CN116144552A - Microbial bacterium EDC-1 and application thereof in-situ remediation of mining area composite contaminated soil - Google Patents
Microbial bacterium EDC-1 and application thereof in-situ remediation of mining area composite contaminated soil Download PDFInfo
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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
- C12N1/205—Bacterial isolates
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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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- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
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- B09C2101/00—In situ
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- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
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- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/30—Organic compounds
- C02F2101/32—Hydrocarbons, e.g. oil
- C02F2101/327—Polyaromatic Hydrocarbons [PAH's]
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Abstract
The invention discloses a microorganism EDC-1 and application thereof in-situ restoration of mining area composite polluted soil. The EDC-1 strain is obtained by screening and separating from the soil of a coal gangue storage yard of a certain coal mine in Shandong, can use naphthalene, phenanthrene, anthracene or pyrene as a unique carbon source and has certain tolerance to heavy metals. The strain screened by the invention has the degradation capability of various PAHs under the heavy metal background, is simple to culture and low in cost, and has a good application prospect in bioremediation of sites with polycyclic aromatic hydrocarbon and heavy metal combined pollution.
Description
Technical Field
The invention belongs to the field of environmental protection, in particular to the field of organic pollutant degradation, and more particularly relates to screening culture and application of a mining area soil combined pollution repairing bacterium escherichia coli EDC-1.
Background
Aromatic compounds are one of the common and persistent pollutants in the environment, being widely-occurring hydrophobic organic pollutants. Complex Polycyclic Aromatic Hydrocarbons (PAHs) and their derivatives often accumulate and coexist in the surrounding soil of coal mines, thermal power plants, and the like. In general, polycyclic aromatic hydrocarbons are relatively stable in soil and are more difficult to degrade than many other organic compounds. The sources of the polycyclic aromatic hydrocarbon are mainly divided into two major categories, namely an artificial source and a natural source, and forest fires, animal and plant and microbial metabolism in the nature are important sources of the polycyclic aromatic hydrocarbon. Industrial production, garbage incineration, incomplete combustion of various fossil fuels such as coal, petroleum and natural gas in the human activity process can release a large amount of polycyclic aromatic hydrocarbon into the environment. Because of the characteristics of carcinogenicity, teratogenicity and mutagenicity, the compound composition constitutes a great potential risk for human health.
Mining of coal mine resources required by industrial production is a main propagation path of polycyclic aromatic hydrocarbon soil pollution. The soil surface layer is polluted in mining area exploitation, transportation, processing and stacking processes, and coal gangue is used as a byproduct of coal exploitation, is an important point of polycyclic aromatic hydrocarbon pollution control in mining area, and is discharged into the environment from a large amount of organic pollutants and heavy metal pollutants.
Microbial degradation is a well-known green repair method, and a relatively perfect research foundation exists in organic pollution repair. As a valuable tool for reducing the concentration and toxicity of contaminants, bioremediation technology is simultaneously low cost, efficient, highly targeted, environmentally friendly, and has a longer lasting effect than other methods currently used to remove contaminants. However, since the mining area soil belongs to a composite pollution system, the conventional bioremediation method has larger limitation in field application, and heavy metals also have toxicity to influence the activity of degrading bacteria. Thus, in addition to the tolerance and degradation capabilities of the microorganism itself, polycyclic aromatic hydrocarbons, heavy metal tolerance is also an important consideration in performing bioremediation.
Disclosure of Invention
In view of the needs of the prior art, the present invention seeks degrading bacteria for effectively degrading organic pollutants such as aromatic compounds.
Therefore, a first object of the present invention is to provide a microorganism strain EDC-1, which is Enterobacter choleraeEnterobacter hormaecheiThe preservation number is CGMCC No.25958, and the China general microbiological culture Collection center (CGMCC) is preserved for 24 days in 10 months of 2022.
