Bioremediation method for degrading chlorinated hydrocarbon through cooperation of microorganisms and colloid coconut shell biochar and application of biomediation method

Abstract

The invention relates to the technical field of environmental pollution remediation, and particularly discloses a bioremediation method for degrading chlorinated hydrocarbon by microorganisms cooperating with colloidal coconut shell biochar and application of the bioremediation method, and the method comprises the steps of preparation of a combined fungicide, preparation of colloidal coconut shell biochar powder, construction of a synergistic degradation system, design of a reaction system and on-site in-situ remediation application. The problems of insufficient microbial activity, unstable electron donor supply and saturated adsorption material in the traditional bioremediation are effectively overcome; according to the method, the repairing process can be always kept in the optimal state; the engineering practicability and reliability of field repair are effectively improved; the method is easy and convenient to operate, low in energy consumption and economical in cost, has high environmental adaptability and long-term remediation effect and conforms to the green remediation concept; the technical scheme is not only suitable for ex-situ remediation verification, but also can be popularized to practical engineering application of complex polluted sites, and has wide market prospects and popularization value.

Description

Technical Field

[0001] The present invention relates to the technical field of environmental pollution remediation, and particularly relates to a bioremediation method for the degradation of chlorinated hydrocarbons by microorganisms in cooperation with colloidal coconut shell biochar and its application. This technology is applicable to the remediation of industrial polluted sites, groundwater pollution, and soil pollution. It can be used for ex-situ remediation verification and is also suitable for large-scale in-situ remediation projects on-site. It has the characteristics of being green and environmentally friendly, low energy consumption, cost-effective, and long-term stable. Background Art

[0002] Chlorinated Hydrocarbons (CHCs) are a class of chlorine-containing organic compounds, mainly including trichloroethylene (TCE), perchloroethylene (PCE), chloroform, etc. They have stable chemical properties, strong volatility and migration properties, and are commonly used solvents and cleaning agents in industrial production. They are also widely used in fields such as dry cleaning and pesticide production. Due to their potential hazards to the environment and human health, chlorinated hydrocarbons have become one of the main pollutants causing soil and groundwater pollution globally.

[0003] Currently, the remediation methods for chlorinated hydrocarbon pollution mainly include four technical routes: physical remediation, chemical remediation, heat treatment, and bioremediation. Physical remediation technologies such as excavation and landfilling, vapor extraction, and air injection can reduce the concentration of pollutants in the short term, but often only transfer the pollutants to other places and cannot achieve fundamental removal. Chemical remediation technologies use redox reactions, such as Fenton's reagent and persulfate oxidation, which can quickly decompose chlorinated hydrocarbons, but consume a large amount of reagents and are prone to producing by-products, causing secondary pollution to the environment. Heat treatment technologies achieve pollutant removal through high-temperature thermal desorption, but have extremely high energy consumption and damage the soil structure. Bioremediation technologies use the metabolic functions of microorganisms to degrade pollutants into non-toxic or low-toxic substances, which have the advantage of being green and environmentally friendly. However, due to problems such as insufficient microbial activity, insufficient supply of electron donors, and environmental condition limitations, the remediation efficiency is often not high.

[0004] In recent years, coconut shell biochar has received extensive attention due to its wide raw material sources, simple preparation process, large specific surface area, and rich pore structure. When used alone, coconut shell biochar can adsorb organic pollutants, but the adsorption effect is easily saturated and it is difficult to achieve regeneration. How to organically combine the advantages of microbial degradation and coconut shell biochar adsorption to construct a synergistic degradation system that can not only enrich pollutants but also provide a stable growth microenvironment for microorganisms has become a key technical problem to be solved urgently. In addition, in practical engineering applications, the lack of on-line monitoring and automatic control technologies in the reaction system often leads to large fluctuations in reaction conditions and affects the remediation effect. Therefore, developing an efficient remediation system integrating microbial dechlorination degradation, biochar adsorption and enrichment, and on-line automatic control has important theoretical significance and engineering application prospects. Summary of the Invention

[0005] The object of the present invention is to provide a bioremediation method for the degradation of chlorinated hydrocarbons by microorganisms in cooperation with colloidal coconut shell biochar and its application. By constructing a synergistic system of combined microbial agents and colloidal coconut shell biochar, defects such as insufficient activity of the microbial community, easy saturation of the adsorption material, and inaccurate reaction control in traditional bioremediation are overcome, so as to achieve efficient, continuous, and long-term degradation of pollutants.

