Microorganism-based high-throughput reductive dehalogenation method

Abstract

The invention provides a microorganism-based high-flux reductive dehalogenation method, relates to the technical field of environmental restoration, and solves the technical problems of long dehalogenation time consumption and only dehalogenation of single halogenated hydrocarbon in the prior art. The dehalogenation method comprises the steps that a culture pore plate and reaction containers are obtained, the number of the reaction containers is the same as that of fixing pore sites, and the reaction containers comprise a plurality of first reaction containers; adding anaerobic water, a methyl viologen solution, a potassium acetate solution, a titanium citrate (III) solution, halogenated hydrocarbon to be degraded and a dehalogenation bacterium solution into each first reaction container; each first reaction container is correspondingly added into one fixing hole position; and in an oxygen-free environment, carrying out heat preservation on the culture pore plate at a first preset temperature for a first preset time so as to carry out dehalogenation treatment on the to-be-degraded halogenated hydrocarbon through the dehalogenation bacteria. According to the invention, by preparing the specific reaction liquid, the dehalogenation of microorganisms can be realized, the dehalogenation environment is stable, and the dehalogenation effect is good.

Description

Technical Field

[0001] The invention relates to the technical field of environmental remediation, and in particular to a high-throughput reduction dehalogenation method based on microorganisms. Background Art

[0002] Microbial reduction dehalogenation of halogenated hydrocarbons refers to the process in which halogenated hydrocarbons can act as electron acceptors for specific microorganisms, and the halogen atoms in halogenated hydrocarbons are reduced and removed through microbial metabolism, thereby converting toxic halogenated hydrocarbons into more harmless or easily decomposable substances. Microbial reduction dehalogenation technology has been widely studied and applied in the field of environmental remediation because of its green, economical and safe characteristics.

[0003] The traditional dehalogenation method is time-consuming and the target substances studied each time are relatively few, resulting in limited research on halogenated hydrocarbons. Secondly, there is currently a lack of complete sets of dehalogenation equipment, which can only dehalogenate a single halogenated hydrocarbon, and cannot achieve batch dehalogenation. Summary of the invention

[0004] The present invention aims to at least solve the technical problem existing in the related art that "the dehalogenation time is long and only a single halogenated hydrocarbon can be dehalogenated".

[0005] To this end, the present invention provides a high-throughput reduction dehalogenation method based on microorganisms.

[0006] The microbial-based high-throughput reduction dehalogenation method provided by the present invention comprises: obtaining a culture well plate, the culture well plate comprising a plurality of fixed well positions; obtaining reaction containers, the number of the reaction containers being the same as the number of the fixed well positions, the reaction containers comprising a plurality of first reaction containers; adding anaerobic water, methyl viologen solution, potassium acetate solution, titanium (III) citrate solution, halogenated hydrocarbons to be degraded and dehalogenating bacteria solution to each first reaction container; wherein the types of halogenated hydrocarbons to be degraded in different first reaction containers are the same or different; adding each first reaction container to a corresponding fixed well position; in an anaerobic environment, keeping the culture well plate warm at a first preset temperature for a first preset time to dehalogenate the halogenated hydrocarbons to be degraded by the dehalogenating bacteria.

[0007] The high-throughput reduction dehalogenation method based on microorganisms provided by the present invention comprises: obtaining a culture well plate, the culture well plate comprises a plurality of fixed well positions; obtaining a reaction container, the reaction container is used for carrying out a dehalogenation reaction, the number of the reaction containers is the same as the number of the fixed well positions, that is, each reaction container can be fixedly installed in a fixed well position, so as to carry out a dehalogenation experiment, the reaction container comprises a plurality of first reaction containers, the first reaction container is also the reaction container used for the degradation reaction of the halogenated hydrocarbon to be degraded by the dehalogenating bacteria, and the specific dehalogenation process is to add anaerobic water, methyl viologen solution, Potassium acetate solution, titanium (III) citrate solution, halogenated hydrocarbons to be degraded and dehalogenating bacteria solution; wherein the types of halogenated hydrocarbons to be degraded in different first reaction containers are the same or different; that is, the present application can realize a degradation reaction of a halogenated hydrocarbon to be degraded, and can also realize the degradation reaction of multiple types of halogenated hydrocarbons to be degraded at the same time, after the reaction solution and the halogenated hydrocarbons to be degraded are added to the first reaction container, each first reaction container is added to a fixed hole position; in an anaerobic environment, the culture plate is kept warm at a first preset temperature for a first preset time to dehalogenate the halogenated hydrocarbons to be degraded by dehalogenating bacteria. The high-throughput reduction dehalogenation method based on microorganisms of the present invention can realize the dehalogenation of microorganisms by preparing a specific reaction solution (that is, a mixed solution of anaerobic water, methyl viologen solution, potassium acetate solution and titanium (III) citrate solution), the dehalogenation environment is stable, and the dehalogenation effect is good. Among them, anaerobic water provides an anaerobic environment to avoid the inhibitory effect of oxygen on dehalogenating bacteria. Methyl viologen acts as an electron transfer medium to help electrons transfer from dehalogenating bacteria to halogenated hydrocarbons, thereby promoting the reduction dehalogenation reaction. Potassium acetate solution is used to maintain the pH value of the reaction system stable and create an environment suitable for the growth and metabolism of dehalogenating bacteria. Titanium citrate (III) is a reducing agent that provides electrons for the reaction, drives the reaction, maintains a low redox potential, thereby improving the efficiency of dehalogenation and saving dehalogenation time. Among them, the dehalogenating bacteria are anaerobic reducing dehalogenating bacteria. In addition, the present application can simultaneously dehalogenate multiple types of halogenated hydrocarbons through culture well plates and reaction vessels with specific structures, greatly improving research efficiency. Since multiple halogenated hydrocarbon reduction experiments are carried out at the same time, random errors caused by differences in reaction solutions, microorganisms, and culture conditions can be avoided. On the other hand, the reaction vessels are independently arranged, which is convenient for replacement in special circumstances to avoid wasting the entire culture well plate.

[0008] In some technical schemes, optionally, the microbial-based high-throughput reductive dehalogenation method also includes: adding an organic solvent to a first reaction container to dissolve the organic compound in the first reaction container in the organic solvent, wherein different halogenated hydrocarbons to be degraded correspond to different organic solvents; separating the organic solvent and the organic compound in the first reaction container to obtain a first mixture; passing the first mixture in the first reaction container into a chromatography-mass spectrometer, and determining the compounds present in the first reaction container by the chromatography-mass spectrometer, and analyzing the degradation products of the halogenated hydrocarbons to be degraded based on the compounds present in the first reaction container.

