1. A method for screening phenanthrene polycyclic aromatic hydrocarbon degrading bacteria is characterized by comprising the following steps: the method for screening the phenanthrene polycyclic aromatic hydrocarbon degrading bacteria comprises the following steps:

(1) taking petroleum-contaminated soil, placing the soil in LB liquid culture medium containing 300mg/L crystal violet, and carrying out shaking culture at 30 ℃ and 170r/min for 24 hours;

(2) diluting the liquid obtained in the step (1) according to concentration gradient, coating the diluted liquid on an LB flat plate containing 300mg/L crystal violet, and carrying out inverted culture at 30 ℃ for 24 hours;

(3) observing the LB flat plate, selecting a single colony with a colorless circle, placing the single colony in an LB liquid culture medium, and carrying out shake culture at 30 ℃ and 170r/min for 16 hours;

(4) detecting the decolorization rate, comprising the following steps:

A1. preparing a blank control sample, wherein the component is that the volume fraction of the PBS concentration is 0.01M, LB liquid culture medium is 10%;

B1. preparing a screening strain sample before treatment, wherein the components of the screening strain sample before treatment are PBS (phosphate buffer solution) concentration of 0.01M and crystal violet concentration of 300mg/L, detecting the absorbance of the screening strain sample before treatment at the wavelength of 578nm by taking the sample in the step A1 as a control, and obtaining the absorbance as an OD (optical density) value before treatment;

C1. preparing a treated screening strain sample, wherein the components are that the concentration of PBS is 0.01M, the concentration of crystal violet is 300mg/L, the volume fraction of the single colony bacterial liquid obtained in the step (3) is 10%, carrying out shake culture at 30 ℃ and 170r/min for 6 days, detecting the absorbance of the treated screening strain sample at the wavelength of 578nm by taking the sample in the step A1 as a contrast every 12 hours, and obtaining the absorbance as the OD value after treatment;

D1. calculating the decolorization rate: percent decolorization (% OD value before untreated-OD value after treatment)/OD value before untreated;

(5) inoculating the single colony bacterial liquid in the step (3) corresponding to the decolorization rate of more than 80% into an LB liquid culture medium, and carrying out shaking culture at 30 ℃ and 170r/min for 16 hours;

(6) centrifugally collecting thalli, repeatedly cleaning cells for at least 3 times by using sterile water, and finally suspending by using 0.01M PBS solution to prepare an bacterial suspension with an OD600 value of 0.5;

(7) detecting the degradation rate, comprising the following steps:

A2. the method for detecting the initial value comprises the following steps: taking an inorganic salt culture medium with the volume of alpha mL and the concentration of phenanthrene being 50mg/L, carrying out ultrasonic extraction for 2 times by using n-hexane, carrying out ultrasonic extraction for 10 minutes, combining 2 times of extracted organic phases, carrying out rotary evaporation to concentrate the organic phases until the volume is 10mL, measuring the light absorption value of phenanthrene at 250nm by using an ultraviolet spectrophotometer, and obtaining the concentration of phenanthrene as the initial value of phenanthrene by referring to a phenanthrene standard curve;

B2. and (3) detecting a residual value by the method: adding the bacterial suspension obtained in the step (6) with the volume fraction of 5% into an inorganic salt culture medium containing 50mg/L phenanthrene, carrying out shaking culture for 6 days, taking a liquid with the volume of a mL every 12 hours, carrying out ultrasonic extraction on the liquid sampled every time for 2 times by using n-hexane, wherein the volume of the n-hexane used every time is 0.5a mL, carrying out ultrasonic extraction for 10 minutes, combining 2 times of extracted organic phases, carrying out rotary evaporation, concentrating the organic phase to a constant volume of 10mL, measuring the light absorption value of the phenanthrene at 250nm by using an ultraviolet spectrophotometer, and obtaining the residual value of the phenanthrene by referring to a standard curve of the phenanthrene;

C2. calculating the degradation rate of phenanthrene: the degradation rate = (initial value of phenanthrene-residual value of phenanthrene)/initial value of phenanthrene;

(8) and determining the strain with the degradation rate of more than 45 percent as the phenanthrene polycyclic aromatic hydrocarbon degrading bacteria.

2. The method for screening phenanthrene polycyclic aromatic hydrocarbon degrading bacteria according to claim 1, wherein the number of the single colonies picked in the liquid LB test tube without the color circle in the step (3) is at least 2, and the decoloring rate is calculated through the step (4).

