JP2022001364A - METHOD FOR EFFECTIVELY REMOVING USEPA PAHs IN AGRICULTURAL LAND SOIL BY ACCLIMATIZING INDIGENOUS DEGRADATION BACTERIUM COMMUNITY - Google Patents

Abstract

To provide a method for effectively removing USEPA PAHs in agricultural land soil by acclimatizing indigenous degradation bacterium community, and to provide an apparatus.SOLUTION: The problem is solved by a method that includes 1) enrichment culture of indigenous degradation bacterium, 2) acclimatization of indigenous degradation bacterium, 3) isolation and purification of indigenous degradation bacterium, 4) selection of predominant indigenous degradation bacterium, and 5) inoculation of indigenous degradation bacterium.SELECTED DRAWING: Figure 1

Description

The present invention relates to a method of effectively removing USEPA PAHs in agricultural land soil by acclimatizing a resident degrading bacterial community.

PAHs are divided into natural resources and artificial resources. Natural resources are mainly derived from the biosynthesis process of land, aquatic plants and microorganisms, and polycyclic aromatic hydrocarbons are also present in natural fires in forests and grasslands, volcanic eruptions and fossil fuels, lignin, and deposits. Artificial resources are mainly various fossil fuels, wood,
Formed by incomplete combustion of paper and other hydrocarbons, or pyrolysis under reducing conditions.

Due to its teratogenic, carcinogenic and mutagenic effects, PAHs can cause many harms to the human body, including respiratory, circulatory and nervous system damage, liver and kidney damage. .. Currently, microbial repair has become an important technique for removing many contaminants, including polycyclic aromatic hydrocarbons.

Indigenous bacteria live in the natural environment around us and are a mixed group of various beneficial microorganisms. Indigenous bacteria are classified into aerobic bacteria and aerobic bacteria, and depending on the bacterial species, yeast and Aspergillus. And actinomycetes. It is divided into bacteria, lactic acid bacteria, spore bacteria, etc. After 3 billion years of evolution
Bacteria can metabolize almost any compound as energy and are considered the ultimate scavenger of nature. Due to its strong adaptability, bacteria are widely used to reduce or repair the risk of polluting the environment.

However, there are various types of indigenous bacteria, and indigenous bacteria that can be used to remove soil from agricultural land must be isolated and acclimatized before they can be obtained.

In view of the above problems, the present invention provides a method for effectively removing USEPA PAHs in agricultural land soil by acclimatizing a resident degrading bacterial community.

The technical solution of the present invention is
How to effectively remove USEPA PAHs in agricultural land soil by acclimatizing resident degrading bacterial communities
1) Prepare a soil sample that is not contaminated with PAHs into a soil diluent, collect the soil diluent, perform aerobic enrichment culture and anaerobic enrichment culture, and obtain an aerobic medium solution and an anaerobic medium solution.
2) Prepare a soil sample contaminated with PAHs into a soil suspension, divide the soil suspension into two parts, inoculate the aerobic medium solution and the anaerobic medium solution according to the gradient concentration, respectively, and culture and proliferate.
Acclimatizing resident degrading bacteria using PAHs as the sole carbon source, and obtaining the conditioned aerobic resident degrading bacterial fluid and anaerobic resident degrading bacterial fluid,
3) Separation and purification of acclimatized aerobic resident degrading bacterial fluid and anaerobic resident degrading bacterial fluid to obtain multiple resident degrading bacteria.
4) Multiple resident degrading bacteria obtained by isolation and purification were used to treat soil extracts containing PAHs, respectively, and the PAHs content of the treated soil was measured using HPLC.
To obtain the predominant resident degrading bacterial solution by selecting and multiplying the soil-degrading bacterium with the highest decomposition rate as the predominant bacterium.
1) Inoculating a predominant resident degrading bacterial solution into a farm soil contaminated with PAHs and using the resident degrading bacteria to reduce the PAHs content in the farm soil.

In one aspect of the present invention, in 1),
Preparation of soil diluent: Add a soil sample uncontaminated with PAHs to the sterilized water next to the flame and add.
The volume ratio of the soil sample not contaminated with PAHs to the sterilized water is 1: 9, and the sterilized water is uniformly vibrated for 10 to 20 minutes using a vibrating device to uniformly sterilize the indigenous decomposed bacteria in the soil. Disperse, leave for 5 to 8 hours to remove the supernatant, centrifuge for 0.4 to 0.5 h with a centrifuge, set the centrifugal rotation speed to 4000-5000 rpm, remove the centrifugal supernatant to obtain a soil diluted solution. The concentration of the soil diluent is 10%, which contributes to the further dilution of the soil diluent, and if operated next to the flame, other bacteria in the air will be mixed into the soil diluent and affect the acclimatization of the indigenous decomposition bacteria. To exert.

In one aspect of the present invention, in 1),
Aerobic enrichment culture: Place the aerobic medium in the open flask, suck up the soil diluent using a sterilization pipette, incubate the aerobic medium, put the open flask in the culture box, and set the temperature of the culture box to 3.
Controlled to a range of 3 to 35 ° C. and vibrated for 18 to 20 hours to obtain an aerobic medium solution, and the aerobic bacteria must be cultured under air conditions. Avoids the predominant soil-degrading bacteria dying from oxygen deficiency by moving to the surface of the broth to obtain oxygen.
Anaerobic accumulation culture: Place the anaerobic medium in the triangular flask, suck up the soil diluent using a sterilization pipette, incubate the anaerobic medium, seal the triangular flask with a bolt plug and put it in the culture box, and the temperature of the culture box. Is controlled in the range of 33 to 35 ° C. and left-cultured for 18 to 20 hours to obtain an anaerobic medium solution, and the anaerobic culture is left-cultured under closed conditions, and vibration adversely affects the growth of anaerobic bacteria.

