Bacterial strain for efficiently degrading benzene series and chlorobenzene pollutants at low temperature and application of bacterial strain in groundwater remediation

Abstract

The invention relates to a bacterial strain capable of efficiently degrading benzene series and chlorobenzene pollutants at low temperature and application of the bacterial strain in groundwater remediation, the bacterial strain is pseudomonas, is named as Pseudomonas asiaticus TJ2-2, is preserved in the China Center for Type Culture Collection, has the preservation number of CCTCC NO: M 20242285, and is preserved in October 21, 2024. Compared with the prior art, the strain provided by the invention can effectively degrade benzene, toluene, ethylbenzene, o-xylene, m-xylene, p-xylene, chlorobenzene, o-dichlorobenzene, m-dichlorobenzene, p-dichlorobenzene and the like at low temperature in a wide pH range, and has the advantages of no accumulation of toxic metabolites, no secondary pollution and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of environmental pollution microbial remediation, and relates to a strain capable of efficiently degrading benzene series and chlorobenzene pollutants at low temperature and application thereof. Background Art

[0002] Benzene series (such as benzene, toluene, ethylbenzene, and xylene, abbreviated as BTEX) and chlorobenzene compounds (such as chlorobenzene and dichlorobenzene, abbreviated as CBs) are characteristic organic pollutants in soil and groundwater at contaminated sites in industries such as fine chemicals, pesticide manufacturing, and ink printing and coatings. These pollutants are easily introduced into groundwater environments due to factors such as equipment leaks, waste liquid seepage, and historical emissions, resulting in complex pollution. BTEX and CBs are currently a key focus of groundwater pollution prevention and control due to their high detection frequency, strong migration potential, and high toxicity. Furthermore, these pollutants are prone to migration and diffusion along groundwater plumes and long-term retention in aquifers, posing a potential threat to ecosystems and human health.

[0003] The main methods for removing benzene series and chlorobenzene pollutants include chemical oxidation / reduction, bioremediation, etc. 2- , ·OH, O3, Pd / TiO2, Pt / TiO2 and other oxidative materials have shown a certain degradation efficiency for BTEX and CBs pollutants. It is reported that S2O8 2- 80% BTEX (150 mg / L) can be degraded within 3 hours, while alkali-activated S2O8 2- Degrading BTEX can increase soil pH and potentially lead to salinization. Bimetallic systems composed of nano-zero-valent iron and other metals can achieve excellent reductive dechlorination effects. Pd / Fe bimetallic nanoparticles have reportedly demonstrated a dechlorination efficiency exceeding 80% for 1,2-dichlorobenzene (40 mg / L). However, in actual groundwater remediation, the oxidizing / reducing agents are difficult to distribute evenly, and incomplete reactions can lead to the accumulation of byproducts such as benzaldehyde and benzene, resulting in secondary contamination. Furthermore, chemical oxidation / reduction remediation of contaminated sites is costly and prone to the accumulation of toxic byproducts.

[0004] Due to its sustainability, low cost, and environmentally friendly nature, bioremediation is currently a major research focus in site remediation using microorganisms to degrade BTEX and chlorobenzenes. Under aerobic conditions, microorganisms can utilize enzymes such as oxygenases and dehydrogenases to degrade BTEX and chlorobenzenes, ultimately producing non-toxic metabolites such as CO2. However, the efficiency and duration of degradation are limited by factors such as groundwater temperature and pH. Groundwater temperatures typically remain around 10-20°C year-round, while existing BTEX and chlorobenzene-degrading bacteria typically have an optimal temperature range of 25-34°C, making them difficult to adapt to.

[0005] For example, CN114292775A discloses Pseudomonas stutzeri YJY21-01, which has the ability to degrade benzene compounds, and its applications. This bacterium can efficiently degrade toluene at 34°C, but its degradation rate is only 50% of that under optimal conditions at 25°C. CN101624576A discloses Mycobacterium cosmeticum byf-4, which has the ability to degrade benzene compounds, and its applications. This bacterium can degrade benzene, toluene, ethylbenzene, and o-xylene at 30°C and a pH of 7.2-7.4, but its BTEX degradation rate also decreases by more than 20% compared to its optimal conditions at low temperatures (20°C) or alkaline conditions (pH = 8). Furthermore, groundwater environments often exhibit complex pollution, making it difficult for existing degrading bacteria to simultaneously degrade multiple benzene compounds and chlorobenzene compounds. CN112251378A systematically studied the efficient degradation of benzene by Corynebacterium AL-5. AL-5 bacteria can completely degrade 100 mg / L of benzene and 80% of toluene within 10 hours at 28 ° C, but does not have the ability to degrade xylene. CN202311191119.7 discloses a highly efficient aromatic hydrocarbon degrading bacterium (Pseudomonassp.BO3-4), which can degrade BTEXS with an initial concentration of 100 mg / L and 10 mg / L of p-dichlorobenzene and chlorobenzene within 96 hours, but cannot degrade o-dichlorobenzene and m-dichlorobenzene. CN118620793A discloses a chlorobenzene pollutant degradation bacterium Pseudomonas putida BS-1 and its application. The bacterium can completely degrade p-dichlorobenzene, chlorobenzene and benzene within 48 hours, but cannot degrade xylene, o-dichlorobenzene and m-dichlorobenzene under conditions where pollutants are the only carbon source. Therefore, for the in situ bioremediation of groundwater in actual contaminated sites, there is an urgent need for degradative bacteria that can adapt to low-temperature environments and efficiently degrade complex pollutants. Summary of the Invention

[0006] The purpose of the present invention is to provide a strain and its application for the low-temperature and high-efficiency degradation of benzene series and chlorobenzene pollutants, so as to achieve the high-efficiency degradation of benzene, toluene, ethylbenzene, o-xylene, m-xylene, p-xylene, chlorobenzene, o-dichlorobenzene, m-dichlorobenzene and p-dichlorobenzene under low-temperature conditions in groundwater.

