fermentation bacteria liquid.

[0016] Preferably, the mass ratio of the EM fermentation bacteria to brown sugar is 1:1.

[0017] Preferably, the volume ratio of the EM fermentation bacteria to water is 1:50.

[0018] Preferably, the static activation is activation by standing still for 24 hours.

[0019] Preferably, the dilution is to add 7 times the volume of water.

[0020] Preferably, the fermentation is carried out for 7 days.

[0021] Preferably, the drying is sun drying or oven drying at 70-100°C.

[0022] Preferably, the fermented product and water are mixed in a ratio of 1:1 by volume.

[0023] Preferably, the shaking is carried out on a shaker at a temperature of 30° C. and a rotation speed of 150 r for 24 h.

[0024] Preferably, when the environment is liquid, the amount of the biostimulant sodium gluconate added is: 1-4g sodium gluconate per 1L of liquid, the amount of the biostimulant sodium citrate added is: 2-6g sodium citrate per 1L of liquid, the amount of the biostimulant peanut cake powder and wheat bran fermentation liquid added is: 1% of the volume of the liquid, and the amount of the biostimulant soybean cake powder and corn straw powder fermentation liquid added is: 1% of the volume of the liquid; more preferably, the amount of the biostimulant sodium gluconate added is: 2g sodium gluconate per 1L of liquid, and the amount of the biostimulant sodium citrate added is: 4g sodium citrate per 1L of liquid.

[0025] When the environment is solid, the amount of the biostimulant sodium gluconate added is: 1-4g of sodium gluconate per 1kg of solid, the amount of the biostimulant sodium citrate added is: 2-6g of sodium citrate per 1kg of solid, the volume mass ratio of the fermentation liquid of the biostimulants peanut cake powder and wheat bran added to the solid is 10mL:100g, and the volume mass ratio of the fermentation liquid of the biostimulants soybean cake powder and corn straw powder added to the solid is 10mL:100g.

[0026] Preferably, the method for promoting the degradation of benzo[a]pyrene by bacteria using biostimulants further comprises the following steps: adding dispersed Pantoea into the environment where benzo[a]pyrene exists; (Pantoea dispersa MSC14, biostimulant to promote the dispersion of Pantoea ( Pantoea dispersa )MSC14 degrades benzo[a]pyrene in the environment.

[0027] Preferably, the degradation of benzo[a]pyrene is carried out at a temperature of 25 to 35° C. and a pH of 6.4 to 8.0; more preferably, the degradation is carried out at a temperature of 30° C. and a pH of 7.2.

[0028] The application of the above-mentioned method of using biostimulants to promote bacterial degradation of benzo[a]pyrene in environmental remediation.

[0029] Furthermore, the environment includes soil and water.

[0030] The present invention has the following advantages and effects compared to the prior art:

[0031] The present invention has discovered a new biostimulant comprising at least one of sodium gluconate, sodium citrate, fermentation broths of peanut meal and wheat bran, and fermentation broths of soybean meal and corn stalk meal. Adding the biostimulant to an environment containing benzo[a]pyrene can promote the degradation of benzo[a]pyrene by bacteria in the environment. The biostimulant is low-cost and highly effective.

[0032] The biostimulant of the present invention can also be used with dispersed Pantoea ( Pantoea dispersa )MSC14 were added to the environment where benzo[a]pyrene was present, and the biostimulant promoted the dispersion of Pantoea ( Pantoea dispersa )MSC14 degrades benzo[a]pyrene in the environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is a comparison chart of the degradation effect of benzo[a]pyrene by dispersed Pantoea MSC14 under the action of various biostimulants.

[0034] Figure 2 This is a comparison of the degradation effects of dispersed Pantoea MSC14 on benzo[a]pyrene under stimulation with different concentrations of sodium citrate.

[0035] Figure 3 This is a comparison of the degradation effects of dispersed Pantoea MSC14 on benzo[a]pyrene under stimulation of different concentrations of sodium gluconate.

[0036] Figure 4 This is a comparison chart of the degradation effect of Pantoea MSC14 on benzo[a]pyrene under the stimulation of fermentation broths of different agricultural and sideline products.