In one aspect of the present invention, in 2),
Preparation of soil suspension: Add PAHs-contaminated soil samples to sterilized water next to the flame and PAH
The volume ratio of the soil sample contaminated with s and the sterilized water is 1: 4, and the vibration device is used for 10 to 20 m.
In uniform vibration to obtain a soil suspension, the concentration of the soil suspension is 20%, the suspension can completely retain PAHs in the soil, and PAHs are the only carbon source for resident degrading bacteria. Familiarize,
High PAHs content can increase acclimatization efficiency,
Inoculation and acclimatization of aerobic medium solution: Divide the 20% concentration soil suspension into three parts, add sterilized water to two of the soil suspensions, and increase their concentrations to 10% and 15%. First, a 10% concentration soil suspension was inoculated into the aerobic medium solution using a sterilization pipette, and the temperature was 33 to 35 ° C.
Incubate for 20 to 24 hours in the culture box of No. 1 to obtain a first-class aerobic conditioned solution, and then incubate the first-class aerobic conditioned solution into a soil suspension having a concentration of 15% using a sterilization pipette. 20 ~ in a culture box at ~ 35 ° C
Incubate for 24 hours to obtain a secondary aerobic conditioned solution, and finally inoculate a 20% concentration soil suspension with the secondary aerobic conditioned solution using a sterilization pipette, and incubate in a culture box at 33 to 35 ° C. After 20 to 24 hours of vibration culture, the aerobic resident degrading bacterial solution was finally obtained, and if the initial concentration of PAHs was too high, it affected the survival of the aerobic resident degrading bacteria, and the soil was adjusted according to the gradient concentration. Gradually increasing the concentration of PAHs in the suspension makes the presence of microbial bacteria other than resident-degrading bacteria in the soil diluent more difficult, and finally all die, leaving only aerobic resident-degrading bacteria. ,
Inoculation and acclimatization of anaerobic medium solution: Divide the 20% concentration soil suspension into three parts, add sterilized water to two of the soil suspensions, and increase their concentrations to 10% and 15%. First, inoculate the anaerobic medium solution with a 10% concentration soil suspension using a sterilization pipette, seal it in a culture box at 33 to 35 ° C., leave it for 20 to 24 hours, and incubate it for 20 to 24 hours. Then, using a sterilization pipette, inoculate a first-class anaerobic conditioned solution into a soil suspension having a concentration of 15%, seal it in a culture box at 33 to 35 ° C., and leave it for 20 to 24 hours for culture. Obtain a grade anaerobic culture medium and finally use a sterilization pipette 2
Incubate a 20% concentration soil suspension with a grade anaerobic incubator, seal in a culture box at 33-35 ° C, and 20
After culturing for ~ 24 hours, the anaerobic resident degrading bacterial solution finally acclimatized was obtained, and if the initial concentration of PAHs was too high, it affected the survival of the anaerobic resident degrading bacteria, and in the soil suspension according to the gradient concentration. P
By gradually increasing the concentration of AHs, the presence of microbial bacteria other than the resident degrading bacteria in the soil diluent becomes more difficult, and finally all of them die, leaving only the anaerobic resident degrading bacteria.

In one aspect of the present invention, in 3),
Specific steps for isolation and purification: Prepare multiple sterilized culture plates and use an inoculation loop to adhere to the aerobic and anaerobic resident degrading bacterial solutions that have been acclimatized, respectively, and sterilize the culture. The sterilized culture plate applied on the plate is left to incubate for 20 to 24 hours, and the above operation is repeated to dilute, apply, and separate multiple times, and the number of indigenously degrading bacteria decreases as the number of dilutions increases. The purified resident-degrading bacteria obtained by gradual purification and separation are stored on an inclined surface, and the resident-degrading bacteria acclimatized by separation and purification are individually separated to obtain higher-purity resident decomposition. Bacteria can be obtained, which is useful for the subsequent selection of predominant soil-degrading bacteria.

In one aspect of the present invention, in 4),
Specific Steps for Treating a Soil Extract Containing PAHs Using Resident Degrading Bacteria: Multiple resident decomposition bacteria obtained by separation and purification in 3) are grown, respectively, and a soil sample containing PAHs is prepared. Prepared into a soil suspension, use HPLC to detect the PAHs content in the soil suspension, inoculate the soil suspension with multiple resident degrading bacteria, each of which has grown, and treat the resident decomposition. The volume ratio of the bacterial culture solution to the soil suspension is 1: 10 to 80, and after 3d treatment, the PAHs content in the soil suspension is detected using HPLC, and the soil with the highest PAHs removal efficiency. Degrading bacteria are selected to increase the efficiency of removing PAHs in the soil.

In one aspect of the invention, the apparatus used in the above method includes HPLC for detecting the PAHs content in the soil, a vibrating apparatus for vibrating the soil diluent and the soil suspension, and soil dilution. A centrifuge for centrifuging the liquid to obtain the supernatant, an open flask for aerobic accumulation culture of the bacterial liquid in the soil, a sterilization pipette for inoculation and liquid removal, and a resident degrading bacterium. A culture box for culturing, a triangular flask for anaerobic accumulation culture of the bacterial solution in the soil, and a sterilization culture for separating and purifying the acclimatized aerobic resident decomposition bacterial solution and anaerobic resident decomposition bacterial solution. With a plate,
Includes an inoculation loop for inoculation to the sterilized culture plate.

In one aspect of the invention, the device
The culture box includes an aerobic culture box for culturing aerobic indigenous degrading bacteria and an aerobic culture box.
An anaerobic culture box placed above the aerobic culture box for culturing anaerobic resident degrading bacteria, and an anaerobic culture box.
The control box arranged on the side of the aerobic culture box and the anaerobic culture box,
A fixing device arranged inside the aerobic culture box and an anaerobic culture box to fix the microbial container, and the fixing device for controlling the internal / external rotation of the two semi-annular fixing arms and the fixing arm, respectively. Including the stepping motor, a pressure sensor is further installed at the connection point between the fixed arm and the stepping motor, and the pressure sensor controls the on / off of the stepping motor according to the pressure.
A temperature control device for controlling the indoor temperature, which is arranged inside the aerobic culture box and the anaerobic culture box, respectively, and the temperature control device are provided on the inner walls of the aerobic culture box and the anaerobic culture box, respectively. Includes heating coil and temperature sensor for monitoring room temperature,
A ventilation device for ventilation and ventilation of the culture box, and the ventilation device includes an air supply device provided at the top of the anaerobic culture box and an exhaust hole provided at the bottom of the rear wall of the aerobic culture box. The air supply device includes an air pump for sucking external air into the culture box and a filtration groove provided at the air supply port of the air pump for filtering dust and microorganisms in the air, and the exhaust air is provided outside the exhaust hole. A filtration membrane for filtering indigenous bacteria is provided,
A controller placed inside the control box and 2 placed on the front side of the control box
Includes two temperature control knobs.

