CN104830720B - Strains A W29 and its purposes and seabed sedimentation oil degradation microbial inoculum suitable for oil degradation - Google Patents
Strains A W29 and its purposes and seabed sedimentation oil degradation microbial inoculum suitable for oil degradation Download PDFInfo
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- CN104830720B CN104830720B CN201510218039.5A CN201510218039A CN104830720B CN 104830720 B CN104830720 B CN 104830720B CN 201510218039 A CN201510218039 A CN 201510218039A CN 104830720 B CN104830720 B CN 104830720B
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Purification Treatments By Anaerobic Or Anaerobic And Aerobic Bacteria Or Animals (AREA)
Abstract
Strains A W29 and its purposes and seabed sedimentation oil degradation microbial inoculum suitable for oil degradation, belong to microbial technology field, the purposes that inventor provides one plant of strains A W29, described strains A W29 for being applied to oil degradation is that the improvement of oil pollution is carried out in a manner of oil degradation.Realized in the form of it can settle microbial inoculum particle.Microbial inoculum particle obtains activation culture material, propagating culture medium processed according to following steps；Fermentation material of the strain system of spreading cultivation suitable for the strain of oil degradation；Mixing granulation, obtain microbial inoculum particle；The present invention filters out the strains A W29 suitable for oil degradation from the indigenous microorganism of ocean first；A kind of submarine oil degradation bacterial agent of research and development, not only makes oil degradation microorganism possess the space lived away from home, while microorganism can obtain the nutrition of sustained release；The degradation bacterial agent possesses settling character, solves caused seabed sedimentation oil pollution and the aqueous desert problem then triggered after oil pollution.
Description
Technical field
The invention belongs to microbial technology field, and in particular to one plant of strains A W29 for being applied to oil degradation
(Bacillus thermotolerans, CGMCC No.10352) and the application using the bacterial strain Marine Environmental Governance field,
More particularly, to the application in the biological treating field of caused oil pollution after oil pollution.
Background technology
There is hair recently as the transport of offshore oil, exploitation, handling, processing and use, during offshore oil incident of leakage
Raw, petroleum pollution in ocean is increasingly sharpened.Destruction of the oil to bio-diversity is serious, including the common various fish of people, bird
Class and other biologies are all received and had a strong impact on, oil can be attached on the fish gill, fish is suffocated, and are suppressed aquatic bird spawning and are incubated
Change, destroy the impermeability of its feather, reduce aquatic product quality；Meanwhile the oil of leakage can form oil film, answering for water body is hindered
Oxygen acts on, and influences halomereid growth, destroys marine ecology balance；Beachscape can be additionally destroyed, influences beach U.S.
Learn value.
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 serving 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
In order to solve the pollution problem that oil leakage triggers, such as the pollution problem of the floating oil on sea；And for example Chemo-Therapy
The secondary pollution triggered is managed, oil pollution and the sea then triggered are settled especially for caused seabed after solving oil pollution
Bottom Desertification, inventor reach out for efficient ocean original inhabitants petroleum microorganism and studied, and have finally given one plant
Suitable for the strains A W29 of oil degradation.Inventor requires to protect its purposes while this bacterial strain is claimed.
Technical scheme is as follows：
One plant of strains A W29 for being applied to oil degradation, the suggestion Classification And Nomenclature of the bacterial strain is Bacillus
thermotolerans.Its 16S rRNA sequence is as shown in SEQ ID No.1.
The described strains A W29 suitable for oil degradation is inventor's one plant of gemma bar isolated from nature
Bacterium.Separate and obtain in the bottom sediment that strains A W29 gathers from Talien New Port oil pollution marine site, picker's name Liu length build,
Picker's contact method 0411-87656046.
The described strains A W29 suitable for 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.10352；
The described strains A W29 suitable for oil degradation form and physiological and biochemical property be：Bacterium colony is circular, smooth complete
Edge, near-transparent；Thalline is circular or oval, the smooth no decorative pattern in surface, 0.6-0.8 μm of size.Gram-positive bacterium, produce bud
Spore, oxidizing ferment, catalase positive, 15-65 DEG C of growth temperature range, 50 DEG C of optimum temperature, pH scopes 5.0-9.0 are most suitable
8.0-8.5, it can be grown in the range of NaCl concentration 0-10%, most suitable NaCl concentration 3%；Amphimicrobian, aerobic and anaerobism feelings
It can be grown under condition.During anaerobism, with anthraquinone 2,6- disulfonic acid (AQDS) is as redox electronics transfer intermediate, NO3-Make
For electron acceptor.
