CN109609414A - One plant of uns-dimethylhydrazine degradation bacteria strains WP52 and its application - Google Patents
One plant of uns-dimethylhydrazine degradation bacteria strains WP52 and its application Download PDFInfo
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Abstract
Cold pseudomonad (Pseudomonas extremaustralis) WP52 is fitted the present invention relates to one plant of uns-dimethylhydrazine that can degrade, belongs to microorganisms technical field.The bacterial strain is stored in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number on June 11st, 2018 are as follows: CGMCC No.15921.Bacterial strain WP52 Suitable sites range of the invention is wide, easily cultivates, all has degradation to basic, normal, high concentration uns-dimethylhydrazine, high-efficient, at low cost, can be applied to the biological prosthetic of uns-dimethylhydrazine polluted-water and soil, achievees the purpose that uns-dimethylhydrazine of thoroughly degrading.
Description
Technical field
The present invention relates to one plant of uns-dimethylhydrazine degradation bacteria strains and its applications, belong to microorganisms technical field.
Background technique
Uns-dimethylhydrazine (UDMH), molecular formula C2H8N2, it is a kind of high-energy fuel, is widely used in guided missile, satellite and flies
In the transmitting of the spacecrafts such as ship.Uns-dimethylhydrazine produces in the careless discharge and emission process during production, transport or storage
Raw a large amount of waste water, can all pollute the environment.There is stringent limitation in China to the safe level of uns-dimethylhydrazine in the environment.
The processing method of uns-dimethylhydrazine mainly has physics, chemistry and biological method in environment.Physics and chemical method exist
Processing is not thorough, at high cost or there are secondary pollution problems.Biological method has many advantages, such as efficient, low consumption, environmentally protective, is
A kind of extensive processing method of application future.Currently, the research report both at home and abroad in relation to uns-dimethylhydrazine biodegrade aspect is less,
Be related to capable of degrading uns-dimethylhydrazine microbial strains it is then less, and mainly around middle low concentration (≤50mg/L) uns-dimethylhydrazine
Biodegrade.The present invention will provide a kind of microbial strains that can have degradation to basic, normal, high concentration uns-dimethylhydrazine
WP52 and its application.
Summary of the invention
The purpose of the present invention is to provide the bacterial strain WP52 of one plant of uns-dimethylhydrazine that can degrade and its applications.
Uns-dimethylhydrazine degradation bacteria strains WP52 provided by the present invention, screening technique is: soil sample near acquisition launching site, addition
It is 28-37 DEG C in temperature into the inorganic salt liquid culture medium of uns-dimethylhydrazine concentration 40-300mg/L, revolving speed 150-200r/
After cultivating 2-7d under the conditions of min, with the scribing line point of beef-protein medium, minimal medium and solid potato culture medium
From, temperature be 28-37 DEG C at cultivate 2-7d.Then, isolated bacterial strain is crossed purifying on above-mentioned solid medium, is obtained
To purifying bacterial strain.Meanwhile using method of dilution butteron on plate, suspension coated plate directly is made with soil sample, separates according to the method described above and pure
Change bacterial strain.
Purifying is obtained into bacterial strain, is inoculated in the inorganic salts that uns-dimethylhydrazine is only nitrogen source, concentration is 50-300mg/L respectively
It is 28-37 DEG C in temperature in fluid nutrient medium, revolving speed is cultivated under the conditions of being 150-200r/min, is periodically sampled, is measured inclined diformazan
Hydrazine content, and calculate degradation rate.The high bacterial strain of degradation rate is chosen, thus screening obtains uns-dimethylhydrazine degradation bacteria strains WP52.
Minimal medium: K2HPO41.000-3.000g/L KH2PO41.000-3.000g/L NaCl 5.000-
8.000g/L MgSO40.200-0.800g/L, CaCl20.010-0.050g/L, FeSO40.005-0.010g/L, H3BO3
0.100-0.200mg/L, ZnSO40.100-0.200mg/L, uns-dimethylhydrazine 40-300mg/L, glucose 0.0-5.000g/L.
Solid medium adds agar 10.0-15.0g/L.
