A chlorobenzene-degrading bacterium and its application in the remediation of contaminated sites

Abstract

The present invention relates to a chlorobenzene degrading bacterium and its application in contaminated site remediation, the degrading bacterium is Pandoraea, named Pandoraea sp.XJJ‑1, preserved in China Center for Type Culture Collection, deposit number is CCTCC NO: M 2021057, the screening method includes taking a contaminated soil sample in a closed serum bottle, obtained after acclimation, separation and purification; XJJ‑1 bacteria can completely degrade 100mg / L of chlorobenzene within 11h, and non-toxic intermediates are metabolically accumulated; the bacteria can tolerate and degrade up to 600mg / L of chlorobenzene and the bacteria have a wide growth temperature range (10°C‑37°C), especially under 10‑15°C low temperature conditions, chlorobenzene can still be efficiently degraded; at the same time, the bacteria can efficiently degrade benzene series such as benzene, toluene, ethylbenzene, and has a wide range of degradation substrate characteristics. Pandoraea sp.XJJ‑1 has a variety of pollutants such as chlorobenzene that can be efficiently and completely degraded at low temperatures, and can play an important role in the remediation of contaminated soil and groundwater such as pesticides and chemicals and in the comprehensive management of sites of polluting enterprises.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial treatment and degradation, and relates to chlorobenzene-degrading bacteria and application thereof in the restoration of contaminated sites. Background Art

[0002] Chlorobenzene is a toxic and hazardous volatile organic compound (VOCs) with three-hazard effects and has been listed as a priority pollutant by the US EPA. Chlorobenzene is an important intermediate and solvent, widely used in industries such as pesticides, plastics and medicines. Chlorobenzene poses a high potential risk and harmful effects to humans and other organisms around contaminated aquatic and terrestrial ecosystems.

[0003] In recent years, the actual site remediation of chlorobenzene compounds has been continuously carried out. Gu Wei et al. used the circulation well technology to remove 94.5%-96.5% of chlorobenzene in groundwater after 28 days. Chi Keyu et al. used the in-situ electrothermal desorption technology to remove chlorobenzene in the soil and groundwater of a retired chemical reagent plant. Xie et al. used water electrolysis technology to remove chlorobenzene pollution in an abandoned chemical plant. However, these technologies cannot meet the site remediation requirements of in-production enterprises. In-production site remediation should avoid affecting normal production, fully guarantee safety, and prevent pollutant rebound. Therefore, it is necessary to choose a safe, efficient and continuous in-situ remediation method. Microbial remediation has become a better choice due to its flexibility, efficiency, effectiveness, economy and eco-friendliness.

[0004] There are many reports at home and abroad on the use of microorganisms to degrade chlorobenzene and chlorobenzene compounds, and a large number of microorganisms with good degradation effects on chlorobenzene have been screened out. Zhang Lili et al. screened Ralstonia piezoelectrica H2 (patent No. CN 101880642 A) from a biological trickling filter, and Ouyang Dujuan et al. screened Rhodococcus pyridinium (patent No. CN110982728 A) from activated sludge. Although these strains can degrade chlorobenzene, there are problems such as low temperature and many types of pollutants causing degradation bacteria to survive and low activity in chlorobenzene-contaminated groundwater such as pesticides and chemical industries, and research and development of chlorobenzene efficient degradation strains with a wide growth temperature range and many degradation substrates have not yet been reported. Summary of the invention

[0005] The purpose of the present invention is to provide a chlorobenzene-degrading bacterium and its application, so as to overcome at least one of the problems that other existing bacteria have slow degradation speed, insufficient removal of low-concentration chlorobenzene pollutants, and easy to cause secondary pollution.

[0006] The purpose of the present invention can be achieved by the following technical solutions:

[0007] On the one hand, the present invention proposes a chlorobenzene-degrading bacterium, which belongs to the genus Pandoraea, named Pandorae asp.XJJ-1, and is deposited in the China Center for Type Culture Collection (address: Wuhan University, Wuhan, China), with a deposit number of CCTCC NO: M 2021057 and a deposit time of January 14, 2021.

