CN106754528A - A kind of Pseudomonas geniculate and its application - Google Patents
A kind of Pseudomonas geniculate and its application Download PDFInfo
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- CN106754528A CN106754528A CN201611244111.2A CN201611244111A CN106754528A CN 106754528 A CN106754528 A CN 106754528A CN 201611244111 A CN201611244111 A CN 201611244111A CN 106754528 A CN106754528 A CN 106754528A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- C12R2001/38—Pseudomonas
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- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
- C02F3/344—Biological treatment of water, waste water, or sewage characterised by the microorganisms used for digestion of mineral oil
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/30—Organic compounds
- C02F2101/32—Hydrocarbons, e.g. oil
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Abstract
The invention belongs to technical field of bioengineering, a kind of Pseudomonas geniculate is specifically disclosed, China Committee for Culture Collection of Microorganisms's common micro-organisms center was preserved on 09 12nd, 2016, preserving number is CGMCC No.13004；It is named as Pseudomonas geniculate (Pseudomonas geniculata) 3.Also disclose the Pseudomonas geniculate (Pseudomonas geniculata) 3 application in degrading crude oil polluted-water.The Pseudomonas geniculate that the present invention is provided is not only easy to large-scale production, and degradation efficiency is high, and environmental suitability is strong.The bacterium can crude oil concentration up to 1mL/100mL crude oil pollution water body in embody preferably degradation effect, realize the repair process of crude oil pollution water body, crude oil pollution waste water it is biological prosthetic in present good application prospect.
Description
Technical field
The present invention relates to technical field of bioengineering, and in particular to a kind of Pseudomonas geniculate and its application.
Background technology
Crude oil is crude oil, is a kind of sticky black that natural organic substance is formed by geology transition
Or dark-brown liquid or solid, underground or seabed are generally present in mostly, and with penetrating odor, main component
It is chain compound that hydrocarbon atom is combined to form, it has greatly harm to water body environment.
Sustain life required minimum dissolved oxygen content about 2mg/L in water environment, after oil or oily waste water enter water body
On the one hand can swim on the water surface, the entrance of blocking oxygen, influence gas exchanges, on the other hand partly lean on oil to provide carbon source numerous
The microorganism for growing can also consume oxygen, cause the shortage of oxygen in water.Anaerobic organism can be made again simultaneously, and breeding makes water body rapidly
Water quality deterioration.The toxicity that compound fragrant hydrocarbon is produced simultaneously also influences the growth of algae, and then food chain is impacted.This
Outward, the toxic component in oil can also influence the growth and breeding of aquatile, it can attach make on the fish gill its death by suffocation or
Influenceing its ovum, polycyclic aromatic hydrocarbon can destroy the internal system of aquatile, when there is the polycyclic aromatic hydrocarbon of extremely low concentration, fish
Class can all produce fish stink, directly affects the normal existence of fish, aquatic diversity of organism in heavy damage water environment.
At present, the recovery technique to the water body of oil pollution mainly has peripheral doses technology, chemical remediation technology, photocatalysis
Recovery technique and bioremediation technology；Bioremediation technology is one has policy, the processing method of economic and technical attraction,
Be considered as most potential technical method, and progressively develop into a kind of economic benefit and environmental benefit it is all good,
Solve the maximally effective means of complex environment pollution problem.
The content of the invention
For problems of the prior art, it is an object of the invention to provide a kind of Pseudomonas geniculate, the strain
Can be used to degrade and repair crude oil pollution water body.
In order to achieve the above object, the present invention is achieved using following technical scheme.
Offer is a kind of to can be used for the Pseudomonas geniculate of degrading crude oil polluted-water, and the strain is directly added into contaminant water
In body, preferable degradation effect is reached, realize the improvement of crude oil pollution.
In order to achieve the above object, the present invention is achieved using following technical scheme.
(1) a kind of Pseudomonas geniculate, was preserved in BeiChen West Road, Chaoyang District, BeiJing City 1 on 09 12nd, 2016
No. 3 China Committee for Culture Collection of Microorganisms's common micro-organisms centers of Institute of Microorganism, Academia Sinica of institute, preservation
Number be CGMCC No.13004；It is named as Pseudomonas geniculate (Pseudomonas geniculata) 3.
