Composite microbial agent and application thereof in repairing or managing and controlling persistent organic pollution site

Abstract

The invention relates to a compound microbial agent and application thereof in repairing or managing and controlling a persistent organic pollution site, the compound microbial agent comprises compound microorganisms, and the compound microorganisms comprise pseudomonas, Bordetella, Island bacteria and Brucella in a ratio of (0.1-10): (0.1-10): (0.5-1): 1. The remediation efficiency of the persistent organic pollution site is improved, and the method is particularly suitable for green and efficient remediation of soil and underground water of the sandy soil site and pollution source reduction risk management and control and has the advantages of being low in cost, convenient to operate and the like, so that the method has important application prospects.

Description

Technical Field

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

[0002] This section aims to provide background or context for the embodiments of the present invention described in the claims. The description herein is not admitted to be prior art merely because it is included in this section.

[0003] Under natural environment, pollutants in groundwater will undergo natural attenuation mechanisms under the action of microorganisms and hydraulics, etc., resulting in a decrease in pollutant concentration. However, its geochemical reaction process is affected by many factors such as environment, hydrology, and geology. Existing studies have shown that microbial degradation plays a major role in the natural attenuation process of organic pollutants. Due to being restricted by environmental condition factors, such as insufficient number of indigenous functional microorganisms, low activity, and slow growth, etc., the natural degradation rate is usually slow. The enhanced attenuation technology (EA) can make up for the shortcoming of slow natural degradation. Adding a microbial inoculant that can degrade the target pollutant to the contaminated site can improve the microbial degradation rate of organic pollutants in groundwater.

[0004] Persistent organic pollutants (POPs pollutants) commonly found in sandy soil, such as organochlorine pesticides, polychlorinated biphenyls, etc., are difficult to be efficiently degraded by traditional repair methods due to their stable chemical properties, low water solubility, and tight binding with soil particles. The physicochemical properties of POPs pollution vary greatly and the degradation difficulty is high. It is difficult for a single bacterial community to degrade efficiently at the same time.

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

[0006] The object of the present invention is to provide a composite microbial inoculant and its application in repairing or controlling persistent organic pollutant sites.

[0007] To achieve the above object, the technical solution adopted by the present invention is: The first aspect of the present invention provides a composite microbial agent, which comprises composite microorganisms. The composite microorganisms comprise Pseudomonas, Bordetella, Dokdonella, and Brucella with a viable cell number ratio of (0.1-10):(0.1-10):(0.5-1):1.

[0008] According to some specific embodiments, the viable cell number ratio 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.

[0009] According to some specific embodiments, the viable cell number ratio of the Bordetella 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 viable cell number ratio of the Dokdonella to the Brucella is 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, 1:1, etc.

[0011] According to some specific embodiments, the viable cell number ratio of the Pseudomonas, the Bordetella, the Dokdonella, and the Brucella is (1-5):(1-5):(0.5-1):1.

[0012] Further, the viable cell number ratio of the Pseudomonas, the Bordetella, the Dokdonella, and the Brucella is (2-3):(2-3):(0.5-1):1.

[0013] According to some specific embodiments, the viable cell number of the composite microorganisms is not less than 1×10 9 CFU / mL.

[0014] According to some specific embodiments, the composite microorganisms are in a liquid form or a solid form.

[0015] Further, the composite microorganisms are microbial fermentation broth, microbial freeze-dried powder, or glycerol bacteria.

[0016] Further, when the composite microorganisms are in a liquid form, the viable cell number of the composite microorganisms is not less than 1×10 9CFU / mL, and the compound microbial inoculant is used directly or after dilution with water. Among them, the compound microorganism in liquid form is a microbial fermentation broth, which is obtained by separately or mixed culturing the Pseudomonas, Bordetella, Dokdonia, and Brucella to obtain a fermentation broth containing these four kinds of bacteria.

[0017] Further, when the compound microorganism is in solid form, before using the compound microbial inoculant, the compound microorganism is activated and cultured until the viable count is not less than 1×10 9 CFU / mL.

[0018] Still further, when the compound microorganism is in solid form, the viable count of the compound microorganism is not less than 2×10 8 CFU / g.

[0019] According to some specific embodiments, the compound microbial inoculant further includes a slow-release oxygen material, and the slow-release oxygen material includes an active component capable of releasing oxygen and an inert component.

