Method for constructing functional bacterium ecological niche in soil to degrade chlorinated hydrocarbon in soil

Abstract

The invention discloses a method for constructing a functional bacterium ecological niche in soil to degrade chlorohydrocarbon in the soil, which comprises the following steps: S1, loading functional bacteria for degrading chlorohydrocarbon in a biochar pore channel, and preparing an active microsphere with a pore channel structure by using calcium alginate as a gelling agent; the functional bacteria comprise sphingosine Finincella; s2, laying the active microspheres prepared in the step S1 on a soil surface lower layer of a chlorohydrocarbon polluted site to form a functional layer with degradation activity, namely a soil active layer, so that functional bacteria obtain ecological niche with competitive capacity with indigenous bacteria; and S3, drilling to the underground water, pumping out the underground water, spraying to the soil surface, filtering along the soil and infiltrating back to the underground water to form an underground water circulating leaching system, and carrying out circulating suction filtration until the pumped underground water reaches the remediation standard. A stable and slowly moving functional bacterium ecological niche can be constructed in soil, and long-acting and stable degradation of chlorohydrocarbon in the soil and underground water is realized.

Description

Technical Field

[0001] The invention belongs to the technical field of soil and groundwater pollution control, and relates to a method for constructing a functional bacterial niche in soil to degrade chlorinated hydrocarbons in the soil; in particular, it relates to a method for constructing a stable and slowly moving functional bacterial niche in soil to degrade high-concentration chlorinated hydrocarbons in the soil in a long-term manner. Background Art

[0002] Due to leakage and emissions from industrial production activities, the soil and groundwater of industrial sites are currently seriously polluted by chlorinated hydrocarbons. For example, chlorinated hydrocarbons in the soil and groundwater of many sites in Putuo District and Baoshan District of Shanghai exceeded the standard; in a phosphate fertilizer plant in Hebei, trichloroethylene, 1,2-dichloroethane, and 1,1,2-trichloroethane in the groundwater exceeded the screening value by 3-4 times. Chlorinated hydrocarbons are generally highly chemically stable and toxic. Soil and groundwater contaminated by chlorinated hydrocarbons in industrial production activities will have a serious impact on the environment and human health, because these chlorinated hydrocarbons can enter the human body through air, water or food chains, thereby accumulating in the human body and increasing the risk of disease.

[0003] Traditional methods for repairing chlorinated hydrocarbon contaminated soil include physical methods and chemical methods. The physical method, based on the idea of ​​separation and extraction, uses technologies such as gas lift, thermal desorption or leaching. However, due to the high density of soil, this type of technology has large construction volume, high cost, and great damage to the soil environment; the chemical method, based on the idea of ​​converting pollutants into non-toxic or low-toxic substances, often adds chemical oxidizing agents such as persulfate, Fenton reagent or reducing agents such as zero-valent iron. Although it can accurately remove various pollutants, it may cause problems such as incomplete degradation of pollutants and secondary pollution. Although traditional physical (thermal desorption) and chemical (oxidant) methods can remove most of the chlorinated hydrocarbons in the soil, they have high energy consumption, high cost of soil repair, and the problems of environmental damage and secondary pollution still exist. Under rainfall erosion, even if the soil that meets the standards is repaired by physical and chemical methods, as long as a trace amount of soil chlorinated hydrocarbon pollutants is washed into the groundwater by rainfall, it will still cause the pollution concentration in the groundwater to exceed the standard. This is also the main reason why groundwater pollution remediation is difficult and always needs to face pollution rebound.

[0004] In addition to physical and chemical methods, biological methods are often used in the field of pollution control. They use the ability of some microorganisms or plants to metabolize organic pollutants to degrade pollutants. They have less secondary pollution and a wide range of applications, but the remediation efficiency is greatly affected by environmental conditions and the remediation time is long. The metabolic activities of microorganisms can effectively degrade chlorinated hydrocarbons to achieve the purpose of eliminating pollutants. However, in the actual process of soil pollution remediation, microbial degradation technology is rarely used. One of the bottlenecks that restrict its application is that soil remediation is different from water treatment and belongs to a static system. After the microbial liquid is applied to the soil system, its mass transfer is blocked, and it cannot fully contact with the pollutants, and the efficiency is very low. Due to this mass transfer limitation, the microbial cells themselves grow relatively slowly, making it difficult to fight against the indigenous bacteria in the soil to obtain a stable ecological niche, and eventually become inactivated, and the remediation effect is unstable. In addition, the soil itself has extremely strong heterogeneity, pollutants are unevenly distributed, and are tightly adsorbed on soil particles, making it more difficult for implanted functional bacteria to use and degrade.

