A method for preparing a multi-shell structured slow-release agent for remediating petroleum-contaminated groundwater

Abstract

This invention relates to the field of preparation of multi-shell structured sustained-release agents, and discloses a method for preparing a multi-shell structured sustained-release agent for remediating petroleum-contaminated groundwater. The method comprises: Step 1, preparing a nutrient matrix: The weight parts of each component in the nutrient matrix are as follows: NaH2PO4 100.0-150.0 parts, K2HPO4 500.0-600.0 parts, NH4H2PO4 50.0-100.0 parts, NaNO3 50.0-100.0 parts, CaCl2 10.0-20.0 parts, MgSO4·7H2O 50.0-100.0 parts, FeSO4·7H2O 5.0-15.0 parts, VB 12 0.1-0.5 parts; Step 2: Preparation of sustained-release agents: including the preparation of single-shell structure sustained-release agents, double-shell structure sustained-release agents, and triple-shell structure sustained-release agents. Beneficial effects: It can efficiently degrade petroleum hydrocarbons in groundwater. Under experimental conditions, the petroleum hydrocarbon degradation effect is increased by more than 40% after stimulating indigenous microorganisms with the sustained-release agent for 10 days.

Description

Technical Field

[0001] This invention relates to a method for preparing a multi-shell structured sustained-release agent, and particularly to a method for preparing a multi-shell structured sustained-release agent for remediating petroleum-contaminated groundwater. Background Technology

[0002] Currently, research on in-situ bioremediation technology for petroleum-contaminated groundwater faces practical challenges due to the relatively low temperature, lack of oxygen, and low nutrient content of groundwater environments. These challenges include the slow metabolism of indigenous microorganisms, the lack of necessary conditions for degradation reactions, and the resulting low degradation efficiency of organic pollutants. How to effectively stimulate the degradation of petroleum hydrocarbons by indigenous microorganisms in groundwater over a long period is a hot research topic in the industry.

[0003] In recent years, slow-release technology has attracted widespread attention in the field of groundwater pollution remediation due to its sustainable characteristics. By altering the binding mechanism of reactive substances with materials, slow-release technology prolongs the release rate of reactive substances, thereby improving their utilization rate. It has the advantages of extending the duration and release rate of reactive components, and can serve as a green, efficient, and sustainable bioremediation stimulation technology for the long-term remediation of oil-contaminated groundwater. Summary of the Invention

[0004] The main objective of this invention is to address the practical problems in in-situ bioremediation technology for petroleum-contaminated groundwater, such as the slow metabolism of indigenous microorganisms and the lack of necessary conditions for degradation reactions, resulting in low degradation efficiency of organic pollutants.

[0005] Another problem of this invention is how to effectively stimulate indigenous microorganisms in groundwater to degrade petroleum hydrocarbons over a long period of time;

[0006] In order to achieve the above objectives and solve the above problems, the present invention provides a method for preparing a multi-shell structured slow-release agent for remediating petroleum-contaminated groundwater.

[0007] The present invention provides a method for preparing a multi-shell structured slow-release agent for remediating petroleum-contaminated groundwater, the method comprising the following steps:

[0008] Step 1: Prepare the nutrient substrate:

[0009] The weight proportions of each component in the nutrient matrix are as follows:

[0010] NaH₂PO₄ 100.0-150.0 parts, K₂HPO₄ 500.0-600.0 parts, NH₄H₂PO₄ 50.0-100.0 parts, NaNO₃ 50.0-100.0 parts, CaCl₂ 10.0-20.0 parts, MgSO₄·7H₂O 50.0-100.0 parts, FeSO₄·7H₂O 5.0-15.0 parts, VB12 0.1-0.5 portions;

[0011] Step 2: Preparation of sustained-release medication:

[0012] The specific steps for preparing a single-shell structured sustained-release drug are as follows:

[0013] Step 1: Mix the nutrient substrate with the sodium alginate solution evenly to obtain the aqueous phase. The mass percentage ratio of sodium alginate to nutrient substrate to water is 1:4:50.

