CN106754485A - One plant can efficient degradation oil bacillus licheniformis and its application - Google Patents
One plant can efficient degradation oil bacillus licheniformis and its application Download PDFInfo
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Abstract
The invention discloses one plant of bacillus licheniformis of energy efficient degradation oil, the entitled bacillus licheniformis of the bacterial strain (Bacillus licheniformis) Yb1, bacterial strain is deposited in " China Committee for Culture Collection of Microorganisms's common micro-organisms center " on the 18th in September in 2015, and preserving number is CGMCC No.11427.The invention also discloses application of the bacterial strain in degraded oil or the application in biological prosthetic oil polluted environment.Experiment display：Bacterial strain of the invention can be using oil as sole carbon source, degraded oil, and degradation rate is close to 43%, indicates its improvement that can be used for the soil of oil pollution or water body, has preferable application prospect and economic worth in terms of oil pollution is eliminated.
Description
Technical field
The present invention relates to a bacillus licheniformis and its application, more particularly to one plant of lichens bud of energy efficient degradation oil
Spore bacillus and its application in biological prosthetic oil polluted environment, belong to biological technical field.
Background technology
Oil as modern industry blood, be that the progress and development of human civilization are made that tremendous contribution.But oil
While tremendous economic interests are brought, huge threat is also caused to ecological environment.In prospecting, exploitation, transport
And during storage, the soil contamination problem caused by oil is a long-standing environmental problem, and oil pollution is caused
Soil physico-chemical property changes, and is unfavorable for crop growth；It is difficult gradually be penetrated into by the part of adsorption by soil in petroleum hydrocarbon
To underground, further polluted underground water source.Therefore the soil pollution caused by oil is administered to be of great practical significance,
Repairing research for oil-polluted soils is also study hotspot (, Hamme etc. in 2003) in current field of environment protection.
The processing method of current soil petrochina pollution can be divided into 3 kinds by its property：Physical method, chemical method and biology
Method.Physical method is including excavating landfill method, air lift blow-off method, electrolysis, washing method and isolation control methods etc.；Chemical method bag
Include oxidizing method, photochemical oxidation method, thermal decomposition method, extraction etc..Mainly this two classes method for using in the past, though
Certain actual effect can be produced, but processing cost is expensive and easily causes serious secondary pollution.In contrast, biological method
First it is that processing cost is low with the advantage that physical method and chemical method are incomparable, the only about materialization of its processing cost
/ 3rd of method；Next to that reparation is simple to operate, can be repaired with original position (2011, Mukherjee and
Bordoloi)；Secondary pollution of the bioanalysis reparation to environment is smaller (, Ron and Rosenberg in 2002) again.
Petroleum hydrocarbon degradation bacterium is to carry out biodegradation using petroleum hydrocarbon as sole carbon source, and produce gas, aliphatic acid and
The general name of one quasi-microorganism of the metabolites such as biosurfactant.Petroleum hydrocarbon degradation bacterium is repaiied in the biology of oil polluted environment
Play the role of in multiple it is important, microbe oil production research field application increasingly extensively (, what good chrysanthemum etc. in 1997).Due to certainly
Microorganism in right boundary possesses aboundresources, many advantages, such as be easy to culture and be stronger to the tolerance of environment, repaiied for biology
Multiple oil polluted environment has broad application prospects.But retrieval finds lichens gemma bar at present about energy efficient degradation oil
Bacterium and its application in biological prosthetic oil polluted environment are also rarely reported.
Bibliography
1.Hamme J.D.V.,Singh A.,Ward O.P.Microbiol Mol Biol Rev,2003,67(4):
503-549.
2.Mukherjee A.K.,Bordoloi N.K.Environ.Sci.Pollut.Res.Int,2011,471:
478.
3.Ron E.Z.,Rosenberg E.Curr.Opin.Biotechnol,2002,249:252.
4. what good chrysanthemum, Wei Dezhou, Zhang Weiqing environmental science progress, 1997,7 (3):110-115.
The content of the invention
In view of the shortcomings of the prior art, the invention solves the problems that problem is to provide one plant of lichens gemma of energy efficient degradation oil
Bacillus and its application in oil polluted environment is repaired.
