CN104774797B - Psychrotropic bacteria BW02 and its purposes and seabed sedimentation oil degradation microbial inoculum with oil degradation function - Google Patents

Psychrotropic bacteria BW02 and its purposes and seabed sedimentation oil degradation microbial inoculum with oil degradation function Download PDF

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CN104774797B
CN104774797B CN201510218062.4A CN201510218062A CN104774797B CN 104774797 B CN104774797 B CN 104774797B CN 201510218062 A CN201510218062 A CN 201510218062A CN 104774797 B CN104774797 B CN 104774797B
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oil degradation
oil
microbial inoculum
activated
degradation
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CN104774797A (en
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刘秋
于基成
潘俊华
闫建芳
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Dalian Minzu University
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Dalian Nationalities University
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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
    • 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
    • A—HUMAN NECESSITIES
    • A62—LIFE-SAVING; FIRE-FIGHTING
    • A62D—CHEMICAL MEANS FOR EXTINGUISHING FIRES OR FOR COMBATING OR PROTECTING AGAINST HARMFUL CHEMICAL AGENTS; CHEMICAL MATERIALS FOR USE IN BREATHING APPARATUS
    • A62D3/00—Processes for making harmful chemical substances harmless or less harmful, by effecting a chemical change in the substances
    • A62D3/02—Processes for making harmful chemical substances harmless or less harmful, by effecting a chemical change in the substances by biological methods, i.e. processes using enzymes or microorganisms
    • 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
    • C02F3/343—Biological treatment of water, waste water, or sewage characterised by the microorganisms used for digestion of grease, fat, oil
    • 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
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • 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
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
    • A—HUMAN NECESSITIES
    • A62—LIFE-SAVING; FIRE-FIGHTING
    • A62D—CHEMICAL MEANS FOR EXTINGUISHING FIRES OR FOR COMBATING OR PROTECTING AGAINST HARMFUL CHEMICAL AGENTS; CHEMICAL MATERIALS FOR USE IN BREATHING APPARATUS
    • A62D2101/00—Harmful chemical substances made harmless, or less harmful, by effecting chemical change
    • A62D2101/20—Organic substances
    • 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

Abstract

Psychrotropic bacteria BW02 and its purposes and seabed sedimentation oil degradation microbial inoculum with oil degradation function, belong to microbial technology field, described bacterial strain BW02 purposes is that the improvement of oil pollution is carried out in a manner of oil degradation, is 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 bacterial strain BW02 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