The Pseudomonas geniculate is cultivated on beef-protein medium, bacterial strain 3 is in the flat of beef extract-peptone
It is in originally faint yellow on plate, with the increase of cell age, color is more and more deeper, finally in yellow.The colony characteristicses are needle point size
Faint yellow bacterium colony, in regular circle shapes, the smooth moistening in surface, neat in edge, after Gram's staining, not easy coloring.Somatic cells
In quarter butt, bar-shaped, size is 0.5 μm of (1-1.5) μ m, is Gram-negative bacteria.
The 16S rDNA sequencing results such as SEQ ID of the Pseudomonas geniculate (Pseudomonas geniculata) 3
Shown in No.1.
(2) a kind of Pseudomonas geniculate (Pseudomonas geniculata) 3 answering in degrading crude oil polluted-water
With.
Compared with prior art, beneficial effects of the present invention are：
The Pseudomonas geniculate that the present invention is provided is not only easy to large-scale production, and degradation efficiency is high, environmental suitability
By force.The Pseudomonas geniculate for being provided can large amount of organic presence under conditions of smooth growth, with crude oil as carbon source, reach compared with
Good degradation effect.The bacterium can embody preferably degradation effect in crude oil concentration reaches the crude oil pollution water body of 1mL/100mL,
Realize the repair process of crude oil pollution water body, crude oil pollution waste water it is biological prosthetic in present good application prospect.
Brief description of the drawings
The present invention is described in further details with specific embodiment below in conjunction with the accompanying drawings.
Fig. 1 is Pseudomonas geniculate of the invention (Pseudomonas geniculata) 3 in beef extract-peptone culture
Colonial morphology figure on base flat board；
Fig. 2 is Pseudomonas geniculate of the invention (Pseudomonas geniculata) 3 thalline under the microscope
Aspect graph, in figure, multiplication factor is 1000 times；
Fig. 3 is the wavelength-absorbance curve figure of I crude oil in the embodiment of the present invention 3；In figure, abscissa is wavelength, unit
It is nm, ordinate is absorbance, and unit is 1；
Fig. 4 is the canonical plotting of I crude oil in the embodiment of the present invention 3；In figure, abscissa is concentration, and unit is mg/L,
Ordinate is absorbance, and unit is 1.
Specific embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
It will be appreciated that the following example is merely to illustrate the present invention, and it is not construed as limiting the scope of the present invention.
Embodiment 1 bacterial strain is isolated and purified
(1) bacterium source
The present invention provides a kind of Pseudomonas geniculate (Pseudomonas geniculata) 3, is isolated from extending oil gathering
In seven inner hole oil recovery field oil gathering station ground crude oil contaminated soils of group, the physicochemical property of soil is：Ammonium nitrogen 15.09ppm, nitre state
Nitrogen 0.72ppm；Nitrogen content 15.81ppm, phosphorus content 82.05ppm, potassium content 17.45ppm, the content of organic matter 1.43%, pH is
7.65。
(2) bacterial strain is isolated and purified
By 10g oil-polluted soils sample add 100mL enriched liquid culture mediums in, 28 DEG C, 130r/min shaking table cultures
7d.After after nutrient solution muddiness, absorption 5mL nutrient solutions are transferred in fresh enriched medium again, identical with above-mentioned condition of culture
Continuous switching enrichment culture 3 times.The above-mentioned bacteria suspensions of 1mL are taken, after being serially diluted with sterilized water, beef extract-peptone is inoculated in respectively
In culture medium, 29 DEG C or so incubated 3d.Wherein, the composition of enriched liquid culture medium is：NaNO31.5g, (NH4)2SO4
1.5g, K2HPO41g, MgSO4·7H2O 0.5g, KCl 0.5g, FeSO4·7H2O 0.01g, CaCl20.002g, distilled water
1000mL, crude oil No. I 1% (volume ratio), pH 7.0.The composition of beef-protein medium is：Beef extract 3.0g, peptone
10.0g, NaCl 5.0g, nutrient agar 15.0g, distilled water 1000mL, pH 7.4-7.6.After flat-plate bacterial colony is efficiently separated, root
According to the difference of colony colour, morphological feature and surface moisture, plate streaking purifies and separates are carried out with transfer needle choosing colony respectively.
