A composite microbial agent and its application in remediation or control of persistent organic polluted sites

Abstract

The present invention relates to a composite microbial agent and its use in the remediation or management of persistent organic polluted sites. The composite microbial agent comprises a composite microorganism comprising Pseudomonas, Bordetella, Dokshima, and Brucella in a ratio of (0.1-10):(0.1-10):(0.5-1):1. This agent improves the remediation efficiency of persistent organic polluted sites and is particularly suitable for green and efficient remediation of soil and groundwater in sandy soil sites and for pollution source reduction risk management. It also offers advantages such as low cost and ease of operation, thus possessing significant application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of biodegradation treatment of contaminated sites, and specifically relates to a composite microbial agent and its application in repairing or controlling persistent organic polluted sites. Background Art

[0002] This section is intended to provide a background or context to the embodiments of the invention that are recited in the claims. No statement herein is admitted to be prior art by virtue of its inclusion in this section.

[0003] Under natural conditions, pollutants in groundwater undergo a natural attenuation mechanism driven by microorganisms and hydraulic forces, resulting in a decrease in pollutant concentrations. However, these geochemical reactions are influenced by numerous factors, including environmental, hydrological, and geological factors. Studies have shown that microbial degradation plays a major role in the natural attenuation of organic pollutants. However, due to environmental constraints, such as insufficient numbers of indigenous functional microorganisms, low activity levels, and slow growth, natural degradation is typically slow. Enhanced attenuation (EA) technology can compensate for this slow natural degradation. By adding microbial agents that degrade target pollutants to contaminated sites, the rate of microbial degradation of organic pollutants in groundwater can be increased.

[0004] Common persistent organic pollutants (POPs) in sandy soils, such as organochlorine pesticides and polychlorinated biphenyls (PCBs), are difficult to effectively degrade using traditional remediation methods due to their stable chemical properties, poor water solubility, and strong binding to soil particles. POPs pollution has diverse physical and chemical properties, making degradation challenging and difficult for a single bacterial community to effectively degrade simultaneously.

[0005] Therefore, it is necessary to develop a composite microbial agent suitable for persistent organic pollution (POPs pollution) in soil and groundwater in sandy soil sites, so as to solve the application bottleneck of existing microorganisms in the restoration or control of persistent organic pollution in soil and groundwater in sandy soil sites, and improve the restoration efficiency of persistent organic pollution sites and the risk control of pollution source reduction. Summary of the Invention

[0006] The purpose of the present invention is to provide a composite microbial agent and its application in the restoration or management of persistent organic polluted sites.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is:

[0008] A first aspect of the present invention provides a composite microbial agent, comprising composite microorganisms, wherein the composite microorganisms include Pseudomonas, Bordetella, Dokdonella, and Brucella at a viable cell count ratio of (0.1-10):(0.1-10):(0.5-1):1.

[0009] According to some specific embodiments, the ratio of the viable count of the Pseudomonas to the Brucella is 0.1:1, 0.5:1, 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, 5.5:1, 6:1, 6.5:1, 7:1, 7.5:1, 8:1, 8.5:1, 9:1, 9.5:1, 10:1, etc.

[0010] According to some specific embodiments, the ratio of the number of viable bacteria of Bordetella to that of Brucella is 0.1:1, 0.5:1, 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, 5.5:1, 6:1, 6.5:1, 7:1, 7.5:1, 8:1, 8.5:1, 9:1, 9.5:1, 10:1, etc.

[0011] According to some specific embodiments, the ratio of the number of live bacteria of the Dokshima bacteria to the Brucella is 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, 1:1, etc.

[0012] According to some specific embodiments, the ratio of the number of live bacteria of the Pseudomonas, Bordetella, Dokshima and Brucella is (1-5): (1-5): (0.5-1): 1.

[0013] Furthermore, the ratio of the number of live bacteria of the Pseudomonas, Bordetella, Dokshima and Brucella is (2-3): (2-3): (0.5~1): 1.

[0014] According to some embodiments, the viable count of the composite microorganism is not less than 1×10 9 CFU / mL.