The bacillus licheniformis of energy efficient degradation oil of the present invention, it is characterised in that：The entitled lichens gemma of the bacterial strain
Bacillus (Bacillus licheniformis) Yb1, bacterial strain is deposited in " Chinese microorganism strain guarantor on the 18th in September in 2015
Hide administration committee's common micro-organisms center ", preserving number is CGMCC No.11427.
The bacillus licheniformis of above-mentioned energy efficient degradation oil, its biological property is：Shaft-like, single, cell is presented
Size is (0.6 μm~0.8 μm) × (1.3 μm~2.0 μm), starts within 20-24 hours to give birth to spore, during 37 DEG C of solid cultures at the beginning of bacterium colony
Phase ovalize, as the extension of incubation time is gradually in irregular, bacterium colony is flat and opaque, rough surface, edge not
Together；Its physiological and biochemical property is：Gram-positive, amphimicrobian, chemoheterotrophic bacteria, optimum growth temperature is 30~37
DEG C, it is 6.5~7.5 to be best suitable for growth pH value, and bio-chemical characteristics result is consistent compared with bacillus licheniformis type strain, in detail
It is shown in Table 1.
Table 1：Yb1 bacterial strains compare with the physiological and biochemical property of Bacillus licheniformis
Note："+" represents that bacterial strain is positive；"-" represents that bacterial strain is negative.
*：Eastern show pearl wonderful English of Cai etc., common bacteria system identification handbook, Science Press, 2001, the first edition, p62-63
The above-mentioned fluid nutrient medium for thalli morphology observation is constituted is：Contain tryptone in per 1000ml deionized waters
17g, soy peptone 3g, sodium chloride 5g, dipotassium hydrogen phosphate 2.5g, glucose 2.5g, and adjust pH 7.5.
The above-mentioned solid medium for thalli morphology observation is constituted is：Contain tryptone in per 1000ml deionized waters
17g, soy peptone 3g, sodium chloride 5g, dipotassium hydrogen phosphate 2.5g, glucose 2.5g, agar powder 15g, and adjust pH 7.5.
The experimental technique of above-mentioned observation of morphological is write with reference to east show pearl, Cai Miaoying etc.《Common bacteria system identification
Handbook》, Science Press, 2001, the first edition, p353-363.
What above-mentioned physiological and biochemical test culture medium and experimental technique were write with reference to east show pearl, Cai Miaoying etc.《Common bacteria system
System identification handbook》, Science Press, 2001, the first edition, p364-398.
Experiment is confirmed：Yb1 bacterial strains of the present invention are trained on the inorganic salts solid medium with oil as sole carbon source
When foster can degraded oil, while producing obvious degraded circle, as a result see Fig. 3.Determine the degradation rate for showing the bacterial strain to oil
Close to 43%.Indicate that it has preferable application prospect in terms of oil-polluted soils or water body is administered.
Its gene order length is to be shown to the result that bacterial strain Yb1 of the present invention determines 16S rRNA gene orders
1419bp, the nucleotide sequence is as shown in SEQ ID NO.1.
By using U.S. Biotechnology Information center (National Center for Biotechnology
Information, NCBI) comparison of BLASTN programs, find the gene order and NCBI notes of Yb1 bacterial strains 16S rRNA of the invention
The gene order of many bacillus licheniformis (Bacillus licheniformis) 16S rRNA of volume has high homology,
Can determine that Yb1 bacterial strains of the present invention are a bacillus licheniformis (Bacillus licheniformis).
Choose the 16S rDNA of tetraploid rice typical bacillus licheniformis (Bacillus licheniformis) high
Sequence is used as reference subject；The software building Yb1 bacterial strains of Mega 5 and reference are used using neighbouring method (Neighbour-Joining)
Systematic evolution tree between bacterial strain, from Pseudomonas aeruginosa as Wai Qun branches.Result is shown in accompanying drawing 4.
It is of the present invention can the basic skills of bacillus licheniformis Yb1 strain improvements of degraded oil be：
The pedotheque of the oil pollution that will be collected SPSS dissolves, and vibration mixing is made soil supension,
Gradient dilution spread plate, quiescent culture under the conditions of 37 DEG C is existing to there is obvious oil degradation to iris out, picking oil degradation circle compared with
Big bacterium colony, line is transferred to solid medium, quiescent culture under the conditions of 37 DEG C, to there is obvious single bacterium colony to produce, through twice
After purification, bacterium is protected in glycerine cold storage.The bacterial strain is deposited in " Chinese microorganism strain preservation conservator on the 18th in September in 2015
Meeting common micro-organisms center ", preserving number is CGMCC No.11427.
