CN108277171A - A kind of ocean degradation bacteria of degrading polyaromatic hydrocarbon and its application - Google Patents
A kind of ocean degradation bacteria of degrading polyaromatic hydrocarbon and its application Download PDFInfo
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- CN108277171A CN108277171A CN201711041115.5A CN201711041115A CN108277171A CN 108277171 A CN108277171 A CN 108277171A CN 201711041115 A CN201711041115 A CN 201711041115A CN 108277171 A CN108277171 A CN 108277171A
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- pahs
- halomonas
- bacterial strain
- polycyclic aromatic
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- 230000015556 catabolic process Effects 0.000 title claims abstract description 29
- 238000006731 degradation reaction Methods 0.000 title claims abstract description 29
- 125000005575 polycyclic aromatic hydrocarbon group Chemical group 0.000 title claims abstract description 28
- 230000000593 degrading effect Effects 0.000 title claims abstract description 12
- 241000894006 Bacteria Species 0.000 title description 17
- 230000001580 bacterial effect Effects 0.000 claims abstract description 28
- 241000206596 Halomonas Species 0.000 claims abstract description 14
- 239000003205 fragrance Substances 0.000 claims abstract description 8
- 241000429668 Halomonas sp. BN3-1 Species 0.000 claims abstract description 6
- 239000002773 nucleotide Substances 0.000 claims abstract description 3
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 3
- 238000004321 preservation Methods 0.000 claims description 2
- YNPNZTXNASCQKK-UHFFFAOYSA-N phenanthrene Chemical compound C1=CC=C2C3=CC=CC=C3C=CC2=C1 YNPNZTXNASCQKK-UHFFFAOYSA-N 0.000 description 21
- 239000007787 solid Substances 0.000 description 11
- GVEPBJHOBDJJJI-UHFFFAOYSA-N fluoranthrene Natural products C1=CC(C2=CC=CC=C22)=C3C2=CC=CC3=C1 GVEPBJHOBDJJJI-UHFFFAOYSA-N 0.000 description 9
- 239000001963 growth medium Substances 0.000 description 9
- 239000002609 medium Substances 0.000 description 9
- 239000000243 solution Substances 0.000 description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 8
- UFWIBTONFRDIAS-UHFFFAOYSA-N Naphthalene Chemical compound C1=CC=CC2=CC=CC=C21 UFWIBTONFRDIAS-UHFFFAOYSA-N 0.000 description 6
- 235000010210 aluminium Nutrition 0.000 description 6
- BBEAQIROQSPTKN-UHFFFAOYSA-N pyrene Chemical compound C1=CC=C2C=CC3=CC=CC4=CC=C1C2=C43 BBEAQIROQSPTKN-UHFFFAOYSA-N 0.000 description 6
- 229910052782 aluminium Inorganic materials 0.000 description 5
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 5
- 238000000034 method Methods 0.000 description 5
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 4
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 4
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 150000001875 compounds Chemical class 0.000 description 4
- 239000008367 deionised water Substances 0.000 description 4
- 229910021641 deionized water Inorganic materials 0.000 description 4
- 230000012010 growth Effects 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 3
- 241000192128 Gammaproteobacteria Species 0.000 description 3
- 239000004411 aluminium Substances 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 244000005700 microbiome Species 0.000 description 3
- 125000003367 polycyclic group Chemical group 0.000 description 3
- 229930003231 vitamin Natural products 0.000 description 3
- 235000013343 vitamin Nutrition 0.000 description 3
- 239000011782 vitamin Substances 0.000 description 3
- 229940088594 vitamin Drugs 0.000 description 3
- 241001156739 Actinobacteria <phylum> Species 0.000 description 2
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- 241001062571 Aurantimonas coralicida Species 0.000 description 2
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 2
- 239000004215 Carbon black (E152) Substances 0.000 description 2
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 2
- 241000027963 Cobetia amphilecti Species 0.000 description 2
