CN102533575B

CN102533575B - Strain capable of degradation of benzo[alpha]pyrene

Info

Publication number: CN102533575B
Application number: CN 201010584918
Authority: CN
Inventors: 程莉蓉; 豆俊峰; 丁爱中; 李帅冉; 杜勇超; 范福强
Original assignee: Beijing Normal University
Current assignee: Beijing Normal University
Priority date: 2010-12-13
Filing date: 2010-12-13
Publication date: 2013-10-02
Anticipated expiration: 2030-12-13
Also published as: CN102533575A

Abstract

The invention discloses a strain capable of degradation of benzo[a]pyrene, and belongs to the technical field of biodegradation treatment. The benzo[a]pyrene degradation strain is identified as Achromobacter sp., and the strain preservation number is CGMCC3684. The strain is prepared into the bacterium suspension with the bacterium content of 1.25*10<8> CFU/ml. With the strain of the present invention, the degradation removal rates of the materials with the initial benzo[a]pyrene concentrations of 0.11 mg/L, 0.34 mg/L, 0.71 mg/L, and 1.19 mg/L are respectively 74.35%, 84.66%, 78.91% and 68.33%.

Description

One strain has the bacterial strain of degradation function to benzo [a] pyrene

Technical field

The invention belongs to the biological degradation processing technology field of polluted soil and water, particularly a strain has the bacterial strain of degradation function to benzo [a] pyrene.

Background technology

(Polycyclic Aromatic Hydrocarbons is the toxic organic pollutant that a class contains two or more phenyl ring PAHs) to polycyclic aromatic hydrocarbons, has strong carcinogenesis, teratogenesis and mutagenesis.PAHs can cause very big harm to ecotope and HUMAN HEALTH by the transfer function of biological accumulation and food chain, has caused various countries environmentalist's great attention.EPA just is defined as priority pollutant in the environment to 16 kinds of PAHs with branch as far back as the eighties, and China is also listed PAHs in the Black List of environmental pollution in.Biological degradation is one of effective means of realize recovering the PAHs contaminate environment.

In general, along with the increase of polycyclic aromatic hydrocarbons phenyl ring quantity, its degradation rate reduces.Therefore, low-molecular-weight polycyclic aromatic hydrocarbons can comparatively fast be degraded in environment, and the time that exists in environment is shorter, and the polycyclic aromatic hydrocarbons of high molecular then is difficult to degraded, and longer-term is present in the environment.Yet owing to lack polycyclic aromatic hydrocarbon-degrading bacteria efficiently, restricted further developing and using of polycyclic aromatic hydrocarbon pollution bioremediation technology.The research of existing microbiological deterioration PAHs generally concentrates on degraded Screening of Bioflocculant-producing Bacteria, the isolation and purification of lower molecular weight PAHs, and less for the microorganism report of efficient degradation high molecular PAHs.Isolation and purification high molecular PAHs efficient degrading bacteria is the key of biological restoration PAHs contaminate environment.

Therefore, need be from the environment that pollutes acclimation and screening go out polycyclic aromatic hydrocarbons (as benzo [a] pyrene) degradation rate microbial strains faster to high molecular, and then they are applied to the biological treating of actual PAHs contaminate environment, this has important meaning for administering and repair polycyclic aromatic hydrocarbon pollution.

Summary of the invention

The invention provides a strain has degradation function to benzo [a] pyrene bacterial strain.Bacterial strain uses therefor is through being accredited as achromobacter Achromobacter sp., be preserved in Chinese common micro-organisms culture presevation administrative center on March 23rd, 2010, this is centered close to Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, and culture presevation number is CGMCC3684.It is 1.25 * 10 that this bacterial strain is made bacteria containing amount ⁸The bacteria suspension of CFU/ml can be used for the degraded of benzo in the environment [a] pyrene.

The step of the described benzo of separation and purification [a] pyrene degradation bacteria is as follows from contaminated soil:

(1) choose petroleum-polluted soil, with its by diameter be positioned over immediately behind the mesh screen of 2mm preserve in 4 ℃ of refrigerators standby.

(2) to the acetone mixing solutions that adds the benzo that 25～30mL concentration is 1g/L [a] pyrene during volume is the open glass bottle of 1000mL, for removing acetone the vial opening was placed 2 days.

(3) in step (2) split shed vial, add 500～600mL basic medium, add the soil 250g in the step (1) after under rotating speed is the condition of 100r/min, stirring 40～50min; Placing temperature then is that 25～30 ℃ environment is to stir the enrichment domestication under the condition of 100r/min to cultivate 30d at rotating speed, regularly adds basic medium in the vial in the training period and remains unchanged with the water and soil ratio of keeping mud system in the vial; Consisting of of basic medium: NH wherein ₄Cl:1.5gL ^-1, KH ₂PO ₄: 1.5gL ^-1, MgCl ₂: 0.25gL ^-1, CaCl ₂2H ₂O:0.10gL ^-1

(4) be that to add 10mL concentration in the Erlenmeyer flask of 100～200mL be the acetone mixing solutions of benzo [a] pyrene of 1g/L to volume, for removing acetone the Erlenmeyer flask opening placed 2 days.