Described strains A W29 purposes is that the improvement of oil pollution is carried out in a manner of oil degradation.
Further, described strains A W29 settles oil for caused seabed after handling oil pollution.
Preferably, the purposes is realized in the form of it can settle microbial inoculum particle.
First technical concept of inventor be：A kind of oil degradation microorganism carrier is researched and developed, can not only be bacterial strain
AW29 provides the space lived away from home, while the carrier can provide slow-release nutrient for strains A W29 and possess settling character.
This purpose of the present invention is realized by following proposal：
Seabed settles oil degradation microbial inoculum, is the strain AW29 that will be suitable for oil degradation fermentation material and the micro- life of oil degradation
The preparing raw material of thing carrier is according to 1:5-15 mass ratio, preferably 1:10 mass ratio mixing after granulation drying and obtain；Further
, it is 10 to make final bacteria suspension concentration10More than cfu/ml is preferred；
The preparing raw material and its proportioning of described oil degradation microbe carrier include：
| Humic acid | 50-80wt% |
| Groundnut meal | 2-5wt% |
| Wheat bran | 2-5wt% |
| Bentonite | 15-39wt% |
| Na2HPO4 | 0.5-0.7wt% |
| KH2PO4 | 0.1-0.2wt% |
| NH4NO3 | 0.2-0.3wt% |
Further, the described strain AW29 suitable for oil degradation fermentation material can use those of ordinary skill in the art
Known any one fermentation process obtains.But using following methods production fermentation material used in inventor, it can obtain
More preferable effect：
Fermentation material suitable for the strain AW29 of oil degradation is that the strain AW29 suitable for oil degradation that will have been activated is cultivated
Material is with propagating culture medium according to 1:5-10 mass ratioes, preferably 1:10 mass ratio mixing；The solid at 20~50 DEG C, preferably 37 DEG C
Fermentation 3~7 days, preferably ferment 5 days, obtain strain AW29 fermentation material, further, make the viable count of final fermentation material
1010More than cfu/g；
Described activate suitable for the strain AW29 composts of oil degradation is to be suitable to oil LB inclined-planes picking is to be activated
In the bacterial strain access activated liquid LB culture mediums of degraded at 25-50 DEG C, preferably 37 DEG C activated and obtained；After obtaining activation
Cell concentration is 1010Cfu/ml compost；The activated liquid LB culture mediums are：Peptone 10g/L, NaCl10g/L, ferment
Female cream 5g/L, pH7.0~7.5；121 DEG C, 30min sterilizings；
Described propagating culture medium solid feed and proportioning：
| Humic acid | 50-80wt% |
| Groundnut meal | 2-5wt% |
| Wheat bran | 3-8wt% |
| Bentonite | 15-37wt% |
The humidity of propagating culture medium finished product preferably 55%.
Further, described granulation drying can be conventionally granulated, but preferably following inventor's uses
Method, more excellent effect can be produced：
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；Sedimentation oil degradation microbial inoculum in seabed is produced after cooling, is protected
Viable count reaches 10 in card seabed sedimentation oil degradation microbial inoculum9cfu/ml；
Preferably, described following current drying, wet granular is specifically entered into Drying by the anterior intermediate bin of dryer
Interior, at the same time hot blast enters Drying in a co-current manner, and in the case where the interior flight 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 beneficial effects of the present invention are：
1. the present invention filters out the strains A W29 suitable for oil degradation from the indigenous microorganism of ocean first；
2. a kind of submarine oil degradation bacterial agent of inventor's research and development, not only makes oil degradation microorganism possess the sky lived away from home
Between, while microorganism can obtain the nutrition of sustained release；The degradation bacterial agent possesses settling character, solves caused sea after oil pollution
Bottom settles oil pollution and the aqueous desert problem then triggered.
Brief description of the drawings
Fig. 1 strains A W29 of the present invention (Bacillus thermotolerans) microbial inoculum particle shape figure；
Fig. 2 strains As W29 (Bacillus thermotolerans) colonial morphology figure；
Fig. 3 strains As W29 SEM observation figure；
Fig. 4 oil degradations AW29 (Bacillus thermotolerans) phylogenetic tree；
The measure design sketch of Fig. 5 strains A W29 oil degradation performances；In figure：A is at addition oil degradation bacterial strain AW29
Design sketch is managed, B is not plus oil degradation bacterial strain AW29 compares design sketch；
The oil degradation impact of performance figure of Fig. 6 microbial inoculum particles；
Trophism Parameter Map of Fig. 7 microbial inoculum carriers to strains A W29.
