A bioremediation method for the degradation of chlorinated hydrocarbons by microorganisms in cooperation with colloidal coconut shell biochar and its application

Abstract

The present invention relates to the technical field of environmental pollution remediation, and specifically discloses a bioremediation method for degrading chlorinated hydrocarbons using microorganisms in collaboration with colloidal coconut shell biochar, and its application. The method comprises: preparing a combined bacterial agent, preparing colloidal coconut shell biochar powder, constructing a collaborative degradation system and designing a reaction system, and on-site in-situ remediation application. The method effectively overcomes the problems of insufficient microbial activity, unstable electron donor supply, and saturation of adsorbent materials in traditional bioremediation. The present invention can always maintain the remediation process in an optimal state, effectively improving the engineering practicality and reliability of on-site remediation. The method is simple to operate, has low energy consumption, and is cost-effective. It also has high environmental adaptability and long-term remediation effects, conforming to the concept of green remediation. The technical solution of the present invention is not only suitable for ex-situ remediation verification, but can also be extended to actual engineering applications in complex contaminated sites, with broad market prospects and promotional value.

Description

Technical Field

[0001] The present invention relates to the field of environmental pollution remediation technology, specifically to a bioremediation method for degrading chlorinated hydrocarbons using microorganisms in conjunction with colloidal coconut shell biochar, and its application. This technology is suitable for remediating industrial contaminated sites, groundwater contamination, and soil pollution. It can be used for both ex situ remediation verification and large-scale in situ remediation projects, and is environmentally friendly, energy-efficient, cost-effective, and long-lasting. Background Art

[0002] Chlorinated hydrocarbons (CHCs) are a class of chlorine-containing organic compounds, primarily including trichloroethylene (TCE), tetrachloroethylene (PCE), and chloroform. They are chemically stable, highly volatile, and mobile. They are commonly used as solvents and cleaning agents in industrial production and are also widely used in dry cleaning and pesticide production. Due to their potential harm to the environment and human health, chlorinated hydrocarbons have become a major global source of soil and groundwater contamination.

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

[0004] In recent years, coconut shell biochar has attracted widespread attention due to its wide source of raw materials, 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 difficult to regenerate. How to organically combine microbial degradation with the adsorption advantages of coconut shell biochar 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 that needs to be solved urgently. In addition, in actual engineering applications, the lack of online monitoring and automatic control technology in the reaction system often leads to large fluctuations in reaction conditions, affecting the remediation effect. Therefore, the development of an efficient remediation system that integrates microbial dechlorination degradation, biochar adsorption enrichment and online automatic control has important theoretical significance and engineering application prospects.‌ Summary of the Invention

[0005] The purpose of the present invention is to provide a bioremediation method for degrading chlorinated hydrocarbons by using microorganisms in collaboration with colloidal coconut shell biochar and its application. By constructing a synergistic system of combined bacterial agents and colloidal coconut shell biochar, the defects of traditional bioremediation, such as insufficient bacterial activity, easy saturation of adsorption materials, and imprecise reaction control, are overcome, thereby achieving efficient, continuous and long-term degradation of pollutants.

[0006] The present invention adopts the following technical solutions:

[0007] A bioremediation method for degrading chlorinated hydrocarbons by using microorganisms in conjunction with colloidal coconut shell biochar comprises the following steps:

[0008] S1. Soil samples were collected from chlorinated hydrocarbon contaminated sites, pretreated with oxygen-free PBS, shaken on a shaker, and then centrifuged to extract the natural microbial flora to obtain a preliminary bacterial solution.

[0009] S2, inoculating the bacterial solution into a pre-configured anaerobic culture medium, and performing multiple subcultures in an anaerobic environment to form a highly active combined bacterial agent;

[0010] S3. Preparation of colloidal coconut shell biochar powder (CAC): The coconut shell is washed, dried, crushed, and then pyrolyzed at 700°C under nitrogen protection for 4 hours to obtain a primary biochar product. After air flow milling and ball milling, an appropriate dispersant is added and mixed uniformly. Subsequently, centrifugation and vacuum freeze drying are performed to obtain a colloidal biochar powder with uniform particle size and high specific surface area.

