CN113957014A - Functional strain with polyhalogenated hydrocarbon degradation and chromium (VI) reduction activities and application thereof - Google Patents
Functional strain with polyhalogenated hydrocarbon degradation and chromium (VI) reduction activities and application thereof Download PDFInfo
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09C—RECLAMATION OF CONTAMINATED SOIL
- B09C1/00—Reclamation of contaminated soil
- B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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Abstract
The invention belongs to the technical field of microbial remediation of organic-inorganic composite contaminated soil, and particularly relates to a functional strain with polyhalogenated hydrocarbon degradation and chromium (VI) reduction activities and application thereof. The degrading bacteria are Stenotrophomonas (Stenotrophoromonas sp.) JWDH2-3, belong to gram-negative bacteria, are preserved in China center for type culture Collection (CCTCC M2021222) at 3 months and 12 days of 2021. The functional strain can be applied to the treatment and restoration of respectively singly polluted soil and composite polluted soil of polyhalogenated hydrocarbon and heavy metal chromium (VI), and has good application prospect.
Description
Technical Field
The invention belongs to the technical field of microbial remediation of organic-inorganic composite contaminated soil, and particularly relates to a functional strain with polyhalogenated hydrocarbon degradation and chromium (VI) reduction activities and application thereof.
Background
With the rapid development of science and technology, the living standard of human materials is continuously improved, however, Volatile Organic Compounds (VOCs) are stored in soil due to the random discharge of chemical production and the improper treatment of sewage, waste gas, garbage and the like. VOCs can be classified into alkanes, aromatics, alkenes, halocarbons, esters, aldehydes, ketones, and the like, depending on their chemical structures. Research shows that VOCs can affect the immune level, the central nervous system and the digestive system of a human body, and cause subjective symptoms of dizziness, weakness, chest distress and the like of the human body, and symptoms of inappetence, nausea and the like. Among the many volatile organic compounds, most polyhalogenated hydrocarbon contaminants have a distinctive odor, have been shown to have triproducidal effects (carcinogenic, teratogenic, mutagenic), can enter the human body through respiratory or skin contact, and are blacklisted as being preferred pollutants by many countries. Therefore, how to green and efficiently repair polyhalogenated hydrocarbon pollutants in the environment becomes one of the focus of research.
Meanwhile, with the development of processing industries such as electroplating, tanning, alloy and the like, the pollution problem caused by heavy metal pollution is more and more concerned by people. Chromium (Cr) is one of the common heavy metal pollutants, and mainly exists in two forms of trivalent (iii) and hexavalent (vi) in the environment. Wherein, chromium (III) can participate in the metabolic process of saccharides and fat of human bodies, is a necessary trace element for human bodies and animals, and chromium (VI) has strong toxicity and can cause serious harm to the health of human bodies. Therefore, the reduction of chromium (VI) to chromium (III) is one of the major routes to remediate chromium metal contamination.
In addition, in the land used in various chemical plant parks, the composite polluted soil in which the VOCs organic pollutants represented by polyhalogenated hydrocarbons and the heavy metal inorganic pollutants represented by chromium coexist is also an important restoration object in the treatment of industrial polluted sites, and the development of a suitable restoration technology and a restoration material which are compatible with both the VOCs organic pollutants and the chromium inorganic pollutants is the key point for the treatment of the polluted soil.
