CN106318891A - Pyridine degradation strain a5, fungicide made from pyridine degradation strain a5 and application thereof - Google Patents
Pyridine degradation strain a5, fungicide made from pyridine degradation strain a5 and application thereof Download PDFInfo
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Abstract
The invention discloses a pyridine degradation strain a5 which is identified as Rhodococcus ruber and has been preserved in China General Microbiological Culture Collection Center, wherein the preservation date is August 31, 2016 and the preservation number is CGMCC No.12919. The invention also discloses an application of the pyridine degradation strain a5 in producing pyridine degradation fungicide. The degradation fungicide can degrade pyridine and can achieve the degradation rate 90% or above of the pyridine remained in soil within a short period of time and the degradation rate 95% or above of the pyridine remained in high-concentration pyridine wastewater. The degradation fungicide provided by the invention can be applied to the general fermentation equipment in fermentation industry for production and has the advantages of low production cost, convenience in use and good removing effect.
Description
Technical field
The present invention relates to environmental pollution microorganism remediation, be specifically related to a pyridine degradation bacterium strain strain and production thereof microbial inoculum and
Application.
Background technology
Industrialization fast phase causes high-volume to use various synthetic chemical substance it is considered to be heteroplasia thing
Matter, causes environmental pollution.At present commercial production all produces and discharges substantial amounts of aromatic compound every day, and the most about 2/
3 is all heterocyclic compound.Heterocyclic compound is widely used in the fields such as industrial solvent, dyestuff, explosive, medicine and pesticide.Pyrrole
Pyridine is exactly the typical nitrogen-containing heterocycle compound of one of which.Pyridine, nitrogen-containing heterocycle compound, in benzene molecular can be regarded as
(CH) compound replaced by N, thus also known as pyridine, colourless or slightly yellow liquid, foul smelling.Pyridine and homologue thereof are present in bone
In tar, coal tar, coal gas, shale oil, oil.Pyridine is widely used in the industries such as chemical industry, medical industry and pesticide producing, tool
Toxic big, teratogenecity, carcinogenecity strong and the feature such as difficult for biological degradation.
The biodegradation of xenobiotic is considered to have one of challenging task.And as the allusion quotation of xenobiotic
Type represents pyridine, and owing to water solublity and diffusibility are preferable, it has also become pollutant common in soil and waste water, biodegradation is
Solve the effective ways of such problem.Biodegradation and physical chemistry degraded are very different, and it is a natural process, is
Microorganism utilizes organic pollution materials to carry out growth and the physiological metabolism process of self.Utilize the micro-life of degraded of screening from environment
Thing can effectively remove the polluter such as heterocycle and phenyl ring class in environment.
Pyridine produces damage to people's physical ability, and it has intense stimulus, when concentration is relatively low, can anaesthetize central nervous system,
Unison higher time, the lighter has glad or sensation of asphyxia, the symptoms such as depression, myasthenia, vomiting followed by occurs, severe one loss of consciousness, big
Urinary incontinence, tonic spasm, blood pressure drops, wrongly taking can be lethal.Long-term suck occur dizzy, have a headache, have a sleepless night, instability of gait and
Digestive tract function is disorderly, hepatorenal damage can occur, also can cause dermatitis.Showing according to correlational study, male is damaged greatly by pyridine,
Long Term Contact can affect spermatozoon activity and then cause male infertility.
The most of common occurrence about the research of pyridine degradable bacteria.It is concentrated mainly on 2 aspects: 1, metabolic pathway, learns
Persons are according to different bacterium or the research of fungus (aerobic, anaerobism) at different conditions, it is proposed that multiple-microorganism degraded pyrrole
Pyridine approach.2, biological reinforced removal effect, directly add in Waste Water Treatment pyridine efficient degrading bacterial strain or immobilization this
A little strain cells realize the removal of pollutant.From 20 century 70s, scholar is separated to some pyridine degradable bacterias one by one,
Wherein most is antibacterial, such as Rhodopseudomonas, pimelobacter sp genus, bacillus, achromobacter, Flavobacterium, chaos
Rhod, Arthrobacter etc..Also there are actinomycetes and fungus, such as white rot fungi.But there is high effect for actual biological restoration
Rarely have appearance, it is within the contemplation of the invention that separate and identify efficient pyridine degradable bacteria, seek the race relation of efficient degrading bacteria, for pyrrole
The biological restoration of pyridine contaminated soil provides theories integration.