[0007] The purpose of the present invention can be achieved by the following technical solutions:

[0008] In one aspect, the present invention provides a strain for efficiently degrading benzene series and chlorobenzene pollutants at low temperatures. The strain is Pseudomonas asiatica TJ2-2, deposited with the China Center for Type Culture Collection under the accession number CCTCC NO: M 20242285 on October 21, 2024. The 16S rDNA sequence of the strain has a GenBank accession number of PV067671.

[0009] Furthermore, the degrading bacteria of the present invention are Gram-negative, lack a capsule, and have regular colony morphology on Luria-Bertani medium, appearing white, raised, opaque, and smooth. 16S rDNA sequencing was performed on the bacteria, and the resulting 16S rDNA sequence is shown in SEQ ID NO. 1. A BLAST comparison of the resulting 16S rDNA sequence revealed that the nucleotide sequence of the 16S rDNA of strain TJ2-2 shared greater than 99% homology with nucleotide sequences of different strains of the genus Pseudomonas.

[0010] The nucleotide sequence of 16S rDNA of Pseudomonas asiatica TJ2-2 provided by the present invention is shown in SEQ ID NO.1:

[0011]

[0012] In another aspect, the present invention also provides a method for screening bacteria for degrading benzene series and chlorobenzene pollutants, comprising the following steps:

[0013] (1) Mixing the basic inorganic salt culture medium and the trace element mixture to obtain the SSDM liquid culture medium;

[0014] (2) Contaminated sludge from a chemical plant in North China was added to a liquid culture medium to prepare a first bacterial mixture. 60 μL of BTEX solution was then injected with a syringe to mix the mixture. After 3-5 days in a constant temperature shaking incubator at 20°C, 10% of the first culture solution was transferred to another fresh liquid culture medium to obtain a second mixture. This process was repeated under the same conditions and repeated inoculation until the nth mixture was obtained, where n = 5-8;

[0015] (3) The obtained sixth mixed solution was diluted and spread on an SSDM solid culture plate, cultured in an incubator at 20°C to 28°C for 5 to 7 days, and streaked and cultured on a Luria-Bertani medium plate to obtain BTEX, o-dichlorobenzene, m-dichlorobenzene, p-dichlorobenzene, and chlorobenzene-degrading bacteria.

[0016] Preferably, in step (1), the inorganic salt components in the basic inorganic salt culture medium at least include: 0.2g / L NH4Cl, 7.95g / L NaCl, 0.77g / L MgCl2·6H2O, 1.05g / L MgSO4·7H2O, 0.076g / LCaCl2, 0.22g / L KCl, 0.01g / L NaHCO3, 0.026g / L NaBr, and 0.25g / L K2HPO4.

[0017] Preferably, 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.

[0018] Preferably, in step (2), the total concentration of BTEX is 100 mg / L (B:T:E:X=1:1:1:1), the concentrations of o-dichlorobenzene, m-dichlorobenzene, p-dichlorobenzene and chlorobenzene solutions are all 10 mg / L, and the solvent is N,N-dimethylformamide.

[0019] As the more preferred amount, in step (2), the volume ratio of the inorganic salt culture medium, the trace element solution, and the BTEX, o-dichlorobenzene, m-dichlorobenzene, p-dichlorobenzene, and chlorobenzene injected by the syringe each time is 10 3 :1:1:1:1:1:1:1.

[0020] Preferably, in step (2), when preparing the first mixed solution, the ratio of the added amount of sludge sample to the liquid culture medium is (4-6) g:20 mL.

[0021] Preferably, in step (2), the culture is carried out under closed conditions at a temperature of 15-20° C., and each culture lasts for 5-7 days.

[0022] Preferably, in step (3), the ingredients in the SSDM solid culture plate include: 0.2g / L NH4Cl, 7.95g / L NaCl, 0.77g / L MgCl2·6H2O, 1.05g / L MgSO4·7H2O, 0.076g / L CaCl2, 0.22g / L KCl, 0.01g / L NaHCO3, 0.026g / L NaBr, 0.25g / L K2HPO4, 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, 0.06g / L H3BO3, 15g / L agar powder.

[0023] The streak separation and culture process is as follows: take 0.1 mL of the nth mixed solution, add it to 0.9 mL of sterilized liquid inorganic salt culture medium and mix well. -1 , and then diluted step by step to 10 -2 , 10 -3 , 10 -4 , 10 -5, and plated onto SSDM solid culture medium plates, with three replicates per gradient. A certain amount of BTEX, o-dichlorobenzene, m-dichlorobenzene, p-dichlorobenzene, and chlorobenzene solution was dripped onto the bottom of the plates, leveraging the volatility of these substances to provide the strains on the plates with a carbon source required for growth. A single colony was then picked with an inoculation loop and isolated on a Luria-Bertani culture medium plate. This process was repeated 2-3 times, and the resulting single bacteria were inoculated into the degradation system to verify degradation ability. If the bacteria were observed to be able to grow using BTEX, o-dichlorobenzene, m-dichlorobenzene, p-dichlorobenzene, or chlorobenzene as the sole carbon source and to reduce the concentrations of BTEX, o-dichlorobenzene, m-dichlorobenzene, p-dichlorobenzene, or chlorobenzene, they were identified as BTEX-, o-dichlorobenzene-, m-dichlorobenzene-, p-dichlorobenzene-, or chlorobenzene-degrading bacteria.