Psychrotropic bacteria BW02 and its purposes and seabed sedimentation stone with oil degradation function Oily degradation bacterial agent
Technical field
The invention belongs to microbial technology field, and in particular to have the psychrotropic bacteria BW02 of oil degradation function (Rhodococcusqingshengii, CGMCC No.10354) 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 Psychrotropic bacteria BW02 with oil degradation function.Inventor requires to protect its purposes while this bacterial strain is claimed Shield.
Technical scheme is as follows：
Psychrotropic bacteria BW02 (Rhodococcus qingshengii) with oil degradation function.Its 16S rRNA sequence Row are as shown in SEQ ID No.1.
The described psychrotropic bacteria BW02 with oil degradation function is isolated from nature one plant of inventor Celebrate sheng, a reed pipe wind instrument Rhodococcus sp.Separate and obtain in the bottom sediment that bacterial strain BW02 gathers from Talien New Port oil pollution marine site, picker's surname Name Liu Changjian, picker's contact method 0411-87656046.
The described psychrotropic bacteria BW02 with oil degradation function 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.10354；
The described psychrotropic bacteria BW02 with oil degradation function form and physiological and biochemical property be：
(1) colony morphology characteristic
Bacterial strain BW02 colonial morphology is：After being cultivated 7 days on LB culture mediums, bacterium colony circular protrusions, yellow pink, surface light Sliding moistening, neat in edge.
(2) strain morphology and its physio-biochemical characteristics
Gram-positive, aerobic bacteria, thalline is shaft-like, 0.8-1.2*0.2-0.3 μm, oxidase negative, nitrate reductase The positive, produce indoles.It can be grown in the range of NaCl concentration 0-20%, most suitable NaCl concentration 3%, the temperature range 4- of growth 30 DEG C, 15-20 DEG C of optimum temperature range, pH scope 4.5-10.5, optimal pH 7.0-8.0, not gelatin hydrolysate, it is impossible to using forming sediment Powder, glucose, lactose, galactolipin, maltose.Sucrose, ribose, fructose and mannose can be utilized.
The described psychrotropic bacteria BW02 with oil degradation function purposes is that oil is carried out in a manner of oil degradation The improvement of 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 BW02 provides the space lived away from home, while the carrier can provide slow-release nutrient for bacterial strain BW02 and possess settling character.
This purpose of the present invention is realized by following proposal：
Seabed settles oil degradation microbial inoculum, is by the fermentation material and oil of the psychrotropic bacteria BW02 with oil degradation function The preparing raw material of degrading microorganism carrier is according to 1:5-15 mass ratio, preferably 1:10 mass ratio mixing after granulation drying and ；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 psychrotropic bacteria BW02 with oil degradation function fermentation material can use this area common Any one fermentation process well known to technical staff obtains.But using following methods production fermentation used in inventor Material, can obtain more preferable effect：
The fermentation material of psychrotropic bacteria BW02 with oil degradation function is the strain suitable for oil degradation that will have been activated BW02 composts are with propagating culture medium according to 1:5-10 mass ratioes, preferably 1:10 mass ratio mixing；In 20~50 DEG C, preferably 37 Solid fermentation 3~7 days at DEG C, preferably ferment 5 days, obtain strain BW02 fermentation material, further, make the viable bacteria of final fermentation material Number 1010More than cfu/g；
The described compost for activating the psychrotropic bacteria BW02 with oil degradation function is to wait to live in LB inclined-planes picking Change in the psychrotropic bacteria BW02 bacterial strains access activated liquid LB culture mediums with oil degradation function at 25-50 DEG C, preferably 37 DEG C Activated and obtained；So as to which cell concentration is 10 after being activated10Cfu/ml compost；The activated liquid LB culture mediums For：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：
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 psychrotropic bacteria BW02 with oil degradation function 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 bacterial strains BW02 colonial morphology figure；
The SEM observation figure of Fig. 2 bacterial strains BW02 thalline；
Fig. 3 bacterial strains BW02 phylogenetic tree；
