CN104830723B - There is the bacterium AW25 of oil degradation and its purposes and bacteria carrier - Google Patents
There is the bacterium AW25 of oil degradation and its purposes and bacteria carrier Download PDFInfo
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- CN104830723B CN104830723B CN201510222043.9A CN201510222043A CN104830723B CN 104830723 B CN104830723 B CN 104830723B CN 201510222043 A CN201510222043 A CN 201510222043A CN 104830723 B CN104830723 B CN 104830723B
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- 230000015556 catabolic process Effects 0.000 title claims abstract description 52
- 238000006731 degradation reaction Methods 0.000 title claims abstract description 52
- 241001501119 bacterium AW25 Species 0.000 title claims abstract description 17
- 241000894006 Bacteria Species 0.000 title claims abstract description 13
- 230000006872 improvement Effects 0.000 claims abstract description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 17
- 239000011780 sodium chloride Substances 0.000 claims description 8
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 claims description 4
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 claims description 4
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 claims description 4
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- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 claims description 2
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- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 claims description 2
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- 235000007201 Saccharum officinarum Nutrition 0.000 claims 1
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- 239000003921 oil Substances 0.000 description 63
- 239000000463 material Substances 0.000 description 28
- 238000000855 fermentation Methods 0.000 description 24
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- 238000001035 drying Methods 0.000 description 18
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- 230000001902 propagating effect Effects 0.000 description 12
- 230000001580 bacterial effect Effects 0.000 description 11
- 238000000034 method Methods 0.000 description 11
- 238000005469 granulation Methods 0.000 description 9
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- 239000002361 compost Substances 0.000 description 7
- QJZYHAIUNVAGQP-UHFFFAOYSA-N 3-nitrobicyclo[2.2.1]hept-5-ene-2,3-dicarboxylic acid Chemical compound C1C2C=CC1C(C(=O)O)C2(C(O)=O)[N+]([O-])=O QJZYHAIUNVAGQP-UHFFFAOYSA-N 0.000 description 6
- 235000003276 Apios tuberosa Nutrition 0.000 description 6
- 244000105624 Arachis hypogaea Species 0.000 description 6
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- 239000000440 bentonite Substances 0.000 description 6
- 229910000278 bentonite Inorganic materials 0.000 description 6
- SVPXDRXYRYOSEX-UHFFFAOYSA-N bentoquatam Chemical compound O.O=[Si]=O.O=[Al]O[Al]=O SVPXDRXYRYOSEX-UHFFFAOYSA-N 0.000 description 6
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- 239000010779 crude oil Substances 0.000 description 4
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 4
- 229910000397 disodium phosphate Inorganic materials 0.000 description 4
- 238000002156 mixing Methods 0.000 description 4
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 4
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 4
- 239000000725 suspension Substances 0.000 description 4
- 239000001888 Peptone Substances 0.000 description 3
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- 241000623259 Pseudomonas indoloxydans Species 0.000 description 3
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- 229940041514 candida albicans extract Drugs 0.000 description 3
- 235000013339 cereals Nutrition 0.000 description 3
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- 108020004465 16S ribosomal RNA Proteins 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
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- 150000002170 ethers Chemical class 0.000 description 2
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- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 206010007269 Carcinogenicity Diseases 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 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
- 208000031320 Teratogenesis Diseases 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- 150000004945 aromatic hydrocarbons Chemical class 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 231100000260 carcinogenicity Toxicity 0.000 description 1
- 230000007670 carcinogenicity Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000001311 chemical methods and process Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000013068 control sample Substances 0.000 description 1
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 description 1
- 229910000366 copper(II) sulfate Inorganic materials 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 239000003599 detergent Substances 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000004945 emulsification Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000007667 floating Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- -1 galactolipin Chemical compound 0.000 description 1
- 230000007886 mutagenicity Effects 0.000 description 1
- 231100000299 mutagenicity Toxicity 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
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- 238000000053 physical method Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 239000013535 sea water Substances 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
- 230000003068 static effect Effects 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 231100000167 toxic agent Toxicity 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 230000001052 transient effect Effects 0.000 description 1
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 description 1
- 229910000368 zinc sulfate Inorganic materials 0.000 description 1
- 239000011686 zinc sulphate 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
- 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
- C12R2001/38—Pseudomonas
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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
- 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
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
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- Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
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- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Water Supply & Treatment (AREA)
- Biodiversity & Conservation Biology (AREA)
- Hydrology & Water Resources (AREA)
- Environmental & Geological Engineering (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Medicinal Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
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- Biomedical Technology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Purification Treatments By Anaerobic Or Anaerobic And Aerobic Bacteria Or Animals (AREA)
Abstract
There is the bacterium AW25 of oil degradation and its purposes and bacteria carrier, belong to microbial technology field, inventor provides one plant of bacterium AW25 with oil degradation, and the bacterium AW25 purposes with oil degradation is that the improvement of oil pollution is carried out in a manner of degraded oil.The purposes is realized in the form of microbial inoculum particle.The present invention filters out AW25 from the indigenous microorganism of ocean first；The microbial inoculum particulate vector of inventor's research and development, manufacture is of low cost, and using effect is good, there is very strong application value.