[0024] In the second aspect, the present invention provides an application of a strain that can efficiently degrade benzene series and chlorobenzene pollutants at low temperature. The degrading bacteria is used to degrade benzene, toluene, ethylbenzene, o-xylene, m-xylene, p-xylene, chlorobenzene, o-dichlorobenzene, m-dichlorobenzene, and p-dichlorobenzene.

[0025] Furthermore, the degradation bacteria are used for in-situ bioremediation of soil and groundwater in sites contaminated by benzene series and / or chlorobenzenes.

[0026] Furthermore, when the degradation bacteria are used to degrade benzene, toluene, ethylbenzene, o-xylene, m-xylene and p-xylene, the working temperature is 10-28°C.

[0027] Furthermore, when the degradation bacteria are used to degrade chlorobenzene, o-dichlorobenzene, m-dichlorobenzene and p-dichlorobenzene, the working temperature is 15-28°C.

[0028] Furthermore, when the degrading bacteria are used to degrade benzene, toluene, ethylbenzene, o-xylene, m-xylene and p-xylene, the concentration of the target pollutants is no more than 200 mg / L.

[0029] Furthermore, when the degrading bacteria are used to degrade chlorobenzene, o-dichlorobenzene, m-dichlorobenzene and p-dichlorobenzene, the concentration of the target pollutants is no more than 10 mg / L.

[0030] Compared with the prior art, the present invention has the following advantages:

[0031] (1) By collecting contaminated sludge from the contaminated site of an existing petrochemical enterprise park, BTEX, o-dichlorobenzene, m-dichlorobenzene, p-dichlorobenzene, and chlorobenzene-degrading bacteria TJ2-2 were screened out. The bacteria were able to degrade these 10 pollutants within 96 hours in a mixed substrate containing 6 benzene series or 4 chlorobenzene series pollutants under low-temperature culture conditions of 15°C.

[0032] (2) The Pseudomonas asiatica TJ2-2 provided by the present invention degrades BTEX, o-dichlorobenzene, m-dichlorobenzene, p-dichlorobenzene, and chlorobenzene, achieving the goal of removing complex pollutants in groundwater at low temperatures without metabolic accumulation of toxic intermediates or the generation of secondary pollutants; the intermediates are completely degraded. This operation has low operating costs, minimal negative impact on the environment, and excellent development and utilization prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is a picture of the culture medium becoming turbid over time during the process of the strain degrading BTEX and four chlorobenzene compounds in a 150 mL serum bottle provided by the present invention;

[0034] Figure 2 The colony morphology and scanning electron micrograph of Pseudomonas asiatica TJ2-2 provided by the present invention;

[0035] Figure 3 A phylogenetic tree of Pseudomonas asiatica TJ2-2 provided by the present invention;

[0036] Figure 4 The growth curve and degradation curve of Pseudomonas asiatica TJ2-2 at 15°C provided by the present invention for degrading benzene series;

[0037] Figure 5 This is a schematic diagram showing the degradation rate of chlorobenzene compounds by the aromatic hydrocarbon-degrading strain Pseudomonas asiatica TJ2-2 provided in the present invention;

[0038] Figure 6 Schematic diagram of the degradation rate of benzene series by Pseudomonas asiatica TJ2-2 at different temperatures provided by the present invention;

[0039] Figure 7 This is a schematic diagram of the degradation rate of benzene series by Pseudomonas asiatica TJ2-2 at different pH values ​​provided by the present invention;

[0040] Figure 8 Schematic diagram of the degradation rate of benzene series by Pseudomonas asiatica TJ2-2 at different initial pollutant concentrations provided by the present invention;

[0041] Figure 9 This is a schematic diagram of the degradation rate of benzene series by Pseudomonas asiatica TJ2-2 at different yeast powder concentrations provided by the present invention;

[0042] Figure 10 The degradation pathways of six aromatic hydrocarbons by Pseudomonas asiatica TJ2-2 provided by the present invention;

[0043] Figure 11 This is a color change diagram of the degradation of an actual groundwater sample by Pseudomonas asiatica TJ2-2 provided by the present invention at 15°C. DETAILED DESCRIPTION

[0044] The present invention is described in detail below with reference to 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.

[0045] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application pertains. The terms used herein in the specification of this application are for the purpose of describing specific embodiments only and are not intended to limit this application.

[0046] The terms "and / or", "or / and", and "and / or" used herein include any one of two or more related listed items, and also include any and all combinations of the related listed items, wherein the any and all combinations include any combination of two related listed items, any more related listed items, or all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical AND" and technical solutions connected by "logical OR".

[0047] In this application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0048] In this application, when referring to numerical ranges, unless otherwise specified, the numerical ranges are considered continuous and include the minimum and maximum values ​​of the range, as well as every value between such minimum and maximum values. Further, when a range refers to an integer, every integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe a feature or characteristic, the ranges can be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges subsumed therein.

[0049] Only certain numerical ranges are specifically disclosed herein. However, any lower limit may be combined with any upper limit to form an unspecified range; and any lower limit may be combined with other lower limits to form an unspecified range, and similarly, any upper limit may be combined with any other upper limit to form an unspecified range. In addition, each individually disclosed point or single value may itself serve as a lower limit or upper limit and be combined with any other point or single value, or with other lower limits or upper limits, to form an unspecified range.

