Preparation method and application of biochar-based microbial inoculum for combined reduction of estrogen

Abstract

The invention provides a preparation method and application of a biochar-based microbial agent for combined reduction of estrogens, belongs to the technical field of microorganisms, and can efficiently degrade various environmental estrogens in a pollution medium environment with various environmental estrogens. According to the preparation method, a charcoal-based microbial inoculum containing sphingomonas JY-1, rhodococcus JY-2 and neo-sphingobacterium is prepared and is used for degrading environmental estrogen in a pollution medium; wherein the preservation number of the sphingomonas sp. JY-1 is CGMCC (China General Microbiological Culture Collection Center) No.33550; the preservation number of the rhodococcus sp. JY-2 is CGMCC (China General Microbiological Culture Collection Center)

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to a preparation method and application of a biochar-based bacterial agent for combined estrogen reduction. Background Art

[0002] Environmental estrogens, as a new type of pollutant, have endocrine disrupting effects. -1 Concentrations of this level can interfere with the endocrine system and nervous system of fish and amphibians and even cause reproductive disorders, leading to species extinction. Livestock and poultry manure is the environmental source of natural steroidal estrogens. As the leachate from the pile of livestock and poultry manure flows downward, some estrogens are adsorbed by the soil and some pollute groundwater. The estrogen remaining in the soil will be absorbed by crops and vegetables, threatening human health and ecological balance.

[0003] In the prior art, microbial technology is often used to repair water bodies and soil contaminated by environmental estrogens. Usually, estrogen-degrading strains or degrading bacterial communities are isolated from contaminated sites, and environmental pressure is continuously applied for domestication, and finally bacteria with estrogen-degrading functions are obtained. However, when there are multiple environmental estrogens in the actual application scenario, the application of functional bacterial communities is often difficult to adapt to complex environmental conditions, and in the face of multiple substrates, the degradation effect of estrogen is far less than the initial effect of separation. Summary of the invention

[0004] The present invention proposes a method for preparing and applying a biochar-based bacterial agent for combined estrogen reduction. The estrogen-degrading bacterial community fixed in the prepared biochar-based bacterial agent includes Sphingomonas JY-1, Rhodococcus JY-2 and Neosphingobacillus, which can degrade a variety of environmental estrogens more efficiently in a polluted medium environment where a variety of environmental estrogens exist.

[0005] In order to achieve the above object, the present invention adopts the following technical solution: In a first aspect, the present invention provides a method for preparing a biochar-based bacterial agent for combined estrogen reduction, comprising: An estrogen-degrading bacterial flora is prepared, wherein the estrogen-degrading bacterial flora includes Sphingomonas JY-1, Rhodococcus JY-2 and Neosphingobacterium; the preservation number of Sphingomonas JY-1 is CGMCC No.33550; the preservation number of Rhodococcus JY-2 is CGMCC No.33549; preparing biochar base; The estrogen-degrading bacterial community is fixed on the biochar base to obtain a biochar-based bacterial agent that can jointly reduce estrogen.

[0006] In one implementation of the first aspect, preparing an estrogen-degrading bacterial flora comprises: Activating and culturing Sphingomonas JY-1, Rhodococcus JY-2 and Neosphingobacterium, respectively, to obtain bacterial liquid of Sphingomonas JY-1, bacterial liquid of Rhodococcus JY-2 and bacterial liquid of Neosphingobacterium; The bacterial liquid of Sphingomonas JY-1, the bacterial liquid of Rhodococcus JY-2 and the bacterial liquid of Neosphingobacterium were mixed to obtain an estrogen-degrading bacterial flora.