The present invention has a beneficial effect. The present invention aerobically and anaerobically cultivates indigenous degrading bacteria in soil diluents, respectively, and gradient acclimatizes soil degrading bacteria using soil suspensions containing PAHs, as well as other soil degrading bacteria. All bacteria are removed, and the species of the acclimatized indigenous degrading bacteria are isolated and purified, the indigenous degrading bacteria having the highest PAHs removal efficiency are selected and propagated, and inoculated into the contaminated soil to decompose PAHs. However, the decomposition efficiency is high.

It is a flowchart of the method and apparatus of this invention. It is a schematic diagram of the structure of the culture box of this invention.

[Explanation of sign]
1 culture box 11 aerobic culture box 12 anaerobic culture box 13 control box 131 temperature control knob 14 fixing device 141 fixing arm 142 stepping motor 143 pressure sensor 15 temperature control device 151 heating coil 152 temperature sensor 16 ventilation device 161 air supply device 1611 Air pump 1612 Filter groove 162 Exhaust hole 1621 Filter membrane

To facilitate understanding of the technical solutions of the invention, the invention is further described below with reference to specific examples and accompanying FIGS. 1-2, wherein the examples are the scope of protection of the invention. Do not configure restrictions on.

Example 1: As shown in FIG. 1, US in agricultural land soil by acclimatizing a resident degrading bacterial community.
How to effectively remove EPA PAHs
1) Prepare a soil sample that is not contaminated with PAHs into a soil diluent, collect the soil diluent, perform aerobic enrichment culture and anaerobic enrichment culture, and obtain an aerobic medium solution and an anaerobic medium solution.
Preparation of soil diluent: Add a soil sample uncontaminated with PAHs to the sterilized water next to the flame and add.
The volume ratio of the soil sample not contaminated with PAHs to the sterilized water is 1: 9, and the sterilized water is uniformly vibrated for 20 minutes using a vibrating device to uniformly disperse the resident degrading bacteria in the soil. ,
Leave it for 8 hours to remove the supernatant, centrifuge for 0.5 hours with a centrifuge, and set the centrifugal rotation speed to 5000.
At rpm, the centrifugal supernatant was taken to obtain the soil diluent, and the concentration of the soil diluent was 10%.
Aerobic enrichment culture: Place the aerobic medium in the open flask, suck up the soil diluent using a sterilization pipette, incubate the aerobic medium, put the open flask in the culture box 1, and set the temperature of the culture box 1. Control to 35 ° C. and incubate for 20 hours to obtain an aerobic medium solution.
Anaerobic enrichment culture: Place the anaerobic medium in the Erlenmeyer flask, suck up the soil diluent using a sterilization pipette, incubate the anaerobic medium, seal the Erlenmeyer flask with a bolt plug, and put it in the culture box 1.
The temperature of the culture box 1 was controlled to 35 ° C., and the cells were left to incubate for 20 hours to obtain an anaerobic medium solution.
2) Prepare a soil sample contaminated with PAHs into a soil suspension, divide the soil suspension into two parts, inoculate the aerobic medium solution and the anaerobic medium solution according to the gradient concentration, respectively, and culture and proliferate.
Acclimatizing resident degrading bacteria using PAHs as the sole carbon source, and obtaining the conditioned aerobic resident degrading bacterial fluid and anaerobic resident degrading bacterial fluid,
Preparation of soil suspension: Add PAHs-contaminated soil samples to sterilized water next to the flame and PAH
The volume ratio of the soil sample contaminated with s to the sterilized water was 1: 4, and the soil suspension was obtained by uniformly vibrating for 20 minutes with a vibrating device, and the concentration of the soil suspension was 20%.
Inoculation and acclimatization of aerobic medium solution: Divide the 20% concentration soil suspension into three parts, add sterilized water to two of the soil suspensions, and increase their concentrations to 10% and 15%. First, a 10% concentration soil suspension was inoculated into the aerobic medium solution using a sterilization pipette, and vibrated in a culture box 1 at 35 ° C. for 24 hours to obtain a first-class aerobic nutrient solution. Then, using a sterilization pipette, inoculate a first-class aerobic nutrient solution into a soil suspension having a concentration of 15%, and vibrate and culture in a culture box 1 at 35 ° C. for 24 hours.
Obtain a grade aerobic nutrient solution, and finally incubate a grade 2 aerobic nutrient solution into a soil suspension having a concentration of 20% using a sterilization pipette, and incubate in a culture box 1 at 35 ° C. for 24 hours, and finally. Obtained an aerobic resident decomposition bacterial solution that has been acclimatized to
Inoculation and acclimatization of anaerobic medium solution: Divide the 20% concentration soil suspension into three parts, add sterilized water to two of the soil suspensions, and increase their concentrations to 10% and 15%. First, inoculate the anaerobic medium solution with a 10% concentration soil suspension using a sterilization pipette, seal in a culture box 1 at 33 to 35 ° C., leave and incubate for 20 to 24 hours, and perform first-class anaerobic acclimation. Obtain the solution, then use a sterilization pipette to inoculate the first-grade anaerobic conditioned solution into a soil suspension with a concentration of 15%, seal in a culture box 1 at 35 ° C., leave and incubate for 24 hours, and second-grade anaerobic. Obtain the conditioned solution, and finally inoculate the secondary anaerobic conditioned solution into a soil suspension having a concentration of 20% using a sterilization pipette, seal in a culture box 1 at 35 ° C., and leave to culture for 24 hours, and finally. Obtained an anaerobic resident decomposition medium acclimatized to
3) Separation and purification of acclimatized aerobic resident degrading bacterial fluid and anaerobic resident degrading bacterial fluid to obtain multiple resident degrading bacteria.
Specific steps for isolation and purification: Prepare multiple sterilized culture plates and use an inoculation loop to adhere to the aerobic and anaerobic resident degrading bacterial solutions that have been acclimatized, respectively, and sterilize the culture. It was applied on a plate, and the applied sterilized culture plate was left to inoculate for 24 hours, and the above operation was repeated to dilute, apply, and separate multiple times. Purified resident degrading bacteria obtained by gradual purification and isolation were stored on an inclined surface.