[0037] Figure 5 This is a picture of peanut cake flour and fermented wheat bran powder.

[0038] Figure 6 This is a powder image of fermented soybean cake powder and corn straw powder.

[0039] Figure 7 These are pictures of the fermentation liquid of peanut cake powder and wheat bran, as well as the fermentation liquid of soybean cake powder and corn straw powder.

[0040] Figure 8 This is a graph showing changes in cell membrane permeability after sodium gluconate and sodium citrate stimulated the degradation of benzo[a]pyrene by dispersed Pantoea MSC14.

[0041] Figure 9 This is a scanning electron micrograph of the degradation of benzo[a]pyrene by dispersed Pantoea stimulated by sodium citrate.

[0042] Figure 10 This is a scanning electron micrograph of the degradation of benzo[a]pyrene by dispersed Pantoea stimulated by sodium citrate.

[0043] Figure 11 This is a scanning electron micrograph of the degradation of benzo[a]pyrene by dispersed Pantoea stimulated by sodium gluconate.

[0044] Figure 12 This is a scanning electron micrograph of the degradation of benzo[a]pyrene by dispersed Pantoea stimulated by sodium gluconate.

[0045] Figure 13This is a transmission electron micrograph of the degradation of benzo[a]pyrene by dispersed Pantoea in an inorganic salt culture medium.

[0046] Figure 14 This is a transmission electron micrograph of the degradation of benzo[a]pyrene by dispersed Pantoea stimulated by sodium citrate.

[0047] Figure 15 This is a transmission electron micrograph of the degradation of benzo[a]pyrene by dispersed Pantoea stimulated by sodium gluconate.

[0048] Figure 16 This is a flow chart of the intermediate metabolites of benzo[a]pyrene degradation by dispersed Pantoea under the stimulation of sodium citrate or sodium gluconate.

[0049] Figure 17 This is a comparison chart of the effects of four biostimulants on stimulating soil microorganisms to degrade benzo[a]pyrene in the soil. DETAILED DESCRIPTION

[0050] The present invention will be described in further detail below in conjunction with Examples and accompanying drawings, but embodiments of the present invention are not limited thereto. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the art. Unless otherwise specified, the reagents and raw materials used in the present invention can be obtained commercially.

[0051] 1. Preparation of fermentation liquid of agricultural and sideline products

[0052] The agricultural and sideline products used in the fermented agricultural products are 30% carbon source and 70% nitrogen source by weight. Carbon sources include rice husk powder, rice straw powder, yellow bamboo powder, corn cob powder, rape straw powder, corn straw powder, and wheat bran. Nitrogen sources include bean dregs, soybean cake powder, peanut cake powder, corn steep liquor, cottonseed cake powder, and rapeseed cake. Agricultural and sideline products and EM bacteria are sourced from Qiangxing Bio's Qiangxing compost starter.

[0053] (1) Preparation of EM bacteria dilution: EM bacteria and brown sugar of equal weight are diluted with warm water (the mass volume ratio of EM bacteria to warm water is 1:50) and left to stand for 24 hours for activation to obtain EM activation solution. The EM activation solution is diluted with 7 times warm water to obtain EM dilution solution. To prepare EM activation solution, first dissolve brown sugar in a small amount of hot water, let it cool, mix it with EM bacteria, and then slowly pour it into water and stir thoroughly. The dilution solution should be prepared immediately before use.

[0054] (2) Mixing EM bacteria dilution and agricultural and sideline products: Mix EM bacteria dilution with agricultural and sideline products in a ratio of 65:100. If the moisture content is too low, add EM bacteria dilution. Mix the fermented materials thoroughly and then add water to mix well. The moisture content of the materials is generally controlled between 50% and 60%. The judgment method is: grab a handful of the mixed materials tightly. If water marks can be seen between the fingers but no water drips, and it falls apart when it is released, it is suitable. If water can be squeezed out and it falls to the ground without falling apart, the moisture content is greater than 75%. Too dry or too wet is not good and should be adjusted.