[0014] Step 2: Put soybean oil into a container, add 2% Span80 and emulsify for 10 minutes to obtain the oil phase for the experiment. The ratio of oil phase to water phase by mass percentage is 3:2.

[0015] Step 3: Slowly add the aqueous phase to the oil phase and stir to form a W / O emulsion;

[0016] Step 4: Slowly add succinic acid solution to the emulsion and stir. Pour it into a separatory funnel and shake for a period of time. The mass percentage ratio of succinic acid to sodium alginate is 1:1.

[0017] Step 5: Let stand to allow the solution to separate into layers;

[0018] Step 6: Remove the lower emulsion, add sesame protein solution and stir. The ratio of sesame protein to sodium alginate to water is 1:1:50 according to the mass percentage.

[0019] Step 7: Centrifuge the suspension to obtain the precipitate;

[0020] Step 8: Wash the precipitate with ethanol and water respectively to obtain a single-shell structured sustained-release agent.

[0021] The specific steps for preparing a double-shell structured sustained-release drug are as follows:

[0022] Step 1: Add the single-shell structure slow-release agent back into the above nutrient matrix solution as the aqueous phase, with the nutrient matrix to water ratio being 2:25 by mass percentage.

[0023] Step 2: The oil phase preparation method is the same as step 2 in the preparation of single-shell structure sustained-release agents. The aqueous phase is slowly added to the oil phase and stirred to form a W / O type emulsion.

[0024] Step 3: Add succinic acid solution to the emulsion and stir, pour into a separatory funnel and shake for a period of time;

[0025] Step 4: Let stand to allow the solution to separate into layers;

[0026] Step 5: Remove the lower emulsion, add 5% chitosan-acetic acid (CS) aqueous solution and stir. The ratio of chitosan to acetic acid to water is 1:1:49.

[0027] Step 6: Centrifuge the suspension to obtain the precipitate;

[0028] Step 7: Wash the precipitate with ethanol and water respectively to obtain a double-shell structured sustained-release drug.

[0029] The specific steps for preparing a three-shell structured sustained-release drug are as follows:

[0030] Step 1: Add the two-shell structure slow-release agent back into the solution of sodium alginate and the above nutrient matrix as the aqueous phase, with the mass percentage ratio of sodium alginate to nutrient matrix to water being 1:4:50.

[0031] Step 2: The oil phase preparation method is the same as step 2 in the preparation of single-shell structure sustained-release agents. The aqueous phase is slowly added to the oil phase and stirred to form a W / O type emulsion.

[0032] Step 3: Add succinic acid solution to the emulsion and stir, pour into a separatory funnel and shake for a period of time;

[0033] Step 4: Let stand to allow the solution to separate into layers;

[0034] Step 5: Remove the lower emulsion, add sesame protein solution and stir. The mass percentage ratio of sesame protein to sodium alginate to water is 1:1:50.

[0035] Step 6: Centrifuge the suspension to obtain the precipitate;

[0036] Step 7: Wash the precipitate with ethanol and water respectively, and dry it to obtain a three-shell structured sustained-release drug.

[0037] The stirring time in the above steps is 20-40 minutes.

[0038] The centrifugation speed in the above steps is 2000-3000 r·min. -1 The centrifugation time should be 15-30 minutes.

[0039] The drying temperature in the above steps is 20-30℃, and the drying time is 24-48h.

[0040] The multi-shell structured slow-release agent prepared by the aforementioned method for remediating petroleum-contaminated groundwater is used to remediate petroleum-contaminated groundwater and degrade petroleum hydrocarbons in the groundwater.