[0007] In an implementation of the first aspect, Sphingomonas JY-1, Rhodococcus JY-2, and Neosphingobacterium are activated and cultured respectively, including: Sphingomonas JY-1 and Rhodococcus JY-2 were inoculated into LB medium for activation and cultured until OD 600 =1.0, to obtain the pre-prepared bacterial solution of Sphingomonas JY-1 and the pre-prepared bacterial solution of Rhodococcus JY-2; The new sphingobacterium was inoculated into R2A medium for activation and cultured until the OD of R2A medium was 600 = 1.0, to obtain the pre-prepared bacterial solution of Neosphingobacterium; The pre-prepared bacterial solution of Sphingomonas JY-1, the pre-prepared bacterial solution of Rhodococcus JY-2 and the pre-prepared bacterial solution of Neosphingobacterium were washed and resuspended to an OD of 600 =1.0, the bacterial solution of Sphingomonas JY-1, the bacterial solution of Rhodococcus JY-2 and the bacterial solution of Neosphingobacterium were obtained; The formula of LB medium is: yeast extract 10 g·L -1 , Tryptone 10 g·L -1 , sodium chloride 10g·L -1 ; The formula of R2A medium is: yeast extract powder 0.5 g·L -1 , peptone 0.5 g·L -1 , Acid hydrolyzed casein 0.5g·L -1 , glucose 0.5 g·L -1 , soluble starch 0.5 g·L -1 , sodium pyruvate 0.3 g·L -1 , Potassium hydrogen phosphate 0.3 g·L -1 , anhydrous magnesium sulfate 0.024 g·L -1 ; The formula of inorganic salt culture medium is: ammonium nitrate 1.5 g·L -1 , potassium hydrogen sulfate 1.9 g·L -1 , potassium dihydrogen sulfate 0.5 g·L -1 , sodium chloride 0.5 g·L -1 , magnesium sulfate heptahydrate 0.2 g·L -1 .

[0008] In an implementation of the first aspect, the volume mixing ratio of the bacterial solution of Sphingomonas JY-1, the bacterial solution of Rhodococcus JY-2, and the bacterial solution of Neosphingobacterium is: 1~1.5:1~1.5:1~2.

[0009] In an implementation of the first aspect, preparing a biochar base includes: pyrolyzing a plant material to obtain a biochar base.

[0010] In an implementation of the first aspect, the ratio of the estrogen-degrading bacteria to the biochar-based fixation bacteria is 10 mL to 30 mL: 1 g.

[0011] In one implementation of the first aspect, the effective viable bacteria count of the combined estrogen-reducing biochar-based bacterial agent is 5.4×10 9 CFU / g ~ 9.6×10 9 CFU / g.

[0012] In a second aspect, the present invention provides the use of the biochar-based bacterial agent for combined estrogen reduction prepared by the method provided in the first aspect in removing environmental estrogens in a contaminated medium.

[0013] In an implementation of the second aspect, the polluted medium is sewage, contaminated soil or livestock and poultry manure.

[0014] In an implementation of the second aspect, the environmental estrogen includes one or more of estriol, estradiol and estrone.

[0015] In one implementation of the second aspect, the application includes: adding a biochar-based bacterial agent for combined estrogen reduction into a polluted medium to degrade environmental estrogens in the polluted medium.

[0016] In an implementation of the second aspect, the input amount of the combined estrogen-reducing biochar-based bacterial agent is 3% to 5% of the mass of the contaminated medium.

[0017] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention not only provides a biochar-based bacterial agent capable of reducing multiple natural estrogens, but can also effectively combine biochar with microbial remediation technology, especially having a good remediation effect on water bodies and livestock and poultry manure contaminated by multiple environmental estrogens; (2) The present invention uses biochar as a carrier to fix the estrogen-degrading functional bacteria on the biochar. Compared with free bacteria, the estrogen degradation effect is better and the pollutant residue is lower. The biochar-based bacterial agent can efficiently degrade a variety of natural estrogens in polluted water and livestock and poultry manure, reduce the ecological risks of residual pollutants, and protect human health. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is a schematic diagram of the removal rate of environmental estrogens in the experimental group, the control group 1 and the control group 2 in Example 16 of the present application; Figure 2 It is a schematic diagram of the removal rate of environmental estrogens in the experimental group, the control group 1 and the control group 2 in Example 17 of the present application; Figure 3 This is a schematic diagram of the removal rate of environmental estrogens by the biochar-based bacterial agent prepared from different plant materials in Experimental Example 1 of the present application; Figure 4 This is a scanning electron microscope image of the biochar-based bacterial agent prepared in Example 1 of the present application; Figure 5 This is a scanning electron microscope image of the biochar-based bacterial agent prepared in Example 2 of the present application; Figure 6 This is a scanning electron microscope image of the biochar-based bacterial agent prepared in Example 3 of the present application; Figure 7 This is a scanning electron microscope image of the biochar-based bacterial agent prepared in Example 4 of the present application; Figure 8 This is a schematic diagram of the removal rate of environmental estrogens by the biochar-based bacterial agent prepared with different bacterial-to-material ratios in Experimental Example 2 of the present application; Fig. 9 It is a schematic diagram of the removal rate of environmental estrogens by the biochar-based bacterial agent prepared with different particle sizes in Experimental Example 3 of the present application; Fig.10 It is a schematic diagram of the removal rate of environmental estrogens by the biochar-based bacterial agent prepared at different fixed times in Experimental Example 4 of the present application; Fig.11 This is a schematic diagram of the removal rate of environmental estrogens by the biochar-based bacterial agent prepared at different fixed temperatures in Experimental Example 4 of the present application. DETAILED DESCRIPTION