[0009] In this technical solution, after dehalogenation, an organic solvent is added to the first reaction container to extract the organic compounds in the first reaction container, and then the organic solvent and the organic compounds are separated, and finally the compounds present in the first reaction container are determined by a chromatogram-mass spectrometer, and finally the degradation products of the halogenated hydrocarbons to be degraded are analyzed based on the compounds present in the first reaction container. For example, in the process of dehalogenation of tetrachloroethylene, after microbial dehalogenation, the organic compounds in the first reaction container are extracted by an organic solvent, and then the mixture is passed into a chromatogram-mass spectrometer. Since the chromatogram-mass spectrometer can separate the mixture and determine the compounds contained in the mixture at the same time, for example, the ethylene compound is detected by the chromatogram-mass spectrometer, indicating that during the dehalogenation of tetrachloroethylene, the degradation product generates ethylene, so the degradation product can be analyzed.

[0010] In some technical schemes, optionally, the reaction container includes multiple second reaction containers, and the number of the second reaction containers is the same as the number of the first reaction containers. The microbial-based high-throughput reduction dehalogenation method also includes: adding anaerobic water, methyl viologen solution, potassium acetate solution, titanium (III) citrate solution and halogenated hydrocarbons to be degraded to each second reaction container; wherein the types of halogenated hydrocarbons to be degraded in different second reaction containers are the same or different, and the halogenated hydrocarbons to be degraded in each second reaction container are the same as the type of halogenated hydrocarbons to be degraded in a first reaction container; adding each second reaction container to a fixed hole position; adding an organic solvent to the second reaction container to dissolve the organic compound in the second reaction container in the organic solvent; separating the organic solvent and the organic compound in the second reaction container to obtain a second mixture; passing the second mixture in the second reaction container into a chromatography-mass spectrometer, and determining the compounds present in the second reaction container by the chromatography-mass spectrometer, and analyzing the degradation conditions of the halogenated hydrocarbons to be degraded based on the compounds present in the first reaction container and the compounds present in the second reaction container.

[0011] In this technical solution, it is equivalent to adding a set of reference experiments without adding dehalogenating bacteria for each type of halogenated hydrocarbon to be degraded, which can determine whether different types of halogenated hydrocarbons will undergo natural degradation, thereby analyzing the degradation conditions of different halogenated hydrocarbons, such as natural degradation or microbial degradation. For example, still taking tetrachloroethylene as an example, if trichloroethylene, dichloroethylene, vinyl chloride or ethylene is produced in the second reaction container after the dehalogenation reaction, it means that tetrachloroethylene can be naturally degraded, because no dehalogenating bacteria solution is added to the second reaction container. If trichloroethylene, dichloroethylene, vinyl chloride or ethylene is not produced in the second reaction container after the dehalogenation reaction, and trichloroethylene, dichloroethylene, vinyl chloride or ethylene is produced in the first reaction container, it means that tetrachloroethylene can only undergo degradation reaction in the presence of dehalogenating bacteria solution, thereby analyzing the degradation conditions of tetrachloroethylene.

[0012] In some technical solutions, optionally, the reaction container further includes a third reaction container and a fourth reaction container, and before the step of adding anaerobic water, methyl viologen solution, potassium acetate solution, titanium (III) citrate solution, halogenated hydrocarbons to be degraded and dehalogenating bacteria solution to each first reaction container, the step further includes: adding anaerobic water, methyl viologen solution, potassium acetate solution, titanium (III) citrate solution and dehalogenating bacteria solution to the third reaction container and the fourth reaction container; adding a first mass of hexachlorobenzene to the third reaction container; in an anaerobic environment, keeping the culture plate warm at a first preset temperature for a first preset time; adding hexane to the third reaction container and the fourth reaction container to dissolve the organic compounds in the third reaction container and the fourth reaction container in the hexane; separating the hexane and organic compounds in the third reaction container. compound to obtain a third mixture; separate the hexane and the organic compound in the fourth reaction container to obtain a fourth mixture; pass the third mixture into a chromatography-mass spectrometer to determine the mass of hexachlorobenzene in the third reaction container by the chromatography-mass spectrometer, recorded as the second mass, and determine whether the target compound exists in the third reaction container; pass the fourth mixture into a chromatography-mass spectrometer to determine whether the target compound exists in the fourth reaction container by the chromatography-mass spectrometer; when the second mass is less than the first mass, the target compound exists in the third reaction container, and the target compound does not exist in the fourth reaction container, add anaerobic water, methyl viologen solution, potassium acetate solution, titanium (III) citrate solution, halogenated hydrocarbons to be degraded and dehalogenating bacteria solution to each first reaction container.

[0013] In this technical solution, before conducting the halogenated hydrocarbon dehalogenation experiment, the feasibility of the solution of this application is first proved by a control experiment of hexachlorobenzene. Specifically, the reaction solution and the dehalogenating bacteria solution are added to the third reaction container and the fourth reaction container, but hexachlorobenzene is not added to the fourth reaction container, and hexachlorobenzene is only added to the third reaction container. In this way, in the process of separating the mixture by chromatography-mass spectrometry, if the mass of hexachlorobenzene in the third reaction container is reduced, and target compounds such as pentachlorobenzene, tetrachlorobenzene, trichlorobenzene, dichlorobenzene, etc. are generated, and there is no target compound in the fourth reaction container, it means that the hexachlorobenzene in the third reaction container has undergone a degradation reaction under the action of dehalogenating bacteria, and hexachlorobenzene is not added to the fourth reaction container, so no degradation reaction will occur in the fourth reaction container, and no target compound will be produced, so that it can be proved that under the environment of this application, dehalogenating bacteria can degrade halogenated hydrocarbons, so as to carry out subsequent degradation experiments of unknown halogenated hydrocarbons. If the target compound also appears in the fourth reaction container, it proves that the solution of this application is not feasible, so there is no need to carry out the subsequent degradation experiment of unknown halogenated hydrocarbons.

[0014] In some technical schemes, optionally, when the halogenated hydrocarbons to be degraded include polychlorinated biphenyls and / or polychlorinated dibenzo-p-dioxins, the organic solvent includes n-hexane and / or toluene; when the halogenated hydrocarbons to be degraded include brominated flame retardants, the organic solvent includes cyclohexane and / or ethyl acetate; when the halogenated hydrocarbons to be degraded include perfluorinated compounds, the organic solvent includes methanol and / or acetonitrile.