[0041] The beneficial effects of this invention are:

[0042] The multi-shell structured biostimulated sustained-release agent for remediating petroleum-contaminated groundwater provided by this invention comprises nano-sized microspheres that readily migrate in groundwater, exhibiting excellent encapsulation and sustained-release properties. This multi-shell structured biostimulated sustained-release agent can efficiently degrade petroleum hydrocarbons in groundwater. Under experimental conditions, the petroleum hydrocarbon degradation effect is increased by more than 40% after stimulating indigenous microorganisms for 10 days. Attached Figure Description

[0043] Figure 1 This is a schematic diagram illustrating the synthesis of the multi-shell structure sustained-release drug described in this invention. Detailed Implementation

[0044] Please see Figure 1 As shown:

[0045] The present invention provides a method for preparing a multi-shell structured slow-release agent for remediating petroleum-contaminated groundwater, the method comprising the following steps:

[0046] Step 1: Prepare the nutrient substrate:

[0047] The weight proportions of each component in the nutrient matrix are as follows:

[0048] NaH₂PO₄ 100.0-150.0 parts, K₂HPO₄ 500.0-600.0 parts, NH₄H₂PO₄ 50.0-100.0 parts, NaNO₃ 50.0-100.0 parts, CaCl₂ 10.0-20.0 parts, MgSO₄·7H₂O 50.0-100.0 parts, FeSO₄·7H₂O 5.0-15.0 parts, VB 12 0.1-0.5 portions;

[0049] Step 2: Preparation of sustained-release medication:

[0050] The specific steps for preparing a single-shell structured sustained-release drug are as follows:

[0051] Step 1: Mix the nutrient substrate with the sodium alginate solution evenly to obtain the aqueous phase. The mass percentage ratio of sodium alginate to nutrient substrate to water is 1:4:50.

[0052] Step 2: Put soybean oil into a container, add 2% Span80 and emulsify for 10 minutes to obtain the oil phase for the experiment. The ratio of oil phase to water phase by mass percentage is 3:2.

[0053] Step 3: Slowly add the aqueous phase to the oil phase and stir to form a W / O emulsion;

[0054] Step 4: Slowly add succinic acid solution to the emulsion and stir. Pour it into a separatory funnel and shake for a period of time. The mass percentage ratio of succinic acid to sodium alginate is 1:1.

[0055] Step 5: Let stand to allow the solution to separate into layers;

[0056] Step 6: Remove the lower emulsion, add sesame protein solution and stir. The ratio of sesame protein to sodium alginate to water is 1:1:50 according to the mass percentage.

[0057] Step 7: Centrifuge the suspension to obtain the precipitate;

[0058] Step 8: Wash the precipitate with ethanol and water respectively to obtain a single-shell structured sustained-release agent.

[0059] The specific steps for preparing a double-shell structured sustained-release drug are as follows:

[0060] Step 1: Add the single-shell structure slow-release agent back into the above nutrient matrix solution as the aqueous phase, with the nutrient matrix to water ratio being 2:25 by mass percentage.

[0061] Step 2: The oil phase preparation method is the same as step 2 in the preparation of single-shell structure sustained-release agents. The aqueous phase is slowly added to the oil phase and stirred to form a W / O type emulsion.

[0062] Step 3: Add succinic acid solution to the emulsion and stir, pour into a separatory funnel and shake for a period of time;

[0063] Step 4: Let stand to allow the solution to separate into layers;

[0064] Step 5: Remove the lower emulsion, add 5% chitosan-acetic acid (CS) aqueous solution and stir. The ratio of chitosan to acetic acid to water is 1:1:49.

[0065] Step 6: Centrifuge the suspension to obtain the precipitate;

[0066] Step 7: Wash the precipitate with ethanol and water respectively to obtain a double-shell structured sustained-release drug.

[0067] The specific steps for preparing a three-shell structured sustained-release drug are as follows:

[0068] Step 1: Add the two-shell structure slow-release agent back into sodium alginate and the above nutrient matrix solution as the aqueous phase, with the mass percentage ratio of sodium alginate to nutrient matrix to water being 1:4:50.

[0069] Step 2: The oil phase preparation method is the same as step 2 in the preparation of single-shell structure sustained-release agents. The aqueous phase is slowly added to the oil phase and stirred to form a W / O type emulsion.