[0050] The temperature parameters in this application, unless otherwise specified, allow for both constant temperature treatment and treatment within a certain temperature range. The constant temperature treatment allows for temperature fluctuations within the precision range of instrument control. Fluctuations within ranges such as ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are permitted.

[0051] Herein, the “suitable” mentioned in “suitable combination”, “suitable method”, “any suitable method”, etc. shall be based on the ability to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0052] In this application, "further", "further", "particularly" and the like are used for descriptive purposes to indicate differences in content, but should not be understood as limiting the scope of protection of this application.

[0053] In this application, the terms "optionally," "optional," and "optional" mean optional or dispensable, i.e., they refer to either option being selected from two parallel options: "with" or "without." If a technical solution contains multiple "optional" clauses, each "optional" clause is independent unless otherwise specified and there are no contradictions or constraints.

[0054] In the description of the application, "multiple" means at least two, such as two, three, etc., unless otherwise clearly and specifically defined.

[0055] Unless otherwise stated, all formulations and tests herein took place at 25°C.

[0056] As used herein, the terms "comprise," "include," "contain," "have," "have," or other variations thereof are intended to encompass non-exclusive inclusions, and no distinction is made between these terms. The term "comprising" means that other steps and ingredients that do not affect the end result may be added. The compositions and methods / processes of the present invention comprise, consist of, and consist essentially of the essential elements and limitations described herein, as well as any additional or optional ingredients, components, steps, or limitations described herein. As used herein, no distinction is made between the terms "efficacy," "performance," "effect," and "efficacy."

[0057] Unless otherwise specified, all embodiments and optional embodiments of the present application can be combined with each other to form a new technical solution. Unless otherwise specified, all technical features and optional technical features of the present application can be combined with each other to form a new technical solution.

[0058] Unless otherwise specified, all steps of the present application can be performed sequentially or randomly, preferably sequentially.

[0059] In particular, the endpoints of the ranges and any values ​​disclosed herein are not limited to the exact ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be considered to be specifically disclosed herein.

[0060] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0061] Example 1 Acquisition of strains

[0062] This example provides a strain of Pseudomonas asiatica TJ2-2, a highly efficient and stable BTEX, o-dichlorobenzene, m-dichlorobenzene, p-dichlorobenzene, and chlorobenzene-degrading bacterium. The strain, designated TJ2-2, is Gram-negative and has a regular, white, convex, opaque, and smooth morphology. 16S rDNA sequencing and whole-genome analysis were performed on the strain. BLAST comparison of the obtained 16S rDNA sequence revealed that the nucleotide sequence of the 16S rDNA of strain TJ2-2 shared greater than 99% identity with nucleotide sequences from different strains of the genus Pseudomonas.

[0063] The strain provided in this example is deposited in the China Center for Type Culture Collection with the accession number CCTCCM20242285 and the deposit date is October 21, 2024.

[0064] The specific screening and acquisition process of the strains is as follows:

[0065] (1) Mixing the basic inorganic salt culture medium and the trace element mixture to obtain the SSDM liquid culture medium;

[0066] (2) Contaminated sludge from a chemical plant in North China was added to a liquid culture medium to prepare a first bacterial mixture. 60 μL of BTEX solution was then injected with a syringe to mix the mixture. After 3-5 days in a constant temperature shaking incubator at 20°C, 10% of the first culture solution was transferred to another fresh liquid culture medium to obtain a second mixture. The culture was continued under the same conditions and the inoculation was repeated until the nth mixture was obtained, where n = 6;

[0067] (3) The obtained sixth mixed solution was diluted and spread on an SSDM solid culture plate, cultured in an incubator at 20°C to 28°C for 5 to 7 days, and streaked and cultured on a Luria-Bertani medium plate to obtain BTEX, o-dichlorobenzene, m-dichlorobenzene, p-dichlorobenzene, and chlorobenzene-degrading bacteria.

[0068] In step (1), the inorganic salt components in the basic inorganic salt culture medium include: 0.2g / L NH4Cl, 7.95g / L NaCl, 0.77g / L MgCl2·6H2O, 1.05g / L MgSO4·7H2O, 0.076g / L CaCl2, 0.22g / LKCl, 0.01g / L NaHCO3, 0.026g / L NaBr, and 0.25g / L K2HPO4.

[0069] 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 / L NiCl2·6H2O, 0.01g / L CuCl2, 0.1g / L Na2MoO4·2H2O, and 0.06g / L H3BO3.

[0070] In step (2), the total concentration of BTEX is 100 g / L (1:1:1:1), the concentrations of o-dichlorobenzene, m-dichlorobenzene, p-dichlorobenzene, and chlorobenzene solutions are all 10 g / L, and the solvent is N,N-dimethylformamide.

[0071] In step (2), the volume ratio of the inorganic salt culture medium, trace element solution, and BTEX, o-dichlorobenzene, m-dichlorobenzene, p-dichlorobenzene, and chlorobenzene injected by syringe each time is 10 3 :1:1:1:1:1:1:1.

[0072] In step (2), when preparing the first mixed solution, the ratio of the added amount of sludge sample to the liquid culture medium is 5g:20mL.

[0073] In step (2), the culture is carried out under closed conditions at a temperature of 18° C., and each culture lasts for 5 days.