[0019] Example 1: This example describes a method for preparing a biochar-based bacterial agent for combined estrogen reduction, including S1-S3.

[0020] S1. preparing estrogen-degrading bacterial flora; The estrogen-degrading bacterial community included Sphingomonas JY-1, Rhodococcus JY-2, and Neosphingobacterium; Among them, Sphingomonas JY-1 and Rhodococcus JY-2 are two new strains of bacteria newly obtained in this application; the above-mentioned new Sphingobacterium adopts existing bacteria; illustratively, the above-mentioned new Sphingobacterium is new Sphingobacterium ES2-1; The above-mentioned Sphingomonas JY-1 is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration Committee, and the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is February 20, 2025. The deposit number given by the depository is CGMCC No. 33550. The classification name of the above-mentioned Sphingomonas JY-1 (also known as biological material) is Sphingomonas, and the Latin name of the classification name is Sphingomonas sp. ; The above-mentioned Rhodococcus JY-2 is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration Committee, and the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. The deposit date is February 20, 2025. The deposit number given by the depository is CGMCC No. 33549. The classification name of the above-mentioned Rhodococcus JY-2 (also known as biological material) is Rhodococcus, and the Latin name of the classification name is Rhodococcus sp .; The above-mentioned new sphingobacterium ES2-1 is deposited in the General Microbiology Center of China Microbiological Culture Collection Administration Committee, and the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. The deposit date is September 1, 2016. The deposit number given by the depository is CGMCC No.12925. The above-mentioned new sphingobacterium ES2-1 is classified and named Novosphingobium Subterraneum The Latin name of this classification is Novosphingobium subterraneum ; The enrichment culture process of Sphingomonas JY-1, Rhodococcus JY-2, and Neosphingobacterium ES2-1 is described in detail below; Taking estriol, estradiol and estrone as target pollutants, activated sludge samples were inoculated with 20 mg·L -1 After 7 days of enrichment culture at 30°C and 150 rpm in an inorganic salt medium containing estriol, estradiol, and estrone, 10% (v / v) of the enrichment culture medium was aspirated and transferred to a fresh medium containing 20 mg·L -1 The culture medium containing estriol, estradiol and estrone was cultured again under the same environmental conditions for 7 days. After three consecutive enrichment cultures, the concentration of remaining natural estrogen in the enrichment solution was determined to preliminarily determine whether there were natural estrogen-degrading bacteria in the enrichment culture solution. 1 mL of the enrichment culture solution that may contain degrading bacteria was taken and concentrated by 10 -1 -10 -6 Continuous dilution, the dilution factor is 10 -3 Up to 10 -6 The dilutions (100 µL each) were evenly spread on the -1 Estriol, estradiol, and estrone were cultured on inorganic salt solid plates at 30°C for more than 36 hours; All colonies with different morphologies were selected, and they were repeatedly streaked and purified for at least three generations. Single colonies were obtained as suspected natural estrogen-degrading bacteria. Subsequently, each single colony was inoculated into R2A or LB liquid culture medium for expansion culture to prepare bacterial suspension. The specific preparation steps were as follows: the proliferation culture of each strain was centrifuged at 6,000 rpm for 10 min, the supernatant was discarded, and the precipitated bacteria were resuspended with fresh inorganic salt culture medium (or sterile water), and centrifuged again under the same conditions; the above steps were repeated twice, and the bacteria were resuspended with fresh