Summary of the invention
Goal of the invention: the problem existed for prior art, invention provides a pyridine degradation bacterium strain strain a5, is used for soil of degrading
The pyridine of residual in earth or water body environment；It is a further object of the present invention to provide the microbial inoculum that this pyridine degradable bacteria produces, this bacterial strain
The degradation bacterial agent of preparation can make the pyridine degradable of residual in soil or water body environment at short notice.
Technical scheme: to achieve these goals, a pyridine degradation bacterium strain strain a5 as described in the present invention, it is identified as red
Rhodococcus fascians (Rhodococcus ruber), has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center,
Address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica, postcode: 100101, preservation time
Being on August 31st, 2016, deposit number is CGMCC NO.12919.This bacterial strain is that inventor has from Anhui Province Wan Bei Pharmaceutical share
The activated sludge of the pharmaceutical factory waste water bio-chemical processing pool of limit company is separated to.Degradation bacteria strains a5 Main Biological is: leather
Lan Shi positive bacteria, after cultivating 2-3 days on the pyridine LB solid medium containing 3000mg/L, bacterium colony surface is red, coarse, have pleat
Wrinkle, in mucus shape.
The bacterial strain a5 of the present invention application in degraded pyridine.
As preferably, described bacterial strain a5 is the application of pyridine in degraded soil or water body environment.
The bacterial strain a5 of the present invention application in producing pyridine degradable microbial inoculum.
A kind of degradation bacterial agent utilizing above-mentioned bacterial strains a5 to produce.
Described degradation bacterial agent, made by following steps:
1) the test tube liquid that degradation bacteria strains a5 cultivates logarithmic (log) phase is inoculated in fermentation by the inoculum concentration of 0.5-1.5% volume ratio
In culture medium, shaken cultivation, to logarithmic (log) phase, prepares fermented bacterium；
2) above-mentioned prepared fermented bacterium is inoculated by the inoculum concentration of 5-10% volume ratio in the culture medium of seed tank, cultivate
To exponential phase, prepare seed liquor；
3) inoculum concentration that seed liquor is pressed 8-10% volume ratio accesses cultivation in the culture medium producing tank；
4) in the incubation of seed tank and production tank, the ventilation of filtrated air is 1:(0.8-1.5), mixing speed
For 180-250rpm, cultivation temperature is 25-32 DEG C, and whole process incubation time is 36-48 hour, and the rear culture fluid that fermented goes out tank
Direct plastic barrel or Packaging Bottle are distributed into liquid agent.
The application in degraded pyridine of the above-mentioned degradation bacterial agent.
As preferably, described degradation bacterial agent is the application of pyridine in degraded soil or water body environment.
Reagent of the present invention and instrument:
Pyridine Pyridine (Shanghai fuzz chemical company), content >=99.5%
Glycerol glycerinum (Pharmaceutical Group Solution on Chemical Reagents in Shanghai company), content >=99.9%
Peptone Peptone (Angel biotech firm)
Yeast extract Yeast extract (Angel biotech firm)
Salt NaCl (Angel biotech firm)
Ammonium sulfate (NH4)2SO4(Shanghai De Chuan biological reagent company)
Potassium dihydrogen phosphate KH2PO4(Shanghai De Chuan biological reagent company)
Dipotassium hydrogen phosphate K2HPO4(Shanghai De Chuan biological reagent company)
Magnesium sulfate heptahydrate MgSO4·7H2O (Shanghai Hua Chen biotech firm)
Agar (Angel biotech firm)
Agar A (Angel biotech firm)
Superclean bench (converge good bio-instruments in Shanghai)
Constant incubator HZQ-X100 (Harbin Dong Lian Electronic Technology Development Co.)