[0008] Furthermore, its Gram staining was negative, the strain morphology had no capsule or bacillus, and the colony morphology on Luria-Bertani medium was regular, yellow, convex, opaque and smooth. At the same time, 16S rDNA sequencing was performed on it, and the measured 16S rDNA sequence was shown in SEQ ID NO.1, and the 16S rDNA sequence was subjected to BLAST comparison, and the comparison results showed that the nucleotide sequence of 16S rDNA of strain XJJ-1 had greater than 99% homology with the nucleotide sequences of different strains of Pandoraea sp.

[0009] On the other hand, the present invention also provides a method for screening chlorobenzene-degrading bacteria, comprising the following steps:

[0010] (1) mixing a basic inorganic salt culture medium, a trace element mixed solution and a chlorobenzene solution to obtain a liquid culture medium;

[0011] (2) Weigh a certain mass of soil sample contaminated by a pesticide factory and place it in a liquid culture medium. After culturing on a constant temperature shaker, obtain a first culture solution containing bacteria. Transfer 10% of the first culture solution (here, volume) to another liquid culture medium. After culturing, obtain a second culture solution containing bacteria. Repeat this process multiple times until an nth culture solution is obtained, where n = 5-8;

[0012] (3) The obtained nth culture solution is spread, diluted, streaked and cultured to obtain chlorobenzene-degrading bacteria.

[0013] Furthermore, in step (1), the inorganic salt components in the basic inorganic salt culture medium at least include: 0.023g / L CaCl2; 0.2g / L MgSO4·7H2O; 2.5g / L (NH4)2SO4; 1.0g / L KH2PO4; 4.5g / L Na2HPO4·12H2O.

[0014] Furthermore, in step (1), the solute components in the trace element mixed solution include at least: 0.15g / LZnSO4·7H2O, 0.26g / L MnSO4·H2O, 0.03g / L CoCl2·6H2O, 4.5g / L FeSO4·7H2O, 0.02g / LNiCl2·6H2O, 0.01g / L CuCl2, 0.1g / L Na2MoO4·2H2O, and 0.06g / L H3BO3.

[0015] Furthermore, in step (1), the solvent used for the chlorobenzene solution is N,N-dimethylformamide, and its concentration is preferably 50 g / L;

[0016] The volume ratio of inorganic salt culture medium, trace element solution and chlorobenzene solution is 10 6 :10 3 :2.

[0017] Further, the amount of soil sample and liquid culture medium obtained from the pesticide factory can be adjusted according to actual requirements. For example, in step (2), when the first culture solution is prepared, the ratio of the amount of soil sample to the amount of liquid culture medium added can be (3-6) g: 30 mL. Similarly, the amount transferred from the first culture solution to the second culture solution can be 1:30 to 1:10 in volume to the first mixed solution, and the present invention is not limited thereto.

[0018] Furthermore, during the entire domestication process of step (2), the tools used can be specifically selected according to the actual situation. The bottle used for screening here is a 150 mL crimp-top serum bottle, which is sealed with an aluminum cap and a gasket, and the added liquid is 20-30 mL. Of course, the present invention is not limited to this.

[0019] Furthermore, in step (2), the acclimation conditions can be specifically selected according to the actual situation. For example, the applicable shaking speed here can be 180 r / min, the culturing process is carried out under closed conditions, the temperature is 28° C., and the culturing time for each time is 2-3 days.

[0020] Furthermore, in step (3), the dilution, coating and separation culture process is specifically as follows: 1 mL of the nth culture solution is taken and diluted step by step with phosphate buffer to 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6, 100 μL of liquid was aspirated from each concentration gradient and transferred to a Luria-Bertani medium plate containing chlorobenzene, and spread and separated using a spreading rod, with 3 parallels for each gradient. After a certain period of culture, a suitable single colony was picked up with an inoculation loop and separated on the Luria-Bertani medium plate, and this was repeated 2-3 times. The obtained single bacteria were inoculated into an inorganic salt medium containing chlorobenzene to verify the degradation ability. If the chlorobenzene concentration was observed to decrease and the bacteria could grow with chlorobenzene as the only carbon source, they were chlorobenzene-degrading bacteria.