[0055] (3) Fermentation: After mixing well, put it into a fresh-keeping box. Leave some ventilation holes on the lid and ferment it at 30℃ for about 7 days. Open the fresh-keeping box every two or three days to stir the material until white mycelium appears. The fermented raw materials are dried in the sun and used as fermented agricultural and sideline products. Figure 5 and Figure 6 shown.

[0056] (4) Preparation of fermentation liquid of agricultural and sideline products: Take the fermentation liquid of agricultural and sideline products and ultrapure water in a weight-to-volume ratio of 1:1 and put them into a centrifuge tube. Shake them in a shaker at 30°C and 150r for 24h. After centrifugation, take the supernatant, which is the fermentation liquid of agricultural and sideline products. Prepare it and use it immediately. Figure 7 The supernatant is the fermentation broth.

[0057] 2. Preparation of culture medium used in the examples:

[0058] LB liquid medium: Weigh 10 g of peptone, 5 g of yeast powder, and 10 g of NaCl, and dissolve them in 1 L of ultrapure water.

[0059] Inorganic salt culture medium: weigh 1g (NH4)2SO4, 1g Na2HPO4, 0.2g KH2PO4, 0.2g MgSO4·7H2O, 0.005g FeCl3·3H2O, 0.001g (NH4)6Mo7O 24 ·4H2O and 0.1g CaCl2·2H2O, and dissolve in 1L ultrapure water, adjust the pH to 7.2; sterilize at 121℃.

[0060] Inorganic salt culture medium with biostimulants added: weigh 1g (NH4)2SO4, 1g Na2HPO4, 0.2g KH2PO4, 0.2g MgSO4·7H2O, 0.005g FeCl3·3H2O, 0.001g (NH4)6Mo7O 24 ·4H2O and 0.1g CaCl2·2H2O, and dissolved in 1L ultrapure water; adding biostimulant at amounts of 0.5, 1, 2, 4, 6, and 8g / L, and adjusting the pH to 7.2; sterilizing at 121°C to obtain inorganic salt culture media with the corresponding addition of 0.5, 1, 2, 4, 6, and 8g / L of biostimulant.

[0061] The biostimulants include sodium gluconate, glucose, sodium citrate, yeast powder, peptone, feather hydrolyzate and corn flour. Sodium gluconate, glucose, sodium citrate, yeast powder, peptone, feather hydrolyzate and corn flour were purchased from Maclean and were of analytical grade.

[0062] Inorganic salt culture medium with fermentation broth of agricultural and sideline products: weigh 1g (NH4)2SO4, 1g Na2HPO4, 0.2g KH2PO4, 0.2g MgSO4·7H2O, 0.005g FeCl3·3H2O, 0.001g (NH4)6Mo7O 24 ·4H2O and 0.1g CaCl2·2H2O, and dissolve in 1L ultrapure water; add agricultural and sideline product fermentation broth at 1% (v / v) and adjust the pH to 7.2; sterilize at 121°C to obtain an inorganic salt culture medium added with agricultural and sideline product fermentation broth.

[0063] 3. Benzo[a]pyrene content determination method: Dissolve a benzo[a]pyrene standard in acetone to prepare samples of varying concentrations. Detect the sample using HPLC and plot a standard curve. Extract the benzo[a]pyrene in the culture medium with dichloromethane and detect it using HPLC. Calculate the benzo[a]pyrene content in the culture medium by substituting the concentration into the standard curve.

[0064] 4. Calculation of degradation rate: Degradation rate on day n = (benzo[a]pyrene content of uninoculated control on day n - benzo[a]pyrene content of inoculated control on day n) / initial benzo[a]pyrene content on day 0

[0065] 5. Calculation of stimulation efficiency: stimulation efficiency = (degradation rate of benzo[a]pyrene with added biostimulant - degradation rate of benzo[a]pyrene in control) / degradation rate of benzo[a]pyrene in control

[0066] Example 1 Sodium citrate promotes the degradation of benzo[a]pyrene by dispersed Pantoea MSC14

[0067] Pantoea dispersa ( Pantoea dispersa )MSC14, with the deposit number GDMCC No: 62680, was deposited on August 3, 2022 at the Guangdong Provincial Microbial Culture Collection Center, Guangdong Provincial Institute of Microbiology, Building 59, 5th Floor, No. 100 Xianlie Middle Road, Guangzhou.