Compared with the traditional physical and chemical remediation method, the microbial remediation has the advantages of environmental friendliness, economy, small environmental influence and the like, and has wide application prospects in the aspect of environmental remediation of polyhalogenated hydrocarbon pollutants and chromium heavy metal pollutants. Recently, polyhalogenated hydrocarbon biodegradation bacteria which have been isolated and identified at home and abroad include Pseudomonas (Pseudomonas), Bacillus (Bacillus), campylobacter (Ancylobacter), Stenotrophomonas (Stenotrophomonas), Xanthobacter (Xanthobacter), Burkholderia (Burkholderia) and the like. Although the above-mentioned degrading bacteria have high degrading efficiency for a single polyhalogenated hydrocarbon, there are few reports of a single bacterium which can degrade multiple polyhalogenated hydrocarbon contaminants simultaneously. In consideration of the complexity of pollutants in water and soil, the development of multifunctional degrading strains with broad spectrum is of great significance to the degradation and remediation of target pollutants. The chromium (VI) reducing bacteria which are separated and identified at home and abroad comprise Pseudomonas (Pseudomonas), Bacillus (Bacillus), Desulfovibrio (Desulfovibrio), Ochrobactrum (Ochrobactrum), lysine Bacillus (Lysinibacillus) and the like. Although the above-mentioned reducing bacteria can reduce chromium (VI), there are few microorganisms which can reduce chromium (VI) and degrade polyhalogenated hydrocarbons at the same time.
Therefore, the functional strains capable of simultaneously degrading or tolerating the polyhalogenated hydrocarbon pollutants and the heavy metal chromium are screened, and the method has great significance for realizing the treatment and restoration of the polyhalogenated hydrocarbon and heavy metal chromium (VI) composite polluted soil and has wide application prospect.
Disclosure of Invention
The invention aims to provide a functional strain with both polyhalogenated hydrocarbon degradation and chromium (VI) reduction activity and application thereof
In order to achieve the purpose, the invention adopts the technical scheme that:
a functional strain with polyhalogenated hydrocarbon degradation and chromium (VI) reduction activity, which is Stenotrophomonas sp JWDH2-3, is preserved in China center for type culture collection (12 months 3) in 2021, and has the preservation number of CCTCC M2021222 and the preservation address of No. 299 of the eight-way zone in Wuchang city, Wuhan, Hubei province.
The application of the functional strain is the application of the strain in degrading polyhalogenated hydrocarbon in polluted soil and removing heavy metal chromium (VI).
The strain is applied to dehalogenation and metabolism of polyhalogenated alkane under the anoxic and anaerobic conditions and mineralization and degradation of polyhalogenated hydrocarbon under the aerobic condition.
The strain reduces and removes chromium (VI) in soil under the anoxic and anaerobic conditions.
The chromium (VI) reduction and the dehalogenation metabolism of the polyhalogenated alkane are carried out under the anoxic and anaerobic conditions, and the suitable oxidation-reduction potential condition is less than or equal to 20-50 mV;
under the aerobic condition, the oxidation-reduction potential condition in the environment suitable for the strain is more than or equal to 150-200 mV, the strain has good tolerance capability to chromium (VI), and the strain can be kept from being killed by the toxic action of the chromium (VI) under the condition;
the polyhalogenated hydrocarbon is one or more of gamma-hexachlorocyclohexane, trichloroethylene, 1, 2-dichloroethane, 1, 2-dichloropropane and 1,1, 2-trichloroethane.
A microbial preparation comprises JWDH2-3 of Stenotrophomonas sp.
The strain is cultured in an inorganic salt culture medium to obtain a culture or a culture bacterial suspension containing the strain.
The application of the microbial inoculum in degrading polyhalogenated hydrocarbon in polluted soil and removing heavy metal chromium (VI).
The invention has the advantages that:
(1) the polyhalogenated hydrocarbon degrading bacterium Stenotrophomonas sp JWDH2-3 provided by the invention can efficiently degrade various polyhalogenated hydrocarbon compounds and has wide polyhalogenated hydrocarbon substrate adaptation characteristics;
(2) the polyhalogenated hydrocarbon degrading bacterium Stenotrophomonas sp JWDH2-3 provided by the invention can simultaneously degrade polyhalogenated hydrocarbon and reduce heavy metal chromium (VI), and has the advantages of two purposes, economy and high efficiency aiming at the treatment of the complex polluted soil of the polyhalogenated hydrocarbon and the heavy metal chromium (VI);
(3) the polyhalogenated hydrocarbon degrading bacterium Stenotrophomonas (Stenotrophoromonas sp.) JWDH2-3 developed in the invention belongs to facultative aerobes, can respectively carry out dehalogenation degradation of polyhalogenated hydrocarbon organic pollutants and reduction of chromium (VI) under the anoxic/anaerobic condition, carry out mineralization and metabolism of multi-channel substituted hydrocarbon organic pollutants under the aerobic condition, and also has tolerance to the highly toxic heavy metal chromium (VI), although the reducing power to chromium (VI) is not significant under anoxic/anaerobic conditions, but still keeps good cell abundance and recovers high-efficiency chromium (VI) reduction activity under the condition of hypoxia or even anaerobic condition, so as to complete the reduction of chromium (VI) while dehalogenating the polyhalogenated hydrocarbon under the condition of low oxygen or anaerobic, and deep mineralization and degradation of polyhalogenated hydrocarbon are carried out under aerobic condition, and the functional activity of the strain is not influenced by the high toxicity of chromium (VI).