Bacterial strain WP52 provided by the present invention is extracted, PCR amplification by 16S rDNA, sequencing, sequence alignment, chadogram
Building, and combining form observation of characteristics determine that the bacterial strain is that Pseudomonas extremaustralis (fits cold false unit cell
Bacterium), and be named as and fit cold pseudomonad WP52 (Pseudomonas extremaustralis WP52).The bacterial strain is in 2018
On June 11, in is stored in China Committee for Culture Collection of Microorganisms's common micro-organisms center (abbreviation CGMCC), deposit number
Are as follows: CGMCC No.15921, depositary institution address are as follows: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, the micro- life of the Chinese Academy of Sciences
Object research institute.
Bacterial strain WP52 in the present invention, crosses on beef extract-peptone solid medium, is placed under the conditions of 28 DEG C -35 DEG C
2-3d is cultivated, colony diameter 0.8-1.3mm, colonial morphology is creamy white, opaque, protuberance, and surface and the smooth of the edge have light
Pool, round, odorless, tasteless.It is bacillus that microscopic morphology, which is viewed as bacterial strain WP52,6-14 μm long, 3-4 μm wide.
Bacterial strain WP52 of the present invention, is inoculated into beef extract-peptone fluid nutrient medium and cultivates, and is 28- in cultivation temperature
35 DEG C, revolving speed 150-180r/min enters growth logarithmic phase for 10 hours, is later stationary phase, under growth starts after 18 hours
Drop.
Bacterial strain WP52 of the present invention, growth Bioclimatic analysis is wide, trains in beef-protein medium, potato glucose
Support base, minimal medium and using uns-dimethylhydrazine to grow in the minimal medium of only nitrogen source.
Bacterial strain WP52 of the present invention, in minimal medium that pH be 3.0-11.0 equal energy wide to pH adaptation range
Growth.
Bacterial strain WP52 provided by the present invention, it is wide using uns-dimethylhydrazine concentration range, can be only nitrogen source in uns-dimethylhydrazine,
It grows, cultivates 2 days, biomass OD in the minimal medium that concentration is 50mg/L-300mg/L600Value is by initial 0.42-
0.48 rises to 1.32-1.59.
Bacterial strain WP52 provided by the present invention all has degradation to the uns-dimethylhydrazine of high, medium and low concentration, when inclined two
When methylhydrazine concentration≤100mg/L, 3d degradation rate can reach 100%.
The acquisition of bacterial strain WP52 and its culture provided by the present invention and degradation to uns-dimethylhydrazine.Bacterial strain WP52 and
Its culture medium, including fermentation liquid, metabolite, object intracellular, object extract intracellular and thallus etc. have drop to uns-dimethylhydrazine
Solve function, can directly using or be made microbial inoculum or preparation, the biology drop for uns-dimethylhydrazine in soil, water body and other environment
Solution.
The beneficial effects of the present invention are:
1. bacterial strain WP52 Bioclimatic analysis provided by the invention is wider, easily cultivates, can be grown in a variety of culture mediums, such as in ox
Meat extract peptone culture medium, potato culture can be grown in minimal medium.Bacterial strain WP52 is adaptable to pH,
PH is that the range of 3.0-11.0 can be grown.
2. bacterial strain WP52 provided by the invention can be grown in the uns-dimethylhydrazine culture solution of high, medium and low concentration, when inclined two
When methylhydrazine is only nitrogen source, concentration is 50mg/L-300mg/L, strain growth is preferable.
3. bacterial strain WP52 provided by the invention, spectrum width of degrading all have degradation to the uns-dimethylhydrazine of high, medium and low concentration and make
With as uns-dimethylhydrazine concentration≤100mg/L, 3d degradation rate is up to 100%.
4, bacterial strain WP52 provided by the invention, bacterial strain and its culture, including fermentation liquid, metabolite liquid, object intracellular
Extracting solution, which must wait, has degradation to uns-dimethylhydrazine.
5. bacterial strain WP52 provided by the invention and its culture, can directly using or microbial inoculum be made be applied to repair by inclined two
Water body and soil of methylhydrazine pollution etc., simple process is at low cost, high-efficient.
Detailed description of the invention
Fig. 1 is the colonial morphology figure of bacterial strain WP52.
Fig. 2 is the micro- scanning electron microscope (SEM) photograph of bacterial strain WP52.
Fig. 3 is the phylogenetic tree of bacterial strain WP52.
Fig. 4 is bacterial strain WP52 growth curve in beef-protein medium.
Fig. 5 is increment of the bacterial strain WP52 in various concentration uns-dimethylhydrazine culture medium.
Fig. 6 is degradation of the bacterial strain WP52 to various concentration uns-dimethylhydrazine.