4) Multiple resident degrading bacteria obtained by isolation and purification were used to treat soil extracts containing PAHs, respectively, and the PAHs content of the treated soil was measured using HPLC.
To obtain the predominant resident degrading bacterial solution by selecting and multiplying the soil-degrading bacterium with the highest decomposition rate as the predominant bacterium.
Specific steps for treating a soil extract containing PAHs using resident-degrading bacteria: Multiple resident-degrading bacteria obtained by separation and purification in 3) are grown, and a soil sample containing PAHs is prepared. Prepared into a soil suspension, use HPLC to detect the PAHs content in the soil suspension, inoculate the soil suspension with multiple indigenously degrading bacteria, each of which has grown, and treat with indigenous degradation. The volume ratio of the bacterial culture solution to the soil suspension was 1:80, and after 3d treatment, HPLC was used to detect the PAHs content in the soil suspension.
5) Inoculating the predominant resident degrading bacterial solution into the agricultural land soil contaminated with PAHs and using the resident degrading bacteria to reduce the PAHs content in the agricultural land soil.
Example 2: Example 2 differs from Example 1 as follows:
1) Prepare a soil sample not contaminated with PAHs as a soil diluent, and aerobic enrichment culture of the soil diluent to obtain an aerobic medium solution.
Preparation of soil diluent: Add a soil sample uncontaminated with PAHs to the sterilized water next to the flame and add.
The volume ratio of the soil sample not contaminated with PAHs to the sterilized water is 1: 9, and the sterilized water is uniformly vibrated for 10 minutes using a vibrating device to uniformly disperse the resident degrading bacteria in the soil. ,
Leave it for 5 hours to remove the supernatant, centrifuge for 0.4 hours with a centrifuge, and set the centrifugal rotation speed to 4000.
At rpm, the centrifugal supernatant was taken to obtain the soil diluent, and the concentration of the soil diluent was 10%.
Aerobic enrichment culture: Place the aerobic medium in the open flask, suck up the soil diluent using a sterilization pipette, incubate the aerobic medium, put the open flask in the culture box 1, and set the temperature of the culture box 1. Control to 33 ° C. and incubate for 18 hours to obtain an aerobic medium solution.
2) Prepare a soil sample contaminated with PAHs into a soil suspension, inoculate the soil suspension into an aerobic medium solution according to the gradient concentration, culture and proliferate, and use PAHs as the sole carbon source to generate resident degrading bacteria. Acclimate and obtain acclimatized aerobic resident decomposition medium,
Preparation of soil suspension: Add PAHs-contaminated soil samples to sterilized water next to the flame and PAH
The volume ratio of the soil sample contaminated with s to the sterilized water was 1: 4, and the soil suspension was obtained by uniformly vibrating for 10 minutes with a vibrating device, and the concentration of the soil suspension was 20%.
Inoculation and acclimatization of aerobic medium solution: Divide the 20% concentration soil suspension into three parts, add sterilized water to two of the soil suspensions, and increase their concentrations to 10% and 15%. First, a 10% concentration soil suspension was inoculated into the aerobic medium solution using a sterilization pipette, and vibrated in a culture box 1 at 33 ° C. for 20 hours to obtain a first-class aerobic nutrient solution. Then, using a sterilization pipette, inoculate a first-class aerobic nutrient solution into a soil suspension having a concentration of 15%, and vibrate and culture in a culture box 1 at 33 ° C. for 20 hours.
Obtain a grade aerobic nutrient solution, and finally incubate a grade 2 aerobic nutrient solution into a soil suspension having a concentration of 20% using a sterilization pipette, and incubate in a culture box 1 at 33 ° C. for 20 hours, and finally. Obtained an aerobic resident decomposition bacterial solution that has been acclimatized to
3) Isolate and purify the acclimatized aerobic resident degrading bacterial solution to obtain multiple resident degrading bacteria.
Specific steps for isolation and purification: Prepare multiple sterilized culture plates and use inoculation loop 9 to tackle and sterilize the aerobic and anaerobic resident degrading bacterial solutions that have been acclimatized, respectively. It was applied on a culture plate, and the applied sterilized culture plate was left to inoculate for 24 hours, and the above operation was repeated to dilute, apply, and separate multiple times, and the number of indigenously degrading bacteria decreased as the number of dilutions increased. , Gradually purified and isolated and obtained purified resident degrading bacteria are stored on the inclined surface respectively.

Experimental Example 1: PAH in soil by a single aerobic resident degrading bacterium and an aerobic / anaerobic complex resident degrading bacterium
Examination of the effect of s on the removal efficiency Experimental target: Obtained by preparing the predominant resident decomposing bacterial solution in Example 1 and preparing the predominant resident decomposing bacterial solution in Example 2, and in it. , The predominant resident degrading bacterial solution of Example 1 contains aerobic resident degrading bacteria and anaerobic resident degrading bacteria, and the predominant resident degrading bacterial solution of Example 2 contains only aerobic resident degrading bacteria. Is included.
^{Experimental conditions: Soil samples of 10 cm 3} were randomly collected from farmland soil contaminated with PAHs, and the soil samples were divided into two parts on average. Two soil samples were inoculated with the predominant resident degrading bacterial solution, and the soil samples were left in a simulated farmland environment in the laboratory, 1 cm after 1d, 3d, 5d and 7d, respectively.
^{A soil sample of 3} is collected and PAHs are detected.
Experimental results: Detection results are shown in Table 1:
Table 1 Comparison table of PAHs removal efficiency by single aerobic degrading bacteria and aerobic / anaerobic compound degrading bacteria

Experimental conclusion: The efficiency of removing PAHs in the soil by the predominant resident degrading bacterial solution containing the aerobic and anaerobic complex resident degrading bacteria of Example 1 is higher, but only the aerobic resident degrading bacteria of Example 2 are used. The efficiency of removing PAHs in soil by the predominantly resident degrading bacterial solution containing is not much different from that of Example 1, and aerobic resident degrading bacteria play a major role in removing PAHs in soil.