(5) go into 50mL basic medium, 0.5mL trace metal liquid and 0.1mL vitamin c solution in the Erlenmeyer flask in the step (4); Consisting of of trace metal liquid: CoCl wherein ₂6H ₂O:35mgL ^-1, CuCl ₂: 0.20mgL ^-1, H ₃BO ₃: 6.0mgL ^-1, MnCl ₂4H ₂O:25mgL ^-1, Na ₂MoO ₄2H ₂O:3.0mgL ^-1, NiCl ₂2H ₂O:2.0mgL ^-1, ZnCl ₂: 2.5mgL ^-1

(6) insert the soil 0.25g of step (3) domestication in the Erlenmeyer flask in the step (5), placing temperature then is that the vibrator that 25～30 ℃ of rotating speeds are 100r/min was cultivated 5～7 days, opens bottleneck every day once to recover the dissolved oxygen in the Erlenmeyer flask; Be in the Erlenmeyer flask after 200～250: 1 ratio changes another over to handle (4) to (5) set by step in the inoculation volume ratio, after the switching number of times is greater than 8, can obtain efficiently to remove the aerobic degradation microbial community of benzo [a] pyrene.

(7) be to add 400mL basic medium, 1.0mL trace metal liquid, 0.1mL vitamin c solution in the Erlenmeyer flask of 500mL to volume, it is standby as liquid nutrient medium to shake up the back.

(8) be to add 50mL liquid nutrient medium and the 0.60g agar of (7) preparation set by step in the Erlenmeyer flask of 150mL to volume, be sterilization 20 minutes in 121 ℃ the pressure kettle in temperature then, when at room temperature being cooled to 65～70 ℃, in Bechtop, be poured in the culture dish that volume is 160mL, for removing the media surface redundant moisture this culture dish placed in Bechtop 2 days.

(9) adding 0.5mL concentration in the culture dish in the step (8) in Bechtop is benzo [a] the pyrene acetone soln of 1g/L, tilt to rotate culture dish back and forth the acetone soln of benzo [a] pyrene is evenly distributed, for the acetone of removing media surface was placed this culture dish 2 days in Bechtop.

(10) being to add 10mL liquid nutrient medium and the 0.25g agar of (7) preparation set by step in the Erlenmeyer flask of 25mL to volume, is sterilization 20 minutes in 121 ℃ the pressure kettle in temperature, is cooled to 38～40 ℃ in Bechtop.

(11) in Bechtop, in the Erlenmeyer flask of step (10), add benzo [a] the pyrene aerobic degradation microbial community solution that 1mL step (6) obtains, shake up the back and draw 1mL and slowly add in the culture dish in the step (9), tilt to rotate culture dish back and forth and make inoculation have the culture medium solution of mixed bacterial evenly to distribute.It is that 25 ℃ incubator cultivate to be observed that culture dish is put into temperature, can find just after 10～15 days that tangible bacterium colony occurs.

(12) bacterium colony in picking step (11) culture dish in Bechtop carries out repeated isolation, 8 of separation and purification can obtain more than the cycle can efficient degradation benzo [a] pyrene pure strain.

Use aforesaid method separation and purification to arrive the microbial strains good to the pyrene degradation property.

Embodiment

Carry out enrichment and the domestication of benzo [a] pyrene degraded microorganism to the petroleum-polluted soil of the interior adding of the open glass bottle of adding benzo [a] pyrene and basic medium, the soil of taming to the interior access of the Erlenmeyer flask that adds benzo [a] pyrene, basic medium, trace metal liquid, vitamin c solution and sorbent material enrichment behind the 30d.Be after 25～30 ℃, rotating speed are to cultivate 5～7 days in the vibrator of 100r/min in temperature, the degradation flora of benzo [a] pyrene can be efficiently removed in the switching that circulates after cycle index is greater than 8 times.Making with benzo [a] pyrene in Bechtop is the agar solid medium bottom platform of sole carbon source and the energy, make the agar solid medium top layer flat board that contains benzo [a] pyrene degradation flora at bottom platform, the culture medium flat plate of making was cultivated in incubator 10～15 days.In Bechtop in the Erlenmeyer flask that adds benzo [a] pyrene, basic medium, trace metal liquid, vitamin c solution and sorbent material the bacterium colony in the access culture medium flat plate, be that 25～30 ℃, rotating speed are to cultivate 5～7 days in the vibrator of 100r/min in temperature.The switching that circulates then, the pure strain of benzo [a] pyrene that after cycle index is greater than 8 times, can obtain degrading.

The bacterial classification achromobacter Achromobacter sp. of degraded benzo [a] pyrene that the utilization separation and purification obtains makes bacteria suspension, and bacteria containing amount is 1.25 * 10 ⁸CFU/ml, the degraded test of the benzo of degrading [a] pyrene.The result shows that bacterial strain achromobacter Achromobactersp. can effectively degrade to benzo [a] pyrene, when the starting point concentration of benzo [a] pyrene was 0.11mg/L, 0.34mg/L, 0.71mg/L, 1.19mg/L, the degraded clearance after 20 days was respectively: 74.35%, 84.66%, 78.91%, 68.33%.

Claims

1. a strain has the bacterial strain of degradation function to benzo [a] pyrene, it is characterized in that, described bacterial strain is through being accredited as achromobacter (Achromobacter sp.), and culture presevation number is CGMCC NO:3684.