Description
Technical field
The invention belongs to microbial technology field, and in particular to have the function of the bacterium AW25 of oil degradation and utilize to be somebody's turn to do
The application in bacterial strain Marine Environmental Governance field, more particularly, to the biological treating field of caused oil pollution after oil pollution
Using.
Background technology
In recent years, sharply increasing due to Crude Oil at Sea transport, offshore production platform and oil port quantity, and various oil
The accident of leakage also occurs again and again, and petroleum pollution in ocean is increasingly sharpened.Since petroleum pollution in ocean will cause two packing spaces impatient
Play declines, contained a variety of aromatic hydrocarbons toxic compounds especially in oil, because it stablizes, persistently and with biology in the environment
Accumulation and enlarge-effect, and some compounds are even more to have stronger carcinogenicity, mutagenicity and teratogenesis.So its is right
The destruction of ecological environment particularly bio-diversity will be fatal.Meanwhile reduction beach environment is used valency by oil pollution
Value, destroy coastal facility, influence the hydrometeorological condition in some areas and reduce the self-purification capacity of ocean.Meanwhile these quilts
The recovery of environment and recovering again for biotic population are polluted, needs very long and difficult process.
At present, oil pollution is directed to, especially large area Oil spills, the emergency measure of generally use is except oil recovery people
Work such as salvages at the physical method, also makes petroleum emulsification using adding detergent, and the method settled rapidly is to recover temporary transient clear in sea
Clean, although this method seems the effect for playing improvement in short-term, under cover crisis behind, it can cause secondary chemistry dirty
Dye.
Therefore, the hardly possible realization of reconstruction that physico-chemical process carries out ecological environment is implemented, biological prosthetic ocean is dirty
Dye the approach for unique feasible.But relative to land, sea-plant is relatively deficient, Marine Pollution it is biological prosthetic also mainly according to
Rely the degradation of marine microorganism, depend particularly on the various indigenous oil degradation microorganisms for being already adapted to environments such as subsea.This
A little oil degradation microorganisms can overcome seabed dark, low temperature, oligotrophy, anoxic in seabed by the use of oil as its sole carbon source
Extreme condition, using oil as food, oil is subjected to decomposition and inversion, so as to change and aqueous desert problem.
The content of the invention
To solve the technical barrier of above-mentioned sea oil pollution processing, of the invention first purpose, which is to provide one plant, to be had
The bacterium AW25 of oil degradation function.Inventor reaches out for efficient ocean original inhabitants petroleum microorganism and is studied, finally
Pseudomonad Bacillus genus strain AW25 (Pseudomonas indoloxydans, CGMCC No.10351) is obtained.Inventor
Require to protect its purposes while this bacterial strain is claimed.Third object of the present invention is to provide a kind of with battalion
Support function carrier, at the same for it is above-mentioned have the function of to agglomerate the nutrition carrier in place of living away from home is provided with the bacterium of degraded oil.
Technical scheme is as follows：
There is the bacterium AW25 of oil degradation, its 16S rRNA sequence is as shown in SEQ ID No.1.
The bacterium AW25 with oil degradation is that inventor is isolated from nature.It is specifically new from Dalian
Separate and obtain in the bottom sediment of port oil pollution marine site collection.5 grams of mud sample is taken to dilute 10 with sterile water5Times, crude oil is equal
It is even to be applied on ASM culture mediums, 15 DEG C of cultures, 7 days isolated bacterial strains.Strains A W25 is adopted from Talien New Port oil pollution marine site
Separate and obtain in the bottom sediment of collection, picker's name Liu length is built, picker's contact method 0411-87656046.
