Microbial agent for degrading trichloroethane as well as enrichment method and application of microbial agent

Abstract

The invention relates to a microbial agent for degrading trichloroethane as well as an enrichment method and application of the microbial agent, and belongs to the technical field of microbial engineering. The invention provides a microbial agent for degrading trichloroethane, which comprises a functional bacterium A and a functional bacterium B. The functional bacterium A and the functional bacterium B have amino acid sequences for coding dehalogenase a and / or dehalogenase b, the amino acid sequence for coding dehalogenase a is shown as SEQ ID NO.3, and the amino acid sequence for coding dehalogenase b is shown as SEQ ID NO.4. The invention further provides a preparation method of the microbial agent for degrading trichloroethane. Experiments find that 1, 1, 2-TCA can be degraded into 1, 2-DCA by the dehalogenase a under the condition that the dehalogenase a and the dehalogenase b exist at the same time, and then the 1, 2-DCA is instantly converted into ethylene by the dehalogenase, so that only a small amount or no toxic intermediate product 1, 2-DCA or VC is generated in the degradation process.

Description

Technical Field

[0001] This invention relates to the field of microbial engineering technology, and in particular to a bacterial agent for degrading trichloroethane, its enrichment method, and its application. Background Technology

[0002] 1,1,2-Trichloroethane (1,1,2-TCA) is a halogenated organic compound with carcinogenic and neurotoxic properties, and is listed as a priority pollutant in many countries. 1,1,2-TCA is widely used as an industrial solvent, metal degreasing agent, and chemical intermediate. Industrial leaks, improper use, and inadequate disposal have led to its entry into groundwater, causing groundwater pollution. 1,1,2-TCA is chemically stable, has a long half-life, and tends to persist in groundwater for extended periods, easily spreading and forming pollution plumes.

[0003] Microbial remediation (bioaugmentation or biostimulation) is an effective means of degrading 1,1,2-TCA, and its core relies on the reductive dechlorination of organohalogenating bacteria (OHRB). Currently, the reductive dechlorination agents discovered domestically and internationally include *Dehalogenimonas*, *Desulfitobacterium*, *Geobacter (Trichlorobacter)*, and *Dehalobacter*. Species of these four genera reductively dechlorinate 1,1,2-TCA through dichloroelimination or hydrogenolysis. The product of *Geobacter* strain IAE's reductive dechlorination of 1,1,2-TCA via dichloroelimination is vinyl chloride (VC). The product of *Desulfitobacterium* strain PR's reductive dechlorination of 1,1,2-TCA via hydrogenolysis is 1,2-dichloroethane (1,2-DCA). VC is listed as a Group 1 carcinogen, 1,2-DCA is listed as a Group 2B carcinogen, and 1,1,2-TCA is listed as a Group 3 carcinogen. This indicates that existing 1,1,2-TCA bacterial agents produce products that are more toxic than the parent material, limiting their promotion and application.

[0004] However, although there are two reductive dechlorination pathways for 1,1,2-TCA, the number of microbial agents that perform reductive dechlorination via dichloroelimination far exceeds those that perform hydrogenolytic reductive dechlorination. Currently, only *Desulfitobacterium* strain PR has been reported as a microbial agent for the hydrogenolytic reductive dechlorination of 1,1,2-TCA. Therefore, existing microbial agent technologies for the reductive dechlorination of 1,1,2-TCA mainly rely on dichloroelimination, with vitamin C (VC) as the toxic product. To eliminate VC products, *Dehalococcoides mccartyi* is often used to further degrade VC products. For example, *Geobacter* strain IAE and *Dehalococcoides mccartyi* are combined to complete the detoxification process of 1,1,2-TCA → VC → ethylene. Although this method can remove the toxic product VC, *Dehalococcoides mccartyi*, as a specific OHRB, has a very narrow ecological niche compared to *Desulfitobacterium*. The dehalococcoides mccartyi reduction of vitamin C to ethylene requires a strictly anaerobic environment, hydrogen as the sole electron donor, and a high vitamin B content. 12 The ideal groundwater environment requires a neutral pH. However, in real-world groundwater conditions, it is difficult to simultaneously meet all these conditions, leading to poor remediation results and significantly prolonged remediation cycles. Therefore, these demanding environmental conditions objectively limit the feasibility of this technology.

[0005] Therefore, there is an urgent need for a trichloroethane degrading bacterial agent that generates low levels or no toxic intermediates during the reductive dechlorination process, has relatively relaxed remediation conditions, and a short remediation cycle. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a bacterial agent for degrading trichloroethane, as well as its enrichment method and application, thereby reducing the occurrence of toxic intermediate products during the degradation of trichloroethane.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] In a first aspect, the present invention provides a bacterial agent for degrading trichloroethane, comprising functional bacteria A and functional bacteria B, wherein functional bacteria A and functional bacteria B have amino acid sequences encoding dehalogenase a and / or dehalogenase b, wherein the amino acid sequence encoding dehalogenase a is shown in SEQ ID NO.3, and the amino acid sequence encoding dehalogenase b is shown in SEQ ID NO.4. The present invention has experimentally demonstrated that when functional bacteria expressing dehalogenase a and dehalogenase b are present simultaneously, trichloroethane can be rapidly degraded into ethylene, with no toxic intermediate products formed during the degradation process, enabling faster, more convenient, and safer degradation of trichloroethane.

[0009] In a preferred embodiment of the bacterial agent for degrading trichloroethane according to the present invention, the 16S rDNA of functional bacteria A and functional bacteria B in the bacterial agent has at least 90% deoxynucleotide sequence identity with SEQ ID NO.5.