[0021] On the other hand, the present invention also proposes an application of chlorobenzene degrading bacteria, specifically, it is applied in the degradation of chlorobenzene. More specifically, the chlorobenzene degrading bacteria is used to repair chlorobenzene contaminated sites, preferably, the chlorobenzene contaminated sites are chlorobenzene contaminated soil or chlorobenzene contaminated groundwater. At the same time, the temperature of the chlorobenzene degrading bacteria when repairing the chlorobenzene contaminated site can be 10 to 37°C. Specifically, it can be 10 to 15°C. In addition, the chlorobenzene degrading bacteria is used to degrade chlorobenzene, benzene, toluene and ethylbenzene.

[0022] The chlorobenzene degrading bacteria provided by the present invention are Pandoraea sp.XJJ-1, the 16S rDNA sequence GenBank accession number is MW453096, and the deposit number is CCTCC No: M 2021057. The screening method comprises: obtaining the chlorobenzene degrading bacteria from the contaminated soil sample around the pesticide factory after acclimation, separation and purification. The present invention extracts contaminated soil from the surrounding of the pesticide factory and screens out the chlorobenzene degrading bacteria XJJ-1 therefrom. The bacteria can not only efficiently degrade chlorobenzene, but also have a good degradation effect on benzene, toluene, ethylbenzene and xylene. The chlorobenzene in the water is degraded by the above-mentioned chlorobenzene degrading bacteria XJJ-1, so as to achieve the purpose of removing pollutants, and the intermediate products are completely degraded. The operation cost is low, the negative impact on the environment is small, and it has a good prospect for development and utilization.

[0023] The beneficial effects of the present invention are mainly reflected in: a strain of chlorobenzene efficient degradation bacteria was screened out from the contaminated soil of a pesticide factory, which has good pH and temperature adaptability, can tolerate up to 600 mg / L chlorobenzene, and can completely degrade 100 mg / L chlorobenzene in 11 hours. At the same time, the bacteria can efficiently degrade benzene series such as benzene, toluene, and ethylbenzene, and has a certain substrate broad spectrum. Compared with the prior art, the Pandoraea sp.XJJ-1 provided by the present invention is an indigenous bacterium of a pesticide factory contaminated site, and has an efficient chlorobenzene degradation ability, which can play an important role in the pollution remediation of pesticide factory sites and the risk control of production enterprise sites. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The picture provided by the present invention shows that the culture medium becomes turbid over time during the process of the strain degrading chlorobenzene in a 150 mL serum bottle.

[0025] Figure 2 This is a colony morphology diagram of a chlorobenzene-degrading strain XJJ-1 provided by the present invention.

[0026] Figure 3 This is a scanning electron microscope image of a chlorobenzene-degrading strain XJJ-1 provided by the present invention.

[0027] Figure 4 The present invention provides a phylogenetic tree of the chlorobenzene-degrading strain XJJ-1.

[0028] Figure 5 This is a degradation characteristic diagram of a chlorobenzene-degrading strain XJJ-1 provided in the present invention.

[0029] Figure 6 The graphs show the growth curve and degradation curve of chlorobenzene degradation by the chlorobenzene-degrading strain XJJ-1 provided in the present invention.

[0030] Figure 7 The HPLC chromatogram of the chlorobenzene degradation process of the chlorobenzene-degrading strain XJJ-1 provided in the present invention.

[0031] Figure 8 This is a diagram of the chlorobenzene metabolic pathway of the chlorobenzene-degrading strain XJJ-1 provided in the present invention. DETAILED DESCRIPTION

[0032] The present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments. This embodiment is implemented based on the technical solution of the present invention, and provides a detailed implementation method and specific operation process, but the protection scope of the present invention is not limited to the following embodiments.

[0033] In particular, the endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0034] In the following examples, unless otherwise specified, raw materials or processing techniques are conventional commercially available raw materials or conventional processing techniques in the art.

[0035] On the one hand, the present invention proposes a chlorobenzene-degrading bacterium, which is Pandora's bacteria, with a strain code of XJJ-1, preserved in the China Center for Type Culture Collection (address: Wuhan University, Wuhan, China), with a preservation number of CCTCCNO: M2021057, and a preservation time of January 14, 2021.

[0036] Furthermore, its Gram staining was negative, the strain morphology had no capsule or bacillus, and the colony morphology on Luria-Bertani medium was regular, yellow, convex, opaque and smooth. At the same time, 16S rDNA sequencing was performed on it, and the measured 16S rDNA sequence was shown in SEQ ID NO.1, and the 16S rDNA sequence was subjected to BLAST comparison, and the comparison results showed that the nucleotide sequence of 16S rDNA of strain XJJ-1 had greater than 99% homology with the nucleotide sequences of different strains of Pandoraea sp.