- 241000100611 Halomonas denitrificans Species 0.000 description 2
- 239000007832 Na2SO4 Substances 0.000 description 2
- 241000192142 Proteobacteria Species 0.000 description 2
- 241000589614 Pseudomonas stutzeri Species 0.000 description 2
- 241000187561 Rhodococcus erythropolis Species 0.000 description 2
- 239000004115 Sodium Silicate Substances 0.000 description 2
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 2
- 241000828391 Thalassospira povalilytica Species 0.000 description 2
- 239000001110 calcium chloride Substances 0.000 description 2
- 229910001628 calcium chloride Inorganic materials 0.000 description 2
- 239000002131 composite material Substances 0.000 description 2
- 229910000397 disodium phosphate Inorganic materials 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 239000003344 environmental pollutant Substances 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- XLYOFNOQVPJJNP-ZSJDYOACSA-N heavy water Substances [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 description 2
- 229930195733 hydrocarbon Natural products 0.000 description 2
- 150000002430 hydrocarbons Chemical class 0.000 description 2
- 229910001629 magnesium chloride Inorganic materials 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 230000010355 oscillation Effects 0.000 description 2
- 238000007747 plating Methods 0.000 description 2
- 231100000719 pollutant Toxicity 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- LXNHXLLTXMVWPM-UHFFFAOYSA-N pyridoxine Chemical compound CC1=NC=C(CO)C(CO)=C1O LXNHXLLTXMVWPM-UHFFFAOYSA-N 0.000 description 2
- 238000005067 remediation Methods 0.000 description 2
- 239000013535 sea water Substances 0.000 description 2
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 235000019795 sodium metasilicate Nutrition 0.000 description 2
- NTHWMYGWWRZVTN-UHFFFAOYSA-N sodium silicate Chemical compound [Na+].[Na+].[O-][Si]([O-])=O NTHWMYGWWRZVTN-UHFFFAOYSA-N 0.000 description 2
- 229910052911 sodium silicate Inorganic materials 0.000 description 2
- 229910052938 sodium sulfate Inorganic materials 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 229910001631 strontium chloride Inorganic materials 0.000 description 2
- AHBGXTDRMVNFER-UHFFFAOYSA-L strontium dichloride Chemical compound [Cl-].[Cl-].[Sr+2] AHBGXTDRMVNFER-UHFFFAOYSA-L 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 239000011573 trace mineral Substances 0.000 description 2
- 235000013619 trace mineral Nutrition 0.000 description 2
- 108020004465 16S ribosomal RNA Proteins 0.000 description 1
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 1
- 241000186361 Actinobacteria <class> Species 0.000 description 1
- 241000186046 Actinomyces Species 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 241001135756 Alphaproteobacteria Species 0.000 description 1
- 206010007269 Carcinogenicity Diseases 0.000 description 1
- 229910021580 Cobalt(II) chloride Inorganic materials 0.000 description 1
- 229910021592 Copper(II) chloride Inorganic materials 0.000 description 1
- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 0.000 description 1
- 241001653918 Halomonas sp. Species 0.000 description 1
- 229910004619 Na2MoO4 Inorganic materials 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 241000589516 Pseudomonas Species 0.000 description 1
- 241001223182 Pseudomonas plecoglossicida Species 0.000 description 1
- 241000507051 Pseudomonas zhaodongensis Species 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 229930003268 Vitamin C Natural products 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 150000001491 aromatic compounds Chemical class 0.000 description 1
- 150000004945 aromatic hydrocarbons Chemical class 0.000 description 1
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 230000007670 carcinogenicity Effects 0.000 description 1