It is of the present invention can efficient degradation oil application of the bacillus licheniformis in degraded oil or biological prosthetic
Application in oil polluted environment.
Wherein：The oil polluted environment preferably refers to the soil of oil pollution or the water of oil pollution.
Specifically, the method that above-mentioned application is related to is：
(1) strain selection：Select bacillus licheniformis (Bacillus licheniformis) Yb1 bacterial strains；
(2) inclined-plane culture activated spawn：Strain is inoculated in slant medium, under the conditions of 35~40 DEG C, static gas wave refrigerator 20
It is~40 hours, standby；
(3) seed culture：The bacterial strain that step (2) is cultivated aseptically connects 1~2 ring in containing 50mL with oese
In the triangular flask (250mL) of liquid seed culture medium, put rotary rpm be 180~220 revs/min, radius of turn be 40mm
On shaking table, 35~40 DEG C are cultivated 18~24 hours, obtain seed liquor；
(4) biological prosthetic oil polluted environment and oil resolution ratio are detected：With the inoculum concentration of 1~5% volume ratio, will plant
Sub- liquid is inoculated in the triangular flask of the minimal medium that 100mg crude oil has been added containing 50mL of simulation oil polluted environment
In (250mL), put rotary rpm be 180~220 revs/min, radius of turn on the shaking table of 40mm, 35~40 DEG C of cultures 8~
10 days；Then with the petroleum hydrocarbon remained in carbon tetrachloride extraction culture medium, reference solution is made with carbon tetrachloride, using infrared spectroscopy
Photometer measures its absorbance at 2930cm-1,2960cm-1,3030cm-1；The culture of Yb1 bacterial strains will not accessed simultaneously
Base is measured under the same conditions as blank, according to People's Republic of China (PRC) state environment protecting standard (HJ
The measure infrared spectrophotometer of 637-2012 water-quality petroleums and animals and plants oils) in the formula listed calculate total stone in sample
The content of petroleum hydrocarbon, draws oil resolution ratio；
Wherein：
Above-mentioned slant medium is constituted：Contain tryptone 17g, soy peptone 3g in per 1000ml deionized waters,
Sodium chloride 5g, dipotassium hydrogen phosphate 2.5g, glucose 2.5g, agar powder 15g, and adjust pH 7.5；
Aforesaid liquid seed culture medium is constituted：Contain tryptone 17g, soy peptone in per 1000ml deionized waters
3g, sodium chloride 5g, dipotassium hydrogen phosphate 2.5g, glucose 2.5g, and adjust pH 7.5.
Above-mentioned minimal medium is constituted：NaNO31.5g/L, (NH4)2SO41.5g/L, K2HPO41g/L,
MgSO40.5g/L, KCl 0.5g/L, FeSO40.01g/L, CaCl20.002g/L, crude oil 5g/L, pH 7.0, distilled water is matched somebody with somebody
System.
In the above method, preferably 37 DEG C ± 0.2 DEG C of step (2), (3), (4) described cultivation temperature.
In the above method, step (2), (3) described incubation time preferably 24 hours.
In the above method, step (4) seed liquor accesses volume ratio preferably 2%.
The bacillus licheniformis (Bacillus licheniformis) of one plant of energy efficient degradation oil disclosed by the invention
Yb1 can be using oil as sole carbon source, degraded oil, and degradation rate is higher, indicate its can be used for the soil of oil pollution or
The improvement of water body, has preferable application prospect and economic worth in terms of oil pollution is eliminated.
Brief description of the drawings
Bacillus licheniformis (Bacillus licheniformis) Yb1 bacterial strains of the present invention are in September 18 in 2015
Day is deposited in " China Committee for Culture Collection of Microorganisms's common micro-organisms center ", and preserving number is CGMCCNo.11427.
Fig. 1 is bacillus licheniformis Yb1 bacterial strains cellular morphology under the microscope.
Fig. 2 is bacillus licheniformis Yb1 bacterial strains gemma form under the microscope.
Fig. 3 is bacterium colony of the bacillus licheniformis Yb1 bacterial strains on the inorganic salts solid medium with oil as sole carbon source
Form.
Fig. 4 is the systematic evolution tree between bacillus licheniformis Yb1 bacterial strains and reference strains.