Drawings
FIG. 1 is a plate-coated colony growth morphology of Stenotrophomonas (Stenotrophoromonas sp.) JWDH2-3 of the present invention.
FIG. 2 is a scanning electron microscope image of the strain morphology of Stenotrophomonas sp JWDH2-3 of the present invention.
FIG. 3 is a phylogenetic analysis diagram of Stenotrophomonas (Stenotrophoromonas sp.) JWDH2-3 according to the present invention.
FIG. 4 is a graph showing the effect of JWDH2-3 on the metabolic degradation characteristics of gamma-hexachlorocyclohexane, trichloroethylene, 1, 2-dichloroethane, 1, 2-dichloropropane and 1,1, 2-trichloroethane according to the present invention.
FIG. 5 is a graph showing the effect of JWDH2-3 on the reduction of chromium (VI) by Stenotrophomonas sp.
FIG. 6 is a graph showing the effect of JWDH2-3 on soil remediation under the condition of complex contamination of polyhalogenated hydrocarbons and chromium (VI) according to the present invention.
Detailed Description
The following will further describe the screening of the functional strains and the application characteristics thereof with reference to the accompanying drawings and examples, but the examples described are not intended to limit the present invention.
The degrading bacteria are Stenotrophomonas (Stenotrophoromonas sp.) JWDH2-3, belong to gram-negative bacteria, are preserved in China center for type culture Collection (CCTCC M2021222) at 3 months and 12 days of 2021. The strain has the capability of simultaneously and rapidly degrading various polyhalogenated hydrocarbons such as gamma-hexachlorocyclohexane, trichloroethylene, 1, 2-dichloroethane, 1, 2-dichloropropane and 1,1, 2-trichloroethane and reducing chromium (VI), has the function of reducing the chromium (VI) into low-toxicity chromium (III), can simultaneously carry out dehalogenation metabolism of polyhalogenated alkane and reduction of the chromium (VI) under the anoxic and anaerobic conditions with the oxidation-reduction potential of less than or equal to 20-50 mV, carries out mineralization degradation of the polyhalogenated hydrocarbons under the aerobic conditions with the oxidation-reduction potential of more than or equal to 150-200 mV, and has good tolerance capability on the chromium (VI). The functional strain can be applied to the treatment and restoration of respectively singly polluted soil and composite polluted soil of polyhalogenated hydrocarbon and heavy metal chromium (VI), and has good application prospect.
