CN106479942A - 一种苏云金芽孢杆菌及其应用 - Google Patents
一种苏云金芽孢杆菌及其应用 Download PDFInfo
- Publication number
- CN106479942A CN106479942A CN201611243613.3A CN201611243613A CN106479942A CN 106479942 A CN106479942 A CN 106479942A CN 201611243613 A CN201611243613 A CN 201611243613A CN 106479942 A CN106479942 A CN 106479942A
- Authority
- CN
- China
- Prior art keywords
- bacillus
- crude oil
- thuringiensiss
- bacillus thuringiensiss
- oil
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 241000193388 Bacillus thuringiensis Species 0.000 title claims abstract description 49
- 239000010779 crude oil Substances 0.000 claims abstract description 33
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 26
- 229940097012 bacillus thuringiensis Drugs 0.000 claims abstract description 16
- 244000005700 microbiome Species 0.000 claims abstract description 5
- 230000000593 degrading effect Effects 0.000 claims abstract description 4
- 108020004465 16S ribosomal RNA Proteins 0.000 claims description 4
- 238000004321 preservation Methods 0.000 claims 1
- 230000015556 catabolic process Effects 0.000 abstract description 13
- 238000006731 degradation reaction Methods 0.000 abstract description 13
- 241000894006 Bacteria Species 0.000 abstract description 12
- 230000000694 effects Effects 0.000 abstract description 4
- 239000002351 wastewater Substances 0.000 abstract description 3
- 230000007613 environmental effect Effects 0.000 abstract description 2
- 238000011031 large-scale manufacturing process Methods 0.000 abstract description 2
- 230000008439 repair process Effects 0.000 abstract description 2
- 239000003921 oil Substances 0.000 description 22
- 230000001580 bacterial effect Effects 0.000 description 11
- 239000002609 medium Substances 0.000 description 10
- 238000002835 absorbance Methods 0.000 description 9
- 239000001888 Peptone Substances 0.000 description 8
- 239000003208 petroleum Substances 0.000 description 8
- 235000015278 beef Nutrition 0.000 description 6
- 239000001963 growth medium Substances 0.000 description 6
- HIXDQWDOVZUNNA-UHFFFAOYSA-N 2-(3,4-dimethoxyphenyl)-5-hydroxy-7-methoxychromen-4-one Chemical compound C=1C(OC)=CC(O)=C(C(C=2)=O)C=1OC=2C1=CC=C(OC)C(OC)=C1 HIXDQWDOVZUNNA-UHFFFAOYSA-N 0.000 description 5
- 239000012153 distilled water Substances 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 5
- RTZKZFJDLAIYFH-UHFFFAOYSA-N ether Substances CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 5
- 238000000034 method Methods 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 239000001301 oxygen Substances 0.000 description 5
- 229910052760 oxygen Inorganic materials 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 241000251468 Actinopterygii Species 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 3