[0005] The invention patent "A modified biochar composite material with embedded bacterial flora and its preparation and application" (application number: 202211633640.7) involves an application of embedding bacterial flora and magnetic biochar into alginate to prepare gel balls and use them in sewage treatment. The encapsulation technology is used to provide a living environment for microorganisms, thereby promoting the removal of pollutants such as COD, BOD, antibiotics and heavy metals in sewage. The gel material can be reused many times, with excellent remediation effect and low cost. However, it uses wastewater as the treatment medium, and wastewater and soil are completely different systems. It only considers the recyclability of the composite material, so it emphasizes the magnetism of biochar, and the substance applied to the soil does not need to consider its recyclability. The bacteria it embeds are a mixed composite flora, which does not have degradation specificity and is difficult to target difficult-to-degrade chlorinated hydrocarbon pollutants. Therefore, this technology is difficult to use for the remediation of chlorinated hydrocarbons in the soil.

[0006] The patent application "Zero-valent iron-biochar-microorganism ternary layered gel beads and their preparation method and application" (application number: 202311702850.1) relates to a method for preparing zero-valent iron-biochar-microorganism ternary layered gel beads and their application in the degradation of chlorinated hydrocarbon contaminated wastewater. When preparing the gel beads, zero-valent iron is added to half of the gel balls by layered dropwise addition, and biochar with microorganisms attached is added to the other half of the gel balls, thereby preparing ternary layered gel beads that can reduce chlorinated hydrocarbons. This technology not only plays the role of zero-valent iron in reducing chlorinated hydrocarbons, but also plays the role of microorganisms in degrading chlorinated hydrocarbons, making its remediation efficiency high. However, this technology is difficult to apply to the treatment of soil and groundwater in industrial sites, because the zero-valent iron therein is unstable in the soil environment and is easily oxidized quickly and loses its reducing effect; in addition, there is also a situation where the material has poor contact with pollutants in the soil static system, resulting in a very low treatment efficiency.

[0007] The invention patent "A method for treating petroleum-contaminated clay foundation with a microbial composite flora" (application number: 201810024716.3) involves a method of mixing a microbial agent with soil and using a microbial composite flora to degrade petroleum hydrocarbons in clay. The agent and soil are mixed evenly in proportion at the remediation site, and injection wells are arranged at the site to provide nutrients for the microorganisms, and detection wells are arranged to test the remediation effect. It directly contacts the agent with the soil, adds a flora with degradation ability to the soil, and provides additional nutrients to the microorganisms, but it does not only promote the growth of functional bacteria, but also the indigenous bacteria will grow in large quantities. It still cannot solve the problem that the functional flora is difficult to fight against the indigenous bacteria, which affects the degradation efficiency and is difficult to avoid secondary pollution.

[0008] As for the degradation and remediation of chlorinated hydrocarbon pollutants in the soil environment, it is difficult to allow the functional bacteria to occupy a stable ecological niche in the soil and establish a stable symbiotic relationship with the indigenous flora by directly adding functional bacteria to the soil due to the strong survival competitiveness of the indigenous flora. Therefore, for many years, the microbial remediation technology of chlorinated hydrocarbon pollutants has not been well applied, and there are few successful engineering cases, especially it is difficult to maintain long-term remediation. In addition, the soil properties of different sites vary greatly. Even if a functional bacterium has a certain degradation ability in a certain piece of soil, after changing the site, its ecological restoration effect cannot be guaranteed. It is difficult to replicate in different sites, and the functional bacteria often fail.

[0009] Through the above analysis, it can be found that there are several main problems in the technology of embedding microorganisms and biochar for soil or groundwater bioremediation: (1) Due to the strong survival competitiveness of indigenous bacteria, it is difficult for the functional bacteria to gain an advantage in the competition with indigenous bacteria, establish a stable ecological niche for the added bacteria, and form a stable bacterial community structure with the soil indigenous bacteria; (2) Soil is a static system. Unlike wastewater and water bodies, it is difficult for microorganisms to form good contact with pollutants in the soil, especially chlorinated hydrocarbon pollutants in deep soil. It is not easy to directly implant functional bacteria into the deep soil system in conventional processes. Based on the above two points, microbial degradation technology has not yet been truly applied in the remediation of contaminated soil-groundwater. Summary of the invention

[0010] In view of the key problems that need to be solved above, the purpose of the present invention is to provide a method for degrading chlorinated hydrocarbons in soil by constructing a functional bacterial niche in the soil. By constructing a dynamic water circulation leaching system and combining biochar-loaded functional bacteria, a stable and slowly moving functional bacterial niche is constructed in the soil, so that the static soil microbial degradation system is transformed into a dynamic system, mass transfer is enhanced, and the proliferation of microorganisms and the full contact between the bacteria and pollutants are promoted; and the foreign functional bacteria are enabled to construct a stable niche in the soil environment, so that they can better compete with the indigenous bacteria in the soil, and at the same time, the functional niche is promoted to slowly spread in the soil, achieving a good repair effect and maintaining long-term degradation stability.