Example 1 isolation and characterization of the polyhalogenated hydrocarbon degrading bacterium Stenotrophomonas (Stenotrophoromonas sp.) JWDH2-3
The soil sample is collected from soil which is compositely polluted by polyhalogenated hydrocarbon pollutants and heavy metal chromium (VI) in an old site of a certain chemical plant area in Shenyang city of Liaoning province for a long time. Taking 10.0g of a contaminated soil sample, adding the contaminated soil sample into 100mL of inorganic salt liquid culture medium containing 20mg/L polyhalogenated hydrocarbon compounds and 20mg/L chromium (VI) to serve as a culture medium screening system, carrying out aerobic enrichment culture on the contaminated soil sample by using a constant-temperature shaking table at 30 ℃ and 160r/min for 10d, transferring the enrichment culture into a serum bottle containing 100mL of the culture medium screening system according to the proportion of 10 wt%, ensuring that the serum bottle is filled with liquid, and carrying out oscillatory anaerobic culture at 30 ℃ and 100r/min for 10d to serve as one-round screening and domestication culture (namely, a first round); then 5mL of enrichment liquid is taken to be added into a new 100mL of inorganic salt culture medium containing polyhalogenated hydrocarbon compounds (50mg/L) and chromium (VI) (50mg/L), shaking aerobic culture is carried out on a constant temperature table at 30 ℃ and 160r/min for 10d, then the enrichment culture is transferred into a serum bottle containing 100mL of the culture medium screening system according to the proportion of 10 wt%, and anaerobic culture is carried out at 30 ℃ and 100r/min for 10 d; and similarly, respectively carrying out a third round of screening and domestication culture according to the steps, wherein each round of screening is respectively subjected to 10d of aerobic screening and 10d of anaerobic screening, the concentrations of the polyhalogenated hydrocarbon compound and the chromium (VI) in the culture medium screening system in the third round of screening and the fourth round of screening are respectively 100mg/L and 200mg/L, the adopted polyhalogenated hydrocarbon compound is a mixture of gamma-hexachlorocyclohexane, trichloroethylene, 1, 2-dichloroethane, 1, 2-dichloropropane and 1,1, 2-trichloroethane, and the concentration of each single compound is uniformly distributed. Ladder for carrying out fourth round of enrichment culture solutionDiluting by 10 degrees1-106Taking 200 mu L of different gradient diluents to respectively carry out plate coating separation on an inorganic salt solid culture medium containing polyhalogenated hydrocarbon compounds (50mg/L) and chromium (VI) (50mg/L), carrying out static culture for 3d at the temperature of 30 ℃, and picking out a single colony grown out to carry out three-stage streak separation and purification on the solid inorganic salt culture medium. A single colony is respectively inoculated into an inorganic salt liquid culture medium containing polyhalogenated hydrocarbon compounds (50mg/L) and chromium (VI) (50mg/L) to carry out anaerobic degradation experiments and aerobic reduction experiments, and the degradation capability of each strain on the polyhalogenated hydrocarbon compounds and the reduction capability on the chromium (VI) are examined. And performing functional verification by using 1, 2-dichloroethane as a degradation experiment substrate. And (3) performing anaerobic degradation experiments in the serum bottle containing the inorganic salt culture medium system, wherein anaerobic and aerobic experimental conditions and culture periods are the same as those of the screening process, the anaerobic degradation period is 5d, and then transferring all the mixture in the serum bottle into a conical flask for aerobic shaking culture, wherein the aerobic degradation period is 5 d.
The inorganic salt liquid culture medium comprises the following components: MgSO (MgSO)4·7H2O 0.2g/L，CaCl2 0.01g/L，FeSO4·7H2O 0.001g/L，Na2HPO4·12H2O 1.51g/L，K2HPO4 0.4g/L，MnSO4·H2O 0.02g/L，NH4NO31.0g/L, solvent is water, pH 7.0. The inorganic salt solid culture medium comprises the following components: MgSO (MgSO)4·7H2O 0.2g/L，CaCl20.01g/L，FeSO4·7H2O 0.001g/L，Na2HPO4·12H2O 1.51g/L，K2HPO4 0.4g/L，MnSO4·H2O 0.02g/L，NH4NO31.0g/L, agar 20g/L, solvent water, pH 7.0.
The culture medium is sterilized at 121 ℃ for 20min for later use.
Selecting a strain JWDH2-3 with highest degradation efficiency on 1, 2-dichloroethane and highest reduction efficiency on chromium (VI) to the inorganic salt solid culture medium plate, carrying out inverted culture at 30 ℃ for 3d in a constant temperature incubator, and observing the colony morphology of the strain as shown in figure 1, wherein the colony is approximately round, smooth, glossy, neat in edge and light yellow. Collecting appropriate amount of cultured bacteria, washing with the inorganic salt liquid culture medium, fixing with 2.5% glutaraldehyde solution for 2 hr, dewatering with ethanol step by step, freeze drying, and observing strain shape with a FEINA Phenom desk type scanning electron microscope as shown in FIG. 2, wherein the thallus cells are rod-shaped and have size of 0.4-0.5 × 1.3-1.6 μm.