[0010] In a preferred embodiment of the bacterial agent for degrading trichloroethane described in this invention, functional bacteria A and B in the bacterial agent belong to *Desulfitobacterium* sp. in biological classification. This invention has discovered that functional bacteria A and B, which belong to *Desulfitobacterium* sp. in biological classification, are non-specific OHRBs and can secrete dehalogenases a and b without demanding culture conditions, thereby achieving rapid degradation of 1,1,2-TCA and reducing the content of toxic intermediate products that occur during the degradation process. Simultaneously, functional bacteria A and B replace specific OHRBs (such as *Dehalogenimonas* sp.), improving the ecological niche of functional bacteria A and B and completely avoiding the occurrence of toxic intermediate products.

[0011] In a preferred embodiment of the bacterial agent for degrading trichloroethane according to the present invention, when functional bacteria A encodes dehalogenase a and functional bacteria B encodes dehalogenase b, the final concentration ratio of functional bacteria A to functional bacteria B in the bacterial agent is functional bacteria A:functional bacteria B = (1-2):1, and the final concentration of functional bacteria B in the bacterial agent is ≥1×10⁻⁶. 5 Cells / L. Dehalogenase b can directly degrade 1,1,2-TCA to VC, but VC cannot be further dechlorinated to ethylene. Therefore, the presence of dehalogenase a is necessary, and the concentration of functional bacteria of dehalogenase a must be equal to or higher than that of functional bacteria of dehalogenase b. Dehalogenase a first degrades 1,1,2-TCA to 1,2-DCA, and then dehalogenase b degrades 1,2-DCA to ethylene. This avoids the formation of VC and can degrade 1,1,2-TCA rapidly, efficiently, and without the appearance of toxic intermediate products.

[0012] In a preferred embodiment of the bacterial agent for degrading trichloroethane according to the present invention, the final concentration ratio of functional bacteria A to functional bacteria B in the bacterial agent is functional bacteria A: functional bacteria B = 1.5:1.

[0013] Secondly, the present invention provides a method for enriching the above-mentioned bacterial agent for degrading trichloroethane, comprising the following steps:

[0014] 1. Enrichment of functional bacteria expressing dehalogenase a:

[0015] A1. Mix the chloroform-contaminated water source with anaerobic mineral salt culture medium and chloroform to form a mixed solution, and then anaerobically culture the mixed solution.

[0016] A2. After the chloroform in the mixture described in step A1 is converted into dichloromethane, the mixture is inoculated into a new anaerobic mineral salt culture medium for subculture to obtain a culture medium.

[0017] A3. After the chloroform in the culture medium described in step A2 is converted to dichloromethane, the culture is passaged according to the operation in step A2. When the relative abundance of Desulfitobacterium sp. in the nth generation culture medium is at least 10 times higher than that in the first generation culture medium, the passage ends and the nth generation culture medium is the enriched functional bacteria expressing dehalogenase a.

[0018] 2. Enrichment of functional bacteria expressing dehalogenase b:

[0019] B1. Mix the water source contaminated with 1,1,2-trichloroethane with anaerobic mineral salt culture medium and 1,1,2-trichloroethane to form a mixed solution, and then anaerobically culture the mixed solution.

[0020] B2. After the 1,1,2-trichloroethane in the mixture described in step B1 is converted into vinyl chloride or ethylene, the mixture is inoculated into a new anaerobic mineral salt culture medium for subculture to obtain a culture medium.

[0021] B3. After the 1,1,2-trichloroethane in the culture medium described in step B2 is converted into vinyl chloride or ethylene, subculture is performed according to the operation in step B2. When the relative abundance of Desulfitobacterium sp. in the nth generation culture medium is at least 10 times higher than that in the first generation culture medium, the subculture ends, and the nth generation culture medium is the enriched functional bacteria expressing dehalogenase b.

[0022] This invention selects chloroform-contaminated water sources as inoculum because such water sources often contain abundant *Desulfitobacterium* sp. populations. The chloroform molecule has three chlorine atoms on a single substrate; this unique molecular structure is currently only known to *Desulfitobacterium* sp. and *Dehalobacter* sp. bacteria, which can reduce chloroform by removing one chlorine atom to produce dichloromethane. Therefore, using chloroform as an electron acceptor allows for the screening of *Desulfitobacterium* sp. and *Dehalobacter* sp. Simultaneously, by combining 16S amplicon high-throughput sequencing or PCR amplification techniques, water sources containing *Desulfitobacterium* sp. populations can be pre-screened. Through specific culture and multiple passages, functional bacteria with high *Desulfitobacterium* sp. abundance and expressing dehalogenase a can be enriched.

[0023] The present invention selects water contaminated with 1,1,2-trichloroethane (1,1,2-TCA) as the inoculum source because there are only two pathways for the reductive dehydrogenation of 1,1,2-TCA. The first is to first dechlorinate it by hydrogenolysis to 1,2-DCA, and then dechlorinate it by dichloroelimination to ethylene. The second is to directly dechlorinate it by dichloroelimination to generate vinyl chloride (VC). The *Desulfitobacterium* sp. population can degrade 1,1,2-TCA via both pathways mentioned above. If the *Desulfitobacterium* sp. population degrades 1,1,2-TCA via the first pathway, then functional bacteria expressing both dehalogenase a and dehalogenase b can be screened. If the *Desulfitobacterium* sp. population degrades 1,1,2-TCA via the second pathway, then functional bacteria expressing dehalogenase b can be screened. Therefore, it is necessary to first confirm whether the water source contaminated with 1,1,2-TCA contains the *Desulfitobacterium* sp. population, and then enrich the *Desulfitobacterium* sp. through multiple passages using specific culture to obtain functional bacteria expressing dehalogenase b.

[0024] This invention screens functional bacteria capable of converting chloroform or 1,1,2-trichloroethane using specific reagents, and further enriches them through continuous subculturing. This increases the relative abundance of functional bacteria expressing dehalogenase a and dehalogenase b in functional bacteria A and B. When the relative abundance of functional bacteria expressing dehalogenase a and dehalogenase b increases to more than 10 times that of the first subculture, it indicates a significant increase in the relative abundance of functional bacteria expressing dehalogenase a and dehalogenase b, enabling the enriched functional bacteria to degrade 1,1,2-trichloroethane more rapidly.