[0011] The objective of the present invention is achieved through the following technical solutions:

[0012] The present invention provides a method for constructing a functional bacterial niche in soil to degrade chlorinated hydrocarbons in the soil, comprising the following steps:

[0013] S1. Loading functional bacteria that degrade chlorinated hydrocarbons into biochar pores, and then using calcium alginate as a gelling agent to prepare the biochar loaded with the functional bacteria into active small spheres with a pore structure; the functional bacteria include Sphingosine Finnish bacteria;

[0014] S2, laying the active small spheres prepared in step S1 on the subsurface layer of the soil in a chlorinated hydrocarbon contaminated site (a soil-groundwater co-contaminated system with a high pollution concentration) to form a soil active layer;

[0015] S3. Extract the groundwater and spray it onto the soil surface. The groundwater will pass through the soil active layer, filter along the soil and seep back into the underground, forming a groundwater circulation leaching system. The system will circulate and filter until the extracted groundwater is restored to meet the standards.

[0016] As some specific embodiments of the present invention, in step S1, the biochar can be prepared by pyrolysis of biomass solid waste, and the biomass solid waste includes at least one of crop straw and livestock manure. The crop straw includes one or more of corn straw, wheat straw, rice straw, etc.

[0017] As some specific embodiments of the present invention, in step S1, the method for preparing biochar specifically includes: crushing and drying the biomass solid waste, pyrolyzing it under anaerobic conditions, grinding it and then sieving it to obtain the biochar.

[0018] Furthermore, the drying temperature may be 60-100°C and the drying time may be 12-24h;

[0019] and / or, when pyrolyzing under anaerobic conditions, heating to 500°C at a heating rate of 10-15°C / min and maintaining for 1-2h;

[0020] And / or, the sieve has a pore size of 2-3 mm.

[0021] As some specific embodiments of the present invention, in step S1, the method for preparing active spheres specifically comprises the following steps:

[0022] S11, adding biochar to the functional bacteria liquid, stirring evenly, then adding sodium alginate powder, stirring to dissolve it, and forming a black gel-like liquid;

[0023] S12. Add the black gel-like liquid (through a peristaltic pump and a hose) dropwise into the calcium chloride solution with stirring. The gel and calcium chloride are cross-linked to form calcium alginate gel balls. After the titration is completed, continue stirring to allow sufficient cross-linking. Soak and wash with deionized water to obtain the alginate gel balls.

[0024] As some specific embodiments of the present invention, in step S11, the method for preparing the functional bacteria liquid includes: expanding and culturing the functional bacteria in R2A culture medium, centrifuging and precipitating the bacteria, resuspending and washing them with phosphate buffer, and diluting to obtain the functional bacteria liquid.

[0025] As some specific embodiments of the present invention, in step S11, biochar is added to the functional bacteria liquid at a mass ratio of 2-4%, and sodium alginate powder is then added at a mass ratio of 1-3%.

[0026] As some specific embodiments of the present invention, in step S12, the volume ratio of the calcium chloride solution to the black gel-like liquid is 1-3:1.

[0027] As some specific embodiments of the present invention, in step S2, when the active small spheres are laid in the subsurface layer of the soil of the chlorinated hydrocarbon contaminated site, the surface soil of the contaminated site needs to be plowed first, the subsurface soil needs to be loosened, and then the active small spheres are evenly mixed with the subsurface soil and laid in the subsurface layer of the soil to form a soil active layer;

[0028] And / or, the thickness of the soil active layer is 5-10 cm.

[0029] As some specific embodiments of the present invention, in step S3, groundwater is extracted by drilling holes, the interval of the holes is 2-4m, and the density of the holes is 0.25-1 hole / m 2 , the radius of the drilling hole can be 8-12cm.

[0030] As some specific embodiments of the present invention, in step S3, the extracted groundwater is sprayed onto the soil surface through a rotary spray water distributor, and the water distributors are set at intervals of 2-4m, or at a density of 0.25-1 water distributor / m 2 The water distribution flow rate is set to about 4 to 8 m per 100 m2 of land. 3 / d, the specific water distribution flow rate needs to be determined according to the actual site soil infiltration rate, that is, when the permeability is strong, the water distribution flow rate is increased, and when the permeability is weak, the water distribution flow rate is reduced to maintain a stable flooding state on the surface.

[0031] As some specific embodiments of the present invention, in step S3, 1-2 g / L sodium 3-hydroxybutyrate is first added to the extracted groundwater before spraying it onto the soil surface. Specifically, the sodium 3-hydroxybutyrate is added in a water distributor.

[0032] As some specific embodiments of the present invention, in step S3, when performing the circulating leaching, the top of the chlorinated hydrocarbon contaminated site is sealed to prevent the chlorinated hydrocarbons from volatilizing in the form of gas.

[0033] As some specific embodiments of the present invention, in step S3, the standard for detecting whether groundwater remediation meets the standard includes at least one of the Soil Environmental Quality - Construction Land Soil Pollution Risk Control Standard (Trial) (GB-36600-2018) and the Groundwater Quality Standard (GB-14848-2017).