The bacterium AW25 with oil degradation has been filed on preservation, and specific preservation information is as follows：
Depositary institution's title：China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC)；
Depositary institution address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica；
Preservation date：On 01 13rd, 2015；
Deposit number：CGMCC No.10351；
Classification And Nomenclature：Pseudomonas indoloxydans
The form with the bacterium AW25 of oil degradation and physiological and biochemical property are：
(1) colony morphology characteristic
The colonial morphology of strains A W25 is：After being cultivated 7 days on LB culture mediums, bacterium colony is translucent colourless, and surface slightly has light
Pool, smooth, colony edge is unintelligible.
(2) strain morphology and its physio-biochemical characteristics
Gram-negative aerobic bacteria, oxidase positive, non-pigment produce, and thalline is rod-shaped, 1.0-1.8*0.2-0.3 μm.
It can be grown in the range of NaCl concentration 0-20%, most suitable NaCl concentration 4%, 10-45 DEG C of the temperature range of growth, is most temperature
28-35 DEG C of scope, pH scope 5.5-10.5, optimal pH 7.5-8.5, gelatin liquefaction, not hydrolysis starch, it is impossible to using glucose,
Sucrose, lactose, galactolipin, maltose, arabinose, fructose, xylose, mannose.Nitrate reductase is negative, produces indoles, production
Raw H2S。
The bacterium AW25 purposes with oil degradation is that oil pollution is carried out in a manner of degraded oil
Administer.
Further, the bacterium AW25 with oil degradation is used to progressively drop the oil for floating on sea
Solution, is carried out at the same time cleaning.
The beneficial effects of the present invention are：
1. the present invention filters out the bacterium AW25 with oil degradation from the indigenous microorganism of ocean first；
2. the microbial inoculum particle of inventor's research and development, manufacture is of low cost, and using effect is good, there is very strong application value.
Brief description of the drawings
The colonial morphology figure of Fig. 1 strains As W25；
The scanning electron microscope observation figure of the thalline of Fig. 2 strains As W25；
The phylogenetic tree of Fig. 3 strains As W25；
The oil degradation performance measurement of Fig. 4 strains As W25；
Fig. 5 strains A W25 microbial inoculums particle shape figures of the present invention.
Embodiment
Technical scheme is further described in a manner of specific implementation below, but the present invention is not with any
Form constrained is in embodiment content.
First, embodiment
Embodiment 1
Seabed settles oil degradation microbial inoculum, is that the preparation of the fermentation material of strain AW25 and oil degradation microbe carrier is former
Material is according to 1:15 mass ratio mixing after granulation drying and obtain；Further, it is 10 to make final bacteria suspension concentration10More than cfu/ml
It is preferred；
The preparing raw material and its proportioning of the oil degradation microbe carrier include：
| Humic acid | 80wt% |
| Groundnut meal | 2wt% |
| Wheat bran | 2wt% |
| Bentonite | 15wt% |
| Na2HPO4 | 0.5wt% |
| KH2PO4 | 0.2wt% |
| NH4NO3 | 0.3wt% |
Further, the fermentation material of the strain AW25 can use known to ordinary skill in the art any one
Fermentation process is planted to obtain.But using following methods production fermentation material used in inventor, more preferable effect can be obtained：
The fermentation material of strain AW25 is the strain AW25 composts and propagating culture medium that will have been activated according to 1:10 mass ratioes,
Solid fermentation 7 days at 50 DEG C, obtain the fermentation material of strain AW25, further, make the viable count 10 of final fermentation material10cfu/g
More than；
The strain AW25 composts are to access activating solution in the LB inclined-planes picking bacterial strain to be activated suitable for oil degradation
In body LB culture mediums at 50 DEG C, preferably 37 DEG C activated and obtained；So that cell concentration is 10 after being activated10The training of cfu/ml
Nutriment；The activated liquid LB culture mediums are：Peptone 10g/L, NaCl 10g/L, yeast extract 5g/L, pH 7.0~7.5；121
DEG C, 30min sterilizings；
The propagating culture medium solid feed and proportioning：
| Humic acid | 80wt% |
| Groundnut meal | 2wt% |
| Wheat bran | 3wt% |
| Bentonite | 15wt% |
The humidity of propagating culture medium finished product preferably 55%.