Fig. 7 is degradation of the bacterial strain WP52 culture component to uns-dimethylhydrazine.
Specific embodiment
Specific embodiment below is to be described in further detail to technical solution of the present invention, but the present invention does not limit to
In act specific embodiment set forth below.
Embodiment 1: the screening and identification of bacterial strain WP52
1, bacterial strain screening
(1) strain isolation and purifying
Soil sample around launching site is acquired, by soil sample according to 5% ratio, being added separately to uns-dimethylhydrazine concentration is 40-
In the inorganic salt liquid culture medium of 300mg/L, being placed in temperature is 28-37 DEG C, and revolving speed cultivates 2- under the conditions of being 150-200r/min
After 7d, separation of crossing in beef extract-peptone, inorganic salts and solid potato culture medium is cultivated at being 28-37 DEG C in temperature
Isolated bacterial strain is continued to continue to draw plate in above-mentioned plate, until obtaining purifying bacterial strain by 2-7d.
At the same time, soil sampling, according to method of dilution butteron on plate, prepare soil sample: physiological saline is the suspension of 1:9, and with giving birth to
Reason salt water continues dilution and obtains 10-1-10-3Suspension shakes 10-30 minutes, 100ul is taken to be respectively coated on uns-dimethylhydrazine content
It is 28-37 in temperature in the minimal medium of 40mg/L, 50mg/L, 100mg/L, 150mg/L, 200mg/L, 300mg/L
2-7d, the bacterial strain grown on daily isolation medium are cultivated under the conditions of DEG C, and is crossed on inorganic salts solid medium, are obtained pure
Change bacterial strain.
By above-mentioned isolation and purification method, obtain in uns-dimethylhydrazine concentration being that 40-300mg/L cultivates basal growth bacterial strain 30
Strain.
(2) uns-dimethylhydrazine degradation bacteria strains screen
By above-mentioned 30 plants of isolated bacterial strains, 12h-48h is activated with beef extract-peptone fluid nutrient medium.By 2%-
10% inoculum concentration be inoculated in respectively uns-dimethylhydrazine be only nitrogen source, concentration 40mg/L, 50mg/L, 100mg/L, 150mg/L,
It is 28-37 DEG C in temperature, shaking speed is 150-200r/min item in the inorganic salt liquid culture medium of 200mg/L, 300mg/L
Under part, 3-4d is cultivated, is sampled daily, measure and calculate each bacterial strain to the degradation rate of uns-dimethylhydrazine, chooses the high bacterial strain of degradation rate,
Screening obtains bacterial strain WP52.
Beef-protein medium: beef extract 5.0g, peptone 10.0g, NaCl 5.0g, distilled water 1L, 121 DEG C go out
Bacterium 15min.Solid medium adds agar 10.0-15.0g.
Potato dextrose medium (PDA): potato 200g, glucose 20g, distilled water 1L, pH are naturally, 115 DEG C go out
Bacterium 30min.Solid medium adds agar 10.0-15.0g.
Minimal medium: K2HPO41.000-3.000g/L KH2PO41.000-3.000g/L NaCl 5.000-
8.000g/L MgSO40.200-0.800g/L, CaCl20.010-0.050g/L, FeSO40.005-0.010g/L, H3BO3
0.100-0.200mg/L, ZnSO40.100-0.200mg/L, uns-dimethylhydrazine 40-300mg/L, glucose 0.0-5.000g/L.
Solid medium adds agar 10.0-15.0g/L.
2, strain morphology and molecular biology identification
The observation of 2.1 colony morphology characteristics
Bacterial strain WP52 crosses on beef extract-peptone solid medium, and 2-3d, bacterial strain are cultivated under the conditions of 28 DEG C -37 DEG C
The colony diameter of WP52 is 0.8-1.3mm, and bacterium colony is creamy white, and is swelled, and surface is smooth, glossy, round, the smooth of the edge, no
It is transparent, odorless, tasteless.The colonial morphology of bacterial strain WP52 is shown in Fig. 1.It is aobvious that bacterial strain WP52 is observed by scanning electron microscope (Phenom Pro)
Titanium miniplate learns that the bacterium is bacillus, 6-14 μm long, 3-4 μm wide, sees Fig. 2.
The molecular biology identification of 2.2 bacterial strain WP52
2.2.1 the extraction of genome
The DNA of bacterial strain WP52 extracts the CTAB/NaCl method that uses, specific as follows:
(1) bacterial strain activates: bacterial strain WP52 is seeded in beef extract-peptone fluid nutrient medium, and 28-37 DEG C, 150-180r/
1-2d is cultivated under the conditions of min.
