CN106244506A - The application of Mo Haiwei bacillus and the method for degraded naphthalene thereof - Google Patents
The application of Mo Haiwei bacillus and the method for degraded naphthalene thereof Download PDFInfo
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Abstract
The present invention relates to application and the method for degraded naphthalene thereof of Mo Haiwei bacillus cereus, belong to environmental microorganism application.The Mo Haiwei bacillus cereus B1811 of the present invention, deposit number is CGMCCNo.12805；The preservation time is on July 21st, 2016；Belong to shaft-like gram negative bacteria, spore can be formed；Its application is degraded naphthalene (NAP)；The degradation rate of naphthalene be may be up to 99.6%.The method of the Mo Haiwei bacillus cereus B1811 degraded naphthalene of the employing present invention is: under conditions of yeast extract exists, Mo Haiwei bacillus cereus B1811 contacts with naphthalene.The present invention degrades the method for naphthalene, compared with the method for antibacterial, fungus degrading naphthalene before the present invention, has new meaning on strain source, and degradation rate is high, and degradation cycle is short, simple to operate.
Description
Technical field
The present invention relates to application and the method for degraded naphthalene thereof of Mo Haiwei bacillus, belong to environmental microorganism application neck
Territory.
Background technology
Naphthalene (naphthalene, NAP) is a representative class organic dirt of important persistency generally existed in environment
Dye thing (persistentorganic pollutants, POPs).Numerous studies are it has been proved that naphthalene has chronic toxicity and cause
Cancer, teratogenesis, mutagenic " three cause " effect, be that in environment, a class is dangerous and need primary study, Ye Shi various countries priority acccess control
Pollutant.Owing to atmospheric sedimentation, sewage irrigation, solid waste landfill seepage, oilfield exploitation and oil product use in a large number
Deng, in causing global range, local soil naphthalene pollutes, and the situation of more serious pollution has occurred in China's many places.Naphthalene is due to property
Matter is stable, be difficult to degrade, in the trend of constantly accumulation in soil environment, serious harm the production of soil and ecological functions,
Agricultural product quality and human health.At present, the method for pollution administration soil mainly has peripheral doses, chemical redemption and biology to repair
Multiple.Wherein bioremediation technology because of have low cost, non-secondary pollution, can the particular advantages such as large-area applications and be increasingly subject to
The attention of people, is one of the most most potential soil restoring technology.
Mo Haiwei bacillus cereus (Bacillus mojavensis) is mainly used in the fermentation of lipase, there are no at present
Close the report utilizing Mo Haiwei bacillus cereus degraded naphthalene.
Summary of the invention
It is an object of the invention to provide the new strains of a kind of naphthalene of can degrading；And the application of this bacterial strain and use this bacterial strain
The method of degraded naphthalene.
Technical scheme
The present invention screens from soil and has isolated the bacterial strain that a strain is new；It is shaft-like gram negative bacteria, spore can be formed；
Identified, this bacterial strain be a kind of Mo Haiwei bacillus cereus (Bacillus mojavensis)；Named Mo Haiwei bacillus cereus
B1811；It is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC)；Deposit number is
CGMCCNo.12805；The preservation time is on July 21st, 2016.
The Mo Haiwei bacillus cereus B1811 of the present invention can degrade naphthalene (NAP)；Mo Haiwei bacillus cereus B1811 is to NAP's
Degradation rate may be up to 96.4-99.6%.
The one of which method of the Mo Haiwei bacillus cereus B1811 degraded NAP of the employing present invention is: deposit at yeast extract
Under the conditions, Mo Haiwei bacillus cereus B1811 with NAP contacts.Wherein, the amount of yeast extract can affect degradation rate.So,
Said method, it is preferred that under conditions of improvement BSM basis salt fluid medium exists, Mo Haiwei bacillus cereus B1811 with
NAP contacts；Described improvement BSM basis salt fluid medium, containing following component in every 1L: yeast extract 3-8g, ammonium sulfate
0.5g, sodium chloride 4.0g, three hypophosphite monohydrate potassium 0.5g, bitter salt 0.4g, distilled water surplus；pH7.0.
