Method for repairing chromium contaminated soil by microorganism in cooperation with natural iron-containing mineral

Abstract

The application discloses a method for repairing chromium contaminated soil by microorganisms and natural iron-containing minerals, which comprises the following steps: mixing the natural iron-containing minerals into the chromium contaminated soil, adding mixed bacteria into the obtained system after standing for several days, standing to carry out a reduction reaction, reducing the high-valence iron and high-valence sulfur in the system into divalent iron and divalent sulfur, and forming a low redox potential environment in the system; adding chromium-reducing bacteria into the system in the low redox potential environment, standing to reduce the hexavalent chromium, obtaining trivalent chromium, and carrying out a mineralization reaction between the trivalent chromium and the divalent iron to obtain a chromitoid product, thereby realizing the repair of the chromium contaminated soil. In the method, the natural iron-containing minerals, the mixed bacteria and the chromium-reducing bacteria cooperatively reduce and mineralize the hexavalent chromium, realize the mineralization and stable repair of the hexavalent chromium, solve the problems of unstable chromium product after traditional repair, easy resolubilization and recontamination and the like, and have the advantages of short repair period, low construction cost and long-acting and stable effect, thereby avoiding the secondary pollution problem in the treatment process.

Description

Technical Field

[0001] This application belongs to the field of chromium-contaminated soil remediation technology, specifically relating to a method for remediating chromium-contaminated soil by microorganisms in synergy with natural iron-containing minerals. Background Technology

[0002] Chromium slag discharged during chromium salt production can easily cause serious pollution to the stockpile and surrounding environment. The toxicity of chromium mainly comes from hexavalent chromium, which is about 100 times more toxic than trivalent chromium. It has strong oxidizing properties, is easily absorbed by organisms, and can cause mutations in organisms. Therefore, the remediation of chromium pollution in soil is urgent.

[0003] Current research has found that microorganisms have the function of accumulating and detoxifying hexavalent chromium, and their treatment of chromium pollution has advantages such as high efficiency, low cost, and minimal environmental impact. Therefore, microbial remediation of chromium pollution has been reported in many studies. However, the microbial remediation of hexavalent chromium still generally suffers from problems such as insufficient electron donors and poor stability of remediation products, making it difficult to continuously improve remediation efficiency. Furthermore, the reduced trivalent chromium is easily re-oxidized to hexavalent chromium by oxidizing substances in the environment, causing the pollutant to "return yellow," which seriously restricts the practical application and long-term stability of this technology. Summary of the Invention

[0004] This application aims to provide a method for the remediation of chromium-contaminated soil by microorganisms in synergy with natural iron-bearing minerals, in order to solve the problems of low remediation efficiency and unstable remediation products in current chromium-contaminated soil remediation methods. It utilizes natural iron-bearing minerals to efficiently provide electron donors, promotes the reduction and mineralization of hexavalent chromium to generate chromite-like products, achieves long-term and stable remediation of chromium-contaminated soil, and avoids the re-dissolution and re-contamination of chromium-contaminated soil after remediation.

[0005] To solve the above-mentioned technical problems, this application is implemented as follows: In a first aspect, embodiments of this application propose a method for the remediation of chromium-contaminated soil by synergistic use of microorganisms and natural iron-containing minerals, the method comprising: Natural iron-containing minerals were mixed into chromium-contaminated soil. After standing for several days, mixed bacteria were added to the resulting system and allowed to stand for a reduction reaction. This reduced the high-valent iron and high-valent sulfur in the system to ferrous iron and ferrous sulfur, and created a low redox potential environment in the system. Chromium-reducing bacteria are added to a system in a low redox potential environment, and hexavalent chromium is reduced by static setting to obtain trivalent chromium. The trivalent chromium then undergoes a mineralization reaction with divalent iron to obtain a chromite-like product, thereby achieving the remediation of the chromium-contaminated soil. The mixed bacteria consist of sulfate-reducing bacteria EM2 and cyclophosphamide.

[0006] Optionally, the sulfate-reducing bacterium EM2 is deposited at the China Center for Type Culture Collection, with accession number CCTCC NO: M2017645; The chromium-reducing bacteria are selected from at least one of Microbacterium GRINML SWG1, Microbacterium GRINML SWG2, and Microbes fincellus GRINML SWG3; The microbacterium GRINML SWG1 is deposited at the China Center for Type Culture Collection, with accession number CCTCC NO: M2021992; The chromium-reducing bacillus GRINML SWG2 is deposited at the China Center for Type Culture Collection, with accession number CCTCCNO: M2021991; The fin-type cellulose microorganism GRINML SWG3 is deposited at the China Center for Type Culture Collection, with accession number CCTCC NO: M2021990.

[0007] Optionally, the method further includes: In the step of static reduction of hexavalent chromium, the redox potential of the reduction system is monitored. When the redox potential is higher than zero, the mixed bacteria are added to the reduction system to restore the redox potential of the reduction system to below zero.

[0008] Optionally, the natural iron-bearing mineral is selected from at least one of natural pyrite, natural magnetite, and natural hematite.

[0009] Optionally, the grain size of the natural iron-bearing mineral is 63.5 μm-254 μm; The amount of the natural iron-containing minerals added accounts for 0.2%-1% of the mass of the chromium-contaminated soil.

[0010] Optionally, the settling period shall satisfy the following conditions: the settling period shall be no less than 3 days and no more than 7 days.

[0011] Optionally, the mixed bacteria are obtained by mixing sulfate-reducing bacteria EM2 and cyclophosphamide in a volume ratio of 1:2 to 2:1 and then incubating them in an incubator at a temperature of 25℃-35℃ for 3-4 days.

[0012] Optionally, the inoculation amount of the mixed bacteria is 5%-40% of the volumetric mass of the chromium-contaminated soil; The reduction reaction takes 3 to 7 days.

[0013] Optionally, the chromium-reducing bacteria are cultured in an oscillator at a temperature of 25℃-35℃ and a rotation speed of 120rpm-180rpm for 10-14 days before being added to the system in a low redox potential environment to reduce hexavalent chromium.