3. The method for screening phenanthrene polycyclic aromatic hydrocarbon-degrading bacteria according to claim 1, wherein a in step (7) has a value of 200.

Background

Polycyclic aromatic hydrocarbons refer to aromatic hydrocarbons containing two or more benzene rings, also called PAHs, which have toxicity, genetic toxicity, mutagenicity and carcinogenicity, and can cause various damages to human body, such as damages to respiratory system, circulatory system and nervous system, and damages to liver and kidney. Are identified as major organic pollutants affecting human health. Because PAHs have good chemical stability and are difficult to decompose through chemical actions such as hydrolysis and the like under natural conditions, except that a small part of PAHs are decomposed by photochemical chemistry, most of the PAHs are mainly slowly disappeared from the environment through a biodegradation way. Therefore, conversion of PAHs into harmless substances by the degradation action of microorganisms is considered to be the best means for removing PAHs in the environment.

At present, people already separate a plurality of bacteria, fungi and actinomycetes with PAHs degrading capability through technologies such as artificial enrichment culture, and the bacteria dominate due to various biochemical adaptability and easy-induced mutant strains; the dye wastewater is treated by a microbial degradation method, so that the method is economical, effective and environment-friendly, and the degradation of the polycyclic aromatic hydrocarbon by using the degradation characteristic of microorganisms can provide bioremediation for wastewater treatment and environmental pollution; however, the separated PAHs degrading bacteria have few high-efficiency strains, and in the experimental process of screening the polycyclic aromatic hydrocarbon degrading bacteria, naphthalene, anthracene, phenanthrene and other substances are conventionally utilized, because the substances have extremely poor solubility in water and are volatile, other organic cosolvents cannot be uniformly added into a culture medium, and some methods adopt a spraying method and a sublimation method, so that the organic solvents pollute the surrounding environment, have toxic action on bacteria, and are complex in operation process and difficult to control.

Disclosure of Invention

In order to solve the technical problems, the invention provides a method for screening phenanthrene polycyclic aromatic hydrocarbon degrading bacteria, which aims to solve the problems that naphthalene, anthracene, phenanthrene and other substances are conventionally utilized in the current experimental process for screening polycyclic aromatic hydrocarbon degrading bacteria, and because the substances have extremely poor solubility in water and are volatile, other organic cosolvents cannot be uniformly added into a culture medium, and some methods adopt a spraying method and a sublimation method, the substances not only pollute the surrounding environment organic solvent, but also have toxic action on bacteria, and the operation process is complex and is not easy to control.

The invention relates to a method for screening phenanthrene polycyclic aromatic hydrocarbon degrading bacteria, which aims at achieving the following specific technical means:

a method for screening phenanthrene polycyclic aromatic hydrocarbon degrading bacteria comprises the following steps:

(1) taking petroleum-contaminated soil, placing the soil in LB liquid culture medium containing 300mg/L crystal violet, and carrying out shaking culture at 30 ℃ and 170r/min for 24 hours;

(2) diluting the liquid obtained in the step (1) according to concentration gradient, coating the diluted liquid on an LB flat plate containing 300mg/L crystal violet, and carrying out inverted culture at 30 ℃ for 24 hours;

(3) observing the LB flat plate in the step (2), picking out a single colony with a colorless circle in a liquid LB test tube, and carrying out shaking culture at 30 ℃ and 170r/min for 16 hours; if no single colony with an achromatic circle appears, re-screening can be performed, and the steps (1) to (2) are repeated, wherein the gradient dilution of the step (2) can dilute different times to be respectively coated on an LB flat plate, so that the single colony with the achromatic circle can be obtained more quickly, and preferably, a plurality of single colonies with the achromatic circle appearing are picked at one time and respectively put into an LB liquid culture medium for subsequent steps, namely, the decolorization rate of each single colony strain picked in the subsequent detection is determined, so that strains with the decolorization rate larger than 80% can be screened quickly.

(4) Detecting the decolorization rate, comprising the following steps:

A1. preparing a blank control sample, wherein the component of the sample is PBS with the concentration of 0.01M, LB and the volume fraction of liquid culture medium is 10 percent; PBS was phosphate buffered saline.