- 230000003321 amplification Effects 0.000 description 3
- 239000001110 calcium chloride Substances 0.000 description 3
- 229910001628 calcium chloride Inorganic materials 0.000 description 3
- 229910052799 carbon Inorganic materials 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- 239000012531 culture fluid Substances 0.000 description 3
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 3
- 229910052564 epsomite Inorganic materials 0.000 description 3
- 230000006872 improvement Effects 0.000 description 3
- 229910017053 inorganic salt Inorganic materials 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 238000009630 liquid culture Methods 0.000 description 3
- 229910052603 melanterite Inorganic materials 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- 238000011084 recovery Methods 0.000 description 3
- VWDWKYIASSYTQR-UHFFFAOYSA-N sodium nitrate Inorganic materials [Na+].[O-][N+]([O-])=O VWDWKYIASSYTQR-UHFFFAOYSA-N 0.000 description 3
- 239000002689 soil Substances 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 2
- ROWKJAVDOGWPAT-UHFFFAOYSA-N Acetoin Chemical compound CC(O)C(C)=O ROWKJAVDOGWPAT-UHFFFAOYSA-N 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 108010000912 Egg Proteins Proteins 0.000 description 2
- 102000002322 Egg Proteins Human genes 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- SIKJAQJRHWYJAI-UHFFFAOYSA-N Indole Chemical compound C1=CC=C2NC=CC2=C1 SIKJAQJRHWYJAI-UHFFFAOYSA-N 0.000 description 2
- 108010080698 Peptones Proteins 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 238000009395 breeding Methods 0.000 description 2
- 230000001488 breeding effect Effects 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000006071 cream Substances 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 229910000041 hydrogen chloride Inorganic materials 0.000 description 2
- IXCSERBJSXMMFS-UHFFFAOYSA-N hydrogen chloride Substances Cl.Cl IXCSERBJSXMMFS-UHFFFAOYSA-N 0.000 description 2
- 230000007062 hydrolysis Effects 0.000 description 2
- 238000006460 hydrolysis reaction Methods 0.000 description 2
- 238000007373 indentation Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 230000000877 morphologic effect Effects 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 239000006916 nutrient agar Substances 0.000 description 2
- 235000019319 peptone Nutrition 0.000 description 2
- 230000035479 physiological effects, processes and functions Effects 0.000 description 2
- 125000005575 polycyclic aromatic hydrocarbon group Chemical group 0.000 description 2
- 238000012797 qualification Methods 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 description 1
- 235000001674 Agaricus brunnescens Nutrition 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 1