(2) it takes 1.5ml culture 12000rpm to be centrifuged 2min, removes supernatant.
(3) 0.5mlTE buffer is added in sediment, blows and beats repeatedly, and 12000rpm is centrifuged 2min, abandons supernatant.
(4) the TE buffer of 0.5ml is added, is allowed to suspend again, the Proteinase K of 30 μ l 10%SDS and 15 μ l is added,
It mixes, in 37 DEG C of incubation 1h.
(5) 100 μ l 5mol/L NaCl are added, mix well, 80 μ l CTAB/NaCl solution are added, mix, 65 DEG C of temperature
Educate 10min.
(6) it is placed in ice and is cooled to room temperature, isometric phenol/chloroform/isoamyl alcohol is added and mixes (25:24:1), it is sufficiently mixed
It is even, 4 DEG C, 10000g, it is centrifuged 10min, takes supernatant into new EP pipe.
(7) isometric chloroform/isoamyl alcohol (24:1) is added, mixes well, 4 DEG C, 10000g, is centrifuged 10min, takes supernatant
Liquid is into new EP pipe.
(8) the pre- cold isopropanol of 0.6-0.8 times of volume and the NaAc (3mol/L) of 1/10 volume is added, is gently mixed, puts
Enter -20 DEG C, precipitate 30-60min, 4 DEG C, 12000rpm is centrifuged 5-10min, abandons supernatant.
(9) with after 70% ethanol washing of 1ml pre-cooling, 4 DEG C, 12000rpm is centrifuged 5min and abandons ethyl alcohol, in clean bench
In it is slightly dry, be dissolved in 20 μ l μ l TE buffers.
(10) it is detected with 1.0% agarose gel electrophoresis.
2.2.2PCR amplification
Using bacterial universal primers 27F (GCATATCAATAAGCGGAGGAAAAG) and 1492R
(GGTCCGTGTTTCAAGACGG), PCR sequence is carried out to the 16SrDNA gene of bacterial strain and expands sequence.PCR amplification is reacted using 50 μ L
System: 5 μ 10 × PCR of L Buffer, 1 μ L dNTP mix, 2 μ L DNA profilings, primer 2 7F4 μ l, 1492R4 μ l, TaqDNA are poly-
0.25 33.75 μ l of μ l, ddH2O of synthase.
PCR reaction condition: 95 DEG C of 5min, circulation primary；95 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 1min are recycled 35 times；72℃
7min.Pcr amplification product is detected with 1.0% agarose gel electrophoresis.
2.2.3 sequencing and phylogenetic tree building
The 16SrDNA sequence that amplification is obtained send Beijing promise match genome research center sequencing, obtains bacterial strain WP52's
16SrDNA gene order is as follows:
By above-mentioned sequencing result and website https: gene order compares in //www.ezbiocloud.net/, finds bacterium
The gene order of the 16SrDNA of strain WP52 and the sequence homology of some bacterial strains in Pseudomonas extremaustralis
Property is 99.78%.According to comparison result, the gene order of the close bacterial strain of similarity is selected in EZbiocloud, is used
Clustalx software carries out sequence alignment, and with MEGA6.0 software building phylogenetic tree, sees Fig. 3.
3, bacterial strain WP52 is accredited as new function bacterial strain
According to bacterial strain WP52 colony morphology characteristic and molecular biology identification as a result, determining that bacterial strain WP52 is to fit cold false unit cell
Bacterium (Pseudomonas extremaustralis), and be named as and fit cold pseudomonad WP52 (Pseudomonas
extremaustralis WP52).The bacterial strain is stored in Chinese microorganism strain preservation conservator on June 11st, 2018
Meeting common micro-organisms center (abbreviation CGMCC), deposit number are CGMCC No.15921.
Currently, there is no literature reported on fit cold pseudomonad (Pseudomonas extremaustralis) to have both at home and abroad
Degradation uns-dimethylhydrazine function, therefore, bacterial strain WP52 are one plant of new function bacterial strains with degradation uns-dimethylhydrazine.
2 bacterial strain WP52 growth characteristics of embodiment
1, bacterial strain WP52 is grown in different medium
Uns-dimethylhydrazine degradation bacteria strains WP52 of the present invention can be grown in a variety of culture mediums, as beef extract-peptone is trained
Supporting can grow in the minimal medium that base, potato culture, minimal medium and uns-dimethylhydrazine are only nitrogen source,
Culture is easy.