[0068] Single colonies of dispersed Pantoea MSC14 were picked and inoculated into LB liquid medium, cultured overnight on a shaker at 37°C and 180 rpm, and the bacteria were collected after centrifugation. The bacteria were washed three times with inorganic salt medium, and then the bacterial suspension was adjusted to OD 600= 1.0. The seed solution was inoculated into an inorganic salt medium supplemented with 1 g / L sodium citrate at a volume of 10% of the medium volume. The culture was incubated at 30°C, 150 rpm, and protected from light for 4 days. Three replicates were set for each single colony sample. The benzo[a]pyrene content after culture was determined, and the degradation rate was calculated.

[0069] Example 2 Sodium gluconate promotes the degradation of benzo[a]pyrene by dispersed Pantoea MSC14

[0070] Single colonies of dispersed Pantoea MSC14 were picked and inoculated into LB liquid medium, cultured overnight on a shaker at 37°C and 180 rpm, and the bacteria were collected after centrifugation. The bacteria were washed three times with inorganic salt medium, and then the bacterial suspension was adjusted to OD 600 = 1.0. The seed solution was inoculated into an inorganic salt medium supplemented with 1 g / L sodium gluconate at a volume of 10% of the medium volume. The culture was incubated at 30°C, 150 rpm, and protected from light for 4 days. Three replicates were set for each single colony sample. The benzo[a]pyrene content after culture was determined, and the degradation rate was calculated.

[0071] Comparative Example 1 Commercially available biostimulants promote the degradation of benzo[a]pyrene by dispersed Pantoea MSC14

[0072] Single colonies of dispersed Pantoea MSC14 were picked and inoculated into LB liquid medium, cultured overnight on a shaker at 37°C and 180 rpm, and the bacteria were collected after centrifugation. The bacteria were washed three times with inorganic salt medium, and then the bacterial suspension was adjusted to OD 600 =1.0 seed solution.

[0073] The seed solution was inoculated in an amount of 10% of the volume of the inorganic salt culture medium (as a blank control), an inorganic salt culture medium supplemented with 1 g / L glucose, an inorganic salt culture medium supplemented with 1 g / L yeast powder, an inorganic salt culture medium supplemented with 1 g / L peptone, an inorganic salt culture medium supplemented with 1 g / L feather hydrolyzate, and an inorganic salt culture medium supplemented with 1 g / L corn flour, respectively. The culture was carried out at 30°C, 150 r / min, and in the dark for 4 days. Three replicates were set for each single colony sample. The content of benzo[a]pyrene after culture was determined, and the degradation rate was calculated.

[0074] The results of Examples 1, 2 and Comparative Example 1 are as follows Figure 1 As shown, the degradation rates of sodium gluconate and sodium citrate were significantly improved compared with the blank control group. The improvement in degradation rate of sodium gluconate and sodium citrate used in Examples 1 and 2 was also significantly better than that of other biostimulants such as glucose, yeast powder, peptone, feather hydrolyzate and corn flour used in Comparative Example 1.

[0075] Example 3

[0076] The experimental steps were the same as in Example 1, except that the inorganic salt medium supplemented with 1 g / L sodium citrate was replaced with inorganic salt medium, inorganic salt medium supplemented with 0.5, 1, 2, 4, and 6 g / L sodium citrate. Figure 2 The optimal concentration of sodium citrate for stimulating Pantoea dispersa MSC14 to degrade benzo[a]pyrene was 1-4 g / L, with the optimal concentration being 2 g / L.

[0077] Example 4

[0078] The experimental steps were the same as in Example 2, except that the inorganic salt medium supplemented with 1 g / L sodium gluconate was replaced with inorganic salt medium, inorganic salt medium supplemented with 1, 2, 4, 6, and 8 g / L sodium gluconate, respectively. Figure 3 The optimal concentration of sodium gluconate for stimulating Pantoea dispersa MSC14 to degrade benzo[a]pyrene was 2-6 g / L, with the optimal concentration being 4 g / L.