[0015] According to some embodiments, the composite microorganism is in liquid form or solid form.

[0016] Furthermore, the composite microorganism is microbial fermentation liquid, microbial freeze-dried powder or glycerol bacteria.

[0017] Furthermore, when the composite microorganism is in liquid form, the viable count of the composite microorganism is not less than 1×10 9CFU / mL, the composite microbial agent is used directly or diluted with water. Wherein, the liquid composite microorganism is a microbial fermentation broth, which is obtained by culturing the Pseudomonas, Bordetella, Dokshima and Brucella separately or in combination to obtain a fermentation broth containing the four bacteria.

[0018] Furthermore, when the composite microorganism is in solid form, the composite microorganism is activated and cultured to a viable count of not less than 1×10 9 CFU / mL.

[0019] Furthermore, when the composite microorganism is in a solid form, the viable count of the composite microorganism is not less than 2×10 8 CFU / g.

[0020] According to some specific embodiments, the composite microbial agent further comprises a slow-release oxygen material, wherein the slow-release oxygen material comprises an active component capable of releasing oxygen and an inert component.

[0021] Furthermore, the active component is calcium peroxide; and the inert component includes bentonite.

[0022] Furthermore, the mass fraction of the active component in the slow-release oxygen material is 55% to 65%, for example, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, etc., and the mass fraction of the inert component in the slow-release oxygen material is 35% to 45%, for example, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, etc.

[0023] Furthermore, the mass ratio of the composite microorganism to the slow-release oxygen material is (2-50):1, for example, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1 1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1, 41:1, 42:1, 43:1, 44:1, 45:1, 46:1, 47:1, 48:1, 49:1 or 50:1.

[0024] Furthermore, the mass ratio of the composite microorganism to the slow-release oxygen material is (20-50):1.

[0025] Furthermore, the mass ratio of the composite microorganism to the slow-release oxygen material is (2-20):1.

[0026] According to some specific embodiments, the composite microbial agent further comprises trace elements.

[0027] Furthermore, the trace elements include Ca 2+ 、Fe 2+ 、Cu 2+ 、Zn 2+ 、Mn 2+ Mg 2+ One or more of .

[0028] Furthermore, the trace elements include 1 to 50 mg of Ca per liter of the composite microorganism. 2+ , 1~50mg Mg 2+ , 1~10mg Zn 2+ and 1~10mg of Mn 2+ .

[0029] The mass ratio of the composite microorganism to the slow-release oxygen material, and the volume of the composite microorganism in the amount of trace elements added, refer to the mass and volume of the composite microorganism when the composite microorganism is sold in liquid form; when the composite microorganism is sold in solid form, the mass ratio refers to the activation and cultivation of the composite microorganism to a viable count of not less than 1×10 9 The mass and volume of the fluid at the CFU / mL.

[0030] A second aspect of the present invention provides a use of the composite microbial agent as described above in the remediation or management of persistent contaminated sites, wherein the persistent contaminated sites include sandy soil and / or groundwater.

[0031] A third aspect of the present invention provides a method for remediating persistent pollution, comprising adding the composite microbial agent described above to silty soil and / or groundwater.

[0032] According to some specific embodiments, the composite microbial agent is added with water to form a slurry, and then the slurry is injected into the sandy soil and / or groundwater through an injection rod; or injected into the sandy soil and / or groundwater through a well; or groundwater is extracted and mixed with the composite microbial agent and re-injected.

[0033] According to some specific embodiments, the slurry is injected into the sandy soil after the sandy soil is pretreated by crushing and screening.

[0034] According to some specific embodiments, the pollutants in the powdery clay soil include one or more of polychlorinated biphenyls, organic pesticides, and dioxins. The organic pesticides may specifically include organochlorine pesticides, and the organochlorine pesticides may include hexachlorobenzene, pentachlorobenzene, and the like.