[0020] Further, the active component is calcium peroxide; the inert component includes bentonite.

[0021] Further, the mass fraction of the active component in the slow-release oxygen material is 55% - 65%, such as 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% - 45%, such as 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, etc.

[0022] Further, the mass ratio of the compound microorganism to the slow-release oxygen material is (2 - 50):1, such as 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, 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.

[0023] Still further, the mass ratio of the compound microorganism to the slow-release oxygen material is (20 - 50):1.

[0024] Further, the mass ratio of the composite microorganism to the slow-release oxygen material is (2 to 20):1.

[0025] According to some specific embodiments, the composite microorganism agent further includes trace elements.

[0026] Further, the trace elements include Ca 2+ , Fe 2+ , Cu 2+ , Zn 2+ , Mn 2+ , Mg 2+ and one or more of them.

[0027] Further, the trace elements include adding 1 to 50 mg of Ca 2+ , 1 to 50 mg of Mg 2+ , 1 to 10 mg of Zn 2+ and 1 to 10 mg of Mn 2+ per liter of the composite microorganism.

[0028] Among them, the mass of the composite microorganism in the mass ratio of the composite microorganism to the slow-release oxygen material, and the volume of the composite microorganism in the addition amount of the trace elements, when the composite microorganism is sold in a liquid form, are the mass and volume of the composite microorganism; when the composite microorganism is sold in a solid form, they are the mass and volume of the liquid when the composite microorganism is activated and cultured to a viable count of not less than 1×10 9 CFU / mL.

[0029] The second aspect of the present invention provides an application of the composite microorganism agent as described above in the repair or control of persistent pollution sites, wherein the persistent pollution sites include sandy soil and / or groundwater.

[0030] The third aspect of the present invention provides a method for repairing persistent pollution, by adding the composite microorganism agent as described above to silty sand soil and / or groundwater.

[0031] According to some specific embodiments, the composite microorganism agent is mixed 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 the groundwater is extracted, mixed with the composite microorganism agent, and then reinjected.

[0032] According to some specific embodiments, after the sandy soil is crushed and screened for pretreatment, the slurry is injected into the sandy soil.

[0033] According to some specific embodiments, the pollutants in the powdery cohesive 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, etc.

[0034] Due to the application of the above technical solutions, the present invention has the following advantages compared with the prior art: The present invention has developed a composite microbial agent suitable for the remediation or control of persistent organic pollution (POPs pollution) sites, especially suitable for the green and efficient remediation of soil and groundwater and the risk control of pollution source reduction in sandy soil sites. It mainly uses the synergistic effect of specific proportions of Pseudomonas, Bordetella, Dokdonella, and Brucella to effectively solve the problem of difficult remediation of persistent organic pollution sites, overcome the problems of low mass transfer efficiency and limited microbial activity of traditional remediation technologies, improve the remediation efficiency of persistent organic pollution in the soil and groundwater of sandy soil sites, achieve green and efficient remediation and risk control of pollution source reduction, and has the advantages of low cost and convenient operation, thus having important application prospects. Specific embodiments

[0035] Since the existing microorganisms have poor effects in remediating or controlling persistent organic pollution 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 pollution (POPs pollution) sites, especially suitable for the green and efficient remediation of soil and groundwater and the risk control of pollution source reduction in sandy soil sites. It mainly uses the synergistic effect of specific proportions of Pseudomonas, Bordetella, Dokdonella, and Brucella, and further combines with slow-release oxygen materials and trace elements to effectively solve the problem of difficult remediation of persistent organic pollution (POPs pollution) sites, overcome the problems of low mass transfer efficiency and limited microbial activity of traditional remediation technologies, improve the remediation efficiency of persistent organic pollutants in the soil and groundwater of sandy soil sites, achieve green and efficient remediation and risk control of pollution source reduction, and has the advantages of low cost and convenient operation, thus having important application prospects.

[0036] Further, in the composite microorganism, the viable count ratio of Pseudomonas, Bordetella, Dokdonella, and Brucella is (0.1~10):(0.1~10):(0.5~1):1. If the addition ratio of each bacterium exceeds the upper limit or is lower than the lower limit, the remediation effect of the composite microbial agent will decrease significantly.

[0037] The principles of the functions of each bacterial community and their synergistic effects in improving the remediation efficiency of persistent organic pollutant sites are as follows: Pseudomonas can initiate the oxidation of polycyclic aromatic hydrocarbons (PAHs) through dioxygenase, generate intermediate products, 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, especially dominant in oxygen-rich surface soil.