Minimal medium: K2HPO41.000-3.000g/L KH2PO41.000-3.000g/L NaCl 5.000-
8.000g/L MgSO40.200-0.800g/L, CaCl20.010-0.050g/L, FeSO40.005-0.010g/L, H3BO3
0.100-0.200mg/L, ZnSO40.100-0.200mg/L, uns-dimethylhydrazine 40-300mg/L, glucose 0.500-5.000g/
L。
2, bacterial strain WP52 growth curve
By bacterial strain WP52 according to 3-5% inoculum concentration, it is seeded to the 100mL equipped with 50mL beef extract-peptone fluid nutrient medium
It is 150-180r/min in revolving speed in triangular flask, under conditions of 28-32 DEG C of cultivation temperature, cultivates 24 hours, samples and survey every 2h
Measure OD600, growth curve is drawn with Excel, sees Fig. 4, is later stationary phase into logarithmic growth phase after strain growth 10h,
16-18d biomass reaches maximum, grows and begins to decline after 18 hours.
3, bacterial strain WP52 is to pH value adaptability
By bacterial strain WP52 according to 3-5% inoculum concentration, be inoculated in respectively uns-dimethylhydrazine content be 100mg/L, pH value 3.0,
It in 5.0,7.0,9.0,11.0 inorganic salt liquid culture medium, is cultivated under the conditions of temperature is 20 DEG C -37 DEG C, finds WP52 in pH
It can be grown in the culture medium for being 3.0,5.0,7.0,9.0,11.0, at the 3rd day of culture, OD600It can achieve 0.90-1.36.
4, bacterial strain WP52 is grown in various concentration uns-dimethylhydrazine culture medium
Bacterial strain activation: bacterial strain WP52 is inoculated in beef extract-peptone fluid nutrient medium, is 28-37 DEG C in temperature, revolving speed
Under the conditions of 160-200r/min, shaken cultivation 12-24h.
Inoculation and culture: by the WP52 bacterium solution of above-mentioned activation, it is inoculated in uns-dimethylhydrazine respectively according to 2%-10% inoculum concentration
Be respectively 50 for only nitrogen source, content, 150,200,250, in the inorganic salt liquid culture medium of 300mg/L, in temperature be 28-37
DEG C, revolving speed is to cultivate 3 days in 160-200r/min isothermal vibration incubator.Daily sampling and measuring OD600, drawing bacterial strain WP52 exists
Uns-dimethylhydrazine is the biomass (OD under only nitrogen source and concentration different condition600), as a result see Fig. 5.
As shown in Figure 5, bacterial strain WP52 is in the inorganic salts culture that uns-dimethylhydrazine is only nitrogen source and concentration is 50-300mg/L
In base, can it grow, at the 2nd day of culture, bacterial strain biomass OD600Value rises to 1.32-1.59 by initial 0.42-0.48,
Illustrate that bacterial strain WP52 can be grown using the uns-dimethylhydrazine of high, medium and low concentration, it is wide using uns-dimethylhydrazine concentration range.
Minimal medium: K2HPO41.000-3.000g/L KH2PO41.000-3.000g/L NaCl 5.000-
8.000g/L MgSO40.200-0.800g/L, CaCl20.010-0.050g/L, FeSO40.005-0.010g/L, H3BO3
0.100-0.200mg/L, ZnSO40.100-0.200mg/L, uns-dimethylhydrazine 40-300mg/L, glucose 0.500-5.000g/
L。
Degradation characteristic of the 3 bacterial strain WP52 of embodiment to uns-dimethylhydrazine
1, degradation of the bacterial strain to various concentration uns-dimethylhydrazine
It is high, medium and low 3 different gradient concentrations that uns-dimethylhydrazine concentration, which is arranged, carries out degradation experiment.Wherein uns-dimethylhydrazine is high
Concentration is >=100mg/L, and middle concentration is < 100mg/L and > 40mg/L, and low concentration is≤40mg/L.
Bacterial strain WP52 activation: method is the same as above-mentioned 4.