[0035] Due to the application of the above technical solution, the present invention has the following advantages compared with the prior art:

[0036] The present invention develops a composite microbial agent suitable for the remediation or control of persistent organic pollutant (POPs) pollution sites, and is particularly suitable for the green and efficient remediation of soil and groundwater in sandy soil sites and the risk reduction control of pollution sources. The composite microbial agent mainly solves the problem of the difficulty of remediation of persistent organic polluted sites through the synergistic effect of specific proportions of Pseudomonas, Bordetella, Dokdonella, and Brucella, overcomes the problems of low mass transfer efficiency and limited microbial activity of traditional remediation technologies, improves the remediation efficiency of persistent organic pollution in soil and groundwater in sandy soil sites, realizes green and efficient remediation and pollution source reduction risk control, and has the advantages of low cost and easy operation, and therefore has important application prospects. DETAILED DESCRIPTION

[0037] Because existing microorganisms are not very effective in remediating or controlling persistent organic pollutants in the soil and groundwater of sandy soil sites, the present invention has developed a composite microbial agent suitable for the remediation of persistent organic pollutants (POPs) contaminated sites, and is particularly suitable for the green and efficient remediation of soil and groundwater in sandy soil sites and the risk reduction and control of pollution sources. The composite microbial agent mainly uses the synergistic effect of specific proportions of Pseudomonas, Bordetella, Dokdonella, and Brucella, and is further combined with slow-release oxygen materials and trace elements. This effectively solves the difficulty of remediating persistent organic pollutants (POPs) contaminated sites, overcomes the problems of low mass transfer efficiency and limited microbial activity of traditional remediation technologies, improves the remediation efficiency of persistent organic pollutants in the soil and groundwater of sandy soil sites, achieves green and efficient remediation and pollution source reduction and risk control, and has the advantages of low cost and easy operation, thus having important application prospects.

[0038] Furthermore, among the composite microorganisms, the ratio of the number of live bacteria of Pseudomonas, Bordetella, Dokshima and Brucella is (0.1~10):(0.1~10):(0.5~1):1. If the addition ratio of each bacteria exceeds the upper limit or is lower than the lower limit, the repair effect of the composite microbial agent will be significantly reduced.

[0039] The functions and synergistic effects of various bacterial communities improve the remediation efficiency of persistent organic polluted sites as follows:

[0040] Pseudomonas can initiate the oxidation of polycyclic aromatic hydrocarbons (PAHs) through dioxygenases, generate intermediates, secrete substances such as rhamnolipids, improve the bioavailability of PAHs, and promote the degradation efficiency of itself and the community; it can degrade PAHs in both aerobic and microaerobic environments, and is particularly dominant in oxygen-rich surface soils.

[0041] Bordetella is good at degrading intermediates after PAHs oxidation (such as catechol and salicylic acid). It can rely on short-chain fatty acids or sugars as co-substrates to activate the dehydrogenase system to assist in the degradation of PAHs. In addition, it has strong tolerance to reactive oxygen species (ROS) produced by PAHs metabolism (such as high catalase activity).

[0042] Dokshima bacteria can degrade PAHs derivatives (such as hydroxylated PAHs) through amination reactions under low nutrient (such as nitrogen-deficient) conditions, and can secrete extracellular polysaccharides (EPS) to adsorb PAHs particles and protect other bacterial communities from toxic inhibition, and can couple PAHs oxidation and nitrate reduction.

[0043] Brucella may convert PAHs into low-toxic derivatives through the glutathione metabolic pathway, provide vitamins or iron carriers, promote the proliferation of degrading bacteria such as Pseudomonas, and participate in the partial oxidation of PAHs through microaerobic respiration in deep soil.