- 206010003497 Asphyxia Diseases 0.000 description 1
- 241000193755 Bacillus cereus Species 0.000 description 1
- 239000004215 Carbon black (E152) Substances 0.000 description 1
- 102000016938 Catalase Human genes 0.000 description 1
- 108010053835 Catalase Proteins 0.000 description 1
- 241000195493 Cryptophyta Species 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 229910002651 NO3 Inorganic materials 0.000 description 1
- 241000772415 Neovison vison Species 0.000 description 1
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- MMDJDBSEMBIJBB-UHFFFAOYSA-N [O-][N+]([O-])=O.[O-][N+]([O-])=O.[O-][N+]([O-])=O.[NH6+3] Chemical compound [O-][N+]([O-])=O.[O-][N+]([O-])=O.[O-][N+]([O-])=O.[NH6+3] MMDJDBSEMBIJBB-UHFFFAOYSA-N 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 150000001491 aromatic compounds Chemical class 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000004166 bioassay Methods 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 239000005018 casein Substances 0.000 description 1
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 1
- 235000021240 caseins Nutrition 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000000205 computational method Methods 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- 230000006866 deterioration Effects 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 235000014103 egg white Nutrition 0.000 description 1
- 210000000969 egg white Anatomy 0.000 description 1
- 239000012259 ether extract Substances 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 238000012215 gene cloning Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 230000003054 hormonal effect Effects 0.000 description 1
- 229930195733 hydrocarbon Natural products 0.000 description 1
- 150000002430 hydrocarbons Chemical class 0.000 description 1
- 125000001183 hydrocarbyl group Chemical group 0.000 description 1
- -1 indentation Substances 0.000 description 1
- PZOUSPYUWWUPPK-UHFFFAOYSA-N indole Natural products CC1=CC=CC2=C1C=CN2 PZOUSPYUWWUPPK-UHFFFAOYSA-N 0.000 description 1
- RKJUIXBNRJVNHR-UHFFFAOYSA-N indolenine Natural products C1=CC=C2CC=NC2=C1 RKJUIXBNRJVNHR-UHFFFAOYSA-N 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 239000005416 organic matter Substances 0.000 description 1
- 210000004681 ovum Anatomy 0.000 description 1
- 239000003973 paint Substances 0.000 description 1
- 230000000149 penetrating effect Effects 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 238000007146 photocatalysis Methods 0.000 description 1
- 230000001699 photocatalysis Effects 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000003672 processing method Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 239000012088 reference solution Substances 0.000 description 1