[0006] The present invention adopts the following technical solutions: A bioremediation method for the degradation of chlorinated hydrocarbons by microorganisms in cooperation with colloidal coconut shell biochar, comprising the following steps: S1. Collect soil samples from a chlorinated hydrocarbon-polluted site, pretreat the soil with anaerobic PBS, shake it with a shaker and then centrifuge to separate, and extract the natural microbial community therein to obtain a preliminary bacterial-containing solution; S2. Inoculate the bacterial-containing solution into a pre-prepared anaerobic culture medium, and perform multiple subcultures in an anaerobic environment to form a highly active combined microbial agent; S3. Prepare colloidal coconut shell biochar powder (CAC). The coconut shell is washed, dried, crushed, and pyrolyzed at 700 °C for 4 hours under nitrogen protection to obtain a primary biochar product. After airflow pulverization and ball milling treatment, an appropriate dispersion liquid is added and mixed evenly, and then centrifugal separation and vacuum freeze-drying are used to obtain colloidal biochar powder with uniform particle size and high specific surface area; S4. Mix the combined microbial agent prepared in step S2 and the colloidal coconut shell biochar powder prepared in step S3 according to a predetermined ratio to form a mixed system with the synergistic action of microorganisms and biochar; S5. Add the mixed system into a sterilized anaerobic reactor, add about 70% by volume of the anaerobic culture medium into the reactor, and introduce a mixed gas containing nitrogen and an appropriate amount of carbon dioxide (flow rate: 0.1 - 0.3 L / min) to ensure that the reactor is always in a stable anaerobic state; S6. A multi-parameter on-line monitoring module is set in the reactor to detect and record key parameters such as pH, temperature, and electron donor concentration in real time, and use a PLC system or an automatic feeding device to automatically regulate the electron donor concentration, so as to ensure that all physical and chemical conditions in the reaction system are maintained in the optimal state and achieve continuous dechlorination and degradation of chlorinated hydrocarbons; S7. In the in-situ bioremediation mode, the above mixed system is fixed into immobilized microspheres with a diameter of 3 - 5 mm by titration method, and evenly distributed in the polluted area through underground injection. At the same time, an underground sensor network is arranged in the remediation area to collect information on pH, temperature, and pollutant concentration in the remediation area in real time, and adjust the subsequent donor supplementation and injection volume accordingly.

[0007] Preferably, in step S1, the operating conditions for shaking on a shaker and then centrifuging are shaking for 10 minutes and centrifuging at 2000 rpm for 5 minutes, to ensure that the microorganisms are fully released and to avoid disturbing excessive precipitation.

[0008] Preferably, in step S2, the steps for preparing the anaerobic medium include: Prepare a 100-fold concentrated salt stock solution, the components of which are sodium chloride, magnesium chloride, potassium dihydrogen phosphate, ammonium chloride, potassium chloride and calcium chloride; Prepare a trace element stock solution, including trace element A stock solution and trace element B stock solution. Trace element A stock solution contains FeCl2·4H2O, CoCl2·6H2O, MnCl2·4H2O, ZnCl2, H3BO3, Na2MoO4·2H2O, NiCl2·6H2O and CuCl2·2H2O, and trace element B stock solution contains Na2WO4·2H2O and NaOH; Prepare a resazurin stock solution and a 1000-fold vitamin solution, adjust the pH to 7.5 with sodium hydroxide, filter and sterilize after purging with nitrogen, and finally mix to obtain the anaerobic medium.

[0009] Preferably, in step S3, the steps for preparing the colloidal coconut shell biochar powder include: After the dried coconut shell is preliminarily crushed, biochar is prepared through a pyrolysis reaction; Using a jet mill, refine the biochar at a rate of 2 g / min under an air flow pressure of 0.8 MPa; Using a ball mill and zirconia grinding beads, set the rotation speed at about 575 rpm and grind for 6 hours, and then obtain the colloidal biochar powder through centrifugation and freeze-drying.

[0010] Preferably, the mixing ratio of the combined bacterium agent to the colloidal coconut shell biochar powder in the mixed system is 1:5.

[0011] Preferably, in step S5, the mixed gas in the anaerobic reactor consists of nitrogen and an appropriate amount of carbon dioxide. Nitrogen is the main component, and the proportion of carbon dioxide is controlled within the range that can ensure the stability of the anaerobic environment and participate in the reaction regulation appropriately. The ratio of nitrogen to carbon dioxide is 95:5, and the gas flow rate is controlled at 0.1 L / min to ensure that the reactor is always in a stable anaerobic state.

[0012] Preferably, in step S6, the on-line monitoring module in the reactor includes a high-precision pH probe, a temperature sensor and an electron donor concentration detector. The PLC system automatically drops 0.1 M NaOH or the corresponding acid solution according to the collected data to regulate the pH value to keep it between 7.0 and 7.5, and at the same time regulates the heating or cooling device to keep the temperature within the range of 28-32°C.

[0013] Preferably, in step S7, the preparation steps of the immobilized microspheres include: Fixing the mixed system into microspheres with a diameter of 3 - 5 mm by titration; After the microspheres are prepared, use underground injection equipment to evenly distribute them in the contaminated soil; Meanwhile, deploy an underground sensor network in the contaminated area to achieve real-time monitoring and dynamic data transmission of the pH, temperature, and pollutant concentration in the remediation area, facilitating on-site operators to adjust the process based on the data.

[0014] Preferably, in step S6, the electron donor is lactic acid or acetate, and its initial concentration is set at 5 - 10 mM, and it is replenished in real time according to the on-line monitoring data by an automatic feeding device.

[0015] Another object of the present invention is to provide an application of the bioremediation method for the degradation of chlorinated hydrocarbons by microorganisms synergistically with colloidal coconut shell biochar as described above in the remediation of chlorinated hydrocarbon-contaminated soil and groundwater.