Vertical pressure steam sterilizer LDZX-50FBS (Harbin Dong Lian Electronic Technology Development Co.)
Table model high speed centrifuge (Shanghai Analytical Instrument Co., Ltd of Jinpeng)
PCR instrument (BIO-RAD Laboratories-Segrate (Milan) Italy)
Gel image analyser (BIO-RAD Laboratories-Segrate (Milan) Italy)
Spectrophotometer UV-2100 (Shanghai the 3rd analytical tool factory)
Trace ultraviolet spectrophotometer UV-1700 (Shimadzu)
Culture medium and condition of culture:
Basal salt media (1000mL ultra-pure water)
Total amount of binder salt culture medium adds about 1.5% agar, 121 DEG C of autoclaving 30min.
LB culture medium (1000mL ultra-pure water)
Peptone 10.0g
Yeast extract 5.0g
NaCl 10.0g
Solid LB media adds about 1.5% agar, 121 DEG C of autoclaving 30min.
When doing Characteristics Detection, use LB culture medium, need test salinity on bacteria growing when affecting, control NaCl amount,
When needing to test pH, then add HCl or NaOH and regulate acid-base value.
Fermentation medium, the culture medium of seed tank, produce tank culture medium prescription identical, be glucose 0.1wt%,
NaCl1.0wt%, peptone 0.5wt%, yeast extract 0.25wt%, solvent is distilled water, pH7.2-7.5.
Beneficial effect: compared by prior art, the separated screening of the present invention obtains pyridine degradable bacterial strain a5
(Rhodococcus ruber a5), the pyridine of residual in degrade soil or water body environment；Degradation bacteria prepared by this bacterial strain
Agent can be degraded pyridine, and this microbial inoculum can make the degradation rate of the pyridine of pedo relict reach more than 90% at short notice, make high concentration
In pyridine waste water, pyridine degradable rate reaches more than 95%, solves residual pyridine to soil water body environment, crops and health
Harm problem.
The degradation bacterial agent of the present invention can produce by the general Zymolysis Equipment of fermentation industry, has production cost low, makes
With convenient, the advantage that removal effect is good, it is suitable for administering the pollution of pyridine in soil and water body environment；The present invention is for protecting ecology
Environment, protection the healthy of human body has great importance.
Accompanying drawing explanation
Fig. 1 is the uv-spectrogram of 3 days degraded pyridines of bacterial strain a5；
Fig. 2 is bacterial strain a5 bacterium colony photo；
Fig. 3 is the systematic evolution tree of bacterial strain a5；
Fig. 4 is the growth collection of illustrative plates under bacterial strain a5 difference salinity；
Fig. 5 is the growth collection of illustrative plates under bacterial strain a5 difference pH；
Fig. 6 is the growth collection of illustrative plates under bacterial strain a6 different temperatures.
Detailed description of the invention
Below in conjunction with drawings and Examples, the invention will be further described.
Embodiment 1
The separation of bacterial strain a5 and qualification:
Give up in the pharmaceutical factory taking from Anhui Wanbei Pharmaceutical Co., Ltd. for being enriched with the enriched medium of pyridine degradable bacterial strain
The activated sludge of water biochemical treatment tank, takes activated sludge 5.0g and adds in basal salt media, add 50mg L-1Pyridine, 30
DEG C, 150rpm cultivates 7d, is transferred in identical culture medium with the inoculum concentration of 5%, continuously switching 3 times, gradient dilution pregnant solution,
Take 10-4-10-7The each 0.1mL of dilution pregnant solution coats added with 100mg L-1The total amount of binder salt culture medium flat board of pyridine
On, after 30 DEG C are cultivated 3d, single colony inoculation that picking grows is in containing 200mg L-1In pyridine basal salt media, 30 DEG C,
180rpm cultivates 3d, verifies the degradation effect of its degraded bacterium solution.