[0070] Step 3: Add succinic acid solution to the emulsion and stir, pour into a separatory funnel and shake for a period of time;

[0071] Step 4: Let stand to allow the solution to separate into layers;

[0072] Step 5: Remove the lower emulsion, add sesame protein solution and stir. The mass percentage ratio of sesame protein to sodium alginate to water is 1:1:50.

[0073] Step 6: Centrifuge the suspension to obtain the precipitate;

[0074] Step 7: Wash the precipitate with ethanol and water respectively, and dry it to obtain a three-shell structured sustained-release drug.

[0075] The stirring time in the above steps is 20-40 minutes.

[0076] In the above steps, the centrifugation speed should be 2000-3000 r / min, and the centrifugation time should be 15-30 min.

[0077] The drying temperature in the above steps is 20-30℃, and the drying time is 24-48h.

[0078] The specific implementation method is as follows:

[0079] Example 1:

[0080] A multi-shell structured biostimulant slow-release agent for remediating petroleum-contaminated groundwater, the preparation steps of which are as follows:

[0081] Preparation of a single-shell structure: Nutrient matrix: NaH2PO4 1.25g, K2HPO4 5g, NH4H2PO4 0.5g, NaNO3 0.5g, CaCl2 125mg, MgSO4·7H2O 0.5g, FeSO4·7H2O 0.1g, VB 12 0.25 mg of ammonia was mixed with a 2% (w / w) sodium alginate solution at room temperature to obtain the aqueous phase for the experiment. 150 mL of soybean oil was placed in a beaker, and 2% Span 80 was added, followed by emulsification for 10 min to obtain the oil phase. The aqueous phase was slowly added to the oil phase, and the mixture was stirred at 1000 rpm for 30 min using a magnetic stirrer to form a W / O emulsion. Succinic acid solution was slowly added and stirred for 30 min. The solution was then poured into a separatory funnel and shaken for a period of time. The solution was then allowed to stand and separate into layers. The lower emulsion was removed, and a 2% (w / w) sesame protein solution was added and stirred for 30 min. The mixture was centrifuged at 2000 rpm for 30 min to obtain the precipitate. The precipitate was washed three times with anhydrous ethanol and ultrapure water to obtain a single-shell structured sustained-release agent.

[0082] Preparation of the two-shell structure: The single-shell structured sustained-release agent was added back to the above-mentioned nutrient matrix solution as the aqueous phase. The aqueous phase was slowly added to the oil phase and stirred for 30 min to form a W / O emulsion. Succinic acid solution was slowly added and stirred for 30 min. The solution was poured into a separatory funnel and shaken for a period of time. Then, the solution was allowed to stand and separate into layers. The lower emulsion was taken out, and chitosan-acetic acid aqueous solution was added and stirred for 30 min. The mixture was centrifuged at 2000 r / min for 30 min to obtain the precipitate. The precipitate was washed three times with anhydrous ethanol and ultrapure water to obtain the double-shell structured sustained-release agent.

[0083] Preparation of the three-shell structure: The two-shell structured sustained-release agent was re-added to the sodium alginate and nutrient matrix solution as the aqueous phase. The aqueous phase was slowly added to the oil phase and stirred for 30 min to form a W / O emulsion. Succinic acid solution was slowly added and stirred for 30 min. The solution was poured into a separatory funnel and shaken for a period of time. Then, the solution was allowed to stand and separate into layers. The lower emulsion was removed, and SPI solution was added and stirred for 30 min. The mixture was centrifuged at 2000 r / min for 30 min to obtain the precipitate. The precipitate was washed with anhydrous ethanol and ultrapure water, respectively, and dried at 30℃ for 24 hours to obtain the three-shell structured sustained-release agent. The encapsulation efficiencies of the first, second, and third shells of the sustained-release agent were 50%, 52.5%, and 65.8%, respectively, indicating good encapsulation effect and theoretically, sustained release for 87 days.