[0014] Optionally, the inoculation amount of the chromium-reducing bacteria is 5%-30% of the volumetric mass of the chromium-contaminated soil.

[0015] Beneficial technical effects: In the embodiments of this application, by first introducing natural iron-bearing minerals as initial electron donors and microbial colonization carriers into chromium-contaminated soil, and then adding a mixed bacterial strain composed of sulfate-reducing bacteria EM2 and cyclophosphamide in stages, the metabolic activities of these bacteria reduce high-valent iron and high-valent sulfur in the system to ferrous iron and ferrous sulfur, thereby constructing and maintaining a stable low redox potential environment in the soil. This effectively solves the problem of continuous electron donor supply in traditional microbial detoxification. Subsequently, chromium-reducing bacteria are added to this reducing environment, enabling them to efficiently reduce hexavalent chromium to trivalent chromium. The resulting trivalent chromium can then undergo an in-situ mineralization reaction with the ferrous iron continuously provided by microbial activity and by natural iron-bearing minerals, thereby generating a highly dense and stable chromite-like product. This solves the problem of easy oxidation and re-dissolution of trivalent chromium precipitates in traditional remediation, achieving long-term and stable remediation of chromium-contaminated soil.

[0016] In the embodiments of this application, the method of microbial synergy with natural iron-containing minerals for remediation of chromium-contaminated soil also has a highly efficient remediation effect on chromium-contaminated soil in high-altitude and cold regions (annual temperature range -26℃ to 25℃). After remediation, the hexavalent chromium-contaminated soil in a certain area of ​​Qinghai Province reached below 2 mg / kg, meeting the relevant control standards, thus providing a new method for the remediation of chromium-contaminated soil in high-altitude and cold regions.

[0017] Additional aspects and advantages of this application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of this application. Attached Figure Description

[0018] The above and / or additional aspects and advantages of this application will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which: Figure 1 This is a flowchart of the method for remediating chromium-contaminated soil using microorganisms in conjunction with natural iron-containing minerals, as proposed in the embodiments of this application. Figure 2 This is a shake-flask experiment effect diagram of the synergistic effect of microorganisms and natural iron-containing minerals on the remediation of chromium-contaminated soil proposed in the embodiments of this application; Figure 3 This is a soil column test effect diagram of the microbial synergistic natural iron-containing mineral remediation of chromium-contaminated soil proposed in the embodiments of this application; Figure 4 This is a diagram of the mineralization products from a soil column test of microbial synergistic natural iron-containing mineral remediation of chromium-contaminated soil, as proposed in the embodiments of this application. Figure 5This is a box test device and effect diagram for the synergistic remediation of chromium-contaminated soil by microorganisms and natural iron-containing minerals, as proposed in the embodiments of this application; Figure 6 This is a diagram of the mineralization products from a box test of microbial synergistic natural iron-containing mineral remediation of chromium-contaminated soil, as proposed in the embodiments of this application. Figure 7 This is a field application effect diagram of the microbial synergistic natural iron-containing mineral remediation of chromium-contaminated soil proposed in the embodiments of this application; Figure 8 This is a diagram of mineralized products from the field application of the microbial synergistic natural iron-containing mineral remediation of chromium-contaminated soil, as proposed in the embodiments of this application. Detailed Implementation

[0019] The embodiments of this application will now be described in detail. Examples of these embodiments are illustrated in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain this application, and should not be construed as limiting this application. All other embodiments obtained by those skilled in the art based on the embodiments of this application without inventive effort are within the scope of protection of this application.

[0020] The terms "first" and "second" in the specification and claims of this application may explicitly or implicitly include one or more of the features. In the description of this application, unless otherwise stated, "multiple" means two or more. Furthermore, "and / or" in the specification and claims indicates at least one of the connected objects, and the character " / " generally indicates that the preceding and following objects are in an "or" relationship.

[0021] In the description of this application, it should be understood that the terms "center", "longitudinal", "lateral", "length", "width", "thickness", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", "clockwise", "counterclockwise", "axial", "radial", "circumferential", etc., indicating the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings, are only for the convenience of describing this application and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of this application.

[0022] In the description of this application, it should be noted that, unless otherwise expressly specified and limited, the terms "installation," "connection," and "linking" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal connection between two components. Those skilled in the art can understand the specific meaning of the above terms in this application based on the specific circumstances.

[0023] In related technologies, the variable valence state elements of natural minerals possess excellent redox properties and can participate in electron transfer processes in microorganisms. Among them, minerals containing oxidized metal elements can act as electron acceptors in microbial electron transfer, while minerals containing reduced metal elements can act as electron donors in the reaction.

[0024] Based on this, the embodiments of this application have found that natural pyrite, magnetite, hematite, etc., can provide electron donors or electron acceptors for chromium-reducing microorganisms, participating in the bioreduction process. This provides nutrients and space for microbial growth and metabolism, improving the reduction efficiency of hexavalent chromium, and also allows for further reaction with the trivalent chromium formed by microbial reduction to form mineralized products, thereby improving the stability of the chromium products.

[0025] Specifically, this application provides a method for the remediation of chromium-contaminated soil by synergistic use of microorganisms and natural iron-containing minerals. Figure 1 This is a flowchart of the method for remediating chromium-contaminated soil using microorganisms in conjunction with natural iron-containing minerals, as proposed in the embodiments of this application. The method specifically includes: Step S1: Mix natural iron-containing minerals into chromium-contaminated soil, let it stand for several days, add mixed bacteria to the resulting system, let it stand to carry out a reduction reaction, reduce the high-valent iron and high-valent sulfur in the system to divalent iron and divalent sulfur, and form a low redox potential environment in the system. It should be noted that in this embodiment, natural iron-containing minerals are first mixed into the chromium-contaminated soil and allowed to stand for several days to introduce a reaction substrate and buffer medium. Natural iron-containing minerals contain iron with variable valence states, which can act as an initial reducing agent to preliminarily reduce and adsorb the high concentration of hexavalent chromium in the soil, thereby reducing the toxicity to subsequently added microorganisms and providing favorable conditions for the colonization of multifunctional microorganisms. Simultaneously, these natural iron-containing mineral particles also serve as a solid-phase carrier, providing a surface for microbial growth and attachment, and the slowly released iron ions provide a reserve of electron donors for the subsequent bioreduction process.