culture medium and the bacterial suspension (OD 600 ) The absorbance was adjusted to 1 ± 0.1 for later use; the bacterial suspension corresponding to each strain was inoculated with 20 mL of 20 mg·L −1 After culturing for 7 days in an inorganic salt medium containing estriol, estradiol and estrone at 150 rpm and 30°C, an equal volume of methanol was added to the solution, ultrasonicated for 30 minutes, filtered through a 0.22 μm organic phase filter, and the residual concentrations of estriol, estradiol and estrone in the solution were detected by liquid chromatography, and the strain with the highest degradation efficiency was selected as the natural estrogen-degrading functional microorganism; finally, Sphingomonas JY-1 and Rhodococcus JY-2 with estradiol degradation ability and Neosphingobacterium with estriol, estradiol and estrone degradation ability were isolated; In this embodiment, an estrogen-degrading bacterial flora is prepared, including S11-S12; S11, activating and culturing Sphingomonas JY-1, Rhodococcus JY-2 and Neosphingobacterium, respectively, to obtain bacterial liquid of Sphingomonas JY-1, bacterial liquid of Rhodococcus JY-2 and bacterial liquid of Neosphingobacterium; Specifically, S11 includes the following contents: Sphingomonas JY-1 and Rhodococcus JY-2 were inoculated into LB medium for activation and cultured until OD 600 =1.0, to obtain the pre-prepared bacterial solution of Sphingomonas JY-1 and the pre-prepared bacterial solution of Rhodococcus JY-2; The new sphingobacterium was inoculated into R2A medium for activation and cultured until the OD of R2A medium was 600 = 1.0, to obtain the pre-prepared bacterial solution of Neosphingobacterium; The pre-prepared bacterial solution of Sphingomonas JY-1, the pre-prepared bacterial solution of Rhodococcus JY-2 and the pre-prepared bacterial solution of Neosphingobacterium were washed and resuspended to an OD of 600 =1.0, the bacterial solution of Sphingomonas JY-1, the bacterial solution of Rhodococcus JY-2 and the bacterial solution of Neosphingobacterium were obtained; The formula of LB medium is: yeast extract 10 g·L -1 , Tryptone 10 g·L-1 , sodium chloride 10g·L -1 ; The formula of R2A medium is: yeast extract powder 0.5 g·L -1 , peptone 0.5 g·L -1 , Acid hydrolyzed casein 0.5g·L -1 , glucose 0.5 g·L -1 , soluble starch 0.5 g·L -1 , sodium pyruvate 0.3 g·L -1 , Potassium hydrogen phosphate 0.3 g·L -1 , anhydrous magnesium sulfate 0.024 g·L -1 ; The formula of inorganic salt culture medium is: ammonium nitrate 1.5 g·L -1 , potassium hydrogen sulfate 1.9 g·L -1 , potassium dihydrogen sulfate 0.5 g·L -1 , sodium chloride 0.5 g·L -1 , magnesium sulfate heptahydrate 0.2 g·L -1 ; S12, mixing the bacterial solution of Sphingomonas JY-1, the bacterial solution of Rhodococcus JY-2 and the bacterial solution of Neosphingobacterium to obtain an estrogen-degrading bacterial flora; wherein the volume mixing ratio of the bacterial solution of Sphingomonas JY-1, the bacterial solution of Rhodococcus JY-2 and the bacterial solution of Neosphingobacterium is 1:1:1; S2, preparing biochar base; S3, fixing the estrogen-degrading bacteria on the biochar base to obtain a biochar-based bacterial agent for combined estrogen reduction; wherein the bacterial material ratio of the estrogen-degrading bacteria to the biochar base for fixation is 20 mL: 1 g; Specifically, S3 includes S31-S33; S31, passing the biochar obtained in S2 through a 60-mesh sieve to obtain a biochar with a particle size of 60 mesh; S32, add estrogen-degrading bacteria into a conical flask at a bacterial-to-material ratio of 1 g biochar: 20 mL bacterial solution, fix the rotation speed at 150 rpm, the fixed temperature at 30 °C, and the fixed time at 3 days during the culture process; S33. After fixation, centrifuge at 10,000 rpm, pour off the supernatant, and dry the precipitate at 30°C aseptically to obtain a biochar-based bacterial agent for combined estrogen reduction.