[0015] In this technical solution, both n-hexane and toluene can be used as organic solvents for polychlorinated biphenyls and polychlorinated dibenzo-p-dioxins, that is, the compounds of polychlorinated biphenyls and polychlorinated dibenzo-p-dioxins after dechlorination can be dissolved in n-hexane or toluene solvents, thereby being separated, and then subsequent quantitative and qualitative analysis can be performed by chromatography-mass spectrometry. Similarly, cyclohexane and ethyl acetate can be used as organic solvents for brominated flame retardants, and methanol and acetonitrile can be used as organic solvents for perfluorinated compounds.

[0016] In some technical solutions, optionally, the polychlorinated biphenyls include one or a combination of the following: 12 H3Cl7、C 12 H4Cl6 and C 12 H7Cl3; Polychlorinated dibenzo-p-dioxins include one or a combination of the following: C 12 H4Cl4O2、C 12 H2Cl6O2 and C 12 Cl8O2; Brominated flame retardants include one or a combination of the following: C 15 H 12 Br4O2、C 12 H2Br8O and C12 H 18 Br6; Perfluorinated compounds include one or a combination of the following: C8HF 15 O2、C9H5F 13 O2 and C8HF 17 O3S.

[0017] In some technical solutions, optionally, the types of halogenated hydrocarbons to be degraded in different first reaction containers are different, and the number of types of halogenated hydrocarbons to be degraded is greater than or equal to 50 and less than or equal to 70.

[0018] In this technical solution, the present application can simultaneously perform degradation experiments on dozens of halogenated hydrocarbons, greatly improving efficiency.

[0019] In some technical schemes, optionally, any fixed hole position has a hole position number, and the types of halogenated hydrocarbons to be degraded in different first reaction containers are different. Before the step of adding each first reaction container to a fixed hole position, it also includes: establishing a corresponding relationship between the hole position number and the corresponding type of halogenated hydrocarbons to be degraded.

[0020] In this technical solution, since the culture well plate of the present application has hundreds of fixed wells and can carry out degradation experiments of 60 halogenated hydrocarbons at the same time, a correspondence between the well number and the corresponding type of halogenated hydrocarbon to be degraded is established, which is convenient for the management of the degradation experiments of dozens of halogenated hydrocarbons and is not easy to cause confusion. That is, the type of halogenated hydrocarbon to be degraded in the reaction container can be determined based on the well number and the corresponding relationship, so as to understand the degradation products and degradation pathways of the halogenated hydrocarbon to be degraded.

[0021] In some technical solutions, optionally, the volume of anaerobic water is 732.5 μL.

[0022] In some technical solutions, optionally, the concentration of methyl viologen in the methyl viologen solution is 10 mmol / L, and the volume of the methyl viologen solution is 125 μL.

[0023] In some technical solutions, optionally, the concentration of potassium acetate in the potassium acetate solution is 1 mol / L, and the volume of the potassium acetate solution is 125 μL.

[0024] In some technical schemes, optionally, the mass fraction of titanium (III) citrate in the titanium (III) citrate solution is 15%, and the volume of the titanium (III) citrate solution is 5 μL.

[0025] In some technical solutions, optionally, the number of fixed wells of the culture well plate is greater than or equal to 300 and less than or equal to 500. For example, it can be 384.

[0026] In some technical solutions, optionally, the first preset temperature is greater than or equal to 25° and less than or equal to 35°, for example, it may be 30°.

[0027] In some technical solutions, optionally, the first preset duration is greater than or equal to 20 hours and less than or equal to 28 hours, for example, it may be 24 hours.

[0028] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which: Figure 1 One of the preparation flow charts of a high-throughput reduction dehalogenation method based on microorganisms according to an embodiment of the present application is shown; Figure 2 A schematic diagram showing the structure of a high-throughput screening device according to an embodiment of the present application is shown; Figure 3 A schematic diagram of the structure of a culture well plate according to an embodiment of the present application is shown; Figure 4 The second preparation flow chart of a high-throughput reduction dehalogenation method based on microorganisms according to an embodiment of the present application is shown.

[0030] in, Figure 2 and Figure 3 The corresponding relationship between the reference numerals and the component names is as follows: 1 high-throughput screening device, 11 culture well plate, 12 reaction container, 13 fixed well position. DETAILED DESCRIPTION

[0031] In order to more clearly understand the above-mentioned purposes, features and advantages of the present application, the present application is further described in detail below in conjunction with the accompanying drawings and specific implementation methods. It should be noted that the embodiments of the present application and the features in the embodiments can be combined with each other without conflict.

[0032] In the following description, many specific details are set forth to facilitate a full understanding of the present application. However, the present application may also be implemented in other ways different from those described herein. Therefore, the scope of protection of the present application is not limited to the specific embodiments disclosed below.

[0033] like Figure 1 As shown, the high-throughput reduction dehalogenation method based on microorganisms provided in this embodiment comprises: S102: Obtain a culture well plate, the culture well plate comprising a plurality of fixed wells; S104: Acquire reaction containers, the number of which is the same as the number of fixed wells, and the reaction containers include a plurality of first reaction containers; S106: adding anaerobic water, methyl viologen solution, potassium acetate solution, titanium (III) citrate solution, halogenated hydrocarbons to be degraded and dehalogenating bacteria solution to each first reaction container; wherein the types of halogenated hydrocarbons to be degraded in different first reaction containers are the same or different; S108: adding each first reaction container to a corresponding fixed hole position; S110: In an anaerobic environment, the culture plate is kept warm at a first preset temperature for a first preset time to dehalogenate the halogenated hydrocarbons to be degraded by dehalogenating bacteria.