[0025] In a preferred embodiment of the enrichment method of the present invention, in step A1, the chloroform-contaminated water source contains Desulfitobacterium sp.;

[0026] In step B1, the water source contaminated with 1,1,2-trichloroethane contains Desulfitobacterium sp.

[0027] In a preferred embodiment of the enrichment method of the present invention, whether the water source contaminated with chloroform and the water source contaminated with 1,1,2-trichloroethane contain Desulfitobacterium sp. can be determined by at least one of the following methods (1) to (2):

[0028] (1) 16S amplicon high-throughput sequencing;

[0029] (2) Detection using PCR amplification technology.

[0030] As a preferred embodiment of the enrichment method of the present invention, when using PCR amplification technology to determine whether Desulfitobacterium sp. is present in water sources contaminated with chloroform and water sources contaminated with 1,1,2-trichloroethane, specific detection primers should be selected for PCR amplification. The nucleotide sequence of the forward primer in the specific detection primer is shown in SEQ ID NO.1, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.2.

[0031] In a preferred embodiment of the enrichment method of the present invention, in step A1, the volume ratio of the chloroform-contaminated water source to the anaerobic mineral salt culture medium is chloroform-contaminated water source: anaerobic mineral salt culture medium = (8-10): (58-60), and the volume ratio of the anaerobic mineral salt culture medium to chloroform is anaerobic mineral salt culture medium: chloroform = (60-62): (0.005-0.007).

[0032] In a preferred embodiment of the enrichment method of the present invention, in step B1, the volume ratio of the water source contaminated with 1,1,2-trichloroethane to the anaerobic mineral salt culture medium is water source contaminated with 1,1,2-trichloroethane: anaerobic mineral salt culture medium = (8-10): (58-60), and the volume ratio of the anaerobic mineral salt culture medium to 1,1,2-trichloroethane is anaerobic mineral salt culture medium: 1,1,2-trichloroethane = (60-62): (0.008-0.010).

[0033] In a preferred embodiment of the enrichment method of the present invention, in steps A1 and B1, the water source includes, but is not limited to, groundwater.

[0034] In a preferred embodiment of the enrichment method of the present invention, in steps A2 and B2, the volume ratio of the mixture to the new anaerobic mineral salt culture medium is mixture: new anaerobic mineral salt culture medium = (5-8):(92-95).

[0035] In a preferred embodiment of the enrichment method described in this invention, in steps A3 and B3, n in the nth generation is an integer ≥2. In practical applications, the number of passages can be determined based on the change in the relative abundance of Desulfitobacterium sp. When the relative abundance of Desulfitobacterium sp. increases significantly, it indicates that Desulfitobacterium sp. has been enriched. Furthermore, the higher the number of passages, the higher the relative abundance of Desulfitobacterium sp., and the better the degradation effect of 1,1,2-TCA. However, considering the time cost, this invention only demonstrates the enrichment effect of 1-5 passages. Those skilled in the art can determine the number of passages based on the relative abundance of Desulfitobacterium sp. in the collected water source (a higher initial relative abundance of Desulfitobacterium sp. can reduce the number of passages; a lower initial relative abundance of Desulfitobacterium sp. can increase the number of passages) in order to enrich functional bacteria with higher relative abundance of Desulfitobacterium sp.

[0036] In a preferred embodiment of the enrichment method of the present invention, in steps A3 and B3, n in the nth generation is an integer from 3 to 5.

[0037] Thirdly, the present invention provides an enzyme for degrading trichloroethane, the enzyme comprising dehalogenase a and / or dehalogenase b;

[0038] The dehalogenase a has at least 90% amino acid sequence identity with SEQ ID NO.3, and the dehalogenase b has at least 90% amino acid sequence identity with SEQ ID NO.4.

[0039] The present invention has discovered through experiments that when dehalogenase a and dehalogenase b are present simultaneously, dehalogenase a can degrade 1,1,2-TCA into 1,2-DCA. Then, 1,2-DCA is instantly converted into ethylene by the dehalogenase, so that only a small amount or no toxic intermediate product 1,2-DCA or VC is produced during the degradation process.

[0040] As a preferred embodiment of the enzyme agent for degrading trichloroethane according to the present invention, the trichloroethane includes at least one of 1,1,1-trichloroethane, 1,1,2-trichloroethane, and 1,2,2-trichloroethane.

[0041] In a preferred embodiment of the enzyme for degrading trichloroethane according to the present invention, the dehalogenase a has at least 95% amino acid sequence identity with SEQ ID NO.3, and the dehalogenase b has at least 95% amino acid sequence identity with SEQ ID NO.4.

[0042] In a preferred embodiment of the enzyme for degrading trichloroethane according to the present invention, the dehalogenase a has at least 97% amino acid sequence identity with SEQ ID NO.3, and the dehalogenase b has at least 97% amino acid sequence identity with SEQ ID NO.4.

[0043] In a preferred embodiment of the enzyme for degrading trichloroethane according to the present invention, the dehalogenase a has at least 99% amino acid sequence identity with SEQ ID NO.3, and the dehalogenase b has at least 99% amino acid sequence identity with SEQ ID NO.4.

[0044] In a preferred embodiment of the enzyme agent for degrading trichloroethane according to the present invention, the ratio of dehalogenase a to dehalogenase b is dehalogenase a: dehalogenase b = (1-2) U: 1 U.

[0045] In a preferred embodiment of the enzyme agent for degrading trichloroethane according to the present invention, the ratio of dehalogenase a to dehalogenase b is dehalogenase a: dehalogenase b = 1.5 U: 1 U.

[0046] Fourthly, the present invention provides the application of the above-mentioned bacterial agent or enzyme agent for degrading trichloroethane in the preparation of products for remediating trichloroethane-containing water sources.