[0026] In some embodiments of this application, the natural iron-bearing mineral is selected from at least one of natural pyrite, natural magnetite, and natural hematite. Natural pyrite, natural magnetite, and natural hematite are widely available, inexpensive, and environmentally friendly. The variable valence iron element abundant in their structure can directly participate in the chemical reduction of high concentrations of hexavalent chromium in the soil during the remediation process, reducing the toxic effects of chromium on subsequent microorganisms, and continuously providing electrons as an electron donor for subsequent microorganisms to reduce hexavalent chromium.

[0027] In some embodiments of this application, natural iron-containing minerals need to be ground to a certain particle size before being added to chromium-contaminated soil; grinding can increase the specific surface area of ​​mineral particles, thereby enhancing their contact efficiency with soil and pollutants, and thus improving their initial adsorption and reduction capacity for hexavalent chromium.

[0028] In some embodiments of this application, the particle size of the natural iron-bearing mineral is 63.5 μm-254 μm; In some embodiments of this application, the particle size of the natural iron-bearing minerals is one or any two of the following: 63.5 μm, 65 μm, 68 μm, 70 μm, 80 μm, 90 μm, 100 μm, 120 μm, 140 μm, 160 μm, 180 μm, 200 μm, 220 μm, 240 μm, and 254 μm. When the natural iron-bearing minerals are ground to a particle size of 63.5 μm-254 μm, not only can the specific surface area be increased to enhance the initial adsorption and reduction capacity for hexavalent chromium and alleviate the toxic effects of high concentrations of chromium on microorganisms, but it can also avoid the impact on the long-term effectiveness of remediation due to excessively fine particles causing soil pore blockage or excessively rapid consumption of minerals.

[0029] In some embodiments of this application, the amount of the natural iron-bearing mineral added accounts for 0.2%-1% of the mass of the chromium-contaminated soil; In some embodiments of this application, the amount of natural iron-bearing minerals added is within the range of 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, and 1% of the mass of chromium-contaminated soil, or any two of these ranges. The addition of 0.2%-1% of natural iron-bearing minerals in this embodiment is to ensure that the initial supply of electron donors and microbial carriers in the system is balanced, to avoid insufficient mineral addition leading to limited initial adsorption and reduction capacity for hexavalent chromium, inability to effectively alleviate the toxicity of high concentrations of chromium to subsequent microorganisms, and to prevent excessive mineral addition from causing damage to the soil pore structure or excessive release of iron, which would inhibit microbial activity.

[0030] In some embodiments of this application, the settling period is specified as follows: the settling period is not less than 3 days and not more than 7 days. A settling time of not less than 3 days and not more than 7 days for natural iron-containing minerals ensures sufficient contact and adsorption / reduction between the minerals and the soil and hexavalent chromium, while also providing sufficient time for the formation of a suitable microenvironment for microbial colonization on the mineral surface. If the settling time is too short, the interaction between the minerals and the soil will be insufficient, resulting in an inability to provide a stable carrier and electron donor source for the subsequent metabolic activities of the mixed bacteria. Conversely, if the settling time is too long, it will lead to mineral surface passivation and excessive consumption of soluble components in the system, thereby weakening the synergistic effect with subsequent microorganisms.

[0031] In some embodiments of this application, the mixed bacteria are composed of sulfate-reducing bacteria EM2 and cyclophosphamide; The sulfate-reducing bacterium EM2 is deposited at the China Center for Type Culture Collection, with accession number CCTCC NO: M2017645. Specifically, the preservation date of sulfate-reducing bacteria EM2 is November 1, 2017, at Wuhan University, China.

[0032] It should also be noted that the mixed bacteria are composed of sulfate-reducing bacteria EM2 and cyclophosphamide; among them, sulfate-reducing bacteria EM2 is sulfate-reducing bacteria, and cyclophosphamide is iron-reducing bacteria.

[0033] In practice, sulfate-reducing bacteria use sulfate ions as electron acceptors to reduce them to sulfur. 2- Iron-reducing bacteria can use ferric iron (Fe3+) naturally occurring in iron-containing minerals or soil as a terminal electron acceptor, reducing it to dissolved ferrous iron (Fe2+) through metabolism. The synergistic metabolic activity of these two microorganisms continuously consumes oxidizing substances in the system while simultaneously generating ferrous iron and sulfur. 2- The presence of highly reducing substances creates a low redox potential environment in the soil microenvironment. This low redox potential environment allows for the efficient reduction of hexavalent chromium and maintains the long-term stability of subsequently generated trivalent chromium, preventing its re-oxidation.

[0034] In some embodiments of this application, the natural iron-containing minerals added in the embodiments of this application are ground. The refined mineral particles can provide more attachment sites and micro-ecological space for sulfate-reducing bacteria and iron-reducing bacteria, which is conducive to the rapid colonization and film formation of microorganisms, promotes the biodissolution and release of iron in the minerals and the electron transfer process, and strengthens the construction of a low redox potential environment.

[0035] In some embodiments of this application, sulfate-reducing bacteria EM2 is selected. This microorganism can survive in chromium-containing environments and gradually reduce hexavalent chromium, exhibiting a certain degree of chromium tolerance. Iron-reducing bacteria cyclophosphamide is selected. In soil systems with added natural iron-containing minerals, this microorganism can utilize the released or inherent trivalent iron in the soil as an electron acceptor to reduce it to dissolved divalent iron. This process is a necessary reactant for subsequent mineralization reactions with trivalent chromium to form chromite-like minerals (FeCr2O4).

[0036] When combined as a mixed bacterial strain, the two can synergistically construct a low redox potential environment. Specifically, sulfate-reducing bacteria EM2 utilizes sulfate ions, while cycloaliphatic bacteria utilize ferric iron. Together, they consume oxidizing substances in the system, thereby accelerating the construction of a low redox potential environment.