[0034] The high-throughput reduction dehalogenation method based on microorganisms provided by the present invention comprises: obtaining a culture well plate, the culture well plate comprises a plurality of fixed well positions; obtaining a reaction container, the reaction container is used for carrying out a dehalogenation reaction, the number of the reaction containers is the same as the number of the fixed well positions, that is, each reaction container can be fixedly installed in a fixed well position, so as to carry out a dehalogenation experiment, the reaction container comprises a plurality of first reaction containers, the first reaction container is also the reaction container used for the degradation reaction of the halogenated hydrocarbon to be degraded by the dehalogenating bacteria, and the specific dehalogenation process is to add anaerobic water, methyl viologen solution, potassium acetate solution, titanium citrate (III) solution, the halogenated hydrocarbon to be degraded into each first reaction container; Degradation of halogenated hydrocarbons and dehalogenating bacteria solution; wherein the types of halogenated hydrocarbons to be degraded in different first reaction containers are the same or different; that is, the present application can realize a degradation reaction of a halogenated hydrocarbon to be degraded, and can also realize the degradation reaction of multiple types of halogenated hydrocarbons to be degraded at the same time, after adding the reaction solution (i.e., a mixed solution of anaerobic water, methyl viologen solution, potassium acetate solution and titanium (III) citrate solution) and the halogenated hydrocarbon to be degraded to the first reaction container, each first reaction container is added to a fixed hole position; in an anaerobic environment, the culture plate is kept warm at a first preset temperature for a first preset time to dehalogenate the halogenated hydrocarbon to be degraded by dehalogenating bacteria. The high-throughput reduction dehalogenation method based on microorganisms of the present invention can realize the dehalogenation of microorganisms by preparing a specific reaction solution, the dehalogenation environment is stable, and the dehalogenation effect is good. Among them, anaerobic water provides an anaerobic environment to avoid the inhibitory effect of oxygen on dehalogenating bacteria. Methyl viologen acts as an electron transfer medium to help electrons transfer from dehalogenating bacteria to halogenated hydrocarbons, thereby promoting the reduction dehalogenation reaction. Potassium acetate solution is used to maintain the pH value of the reaction system stable and create an environment suitable for the growth and metabolism of dehalogenating bacteria. Titanium citrate (III) is a reducing agent that provides electrons for the reaction, drives the reaction, maintains a low redox potential, thereby improving the efficiency of dehalogenation and saving dehalogenation time. Among them, the dehalogenating bacteria are anaerobic reducing dehalogenating bacteria. In addition, the present application can simultaneously dehalogenate multiple types of halogenated hydrocarbons through culture well plates and reaction vessels with specific structures, greatly improving research efficiency. Since multiple halogenated hydrocarbon reduction experiments are carried out at the same time, random errors caused by differences in reaction solutions, microorganisms, and culture conditions can be avoided. On the other hand, the reaction vessels are independently arranged, which is convenient for replacement in special circumstances to avoid wasting the entire culture well plate.

[0035] In some embodiments, optionally, the microbial-based high-throughput reductive dehalogenation method further includes: adding an organic solvent to a first reaction container to dissolve the organic compound in the first reaction container in the organic solvent, wherein different halogenated hydrocarbons to be degraded correspond to different organic solvents; separating the organic solvent and the organic compound in the first reaction container to obtain a first mixture; passing the first mixture in the first reaction container into a chromatography-mass spectrometer, and determining the compounds present in the first reaction container by the chromatography-mass spectrometer, and analyzing the degradation products of the halogenated hydrocarbons to be degraded based on the compounds present in the first reaction container.

[0036] In this embodiment, after dehalogenation, an organic solvent is added to the first reaction container to extract the organic compound in the first reaction container, and then the organic solvent and the organic compound are separated, and finally the compounds present in the first reaction container are determined by a chromatogram-mass spectrometer, and finally the degradation products of the halogenated hydrocarbon to be degraded are analyzed based on the compounds present in the first reaction container. For example, in the process of dehalogenation of tetrachloroethylene, after microbial dehalogenation, the organic compound in the first reaction container is extracted by an organic solvent, and then the mixture is passed into a chromatogram-mass spectrometer. Since the chromatogram-mass spectrometer can separate the mixture and determine the compounds contained in the mixture at the same time, for example, the ethylene compound is detected by the chromatogram-mass spectrometer, indicating that during the dehalogenation of tetrachloroethylene, the degradation product generates ethylene, so that the degradation product can be analyzed.

[0037] In some embodiments, optionally, the step of adding an organic solvent to the first reaction container specifically includes: adding an organic solvent to the first reaction container and shaking the container at a rotation speed of 800 rpm for a first preset time. This can increase the amount of organic compound dissolved, thereby ensuring more accurate experimental data during subsequent quantitative analysis.

[0038] In some embodiments, optionally, the reaction container includes multiple second reaction containers, the number of the second reaction containers is the same as the number of the first reaction containers, and the microbial-based high-throughput reduction dehalogenation method further includes: adding anaerobic water, methyl viologen solution, potassium acetate solution, titanium (III) citrate solution and halogenated hydrocarbons to be degraded to each second reaction container; wherein the types of halogenated hydrocarbons to be degraded in different second reaction containers are the same or different, and the halogenated hydrocarbons to be degraded in each second reaction container are the same as the type of halogenated hydrocarbons to be degraded in a first reaction container; adding each second reaction container to a fixed hole position; adding an organic solvent to the second reaction container to dissolve the organic compound in the second reaction container in the organic solvent; separating the organic solvent and the organic compound in the second reaction container to obtain a second mixture; passing the second mixture in the second reaction container into a chromatography-mass spectrometer, and determining the compounds present in the second reaction container by the chromatography-mass spectrometer, and analyzing the degradation conditions of the halogenated hydrocarbons to be degraded based on the compounds present in the first reaction container and the compounds present in the second reaction container.

[0039] In this embodiment, it is equivalent to adding a group of reference experiments without adding dehalogenating bacteria for each type of halogenated hydrocarbon to be degraded, which can determine whether different types of halogenated hydrocarbons will undergo natural degradation, thereby analyzing the degradation conditions of different halogenated hydrocarbons, such as natural degradation or microbial degradation. For example, still taking tetrachloroethylene as an example, if trichloroethylene, dichloroethylene, vinyl chloride or ethylene is produced in the second reaction container after the dehalogenation reaction, it means that tetrachloroethylene can be naturally degraded, because no dehalogenating bacteria solution is added to the second reaction container. If trichloroethylene, dichloroethylene, vinyl chloride or ethylene is not produced in the second reaction container after the dehalogenation reaction, and trichloroethylene, dichloroethylene, vinyl chloride or ethylene is produced in the first reaction container, it means that tetrachloroethylene can only undergo degradation reaction in the presence of dehalogenating bacteria solution, thereby analyzing the degradation conditions of tetrachloroethylene.