[0016] Compared with the prior art, the present invention has the following beneficial effects: 1. Effectively overcome the problems of insufficient microbial activity, unstable supply of electron donors, and saturation of adsorption materials in traditional bioremediation, and achieve continuous and rapid dechlorination and degradation of chlorinated hydrocarbons; 2. Through the on-line monitoring and automatic control system, key parameters such as pH, temperature, and electron donor concentration are collected in real time, and compensation adjustment can be quickly carried out when the reaction conditions fluctuate, so that the remediation process always remains in the optimal state; 3. The immobilized microsphere technology combined with the underground sensor network realizes the dynamic monitoring and feedback control of in-situ remediation on-site, greatly improving the engineering practicability and reliability of on-site remediation; 4. This method is easy to operate, has low energy consumption, is cost-effective, and at the same time has high environmental adaptability and long-term remediation effects, meeting the concept of green remediation; 5. The technical solution of the present invention is not only applicable to off-site remediation verification, but also can be extended to the actual engineering application of complex contaminated sites, with broad market prospects and popularization value. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is a flowchart of a bioremediation method for the degradation of chlorinated hydrocarbons by microorganisms synergistically with colloidal coconut shell biochar provided by the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0018] The following describes the technical solutions in the embodiments of the present invention clearly and completely with reference to specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of them. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0019] An embodiment of the present invention provides a bioremediation method for the degradation of chlorinated hydrocarbons by microorganisms in cooperation with colloidal coconut shell biochar, including the following steps: S1. Collect soil samples from the chlorinated hydrocarbon contaminated site, pretreat the soil with anaerobic PBS, shake it with a shaker and then centrifuge to separate, and extract the natural microbial flora therein to obtain a preliminary bacterial-containing solution; S2. Inoculate the bacterial-containing solution into a pre-prepared anaerobic medium, and perform multiple subculture in an anaerobic environment to form a highly active combined microbial agent; S3. Prepare colloidal coconut shell biochar powder (CAC). Select Hainan coconut shell as the raw material, wash, dry, and crush it, and then pyrolyze it at 700 °C for 4 hours under nitrogen protection to obtain a primary biochar product. After airflow pulverization and ball milling treatment, add an appropriate dispersant and mix evenly, and then use centrifugal separation and vacuum freeze-drying to obtain colloidal biochar powder with uniform particle size and high specific surface area; S4. Mix the combined microbial agent prepared in step S2 and the CAC colloidal coconut shell biochar powder prepared in step S3 according to a predetermined ratio to form a mixed system in which microorganisms and biochar act synergistically, and the mass ratio of the microbial agent to the biochar is 1:5; S5. Add the mixed system into a sterilized anaerobic reactor with a volume of about 3 L. Add about 70% of the anaerobic medium by volume into the reactor, and introduce a mixed gas of nitrogen and an appropriate amount of carbon dioxide (flow rate is 0.1 - 0.3 L / min) to ensure that the reactor is always in a stable anaerobic state; S6. A multi-parameter on-line monitoring module is set in the reactor to detect and record key parameters such as pH, temperature, and electron donor concentration in real time, and use a PLC system or an automatic feeding device to automatically control the electron donor concentration, so as to ensure that all physical and chemical conditions in the reaction system are maintained in the optimal state, and realize the continuous dechlorination and degradation of chlorinated hydrocarbons; S7. In the in-situ remediation mode, the above mixed system is fixed into immobilized microspheres with a diameter of 3 - 5 mm by titration method, and evenly distributed in the contaminated area by underground injection. At the same time, an underground sensor network is arranged in the remediation area to collect information on pH, temperature, and pollutant concentration in the remediation area in real time, and adjust the subsequent donor addition and injection volume accordingly.

[0020] In step S1 of this embodiment, the operating conditions for shaking with a shaker and then centrifuging are shaking for 10 - 12 minutes and centrifuging at 2000 rpm for 5 - 8 minutes, so as to ensure the full release of microorganisms and avoid disturbing excessive precipitation.

[0021] In step S2 of this embodiment, the steps for preparing the anaerobic medium include: Prepare 100 - fold concentrated salt stock solution, whose components are sodium chloride, magnesium chloride, potassium dihydrogen phosphate, ammonium chloride, potassium chloride and calcium chloride; Prepare trace element stock solutions, including trace element A stock solution and trace element B stock solution. Trace element A stock solution contains FeCl2·4H2O, CoCl2·6H2O, MnCl2·4H2O, ZnCl2, H3BO3, Na2MoO4·2H2O, NiCl2·6H2O and CuCl2·2H2O, and trace element B stock solution contains Na2WO4·2H2O and NaOH; Prepare resazurin mother liquor and 1000 - fold vitamin solution, adjust the pH to 7.5 with sodium hydroxide, filter and sterilize after purging with nitrogen, and finally mix to obtain the anaerobic medium.

[0022] In step S3 of this embodiment, the steps for preparing the colloidal coconut shell biochar powder include: After the dry coconut shell is preliminarily crushed, biochar is obtained through pyrolysis reaction; Using a jet mill, under a gas pressure of 0.8 MPa, the biochar is refined at a rate of 2 g / min; Using a ball mill and zirconia grinding beads, set the rotation speed at 550 - 600 rpm and grind for 6 hours, and then obtain the colloidal biochar powder through centrifugation and freeze - drying treatment.

[0023] In the mixed system of this embodiment, the mixing ratio of the consortium to the colloidal coconut shell biochar powder is 1:5. This ratio can give full play to the microbial degradation function and the adsorption and pre - enrichment effect of biochar, thereby significantly improving the dechlorination degradation efficiency of chlorinated hydrocarbons.

[0024] In step S5 of this embodiment, the mixed gas in the anaerobic reactor consists of nitrogen and an appropriate amount of carbon dioxide. Nitrogen is the main component, and the proportion of carbon dioxide is controlled within the range that can ensure the stability of the anaerobic environment and participate in reaction regulation appropriately. The ratio of nitrogen to carbon dioxide is 95:5, and the gas flow rate is controlled at 0.1 L / min to ensure that the reactor is always in a stable anaerobic state.