[0038] Bordetella is good at degrading the intermediates after PAHs oxidation (such as catechol, salicylic acid), can rely on short-chain fatty acids or sugars as cosubstrates to activate the dehydrogenase system to assist in PAHs degradation, and in addition has strong tolerance to reactive oxygen species (ROS) generated by PAHs metabolism (such as high catalase activity).

[0039] Dokdonia can degrade PAHs derivatives (such as hydroxylated PAHs) through amination reaction under low-nutrient conditions (such as nitrogen-poor), and can secrete extracellular polysaccharides (EPS) to adsorb PAHs particles and protect other bacterial communities from toxicity inhibition, and can couple PAHs oxidation with nitrate reduction.

[0040] Brucella may transform PAHs into low-toxic derivatives through the glutathione metabolic pathway, supply vitamins or siderophores, promote the proliferation of degradation bacteria such as Pseudomonas, and participate in the partial oxidation of PAHs through microaerobic respiration in deep soil.

[0041] Synergistic mechanism The composite microbial inoculant provided by the present invention can achieve efficient degradation of PAHs from emulsification, ring opening to complete mineralization through strategies of spatial differentiation, metabolic complementarity and environmental adaptation during the degradation process of typical POPs pollutants such as polycyclic aromatic hydrocarbons (PAHs). First, Pseudomonas secretes surfactants (such as rhamnolipids) to promote the dissolution of PAHs in soil, improve the bioavailability of PAHs, and secretes dioxygenase to initiate hydroxylation reactions to convert PAHs into intermediates such as catechol and salicylic acid. Bordetella can degrade PAHs oxidation intermediates such as catechol and salicylic acid and completely mineralize them into CO2 and H2O through the β-ketoadipic acid pathway. In addition, Brucella forms a biofilm by secreting extracellular polymers, assisting Pseudomonas and Bordetella in in-situ colonization to form stable biofilm colonies, ensuring an adequate amount of microorganisms with pollutant degradation functions, facilitating the formation of a degradation microenvironment, and enhancing the stress resistance of the microbial community. In addition, this inoculant has good environmental adaptability. Under aerobic conditions, Pseudomonas and Bordetella can cooperate with each other to completely mineralize PAHs. Under microaerobic conditions, Bordetella can also effectively utilize nitrate in the soil as an electron acceptor with Dokdonia to jointly degrade methylated PAHs. Under anaerobic conditions, Dokdonia degrades low-ring PAHs (such as naphthalene) by using ferric iron and sulfate as electron acceptors and releases organic acids. This inoculant has a wide range of environmental pH adaptability. In particular, the extracellular polymers secreted by Pseudomonas and Dokdonia can protect other functional bacteria to better adapt to acidic or alkaline environments.

[0042] Summary of the synergistic mechanism: The following Table 1 is a summary of the synergistic mechanisms of each microbial community in the inoculant provided by the present invention:

[0043] Slow-release oxygen material The present invention provides a composite microbial inoculant specifically for the degradation of organic pollutants. This inoculant is composed of a composite of multiple highly efficient degradation strains and can effectively degrade organic pollutants in groundwater and soil under aerobic conditions. In order to ensure that the inoculant 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 for the degradation rate of organic pollutants. Excessive slow-release oxygen material may lead to too fast an oxygen release rate, affecting the adaptability and survival of the inoculant, while insufficient oxygen supply may inhibit the metabolic activity of the inoculant and reduce the degradation efficiency. Therefore, in the present invention, the slow-release oxygen material is added so that the inoculant can maintain a suitable aerobic state in different polluted environments and promote the efficient degradation of organic pollutants by microorganisms.

[0044] Active component: CaO2 is the most commonly used oxygen-releasing agent material at present. Calcium peroxide is inexpensive, easy to obtain, and its reaction products are pollution-free. From the perspective of oxygen release, when CaO2 is added to water, an immediate reaction occurs, rapidly increasing the dissolved oxygen concentration in the water to a very high level (reaction formula: 2CaO2 + 2H2O → 2Ca(OH)2 + O2↑). This easily leads to over-addition and a lack of persistence in controlling the oxygen release rate.