[0040] In some embodiments, optionally, the reaction container further includes a third reaction container and a fourth reaction container, and before the step of adding anaerobic water, methyl viologen solution, potassium acetate solution, titanium (III) citrate solution, halogenated hydrocarbons to be degraded and dehalogenating bacteria solution to each first reaction container, the step further includes: adding anaerobic water, methyl viologen solution, potassium acetate solution, titanium (III) citrate solution and dehalogenating bacteria solution to the third reaction container and the fourth reaction container; adding a first mass of hexachlorobenzene to the third reaction container; in an anaerobic environment, keeping the culture plate warm at a first preset temperature for a first preset time; adding hexane to the third reaction container and the fourth reaction container to dissolve the organic compounds in the third reaction container and the fourth reaction container in the hexane; separating the hexane and organic compounds in the third reaction container compound to obtain a third mixture; separate the hexane and the organic compound in the fourth reaction container to obtain a fourth mixture; pass the third mixture into a chromatography-mass spectrometer to determine the mass of hexachlorobenzene in the third reaction container by the chromatography-mass spectrometer, recorded as the second mass, and determine whether the target compound exists in the third reaction container; pass the fourth mixture into a chromatography-mass spectrometer to determine whether the target compound exists in the fourth reaction container by the chromatography-mass spectrometer; when the second mass is less than the first mass, and the target compound exists in the third reaction container but not in the fourth reaction container, add anaerobic water, methyl viologen solution, potassium acetate solution, titanium (III) citrate solution, halogenated hydrocarbons to be degraded and dehalogenating bacteria solution to each first reaction container.

[0041] In this embodiment, before the halogenated hydrocarbon dehalogenation experiment is carried out, the feasibility of the scheme of the present application is first proved by the control experiment of hexachlorobenzene. Specifically, the reaction solution and the dehalogenating bacteria solution are added to the third reaction container and the fourth reaction container, but hexachlorobenzene is not added to the fourth reaction container, and hexachlorobenzene is only added to the third reaction container. In this way, in the process of separating the mixture by chromatography-mass spectrometry, if the mass of hexachlorobenzene in the third reaction container is reduced, and target compounds such as pentachlorobenzene, tetrachlorobenzene, trichlorobenzene, dichlorobenzene, etc. are generated, and there is no target compound in the fourth reaction container, it means that the hexachlorobenzene in the third reaction container has undergone a degradation reaction under the action of dehalogenating bacteria, and hexachlorobenzene is not added in the fourth reaction container, so no degradation reaction will occur in the fourth reaction container, and no target compound will be produced, so that it can be proved that under the environment of the present application, dehalogenating bacteria can degrade halogenated hydrocarbons, so as to carry out subsequent unknown degradation experiments of halogenated hydrocarbons. If the target compound also appears in the fourth reaction container, it is proved that the scheme of the present application is not feasible, so there is no need to carry out the subsequent unknown degradation experiments of halogenated hydrocarbons.

[0042] In some embodiments, optionally, when the halogenated hydrocarbons to be degraded include polychlorinated biphenyls and / or polychlorinated dibenzo-p-dioxins, the organic solvent includes n-hexane and / or toluene; when the halogenated hydrocarbons to be degraded include brominated flame retardants, the organic solvent includes cyclohexane and / or ethyl acetate; when the halogenated hydrocarbons to be degraded include perfluorinated compounds, the organic solvent includes methanol and / or acetonitrile.

[0043] In this embodiment, both n-hexane and toluene can be used as organic solvents for polychlorinated biphenyls and polychlorinated dibenzo-p-dioxins, that is, the compounds of polychlorinated biphenyls and polychlorinated dibenzo-p-dioxins after dechlorination can be dissolved in n-hexane or toluene solvents, thereby being separated, and then subsequent quantitative and qualitative analysis can be performed by chromatography-mass spectrometry. Similarly, cyclohexane and ethyl acetate can be used as organic solvents for brominated flame retardants, and methanol and acetonitrile can be used as organic solvents for perfluorinated compounds.

[0044] In some embodiments, the polychlorinated biphenyls may include one or a combination of the following: 12 H3Cl7、C 12 H4Cl6 and C 12 H7Cl3; Polychlorinated dibenzo-p-dioxins include one or a combination of the following: C 12 H4Cl4O2、C 12 H2Cl6O2 and C 12 Cl8O2; Brominated flame retardants include one or a combination of the following: C 15 H 12 Br4O2、C 12 H2Br8O and C 12 H 18 Br6; Perfluorinated compounds include one or a combination of the following: C8HF 15 O2、C9H5F 13 O2 and C8HF 17 O3S.

[0045] In some embodiments, optionally, the types of halogenated hydrocarbons to be degraded in different first reaction containers are different, and the types of halogenated hydrocarbons to be degraded are greater than or equal to 50 and less than or equal to 70.

[0046] In this embodiment, the present application can simultaneously perform degradation experiments on dozens of halogenated hydrocarbons, greatly improving efficiency.

[0047] In some embodiments, optionally, any fixed hole position has a hole position number, and the types of halogenated hydrocarbons to be degraded in different first reaction containers are different. Before the step of adding each first reaction container to a fixed hole position, it also includes: establishing a corresponding relationship between the hole position number and the corresponding type of halogenated hydrocarbons to be degraded.

[0048] In this embodiment, since the culture well plate of the present application has hundreds of fixed wells and can carry out degradation experiments of 60 halogenated hydrocarbons at the same time, a correspondence between the well number and the corresponding type of halogenated hydrocarbon to be degraded is established, which is convenient for the management of the degradation experiments of dozens of halogenated hydrocarbons and is not easy to cause confusion. That is, the type of halogenated hydrocarbon to be degraded in the reaction container can be determined based on the well number and the corresponding relationship, so as to understand the degradation products and degradation pathways of the halogenated hydrocarbon to be degraded.

[0049] In some embodiments, optionally, the volume of anaerobic water is 732.5 μL.

[0050] In some embodiments, optionally, the concentration of methyl viologen in the methyl viologen solution is 10 mmol / L, and the volume of the methyl viologen solution is 125 μL.

[0051] In some embodiments, optionally, the concentration of potassium acetate in the potassium acetate solution is 1 mol / L, and the volume of the potassium acetate solution is 125 μL.

[0052] In some embodiments, optionally, the mass fraction of titanium (III) citrate in the titanium (III) citrate solution is 15%, and the volume of the titanium (III) citrate solution is 5 μL.

[0053] In some embodiments, optionally, the number of fixed wells of the culture well plate is greater than or equal to 300 and less than or equal to 500. For example, it can be 384.