Concrete, it is that NAP is added improvement BSM basis salt fluid medium；By Mo Haiwei bacillus cereus B1811 seed
Liquid is inoculated in improvement BSM basis salt fluid medium, and under conditions of 150-200 rpm, 30-35 DEG C, isothermal vibration is cultivated.
Said method, it is preferred that in the salt fluid medium of improvement BSM basis, the content of yeast extract is 6g/L.
Said method, it is preferred that condition of culture is rotating speed 175rpm, temperature 30 DEG C, under other identical conditions, this
Under part, the degradation rate of bacterium B1811 is the highest.
Said method, Mo Haiwei bacillus cereus B1811 seed liquor connects relative to improvement BSM basis salt fluid medium
Plant the change of amount, the NAP degradation rate in the unit interval is had a significant effect；Under normal circumstances, inoculum concentration is the most, in the unit interval
Degradation rate is the highest；But final degradation rate (degradation rate when NAP content reaches stable in fermentation liquid) is had not significant impact.
Said method, described Mo Haiwei bacillus cereus B1811 seed liquor is to be cultivated by Mo Haiwei bacillus cereus B1811 to obtain
's.Under conditions of obtaining Mo Haiwei bacillus cereus B1811, those skilled in the art i.e. can obtain not sea by routine operation
Prestige bacillus cereus B1811 seed liquor.
In the present invention, for the percentage ratio of the content of certain composition, if not otherwise specified, refer both to percentage by weight
(w/w).
Technical term explanation used by the present invention: rpm is Speed unit, and 1 rpm refers to that rotation per minute is gone around.
Beneficial effect:
(1) separation and Culture goes out the Mo Haiwei bacillus cereus B1811 of naphthalene of can degrading first；
(2) Mo Haiwei bacillus cereus B1811 is up to 99.6% to the degradation rate of naphthalene；
(3) present invention degrades the method for naphthalene, compared with the method for antibacterial, fungus degrading naphthalene before the present invention, on strain is originated
Having new meaning, it is notable to the degradation effect of naphthalene, and degradation cycle is short, simple to operate.
Preservation illustrates:
Depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center
Address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica
Preservation date: on July 21st, 2016
Deposit number: CGMCCNo.12805
Classification And Nomenclature: Mo Haiwei bacillus cereus (Bacillus mojavensis).
Accompanying drawing explanation
Fig. 1 be heretofore described Mo Haiwei bacillus cereus B1811 microscope under morphological characteristic；In Fig. 1, Mo Haiwei bud
Spore bacillus B1811 is shaft-like gram negative bacteria, can form spore；
Fig. 2 is the standard curve of light absorption value (the OD)-naphthalene concentration using ultraviolet spectrometry light photometry to obtain；
Fig. 3 is the 16S rDNA sequential system developmental analysis of Mo Haiwei bacillus cereus B1811 and relevant kind；
Fig. 4 is the recA sequential system developmental analysis of Mo Haiwei bacillus cereus B1811 and relevant kind.
Detailed description of the invention
In following embodiment, if no special instructions, it is method commonly used in the art；Agents useful for same or the unreceipted production of instrument
Manufacturer person, be can by city available from conventional products.
The qualification of embodiment 1 bacterial strain B1811
Bacterial strain B1811 is isolatable from soil, and its form, as it is shown in figure 1, belong to shaft-like gram negative bacteria, can form spore.Carry
Take its genomic DNA, and utilize 16s rDNA and BCR1 primer to carry out polymerase chain reaction for this genomic DNA
(Polymerase Chain Reaction, PCR) is to breed specific DNA sequence, and utilizes American National Biotechnology Information
Center (National Center for Biotechnology Information is called for short NCBI) data base carries out bacterial strain ratio
Right.