[0025] In step S6 of this embodiment, the on-line monitoring module in the reactor includes a high-precision pH probe, a temperature sensor, and an electron donor concentration detector. The PLC system automatically drops 0.1 M NaOH or the corresponding acid solution according to the collected data to adjust the pH value to keep it between 7.0 and 7.5, and at the same time adjusts the heating or cooling device to keep the temperature within the range of 28-32 °C.

[0026] In step S7 of this embodiment, the preparation steps of the immobilized microspheres include: Fix the mixed system into microspheres with a diameter of 3-5 mm by titration; After the microspheres are prepared, use underground injection equipment to evenly distribute them in the contaminated soil; At the same time, deploy an underground sensor network in the contaminated area to realize real-time monitoring and dynamic data transmission of the pH, temperature, and pollutant concentration in the remediation area, so as to facilitate on-site operators to adjust the process according to the data.

[0027] In step S6 of this embodiment, the electron donor is lactic acid or acetate, and its initial concentration is set at 5-10 mM. According to the on-line monitoring data, it is replenished in real time by an automatic feeding device to ensure that the electron donor concentration is maintained at the optimal level, so as to promote the dechlorination and degradation reactions of chlorinated hydrocarbons.

[0028] In this embodiment, the multi-parameter on-line monitoring and automatic control system can collect the data in the reactor in real time, and perform feedback regulation on the lactic acid donor feeding, pH adjustment, and temperature control through the PLC system to realize a continuous and stable dechlorination and degradation process, ensuring that the system can quickly respond and adjust to the best state when the reactant concentration fluctuates.

[0029] Another object of the present invention is to provide an application of the biological remediation method for degrading chlorinated hydrocarbons by microorganisms in cooperation with colloidal coconut shell biochar as described above in the remediation of chlorinated hydrocarbon-contaminated soil and groundwater. It can not only achieve fine control in an ex-situ remediation reactor, but also be suitable for large-scale on-site remediation, and has the technical advantages of simple operation, environmental friendliness, low energy consumption, low cost, and long-term stability.

[0030] To facilitate those skilled in the art to better understand the technical solution of the present invention, the following specific embodiments of the present invention are given: Example 1, please refer to Figure 1 .

[0031] I. Preparation of the combined microbial agent 1. Collect 5 grams of soil samples from the chlorinated hydrocarbon-contaminated site, place them in a sterile centrifuge tube, and add 10 mL of anaerobic PBS buffer solution.

[0032] 2. Place the sample on a shaker and shake it at 100 rpm for 10 minutes to ensure that the microorganisms in the soil are fully dissolved.

[0033] 3. After shaking, centrifuge at 2000 rpm for 5 minutes, separate and collect the supernatant as the preliminary bacterial-containing solution.

[0034] 4. Inoculate the preliminary bacterial-containing solution into the previously prepared anaerobic culture medium. The formula of this anaerobic culture medium includes: 100-fold concentrated salt stock solution (sodium chloride, magnesium chloride, potassium dihydrogen phosphate, ammonium chloride, potassium chloride, and calcium chloride), trace element A stock solution (containing FeCl2·4H2O, CoCl2·6H2O, MnCl2·4H2O, ZnCl2, H3BO3, Na2MoO4·2H2O, NiCl2·6H2O, and CuCl2·2H2O), trace element B stock solution (Na2WO4·2H2O and NaOH), resazurin mother liquor, and 1000-fold vitamin solution. In the finally prepared 1 L culture medium, the concentrations of each component are as follows: Macronutrient salts (diluted from 10 mL of 100× concentrated salt stock solution): NaCl 1 g·L -1 , MgCl2·6H2O 0.5 g·L -1 , KH2PO4 0.2 g·L -1 , NH4Cl 0.3 g·L -1 , KCl 0.3g·L -1 , CaCl2·2H2O 0.015 g·L -1 ; Trace element A (1 mL·L -1 ): 25% HCl 0.01 mL·L -1 , FeCl2·4H2O 1.5 mg·L -1 , CoCl2·6H2O 0.19 mg·L -1 , MnCl2·4H2O 0.1 mg·L -1 , ZnCl2 0.07 mg·L -1 , H3BO3 0.006 mg·L -1 , Na2MoO4·2H2O 0.036 mg·L -1 , NiCl2·6H2O 0.024mg·L -1 , CuCl2·2H2O 0.002 mg·L -1 ; Trace element B (1 mL·L -1 ): Na2WO4·2H2O 0.008 mg·L -1 , NaOH 0.5 mg·L -1 ; Resazurin (0.1% mother liquor 0.25 mL·L -1 ): 0.25 mg·L -1; Carbon source and reducing agent: NaHCO3 30 mM (2.52 g·L -1 ), L-cysteine 0.2 mM (0.0242 g·L -1 ), Na2S·9H2O 0.2 mM (0.0480 g·L -1 ); Vitamins: biotin 20 μg·L -1 , folic acid 20 μg·L -1 , pyridoxine hydrochloride 100 μg·L -1 , riboflavin 50 μg·L -1 , thiamine 50 μg·L -1 , nicotinic acid 50 μg·L -1 , pantothenic acid 50 μg·L -1 , vitamin B 12 50 μg·L -1 , p-aminobenzoic acid 50 μg·L -1 and lipoic acid 50 μg·L -1 .