- 238000005067 remediation Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 239000004575 stone Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
- 238000000870 ultraviolet spectroscopy Methods 0.000 description 1
Classifications
-
- 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
- C12R2001/07—Bacillus
- C12R2001/075—Bacillus thuringiensis
-
- C—CHEMISTRY; METALLURGY
- 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
-
- 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
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Microbiology (AREA)
- Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Hydrology & Water Resources (AREA)
- Biodiversity & Conservation Biology (AREA)
- Water Supply & Treatment (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Environmental & Geological Engineering (AREA)
- Virology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Medicinal Chemistry (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明属于生物工程技术领域，具体公开了一种苏云金芽孢杆菌，于2016年09月12日保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏号为CGMCC No.13005；命名为苏云金芽孢杆菌(Bacillus thuringiensis)3‑1。还公开了所述苏云金芽孢杆菌(Bacillus thuringiensis)3‑1在降解原油污染水体中的应用。本发明提供的苏云金芽孢杆菌不但易于规模化生产，而且降解效率高，环境适应性强。该菌能在原油浓度达1mL/100mL的原油污染水体中体现出较优的降解效果，实现原油污染水体的修复处理，在原油污染废水的生物修复中展现了良好的应用前景。
Description
技术领域
本发明涉及生物工程技术领域，具体涉及一种苏云金芽孢杆菌及其应用。
背景技术
原油是未经加工的石油，是天然有机物质经过地质变迁而形成的一种粘稠的黑色或深棕色的液体或固体，一般情况下大多存在于地下或海底，且带有刺激性气味，主要成分是碳氢原子结合形成的链状化合物，其对水体环境具有极大的危害。
水环境中维持生命所需的最小溶解氧量约为2mg/L，石油或含油废水排入水体后一方面会漂浮在水面上，阻隔氧气的进入，影响气体交换，另一方面部分靠石油提供碳源繁殖的微生物也会消耗氧气，导致水中溶解氧的缺乏。同时又会使厌氧生物迅速繁殖使水体水质恶化。同时芳香烃类化合物产生的毒性也影响藻类的生长，进而对食物链造成影响。此外，石油中的有毒成分也会影响水生生物的生长繁殖，它会沾附在鱼鳃上使其窒息死亡或影响其卵化，多环芳烃会破坏水生生物的内分泌系统，当存在极低的浓度的多环芳烃时，鱼类都会产生鱼臭味，直接影响了鱼类的正常生存，严重破坏了水环境中水生生物的多样性。
目前，对石油污染的水体的修复技术主要有物理修复技术、化学修复技术、光催化修复技术和生物修复技术；生物修复技术是一个具有政策、经济和技术吸引力的处理方法，被认为是最具发展潜力的技术方法，并且逐步发展成为一种经济效益和环境效益俱佳的、解决复杂环境污染问题的最有效的手段。
发明内容
针对现有技术中存在的问题，本发明的目的在于提供一种可用于降解原油污染水体的苏云金芽孢杆菌，将该菌种直接投加入污染水体中，达到较好的降解效果，实现原油污染的治理。
为了达到上述目的，本发明采用以下技术方案予以实现。
(一)一种苏云金芽孢杆菌，已于2016年09月12日保藏于北京市朝阳区北辰西路1号院3号中国科学院微生物研究所的中国微生物菌种保藏管理委员会普通微生物中心，保藏号为CGMCC No.13005；命名为苏云金芽孢杆菌(Bacillus thuringiensis)3-1。
将所述苏云金芽孢杆菌在牛肉膏蛋白胨培养基上培养，菌株3-1在牛肉膏蛋白胨的平板上呈圆形，边缘呈锯齿状，中间凸起，乳黄色，表面光滑，湿润。菌体为短杆状，大小为(1.2-1.5)μm×(3-5)μm，革兰氏染色呈现阳性。
(二)一种苏云金芽孢杆菌(Bacillus thuringiensis)3-1在降解原油污染水体中的应用。
与现有技术相比，本发明的有益效果为：
本发明提供的苏云金芽孢杆菌不但易于规模化生产，而且降解效率高，环境适应性强。所提供的苏云金芽孢杆菌能在大量有机物存在的条件下顺利生长，以原油为碳源，达到较好的降解效果。该菌能在原油浓度达1mL/100mL的原油污染水体中体现出较优的降解效果，实现原油污染水体的修复处理，在原油污染废水的生物修复中展现了良好的应用前景。
附图说明
下面结合附图和具体实施例对本发明做进一步详细说明。
图1为本发明的苏云金芽孢杆菌(Bacillus thuringiensis)3-1在牛肉膏蛋白胨培养基平板上的菌落形态图；
图2为本发明的苏云金芽孢杆菌(Bacillus thuringiensis)3-1在显微镜下的的菌体形态图，图中，放大倍数为4000倍；
图3为本发明实施例3中I号原油的波长-吸光度曲线图；图中，横坐标为波长，单位为nm，纵坐标为吸光度，单位为1；
图4为本发明实施例3中I号原油的标准曲线图；图中，横坐标为浓度，单位为mg/L，纵坐标为吸光度，单位为1。
