Compound microbial agent and application thereof in repairing or controlling chlorinated hydrocarbon polluted sites

Abstract

The invention relates to a compound microbial agent and application thereof in repairing or controlling chlorinated hydrocarbon contaminated sites, the compound microbial agent comprises compound microorganisms, and the compound microorganisms comprise Gordonia, pseudomonas, pseudoxanthomonas and geomonas in a viable count ratio of 100: (25-600): (10-900): (10-500). The method effectively solves the problem that the chlorohydrocarbon contaminated site is difficult to repair, overcomes the problems of low mass transfer efficiency, limited microbial activity and the like of the traditional repair technology, improves the repair and risk management and control efficiency of the chlorohydrocarbon contaminated site, and reduces the construction cost. The method is especially suitable for green and efficient remediation of soil and underground water in silt soil sites, pollution source reduction and risk management and control, has the advantages of low cost, convenience in operation and the like, and 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 chlorinated hydrocarbon contaminated 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 by including it in this section.

[0003] In the 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 research has shown that microbial degradation plays a major role in the natural attenuation process of organic pollutants. Due to restrictions 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 (EA) technology 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 increase the microbial degradation rate of organic pollutants in groundwater.

[0004] Chlorinated hydrocarbons are a class of organic compounds with high chemical activity and a wide variety. Due to their excellent chemical properties, they are widely used in the industrial field, including as chemical raw materials, intermediates, and organic solvents, and are commonly used in fields such as aircraft engine manufacturing, automotive parts manufacturing, electronic component manufacturing, and clothing degreasing. However, the extensive use of chlorinated hydrocarbons has led to their widespread presence in the environment, especially being frequently detected in soil and groundwater, becoming an important environmental pollution source. DNAPL (dense non-aqueous phase liquid) has the characteristics of high density and low solubility, and is prone to forming a long-term pollution source. The pollution of groundwater by DNAPL is a major problem in the current environmental remediation field.

[0005] Currently, the treatment methods for chlorinated hydrocarbon pollution mainly include chemical oxidation method, divalent iron-catalyzed hydrogen peroxide decomposition method, and in-situ soil thermal desorption method, etc. Although these methods can remove chlorinated hydrocarbon pollution to a certain extent, they also have obvious limitations. For example, the chemical oxidation method may introduce new chemical substances, resulting in secondary pollution; the divalent iron-catalyzed hydrogen peroxide decomposition method may change the chemical properties of the soil and damage the soil ecological environment; while the in-situ soil thermal desorption method requires high energy consumption and may cause irreversible damage to the soil structure.

[0006] Microbial remediation or control technology has advantages such as low cost and environmental friendliness, and is considered one of the important development directions for future pollution treatment. However, there are relatively few reports on microbial remediation or control of chlorinated hydrocarbon pollution at present, and a mature technical system has not yet been formed. Therefore, developing an efficient, environmentally friendly and targeted microbial remediation or control technology to solve the soil and / or groundwater pollution problems in chlorinated hydrocarbon contaminated sites has important practical significance and broad application prospects. Summary of the Invention

[0007] The purpose of the present invention is to provide a composite microbial agent and its application in remediating or controlling chlorinated hydrocarbon contaminated sites.

[0008] To achieve the above purpose, the technical solution adopted by the present invention is: The first aspect of the present invention provides a composite microbial agent, which includes composite microorganisms, and the composite microorganisms include Gordonia, Pseudomonas, Pseudoxanthomonas, and Terrimonas with a viable cell number ratio of 100:(25 - 600):(10 - 900):(10 - 500).

[0009] According to some specific embodiments, the viable cell number ratio of the Gordonia bacterium to the Pseudomonas bacterium is 100:25, 100:30, 100:35, 100:40, 100:45, 100:50, 100:55, 100:60, 100:65, 100:70, 100:75, 100:80, 100:85, 100:90, 100:95, 100:100, 100:105, 100:110, 100:115, 100:120, 100:125, 100:130, 100:135, 100:140, 100:145, 100:150, 100:155, 100:160, 100:165, 100:170, 100:175, 100:180, 100:185, 100:190, 100:195, 100:200, 100:205, 100:210, 100:215, 100:220, 100:225, 100:230, 100:235, 100:240, 100:245, 100:250, 100:255, 100:260, 100:265, 100:270, 100:275, 100:280, 100:285, 100:290, 100:295, 100:300, 100:305, 100:310, 100:315, 100:320, 100:325, 100:330, 100:335, 100:340, 100:345, 100:350, 100:355, 100:360, 100:365, 100:370, 100:375, 100:380, 100:385, 100:390, 100:395, 100:400, 100:405, 100:410, 100:415, 100:420, 100:425, 100:430, 100:435, 100:440, 100:445, 100:450, 100:455, 100:460, 100:465, 100:470, 100:475, 100:480, 100:485, 100:490, 100:495, 100:500, 100:505, 100:510, 100:515, 100:520, 100:525, 100:530, 100:535, 100:540, 100:545, 100:550, 100:555, 100:560, 100:565, 100:570, 100:575, 100:580, 100:585, 100:590, 100:595, 100:600, etc.