In basal salt media containing pyridine, whether the degraded of pyridine, can be by observing culture fluid by clarifying muddy the showing of change
As tentatively judging.
The verification method of degradation effect: use UV-1700 trace ultraviolet spectrophotometer to measure the mass concentration of pyridine.Past
Adding the dichloromethane of equivalent volumes in degraded bacterium solution to be measured, acutely vibrate 5-10min, then stands until aqueous phase and organic facies
Being layered completely, draw upper strata aqueous phase, retain organic facies, upper strata aqueous phase extracts with isopyknic dichloromethane again, twice
The organic facies arrived is after the moisture that anhydrous sodium sulfate removes residual, at wavelength 200-350nm on ultraviolet-visible spectrophotometer
In the range of be scanned.Pyrrole in degradation solution is judged by the height of pyridine characteristic absorption peak-to-peak value at 200nm-350nm
The height of pyridine concentration.
From pregnant solution, be separated to 1 pyridine degradation bacterium strain, named a5, this bacterium in 3d to 5000mg L-1Pyridine
Degraded uv-spectrogram, as shown in Figure 1.Wherein it is processed as in basal salt media, add final concentration of 5000mg L-1Pyrrole
Pyridine, is accessed the bacterial strain a5 bacterium solution of logarithmic (log) phase by the inoculum concentration of 1% volume ratio；Comparison is for adding final concentration in basal salt media
For 5000mg L-1Pyridine, inoculate the bacterial strain a5 bacterium solution of logarithmic (log) phase of inactivation by the inoculum concentration of 1% volume ratio, result shows,
The process 3d adding bacterial strain a5 is interior to 5000mg L-1The degradation rate of pyridine reaches more than 90%.
After bacterial strain a5 grows 3d on solid LB media, bacterium colony surface is red, coarse, have fold, in mucus shape, such as figure
Shown in 2.
With the genomic DNA of bacterial strain a5 as template, carry out PCR amplification with bacterial 16 S rRNA gene order universal primer,
Obtain the 16S rDNA gene order of a length of 1388bp, as shown in SEQ ID No:1.EzTaxon data base
(www.ezbiocloud.net) carrying out Blast in, result shows bacterial strain a5 and Rhodococcus ruber (Rhodococcus ruber) bacterium
The homology of strain is nearest, with type strain and Rhodococcus ruber DSM 43338T(X80625) homology reaches
99.86%, set up phylogenetic tree as it is shown on figure 3, combining form and physiological and biochemical property, bacterial strain a6 is initially identified as crimson
Coccus (Rhodococcus ruber), named Rhodococcus ruber a5 (Rhodococcus ruber a5).This bacterial strain a5 is delivered
It is positioned at BeiJing, China, in China Committee for Culture Collection of Microorganisms's common micro-organisms of Institute of Microorganism, Academia Sinica
The heart (being called for short CGMCC) preservation, the preservation time is on August 31st, 2016, and deposit number is CGMCC NO.12919.
Embodiment 2
Different salinity bacterial strain a5 growth quantitative analyses:
Take 5 clean tube and be separately added into 2mL LB culture fluid, with salinity 0%, 1%, 2%, 3%, 4%, probe into not
With the salinity growth effect to pyridine degradable bacteria a5.Condition of culture is 30 DEG C, 150r/min constant-temperature table shaken cultivation, cultivates
By spectrophotometric determination optical density of culture fluid when 600nm after 24h.Result as shown in Figure 4, when salinity is at 0-1%
A5 can grow, and increases salinity in this range the least to the growth effect of bacterium, and reaches summit when 1%.But with
The further raising of salinity, a5 growth all presents downward trend. there is no bacteria growing when salinity reaches 4%.