- 231100000260 carcinogenicity Toxicity 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- ORTQZVOHEJQUHG-UHFFFAOYSA-L copper(II) chloride Chemical compound Cl[Cu]Cl ORTQZVOHEJQUHG-UHFFFAOYSA-L 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 230000004907 flux Effects 0.000 description 1
- 238000002290 gas chromatography-mass spectrometry Methods 0.000 description 1
- 229910001385 heavy metal Inorganic materials 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 230000007886 mutagenicity Effects 0.000 description 1
- 231100000299 mutagenicity Toxicity 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 230000002085 persistent effect Effects 0.000 description 1
- -1 phenanthrene compound Chemical class 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- RADKZDMFGJYCBB-UHFFFAOYSA-N pyridoxal hydrochloride Natural products CC1=NC=C(CO)C(C=O)=C1O RADKZDMFGJYCBB-UHFFFAOYSA-N 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000004062 sedimentation Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 239000011684 sodium molybdate Substances 0.000 description 1
- TVXXNOYZHKPKGW-UHFFFAOYSA-N sodium molybdate (anhydrous) Chemical compound [Na+].[Na+].[O-][Mo]([O-])(=O)=O TVXXNOYZHKPKGW-UHFFFAOYSA-N 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 235000019158 vitamin B6 Nutrition 0.000 description 1
- 239000011726 vitamin B6 Substances 0.000 description 1
- 235000019154 vitamin C Nutrition 0.000 description 1
- 239000011718 vitamin C Substances 0.000 description 1
- 229940011671 vitamin b6 Drugs 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 0.000 description 1
- 239000011592 zinc chloride Substances 0.000 description 1
Classifications
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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
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
-
- A—HUMAN NECESSITIES
- A62—LIFE-SAVING; FIRE-FIGHTING
- A62D—CHEMICAL MEANS FOR EXTINGUISHING FIRES OR FOR COMBATING OR PROTECTING AGAINST HARMFUL CHEMICAL AGENTS; CHEMICAL MATERIALS FOR USE IN BREATHING APPARATUS
- A62D3/00—Processes for making harmful chemical substances harmless or less harmful, by effecting a chemical change in the substances
- A62D3/02—Processes for making harmful chemical substances harmless or less harmful, by effecting a chemical change in the substances by biological methods, i.e. processes using enzymes or microorganisms
-
- A—HUMAN NECESSITIES
- A62—LIFE-SAVING; FIRE-FIGHTING
- A62D—CHEMICAL MEANS FOR EXTINGUISHING FIRES OR FOR COMBATING OR PROTECTING AGAINST HARMFUL CHEMICAL AGENTS; CHEMICAL MATERIALS FOR USE IN BREATHING APPARATUS
- A62D2101/00—Harmful chemical substances made harmless, or less harmful, by effecting chemical change
- A62D2101/20—Organic substances
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- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Microbiology (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Toxicology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Business, Economics & Management (AREA)
- Emergency Management (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Purification Treatments By Anaerobic Or Anaerobic And Aerobic Bacteria Or Animals (AREA)
Abstract
The present invention relates to a kind of Halomonas bacterial strain Halomonas sp.BN3 1, it is characterised in that：Its nucleotide sequence is as shown in SEQ ID NO.1, compared with prior art, the invention has the advantages that：The speed of its degrading polycyclic aromatic hydrocarbons of the Halomonas bacterium of the present invention, Halomonas bacterium culture one week in 100ml culture mediums (luxuriant and rich with fragrance solubility is 100mg/L), bacterial strain BN1 1, BF1 1, BN3 1 are respectively 30%, 33%, 74% to luxuriant and rich with fragrance degradation rate, and wherein bacterial strain BN3 1 has higher degradation rate.
Description
Technical field
The invention belongs to microorganism remediation polluted soils and water body technical field, more particularly to a kind of degradable polycyclic virtue
The degradation bacteria of hydrocarbon and its application.