[0037] On the other hand, the present invention also provides a method for screening chlorobenzene-degrading bacteria, comprising the following steps:

[0038] (1) mixing a basic inorganic salt culture medium, a trace element mixed solution and a chlorobenzene solution to obtain a liquid culture medium;

[0039] (2) Weigh a certain mass of soil sample contaminated by a pesticide factory and place it in a liquid culture medium. After culturing in a constant temperature shaker, obtain a first culture solution containing bacteria. Transfer 10% of the first culture solution to another liquid culture medium. After culturing, obtain a second culture solution containing bacteria. Repeat this process multiple times until an nth culture solution is obtained, where n = 5-8;

[0040] (3) The obtained nth culture solution is spread, diluted, streaked and cultured to obtain chlorobenzene-degrading bacteria.

[0041] In some specific embodiments, in step (1), the inorganic salt components in the basic inorganic salt culture medium at least include: 0.023g / L CaCl2; 0.2g / L MgSO4·7H2O; 2.5g / L (NH4)2SO4; 1.0g / L KH2PO4; 4.5g / L Na2HPO4·12H2O.

[0042] In some specific embodiments, in step (1), the solute components in the trace element mixed solution include at least: 0.15g / L ZnSO4·7H2O, 0.26g / L MnSO4·H2O, 0.03g / L CoCl2·6H2O, 4.5g / L FeSO4·7H2O, 0.02g / L NiCl2·6H2O, 0.01g / L CuCl2, 0.1g / L Na2MoO4·2H2O, and 0.06g / L H3BO3.

[0043] In some specific embodiments, in step (1), the solvent used for the chlorobenzene solution is N,N-dimethylformamide, and its concentration is preferably 50 g / L;

[0044] The volume ratio of inorganic salt culture medium, trace element solution and chlorobenzene solution is 10 6 :10 3 :2.

[0045] In some specific embodiments, the amount of soil sample and liquid culture medium obtained from the pesticide factory can be adjusted according to actual requirements. For example, in step (2), when the first mixed solution is prepared, the ratio of the amount of soil sample to the amount of liquid culture medium added can be (3-6) g: 30 mL. Similarly, the amount transferred from the first mixed solution to the second mixed solution can be 1:30 to 1:10 in volume to the first mixed solution, and the present invention is not limited thereto.

[0046] In some specific embodiments, during the entire acclimation process of step (2), the tools used can be specifically selected according to actual conditions. The bottle used for screening here is a 150 mL crimp-top serum bottle, sealed with an aluminum cap and a gasket, wherein the added liquid is 20-30 mL. Of course, the present invention is not limited to this.

[0047] In some specific embodiments, in step (2), the acclimation conditions can be specifically selected according to the actual situation. For example, the applicable shaking speed here can be 180r / min, the culture process is carried out under closed conditions, the temperature is 28°C, and the culture time for each time is 2-3 days.

[0048] In some specific embodiments, in step (3), the dilution, coating and separation culture process is specifically as follows: taking 1 mL of the nth culture solution, diluting it stepwise with phosphate buffer to 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 100 μL of liquid was aspirated from each concentration gradient and transferred to a Luria-Bertani medium plate containing chlorobenzene, and spread and separated using a spreading rod, with 3 parallels for each gradient. After a certain period of culture, a suitable single colony was picked up with an inoculation loop and separated on the Luria-Bertani medium plate, and this was repeated 2-3 times. The obtained single bacteria were inoculated into an inorganic salt medium containing chlorobenzene to verify the degradation ability. If the chlorobenzene concentration was observed to decrease and the bacteria could grow with chlorobenzene as the only carbon source, they were chlorobenzene-degrading bacteria.

[0049] On the other hand, the present invention also proposes the use of chlorobenzene-degrading bacteria in degrading chlorobenzene.