[0010] According to some specific embodiments, the viable cell number ratio of the Gordonia bacterium to the Pseudomonas bacterium is 100:10, 100:15, 100:20, 100:25, 100:30, 100:35, 100:40, 100:45, 100:50, 100:55, 100:60, 100:65, 100:70, 100:75, 100:80, 100:85, 100:90, 100:95, 100:100, 100:105, 100:110, 100:115, 100:120, 100:125, 100:130, 100:135, 100:140, 100:145, 100:150, 100:155, 100:160, 100:165, 100:170, 100:175, 100:180, 100:185, 100:190, 100:195, 100:200, 100:205, 100:210, 100:215, 100:220, 100:225, 100:230, 100:235, 100:240, 100:245, 100:250, 100:255, 100:260, 100:265, 100:270, 100:275, 100:280, 100:285, 100:290, 100:295, 100:300, 100:305, 100:310, 100:315, 100:320, 100:325, 100:330, 100:335, 100:340, 100:345, 100:350, 100:355, 100:360, 100:365, 100:370, 100:375, 100:380, 100:385, 100:390, 100:395, 100:400, 100:405, 100:410, 100:415, 100:420, 100:425, 100:430, 100:435, 100:440, 100:445, 100:450, 100:455, 100:460, 100:465, 100:470, 100:475, 100:480, 100:485, 100:490, 100:495, 100:500, 100:505, 100:510, 100:515, 100:520, 100:525, 100:530, 100:535, 100:540, 100:545, 100:550, 100:555, 100:560, 100:565, 100:570, 100:575, 100:580, 100:585, 100:590, 100:595, 100:600, 100:605, 100:610, 100:615, 100:620,100:625, 100:630, 100:635, 100:640, 100:645, 100:650, 100:655, 100:660, 100:665, 100:670, 100:675, 100:680, 100:685, 100:690, 100:695, 100:700, 100:705, 100:710, 100:715, 100:720, 100:725, 100:730, 100:735, 100:740, 100:745, 100:750, 100:755, 100:760, 100:765, 100:770, 100:775, 100:780, 100:785, 100:790, 100:795, 100:800, 100:805, 100:810, 100:815, 100:820, 100:825, 100:830, 100:835, 100:840, 100:845, 100:850, 100:855, 100:860, 100:865, 100:870, 100:875, 100:880, 100:885, 100:890, 100:895, 100:900, etc.

[0011] According to some specific embodiments, the viable cell number ratio of the Gordonia to the Terrimonas is 100:10, 100:15, 100:20, 100:25, 100:30, 100:35, 100:40, 100:45, 100:50, 100:55, 100:60, 100:65, 100:70, 100:75, 100:80, 100:85, 100:90, 100:95, 100:100, 100:105, 100:110, 100:115, 100:120, 100:125, 100:130, 100:135, 100:140, 100:145, 100:150, 100:155, 100:160, 100:165, 100:170, 100:175, 100:180, 100:185, 100:190, 100:195, 100:200, 100:205, 100:210, 100:215, 100:220, 100:225, 100:230, 100:235, 100:240, 100:245, 100:250, 100:255, 100:260, 100:265, 100:270, 100:275, 100:280, 100:285, 100:290, 100:295, 100:300, 100:305, 100:310, 100:315, 100:320, 100:325, 100:330, 100:335, 100:340, 100:345, 100:350, 100:355, 100:360, 100:365, 100:370, 100:375, 100:380, 100:385, 100:390, 100:395, 100:400, 100:405, 100:410, 100:415, 100:420, 100:425, 100:430, 100:435, 100:440, 100:445, 100:450, 100:455, 100:460, 100:465, 100:470, 100:475, 100:480, 100:485, 100:490, 100:495, 100:500, etc.