Further, the granulation drying can be conventionally granulated, but preferably following inventor's uses
Method, can produce more preferably effect：
Granulation drying is to add mixture into mixer and add binding agent stirring, and adding water to be sufficiently mixed makes water content low
In 25wt%, extruder grain, below 10min is dried in less than 80 DEG C following currents；Up to seabed sedimentation oil degradation microbial inoculum after cooling, protect
Viable count reaches 10 in card seabed sedimentation oil degradation microbial inoculum9cfu/ml；
Preferably, following current drying, specifically enters Drying by wet granular by the intermediate bin of dryer front portion
Interior, at the same time hot wind enters Drying in a co-current manner, and in the case where the interior shovelling plate of rotary barrel drives, material is taken up, and is formed
Divide the material curtain for spreading uniform comparatively dense；
Preferably, the preparing raw material of oil degradation microbe carrier is the fine powder below 20 mesh；
Preferably, the preparing raw material of propagating culture medium is the fine powder below 20 mesh.
The dryer can select any one commercially available dryer that can complete baking step, but preferably following factory
The dryer of the model of family's production：Zhangqiu Yulong Machine Co., Ltd., following current roller dryer GHG ф 1.0*12*1.
Embodiment 2
Seabed settles oil degradation microbial inoculum, is that the preparation of the fermentation material of strain AW25 and oil degradation microbe carrier is former
Material is according to 1:5 mass ratio mixing after granulation drying and obtain；Further, it is 10 to make final bacteria suspension concentration10More than cfu/ml
It is preferred；
The preparing raw material and its proportioning of the oil degradation microbe carrier include：
| Humic acid | 53.1wt% |
| Groundnut meal | 5wt% |
| Wheat bran | 2wt% |
| Bentonite | 39wt% |
| Na2HPO4 | 0.5wt% |
| KH2PO4 | 0.2wt% |
| NH4NO3 | 0.2wt% |
Further, the fermentation material of the strain AW25 can use known to ordinary skill in the art any one
Fermentation process is planted to obtain.But using following methods production fermentation material used in inventor, more preferable effect can be obtained：
The fermentation material of strain AW25 is the strain AW25 composts and propagating culture medium that will have been activated according to 1:5 mass ratioes mix
Close；Solid fermentation 3 days at 20 DEG C, preferably ferment 5 days, obtain the fermentation material of strain AW25, further, make final fermentation material
Viable count 1010More than cfu/g；
The strain AW25 composts that activated are accessed in the LB inclined-planes picking bacterial strain to be activated suitable for oil degradation
Activated and obtained at 25 DEG C in activated liquid LB culture mediums；So that cell concentration is 10 after being activated10The culture of cfu/ml
Material；The activated liquid LB culture mediums are：Peptone 10g/L, NaCl 10g/L, yeast extract 5g/L, pH 7.0~7.5；121
DEG C, 30min sterilizings；
The propagating culture medium solid feed and proportioning：
| Humic acid | 55wt% |
| Groundnut meal | 5wt% |
| Wheat bran | 3wt% |
| Bentonite | 37wt% |
The humidity of propagating culture medium finished product preferably 55%.
Further, the granulation drying can be conventionally granulated, but preferably following inventor's uses
Method, can produce more preferably effect：
Granulation drying is to add mixture into mixer and add binding agent stirring, and adding water to be sufficiently mixed makes water content low
In 25wt%, extruder grain, below 10min is dried in less than 80 DEG C following currents；Up to seabed sedimentation oil degradation microbial inoculum after cooling, protect
Viable count reaches 10 in card seabed sedimentation oil degradation microbial inoculum9cfu/ml；
Preferably, following current drying, specifically enters Drying by wet granular by the intermediate bin of dryer front portion
Interior, at the same time hot wind enters Drying in a co-current manner, and in the case where the interior shovelling plate of rotary barrel drives, material is taken up, and is formed
Divide the material curtain for spreading uniform comparatively dense；
Preferably, the preparing raw material of oil degradation microbe carrier is the fine powder below 20 mesh；
Preferably, the preparing raw material of propagating culture medium is the fine powder below 20 mesh.