Background technology
Polycyclic aromatic hydrocarbon (Polycyclic aromatic hydrocarbons, PAHs) is generally existing in a kind of environment
Organic pollution.It is strong to environment and the public due to it with toxicity, to mutation, to mutagenicity, carcinogenicity and characteristic difficult to degrade
Health causes huge harm.Permitted various harm to environment and the mankind with polycyclic aromatic hydrocarbon gradually to be understood, it is now more next
More researchers starts the PAHs being dedicated in the how significantly more efficient removal environment of research.Polycyclic aromatic hydrocarbon in the natural environment
It is degraded by absorption, volatilization, light degradation and chemical oxidation.Ocean is that the important of PAHs collects ground, in land and air
PAHs can enter the contingencies such as ocean, while leaked offshore oil also by entering the modes such as extra large runoff, atmospheric sedimentation and can make
It is polluted at PAHs.It can be re-released into environment again in addition, PAHs can also be acted on by settling flux, to cause " secondary dirt
Dye ".Due to the higher lipophilicitys of PAHs, it is easily transferred in organism and deposit into the PAHs in marine environment, and lead to
It crosses food chain and enters human body, there is prodigious potential hazard to human health and ecological environment.Due to the above-mentioned harm of PAHs,
Environmental Protection Agency USA (US EPA) is just classified as 16 kinds of not branched PAHs in environment early in the 1980s and preferentially controls
Pollutant is managed, and PAHs is also included in priority monitoring and the environmental pollution blacklist of control by Ministry of Environmental Protection's door.
It is numerous studies have shown that during microbial degradation in the Transport And Transformation of pollutant or even is finally removed from environment
It occupies an important position, is PAHs and the most important approach of heavy metals removal in environment.Meanwhile microorganism remediation have it is mild,
Efficient and economic feature.But the resources reserve of existing polycyclic aromatic hydrocarbons (PAH) degradation bacteria strains is also very limited, seriously limits environment dirt
Contaminate the further development of object PAHs bioremediation technologies.
Invention content
A technical problem to be solved by this invention be for the prior art present situation provide it is a kind of can effectively degrade it is more
The degradation bacteria of cycloaromatics.
Another technical problem to be solved by this invention is the above-mentioned drop of a kind of utilization of present situation offer for the prior art
Solve application of the bacterium in degrading polycyclic aromatic hydrocarbons.
Technical solution is used by the present invention solves above-mentioned technical problem：Halomonas bacterial strain Halomonas
Sp.BN3-1, it is characterised in that：Its nucleotide sequence is as shown in SEQ ID NO.1.
The bacterial strain is one plant of Halophilic Bacterium strain, and salinity growth scope is 0-20% (the most suitable growth ranging from 7-
10%), therefore its PAHs that can degrade in the higher environment of salinity, to achieve the effect that remove PAHs from environment.
Further, the deposit number of the Halomonas bacterial strain Halomonas sp.BN3-1 is CGMCC
NO.14838。
The present invention provides a kind of Halomonas bacterial strain Halomonas sp.BN3- to solve second technical problem
1 application in degrading polycyclic aromatic hydrocarbons, it is characterised in that：70%~74% can reach to the luxuriant and rich with fragrance degradation efficiency in polycyclic aromatic hydrocarbon.
Compared with prior art, the invention has the advantages that：Its polycyclic virtue of degrading of the Halomonas bacterium of the present invention
The speed of hydrocarbon, the Halomonas bacterium culture one week, bacterial strain in 100ml culture mediums (luxuriant and rich with fragrance solubility is 100mg/L)
BN1-1, BF1-1, BN3-1 are respectively 30%, 33%, 74% to luxuriant and rich with fragrance degradation rate, and wherein bacterial strain BN3-1 has higher degradation
Rate.
Preservation explanation
1, Halomonas bacterial strain BN3-1, Classification And Nomenclature Halomonas submarina, on October 27th, 2017,
It is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), depositary institution address is：Beijing
The institute 3 of Chaoyang District North Star West Road 1, deposit number are CGMCC NO.14838.
Description of the drawings
Fig. 1 is bacterial strain BN3-1 colonial morphology figures in the present invention.
Fig. 2 is the system and device figure that sublimed method prepares PAHs solid mediums in the present invention.
Specific implementation mode
Below by way of in conjunction with attached drawing, sequence table and embodiment, the invention will be further described.