(1) 16S rDNA sequence analysis:
16s rDNA forward primer 27F:5 '-AGA GTT TGA TCC TGG CTC AG-3 ', as shown in SEQ ID NO.1；
16s rDNA reverse primer 1541R:5 '-AAG GAG GTG ATC CAG CC-3 ', as shown in SEQ ID NO.2；
Amplification gained 16S rDNA sequence is as shown in SEQ ID NO:3, and sequence is 1461bp.By this amplification gained 16S
RDNA sequence compares with the gene order of the related strain in GenBank data base, carries out sequence ratio with MEGA4.1 software
Right, use and face a connection method phylogenetic tree construction, through 1000 stochastic sampling, calculate Bootstrap value, constructed system
Grow tree such as Fig. 3.Figure is grown tree node and only shows that Bootstrap value is more than 50% numerical value, upper target " T " intermediate scheme bacterium
Strain.Bacterial strain " B1811 " and type strain Bacillus mojavensis RO-H-1T/JH600280 can be found from NCBI
Homology reaches 99.8%.
(2) gyrB gene sequencing
GyrB-34F gene forward primer: 5'-ggTgTWRgKgCNgTCgTAAACg-3', as shown in SEQ ID NO.4；
GyrB-977R gene reverse primer: 5'-CCSgCAgARTCACCCTCTACg-3', as shown in SEQ ID NO.5；
Amplification gained gyrB gene order is as shown in SEQ ID NO:6, and sequence is 887bp.By this amplification gained gyrB base
Because the gene order of sequence with the related strain in GenBank data base compares, carry out sequence ratio with MEGA4.1 software
Right, use and face a connection method phylogenetic tree construction, through 1000 stochastic sampling, calculate Bootstrap value, constructed system
Grow tree such as Fig. 4.Figure is grown tree node and only shows that Bootstrap value is more than 50% numerical value, upper target " T " intermediate scheme bacterium
Strain.Bacterial strain " B1811 " and type strain can be found from NCBIB.malacitensisCECT 5687 (DQ903179) is same
Source property reaches 99.5%.
Therefore can be determined that B1811 is a kind of brand-new Mo Haiwei bacillus cereus.After identifying and confirming strain belonging to it,
Mo Haiwei bacillus cereus B1811 is preserved in the common micro-life of China Committee for Culture Collection of Microorganisms on July 21st, 2016
Thing center；Deposit number is CGMCCNo.12805；This biomaterial survival test is tested and passes through this test.
Prepared by embodiment 2 Mo Haiwei bacillus cereus B1811 liquid submerged culture liquid
(1) slant culture: Mo Haiwei bacillus cereus B1811 is inoculated on slant medium, cultivates 48 hours at 40 DEG C, obtains tiltedly
Face bacterial strain.Slant medium used, the composition contained in every 1L is: glucose 2.0g, peptone 1.0g, yeast extract
0.5g, agar 1.8g, distilled water surplus, pH is neutral, 115 DEG C of sterilizing 20 min.
(2) inclined-plane inoculation is taken in sterilized seed culture medium, in 175 rpm, constant temperature shake under conditions of 30 DEG C
Swing cultivation 24h, obtain seed culture fluid；Described seed culture medium, the composition contained in every 1L is: ammonium sulfate 0.5g, sodium chloride
4.0g, three hypophosphite monohydrate potassium 0.5g, bitter salt 0.4g, agar powder 15g, yeast extract 5g, distilled water surplus,
PH7.0,121 DEG C of sterilizing 25min.
(3) NAP of 5.0mg is added shaking of the improvement BSM basis salt fluid medium containing 50mL with sterile working
In Ping, the seed culture fluid of Mo Haiwei bacillus cereus B1811 is inoculated in shaking flask with the inoculum concentration of 1mL, 175r/min, 30
Under conditions of DEG C, isothermal vibration cultivates 72h；Described improvement BSM basis salt fluid medium, the composition contained in every 1L is: ferment
More than female extract 5.0g, ammonium sulfate 0.5g, sodium chloride 4.0g, three hypophosphite monohydrate potassium 0.5g, bitter salt 0.4g, distilled water
Amount, pH7.0,121 DEG C of sterilizing 15min.