The dryer can select any one commercially available dryer that can complete baking step, but preferably following factory
The dryer of the model of family's production：Zhangqiu Yulong Machine Co., Ltd., following current roller dryer GHG ф 1.0*12*1.
Embodiment 3
Seabed settles oil degradation microbial inoculum, is that the preparation of the fermentation material of strain AW25 and oil degradation microbe carrier is former
Material is according to 1:10 mass ratio mixing after granulation drying and obtain；Further, it is 10 to make final bacteria suspension concentration10More than cfu/ml
It is preferred；
The preparing raw material and its proportioning of the oil degradation microbe carrier include：
| Humic acid | 75wt% |
| Groundnut meal | 3wt% |
| Wheat bran | 3wt% |
| Bentonite | 18wt% |
| Na2HPO4 | 0.5wt% |
| KH2PO4 | 0.2wt% |
| NH4NO3 | 0.3wt% |
Further, the fermentation material of the strain AW25 can use known to ordinary skill in the art any one
Fermentation process is planted to obtain.But using following methods production fermentation material used in inventor, more preferable effect can be obtained：
The fermentation material of strain AW25 is the strain AW25 composts and propagating culture medium that will have been activated according to 1:8 mass ratioes mix
Close；Solid fermentation 5 days at 37 DEG C, obtain the fermentation material of strain AW25, further, make the viable count of final fermentation material
1010More than cfu/g；
The strain AW25 composts that activated are accessed in the LB inclined-planes picking bacterial strain to be activated suitable for oil degradation
Activated and obtained for 37 DEG C in activated liquid LB culture mediums；So that cell concentration is 10 after being activated10The compost of cfu/ml；
The activated liquid LB culture mediums are：Peptone 10g/L, NaCl 10g/L, yeast extract 5g/L, pH 7.0~7.5；121℃,
30min sterilizes；
The propagating culture medium solid feed and proportioning：
| Humic acid | 74wt% |
| Groundnut meal | 4wt% |
| Wheat bran | 7wt% |
| Bentonite | 15wt% |
The humidity of propagating culture medium finished product preferably 55%.
Further, the granulation drying can be conventionally granulated, but preferably following inventor's uses
Method, can produce more preferably effect：
Granulation drying is to add mixture into mixer and add binding agent stirring, and adding water to be sufficiently mixed makes water content low
In 25wt%, extruder grain, below 10min is dried in less than 80 DEG C following currents；Up to seabed sedimentation oil degradation microbial inoculum after cooling, protect
Viable count reaches 10 in card seabed sedimentation oil degradation microbial inoculum9cfu/ml；
Preferably, following current drying, specifically enters Drying by wet granular by the intermediate bin of dryer front portion
Interior, at the same time hot wind enters Drying in a co-current manner, and in the case where the interior shovelling plate of rotary barrel drives, material is taken up, and is formed
Divide the material curtain for spreading uniform comparatively dense；
Preferably, the preparing raw material of oil degradation microbe carrier is the fine powder below 20 mesh；
Preferably, the preparing raw material of propagating culture medium is the fine powder below 20 mesh.
The dryer can select any one commercially available dryer that can complete baking step, but preferably following factory
The dryer of the model of family's production：Zhangqiu Yulong Machine Co., Ltd., following current roller dryer GHG ф 1.0*12*1.
2nd, bacterial strain strains A W25 oil degradation performance measurements
(1) conical flask of 150ml adds 121 DEG C of sterilizing 30min of ASM culture mediums 50ml/ bottles；ASM culture medium prescriptions：
NaCl：30g, NH4NO3：1g, KH2PO4：0.2245g, Na2HPO4·12H2O：5g, natural sea-water 1000ml, it is micro
Solution：10ml, pH 7.5.121 DEG C of 30min of sterilising temp.
Micro solution formula：
| CaCl2 | FeCl.6H2O | CuSO4 | MnCl.4H2O | ZnSO4·7H2O | Distilled water |
| 2mg | 50mg | 0.5mg | 0.5mg | 10mg | 1000ml |
(2) 250ul crude oil and micro solution 0.5ml will be added in the culture medium for bacterium of having gone out.
(3) cell concentration 10 of bacterial strain is controlled8Cfu/ml, is separately added into 1ml bacteria suspensions in conical flask.