Inoculation and degradation: activation bacterium solution is pressed into 2-10% inoculum concentration, being inoculated in uns-dimethylhydrazine respectively is only nitrogen source, concentration
Respectively in the inorganic salt liquid culture medium of 40mg/L, 60mg/L, 80mg/L, 100mg/L, 120mg/L, being subsequently placed in temperature is
28-37 DEG C, revolving speed is to cultivate 3-4 days in the shaking table of 160-200r/min.Sampling daily, discovery bacterial strain WP52 are trained in each concentration
Supporting can grow in base.According to GB/T14376-2017, uns-dimethylhydrazine content in culture medium is measured, and based on following formula (1)
Calculate degradation rate.
Y=(X0-Xn)/X0* 100% (1)
Wherein Y is uns-dimethylhydrazine degradation rate %, X0For the 0th day uns-dimethylhydrazine concentration, Xn was n-th day uns-dimethylhydrazine concentration.
Bacterial strain WP52 is shown in Fig. 6 to the degradation results of various concentration uns-dimethylhydrazine.It will be appreciated from fig. 6 that WP52 is 40- to concentration
The uns-dimethylhydrazine of 120mg/L all has efficient degradation effect.2nd day is the inclined diformazan of 40,60,80,100 and 120mg/L to concentration
The degradation rate of hydrazine can reach 90% or more, and for concentration≤100mg/L uns-dimethylhydrazine, degradation rate can reach 100% within the 3rd day；
It is 120mg/L experimental group for concentration, 3d degradation rate can reach 97-99%, and 4d can be degradable.
Minimal medium: K2HPO41.000-3.000g/L KH2PO41.000-3.000g/L NaCl 5.000-
8.000g/L MgSO40.200-0.800g/L, CaCl20.010-0.050g/L, FeSO40.005-0.010g/L, H3BO3
0.100-0.200mg/L, ZnSO40.100-0.200mg/L, uns-dimethylhydrazine 40-300mg/L, glucose 0.500-5.000g/
L。
2, the degradation of bacterial strain WP52 and its culture to uns-dimethylhydrazine
Strain culturing: bacterial strain WP52 is inoculated in beef extract fluid nutrient medium, is 28-37 DEG C in temperature, revolving speed is
12h-72h is cultivated under 160-200r/min oscillating condition, obtains fermentation liquid A.Take fermentation liquid 2-3mL, 5000-9000r/min,
Centrifugation (or using filter method), collects supernatant, obtains metabolite liquid B under the conditions of 4 DEG C.At the same time, acquisition centrifugation (or
Filtering) sediment, obtain thallus.By oscillation washing repeatedly, 5000-9000r/ in the phosphate buffer of thallus merging 0.05M
It is centrifuged 10min under the conditions of min, takes precipitating thallus, 0.05M phosphate buffer is added, bacteria suspension is made, with JY92- II N ultrasound
Wave biomixer (Ningbo Xin Yi ultrasonic device Co., Ltd) interval is crushed somatic cells 8-12min (300W, 1S/4S), so
Afterwards under the conditions of 4 DEG C, 5000-9000r/min is centrifuged 10min, takes supernatant, obtains object intracellular and its object extracting solution C intracellular.
According to 2-10% inoculum concentration, bacterial strain fermentation liquor A, metabolite liquid B and object extracting solution C intracellular are inoculated into partially respectively
Dimethylhydrazine content is 3 repetitions of each experimental setup in 100mg/L minimal medium.Inoculation is placed on 28-37 DEG C, 160-
In 200r/min isothermal vibration incubator, cultivate 3 days.Sampling daily measures uns-dimethylhydrazine according to GB/T14376-2017 method
Content calculates uns-dimethylhydrazine degradation rate according to preceding formula (1), obtains in bacterial strain WP52 culture main component to uns-dimethylhydrazine
Degradation rate, see Fig. 7.
It, can inclined to 100mg/L two the results show that A, B, component C in bacterial strain WP52 culture, contain active material
Methylhydrazine is degraded.Bacterial strain WP52 and its culture, including fermentation liquid, metabolite, object intracellular, object extract intracellular and bacterium
Body etc. has degradation function to uns-dimethylhydrazine, can directly using or be prepared into microbial inoculum or preparation, for soil, water body and
The degradation treatment of uns-dimethylhydrazine in other environment.