By inoculation in inclined-plane, 4 DEG C of refrigerator cold-storages are standby.
(3) molten scraper ring method screening oil degradation bacteria
Above-mentioned strain is inoculated on oil-containing inorganic salts solid medium, is placed in 37 DEG C of incubators and is cultivated 10-15d, seen
Examine and whether there is thermophilic scraper ring, and the oil degradation energy of bacterial strain is primarily determined that according to the ratio of thermophilic scraper ring diameter (D) and colony diameter (d)
Power；Wherein, the composition of oil-containing inorganic salts solid medium is NaNO31.5g, (NH4)2SO41.5g, K2HPO41g, MgSO4·
7H2O 0.5g, KCl 0.5g, FeSO4·7H2O 0.01g, CaCl20.002g, distilled water 1000mL, No. I 1% (volume of crude oil
Than).The oil degradation bacteria of preliminary screening is inoculated in slant medium, 4 DEG C of refrigerator cold-storages are standby.
The identification of the bacterial strain of embodiment 2
(1) morphological feature of bacterial strain
As shown in figure 1, the Pseudomonas geniculate is seen after 30 DEG C of incubated 24h on beef-protein medium
Colonial morphology is examined, originally bacterial strain 3 is in faint yellow, and with the increase of cell age, color is more and more deeper, finally in yellow.The bacterium colony is special
The faint yellow bacterium colony for needle point size is levied, in regular circle shapes, the smooth moistening in surface, neat in edge after Gram's staining, is difficult
Color.As shown in Fig. 2 somatic cells are in quarter butt, bar-shaped, size is 0.5 μm of (1-1.5) μ m, is Gram-negative bacteria.
(2) biochemical characteristic
According to《Primary Jie Shi Bacteria Identifications handbook》With《Common bacteria system identification handbook》Pseudomonas geniculate is given birth to
Reason biochemical identification, qualification result is as shown in table 1.
The bacterial strain Physiology and biochemistry qualification result of the Pseudomonas geniculate of table 1 (Pseudomonas geniculata) 3
| Index | Characteristic |
| Gram's staining | — |
| Glucose sugar | + |
| Maltose | + |
| Starch Hydrolysis | — |
| M.P (methyl red) is tested | — |
| Hydrogen sulfide | — |
| Nitrate reduction | — |
| Oxidizing ferment | + |
| Indoles | + |
| V.P (acetyl methyl carbinol) is tested | — |
| Catalase | + |
| Catalase | + |
| Gelatin liquefaction | + |
Note："-" represents that reaction is feminine gender；"+" represents that reaction is the positive
(3) the 16S rDNA identifications of bacterial strain
With DNA extraction kit extract Pseudomonas geniculate 3 genomic DNA, then carry out 16S rDNA gene clonings,
Sequencing, its 16S rDNA sequencing result is specific as follows as shown in SEQ ID No.1：
Blast comparisons are carried out in GenBank afterwards, are as a result shown, with its sequence similarity reach 98.56% be it is curved
Bent pseudomonad (Pseudomonas geniculata), is attributed to unit cell mushroom and is named as Pseudomonas geniculate
(Pseudomonas geniculata)3。
The Pseudomonas geniculate of embodiment 3 (Pseudomonas geniculata) 3 degraded pair crude oil pollution water body
Test method：
(1) preparation of the bacteria suspension of Pseudomonas geniculate 3
Aseptically, picking isolates and purifies the oil degradation bacteria of acquisition, is inoculated in the beef extract-peptone liquid of 100mL
In body culture medium, shaking table Amplification Culture culture 48h；Wherein, the composition of beef extract-peptone fluid nutrient medium is：Beef extract
3.0g, peptone 10.0g, NaCl 5.0g, nutrient agar 15.0g, distilled water 1000mL, pH 7.4-7.6.PH value 1mol/L
NaOH or 1mol/L hydrogen chloride be adjusted to required value.Medium sterilization condition is：Temperature is 121~126 DEG C, and the time is
25min。
(2) measure of I crude oil maximum absorption wavelength
Oil and products thereof has certain Absorption Characteristics in ultraviolet region, the aromatic compound with phenyl ring in petroleum component
Object wave generally 250~260nm long, and the compound wavelength for carrying conjugated double bond is general between 215~230nm, there is research
Show, when being determined using ultraviolet spectrometry light method, 255nm or so is crude oil and the range of choice of mink cell focus, 225nm or so with for
The range of choice of the oil product of light oil and oil plant.Under 100mg/L concentration, according to the wavelength and absorbance data that obtain, paint
System is for trying the wavelength-absorbance curve of I crude oil as shown in figure 3, wherein, I crude oil picks up from the inner hole of extension Petroleum Group seven and adopts
Oily field oil gathering station, density is 0.65g/mL.