Different pH bacterial strain a5 growth quantitative analyses:
Take 7 clean tube and be separately added into 2mL LB culture fluid, regulate its pH and make it be divided into 4,5,6,7,8,9,10 7
Gradient is to probe into the different pH growth effect to pyridine degradable bacteria a5, and condition of culture is 30 DEG C, the cultivation of 150r/min constant-temperature table,
Spectrophotometric determination optical density of culture fluid when 600nm is used after cultivating 24h.Result as it is shown in figure 5, a5 pH be 6-9 it
Between growing way preferable, slightly reduce when pH is 7, but a5 general trend all present ascendant trend from 6-8, reaches peak when pH is 8
Value, when pH continues to improve, bacteria growing is reducing tendency, when pH arrives 10, does not substantially have the growth of bacterium.
Different temperatures bacterial strain a5 grows quantitative analysis:
Take 5 clean tube and be separately added into 2ml LB culture fluid, with 15 DEG C, 20 DEG C, 30 DEG C, 37 DEG C, 42 DEG C probe into not
The synthermal growth effect to pyridine degradable bacteria a5, condition of culture is 30 DEG C, 150r/min, the vibration training of above-mentioned different temperatures shaking table
Support, after cultivating 24h, use spectrophotometric determination optical density of culture fluid when 600nm.As shown in Figure 6, a5 in temperature is result
Growing vigorous between 30-37 DEG C, near especially 30 DEG C, growth arrives peak, all presents rising from the latter two bacteria growings of 20 DEG C and becomes
Gesture, in falling tendency after 30 DEG C, arrives 42 DEG C of asepsis growths.
Embodiment 3
The preparation of the degradation bacterial agent of bacterial strain a5
1) degradation bacteria strains a5 cultivates the test tube liquid of logarithmic (log) phase be inoculated in 100mL by the inoculum concentration of 0.5% volume ratio and send out
In ferment culture medium, shaken cultivation, to logarithmic (log) phase, prepares fermented bacterium；
2) above-mentioned prepared fermented bacterium is inoculated into liquid amount by the inoculum concentration of 5% (v/v, on the basis of culture volume)
Be 70% (on the basis of fermenter volume, lower with) 500 liters of seed tanks culture medium in cultivate, cultivate to exponential phase,
Prepare seed liquor；
3) by the inoculum concentration of 9% (v/v, on the basis of culture volume, lower same), seed liquor being accessed liquid amount is 70%
Produce tank culture medium in cultivate.
4) in the incubation of seed tank and production tank, the ventilation of filtrated air is 1:1.5, and mixing speed is
250rpm, cultivation temperature is 25 DEG C, and whole technological process incubation time is 36 hours, and after fermentation ends, thalline quantity reaches 10
Hundred million/more than mL.The rear culture fluid that fermented goes out tank and is directly distributed into liquid agent with plastic barrel.
Embodiment 4
The preparation of the degradation bacterial agent of bacterial strain a5
1) degradation bacteria strains a5 cultivates the test tube liquid of logarithmic (log) phase be inoculated in 100mL by the inoculum concentration of 1.5% volume ratio and send out
In ferment culture medium, shaken cultivation, to logarithmic (log) phase, prepares fermented bacterium；
2) above-mentioned prepared fermented bacterium is inoculated into liquid amount by the inoculum concentration of 8% (v/v, on the basis of culture volume)
Be 70% (on the basis of fermenter volume, lower with) 500 liters of seed tanks culture medium in cultivate, cultivate to exponential phase,
Prepare seed liquor；
3) by the inoculum concentration of 8% (v/v, on the basis of culture volume, lower same), seed liquor being accessed liquid amount is 70%
Produce tank culture medium in cultivate.
4) in the incubation of seed tank and production tank, the ventilation of filtrated air is 1:0.8, and mixing speed is
180rpm, cultivation temperature is 32 DEG C, and whole technological process incubation time is 48 hours, and after fermentation ends, thalline quantity reaches 10
Hundred million/more than mL.The rear culture fluid that fermented goes out tank and is directly distributed into liquid agent with plastic barrel.