Specific embodiment
Present invention is elaborated and is understood below by the specific embodiment for being given and deeply, but following embodiments
Only the present invention is explained rather than limiting the scope of the present invention.
Embodiment 1：The screening of the bacillus licheniformis of energy efficient degradation oil
The pedotheque of the oil pollution that 1g is collected is taken, is put into 250ml triangular flasks, be added thereto to the aseptic lifes of 9ml
Reason salt solution, vibration mixing is made soil supension, and doubling dilution is carried out by 10 times of concentration gradients.Taking 100 μ l dilution factors respectively is
10-3、10-4、10-5With 10-6Soil supension, be added drop-wise on the inorganic salts solid medium with oil as sole carbon source, with coating
Rod smears uniform, quiescent culture 7 days under the conditions of 37 DEG C.
Treat that obvious oil degradation is irised out now, with the bacterium colony that oese picking oil degradation circle is larger, in solid culture
Rule on base, the quiescent culture under the conditions of 37 DEG C is produced to there is obvious single bacterium colony.Switching line is twice.
With oese picking single bacterium colony, it is transferred in the test tube equipped with 8ml fluid nutrient mediums, in 37 DEG C, 180rpm conditions
Lower shaken cultivation is overnight.- 80 DEG C are placed to the bacterium solution for being separately added into 200 μ l glycerine and 800 μ l in cold storage pipe, after being well mixed to surpass
Preserved in low temperature refrigerator, strain number is Yb1.
Above-mentioned SPSS is constituted：0.85%NaCl solution.
Above-mentioned inorganic salts solid medium is constituted：NaNO31.5g/L, (NH4)2SO41.5g/L, K2HPO41g/L,
MgSO40.5g/L, KCl 0.5g/L, FeSO40.01g/L, CaCl20.002g/L, agar powder 15g/L, crude oil 5g/L, pH
7.0, distilled water is prepared.
Above-mentioned solid medium is constituted：Contain tryptone 17g, soy peptone 3g in per 1000ml deionized waters,
Sodium chloride 5g, dipotassium hydrogen phosphate 2.5g, glucose 2.5g, agar powder 15g, and adjust pH 7.5.
Embodiment 2：Yb1 strain morphologies are observed and physiological and biochemical property identification
The form of Yb1 bacterial strains is observed using the oil mirror of Nikon inverted microscope.
Yb1 bacterial strain physiological and biochemical properties authentication method is write with reference to east show pearl, Cai Miaoying etc.《Common bacteria system identification
Handbook》(Science Press, 2001, the first edition), qualification test cultivation temperature is set to 37 DEG C.Simultaneously analyze Yb1 bacterial strains with
Oil for sole carbon source inorganic salts cultured on solid medium when optimum temperature be best suitable for grow pH value.
The biological property of Yb1 bacterial strains is：It is presented shaft-like, single, cell size is (0.6 μm~0.8 μm) × (1.3 μm
~2.0 μm), start within 20-24 hours to give birth to spore, bacterium colony initial stage ovalize during 37 DEG C of solid cultures, with prolonging for incubation time
It is long gradually in irregular, bacterium colony is flat and opaque, rough surface, edge are uneven.(see Fig. 1)
The physiological and biochemical property of Yb1 bacterial strains is：Gram-positive, amphimicrobian, chemoheterotrophic bacteria, glucose fermentation
Acid is produced, catalase test, VP experiments, citrate are presented positive using the experiment of experiment, Starch Hydrolysis and gelatin hydrolysis, hair
The experiment of ferment glucose aerogenesis, PD experiment, methyl red test are presented negative.Optimum growth temperature is 30~37
DEG C, it is 6.5~7.5 to be best suitable for growth pH value, and bio-chemical characteristics result is consistent compared with bacillus licheniformis type strain, in detail
It is shown in Table 1.
Table 1：Yb1 bacterial strains compare with the physiological and biochemical property of Bacillus licheniformis
Note："+" represents that bacterial strain is positive；"-" represents that bacterial strain is negative.
*：Eastern show pearl wonderful English of Cai etc., common bacteria system identification handbook, Science Press, 2001, the first edition, on p62-63
The fluid nutrient medium stated for thalli morphology observation constitutes and is：Contain tryptone 17g, soybean egg in per 1000ml deionized waters
White peptone 3g, sodium chloride 5g, dipotassium hydrogen phosphate 2.5g, glucose 2.5g, and adjust pH 7.5.