[0079] Example 5 Peanut cake powder and wheat bran fermentation broth promotes the degradation of benzo[a]pyrene by dispersed Pantoea MSC14

[0080] The fermentation liquid of peanut cake powder and wheat bran was prepared by using 30% by mass of carbon source (wheat bran) and 70% by mass of nitrogen source (peanut cake powder) according to the preparation method of the fermentation liquid of agricultural and sideline products.

[0081] The experimental steps were the same as those in Example 1, except that the inorganic salt medium supplemented with 1 g / L sodium citrate was replaced with an inorganic salt medium supplemented with fermentation broth containing peanut meal and wheat bran.

[0082] Example 6: Fermentation broth of soybean cake powder and corn straw powder promotes the degradation of benzo[a]pyrene by dispersed Pantoea MSC14

[0083] A fermentation liquid of soybean cake powder and corn straw powder was prepared using 30% by mass of a carbon source (corn straw powder) and 70% by mass of a nitrogen source (soybean cake powder) according to the above-mentioned method for preparing a fermentation liquid of agricultural and sideline products.

[0084] The experimental steps were the same as those in Example 1, except that the inorganic salt medium supplemented with 1 g / L sodium citrate was replaced with an inorganic salt medium supplemented with fermentation broth containing soybean cake powder and corn stalk powder.

[0085] Comparative Example 2: Agricultural and sideline product fermentation broth promotes the degradation of benzo[a]pyrene by dispersed Pantoea MSC14

[0086] According to the above-mentioned method for preparing fermentation liquid of agricultural and sideline products, fermentation liquid of rapeseed cake and rice straw powder, fermentation liquid of cottonseed cake powder and rice straw powder, fermentation liquid of rapeseed cake and corn straw powder, fermentation liquid of cottonseed cake powder and corn straw powder, fermentation liquid of cottonseed cake powder and wheat bran, fermentation liquid of cottonseed cake powder and rice husk powder, fermentation liquid of soybean cake powder and rice straw powder, fermentation liquid of soybean cake powder and wheat bran, fermentation liquid of rapeseed cake and wheat bran, fermentation liquid of peanut cake powder and corn straw powder, fermentation liquid of peanut cake powder and rice husk powder, fermentation liquid of peanut cake powder and rice straw powder, and fermentation liquid of soybean cake powder and rice husk powder were prepared.

[0087] The experimental steps were the same as those in Example 1, except that the inorganic salt culture medium supplemented with 1 g / L sodium citrate was replaced with the inorganic salt culture medium supplemented with the above-mentioned agricultural and sideline product fermentation broth.

[0088] The results of Examples 5, 6 and Comparative Example 2 are as follows Figure 4 The fermentation liquids of peanut cake powder and wheat bran, as well as soybean cake powder and corn stalk powder, have significantly better promoting effects than the fermentation liquids of other agricultural and sideline products.

[0089] Example 7 Study on the Mechanism of Sodium Citrate and Sodium Gluconate Promoting Bacterial Degradation of Benzo[a]pyrene

[0090] (1) Effects of sodium citrate and sodium gluconate on the pH value of the culture medium

[0091] After four days of culture using the methods of Examples 1 and 2, a pH meter measured the pH of the culture medium supplemented with sodium citrate to be 8-9, while the pH of the culture medium supplemented with sodium gluconate was 4-5. While the use of sodium citrate and sodium gluconate as biostimulants increased the degradation rate of benzo[a]pyrene, the pH values of the culture medium in the two experimental groups were not similar, indicating that the two biostimulants did not promote the degradation of benzo[a]pyrene by regulating the pH of the environment.

[0092] (2) Effects of sodium citrate and sodium gluconate on cell membrane permeability

[0093] According to the inoculation method described in Example 1, dispersed Pantoea were cultured in an inorganic salt medium for 12 h. Subsequently, the cells were collected by centrifugation (6000×g) for 10 min, washed three times with 0.01 M PBS (pH 7.2), and resuspended (10 9CFU / mL) in an inorganic salt medium. 2 mL of bacterial suspension, 18 mL of biostimulant (2 g / L sodium citrate solution or 4 g / L sodium gluconate solution) and 1 mL of ONPG (30 mM) were mixed. Samples without sodium citrate or sodium gluconate concentrate (2 mL of bacterial suspension, 18 mL of ultrapure water and 1 mL of ONPG) were used as blank controls. After incubation at 30 ° C for 12 hours, the culture was centrifuged (12,000 × g) for 10 minutes, and the production of o-nitrophenol (ONP) was measured at 415 nm by a UV spectrophotometer. The release of β-galactosidase was evaluated based on the productivity of o-nitrophenol (ONP) and calculated using the following formula:

[0094]

[0095] where A415 (A415 = A415,i - A415,0) is the absorbance caused by the surfactant; ξ is the extinction coefficient of ONP (4.86 cm / mM); ν, ν0, and t are the volume of the experimental group (mL), the volume of the control group (mL), and the reaction time (h), respectively; and d is the optical path length of the cuvette (cm).

[0096] The results are as follows Figure 8 As shown, after sodium citrate and sodium gluconate stimulated the degradation of benzo[a]pyrene in Pantoea dispersa, cell membrane permeability increased. Sodium citrate had a stronger effect on Pantoea dispersa cell membrane permeability than sodium gluconate. This enhanced cell membrane permeability facilitates the entry of benzo[a]pyrene into the bacterial cell membrane, enabling the bacteria to utilize benzo[a]pyrene more quickly, thus achieving the desired degradation effect.

[0097] (3) Effects of sodium citrate and sodium gluconate on bacterial morphology

[0098] After culturing according to Examples 1 and 2, the cells were centrifuged at 4000 rpm and 4°C for 10 minutes, washed twice with 0.01M PBS (pH 7.2), fixed for 24 hours with 2.5% glutaraldehyde (v / v) electron microscopy fixative, and then rinsed 2-3 times with 0.01M PBS (pH 7.2). Dehydrated for 5 minutes with 40%, 70%, 90%, and 100% ethanol, respectively. After dehydration, the cells were replaced with a 1:1 ethanol:tert-butanol (v / v) solution and then 100% tert-butanol solution for 20 minutes. The sample was added dropwise onto a 5*5mm coverslip, frozen at -80°C, and freeze-dried for at least 24 hours. The sample was placed in a vacuum coating machine, gold was spray-coated on the sample surface, and then the sample was removed and observed under a scanning electron microscope.

[0099] The scanning electron microscopy results of bacteria after sodium citrate stimulation are as follows Figure 9 and Figure 10 As shown in the figure, the SEM results of bacteria after sodium gluconate stimulation are as follows Figure 11 and Figure 12 As shown, under stimulation with sodium citrate and sodium gluconate, bacteria aggregated. This agglomeration is caused by biofilm formation, and the absence of flagella facilitates aggregation and biofilm formation. Low nutrient levels or toxic stress enhance bacterial attachment and biofilm formation, while also increasing bacterial survival within the biofilm. Aggregate formation helps bacteria withstand environmental stress. Scanning electron micrographs show numerous white "threads," representing bacterial pili. These pili are found in a small number of Gram-negative bacteria. Once a bacterium develops drug resistance, it can use these pili to transfer DNA to other individuals, thereby conferring drug resistance and rapidly increasing the number of bacteria that develop resistance. This explains why Pantoea dispersa can rapidly adapt to environments containing benzo[a]pyrene and degrade it under stimulation with sodium citrate and sodium gluconate.

[0100] (4) Effects of sodium citrate and sodium gluconate on benzo[a]pyrene transport

[0101] After culturing according to the blank control group of Examples 1 and 2 and Comparative Example 1, the cells were centrifuged at 4000 r / m for 10 min at 4°C, washed twice with 0.01M PBS (pH 7.2), fixed for 24 h with 2.5% glutaraldehyde electron microscopy fixative, and then rinsed 2-3 times with 0.01M PBS (pH 7.2). The cells were centrifuged at 4000 r / m for 10 min, and the cells were collected and suspended in 0.01M PBS (pH 7.2). The cell concentration was adjusted to 10 8 ~10 9 cfu / ml. Mix equal amounts of bacterial suspension with equal amounts of 2% sodium phosphotungstate aqueous solution to create a mixed bacterial suspension. Use a pipette tip to draw up the mixed bacterial suspension and drop it onto a copper mesh.