Sequence table
<110>Beijing Institute of Technology
<120>one plants of uns-dimethylhydrazine degradation bacteria strains WP52 and its applications
<130> 1
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1354
<212> DNA
<213> Pseudomonas extremaustralis
<400> 1
tgcttctctt gagagcggcg gacgggtgag taatgcctag gaatctgcct ggtagtgggg 60
gataacgttc ggaaacggac gctaataccg catacgtcct acgggagaaa gcaggggacc 120
ttcgggcctt gcgctatcag atgagcctag gtcggattag ctagttggtg aggtaatggc 180
tcaccaaggc gacgatccgt aactggtctg agaggatgat cagtcacact ggaactgaga 240
cacggtccag actcctacgg gaggcagcag tggggaatat tggacaatgg gcgaaagcct 300
gatccagcca tgccgcgtgt gtgaagaagg tcttcggatt gtaaagcact ttaagttggg 360
aggaagggca gttacctaat acgtgattgt tttgacgtta ccgacagaat aagcaccggc 420
taactctgtg ccagcagccg cggtaataca gagggtgcaa gcgttaatcg gaattactgg 480
gcgtaaagcg cgcgtaggtg gttagttaag ttggatgtga aatccccggg ctcaacctgg 540
gaactgcatt caaaactgac tgactagagt atggtagagg gtggtggaat ttcctgtgta 600
gcggtgaaat gcgtagatat aggaaggaac accagtggcg aaggcgacca cctggactga 660
tactgacact gaggtgcgaa agcgtgggga gcaaacagga ttagataccc tggtagtcca 720
cgccgtaaac gatgtcaact agccgttggg agccttgagc tcttagtggc gcagctaacg 780
cattaagttg accgcctggg gagtacggcc gcaaggttaa aactcaaatg aattgacggg 840
ggcccgcaca agcggtggag catgtggttt aattcgaagc aacgcgaaga accttaccag 900
gccttgacat ccaatgaact ttctagagat agattggtgc cttcgggaac attgagacag 960
gtgctgcatg gctgtcgtca gctcgtgtcg tgagatgttg ggttaagtcc cgtaacgagc 1020
gcaacccttg tccttagtta ccagcacgtt atggtgggca ctctaaggag actgccggtg 1080
acaaaccgga ggaaggtggg gatgacgtca agtcatcatg gcccttacgg cctgggctac 1140
acacgtgcta caatggtcgg tacagagggt tgccaagccg cgaggtggag ctaatcccac 1200
aaaaccgatc gtagtccgga tcgcagtctg caactcgact gcgtgaagtc ggaatcgcta 1260
gtaatcgcga atcagaatgt cgcggtgaat acgttcccgg gccttgtaca caccgcccgt 1320
cacaccatgg gagtgggttg caccagaagt agct 1354
Claims (10)
1. one plant of uns-dimethylhydrazine degradation bacteria strains WP52 and its application, it is characterised in that: through observation of morphological characteristics with molecular biosciences
Identification is learned, determines that bacterial strain WP52 is Pseudomonas extremaustralis (fitting cold pseudomonad), and be named as and fit cold vacation
Monad WP52 (Pseudomonas extremaustralis WP52)；During the bacterial strain has been stored on June 11st, 2018
State's Microbiological Culture Collection administration committee common micro-organisms center (CGMCC), deposit number are as follows: CGMCC No.15921, preservation
Unit address are as follows: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica.
2. one plant of uns-dimethylhydrazine degradation bacteria strains WP52 according to claim 1, it is characterised in that: the screening side bacterial strain WP52
Method, which comprises acquisition soil sample is added in the inorganic salt liquid culture medium of uns-dimethylhydrazine concentration 40-300mg/L,
Temperature is 28-37 DEG C, after revolving speed cultivates 2-7d under the conditions of being 150-200r/min, with beef-protein medium, inorganic salts
Culture medium and solid potato culture medium scribing line separation, cultivate 2-7d at being 28-37 DEG C in temperature, then, by isolated bacterium
Strain is crossed on above-mentioned solid medium and is purified, and purifying bacterial strain is obtained；Meanwhile using method of dilution butteron on plate, directly it is made of soil sample
Suspension coated plate, scribing line isolates and purifies bacterial strain according to the method described above, obtains purifying bacterial strain；
Purifying is obtained into bacterial strain, is inoculated in the inorganic salt liquid that uns-dimethylhydrazine is only nitrogen source, concentration is 50-300mg/L respectively
It is 28-37 DEG C in temperature in culture medium, revolving speed is cultivated under the conditions of being 150-200r/min, and uns-dimethylhydrazine content is measured by sampling in the phase,
And degradation rate is calculated, the high bacterial strain of degradation rate is chosen, screening obtains bacterial strain WP52；
Minimal medium: K2HPO41.000-3.000g/L KH2PO41.000-3.000g/L NaCl 5.000-8.000g/
L, MgSO40.200-0.800g/L, CaCl20.010-0.050g/L, FeSO40.005-0.010g/L, H3BO3 0.100-
0.200mg/L, ZnSO40.100-0.200mg/L, uns-dimethylhydrazine 40-300mg/L, glucose 0.0-5.000g/L, solid training
It supports base and adds agar 10.0-15.0g/L.