[0011] S4, mixing the combined bacterial agent prepared in step S2 and the colloidal coconut shell biochar powder prepared in step S3 in a predetermined ratio to form a mixed system in which microorganisms and biochar act synergistically;

[0012] S5. Add the mixed system to a sterilized anaerobic reactor, add about 70% of the volume of anaerobic culture medium into the reactor, and introduce a mixed gas containing nitrogen and an appropriate amount of carbon dioxide (flow rate of 0.1-0.3 L / min) to ensure that the reactor is always in a stable anaerobic state;

[0013] S6. A multi-parameter online monitoring module is installed in the reactor to detect and record key parameters such as pH, temperature, and electron donor concentration in real time, and the electron donor concentration is automatically regulated by a PLC system or an automatic feeding device to ensure that the physical and chemical conditions in the reaction system are maintained at the optimal state, thereby achieving continuous dechlorination and degradation of chlorinated hydrocarbons;

[0014] S7. In the on-site in-situ remediation mode, the mixed system is fixed into immobilized microspheres with a diameter of 3 to 5 mm by titration method, and evenly distributed in the contaminated area by underground injection. At the same time, an underground sensor network is deployed in the remediation area to collect real-time information on pH, temperature and pollutant concentration in the remediation area, and adjust the subsequent donor addition and injection volume accordingly.

[0015] Preferably, in step S1, the operating conditions for shaking on a shaker followed by centrifugation are shaking for 10 minutes and centrifuging at 2000 rpm for 5 minutes, so as to ensure that the microorganisms are fully released and avoid excessive sedimentation disturbance.

[0016] Preferably, in step S2, the step of preparing the anaerobic culture medium includes:

[0017] Prepare 100 times concentrated salt solution, which contains sodium chloride, magnesium chloride, potassium dihydrogen phosphate, ammonium chloride, potassium chloride and calcium chloride;

[0018] Prepare trace element stock solutions, 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;

[0019] The resazurin mother solution and 1000-fold vitamin solution were prepared, the pH was adjusted to 7.5 with sodium hydroxide, the mixture was filtered and sterilized after nitrogen purge, and finally mixed to prepare an anaerobic culture medium.

[0020] Preferably, in step S3, the step of preparing colloidal coconut shell biochar powder comprises:

[0021] After the dried coconut shells are initially crushed, biochar is produced through pyrolysis reaction;

[0022] The biochar was finely divided using a jet mill at a rate of 2 g / min under an air flow pressure of 0.8 MPa.

[0023] A ball mill with zirconium dioxide grinding beads was used, and the speed was set at about 575 rpm. After grinding for 6 hours, the colloidal biochar powder was obtained by centrifugation and freeze-drying.

[0024] Preferably, the mixing ratio of the combined bacterial agent and the colloidal coconut shell biochar powder in the mixed system is 1:5.

[0025] Preferably, in step S5, the mixed gas in the anaerobic reactor consists of nitrogen and an appropriate amount of carbon dioxide, wherein nitrogen is the main component and the proportion of carbon dioxide is controlled within a range that ensures the stability of the anaerobic environment while appropriately participating in the reaction regulation. 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.

[0026] Preferably, in step S6, the online monitoring module in the reactor includes a high-precision pH probe, a temperature sensor and an electron donor concentration detector. The PLC system automatically adds 0.1 M NaOH or 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 to 32°C.

[0027] Preferably, in step S7, the preparation step of the immobilized microspheres comprises:

[0028] The mixed system is fixed into microspheres with a diameter of 3 to 5 mm by titration;

[0029] After the microspheres are prepared, they are evenly placed in the contaminated soil using underground injection equipment;

[0030] At the same time, an underground sensor network is deployed in the contaminated area to realize real-time monitoring and dynamic data transmission of pH, temperature and pollutant concentration in the remediation area, making it easier for on-site operators to adjust the process based on the data.

[0031] Preferably, in step S6, the electron donor is lactic acid or acetate, and its initial concentration is set at 5-10 mM, and is replenished in real time using an automatic feeding device based on online monitoring data.

[0032] Another object of the present invention is to provide an application of the above-mentioned bioremediation method of degrading chlorinated hydrocarbons by microorganisms in collaboration with colloidal coconut shell biochar in the remediation of chlorinated hydrocarbon-contaminated soil and groundwater.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] 1. It effectively overcomes the problems of insufficient microbial activity, unstable electron donor supply, and saturated adsorption materials in traditional bioremediation, achieving continuous and rapid dechlorination and degradation of chlorinated hydrocarbons;

[0035] 2. Through online monitoring and automatic control systems, key parameters such as pH, temperature, and electron donor concentration are collected in real time, enabling rapid compensation adjustments when reaction conditions fluctuate, keeping the repair process at its optimal state.