[0012] According to some specific embodiments, the viable cell number ratio of the Gordonia, the Pseudomonas, the Xanthomonas and the Terrimonas is 100:(50 - 300):(20 - 600):(10 - 300).

[0013] Further, the viable cell numbers of the Gordonia, the Pseudomonas, the Pseudoxanthomonas, and the Terrabacter are in a ratio of 100:(80 - 150):(30 - 300):(15 - 200).

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

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

[0016] Further, the composite microorganism is a microbial fermentation broth, a microbial freeze-dried powder, or a glycerol bacterium.

[0017] Further, when the composite microorganism is in a liquid form, the viable cell number of the composite microorganism is not less than 1×10 9 CFU / mL, and the composite microorganism agent is used directly or after dilution with water. Among them, the liquid-form composite microorganism is a microbial fermentation broth, which is obtained by separately or mixedly culturing the Gordonia, the Pseudomonas, the Pseudoxanthomonas, and the Terrabacter to obtain a fermentation broth containing these four bacteria.

[0018] Further, when the composite microorganism is in a solid form, before using the composite microorganism agent, the composite microorganism is activated and cultured until the viable cell number is not less than 1×10 9 CFU / mL.

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

[0020] According to some specific embodiments, 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.

[0021] Further, the active component includes calcium peroxide; the inert component includes bentonite.

[0022] Further, the mass fraction of the active component in the slow-release oxygen material is 40%-60%, such as 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, etc., and the mass fraction of the inert component in the slow-release oxygen material is 40%-60%, such as 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, etc.

[0023] Further, the mass ratio of the composite 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.

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

[0025] Still further, 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 microorganism inoculant further includes trace elements.

[0027] Further, the trace elements include Ca 2+ , Fe 2+ , Mn 2+ , Mg 2+ or one or more of them.

[0028] Further, the trace elements include adding 1-100 mg of Ca 2+ , 1-100 mg of Mg 2+ , 1-50 mg of Fe 2+ and 1-5 mg of Mn 2+ per liter of the composite microorganism.

[0029] 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 element, 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 obtained by activating and culturing the composite microorganism until the viable count is not less than 1×10 9 CFU / mL.

[0030] The second aspect of the present invention provides an application of the composite microorganism agent as described above in the repair or control of chlorinated hydrocarbon contaminated sites, wherein the chlorinated hydrocarbon contaminated sites include soil and / or groundwater.

[0031] The third aspect of the present invention provides a method for repairing or controlling chlorinated hydrocarbon contaminated sites by adding the composite microorganism agent as described above to soil and / or groundwater.

[0032] 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 soil and / or groundwater under high pressure through an injection rod, or injected into the soil and / or groundwater through a well; or the groundwater is extracted, mixed with the composite microorganism agent, and then reinjected.

[0033] According to some specific embodiments, the pollutants in the chlorinated hydrocarbon contaminated sites include one or more of 1,2-dichloroethane, chloroform, polychlorinated biphenyls, polycyclic aromatic hydrocarbons, and organochlorine pesticides, as well as pollutants existing in the form of dense non-aqueous phase liquid (DNAPL).

[0034] According to some specific embodiments, the soil includes silty soil.

[0035] 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 microorganism agent suitable for the repair or control of chlorinated hydrocarbon contaminated sites. It mainly through the synergistic effect of Gordonia, Pseudomonas, Pseudoxanthomonas, and Solibacter in specific proportions, effectively solves the problems of difficult repair or high control degree of chlorinated hydrocarbon contaminated sites, overcomes the problems of low mass transfer efficiency and limited microbial activity in traditional repair or control technologies, improves the repair and risk control efficiency of soil and groundwater (especially DNAPL in groundwater) in chlorinated hydrocarbon contaminated sites, realizes green and efficient repair, source reduction, and risk control, and has the advantages of low cost and convenient operation. Therefore, it has important application prospects. Specific Embodiments