Example 3: Example 3 differs from Example 1 as follows:
1) Prepare a soil sample that is not contaminated with PAHs into a soil diluent, and anaerobic enrichment culture of the soil diluent to obtain an anaerobic medium solution.
Preparation of soil diluent: Add a soil sample uncontaminated with PAHs to the sterilized water next to the flame and add.
The volume ratio of the soil sample not contaminated with PAHs to the sterilized water is 1: 9, and the sterilized water is uniformly vibrated for 10 to 20 minutes using a vibration device to uniformly sterilize the indigenous decomposed bacteria in the soil. Disperse and leave for 5 hours to remove the supernatant, centrifuge for 0.4h with a centrifuge, and set the centrifugation speed to 4.
At 000 rpm, take the centrifugal supernatant to obtain the soil diluent, and the concentration of the soil diluent is 10%.
And
Anaerobic enrichment culture: Place the anaerobic medium in the Erlenmeyer flask, suck up the soil diluent using a sterilization pipette, incubate the anaerobic medium, seal the Erlenmeyer flask with a bolt plug, and put it in the culture box 1.
The temperature of the culture box 1 was controlled to 33 ° C., and the cells were left to incubate for 18 hours to obtain an anaerobic medium solution.
2) Prepare a soil sample contaminated with PAHs into a soil suspension, inoculate the soil suspension into an anaerobic medium solution according to the gradient concentration, culture and proliferate, and acclimate the resident degrading bacteria using PAHs as the sole carbon source. And obtain the acclimatized anaerobic resident decomposition bacterial solution,
Preparation of soil suspension: Add PAHs-contaminated soil samples to sterilized water next to the flame and PAH
The volume ratio of the soil sample contaminated with s to the sterilized water was 1: 4, and the soil suspension was obtained by uniformly vibrating for 10 minutes with a vibrating device, and the concentration of the soil suspension was 20%.
Inoculation and acclimatization of anaerobic medium solution: Divide the 20% concentration soil suspension into three parts, add sterilized water to two of the soil suspensions, and increase their concentrations to 10% and 15%. First, inoculate the anaerobic medium solution with a 10% concentration soil suspension using a sterilization pipette, seal it in a culture box 1 at 33 ° C., leave it for 20 hours, and obtain a first-class anaerobic acclimatization solution. Then, using a sterilization pipette, inoculate a 15% concentration soil suspension with a first-grade anaerobic conditioned solution, seal it in a culture box 1 at 33 ° C., leave it for 20 hours, and obtain a second-grade anaerobic conditioned solution. Finally, use a sterilization pipette to add 20% of the second grade anaerobic conditioned solution.
Incubate in a soil suspension of a concentration, seal in a culture box 1 at 33 ° C., leave and incubate for 20 hours, and finally obtain an anaerobic resident decomposition bacterial solution that has been acclimatized.
3) Isolate and purify the acclimatized anaerobic resident degrading bacterial solution to obtain multiple resident degrading bacteria.
Specific steps for isolation and purification: Prepare multiple sterilized culture plates and use inoculation loop 9 to tackle and sterilize the aerobic and anaerobic resident degrading bacterial solutions that have been acclimatized, respectively. It was applied on a culture plate, and the applied sterilized culture plate was left to inoculate for 20 hours, and the above operation was repeated to dilute, apply, and separate multiple times, and the number of indigenously degrading bacteria decreased as the number of dilutions increased. , Gradually purified and isolated and obtained purified resident degrading bacteria are stored on the inclined surface respectively.

Experimental example 2: PAH in soil by a single anaerobic resident degrading bacterium and an aerobic / anaerobic compound resident degrading bacterium
Examination of the effect of s on the removal efficiency Experimental target: Obtained by preparing the predominant resident decomposing bacterial solution in Example 1 and preparing the predominant resident decomposing bacterial solution in Example 3, and in it. The predominant resident degrading bacterial solution of Example 1 contains aerobic resident degrading bacteria and anaerobic resident degrading bacteria, and the predominant resident degrading bacterial solution of Example 3 contains only anaerobic resident degrading bacteria. Is done.
^{Experimental conditions: Soil samples of 10 cm 3} were randomly collected from farmland soil contaminated with PAHs, and the soil samples were divided into two parts on average. Two soil samples were inoculated with the predominant resident degrading bacterial solution, and the soil samples were left in a simulated farmland environment in the laboratory, 1c after passing 1d, 3d, 5d and 7d, respectively.
The soil sample of m ³ were collected perform the PAHs detected.
Experimental results: Detection results are shown in Table 2:
Table 2 Comparison table of PAHs removal efficiency by single anaerobic degrading bacteria and aerobic / anaerobic compound degrading bacteria

Experimental conclusion: The efficiency of removing PAHs in the soil by the predominant resident degrading bacterial solution containing the aerobic / anaerobic complex resident degrading bacterium of Example 1 is high, and the predominant anaerobic resident degrading bacterium of Example 3 is contained. The efficiency of removing PAHs in soil by the resident degrading bacterial solution is low, the role of anaerobic resident degrading bacteria is not so great in removing PAHs in soil, and a large amount of cost and time are required for preparing anaerobic resident degrading bacteria. In consideration of the need, it is sufficient to prepare only aerobic resident decomposition bacterial solution to remove PAHs in the soil for practical application, and aerobic resident decomposition is required when higher removal efficiency is required. A resident degrading bacterial solution containing bacteria and anaerobic resident degrading bacteria may be prepared.
Example 4: Example 4 differs from Example 1 as follows:
2) Without separating and purifying the aerobic resident degrading bacteria and anaerobic resident degrading bacteria conditioned in 2), the aerobic resident degrading bacterial solution and anaerobic resident degrading bacterial solution conditioned in 2) remain as they are. Each is treated with a soil extract containing PAHs, the decomposition rate of the treated soil PAHs is detected, the resident decomposition bacterial solution with the highest decomposition rate is selected and propagated, and the dominant resident decomposition bacterial solution is predominant. To get.