[0036] 3. Immobilized microsphere technology combined with an underground sensor network enables dynamic monitoring and feedback control of in-situ remediation, greatly improving the engineering practicality and reliability of on-site remediation.

[0037] 4. This method is easy to operate, has low energy consumption, is cost-effective, and has high environmental adaptability and long-term restoration effects, conforming to the concept of green restoration;

[0038] 5. The technical solution of the present invention is not only applicable to ex situ remediation verification, but can also be extended to actual engineering applications in complex contaminated sites, and has broad market prospects and promotion value. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 The present invention provides a flow chart of a bioremediation method for degrading chlorinated hydrocarbons by using microorganisms in collaboration with colloidal coconut shell biochar. DETAILED DESCRIPTION

[0040] The following is a clear and complete description of the technical solutions in the embodiments of the present invention in conjunction with specific embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0041] The present invention provides a bioremediation method for degrading chlorinated hydrocarbons by using microorganisms in collaboration with colloidal coconut shell biochar, comprising the following steps:

[0042] S1. Soil samples were collected from chlorinated hydrocarbon contaminated sites, pretreated with oxygen-free PBS, shaken on a shaker, and then centrifuged to extract the natural microbial flora to obtain a preliminary bacterial solution.

[0043] S2, inoculating the bacterial solution into a pre-configured anaerobic culture medium, and performing multiple subcultures in an anaerobic environment to form a highly active combined bacterial agent;

[0044] S3. Preparation of colloidal coconut shell biochar powder (CAC), wherein Hainan coconut shells are used as raw materials, which are washed, dried, crushed, and then pyrolyzed at 700°C under nitrogen protection for 4 hours to obtain a primary biochar product. After air flow milling and ball milling, an appropriate dispersant is added and mixed uniformly. Subsequently, centrifugation and vacuum freeze drying are performed to obtain colloidal biochar powder with uniform particle size and high specific surface area;

[0045] S4, mixing the combined microbial agent prepared in step S2 with the CAC colloidal coconut shell biochar powder prepared in step S3 in a predetermined ratio to form a mixed system in which microorganisms and biochar act synergistically, wherein the mass ratio of the microbial agent to the biochar is 1:5;

[0046] S5. Add the mixed system to a sterilized anaerobic reactor with a volume of about 3 L. Add about 70% of the volume of anaerobic culture medium into the reactor, and introduce a mixed gas containing nitrogen and an appropriate amount of carbon dioxide (flow rate of 0.1-0.3 L / min) to ensure that the reactor is always in a stable anaerobic state;

[0047] S6. A multi-parameter online monitoring module is installed in the reactor to detect and record key parameters such as pH, temperature, and electron donor concentration in real time, and the electron donor concentration is automatically regulated by a PLC system or an automatic feeding device to ensure that the physical and chemical conditions in the reaction system are maintained at the optimal state, thereby achieving continuous dechlorination and degradation of chlorinated hydrocarbons;

[0048] S7. In the on-site in-situ remediation mode, the mixed system is fixed into immobilized microspheres with a diameter of 3 to 5 mm by titration method, and evenly distributed in the contaminated area by underground injection. At the same time, an underground sensor network is deployed in the remediation area to collect real-time information on pH, temperature and pollutant concentration in the remediation area, and adjust the subsequent donor addition and injection volume accordingly.

[0049] In step S1 of this embodiment, the operating conditions for centrifugal separation after shaking on a shaker are shaking for 10-12 minutes and centrifuging at 2000 rpm for 5-8 minutes to ensure sufficient release of microorganisms and avoid excessive sedimentation disturbance.

[0050] In step S2 of this embodiment, the step of configuring the anaerobic culture medium includes:

[0051] Prepare 100 times concentrated salt solution, which contains sodium chloride, magnesium chloride, potassium dihydrogen phosphate, ammonium chloride, potassium chloride and calcium chloride;

[0052] Prepare trace element stock solutions, 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;

[0053] The resazurin mother solution and 1000 times vitamin solution were prepared, the pH was adjusted to 7.5 with sodium hydroxide, the mixture was filtered and sterilized after nitrogen purge, and finally mixed to prepare an anaerobic culture medium.

[0054] In step S3 of this embodiment, the steps of preparing colloidal coconut shell biochar powder include:

[0055] After the dried coconut shells are initially crushed, biochar is produced through pyrolysis reaction;

[0056] The biochar was finely divided using a jet mill at a rate of 2 g / min under an air flow pressure of 0.8 MPa.