[0102] After fixing for 3 to 5 minutes, remove the remaining water with filter paper. After the sample is dry, observe it under a transmission electron microscope.

[0103] The results of the control group (no biostimulant added) are as follows Figure 13 As shown, the results of sodium citrate stimulation are as follows Figure 14 As shown, the results of sodium gluconate stimulation are as follows Figure 15 As shown, sodium citrate and sodium gluconate stimulated the cells to produce black granular material, which is benzo[a]pyrene added to the culture medium. This indicates that the biostimulants enabled the bacteria to transport benzo[a]pyrene into the body, thereby achieving degradation. The control group showed less black granular material, suggesting that sodium citrate and sodium gluconate enhance the permeability of bacterial cell membranes to benzo[a]pyrene particles.

[0104] (5) Bacterial degradation and transport pathways of benzo[a]pyrene

[0105] After culturing according to Examples 1 and 2, the culture medium was acidified to pH 2.0 with hydrochloric acid, and then extracted with ethyl acetate. After removing moisture with anhydrous sodium sulfate, the ethyl acetate was evaporated with a rotary evaporator, and 2 ml of n-hexane was added to dissolve benzo[a]pyrene. The sample was then filtered through an organic filter membrane to prepare a sample, and the sample components were detected by gas chromatography-mass spectrometry. Upon testing, the samples contained substances such as benzanthracene, anthracene, pyrene, phenanthrene, and phthalic acid derivatives. Therefore, under the action of the biostimulant, there are two possible pathways for the degradation of benzo[a]pyrene by dispersed Pantoea MSC14: 1. Benzo[a]pyrene is degraded to pyrene, then to phenanthrene, and then to phthalic acid, entering the tricarboxylic acid cycle. 2. Benzo[a]pyrene is degraded to benzanthracene, then to anthracene, and then to phthalic acid, entering the tricarboxylic acid cycle. The metabolic flow chart is as follows: Figure 16 shown.

[0106] Example 12: Effects of Four Biostimulants on the Degradation of Benzo[a]pyrene by Soil Bacteria in Benzo[a]pyrene-Contaminated Soil

[0107] Soil was collected from farmlands, parks, flower beds, and grasslands, air-dried in the shade, and then passed through a 100-mesh sieve after removing stones and fallen leaves. Mix thoroughly, then add a solution of benzo[a]pyrene in acetone to the soil and stir thoroughly to achieve an initial benzo[a]pyrene concentration of 30 mg / kg. Five equal portions of benzo[a]pyrene-contaminated soil were collected: one portion served as a control, and the other four portions were supplemented with 2 g / kg sodium citrate, 4 g / kg sodium gluconate, 10% (mL / g) peanut meal and wheat bran fermentation liquid, and 10% (mL / g) soybean meal and corn stalk fermentation liquid, respectively. The five soil portions were maintained at a moisture content of 30%. The five soil portions were incubated in a 30°C incubator in the dark for 7 days. After incubation, the benzo[a]pyrene was extracted with dichloromethane, and the benzo[a]pyrene content was determined by high-performance liquid chromatography (HPLC), and the degradation rate was calculated.

[0108] Effects of four biostimulants on the degradation of benzo[a]pyrene by soil microorganisms in nature Figure 17 As shown in the results, compared with the control, the four biostimulants have a promoting effect on the degradation of benzo[a]pyrene by other microorganisms in the natural environment. Therefore, these four biostimulants can be applied to actual contaminated soil sites to repair benzo[a]pyrene-contaminated soil.