Experimental Example 3: Examination of the effect of isolation and purification of acclimatized resident degrading bacteria on the removal efficiency of PAHs in soil Experimental target: Obtained by preparing the predominant resident degrading bacterial solution in Example 1, Example The predominant resident decomposing bacterial solution was prepared in No. 4, and the aerobic resident degrading bacterial solution and the anaerobic anaerobic bacterial solution that had been acclimated for the predominant resident decomposing bacterial solution of Example 1 were used as the decomposing bacterial solution. After isolation and purification, the predominantly degrading bacterium is selected, and for the predominant resident degrading bacterial solution of Example 4, the aerobic resident degrading bacterial solution and the anaerobic resident degrading bacterial solution that have been acclimatized are isolated and purified. Use as it is without.
^{Experimental conditions: Soil samples of 10 cm 3} were randomly collected from farmland soil contaminated with PAHs, and the soil samples were divided into two parts on average, and the predominant resident decomposition bacterial solution of Example 1 and Example 4 were used, respectively. Two soil samples were inoculated with the predominant resident degrading bacterial solution, and the soil samples were left in a simulated farmland environment in the laboratory, 1c after passing 1d, 3d, 5d and 7d, respectively.
The soil sample of m ³ were collected perform the PAHs detected.
Experimental results: Detection results are shown in Table 3:
Table 3 Comparison table of soil PAHs removal efficiency of isolation and purification of resident degrading bacteria

Experimental conclusion: For the predominant resident degrading bacterial solution of Example 1, in order to select the predominant degrading bacteria after separating and purifying the conditioned aerobic resident degrading bacterial solution and the anaerobic resident degrading bacterial solution, in the soil. PAHs removal efficiency is significantly higher than in Example 4, and if the acclimatized aerobic resident degrading bacterial solution and anaerobic normal are separated and purified with the degrading bacterial solution, predominant soil-degrading bacteria can be obtained, which is predominant. Soil-degrading bacteria are grown to remove PAHs in the soil, and the effect is better.
Example 5: Example 5 differs from Example 1 as follows:
4) Multiple indigenously degrading bacteria obtained by separation and purification in 3) are directly grown to obtain a indigenously degrading bacterial solution.
(5) The resident degrading bacterial solution is inoculated into the agricultural land soil contaminated with PAHs, and the resident degrading bacteria are used to reduce the PAHs content in the agricultural land soil.
Experimental Example 4: Examination of the effect of selection of predominant soil-degrading bacteria on the removal efficiency of PAHs in soil Experimental target: Obtained by preparing a predominant resident-degrading bacterial solution in Example 1 and resident in Example 5. A soil-degrading bacterium obtained by preparing a degrading bacterial solution, and the soil-degrading bacterium having the highest decomposition rate after separation and purification in Example 1 was selected as the predominant bacterial growth to obtain a predominant resident-degrading bacterial solution. In Example 5, a plurality of resident degrading bacteria obtained by separation and purification are directly grown to obtain a resident degrading bacterial solution.
^{Experimental conditions: Soil samples of 10 cm 3} were randomly collected from farmland soil contaminated with PAHs, and the soil samples were divided into two parts on average. Two soil samples were inoculated with the predominant resident degrading bacterial solution, and the soil samples were left in a simulated farmland environment in the laboratory, 1c after passing 1d, 3d, 5d and 7d, respectively.
The soil sample of m ³ perform the collected PAHs detected.
Experimental results: Detection results are shown in Table 4:
Table 4 Comparison table of soil PAHs removal efficiency for selection of predominantly degrading bacteria

Experimental conclusion: For the predominant resident degrading bacterial solution of Example 1, the soil-degrading bacterium having the highest decomposition rate after isolation and purification was selected as the predominant bacterium and propagated, and the efficiency of removing PAHs in the soil was higher. In Example 5, a plurality of resident degrading bacteria obtained by separation and purification were directly propagated to obtain a resident degrading bacterial solution, and the efficiency of removing PAHs in soil was relatively low, and PAHs in soil were obtained.
For s removal, the removal efficiency of the predominant resident degrading bacteria is higher.
Unless otherwise stated, all materials and reagents used in the Examples are either conventionally used or commercially available in the art.
Example 6: In this example, the structure of the apparatus used in the above-mentioned Examples 1 to 5 will be described, HPLC for detecting the PAHs content in the soil, a soil diluent and a soil suspension. A vibrating device for vibration treatment, a centrifuge for centrifuging the soil diluent to obtain the supernatant, an open flask for aerobic accumulation and culture of the bacterial solution in the soil, and inoculation and liquid removal. A sterilization pipette for sterilization, a culture box 1 for culturing resident degrading bacteria, a triangular flask for anaerobic accumulation culture of bacterial liquor in soil, and aerobic resident degrading bacterial liquor and anaerobic resident. Includes a sterilized culture plate for separating and purifying the degrading bacterial fluid and an inoculation loop for inoculation into the sterilized culture plate.