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 the strain AW29 that will be suitable for oil degradation fermentation material and the micro- life of oil degradation
The preparing raw material of thing carrier is according to 1:15 mass ratio mixing after granulation drying and obtain；Further, final bacteria suspension concentration is made
For 1010More than cfu/ml is preferred；
The preparing raw material and its proportioning of described 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 described strain AW29 suitable for oil degradation fermentation material can use those of ordinary skill in the art
Known any one fermentation process obtains.But using following methods production fermentation material used in inventor, it can obtain
More preferable effect：
Fermentation material suitable for the strain AW29 of oil degradation is that the strain AW29 suitable for oil degradation that will have been activated is cultivated
Material is with propagating culture medium according to 1:10 mass ratioes, solid fermentation 7 days at 50 DEG C, strain AW29 fermentation material is obtained, further,
Make the viable count 10 of final fermentation material10More than cfu/g；
Described activate suitable for the strain AW29 composts of oil degradation is to be suitable to oil LB inclined-planes picking is to be activated
In the bacterial strain access activated liquid LB culture mediums of degraded at 50 DEG C, preferably 37 DEG C activated and obtained；So as to bacterium after being activated
Bulk concentration is 1010Cfu/ml compost；The activated liquid LB culture mediums are：Peptone 10g/L, NaCl10g/L, yeast extract
5g/L, pH7.0~7.5；121 DEG C, 30min sterilizings；
Described 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, described granulation drying can be conventionally granulated, but preferably following inventor's uses
Method, more excellent effect can be produced：
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；Sedimentation oil degradation microbial inoculum in seabed is produced after cooling, is protected
Viable count reaches 10 in card seabed sedimentation oil degradation microbial inoculum9cfu/ml；
Preferably, described following current drying, wet granular is specifically entered into Drying by the anterior intermediate bin of dryer
Interior, at the same time hot blast enters Drying in a co-current manner, and in the case where the interior flight 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.
Described 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 the strain AW29 that will be suitable for oil degradation fermentation material and the micro- life of oil degradation
The preparing raw material of thing carrier is according to 1:5 mass ratio mixing after granulation drying and obtain；Further, the final bacteria suspension concentration is made to be
1010More than cfu/ml is preferred；
The preparing raw material and its proportioning of described 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 described strain AW29 suitable for oil degradation fermentation material can use those of ordinary skill in the art
Known any one fermentation process obtains.But using following methods production fermentation material used in inventor, it can obtain
More preferable effect：
Fermentation material suitable for the strain AW29 of oil degradation is that the strain AW29 suitable for oil degradation that will have been activated is cultivated
Material is with propagating culture medium according to 1:5 mass ratioes mix；Solid fermentation 3 days at 20 DEG C, preferably ferment 5 days, obtain strain AW29's
Fermentation material, further, make the viable count 10 of final fermentation material10More than cfu/g；
Described activate suitable for the strain AW29 composts of oil degradation is to be suitable to oil LB inclined-planes picking is to be activated
Activated and obtained at 25 DEG C in the bacterial strain access activated liquid LB culture mediums of degraded；So as to which cell concentration is after being activated
1010Cfu/ml compost；The activated liquid LB culture mediums are：Peptone 10g/L, NaCl10g/L, yeast extract 5g/L,
PH7.0~7.5；121 DEG C, 30min sterilizings；
Described 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, described granulation drying can be conventionally granulated, but preferably following inventor's uses
Method, more excellent effect can be produced：
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；Sedimentation oil degradation microbial inoculum in seabed is produced after cooling, is protected
Viable count reaches 10 in card seabed sedimentation oil degradation microbial inoculum9cfu/ml；
Preferably, described following current drying, wet granular is specifically entered into Drying by the anterior intermediate bin of dryer
Interior, at the same time hot blast enters Drying in a co-current manner, and in the case where the interior flight 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.