Embodiment 1
(1) acquisition of sample：
Mud sample is No. six scientific investigation Chuan29Ci sections of in October, 2013 Chinese Sea in Eastern Pacific combined area WBC1306 (warps
Spend W154.235, latitude N10.016) erect-position point acquisition deposit mud column.
(2) dissolving of PAHs：
The acetone soln of polycyclic aromatic hydrocarbon compounds phenanthrene, naphthalene, pyrene, fluoranthene is added in the triangular flask of high pressure sterilization processing, it will
It is placed volatilizees to acetone soln completely overnight with 37 DEG C of shaking table shake denier, and bacteria culture media I is added, makes polycyclic aromatic hydrocarbon compounds
Naphthalene, phenanthrene, pyrene, fluoranthene ultimate density are respectively 200mg/L, 100mg/L, 50mg/L and 20mg/L.
Bacteria culture media I is：Ironic citrate 0.10g, NaCl 19.45g, MgCl2 5.90g、Na2SO4 3.24g、
CaCl2 1.80g、KCl 0.55g、NaHCO3 0.16g、KBr 0.08g、SrCl2 34.00mg、H3BO322.00mg, sodium metasilicate
4.00mg、NaF 2.40mg、(NH4)NO3 1.60mg、Na2HPO48.00mg, 1ml trace element, 1ml vitamins are compound molten
Liquid, deionized water 1L, pH7.2.1.5% agar powder is then added if solid medium.
The group of wherein trace element solution becomes:CoCl2·6H2O 35mg、CuCl2 0.20mg、H3BO3 6.0mg、
MnCl·4H2O 25mg、Na2MoO4·2H2O 3.0mg、NiCl·2H2O 2.0mg、ZnCl22.5mg, deionized water 1L.
The group of vitamin composite solution becomes：Vitamin B6 1.0mg, vitamin C 1.5mg, deionized water 1L.
(3) enrichment of PAHs degradation bacterias
Each erect-position surface deposit takes 5g to be placed in sterile 50ml centrifuge tubes, is placed in 50ml centrifuge tubes, is added 40
Ml is placed on 4 DEG C of refrigerator overnights without Herba Artemisiae Scopariae seawater, after oscillation mixing 10min makes thalline recover.Each sample takes 10ml supernatants
It adds in 100ml PAHs enriched mediums I, wherein polycyclic aromatic hydrocarbon compounds are respectively that phenanthrene, naphthalene, pyrene, fluoranthene its content are respectively
200mg/L, 100mg/L, 50mg/L, 20mg/L, 16 DEG C, 160rpm/min, be protected from light enrichment culture to culture medium and become cloudy (one week
Left and right).Enrichment culture liquid is transferred to according to 2% ratio in fresh phase enriched medium, equal conditions continue enrichment culture 5
It is secondary.The step is repeated 5 times until microbial population is stablized.Above-mentioned enrichment culture liquid 1ml is taken respectively, is distinguished with no Herba Artemisiae Scopariae seawater dilute
It releases to 10-3、10-4、10-5Three gradients, are coated on 2216 tablets, are placed in 16 DEG C of constant temperature incubation carton upside down cultures two weeks.
(4) PAHs degradation bacterias are isolated and purified, are identified
The different single bacterium colony scribing line of picking colony morphological feature is cultivated on fresh culture II plates, 16 DEG C of culture 15d, weight
Multiple scribing line 3 generations of purifying.The isolated strains of purifying carry out the bacterial strains identification such as 16S rRNA gene orders sequencing.
Medium ii：Peptone 5.00g, yeast powder 1.00g, ironic citrate 0.10g, NaCl 19.45g, MgCl2 5.90
g、Na2SO4 3.24g、CaCl2 1.80g、KCl 0.55g、NaHCO3 0.16g、KBr 0.08g、SrCl2 34.00mg、
H3BO322.00mg, sodium metasilicate 4.00mg, NaF 2.40mg, (NH4)NO3 1.60mg、Na2HPO4The micro member of 8.00mg, 1ml
Plain solution, 1ml vitamins composite solution, deionized water 1L, pH7.2.