[0047] Fifthly, the present invention provides the application of the above-mentioned bacterial agents or enzymes for degrading trichloroethane in the remediation of trichloroethane-containing water sources.

[0048] Sixthly, this invention provides a method for remediating trichloroethane-containing water sources. The method involves adding the aforementioned trichloroethane-degrading bacterial agent or enzyme agent to the water source to be remediated, and then anaerobically culturing it at 20-35°C for 5-11 days to obtain the remediated water source. Tests conducted in simulated groundwater containing 1,1,2-TCA revealed that 1,1,2-TCA was completely degraded on the 8th day after the addition of the bacterial agent, with no toxic intermediate products generated. This achieves rapid degradation of trichloroethane while avoiding the formation of toxic intermediate products.

[0049] In a preferred embodiment of the method for remediating trichloroethane-containing water sources according to the present invention, the pH value of the water source to be remediated is 6.5-7.5.

[0050] In a preferred embodiment of the method for repairing trichloroethane-containing water sources according to the present invention, the water source to be repaired contains an electron donor.

[0051] In a preferred embodiment of the method for remediating trichloroethane-containing water sources according to the present invention, the electron donor includes, but is not limited to, at least one of lactate, pyruvate, formate, ethanol, and hydrogen.

[0052] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0053] (1) The present invention discovered through experiments that when dehalogenase a and dehalogenase b are present at the same time, dehalogenase a can degrade 1,1,2-TCA into 1,2-DCA. Then, 1,2-DCA is instantly converted into ethylene by the dehalogenase, so that only a small amount or no toxic intermediate product 1,2-DCA or VC is produced during the degradation process.

[0054] (2) This invention discovers that functional bacteria A and B, which belong to Desulfitobacterium sp. in biological classification, are non-specific OHRBs. They can secrete dehalogenase a and dehalogenase b without harsh culture conditions, thereby achieving rapid degradation of 1,1,2-TCA and reducing the content of toxic intermediate products that appear during the degradation process. At the same time, functional bacteria A and B replace specific OHRBs (such as Dehalogenimonas sp., etc.), improve the ecological niche of functional bacteria A and B, and completely avoid the occurrence of toxic intermediate products. Attached Figure Description

[0055] Figure 1 This is a traditional strategy for degrading 1,1,2-TCA;

[0056] Figure 2 This invention relates to a strategy for degrading 1,1,2-TCA. Detailed Implementation

[0057] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0058] Unless otherwise specified, all other materials and reagents used in the examples, comparative examples, and effect examples are commercially available.

[0059] The anaerobic mineral salt culture medium formulations described in the following examples, comparative examples, and effect examples are as follows: per 1L, there are 1g NaCl, 0.5g MgCl2·6H2O, 0.2g KH2PO4, 3g NH4Cl, 0.3g KCl, 0.015g CaCl2·2H2O, 2.52g NaHCO3, 0.024g L-cysteine, 0.048g Na2S·9H2O, 0.078g dithiothreitol, 2.3g tris(hydroxymethyl)aminoethanesulfonic acid, 1.47mL of 60% DL-lactic acid sodium solution, 0.05mg vitamin B12, 1mL of trace elements, and pH = 6.5-7.5. Each 1L of trace elements contains 10mL of HCl (25% by mass), 1.5g of FeCl2·4H2O, 0.19g of CoCl2·6H2O, 0.1g of MnCl2·4H2O, 0.07g of ZnCl2, 0.006g of H3BO3, 0.036g of Na2MoO4·4H2O, 0.024g of NiCl2·6H2O, and 0.002g of CuCl2·2H2O.

[0060] In the following examples, comparative examples, and effect examples, the deoxynucleotide sequence described in SEQ ID NO.1 is GTGGGGATGACGTCAAATCA;

[0061] The deoxynucleotide sequence described in SEQ ID NO.2 is CATGTTCTCGAGTTGCAGAGAA;

[0062] The amino acid sequence set forth in SEQ ID NO.3 is MEKDKSNNDKPATKINRRQFLKFGAGASSGIAIAVATTALGGKSLIDPKQANAGTVKELDELPFDIPADYKPFTNQRNIFGQAILGVPEPLALVERFDEVRWNGWQTDGSPAFTVLDGAAARASFAVDYYFNGENSACRANKGFFEWHPKVPELNFKWGDPERNIHSPGVKSAEEGTMAVKRMARFFGAAKAGIAPFDKRWVFTETAAFVKTPEGEDLKFIPPDFGFEPKHVISMIIPQSLEGVKCAPSFLGSAEYGLSFAQIGYAAFGLSMFIKDLGYHAVPIGSDSALAIPIAIQAGLGEYSRSGQLITPEFGSNVRLCEVFTDMPLNHDKPISFGVTEFCKTCKKCAEECPPQAISYEDPTIDGPCGQMHNSGIKRWYVDPVKCFEFWSRDNVRNCCGACIVACPFTKPEAWHHTLIRSLVGAPVITPFMKDMDDIFGYGKPNDEKAIADWWK;

[0063] The amino acid sequence set forth in SEQ ID NO.4 is as follows: MGEINRRNFLKASMLGAAAAAVASASVVKGVVSPLVADAADIVAPITETSEFPYKVDAKYQRYNRMKCFFEKIFDPEENKTPIKFHNDDVSKITGKKDTGKDLPTLNAERLGIKGRPATHTETGVLWLGAHTGVMPHLRNLSSKETGNTLLDYALQAGVWAVEFDFHGFNATDGGPGTVITPYPINPMTNEIANEPVVVPGLYNWDNIDVESVRQQGQQWKFKSKEEASKIVKKAACFMGADLVGIAPYDERWTYSTWCRKIAKPWKMPNGRTKLWSWDLPKALSGGGVEVFGHAKFEPDWEKYAGFKPKSVIVFVLEEDYEALRTSPSVIANAATGKDYSSLGGVSYKIAVFLRKLGYYAAPSGNDTGLNVPMAVQAGLGEAGRNGLLITQKFGPRNRIAKVYTDLELAPDKPRKFGVREFCRLCKKCADACPAQAISHETDPKVLQPEDCEESENPYTEKWHVDSKRCGSYWAYNGGLCANCIAVCSYNKIENWNHDVARIATQIPLLQDAARKFDEWFGYGGPVDPDERLASGYVQNMVKDFWNNPESIKQ;

[0064]

[0065] In the following examples, comparative examples, and effect examples, compounds such as chloroform, dichloromethane, 1,1,2-TCA, vinyl chloride, and ethylene can all be detected by gas chromatography (GC). Qualitative and quantitative analysis can be performed using the external standard method. The chromatographic column used is an Agilent HP-5.