[0035] 5. Adjust the pH of the medium to 7.5. After deoxygenation by purging with nitrogen, perform aseptic filtration to ensure that the medium is in a stable anaerobic state.

[0036] 6. At 30 °C, subculture the inoculated medium multiple times until a highly active consortium with a dechlorination rate ≥ 0.20 mmol·L -1 ·d -1 (corresponding to a PCE removal rate ≥ 90%) and a culture broth OD 600 ≥ 0.8 is obtained. During the cultivation process, use an on-line monitoring device to record parameters such as pH and temperature in real time to ensure the stable growth and activity maintenance of the microbial community.

[0037] II. Preparation of colloidal coconut shell biochar powder 1. Select Hainan coconut shells. After thorough cleaning with running water, dry them in an oven at 105 °C for 24 hours to make the raw materials completely dry.

[0038] 2. Obtain coarse powder by mechanically crushing the dried coconut shells. First, pass through a 40-mesh (about 425 μm) sieve to remove large impurities; then pass through an 80-mesh (about 180 μm) sieve to obtain a uniform coarse powder with a concentrated particle size distribution suitable for pneumatic pulverization and pyrolysis, laying a foundation for subsequent biochar refinement and activation.

[0039] 3. Under nitrogen protection, place the coarse powder in a muffle furnace and pyrolyze it at 700 °C for 4 hours to obtain preliminary biochar.

[0040] 4. The preliminary biochar was processed by air flow mill, and the air flow pressure was controlled at 0.8MPa and the feed rate was about 2g / min to refine it into uniform powder.

[0041] 5. Prepare a suspension with a solid-liquid ratio of 1:5 (w / v) by mixing the dried biochar powder with Triton X-100 (non-ionic surfactant, CAS 9002-93-1): add 25 mL of 0.1% (w / v) Triton X-100 aqueous solution for every 5 g of biochar; place the suspension in a ball mill, add zirconium dioxide grinding beads, and grind it at 575 rpm for 6 h to obtain coconut shell biochar powder with finer particle size and more stable colloidal state.

[0042] 6. After centrifugal separation, the suspended matter after ball milling is dried using a vacuum freeze drying device to finally obtain colloidal coconut shell biochar powder (CAC) with uniform particle size and high specific surface area.

[0043] 3. Construction of synergistic degradation reaction system 1. The prepared combined bacterial agent and colloidal coconut shell biochar powder were fully mixed in a mass ratio of 1:5 to form a mixed system.

[0044] 2. Place the mixed system in an anaerobic reactor, and add about 70% volume of anaerobic culture medium into the reactor in advance.

[0045] 3. Connect a mixed gas containing nitrogen and an appropriate amount of carbon dioxide, with a nitrogen to carbon dioxide ratio of 95:5 (nitrogen is dominant, gas flow rate is 0.1L / min), to ensure that the reactor is continuously maintained in a strictly anaerobic state.

[0046] 4. Configure the online monitoring module, install high-precision pH probe, temperature sensor and donor detector, and realize data acquisition and automatic control through the PLC system. Automatically add 0.1 M NaOH or acid solution to keep the pH between 7.0 and 7.5, and control the reaction temperature at 30℃.

[0047] 5. During the reaction, lactic acid was used as an electron donor with an initial concentration of 8 mM, and was dynamically added through an automatic feeding device according to real-time detection data to ensure that the microbial degradation reaction was continuous and stable.

[0048] 6. GC-MS was used to detect the concentrations of PCE, TCE and intermediate products in the reaction system at regular intervals to verify the synergistic degradation effect. The experimental results showed that after 5 days of continuous operation, the degradation rate of tetrachloroethylene in the system exceeded 95%, and the concentration of intermediate products was significantly reduced, proving that this method has good dechlorination degradation performance.

[0049] IV. On-site in-situ repair technology solution (1)For an actual contaminated site, the above-mentioned co-degradation system is further processed, and the mixture is fixed into immobilized microspheres with a diameter of about 3-5 mm by titration. The immobilized microspheres have the advantages of good mechanical strength and long-term maintenance of microbial activity, which is convenient for uniform distribution in the contaminated area.

[0050] (2)The immobilized microspheres are evenly injected into the contaminated soil layer by using underground injection equipment. At the same time, an underground sensor network is arranged in the repair area to monitor the pH, temperature and chlorinated hydrocarbon concentration in the injection area in real time.

[0051] (3)The real-time data collected by the sensors is transmitted to the monitoring center through a wireless data transmission system. The on-site operators can adjust the dosage of the electron donor or supplement the immobilized microspheres in a timely manner based on this to ensure that the repair process is always maintained in the optimal state.

[0052] (4)This in-situ remediation plan has the advantages of simple construction operation, timely data feedback, and stable remediation effect, and is suitable for the treatment of industrial contaminated sites and contaminated sites in complex geological environments.