Embodiment 2
(1) sublimed method prepares PAHs solid mediums:
Due to PAHs compound strong-hydrophobicities, the physical characteristic that can only be dissolved in organic solvent causes polycyclic aromatic hydrocarbon can not
It is added in solid medium with usual way.For this purpose, being made using the physical characteristic that polycyclic aromatic hydrocarbon compounds easily distil
Polycyclic aromatic hydrocarbon solid medium.
Steps are as follows for specific method：
(1) plate of two aluminums is first made, the bore of plate is identical as culture dish bottom cover diameter, can just detain
Above aluminium dish.
(2) prepare a heated at constant temperature instrument；
Constent temperature heater is positioned in draught cupboard, and the plate of aluminum is placed and preheats half an hour right over it.
(3) suitable polycyclic aromatic hydrocarbon compounds are taken uniformly to be laid in the bottom of aluminium dish, and by the tablet containing culture medium I
It is placed on above aluminium dish, while another plate is filled into ice and is placed on the tablet back side, until media surface plating last layer is more
Aromatic compound.
Above-mentioned whole operation process need to be completed in draught cupboard.The heating temperature that common polycyclic aromatic hydrocarbon compounds distil
Degree is that PAHs compounds can be kept to remain solid state in this way less than 5 DEG C of the melting point compound.
(plate of aluminum corresponds to label -1 to the device of the system in Fig. 1, and culture dish corresponds to label -2, constant temperature as shown in Figure 2
Heating instrument corresponds to label -3, and culture medium I corresponds to label -4, and polycyclic aromatic hydrocarbon compounds correspond to label -5, and ice cube corresponds to label -6).
Embodiment 3
Preliminary judgement is carried out to the ability of strains for degrading PAHs using PAHs phenanthrene solid medium
Solid medium I is prepared, plating one layer of phenanthrene compound on the surfaces culture medium I using sublimed method obtains luxuriant and rich with fragrance solid culture
Base.The inoculation of enrichment acquisition before is placed in shaking table 160rpm in II fluid nutrient mediums and is protected from light culture (25 DEG C) 48h.Take high pressure
The scraps of paper for a diameter of 5mm that sterilization treatment is crossed, which are attached to after being bedewed in bacterium solution on PAHs culture mediums, is placed in 25 DEG C of constant temperature trainings
It supports and is cultivated in case.According to bacterial strain whether in PAHs cultured on solid medium and periphery of bacterial colonies transparent circle, the preliminary judgement bacterium
The ability of strain degradation PAHs.If growth, illustrate there is tolerance then to illustrate if periphery of bacterial colonies has transparent circle appearance PAHs
The bacterial strain has PAHs degradation capabilities, whereas if periphery of bacterial colonies does not have transparent circle appearance, then it is polycyclic to illustrate that the bacterial strain does not have
Aromatic hydrocarbon degradation capability, and the degradation capability size of bacterial strain is judged according to the size of degradation circle, i.e. transparent circle size with degrade
Ability is directly proportional.