[0054] More specifically, if Figure 2As shown, the culture well plate 11 of the present application is composed of 4 small well plates, and the number of fixed well positions 13 of the culture well plate 11 is 384, that is, the number of fixed well positions 13 of each small well plate is 96. Then for each small well plate, there are also 96 reaction containers 12 that match it. Among these 96 reaction containers 12, there are 3 third reaction containers, 3 fourth reaction containers, 45 first reaction containers and 45 second reaction containers, so that anaerobic water, methyl violet solution, potassium acetate solution, titanium citrate (III) solution and dehalogenating bacteria solution are added to the 3 third reaction containers, and anaerobic water, methyl violet solution, potassium acetate solution, titanium citrate (III) solution, dehalogenating bacteria solution and hexachlorobenzene are added to the 3 fourth reaction containers, so that through the comparative experiment of the 3 third reaction containers and the 3 fourth reaction containers, it is verified that the reaction liquid components of the present application meet the degradation environment of microorganisms. On this basis, 15 different types of halogenated hydrocarbons are added to 45 first reaction containers, and 3 portions of each halogenated hydrocarbon are added, that is, three groups of parallel experiments are performed for each halogenated hydrocarbon to be degraded, so that the degradation process of 15 halogenated hydrocarbons can be analyzed at the same time, and their degradation products can be determined. In addition, 15 different types of halogenated hydrocarbons corresponding to the first reaction container are added to 45 second reaction containers, and 3 portions of each halogenated hydrocarbon are added, but the second reaction container does not add a dehalogenating bacteria solution, which is mainly used to analyze whether 15 different types of halogenated hydrocarbons will degrade naturally, thereby analyzing the degradation conditions and degradation products of 15 different types of halogenated hydrocarbons. It can be seen that for each small well plate, 15 different types of halogenated hydrocarbons can be analyzed at the same time, so for the culture well plate 11, 15×4=60 different types of halogenated hydrocarbons can be analyzed at the same time, thereby realizing batch experiments and greatly improving experimental efficiency.

[0055] In some embodiments, optionally, the first preset temperature is greater than or equal to 25° and less than or equal to 35°, for example, it may be 30°.

[0056] In some embodiments, optionally, the first preset duration is greater than or equal to 20 hours and less than or equal to 28 hours, for example, it may be 24 hours.

[0057] Another embodiment of the present invention provides a high-throughput reduction dehalogenation method based on microorganisms.

[0058] It is important to understand that the microbial reduction dehalogenation of halogenated hydrocarbons refers to the process in which halogenated hydrocarbons can act as electron acceptors for specific microorganisms (such as organohalide-respiring bacteria, OHRB, Organohalide-Respiring-Bacteria) to reduce and remove the halogen atoms in halogenated hydrocarbons through microbial metabolism, thereby converting toxic halogenated hydrocarbons into more harmless or easily decomposable substances. Microbial reduction dehalogenation technology has been widely studied and applied in the field of environmental remediation because of its green, economical and safe characteristics.

[0059] Existing testing methods for microbial reduction of halogenated hydrocarbons include biosensor technology, gene chip screening and traditional laboratory culture technology: (1) Biosensor technology: Genetic engineering technology is used to construct biosensors to monitor the metabolic activities of microorganisms in the process of degrading halogenated hydrocarbons, thereby evaluating the biodegradability of halogenated hydrocarbons, such as gene reporter constructs and microbial fuel cell-type biotoxicity sensors. Gene reporter constructs are cloned by the promoter of the "sentinel" gene and used to drive the expression of the gene reporter construct. They can be used as early warning biosensors to monitor the metabolism of halogenated hydrocarbons. The messenger RNA expressed at a higher level by these "sentinel" genes under stress can be used as an indicator for monitoring the degradation of halogenated hydrocarbons; microbial fuel cell-type biotoxicity sensors can be used to detect the toxicity of pollutants such as pesticides. By monitoring the change in current, the effect of pollutants on microbial activity can be evaluated, and then it can be determined whether the pollutants are degraded by microorganisms. This sensor can be used to monitor the anaerobic reduction degradation of halogenated hydrocarbons. Although biosensors have high sensitivity and selectivity for the detection of trace substances and weak signals, and can achieve efficient and accurate detection of target substances, they have limited adaptability to different substances and require special design and adjustment to achieve detection of target substances. In addition, the sensitivity and detection range of biosensors are limited by the characteristics of the organism itself. Different substances may require different sensors for detection, which increases the cost of research and development and application, resulting in limited popularity in practical applications.

[0060] (2) Gene chip technology: By fixing a large number of predetermined nucleic acid probes on a solid phase carrier and hybridizing the probes with fluorescently labeled target samples, rapid detection of target genes can be achieved. It can also be used to screen functional genes related to the biodegradation of organic pollutants, reveal the expression pattern and regulatory network of degradation-related genes under pollution conditions, and further analyze the degradation ability and degradation pathway of microorganisms. Gene chip is currently the only high-throughput technology that can simultaneously identify and quantify microbial functional genes. It has the characteristics of high density and high parallelization, but it can only focus on known genes and cannot reveal the discovery of unknown genes and new functions. It also has high R&D costs.

[0061] (3) Laboratory culture and degradation experiments: First, an anaerobic culture medium needs to be prepared and a carbon source and an electron donor are added to the culture medium. Then, the halogenated hydrocarbon monomer or mixture to be studied is added to the culture medium and cultured, or directly enriched based on the sediment or mud of the contaminated site. This traditional microbial reduction and dehalogenation method often takes several months in the experimental cycle, and in order to eliminate the influence of synergistic effects, only a few degradation targets can be studied in one culture. Therefore, this long experimental cycle limits the research efficiency. Currently, only more than 200 halogenated hydrocarbons have been studied for microbial reduction and dehalogenation. Many potential halogenated hydrocarbon pollutants have not been fully studied and treated, which is not conducive to rapid response to the control needs of environmental pollutants.

[0062] In summary, existing research technologies are limited by the characteristics of different substances, and have high R&D costs and limited applicability. Traditional methods have a long experimental cycle and can only study a small number of target objects in one experiment.

[0063] High-throughput technology is a modern scientific technology that can process a large number of samples at the same time, reduce errors caused by different batch operations, improve the accuracy of results and screening efficiency, and reduce research costs. It is widely used in the field of environment. In the field of environmental monitoring, it can be used for screening of organic pollutants, such as high-throughput screening for traceability analysis of drug metabolites in urban wastewater, screening of new pollutants in environmental media, etc. In the field of microorganisms, high-throughput sequencing technology can be used for atmospheric pollution control and air quality assessment, and is also used for natural water material circulation and pollution control and environmental remediation of secondary water bodies, as well as changes in microbial diversity in soil, functional strain construction and screening, etc. High-throughput technology plays an important role in many environmental fields such as environmental monitoring, pollution control, and microbial research, and can identify and evaluate pollutants and microorganisms more quickly and efficiently.