[0036] In the prior art, there are relatively few reports on the microbial remediation or control of chlorinated hydrocarbon pollution, and a mature technical system has not yet been formed. In addition, the remediation or control effect of microbial remediation or control technology on the removal of chlorinated hydrocarbon pollution and the pollution existing in the form of DNAPL in silty soil and groundwater is not good. Therefore, the present invention develops a composite microbial agent suitable for the remediation or control of chlorinated hydrocarbon pollution sites. It mainly through the synergistic effect of Gordonia, Pseudomonas, Pseudoxanthomonas, and Terrimonas in specific proportions, combined with slow-release oxygen materials and trace elements, effectively solves the problem of the large difficulty in the remediation or control of chlorinated hydrocarbon pollution sites, overcomes the problems of low mass transfer efficiency and limited microbial activity of traditional remediation or control technologies, improves the remediation and risk control efficiency of soil (especially silty soil) and groundwater (especially DNAPL in groundwater) in chlorinated hydrocarbon pollution sites, realizes green and efficient remediation, source reduction, and risk control, and has the advantages of low cost and convenient operation. Therefore, it has important application prospects.

[0037] Furthermore, in the composite microorganism, the viable cell number ratio of Gordonia, Pseudomonas, Pseudoxanthomonas, and Terrimonas is 100:(25 - 600):(10 - 900):(10 - 500). If the addition ratio of each bacterium exceeds the upper limit or is lower than the lower limit, the remediation or control effect of the composite microbial agent will decrease significantly.

[0038] The functions of each microbial community and the principle of the synergistic effect in improving the remediation or control efficiency of chlorinated hydrocarbon pollution sites are as follows: Gordonia gradually dechlorinates highly chlorinated hydrocarbons to low-chlorinated products through dehalogenase, and produces glycolipid biosurfactants (such as gordonamycin), alleviating the adsorption and locking of chlorinated hydrocarbons by soil particles. Under anoxic or microoxic conditions, exogenous electron donors (such as lactic acid, hydrogen) can be used to drive dechlorination.

[0039] Pseudomonas oxidatively degrades low-chlorinated hydrocarbons through dioxygenase.

[0040] Pseudoxanthomonas can secrete extracellular polysaccharides (EPS) to form a biofilm, which wraps the microbial community and provides a microoxic / anoxic interface, protecting the dechlorinating bacteria from oxidative stress. And through quinone electron shuttles (such as menaquinone) to mediate interspecies electron transfer, supporting the reductive dechlorination reaction of Gordonia. It is active in the oxidation-anoxic transition zone of silty sand soil, coordinating the connection between aerobic and anaerobic metabolism.

[0041] Terrimonas can degrade complex organic substances in soil (such as cellulose, humus), release small molecule carbon sources (such as glucose, acetic acid), provide electron donors for other functional bacteria, secrete EPS to promote the aggregation of silty sand soil, increase porosity, improve the diffusion of DNAPL and contact with microorganisms, maintain the carbon-energy balance of other functional bacteria, and prevent the stagnation of dechlorination caused by insufficient electron donors.

[0042] Synergistic Mechanism In the context of chlorinated hydrocarbon pollution in soil, the composite microbial agent provided by the present invention can form a continuous degradation path from oxidation to reduction through functional microbial metabolic complementarity and spatial collaboration, which can effectively reduce the persistent pollution of chlorinated hydrocarbons. First, Pseudomonas consumes oxygen and oxidizes low-chlorinated hydrocarbons into non-toxic intermediates such as carboxylic acids by secreting dioxygenases, and gradually mineralizes these intermediates into CO2 and H2O. Secondly, Gordonia produces glycolipid biosurfactants (such as Gordoniamycin) on the one hand, which can emulsify high-chlorinated hydrocarbons and alleviate the adsorption and locking effect of soil particles on chlorinated hydrocarbons. On the other hand, it gradually dechlorinates high-chlorinated hydrocarbons into low-chlorinated products by secreting dehalogenases. In addition, this bacterial agent has good environmental adaptability. Pseudomonas is more active under aerobic conditions, and can consume oxygen through the process of oxidizing low-chlorinated hydrocarbons, creating micro-oxygen / anoxic conditions for Gordonia with dechlorination. Under micro-oxic and anoxic conditions, Pseudo-xanthomonas, as the core flora of biofilm, secretes extracellular polysaccharides (EPS) to form biofilm, protects Gordonia flora from oxidative stress, and ensures sufficient microbial biomass for pollutant degradation. In addition, on the one hand, Terrestrial Mononas can degrade complex organic matter (such as cellulose and humus) in the soil, release small molecular carbon sources (such as glucose and acetic acid), provide electron donors for the dechlorination process of Gordonia, and prevent dechlorination stagnation caused by insufficient electron donors; on the other hand, Terrestrial Mononas can also use the byproducts produced by Gordonia dechlorination to continue mineralization to maintain the carbon cycle in the entire degradation process. This bacterial agent has a wide range of environmental pH adaptability, especially the biofilm formation of Pseudo-xanthomonas, which can protect other functional bacteria from better adapting to acidic or alkaline environments.