[0109] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. Biostimulants promote the dispersion of Pantoea ( Pantoea dispersa ) A method for degrading benzo[a]pyrene using MSC14, characterized in that: The following steps are involved: Adding biostimulants and dispersed Pantoea to the environment where benzo[a]pyrene is present Pantoea dispersa MSC14, biostimulant to promote the dispersion of Pantoea ( Pantoea dispersa MSC14 degrades benzo[a]pyrene; The biostimulant is at least one of a fermentation liquid of peanut meal and wheat bran, a fermentation liquid of soybean meal and corn stalk meal, sodium gluconate and sodium citrate; The dispersed Pantoea ( Pantoea dispersa )MSC14, deposit number is GDMCC No: 62680, and was deposited on August 3, 2022 at the Guangdong Provincial Microbial Culture Collection Center, Guangdong Provincial Institute of Microbiology, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

2. The method according to claim 1, wherein: The preparation method of the fermentation liquid of described peanut cake powder and wheat bran is as follows: Adding EM fermentation bacteria liquid to peanut cake powder and wheat bran, fermenting and drying; mixing the dried fermented product with water and shaking in a culture bottle, centrifuging and collecting the supernatant to obtain the fermentation liquid of peanut cake powder and wheat bran; The preparation method of the fermentation liquid of soybean cake powder and corn straw powder is as follows: EM fermentation bacteria liquid is added to soybean cake powder and corn straw powder, and the mixture is dried after fermentation; the dried fermented product is mixed with water and shaken in a culture bottle, and the supernatant is collected after centrifugation to obtain fermentation liquid of soybean cake powder and corn straw powder.

3. The method according to claim 2, wherein: The EM fermentation bacteria liquid is prepared by the following preparation method: After mixing EM fermentation bacteria, brown sugar and water, the mixture is left to stand for activation, and then diluted to obtain EM fermentation bacteria liquid; The mass ratio of the EM fermentation bacteria to brown sugar is 1:1; The volume ratio of the EM fermentation bacteria to water is 1:50; The static activation is activated by standing for 24 hours; The dilution is to add 7 times the volume of water; The fermentation lasts for 7 days.

4. The method according to claim 2, wherein: The drying is sun drying or oven drying at 70-100°C; The fermented product and water are mixed in a ratio of 1:1 by volume; The shaking is carried out on a shaker at a temperature of 30° C. and a rotation speed of 150 r for 24 h.

5. The method according to claim 1, wherein: When the environment is liquid, the amount of the biostimulant sodium gluconate added is: 1-4g of sodium gluconate per 1L of liquid, the amount of the biostimulant sodium citrate added is: 2-6g of sodium citrate per 1L of liquid, the amount of the biostimulant peanut cake powder and wheat bran fermentation liquid added is: 1% of the volume of the liquid, and the amount of the biostimulant soybean cake powder and corn straw powder fermentation liquid added is: 1% of the volume of the liquid; When the environment is solid, the amount of the biostimulant sodium gluconate added is: 1-4 g of sodium gluconate per 1 kg of solid, the amount of the biostimulant sodium citrate added is: 2-6 g of sodium citrate per 1 kg of solid, the volume mass ratio of the fermentation liquid of the biostimulants peanut cake powder and wheat bran to the solid is 10 mL:100 g, and the volume mass ratio of the fermentation liquid of the biostimulants soybean cake powder and corn straw powder to the solid is 10 mL:100 g; The benzo[a]pyrene degradation process is carried out under the conditions of a temperature of 25 to 35° C. and a pH value of 6.4 to 8.

0.

6. The method according to claim 5, characterized in that: When the environment is liquid, 2 g of sodium gluconate is added per 1 L of liquid, and the amount of the biostimulant sodium citrate added is: 4 g of sodium citrate is added per 1 L of liquid; When the environment is solid, the amount of the biostimulant sodium gluconate added is: 2 g of sodium gluconate per 1 kg of solid, and the amount of the biostimulant sodium citrate added is: 4 g of sodium citrate per 1 kg of solid; The benzo[a]pyrene degradation is carried out under the conditions of a temperature of 30° C. and a pH value of 7.

2.

7. The biostimulant according to any one of claims 1 to 6 promotes the dispersion of Pantoea ( Pantoea dispersa ) Application of the method for degrading benzo[a]pyrene by MSC14 in environmental remediation, characterized in that: The dispersed Pantoea ( Pantoea dispersa )MSC14, deposit number is GDMCC No: 62680, and was deposited on August 3, 2022 at the Guangdong Provincial Microbial Culture Collection Center, Guangdong Provincial Institute of Microbiology, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

8. The use according to claim 7, characterized in that: The environment is soil or water.

Images