[0037] In some embodiments of this application, the mixed bacteria are obtained by mixing sulfate-reducing bacteria EM2 and cyclophosphamide in a volume ratio of 1:2 to 2:1 and then culturing them in an incubator at a temperature of 25℃-35℃ for 3-4 days. In some embodiments of this application, the mixed bacteria are obtained by mixing sulfate-reducing bacteria EM2 and cyclophosphamide in a volume ratio of 1:2, 3:4, 1:1, 3:2, 2:1 or any two of them and then incubating them in an incubator at a temperature of 25℃-35℃ for 3-4 days. In this embodiment, sulfate-reducing bacteria EM2 and cyclophosphamide were mixed in a volume ratio and cultured for 3 to 4 days within a suitable temperature range of 25℃-35℃. This ensured that both microorganisms in the mixed bacteria reached the logarithmic growth phase with vigorous metabolic activity, and that the bacterial population ratio remained stable, maximizing synergistic effects. The culture temperature range closely matched the common growth requirements of the two strains, avoiding metabolic stagnation caused by low temperatures and preventing bacterial inactivation or imbalance caused by high temperatures. The 3-4 day culture period ensured that the bacterial population density and activity reached the optimal inoculation state. When the optimized cultured mixed bacterial solution was inoculated into a soil system containing added natural iron-containing minerals, it rapidly initiated metabolic activity, efficiently utilizing the iron released from the minerals and the inherent iron and sulfur components in the soil. It reduced ferrous iron and ferrous sulfur to ferrous iron and ferrous sulfur, thereby constructing and maintaining a low redox potential environment for a long period. This provided a stable guarantee for the continued action of chromium-reducing bacteria and the mineralization reaction of trivalent chromium and ferrous iron.

[0038] In some embodiments of this application, the inoculation amount of the mixed bacteria is 5%-40% of the volumetric mass of the chromium-contaminated soil; The reduction reaction takes 3-7 days; The inoculation amounts of the mixed bacteria were 5%, 8%, 10%, 12%, 15%, 16%, 18%, 20%, 24%, 26%, 28%, 30%, 32%, 36%, 38%, and 40% of the volumetric mass of the chromium-contaminated soil. In practice, insufficient inoculation or inadequate reaction time will lead to insufficient reduction capacity, failing to provide a sufficient reducing field for the subsequent action of chromium-reducing bacteria. Conversely, excessive inoculation will inhibit activity due to excessive competition among bacterial communities or accumulation of metabolic byproducts, while excessively long reaction time will cause the established reducing environment to experience a potential rebound due to the consumption of electron donors. Therefore, in this embodiment, the inoculation amount of mixed bacteria is set to 5%-40% of the volumetric mass of chromium-contaminated soil, and the reduction reaction time is controlled to 3-7 days. This ensures that sulfate-reducing bacteria and iron-reducing bacteria in the system colonize the soil and mineral surfaces with sufficient community density in the initial stage, effectively utilizing the iron source released from natural iron-containing minerals and the inherent iron and sulfur components in the soil to reduce ferrous iron and ferrous sulfur to ferrous iron and ferrous sulfur, thus constructing and stabilizing a low redox potential environment for the system.

[0039] Step S2: Add chromium-reducing bacteria to the system in a low redox potential environment, allow it to stand and reduce hexavalent chromium to obtain trivalent chromium, and the trivalent chromium reacts with the divalent iron to obtain chromite-like products, thereby achieving the remediation of the chromium-contaminated soil; It should be noted that chromium-reducing bacteria need to be added to the system in a low redox potential environment because, under this low potential environment, chromium-reducing bacteria can reduce soluble, highly toxic hexavalent chromium to less toxic trivalent chromium with higher efficiency and lower metabolic cost.

[0040] In some embodiments of this application, the mineralization reaction between trivalent chromium and divalent iron to obtain chromite-like products is a key step in achieving long-term stability. The trivalent chromium produced by chromium-reducing bacteria chemically tends to co-precipitate with the divalent iron accumulated in large quantities by previous microbial activity in the system. Under continuous reducing conditions, it gradually dehydrates and recrystallizes to form chromite-like products with a spinel structure. In this mineral phase, chromium is firmly bound within the crystal lattice, has extremely low solubility, and its antioxidant capacity far exceeds that of amorphous chromium hydroxide precipitate. Therefore, it can prevent the "yellowing" rebound of chromium pollutants in the soil after remediation, achieving long-term stable remediation.

[0041] In some embodiments of this application, the chromium-reducing bacteria are selected from at least one of Microbacterium GRINML SWG1, Microbacterium GRINML SWG2, and Microbes fimbriae GRINML SWG3; The microbacterium GRINML SWG1 is deposited at the China Center for Type Culture Collection, with accession number CCTCC NO: M2021992; The chromium-reducing bacillus GRINML SWG2 is deposited at the China Center for Type Culture Collection, with accession number CCTCCNO: M2021991; The fin-type cellulose microorganism GRINML SWG3 is deposited at the China Center for Type Culture Collection, with accession number CCTCC NO: M2021990.

[0042] Specifically, the preservation date of the microbacterium GRINML SWG1 was August 6, 2021, at Wuhan University, China. The preservation date of the chromium-reducing bacillus GRINML SWG2 was August 6, 2021, at Wuhan University, China. The preservation date of the Finnigra GRINML SWG3 was August 6, 2021, at Wuhan University, China.

[0043] In this embodiment, at least one of Microbacterium GRINML SWG1, Microbacterium chromium-reducing GRINML SWG2, and Microbacterium fibrinolyticum GRINML SWG3 was selected as the chromium-reducing bacteria because these microorganisms have natural tolerance to high concentrations of hexavalent chromium and efficient reduction capabilities. They can rapidly colonize and continuously metabolize in the low redox potential environment constructed by the pre-mixed bacteria, reducing hexavalent chromium in the soil to trivalent chromium. Furthermore, different strains can form a synergistic effect through complementary metabolic pathways and niche distribution, enhancing their adaptability to complex soil environments and the stability of the reduction process, ensuring that the hexavalent chromium reduction reaction can proceed efficiently in different microdomains.