[0045] Calcium peroxide is used as the active component, and the suspension adsorption material bentonite is used as a carrier to disperse calcium peroxide particles, slow down the oxygen release rate, and enhance the stability of the slurry. Bentonite forms a stable crack network through adsorption and swelling, preventing soil closure, improving the pore structure of cohesive soils, enhancing permeability, and at the same time wrapping CaO2 particles to slow down their reaction rate with water, which can increase the dissolved oxygen level in the soil and promote the growth and metabolism of aerobic microorganisms.

[0046] Through adsorption and isolation, bentonite restricts the direct contact between calcium peroxide and water, forms a local microenvironment, gradually releases oxygen, and promotes the metabolism of aerobic microorganisms. The ion exchange capacity of bentonite can neutralize Ca(OH)2 produced by the hydrolysis of calcium peroxide, alleviating the inhibition of pH increase on microbial activity.

[0047] During the research process of this invention, in combination with the remediation requirements of different polluted sites, the mass ratio of the composite microorganism to the slow-release oxygen material is determined. For example, in an environment with a low concentration of target pollutants and a slow groundwater flow rate, the appropriate mass ratio of the composite microorganism to the slow-release oxygen material for dosing can be (20 - 50):1 to ensure that the oxygen release rate matches the microbial metabolic requirements; while in a site with a high pollution load or a fast groundwater flow rate, the consumption rate of the slow-release oxygen material is relatively high, and usually, the dosing amount of the slow-release oxygen material needs to be increased, and the ratio can be adjusted to (2 - 20):1 to ensure a long-term stable oxygen supply.

[0048] Among them, the mass ratio of the composite microorganism to the slow-release oxygen material for dosing is calculated based on the mass of the composite microorganism fermentation broth or the bacterium solution after activation and cultivation.

[0049] The dosing ratio of the composite microorganism to the slow-release oxygen material described in this invention is optimized, enabling the microbial agent to efficiently adapt to and degrade the target pollutants in the organic polluted environment, while maintaining good environmental stability and remediation effect. In practical applications, the dosing ratio of the microbial agent to the oxygen-releasing material can be appropriately adjusted according to the specific conditions of the polluted site to achieve the best remediation efficiency and economic feasibility.

[0050] Trace element addition The present invention further adds trace elements essential for the growth of microorganisms, further promoting the reproduction and metabolism of microorganisms, thereby further enhancing the efficient adaptation of the microbial agent to the organic polluted environment and degrading target pollutants, while maintaining good environmental stability and remediation effect.

[0051] In one embodiment, when the composite microbial agent of the present invention is used, it is formulated into a slurry with water. After the sandy soil is crushed and screened for pretreatment, the slurry is injected through an injection rod. Bentonite forms a stable crack network under high pressure, preventing the soil from closing, while wrapping calcium peroxide particles and slowing down their reaction rate with water, prolonging the oxygen release period. Thus, the present invention can achieve the efficient remediation of persistent organic pollutants in sandy soil through a simple and convenient method, and has the advantage of low cost, so it has important application prospects.

[0052] Of course, the use method of the composite microbial agent of the present invention is not limited to the above method. For example, methods such as injection through well construction and reinjection after mixing the microbial agent into the groundwater extraction can also be used.

[0053] In one embodiment, when preparing the slurry, the slurry concentration needs to consider both fluidity and slow-release effect. The sandy soil preferably adopts a higher solid-liquid ratio, and the high dispersibility of bentonite in the slurry can prevent the settlement of CaO2 particles. Further, for example, the mass ratio of the slow-release oxygen material to water is 1:3 to 1:6.

[0054] In the following text, only some exemplary embodiments are simply described. As those skilled in the art can recognize, the described embodiments can be modified in various different ways without departing from the spirit or scope of the embodiments of the present invention. Therefore, the description is considered to be exemplary in nature rather than restrictive.

[0055] All features disclosed in the present invention, or all steps in the disclosed methods or processes, except for mutually exclusive features or steps, can be combined in any way.

[0056] To make the purpose, technical solution, 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 embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Unless otherwise stated, they can all be replaced by other equivalent or similar-purpose alternative features. Unless otherwise stated, each feature is only an example in a series of equivalent or similar features. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to 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 claimed invention, but merely represents the selected embodiments of the present invention.

[0057] Unless otherwise specified, the terms used in the present invention generally have the meanings commonly understood by those of ordinary skill in the art. The implementation conditions adopted in the examples can be further adjusted according to different requirements of specific use, and the implementation conditions not specified are conventional conditions in this industry. The technical features involved in each implementation manner of the present invention can be combined with each other as long as they do not conflict with each other.