[0084] The enrichment culture of degrading bacteria used petroleum hydrocarbon-contaminated groundwater in Northeast China as the inoculum source. The inorganic salt culture medium formula (g / L) used was: NaCl 5g, KH2PO4 2g, NaNO3 2g, (NH4)2SO4 1g, MgSO4·7H2O 0.35g, agar powder 20g, and trace element solution 1ml, of which the trace element solution formula (g / L) was: FeSO4 0.56g, CuCl2 0.038g, ZnCl 0.15g, MnSO4·H2O 0.02g, CaCl2 0.015g, Na2MoO4·2H2O 0.015g, and H3BO3 0.005g. The enrichment process followed the "Technical Specification for Microbial Remediation of Chemically Contaminated Soil" (HG / T20719-20XX): 5 mL of groundwater sample was inoculated into 100 mL of sterilized inorganic salt culture medium and cultured under shaking conditions at 10℃ and 100 rpm in the dark. After one week, 5 mL of the enriched solution was transferred to freshly sterilized culture medium of the same formulation for subculturing. To achieve targeted acclimatization and efficient screening of the microbial community, a "low-concentration to high-concentration transition" culture method was adopted during the enrichment process (1, 10, 50, 100, 250, 500, and 1000 μg / L), with multiple rounds of subculturing to ultimately obtain a complex microbial community (at the phylum level) with highly efficient degradation capabilities for petroleum hydrocarbons: Firmicutes, Bacteroides, Actinomycetes, and Proteobacteria.

[0085] A 1 mg / L concentration of petroleum pollutants was prepared from groundwater from a petroleum-contaminated site. 40 mL of the solution was transferred to a sterile brown headspace vial, and 1 mL of the aforementioned degrading bacteria was inoculated. 5 mg of the slow-release agent granules prepared in Example 1 was added, and the vial was sealed and placed under conditions of 10°C, 120 r / min, and protected from light. The concentration of petroleum hydrocarbons was measured, and the growth and changes of the indigenous microbial community were studied.

[0086] Experimental results show that after adding the multi-shell structure sustained-release agent of this invention, the degradation effect of petroleum hydrocarbons increases with time. The petroleum hydrocarbon degradation rate at 10 days was 56.3%, while the degradation rate of the control group without the sustained-release agent was 40.2%, representing a 40% increase in degradation efficiency. Microorganisms grew rapidly from 0-1 day, and OD... 600 The value increased rapidly, after which the microbial growth rate slowed down, and the microbial OD value at 1 day was [value missing]. 600 The value was 0.107, and the microbial OD value at 10 days was... 600 The value is 0.143.

[0087] Example 2:

[0088] A multi-shell structured biostimulant slow-release agent for remediating petroleum-contaminated groundwater, the preparation steps of which are as follows:

[0089] Preparation of a single-shell structure: Nutrient matrix: NaH2PO4 1.25g, K2HPO4 5g, NH4H2PO4 0.5g, NaNO3 0.5g, CaCl2 125mg, MgSO4·7H2O 0.5g, FeSO4·7H2O 0.1g, VB 12 0.25 mg of ammonia was mixed with a 2% (w / w) sodium alginate solution at room temperature to obtain the aqueous phase for the experiment. 150 mL of soybean oil was placed in a beaker, and 2% Span 80 was added, followed by emulsification for 10 min to obtain the oil phase. The aqueous phase was slowly added to the oil phase, and the mixture was stirred at 1000 rpm for 25 min using a magnetic stirrer to form a W / O emulsion. Succinic acid solution was slowly added and stirred for 25 min. The solution was then poured into a separatory funnel and shaken for a period of time. The solution was then allowed to stand and separate into layers. The lower emulsion was removed, and a 2% (w / w) sesame protein solution was added and stirred for 25 min. The mixture was centrifuged at 3000 rpm for 20 min to obtain the precipitate. The precipitate was washed three times with anhydrous ethanol and ultrapure water to obtain a single-shell structured sustained-release agent.