Example 7: Different from Example 1 as follows,
As shown in FIG. 2, the culture box 1 includes an aerobic culture box 11 for culturing aerobic indigenous degrading bacteria and an aerobic culture box 11.
An anaerobic culture box 1 placed above the aerobic culture box 11 for culturing anaerobic resident degrading bacteria.
2 and
The control box 13 arranged on the side of the aerobic culture box 11 and the anaerobic culture box 12,
The fixing device 14 for fixing the microbial container arranged inside the aerobic culture box 11 and the anaerobic culture box 12, respectively, and the fixing device 14 are two semi-annular fixing arms 141 and a fixing arm 1.
Includes a stepping motor 142 to control the internal and external rotation of 41, fixed arm 141
A pressure sensor 143 is further provided at the connection point between the stepping motor 142 and the pressure sensor 143, and the pressure sensor 143 controls the on / off of the stepping motor 142 according to the pressure.
The temperature control device 15 is arranged inside the aerobic culture box 11 and the anaerobic culture box 12 to control the indoor temperature, respectively, and the temperature control device 15 is the aerobic culture box 11 and the anaerobic culture box 12. It includes a heating coil 151 provided on the inner wall and a temperature sensor 152 for monitoring the room temperature.
The aeration device 16 for aeration and ventilation of the culture box 1 and the aeration device 16 are provided at the lower part of the rear wall of the air supply device 161 provided at the top of the anaerobic culture box 12 and the aerobic culture box 11. Exhaust hole 1
The air supply device 161 includes an air pump 1611 for sucking external air into the culture box 1, and a filtration groove 1 provided at the air supply port of the air pump 1611 for filtering dust and microorganisms in the air.
Filtration membrane 162 containing 612 and for filtering indigenous bacteria in the exhaust air outside the exhaust hole 162
1 is provided,
It includes a controller arranged inside the control box 13 and two temperature control knobs 131 arranged on the front surface of the control box 13.

Experimental Example 5: Examination of the difference between the culture box provided by Example 7 and the normal culture box Experimental target: The culture box in Example 6 and the culture box 1 in Example 7 can satisfy the requirements for use of the invention. As far as possible, the culture box in Example 6 can be a commercially available conventional culture box.
Experimental conditions: Ten operators were voluntarily recruited, and each operator used the culture boxes provided in Examples 6 and 7, and predominantly resident degradation according to the method provided in Example 1. Bacterial fluid was prepared and the usability of 10 operators was examined.
Experimental results: Responses from 10 operators:
For the commercially available normal culture boxes in Example 6, at least two culture boxes are required to independently cultivate aerobic and anaerobic resident degrading bacteria, and simultaneous culture is achieved, and one culture box is predominant. Since it has a serious effect on the preparation progress of the indigenous decomposition bacterial solution and is prepared in the laboratory, the content and types of microorganisms in the air are high, and other bacteria grow in the culture box during the culture process. There are situations that affect the purity of the predominantly resident degrading bacterial solution,
With respect to the culture box provided by Example 7, aerobic and anaerobic resident degrading bacteria can be cultured at the same time, the culture time does not need to be recorded individually, and the workload of the operator can be reduced.
Since the culture box provided by is capable of filtering microorganisms in the air, there is no adhesion or growth of other bacteria in the culture box, and the predominant resident decomposition bacterial solution obtained by preparation has a high purity of 99%. In addition, resident degrading bacteria do not escape to the air and do not affect other projects in the laboratory.
Experimental conclusion: The culture box provided by Example 7 simultaneously cultivates aerobic and anaerobic resident degrading bacteria, can reduce the workload of the operator, and by filtering the microorganisms in the outside air, the resident degrading bacteria can be cultivated. By ensuring the preparation purity and filtering the microorganisms in the air discharged from the culture box, it is possible to prevent the resident degrading bacteria from escaping to the outside air and affecting other effects.

Claims (8)