[0058] In the present invention, operations without special instructions are carried out at room temperature. The raw materials in this application can be obtained commercially 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 trace elements such as Ca 2+ 、Fe 2+ 、Cu 2+ 、Zn 2+ 、Mn 2+ 、Mg 2+ are added in the form of corresponding sulfates.

[0059] In the present invention, Pseudomonas can be, for example, Pseudomonas sp. with the number BMZ339652 from Mingzhou Biology, Pseudomonas sp. with the numbers TS278212, TS278215, TS278216, TS278217, TS278218, TS278220, TS278221, TS278222, TS278223, TS278224, etc. from Testo Biology, and Pseudomonas sp. with the product number HZB112253 from Huizao Biology, etc.

[0060] Bordetella can be, for example, Bordetella with the number BMZ066045 from Mingzhou Biology, Bordetella with the number TS341362 from Testo Biology, and Bordetella with the product number HZB113876 from Huizao Biology, etc.

[0061] Dokdonella can be, for example, the type strain DC - 3T of Dokdonella kunshanensis preserved in the China Center for Type Culture Collection (CCTCC), with the preservation number CCTCC AB 2011179T.

[0062] Brucella can be, for example, Brucella with the number BMZ116694 from Mingzhou Biology.

[0063] In the present invention, the fermentation broth of each strain can be cultured according to the conventional methods in the art to obtain a viable cell count of not less than 1×10 9Fermentation broth of each strain at CFU / mL. For example, first activate and culture the strain, then culture the seed liquid, collect the seed liquid in the logarithmic growth phase and inoculate it into a fermentation barrel containing a liquid medium at an inoculation ratio of 10%, shake well to mix evenly, and connect an aeration device to aerate the inside of the fermentation barrel, and expand the culture at room temperature for 32 - 48 h. Regularly sample and monitor its microbial biomass to ensure the growth of the strain. After the fermentation culture is completed, concentrate the fermentation broth, and determine the bacterial content by the plate colony counting method to obtain a viable bacteria count of not less than 1×10 9 Fermentation broth of each strain at CFU / mL.

[0064] Example 1: Prepare a composite microbial fermentation broth: Mix the fermentation broths of each strain according to the ratio of viable bacteria counts of Pseudomonas, Bordetella, Dokdoella, and Brucella of 10:10:1:1 to obtain a composite microbial fermentation broth; Prepare a slow-release oxygen material: Dry-mix calcium peroxide and bentonite in a mass ratio of 6:4, and mix evenly to obtain a slow-release oxygen material.

[0065] Prepare a slurry: Mix the composite microbial fermentation broth, the slow-release oxygen material, trace elements and clear water, and stir evenly to make a slurry. Among them, the dosing mass ratio of the composite microbial fermentation broth to the slow-release oxygen material is 20:3; the dosing mass ratio of the slow-release oxygen material to clear water is 1:6; the dosing amount of trace elements is 20 mg Ca added per liter of the composite microbial fermentation broth 2+ 、20 mg Mg 2 + 、5 mg Zn 2+ and 5 mg Mn 2+ .

[0066] Example 2: It is basically the same as Example 1, except that the ratio of viable bacteria counts of Pseudomonas, Bordetella, Dokdoella, and Brucella in the composite microbial fermentation broth is 2.5:2.5:0.5:1.

[0067] Example 3: It is basically the same as Example 1, except that the ratio of viable bacteria counts of Pseudomonas, Bordetella, Dokdoella, and Brucella in the composite microbial fermentation broth is 0.1:0.1:0.5:1.

[0068] Example 4: It is basically the same as Example 1, except that the ratio of viable bacteria counts of Pseudomonas, Bordetella, Dokdoella, and Brucella in the composite microbial fermentation broth is 1:1:1:1.

[0069] Comparative Example 1: It is basically the same as Example 1, except that the ratio of viable bacteria counts of Pseudomonas, Bordetella, Dokdoella, and Brucella in the composite microbial fermentation broth is 15:15:1:1.

[0070] Comparative Example 2: It is basically the same as Example 1, except that the viable cell numbers ratio of Pseudomonas, Bordetella, Dokdonia, and Brucella in the composite microbial fermentation broth is 1:1:6:12.