The strain is separated from the soil of a coal mine gangue storage yard in Xintai city of Shandong province. Strain EDC-1 is a gram-negative bacterium, and the colony on the LB solid plate is round, milky, raised, smooth, neat and opaque. The EDC-1 of the enterobacter cholerae has an optimal growth temperature of 35-40 ℃ and an optimal pH value of 7-7.5.
The enterobacter cholerae strain (Enterobacter hormaechei) EDC-1 is first enriched and screened out in a coal gangue storage yard in a coal mine area.
The invention provides an application of the microorganism EDC-1 in the remediation of water or soil polluted by polycyclic aromatic hydrocarbon and/or heavy metal.
Specifically, the polycyclic aromatic hydrocarbon is selected from naphthalene, phenanthrene, anthracene and/or pyrene; the polycyclic aromatic hydrocarbon is selected from one or more of NAP, PHE, ANT, PYR, baP, bghiP; the heavy metals are cadmium, chromium, antimony and other heavy metals. More preferably, the bacterial suspension of the combined pollution repair bacteria is applied to the soil.
Preferably, the degradation efficiency is highest when the concentration of the pollutant PHE is 50 mg/L; in a liquid culture medium system, heavy metal Cd 2+ The degradation rate is maintained above 30% when the concentration is less than or equal to 10 mg/L; in a soil system, heavy metal Cd 2+ The metabolic capacity is not obviously inhibited when the concentration is less than or equal to 20mg/L, and the degradation rate is more than 40%.
Further, degradation is detected using High Performance Liquid Chromatography (HPLC); the chromatographic condition of the detection is that a C18 analytical column is used, the mobile phase is ultrapure water, the ratio of the ultrapure water to the chromatographic grade methanol is 20:80; the flow rate is 1mL/min, and the column temperature is 30 ℃; the detection wavelength was 254nm. Further, high Performance Liquid Chromatography (HPLC) was used to detect degradation of contaminants.
The invention also provides a degrading bacterial agent containing the microbial strain EDC-1 for degrading polycyclic aromatic hydrocarbon. Preferably, the microbial strain EDC-1 is present in the form of a bacterial suspension.
The preparation method of the bacterial suspension comprises the following steps:
s1, after a solid inorganic salt culture medium containing polycyclic aromatic hydrocarbon mother liquor is coated, culturing at 35-40 ℃ for about 48-72 hours, and observing complete single colonies;
s2, picking single colony, inoculating into TSB liquid culture medium, shake culturing at 150-180rpm at 35-40deg.C, and making OD 600 Culture medium=0.5-1.5;
s3, inoculating the strain into a new fermentation culture medium, and carrying out shaking fermentation at 150-180rpm for 48-96 hours at the temperature of 35-40 ℃ to obtain a culture solution, namely the degradation microbial inoculum for degrading the polycyclic aromatic hydrocarbon.
In a specific embodiment, the fermentation medium comprises 10g/L glucose, 15g/L yeast powder, 10g/L peptone and K 2 HPO 4 15g/L, distilled water 1L.
Preferably, the number of cells in the bacterial suspension is not less than 1.0X10 10 CFU/ml, preferably 1.0X10 10 CFU/ml to 1.0X10 12 CFU/ml。
The invention also provides a preparation method of the polycyclic aromatic hydrocarbon degrading bacterial agent of the strain EDC-1, which comprises the following steps:
(1) After the solid inorganic salt culture medium containing the polycyclic aromatic hydrocarbon mother liquor is coated, the culture is carried out at 35-40 ℃, and after 48-72 and h are cultivated, complete single colonies (for example, with the diameter of 1-3 mm) are seen;
(2) Inoculating single colony into TSB liquid culture medium, shake culturing at 35-40deg.C at 150-180rpm, loading into sterile centrifuge tube, centrifuging at 4000-5000rpm for 10-15min, removing supernatant, and pouring sterilized normal saline to obtain OD 600 Bacterial suspension=0.5-1.5 (preferably 1);
(3) According to the inoculation amount of 10 percent, inoculating the strain into a new fermentation culture medium, and then carrying out shaking fermentation for 48-96 hours (for example, 72 hours) at the temperature of 35-40 ℃ at the speed of 150-180rpm to obtain a culture solution, namely the degradation microbial inoculum for degrading the polycyclic aromatic hydrocarbon.