[0043] Summary of synergistic mechanism: Table 1 below is a summary of the synergistic mechanism of each bacterial community in the bacterial agent provided by the present invention:

[0044] Slow-release oxygen materials The present invention provides a composite microbial agent specifically used for the degradation of organic pollutants. The agent is composed of a plurality of 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.

[0045] 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 equation: 2CaO2 + 2H2O → 2Ca(OH)2 + O2↑). This easily leads to over-addition and a lack of persistence in controlling the oxygen release rate.

[0046] 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 fracture network through adsorption and swelling, preventing soil closure, improving the pore structure of the soil, 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.

[0047] 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 microbial activity caused by the increase in pH.

[0048] During the research process of the present invention, in combination with the remediation or control 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 added can be (20 - 50):1 to ensure that the oxygen release rate matches the metabolic requirements of the microorganisms; 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 dosage 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 and stable oxygen supply.

[0049] Among them, the mass ratio of the composite microorganism to the slow-release oxygen material added is calculated based on the mass of the composite microorganism fermentation broth or the activated and cultured bacterial solution.

[0050] The addition ratio of the composite microorganism to the slow-release oxygen material described in the present invention is optimized, enabling the bacterial agent to efficiently adapt to and degrade the target pollutants in the organic polluted environment, while maintaining good environmental stability and remediation and risk control effects. In practical applications, the addition ratio of the bacterial agent to the oxygen-releasing material can be appropriately adjusted according to the specific conditions of the polluted site to achieve the best remediation and risk control efficiency and economic feasibility.

[0051] Trace element addition The present invention further adds trace elements essential for microbial growth, 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 the effects of remediation and risk control.

[0052] When the composite microbial agent of the present invention is used, it is formulated into a slurry with water and the slurry is injected into the target formation under high pressure through an injection rod. Fracture cracks are formed in the soil by the pressure, and the slurry diffuses along the cracks and fills the pores, expanding the contact area. Bentonite forms a stable crack network under high pressure, preventing the soil from closing, and at the same time wrapping the calcium peroxide particles, slowing down their reaction rate with water and extending their oxygen release period. Thus, the present invention can achieve the efficient remediation of soil and groundwater, source reduction and risk control in chlorinated hydrocarbon contaminated sites through a simple and convenient method, and has the advantage of low cost, so it has important application prospects. Of course, the use method of the composite microbial agent of the present invention is not limited to the above method, and methods such as injection through well construction and reinjection after mixing the microbial agent into the groundwater extraction can also be used.

[0053] Further, when formulating the slurry, the slurry concentration needs to take into account both fluidity and slow-release effect. For silty sandy soil, a lower solid-liquid ratio is preferably adopted to improve fluidity and reduce pumping resistance. 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 added is 1:5 - 1:8.

[0054] In the following, 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 and not 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 objectives, 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 embodiments. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. Unless otherwise stated, they can all be replaced by other equivalent or alternative features with similar objectives. Unless otherwise stated, each feature is only an example in a series of equivalent or similar features. Based on the embodiments in 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 selected embodiments of the present invention.

[0057] The terms used in the present invention generally have the meanings commonly understood by those of ordinary skill in the art unless otherwise specified. The implementation conditions adopted in the examples can be further adjusted according to different requirements of specific uses, and the implementation conditions not specified are conventional conditions in the industry. The technical features involved in each embodiment 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+ 、Mn 2+ 、Mg 2+ etc. are added in the form of corresponding sulfates.

[0059] In the present invention, Gordonia spp. can be, for example, Gordonia spp. with the number TS277327 or TS277564 from Testo Biotechnology and Gordonia spp. with the product number HZB358936 from Gracilaria Biotechnology, etc.

[0060] Pseudomonas spp. can be, for example, Pseudomonas spp. with the number BMZ339652 from Mingzhou Biotechnology, Pseudomonas spp. with the numbers TS278212, TS278215, TS278216, TS278217, TS278218, TS278220, TS278221, TS278222, TS278223, TS278224, etc. from Testo Biotechnology, and Pseudomonas spp. with the product number HZB112253 from Gracilaria Biotechnology, etc.