(3) drafting of I crude oil standard curve
Take 0 respectively, 2,2.5,5,6,7mg I crude oil in 50mL colorimetric cylinders, use petroleum ether constant volume, concentration point now
Not Wei 0,40,50,100,120,140mg/L, under 258nm wavelength, with petroleum ether as reference solution, the suction of bioassay standard concentration
Luminosity, abscissa is concentration (mg/L), and ordinate is absorbance, draws the mark of concentration (the mg/L)-absorbance (A) of I crude oil
Directrix curve is as shown in Figure 4.
(4) measure of oil degradation rate
The bacteria suspension 3mL of Pseudomonas geniculate 3 of Amplification Culture is taken, is inoculated in 100mL oil-containing inorganic salt liquid culture mediums,
The composition of oil-containing minimal medium is：NaNO31.5g, (NH4)2SO41.5g, K2HPO41g, MgSO4·7H2O 0.5g,
KCl 0.5g, FeSO4·7H2O 0.01g, CaCl20.002g, distilled water 1000mL, crude oil 1.5mL, pH 7.0；At 30 DEG C,
7d is cultivated in 120rpm thermostatic control oscillator vibrations.Nutrient solution uses 5:The sulfuric acid solution regulation pH value of 1 (V/V) is 2-3, adds methyl alcohol
2-4mL, is extracted three times with (60-90 DEG C) of the petroleum ether of 3 × 15mL, and extract is transferred to 50mL capacity after anhydrous sodium sulfate drying
Bottle and constant volume.
By petroleum ether extraction liquid after being serially diluted, with petroleum ether as reference, by its phase of determined by ultraviolet spectrophotometry
The absorbance answered, the standard curve of concentration (the mg/L)-absorbance according to I crude oil, determines petroleum ether extract respective sample
Concentration, according to concentration calculate degradation efficiency.
The computational methods of oil degradation rate refer to below equation：
η (%)=(1-Ct/Co) × 100%
Wherein：η-oil degradation rate；Crude oil concentration in the presence of Ct- bacterial strains；Co- compares crude oil concentration.
It is 71.24% to calculate Pseudomonas geniculate 3-1 to the degradation rate of crude oil pollution water body according to above-mentioned computing formula.
Although the present invention is described in detail with a general description of the specific embodiments in this specification,
But on the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.
Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, belong to claimed model
Enclose.