[0050] The chlorobenzene degrading bacteria provided by the present invention are Pandoraea sp.XJJ-1, the 16S rDNA sequence GenBank accession number is MW453046, and the deposit number is CCTCC No: M 2021057. The screening method comprises: obtaining the chlorobenzene degrading bacteria from the contaminated soil sample around the pesticide factory after acclimatization, separation and purification. The present invention extracts contaminated soil from the surrounding of the pesticide factory and screens out the chlorobenzene degrading bacteria XJJ-1 therefrom. The chlorobenzene in the water is degraded by the chlorobenzene degrading bacteria XJJ-1, so as to achieve the purpose of removing pollutants, and the intermediate products are completely degraded. The operation cost is low, the negative impact on the environment is small, and it has a good prospect for development and utilization.

[0051] The above implementation is described in more detail below in conjunction with specific examples.

[0052] Embodiment 1:

[0053] Acquisition of strains

[0054] Contaminated soil was collected from a pesticide factory and packed in brown bottles and transported back to the laboratory. The soil was added to the culture medium to prepare a mixture. Through screening and domestication, a highly efficient chlorobenzene-degrading bacteria was obtained, and the strain had stable propagation characteristics.

[0055] The strain was Gram-negative, and the strain morphology was non-capsular and bacilli. The colonies on Luria-Bertani agar plates were regular, yellow, raised, opaque and smooth.

[0056] The 16S rDNA sequence was sequenced and the measured 16S rDNA sequence is shown in SEQ ID NO.1. The 16S rDNA sequence was compared by BLAST. The comparison results showed that the nucleotide sequence of 16S rDNA of strain XJJ-1 had greater than 99% homology with the nucleotide sequences of different strains of Pandoraea sp.

[0057] This strain was deposited in the China Center for Type Culture Collection in January 2021, with strain code: XJJ-1 and preservation number: CCTCC NO: M 2021057.

[0058] The specific process of obtaining the strain is as follows:

[0059] (1) mixing a basic inorganic salt culture medium, a trace element mixed solution and a benzene solution to obtain a liquid culture medium;

[0060] (2) Weigh a certain mass of soil sample contaminated by a pesticide factory and place it in a liquid culture medium. After culturing on a constant temperature shaker, a first culture solution containing bacteria is obtained. 10% of the first culture solution is transferred to another liquid culture medium. After culturing, a second culture solution containing bacteria is obtained. This process is repeated multiple times until a fifth culture solution is obtained.

[0061] (3) The fifth culture solution is spread, diluted, streaked and cultured to obtain chlorobenzene-degrading bacteria.

[0062] In step (1), the inorganic salt components in the basic inorganic salt culture medium include: 0.023g / L CaCl2, 0.2g / L MgSO4·7H2O, 2.5g / L (NH4)2SO4, 1.0g / L KH2PO4, and 4.5g / L Na2HPO4·12H2O.

[0063] In step (1), the solute components in the trace element mixed solution include: 0.15g / L ZnSO4·7H2O, 0.26g / L MnSO4·H2O, 0.03g / L CoCl2·6H2O, 4.5g / L FeSO4·7H2O, 0.02g / LNiCl2·6H2O, 0.01g / L CuCl2, 0.1g / L Na2MoO4·2H2O, and 0.06g / L H3BO3.

[0064] In step (1), the solvent used for the chlorobenzene solution (50 g / L) is N,N-dimethylformamide.

[0065] The inorganic salt medium, trace element solution and chlorobenzene solution described in step (1) are added in a volume ratio of 10 6 :10 3 :2.

[0066] In step (2), when preparing the first mixed solution, the ratio of the added amount of soil sample to the liquid culture medium is (3-6) g: 30 mL.

[0067] During the entire acclimation process of step (2), the tool used is a 150 mL crimp-top serum bottle, which is sealed with an aluminum cap and a gasket, and the liquid added is generally 20-30 mL.

[0068] In step (2), the acclimation condition can be 180 rpm, the culture process is carried out under closed conditions, the temperature is 28° C., and the time of each culture is about 2-3 days.