[0021] Example 2: The difference between this example and Example 1 is that in S2, the pyrolysis material is corn stalks.

[0022] Example 3: The difference between this example and example 1 is that in S2, the pyrolysis material is coconut shell.

[0023] Example 4: The difference between this example and Example 1 is that in S2, the pyrolysis material is reed.

[0024] Example 5: The difference between this example and Example 1 is that in S3, the ratio of the estrogen-degrading bacteria to the biochar-based fixation bacteria is 1 g: 10 mL.

[0025] Example 6: The difference between this example and Example 1 is that in S3, the ratio of the estrogen-degrading bacteria to the biochar-based fixation bacteria is 1 g: 30 mL.

[0026] Example 7: The difference between this example and Example 1 is that in S31, the biochar is sieved through a 100-mesh sieve.

[0027] Example 8: The difference between this example and Example 1 is that in S31, the biochar is sieved through a 20-mesh sieve.

[0028] Embodiment 9: The difference between this embodiment and embodiment 1 is that in S32, the fixed time is 1 day.

[0029] Embodiment 10: The difference between this embodiment and embodiment 1 is that in S32, the fixed time is 2 days.

[0030] Embodiment 11: The difference between this embodiment and embodiment 1 is that in S32, the fixed temperature is 40°C.

[0031] Embodiment 12: The difference between this embodiment and embodiment 1 is that in S33, the fixed temperature is 50°C.

[0032] Example 13: The difference between this example and Example 1 is that in S12, the volume mixing ratio of the bacterial solution of Sphingomonas JY-1, the bacterial solution of Rhodococcus JY-2 and the bacterial solution of Neosphingobacterium is 1.25:1.25:1.5.

[0033] Example 14: The difference between this example and Example 1 is that in S12, the volume mixing ratio of the bacterial solution of Sphingomonas JY-1, the bacterial solution of Rhodococcus JY-2 and the bacterial solution of Neosphingobacterium is 1.5:1.5:2.

[0034] Example 15: This example is an application of a biochar-based bacterial agent for combined estrogen reduction. The biochar-based bacterial agent prepared by the method provided in Example 1 is used to degrade environmental estrogens in polluted media; It can be understood that the polluted medium can be sewage, or polluted soil or livestock and poultry manure; environmental estrogens include one or more of estriol, estradiol and estrone; the application method can be: adding the biochar-based bacterial agent for combined estrogen reduction into the polluted medium to degrade the environmental estrogens in the polluted medium; environmental estrogens can include one or more of estriol, estradiol and estrone; Specifically, in this embodiment, sewage was selected as the contaminated medium, and the biochar-based bacterial agent prepared in Example 1 was added to the contaminated medium at a dosage of 5% by mass, and cultured at 150 rpm and 30° C. for 5 days; On this basis, two control groups (control group 1 and control group 2) are also set up in this embodiment; wherein, the difference between control group 1 and the above application is that the free estrogen-degrading bacterial community obtained by configuring S1 in embodiment 1 is added to the contaminated medium at a dosage of 5% by mass; the difference between control group 2 and the above application is that the biochar obtained by S2 in embodiment 1 is added to the contaminated medium at a dosage of 5% by mass; The above application was used as the experimental group. After 5 days, the content of environmental estrogen in the experimental group, control group 1 and control group 2 was measured, and the removal rate of environmental estrogen in the experimental group, control group 1 and control group 2 was calculated as follows: Figure 1 As shown; wherein, E3 represents estriol, E2 represents estradiol, and E1 represents estrone; The method for determining the content of the above environmental estrogens is as follows: add 40 mL of chromatographic pure methanol to 20 mL of sample, ultrasonically vibrate for 30 min, filter through a 0.22 μm organic phase filter, and store in a brown liquid phase vial at -20°C; the liquid phase detection conditions are as follows: Inertsil ODS-SP-C 18 Reversed phase column (120 mm × 4.6 mm, 5 μm), mobile phase acetonitrile: water = 40:60, injection volume 20 μL, flow rate 0.8 mL min -1 , column temperature was 40°C; fluorescence detector excitation wavelength: 280 nm, emission wavelength: 310 nm.