Described 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 the strain AW29 that will be suitable for oil degradation fermentation material and the micro- life of oil degradation
The preparing raw material of thing carrier is according to 1:10 mass ratio mixing after granulation drying and obtain；Further, final bacteria suspension concentration is made
For 1010More than cfu/ml is preferred；
The preparing raw material and its proportioning of described 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 described strain AW29 suitable for oil degradation fermentation material can use those of ordinary skill in the art
Known any one fermentation process obtains.But using following methods production fermentation material used in inventor, it can obtain
More preferable effect：
Fermentation material suitable for the strain AW29 of oil degradation is that the strain AW29 suitable for oil degradation that will have been activated is cultivated
Material is with propagating culture medium according to 1:8 mass ratioes mix；Solid fermentation 5 days at 37 DEG C, strain AW29 fermentation material is obtained, further
, make the viable count 10 of final fermentation material10More than cfu/g；
Described activate suitable for the strain AW29 composts of oil degradation is to be suitable to oil LB inclined-planes picking is to be activated
37 DEG C are activated and are obtained in the bacterial strain access activated liquid LB culture mediums of degraded；So as to which cell concentration is after being activated
1010Cfu/ml compost；The activated liquid LB culture mediums are：Peptone 10g/L, NaCl10g/L, yeast extract 5g/L,
PH7.0~7.5；121 DEG C, 30min sterilizings；
Described 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, described granulation drying can be conventionally granulated, but preferably following inventor's uses
Method, more excellent effect can be produced：
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；Sedimentation oil degradation microbial inoculum in seabed is produced after cooling, is protected
Viable count reaches 10 in card seabed sedimentation oil degradation microbial inoculum9cfu/ml；
Preferably, described following current drying, wet granular is specifically entered into Drying by the anterior intermediate bin of dryer
Interior, at the same time hot blast enters Drying in a co-current manner, and in the case where the interior flight 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.
Described 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.
Performance test
First, strains A W29 oil degradations performance measurement
1) 150ml conical flask adds 121 DEG C of sterilizing 30min of ASM culture medium 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, pH7.5.121 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 controlled9Cfu/ml, 1ml bacteria suspensions are separately added into conical flask；
4) it is control to be 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；It is as shown in Figure 5 to compare effect.
6) assay method of petroleum degradation rate：
A:Above-mentioned cultured conical flask is taken out and adds petroleum ether 20ml and pours into 15min is stood in separatory funnel.
B:Open separatory funnel to come out aqueous phase separation, leave residual petroleum, 1000r/15min is centrifuged after trim, by liquid
Body pours into volumetric flask.
C:Aqueous phase isolated in B is poured into 15ml petroleum ethers again and carries out 2 extraction standing 10min repetition B work.
D:Take 10ml petroleum ethers to clean separatory funnel, and be conducted into volumetric flask.
E：Volumetric flask liquid capacity-fixed, it is to be measured.
The assay method of petroleum degradation rate：Ultraviolet spectrophotometry (wavelength 225nm) measure degradation rate defines as the following formula：
C0It is control sample light absorption value, C1It is determination sample light absorption value.Degradation rate is the average value of three samples.
Strains A W29 petroleum degradation rate
The measure of the strains A W29 oil degradation performances of table 1
avg:Represent average value.
2nd, the oil degradation performance measuring and evaluating of strains A W29 microbial inoculums particle
The viable count prepared is taken to reach 10 respectively9Cfu/ml microbial inoculum particle 0.2g, 0.5g, 1g, 2g, 4g, adds respectively
Adding to and be fitted into the sterile triangular flask of the not petroliferous ASM culture mediums of 100mL, add 0.5mL sterilizing oil, mixing shakes up,
After 15 DEG C of static gas wave refrigerator 9d,
Determine petroleum degradation rate.The culture mediums of ASM containing oil to be added without microbial inoculum particle are used as control.
Influence (petroleum degradation rate %) of the oil degradation bacterial strain AW29 microbial inoculum particle additions of table 2 to oil degradation performance
Degradation effect is as shown in Figure 6.
3rd, trophism of the microbial inoculum carrier to strains A W29
Test method：
Microbial inoculum particle 1800g made from the method for Example 1, is divided into 18 parts, every part of 100g, is placed in pressure-resistant anaerobism training
Support in bottle, every portion of microbial inoculum adds natural sea-water 200ml, first vacuumizes pressure-resistant Anaerobic culturel bottle, and nitrogen is filled with after discharging air
Gas, pressure is reached 1 atmospheric pressure, then pressure-resistant Anaerobic culturel bottle is placed in 15 DEG C of low temperature incubators and cultivated, respectively at the 1st
Week takes weekly 3 bottles in-the 5 week, and viable count counting is carried out on LB culture mediums according to gradient dilution method, calculates weekly strains A W29's
Breeding situation.Compared with the viable count in original microbial inoculum.
Experimental result
As shown in fig. 7, with the extension of incubation time, AW29 viable counts are gradually in rising trend, tend to during to 4th week
It is stable, illustrate that microbial inoculum carrier can provide the nutrition needed for growth for strains A W29.