[0044] Synergistic mechanism

[0045] The composite microbial agent provided by the present invention can achieve efficient degradation of typical POPs pollutants, such as polycyclic aromatic hydrocarbons (PAHs), from emulsification and ring opening to complete mineralization through spatial differentiation, metabolic complementarity, and environmental adaptation strategies. First, Pseudomonas promotes the dissolution of PAHs in soil by secreting surfactants (such as rhamnolipids), increasing their bioavailability. It also secretes dioxygenases to initiate hydroxylation reactions, converting PAHs into intermediates such as catechol and salicylic acid. Bordetella can degrade PAH oxidation intermediates such as catechol and salicylic acid, completely mineralizing them into CO2 and H2O via the β-ketoadipate pathway. Furthermore, Brucella secretes extracellular polymers to form a biofilm, which assists Pseudomonas and Bordetella in the in situ colonization and formation of stable biofilm colonies. This ensures a sufficient number of microorganisms capable of pollutant degradation, fosters a degradation microenvironment, and enhances the stress resistance of the bacterial community. Furthermore, this inoculant exhibits excellent environmental adaptability. Under aerobic conditions, Pseudomonas and Bordetella synergistically degrade PAHs completely. Under microaerobic conditions, Bordetella and Dokshima bacteria effectively utilize soil nitrate as electron acceptors to jointly degrade methylated PAHs. Under anaerobic conditions, Dokshima bacteria utilize trivalent iron and sulfate as electron acceptors to degrade low-ring PAHs (such as naphthalene), releasing organic acids. This inoculant has a wide pH range of adaptability, and the extracellular polymers secreted by Pseudomonas and Dokshima bacteria, in particular, protect other functional bacteria from adapting to acidic or alkaline environments.

[0046] Summary of the synergy mechanism:

[0047] Table 1 below summarizes the synergistic mechanisms of the various bacterial groups in the microbial preparation provided by the present invention:

[0048]

[0049] Slow-release oxygen materials

[0050] The present invention provides a composite microbial agent specifically for the degradation of organic pollutants. The agent is composed of a composite of multiple high-efficiency degradation strains and can effectively degrade organic pollutants in groundwater and soil under aerobic conditions. In order to ensure that the agent maintains a high degradation activity during the remediation process, a slow-release oxygen material is further used in combination to provide a continuous and stable oxygen source to ensure that the microorganisms are in a suitable metabolic state. The oxygen supply level is crucial to the degradation rate of organic pollutants. Excessive slow-release oxygen materials may cause the oxygen release rate to be too fast, affecting the adaptability and survival of the agent, while insufficient oxygen supply may inhibit the metabolic activity of the agent and reduce the degradation efficiency. Therefore, in the present invention, slow-release oxygen materials are added to enable the agent to maintain a suitable aerobic state under different pollution environments and promote the efficient degradation of organic pollutants by microorganisms.

[0051] Active ingredient: CaO2 is currently the most commonly used oxygen-releasing agent. Calcium peroxide is inexpensive, easy to produce, and its reaction products are non-polluting. Regarding oxygen release, when CaO2 is added to water, an immediate reaction occurs, rapidly raising the dissolved oxygen concentration to a very high level (reaction equation: 2CaO2 + 2H2O → 2Ca(OH)2 + O2↑). This can easily lead to overdosing and a lack of consistent control over the oxygen release rate.

[0052] Calcium peroxide is used as the active component, and the suspended adsorption material bentonite is used as a carrier to disperse the calcium peroxide particles, slow down the release rate of oxygen, and enhance the stability of the slurry. Bentonite forms a stable crack network through adsorption and expansion, prevents soil closure, improves the pore structure of silty clay soil, and increases permeability. At the same time, it wraps CaO2 particles, slows down their reaction rate in contact with water, increases the dissolved oxygen level in the soil, and promotes the growth and metabolism of aerobic microorganisms.

[0053] Bentonite, through its adsorption and isolation properties, limits direct contact between calcium peroxide and water, creating a localized microenvironment that gradually releases oxygen and promotes aerobic microbial metabolism. Bentonite's ion exchange capacity neutralizes the Ca(OH)2 produced by the hydrolysis of calcium peroxide, alleviating the inhibitory effect of elevated pH on microbial activity.

[0054] During the research process for this invention, the mass ratio of composite microorganisms to slow-release oxygen materials was determined based on the remediation requirements of different contaminated sites. For example, in environments with low target pollutant concentrations and slow groundwater flow rates, an appropriate mass ratio of composite microorganisms to slow-release oxygen materials may be (20-50):1 to ensure that the oxygen release rate matches the microbial metabolic needs. In sites with high pollution loads or rapid groundwater flow rates, the slow-release oxygen material is consumed at a higher rate, and the dosage of slow-release oxygen materials is generally increased, with the ratio adjusted to (2-20):1 to ensure a long-term, stable oxygen supply.