Specifically, the inorganic salt culture medium is Na 2 HPO 4 2800mg，(NH 4 ) 2 SO 4 500mg, CuCl 2 ·2H 2 O 0.001mg, H 3 BO 3 0.03mg, FeSO 4 ·7H 2 O 0.2mg, MnCl 2 ·4H 2 O 0.003mg, NiCl 2 ·6H 2 O 0.002mg, KH 2 PO 4 1000mg, Na 2 EDTA 0.5mg, CoCL 2 ·6H 2 O 0.02mg, ZnSO 4 ·7H 2 O 0.01mg, Na 2 MoO 4 ·2H 2 O0.003 mg and distilled water 1L.
The TSB culture medium is tryptone 15.0g, soytone 5.0g, sodium chloride 5.0g, K 2 HPO 4 2.5g, distilled water 1.0L, pH7.1-7.5.
The physiological saline is 9.0g of NaCl, 1L of distilled water and pH 7.0-7.5.
The fermentation medium is 10.0g of glucose, 15.0g of yeast powder, 10g.0 g of peptone and K 2 HPO 4 15.0g of distilled water 1.0L and pH 7.2.+ -. 0.2.
The invention enriches and screens the enterobacter cholerae strain from the coal gangue storage yard soil sample of the coal mine factory for the first time, the strain has better metabolism capability in neutral and weak alkaline environments, has good degradation capability to typical polycyclic aromatic hydrocarbon NAP, PHE, ANT, PYR, baP, bghiP and the like, and simultaneously has heavy metal Cd 2+ Has better tolerance capability in Cd 2+ In the soil with the concentration of 20mg/kg, 45% degradation rate is realized, which shows that the strain EDC-1 not only can repair the polycyclic aromatic hydrocarbon polluted water body, but also can be used in the soil with heavy metal and polycyclic aromatic hydrocarbon combined pollution, and the degradation of the polycyclic aromatic hydrocarbon under different environments is realized.
Drawings
FIG. 1 shows the morphology of EDC-1 plate colonies of the invention;
FIG. 2 is an EDC-1 phylogenetic tree based on 16S rRNA gene sequence homology;
FIG. 3 is a graph showing the degradation kinetics of EDC-1 versus phenanthrene according to the present invention;
FIG. 4 shows the degradation ability of EDC-1 of the present invention to phenanthrene of different concentrations;
FIG. 5 shows the degradation ability of EDC-1 of the present invention to phenanthrene at different temperatures;
FIG. 6 shows the degradation ability of EDC-1 of the present invention to phenanthrene at different pH values;
FIG. 7 shows the degradation capacity of EDC-1 of the present invention for 6 different exemplary polycyclic aromatic hydrocarbons;
FIG. 8 shows the degradation ability of EDC-1 of the present invention to phenanthrene at different Cd2+ concentrations.
FIG. 9 shows the degradation ability of EDC-1 of the present invention to phenanthrene in soil with different Cd2+ concentrations.
Biological material preservation information
The microorganism strain EDC-1 has a preservation number of CGMCC NO.25958 and is classified and named as enterobacter choleraeEnterobacter hormaecheiThe microbial strain is preserved in China general microbiological culture Collection center (CGMCC) for 10 months and 24 days in 2022, and the preservation unit address is: no. 1 and No. 3 of the north cinquefoil of the morning sun area of beijing city.
Description of the embodiments
The invention is further described below with reference to the drawings and specific examples.