The PRELIMINARY RESULTS of 4 degrading polycyclic aromatic hydrocarbons efficiency of embodiment
36 plants of bacteriums are obtained altogether by above-mentioned enrichment, separation method.It is under the jurisdiction of Proteobacteria (Proteobacteria),
Actinomyces door (Actinobacteria), is under the jurisdiction of α-deformation Gammaproteobacteria (Alpha proteobacteria), γ-deformation Gammaproteobacteria
(Gamma proteobacteria), Actinomycetes (Actinobacteria) its be under the jurisdiction of belong to 6 belong to 8 kinds, wherein wrapping
Include Aurantimonas coralicida, Thalassospira povalilytica, Pseudomonas stutzeri,
Pseudomonas zhaodongensis、Cobetia amphilecti、Rhodococcus erythropolis、
Halomonas denitrificans、Pseudomonas plecoglossicida。
It is enclosed according to bacterial strain whether PAHs phenanthrene cultured on solid medium situations and periphery of bacterial colonies have degradation to enclose and degrade
Size come the preliminary power for judging strains for degrading PAHs abilities, the judgement of erect-position WBC1306 erect-position strains for degrading capacity of water
As a result：
Note：Representative is not grown ,+represent and have bacterium colony growth, ++ it is weak to represent degradability, +++ it is good to represent degradability
Upper table show bacterial strain Thalassospira povalilytica in erect-position WBC1306, Pseudomonas,
Zhaodongensis, Halomonas denitrificans, Cobetia amphilecti are good to the degradation capability of PAHs phenanthrene
It is good, strain Pseudomonas stutzeri, Rhodococcus erythropolis, Aurantimonas coralicida
It is then weaker to the degradation capability of PAHs phenanthrene；The erect-position isolated strains all have tolerance effect and degradation capability to PAHs phenanthrene.Bacterial strain
BN3-1 colonial morphologies become khaki for round, low convex, moistening, the milky of color from the beginning to later stage, such as Fig. 1 institutes
Show.
The measurement of embodiment 5 bacterial strain BN3-1, BN1-1, BF1-1 to luxuriant and rich with fragrance degradation efficiency
Logarithmic growth phase bacterial strain BN3-1, BN1-1, BF1-1 culture solution is inoculated into the culture medium I of 100ml phenanthrene containing 100mg
In do experimental group, if inoculated and cultured liquid is not control group.It is surplus that shake culture extraction in one week is protected from light in 160rpm, 28 DEG C of shaking tables
Remaining PAHs distinguishes the residual rate of polycyclic aromatic hydrocarbon compounds in determination experiment group and control group, then utilizes test combinations control group
Residual rate calculate degradation efficiency.30ml CHCl are added into culture medium for the extraction of residue PAHs in culture medium3Extraction is not dropped
The PAHs of solution, 200rpm shaking table vibrate 30min, are then ultrasonically treated 20min, then add 30ml CHCl3, 200rpm shakes
Then bed extracts the CHCl of about 60ml after persistent oscillation 30min with separatory funnel3, use CHCl3Calmly molten to arrive 100ml, mixing accurately takes
It is fixed it is molten after solution 1ml, suitable anhydrous Na is added2SO4(in advance at 150 DEG C, toasting 2hours) dehydration, 12000rpm, centrifugation
5min, the then accurate dilution (remembeing extension rate, on one side last result of calculation) for removing certain volume and carrying out suitable multiple.It connects
It, the extract liquor after taking 1ul to dilute carries out GC-MS analyses, and it is bent to bring the peak area of certain obtained PAHs into corresponding standard
In line, the concentration of this kind of PAHs is found out.It carries out repeating to test three times, results are averaged, and BN1-1, BF1-1, BN3-1 are to phenanthrene
Degradation rate is respectively 30%, 33%, 74%.Degradation efficiency=(control group polycyclic aromatic hydrocarbon total amount-experimental group polycyclic aromatic hydrocarbon content)/
Control group PAHs contents * 100%.