The total DNA of the strain JWDH2-3 was extracted and PCR-amplified using bacterial 16S rDNA amplification universal primers 8F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3'). The PCR reaction conditions are 94 ℃ for 5min, 94 ℃ for 1min, 55 ℃ for 1min, 72 ℃ for 2min, 35 cycles and 72 ℃ for 15 min. The sequencing result of the PCR product is analyzed by Blastn comparison, and the obtained strain has higher homology with Stenotrophomonas sp. The construction of a phylogenetic tree is shown in FIG. 3.
>Stenotrophomonas sp.JWDH2-3
GGCAGCACAGAGGAGCTTGCTCCTTGGGTGGCGAGTGGCGGACGGGTGAGGAATACATCGGAATCTACTCTGTCGTGGGGGATAACGTAGGGAAACTTACGCTAATACCGCATACGACCTACGGGTGAAAGCAGGGGACCTTCGGGCCTTGCGCGATTGAATGAGCCGATGTCGGATTAGCTAGTTGGCGGGGTAAAGGCCCACCAAGGCGACGATCCGTAGCTGGTCTGAGAGGATGATCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGCAAGCCTGATCCAGCCATACCGCGTGGGTGAAGAAGGCCTTCGGGTTGTAAAGCCCTTTTGTTGGGAAAGAAATCCAGCTGGCTAATACCCGGTTGGGATGACGGTACCCAAAGAATAAGCACCGGCTAACTTCGTGCCAGCAGCCGCGGTAATACGAAGGGTGCAAGCGTTACTCGGAATTACTGGGCGTAAAGCGTGCGTAGGTGGTCGTTTAAGTCCGTTGTGAAAGCCCTGGGCTCAACCTGGGAACTGCAGTGGATACTGGGCGACTAGAGTGTGGTAGAGGGTAGCGGAATTCCTGGTGTAGCAGTGAAATGCGTAGAGATCAGGAGGAACATCCATGGCGAAGGCAGCTACCTGGACCAACACTGACACTGAGGCACGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCCTAAACGATGCGAACTGGATGTTGGGTGCAATTTGGCACGCAGTATCGAAGCTAACGCGTTAAGTTCGCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGTATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTGGCCTTGACATGTCGAGAACTTTCCAGAGATGGATTGGTGCCTTCGGGAACTCGAACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCCTTAGTTGCCAGCACGTAATGGTGGGAACTCTAAGGAGACCGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGGCCAGGGCTACACACGTACTACAATGGTAGGGACAGAGGGCTGCAAGCCGGCGACGGTAAGCCAATCCCAGAAACCCTATCTCAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGCAGATCAGCATTGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTTTGTTGCACCAGAAGCAGGTAGCTTAAC
By combining the analysis and identification results, the screened facultative aerobic strain JWDH2-3 with high-efficiency polyhalogenated hydrocarbon degradation and chromium (VI) reduction functions is Stenotrophomonas (Stenotrophoromonas sp.) which is preserved in China center for type culture collection in 3, 12 and 2021, the preservation number is CCTCC M2021222, and the preservation address is No. 299 of eight paths in Wuchang region in Wuhan city, Hubei province.
Example 2 degradation experiments of polyhalogenated hydrocarbons by Stenotrophomonas (Stenotrophoromonas sp.) JWDH2-3
Single colonies of Stenotrophomonas (Stenotrophoromonas sp.) JWDH2-3 were picked up on a slant medium, inoculated into beef extract peptone medium, and subjected to shaking culture at 30 ℃ and 160r/min for 48 hours. Collecting Stenotrophomonas (Stenotrophoromonas sp.) JWDH2-3 culture solution, centrifuging at 25 deg.C for 10min at 5000r/min, discarding supernatant, collecting thallus, washing thallus twice with inorganic salt culture medium as described in example 1, and re-suspending thallus with inorganic salt liquid culture medium to obtain suspension, OD600About 1.5, and making into Stenotrophomonas (Stenotrophoromonas sp.) JWDH2-3 seed solution.