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述，但是本领域的技术人员将会理解，下列实施例仅用于说明本发明，而不应视为限制本发明的范围。
实施例1菌株的分离纯化
(1)菌株来源
本发明提供一种苏云金芽孢杆菌(Bacillus thuringiensis)3-1，分离自延长石油集团的七里洞采油场集油站落地原油污染土壤中，土壤的理化性质为：铵态氮15.09ppm，硝态氮0.72ppm；氮含量15.81ppm，磷含量82.05ppm，钾含量17.45ppm，有机质含量1.43％，pH为7.65。
(2)菌株的分离纯化
将10g石油污染土壤样品加入100mL富集液体培养基中，28℃、130r/min摇床培养7d。待培养液混浊后，吸取5mL培养液重新转接入新鲜富集培养基中，与上述培养条件相同连续转接富集培养3次。取1mL上述菌悬液，用无菌水系列稀释后，分别接种于牛肉膏蛋白胨培养基中，29℃左右恒温培养3d。其中，富集液体培养基的成分为：NaNO3 1.5g，(NH4)2SO41.5g，K2HPO4 1g，MgSO4·7H2O 0.5g，KCl 0.5g，FeSO4·7H2O 0.01g，CaCl2 0.002g，蒸馏水1000mL，原油I号1％(体积比)，pH 7.0。牛肉膏蛋白胨培养基的成分为：牛肉膏3.0g，蛋白胨10.0g，NaCl 5.0g，营养琼脂15.0g，蒸馏水1000mL，pH 7.4-7.6。待平板菌落有效分离后，根据菌落颜色、形态特征及表面湿度的不同，分别用接种针挑选菌落进行平板划线纯化分离。将菌株接种于斜面，4℃冰箱冷藏备用。
(3)溶油圈法筛选石油降解菌
将上述纯菌菌种接种于含油无机盐固体培养基上，置于37℃培养箱中培养10-15d，观察有无嗜油圈，并根据嗜油圈直径(D)与菌落直径(d)的比值初步确定菌株的石油降解能力；其中，含油无机盐固体培养基的成分为：NaNO3 1.5g，(NH4)2SO4 1.5g，K2HPO4 1g，MgSO4·7H2O 0.5g，KCl 0.5g，FeSO4·7H2O 0.01g，CaCl2 0.002g，蒸馏水1000mL，原油1.5mL，pH 7.0，pH值用1mol/L的氢氧化钠或1mol/L氯化氢调至要求值。将初步筛选的石油降解菌接种于斜面培养基，4℃冰箱冷藏备用。
实施例2菌株的鉴定
(1)菌株的形态特征
如图1所示，将所述苏云金芽孢杆菌在牛肉膏蛋白胨培养基上30℃恒温培养24h后观察菌落形态，菌株3-1在牛肉膏蛋白胨的平板上呈圆形，边缘呈锯齿状，中间凸起，乳黄色，表面光滑，湿润。如图2所示，菌体为短杆状，大小为(1.2-1.5)μm×(3-5)μm。
(2)生化特性
根据《伯杰氏细菌鉴定手册》和《常见细菌系统鉴定手册》对苏云金芽孢杆菌进行生理生化鉴定，鉴定结果如表1所示。
表1苏云金芽孢杆菌3-1菌株的生理生化鉴定结果
| 指标 | 特性 |
| 革兰氏染色 | + |
| 葡糖糖 | + |
| 蔗糖 | + |
| 淀粉水解 | + |
| 酪素水解 | + |
| M.P(甲基红)试验 | + |
| 硝酸盐还原 | + |
| 产吲哚 | — |
| V.P(乙酰甲基甲醇)试验 | + |
| 过氧化氢酶 | + |
| 明胶水解 | + |
注：“—”表示反应为阴性；“+”表示反应为阳性
(3)菌株的16S rDNA鉴定
用DNA提取试剂盒提取苏云金芽孢杆菌(Bacillus thuringiensis)3的基因组DNA，然后进行16S rDNA基因克隆、测序，其16S rDNA测序结果如SEQ ID No.1所示，具体如下：
之后在GenBank中进行Blast比对，结果显示，与其序列相似度达到98.56％均为苏云金芽孢杆菌(Bacillus thuringiensis)，将其归属为单胞菌类并命名为苏云金芽孢杆菌(Bacillus thuringiensis)3-1。
实施例3苏云金芽孢杆菌(Bacillus thuringiensis)3-1对原油污染水体的降解
(1)苏云金芽孢杆菌3-1菌悬液的制备
在无菌条件下，挑取分离纯化获得的石油降解菌，接种于100mL的牛肉膏蛋白胨液体培养基中，摇床扩大培养48h；其中，牛肉膏蛋白胨液体培养基的成分是：牛肉膏3.0g，蛋白胨10.0g，NaCl 5.0g，营养琼脂15.0g，蒸馏水1000mL，pH 7.4-7.6。pH值用1mol/L的氢氧化钠或1mol/L氯化氢调至要求值。培养基灭菌条件是：温度为121～126℃，时间为25min。
(2)I号原油最大吸收波长的测定
石油及其产品在紫外光区有一定的吸收特征，石油组分中带有苯环的芳香族化合物波长一般为250～260nm，而带有共轭双键的化合物波长一般在215～230nm之间，有研究表明，在使用紫外分光光法测定时，255nm左右为原油和重质油的选择范围，225nm左右与为轻质油及炼油厂的油品的选择范围。在100mg/L浓度下，依据获得的波长与吸光度数据，绘制供试I号原油的波长-吸光度曲线如图3所示，其中，I号原油采自延长石油集团七里洞采油场集油站，密度为0.65g/mL。
(3)I号原油标准曲线的绘制
分别取0、2、2.5、5、6、7mg I号原油于50mL比色管中，用石油醚定容，此时的浓度分别为0、40、50、100、120、140mg/L，在258nm波长下，以石油醚为参比溶液，测定标准浓度的吸光度。横坐标为浓度(mg/L)，纵坐标为吸光度，绘制I号原油的浓度(mg/L)-吸光度(A)的标准曲线如图4所示。
(4)原油降解率的测定
取扩大培养的苏云金芽孢杆菌3-1菌液3mL，接种于100mL含油无机盐液体培养基中，含油无机盐培养基的成分是：NaNO3 1.5g，(NH4)2SO4 1.5g，K2HPO4 1g，MgSO4·7H2O0.5g，KCl 0.5g，FeSO4·7H2O 0.01g，CaCl2 0.002g，蒸馏水1000mL，原油1.5mL，pH 7.0；在30℃，120rpm恒温水浴振荡器中培养7d。培养液用5:1(V/V)的硫酸溶液调节pH值为2-3，加入甲醇2-4mL，用3×15mL的石油醚(60-90℃)萃取三次，萃取液经无水硫酸钠干燥后转入50mL容量瓶并定容。
将石油醚萃取液经系列稀释后，以石油醚为参比，通过紫外分光光度法测定其相应的吸光度，根据浓度(mg/L)-吸光度的标准曲线，确定石油醚提取液相应样品的浓度，依据浓度计算降解效率。
原油降解率的计算方法参考以下公式：
η(％)＝(1-Ct/Co)×100％
