CN101974442A

CN101974442A - Pyrene degrading bacterium and application thereof

Info

Publication number: CN101974442A
Application number: CN2010101158211A
Authority: CN
Inventors: 豆俊峰; 杜勇超; 丁爱中; 程莉蓉; 范福强; 陈海英
Original assignee: Beijing Normal University
Current assignee: Beijing Normal University
Priority date: 2010-03-02
Filing date: 2010-03-02
Publication date: 2011-02-16
Anticipated expiration: 2030-03-02
Also published as: CN101974442B

Abstract

The invention discloses a pyrene degrading bacterium and application thereof, and belongs to the technical field of biological degradation treatment. The pyrene degrading bacterium is authenticated as an achromobacter (Achromobacter sp.), and the strain collection number is CGMCC3637. The Genbank accession number of a 16S rDNA of the strain is EU220009.1. The strain is prepared into a bacterial suspension of which the bacterium content is 1.25*10<8>CFU/ml, and the degradation removal rates to the pyrenes of which the initial concentrations are 0.11mg/L, 0.26mg/L, 0.52mg/L and 1.14mg/L respectively after 7 days are 92.65%, 91.46%, 29% and 89.67% respectively.

Description

One strain pyrene degradation bacteria and application thereof

Technical field

The invention belongs to the biological degradation processing technology field of polluted soil and water, particularly a strain pyrene degradation bacteria and an application thereof.

Background technology

Polycyclic aromatic hydrocarbons (Polycyclic Aromatic Hydrocarbons, PAHs) be that a class contains two or more phenyl ring, condensed ring type compound so that wire, horn shape or bunch shape are arranged has characteristics such as hydrophobicity, low solubility, low-steam pressure, high-melting-point and boiling point, octanol-water partition coefficient height.Polycyclic arene compound has intensive carcinogenesis, teratogenesis and mutagenesis, and in general, the lower molecular weight PAHs of 2～3 rings has significant acute toxicity, and some high-molecular weight PAHs has the potential carinogenicity.Owing to have the big π bonding electron of conjugation system in the PAHs molecule, therefore has advantages of higher stability, its photolysis and biological degradation are slow, can be in environment by various surrounding medium long-distance migrations and long-term existence, by environment accumulate, mode such as biological accumulation, bio-transformation or chemical reaction impairs one's health and environment, caused various countries environmentalist's great attention.EPA just is defined as priority pollutant in the environment to 16 kinds with ramose PAHs 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, and the common degraded PAHs functional microorganism that has has Rhodopseudomonas (Pseudomonas), Sphingol single-cell belongs to (Sphingomonas), Rhod (Rhodococcus), Mycobacterium (Mycobacterium), bud pole Pseudomonas (Bacillus), Flavobacterium (Flavobacterium), Aeromonas (Aeromonas), Beijerinckia (Beijernckia), corynebacterium (Corynebaeterium), cyanobacteria (Cyanobacteria), micrococcus sp (Micrococcus), Nocardia (Nocardia) and Vibrio (Vibrio) etc.

Yet, restricted further developing and using of polycyclic aromatic hydrocarbon pollution bioremediation technology owing to lack polycyclic aromatic hydrocarbon-degrading bacteria efficiently.Therefore, need be from the environment that pollutes acclimation and screening go out degrading polycyclic aromatic hydrocarbons speed microbial strains faster, 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 pyrene degradation bacteria and an application thereof.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 1st, 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 CGMCC3637.The Genbank accession number of this bacterial strain 16S rDNA is EU220009.1, and 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 pyrene in the environment.

The step of the described pyrene degradation bacteria of separation and purification 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) in volume is the open glass bottle of 1000mL, add the acetone mixing solutions that 25～30mL concentration is the pyrene of 1g/L, the vial opening was placed 2 days for removing acetone.

(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 training period and remain unchanged with the water and soil ratio of keeping mud system in the vial in 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 the pyrene of 1g/L to volume, the Erlenmeyer flask opening placed 2 days for removing acetone.

(5) go into 50mL basic medium, 0.5mL trace metal liquid and 0.1mL vitamin c solution in the Erlenmeyer flask in 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 that step (3) is tamed in the Erlenmeyer flask in 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; In the inoculation volume ratio is in the Erlenmeyer flask after 200～250: 1 ratio changes another over to handle (4) to (5) set by step, can obtain efficiently to remove the aerobic degradation microbial community of pyrene after the switching number of times is greater than 8.

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

(8) in being the Erlenmeyer flask of 150mL, volume adds the 50mL liquid nutrient medium and the 0.60g agar of (7) preparation set by step, 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, this culture dish placed in Bechtop 2 days for removing the media surface redundant moisture.

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

(10) adding the 10mL liquid nutrient medium and the 0.25g agar of (7) preparation set by step in volume is the Erlenmeyer flask of 25mL, 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 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 that the culture medium solution uniform distribution of mixed bacterial be arranged.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 the efficient degradation pyrene pure strain.

The invention has the beneficial effects as follows that described method can separation and purification arrive the microbial strains good to the pyrene degradation property.

Embodiment

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

The bacterial classification Achromobacter sp. of the degraded 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 pyrene of degrading.The result shows that strains A chromobacter sp. can effectively degrade to pyrene, when the starting point concentration of pyrene was 0.11mg/L, 0.26mg/L, 0.52mg/L, 1.14mg/L, the degraded clearance after 7 days was respectively: 92.65%, 91.46%, 88.29%, 89.67%.

Claims

1. a strain pyrene degradation bacteria is characterized in that, described bacterial strain is through being accredited as Achromobacter sp., and culture presevation number is CGMCC3615.

2. according to the described pyrene degradation bacteria of claim 1, it is characterized in that the Genbank accession number of this bacterial strain 16S rDNA is EU220009.1.

3. the application of the described pyrene degradation bacteria of claim 1 is characterized in that, the bacteria containing amount of bacteria suspension is 1.25 * 10 ⁸CFU/ml.