Inoculating the prepared seed liquid into an inorganic salt liquid culture medium, and respectively adding polyhalogenated hydrocarbon compounds gamma-hexachlorocyclohexane, trichloroethylene, 1, 2-dichloroethane, 1, 2-dichloropropane and 1,1, 2-trichloroethane, wherein the concentration of the five polyhalogenated hydrocarbon compounds is 50 mg/L. Controlling the inoculation amount of Stenotrophomonas (Stenotrophoromonas sp.) JWDH2-3 to be 8% (v/v), performing anaerobic degradation culture for 48h at 30 ℃ and 100rpm/min in a serum bottle, monitoring the oxidation-reduction potential in a culture system in the serum bottle to be 28 +/-5 mV by adopting a miniature oxidation-reduction potential probe, transferring all the culture into a conical flask with a plug, and performing shake culture for 48h at 30 ℃ and 160rpm/min, wherein the oxidation-reduction potential in the culture system is 195 +/-8 mV; and then extracting residual polyhalogenated aromatic hydrocarbon by using normal hexane by adopting a liquid phase extraction method, performing product analysis by adopting a gas chromatograph after the volume of an extraction product is constant, and detecting the residual quantity of the polyhalogenated hydrocarbon in the culture solution. The sample was top loaded using a top air autosampler with an HP-5MS column (30m 0.32mm 0.5 μm), programmed to an initial column temperature of 40 deg.C, held for 2min, further raised to 90 deg.C at 8 deg.C/min, held for 4min, further raised to 200 deg.C at 6 deg.C/min, held for 15min, with a sample injector temperature of 250 deg.C, a FID detector temperature of 230 deg.C, sample loading set to 1 μ L, and a carrier gas (high purity nitrogen) flow of 18 mL/min.
As shown in FIG. 4, under the above experimental conditions, after 72 hours of degradation culture, the removal rates of 1, 2-dichloroethane, 1, 2-dichloropropane and 1,1, 2-trichloroethane were all 90% or more, and the removal rates of γ -hexachlorocyclohexane and trichloroethylene were 64.28% and 35.29%. Experimental results show that the Stenotrophomonas (Stenotrophoromonas sp.) JWDH2-3 has a wide substrate metabolism spectrum of polyhalogenated hydrocarbons, and therefore the potential bioremediation application potential of the polyhalogenated hydrocarbon polluted soil is reflected.
Example 3 reduction experiment of Stenotrophomonas (Stenotrophoromonas sp.) JWDH2-3 on chromium (VI)
Slant-preserved Stenotrophomonas (Stenotrophomonas sp.) JWDH2-3 was subjected to activation culture, and the seed broth culture process was as described in example 2.
Inoculating the seed liquid into inorganic salt liquid culture medium in anaerobic serum bottle, adding potassium dichromate (K)2Cr2O7) The method is added into a culture medium until the final concentration of chromium (VI) is 250mg/L, simultaneously sodium lactate is added into an inorganic salt culture medium, the addition amount of the sodium lactate accounts for 60 percent (w/v) of the culture medium, the inoculation amount of a Stenotrophomonas sp JWDH2-3 seed solution is controlled to be 10 percent, the culture is carried out for 48 hours at the constant temperature of 30 ℃ and 100rpm/min, and the redox potential in a culture system in a serum bottle is monitored to be 43 +/-5 mV by adopting a miniature redox potential probe. Then, detecting the residual quantity by adopting a diphenylcarbazide spectrophotometry (GB7467-87) according to the content of chromium (VI) in the reaction system; under acidic condition, the solution of chromium (VI) and dibenzoyl dihydrazide is left to stand for 10min, and then the light absorption value is measured at 540nm wavelength.