[0034] In order to enhance the application effect of the contaminated soil microbial remediation technology, the present invention proposes a method of utilizing groundwater "extraction-leaching" and cyclic reciprocating, extracting groundwater contaminated by chlorinated hydrocarbons and spraying it evenly on the active layer, allowing the chlorinated hydrocarbons to be adsorbed and fixed by gel balls in the active layer and degraded by functional microorganisms, while the purified groundwater continues to infiltrate, washing the chlorinated hydrocarbons deep in the soil into the groundwater, and then being extracted again to the active layer for degradation, thereby achieving cyclic leaching of chlorinated hydrocarbons. Through this system, groundwater leaching flows through the soil, which can in situ strengthen the mass transfer of the soil-groundwater system, and by additionally applying sodium 3-hydroxybutyrate, the implanted functional bacteria can better obtain nutrients to achieve proliferation and can more fully contact with pollutants; at the same time, the mechanical flushing effect of the water flow can desorb pollutant molecules from soil particles (pollutant molecules are usually tightly adsorbed in the soil colloidal particle system), which is more conducive to increasing their potential for utilization by microorganisms. Every time groundwater leaches through the soil, it carries organic pollutants with it, and the pollutants are also efficiently degraded during the movement, which can greatly enhance the efficiency of microorganisms in situ degradation of soil pollutants.

[0035] Functional microorganisms and biochar are co-encapsulated into calcium alginate gel balls, and the gel balls and the internal biochar provide shelter for the functional bacteria, promoting niche competition between the functional bacteria and indigenous bacteria, thereby building a functional bacterial community with the ability to degrade chlorinated hydrocarbons in the soil. The chlorinated hydrocarbons are both adsorbed and fixed by the gel balls and degraded by the functional microorganisms. The soil moisture is maintained through the circulation and leaching of groundwater, and the activity of the bacteria is maintained. In addition, the water flow is conducive to carrying the functional bacteria in the gel balls to diffuse outward, so that the functional bacteria can slowly diffuse in the soil system with small spheres as the niche, maintaining a long-term degradation stability, and realizing low-cost remediation of soil contaminated with high-concentration chlorinated hydrocarbons.

[0036] Furthermore, through the groundwater circulation leaching system, combined with the gel balls formed by functional microorganisms and biochar, a stable and slowly moving functional bacterial niche is constructed in the soil. With the action of groundwater flow, it slowly diffuses in the soil and degrades chlorinated hydrocarbons in multiple locations in the soil, which can achieve long-term degradation of chlorinated hydrocarbons in the soil and has degradation stability.

[0037] The invention patent "Integrated remediation method for heavy metal contaminated soil and groundwater" (application number: 201310278001.8) applies heavy metal stabilizers to the contaminated soil in proportion, and at the same time extracts the contaminated groundwater, and evenly sprays or irrigates the contaminated soil with the stabilizer. The heavy metals in the groundwater are intercepted by the soil and undergo adsorption, precipitation, ion exchange, etc. with the stabilizer together with the original heavy metals in the soil, so that the heavy metals are stabilized, and the remediated groundwater infiltrates back into the ground, thereby achieving the effect of simultaneously remediating the contaminated soil and groundwater. Compared with the traditional method of remediating soil and groundwater separately, the patented method can simultaneously remediate the heavy metals in the contaminated groundwater and soil in a simpler system, achieving the purpose of integrated remediation. It has the characteristics of simple treatment process and low cost. The significant difference between the present invention and the patent is that the target pollutants are completely different. The target pollutants of the patent are heavy metals, so the selected materials need to have an immobilization or stabilization effect on the heavy metals, which can be some chemical agents, the purpose of which is to stably fix the heavy metals in the soil; while the present invention targets organic pollutants, which need to be degraded and eliminated by microorganisms. Therefore, the purpose of groundwater circulation leaching is to enhance the growth and activity of bacteria, and to enable pollutants to flow and better contact with bacteria in the soil profile, so that they can be completely degraded and removed. Therefore, the groundwater circulation leaching technology of the present invention combined with gel balls that embed biochar and functional bacteria can well achieve this purpose.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] (1) Innovatively loading functional bacteria onto biochar and fixing them through the gelation of calcium alginate can create a microhabitat with a special structure for functional bacteria with degradation functions, which can not only transmit oxygen, but also slowly dissolve small molecular organic carbon and nutrients from the biochar material. At the same time, it also has certain pores to facilitate the slow release of bacteria after proliferation. This makes the protected chlorinated hydrocarbon degrading functional microorganisms (pure bacteria) have a strong niche competitiveness and can compete with soil flora for living space and nutrients. Otherwise, foreign pure bacteria implanted in the soil can hardly survive in the soil;

[0040] (2) When groundwater is extracted and sprayed onto the active layer, the biochar in the active layer plays an adsorption role while the functional bacteria play a degradation role, thereby removing most of the chlorinated hydrocarbons;

[0041] (3) Circulating extraction and spraying of groundwater increases hydraulic action, promotes the release of chlorinated hydrocarbons in deep soil, and allows functional microorganisms and chlorinated hydrocarbons to have better contact, thereby accelerating the microbial degradation of chlorinated hydrocarbons in soil and groundwater;

[0042] (4) Extracting chlorinated hydrocarbons from groundwater and spraying them onto the active layer, the pollutants in the groundwater are gathered in the active layer and degraded. At the same time, the infiltration of groundwater helps the surface functional bacteria to spread to the deep soil, thereby degrading pollutants at more locations. On the other hand, it can also bring out the chlorinated hydrocarbons in the pores of the deep soil. Multiple cycles can achieve integrated restoration of soil and groundwater.