[0074] In step (3), the ingredients in the SSDM solid culture plate include: 0.2g / L NH4Cl, 7.95g / LNaCl, 0.77g / L MgCl2·6H2O, 1.05g / L MgSO4·7H2O, 0.076g / L CaCl2, 0.22g / L KCl, 0.01g / LNaHCO3, 0.026g / L NaBr, 0.25g / L K2HPO4, 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 / LNa2MoO4·2H2O, 0.06g / L H3BO3, 15g / L agar powder.

[0075] The separation and culture process is as follows: take 0.1 mL of the nth mixed solution, add it to 0.9 mL of sterilized liquid inorganic salt culture medium and mix well. -1 , and then diluted step by step to 10 -2 , 10 -3 , 10 -4 , 10 -5 , and spread it on SSDM solid culture medium plates, with 3 parallels for each gradient, and add a certain amount of BTEX, o-dichlorobenzene, m-dichlorobenzene, p-dichlorobenzene, and chlorobenzene solution to the bottom of the plate, using the volatility of these substances to increase the carbon source required for the growth of the strains on the plate.

[0076] Then use an inoculation loop to pick up a single colony and isolate it on a Luria-Bertani medium plate. Repeat this process 2-3 times and inoculate the obtained single bacteria into the degradation system to verify the degradation ability. If it is observed that the bacteria can use BTEX, o-dichlorobenzene, m-dichlorobenzene, p-dichlorobenzene, and chlorobenzene as the sole carbon source for growth and the concentrations of BTEX, o-dichlorobenzene, m-dichlorobenzene, p-dichlorobenzene, and chlorobenzene decrease, then it is a BTEX, o-dichlorobenzene, m-dichlorobenzene, p-dichlorobenzene, and chlorobenzene-degrading bacteria. The colony morphology of TJ2-2 is as follows: Figure 2 As shown, the morphology is regular, white, convex, opaque and smooth. The 16S rDNA sequence was subjected to BLAST comparison, and the comparison results showed that the nucleotide sequence of the 16S rDNA of strain TJ2-2 had greater than 99% homology with the nucleotide sequences of different strains of Pseudomonas sp. Figure 3The results showed that strain TJ2-2 had a close relationship with other Pseudomonas species and was in the same branch of the developmental tree as Pseudomonas plecoglossicida GSN22 (KF815701.1), indicating that the strain had a close relationship with other species.

[0077] Example 2 Study on the degradation of BTEX by strain TJ2-2

[0078] Place 20 mL of an inorganic salt culture medium containing 100 mg / L BTEX (benzene, toluene, ethylbenzene, and xylene in a 1:1:1:1 volume ratio, with a 1:1:1 molar ratio of o-xylene:m-xylene:p-xylene in the xylenes) in a 150 mL serum bottle and inoculate the culture using the same procedures as in Example 1. The bottle was sealed with a cap to prevent volatilization of BTEX and incubated at 15°C, 150 rpm, and in the dark. Samples were taken periodically to determine the residual BTEX concentration.

[0079] Figure 1 a is a picture showing that the culture medium becomes turbid over time during the process of BTEX degradation by the strain in Example 2.

[0080] Figure 4 The results showed that at a temperature of 15°C, Pseudomonas asiatica TJ2-2 could completely degrade benzene, toluene and ethylbenzene with an initial mixed concentration of 100 mg / L within 4 days, and the degradation rate of m / p-xylene and o-xylene was over 77%, indicating that it can adapt to the actual groundwater temperature and maintain its BTEX degradation activity.

[0081] At 15°C, with benzene derivatives as the sole carbon source, the chlorobenzene-degrading bacterium Pseudomonas putida BS-1 disclosed in CN118620793A was used. This bacterium was able to completely degrade 25 mg / L of toluene and ethylbenzene, with a degradation rate of up to 53.5% for benzene. However, it was almost impossible to degrade meta- / para-xylene and o-xylene.

[0082] Example 3 Study on the degradation of CBs by strain TJ2-2

[0083] In order to investigate the degradation ability of strain TJ2-2 for p-chlorobenzene and dichlorobenzene composite pollutants, the bacterial suspension prepared by strain TJ2-2 was added to the basic inorganic salt medium containing 10 mg / L chlorobenzene and 30 mg / L dichlorobenzene (the mass ratio of o-dichlorobenzene, p-dichlorobenzene, and m-dichlorobenzene was: o-DCB:p-DCB:m-DCB=1:1:1, w / w), respectively. The initial OD 600nmThe initial conditions were set to pH 8.0, temperature 15°C, and shaker speed 150 rpm. After 96 h, 10 mL of culture medium was taken from the degradation system and placed in a headspace bottle. The residual concentrations of chlorobenzene and dichlorobenzene were determined using a headspace gas chromatograph, and the OD was determined using a UV spectrophotometer. 600nm .

[0084] Figure 1 b is a picture showing that the culture medium becomes turbid over time during the process of the strain degrading CBs in Example 3.

[0085] The results show that Figure 5 This is a schematic diagram showing the degradation rate of chlorobenzene compounds by the aromatic hydrocarbon degrading strain Pseudomonas asiatica TJ2-2 provided in the present invention. TJ2-2 has a degradation effect on four chlorobenzene compounds within 96 hours at 15°C, and can completely degrade 10 mg / L of chlorobenzene with a degradation rate of 100%. 600nm In the degradation system of 30 mg / L dichlorobenzene, strain TJ2-2 could degrade 86.7% of p-dichlorobenzene, 45.1% of o-dichlorobenzene and 24.5% of m-dichlorobenzene within 96 hours.