[0055] The mass ratio of the composite microorganisms to the slow-release oxygen material is calculated based on the mass of the composite microorganism fermentation liquid or the bacterial liquid after activation and culture.

[0056] The optimized dosing ratio of the composite microorganisms and slow-release oxygen-releasing materials described in this invention enables the inoculum to efficiently adapt to and degrade target pollutants in organically contaminated environments, while maintaining good environmental stability and remediation effectiveness. In practical applications, the dosing ratio of the inoculum to oxygen-releasing materials can be appropriately adjusted based on the specific conditions of the contaminated site to achieve optimal remediation efficiency and economic feasibility.

[0057] Trace element addition

[0058] The present invention further adds trace elements necessary for microbial growth, further promoting microbial reproduction and metabolism, thereby further improving the efficient adaptation of the bacterial agent in an organically polluted environment and degrading target pollutants, while maintaining good environmental stability and remediation effects.

[0059] In one embodiment, the composite microbial agent of the present invention is prepared into a slurry with water. After pre-treatment by crushing and screening sandy soil, the slurry is injected via an injection rod. Bentonite forms a stable crack network under high pressure, preventing soil closure. It also coats calcium peroxide particles, slowing their reaction with water and prolonging the oxygen release cycle. Thus, the present invention can achieve efficient remediation of persistent organic pollutants in sandy soils through a simple and convenient method, with the advantage of low cost, and thus has significant application prospects.

[0060] Of course, the method of using the composite microbial agent of the present invention is not limited to the above method. For example, it can be used by injecting through well construction, extracting groundwater and mixing it with the agent for reinjection, etc.

[0061] In one embodiment, when preparing the slurry, the slurry concentration must balance fluidity and slow-release effect. For sandy soil, a higher solid-to-liquid ratio is preferred. The high dispersibility of bentonite in the slurry can prevent CaO2 particle sedimentation. Furthermore, for example, the weight ratio of slow-release oxygen material to water is 1:3 to 1:6.

[0062] Hereinafter, only certain exemplary embodiments are briefly described. As will be appreciated by those skilled in the art, the described embodiments may be modified in various ways without departing from the spirit or scope of the embodiments of the present invention. Therefore, the description is to be regarded as illustrative in nature and not restrictive.

[0063] All features disclosed in the present invention, or all steps in the methods or processes disclosed, except for mutually exclusive features or steps, may be combined in any manner.

[0064] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with specific examples. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments, and unless otherwise stated, they can be replaced by other equivalent or alternative features with similar purposes. Unless otherwise stated, each feature is just an example of a series of equivalent or similar features. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. Therefore, the detailed description of the embodiments of the present invention provided below is not intended to limit the scope of the invention for which protection is claimed, but merely represents selected embodiments of the present invention.

[0065] Unless otherwise specified, the terms used in this invention generally have the meanings commonly understood by those skilled in the art. The implementation conditions used in the examples may be further adjusted according to the specific requirements of the application. Unspecified implementation conditions are conventional conditions in the industry. The technical features involved in the various embodiments of the present invention may be combined with each other as long as they do not conflict with each other.

[0066] In the present invention, the operation without special instructions is carried out at room temperature. The raw materials in this application can be purchased from the market or prepared by conventional methods in the prior art. Among them, the purity of calcium peroxide is 75%, the average particle size of bentonite is 200-500 mesh, and the Ca 2+ 、Fe 2+ 、Cu 2+ 、Zn 2+ 、Mn 2+ Mg 2+ Trace elements such as sulfuric acid are added in the form of corresponding sulfates.