[0043] (5) Through the groundwater circulation leaching system, combined with the gel balls formed by functional microorganisms and biochar, a stable and slowly moving functional bacteria niche is constructed in the soil. With the action of groundwater flow, it slowly diffuses in the soil and degrades chlorinated hydrocarbons at multiple locations in the soil, which can achieve long-term degradation of chlorinated hydrocarbons in the soil and has degradation stability;

[0044] (6) The method of the present invention can be applied to the degradation of high-concentration chlorinated hydrocarbons in soil, and has a high efficiency in degrading chlorinated hydrocarbons in soil with a concentration of 560 ppm or more. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Other features, objects and advantages of the present invention will become more apparent from the detailed description of non-limiting embodiments made with reference to the following drawings:

[0046] Figure 1 The flow chart of the preparation of gel balls for embedding biochar and functional bacteria of the present invention;

[0047] Figure 2 It is a schematic diagram of the soil active layer and groundwater circulation leaching system of the present invention;

[0048] Figure 3Schematic diagram of the working principle of the soil active layer and groundwater circulation leaching system to achieve integrated soil-groundwater remediation;

[0049] Figure 4 This is an experimental diagram of the adsorption capacity of 2,4,6-trichlorophenol by calcium alginate gel balls embedded with biochar;

[0050] Figure 5 The graph is a graph showing the change of the content of 2,4,6-trichlorophenol (TCP) in the groundwater of each control group in Example 3 with the number of cycles;

[0051] Figure 6 The graph is a graph showing the change of 2,4,6-trichlorophenol (TCP) content in the soil of each control group in Example 3 with the number of cycles;

[0052] Figure 7 This is a diagram of the bacterial community structure in the calcium alginate gel balls of control groups ③ and ⑤ in Example 4 after 90 days of constructing the soil active layer;

[0053] Figure 8 This is a diagram of the soil flora structure of the four control groups in Example 4. DETAILED DESCRIPTION

[0054] The present invention is described in detail below in conjunction with specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, several variations and improvements may be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0055] Soil contaminated by chlorinated hydrocarbons: Since the soil left behind by relocated industrial enterprises often has the risk of exceeding the standard of pollutants, according to the provisions of my country's Soil Pollution Investigation and Risk Assessment (GB36600-2018), any soil (Class I or Class II) with pollutants exceeding the screening value is contaminated soil. A large amount of survey data shows that chlorinated hydrocarbon pollution is very common in industrial sites, especially tetrachloroethylene, trichloroethylene, dichloromethane, trichloropropane, etc. These volatile substances have very low solubility in water and high density. They sink through the vadose zone under the action of gravity and capillary force in the form of heavy non-aqueous phase liquid (DNAPL), slowly dissolve in groundwater and migrate vertically downward to the deep fracture aquifer to form a stable pollution plume, causing simultaneous pollution of soil and groundwater.

[0056] In situ biodegradation: refers to the use of microorganisms in situ to degrade organic pollutants in the soil in soil pollution remediation technology, without the need to excavate the soil and place it in some equipment or containers to remove pollutants.

[0057] Biochar: Biochar is an aromatized carbon material prepared by pyrolyzing waste biomass, such as agricultural and forestry waste, livestock and poultry manure, etc. under oxygen-limited conditions. It has a certain pore structure and specific surface area and is often used as an environmentally friendly functional material for water, soil and air pollution control.

[0058] Pollution rebound: refers to the phenomenon that after environmental protection measures are taken, the pollution that was expected to be reduced actually increases in the later period. This phenomenon usually occurs when technical or policy measures are taken to reduce a certain type of pollution, but the subsequent adverse effects or incomplete repairs cause the original pollutant reduction effect to deteriorate, and the pollutants are released again, resulting in an increase in concentration.

[0059] Active layer: in the present invention, it refers to the soil layer with higher functional microbial activity. Because gel balls encapsulating functional bacteria are added, it has the dual functions of adsorbing and degrading pollutants.

[0060] Ecological niche: refers to the specific role and position that a species or individual occupies in an ecosystem. The niche is determined by the species' lifestyle, how it uses resources, and its interactions with other organisms. For example, some microorganisms may seek specific substrates in water or soil for decomposition, releasing nutrients that affect the growth and distribution of other microbial communities. Understanding the niche helps predict the response of microorganisms to environmental changes, assess the stability of microbial communities, and develop biotechnologies such as bioremediation and biocontrol.

[0061] Bioremediation technology: refers to a technical strategy to improve polluted environments by introducing specific biological species or enhancing existing biological communities. Its core concept is to use certain technical means to enhance the metabolic activities and biochemical reaction capabilities of organisms, repair and degrade harmful chemicals in soil, water or air, and thus restore the natural functions and health of the environment.