其中：η-原油降解率；Ct-菌株存在下原油浓度；Co-对照原油浓度。
按照上述计算公式计算得苏云金芽孢杆菌(Bacillus thuringiensis)对原油污染水体的降解率为70.46％。
虽然，本说明书中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
SEQUENCE LISTING
<110> 长安大学
<120> 一种苏云金芽孢杆菌及其应用
<130> 2016
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1404
<212> DNA
<213> 苏云金芽孢杆菌（Bacillus thuringiensis）3-1
<400> 1
TAAGAGCTTG CTCTTATGAA GTTAGCGGCG GACGGGTGAG TAACACGTGG GTAACCTGCC 60
CATAAGACTG GGATAACTCC GGGAAACCGG GGCTAATACC GGATAACATT TTGAACTGCA 120
TGGTTCGAAA TTGAAAGGCG GCTTCGGCTG TCACTTATGG ATGGACCCGC GTCGCATTAG 180
CTAGTTGGTG AGGTAACGGC TCACCAAGGC AACGATGCGT AGCCGACCTG AGAGGGTGAT 240
CGGCCACACT GGGACTGAGA CACGGCCCAG ACTCCTACGG GAGGCAGCAG TAGGGAATCT 300
TCCGCAATGG ACGAAAGTCT GACGGAGCAA CGCCGCGTGA GTGATGAAGG CTTTCGGGTC 360
GTAAAACTCT GTTGTTAGGG AAGAACAAGT GCTAGTTGAA TAAGCTGGCA CCTTGACGGT 420
ACCTAACCAG AAAGCCACGG CTAACTACGT GCCAGCAGCC GCGGTAATAC GTAGGTGGCA 480
AGCGTTATCC GGAATTATTG GGCGTAAAGC GCGCGCAGGT GGTTTCTTAA GTCTGATGTG 540
AAAGCCCACG GCTCAACCGT GGAGGGTCAT TGGAAACTGG GAGACTTGAG TGCAGAAGAG 600
GAAAGTGGAA TTCCATGTGT AGCGGTGAAA TGCGTAGAGA TATGGAGGAA CACCAGTGGC 660
GAAGGCGACT TTCTGGTCTG TAACTGACAC TGAGGCGCGA AAGCGTGGGG AGCAAACAGG 720
ATTAGATACC CTGGTAGTCC ACGCCGTAAA CGATGAGTGC TAAGTGTTAG AGGGTTTCCG 780
CCCTTTAGTG CTGAAGTTAA CGCATTAAGC ACTCCGCCTG GGGAGTACGG CCGCAAGGCT 840
GAAACTCAAA GGAATTGACG GGGGCCCGCA CAAGCGGTGG AGCATGTGGT TTAATTCGAA 900
GCAACGCGAA GAACCTTACC AGGTCTTGAC ATCCTCTGAA AACCCTAGAG ATAGGGCTTC 960
TCCTTCGGGA GCAGAGTGAC AGGTGGTGCA TGGTTGTCGT CAGCTCGTGT CGTGAGATGT 1020
TGGGTTAAGT CCCGCAACGA GCGCAACCCT TGATCTTAGT TGCCATCATT AAGTTGGGCA 1080
CTCTAAGGTG ACTGCCGGTG ACAAACCGGA GGAAGGTGGG GATGACGTCA AATCATCATG 1140
CCCCTTATGA CCTGGGCTAC ACACGTGCTA CAATGGACGG TACAAAGAGC TGCAAGACCG 1200
CGAGGTGGAG CTAATCTCAT AAAACCGTTC TCAGTTCGGA TTGTAGGCTG CAACTCGCCT 1260
ACATGAAGCT GGAATCGCTA GTAATCGCGG ATCAGCATGC CGCGGTGAAT ACGTTCCCGG 1320
GCCTTGTACA CACCGCCCGT CACACCACGA GAGTTTGTAA CACCCGAAGT CGGTGGGGTA 1380
ACCTTTTTGG AGCCAGCCGC CTAA 1404
Claims (3)
1.一种苏云金芽孢杆菌，其特征在于，于2016年09月12日保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏号为CGMCC No.13005；命名为苏云金芽孢杆菌(Bacillusthuringiensis)3-1。
2.根据权利要求1所述的苏云金芽孢杆菌，其特征在于，所述苏云金芽孢杆菌(Bacillus thuringiensis)3-1的16S rDNA序列如SEQ ID No.1所示。
3.权利要求1或2所述的苏云金芽孢杆菌(Bacillus thuringiensis)3-1在降解原油污染水体中的应用。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611243613.3A CN106479942B (zh) | 2016-12-29 | 2016-12-29 | 一种苏云金芽孢杆菌及其应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611243613.3A CN106479942B (zh) | 2016-12-29 | 2016-12-29 | 一种苏云金芽孢杆菌及其应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106479942A true CN106479942A (zh) | 2017-03-08 |
| CN106479942B CN106479942B (zh) | 2019-07-02 |
Family
ID=58285953