[0067] In the present invention, Pseudomonas can be, for example, Pseudomonas genus numbered BMZ339652 from Mingzhou Bio, Pseudomonas genus numbered TS278212, TS278215, TS278216, TS278217, TS278218, TS278220, TS278221, TS278222, TS278223, TS278224, etc. from Testo Bio, and Pseudomonas genus numbered HZB112253 from Gray Algae Bio.

[0068] Bordetella can be, for example, Bordetella with the product number BMZ066045 from Mingzhou Biotechnology, Bordetella with the product number TS341362 from Testo Biotechnology, and Bordetella with the product number HZB113876 from Huizai Biotechnology.

[0069] For example, Dokdonella kunshanensis can be derived from the type strain DC-3T of Dokdonella kunshanensis deposited in the China Center for Type Culture Collection (CCTCC), with the deposit number being CCTCC AB 2011179T.

[0070] Brucella can be obtained from, for example, Brucella BMZ116694 from Mingzhou Biotechnology.

[0071] In the present invention, the fermentation broth of each strain can be cultured according to conventional methods in the art to obtain a viable cell count of not less than 1×10 9CFU / mL of fermentation broth of each strain. For example, the strain is first activated and then the seed liquid is cultured. The seed liquid in the logarithmic growth period is collected and inoculated into a fermentation barrel containing liquid culture medium at a 10% inoculation ratio. The mixture is fully shaken to mix evenly, and an aeration device is connected to the fermentation barrel for aeration. The fermentation is expanded and cultured at room temperature for 32 to 48 hours. Samples are taken regularly to monitor the microbial biomass to ensure the growth of the strain. After the fermentation culture is completed, the fermentation broth is concentrated and the bacterial content is determined by the plate colony count method to obtain a viable bacterial count of not less than 1×10 9 CFU / mL of fermentation broth of each strain.

[0072] Example 1: Preparation of composite microbial fermentation broth: According to the ratio of the number of live bacteria of Pseudomonas, Bordetella, Dokshima and Brucella of 10:10:1:1, the fermentation broths of each strain were mixed to obtain composite microbial fermentation broth;

[0073] Preparation of slow-release oxygen material: dry-mix calcium peroxide and bentonite in a mass ratio of 6:4, and mix well to obtain slow-release oxygen material.

[0074] Prepare slurry: Mix the composite microbial fermentation liquid, slow-release oxygen material and trace elements with clean water and stir evenly to make slurry. The mass ratio of composite microbial fermentation liquid to slow-release oxygen material is 20:3; the mass ratio of slow-release oxygen material to clean water is 1:6; the amount of trace elements added is 20mg Ca per liter of composite microbial fermentation liquid. 2+ 、20mg Mg 2 + , 5 mg Zn 2+ and 5 mg Mn 2+ .

[0075] Example 2: is basically the same as Example 1, except that the ratio of the number of live bacteria of Pseudomonas, Bordetella, Dokshima and Brucella in the composite microbial fermentation broth is 2.5:2.5:0.5:1.

[0076] Example 3: is basically the same as Example 1, except that the ratio of the number of live bacteria of Pseudomonas, Bordetella, Dokshima and Brucella in the composite microbial fermentation broth is 0.1:0.1:0.5:1.

[0077] Example 4: is basically the same as Example 1, except that the ratio of the number of live bacteria of Pseudomonas, Bordetella, Dokshima and Brucella in the composite microbial fermentation broth is 1:1:1:1.

[0078] Comparative Example 1: is basically the same as Example 1, except that the ratio of the number of live bacteria of Pseudomonas, Bordetella, Dokshima and Brucella in the composite microbial fermentation broth is 15:15:1:1.

[0079] Comparative Example 2: is basically the same as Example 1, except that the ratio of the number of live bacteria of Pseudomonas, Bordetella, Dokshima and Brucella in the composite microbial fermentation broth is 1:1:6:12.

[0080] Comparative Example 3: The same as Example 1, except that the composite microbial fermentation broth contains Pseudomonas and Bordetella at a viable cell count ratio of 1:1.

[0081] Comparative Example 4 is basically the same as Example 1, except that the composite microbial fermentation broth contains Pseudomonas, Bordetella and Dokshima bacteria at a live cell count ratio of 1:1:1.