[0053] The specific application of in-situ remediation is as follows: (I)On-site investigation and plan formulation 1. Conduct detailed groundwater and soil sampling on an industrial contaminated site to detect the concentration of tetrachloroethylene, pH value, temperature and hydrogeological conditions in the area. First, use a handheld GPS locator to calibrate each sampling point in the contaminated site, select representative points, and consider the terrain, geological structure and groundwater flow direction to ensure the representativeness of the samples. For soil sampling, use sampling instruments determined through prior geological and hydrogeological surveys to conduct layered sampling at different depths, shallow layer (0-2 m), middle layer (2-10 m) and deep layer (10-15 m), to ensure a comprehensive reflection of the distribution of pollutants in the entire soil layer. During the operation, first wipe the instruments with disinfectant, then collect the samples. After sampling, put the samples into sterile containers marked with the sampling point number, depth, date and temperature, and ensure that the samples are quickly sent to the laboratory under refrigerated conditions. For groundwater sampling, it is necessary to pump out the interfering water in advance with a low-flow pump before sampling. After the water quality is stable, collect samples from the groundwater well. At the same time, measure key parameters such as the on-site pH value, temperature, dissolved oxygen, conductivity, etc., and conduct immediate detection using a portable on-site water quality analyzer. The water samples are also clearly marked in airtight and low-temperature storage containers to ensure the integrity of the samples. All sampling processes need to record detailed operation logs, including the disinfection method of sampling instruments, sampling time, on-site environmental conditions and abnormal situations encountered, to provide a basis for subsequent data comparison and quality control.

[0054] 2. Adjust the preparation parameters of the combined microbial agent and CAC colloidal biochar according to the on-site environmental data to ensure that the remediation agent is suitable for the actual on-site environment. First, fine-tune the salt content, trace elements, and temperature conditions in the culture medium based on the hydrogeological data and soil physicochemical indicators collected on-site. For example, if the sampling data shows that the soil pH is acidic or alkaline, the initial pH value in the anaerobic culture medium should be appropriately adjusted (e.g., from 7.5 to 7.2 or 7.8) to match the on-site environment, enabling the microbial community to quickly adapt after injection. In addition, if the on-site temperature is generally lower than the laboratory culture temperature, the culture temperature can be adjusted to the outdoor environmental temperature range (e.g., 28 - 30 °C) during subculture, and the subculture time can be extended to ensure that the growth rate and activity of the microbial community are consistent with the on-site environment. Regarding the colloidal coconut shell biochar powder (CAC), if the organic matter content in the soil is high or the soil particle size is coarser, the pyrolysis temperature and ball milling process can be appropriately optimized, and the ball milling time and dispersion liquid ratio can be adjusted to prepare biochar powder with a particle size more suitable for the soil structure and higher porosity, thereby improving the adsorption capacity for target pollutants. During the entire parameter adjustment process, small-scale on-site tests or simulation tests should also be used to fine-tune the ratio and pretreatment method of the combined microbial agent and CAC by real-time monitoring of pollutant concentration changes, pH value, and microbial activity to ensure that the remediation agent can achieve the best degradation effect on-site.

[0055] 3. Develop an in-situ remediation plan for the site, including the preparation of immobilized microspheres, the selection of injection equipment, and the layout plan of the underground sensor network. Engineering and technical personnel need to fully investigate the actual situation of the polluted site and design the overall remediation process based on the site characteristics. First, after a detailed assessment of the soil layer structure, aquifer distribution, underground water flow direction, and pollutant concentration gradient of the site, develop the preparation process of immobilized microspheres, the selection plan of injection equipment, and the layout plan of the underground sensor network. The process parameters, equipment models, and layout positions of each link need to be clearly defined in the plan. For example, in the preparation of immobilized microspheres, the composition of the titrant, titration speed, curing temperature, and humidity control standards need to be determined; for the injection equipment, a suitable underground injection pump or injection system should be selected according to the soil layer hardness, porosity, and water content; while the layout of the underground sensor network needs to comprehensively consider the density of monitoring points, transmission distance, and interference problems of wireless signals to ensure stable and reliable data transmission. All plans have been verified by small-scale on-site tests to ensure that the overall remediation measures are practical and the operation process is reliable.

[0056] (2) Preparation and injection of immobilized microspheres 1. Fix the above mixed system into immobilized microspheres with a diameter of about 3 - 5 mm by titration under aseptic conditions. During the fixation process, pay strict attention to controlling the temperature, humidity and pH value to ensure that the microbial activity in the microspheres is not affected. First, use titration to make the mixed combined inoculant and CAC colloid biochar system into immobilized microspheres with a diameter of about 3 - 5 mm in an aseptic operating room. In this process, a titration device that has been sterilized by high temperature should be selected to ensure an aseptic operating environment. The operator needs to carry out the titration operation under constant temperature and humidity conditions (for example, the temperature is controlled at 25 - 28 °C and the humidity is controlled at 50% - 60%), and strictly monitor the pH value of the solution during the solidification process through an online pH monitoring instrument to keep it within the optimal activity range suitable for microorganisms (such as 7.0 - 7.5). During the titration process, it is recommended to carry out prefabrication in batches, and perform activity detection after each batch is prepared to ensure that the microbial activity in the prepared microspheres does not decrease significantly, so as to ensure its degradation ability during the subsequent injection and repair process.

[0057] 2. Use an underground injection device to evenly inject the immobilized microspheres into the contaminated soil layer. The injection depth and uniformity are determined according to the on-site soil conditions. When choosing the injection device, the structural characteristics of the soil layer and the specific on-site conditions should be considered. Commonly used devices include hydraulic injection pumps or electric injection machines, which are required to have the function of accurately controlling the injection rate and injection pressure. During operation, first establish an injection well in advance through drilling or excavation according to the soil conditions. The wellhead and the wellbore need to be aseptically treated to prevent secondary pollution. During the injection process, the device displays the injection depth, injection rate and injection pressure data in real time. The operator adjusts the device parameters in a timely manner according to the on-site feedback information to ensure that the immobilized microspheres are evenly distributed in the contaminated soil layer, and at the same time avoid local overdosage or equipment blockage. The injection depth is usually determined according to the soil layer thickness and the groundwater level, and strives to cover the target contaminated area, and conduct on-site detection to confirm the uniformity after the injection is completed.