[0057] A ball mill with zirconium dioxide grinding beads was used, and the rotation speed was set at 550-600 rpm for 6 hours, and then the colloidal biochar powder was obtained by centrifugation and freeze-drying.

[0058] In the mixed system of this embodiment, the mixing ratio of the combined bacterial agent and the colloidal coconut shell biochar powder is 1:5. This ratio can fully exert the microbial degradation function and the biochar adsorption pre-enrichment effect, thereby significantly improving the dechlorination efficiency of chlorinated hydrocarbons.

[0059] In step S5 of this embodiment, the mixed gas in the anaerobic reactor consists of nitrogen and an appropriate amount of carbon dioxide, wherein nitrogen is the main component and the proportion of carbon dioxide is controlled within a range that ensures the stability of the anaerobic environment while appropriately participating in the reaction regulation. 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.

[0060] In step S6 of this embodiment, the online monitoring module in the reactor includes a high-precision pH probe, a temperature sensor, and an electron donor concentration detector. The PLC system automatically adds 0.1 M NaOH or a corresponding acid solution based on the collected data to adjust the pH value to maintain it between 7.0 and 7.5, and simultaneously controls the heating or cooling device to maintain the temperature within the range of 28 to 32°C.

[0061] In step S7 of this embodiment, the preparation steps of the immobilized microspheres include:

[0062] The mixed system is fixed into microspheres with a diameter of 3 to 5 mm by titration;

[0063] After the microspheres are prepared, they are evenly placed in the contaminated soil using underground injection equipment;

[0064] At the same time, an underground sensor network is deployed in the contaminated area to realize real-time monitoring and dynamic data transmission of pH, temperature and pollutant concentration in the remediation area, making it easier for on-site operators to adjust the process based on the data.

[0065] In step S6 of this embodiment, the electron donor is lactic acid or acetate, and its initial concentration is set at 5-10 mM. Based on the online monitoring data, it is replenished in real time using an automatic feeding device to ensure that the electron donor concentration is maintained at an optimal level, thereby promoting the dechlorination and degradation reactions of chlorinated hydrocarbons.

[0066] In this embodiment, the multi-parameter online monitoring and automatic control system can collect data in the reactor in real time and perform feedback adjustment on the lactic acid donor addition, pH adjustment and temperature control through the PLC system to achieve a continuous and stable dechlorination degradation process, ensuring that the system responds quickly and adjusts to the optimal state when the reactant concentration fluctuates.

[0067] Another object of the present invention is to provide a bioremediation method for degrading chlorinated hydrocarbons by microorganisms in collaboration with colloidal coconut shell biochar as described above, and to apply the method in the remediation of chlorinated hydrocarbon-contaminated soil and groundwater. The method can achieve fine control in an ex situ remediation reactor and is also suitable for large-scale on-site remediation. The method has the technical advantages of simple operation, environmental friendliness, low energy consumption, low cost, and long-term stability.

[0068] In order to facilitate those skilled in the art to better understand the technical solutions of the present invention, specific embodiments of the present invention are given as follows:

[0069] Example 1, please refer to Figure 1 .

[0070] 1. Preparation of combined bacterial agents

[0071] 1. Collect 5 g of soil sample from a chlorinated hydrocarbon contaminated site, place it in a sterile centrifuge tube, and add 10 mL of oxygen-free PBS buffer.

[0072] 2. Place the sample on a shaker at 100 rpm for 10 minutes to ensure that the microorganisms in the soil are fully dissolved.

[0073] 3. After shaking, centrifuge at 2000 rpm for 5 minutes, separate and collect the supernatant as the initial bacterial solution.

[0074] 4. Inoculate the initial bacterial suspension into the pre-prepared anaerobic medium. This anaerobic medium consists of: a 100x concentrated salt solution (sodium chloride, magnesium chloride, potassium dihydrogen phosphate, ammonium chloride, potassium chloride, and calcium chloride), a trace element A solution (containing FeCl2·4H2O, CoCl2·6H2O, MnCl2·4H2O, ZnCl2, H3BO3, Na2MoO4·2H2O, NiCl2·6H2O, and CuCl2·2H2O), a trace element B solution (Na2WO4·2H2O and NaOH), a resazurin stock solution, and a 1000x vitamin solution. The final concentrations of each component in 1 L of medium are as follows: Macrosalts (diluted from 10 mL of the 100x concentrated salt 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 0.3 g·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 , ZnCl20.07 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% stock solution 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 , niacin 50 μg·L -1 , pantothenic acid 50 μg·L -1 , vitamin B 12 50 μg·L -1 , para-aminobenzoic acid 50 μg·L -1 and lipoic acid 50 μg·L -1 .