[0035] Depend on Figure 1 It can be seen that the experimental group, control group 1 and control group 2 can all reduce environmental estrogens in the water body, especially the addition of biochar-based bacterial agents in the experimental group has a more prominent degradation effect on the three natural estrogens. It is speculated that biochar first adsorbs estrogens in the water body, some estrogens are adsorbed on the surface of biochar, and some estrogens are bound by biochar. At the same time, some free bacteria loaded on the biochar base are slowly released in the water to degrade estrogens, and the biofilm formed on the biochar also plays an adsorption and degradation role.

[0036] Example 16: This example is an application of a biochar-based bacterial agent for combined estrogen reduction. The biochar-based bacterial agent prepared by the method provided in Example 1 is used to degrade environmental estrogens in polluted media; Specifically, in this example, livestock and poultry manure was selected as the contaminated medium, and the biochar-based bacterial agent prepared in Example 1 was added to the contaminated medium at a dosage of 5%. After being fully stirred, it was stored at 30° C. and protected from light. Sterile water was sprayed every 4 days until the surface was moistened and the compost was fully turned to maintain an aerobic state. Samples were taken after 28 days. On this basis, two control groups (control group 1 and control group 2) are also set up in this embodiment; wherein, the difference between control group 1 and the above application is that the free estrogen-degrading bacterial community obtained by configuring S1 in embodiment 1 is added to the contaminated medium at a dosage of 5%; the difference between control group 2 and the above application is that the biochar obtained by S2 in embodiment 1 is added to the contaminated medium at a dosage of 5%; The above application was used as the experimental group. After 28 days, the content of environmental estrogens in the experimental group, control group 1 and control group 2 was measured, and the removal rates of environmental estrogens in the experimental group, control group 1 and control group 2 were calculated as follows: Figure 2 As shown; Determination of estrogen in livestock and poultry feces: Accurately weigh 1.00 g of freeze-dried sample, add 20 mL of ethyl acetate, and ultrasonically vibrate for 30 min at 4000 r / min. -1 Centrifuge for 20 min, transfer the supernatant to a clean headspace bottle, repeat the operation once, combine the two extracts, blow nitrogen to dryness, reconstitute with 1 mL of methanol, add pure water to make up to 20 mL, and then pass through C 18 The solid phase extraction column was eluted with 20 mL of a mixture of methanol and ethyl acetate in a ratio of 1:1, blown to dryness with nitrogen, and finally re-dissolved with 1 mL of methanol and filtered through a 0.22 μm organic phase filter membrane, and stored in a brown liquid phase vial at -20°C.

[0037] Depend on Figure 2 It can be seen that compared with control group 1 and control group 2, the addition of biochar-based bacterial agents in the experimental group has a more prominent degradation effect on the three natural estrogens. It is speculated that biochar as a carrier avoids direct contact between the bacterial flora and the external environment, buffers the mutual interference between the environment and the bacterial flora, and enables the bacterial flora to slowly adapt to the environment. In the process of pollutants in livestock and poultry manure being adsorbed by biochar, the bacterial flora gradually degrades and thus effectively exerts its degradation efficiency in the long term; from the comparison between control group 1 and control group 2, it can be seen that the addition of biochar and bacterial flora can also degrade estrogen. The combination of the two fully integrates the advantages of biochar adsorption characteristics and rapid and thorough microbial degradation.

[0038] Experimental Example 1: This experimental example is based on the biochar-based bacterial agent prepared by the methods provided in Examples 1 to 4, and explores the effect of biochar obtained by pyrolysis of different plant materials on the biochar-based bacterial agent.

[0039] Specifically, the environmental estrogen was degraded by using the biochar-based bacterial agent prepared by the methods provided in Examples 1, 2, 3 and 4, and the removal rate of environmental estrogen was used to determine the effects of different ratios of estrogen-degrading bacterial communities to biochar-based fixed bacterial materials on the removal of estrogen by the biochar-based bacterial agent.