Example 1: isolation and characterization of EDC-1
1. Sample source
Soil is collected from a mountain eastern coal mine gangue storage yard polluted site, high-concentration phenanthrene is used as a carbon source for long-term domestication, and high-efficiency phenanthrene degrading bacteria are obtained through multiple screening, separation and purification.
2. Culture medium
2.1 inorganic salt Medium
The inorganic salt culture medium is used for sample microorganism culture and polycyclic aromatic hydrocarbon degradation experiment under the condition of pure bacteria and microbial inoculum. The formulation of the medium is shown in Table 1. The preparation method comprises adding the above materials into distilled water, stirring, adjusting pH to 7.2-7.5, and sterilizing.
TABLE 1 inorganic salt Medium formulation
2.2 nutrient Medium
The liquid nutrient medium types and compositions used in this experiment are shown in tables 2 and 3. The corresponding solid culture medium is prepared by adding 12-15g/L agar powder into the original culture medium. If the culture conditions of the strain are not particularly specified, the pH of the medium is adjusted to 7.1 to 7.5. The preparation method comprises adding the above components into water, stirring, mixing, sterilizing at 121deg.C for 15 min.
TABLE 2 TSB Medium composition
TABLE 3 fermentation Medium composition
3. Domestication, screening and isolation of strains
Adding the collected polluted soil into the sterilized inorganic salt culture medium, wherein the mass ratio of the two is 1:10, and culturing for 5d at the temperature of between 35 and 40 ℃ at the speed of between 150 and 180 rpm. Then sequentially inoculating the strain into a strain with the concentration of 10 mg.L -1 、50mg·L -1 、100mg·L -1 In the phenanthrene inorganic salt culture medium, the inoculation proportion is 10%, and the culture medium is placed in a 35 ℃ incubator for light-shielding shake culture after each inoculation.
The strain domestication is to use phenanthrene as the only carbon source in an inorganic salt culture medium, and 5d is one domestication period. 10% of the inoculum size was transferred to fresh medium of a relatively higher concentration system after the end of the cycle and the procedure was repeated three times.
The culture samples obtained above were diluted and subjected to a coating separation, and the samples were separated with an inorganic salt medium containing 100mg/L of phenanthrene. Culturing the coated sample at 35deg.C in dark for 2-4d, picking different single colonies according to the shape, size, color, transparency, etc., and streaking and purifying on nutrient medium plate. And picking and storing the purified single colony.
4. Identification of strains
And (5) carrying out preliminary identification on the strain according to the gram staining reaction result and morphological characteristics of the strain. The morphology of the plate colonies of strain EDC-1 is shown in FIG. 1. The bacterial colony on the LB solid plate is round, milky, raised, smooth, tidy and opaque, and has the main biological characteristics of gram negative, positive catalase reaction, negative oxidase reaction and negative amylase hydrolysis.
Identification of Strain 16S rDNA, in which single colony is taken and cleaved as a template for PCR, and a primer 27F primer is selected from AGAGTTTGATCMTGGCTCAG and 1492R is TACGGYTACCTTGTTACGACTT. The PCR reaction conditions were pre-changed to 5min at 94 ℃, denatured for 30s at 94 ℃, annealed for 30s at 54 ℃, extended for 30s at 72 ℃,24 cycles, and finally extended for 10min at 72 ℃. The 16S rRNA gene sequence is shown as SEQ ID NO. 1. The sequence was aligned with NCBI library, showing that the homology of strain EDC-1 with Enterobacter cholerae reached 99.15%, and the strain was determined to be Enterobacter. The phylogenetic tree is constructed as shown in fig. 2.
Example 2: EDC-1 polycyclic aromatic hydrocarbon degradation experiment
1. Preparation of microbial inoculum
(1) Picking single colony purified in plate, inoculating into TSB liquid culture medium, shake culturing at 35deg.C and 180rpm to obtain OD 600 Bacterial suspension=1;
(2) The bacterial suspension is inoculated into a new fermentation culture medium according to the inoculation amount of 10 percent, and is subjected to shaking fermentation for 72 hours at 180rpm at 35 ℃, and the concentration of the fermentation liquid is detected to be 10 9-11 CFU/ml, the obtained fermentation broth is EDC-1 liquid microbial inoculum.