[0066] In the following embodiments, the methods for extracting dehalogenase a and dehalogenase b from functional bacteria A and functional bacteria B, respectively, can be carried out with reference to the protein extraction methods disclosed in the prior art, such as those in "Principles and Techniques of Biochemical Experiments (Third Edition)" by Wang Jingyan et al. and "Protein Purification Guide (Chinese Second Edition)" edited by RR Burgess.

[0067] Example 1

[0068] Example 1 provides a microbial agent for degrading trichloroethane and its enrichment method. The microbial agent for degrading trichloroethane includes functional bacteria A expressing dehalogenase a and functional bacteria B expressing dehalogenase b. The enrichment method includes the following steps:

[0069] 1. Enrichment of functional bacteria A expressing dehalogenase a.

[0070] 1.1 Groundwater contaminated with chloroform was collected, and 16S amplicon high-throughput sequencing analysis confirmed the presence of Desulfitobacterium sp. in the groundwater. The species and their relative abundances obtained from the groundwater analysis are shown in Table 1.

[0071] 1.2 Add 10 mL of the chloroform-contaminated groundwater described in step 1.1 to a 110 mL anaerobic serum bottle containing 60 mL of anaerobic mineral salt culture medium to obtain a mixture. The 60 mL of anaerobic mineral salt culture medium also contains 5 μL of chloroform. The mixture is anaerobic cultured at 30 °C, and the chloroform conversion is detected every 2 days.

[0072] 1.3 After chloroform was detected to have converted to dichloromethane in the mixture described in step 1.2, it was passaged and inoculated into a new anaerobic mineral salt medium (containing the chloroform described in step 1.2 at a 5% volume ratio, with the ratio unchanged). The medium was then anaerobically cultured according to the method in step 1.2 to obtain the culture medium. When chloroform was detected to have converted to dichloromethane in the culture medium, passaged again according to the above procedure. 16S amplicon high-throughput sequencing was performed on the culture media from generation 1 to 5. The relative abundance of Desulfitobacterium sp. in each generation of culture medium is shown in Table 2. When cultured to generation 4, the relative abundance of Desulfitobacterium sp. was 10 times higher than that of generation 1, while the relative abundance of Desulfitobacterium sp. in generation 5 was significantly higher than that of generation 1. The relative abundance of sp. increased by about 14 times compared to the previous generation. Therefore, both the 4th and 5th generation culture media can be used as functional bacteria A for expressing dehalogenase after enrichment. However, in order to achieve better degradation of 1,1,2-TCA, the 5th generation culture media was selected as the functional bacteria A for expressing dehalogenase after enrichment as described in this embodiment.

[0073] Table 1. Top 10 genera with the highest relative abundance in chloroform-contaminated groundwater.

[0074] Genus name Relative abundance (×100%) Desulfitobacterium 0.11494032 Dehalobacter 0.10219834 Acetobacterium 0.04998775 vadinBA26 0.03890853 Desulfovibrio 0.03778836 Trichococcus 0.03637064 Desulfosporosinus 0.02704169 Saccharimonadales 0.02215843 Dehalobacterium 0.02025064 Sphaerochaeta 0.01955053

[0075] Table 2. Relative abundance of Desulfitobacterium sp. in different generations of culture medium.

[0076] Cultivating Algebra Relative abundance (×100%) Generation 1 0.01045498 2nd generation 0.04942580 3rd generation 0.06413961 4th generation 0.11764400 5th generation 0.14259521

[0077] 2. Enrichment of functional bacteria B expressing dehalogenase b.

[0078] 2.1 Groundwater contaminated with 1,1,2-TCA was collected, and 16S amplicon high-throughput sequencing analysis confirmed the presence of Desulfitobacterium sp. in the groundwater. The species and their relative abundances obtained from the groundwater analysis are shown in Table 3.

[0079] 2.2 Add 10 mL of the 1,1,2-TCA-contaminated groundwater described in step 2.1 to a 110 mL anaerobic serum bottle containing 60 mL of anaerobic mineral salt culture medium to obtain a mixture. The 60 mL of anaerobic mineral salt culture medium also contains 8 μL of 1,1,2-TCA. The mixture is anaerobic cultured at 30 °C, and the conversion of 1,1,2-TCA is detected every 2 days.

[0080] 2.3 When 1,1,2-TCA is detected to have converted to vinyl chloride or ethylene in the mixture described in step 2.2, it is passaged and inoculated into a new anaerobic mineral salt medium (containing the 1,1,2-TCA described in step 2.2 at a volume ratio of 5%). The medium is then anaerobically cultured according to the method in step 2.2 to obtain a culture solution. When 1,1,2-TCA is detected to have converted to vinyl chloride or ethylene in the culture solution, passage is continued according to the above operation. When passaged to the 5th generation, the 5th generation culture solution is selected as the enriched functional bacterium B expressing dehalogenase b.

[0081] Table 3. Top 10 genera with the highest relative abundance of species in groundwater contaminated with 1,1,2-TCA.