[0082] Comparative Example 5: is basically the same as Example 1, except that the composite microbial fermentation broth contains Pseudomonas, Bordetella and Brucella at a viable cell count ratio of 1:1:1.

[0083] Comparative Example 6: Basically the same as Example 1, the only difference is that the microorganisms in the composite microbial fermentation liquid are commercially available polycyclic aromatic hydrocarbon microbial composite bacterial agents, and the main functional bacteria are Bacillus, lignin bacteria, etc. The microbial agent is a soil remediation agent (polycyclic aromatic hydrocarbon degradation composite bacteria) from Jiangxi Enyang Biotechnology Co., Ltd. After being activated according to the method provided by the manufacturer, it is prepared into a slurry according to the method of Example 1 for use.

[0084] Experimental Example 1: The soil of a contaminated plot is primarily sandy, and its characteristic pollutants are the representative PAHs naphthalene and benzo[a]pyrene, typical POPs. Multiple samples of sandy soil were collected from this plot. After pretreatment, such as crushing and screening, the soil was then injected with the slurries prepared in the above examples and comparative examples. Four liters of slurry were injected per cubic meter of sandy soil. The soil was then piled into a biopile. Moisture and temperature conditions were controlled to maintain a suitable environment for microbial growth. Soil moisture was maintained between 10% and 30%, and the curing temperature was maintained between 25°C and 30°C. The degradation rates of naphthalene and benzo[a]pyrene were measured at different testing times. Table 2 below shows the degradation rates of the PAH naphthalene at different testing times, in %. Table 3 shows the degradation rates of benzo[a]pyrene at different testing times, in %. Among them, the PAHs naphthalene content and benzo[a]pyrene content are determined according to the "Determination of Semi-volatile Organic Compounds in Soil and Sediment by Gas Chromatography-Mass Spectrometry" (HJ834-2017).

[0085]

[0086]

[0087] In the above table, “ / ” means not detected.

[0088] As can be seen from Table 2 and Table 3, the degradation rates of naphthalene and benzo[a]pyrene in the Examples are higher than those in the Comparative Examples, among which Example 2 has the best effect.

[0089] Test Example 2: The main component of the slow-release oxygen material in this test example is calcium peroxide with a purity of 75%, which reacts with water to release oxygen. The slow-release material is dry-mixed with calcium peroxide and bentonite in a mass ratio of 6:4. The slow-release oxygen material is then mixed with clean water in a mass ratio of 1:6 and stirred to form a homogeneous suspension slurry. Sodium sulfite is added as a reducing agent according to the saturated or supersaturated dissolved oxygen content in the water to consume oxygen. The oxygen-release capacity of the added calcium peroxide is verified by comparison with a pure water blank sample. After 30 days of continuous testing, the sample containing calcium peroxide can still maintain a stable oxygen release capacity after consuming a certain amount of sodium sulfite daily, and the dissolved oxygen in the sample is maintained at 8-14 mg / L.

[0090] Test Cases 3-6: Polychlorinated biphenyl (PCB) contamination of sandy soil at a petrochemical site in Shaanxi Province. Based on the site's environmental investigation and risk assessment, groundwater contamination was identified, exceeding acceptable health risks. The primary contaminant in the site was PCBs (total amount). The remediation process employed was a biopile remediation process, with the goal of reducing the total PCB content in the contaminated soil to less than 0.38 mg / kg.

[0091] The composite microbial agent of the present invention is used to evaluate the repair effect of site soil.

[0092] A slurry was prepared according to the protocol of Example 2. After pretreatment of the contaminated soil by crushing and screening, the slurries prepared in the above examples and comparative examples were injected into sandy soil. Four liters of slurry were injected per cubic meter of sandy soil. The soil was then piled into a biopile. Moisture and temperature conditions were regulated to maintain the required environment for microbial growth. Soil moisture was controlled between 10% and 30%, and the curing temperature was controlled between 25°C and 30°C. Sampling was performed every seven days during the curing process, and the curing period was 35 days. The degradation rate of polychlorinated biphenyls (PCBs) was measured at different times. Table 4 shows the degradation rate of PCBs at different test times, in %. PCBs were measured according to the "Determination of Polychlorinated Biphenyls in Soil and Sediment by Gas Chromatography-Mass Spectrometry" (HJ743-2015).