2. PHE degradation by microbial inoculum
Inoculating 2mL of the prepared EDC-1 liquid microbial inoculum into 50mL of inorganic salt culture medium containing 50mg/L of phenanthrene, placing the culture medium into a constant temperature shaking table at 35 ℃ for culture, setting 3 groups of repeated experiments without inoculating microbial inoculum in a control group, and measuring PHE concentration and OD in the culture medium every day 600 。
20mL of chromatographically pure hexane was added to the conical flask of the medium, and after sealing, shaking at 200rpm for 30min, then liquid-liquid extraction was performed through a separating funnel, and the n-hexane was separated and collected, and repeated twice. All the collected n-hexane extract is processed by a nitrogen blowing instrumentConcentrating by nitrogen blowing, taking 1mL of nitrogen blowing to near dryness after the volume is fixed to 10mL, passing through a 0.22 mu m filter membrane by using chromatographic pure methanol after the volume is fixed to 1mL, filling into a brown liquid phase vial, and measuring the concentration of the polycyclic aromatic hydrocarbon by adopting a method of a high performance liquid chromatograph (HJ 478-2009). OD (optical density) 600 In the measurement of (2), 1mL of the solution is taken and zeroed by an initial phenanthrene-containing inorganic salt culture medium when the ultraviolet spectrophotometer is adjusted to 600nm, and then a sample is measured. The degradation kinetic curve and the growth curve of phenanthrene after the microbial inoculum is added are shown in figure 3, the microbial density of the culture medium reaches the highest at 3d, and the microbial density is degraded to below 25mg/L after 7 d.
3. PHE degradation under different conditions in liquid medium
2mL of the prepared EDC-1 liquid microbial inoculum was inoculated into 50mL of an inorganic salt medium containing 25mg/L, 50mg/L, 100mg/L and 150mg/L of phenanthrene, and the medium was placed in a constant temperature shaker at 35℃for 180rpm for cultivation, after 7 days, PHE concentration in the medium was measured, and the measurement was repeated 3 times. The degradation rate of the EDC-1 microbial inoculum at different PHE concentrations is shown as figure 4, and the degradation rate is highest and reaches 51% when the PHE concentration is 50 mg/L.
2mL of the prepared EDC-1 liquid microbial inoculum was inoculated into 50mL of an inorganic salt culture medium containing 50mg/L of phenanthrene, the culture medium was placed in a constant temperature shaker at 20 ℃, 25 ℃, 30 ℃,35 ℃ and 40 ℃ for culture at 180rpm, PHE concentration in the culture medium was measured after 7 days, and the measurement was repeated 3 times. The degradation rate of PHE at different temperatures is shown in figure 5, the degradation effect of the microbial inoculum is good at 35-40 ℃, and the degradation rate is maintained at 43% -51%. Then further carrying out shaking culture on the microbial inoculum at 36 ℃, 37 ℃, 38 ℃ and 39 ℃ and the degradation effect is best at 37 ℃ after 7d, reaching 54%.
50mL of inorganic salt culture medium containing 50mg/L of phenanthrene is respectively adjusted to pH values of 5, 6, 7, 8 and 9, 2mL of prepared EDC-1 liquid microbial inoculum is inoculated into the culture medium, the culture medium is placed in a constant temperature shaking table at 35 ℃ for 180rpm for culture, PHE concentration in the culture medium is measured after 7 days, and the measurement is repeated for 3 times. The degradation rate of the phenanthrene under different pH values is shown in figure 6, and when the pH value of the culture medium is between 7 and 8, the degradation rate of the microbial inoculum is the highest, and the degradation rate is kept between 33 and 51 percent.