[0082] Genus name Relative abundance (×100%) Dehalobacter 0.09762147 Thermincola 0.07426888 vadinBA26 0.06568019 OPB41 0.05547696 Thermovirga 0.04672182 Candidatus_Colwellbacteria 0.04429168 Candidatus_Falkowbacteria 0.02566621 Geobacter 0.02553305 Trichlorobacter 0.02002364 Sedimentibacter 0.01952429 Desulfosporosinus 0.01944107 Desulfitobacterium 0.01925798

[0083] High-throughput sequencing was performed on the enriched functional bacteria A and B to determine that functional bacteria A can express dehalogenase a and functional bacteria B can express dehalogenase b. The amino acid sequence of dehalogenase a is shown in SEQ ID NO.3, the amino acid sequence of dehalogenase b is shown in SEQ ID NO.4, and the 16S rDNA sequence of functional bacteria A is shown in SEQ ID NO.5.

[0084] The trichloroethane-degrading bacterial agent comprises the enriched functional bacteria A and B, wherein the final concentration ratio of functional bacteria A to functional bacteria B is 1.5:1, and the final concentration of functional bacteria B in the bacterial agent is 1×10⁻⁶. 5 per L.

[0085] Example 2

[0086] Example 2 provides a microbial agent for degrading trichloroethane and its enrichment method. The microbial agent for degrading trichloroethane includes functional bacteria A expressing dehalogenase a and functional bacteria B expressing dehalogenase b. The enrichment method is similar to that in Example 1, with the following differences:

[0087] Replace step 1.1 with: perform PCR amplification on the collected chloroform-contaminated groundwater, using primers as shown in SEQ ID NO.1-2. If a band of about 1200bp is obtained, it indicates that the chloroform-contaminated groundwater contains Desulfitobacterium sp., and use Desulfitobacterium sp. enrichment bacteria A for chloroform-contaminated groundwater.

[0088] Replace step 2.1 with: perform PCR amplification on the collected groundwater contaminated with 1,1,2-TCA, using primers as shown in SEQ ID NO.1-2. If a band of approximately 1200bp is obtained, it indicates that the groundwater contaminated with 1,1,2-TCA contains Desulfitobacterium sp., and use chloroform-contaminated groundwater enrichment bacteria B containing Desulfitobacterium sp.

[0089] The remaining steps and parameters remain unchanged.

[0090] Example 3

[0091] Example 3 provides a bacterial agent for degrading trichloroethane and its enrichment method. The enrichment method of the bacterial agent is the same as that of Example 1. The bacterial agent includes functional bacteria A and functional bacteria B enriched in Example 1. The final concentration ratio of functional bacteria A to functional bacteria B is 1:1, and the final concentration of functional bacteria B in the bacterial agent is 1 × 10⁻⁶. 5 per L.

[0092] Example 4

[0093] Example 4 provides a bacterial agent for degrading trichloroethane and its enrichment method. The enrichment method of the bacterial agent is the same as that of Example 1. The bacterial agent includes functional bacteria A and functional bacteria B enriched in Example 1. The final concentration ratio of functional bacteria A to functional bacteria B is 2:1. The final concentration of functional bacteria B in the enzyme agent is 1 × 10⁻⁶. 5 per L.

[0094] Example 5

[0095] Example 5 provides an enzyme for degrading trichloroethane, the enzyme comprising dehalogenase a and dehalogenase b, wherein the final concentration ratio of dehalogenase a to dehalogenase b in the enzyme is dehalogenase a: dehalogenase b = 1.5 U: 1 U;

[0096] The amino acid sequence of the dehalogenase a is shown in SEQ ID NO.3, and the amino acid sequence of the dehalogenase b is shown in SEQ ID NO.4;

[0097] The dehalogenase a and dehalogenase b can be obtained by expanding the culture of functional bacteria A and functional bacteria B in anaerobic mineral salt medium, then physically disrupting and centrifuging the bacterial solution, and the resulting supernatant is dehalogenase a or dehalogenase b.

[0098] Example 6

[0099] Example 6 provides an enzyme for degrading trichloroethane, the enzyme comprising dehalogenase a and dehalogenase b, wherein the final concentration ratio of dehalogenase a to dehalogenase b in the enzyme is dehalogenase a: dehalogenase b = 1U: 1U;

[0100] The amino acid sequence of the dehalogenase a is shown in SEQ ID NO.3, and the amino acid sequence of the dehalogenase b is shown in SEQ ID NO.4;

[0101] The dehalogenase a and dehalogenase b can be obtained by expanding the culture of functional bacteria A and functional bacteria B in anaerobic mineral salt medium, then physically disrupting and centrifuging the bacterial solution, and the resulting supernatant is dehalogenase a or dehalogenase b.

[0102] Comparative Example 1

[0103] Comparative Example 1 provides a bacterial agent for degrading trichloroethane and its enrichment method. The enrichment method is the same as that in Example 1. The bacterial agent includes functional bacteria A enriched in Example 1, with a cell concentration of 1.5 × 10⁻⁶. 5 per L.

[0104] Comparative Example 2

[0105] Comparative Example 2 provides a bacterial agent for degrading trichloroethane and its enrichment method. The enrichment method is the same as that in Example 1. The bacterial agent includes functional bacteria B enriched in Example 1, with a cell concentration of 1 × 10⁻⁶. 5 per L.

[0106] Comparative Example 3

[0107] Comparative Example 3 provides a bacterial agent for degrading trichloroethane and its enrichment method. The enrichment method is the same as that in Example 1, and the composition of the bacterial agent is similar to that in Example 1. The difference is that the concentration ratio of functional bacteria A and functional bacteria B is functional bacteria A: functional bacteria B = 1:4. Functional bacteria A and functional bacteria B are enriched by the method described in Example 1, and the concentration of functional bacteria B is 1 × 10⁻⁶. 5 per L.