[0062] Example 1 Preparation of gel spheres encapsulating biochar and functional bacteria

[0063] like Figure 1 The figure shows a flow chart of the preparation of gel balls (i.e., active small spheres) encapsulating biochar and functional bacteria according to the present invention. The process specifically includes the following steps:

[0064] (1) The corn stalks were crushed and dried in an oven at 60 °C for 12 h, then pyrolyzed at 500 °C for 2 h in a nitrogen atmosphere at a heating rate of 10 °C / min, and then ground and sieved through a 2 mm sieve to obtain biochar.

[0065] (2) A pure bacterial strain with chlorinated hydrocarbon degradation ability, Sphingomonas fennica K101 (Sphingomonas fennica K101, purchased from the China Microbiological Culture Collection Administration Center) was used to 10427), cultured in sterile R2A medium (purchased from HopeBio, catalog number HB0167-2, composition shown in Table 1, pH value 7.2±0.2) at 25°C and at 6000 r min -1 The cells were centrifuged and precipitated, resuspended and washed with phosphate buffer, and diluted with water to obtain the OD 600 The functional bacterial solution is 6. The actual test OD 600 Take 0.5 mL of bacterial solution, add 4.5 mL of phosphate buffer, and then place it in a spectrophotometer for testing.

[0066] Table 1 R2A (HB0167-2) culture medium components

[0067]

[0068]

[0069] (3) Add corn straw biochar to the functional bacteria solution in step (2) at a mass ratio of 2%, stir evenly, then add sodium alginate powder at a mass ratio of 2%, stir several times to allow the sodium alginate to fully dissolve in the functional bacteria solution, and finally form a black gel-like liquid.

[0070] (4) The black gel-like liquid obtained in step (3) is dripped into a 2% calcium chloride solution drop by drop through a peristaltic pump and a hose with an inner diameter of 2 mm. During the dripping process, the calcium chloride solution is stirred with a magnetic stirring rotor to allow the gel to quickly cross-link with the calcium chloride to form calcium alginate gel balls. During the titration process, the amount of 2% calcium chloride solution used is twice the volume of the black gel-like liquid. After the titration is completed, stirring is continued for 30 minutes to allow the gel balls to fully cross-link, and then soaked and washed with deionized water for 3-4 times to obtain calcium alginate gel balls that encapsulate biochar and functional bacteria, i.e., active small spheres.

[0071] Example 2 Construction of soil active layer and groundwater circulation leaching system

[0072] Figure 2 The present invention is a schematic diagram of the integrated remediation of chlorinated hydrocarbons in soil and groundwater by using a soil active layer constructed by calcium alginate gel balls (i.e., active small spheres) embedded with biochar and functional bacteria and a groundwater circulation leaching system.

[0073] (1) The surface soil of the contaminated site is plowed, the subsurface soil is loosened, the active small spheres prepared in Example 1 are mixed evenly with the subsurface soil and laid on the subsurface soil, thereby implanting the active spheres in the soil of the contaminated site to form a soil active layer, and the thickness of the active layer can be set to be about 5-10 cm. Loosening the soil can make it easier for groundwater to flow in the later stage and avoid local water accumulation.

[0074] (2) At the same time, drilling holes are drilled at intervals of 2-4 m on the site, with the density of holes determined by the soil permeability (ranging from 0.25-1 hole / m 2 ), drill to the contaminated groundwater layer, keep the drilling radius about 10cm, install the pump, and install the rotary spray water distributor at intervals of 2-4 meters. The setting density of the water distributor is 0.25-1 water distributor / m 2 , and then seal the top of the entire site to prevent chlorinated hydrocarbons from volatilizing in the form of gas.

[0075] (3) When the repair starts, use a pump to extract the groundwater and spray it evenly onto the active layer through a water distributor. Add 1g / L of sodium 3-hydroxybutyrate required for the growth of Sphingosine Finnish bacteria K101 into the water distributor. For the carbon source required by the pure bacteria used in the present invention, if the strain is changed, the carbon source needs to be replaced at the same time. During the entire repair process, try to keep the active layer in a saturated water state, that is, the surface groundwater level just covers the active layer, which is more conducive to the adsorption of pollutants by the gel balls and the degradation of pollutants by the functional bacteria. The water distribution flow rate is set to a water distribution flow rate of about 4 to 8m per 100 square meters of land. 3 / d, the specific water distribution flow rate needs to be determined according to the actual site soil infiltration rate, that is, when the permeability is strong, the water distribution flow rate is increased, and when the permeability is weak, the water distribution flow rate is reduced to maintain a stable flooding state on the surface.

[0076] (4) Circulate until the groundwater is remediated to meet the standards.