[0061] Pseudoxanthomonas, for example, can be Pseudoxanthomonas with the catalog number HZB129563 from Gracilaria biota, Pseudoxanthomonas with the numbers BMZ147593 and BMZ147591 from Mingzhou biota, and Pseudoxanthomonas with the number TS326425 from Taisituo biota, etc.

[0062] Terrabacter, for example, can be the genus Terrabacter with the catalog number bio-097242 from Biovector Science Lab, Terrabacter with the number TS278761 from Taisituo biota, Terrabacter with the number BMZ133864 from Mingzhou biota, and Terrabacter with the catalog number HZB133139 from Gracilaria biota, etc.

[0063] In the present invention, the fermentation broth of each strain can be cultured according to the conventional methods in the art to obtain the fermentation broth of each strain with the viable count not less than 1×10 9 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 tank containing a liquid medium at an inoculation ratio of 10%, shake it well to make it evenly mixed, and connect an aeration device to aerate the inside of the fermentation tank, and expand the culture at room temperature for 2 - 3 days. Regularly take samples to monitor the 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 the fermentation broth of each strain with the viable count not less than 1×10 9 CFU / mL.

[0064] The preparation method of the slurry in Example 1 includes the following steps: Prepare a composite microbial fermentation broth: Mix the fermentation broths of each strain according to the ratio of the viable count of Gordonia, Pseudomonas, Pseudoxanthomonas, and Terrabacter being 100:600:10:10 to obtain a composite microbial fermentation broth; Prepare a slow-release oxygen material: Dry-mix calcium peroxide and bentonite in a mass ratio of 1:1, and mix them 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 mass ratio of the composite microbial fermentation broth to the slow-release oxygen material added is 5:1; the mass ratio of the slow-release oxygen material to the clear water added is 1:5; the addition amount of trace elements is 50 mg Ca added per liter of the composite microbial fermentation broth 2+ 、50 mg Mg 2 + 、20 mg Fe 2+ and 3 mg Mn 2+ .

[0066] The preparation method of the slurry in Example 2 includes the following steps: It is basically the same as Example 1, except that the viable cell number ratio of Gordonia, Pseudomonas, Pseudoxanthomonas, and Terrimonas in the composite microbial fermentation broth is 100:100:40:20.

[0067] The preparation method of the slurry in Example 3 includes the following steps: It is basically the same as Example 1, except that the viable cell number ratio of Gordonia, Pseudomonas, Pseudoxanthomonas, and Terrimonas in the composite microbial fermentation broth is 100:25:10:10.

[0068] The preparation method of the slurry in Example 4 includes the following steps: It is basically the same as Example 1, except that the viable cell number ratio of Gordonia, Pseudomonas, Pseudoxanthomonas, and Terrimonas in the composite microbial fermentation broth is 1:1:1:1.

[0069] The preparation method of the slurry in Example 5 includes the following steps: It is basically the same as Example 1, except that the viable cell number ratio of Gordonia, Pseudomonas, Pseudoxanthomonas, and Terrimonas in the composite microbial fermentation broth is 100:100:900:500.

[0070] The preparation method of the slurry in Comparative Example 1 includes the following steps: It is basically the same as Example 1, except that the viable cell number ratio of Gordonia, Pseudomonas, Pseudoxanthomonas, and Terrimonas in the composite microbial fermentation broth is 100:700:10:10.

[0071] The preparation method of the slurry in Comparative Example 2 includes the following steps: It is basically the same as Example 1, except that the viable cell number ratio of Gordonia, Pseudomonas, Pseudoxanthomonas, and Terrimonas in the composite microbial fermentation broth is 100:20:10:10.

[0072] The preparation method of the slurry in Comparative Example 3 includes the following steps: It is basically the same as Example 1, except that the viable cell number ratio of Gordonia, Pseudomonas, Pseudoxanthomonas, and Terrimonas in the composite microbial fermentation broth is 100:100:1000:600.

[0073] The preparation method of the slurry in Comparative Example 4 includes the following steps: It is basically the same as Example 1, except that the composite microbial fermentation broth contains Gordonia and Pseudomonas with a viable cell number ratio of 1:1.