[0040] The removal rates of environmental estrogens by the biochar-based bacterial agents prepared by the methods provided in Examples 1, 2, 3 and 4 are as follows: Figure 3 As shown by Figure 3 It can be seen that the degradation effects of Examples 2 to 4 on the three natural estrogens in 5 days are all lower than that of Example 1.

[0041] The above degradation method is described in detail below.

[0042] The biochar-based bacterial agent prepared by the methods provided in Examples 1, 2, 3 and 4 was added at a dosage of 5% to 20 mL of 5 mg·L -1 The inorganic salt culture medium of estriol, estradiol and estrone was cultured at 150 rpm and 30°C for 5 days, and the removal rates of estriol, estradiol and estrone by the above three biochar-based bacterial agents were determined.

[0043] Furthermore, the biochar-based bacterial agents prepared in Examples 1 to 4 were scanned, and the obtained scanning electron microscope images are as follows: Figure 4 As shown. Among them, Figure 4 This is a scanning electron microscope image of the biochar-based bacterial agent prepared in Example 1; Figure 5 This is a scanning electron microscope image of the biochar-based bacterial agent prepared in Example 2; Figure 6 This is a scanning electron microscope image of the biochar-based bacterial agent prepared in Example 3; Figure 7 This is a scanning electron microscope image of the biochar-based bacterial agent prepared in Example 4.

[0044] The effective live bacteria on the biochar-based bacterial agents prepared in Examples 1 to 4 were counted respectively. The number of effective live bacteria loaded on the biochar-based bacterial agent in Example 1 (the pyrolyzed plant material is bamboo) is 9.6×10 9 CFU / g, the effective number of live bacteria loaded on the biochar-based bacterial agent of Example 2 (the pyrolyzed plant material is corn stalks) is 8.2×10 9 CFU / g, the effective number of viable bacteria loaded on the biochar-based bacterial agent of Example 3 (the pyrolyzed plant material is coconut shell) is 5.4×109 CFU / g, the effective number of viable bacteria loaded on the biochar-based bacterial agent of Example 4 was 7.4×10 9 CFU / g.

[0045] By comparing the electron microscope images and the effective live bacteria counts, it can be seen that the number of effective live bacteria loaded on the biochar-based bacterial agent of Example 1 is the largest, which means that the biochar-based bacterial agent prepared using bamboo as the material not only has the largest number of effective live bacteria, but also that bacteria are observed to secrete extracellular polymers, which is conducive to the formation of biofilms and can effectively improve the removal of organic pollutants.

[0046] Experimental Example 2: This experimental example is based on the biochar-based bacterial agent prepared by the methods provided in Examples 1, 5 and 6, and explores the effects of different ratios of estrogen-degrading bacterial communities to biochar-based fixed bacterial materials on the removal of environmental estrogens by the biochar-based bacterial agent.

[0047] Specifically, the biochar-based bacterial agent prepared by the methods provided in Examples 1, 5, and 6 was used to degrade environmental estrogens, and the removal rate of environmental estrogens was used to determine the effect of different ratios of estrogen-degrading bacterial communities to biochar-based fixed bacterial materials on the removal of environmental estrogens by the biochar-based bacterial agent. The above-mentioned degradation method is described in Experimental Example 1. The removal rate of environmental estrogens is as follows: Figure 8 As shown, in Example 5, a too small bacterial-to-feed ratio will result in a small number of fixed bacteria, while in Example 6, a too large bacterial-to-feed ratio will result in too high a cost, and the 5-day degradation effects of the biochar-based bacteria in Example 5 and Example 6 on the three environmental estrogens are lower than those of the biochar-based bacteria in Example 1, so 20 mL: 1 g is the optimal bacterial-to-feed ratio.

[0048] Experimental Example 3: This experimental example is based on the biochar-based bacterial agent prepared by the methods provided in Examples 1, 7 and 8, and explores the effect of the biochar-based particle size on the effect of the biochar-based bacterial agent in removing environmental estrogens.