Sequence table
<110>Wanli College, Zhejiang
<120>A kind of ocean degradation bacteria of degrading polyaromatic hydrocarbon and its application
<130> Not published yet
<141> 2017-10-30
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1558
<212> DNA
<213> Halomonas sp.BN3-1
<400> 1
ttgcatgcct gcaggtcgac gattagagtt tgatcctggc tcagattgaa cgctggcggc 60
aggcctaaca catgcaagtc gagcggaaac gatcctagct tgctaggagg cgtcgagcgg 120
cggacgggtg agtaatgcat aggaatctgc ccggtagtgg gggataacct ggggaaaccc 180
aggctaatac cgcatacgcc ctacggggga aagcagggga tcttcggacc ttgcgctatc 240
ggatgagcct atgtcggatt agctagttgg tgaggtaacg gctcaccaag gctgcgatcc 300
gtagctggtc tgagaggatg atcagccaca tcgggactga gacacggccc gaactcctac 360
gggaggcagc agtggggaat attggacaat gggcgaaagc ctgatccagc catgccgcgt 420
gtgtgaagaa ggccttcggg ttgtaaagca ctttcagtga ggaagaaagc cttgaggtta 480
ataccctcga gggaggacat cactcacaga agaagcaccg gctaactccg tgccagcagc 540
cgcggtaata cggagggtgc gagcgttaat cggaattact gggcgtaaag cgcgcgtagg 600
cggcttgata agccggttgt gaaagccccg ggctcaacct gggaacggca tccggaactg 660
tcaggctaga gtgcaggaga ggaaggtaga attcccggtg tagcggtgaa atgcgtagag 720
atcgggagga ataccagtgg cgaaggcggc cttctggact gacactgacg ctgaggtgcg 780
aaagcgtggg tagcaaacag gattagatac cctggtagtc cacgccgtaa acgatgtcga 840
ctagccgttg ggctccttga gagctttgtg gcgcagttaa cgcgataagt cgaccgcctg 900
gggagtacgg ccgcaaggtt aaaactcaaa tgaattgacg ggggcccgca caagcggtgg 960
agcatgtggt ttaattcgat gcaacgcgaa gaaccttacc tacccttgac atcgagagaa 1020
ctttccagag atggattggt gccttcggga actctcagac aggtgctgca tggctgtcgt 1080
cagctcgtgt tgtgaaatgt tgggttaagt cccgtaacga gcgcaaccct tgtccctatt 1140
tgccagcgat tcggtcggga actctaggga gactgccggt gacaaaccgg aggaaggtgg 1200
ggacgacgtc aagtcatcat ggcccttacg ggtagggcta cacacgtgct acaatggccg 1260
gtacaatggg ttgcgaatcc gcgaggtgga gctaatccca taaagccggt ctcagtccgg 1320
atcggagtct gcaactcgac tccgtgaagt cggaatcgct agtaatcgtg aatcagaatg 1380
tcacggtgaa tacgttcccg ggccttgtac acaccgcccg tcacaccatg ggagtggact 1440
gcaccagaag tggttagcct aacttcggag ggcgatcacc acggtgtggt tcatgactgg 1500
ggtgaagtcg taacaaggta gccgtaatct ctagaggatc cccgggtacc gagctcga 1558
Claims (3)
1. a kind of Halomonas bacterial strain Halomonas sp.BN3-1, it is characterised in that：Its nucleotide sequence such as SEQ ID
Shown in NO.1.
2. according to the Halomonas of Halomonas bacterial strain described in claim 1 sp.BN3-1, it is characterised in that：Its preservation
Number is CGMCC NO.14838.
3. a kind of applications of Halomonas bacterial strain Halomonas sp.BN3-1 in degrading polycyclic aromatic hydrocarbons, feature exist
In：70%~74% can reach to the luxuriant and rich with fragrance degradation efficiency in polycyclic aromatic hydrocarbon.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101838616A (en) * | 2009-03-17 | 2010-09-22 | 清华大学 | Halomonas capable of degrading polyaromatic hydrocarbon and application thereof |
| US10478652B2 (en) * | 2017-12-15 | 2019-11-19 | King Fadh University Of Petroleum And Minerals | Method for biodegrading high molecular weight polycyclic aromatic hydrocarbon pyrenes with halophilic bacteria |
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| CN101838616A (en) * | 2009-03-17 | 2010-09-22 | 清华大学 | Halomonas capable of degrading polyaromatic hydrocarbon and application thereof |
| US10478652B2 (en) * | 2017-12-15 | 2019-11-19 | King Fadh University Of Petroleum And Minerals | Method for biodegrading high molecular weight polycyclic aromatic hydrocarbon pyrenes with halophilic bacteria |
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| CN111268810A (en) * | 2020-03-20 | 2020-06-12 | 微米环创生物科技(北京)有限公司 | Nitrogen and phosphorus removal microbial community and application thereof |
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