[0074] The preparation method of the slurry in Comparative Example 5 includes the following steps: It is basically the same as Example 1, except that the compound microbial fermentation broth contains Gordonia, Pseudomonas, and Pseudoxanthomonas with a viable cell number ratio of 1:1:1.

[0075] The preparation method of the slurry of Comparative Example 6 includes the following steps: It is basically the same as Example 1, except that the compound microbial fermentation broth contains Gordonia, Pseudomonas, and Solimonas with a viable cell number ratio of 1:1:1.

[0076] The preparation method of the slurry of Comparative Example 7 includes the following steps: It is basically the same as Example 1, except that the compound microbial fermentation broth uses the diluted soil remediation agent bacterial solution. The soil remediation agent comes from the soil remediation agent of Jiangxi Tiaoshui Rensheng Ecological Environment Engineering Co., Ltd., and the soil remediation agent is diluted with water at a ratio of 1:10 to form the soil remediation agent bacterial solution.

[0077] Test Example 1: The soil quality of a polluted site is mainly silty sand. The characteristic pollutants in the soil are 1,2-dichloroethane and chloroform, and the highest pollution concentrations are 120 mg / kg and 13.1 mg / kg respectively, which is a typical chlorinated hydrocarbon DNAPL polluted site. The soil of this site was collected respectively, and the slurries prepared in the above examples and comparative examples were injected into the soil respectively. The degradation rates of 1,2-dichloroethane and chloroform at different times were detected. Among them, 4 L of slurry was injected into every 1 cubic meter of soil. The following Table 2 shows the degradation rate results of 1,2-dichloroethane at different detection times, with the unit of %. Table 3 shows the degradation rate results of chloroform at different detection times, with the unit of %. Among them, the determination methods of the 1,2-dichloroethane content and the chloroform content are: Purge and Trap / Gas Chromatography-Mass Spectrometry HJ605-2011.

[0078]

[0079]

[0080] In the above table, " / " means not detected.

[0081] As can be seen from Table 2 and Table 3, the degradation rates of 1,2-dichloroethane and chloroform in each example are higher than those in each comparative example. Among them, the effect of Example 2 is the best. In Comparative Example 1, due to the too high addition amount of Pseudomonas, and the addition amount of Pseudomonas in Comparative Example 2 is too low, which is not conducive to the synergistic effect of Gordonia and Pseudomonas, and thus leads to a decline in the repair effect. Although the addition amount and addition ratio of Gordonia and Pseudomonas in Comparative Example 3 are appropriate, the addition amount of Pseudoxanthomonas and Solimonas in it is too high, resulting in a competitive relationship between Pseudoxanthomonas and Solimonas and Gordonia and Pseudomonas, and thus leading to a decline in the repair effect.

[0082] Test Example 2: The slow-release oxygen material and clear water were mixed at a mass ratio of 1:8, and stirred to form a homogeneous suspension slurry. Sodium sulfite was added as a reducing agent to consume oxygen according to the saturated or supersaturated dissolved oxygen content in the 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 consuming a certain amount of sodium sulfite every day, a stable oxygen release capacity could still be maintained, and the dissolved oxygen in the sample was maintained at 8-14 mg / L.

[0083] Test Example 3: A certain chemical pollution site in Jiangsu was historically a chemical raw material production enterprise. According to the calculated risk control value and the Class IV water quality limit of the "Groundwater Quality Standard" (GB / T 14848-2017), it was finally determined that the risk of unconfined groundwater was unacceptable, and the pollutant control target value was chloroform, and the pollution depth was 12 m (silty sand layer). Considering economy and stability, the composite microbial agent of the present invention was used to repair and control the groundwater of the site.

[0084] An existing and exceeding-standard monitoring well in the site was selected, and the slurry was prepared according to the scheme of Example 2. The slurry was injected into the existing and exceeding-standard soil in the site through the injection rod of the monitoring well at a high pressure, and applied at a rate of 2 L of slurry per cubic meter of silty sand soil. Fracture fissures were formed in the soil by pressure, and the slurry diffused along the fissures and filled the pores, expanding the contact area to ensure that it reached the polluted area. New monitoring wells were built within the range of 1 m - 5 m horizontal distance from the injection well, and groundwater samples were collected for monitoring. A control group (the control group was the soil without injected slurry) was set up in the plot to explore the influence of the microbial agent on the change of pollutant concentration in this area. The monitored pollutant was chloroform. Within the degradation time of 90 d, the chloroform concentration decreased by 67.1% - 94.3% respectively within the monitoring range of 1 m - 5 m from the injection well. The specific results are shown in Table 4 below. Among them, the determination method of chloroform content was: purge and trap / gas chromatography-mass spectrometry HJ605-2011.