SEQUENCE LISTING
<110>Chang An University
<120>A kind of Pseudomonas geniculate and its application
<130> 2016
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1319
<212> DNA
<213>Pseudomonas geniculate（Pseudomonas geniculata）3
<400> 1
AGCACAGAGG AGCTTGCTCC TTGGGTGGCG AGTGGCGGAC GGGTGAGGAA TACATCGGAA 60
TCTACTCTGT CGTGGGGGAT AACGTAGGGA AACTTACGCT AATACCGCAT ACGACCTACG 120
GGTGAAAGCA GGGGACCTTC GGGCCTTGCG CGATTGAATG AGCCGATGTC GGATTAGCTA 180
GTTGGCGGGG TAAAGGCCCA CCAAGGCGAC GATCCGTAGC TGGTCTGAGA GGATGATCAG 240
CCACACTGGA ACTGAGACAC GGTCCAGACT CCTACGGGAG GCAGCAGTGG GGAATATTGG 300
ACAATGGGCG CAAGCCTGAT CCAGCCATAC CGCGTGGGTG AAGAAGGCCT TCGGGTTGTA 360
AAGCCCTTTT GTTGGGAAAG AAATCCAGCT GGCTAATACC CGGTTGGGAT GACGGTACCC 420
AAAGAATAAG CACCGGCTAA CTTCGTGCCA GCAGCCGCGG TAATACGAAG GGTGCAAGCG 480
TTACTCGGAA TTACTGGGCG TAAAGCGTGC GTAGGTGGTC GTTTAAGTCC GTTGTGAAAG 540
CCCTGGGCTC AACCTGGGAA CTGCAGTGGA TACTGGGCGA CTAGAGTGTG GTAGAGGGTA 600
GCGGAATTCC TGGTGTAGCA GTGAAATGCG TAGAGATCAG GAGGAACATC CATGGCGAAG 660
GCAGCTACCT GGACCAACAC TGACACTGAG GCACGAAAGC GTGGGGAGCA AACAGGATTA 720
GATACCCTGG TAGTCCACGC CCTAAACGAT GCGAACTGGA TGTTGGGTGC AATTTGGCAC 780
GCAGTATCGA AGCTAACGCG TTAAGTTCGC CGCCTGGGGA GTACGGTCGC AAGACTGAAA 840
CTCAAAGGAA TTGACGGGGG CCCGCACAAG CGGTGGAGTA TGTGGTTTAA TTCGATGCAA 900
CGCGAAGAAC CTTACCTGGC CTTGACATGT CGAGAACTTT CCAGAGATGG ATGGGTGCCT 960
TCGGGAACTC GAACACAGGT GCTGCATGGC TGTCGTCAGC TCGTGTCGTG AGATGTTGGG 1020
TTAAGTCCCG CAACGAGCGC AACCCTTGTC CTTAGTTGCC AGCACGTAAT GGTGGGAACT 1080
CTAAGGAGAC CGCCGGTGAC AAACCGGAGG AAGGTGGGGA TGACGTCAAG TCATCATGGC 1140
CCTTACGGCC AGGGCTACAC ACGTACTACA ATGGTAGGGA CAGAGGGCTG CAAGCCGGCG 1200
ACGGTAAGCC AATCCCAGAA ACCCTATCTC AGTCCGGATT GGAGTCTGCA ACTCGACTCC 1260
ATGAAGTCGG AATCGCTAGT AATCGCAGAT CAGCATTGCT GCGGTGAATA CGTTCCCGG 1319
Claims (3)
1. a kind of Pseudomonas geniculate, it is characterised in that be preserved in Chinese microorganism strain preservation pipe on 09 12nd, 2016
Reason committee common micro-organisms center, preserving number is CGMCC No.13004；It is named as Pseudomonas geniculate (Pseudomonas
geniculata)3。
2. Pseudomonas geniculate according to claim 1, it is characterised in that the Pseudomonas geniculate (Pseudomonas
Geniculata) 3 16S rDNA sequences are as shown in SEQ ID No.1.
3. Pseudomonas geniculate (Pseudomonas geniculata) 3 described in claim 1 or 2 is in degrading crude oil contaminant water
Application in body.
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Cited By (2)
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| CN114231471A (en) * | 2022-02-21 | 2022-03-25 | 江苏朴厚环境工程有限公司 | Chemical solvent degrading bacterium pseudomonas flexuosa PH-2 and application thereof in chemical wastewater treatment |
| CN114477471A (en) * | 2022-02-16 | 2022-05-13 | 杭州秀川科技有限公司 | Method for treating emamectin benzoate amination wastewater by using composite flora |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114477471A (en) * | 2022-02-16 | 2022-05-13 | 杭州秀川科技有限公司 | Method for treating emamectin benzoate amination wastewater by using composite flora |
| CN114231471A (en) * | 2022-02-21 | 2022-03-25 | 江苏朴厚环境工程有限公司 | Chemical solvent degrading bacterium pseudomonas flexuosa PH-2 and application thereof in chemical wastewater treatment |
| CN114231471B (en) * | 2022-02-21 | 2022-05-03 | 江苏朴厚环境工程有限公司 | Chemical solvent degrading bacterium pseudomonas flexuosa PH-2 and application thereof in chemical wastewater treatment |
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