The above-mentioned solid medium for thalli morphology observation is constituted is：Contain tryptone in per 1000ml deionized waters
17g, soy peptone 3g, sodium chloride 5g, dipotassium hydrogen phosphate 2.5g, glucose 2.5g, agar powder 15g, and adjust pH 7.5.
Yb1 bacterial strains when being cultivated on the inorganic salts solid medium with oil as sole carbon source can degraded oil, while
Obvious degraded circle is produced, Fig. 3 is as a result seen.
The entitled bacillus licheniformis of Yb1 bacterial strains (Bacillus licheniformis) Yb1, bacterial strain is in September, 2015
It is deposited in " China Committee for Culture Collection of Microorganisms's common micro-organisms center " within 18th, preserving number is CGMCCNo.11427.
Implementation method 3：The 16S rRNA identified for genes of Yb1 bacterial strains
According to the operating instruction of TIANGEN bacterial genomes extracts kit (TianGen companies, article No. DP302-02),
The total genomic dna of the Yb1 bacterial strains of extraction separation and purification.The total genomic dna of extraction uses 0.8% agarose gel electrophoresis
Detected, take 3.0 μ l total genomic dnas samples and 0.6 μ 6 × Loading of l Buffer are sufficiently mixed uniform rear decanting point
In sample hole, Marker selects λ-Hind III DNA Marker, electrophoretic voltage 120V, electrophoresis time 35 minutes.Drawn from upstream
Thing 27F (5 ' -3 '：) and anti-sense primer 1492R (5 ' -3 ' AGAGTTTGATCCTGGCTCAG：GGTTACCTTGTTACGACTT) expand
Increase the 16S rRNA genes of isolated strains, PCR reaction systems and condition are as shown in the table：
PCR primer detected using 1.2% agarose gel electrophoresis, take 3.0 μ l PCR primers and 0.6 μ l 6 ×
Loading Buffer are sufficiently mixed in uniform rear injection loading wells, and Marker selects DL 2000DNA Marker, electrophoretic voltage
120V, electrophoresis time 30 minutes.According to TIANGEN Ago-Gels QIAquick Gel Extraction Kit (TianGen companies, article No. DP209-02)
Operating instruction, purifying recovery is carried out to PCR primer, the PCR primer after recovery is examined with 1.2% agarose gel electrophoresis
Survey.Electrophoresis method is ibid.PCR primer after purifying recovery is sent to sequencing company and is sequenced.
16S rRNA gene orders obtained by sequencing are delivered to ncbi database (http://
Www.ncbi.nlm.nih.gov BLASTN comparisons are carried out in).Result shows the 16S rRNA gene orders and NCBI of the bacterial strain
The homology of the 16S rRNA gene orders of other clothing bacillus is very high in database, and the similitude of indivedual bacterial strains is even high
Up to 100%.Tetraploid rice typical Bacillus licheniformis 16S rRNA gene orders high are chosen as ginseng
Compare as；Using neighbouring method (Neighbour-Joining) between the software building Yb1 bacterial strains of Mega 5 and reference strains
Systematic evolution tree, from Pseudomonas aeruginosa as Wai Qun branches.Result is shown in Fig. 4.
Embodiment 4：Application process step of the Yb1 bacterial strains in biological prosthetic oil pollution be：
(1) strain selection：Select bacillus licheniformis (Bacillus licheniformis) Yb1 bacterial strains；
(2) inclined-plane culture activated spawn：Strain is inoculated in slant medium, under the conditions of 37 DEG C, static gas wave refrigerator 40 hours,
It is standby；
(3) seed culture：The bacterial strain that step (2) is cultivated aseptically connects 2 rings in containing 50mL liquid with oese
In the triangular flask (250mL) of body seed culture medium, put that rotary rpm is 180~220 revs/min, radius of turn is for 40mm's shakes
On bed, 37 DEG C are cultivated 24 hours, obtain seed liquor；
(4) biological prosthetic oil polluted environment and oil resolution ratio are detected：With the inoculum concentration of 2% volume ratio, by seed
Liquid is inoculated in the triangular flask of the minimal medium that 100mg crude oil has been added containing 50mL of simulation oil polluted environment
In (250mL), put on the shaking table that rotary rpm is 200 revs/min, radius of turn is 40mm, 37 DEG C are cultivated 8~10 days；
In media transfer to separatory funnel, 10ml carbon tetrachloride vibration 3min will be added, and period arranges through normally open cock
Gas.After stratification, lower floor's organic phase is transferred in the conical flask for having added 3g anhydrous sodium sulfates in advance, shake is removed for several times
The moisture of residual.If anhydrous sodium sulfate crystallizes blocking, it is necessary to add anhydrous sodium sulfate, stand.Make reference with carbon tetrachloride molten
Liquid, its absorbance is measured using infrared spectrophotometer at 2930cm-1,2960cm-1,3030cm-1.Setting 3 is parallel
Experiment, takes 3 groups of average values of experimental data, while calculating positive negative error.The culture medium of Yb1 bacterial strains will not accessed in the same terms
Under processed, as blank.According to People's Republic of China's state environment protecting standard (HJ 637-2012 water quality stones
The measure infrared spectrophotometer of oils and animals and plants oils) in the formula listed calculate the content of total petroleum hydrocarbon in sample.