[0085]

[0086] In summary, the present invention has developed a composite microbial agent suitable for the repair or control of chlorinated hydrocarbon contaminated sites. It mainly through the synergistic effect of specific proportions of Gordonia, Pseudomonas, Pseudoxanthomonas, and Solimonas, effectively solves the difficult problems of the repair or control of chlorinated hydrocarbon contaminated sites, overcomes the problems of low mass transfer efficiency and limited microbial activity of traditional repair or control technologies, improves the repair and risk control efficiency of soil and groundwater (especially DNAPL in groundwater) in chlorinated hydrocarbon contaminated sites, realizes green and efficient repair, source reduction and risk control, and has the advantages of low cost and convenient operation. Therefore, it has an important application prospect.

[0087] The above embodiments are only for illustrating the technical concept and features of the present invention, and the purpose is to enable those skilled in the art to understand the content of the present invention and implement it accordingly. It is not intended 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 inoculum, characterized in that: It includes composite microorganisms, and the composite microorganisms include Gordonia, Pseudomonas, Pseudoxanthomonas, and Terrimonas with a viable cell number ratio of 100:(25 - 600):(10 - 900):(10 - 500).

2. The composite microbial inoculum according to claim 1, characterized in that: The viable cell number ratio of the Gordonia, the Pseudomonas, the Pseudoxanthomonas, and the Terrimonas is 100:(50 - 300):(20 - 600):(10 - 300).

3. The composite microbial inoculant according to claim 2, characterized in that: The viable cell number ratio of the Gordonia, the Pseudomonas, the Pseudoxanthomonas, and the Terrimonas is 100:(80 - 150):(30 - 300):(15 - 200).

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 inoculum according to claim 5, wherein: The composite microorganisms are microbial fermentation broth, microbial freeze-dried powder, or glycerol bacteria.

7. The composite microbial inoculum according to claim 5, wherein: 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 compound microbial inoculum according to claim 1, wherein: The composite microbial 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 inoculum according to claim 8, characterized in that: The active component includes calcium peroxide; the inert component includes bentonite.

10. The composite microbial inoculant according to claim 8, wherein: The mass fraction of the active component in the slow-release oxygen material is 40% - 60%, and the mass fraction of the inert component in the slow-release oxygen material is 40% - 60%.

11. The compound microbial inoculum according to claim 8, characterized in that: 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 inoculum according to claim 1, characterized in that: The composite microbial agent further includes trace elements.

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

15. The composite microbial inoculum according to claim 13, characterized in that: The trace elements include 1 - 100 mg of Ca added per liter of the composite microorganism 2+ , 1 - 100 mg of Mg 2+ , 1 - 50 mg of Fe 2+ and 1 - 5 mg of Mn 2+ .

16. Use of a composite microbial inoculant according to any one of claims 1 to 15 in the remediation or control of chlorinated hydrocarbon contaminated sites, wherein, The chlorinated hydrocarbon contaminated site includes soil and / or groundwater.

17. A method for repairing or controlling a chlorinated hydrocarbon contaminated site, characterized in that: By adding the composite microbial agent as described in any one of claims 1 to 15 to the soil and / or groundwater.

18. The method for repairing or controlling a chlorinated hydrocarbon contaminated site according to claim 17, wherein: The composite microbial agent is formulated into a slurry by adding water, and then the slurry is injected into the soil and / or groundwater under high pressure through an injection rod, or injected into the soil and / or groundwater through a well; or the groundwater is extracted and then mixed with the composite microbial agent and reinjected.

19. The method for repairing or controlling a chlorinated hydrocarbon contaminated site according to claim 17, characterized in that: The pollutants in the chlorinated hydrocarbon contaminated site include one or more of 1,2-dichloroethane, chloroform, polychlorinated biphenyls, polycyclic aromatic hydrocarbons, organochlorine pesticides, and pollutants existing in the form of dense non-aqueous phase liquids.

20. The method for repairing or controlling a chlorinated hydrocarbon contaminated site according to claim 17, wherein: The soil includes silty soil.

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