Annex：
Sequence table
SEQ ID No.1 (strains A W29 (Bacillus thermotolerans) 16S rRNA sequences)：
CTTCAGCGGCGGACGGGTGAGTAACACGTGGGCAACCTGCCTGTAAGACTGGGATAACTTCGGGAAACC
GGAGCTAATACCGGATAATCCTTTCCTTCACATGGAGGGAAGCTGAAAGACGGTTTCGGCTGTCACTTACAGATGGG
CCCGCGGCGCATTAGCTAGTTGGTGGGGTAACGGCCCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCG
GCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTC
TGACGGAGCAACGCCGCGTGAGCGAAGAAGGCCTTCGGGTCGTAAAGCTCTGTTGTCAGGGAAGAACAAGTACCGGA
GTAACTGCCGGTACCTTGACGGTACCTGACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAG
GTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGCGCGCGCAGGCGGCCTCTTAAGTCTGATGTGAAAGCCCACGG
CTCAACCGTGGAGGGTCATTGGAAACTGGGGGGCTTGAGTGCAGAAGAGGAGAGCGGAATTCCACGTGTAGCGGTGA
AATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGGCTCTCTGGTCTGTAACTGACGCTGAGGCGCGAAAGCG
TGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGTTTCCGCC
CTTTAGTGCTGCAGCAAACGCATTAAGCACTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAAGGAATTGAC
GGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTC
TGACACTCCTGGAGACAGGACGTTCCCCTTCGGGGGACAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTC
GTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGG
TGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACG
TGCTACAATGGATGGTACAAAGGGCAGCGAAGCCGCGAGGTGAAGCCAATCCCATAAAACCATTCTCAGTTCGGATT
GCAGGCTGCAACTCGCCTGCATGAAGCCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCG
GGCCTTGTACACACCGCCCGTCACACCACGGGAGTTTGTAACACCCGAAGTC
<110>Dalian Nationality College
<120>Strains A W29 and its purposes and seabed sedimentation oil degradation microbial inoculum suitable for oil degradation
<160> 1
<210> 1
<211> 1353
<212> rDNA
<213> Bacillus thermotolerans
<400> 1
CTTCAGCGGCGGACGGGTGAGTAACACGTGGGCAACCTGCCTGTAAGACTGGGATAACTTCGGGAAACC
GGAGCTAATACCGGATAATCCTTTCCTTCACATGGAGGGAAGCTGAAAGACGGTTTCGGCTGTCACTTACAGATGGG
CCCGCGGCGCATTAGCTAGTTGGTGGGGTAACGGCCCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCG
GCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTC
TGACGGAGCAACGCCGCGTGAGCGAAGAAGGCCTTCGGGTCGTAAAGCTCTGTTGTCAGGGAAGAACAAGTACCGGA
GTAACTGCCGGTACCTTGACGGTACCTGACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAG
GTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGCGCGCGCAGGCGGCCTCTTAAGTCTGATGTGAAAGCCCACGG
CTCAACCGTGGAGGGTCATTGGAAACTGGGGGGCTTGAGTGCAGAAGAGGAGAGCGGAATTCCACGTGTAGCGGTGA
AATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGGCTCTCTGGTCTGTAACTGACGCTGAGGCGCGAAAGCG
TGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGTTTCCGCC
CTTTAGTGCTGCAGCAAACGCATTAAGCACTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAAGGAATTGAC
GGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTC
TGACACTCCTGGAGACAGGACGTTCCCCTTCGGGGGACAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTC
GTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGG
TGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACG
TGCTACAATGGATGGTACAAAGGGCAGCGAAGCCGCGAGGTGAAGCCAATCCCATAAAACCATTCTCAGTTCGGATT
GCAGGCTGCAACTCGCCTGCATGAAGCCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCG
GGCCTTGTACACACCGCCCGTCACACCACGGGAGTTTGTAACACCCGAAGTC
Claims (3)
1. one plant of strains A W29 for being applied to oil degradation, it is characterised in that its deposit number is CGMCC No.10352, its
16SrRNA sequences are as shown in SEQ ID No.1；
Described one plant is applied to the strains A W29 of oil degradation, form and physiological and biochemical property：Bacterium colony is circular, smooth full edge,
Near-transparent；Thalline is circular or oval, the smooth no decorative pattern in surface, 0.6-0.8 μm of size, gram-positive bacterium, produces gemma,
Oxidizing ferment, catalase positive, 15-65 DEG C of growth temperature range, 50 DEG C of optimum temperature, pH scope 5.0-9.0, optimal pH
8.0-8.5, it can be grown in the range of NaCl concentration 0-10%, most suitable NaCl concentration 3%；Amphimicrobian, aerobic and anaerobism feelings
It can be grown under condition.