B1. Preparing a screening strain sample before treatment, wherein the components of the sample are PBS (phosphate buffer solution) concentration of 0.01M and crystal violet concentration of 300mg/L, detecting the absorbance of the crystal violet of the screening strain sample before treatment at a wavelength of 578nm by taking the sample in the step A1 as a control, and obtaining the absorbance as an OD (optical density) value before treatment;

C1. preparing a treated screening strain sample, wherein the components of the sample are PBS (phosphate buffer solution) concentration of 0.01M, crystal violet concentration of 300mg/L and single colony bacterial liquid volume fraction of 10% in the step (3), performing shake culture at 30 ℃ and 170r/min for 6 days, detecting the absorbance of the treated screening strain sample at the wavelength of 578nm by taking the sample in the step A1 as a control every 12 hours, and obtaining the absorbance as a treated OD value;

D1. calculating the decolorization rate: percent decolorization (% OD value before untreated-OD value after treatment)/OD value before untreated;

(5) inoculating the single colony bacterial liquid in the step (3) corresponding to the decolorization rate of more than 80% into an LB liquid culture medium, and carrying out shaking culture at 30 ℃ and 170r/min for 16 hours; a plurality of strains with the decolorization rate of more than 80 percent can be selected for subsequent experiments, so that strains with the phenanthrene degradation rate of more than 45 percent can be screened more quickly, and strains with higher phenanthrene degradation rate can be screened.

(6) The cells were collected by centrifugation, washed repeatedly with sterile water at least 3 times, and finally suspended with 0.01M PBS solution to prepare a suspension with an OD600 of 0.5.

(7) Detecting the degradation rate, comprising the following steps:

A2. the method for detecting the initial value comprises the following steps: taking an inorganic salt culture medium with the volume of alpha mL and the phenanthrene of 50mg/L, carrying out ultrasonic extraction for 2 times by using n-hexane, carrying out ultrasonic extraction for 10 minutes, combining 2 times of extracted organic phases, carrying out rotary evaporation to concentrate the organic phases until the volume is 10mL, measuring the absorbance of the phenanthrene at 250nm by using an ultraviolet spectrophotometer, and obtaining the concentration of the phenanthrene as an initial value of the phenanthrene by referring to a standard curve of the phenanthrene;

B2. and (3) detecting a residual value by the method: adding the bacterial suspension obtained in the step (6) with the volume fraction of 5% into an inorganic salt culture medium containing 50mg/L of phenanthrene, carrying out shaking culture for 6 days, taking a mL liquid every 12 hours, carrying out ultrasonic extraction on the liquid sampled every time for 2 times by using n-hexane, carrying out ultrasonic extraction for 10 minutes, combining 2 times of extracted organic phases, carrying out rotary evaporation, concentrating the organic phase to a constant volume of 10mL, measuring the light absorption value of the phenanthrene at 250nm by using an ultraviolet spectrophotometer, and obtaining the residual value of the phenanthrene by referring to a standard curve of the phenanthrene;

C2. calculating the degradation rate of phenanthrene: percent degradation (% phenanthrene initial value-residual value of phenanthrene)/phenanthrene initial value.

The inorganic salt medium can be configured in the following composition to the required volume of the amount: (NH)₄)₂SO₄ 1.5g、NaC1 0.5g、KH₂PO₄ 1.5g、K₂HPO₄ 0.5g、MgSO₄·7H₂O 0.2g、FeSO₄·7H₂O 0.05g、CaCl₂0.05g, distilled water 1000mL, pH adjusted to 7.0, and moist heat sterilized at 121 ℃ for 30 minutes.

The a mL represents a certain volume, and a is used for representing that the volumes taken in the A2 step and the B2 step are the same and are a mL, and preferably, the value of a is 200.

The method for obtaining the standard curve of the phenanthrene comprises the following steps: cyclohexane is used as a solvent, phenanthrene standard solutions with different concentrations are accurately prepared, an ultraviolet spectrophotometer is used for measuring the light absorption value of phenanthrene at the wavelength of 250nm, and a phenanthrene standard curve is drawn according to the result.

(8) And determining the strain with the degradation rate of more than 45 percent as the phenanthrene polycyclic aromatic hydrocarbon degrading bacteria.