[0064] The present application proposes a method that can simultaneously simulate the reduction and dehalogenation of multiple halogenated hydrocarbons by microorganisms and understand their reaction pathways.

[0065] The entire experimental operation process was carried out in an anaerobic glove box. Figure 2 and Figure 3 As shown, first, a high-throughput screening device 1 is used to add dehalogenating bacteria cells to a reaction vessel 12 containing a halogenated hydrocarbon reaction solution. The dehalogenase in the outer membrane of the dehalogenating bacteria cells can react rapidly with halogenated hydrocarbons in the reaction solution environment to dehalogenate. After 24 hours of culture, an organic solvent is added to extract the culture sample, and the type and content of its degradation products are qualitatively and quantitatively determined by chromatography-mass spectrometry to analyze its reaction path. This method can quickly and massively screen out halogenated hydrocarbons with microbial reduction and degradation advantages, and can determine and analyze their degradation products and degradation pathways, thereby helping to formulate effective remediation strategies for contaminated sites.

[0066] In more detail, the present invention provides a method for rapidly and systematically screening halogenated hydrocarbons that can be degraded by microorganisms and analyzing their degradation pathways, wherein a high-throughput screening device 1 comprises a reaction vessel 12 with a volume of 2 mL for reacting halogenated hydrocarbons with dehalogenating bacteria cells and a high-throughput culture plate 11 with 384 wells, both of which are made of quartz material.

[0067] like Figure 4 As shown, the method of the present application specifically includes the following steps: S402: Preparation of experimental drugs and equipment.

[0068] Specifically, anaerobic water, 10 mmol / L methyl viologen, 1 mol / L potassium acetate solution, 15% titanium (III) citrate and 50 mmol / L halogenated hydrocarbon as an electron acceptor were prepared according to the experimental requirements.

[0069] S404: dehalogenation reaction.

[0070] Specifically, 732.5 μL of anaerobic water, 125 μL of methyl viologen, 125 μL of potassium acetate solution, 5.0 μL of titanium (III) citrate and 12.5 μL of halogenated hydrocarbons were added to the reaction vessel 12, and then 125 μL of dehalogenating bacteria cells were added. All operations were performed in an anaerobic glove box. A negative control without a halogenated hydrocarbon substrate and a positive control of hexachlorobenzene, a known degradable halogenated hydrocarbon, were set up in each batch of experiments, and a chemical control without microorganisms was set up in each halogenated hydrocarbon sample. Each sample was run in parallel 3 times, so 60 halogenated hydrocarbons could be tested for each batch of samples. Cultured at 30°C for 24 hours.

[0071] S406: Extraction.

[0072] Specifically, 400 μL of an organic solvent was added to the sample after the culture was completed, mixed, and extracted at 800 rpm for 24 hours in a shaker to purify and concentrate.

[0073] S408: Determine the product structure and concentration using chromatography-mass spectrometry.

[0074] Specifically, the product solution obtained by extraction is subjected to a chromatography-mass spectrometer to perform qualitative and quantitative analysis on the product to determine the chemical structure and content of the product.

[0075] S410: Analyze the reaction pathway.

[0076] In the embodiments of the present invention, the degradation reaction pathway of halogenated hydrocarbons under the action of dehalogenating bacteria cells is comprehensively analyzed based on the product information measured by chromatography-mass spectrometry and combined with existing research results related to microbial metabolic pathways.

[0077] The method provided in this embodiment has the following beneficial effects: The optimized reaction liquid components can make the reduction dehalogenation reaction proceed quickly, which greatly reduces the culture time compared with the traditional culture method, thereby improving the efficiency of scientific research; and the reaction conditions are applicable to all halogenated hydrocarbons. Compared with biosensor technology and gene chips that can only detect specific compounds, this method has a wider applicability and low research cost. After setting up negative controls, positive controls and chemical controls respectively, the experiment can still carry out 60 kinds of halogenated hydrocarbon microbial degradation experiments at the same time, which further improves the rate of scientific research. The cultured samples are then extracted, and the structure and concentration of the products are analyzed by chromatography-mass spectrometry. The degradation experiments and detection analyses of multiple halogenated hydrocarbons are carried out simultaneously, which can also avoid systematic errors and random errors caused by differences in reaction conditions and instrument status of different batches of experiments.

[0078] The high-throughput reduction dehalogenation method provided by the present invention aims to improve the screening rate of halogenated hydrocarbons that can be reduced by microorganisms. The main improvements are as follows: (1) The reaction vessel is set up independently, which is convenient for replacement in special circumstances and avoids wasting the entire culture plate; (2) The design of the culture well plate is conducive to fixing the reaction vessel and avoiding contamination caused by tipping over the reaction vessel; (3) The culture plate has 384 wells, which can simultaneously carry out degradation experiments on 60 halogenated hydrocarbons, greatly improving research efficiency; (4) The culture plate numbers are clear, making it easy to record experimental samples; (5) 60 halogenated hydrocarbons simulated microbial reduction experiments were carried out simultaneously, which can avoid random errors caused by differences in reaction solutions, microorganisms, and culture conditions; (6) The detachable design between the reaction vessel and the culture plate facilitates the arrangement of different samples and facilitates detection and analysis; (7) The overall device is simple in design, easy to assemble and disassemble, and convenient to operate in the anaerobic glove box; (8) This method has a wide range of applications, requires less material for the experiment, and is highly economical.

[0079] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "illustrative embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present application. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

[0080] Although the embodiments of the present application have been shown and described, those skilled in the art will appreciate that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present application, and that the scope of the present application is defined by the claims and their equivalents.

Claims

1. A high-throughput reduction dehalogenation method based on microorganisms, characterized in that: include: Obtaining a culture well plate, wherein the culture well plate comprises a plurality of fixed well positions; Obtaining reaction containers, the number of which is the same as the number of the fixed holes, and the reaction containers include a plurality of first reaction containers; Adding anaerobic water, methyl viologen solution, potassium acetate solution, titanium (III) citrate solution, halogenated hydrocarbons to be degraded and dehalogenating bacteria solution to each of the first reaction containers; wherein the types of halogenated hydrocarbons to be degraded in different first reaction containers are the same or different; Adding each of the first reaction containers into a corresponding fixed hole; In an anaerobic environment, the culture well plate is kept warm at a first preset temperature for a first preset time period to dehalogenate the halogenated hydrocarbons to be degraded by dehalogenating bacteria.