[0108] Example 1

[0109] To verify whether the bacterial agent for degrading 1,1,2-trichloroethane (hereinafter referred to as 1,1,2-TCA) can completely degrade 1,1,2-TCA without producing intermediate products, 1,1,2-TCA was added to an anaerobic mineral salt culture medium, and the bacterial agents obtained in Examples 1, 3-4, and Comparative Examples 1-3 were inoculated respectively. The contents of 1,1,2-TCA, VC, and 1,2-DCA were determined by anaerobic culture. The specific scheme is as follows:

[0110] The bacterial agents obtained in Examples 1, 3-4, and Comparative Examples 1-3 were inoculated at a rate of 10% v / v into 10 mL of anaerobic mineral salt medium containing 5.374 μmol 1,1,2-TCA. This anaerobic mineral salt medium also contained 10 mM lactate and 30 mM sodium bicarbonate, with a pH of 7.0. The medium was anaerobically cultured at 30°C for 11 days. The contents of 1,1,2-TCA, VC, and 1,2-DCA in the medium were detected by GC at 0, 4, 7, and 11 days after inoculation. The results are shown in Table 4.

[0111] Table 4. Degradation effect of different bacterial agents on trichloroethane

[0112]

[0113]

[0114] like Figure 1 As shown, both dehalogenase a and dehalogenase b react with 1,1,2-TCA to degrade it. Dehalogenase a can degrade 1,1,2-TCA to 1,2-DCA, while dehalogenase b can degrade 1,1,2-TCA to VC. Dehalogenase a and dehalogenase b can further dehalogenate with 1,2-DCA or VC to form ethylene.

[0115] As shown in Table 4, the degradation of 1,1,2-TCA by functional bacteria A or B alone produced a large amount of toxic intermediates (1,2-DCA or VC), and after 11 days of culture, 1,1,2-TCA could not be degraded into ethylene. However, when functional bacteria A and B were combined to degrade 1,1,2-TCA, it was found that 1,1,2-TCA could be degraded into ethylene without the residue of toxic intermediates (such as...). Figure 2 As shown in the figure, this indicates that only when the microbial agent can encode dehalogenase a and dehalogenase b can it achieve rapid degradation of 1,1,2-TCA into ethylene without any toxic intermediate product residues. Meanwhile, when the concentration of functional bacteria B is higher than that of functional bacteria A, functional bacteria B will convert 1,1,2-DCA into VC faster than functional bacteria A. VC cannot be dechlorinated into ethylene, and the VC content is higher than that of 1,2-DCA. A large number of toxic intermediate products are generated during the degradation of 1,1,2-DCA. This indicates that only when the concentration ratio of functional bacteria A to functional bacteria B is similar or the concentration of functional bacteria A is higher than that of functional bacteria B can 1,1,2-DCA be converted into 1,2-DCA and then degraded into ethylene. Furthermore, when the cell concentration ratio of functional bacteria A to functional bacteria B is functional bacteria A:functional bacteria B = (1-2):1, the microbial agent exhibits superior 1,1,2-TCA degradation performance.

[0116] Example 2

[0117] To verify whether the bacterial agent for degrading trichloroethane of the present invention still has the effect of immediate degradation of trichloroethane and non-toxic intermediate product release in water sources containing trichloroethane, an anaerobic mineral salt culture medium containing 107.49 μmol 1,1,2-TCA was used to simulate real-world groundwater containing trichloroethane. The bacterial agent obtained in Example 1 was inoculated into 100 mL of anaerobic mineral salt culture medium containing 1.0749 μmol / mL 1,1,2-TCA at an inoculation rate of 10 v / v%, and anaerobic cultured at 30°C for 11 days. During this period, the contents of 1,1,2-TCA, VC, and 1,2-DCA in the anaerobic mineral salt culture medium were detected by GC at 0, 4, 7, and 11 days after inoculation. The results are shown in Table 5.

[0118] Table 5. Determination of the efficacy of the bacterial agent obtained in Example 1 on the degradation of trichloroethane in a simulated trichloroethane-containing water source.

[0119]

[0120]

[0121] As shown in Table 5, in simulated groundwater containing trichloroethane, the bacterial agent began to degrade 1,1,2-TCA with increasing anaerobic culture time. On day 1 after inoculation, most of the degraded 1,1,2-TCA was degraded to ethylene, and a small amount was degraded to vitamin C. On day 4 after inoculation, a large amount of 1,1,2-TCA was degraded to ethylene, and a small amount was degraded to 1,2-DCA. On day 8 after inoculation, 1,1,2-TCA was completely degraded, and no toxic intermediate products (vitamin C, 1,2-DCA) were formed at this time. This is because functional bacteria A reacted with 1,1,2-TCA under the action of dehalogenase a. The moment 1,2-DCA is generated, the dehalogenase b of functional bacteria B undergoes a reductive dehalogenation reaction to form ethylene, which rapidly degrades the toxic intermediates in the degradation process of 1,1,2-TCA, achieving the degradation of 1,1,2-TCA without the appearance of toxic intermediates. However, since other intermediates besides toxic intermediates VC and 1,2-DCA will appear in the degradation process of 1,1,2-TCA, it takes 11 days after inoculation for all 1,1,2-TCA to be degraded into ethylene (the degradation rate is 99.993%).

[0122] Furthermore, 1,2-DCA could not be detected on day 8 because its content was below the instrument's minimum detection limit, and therefore was labeled as 0.

[0123] Example 7

[0124] Example 7 provides a method for remediating water sources containing trichloroethane, comprising the following steps:

[0125] The bacterial agent obtained in Example 1 was inoculated into the water source to be repaired at an inoculation rate of 10 v / v%, and anaerobic cultured at 30°C for 11 days to obtain the repaired water source, wherein the water source to be repaired contains at least 30 mM of electron donor.

[0126] Example 8

[0127] Example 8 provides a method for remediating water sources containing trichloroethane. This method is similar to that of Example 7, except that the bacterial agent obtained in Example 1 is replaced with the bacterial agent obtained in Example 3, while the remaining steps and parameters remain unchanged.