50mL of inorganic salt culture medium containing NAP 50mg/L, PHE mg/L, ANT 50mg/L, PYR mg/L, baP 20mg/L, bghiP mg/L is prepared, 2mLEDC-1 microbial inoculum is inoculated to the culture medium and placed in a constant temperature shaking table at 35 ℃ for 180rpm for culture, the concentration of polycyclic aromatic hydrocarbon in the culture medium is measured after 7 days, and the measurement is repeated for 3 times. The degradation rate of EDC-1 to six polycyclic aromatic hydrocarbons is shown in figure 7, and the degradation rates of six polycyclic aromatic hydrocarbons are NAP 86.7%, PHE 50.6%, ANT 35.7%, PYR 37.1%, baP45.9% and BghiP53.6% respectively.
50mL of inorganic salt culture medium containing 50mg/L of phenanthrene is prepared, and 0.8mL, 1.6mL, 3.2mL and 4.8mL of CdCl with the concentration of 10g/L are respectively added into each culture medium 2 Solution, which is prepared into Cd 2+ The concentration is 5mg/L, 10mg/L, 20mg/L and 40mg/L, then 2mLEDC-1 microbial inoculum is added into each culture medium, and the culture is carried out by placing the culture medium in a constant temperature shaking table at 35 ℃ at 180rpm, after 7 days, PHE concentration in the culture medium is measured, and the measurement is repeated for 3 times. EDC-1 at different Cd 2+ The degradation rate at concentration is shown in FIG. 8, at Cd 2+ When the concentration is less than or equal to 5mg/L, the degradation efficiency of polycyclic aromatic hydrocarbon of the microbial inoculum is slightly improved, the degradation rate is up to 56.7%, and when Cd is generated 2+ When the concentration reaches 40mg/L, the PHE degradation rate can still be maintained above 20 percent.
4. Degradation of PHE in contaminated soil under different conditions
Experiment setting: the soil is screened by a 10-mesh sieve and then is uniformly mixed. 20mg PHE is dissolved in 10mL of acetone and added into 100g of soil to prepare 200mg/kg of PHE contaminated soil subspecies, 1.6mL of CdCl with the concentration of 10g/L is added 2 The solution was added to 10g of soil to prepare 1g/kg of Cd 2+ Contaminated soil subspecies. Adding 86-90g of soil, 10g of PHE contaminated soil subspecies and 1-4gCd into a beaker 2+ The contaminated soil subspecies are evenly mixed to prepare PHE with concentration of 20mg/kg and Cd 2+ The concentration of the contaminated soil was 10mg/kg, 20mg/kg, 40 mg/kg. Adding 5mLEDC-1 microbial inoculum into 100g of polluted soil, uniformly mixing to ensure that the water content is kept at 20%, placing the mixture in a constant temperature incubator at 35 ℃ for light-shielding cultivation for 20 days, and taking a soil sample at intervals of 10 days to determine the PHE concentration.
Determination of PHE concentration in soil: and extracting PAHs in the soil by referring to the high performance liquid chromatography for determining polycyclic aromatic hydrocarbon in soil and sediment of national environmental protection standard of the people's republic of China. Weighing 5g of soil sample, freeze-drying, grinding, sieving with a 60-mesh sieve, adding 15mL of acetone-n-hexane (1:1) mixed solution, sealing, soaking for 8 hours in dark place, performing ultrasonic treatment for 30min, centrifuging, pouring out the solution, and repeating extraction for 3 times. The extract was mixed and concentrated to about 1mL by nitrogen blowing and purified using a magnesium silicate solid phase extraction column. The purified eluent is concentrated to near dryness again by nitrogen blowing, and about 3mL of methanol is added, repeated three times, and finally the volume is fixed to 1mL. The sample is measured by a high performance liquid chromatograph, the sample injection amount is 10 mu L, the column temperature is 35 ℃, the mobile phase is selected from methanol and water B (80:20), and the detection wavelength is selected from 252nm.