As shown in fig. 5, under the above experimental conditions, the removal rate of chromium (vi) after 48 hours of treatment reached 91.63%. Therefore, the Stenotrophomonas sp JWDH2-3 has high-efficiency chromium (VI) reducing capability under anaerobic conditions and has potential application potential of reducing and repairing chromium (VI) in polluted soil.
Example 4 Stenotrophomonas sp JWDH2-3 evaluation of soil remediation Effect under Complex contamination conditions of polyhalogenated hydrocarbons and chromium (VI)
Activated culture is carried out on Stenotrophomonas (Stenotrophoromonas sp.) JWDH2-3 preserved on a slant, 200mL strain seed liquid is prepared according to the method described in example 2, the seed liquid is inoculated into 800g soil compositely polluted by polyhalogenated hydrocarbon and chromium (VI), the soil compositely polluted by the polyhalogenated hydrocarbon pollutant and heavy metal chromium (VI) in the old site of a certain chemical plant area in Shenyang city of Liaoning province, and the pollutants to be concerned in the soil are detected to be gamma-hexachlorocyclohexane (32mg/kg), trichloroethylene (12mg/kg), 1, 2-dichloroethane (46mg/kg), 1, 2-dichloropropane (31mg/kg), 1, 2-trichloroethane (24mg/kg) and chromium (VI) (42 mg/kg). Mixing the seed liquid with the contaminated soil, loading the mixture into a serum bottle with a plug, and injecting N2The method comprises the steps of evacuating air in a serum bottle, performing light-shielding standing culture for 20 days at the temperature of 28-30 ℃, and monitoring the oxidation-reduction potential in a culture system in the serum bottle to be 21 +/-8 mV by using a miniature oxidation-reduction potential probe; and then transferring the soil mixture into an open experiment box, and performing light-shielding standing culture for 20 days at the temperature of 28-30 ℃ on the premise of covering a plastic film with holes to prevent the soil system from being rapidly dehydrated, wherein the oxidation-reduction potential in the culture system is 231 +/-4 mV.
After 20 days of anaerobic treatment and 20 days of aerobic treatment, the degradation experiment is carried out to carry out solid-phase extraction and residual quantity measurement on residual components of five kinds of polyhalogenated aromatic hydrocarbon and heavy metal chromium (VI) in soil. In the same manner as in example 2, solid-phase extraction and gas chromatography headspace sample injection measurement were performed using n-hexane. The heavy metal chromium (VI) is subjected to residual detection by adopting a dibenzoyl dihydrazide spectrophotometry (GB 7467-87).
As shown in fig. 6, under the above experimental conditions of the soil remediation treatment, the removal rates of γ -hexachlorocyclohexane, trichloroethylene, 1, 2-dichloroethane, 1, 2-dichloropropane and 1,1, 2-trichloroethane in the soil were 62.97%, 36.03%, 98.68%, 96.12% and 95.89%, respectively, while the reduction rates of chromium (vi) were 90.89%, 91.43%, 91.89%, 91.52% and 91.22%, respectively. Therefore, the Stenotrophomonas sp JWDH2-3 provided by the invention can simultaneously complete the treatment process of efficient degradation of organic polyhalogenated hydrocarbon and reduction of high-toxicity chromium (VI) in the soil environment compositely polluted by the polyhalogenated hydrocarbon and the chromium (VI), thereby showing the good application effect of treating and repairing the soil compositely polluted by the polyhalogenated hydrocarbon and the chromium (VI).