[0075] 5. Adjust the pH of the culture medium to 7.5, deoxygenate it with nitrogen purge, and then sterile filter it to ensure that the culture medium is in a stable anaerobic state.

[0076] 6. Subculture the inoculated culture medium several times at 30°C until a dechlorination rate of ≥ 0.20 mmol·L is achieved. -1 ·d -1 (corresponding to PCE removal rate ≥90%) and culture medium OD 600 Highly active combined bacterial agent with a pH of ≥ 0.8. During the culture process, online monitoring equipment records parameters such as pH and temperature in real time to ensure stable growth and activity of the bacterial population.

[0077] 2. Preparation of colloidal coconut shell biochar powder

[0078] 1. Select Hainan coconut shells, wash them thoroughly with running water, and then dry them in an oven at 105℃ for 24 hours to make the raw materials completely dry.

[0079] 2. The dried coconut shell is mechanically crushed to obtain a coarse powder, which is first passed through a 40-mesh (about 425 μm) sieve to remove large impurities; then passed through an 80-mesh (about 180 μm) sieve to obtain a uniform coarse powder with a concentrated particle size distribution suitable for airflow crushing and pyrolysis, laying the foundation for subsequent biochar refinement and activation.

[0080] 3. Under nitrogen protection, the coarse powder was placed in a muffle furnace and pyrolyzed at 700℃ for 4 hours to obtain preliminary biochar.

[0081] 4. The preliminary biochar was processed by air flow mill, controlling the air flow pressure at 0.8 MPa and the feed rate at about 2 g / min to refine it into a uniform powder.

[0082] 5. Prepare a suspension of dry biochar powder with Triton X-100 (a nonionic surfactant, CAS 9002-93-1) at a solid-to-liquid ratio of 1:5 (w / v): add 25 mL of 0.1% (w / v) Triton X-100 aqueous solution for every 5 g of biochar. Mill the suspension in a ball mill with zirconium dioxide beads at 575 rpm for 6 h to obtain a finer particle size and more stable colloidal coconut shell biochar powder.

[0083] 6. After centrifugation, the milled suspension is dried using a vacuum freeze-drying device to obtain colloidal coconut shell biochar powder (CAC) with uniform particle size and high specific surface area.

[0084] 3. Construction of synergistic degradation reaction system

[0085] 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.

[0086] 2. Place the mixed system in an anaerobic reactor, and add about 70% volume of anaerobic culture medium into the reactor in advance.

[0087] 3. Introduce 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 continues to maintain a strict anaerobic state.

[0088] 4. Configure an online monitoring module, install a high-precision pH probe, temperature sensor, and donor detector, and implement data acquisition and automatic control through a PLC system. Automatically add 0.1 M NaOH or acid to maintain the pH between 7.0 and 7.5, while controlling the reaction temperature at 30°C.

[0089] 5. During the reaction, lactic acid was used as an electron donor with an initial concentration of 8 mM. Lactic acid was added dynamically based on real-time detection data through an automatic feeding device to ensure that the microbial degradation reaction proceeded continuously and stably.

[0090] 6. GC-MS was used to regularly monitor the concentrations of PCE, TCE, and intermediates in the reaction system to verify the synergistic degradation effect. The results showed that after five days of continuous operation, the degradation rate of tetrachloroethylene in the system exceeded 95%, and the concentration of intermediates was significantly reduced, demonstrating the excellent dechlorination performance of this method.

[0091] IV. On-site in-situ repair technology plan

[0092] (1) For actual contaminated sites, the above-mentioned synergistic degradation system was further processed and the mixture was fixed into immobilized microspheres with a diameter of about 3 to 5 mm by titration. The immobilized microspheres have the advantages of good mechanical strength and long-term maintenance of microbial activity, which facilitates uniform distribution in the contaminated area.

[0093] (2) Use underground injection equipment to evenly inject the immobilized microspheres into the contaminated soil layer. At the same time, deploy an underground sensor network in the remediation area to monitor the pH, temperature and chlorinated hydrocarbon concentration of the injection area in real time.

[0094] (3) The real-time data collected by the sensor is transmitted to the monitoring center through a wireless data transmission system. On-site operators can adjust the amount of electron donor added or add immobilized microspheres accordingly to ensure that the repair process is always maintained in the optimal state.