[0071] Comparative Example 3: It is basically the same as Example 1, except that the composite microbial fermentation broth contains Pseudomonas and Bordetella with a viable cell numbers ratio of 1:1.

[0072] Comparative Example 4: It is basically the same as Example 1, except that the composite microbial fermentation broth contains Pseudomonas, Bordetella, and Dokdonia with a viable cell numbers ratio of 1:1:1.

[0073] Comparative Example 5: It is basically the same as Example 1, except that the composite microbial fermentation broth contains Pseudomonas, Bordetella, and Brucella with a viable cell numbers ratio of 1:1:1.

[0074] Comparative Example 6: It is basically the same as Example 1, except that the microorganisms in the composite microbial fermentation broth are a commercially available polycyclic aromatic hydrocarbon microbial complex agent, and the main functional bacteria are Bacillus and lignin bacteria. This microbial complex agent is a soil remediator (polycyclic aromatic hydrocarbon degrading complex strains) from Jiangxi Enyang Biotechnology Co., Ltd. After being activated according to the method provided by the manufacturer, it is formulated into a slurry according to the method of Example 1 for use.

[0075] Test Example 1: The soil quality of a polluted site is mainly sandy soil, and its characteristic pollutants are representative PAHs naphthalene and benzo[a]pyrene, which are typical POPs pollutants. Multiple samples of sandy soil from this site were collected. After pretreatment of the polluted soil such as crushing and screening, the slurries prepared in the above Examples and Comparative Examples were respectively injected into the sandy soil. Among them, 4L of slurry was injected into every 1 cubic meter of sandy soil, and it was piled up into a biopile. Conditions such as moisture and temperature were regulated to maintain the environment required for microbial growth. The soil humidity was controlled at 10% - 30%, and the curing temperature was controlled at 25℃ - 30℃. The degradation rates of naphthalene and benzo[a]pyrene at different times were detected. The following Table 2 shows the degradation rate results of PAHs naphthalene at different detection times, with the unit of %. Table 3 shows the degradation rate results of benzo[a]pyrene at different detection times, with the unit of %. Among them, the contents of PAHs naphthalene and benzo[a]pyrene were specifically determined according to "Soil and Sediments - Determination of Semi-volatile Organic Compounds - Gas Chromatography-Mass Spectrometry" (HJ834-2017).

[0076]

[0077]

[0078] In the above table, " / " indicates not detected.

[0079] 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 each comparative example, among which, the effect of Example 2 is the best.

[0080] Test Example 2: In this test example, the main component of the slow-release oxygen material is calcium peroxide with a purity of 75%, which can react with water to release oxygen. The slow-release material is dry-mixed by calcium peroxide and bentonite in a mass ratio of 6:4. The slow-release oxygen material is mixed with clear water in a dosing mass ratio of 1:6, and stirred to form a homogeneous suspension slurry. Sodium sulfite is added as a reducing agent to consume oxygen according to the saturated or supersaturated dissolved oxygen content in water, and compared with the pure water blank sample to verify the oxygen release capacity of the added calcium peroxide. After 30 days of continuous test records, in the sample added with calcium peroxide, after a certain amount of sodium sulfite is consumed every day, a stable oxygen release capacity can still be maintained, and the dissolved oxygen in the sample is maintained at 8-14 mg / L.

[0081] Test Examples 3-6: Polychlorinated biphenyl pollution in sandy soil at a petrochemical site in Shaanxi Province. According to the results of site environmental investigation and risk assessment, there is groundwater pollution exceeding the acceptable health risk. The main pollutant in the plot is polychlorinated biphenyls (total amount). The adopted remediation process is the biopile remediation process, and the remediation goal is: the content of polychlorinated biphenyls (total amount) in the soil is less than 0.38 mg / kg after the contaminated soil is remediated.

[0082] The composite microbial inoculant of the present invention is used to evaluate the remediation effect of the site soil.

[0083] Prepare the slurry according to the scheme of Example 2. After the contaminated soil is pretreated by crushing, screening, etc., the slurries prepared in the above examples and comparative examples are respectively injected into the sandy soil. Among them, 4L of slurry is injected into every 1 cubic meter of sandy soil, and it is piled up into a biopile. Control conditions such as moisture and temperature to maintain the environment required for microbial growth. The soil humidity is controlled at 10%-30%, and the curing temperature is controlled at 25°C-30°C. Sampling and detection are carried out every 7 days during the curing process. The curing period is 35 days, and the degradation rate of polychlorinated biphenyls at different times is detected. Table 4 shows the results of the degradation rate of polychlorinated biphenyls at different detection times, in %. Among them, polychlorinated biphenyls are specifically determined according to "Soil and Sediment - Determination of Polychlorinated Biphenyls - Gas Chromatography-Mass Spectrometry" (HJ743-2015).