[0044] In some embodiments of this application, the chromium-reducing bacteria are cultured for 10-14 days in an oscillator at a temperature of 25℃-35℃ and a rotation speed of 120rpm-180rpm before being added to the system in a low redox potential environment to reduce hexavalent chromium.

[0045] In this embodiment, chromium-reducing bacteria are cultured at 25℃-35℃ and 120rpm-180rpm for 10 to 14 days to fully activate microbacteria GRINML SWG1, chromium-reducing bacteria GRINML SWG2 and / or fenestrated microbes GRINMLSWG3 and reach a stable growth period with vigorous metabolic activity, with the bacterial population density and enzyme activity at their best. When the chromium-reducing bacterial solution obtained through cultivation is inoculated into a low redox potential environment constructed by the previous mixed bacteria, it can quickly adapt to and efficiently initiate the reduction metabolism of hexavalent chromium. It makes full use of the existing reducing atmosphere and divalent iron in the system to reduce hexavalent chromium to trivalent chromium. The trivalent chromium generated then undergoes an in-situ mineralization reaction with the divalent iron continuously supplied in the system to generate stable chromite-like products, thus achieving functional continuity and long-term stability of the entire remediation process.

[0046] In some embodiments of this application, the inoculation amount of the chromium-reducing bacteria is 5%-30% of the volumetric mass of the chromium-contaminated soil.

[0047] In some embodiments of this application, the inoculation amount of chromium-reducing bacteria is one or any two of the following values: 5%, 7%, 9%, 12%, 15%, 18%, 20%, 22%, 26%, 28%, and 30% of the volumetric mass of the chromium-contaminated soil.

[0048] In this embodiment, the inoculation amount of chromium-reducing bacteria is set to 5%-30% of the volume mass of chromium-contaminated soil. This ensures that sufficient functional strains can be evenly distributed and effectively colonized on the soil and mineral surfaces, forming a continuous reduction capacity for hexavalent chromium. This inoculation amount range ensures that the bacterial community density is sufficient to cover the hexavalent chromium contamination sites in different micro-domains of the soil, avoiding incomplete reduction or delayed reaction due to insufficient inoculation amount. It also prevents problems such as competitive inhibition between bacterial communities, accumulation of metabolic byproducts, or excessive nutrient consumption caused by excessive inoculation.

[0049] In some embodiments of this application, the method further includes: In the step of static reduction of hexavalent chromium, the redox potential of the reduction system is monitored. When the redox potential is higher than zero, the mixed bacteria are added to the reduction system to restore the redox potential of the reduction system to below zero.

[0050] It should be noted that the reduction system is the soil system after adding chromium-reducing bacteria in step S2; Redox potential (RPP) characterizes the overall redox state of a soil system. In chromium pollution remediation systems, the potential determines the direction and extent of the reduction of hexavalent chromium to trivalent chromium, as well as whether the generated trivalent chromium can exist stably. Only when the system is in a low potential (reduced state) does hexavalent chromium possess the thermodynamic driving force for reduction, and only then can trivalent chromium exist in a stable form without being re-oxidized. Therefore, using RPP as a monitoring indicator can directly and in real-time reflect whether the reduction capacity of the reduction system meets the remediation requirements.

[0051] In this embodiment, a redox potential above zero potential is set as the initiation threshold. Zero potential (relative to the standard hydrogen electrode) is a critical node in the transition of the system from weak reduction to weak oxidation. When the potential of the reduction system is above zero potential, it indicates that the reducing capacity of the system has significantly decreased, and the generated trivalent chromium faces the risk of re-oxidation. Therefore, using zero potential as the critical value for triggering supplementation allows for timely intervention before the system deteriorates severely, ensuring the continuity and stability of the repair process.

[0052] In some embodiments of this application, when the redox potential of the reduction system is higher than zero, a mixed bacterial culture consisting of sulfate-reducing and iron-reducing bacteria is added instead of directly adding chromium-reducing bacteria. This is because the mixed bacteria are the main microorganisms that construct and maintain a low redox potential environment, and the fundamental reason for the potential rise lies in the reducing substances (such as Fe) in the system. 2+ S 2- The consumption rate of bacteria (such as chromium-reducing bacteria) exceeded the rate of microbial regeneration. In the entire remediation process, mixed bacteria are responsible for creating a reducing field, while chromium-reducing bacteria are responsible for reducing hexavalent chromium. When the potential increases, it indicates that the strength of the reducing field is insufficient to support subsequent reactions. Therefore, it is necessary to prioritize restoring the ability to construct the reducing field rather than directly enhancing the reduction process of hexavalent chromium.

[0053] The added mixed bacteria not only do not compete with the existing chromium-reducing bacteria, but also create more favorable thermodynamic conditions for the continued action of the chromium-reducing bacteria by reconstructing a low-potential environment.

[0054] In the embodiments of this application, by intermittently supplementing the mixed bacteria, the generation rate and consumption rate of reducing substances in the system can be rebalanced, thereby achieving self-maintenance of the reducing capacity and ensuring that the reducing capacity does not break down throughout the entire repair cycle, thus ensuring the continuity and long-term stability of the repair process.

[0055] In some embodiments of this application, remediation is considered successful when hexavalent chromium in the soil reaches the standard and chromite minerals are detected. Subsequently, the crystallinity of chromite minerals can be tested periodically.

[0056] To enable those skilled in the art to better understand this application, the following embodiments will be used to provide a detailed description of a method for the remediation of chromium-contaminated soil by microorganisms in synergy with natural iron-containing minerals.