[0093]

[0094] As can be seen from the above table, after degradation by the composite microbial agent, the PCB removal rate reached 95% to 96.5%, and all contaminated soil was repaired to meet the standards.

[0095] The above embodiments are intended only to illustrate the technical concepts and features of the present invention. Their purpose is to enable those skilled in the art to understand the contents of the present invention and implement them accordingly. They are not intended to limit the scope of protection of the present invention. Any equivalent changes or modifications made in accordance with the spirit of the present invention are intended to be covered by the scope of protection of the present invention.

Claims

1. A composite microbial agent, characterized in that: The invention comprises a composite microorganism, wherein the composite microorganism is Pseudomonas, Bordetella, Dokshima bacteria and Brucella with a viable cell count ratio of (2-3): (2-3): (0.5~1): 1, wherein the Dokshima bacteria is derived from the model strain DC-3T of Kunshan Dokshima bacteria preserved in the China Center for Type Culture Collection, with the preservation number being CCTCCAB 2011179T.

2. The composite microbial agent according to claim 1, characterized in that: The viable count of the composite microorganisms is not less than 1×10 9 CFU / mL.

3. The composite microbial agent according to claim 1, characterized in that: The composite microorganism is in liquid form or solid form.

4. The composite microbial agent according to claim 3, characterized in that: The composite microorganism is microbial fermentation liquid, microbial freeze-dried powder or glycerol bacteria.

5. The composite microbial agent according to claim 3, characterized in that: When the composite microorganism is in liquid form, the viable count of the composite microorganism is not less than 1×10 9 CFU / mL, the composite microbial agent is used directly or diluted with water; when the composite microorganism is in solid form, the composite microorganism is activated and cultured to a viable count of not less than 1×10 9 CFU / mL.

6. The composite microbial agent according to claim 1, characterized in that: The composite microbial agent also includes a slow-release oxygen material, which includes an active component capable of releasing oxygen and an inert component. The active component is calcium peroxide, and the inert component is bentonite. The mass fraction of the active component in the slow-release oxygen material is 55% to 65%, and the mass fraction of the inert component in the slow-release oxygen material is 35% to 45%.

7. The composite microbial agent according to claim 6, characterized in that: The mass ratio of the composite microorganism to the slow-release oxygen material is (2-50):

1.

8. The composite microbial agent according to claim 7, characterized in that: The mass ratio of the composite microorganism to the slow-release oxygen material is (20-50):

1.

9. The composite microbial agent according to claim 7, characterized in that: The mass ratio of the composite microorganism to the slow-release oxygen material is (2-20):

1.

10. The composite microbial agent according to claim 1, characterized in that: The composite microbial agent also includes trace elements.

11. The composite microbial agent according to claim 10, characterized in that: The trace elements include Ca 2+ 、Fe 2+ 、Cu 2+ 、Zn 2+ 、Mn 2+ Mg 2+ One or more of .

12. The composite microbial agent according to claim 10, characterized in that: The trace elements include 1-50 mg of Ca per liter of the composite microorganism. 2+ , 1~50mg Mg 2+ , 1~10mg Zn 2+ and 1~10mg of Mn 2+ .

13. Use of the composite microbial agent according to any one of claims 1 to 12 in the remediation or management of persistent contaminated sites, wherein: The persistent contaminated site is sandy soil.

14. A method for remediating a persistent contaminated site, characterized by: The composite microbial agent according to any one of claims 1 to 12 is added to sandy soil, the composite microbial agent is added with water to form a slurry, and then the slurry is injected into the sandy soil through an injection rod; or injected into the sandy soil through a well.

15. The repair method according to claim 14, characterized in that: The pollutants in the sandy soil include one or more of polychlorinated biphenyls, naphthalene, and benzo[a]pyrene.

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