Embodiment 3 NAP standard curve is formulated and assay
(1) with normal hexane be solvent preparation 400mg/L naphthalene mother solution, respectively dilution 20 times, 25 times, 30 times, 35 times, 40
Times, 80 times, 100 times, 200 times with standby, the naphthalene solution taking 2.5mL difference extension rate is placed in 3mL quartz colorimetric utensil,
Measure the light absorption value under 275nm wavelength respectively with ultraviolet spectrophotometer, and with light absorption value as vertical coordinate, the concentration of naphthalene is as horizontal stroke
Coordinate draws standard curve；Standard curve is as shown in Figure 2；And then acquisition light absorption value-concentration equation: y=0.0424x-0.0033, x
For the concentration of naphthalene, y is light absorption value.
(2) fermentation liquid taking 2.5mL embodiment 1 acquisition is placed in 3mL quartz colorimetric utensil, with ultraviolet spectrophotometer respectively
Measure the light absorption value under 275nm wavelength；Then the concentration of naphthalene is calculated according to light absorption value-concentration equation.
(3) computing formula of degradation rate: degradation rate %=(C0-C)/C0*100%, C0 is for use Mo Haiwei bacillus cereus
NAP mass concentration (mg/L) (i.e. mixed liquor in shaking flask before embodiment 1 step (3) fermentation culture before B1811 degraded
NAP mass concentration 89.29mg/L) in, C is the NAP residual concentration (mg/L) in fermentation liquid.It is computed, the degraded of embodiment 1
Rate is 98.1%.
The optimization of embodiment 4 Mo Haiwei bacillus cereus B1811 degraded naphthalene condition (inoculum concentration)
Seed liquor inoculum concentration in embodiment 2 step (3) is changed into successively 2,3,4,5,6mL, other operations are with embodiment 1.Press
According to the NAP concentration of the step measurements fermentation liquid of embodiment 3, and then calculate the degradation rate of NAP.Under the conditions of different vaccination amount, NAP
Degradation rate is as shown in table 1.
The embodiment 5 Mo Haiwei bacillus cereus B1811 degraded naphthalene condition (yeast of improvement BSM basis salt fluid medium
The content of extract) optimization
The content of the yeast extract of improvement BSM basis used salt fluid medium in embodiment 2 step (3) is revised successively
Be 3,4,5,6,7,8g/L；Other operations are with embodiment 2.According to the NAP concentration of the step measurements fermentation liquid of embodiment 3, and then
Calculate the degradation rate of NAP.Under the conditions of the content of different yeast extracts, NAP degradation rate is as shown in table 2.
Table 1
| Seed liquor inoculum concentration (mL) | NAP degradation rate (%) |
| 0 | 0 |
| 1 | 98.1 |
| 2 | 98.3 |
| 3 | 98.9 |
| 4 | 99.2 |
| 5 | 99.5 |
| 6 | 99.5 |
；
Table 2
| The content (g/L) of yeast extract | NAP degradation rate (%) |
| 0 | 0 |