Different Cd 2+ The degradation rate of phenanthrene in the concentration soil is shown in FIG. 9, and the concentration soil Cd is shown in the soil 2+ When the concentration is less than or equal to 20mg/kg, the activity of the microbial inoculum is not obviously inhibited, and the degradation rate of PHE in soil is maintained at 45-63%; when the soil Cd 2+ When the concentration reaches 40mg/kg, the metabolic activity is inhibited, and the degradation rate is about 29%.
The foregoing is merely a preferred embodiment of the present invention, and it should be noted that the above-mentioned preferred embodiment should not be construed as limiting the invention, and the scope of the invention should be defined by the appended claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention and these modifications and adaptations are intended to be comprehended within the scope of the invention.
Claims (10)
1. A microbial strain EDC-1 belongs to Enterobacter hormaechei, and has a preservation number of CGMCC No.25958, and is preserved in China general microbiological culture Collection center.
2. Use of the microbial bacteria EDC-1 according to claim 1 in the remediation of polycyclic aromatic hydrocarbon contaminated and/or heavy metal contaminated water or soil; the polycyclic aromatic hydrocarbon is selected from naphthalene, phenanthrene, anthracene and/or pyrene.
3. The use according to claim 2, wherein the polycyclic aromatic hydrocarbon is selected from one or more of NAP, PHE, ANT, PYR, baP, bghiP; the heavy metals are cadmium, chromium and antimony heavy metals.
4. The method according to claim 3, wherein the bacterial suspension of the microbial strain EDC-1 is applied to a water or soil contaminated by polycyclic aromatic hydrocarbons and/or heavy metals.
5. A polycyclic aromatic hydrocarbon degrading bacterial agent containing the microbial strain EDC-1 as described in claim 1.
6. The degrading bacterial preparation for degrading polycyclic aromatic hydrocarbon according to claim 5, wherein the microbial strain EDC-1 exists in the form of bacterial suspension.
7. The degrading bacterial agent for degrading polycyclic aromatic hydrocarbon according to claim 6, wherein the bacterial suspension preparation method is as follows:
s1, coating a solid inorganic salt culture medium containing polycyclic aromatic hydrocarbon mother liquor with the microbial strain EDC-1, and culturing at 35-40 ℃ for 48-72 hours until complete single colony is seen;
s2, picking single colony, inoculating into tryptone soybean broth TSB liquid culture medium, shake culturing at 35-40deg.C at 150-180rpm to obtain OD 600 Bacterial suspension=0.5-1.5;
s3, inoculating the strain into a new fermentation culture medium, and carrying out shaking fermentation at 150-180rpm for 48-96 hours at the temperature of 35-40 ℃ to obtain a culture solution, namely the degradation microbial inoculum for degrading the polycyclic aromatic hydrocarbon.
8. The degrading bacterial preparation for degrading polycyclic aromatic hydrocarbon according to claim 7, wherein the tryptone soybean broth TSB liquid culture medium formula is tryptone 15.0g/L, soytone 5.0g/L, sodium chloride 5.0g/L, K 2 HPO 4 2.5g/L, distilled water 1.0L, pH 7.3+ -0.2; the formula of the fermentation medium comprises 10g/L glucose, 15g/L yeast powder, 10g/L peptone and K 2 HPO 4 15g/L, distilled water 1L, pH 7.2.+ -. 0.2.
9. The degrading bacterial agent for degrading polycyclic aromatic hydrocarbon according to claim 8, wherein the number of bacterial cells in the degrading bacterial agent is not less than 1.0x10 10 CFU/ml。
10. The degrading bacterial agent for degrading polycyclic aromatic hydrocarbon according to claim 9, wherein the number of bacterial cells in the degrading bacterial agent is 1.0 x 10 10 CFU/ml to 1.0X10 12 CFU/ml。
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