Result shows, after accessing Yb1 bacterial strains, in culture medium the content of total petroleum hydrocarbon drop to 57 from initial 100mg ±
4mg, the clearance of oil is close to 43%.
Above-mentioned slant medium is constituted：Contain tryptone 17g, soy peptone 3g in per 1000ml deionized waters,
Sodium chloride 5g, dipotassium hydrogen phosphate 2.5g, glucose 2.5g, agar powder 15g, and adjust pH 7.5.
Aforesaid liquid seed culture medium is constituted：Contain tryptone 17g, soy peptone in per 1000ml deionized waters
3g, sodium chloride 5g, dipotassium hydrogen phosphate 2.5g, glucose 2.5g, and adjust pH 7.5.
Above-mentioned minimal medium is constituted：NaNO31.5g/L, (NH4)2SO41.5g/L, K2HPO41g/L,
MgSO40.5g/L, KCl 0.5g/L, FeSO40.01g/L, CaCl20.002g/L, pH 7.0, distilled water is prepared.
Sequence table
<110>Shandong University
<120>One plant can efficient degradation oil bacillus licheniformis and its application
<141> 2016-11-21
<160> 1
<210> 1
<211> 1419
<212> rRNA
<213>Bacillus licheniformis（Bacillus licheniformis）
<221>Bacillus licheniformis（Bacillus licheniformis）Yb1 CGMCC No.11427
<222>（1）…（1419）
<400> 1
tcggcggctg gctccaaagg ttacctcacc gacttcgggt gttacaaact ctcgtggtgt 60
gacgggcggt gtgtacaagg cccgggaacg tattcaccgc ggcatgctga tccgcgatta 120
ctagcgattc cagcttcacg cagtcgagtt gcagactgcg atccgaactg agaacagatt 180
tgtgggattg gcttagcctc gcggcttcgc tgccctttgt tctgcccatt gtagcacgtg 240
tgtagcccag gtcataaggg gcatgatgat ttgacgtcat ccccaccttc ctccggtttg 300
tcaccggcag tcaccttaga gtgcccaact gaatgctggc aactaagatc aagggttgcg 360
ctcgttgcgg gacttaaccc aacatctcac gacacgagct gacgacaacc atgcaccacc 420
tgtcactctg cccccgaagg ggaagcccta tctctagggt tgtcagagga tgtcaagacc 480
tggtaaggtt cttcgcgttg cttcgaatta aaccacatgc tccaccgctt gtgcgggccc 540
ccgtcaattc ctttgagttt cagtcttgcg accgtactcc ccaggcggag tgcttaatgc 600
gtttgctgca gcactaaagg gcggaaaccc tctaacactt agcactcatc gtttacggcg 660
tggactacca gggtatctaa tcctgttcgc tccccacgct ttcgcgcctc agcgtcagtt 720
acagaccaga gagtcgcctt cgccactggt gttcctccac atctctacgc atttcaccgc 780
tacacgtgga attccactct cctcttctgc actcaagttc cccagtttcc aatgaccctc 840
cccggttgag ccgggggctt tcacatcaga cttaagaaac cgcctgcgcg cgctttacgc 900
ccaataattc cggacaacgc ttgccaccta cgtattaccg cggctgctgg cacgtagtta 960
gccgtggctt tctggttagg taccgtcaag gtgccgccct attcgaacgg tacttgttct 1020
tccctaacaa cagagtttta cgatccgaaa accttcatca ctcacgcggc gttgctccgt 1080
cagactttcg tccattgcgg aagattccct actgctgcct cccgtaggag tctgggccgt 1140
gtctcagtcc cagtgtggcc gatcaccctc tcaggtcggc tacgcatcgt cgccttggtg 1200
agccgttacc tcaccaacta gctaatgcgc cgcgggtcca tctgtaagtg gtagctaaaa 1260
gccacctttt atgattgaac catgcggttc aatcaagcat ccggtattag ccccggtttc 1320
ccggagttat cccagtctta caggcaggtt acccacgtgt tactcacccg tccgccgctg 1380
acctaaggga gcaagctccc gtcggtccgc tcgactgca 1419
Claims (5)
1. one plant can efficient degradation oil bacillus licheniformis, it is characterised in that：The entitled bacillus licheniformis of the bacterial strain
(Bacillus licheniformis) Yb1, bacterial strain is deposited in " Chinese microorganism strain preservation pipe on 18th in September in 2015
Reason committee common micro-organisms center ", preserving number is CGMCC No.11427.