| 3 | 96.4 |
| 4 | 96.7 |
| 5 | 98.1 |
| 6 | 99.6 |
| 7 | 99.2 |
| 8 | 99.2 |
<110>Beijing Technology and Business University
<120>Mo Haiwei bacillus application and degraded naphthalene method
<160>6
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AGAGTTTGAT CCTGGCTCAG 20
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AAGGAGGTGA TCCAGCC 17
<210>3
<211>1461
<212>DNA
<213>synthetic
<400>3
ATACATGCAG TCGAGCGGAC AGATGGGAGC TTGCTCCCTG ATGTTAGCGG CGGACGGGTG 60
AGTAACACGT GGGTAACCTG CCTGTAAGAC TGGGATAACT CCGGGAAACC GGGGCTAATA 120
CCGGATGCTT GTTTGAACCG CATGGTTCAA ACATAAAAGG TGGCTTCGGC TACCACTTAC 180
AGATGGACCC GCGGCGCATT AGCTAGTTGG TGAGGTAATG GCTCACCAAG GCAACGATGC 240
GTAGCCGACC TGAGAGGGTG ATCGGCCACA CTGGGACTGA GACACGGCCC AGACTCCTAC 300
GGGAGGCAGC AGTAGGGAAT CTTCCGCAAT GGACGAAAGT CTGACGGAGC AACGCCGCGT 360
GAGTGATGAA GGTTTTCGGA TCGTAAAGCT CTGTTGTTAG GGAAGAACAA GTACCGTTCG 420
AATAGGGCGG TACCTTGACG GTACCTAACC AGAAAGCCAC GGCTAACTAC GTGCCAGCAG 480
CCGCGGTAAT ACGTAGGTGG CAAGCGTTGT CCGGAATTAT TGGGCGTAAA GGGCTCGCAG 540
GCGGTTCCTT AAGTCTGATG TGAAAGCCCC CGGCTCAACC GGGGAGGGTC ATTGGAAACT 600
GGGGAACTTG AGTGCAGAAG AGGAGAGTGG AATTCCACGT GTAGCGGTGA AATGCGTAGA 660
GATGTGGAGG AACACCAGTG GCGAAGGCGA CTCTCTGGTC TGTAACTGAC GCTGAGGAGC 720
GAAAGCGTGG GGAGCGAACA GGATTAGATA CCCTGGTAGT CCACGCCGTA AACGATGAGT 780
GCTAAGTGTT AGGGGGTTTC CGCCCCTTAG TGCTGCAGCT AACGCATTAA GCACTCCGCC 840
TGGGGAGTAC GGTCGCAAGA CTGAAACTCA AAGGAATTGA CGGGGGCCCG CACAAGCGGT 900
GGAGCATGTG GTTTAATTCG AAGCAACGCG AAGAACCTTA CCAGGTCTTG ACATCCTCTG 960
ACAATCCTAG AGATAGGACG TCCCCTTCGG GGGCAGAGTG ACAGGTGGTG CATGGTTGTC 1020
GTCAGCTCGT GTCGTGAGAT GTTGGGTTAA GTCCCGCAAC GAGCGCAACC CTTGATCTTA 1080
GTTGCCAGCA TTCAGTTGGG CACTCTAAGG TGACTGCCGG TGACAAACCG GAGGAAGGTG 1140
GGGATGACGT CAAATCATCA TGCCCCTTAT GACCTGGGCT ACACACGTGC TACAATGGAC 1200
AGAACAAAGG GCAGCGAAAC CGCGAGGTTA AGCCAATCCC ACAAATCTGT TCTCAGTTCG 1260
GATCGCAGTC TGCAACTCGA CTGCGTGAAG CTGGAATCGC TAGTAATCGC GGATCAGCAT 1320
GCCGCGGTGA ATACGTTCCC GGGCCTTGTA CACACCGCCC GTCACACCAC GAGAGTTTGT 1380
AACACCCGAA GTCGGTGAGG TAACCTTTAT GGAGCCAGCC GCCGAAGGTG GGACAGATGA 1440
TTGGGGTGAA GTCGTAACAA G 1461
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ggTgTWRgKg CNgTCgTAAA Cg 22
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CCSgCAgART CACCCTCTAC g 21
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AACGCGTTAT CAACAGAGCT TGATGTGACT GTTCACCGTG ACGGAAAAAT CCATCGCCAA 60
GTCTATAACC GCGGTGTCCC GGTTTCTGAT CTTGAGGTTA TTGGCGAAAC GGATCATACA 120
GGAACGACTA CACACTTTGT TCCAGATCCT GAAATCTTCA CGGAAACAAC TGAGTATGAA 180
TATGATTTGC TTGCTAACCG TGTTCGCGAA CTAGCCTTTT TGACAAAAGG CGTAAACATC 240
ACGATTGAAG ATAAACGTGA AGGCCAAGAG CGCAAAAATG AGTATCATTA CGAAGGCGGA 300
ATTAAAAGCT ATGTAGAGTA TTTAAACCGC TCCAAAGAAG TTGTCCATGA AGAGCCGATT 360
TATATTGAAG GCGAAAAGGA CGGCATTACG GTTGAAGTCG CTCTGCAATA CAATGACAGC 420
TACACAAGCA ATATTTACTC ATTTACAAAC AATATCAACA CGTACGAAGG CGGTACCCAC 480
GAAGCCGGTT TTAAAACGGG GCTGACTCGT GTCATCAATG ATTACGCCAG AAAAAAAGGA 540