Sequence listing
<110> Liaoning Zhongbo ecological Environment technology Co., Ltd
<120> a functional strain with polyhalogenated hydrocarbon degradation and chromium (VI) reduction activity and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1384
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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ggcagcacag aggagcttgc tccttgggtg gcgagtggcg gacgggtgag gaatacatcg 60
gaatctactc tgtcgtgggg gataacgtag ggaaacttac gctaataccg catacgacct 120
acgggtgaaa gcaggggacc ttcgggcctt gcgcgattga atgagccgat gtcggattag 180
ctagttggcg gggtaaaggc ccaccaaggc gacgatccgt agctggtctg agaggatgat 240
cagccacact ggaactgaga cacggtccag actcctacgg gaggcagcag tggggaatat 300
tggacaatgg gcgcaagcct gatccagcca taccgcgtgg gtgaagaagg ccttcgggtt 360
gtaaagccct tttgttggga aagaaatcca gctggctaat acccggttgg gatgacggta 420
cccaaagaat aagcaccggc taacttcgtg ccagcagccg cggtaatacg aagggtgcaa 480
gcgttactcg gaattactgg gcgtaaagcg tgcgtaggtg gtcgtttaag tccgttgtga 540
aagccctggg ctcaacctgg gaactgcagt ggatactggg cgactagagt gtggtagagg 600
gtagcggaat tcctggtgta gcagtgaaat gcgtagagat caggaggaac atccatggcg 660
aaggcagcta cctggaccaa cactgacact gaggcacgaa agcgtgggga gcaaacagga 720
ttagataccc tggtagtcca cgccctaaac gatgcgaact ggatgttggg tgcaatttgg 780
cacgcagtat cgaagctaac gcgttaagtt cgccgcctgg ggagtacggt cgcaagactg 840
aaactcaaag gaattgacgg gggcccgcac aagcggtgga gtatgtggtt taattcgatg 900
caacgcgaag aaccttacct ggccttgaca tgtcgagaac tttccagaga tggattggtg 960
ccttcgggaa ctcgaacaca ggtgctgcat ggctgtcgtc agctcgtgtc gtgagatgtt 1020
gggttaagtc ccgcaacgag cgcaaccctt gtccttagtt gccagcacgt aatggtggga 1080
actctaagga gaccgccggt gacaaaccgg aggaaggtgg ggatgacgtc aagtcatcat 1140
ggcccttacg gccagggcta cacacgtact acaatggtag ggacagaggg ctgcaagccg 1200
gcgacggtaa gccaatccca gaaaccctat ctcagtccgg attggagtct gcaactcgac 1260
tccatgaagt cggaatcgct agtaatcgca gatcagcatt gctgcggtga atacgttccc 1320
gggccttgta cacaccgccc gtcacaccat gggagtttgt tgcaccagaa gcaggtagct 1380
taac 1384
Claims (8)
1. A functional strain with polyhalogenated hydrocarbon degradation and chromium (VI) reduction activities, which is characterized in that: the strain is Stenotrophomonas (Stenotrophormonas sp.) JWDH2-3, which has been preserved in China center for type culture Collection at 12.3.2021, with the preservation number of CCTCC M2021222 and the preservation address of No. 299 in Wuhan city, Hubei province.
2. Use of a functional strain according to claim 1, characterized in that: the strain is applied to degrading polyhalogenated hydrocarbon in polluted soil and removing heavy metal chromium (VI).
3. Use of a functional strain according to claim 2, characterized in that: the strain is applied to dehalogenation and metabolism of polyhalogenated alkane under the anoxic and anaerobic conditions and mineralization and degradation of polyhalogenated hydrocarbon under the aerobic condition.
4. Use of a functional strain according to claim 2, characterized in that: the strain reduces and removes chromium (VI) in soil under the anoxic and anaerobic conditions.
5. Use of a functional strain according to claim 2, characterized in that: the polyhalogenated hydrocarbon is one or more of gamma-hexachlorocyclohexane, trichloroethylene, 1, 2-dichloroethane, 1, 2-dichloropropane and 1,1, 2-trichloroethane.
6. A microbial inoculum, which is characterized in that: JWDH2-3 comprising Stenotrophomonas sp according to claim 1.
7. The microbial inoculum according to claim 6, wherein: the strain is cultured in an inorganic salt culture medium to obtain a culture or a culture bacterial suspension containing the strain.
8. The application of the microbial inoculum is characterized in that: the microbial inoculum is applied to degrading polyhalogenated hydrocarbon in polluted soil and removing heavy metal chromium (VI).
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