3. one plant of uns-dimethylhydrazine degradation bacteria strains WP52 according to claim 1, it is characterised in that: bacterial strain WP52 growth and benefit
Wide with uns-dimethylhydrazine concentration range, bacterial strain WP52 can be grown in a variety of culture mediums, such as beef-protein medium, Ma Ling
Potato dextrose culture-medium, minimal medium, and in the nothing that uns-dimethylhydrazine is only nitrogen source, concentration is 40mg/L-300mg/L
It is grown in machine salt culture medium；Bacterial strain WP52 can be grown in the minimal medium that pH value is 3.0-11.0, adapt to model to pH value
Enclose width.
4. one plant of uns-dimethylhydrazine degradation bacteria strains WP52 according to claim 1, it is characterised in that: the culture of bacterial strain WP52
Object, signified culture include fermentation liquid, metabolite, object intracellular, object extracting solution intracellular and thallus.
5. one plant of uns-dimethylhydrazine degradation bacteria strains WP52 according to claim 1, it is characterised in that: bacterial strain WP52 culture is
The bacteria agent of active component is including at least one kind of in fermentation liquid, metabolite, object intracellular, object extracting solution intracellular and thallus
The bacteria agent of active component.
6. the application of one plant of uns-dimethylhydrazine degradation bacteria strains WP52 according to claim 1, it is characterised in that: bacterial strain WP52 exists
Application in uns-dimethylhydrazine degradation.
7. the application of bacterial strain WP52 according to claim 6, it is characterised in that: bacterial strain WP5 bacterial strain is applied to uns-dimethylhydrazine
Degradation, application method: (1) bacterial strain activates: it is activated with beef-protein medium, is 28-37 DEG C in temperature, revolving speed 160-
Under the conditions of 200r/min, 12h-24h is cultivated；(2) inoculation and degradation: activation bacterium solution is taken, by 2-10% inoculum concentration, is inoculated in respectively
Uns-dimethylhydrazine is that only nitrogen source, content are respectively in the inorganic salt liquid culture medium of 40-120mg/L, in temperature be 28-37 DEG C,
Revolving speed is to cultivate in 160-200r/min shaking table, period sampling measuring uns-dimethylhydrazine content；Inclined for concentration≤100mg/L two
Methylhydrazine, degradation rate can reach 100% within the 3rd day；
Inorganic salt liquid culture medium: K2HPO4 1.000-3.000g/L, KH2PO4 1.000-3.000g/L, NaCl 5.000-
8.000g/L, MgSO4 0.200-0.800g/L, CaCl2 0.010-0.050g/L, FeSO4 0.005-0.010g/L, H3BO3
0.100-0.200mg/L, ZnSO4 0.100-0.200mg/L, uns-dimethylhydrazine 40-300mg/L, glucose 0.500-5.000g/
L。
8. the application of bacterial strain WP52 according to claim 6, it is characterised in that: bacterial strain WP52 and its culture are applied to partially
The degradation of Dimethylhydrazine；Signified culture includes in fermentation liquid, metabolite, object intracellular, object extracting solution intracellular and its thallus
At least one, the degradation applied to uns-dimethylhydrazine.
9. the application of bacterial strain WP52 according to claim 6, it is characterised in that: bacterium made of bacterial strain WP52 and its culture
Agent or preparation, the degradation applied to uns-dimethylhydrazine；Signified culture includes that fermentation liquid, metabolite, object intracellular, object intracellular mention
Take at least one of liquid and thallus.
10. one plant of uns-dimethylhydrazine degradation bacteria strains WP52 according to claim 1 and its application, it is characterised in that: bacterial strain and
Its culture, including fermentation liquid, metabolite, object intracellular, object extracting solution and thallus intracellular preparation method, pass through following steps
Obtain: strain culturing, centrifugation or filtering, the operating process such as bacterial cell disruption, extraction obtain.
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