[0095] (4) This on-site remediation solution has the advantages of simple construction and operation, timely data feedback, and stable remediation effect. It is suitable for pollution control in industrial contaminated sites and complex geological environments.

[0096] The specific applications of on-site in-situ repair are as follows:

[0097] (1) On-site investigation and plan formulation

[0098] 1. Detailed groundwater and soil sampling was conducted at a contaminated industrial site to measure tetrachloroethylene concentrations, pH, temperature, and hydrogeological conditions within the area. First, a handheld GPS locator was used to calibrate sampling points within the contaminated site. Representative locations were selected, taking into account topography, geological structure, and groundwater flow to ensure representative samples. For soil sampling, sampling instruments, pre-qualified by geological and hydrological surveys, were used. Sampling was conducted at different depths: shallow (0-2 meters), mid-layer (2-10 meters), and deep (10-15 meters). This ensured a comprehensive representation of the contaminant's distribution throughout the soil layer. Instruments were wiped with disinfectant before collecting samples. After sampling, samples were placed in sterile containers labeled with the sampling site number, depth, date, and temperature. Samples were refrigerated and promptly transported to the laboratory. Groundwater sampling was performed using a low-flow pump to remove any interfering water. After the water stabilized, samples were collected from the groundwater well. Key parameters, such as pH, temperature, dissolved oxygen, and electrical conductivity, were measured on-site. A portable water quality analyzer was used for immediate testing. Water samples are also clearly labeled in sealed, cryogenically stored containers to ensure sample integrity. A detailed log is maintained for all sampling processes, including sampling equipment disinfection methods, sampling time, on-site environmental conditions, and any abnormalities encountered, to provide a basis for subsequent data comparison and quality control.

[0099] 2. Adjust the preparation parameters of the combined inoculum and CAC colloidal biochar based on field environmental data to ensure the remediation agent is compatible with the actual field 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 indicates that the soil pH is acidic or alkaline, the initial pH value in the anaerobic culture medium should be appropriately adjusted (for example, from 7.5 to 7.2 or 7.8) to match the field environment and enable the bacterial community to quickly adapt after injection. In addition, if the field temperature is generally lower than the laboratory culture temperature, the culture temperature can be adjusted to the outdoor ambient temperature range (for example, 28-30°C) during the subculture process, and the subculture time can be extended to ensure that the growth rate and activity of the bacterial community are consistent with the field environment. Regarding colloidal coconut shell biochar (CAC) powder, if the soil has a high organic matter content or coarse soil particle size, it is possible to consider optimizing the pyrolysis temperature and ball milling process, adjusting the ball milling time and the dispersion ratio to produce a biochar powder with a particle size more suitable for the soil structure and a higher porosity, thereby improving the adsorption capacity of target pollutants. The entire parameter adjustment process should also be supported by small-scale field trials or simulation tests. By monitoring changes in pollutant concentrations, pH values, and microbial activity in real time, the ratio of the combined bacterial agent to CAC and the pretreatment method should be fine-tuned to ensure that the remediation agent can achieve optimal degradation results on site.

[0100] 3. Develop an on-site in-situ remediation plan, including the preparation of immobilized microspheres, the selection of injection equipment, and the deployment of an underground sensor network. Engineering technicians must thoroughly investigate the actual conditions of the contaminated site and design an overall remediation process based on the site's characteristics. First, after a detailed assessment of the site's soil structure, aquifer distribution, groundwater flow patterns, and pollutant concentration gradients, a plan for the preparation of immobilized microspheres, the selection of injection equipment, and the deployment of an underground sensor network must be developed. The plan must clearly define the process parameters, equipment models, and deployment locations for each step. For example, in the preparation of immobilized microspheres, the titrant composition, titration rate, curing temperature, and humidity control standards must be determined. Injection equipment should select an appropriate underground syringe pump or injection system based on soil hardness, porosity, and moisture content. The deployment of the underground sensor network must comprehensively consider the density of monitoring points, transmission distance, and wireless signal interference to ensure stable and reliable data transmission. All plans must undergo small-scale field validation to ensure the feasibility of the overall remediation measures and the authenticity of the operational procedures.