[0086] Under the same test conditions, Pseudomonas putida BS-1 was able to degrade 96.5% of chlorobenzene at 15°C using chlorobenzene as its sole carbon source, with degradation rates reaching up to 60.7% for p-dichlorobenzene. However, it was almost unable to degrade o-dichlorobenzene and m-dichlorobenzene. Meanwhile, the chlorobenzene-degrading bacterium Pandoraea sp. XJJ-1 disclosed in CN115873755B was able to degrade 84.8% of chlorobenzene at an initial concentration of 100 mg / L at 15°C, but was unable to degrade dichlorobenzene.

[0087] Example 4 Study on the degradation characteristics of strain TJ2-2

[0088] After TJ2-2 was expanded in Luria-Bertani medium, it was centrifuged at 8000 rpm for 5 min and resuspended in 0.9% saline to wash away the yeast powder and other carbon sources. This was repeated twice and the total volume remained unchanged. The absorbance was measured at a wavelength of 600 nm and added to a 150 mL serum bottle containing 20 mL of SSDM medium containing BTEX. The initial OD value in the degradation system was 0.04. 600nm To prevent the volatilization of BTEX, the culture was sealed with a lid and incubated at 150 rpm in the dark. The temperature, pH, initial pollutant concentration, and yeast powder concentration were varied. Samples were taken after 96 h, and the residual BTEX concentration was determined by headspace gas chromatography.

[0089] 1. Different temperatures

[0090] Degradation conditions: pH 8.0, 100 mg / L BTEX (B:T:E:X = 1:1:1:1, i.e., the volume ratio of benzene, toluene, ethylbenzene, and xylene is 1:1:1:1); temperature: 10°C, 15°C, 20°C, and 28°C;

[0091] The results show that: Figure 6 This diagram shows the effect of different temperatures on the degradation rate of benzene series by the aromatic hydrocarbon-degrading strain Pseudomonasasiatica TJ2-2, provided in the present invention. Strain TJ2-2 exhibits excellent degradation of benzene, toluene, ethylbenzene, and xylene at temperatures between 15°C and 28°C, with degradation rates exceeding 99.0% for each of these, and over 77% for xylene. At temperatures as low as 10°C, the strain also exhibits excellent degradation of benzene, toluene, and ethylbenzene, with degradation rates reaching 31.0% to 45.4%. It also exhibits some degradation of m-xylene, p-xylene, and o-xylene, demonstrating excellent low-temperature adaptability.

[0092] 2. Different pH:

[0093] Degradation conditions: 100 mg / L BTEX (B:T:E:X=1:1:1:1); temperature: 15°C; pH: 6.0, 7.0, 8.0, 9.0;

[0094] The results show that: Figure 7 A schematic diagram shows the effect of different pH values ​​on the degradation of BTEX by the aromatic hydrocarbon-degrading strain Pseudomonasasiatica TJ2-2, provided in the present invention. Strain TJ2-2 showed a strong degradation of BTEX across a pH range of 6.0-9.0, with the highest degradation efficiency at a weakly alkaline pH of 8.0. It was able to completely degrade benzene, toluene, and ethylbenzene, with degradation rates exceeding 77.0% for o-, m-, and p-xylenes.

[0095] 3. Different initial pollutant concentrations

[0096] Degradation conditions: pH 8.0; temperature: 15°C; 50 / 100 / 150 / 200 mg / L BTEX (B:T:E:X=1:1:1:1);

[0097] The results show that: Figure 8 A schematic diagram shows the effect of different initial pollutant concentrations on the degradation rate of BTEX by the aromatic hydrocarbon-degrading strain Pseudomonas asiatica TJ2-2, provided in the present invention. Strain TJ2-2 was able to degrade a BTEX mixture in the 50mg / L-200mg / L concentration range, with degradation rates of 95.8% to 100% for benzene, toluene, and ethylbenzene within the 50mg / L-150mg / L range.

[0098] 4. Different yeast powder concentrations

[0099] Degradation conditions: pH 8.0; temperature: 15°C; 100 mg / L BTEX (B:T:E:X=1:1:1:1); yeast powder concentration: 0 / 10 / 30 / 50 mg / L.

[0100] The results show that: Figure 9 A schematic diagram shows the effect of different yeast powder concentrations on the degradation of BTEX by the aromatic hydrocarbon-degrading strain Pseudomonasasiatica TJ2-2, provided in the present invention. Compared to the no yeast addition group, the addition of 10mg / L to 50mg / L yeast powder significantly enhanced the degradation of BTEX by strain TJ2-2. After 96 hours of incubation, the 10mg / L yeast powder group increased the degradation of m- / p-xylene and o-xylene by 6.29% and 12.96%, respectively. The 30mg / L to 50mg / L yeast powder groups increased the degradation of all six BTEX compounds by strain TJ2-2 to 100%, demonstrating that the appropriate addition of an additional carbon source facilitates pollutant degradation by TJ2-2.

[0101] Example 5 Study on the metabolic pathway of benzene series by strain TJ2-2

[0102] 20 mL of SSDM medium was placed in a 150 mL serum bottle and the washed cells were resuspended in 20 mL of SSDM medium without carbon source. The initial OD 600nm The concentration of the six benzene derivatives was 0.2. The culture medium was sealed, and six benzene derivatives were added to a final mixed concentration of 100 mg / L. Samples were collected every 24 hours for a total of 96 hours. After extraction with an equal volume of ethyl acetate, the extracts were combined, dried over anhydrous sodium sulfate, evaporated to dryness using a rotary evaporator, reconstituted with acetonitrile, and filtered through a 0.22 μm organic solvent-resistant filter. The sample was derivatized by silanization using N, O-bis(trimethylsilyl)trifluoroacetamide as the derivatization reagent. 750 μL of the concentrate was reacted with 450 μL of the derivatization reagent at 60°C for 1 hour to obtain the TMS derivative.