[0058] 3. Install an underground sensor network around the injection area to monitor the pH, temperature and chlorinated hydrocarbon concentration in the soil layer in real time, and transmit the data to the monitoring center through a wireless transmission system. The layout plan of the sensor network should include multi-parameter detection devices, such as high-precision pH probes, temperature sensors and chlorinated hydrocarbon concentration detectors. These devices form a monitoring network covering the entire repair area through monitoring points buried at different depths and different positions. Before installation, it is necessary to determine the location and quantity of the monitoring points through geological exploration to ensure that each key area is covered by monitoring. All sensor data is transmitted to the on-site monitoring center in real time through a wireless transmission module. The monitoring system has an automatic data recording and alarm function. Once abnormal parameters are detected, the system will immediately prompt the on-site technical personnel to take remedial measures. The entire transmission system should also be equipped with a backup power supply and signal relay equipment to ensure uninterrupted data transmission under harsh on-site environments, so as to provide reliable data support for the dynamic regulation of the repair process.

[0059] (III) On-site data collection and feedback control 1. Underground sensors are used to collect environmental parameter data in real time, and remote monitoring is achieved through wireless data transmission. During specific operations, various sensors are buried in each key repair area, including high-precision pH probes, temperature sensors, humidity sensors, and special chlorinated hydrocarbon concentration detectors. All sensors are pre-calibrated on-site and reasonable sampling frequencies are set to ensure the timeliness and accuracy of data. The data signals collected by the sensors are transmitted to the remote monitoring center via the on-site wireless data transmission module through a low-power remote transmission protocol. The monitoring center is equipped with a data storage and analysis system, which can automatically summarize, trend-analyze, and alarm for anomalies in the data of each monitoring point, thus realizing real-time remote monitoring of the entire repair process.

[0060] 2. Based on the real-time data feedback from the sensor network, on-site operators can dynamically adjust the key parameters in the repair system. Specifically, when the monitoring center shows that the electron donor concentration is lower than the preset optimal value, the system will automatically prompt the operator to add lactic acid or acetate, and at the same time record the specific amount added; if the monitoring data indicates that the injection density of the microspheres in the repair area is insufficient or unevenly distributed, the operator can adjust the injection quantity of the immobilized microspheres according to the data feedback and use the underground injection equipment to adjust the injection depth to ensure that the repair agent can be evenly diffused throughout the contaminated layer. During the entire dynamic adjustment process, the operator relies on the on-site real-time data chart and the automatic control system, and through the preset control strategies (such as PID control or fuzzy logic regulation), precisely operates the feeding system, injection equipment, and depth control module to ensure that the reaction system always operates under the best degradation state.

[0061] 3. After three consecutive months of on-site monitoring, the data recorded by the monitoring center shows that the concentration of tetrachloroethylene in the contaminated area shows a gradually decreasing trend and continues to decline within a certain period until it reaches below the national environmental safety standard. At the same time, the online monitoring data also shows that the pH value, temperature, and electron donor concentration in the repair area are all maintained within the preset range, indicating that the microbial degradation reaction is in a stable operating state. The on-site regular sampling test data is highly consistent with the wireless monitoring data, further verifying that this technical solution has good degradation effects and stability in practical engineering applications, providing solid data support and practical basis for large-scale promotion.

[0062] In summary, the present invention realizes the efficient, continuous, and long-term dechlorination and degradation of chlorinated hydrocarbon pollutants by constructing a synergistic degradation system of combined bacterial agents and colloidal coconut shell biochar, and combining advanced on-line monitoring and automatic regulation technologies. The technical solution of the present invention not only achieved remarkable results in the off-site verification stage, but also showed stable repair performance in the actual field application, and has extremely high engineering popularization and application value. All the above-mentioned public contents shall be regarded as a full disclosure of the present invention, and any modification, equivalent replacement or combination thereof shall fall within the protection scope of the present invention.

[0063] For those skilled in the art, it is obvious that the present invention is not limited to the details of the above-mentioned exemplary embodiments, and the present invention can be implemented in other specific forms without departing from the spirit or basic characteristics of the present invention. Therefore, from any point of view, the embodiments should be regarded as exemplary and non-limiting. The scope of the present invention is defined by the appended claims rather than the above description. Therefore, it is intended to include all changes falling within the meaning and scope of the equivalent elements of the claims in the present invention.