[0128] Example 9

[0129] Example 9 provides a method for remediating water sources containing trichloroethane. This method is similar to that of Example 7, except that the bacterial agent obtained in Example 1 is replaced with the bacterial agent obtained in Example 4, while the remaining steps and parameters remain unchanged.

[0130] Example 10

[0131] Example 10 provides a method for remediating water sources containing trichloroethane. This method is similar to that in Example 7, except that the anaerobic culture temperature is adjusted to 35°C, while the other steps and parameters remain unchanged.

[0132] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A bacterial agent for degrading trichloroethane, characterized in that, It includes functional bacteria A and functional bacteria B, which have amino acid sequences encoding dehalogenase a and / or dehalogenase b, wherein the amino acid sequence encoding dehalogenase a is shown in SEQ ID NO.3 and the amino acid sequence encoding dehalogenase b is shown in SEQ ID NO.

4.

2. The bacterial agent for degrading trichloroethane as described in claim 1, characterized in that, When functional bacteria A encodes dehalogenase a and functional bacteria B encodes dehalogenase b, the final concentration ratio of functional bacteria A to functional bacteria B in the bacterial agent is functional bacteria A:functional bacteria B = (1-2):1, and the initial concentration of functional bacteria B in the bacterial agent is ≥1×10⁻⁶. 5 per L.

3. The bacterial agent for degrading trichloroethane as described in claim 1, characterized in that, The 16S rDNA of functional bacteria A and functional bacteria B in the bacterial agent has at least 90% deoxynucleotide sequence identity with SEQ ID NO.

5.

4. The enrichment method for the bacterial agent degrading trichloroethane as described in any one of claims 1-3, characterized in that, Includes the following steps:

1. Enrichment of functional bacteria expressing dehalogenase a: A1. Mix the chloroform-contaminated water source with anaerobic mineral salt culture medium and chloroform to form a mixed solution, and then anaerobically culture the mixed solution. A2. After the chloroform in the mixture described in step A1 is converted into dichloromethane, the mixture is inoculated into a new anaerobic mineral salt culture medium for subculture to obtain a culture medium. A3. After the chloroform in the culture medium described in step A2 is converted to dichloromethane, the culture is passaged according to the operation in step A2. When the relative abundance of Desulfitobacterium sp. in the nth generation culture medium is at least 10 times higher than that in the first generation culture medium, the passage ends and the nth generation culture medium is the enriched functional bacteria expressing dehalogenase a.

2. Enrichment of functional bacteria expressing dehalogenase b: B1. Mix the water source contaminated with 1,1,2-trichloroethane with anaerobic mineral salt culture medium and 1,1,2-trichloroethane to form a mixed solution, and then anaerobically culture the mixed solution. B2. After the 1,1,2-trichloroethane in the mixture described in step B1 is converted into vinyl chloride or ethylene, the mixture is inoculated into a new anaerobic mineral salt culture medium for subculture to obtain a culture medium. B3. After the 1,1,2-trichloroethane in the culture medium described in step B2 is converted into vinyl chloride or ethylene, subculture is performed according to the operation in step B2. When the relative abundance of Desulfitobacterium sp. in the nth generation culture medium is at least 10 times higher than that in the first generation culture medium, the subculture ends, and the nth generation culture medium is the enriched functional bacteria expressing dehalogenase b.

5. The enrichment method as described in claim 4, characterized in that, Includes at least one of the following (A) to (E): (A) In step A1, the chloroform-contaminated water source contains Desulfitobacterium sp.; (B) In step B1, the water source contaminated with 1,1,2-trichloroethane contains Desulfitobacterium sp.; (C) In step A1, the volume ratio of the chloroform-contaminated water source to the anaerobic mineral salt culture medium is chloroform-contaminated water source: anaerobic mineral salt culture medium = (8-10): (58-60), and the volume ratio of the anaerobic mineral salt culture medium to chloroform is anaerobic mineral salt culture medium: chloroform = (60-62): (0.005-0.007); (D) In ​​step B1, the volume ratio of the water source contaminated with 1,1,2-trichloroethane to the anaerobic mineral salt culture medium is water source contaminated with 1,1,2-trichloroethane: anaerobic mineral salt culture medium = (8-10): (58-60), and the volume ratio of the anaerobic mineral salt culture medium to 1,1,2-trichloroethane is anaerobic mineral salt culture medium: 1,1,2-trichloroethane = (60-62): (0.008-0.010); (E) In steps A2 and B2, the volume ratio of the mixture to the new anaerobic mineral salt culture medium is mixture: new anaerobic mineral salt culture medium = (5-8):(92-95).

6. An enzyme agent for degrading trichloroethane, characterized in that, The enzymes include dehalogenase a and dehalogenase b; The dehalogenase a has at least 90% amino acid sequence identity with SEQ ID NO.3, and the dehalogenase b has at least 90% amino acid sequence identity with SEQ ID NO.

4.

7. The bacterial agent for degrading trichloroethane as described in any one of claims 1-3 or the enzyme agent for degrading trichloroethane as described in claim 6, used in the preparation of products for remediating water sources containing trichloroethane.

8. The bacterial agent for degrading trichloroethane as described in any one of claims 1-3 or the enzyme agent for degrading trichloroethane as described in claim 6, used in the remediation of trichloroethane-containing water sources.

9. A method for remediating water sources containing trichloroethane, characterized in that, Add the bacterial agent for degrading trichloroethane according to any one of claims 1-3 or the enzyme agent for degrading trichloroethane according to claim 6 to the water source to be repaired, and anaerobic culture at 20-35℃ for 5-11 days to obtain the repaired water source.

10. The method for remediating a trichloroethane-containing water source as described in claim 9, characterized in that, Includes at least one of the following (Ⅰ) to (Ⅱ): (I) The pH value of the water source to be repaired is 6.5-7.5; (II) The water source to be repaired contains an electron donor.

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