[0069] In step (3), the dilution, coating and separation culture process is as follows: take 1 mL of the nth culture solution and dilute it step by step with phosphate buffer to 10 -1 , 10 -2 , 10 -3, 10 -4 , 10 -5 , 10 -6 , aspirate 100 μL of liquid from each concentration gradient and transfer it to a Luria-Bertani medium plate containing chlorobenzene, and use a spreading rod for spreading and separation, with 3 parallels for each gradient. After culturing for a certain period of time, use an inoculation loop to pick up suitable single colonies and separate them on the Luria-Bertani medium plate. Repeat this 2-3 times, and inoculate the obtained single bacteria into an inorganic salt medium containing chlorobenzene to verify the degradation ability. If the chlorobenzene concentration is observed to decrease and the bacteria can grow with chlorobenzene as the only carbon source, they are chlorobenzene-degrading bacteria. The colony morphology is as follows Figure 2 As shown in Figure 1, the morphology is regular, yellow, slightly convex, opaque and smooth. The main morphology of XJJ-1 bacteria is as follows Figure 3 As shown, XJJ-1 bacteria are rod-shaped and have no flagella. Figure 4 The results show the relationship between XJJ-1 and other bacteria of the genus Pandora. XJJ-1 and I3-10 are in the same branch of the developmental tree, indicating that they have a close relationship.

[0070] Embodiment 2:

[0071] Study on the degradation characteristics of bacterial strains

[0072] 20mL of inorganic salt medium containing chlorobenzene was placed in a 150mL serum bottle. After XJJ-1 was expanded and cultured in Luria-Bertani medium, it was centrifuged at 8000rpm for 5min and resuspended with 1% inorganic salt medium. Carbon sources such as yeast powder were washed away. After repeating twice, the total volume remained unchanged. The absorbance value was measured at a wavelength of 600nm to ensure that the initial absorbance value in the degradation system was 0.05. To prevent chlorobenzene from volatilizing, the bottle was sealed and cultured at 28℃, 180rpm, and in the dark under shaking conditions. The chlorobenzene concentration, pH value, yeast powder concentration, and temperature were changed respectively. Samples were taken after 12h to determine the residual concentration of chlorobenzene.

[0073] Degradation characteristics results such as Figure 4 As shown in the data, XJJ-1 can tolerate 600 mg / L chlorobenzene and completely degrade 100 mg / L chlorobenzene within 12 h. XJJ-1 can grow well in the pH range of 6-9 and degrade 100 mg / L chlorobenzene. The addition of a small amount of yeast powder can promote the degradation of chlorobenzene, and it can also efficiently degrade chlorobenzene at a low temperature of 15 °C.

[0074] Embodiment 3:

[0075] Study on the broad spectrum of substrates for the degradation of benzene derivatives by strains

[0076] Add 20 mL of inorganic salt culture medium to a 150 mL serum bottle, add the cell suspension cultured to the logarithmic growth phase, so that the initial absorbance is 0.05. Add 50 mg / L of benzene, toluene, and ethylbenzene to the culture medium, seal it with a lid to prevent the volatilization of benzene series, shake and culture at 28°C, 180 rpm, and avoid light. Take samples after 12 hours to determine the residual concentration of benzene series.

[0077] The degradation effect of strain XJJ-1 on benzene derivatives is shown in Table 1. The strain can use benzene, toluene and ethylbenzene as the sole carbon source and can remove 100%, 77% and 83% of benzene, toluene and ethylbenzene respectively within 12 hours.

[0078] Table 1 Degradation effect of Pandoraea sp.XJJ-1 on different substrates

[0079]

[0080] Embodiment 4:

[0081] Study on the degradation of high concentration chlorobenzene in groundwater by bacterial strains and the changes of its intermediate products

[0082] The XJJ-1 strain was grown overnight in Luria-Bertani medium containing 100 mg / L chlorobenzene. The washed cells were resuspended in an inorganic salt medium without a carbon source, and the bacterial suspension was added to 20 mL of chlorobenzene-contaminated groundwater from a pesticide factory, and chlorobenzene was added to 100 mg / L. The initial absorbance at a wavelength of 600 nm was 0.05. Depending on the situation, samples were taken at intervals to determine the residual concentration of chlorobenzene and the distribution of products, the cells were removed by centrifugation, and the supernatant was extracted three times with an equal volume of ethyl acetate. The combined extracts were dried over anhydrous sodium sulfate and evaporated to dryness by a nitrogen stream. The concentrated sample was then redissolved in acetonitrile and filtered through a solvent-resistant filter with a pore size of 0.22 μm. It was analyzed using a Shimadzu HPLC-20A with Eclipse XDB-C18 (5 μm, 4.6×250 mm). The mobile phase consisted of CH3OH / 0.1% acetic acid at a flow rate of 1 mL / min.