[0064] In addition, it should be understood that although this specification is described according to the embodiments, not every embodiment only contains an independent technical solution. This narrative way of the specification is only for clarity. Those skilled in the art should regard the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A bioremediation method for degrading chlorinated hydrocarbons by microorganisms in cooperation with colloidal coconut shell biochar, characterized in that, It includes the following steps: S1. Collect soil samples from the chlorinated hydrocarbon contaminated site, pretreat the soil with anaerobic PBS, shake it using a shaker and then centrifuge to separate, and extract the natural microbial flora therein to obtain a preliminary bacterial solution; S2. Inoculate the bacterial solution into a pre-prepared anaerobic medium, and perform multiple subculture under anaerobic conditions to form a combined microbial agent; S3. Prepare colloidal coconut shell biochar powder. The coconut shell is washed, dried, crushed, and then pyrolyzed under nitrogen protection to obtain a primary biochar product. After airflow pulverization and ball milling treatment, a dispersion liquid is added and mixed evenly, and then centrifugal separation and vacuum freeze-drying are used to obtain the colloidal coconut shell biochar powder; S4. Mix the combined microbial agent prepared in step S2 and the colloidal coconut shell biochar powder prepared in step S3 according to a predetermined ratio to form a mixed system with the synergistic effect of microorganisms and biochar; S5. Add the mixed system into a sterilized anaerobic reactor, add anaerobic medium into the reactor, and ensure that the reactor is always in a stable anaerobic state; S6. A multi-parameter on-line monitoring module is set in the reactor to detect and record key parameters in real time, and automatically regulate the concentration of the electron donor. The key parameters include pH, temperature, and the concentration of the electron donor; S7. In the in-situ remediation mode on site, the mixed system is fixed into immobilized microspheres with a diameter of 3-5 mm by titration method, and evenly distributed in the contaminated area through underground injection. At the same time, an underground sensor network is arranged in the remediation area to collect information on pH, temperature, and pollutant concentration in the remediation area in real time, and accordingly adjust the subsequent donor supplementation and injection volume.

2. The bioremediation method for degrading chlorinated hydrocarbons by microorganisms and colloidal coconut shell biochar according to claim 1, characterized in that, In step S1, the operating conditions for shaking using a shaker and then centrifuging are shaking for 10-12 minutes and centrifuging at 2000 rpm for 5-8 minutes.

3. A bioremediation method for degrading chlorinated hydrocarbons by microorganisms in cooperation with colloidal coconut shell biochar according to claim 1, characterized in that, In step S2, the steps for preparing the anaerobic medium include: Prepare 100-fold concentrated salt stock solution, the components of which are sodium chloride, magnesium chloride, potassium dihydrogen phosphate, ammonium chloride, potassium chloride, and calcium chloride; Prepare trace element stock solution, including trace element A stock solution and trace element B stock solution. The trace element A stock solution contains FeCl2·4H2O, CoCl2·6H2O, MnCl2·4H2O, ZnCl2, H3BO3, Na2MoO4·2H2O, NiCl2·6H2O, and CuCl2·2H2O, and the trace element B stock solution contains Na2WO4·2H2O and NaOH; Prepare resazurin mother liquor and 1000-fold vitamin solution, adjust the pH to 7.5 using sodium hydroxide, filter and sterilize after purging with nitrogen, and finally mix to obtain the anaerobic medium.

4. A bioremediation method for degrading chlorinated hydrocarbons by microorganisms in cooperation with colloidal coconut shell biochar according to claim 1, characterized in that, In step S3, the steps for preparing the colloidal coconut shell biochar powder include: After the dried coconut shell is preliminarily crushed, biochar is obtained through pyrolysis reaction; Using an airflow pulverizer, under an airflow pressure of 0.8 MPa, the biochar is refined at a rate of 2 g / min; Using a ball mill and zirconia grinding beads, set the rotation speed at 550-600 rpm and grind for 6 hours, and then obtain the colloidal biochar powder through centrifugation and freeze-drying treatment.

5. A bioremediation method for degrading chlorinated hydrocarbons by microorganisms synergistically with colloidal coconut shell biochar according to claim 1, characterized in that, In step S4, the mixing ratio of the combined inoculant to the colloidal coconut shell biochar powder in the mixed system is 1:

5.

6. The bioremediation method for degrading chlorinated hydrocarbons by microorganisms in cooperation with colloidal coconut shell biochar according to claim 1, characterized in that, In step S5, the mixed gas in the anaerobic reactor consists of nitrogen and carbon dioxide, and the ratio of nitrogen to carbon dioxide is 95:5, and the gas flow rate is controlled at 0.1 L / min.

7. A bioremediation method for degrading chlorinated hydrocarbons by microorganisms in cooperation with colloidal coconut shell biochar according to claim 1, characterized in that, In step S6, the on-line monitoring module in the reactor includes a high-precision pH probe, a temperature sensor and an electron donor concentration detector. The PLC system automatically regulates the pH value according to the collected data to keep it between 7.0 and 7.5, and at the same time regulates the heating or cooling device to keep the temperature in the range of 28-32 °C.

8. A bioremediation method for degrading chlorinated hydrocarbons by microorganisms in cooperation with colloidal coconut shell biochar according to claim 1, characterized in that, In step S7, the preparation steps of the immobilized microspheres include: Fixing the mixed system into microspheres with a diameter of 3-5 mm by titration; After the microspheres are prepared, they are evenly placed in the contaminated soil by using underground injection equipment; At the same time, an underground sensor network is arranged in the contaminated area to realize real-time monitoring and dynamic data transmission of the pH, temperature and pollutant concentration in the repair area, so as to facilitate on-site operators to adjust the process according to the data.

9. A bioremediation method for degrading chlorinated hydrocarbons by microorganisms in cooperation with colloidal coconut shell biochar according to claim 1, characterized in that, In step S6, the electron donor is lactic acid or acetate, and its initial concentration is set at 5-10 mM, and it is supplemented in real time by using an automatic feeding device according to the on-line monitoring data.

10. An application of a bioremediation method for degrading chlorinated hydrocarbons by microorganisms and colloidal coconut shell biochar according to any one of claims 1-9 in the remediation of chlorinated hydrocarbon-contaminated soil and groundwater.

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