2. the one plant as claimed in claim 1 strains A W29 for being applied to oil degradation is carrying out seabed in a manner of oil degradation
The purposes of the improvement of oil pollution.
It is the strain that one plant described in claim 1 is applied to oil degradation 3. a kind of seabed settles oil degradation microbial inoculum
AW29 fermentation material is with the preparing raw material of oil degradation microbe carrier according to 1:5-15 mass ratio mixing after granulation drying and
；
The preparing raw material and its proportioning of described oil degradation microbe carrier include：
Fermentation material suitable for the strain AW29 of oil degradation be will activate strain AW29 composts suitable for oil degradation with
Propagating culture medium is according to 1:5-10 mass ratioes mix；The solid fermentation 3~7 days at 20~50 DEG C and strain AW29 fermentation
Material；
Described activate suitable for the strain AW29 composts of oil degradation is to be suitable to oil degradation LB inclined-planes picking is to be activated
Bacterial strain access activated liquid LB culture mediums in activated and obtained at 25-50 DEG C；So as to which cell concentration is after being activated
1010Cfu/ml compost；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；
Described propagating culture medium solid feed and proportioning：
Described granulation drying is to add mixture into mixer and add binding agent stirring, and adding water to be sufficiently mixed makes water content
Less than 25wt%, extruder grain, below 10min is dried in less than 80 DEG C following currents；Microbial inoculum is produced after cooling, ensures viable count in microbial inoculum
Reach 109cfu/ml；
Described following current drying is to enter wet granular in Drying by the anterior intermediate bin of dryer, at the same time hot blast with
Concurrent enters Drying, and in the case where the interior flight of rotary barrel drives, material is taken up, and is formed to divide and spreads uniform comparatively dense
Expect curtain；
The preparing raw material of described oil degradation microbe carrier is the fine powder below 20 mesh；
The preparing raw material of the propagating culture medium is the fine powder below 20 mesh.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101597576A (en) * | 2009-06-30 | 2009-12-09 | 山东省科学院生物研究所 | A kind of solid microbial agent to remedy soil contaminated by petroleum and its production and application |
| CN101603018A (en) * | 2009-07-07 | 2009-12-16 | 南开大学 | Degraded oil, restoring petroleum polluted soil ecology bacteria preparation and preparation method thereof |
| CN101654662A (en) * | 2009-09-15 | 2010-02-24 | 北京大学 | Method for decomposing petroleum hydrocarbon and special bacterial strain thereof |
| WO2013007398A1 (en) * | 2011-07-13 | 2013-01-17 | BIOLAND, Ltd | Biological product for clearing of water, industrial wastewater and soil from chemicals, which are resistant to degradation and method for using the same |
| CN103613449A (en) * | 2013-11-08 | 2014-03-05 | 苏州仁成生物科技有限公司 | Fertilizer with high slow release fertilizer efficiency produced by secondary fermentation and preparation method thereof |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101597576A (en) * | 2009-06-30 | 2009-12-09 | 山东省科学院生物研究所 | A kind of solid microbial agent to remedy soil contaminated by petroleum and its production and application |
| CN101603018A (en) * | 2009-07-07 | 2009-12-16 | 南开大学 | Degraded oil, restoring petroleum polluted soil ecology bacteria preparation and preparation method thereof |
| CN101654662A (en) * | 2009-09-15 | 2010-02-24 | 北京大学 | Method for decomposing petroleum hydrocarbon and special bacterial strain thereof |
| WO2013007398A1 (en) * | 2011-07-13 | 2013-01-17 | BIOLAND, Ltd | Biological product for clearing of water, industrial wastewater and soil from chemicals, which are resistant to degradation and method for using the same |
| CN103613449A (en) * | 2013-11-08 | 2014-03-05 | 苏州仁成生物科技有限公司 | Fertilizer with high slow release fertilizer efficiency produced by secondary fermentation and preparation method thereof |
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| Bacillus thermotolerans sp. nov., a thermophilic bacterium capable of reducing humus;Guiqin Yang,et al.;《International Journal of Systematic and Evolutionary Microbiology》;20131001;第63卷;第3672–3678页 * |
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