[0101] (II) Preparation and injection of immobilized microspheres

[0102] 1. The mixed system is titrated under sterile conditions to form immobilized microspheres approximately 3-5 mm in diameter. During the titration process, strict control of temperature, humidity, and pH is crucial to ensure that microbial activity within the microspheres is not compromised. First, the combined microbial agent and CAC colloidal biochar system are titrated to form immobilized microspheres approximately 3-5 mm in diameter within a sterile operating room. High-temperature sterilized titration equipment must be used to ensure a sterile operating environment. The titration should be performed under constant temperature and humidity conditions (e.g., 25-28°C and 50-60% humidity). The pH of the solution should be strictly monitored during the curing process using an online pH monitor to maintain it within the optimal activity range for microbial adaptation (e.g., 7.0-7.5). During the titration process, it is recommended to pre-prepare the microspheres in batches, with activity testing performed after each batch to ensure that the microbial activity within the prepared microspheres does not decrease significantly, thereby ensuring their degradation capacity during the subsequent injection remediation process.

[0103] 2. Use underground injection equipment to evenly inject the immobilized microspheres into the contaminated soil layer. The injection depth and uniformity are determined based on the on-site soil conditions. The selection of injection equipment should take into account the structural characteristics of the soil layer and the specific conditions of the site. Commonly used equipment includes hydraulic injection pumps or electric injection machines, which require precise control of the injection rate and pressure. During operation, an injection well is pre-established by drilling or excavation according to the soil conditions. The wellhead and wellbore must be sterilized to prevent secondary contamination. During the injection process, the equipment displays the injection depth, injection rate, and injection pressure in real time. Operators adjust equipment parameters based on field feedback to ensure uniform distribution of the immobilized microspheres within the contaminated soil layer while avoiding localized overfilling and equipment blockage. The injection depth is typically determined based on soil thickness and groundwater level, aiming to cover the target contaminated area. After the injection is completed, on-site testing is performed to confirm uniformity.

[0104] 3. Deploy an underground sensor network around the injection area to monitor the pH, temperature and chlorinated hydrocarbon concentration in the soil in real time, and feed the data back to the monitoring center through a wireless transmission system. The sensor network deployment plan should include multi-parameter detection equipment, such as high-precision pH probes, temperature sensors and chlorinated hydrocarbon concentration detectors. These devices are buried at monitoring points at different depths and locations to form a monitoring network covering the entire remediation area. Before deployment, geological surveys are required to determine the location and number of monitoring points to ensure that every 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 is equipped with automatic data recording and alarm functions. Once a parameter abnormality is detected, the system will immediately prompt on-site technicians 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 in harsh on-site environments, thereby providing reliable data support for the dynamic regulation of the remediation process.

[0105] (3) On-site data collection and feedback control

[0106] 1. Use underground sensors to collect environmental parameter data in real time and realize remote monitoring through wireless data transmission. In specific operations, a variety of sensors are buried in each key remediation area, including high-precision pH probes, temperature sensors, humidity sensors and dedicated chlorinated hydrocarbon concentration detectors. All sensors are pre-calibrated on-site and set with a reasonable sampling frequency to ensure the timeliness and accuracy of the data. The data signals collected by the sensors are transmitted to the remote monitoring center through the on-site wireless data transmission module via a low-power remote transmission protocol. The monitoring center is equipped with a data storage and analysis system that can automatically summarize, trend and alarm the data of each monitoring point, thereby realizing real-time remote monitoring of the entire remediation process.

[0107] 2. Based on the real-time data fed back by the sensor network, on-site operators can dynamically adjust key parameters in the remediation 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 record the specific amount added. If the monitoring data shows that the microsphere injection density in the remediation area is insufficient or unevenly distributed, the operator can adjust the injection quantity of immobilized microspheres based on the data feedback and use the underground injection equipment to adjust the injection depth to ensure that the remediation 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 charts and automatic control system, and uses preset control strategies (such as PID control or fuzzy logic adjustment) to accurately operate the feeding system, injection equipment and depth control module to ensure that the reaction system always operates in the optimal degradation state.

[0108] 3. After three consecutive months of on-site monitoring, data recorded by the monitoring center indicated that the concentration of tetrachloroethylene in the contaminated area showed a gradual downward trend, continuing to decline over time until it reached below national environmental safety standards. Simultaneously, online monitoring data also showed that the pH, temperature, and electron donor concentration within the remediation area remained within the preset ranges, indicating that the microbial degradation reaction was operating stably. The high consistency between the regular on-site sampling data and the wireless monitoring data further validated the excellent degradation effectiveness and stability of this technical solution in actual engineering applications, providing solid data support and practical basis for large-scale promotion.