[0090] Preparation of the two-shell structure: The single-shell structured sustained-release agent was added back to the above-mentioned nutrient matrix solution as the aqueous phase. The aqueous phase was slowly added to the oil phase and stirred for 25 min to form a W / O emulsion. Succinic acid solution was slowly added and stirred for 25 min. The solution was poured into a separatory funnel and shaken for a period of time. Then, the solution was allowed to stand and separate into layers. The lower emulsion was taken out, and chitosan-acetic acid aqueous solution was added and stirred for 25 min. The mixture was centrifuged at 3000 r / min for 20 min to obtain the precipitate. The precipitate was washed three times with anhydrous ethanol and ultrapure water to obtain the double-shell structured sustained-release agent.

[0091] Preparation of the three-shell structure: The two-shell structured sustained-release agent was re-added to the sodium alginate and nutrient matrix solution as the aqueous phase. The aqueous phase was slowly added to the oil phase and stirred for 25 min to form a W / O emulsion. Succinic acid solution was slowly added and stirred for 25 min. The solution was poured into a separatory funnel and shaken for a period of time. Then, the solution was allowed to stand and separate into layers. The lower emulsion was removed, and SPI solution was added and stirred for 25 min. The mixture was centrifuged at 3000 r / min for 20 min to obtain the precipitate. The precipitate was washed with anhydrous ethanol and ultrapure water, respectively, and dried at room temperature for 48 hours to obtain the three-shell structured sustained-release agent. The encapsulation efficiencies of the first, second, and third shells of the sustained-release agent were 61.2%, 55.3%, and 58.5%, respectively, indicating good encapsulation effect and theoretically, sustained release for 75 days.

[0092] A 2 mg / L concentration of petroleum pollutants was prepared from groundwater at a petroleum-contaminated site. 40 mL of the solution was transferred to a sterile brown headspace vial, and 1 mL of the aforementioned degrading bacteria was inoculated. 5 mg of the slow-release agent granules prepared in Example 2 was added, and the vial was sealed and placed under conditions of 10°C, 120 r / min, and protected from light. Water samples were taken at different time points to determine the concentration of petroleum hydrocarbons and to study the growth changes of the indigenous microbial community.

[0093] Experimental results show that after adding the multi-shell structure sustained-release agent of this invention, the degradation effect of petroleum hydrocarbons increases with time. The petroleum hydrocarbon degradation rate at 10 days was 57.5%, while the degradation rate of the control group without the sustained-release agent was 40.2%, representing a 43% increase in degradation efficiency compared to the control group. Microorganisms grew rapidly from 0-1 day, and OD... 600 The value increased rapidly, after which the microbial growth rate slowed down, and the microbial OD value at 1 day was [value missing]. 600 The value was 0.109, and the microbial OD value at 10 days was... 600 The value is 0.140.

[0094] Example 3:

[0095] A multi-shell structured biostimulant slow-release agent for remediating petroleum-contaminated groundwater, the preparation steps of which are as follows:

[0096] Preparation of a single-shell structure: Nutrient matrix: NaH2PO4 1.25g, K2HPO4 5g, NH4H2PO4 0.5g, NaNO3 0.5g, CaCl2 125mg, MgSO4·7H2O 0.5g, FeSO4·7H2O 0.1g, VB 12 0.25 mg of ammonia was mixed with a 2% (w / w) sodium alginate solution at room temperature to obtain the aqueous phase for the experiment. 150 mL of soybean oil was placed in a beaker, and 2% Span 80 was added, followed by emulsification for 10 min to obtain the oil phase. The aqueous phase was slowly added to the oil phase, and the mixture was stirred at 1000 rpm for 40 min using a magnetic stirrer to form a W / O emulsion. Succinic acid solution was slowly added and stirred for 40 min. The solution was then poured into a separatory funnel and shaken for a period of time. The solution was then allowed to stand and separate into layers. The lower emulsion was removed, and a 2% (w / w) sesame protein solution was added and stirred for 40 min. The mixture was centrifuged at 2500 rpm for 15 min to obtain the precipitate. The precipitate was washed three times with anhydrous ethanol and ultrapure water to obtain a single-shell structured sustained-release agent.