CTCATAAAAG AAAATGATCC AAACTTAAGC GGAGATGATG TGAGAGAAGG GCTTACCGCG 600
ATTATCTCGA TCAAACACCC GGATCCGCAG TTCGAAGGCC AAACGAAAAC AAAACTAGGC 660
AACTCAGAGG CGCGGACGAT CACAGATACG TTATTTTCTG CTGCGTTGGA AACCTTTATG 720
CTGGAAAATC CAGATGCGGC CAAAAAAATC GTTGACAAAG GCTTAATGGC AGCAAGAGCA 780
AGAATGGCTG CGAAAAAAGC GCGTGAATTA ACGCGCCGCA AAAGTGCTTT GGAGATTTCA 840
AACCTTCCTG GTAAATTAGC GGACTGCTCT TCGAAAGACC CGAGCAT 887
Claims (7)
1. Mo Haiwei bacillus cereus (Bacillus mojavensis) a kind of purposes of B1811, it is used for naphthalene of degrading；Described not sea
Prestige bacillus cereus B1811 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), and preservation is compiled
Number being CGMCCNo.12805, the preservation time is on July 21st, 2016.
2. one kind uses the method for Mo Haiwei bacillus cereus B1811 degraded naphthalene described in claim 1, it is characterised in that at yeast
Under conditions of extract exists, Mo Haiwei bacillus cereus B1811 contacts with naphthalene.
Method the most according to claim 2, it is characterised in that in the condition that improvement BSM basis salt fluid medium exists
Under, Mo Haiwei bacillus cereus B1811 contacts with naphthalene；Described improvement BSM basis salt fluid medium, containing following one-tenth in every 1L
Point: yeast extract 3-8g, ammonium sulfate 0.5g, sodium chloride 4.0g, three hypophosphite monohydrate potassium 0.5g, bitter salt 0.4g, steam
Distilled water surplus；pH7.0.
Method the most according to claim 3, it is characterised in that be that naphthalene is added improvement BSM basis salt fluid medium；
Mo Haiwei bacillus cereus B1811 seed liquor is inoculated in improvement BSM basis salt fluid medium, at 150-200 rpm, 30-35
Under conditions of DEG C, isothermal vibration is cultivated.
5. according to the method described in claim 2,3 or 4, it is characterised in that condition of culture is rotating speed 175rpm, temperature 30 DEG C.
Method the most according to claim 5, it is characterised in that yeast extract in the salt fluid medium of improvement BSM basis
Content be 6g/L.
Method the most according to claim 6, it is characterised in that described Mo Haiwei bacillus cereus B1811 seed liquor is by not
Sea prestige bacillus cereus B1811 cultivates acquisition.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110628661A (en) * | 2019-07-05 | 2019-12-31 | 哈尔滨师范大学 | Bacillus mojavensis CJX-61 and application thereof |
| CN113980872A (en) * | 2021-12-08 | 2022-01-28 | 中国矿业大学 | Bacillus mojavensis strain for producing lipopeptide biosurfactant |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110628661A (en) * | 2019-07-05 | 2019-12-31 | 哈尔滨师范大学 | Bacillus mojavensis CJX-61 and application thereof |
| CN113980872A (en) * | 2021-12-08 | 2022-01-28 | 中国矿业大学 | Bacillus mojavensis strain for producing lipopeptide biosurfactant |
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