[0109] In summary, the present invention achieves efficient, continuous, and long-term dechlorination and degradation of chlorinated hydrocarbon pollutants by constructing a combined bacterial agent and colloidal coconut shell biochar synergistic degradation system, and combining advanced online monitoring and automatic control technology. The technical solution of the present invention not only achieved remarkable results in the ex situ verification stage, but also showed stable repair performance in actual field applications, and has extremely high engineering promotion and application value. All of the above disclosures should be regarded as full disclosure of the present invention, and their modifications, equivalent replacements or combinations should fall within the scope of protection of the present invention.

[0110] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the invention can be embodied in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as illustrative and non-restrictive, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations that come within the meaning and range of equivalents of the claims be embraced therein.

[0111] In addition, it should be understood that although this specification is described in terms of implementation methods, not every implementation method contains only one independent technical solution. This narrative method of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

Claims

1. A bioremediation method for degrading chlorinated hydrocarbons by using microorganisms in collaboration with colloidal coconut shell biochar, characterized in that: The following steps are involved: S1. Soil samples were collected from chlorinated hydrocarbon contaminated sites, pretreated with oxygen-free PBS, shaken on a shaker, and then centrifuged to extract the natural microbial flora to obtain a preliminary bacterial solution. S2, inoculating the bacterial solution into a pre-configured anaerobic culture medium, and performing multiple subcultures in an anaerobic environment to form a combined bacterial agent; S3, preparing colloidal coconut shell biochar powder, the coconut shell is washed, dried, crushed, and then pyrolyzed under nitrogen protection to obtain a primary biochar product, which is then subjected to air flow milling and ball milling, and then added to the dispersion solution and mixed evenly, followed by centrifugal separation and vacuum freeze drying to obtain colloidal coconut shell biochar powder; S4, mixing the combined bacterial agent prepared in step S2 and the colloidal coconut shell biochar powder prepared in step S3 in a predetermined ratio to form a mixed system in which microorganisms and biochar act synergistically; S5. Add the mixed system to a sterilized anaerobic reactor, add anaerobic culture medium into the reactor, and ensure that the reactor is always in a stable anaerobic state; S6. A multi-parameter online monitoring module is installed in the reactor to detect and record key parameters in real time and automatically control the electron donor concentration. The key parameters include pH, temperature and electron donor concentration; S7. In the on-site in-situ remediation mode, the mixed system is fixed into immobilized microspheres with a diameter of 3 to 5 mm by titration, and evenly distributed in the contaminated area by underground injection. At the same time, an underground sensor network is deployed in the remediation area to collect real-time information on pH, temperature and pollutant concentration in the remediation area, and adjust the subsequent donor addition and injection volume accordingly; In step S6, the electron donor is lactic acid or acetate, and its initial concentration is set at 5-10 mM. It is replenished in real time using an automatic feeding device based on online monitoring data.

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

3. The bioremediation method for degrading chlorinated hydrocarbons by using microorganisms in collaboration with colloidal coconut shell biochar according to claim 1, characterized in that: In step S2, the step of configuring the anaerobic culture medium includes: Prepare 100 times concentrated salt solution, which contains 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, 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; The resazurin mother solution and 1000 times vitamin solution were prepared, the pH was adjusted to 7.5 with sodium hydroxide, the mixture was filtered and sterilized after nitrogen purge, and finally mixed to prepare an anaerobic culture medium.

4. The bioremediation method for degrading chlorinated hydrocarbons by using microorganisms in collaboration with colloidal coconut shell biochar according to claim 1, characterized in that: In step S3, the steps of preparing colloidal coconut shell biochar powder include: After the dried coconut shells are initially crushed, biochar is produced through pyrolysis reaction; The biochar was finely divided using a jet mill at a rate of 2 g / min under an air flow pressure of 0.8 MPa. A ball mill with zirconium dioxide grinding beads was used, and the rotation speed was set at 550-600 rpm for 6 hours, and then the colloidal biochar powder was obtained by centrifugation and freeze-drying.

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

5.

6. The bioremediation method for degrading chlorinated hydrocarbons by using microorganisms in collaboration 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, the ratio of nitrogen to carbon dioxide is 95:5, and the gas flow rate is controlled at 0.1 L / min.

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

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

9. Use of the bioremediation method of any one of claims 1 to 8 for degrading chlorinated hydrocarbons by using microorganisms in collaboration with colloidal coconut shell biochar in the remediation of soil and groundwater contaminated by chlorinated hydrocarbons.

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