[0057] Example 1 A method for the synergistic remediation of chromium-contaminated soil by microorganisms and natural iron-containing minerals is provided, specifically including: (1) Grind the natural iron-containing minerals composed of 20% natural pyrite and 80% natural magnetite to a particle size of not less than 63.5 μm and not more than 150 μm, and then add them to the chromium-contaminated soil. The amount of natural pyrite added is 1% of the mass of the chromium-contaminated soil. Mix and stir evenly, and let stand for 3-7 days to react. (2) Mix sulfate-reducing bacteria EM2 and cyclophosphamide in a volume ratio of 1:2-2:1 and inoculate into a special culture medium. Incubate at 30°C for 3-4 days to obtain mixed bacteria. (3) After the chromium-contaminated soil with added natural pyrite in step (1) has been left to stand for 3-7 days, the mixed bacteria cultured in step (2) is inoculated into the chromium-contaminated soil and left to stand for another 3-7 days for reduction reaction. The amount of mixed bacteria inoculated is 5% of the volume mass of the chromium-contaminated soil. During this process, the high-valence iron and sulfur in the soil and iron-containing minerals are reduced to divalent iron and divalent sulfur, forming a low redox potential environment that is conducive to the efficient reduction of hexavalent chromium to trivalent chromium and the maintenance of the stability of trivalent chromium. (4) Inoculate the chromium-reducing bacteria (composed of microbacterium GRINML SWG1, chromium-reducing bacteria GRINML SWG2 and fenbac microbacterium GRINML SWG3 in a volume ratio of 1:1:1) into a special culture medium and culture it in a shaker at 30°C and 150 rpm for 10-14 days. (5) After the chromium-contaminated soil with mixed bacteria added in step (3) is allowed to stand for 3-7 days for reduction reaction, the chromium-reducing bacteria cultured in step (4) are inoculated into the chromium-contaminated soil in a low redox potential environment and allowed to stand for reaction. The amount of chromium-reducing bacteria inoculated is 5% of the volume mass of the chromium-contaminated soil. During this process, the chromium-reducing bacteria further reduce the remaining hexavalent chromium to obtain trivalent chromium, and the generated trivalent chromium aggregates with divalent iron to form nucleation mineralization. (6) Regularly monitor the hexavalent chromium content and redox potential in the soil. When the potential is below zero, promptly add mixed bacteria to the reduction system until the hexavalent chromium in the soil is completely repaired and chromite-like products are detected. Subsequently, the crystallinity of chromite minerals can be tested regularly.

[0058] Experimental Example 1 To verify the effectiveness of adding natural iron-containing minerals, mixed bacteria, and chromium-reducing bacteria in the method for remediating chromium-contaminated soil using microorganisms in synergistic remediation with natural iron-containing minerals provided in Example 1, this application embodiment conducted a shake-flask experiment on the synergistic remediation of chromium-contaminated soil using microorganisms and natural iron-containing minerals, specifically including: (1) Take 50.0 g of hexavalent chromium contaminated soil with an initial concentration of 137.56 mg / kg and place it in a 150 mL serum bottle. Add 50 mL of sterile basic salt culture medium to form a mud system with a water-to-soil ratio of 1:1. (2) The experiment was set up with seven treatments: the first group was a blank control consisting only of chromium-contaminated soil and culture medium; The second group is a mineral control with 0.5 g (i.e., 1% of soil dry weight) of sterilized natural iron-containing minerals (containing 20% ​​pyrite and 80% magnetite, with a particle size <150 μm). The third group was an aerobic control group with chromium-reducing bacteria (composed of microbacterium GRINML SWG1, chromium-reducing bacteria GRINML SWG2 and microbacterium fibrillariae GRINML SWG3 mixed in a volume ratio of 1:1:1) added at a 5% (v / w) inoculation amount, and the bottle mouth was covered with a breathable membrane; The fourth group was an anaerobic control group with a mixture of sulfate-reducing bacteria (sulfate-reducing bacteria EM2) and iron-reducing bacteria (cyclobacterium lipophilia) added at the same inoculation amount. The bottle mouth was sealed with butyl rubber stoppers and filled with nitrogen. The fifth group was a two-strain control group that was simultaneously inoculated with the above mixed bacteria and chromium-reducing bacteria (5% each) but without the addition of natural iron-containing minerals. In the early stage of culture (days 0-5), the culture was carried out aerobically with a breathable membrane. On day 5, the culture was changed to be sealed with a rubber stopper to switch to anaerobic conditions until the end of the experiment. Groups 6, 7, and 8 were the core experimental groups. The treatment was the same as that of Group 5, but sterilized natural iron-containing mineral powders of three gradients were added respectively: low (0.25 g, 0.5%), medium (0.5 g, 1%), and high (1 g, 0.2%).

[0059] (3) All groups were placed in a shaker at 30°C and 150 rpm and cultured in the dark for 35 days. Samples were taken on day 0, day 5, day 10, day 20 and day 35 to measure key indicators.

[0060] Figure 2 This is a shake-flask experiment effect diagram of the synergistic effect of microorganisms and natural iron-containing minerals on the remediation of chromium-contaminated soil, as proposed in the embodiments of this application. Figure 2 As shown, after 35 days of cultivation, the concentration of hexavalent chromium in the blank control group showed no significant change. However, the concentration of hexavalent chromium in the mineral-only group (Group 2) decreased to 122.78 mg / kg, with a removal rate of 10.75%. The concentrations of hexavalent chromium in the aerobic bacteria-only group (Group 3) and the anaerobic bacteria-only group (Group 4) decreased to 57.14 mg / kg and 72.72 mg / kg, respectively, with removal rates of 58.47% and 47.14%. The multifunctional bacteria group without added minerals (Group 5, corresponding to…)… Figure 2 The addition of a mixture of bacteria and minerals (such as those from the sixth group) showed a synergistic effect, significantly reducing the concentration of hexavalent chromium to 15.46 mg / kg, with a removal rate of 88.76%. Figure 2 (Middle double bacteria low iron mineral), middle (seventh group, corresponding) Figure 2 (Middle double bacteria, iron minerals), high (Group 8, corresponding to) Figure 2The remediation effect of the core experimental group (containing hematite and high-iron minerals) was further enhanced, with the final concentrations of hexavalent chromium decreasing to 9.05 mg / kg, 3.95 mg / kg, and 4.33 mg / kg, respectively, corresponding to removal rates of 93.42%, 97.13%, and 96.85%. This indicates that multifunctional bacterial communities can reduce hexavalent chromium more efficiently than single bacterial species, and the addition of natural iron-containing minerals can significantly enhance this bioremediation process, especially the promoting effect of hematite at medium to low addition levels (0.5%-1%). However, excessively high mineral addition levels (2%) may lead to soil aggregation or changes in pore structure, slightly inhibiting microbial activity, resulting in a non-linear increase in remediation efficiency with dosage.