(4) compareed with being not added with the blank ASM culture mediums (with the addition of 250ul crude oil) of bacterium.
(5) 15 DEG C of quiescent culture 7d, experiment is in triplicate.
(6) assay method of petroleum degradation rate：Using extraction and the method for colorimetric:
A:The nutrient solution in each conical flask of bacterial strain is not added the addition bacterial strain of above-mentioned culture 7d and, is separately added into oil
After ether 20ml, pour into separatory funnel and fully mix, then stand 15min.
B:Separatory funnel lower switches after layering are opened, aqueous phase separation is come out, leaves residual petroleum, is centrifuged
Processing, centrifugal speed 1000r/min, centrifuges 15min, then pours into supernatant in 50ml volumetric flasks.
C:Water isolated in B is mutually poured into 15ml petroleum ethers again, then carries out the 2nd extraction, 15min is stood, repeats B
Program.
D:Finally, take 10ml petroleum ethers to clean separatory funnel, and all supernatants and separatory funnel cleaning solution are all led
Enter in above-mentioned 50ml volumetric flasks.
E：By volumetric flask liquid capacity-fixed to 50ml.Then the solution to be measured as colorimetric estimation.
(7) spectrophotometric colo measures
The detection method of petroleum degradation rate：Ultraviolet spectrophotometry (wavelength 225nm) measure degradation rate defines as the following formula：
C0It is remaining oil, C in control sample1It is remaining oil in determination sample.
The petroleum degradation rate of strains A W25
3rd, the oil degradation performance measuring and evaluating of strains A W25 microbial inoculums particle
The viable count prepared is taken to reach 10 respectively9Microbial inoculum particle 0.2g, 0.5g, 1g, 2g, 4g of cfu/ml, adds respectively
Adding to and be fitted into the sterile triangular flask of the not petroliferous ASM culture mediums of 100mL, add the sterilizing oil of 0.5mL, mixing shakes up,
After 15 DEG C of static gas wave refrigerator 9d, petroleum degradation rate is measured.The culture mediums of ASM containing oil to be added without microbial inoculum particle are used as control.
Influence (petroleum degradation rate %) of the 2 oil degradation bacterial strain AW25 microbial inoculum particle additive amounts of table to oil degradation performance
Annex：
Sequence table
SEQ ID No.1 (the 16S rRNA nucleotide sequences of strains A W25)：
AGCTTGCTCCTGGATTCAGCGGCGGACGGGTGAGTAATGCCTAGGAATCTGCCTGGTAGTGGGGGATAA
CGTTCCGAAAGGAACGCTAATACCGCGTACGTCCTACGGGAGAAAGCAGGGGACCTTCGGGCCTTGCGCTATCAGAT
GAGCCTAGGTCGGATTAGCTAGTTGGTGAGGTAATGGCTCACCAAGGCGACGATCCGTAACTGGTCTGAGAGGATGA
TCAGTCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGAAA
GCCTGATCCAGCCATGCCGCGTGTGTGAAGAAGGTCTTCGGATTGTAAAGCACTTTAAGTTGGGAGGAAGGGCTGTA
GGCTAATATCCTGCGGTTTTGACGTTACCAACAGAATAAGCACCGGCTAACTTCGTGCCAGCAGCCGCGGTAATACG
AAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCGCGTAGGTGGTTCGTTAAGTTGGATGTGAAAGCCCC
GGGCTCAACCTGGGAACTGCATCCAAAACTGGCGAGCTAGAGTACGGTAGAGGGTAGTGGAATTTCCTGTGTAGCGG
TGAAATGCGTAGATATAGGAAGGAACACCAGTGGCGAAGGCGACTACCTGGACTGATACTGACACTGAGGTGCGAAA
GCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCAACTAGCCGTTGGAATCCTTGA
GATTTTAGTGGCGCAGCTAACGCATTAAGTTGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTG
ACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCTGGCCTTGACATGC
TGAGAACTTTCCAGAGATGGATTGGTGCCTTCGGGAACTCAGACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTC
GTGAGATGTTGGGTTAAGTCCCGTAACGAGCGCAACCCTTGTCCTTAGTTACCAGCACGTTATGGTGGGCACTCTAA
GGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGGCCAGGGCTACACA
CGTGCTACAATGGTCGGTACAAAGGGTTGCCAAGCCGCGAGGTGGAGCTAATCCCATAAAACCGATCGTAGTCCGGA
TCGCAGTCTGCAACTCGACTGCGTGAAGTCGGAATCGCTAGTAATCGTGAATCAGAATGTCACGGTGAATACGTTCC
CGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCTCCAGAAGTAGCTAGTCT
<110>Dalian Nationality College
<120>There is the bacterium AW25 of oil degradation and its purposes and bacteria carrier
<160> 1
<210> 1
<211> 1363
<212> rDNA
<213> Pseudomonas indoloxydans
<400> 1
AGCTTGCTCCTGGATTCAGCGGCGGACGGGTGAGTAATGCCTAGGAATCTGCCTGGTAGTGGGGGATAA
CGTTCCGAAAGGAACGCTAATACCGCGTACGTCCTACGGGAGAAAGCAGGGGACCTTCGGGCCTTGCGCTATCAGAT
GAGCCTAGGTCGGATTAGCTAGTTGGTGAGGTAATGGCTCACCAAGGCGACGATCCGTAACTGGTCTGAGAGGATGA
TCAGTCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGAAA
GCCTGATCCAGCCATGCCGCGTGTGTGAAGAAGGTCTTCGGATTGTAAAGCACTTTAAGTTGGGAGGAAGGGCTGTA
GGCTAATATCCTGCGGTTTTGACGTTACCAACAGAATAAGCACCGGCTAACTTCGTGCCAGCAGCCGCGGTAATACG
AAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCGCGTAGGTGGTTCGTTAAGTTGGATGTGAAAGCCCC
GGGCTCAACCTGGGAACTGCATCCAAAACTGGCGAGCTAGAGTACGGTAGAGGGTAGTGGAATTTCCTGTGTAGCGG
TGAAATGCGTAGATATAGGAAGGAACACCAGTGGCGAAGGCGACTACCTGGACTGATACTGACACTGAGGTGCGAAA
GCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCAACTAGCCGTTGGAATCCTTGA
GATTTTAGTGGCGCAGCTAACGCATTAAGTTGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTG
ACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCTGGCCTTGACATGC
TGAGAACTTTCCAGAGATGGATTGGTGCCTTCGGGAACTCAGACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTC
GTGAGATGTTGGGTTAAGTCCCGTAACGAGCGCAACCCTTGTCCTTAGTTACCAGCACGTTATGGTGGGCACTCTAA
GGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGGCCAGGGCTACACA
CGTGCTACAATGGTCGGTACAAAGGGTTGCCAAGCCGCGAGGTGGAGCTAATCCCATAAAACCGATCGTAGTCCGGA
TCGCAGTCTGCAACTCGACTGCGTGAAGTCGGAATCGCTAGTAATCGTGAATCAGAATGTCACGGTGAATACGTTCC
CGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCTCCAGAAGTAGCTAGTCT
Claims (2)
1. there is the bacterium AW25 of oil degradation, it is characterised in that its deposit number is CGMCC No.10351, its
16SrRNA sequences are as shown in SEQ ID No.1；
Its form and physiological and biochemical property are as follows：
(1) colony morphology characteristic
The colonial morphology of strains A W25 is：After being cultivated 7 days on LB culture mediums, bacterium colony is translucent colourless, and surface slightly has gloss, light
Sliding, colony edge is unintelligible；
(2) strain morphology and its physio-biochemical characteristics
Gram-negative aerobic bacteria, oxidase positive, non-pigment produce, and thalline is rod-shaped, 1.0-1.8*0.2-0.3 μm,
It can be grown in the range of NaCl concentration 0-20%, most suitable NaCl concentration 4%, 10-45 DEG C of the temperature range of growth, is most temperature model
Enclose 28-35 DEG C, pH scope 5.5-10.5, optimal pH 7.5-8.5, gelatin liquefaction, not hydrolysis starch, it is impossible to utilize glucose, sugarcane
Sugar, lactose, galactolipin, maltose, arabinose, fructose, xylose, mannose, nitrate reductase is negative, produces indoles, produces
H2S。
2. the bacterium AW25 with oil degradation as claimed in claim 1 carries out submarine oil in a manner of degraded oil
The purposes of the improvement of pollution.
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