Embodiment 5
The preparation of the degradation bacterial agent of bacterial strain a5
1) the test tube liquid that degradation bacteria strains a5 cultivates logarithmic (log) phase is inoculated in 100mL fermentation by the inoculum concentration of 1% volume ratio
In culture medium, shaken cultivation, to logarithmic (log) phase, prepares fermented bacterium；
2) above-mentioned prepared fermented bacterium is inoculated into dress liquid by the inoculum concentration of 10% (v/v, on the basis of culture volume)
Amount be 70% (on the basis of fermenter volume, lower with) 500 liters of seed tanks culture medium in cultivate, cultivate to logarithmic growth
Phase, prepare seed liquor；
3) by the inoculum concentration of 10% (v/v, on the basis of culture volume, lower same), seed liquor being accessed liquid amount is 70%
Produce tank culture medium in cultivate.
4) in the incubation of seed tank and production tank, the ventilation of filtrated air is 1:1.2, and mixing speed is
220rpm, cultivation temperature is 30 DEG C, and whole technological process incubation time is 42 hours, and after fermentation ends, thalline quantity reaches 10
Hundred million/more than mL.The rear culture fluid that fermented goes out tank and is directly distributed into liquid agent with plastic barrel.
Test example 1
Bacterial strain a5 degradation bacterial agent is to the Degrading experiment of pyridine in soil:
Take vegetable garden soil as examination soil sample.Soil sample is crossed 2mm sieve, takes a certain amount of pyridine soluble in water, it is uniform
Admix in 1000g soil, make pyridine final concentration in soil be 3000mg kg-1, prepare 9 parts of identical soil samples, by embodiment
3, the bacterial strain a5 degradation bacterial agent of 4 and 5 preparations, the inoculum concentration (volume/weight) with 5% is respectively connected to above-mentioned 1000g soil
In, each do three parallel laboratory tests, cultivate in 30 DEG C of constant temperature dark culturing casees, if the identical soil of inoculum is not as right
According to, the water-holding capacity of period soil is maintained at 60%, after cultivating 5 days, utilizes high-performance liquid chromatogram determination residual quantity.Measurement result is
The degradation bacterial agent of embodiment 3,4 and 5 in 5 days to soil in pyridine degradable rate respectively reach 92.7%, 94.5%, 97.3%.
Test example 2
Bacterial strain a5 degradation bacterial agent is to the Degrading experiment of pyridine in waste water:
The waste water containing pyridine in the waste water bio-chemical processing pool of pharmaceutical factory is taked to be as sample, the pyridine final concentration in waste water
5000mg·L-1.Waste water is divided into 9 parts, and every part of waste water is 5L, bacterial strain a5 degradation bacterial agent embodiment 3,4 and 5 prepared, with
The inoculum concentration (volume ratio) of 5% is respectively connected in above-mentioned 5L waste water, each does three parallel laboratory tests, in room temperature dark culturing,
If the identical waste water of inoculum is not as comparison, after cultivating 5 days, utilize high-performance liquid chromatogram determination residual quantity.Measurement result is
The degradation bacterial agent of embodiment 3,4 and 5 in 5 days to waste water in pyridine degradable rate respectively reach 95.7%, 96.5%, 98.3%.