[0083] Figure 6 It shows that chlorobenzene is rapidly degraded within the first 11 h, and intermediates are rapidly generated (such as Figure 7 The intermediate product has the largest accumulation at the 9th hour. By the 11th hour, chlorobenzene has been completely degraded, and the amount of the intermediate product is also decreasing, indicating that XJJ-1 can completely degrade chlorobenzene without the generation of secondary pollutants. Figure 1The phenomenon of turbidity in the culture medium at different times is shown, which is mainly caused by the growth and reproduction of microorganisms using chlorobenzene. In order to better illustrate the advantages of XJJ-1 of the present invention, the degradation characteristics of several degradation bacteria are compared. It can be seen from Table 1 that the degradation rate of XJJ-1 is relatively fast, which is 9.09 mg / (L·h), which has certain advantages.

[0084] Table 2 Comparison of degradation characteristics of several chlorobenzene-degrading bacteria

[0085]

[0086] Embodiment 5:

[0087] Study on metabolic pathways of strains

[0088] The culture media in the five serum bottles of the degradation system with chlorobenzene as the sole carbon source were combined, extracted three times with equal volumes of ethyl acetate, evaporated to dryness with nitrogen flow, and then the concentrated sample was redissolved in acetonitrile and filtered through a 0.22μm nylon filter. The Agilent 7890A-5975C gas chromatograph-mass spectrometer with an HP-5MS column was used for analysis, and the mass spectrometry source selected the EI source. The XJJ-1 strain was grown overnight in Luria-Bertani medium containing 100mg / L chlorobenzene, collected and centrifuged, and then crushed by an ultrasonic crusher (crushing 4 times, 30s each time, 2min interval). The obtained cell crushed material was centrifuged at 4℃, 8000rpm for 30min, and the supernatant was collected as the crude enzyme solution. The enzyme activity reaction system was constructed: catechol, crude cell enzyme solution and phosphate buffer, and the activity of catechol dioxygenase was measured at a wavelength of 260nm.

[0089] The metabolic pathways are speculated to be Figure 8 Combined with GC-MS and enzyme activity analysis, the metabolic pathway of chlorobenzene is inferred as follows: chlorobenzene is monohydroxylated twice under the action of chlorobenzene monooxygenase to form the key intermediate 3-chlorocatechol, which is then ring-opened at the ortho position under the action of catechol dioxygenase to generate 2-chloromuconic acid, which is then gradually dechlorinated and reduced under the action of related enzymes to form β-ketoadipic acid, and finally enters the biological tricarboxylic acid cycle.

[0090] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. A chlorobenzene-degrading bacterium, characterized in that: It belongs to the genus Pandoraea, named Pandoraea sp.XJJ-1, deposited in the China Center for Type Culture Collection, with the preservation number CCTCC NO: M 2021057, and the preservation time is January 14, 2021.

2. A chlorobenzene-degrading bacterium according to claim 1, characterized in that: Its Gram stain was negative, the strain morphology was non-capsular and bacilli, and the colonies on Luria-Bertani medium were regular, yellow, raised, opaque, and smooth.

3. The use of a chlorobenzene-degrading bacterium according to claim 1, characterized in that: The chlorobenzene-degrading bacteria are used for remediating chlorobenzene-contaminated sites.

4. The use of a chlorobenzene-degrading bacterium according to claim 3, characterized in that: The chlorobenzene contaminated site is chlorobenzene contaminated soil or chlorobenzene contaminated groundwater.

5. The use of a chlorobenzene-degrading bacterium according to claim 3, characterized in that: The temperature when the chlorobenzene-degrading bacteria repairs the chlorobenzene-contaminated site is 10 to 37°C.

6. The use of a chlorobenzene-degrading bacterium according to claim 5, characterized in that: The temperature when the chlorobenzene-degrading bacteria repairs the chlorobenzene-contaminated site is 10-15°C.

7. The use of a chlorobenzene-degrading bacterium according to claim 1, characterized in that: The chlorobenzene-degrading bacteria are used for degrading chlorobenzene, benzene, toluene and ethylbenzene.

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