Compared with the prior art, the invention has the following beneficial effects:

the method utilizes crystal violet, which is a common triphenylmethane dye, and because of the chemical functional group of triphenylmethane, the triphenylmethane has high chemical stability and low biodegradability, although the triphenylmethane belongs to one of polycyclic aromatic hydrocarbons, the toxicity of the triphenylmethane is much lower than that of fused ring aromatic hydrocarbons, in the screening experiment process, the crystal violet is used for screening, so that the toxicity to the environment can be reduced, and the crystal violet is easily soluble in water, has color, can be gradually decolored when being degraded, and can be directly observed by naked eyes for degradation efficiency; when an observed sample can be converted from purple to light purple and is finally colorless, the bacterium can degrade crystal violet and has the effect of degrading polycyclic aromatic hydrocarbons.

Drawings

FIG. 1 is a photograph showing the decolorization of crystal violet by a single colony strain selected according to the present invention;

FIG. 2 is a graph showing the decolorization rate of crystal violet by one of the single colony strains selected in the present invention;

FIG. 3 is a graph showing the degradation curve of phenanthrene substances degraded by the bacteria screened in the present invention;

FIG. 4 is an electrophoretogram of the bacteria screened by the present invention after PCR amplification.

Detailed Description

The following examples further describe embodiments of the present invention in detail. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

Example (b):

firstly, screening phenanthrene polycyclic aromatic hydrocarbon degrading bacteria, comprising the following steps:

(1) taking 1g of soil near a filling station to culture in 100mL of LB liquid culture medium containing 300mg/L of crystal violet at 30 ℃ for 24 hours under shaking at 170 r/min;

(2) diluting 100 mu L of the liquid in the step (1) according to a concentration gradient, coating 50 mu L of the diluted liquid on an LB flat plate containing 300mg/L of crystal violet, and carrying out inverted culture at 30 ℃ for 24 hours;

(3) observing the LB flat plate in the step (2), picking 2 single colonies with colorless circles, putting each single colony into a liquid LB test tube, and carrying out shake culture at 30 ℃ and 170r/min for 16 hours;

(4) detecting the decolorization rate, comprising the following steps:

A1. preparing 15mL of a blank control sample, wherein the volume fraction of the liquid culture medium with the PBS concentration of 0.01M, LB in the sample is 10 percent, namely the added LB liquid culture medium is 1.5 mL;

B1. preparing 15mL of a screening strain sample before treatment, wherein the concentration of PBS in the sample is 0.01M, the concentration of crystal violet in the sample is 300mg/L, detecting the absorbance of the screening strain sample before treatment at the wavelength of 578nm by taking the sample in the step A1 as a control, and obtaining the absorbance as the OD value before treatment;

C1. preparing 15mL of a treated screening strain sample, wherein the concentration of PBS in the sample is 0.01M, the concentration of crystal violet in the sample is 300mg/L, and the volume fraction of the single-colony bacterial liquid in the step (3) is 10%, namely the added single-colony bacterial liquid in the step (3) is 1.5mL, carrying out shake culture at 30 ℃ and 170r/min for 6 days, detecting the absorbance of the treated screening strain sample at the wavelength of 578nm by taking the sample in A1 as a control every 12 hours, and obtaining the absorbance as the OD value after treatment; 2 single colonies were picked in step (3), and the number of the selected strain samples prepared here was also 2.

D1. Calculating the decolorization rate: percent decolorization is (OD value before untreated-OD value after treatment)/OD value before untreated.

The decolorization effect is shown in figure 1, the first test tube on the left is the control, the middle test tube and the rightmost test tube are single colony test tubes with 2 colorless circles, the color of the control is purple after 5 days, the decolorization is not realized, and the single colony test tube is decolorized. Calculating the decolorization rate, wherein the decolorization rate of crystal violet of 2 single bacterial colony strains reaches 80% in 5 days; FIG. 2 shows that the destaining rate of one of the single colonies reached 80% at a faster time (around 72 hours) and the strain was used for the next experiment.