Fig. 4 bacterial strains BW02 oil degradation performance measurement design sketch；
In figure：A is addition oil degradation bacterial strain BW02 treatment effect figures, and B is not plus oil degradation bacterial strain BW02 is compareed Design sketch；
Fig. 5 is trophism figure of the microbial inoculum carrier to bacterial strain BW02；
Fig. 6 bacterial strain BW02 oil degradations microbial inoculum 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 strain BW02 fermentation material 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 described oil degradation microbe carrier include：
Further, described strain BW02 fermentation material 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：
Strain BW02 fermentation material is the strain BW02 composts and propagating culture medium that will have been activated according to 1:10 mass ratioes, Solid fermentation 7 days at 50 DEG C, strain BW02 fermentation material is obtained, further, make the viable count 10 of final fermentation material10cfu/g More than；
The described strain BW02 composts that activated are accessed in the LB inclined-planes picking bacterial strain to be activated suitable for oil degradation In activated liquid LB culture mediums at 50 DEG C, preferably 37 DEG C activated and obtained；So as to which cell concentration is 10 after being activated10cfu/ Ml compost；The activated liquid LB culture mediums are：Peptone 10g/L, NaCl 10g/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, adds water abundant
Mixing makes water content be less than 25wt%, extruder grain, and below 10min is dried in less than 80 DEG C following currents；Produced after cooling Seabed settles oil degradation microbial inoculum, ensures that viable count reaches 10 in 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 that the preparation of strain BW02 fermentation material 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 described oil degradation microbe carrier include：
Further, described strain BW02 fermentation material 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：
Strain BW02 fermentation material is the strain BW02 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 strain BW02 fermentation material, further, make final fermentation material Viable count 1010More than cfu/g；
Described strain BW02 composts are in the BW02 bacterial strains access activated liquid LB cultures to be activated of LB inclined-planes picking Activated and obtained at 25 DEG C in base；So as to which cell concentration is 10 after being activated10Cfu/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：
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 that the preparation of strain BW02 fermentation material 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 described oil degradation microbe carrier include：
Further, described strain BW02 fermentation material 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：
Fermentation material suitable for the strain BW02 of oil degradation is the strain BW02 composts and propagating culture medium that will have been activated According to 1:8 mass ratioes mix；Solid fermentation 5 days at 37 DEG C, strain BW02 fermentation material is obtained, further, make finally to ferment The viable count 10 of material10More than cfu/g；
The good strain BW02 composts of described activation are in LB inclined-planes picking BW02 bacterial strains access activated liquid LB to be activated 37 DEG C are activated and are obtained in culture medium；So as to which cell concentration is 10 after being activated10Cfu/ml compost；The activating solution Body 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：
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.
2nd, bacterial strain BW02 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, 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 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 4 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.
The measure of the bacterial strain BW02 petroleum degradation rates of table 1
3rd, the oil degradation performance measuring and evaluating of bacterial strain BW02 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, petroleum degradation rate is determined.The culture mediums of ASM containing oil to be added without microbial inoculum particle are used as control.
Experimental result
With the extension of incubation time, BW02 viable counts are gradually in rising trend, tend towards stability during to 4th week, illustrate bacterium Agent carrier can provide the nutrition needed for growth for bacterial strain BW02.