SEQUENCE LISTING
<110>Anhui Normal University
<120>one pyridine degradation bacterium strain strain a5 and the microbial inoculum of production thereof and application
<130> 1
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1388
<212> DNA
<213>Rhodococcus ruber (Rhodococcus ruber)
<400> 1
atgcacgtag aacgatgaag cccagcttgc tgggtggatt agtggcgaac gggtgagtaa 60
cacgtgggtg atctgccctg cacttcggga taagcctggg aaactgggtc taataccgga 120
taggacctcg ggatgcatgt tccggggtgg aaaggttttc cggtgcagga tgggcccgcg 180
gcctatcagc ttgttggtgg ggtaacggcc caccaaggcg acgacgggta gccggcctga 240
gagggcgacc ggccacactg ggactgagac acggcccaga ctcctacggg aggcagcagt 300
ggggaatatt gcacaatggg cgcaagcctg atgcagcgac gccgcgtgag ggatgacggc 360
cttcgggttg taaacctctt tcagtaccga cgaagcgcaa gtgacggtag gtacagaaga 420
agcaccggcc aactacgtgc cagcagccgc ggtaatacgt agggtgcgag cgttgtccgg 480
aattactggg cgtaaagagc tcgtaggcgg tttgtcgcgt cgtctgtgaa aacccgcagc 540
tcaactgcgg gcttgcaggc gatacgggca gacttgagta ctgcagggga gactggaatt 600
cctggtgtag cggtgaaatg cgcagatatc aggaggaaca ccggtggcga aggcgggtct 660
ctgggcagta actgacgctg aggagcgaaa gcgtgggtag cgaacaggat tagataccct 720
ggtagtccac gccgtaaacg gtgggcgcta ggtgtgggtt tccttccacg ggatccgtgc 780
cgtagctaac gcattaagcg ccccgcctgg ggagtacggc cgcaaggcta aaactcaaag 840
gaattgacgg gggcccgcac aagcggcgga gcatgtggat taattcgatg caacgcgaag 900
aaccttacct gggtttgaca tacaccggac cgccccagag atggggtttc ccttgtggtc 960
ggtgtacagg tggtgcatgg ctgtcgtcag ctcgtgtcgt gagatgttgg gttaagtccc 1020
gcaacgagcg caacccttgt cctgtgttgc cagcacgtaa tggtggggac tcgcaggaga 1080
ctgccggggt caactcggag gaaggtgggg acgacgtcaa gtcatcatgc cccttatgtc 1140
cagggcttca cacatgctac aatggccggt acagagggct gcgataccgc gaggtggagc 1200
gaatccctta aagccggtct cagttcggat cggggtctgc aactcgaccc cgtgaagtcg 1260
gagtcgctag taatcgcaga tcagcaacgc tgcggtgaat acgttcccgg gccttgtaca 1320
caccgcccgt cacgtcatga aagtcggtaa cacccgaagc cggtggccta acccctcgtg 1380
ggagggag 1388
Claims (8)
1. a pyridine degradation bacterium strain strain a5, is identified as Rhodococcus ruber (Rhodococcus ruber), has been preserved in Chinese micro-life
Thing culture presevation administration committee's common micro-organisms center, the preservation time is on August 31st, 2016, and deposit number is CGMCC
NO.12919。
2. the application in degraded pyridine of the bacterial strain a5 described in claim 1.
Application the most according to claim 2, it is characterised in that bacterial strain a5 degraded soil or water body environment in pyridine should
With.
4. the application in producing pyridine degradable microbial inoculum of the bacterial strain a5 described in claim 1.
5. one kind utilizes the degradation bacterial agent that bacterial strain a5 described in claim 1 produces.
Degradation bacterial agent the most according to claim 5, it is characterised in that made by following steps:
1) the test tube liquid that degradation bacteria strains a5 cultivates logarithmic (log) phase is inoculated in fermentation culture by the inoculum concentration of 0.5-1.5% volume ratio
In base, shaken cultivation, to logarithmic (log) phase, prepares fermented bacterium；
2) above-mentioned prepared fermented bacterium is inoculated by the inoculum concentration of 5-10% volume ratio in the culture medium of seed tank, cultivate to right
Number trophophase, prepares seed liquor；
3) inoculum concentration that seed liquor is pressed 8-10% volume ratio accesses cultivation in the culture medium producing tank；
4) in the incubation of seed tank and production tank, the ventilation of filtrated air is 1:(0.8-1.5), mixing speed is
180-250rpm, cultivation temperature is 25-32 DEG C, and whole process incubation time is 36-48 hour, and it is straight that the rear culture fluid that fermented goes out tank
Connect and be distributed into liquid agent by plastic barrel or Packaging Bottle.
7. the application in degraded pyridine of the degradation bacterial agent described in claim 5.
Application the most according to claim 7, it is characterised in that described degradation bacterial agent is pyrrole in degraded soil or water body environment
The application of pyridine.
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