2. described in claim 1 can efficient degradation oil application of the bacillus licheniformis in degraded oil or biological prosthetic
Application in oil polluted environment.
3. application as claimed in claim 2, it is characterised in that：The oil polluted environment refers to the soil or stone of oil pollution
The water of oily pollution.
4. application as claimed in claim 2 or claim 3, it is characterised in that the method that the application is related to is：
(1) strain selection：Select bacillus licheniformis (Bacillus licheniformis) Yb1 bacterial strains；
(2) inclined-plane culture activated spawn：Strain is inoculated in slant medium, under the conditions of 35~40 DEG C, static gas wave refrigerator 20~40
Hour, it is standby；
(3) seed culture：The bacterial strain that step (2) is cultivated aseptically connects 1~2 ring in containing 50mL liquid with oese
In the triangular flask of seed culture medium, put on the shaking table that rotary rpm is 180~220 revs/min, radius of turn is 40mm, 35~
40 DEG C are cultivated 18~24 hours, obtain seed liquor；
(4) biological prosthetic oil polluted environment and oil resolution ratio are detected：With the inoculum concentration of 1~5% volume ratio, by seed liquor
It is inoculated in the triangular flask of the minimal medium that 100mg crude oil has been added containing 50mL of simulation oil polluted environment, puts rotation
Speed of walking around is 180~220 revs/min, radius of turn on the shaking table of 40mm, 35~40 DEG C are cultivated 8~10 days；Then tetrachloro is used
Change the petroleum hydrocarbon remained in carbon extraction culture medium, reference solution is made with carbon tetrachloride, using infrared spectrophotometer in 2930cm-
1st, its absorbance is measured at 2960cm-1,3030cm-1；The culture medium of Yb1 bacterial strains will not accessed as blank simultaneously,
It is measured under the same conditions, according in the formula calculating sample listed in People's Republic of China's state environment protecting standard
The content of total petroleum hydrocarbon, draws oil resolution ratio；
Wherein：
Above-mentioned slant medium is constituted：Contain tryptone 17g, soy peptone 3g, chlorination in per 1000ml deionized waters
Sodium 5g, dipotassium hydrogen phosphate 2.5g, glucose 2.5g, agar powder 15g, and adjust pH 7.5；
Aforesaid liquid seed culture medium is constituted：Contain tryptone 17g, soy peptone 3g in per 1000ml deionized waters,
Sodium chloride 5g, dipotassium hydrogen phosphate 2.5g, glucose 2.5g, and adjust pH 7.5.
Above-mentioned minimal medium is constituted：NaNO31.5g/L, (NH4)2SO41.5g/L, K2HPO41g/L, MgSO4 0.5g/
L, KCl 0.5g/L, FeSO40.01g/L, CaCl20.002g/L, crude oil 5g/L, pH 7.0, distilled water are prepared.
5. application as claimed in claim 4, it is characterised in that：Step (2), (3), (4) described cultivation temperature are 37 DEG C ± 0.2
℃。
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| RU2808248C1 (en) * | 2023-03-17 | 2023-11-28 | федеральное государственное бюджетное образовательное учреждение высшего образования "Санкт-Петербургский горный университет императрицы Екатерины II" | Composition for bioremediation of soil contaminated with oil and petroleum products |
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