1) A step of preparing a soil sample not contaminated with PAHs into a soil diluted solution, collecting the soil diluted solution, performing aerobic enrichment culture and anaerobic enrichment culture, and obtaining an aerobic medium solution and an anaerobic medium solution. ,
2) Prepare a soil sample contaminated with PAHs into a soil suspension, divide the soil suspension into two parts, inoculate the aerobic medium solution and the anaerobic medium solution according to the gradient concentration, respectively, and culture and proliferate.
Steps to acclimate resident degrading bacteria using PAHs as the sole carbon source and obtain the conditioned aerobic resident degrading bacterial fluid and anaerobic resident degrading bacterial fluid,
3) A step of separating and purifying the acclimatized aerobic resident degrading bacterial solution and the anaerobic resident degrading bacterial solution to obtain a plurality of resident degrading bacteria, and
4) Multiple resident degrading bacteria obtained by isolation and purification were used to treat soil extracts containing PAHs, respectively, and the PAHs content of the treated soil was measured using HPLC.
The step of selecting the soil-degrading bacterium with the highest decomposition rate as the predominant bacterium and growing it to obtain the predominant resident degrading bacterial solution, and
5) Inoculating the predominant resident degrading bacterial solution into the farmland soil contaminated with PAHs and using the resident degrading bacteria to reduce the PAHs content in the farmland soil.
A method for effectively removing USEPA PAHs in agricultural land soil by acclimatizing a resident degrading bacterial community containing. In step 1)
Preparation of soil diluent: Add a soil sample uncontaminated with PAHs to the sterilized water next to the flame and add.
The volume ratio of the soil sample not contaminated with PAHs to the sterilized water is 1: 9, and the sterilized water is uniformly vibrated for 10 to 20 minutes using a vibrating device to uniformly sterilize the indigenous decomposed bacteria in the soil. Disperse, leave for 5 to 8 hours to remove the supernatant, centrifuge for 0.4 to 0.5 h with a centrifuge, set the centrifuge to 4000-5000 rpm, remove the centrifugal supernatant to obtain a soil diluted solution. The method according to claim 1, wherein the concentration of the soil diluent is 10%. In step 1)
Aerobic enrichment culture: Place the aerobic medium in an open flask, suck up the soil diluent using a sterilization pipette, incubate the aerobic medium, place the open flask in the culture box (1), and place the open flask in the culture box (1).
) Is controlled in the range of 33 to 35 ° C. and vibrated for 18 to 20 hours to obtain an aerobic medium solution.
Anaerobic enrichment culture: Place an anaerobic medium in a triangular flask, suck up the soil diluent using a sterilization pipette, incubate the anaerobic medium, seal the triangular flask with a bolt plug, put it in the culture box (1), and culture. The method according to claim 1, wherein the temperature of the box (1) is controlled in the range of 33 to 35 ° C., and the culture is left to stand for 18 to 20 hours to obtain an anaerobic medium solution. In step 2)
Preparation of soil suspension: Add PAHs-contaminated soil samples to sterilized water next to the flame and PAH
The volume ratio of the soil sample contaminated with s and the sterilized water is 1: 4, and the vibration device is used for 10 to 20 m.
In uniform vibration to obtain the soil suspension, the concentration of the soil suspension is 20%,
Inoculation and acclimatization of aerobic medium solution: Divide the 20% concentration soil suspension into three parts, add sterilized water to two of the soil suspensions, and increase their concentrations to 10% and 15%. First, a 10% concentration soil suspension was inoculated into the aerobic medium solution using a sterilization pipette, and the temperature was 33 to 35 ° C.
Incubate for 20 to 24 hours in the culture box (1) of the above to obtain a first-class aerobic conditioned solution, and then incubate the first-class aerobic conditioned solution into a soil suspension having a concentration of 15% using a sterilization pipette. Incubator at 33-35 ° C (
Incubate for 20 to 24 hours in 1) to obtain a second-grade aerobic conditioned solution, and finally inoculate the second-grade aerobic conditioned solution into a 20% concentration soil suspension using a sterilization pipette, and 33 to 33 to 2 in the culture box (1) at 35 ° C.
Vibration-cultured for 0 to 24 hours, and finally obtained the aerobic resident decomposition bacterial solution that had been acclimatized.
Inoculation and acclimatization of anaerobic medium solution: Divide the 20% concentration soil suspension into three parts, add sterilized water to two of the soil suspensions, and increase their concentrations to 10% and 15%. After adjustment, first inoculate a 10% concentration soil suspension into an anaerobic medium solution using a sterilization pipette, seal it in a culture box (1) at 33 to 35 ° C., leave it for 20 to 24 hours, and incubate it to the first grade. Obtain the anaerobic conditioned solution, then inoculate the first grade anaerobic conditioned solution into the soil suspension at a concentration of 15% using a sterilization pipette, and inoculate the culture box at 33-35 ° C.
Closed in 1) and left to culture for 20 to 24 hours to obtain a secondary anaerobic conditioned solution, and finally inoculate the secondary anaerobic conditioned solution into a 20% concentration soil suspension using a sterilization pipette, 33. Incubation box at ~ 35 ° C (
The method according to claim 1, wherein the anaerobic resident decomposing bacterial solution is finally obtained after being hermetically sealed in 1) and left to culture for 20 to 24 hours. In step 3)
Specific steps for isolation and purification: Prepare multiple sterilized culture plates and use an inoculation loop to adhere to the aerobic and anaerobic resident degrading bacterial solutions that have been acclimatized, respectively, and sterilize the culture. The sterilized culture plate applied on the plate is left to inoculate for 20 to 24 hours, and the above operation is repeated to dilute, apply, and separate multiple times, and the number of indigenously degrading bacteria decreases as the number of dilutions increases. The method according to claim 1, wherein each of the purified resident degrading bacteria obtained by gradually purifying and separating is stored on an inclined surface. In step 4)
Specific steps for treating soil extracts containing PAHs using resident-degrading bacteria: Multiple resident-degrading bacteria obtained by separation and purification in step 3) are grown, and PAHs are grown.
Soil samples containing s are prepared in soil suspensions and PAHs in soil suspensions are used using HPLC.
The s content was detected, and a plurality of indigenously degrading bacteria that grew respectively were inoculated into the soil suspension for treatment, and the volume ratio of the indigenously degrading bacterial culture solution to the soil suspension was 1: 10 to 80. After 3d processing, H
The method of claim 1, wherein the PLC is used to detect the PAHs content in the soil suspension. HPLC for detecting PAHs content in soil, a vibrating device for vibration treatment of soil diluent and soil suspension, and a centrifuge for centrifuging the soil diluent to obtain a supernatant. An open flask for aerobic accumulation and culture of bacterial fluid in soil, a sterilization pipette for inoculation and fluid removal, a culture box (1) for culturing resident degrading bacteria, and bacterial fluid in soil. For inoculation into the sterilized culture plate, a triangular flask for anaerobic accumulation culture, a sterilized culture plate for separating and purifying the acclimatized aerobic resident decomposition bacterial solution and the anaerobic resident decomposition bacterial solution, and the sterilized culture plate. The device used in the method according to claim 1-5, comprising the inoculation loop of. The culture box (1) is
An aerobic culture box (11) for culturing aerobic indigenous degrading bacteria and
An anaerobic culture box (12) arranged above the aerobic culture box (11) for culturing anaerobic resident degrading bacteria, and
A control box (1) arranged on the side of the aerobic culture box (11) and the anaerobic culture box (12).
3) and
The fixing device (14) arranged inside the aerobic culture box (11) and the anaerobic culture box (12) for fixing the microbial container, and the fixing device (14) are two semi-annular fixing arms, respectively. (1
A stepping motor (41) for controlling the internal and external rotation of the fixed arm (141) and the fixed arm (141).
142) is included, a pressure sensor (143) is further provided at the connection point between the fixed arm (141) and the stepping motor (142), and the pressure sensor (143) turns the stepping motor (142) on / off according to the pressure. Control and
The temperature control device (15) arranged inside the aerobic culture box (11) and the anaerobic culture box (12) for controlling the indoor temperature, and the temperature control device (15) are the aerobic culture boxes, respectively. It includes a heating coil (151) provided on the inner wall of the body (11) and the anaerobic culture box (12) and a temperature sensor (152) for monitoring the room temperature.
The aeration device (16) for aeration and ventilation of the culture box (1), and the aeration device (16) is an air supply device (161) and an aerobic culture box provided at the top of the anaerobic culture box (12). Including an exhaust hole (162) provided in the lower part of the rear wall of (11), the air supply device (161) includes an air pump (1611) for sucking external air into the culture box (1) and an air pump (1611). The exhaust hole (1) includes a filtration groove (1612) provided in the air supply port for filtering dust and microorganisms in the air.
A filtration membrane (1621) for filtering indigenous bacteria in the exhaust air is provided on the outside of 62).
The device of claim 7, comprising a controller disposed inside the control box (13) and a temperature control knob (131) disposed on the front surface of the control box (13).

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