[0049] Specifically, the biochar-based bacterial agent prepared by the methods provided in Examples 1, 7, and 8 was used to degrade environmental estrogens, and the effect of the biochar-based particle size on the biochar-based bacterial agent's estrogen removal effect was determined by the removal rate of environmental estrogens. The above-mentioned degradation method refers to the relevant description of Experimental Example 1. The removal rate of environmental estrogens is as follows: Fig. 9 As shown, in Example 7, the biochar particle size is too fine, which is not conducive to the recovery of the bacterial agent after water treatment, while in Example 8, the biochar particle size is too large, the specific surface area is insufficient, the number of attached bacteria is insufficient, and the estrogen reduction effect is affected. Therefore, the biochar-based particle size of 60 mesh is the best particle size.

[0050] Experimental Example 4: This experimental example is based on the biochar-based bacterial agent prepared by the methods provided in Examples 1, 9 and 10, and explores the effect of fixed time on the effect of the biochar-based bacterial agent in removing environmental estrogens.

[0051] Specifically, the biochar-based bacterial agents prepared by the methods provided in Examples 1, 9 and 10 were used to degrade environmental estrogens, and the effect of fixed time on the effect of biochar-based bacterial agents in removing estrogens was determined by the removal rate of environmental estrogens. The above degradation method refers to the relevant description of Experimental Example 1. The removal rate of environmental estrogens is as follows: Fig.10 As shown in the figure, as the immobilization time increases, the bacterial community can attach to the biochar matrix more effectively, improving the efficiency of environmental estrogen degradation, so 3 days of immobilization time is the optimal immobilization time.

[0052] Experimental Example 5: This experimental example is based on the biochar-based bacterial agent prepared by the methods provided in Examples 1, 11 and 12, and explores the effect of fixed temperature on the effect of the biochar-based bacterial agent in removing estrogen.

[0053] Specifically, the biochar-based bacterial agents prepared by the methods provided in Examples 1, 11, and 12 were used to degrade environmental estrogens, and the effect of fixed temperature on the removal of estrogen by the biochar-based bacterial agents was determined by the removal rate of environmental estrogens. Fig.11 As shown in the figure, with the increase of immobilization temperature, the number of effective bacteria in the biochar-based agent decreased and the degradation efficiency of natural estrogen decreased, so the optimal immobilization temperature was 30℃.

[0054] The various embodiments in this specification are described in a progressive manner, and the same or similar parts between the various embodiments can be referenced to each other, and each embodiment focuses on the differences from other embodiments.

[0055] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A method for preparing a biochar-based bacterial agent for combined estrogen reduction, characterized in that: include: preparing an estrogen-degrading bacterial flora, wherein the estrogen-degrading bacterial flora comprises Sphingomonas JY-1, Rhodococcus JY-2 and Neosphingobacterium; The deposit number of the Sphingomonas JY-1 is CGMCC No.33550; the deposit number of the Rhodococcus JY-2 is CGMCC No.33549; preparing biochar base; The estrogen-degrading bacterial community is fixed on the biochar base to obtain a biochar-based bacterial agent for combined estrogen reduction.

2. The method according to claim 1, characterized in that The estrogen-degrading bacterial flora is prepared, comprising: Activating and culturing Sphingomonas JY-1, Rhodococcus JY-2 and Neosphingobacterium, respectively, to obtain a bacterial solution of Sphingomonas JY-1, a bacterial solution of Rhodococcus JY-2 and a bacterial solution of Neosphingobacterium; The bacterial liquid of the Sphingomonas JY-1, the bacterial liquid of the Rhodococcus JY-2 and the bacterial liquid of the Neosphingobacterium are mixed to obtain the estrogen-degrading bacterial flora.

3. The method according to claim 2, characterized in that The volume ratio of the bacterial solution of Sphingomonas JY-1, the bacterial solution of Rhodococcus JY-2 and the bacterial solution of Neosphingobacterium is 1-1.5:1-1.5:1-2.

4. The method according to claim 1, characterized in that The preparation of biochar comprises: The biochar base is obtained after pyrolysis of plant material.

5. The method according to claim 1, characterized in that The ratio of the estrogen-degrading bacterial community to the biochar-based fixed bacterial material is 10-30 mL: 1 g.

6. Use of the combined estrogen-reducing biochar-based bacterial agent prepared by the method according to any one of claims 1 to 5 in removing environmental estrogens from contaminated media.

7. The use according to claim 6, characterized in that The environmental estrogen includes one or more of estriol, estradiol and estrone.

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