[0097] Preparation of the two-shell structure: The single-shell structured sustained-release agent was added back to the above-mentioned nutrient matrix solution as the aqueous phase. The aqueous phase was slowly added to the oil phase and stirred for 40 min to form a W / O emulsion. Succinic acid solution was slowly added and stirred for 40 min. The solution was poured into a separatory funnel and shaken for a period of time. Then, the solution was allowed to stand and separate into layers. The lower emulsion was taken out, and chitosan-acetic acid aqueous solution was added and stirred for 40 min. The mixture was centrifuged at 2500 r / min for 15 min to obtain the precipitate. The precipitate was washed three times with anhydrous ethanol and ultrapure water to obtain the double-shell structured sustained-release agent.

[0098] Preparation of the three-shell structure: The two-shell structured sustained-release agent was re-added to the sodium alginate and nutrient matrix solution as the aqueous phase. The aqueous phase was slowly added to the oil phase and stirred for 40 min to form a W / O emulsion. Succinic acid solution was slowly added and stirred for 40 min. The solution was poured into a separatory funnel and shaken for a period of time. Then, the solution was allowed to stand and separate into layers. The lower emulsion was removed, and SPI solution was added and stirred for 40 min. The mixture was centrifuged at 2500 r / min for 15 min to obtain the precipitate. The precipitate was washed with anhydrous ethanol and ultrapure water, respectively, and dried at room temperature for 36 hours to obtain the three-shell structured sustained-release agent. The encapsulation efficiencies of the first, second, and third shells of the sustained-release agent were 53.4%, 60.3%, and 64.7%, respectively, indicating good encapsulation effect. Theoretically, the sustained release can last for 95 days.

[0099] A 5 mg / L concentration of petroleum pollutants was prepared from groundwater at a petroleum-contaminated site. 40 mL of the solution was transferred to a sterile brown headspace vial, and 1 mL of the aforementioned degrading bacteria was inoculated. 5 mg of the slow-release agent granules prepared in Example 3 was added, and the vial was sealed and placed under conditions of 10°C, 120 r / min, and protected from light. Water samples were taken at different time points to determine the concentration of petroleum hydrocarbons and to study the growth changes and microbial community structure of the indigenous microbial community.

[0100] Experimental results show that after adding the multi-shell structure slow-release agent of this invention, the degradation effect of petroleum hydrocarbons increases with time. The petroleum hydrocarbon degradation rate at 10 days is 62.3%, while the degradation rate of the control group without the slow-release agent is 40.2%, representing a 55% increase in degradation efficiency compared to the control group. Microorganisms grow rapidly from 0-1 day, and OD... 600 The value increased rapidly, after which the microbial growth rate slowed down, and the microbial OD value at 1 day was [value missing]. 600 The value was 0.145, and the microbial OD value at 10 days was... 600 The value was 0.228. After the action of the sustained-release agent, the abundance of petroleum-degrading bacteria increased significantly, with the abundance of Clostridium botulinum, Acinetobacter spp., Pseudomonas spp., and Acinetobacter spp. increasing by 1.35%-23.07%.

Claims

1. A method for preparing a multi-shell structured slow-release agent for remediating petroleum-contaminated groundwater, characterized in that: The steps include the following: Step 1: Prepare the nutrient substrate: The weight proportions of each component in the nutrient matrix are as follows: NaH₂PO₄ 100.0-150.0 parts, K₂HPO₄ 500.0-600.0 parts, NH₄H₂PO₄ 50.0-100.0 parts, NaNO₃ 50.0-100.0 parts, CaCl₂ 10.0-20.0 parts, MgSO₄·7H₂O 50.0-100.0 parts, FeSO₄·7H₂O 5.0-15.0 parts, VB 12 0.1-0.5 portions; The second step is the preparation of sustained-release drugs: sustained-release drugs include the preparation of single-shell structure sustained-release drugs, double-shell structure sustained-release drugs, and triple-shell structure sustained-release drugs. The specific steps for preparing a single-shell structured sustained-release drug are as follows: Step 1: Mix the nutrient substrate with the sodium alginate solution evenly to obtain the aqueous phase. The mass percentage ratio of sodium alginate to nutrient substrate to water is 1:4:

50. Step 2: Put soybean oil into a container, add 2% Span 80 and emulsify for 10 minutes to obtain the oil phase. The ratio of oil phase to water phase by mass percentage is 3:

2. Step 3: Add the aqueous phase to the oil phase and stir to form a W / O emulsion; Step 4: Add succinic acid solution to the emulsion and stir. Pour it into a separatory funnel and shake for a period of time. The mass percentage ratio of succinic acid to sodium alginate is 1:

1. Step 5: Let stand to allow the solution to separate into layers; Step 6: Remove the lower emulsion, add sesame protein solution and stir. The ratio of sesame protein to sodium alginate to water is 1:1:50 according to the mass percentage. Step 7: Centrifuge the suspension to obtain the precipitate; Step 8: Wash the precipitate with ethanol and water respectively to obtain a single-shell structured sustained-release drug. The specific steps for preparing a double-shell structured sustained-release drug are as follows: Step 1: Add the single-shell structured slow-release agent to the nutrient matrix solution as the aqueous phase, with the nutrient matrix to water ratio being 2:25 by mass percentage. Step 2: Put soybean oil into a container, add 2% Span 80 and emulsify for 10 min to obtain the experimental oil phase. The ratio of oil phase to water phase by mass percentage is 3:

2. Add the water phase to the oil phase and stir to form a W / O type emulsion. Step 3: Add succinic acid solution to the emulsion and stir, pour into a separatory funnel and shake for a period of time; Step 4: Let stand to allow the solution to separate into layers; Step 5: Remove the lower emulsion, add 5% chitosan-acetic acid aqueous solution and stir. The ratio of chitosan to acetic acid to water is 1:1:

49. Step 6: Centrifuge the suspension to obtain the precipitate; Step 7: Wash the precipitate with ethanol and water respectively to obtain a double-shell structured sustained-release drug. The specific steps for preparing a three-shell structured sustained-release drug are as follows: Step 1: Add the two-shell structure slow-release agent to sodium alginate and nutrient matrix solution as the aqueous phase. The mass percentage ratio of sodium alginate to nutrient matrix to water is 1:4:

50. Step 2: Put soybean oil into a container, add 2% Span 80 and emulsify for 10 minutes to obtain the oil phase. The ratio of oil phase to water phase by mass percentage is 3:

2. Add the water phase to the oil phase and stir to form a W / O type emulsion. Step 3: Add succinic acid solution to the emulsion and stir, pour into a separatory funnel and shake for a period of time; Step 4: Let stand to allow the solution to separate into layers; Step 5: Remove the lower emulsion, add sesame protein solution and stir. The mass percentage ratio of sesame protein to sodium alginate to water is 1:1:

50. Step 6: Centrifuge the suspension to obtain the precipitate; Step 7: Wash the precipitate with ethanol and water respectively, and dry it to obtain a three-shell structured sustained-release drug.

2. The method for preparing a multi-shell structured slow-release agent for remediating petroleum-contaminated groundwater according to claim 1, characterized in that: The stirring time is 20-40 minutes.

3. The method for preparing a multi-shell structured slow-release agent for remediating petroleum-contaminated groundwater according to claim 1, characterized in that: The centrifugation speed is 2000-3000 r / min, and the centrifugation time is 15-30 min.

4. The method for preparing a multi-shell structured slow-release agent for remediating petroleum-contaminated groundwater according to claim 1, characterized in that: The drying temperature is 20-30 ℃, and the drying time is 24-48 h.

5. The multi-shell structured slow-release agent for remediating petroleum-contaminated groundwater prepared by the preparation method according to any one of claims 1-4, is used for remediating petroleum-contaminated groundwater and degrading petroleum hydrocarbons in the groundwater.

Images