(5) Inoculating the single colony bacterial liquid screened in the last step into an LB liquid culture medium, and carrying out shaking culture at 30 ℃ and 170r/min for 16 hours;

(6) centrifugally collecting thalli, repeatedly cleaning cells for at least 3 times by using sterile water, and finally suspending by using 0.01M PBS solution to prepare an bacterial suspension with an OD600 value of 0.5;

(7) detecting the degradation rate, comprising the following steps:

A2. the method for detecting the initial value comprises the following steps: taking an inorganic salt culture medium with the volume of 200mL and the concentration of phenanthrene being 50mg/L, carrying out ultrasonic extraction for 2 times by using n-hexane, wherein the volume of n-hexane used for each time is 100mL, carrying out ultrasonic extraction for 10min, combining 2 times of extracted organic phases, carrying out rotary evaporation, concentrating the organic phases to a constant volume of 10mL, measuring the light absorption value of phenanthrene at 250nm by using an ultraviolet spectrophotometer, and obtaining the concentration of phenanthrene as the initial value of phenanthrene by referring to a phenanthrene standard curve;

B2. and (3) detecting a residual value by the method: adding the bacterial suspension obtained in the step (6) with the volume fraction of 5% into an inorganic salt culture medium containing 50mg/L of phenanthrene, carrying out shake culture for 6 days, taking liquid with the volume of 200mL every 12 hours, carrying out ultrasonic extraction on the liquid sampled every time for 2 times by using n-hexane, carrying out ultrasonic extraction for 10min when the volume of the n-hexane used every time is 100mL, combining 2 times of extracted organic phases, carrying out rotary evaporation to concentrate the organic phases to a constant volume of 10mL, measuring the light absorption value of the phenanthrene at 250nm by using an ultraviolet spectrophotometer, and obtaining the residual value of the phenanthrene by referring to a standard curve of the phenanthrene;

C2. calculating the degradation rate of phenanthrene: percent degradation (% phenanthrene initial value-residual value of phenanthrene)/phenanthrene initial value.

FIG. 3 is a graph of degradation rate, the degradation rate can reach more than 45% in 5 days, and the strain is determined to be the phenanthrene polycyclic aromatic hydrocarbon degrading bacteria.

The inorganic salt culture medium comprises the following components: (NH4)₂SO4 1.5g、NaC1 0.5g、KH₂PO4 1.5g、K₂HPO₄ 0.5g、MgSO₄·7H₂O 0.2g、FeSO₄·7H₂O 0.05g、CaCl₂0.05g, distilled water 1000mL, pH adjusted to 7.0, and moist heat sterilized at 121 ℃ for 30 minutes.

Secondly, PCR sequencing detection of strains

Inoculating the polycyclic aromatic hydrocarbon degrading bacteria determined in the previous part into LB liquid culture medium, and performing shake culture at 30 ℃ and 170r/min for 16 hours. The cells were collected by centrifugation, and genomic DNA was extracted and used as a DNA template for PCR amplification of the 16S rRNA sequence.

The 16S rRNA gene sequence of this strain was PCR-amplified using bacterial 16S rRNA gene universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACGGCTACCTTGTTACGACTT-3').

The PCR mixture (50mL) contained 10mM Tris-HCl (pH 8.3), 50mM KCl, 1.5mM MgCl₂Each deoxynucleoside triphosphate was used at a concentration of 200. mu.M, 50pmol of a primer, 2.5U of Taq DNA polymerase, and about 20ng of a DNA template. PCR amplification was performed, the procedure was as follows: denaturation at 94 ℃ for 1 min; followed by 25 cycles of 95 ℃ for 1 minute, 55 ℃ for 1 minute, and 72 ℃ for 1 minute; finally extension at 72 ℃ for 10 min. The PCR products were detected on a 1% agarose gel as shown in FIG. 4.

And recovering the amplification product gel, sending the amplification product gel to a sequencing company for sequencing to obtain a gene sequence, comparing and analyzing the gene sequence with a gene sequence stored in a NCBI database GenBank, finding that the similarity of the strain and the Comamonas testosteroni is higher, and preliminarily determining the strain to be the Comamonas testosterone.

The specific use mode and function of the embodiment are as follows:

the method utilizes crystal violet, which is a common triphenylmethane dye, and the triphenylmethane with a chemical functional group has high chemical stability and low biodegradability, although the triphenylmethane belongs to one of polycyclic aromatic hydrocarbons, the toxicity is much lower than that of fused ring aromatic hydrocarbons.

When an observed sample can be converted from purple to light purple and is finally colorless, the bacterium can degrade crystal violet and has the effect of degrading polycyclic aromatic hydrocarbons.

The embodiments of the present invention have been presented for purposes of illustration and description, and are not intended to be exhaustive or limited to the invention in the form disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art. The embodiment was chosen and described in order to best explain the principles of the invention and the practical application, and to enable others of ordinary skill in the art to understand the invention for various embodiments with various modifications as are suited to the particular use contemplated.