[0077] Figure 3The principle of realizing soil-groundwater integrated remediation through the soil active layer groundwater circulation system constructed by the present invention. A soil active layer with both adsorption performance and microbial degradation ability is artificially constructed in the subsurface layer of the contaminated soil, and then the contaminated groundwater is pumped above the soil and leached through the soil active layer; the pollutants in the groundwater enter the soil active layer with the groundwater, and the gel balls embedded with biochar and functional bacteria in the active layer adsorb and degrade chlorinated hydrocarbons, the circulating leaching of groundwater maintains soil moisture, and the external carbon source leached with the groundwater provides nutrition to the functional bacteria to maintain the activity of the bacteria; under the action of hydraulic power, the pollutants in the soil that are not adsorbed and degraded are transferred to the groundwater, and then pumped to the active layer, and the cycle operation is repeated until the detection concentration of the remediation target pollutants in the soil and groundwater is lower than the control value specified in the current national standards [Soil Environmental Quality-Construction Land Soil Pollution Risk Control Standard (GB-36600-2018) and Groundwater Quality Standard (GB-14848-2017)]. The water flow is conducive to carrying the functional bacteria in the gel ball outward, so that the functional bacteria can slowly diffuse in the soil system with the small sphere as the ecological niche. The functional bacteria can use the small sphere as the base and slowly diffuse to the deeper soil system outside under the action of water. That is, the gel ball does not move, and the functional bacteria continue to multiply in the small sphere and diffuse into the soil with the water flow, thereby maintaining long-term degradation stability.

[0078] Example 3 Verification of the Degradation Capacity of 2,4,6-Trichlorophenol (TCP) in Soil and Groundwater

[0079] Before designing the microbial remediation process of the present invention, it is necessary to first verify the adsorption effect of the gel balls encapsulating the bacteria on pollutants, because the raw materials such as biochar and gel balls used in the design principle of the present invention have a certain fixation ability for pollutants. This is a basic experiment before starting the degradation ability verification experiment. Figure 4 The adsorption capacity of biochar-encapsulated calcium alginate gel balls for 2,4,6-trichlorophenol (TCP) was demonstrated. It can be seen that both the gel balls and biochar have significant adsorption effects on pollutants, which meets the basic principle requirements of the present invention, that is, they have a certain retention capacity for pollutants.

[0080] Several control experiments were set up in the experiment to verify the integrated repair capability, such as Figure 5 shown.

[0081] Control group ①: No functional bacteria were added to the soil;

[0082] Control group ②: Sphingosine Finnish bacteria K101 was directly applied to the subsurface layer of the soil by tillage;

[0083] Control group ③: Sphingosine Finnish bacteria K101 was directly embedded in calcium alginate into gel balls (without biochar, the remaining steps were the same as in Example 1), and applied to the same position in the soil;

[0084] Control group ④: Sphingosine Finnish bacteria K101 was simply mixed with biochar (without being embedded into gel balls with calcium alginate, and the remaining steps were the same as in Example 1) and then applied to the subsurface layer of the soil;

[0085] Control group ⑤: According to the method of Example 1, Sphingosine Finnish bacteria K101 and biochar were co-encapsulated to form calcium alginate gel balls, which were then applied to the same position of the soil.

[0086] The above-mentioned control groups and experimental groups were constructed, and the groundwater circulation leaching system was set up in the same manner as in Example 2 to circulate and leach the soil, and the TCP content of the groundwater extracted after each cycle was tested. Figure 5 As shown, the control group ② in which the bacteria are directly applied to the soil active layer can take effect quickly in the first cycle, but its ability to degrade TCP in the soil decreases significantly in the second cycle, and as the number of cycles increases, the degradation of TCP in the soil gradually slows down, which is the same as the control groups ③ and ④, and the degradation effect is not much different from the control group ① without applying functional bacteria. While the control group ⑤, that is, Example 1 of the present invention, the functional bacteria and biochar are co-embedded in calcium alginate gel balls, and after the active layer is constructed in the soil, although it is not obviously effective in the first cycle, as the number of cycles increases, its degradation effect is significantly enhanced, and the difference with the control groups ①-④ gradually increases, until after 11 cycles, the content of TCP in the extracted groundwater is close to 0. Therefore, the experimental results strongly prove that the present invention has the highest soil and groundwater TCP removal ability compared with other conditions.

[0087] according to Figure 6 The TCP concentration in the soil decreased rapidly after the first groundwater circulation leaching, from nearly 700 mg kg -1 Reduced to 100-200 mg kg -1 , and then the volatility gradually decreases with the increase of the number of cycles, proving that the soil contaminated by high-concentration TCP can be quickly repaired to the standard by the process designed by the present invention. The reason for the fluctuation of pollutant concentration in the soil is that with the progress of dynamic cycle leaching, the pollutants undergo frequent redistribution processes in groundwater and soil. Thanks to this redistribution of pollutants, they are more fully in contact with degrading bacteria, greatly increasing the degradation efficiency. They are not only captured and degraded in the constructed soil active layer, but also degraded by functional bacteria diffused into the soil profile.