[0061] Experimental Example 2 To verify the effectiveness of adding natural iron-containing minerals, mixed bacteria, and chromium-reducing bacteria in the method for remediating chromium-contaminated soil using microorganisms in synergistic remediation with natural iron-containing minerals provided in Example 1, and to detect the final product, chromium-iron minerals, this application embodiment conducted a soil column test on microorganisms synergistically remediating chromium-contaminated soil using natural iron-containing minerals, specifically including: (1) A small-scale soil column remediation test was conducted at a simulated site with a chromium-contaminated soil remediation depth of 50 cm.

[0062] (2) Five remediation conditions were set up, including blank control, conventional chemical remediation (FeSO4), mineral remediation, microbial remediation, and microbial synergistic mineral remediation. The FeSO4 / mineral addition amount was 1%, and 30-50 mL of culture medium was added every 5-7 days. The control group was supplemented with deionized water. Soil samples were taken regularly to monitor the remediation effect of hexavalent chromium.

[0063] Figure 3 This is a soil column test result diagram of the microbial synergistic natural iron-containing mineral remediation of chromium-contaminated soil proposed in the embodiments of this application. Figure 4 This is a diagram of the mineralization products from a soil column test for the synergistic remediation of chromium-contaminated soil by microorganisms and natural iron-bearing minerals, as proposed in the embodiments of this application. Figure 3 As shown, after one year of remediation, the remediation effects were: microbial synergistic mineral remediation > microbial remediation > mineral remediation > chemical remediation > control. In the early stages of remediation, chemical remediation showed better results, but as the remediation time increased, the chemical remediation group gradually experienced re-dissolution, indicating that conventional chemical remediation can lead to secondary pollution. In the later stages of remediation, microbial synergistic mineral remediation was particularly effective, reducing hexavalent chromium from 53.85 mg / kg to undetectable levels, achieving a 100% remediation efficiency for hexavalent chromium.

[0064] like Figure 4 As shown, the hexavalent chromium product was detected in the remediated soil, and only the microbial synergistic mineral remediation group produced FeCr2O4 as the hexavalent chromium product.

[0065] Experiment Example 3 To verify the effectiveness of adding natural iron-containing minerals, mixed bacteria, and chromium-reducing bacteria (composed of *Microbacterium GRINML SWG1*, *Microbacterium chromium-reducing* GRINML SWG2, and *Microbacterium fibrillati* GRINML SWG3 in a 1:1:1 volume ratio) in the method for remediating chromium-contaminated soil using microorganisms in synergistic interaction with natural iron-containing minerals provided in Example 1, this application embodiment conducted a chamber test on the remediation of chromium-contaminated soil using microorganisms in synergistic interaction with natural iron-containing minerals, specifically including: (1) Expand the column experiment and fully simulate the remediation site. Build a 1 m×1 m×1 m box and integrate an intelligent and controllable spraying device to monitor soil parameters in real time, realize the dynamic spraying plan of microbial liquid, and carry out the microbial synergistic mineral remediation expansion experiment.

[0066] (2) Microbial remediation test group and microbial synergistic mineral remediation test group were set up respectively. The mineral addition amount was 1%, and the bacterial solution was added every 5-7 days until the soil moisture reached 10%. Soil samples were collected regularly, and the remediation effect of hexavalent chromium was monitored and tracked in the long term.

[0067] Figure 5 This is a box-type test device and its effect diagram for the synergistic remediation of chromium-contaminated soil by microorganisms and natural iron-containing minerals, as proposed in the embodiments of this application. Figure 6 This is a diagram of the mineralization products from a box test of microbial synergistic natural iron-containing mineral remediation of chromium-contaminated soil, as proposed in the embodiments of this application. Figure 5 and Figure 6 As shown, in the initial remediation phase (0-20 days), both microbial remediation and microbial synergistic mineral remediation rapidly reduced the hexavalent chromium content in the soil, with little difference in their effects. Around 40 days of remediation, the difference in remediation effects between the two gradually increased; around 60 days of remediation, microbial synergistic mineral remediation was significantly more effective than microbial remediation. After one year of remediation, the hexavalent chromium content in the microbial group and the synergistic group decreased from 118.5 mg / kg to 11.2 mg / kg and 1.86 mg / kg, respectively.

[0068] Experiment Example 4 To verify the effectiveness of adding natural iron-containing minerals, mixed bacteria, and chromium-reducing bacteria in the method for remediating chromium-contaminated soil using microorganisms in synergistic remediation with natural iron-containing minerals provided in Example 1, this application embodiment conducted a field application of microorganisms synergistically remediating chromium-contaminated soil using natural iron-containing minerals, specifically including: (1) A control group and a microbial remediation group were set up at a chromium-contaminated site in Qinghai Province (corresponding to...). Figure 7 (Medium biota), microbial synergistic mineral remediation group (corresponding) Figure 7 (China Collaborative Zone) (2) The control group was sprayed with clean water; the different microbial agents in the microbial remediation group were applied by spraying them in staggered shifts through the same pipeline; the minerals in the microbial synergistic mineral remediation group were applied by mixing them with the topsoil, with an application rate of 1%. Microbial agents / deionized water were sprayed every 7 days, 15 days and 30 days before, during and after remediation, respectively, until the soil moisture reached 10%, and the hexavalent chromium content in the soil was tested regularly.

[0069] Figure 7 These are field application effect diagrams of the microbial synergistic natural iron-containing mineral remediation of chromium-contaminated soil proposed in the embodiments of this application. Figure 8 This is a diagram of the mineralization products from the field application of the microbial synergistic natural iron-containing mineral remediation of chromium-contaminated soil, as proposed in the embodiments of this application. Figure 7 As shown, hexavalent chromium leaching and yellowing were observed in both the surface and deep soil layers of the control area. Compared to the control area, the multifunctional bacteria in the microbial remediation area worked synergistically, with a remediation cycle of approximately 4 months. In the microbial-mineral synergistic remediation area, the soil hexavalent chromium content first decreased to the remediation target value of 5.7 mg / kg, with a remediation cycle of approximately 2 months. It is worth emphasizing that the microbial-mineral synergistic effect continued from December to April of the following year (the soil freezing period), indicating that the functional microorganisms and technologies used in the field effectively maintained the activity and sustained function of the microbial community in the high-altitude and cold environment.