2. The high-throughput reduction dehalogenation method based on microorganisms according to claim 1, characterized in that: Also includes: Adding an organic solvent into the first reaction container to dissolve the organic compound in the first reaction container in the organic solvent, wherein different halogenated hydrocarbons to be degraded correspond to different organic solvents; separating the organic solvent and the organic compound in the first reaction container to obtain a first mixture; The first mixture in the first reaction container is introduced into a chromatography-mass spectrometer, and the compounds present in the first reaction container are determined by the chromatography-mass spectrometer, and based on the compounds present in the first reaction container, the degradation products of the halogenated hydrocarbon to be degraded are analyzed.

3. The high-throughput reduction dehalogenation method based on microorganisms according to claim 2, characterized in that: The reaction container includes a plurality of second reaction containers, the number of the second reaction containers is the same as the number of the first reaction containers, and the high-throughput reduction dehalogenation method based on microorganisms further includes: Adding anaerobic water, methyl viologen solution, potassium acetate solution, titanium (III) citrate solution and halogenated hydrocarbons to be degraded into each of the second reaction containers; wherein the types of halogenated hydrocarbons to be degraded in different second reaction containers are the same or different, and the halogenated hydrocarbons to be degraded in each of the second reaction containers are the same as the type of halogenated hydrocarbons to be degraded in one of the first reaction containers; Add each of the second reaction containers into a corresponding fixed hole; adding an organic solvent into the second reaction container so that the organic compound in the second reaction container is dissolved in the organic solvent; separating the organic solvent and the organic compound in the second reaction container to obtain a second mixture; The second mixture in the second reaction container is introduced into a chromatography-mass spectrometer, and the compounds present in the second reaction container are determined by the chromatography-mass spectrometer. Based on the compounds present in the first reaction container and the compounds present in the second reaction container, the degradation conditions of the halogenated hydrocarbon to be degraded are analyzed.

4. The high-throughput reduction dehalogenation method based on microorganisms according to claim 1, characterized in that: The reaction container further comprises a third reaction container and a fourth reaction container. Before the step of adding anaerobic water, methyl viologen solution, potassium acetate solution, titanium (III) citrate solution, halogenated hydrocarbons to be degraded and dehalogenating bacteria solution to each of the first reaction containers, the microbial-based high-throughput reduction dehalogenation method further comprises: Adding anaerobic water, methyl viologen solution, potassium acetate solution, titanium (III) citrate solution and dehalogenating bacteria solution into the third reaction container and the fourth reaction container; adding a first mass of hexachlorobenzene to the third reaction vessel; In an anaerobic environment, keeping the culture well plate warm at a first preset temperature for a first preset time; Adding hexane to the third reaction container and the fourth reaction container to dissolve the organic compound in the third reaction container and the fourth reaction container in the hexane; separating the hexane and the organic compound in the third reaction container to obtain a third mixture; separating the hexane and the organic compound in the fourth reaction container to obtain a fourth mixture; Passing the third mixture into a chromatography-mass spectrometer to determine the mass of hexachlorobenzene in the third reaction container by the chromatography-mass spectrometer, recorded as the second mass, and determining whether the target compound exists in the third reaction container; Passing the fourth mixture into a chromatography-mass spectrometer to determine whether the target compound exists in the fourth reaction container through the chromatography-mass spectrometer; When the second mass is less than the first mass, the target compound exists in the third reaction container, and the target compound does not exist in the fourth reaction container, anaerobic water, methyl viologen solution, potassium acetate solution, titanium (III) citrate solution, halogenated hydrocarbons to be degraded and dehalogenating bacteria solution are added to each of the first reaction containers.

5. The high-throughput reduction dehalogenation method based on microorganisms according to claim 2, characterized in that: In the case where the halogenated hydrocarbons to be degraded include polychlorinated biphenyls and / or polychlorinated dibenzo-p-dioxins, the organic solvent includes n-hexane and / or toluene; In the case where the halogenated hydrocarbon to be degraded includes a brominated flame retardant, the organic solvent includes cyclohexane and / or ethyl acetate; In the case where the halogenated hydrocarbon to be degraded includes a perfluorinated compound, the organic solvent includes methanol and / or acetonitrile.

6. The high-throughput reduction dehalogenation method based on microorganisms according to claim 5, characterized in that: The polychlorinated biphenyls include one or a combination of the following: 12 H3Cl7、C 12 H4Cl6 and C 12 H7Cl3; The polychlorinated dibenzo-p-dioxin includes one or a combination of the following: 12 H4Cl4O2、C 12 H2Cl6O2 and C 12 Cl8O2; The brominated flame retardant includes one or a combination of the following: 15 H 12 Br4O2、C 12 H2Br8O and C 12 H 18 Br6; The perfluorinated compound includes one or a combination of the following: C8HF 15 O2、C9H5F 13 O2 and C8HF 17 O3S.

7. The high-throughput reduction dehalogenation method based on microorganisms according to claim 1, characterized in that: The types of halogenated hydrocarbons to be degraded in different first reaction containers are different, and the types of halogenated hydrocarbons to be degraded are greater than or equal to 50 and less than or equal to 70.

8. The high-throughput reduction dehalogenation method based on microorganisms according to claim 1, characterized in that: Each of the fixed holes has a hole number, and the types of halogenated hydrocarbons to be degraded in different first reaction containers are different. Before the step of adding each first reaction container to a fixed hole, the microbial-based high-throughput reduction dehalogenation method further includes: Establish a corresponding relationship between the pore position number and the corresponding type of halogenated hydrocarbon to be degraded.

9. The high-throughput reduction dehalogenation method based on microorganisms according to claim 1, characterized in that: The volume of the anaerobic water is 732.5 μL; and / or The concentration of methyl viologen in the methyl viologen solution is 10 mmol / L, and the volume of the methyl viologen solution is 125 μL; and / or The concentration of potassium acetate in the potassium acetate solution is 1 mol / L, and the volume of the potassium acetate solution is 125 μL; and / or The mass fraction of titanium (III) citrate in the titanium (III) citrate solution is 15%, and the volume of the titanium (III) citrate solution is 5 μL.

10. The high-throughput reduction dehalogenation method based on microorganisms according to claim 1, characterized in that: The number of fixed wells of the culture well plate is greater than or equal to 300 and less than or equal to 500; and / or The first preset temperature is greater than or equal to 25° and less than or equal to 35°; and / or The first preset duration is greater than or equal to 20 hours and less than or equal to 28 hours.

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