[0088] Example 4 Verification of bacterial abundance in soil

[0089] The control group ⑤ of Example 3 (i.e., the active small spheres in Example 1) and the control group ③ of Example 3 (i.e., the functional bacteria were directly embedded in the calcium alginate gel spheres without adding biochar) were respectively placed in the active layer of the soil contaminated by chlorinated hydrocarbons. After 90 days of cycle leaching, the bacterial community structure in the gel spheres was detected. The results are as follows: Figure 7 As shown, the abundance of functional bacteria (Sphingomonas fennica K101) in the two comparative experiments is higher, indicating that it is difficult for the indigenous soil flora to "invade the territory of functional bacteria", and the use of calcium alginate gel balls can keep the functional bacteria alive. After 90 days of operation of the circulating leaching process, the content of functional bacteria in the gel balls of Example 1 (control group ⑤) was significantly higher than that of the control group ③, proving that the technology of the present invention, i.e., the use of calcium alginate to simultaneously embed biochar and functional bacteria, can better maintain the survival of functional bacteria and enhance the competitiveness of functional bacteria with other indigenous bacteria in the ecological niche.

[0090] Figure 8 It is shown that after the circulating leaching remediation process constructed according to Example 1, Example 2 and Example 3 was run for 90 days, the corresponding bacterial community structure in the soil of control group ②, control group ③, control group ④ and control group ⑤ were each between 10 cm and 12 cm below the active layer. It can be analyzed and concluded that the biochar-embedded functional bacterial microspheres prepared by the technology of the present invention maximize the protection of the functional degradation bacteria "Sphingosine Finnish Bacteria K101", which is still the dominant bacteria in the microspheres after a long period of operation; while the pure bacteria directly implanted into the soil without being embedded have formed a bacterial community with the indigenous soil bacteria, and its abundance has dropped to a very low level.

[0091] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art may make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A method for constructing a functional bacterial niche in soil to degrade chlorinated hydrocarbons in soil, characterized in that: The steps include: S1. Loading functional bacteria that degrade chlorinated hydrocarbons into biochar pores, and then using calcium alginate as a gelling agent to prepare the biochar loaded with the functional bacteria into active small spheres with a pore structure; the functional bacteria include Sphingosine Finnish bacteria; S2, laying the active small spheres obtained in step S1 on the subsurface layer of the soil in the chlorinated hydrocarbon contaminated site to form a soil active layer; S3. Extract the groundwater and spray it onto the soil surface. The groundwater will pass through the soil active layer, filter along the soil and seep back into the underground, forming a groundwater circulation leaching system. The system will circulate and filter until the extracted groundwater is restored to meet the standards.

2. The method according to claim 1, characterized in that In step S1, the biochar is prepared by pyrolysis of biomass solid waste, and the biomass solid waste includes at least one of crop straw and livestock manure.

3. The method according to claim 1 or 2, characterized in that: In step S1, the method for preparing biochar specifically comprises: crushing and drying the biomass solid waste, pyrolyzing it under anaerobic conditions, grinding it and then sieving it to obtain the biochar.

4. The method according to claim 3, characterized in that: The drying temperature is 60-100°C and the drying time is 12-24h; and / or, when pyrolyzing under anaerobic conditions, heating to 500°C at a heating rate of 10-15°C / min and maintaining for 1-2h; And / or, the sieve has a pore size of 2-3 mm.

5. The method according to claim 1, characterized in that In step S1, the method for preparing active spheres specifically comprises the following steps: S11, adding biochar to the functional bacteria liquid, stirring evenly, then adding sodium alginate powder, stirring to dissolve it, and forming a black gel-like liquid; S12. Add the black gel-like liquid dropwise into the calcium chloride solution with stirring. The gel and calcium chloride are cross-linked to form calcium alginate gel balls. After the titration is completed, continue stirring to allow sufficient cross-linking. Soak and wash with deionized water to obtain the alginate gel balls.

6. The method according to claim 5, characterized in that In step S11, biochar is added to the functional bacteria liquid at a mass ratio of 2-4%, and sodium alginate powder is then added at a mass ratio of 1-3%.

7. The method according to claim 5, characterized in that In step S12, the volume ratio of the calcium chloride solution to the black gel-like liquid is 1-3:

1.

8. The method according to claim 1, characterized in that: In step S2, when the active small spheres are laid in the subsurface soil layer of the chlorinated hydrocarbon contaminated site, the surface soil of the contaminated site needs to be plowed first, the subsurface soil needs to be loosened, and then the active small spheres are evenly mixed with the subsurface soil and laid in the subsurface soil layer to form a soil active layer; And / or, the thickness of the soil active layer is 5-10 cm.

9. The method according to claim 1, characterized in that: In step S3, groundwater is extracted by drilling holes, the interval between the holes is 2-4 m, and the density of the holes is 0.25-1 holes / m 2 , the radius of the drilling hole is 8-12cm; And / or, the pumped groundwater is sprayed onto the soil surface through a rotary spray distributor, with the distributors set at intervals of 2-4m or at a density of 0.25-1 distributor / m 2 .

10. The method according to claim 1, characterized in that In step S3, 1-2 g / L sodium 3-hydroxybutyrate is first added to the extracted groundwater before being sprayed onto the soil surface; And / or, in step S3, when performing the circulating leaching, the top of the chlorinated hydrocarbon contaminated site is sealed.

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