[0070] like Figure 8 As shown, the detection of hexavalent chromium products in the remediated soil revealed that stable FeCr2O4 products were only generated in the microbial synergistic mineral remediation zone. In summary, compared to single microbial remediation, microbial synergistic mineral remediation shortens the cycle by 50%, and the hexavalent chromium in the soil remains stable and does not dissolve back after 2 years of remediation.

[0071] In summary, the method for remediating chromium-contaminated soil using microorganisms in synergistic with natural iron-bearing minerals provided in this application, by combining natural iron-bearing minerals with chromium-reducing bacteria, sulfate-reducing bacteria, and iron-reducing bacteria, and applying this combination to the reduction and mineralization treatment of chromium-contaminated soil, can generate stable chromite-like products. The long-term remediation effectiveness of this method has been verified through field application under extreme climatic conditions. This invention not only effectively solves the problems of insufficient electron donors and poor stability of remediation products in traditional microbial remediation of hexavalent chromium, but also provides a new approach for the long-term and stable remediation of chromium-contaminated soil, and has broad application prospects.

[0072] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "illustrative embodiment," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0073] Although embodiments of this application have been shown and described, those skilled in the art will understand that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of this application, the scope of which is defined by the claims and their equivalents.

Claims

1. A method for the remediation of chromium-contaminated soil by microorganisms in synergistic action with natural iron-containing minerals, characterized in that, The method includes: Natural iron-containing minerals were mixed into chromium-contaminated soil. After standing for several days, mixed bacteria were added to the resulting system and allowed to stand for a reduction reaction. This reduced the high-valent iron and high-valent sulfur in the system to ferrous iron and ferrous sulfur, and created a low redox potential environment in the system. Chromium-reducing bacteria are added to a system in a low redox potential environment, and hexavalent chromium is reduced by static setting to obtain trivalent chromium. The trivalent chromium then undergoes a mineralization reaction with divalent iron to obtain a chromite-like product, thereby achieving the remediation of the chromium-contaminated soil. The mixed bacteria consist of sulfate-reducing bacteria EM2 and cyclophosphamide. The sulfate-reducing bacteria use sulfate as an electron acceptor to reduce it to S. 2- The cyclophospholipids use the ferric iron inherent in the natural iron-containing minerals or soil as the terminal electron acceptor, reducing it to dissolved ferrous iron through metabolism; the synergistic metabolic activities of the two microorganisms continuously consume oxidizing substances in the system, while continuously generating ferrous iron and sulfur. 2- This creates a low redox potential environment in the soil microenvironment; The trivalent chromium produced by the chromium-reducing bacteria tends to co-precipitate with the divalent iron accumulated in large quantities by the previous microbial activities in the system, and gradually dehydrates and recrystallizes under continuous reducing conditions to form a spinel-like chromite. In the step of static reduction of hexavalent chromium, the redox potential of the reduction system is monitored. When the redox potential is higher than zero, the mixed bacteria are added to the reduction system to restore the redox potential of the reduction system to below zero. The amount of the natural iron-containing minerals added accounts for 0.2%-1% of the mass of the chromium-contaminated soil; The inoculation amount of the mixed bacteria is 5%-40% of the volumetric mass of the chromium-contaminated soil; The inoculation amount of the chromium-reducing bacteria is 5%-30% of the volumetric mass of the chromium-contaminated soil.

2. The method for remediating chromium-contaminated soil by microorganisms in synergistic effect with natural iron-containing minerals according to claim 1, characterized in that, The sulfate-reducing bacterium EM2 is deposited at the China Center for Type Culture Collection, with accession number CCTCC NO: M2017645. The chromium-reducing bacteria are selected from at least one of Microbacterium GRINML SWG1, Microbacterium GRINML SWG2, and Microbes fincellus GRINML SWG3; The microbacterium GRINML SWG1 is deposited at the China Center for Type Culture Collection, with accession number CCTCCNO: M2021992. The chromium-reducing bacillus GRINML SWG2 is deposited at the China Center for Type Culture Collection, with accession number CCTCC NO: M2021991; The fin-type cellulose microorganism GRINML SWG3 is deposited at the China Center for Type Culture Collection, with accession number CCTCCNO: M2021990.

3. The method for remediating chromium-contaminated soil by microorganisms in synergistic effect with natural iron-containing minerals according to claim 1, characterized in that, The natural iron-bearing mineral is selected from at least one of natural pyrite, natural magnetite, and natural hematite.

4. The method for remediating chromium-contaminated soil by microorganisms in synergistic effect with natural iron-containing minerals according to claim 1 or 3, characterized in that, The grain size of the natural iron-bearing mineral is 63.5 μm-254 μm.

5. The method for remediating chromium-contaminated soil by microorganisms in synergistic effect with natural iron-containing minerals according to claim 1, characterized in that, The required number of days for settling is: no less than 3 days and no more than 7 days.

6. The method for remediating chromium-contaminated soil by microorganisms in synergistic effect with natural iron-containing minerals according to claim 1, characterized in that, The mixed bacteria were obtained by mixing sulfate-reducing bacteria EM2 and cyclophosphamide in a volume ratio of 1:2 to 2:1 and then incubating them in an incubator at a temperature of 25℃-35℃ for 3-4 days.

7. The method for remediating chromium-contaminated soil by microorganisms in synergistic effect with natural iron-containing minerals according to claim 1, characterized in that, The reduction reaction takes 3 to 7 days.

8. The method for remediating chromium-contaminated soil by microorganisms in synergistic effect with natural iron-containing minerals according to claim 1, characterized in that, The chromium-reducing bacteria were cultured for 10-14 days in a shaker at 25℃-35℃ and 120rpm-180rpm before being added to the system in a low redox potential environment to reduce hexavalent chromium.

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