[0103] Qualitative analysis was performed using gas chromatography-mass spectrometry, and the mass spectrometry data were compared with the NIST standard database. A total of 27 metabolites were detected, including phenol derivatives (11.505 min), catechol derivatives (19.467 min), 1,2,4-trihydroxybenzene derivatives (11.577 min), 3,4-dihydroxybutyric acid derivatives (10.126 min), 4-hydroxymethylphenol derivatives (10.489 min), 3,4-dihydroxybenzaldehyde derivatives (22.071 min), o-cresol derivatives (7.217 min), m-cresol derivatives (7.307 min), 3-methylcatechol derivatives (9.795 min), benzyl alcohol derivatives (7.456 min), benzoic acid derivatives (8.402 min), 3,5-dihydroxybenzoic acid derivatives (26.927 min), 2-hydroxyvaleric acid derivatives (7.929 min), 2-oxopentanoic acid derivatives (7.930 min), and 1,2,4-trihydroxybenzaldehyde derivatives (11.577 min). Biological (7.968min), (3Z,5Z)-4,6-dihydroxy-3,5-heptadienoic acid derivatives (30.865min), (2E,4Z)-4-hydroxy-6-oxo-2,4-heptadienoic acid derivatives (26.557min), phenylacetic acid derivatives (8.875min), o-ethylphenol derivatives (7.904min), 2-ethylhydroquinone derivatives (10.379min), 2,5-dihydroxyacetophenone derivatives (11.571min), 1-phenyl-1-ethanol derivatives (7.307min), 4-methylbenzyl alcohol derivatives (8.046min), p-methylbenzoic acid derivatives (9.374min), o-tolylmethanol derivatives (8.377min), o-tolylbenzoic acid derivatives (9.141min), 2,3-dimethylresorcinol derivatives (10.968min), m-tolylmethanol derivatives (8.338min).

[0104] Whole genome analysis of strain TJ2-2

[0105] The strain TJ2-2 was cultured in SSDM medium containing 100 mg / L BTEX to the logarithmic growth phase, the bacterial liquid was collected, and the bacteria were collected by centrifugation at 10,000 rpm for 5 minutes. The DNA of the bacterial colony was extracted according to the instructions of the soil DNA extraction kit (MP BIO FastDNA Spin Kit for Soil). The genomic DNA was transported to Guangdong Meige Gene Technology Co., Ltd. at low temperature. The total amount of DNA was detected using a fluorescent dye (Quant-iT PicoGreen dsDNA Assay Kit), and the genome concentration and purity were tested using 1% agarose gel electrophoresis and an ultra-micro spectrophotometer to determine whether the genome met the library experiment requirements. The experimental process was performed in accordance with the standard protocol provided by Oxford Nanopore Technologies (ONT), including sample quality detection, library construction, library quality detection, and library sequencing. Total DNA from the samples was sequenced using the Illumina second-generation high-throughput sequencing platform. Genome assembly was performed using validated data after sample quality control. MECAT2 was used for assembly of pure third-generation PacBio data (https: / / github.com / xiaochuanle / MECAT2), while Unicycler was used for assembly of data containing both second- and third-generation data (https: / / github.com / rrwick / Unicycler). The amino acid sequences of the coding genes were annotated and aligned against the Non-Redundant (NR), Clusters of Orthologous Groups of proteins (COG), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Swiss Prot databases to obtain functional prediction information for the coding genes.

[0106] Whole-genome analysis revealed that the genome of strain TJ2-2 consists of a circular chromosome and three plasmids. Degradation genes are located on the chromosome and convert BTEX, chlorobenzene, o-dichlorobenzene, m-dichlorobenzene, and p-dichlorobenzene into non-toxic substances by encoding the aromatic dioxygenase tod (C1C2BADEJST), catechol 1,2-dioxygenase catA, and benzoate 1,2-dioxygenase benA. Furthermore, genes encoded on the chromosome of TJ2-2 for cold shock proteins (cspD and cspA), a toluene efflux pump (ttgFEDTA), and genes involved in unsaturated fatty acid synthesis (fabDGFABHVRZ) work synergistically to help TJ2-2 maintain degradation activity in groundwater environments subjected to stress conditions such as low temperatures and combined pollution. Furthermore, genes on the chromosome are typically stably inherited by offspring during cell division and are not easily lost due to environmental stress or a lack of sustained selective pressure. This ensures that strain TJ2-2 maintains its degradation function in contaminated sites for a long time, avoiding the problem of degradation ability attenuation due to plasmid loss.