[0084]

[0085] As can be seen from the above table, after degradation by the composite microbial inoculant, the removal rate of polychlorinated biphenyls reaches 95%-96.5%, and all the contaminated soil is repaired to meet the standards.

[0086] The above embodiments are only used to illustrate the technical concept and features of the present invention. The purpose is to enable those skilled in the art to understand the content of the present invention and implement it accordingly, and it should not be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention.

Claims

1. A composite microbial inoculant, characterized in that: It includes composite microorganisms, and the composite microorganisms include Pseudomonas, Bordetella, Dokdoella, and Brucella with the viable count ratio of (0.1~10):(0.1~10):(0.5~1):

1.

2. The composite microbial inoculum according to claim 1, wherein: The viable count ratio of the Pseudomonas, the Bordetella, the Dokdoella, and the Brucella is (1-5):(1-5):(0.5~1):

1.

3. The composite microbial inoculant according to claim 2, wherein: The viable count ratio of the Pseudomonas, the Bordetella, the Dokdoella, and the Brucella is (2-3):(2-3):(0.5~1):

1.

4. The composite microbial inoculum according to any one of claims 1 to 3, characterized in that: The viable count of the composite microorganism is not less than 1×10 9 CFU / mL.

5. The composite microbial inoculum according to any one of claims 1 to 3, characterized in that: The composite microorganisms are in liquid form or solid form.

6. The composite microbial inoculant according to claim 5, characterized in that: The composite microorganisms are microbial fermentation broth, microbial freeze-dried powder, or glycerol bacteria.

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

8. The composite microbial inoculant according to claim 1, characterized in that: The composite microorganism agent further includes a slow-release oxygen material, and the slow-release oxygen material includes an active component capable of releasing oxygen and an inert component.

9. The composite microbial inoculant according to claim 8, characterized in that: The active component is calcium peroxide; the inert component includes bentonite.

10. The composite microbial inoculum according to claim 8, characterized in that: The mass fraction of the active component in the slow-release oxygen material is 55%~65%, and the mass fraction of the inert component in the slow-release oxygen material is 35%~45%.

11. The compound microbial inoculum according to claim 8, wherein: The mass ratio of the composite microorganisms to the slow-release oxygen material is (2~50):

1.

12. The composite microbial inoculum according to claim 11, wherein: The mass ratio of the composite microorganisms to the slow-release oxygen material is (20~50):1, or the mass ratio of the composite microorganisms to the slow-release oxygen material is (2~20):

1.

13. The composite microbial inoculant according to claim 1, wherein: The composite microorganism agent further includes trace elements.

14. The composite microbial inoculum according to claim 13, characterized in that: The trace elements include Ca 2+ , Fe 2+ , Cu 2+ , Zn 2+ , Mn 2+ , Mg 2+ or one or more of them.

15. The composite microbial inoculum according to claim 13, wherein: The trace elements include 1 to 50 mg of Ca added per liter of the composite microorganism 2+ , 1 to 50 mg of Mg 2+ , 1 to 10 mg of Zn 2+ and 1 to 10 mg of Mn 2+ .

16. Use of a composite microbial inoculum as described in any one of claims 1 to 15 in the remediation or control of persistent contaminated sites, wherein, The persistent pollution site includes sandy soil and / or groundwater.

17. A method for remediating a persistently polluted site, characterized in that: By adding the composite microorganism agent as described in any one of claims 1 to 15 to the sandy soil and / or groundwater.

18. The repair method according to claim 17, characterized in that: The composite microorganism agent is prepared into a slurry by adding water, 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 the groundwater is extracted, mixed with the composite microorganism agent, and then re-injected.

19. The repair method according to claim 18, characterized in that: After the sandy soil is crushed and screened for pretreatment, the slurry is injected into the sandy soil.

20. The repair method according to claim 17, wherein: The pollutants in the sandy soil include one or more of polychlorinated biphenyls, organic pesticides, and dioxins.

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