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201611243613.3A Active CN106479942B (zh) | 2016-12-29 | 2016-12-29 | 一种苏云金芽孢杆菌及其应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106479942B (zh) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112760268A (zh) * | 2021-02-09 | 2021-05-07 | 黑龙江大学 | 一株耐紫外光芽孢杆菌及其应用 |
| CN114672444A (zh) * | 2022-05-13 | 2022-06-28 | 福州大学 | 一株苏云金芽孢杆菌及其在不饱和烯烃加氢还原中的应用 |
-
2016
- 2016-12-29 CN CN201611243613.3A patent/CN106479942B/zh active Active
Non-Patent Citations (1)
| Title |
|---|
| MARWAH THAMER ET AL.: "Biodegradation of Kirkuk light crude oil by Bacillus thuringiensis，Northern of Iraq", 《NATURAL SCIENCE》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112760268A (zh) * | 2021-02-09 | 2021-05-07 | 黑龙江大学 | 一株耐紫外光芽孢杆菌及其应用 |
| CN112760268B (zh) * | 2021-02-09 | 2022-11-18 | 黑龙江大学 | 一株耐紫外光芽孢杆菌及其应用 |
| CN114672444A (zh) * | 2022-05-13 | 2022-06-28 | 福州大学 | 一株苏云金芽孢杆菌及其在不饱和烯烃加氢还原中的应用 |
| CN114672444B (zh) * | 2022-05-13 | 2023-03-10 | 福州大学 | 一株苏云金芽孢杆菌及其在不饱和烯烃加氢还原中的应用 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106479942B (zh) | 2019-07-02 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107201328B (zh) | 一株耐受磺胺类抗生素毒性的无色杆菌及其应用 | |
| CN102154170A (zh) | 微囊藻毒素降解菌株及利用其降解mc-lr的方法 | |
| CN114107092B (zh) | 一株降解邻苯二甲酸酯的植物内生菌戈登氏菌l191及其应用 | |
| CN106635908A (zh) | 一种海洋石油降解菌、菌剂及其应用 | |
| Zhu et al. | Fermentative hydrogen production by the new marine Pantoea agglomerans isolated from the mangrove sludge | |
| CN102533562A (zh) | 米根霉ty gf1菌株及其在降解餐饮废水中高浓度油脂的应用 | |
| CN106635909A (zh) | 一种原油降解混合菌、菌剂及其应用 | |
| CN106635910A (zh) | 一种缺陷短波单胞菌、菌剂及其应用 | |
| CN110283743A (zh) | 降解植物根系分泌物中化感物质的降解菌及其应用 | |
| CN105018392A (zh) | 一株长链烷烃降解菌及其应用 | |
| CN106479942B (zh) | 一种苏云金芽孢杆菌及其应用 | |
| CN109593687A (zh) | 一株假单胞菌及其在石油降解中的应用 | |
| CN104152373B (zh) | 一种可高效降解二甲戊乐灵的菌株及其应用 | |
| CN101531974A (zh) | 一株高效降解原油的北极细菌菌株及其应用 | |
| CN106754528A (zh) | 一种弯曲假单胞菌及其应用 | |
| CN106434408B (zh) | 一种具有降解溴代阻燃剂功能的柠檬酸杆菌y3及其应用 | |
| CN104694439A (zh) | 一种降解原油细菌及其应用 | |
| CN104694435B (zh) | 一株具有三氮唑降解功能的申氏杆菌及其应用 | |
| CN103773714A (zh) | 一种光合细菌在海水养殖水质改善中的应用 | |
| CN103710284A (zh) | 具有氨氮降解能力的菌合剂及其应用 | |
| CN104450543B (zh) | 一种本源微生物修复复合制剂及其应用 | |
| CN103525710B (zh) | 草酸青霉bam-1及其分离纯化方法与应用 | |
| CN102864086B (zh) | 一株降解多环芳烃的菌株及其在土壤修复中的应用 | |
| CN102965298B (zh) | 赖氨酸芽孢杆菌及其降解mc-lr的方法 | |
| CN102533595B (zh) | 一株1,2-二氯乙烷降解菌及其应用 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20210922 Address after: 710021 room 1813, the first building of the old third session, No. 60, Weiyang Road, Weiyang District, Xi'an City, Shaanxi Province Patentee after: Shaanxi Aomei Environmental Protection Technology Co.,Ltd. Address before: 710064 No. 33, South Second Ring Road, Shaanxi, Xi'an Patentee before: CHANG'AN University |
|
| TR01 | Transfer of patent right |