[0107] Combined with the whole genome analysis of the strain, the toluene dioxygenase (TOD) in strain TJ2-2 has a similarity of 99% with that of Pseudomonasputida KL47. TOD is composed of multiple functional protein subunits, which mainly catalyzes the initial oxidation step of aromatic compounds by introducing two oxygen atoms on the benzene ring to form a cis-dihydroxy intermediate, which then enters the downstream aromatic degradation pathway, such as the phthalate pathway or the ring-opening metabolic pathway. Therefore, it is speculated that strain TJ2-2 degrades benzene to produce phenol through dioxygenase, and produces catechol under the action of dehydrogenase, and further undergoes ring-opening cleavage to form small molecular compounds such as 3,4-dihydroxybutyric acid under the action of catechol 2,3-dioxygenase; using dioxygenase and dehydrogenase, toluene can be converted into dihydroxylated products such as 3-methylcatechol, 3,4-dihydroxybenzaldehyde, and 3,5-dihydroxybenzoic acid, and is ring-opened and cleaved to (3Z,5-dihydroxybenzoic acid) under the action of catechol 2,3-dioxygenase. Z)-4,6-dihydroxyhept-3,5-dienoic acid and 2-hydroxyvaleric acid; ethylbenzene can generate 1-phenylethanol, 2,5-dihydroxyacetophenone and phenylacetic acid under the action of dioxygenase and dehydrogenase, and then enter the downstream metabolic pathway using ring-opening enzyme; toluene dioxygenase can convert xylene into 4-methylbenzyl alcohol, o-tolyl methanol, 2,3-dimethylresorcinol and m-tolyl methanol, and generate p-toluic acid and o-toluic acid under the action of dehydrogenase, and then open the ring and enter the downstream pathway. Figure 10 shown.

[0108] Example 6: Small test of strain TJ2-2 on actual contaminated groundwater at 15°C

[0109] We collected groundwater samples from a contaminated site at a petrochemical enterprise and conducted a small-scale laboratory test. Testing revealed that benzene and m / p-xylene exceeded the permitted levels, with concentrations of 52.26 mg / L and 4.43 mg / L, respectively, and a pH of 7.52. The groundwater temperature at the site was 15.1°C. The incubator simulated the actual groundwater temperature (15°C) and set the incubation conditions to: 150 rpm, OD 600nm =0.2, water sample volume: 20mL.

[0110] The results show that: Figure 11 This image shows the color change of a groundwater sample degraded by Pseudomonas asiatica TJ2-2 at 15°C. TJ 2-2 was able to degrade a combined pollutant of benzene and xylene under low-temperature conditions. The sample's color changed from pale white to pale yellow after two days, and then to pale yellowish brown after three days. The degradation rate for both benzene and m- / p-xylene reached 100%. This demonstrates that the TJ 2-2 bacterial agent has excellent degradation efficacy in groundwater at low temperatures, capable of degrading over 50 mg / L of combined pollutants. It can be used as a biomaterial for in situ bioremediation of contaminated groundwater.

[0111] The above description of the embodiments is intended to facilitate understanding and use of the invention by those skilled in the art. It will be apparent that those skilled in the art can readily make various modifications to these embodiments and apply the general principles described herein to other embodiments without requiring inventive effort. Therefore, the present invention is not limited to the above-described embodiments. 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 strain capable of efficiently degrading benzene series and chlorobenzene pollutants at low temperature, characterized in that: It is a Pseudomonas, named Pseudomonas asiatica TJ2-2, deposited in the China Center for Type Culture Collection, with the deposit number CCTCC NO: M20242285, and the deposit date is October 21, 2024.

2. The use of a strain for degrading benzene series and chlorobenzene pollutants at low temperature and high efficiency as claimed in claim 1, characterized in that: The degradation bacteria are used to degrade benzene, toluene, ethylbenzene, o-xylene, m-xylene, p-xylene, chlorobenzene, o-dichlorobenzene, m-dichlorobenzene and p-dichlorobenzene.

3. The use of a strain for degrading benzene series and chlorobenzene pollutants at low temperature and high efficiency according to claim 2, characterized in that: The degradation bacteria are used for in-situ remediation of groundwater in sites contaminated by benzene series and / or chlorobenzenes.

4. The use of a strain for degrading benzene series and chlorobenzene pollutants at low temperature and high efficiency according to claim 2, characterized in that: When the degradation bacteria are used to degrade benzene, toluene, ethylbenzene, o-xylene, m-xylene and p-xylene, the working temperature is 10-28°C.

5. The use of a strain for degrading benzene series and chlorobenzene pollutants at low temperature and high efficiency according to claim 2, characterized in that: When the degradation bacteria are used to degrade chlorobenzene, o-dichlorobenzene, m-dichlorobenzene and p-dichlorobenzene, the working temperature is 15-28°C.

6. The use of a strain for degrading benzene series and chlorobenzene pollutants at low temperature and high efficiency according to claim 2, characterized in that: When the degradation bacteria are used to degrade benzene, toluene, ethylbenzene, o-xylene, m-xylene and p-xylene, the concentration of a single target pollutant is no more than 200 mg / L.

7. The use of a strain for degrading benzene series and chlorobenzene pollutants at low temperature and high efficiency according to claim 2, characterized in that: When the degrading bacteria are used to degrade chlorobenzene, o-dichlorobenzene, m-dichlorobenzene and p-dichlorobenzene, the concentration of a single target pollutant is no more than 10 mg / L.

8. The use of a strain for degrading benzene series and chlorobenzene pollutants at low temperature and high efficiency according to claim 2, characterized in that: When the degradation bacteria are used to degrade benzene, toluene, ethylbenzene, o-xylene, m-xylene and p-xylene, the pH value is 6.0-9.

0.

9. The use of a strain for degrading benzene series and chlorobenzene pollutants at low temperature and high efficiency according to claim 2, characterized in that: When the degrading bacteria are used to degrade chlorobenzene, o-dichlorobenzene, m-dichlorobenzene and p-dichlorobenzene, the pH value is 8.0.

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