Influence (petroleum degradation rate %) of the oil degradation bacterial strain BW02 microbial inoculum particle additions of table 2 to oil degradation performance
Degradation effect is as shown in Figure 4.
4th, trophism of the microbial inoculum carrier to bacterial strain BW02
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 bacterial strain BW02's Breeding situation.Compared with the viable count in original microbial inoculum.
Experimental result
As shown in figure 5, with the extension of incubation time, BW02 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 bacterial strain BW02.
Annex：
Sequence table
SEQ ID No.1 (bacterial strain BW02 (Rhodococcusqingshengii) 16S rRNA sequences)：
ACGAGCGGCGAACGGGTGACTAACACGTGGGTGATCTGCCCTCCACTTCGGGATAAGCCTGGCAAACTG GGTCTAATACCGCATATGACCTCCTATCGCATGGTGCGTGGTGGAAAGATTTATCGCTGCAGGATGGGCCCGCGGCC TATCAGCTTGTTGGTGGCGTAATGGCCTACCAAGGCGACGACGGGTAGCCGACCTGAGAGGGTGACCGGCCACACTG GGACTGACACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGAAAGCCTGATGCAGC GACGCCGCGTGAGCGATGACGGCCTTCGGGTTCTAAACCTCTTTCAGCAGGGACGAAGCGCAAGTGACGGTACCTGC AGAAGAAGCACCGGCTAACTACGTGCCAGCAGCCGCGCTAATACGTAGGGTGCAAGCGTTGTCCGGAATTACTGGGC GTAAAGAGTTCGTAGGCGGTTTGTCGCGTCGTTTGTGAAAACCAGCAGCTCAACTGCTCGCTTGCAGGCGATACGGG CAGACTTGAGTACTGCACGGGAGACTGGAATTCCTGGTCTAGCGGTGAAATGCGCAGATATCAGGAGGAACACCGGT GGCGAAGGCGGGTCTCTGGGCAGTAACTGACGCTGAGGAACGAAAGCGTGGGTAGCGAACAGGATTAGATACCCTGC TAGTCCACGCCGTAAACGGTGGGCCCTAGGTGTGGGTTCCTTCCACGGAATCCGTGCCGTACCTAACGCATTAAGCG CCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGCGGCGCGCACAAGCGGCGGAGCATGTG GATTAATTCGATGCAACGCGAAGAACCTTACCTGGGTTTGACATATACCGGATAGCTGCAGAGATGTGGCCCGCCTT GTGGTCGGTATACAGGTGGTGCATGGCTGACGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCGGCAACGAGCGC AACGCCTATCTTATGTTGCCAGCACGTTATGGTGGGGACTCGTAAGAGACTGCCGGGGTCAACTCAGAGGAAGGTGG GGACGACGTCAACTCATCATGCCCCTTATGTCCAGGGCTTCACACATGCTACAATGGCCAGTACAGAGGGCTGCGAG ACCGTGAGGTGGAGCGAATCCCTTAAAGCTGGTCTCAGTTCGGATCGGGGTCTGCAACTCGACCCCGTGAAGTCGGA GTCGCTAGTAATCGCAGATCAGCAACGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACGTCATG AAAGTCGGTAACACCC
<110>Dalian Nationality College
<120>Psychrotropic bacteria BW02 and its purposes and seabed sedimentation oil degradation microbial inoculum with oil degradation function
<140> 2015102180624
<141> 2015-04-30
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1317
<212> DNA
<213> Rhodococcus qingshengii
<400> 1
acgagcggcg aacgggtgac taacacgtgg gtgatctgcc ctccacttcg ggataagcct 60
ggcaaactgg gtctaatacc gcatatgacc tcctatcgca tggtgcgtgg tggaaagatt 120
tatcgctgca ggatgggccc gcggcctatc agcttgttgg tggcgtaatg gcctaccaag 180
gcgacgacgg gtagccgacc tgagagggtg accggccaca ctgggactga cacacggccc 240
agactcctac gggaggcagc agtggggaat attgcacaat gggcgaaagc ctgatgcagc 300
gacgccgcgt gagcgatgac ggccttcggg ttctaaacct ctttcagcag ggacgaagcg 360
caagtgacgg tacctgcaga agaagcaccg gctaactacg tgccagcagc cgcgctaata 420
cgtagggtgc aagcgttgtc cggaattact gggcgtaaag agttcgtagg cggtttgtcg 480
cgtcgtttgt gaaaaccagc agctcaactg ctcgcttgca ggcgatacgg gcagacttga 540
gtactgcacg ggagactgga attcctggtc tagcggtgaa atgcgcagat atcaggagga 600
acaccggtgg cgaaggcggg tctctgggca gtaactgacg ctgaggaacg aaagcgtggg 660
tagcgaacag gattagatac cctgctagtc cacgccgtaa acggtgggcc ctaggtgtgg 720
gttccttcca cggaatccgt gccgtaccta acgcattaag cgccccgcct ggggagtacg 780
gccgcaaggc taaaactcaa aggaattgac ggcggcgcgc acaagcggcg gagcatgtgg 840
attaattcga tgcaacgcga agaaccttac ctgggtttga catataccgg atagctgcag 900
agatgtggcc cgccttgtgg tcggtataca ggtggtgcat ggctgacgtc agctcgtgtc 960
gtgagatgtt gggttaagtc cggcaacgag cgcaacgcct atcttatgtt gccagcacgt 1020
tatggtgggg actcgtaaga gactgccggg gtcaactcag aggaaggtgg ggacgacgtc 1080
aactcatcat gccccttatg tccagggctt cacacatgct acaatggcca gtacagaggg 1140
ctgcgagacc gtgaggtgga gcgaatccct taaagctggt ctcagttcgg atcggggtct 1200
gcaactcgac cccgtgaagt cggagtcgct agtaatcgca gatcagcaac gctgcggtga 1260
atacgttccc gggccttgta cacaccgccc gtcacgtcat gaaagtcggt aacaccc 1317

Claims (4)

1. the psychrotropic bacteria BW02 with oil degradation function, it is characterised in that its deposit number is CGMCC No.10354, its 16SrRNA sequences are as shown in SEQ ID No.1.
2. there is the psychrotropic bacteria BW02 of oil degradation function, form and physiological and biochemical property as claimed in claim 1：
(1) colony morphology characteristic
Bacterial strain BW02 colonial morphology is：After being cultivated 7 days on LB culture mediums, bacterium colony circular protrusions, yellow pink, surface is smooth wet Profit, neat in edge；
(2) strain morphology and its physio-biochemical characteristics
Gram-positive, aerobic bacteria, thalline is shaft-like, 0.8-1.2*0.2-0.3 μm, oxidase negative, nitrate reductase enzyme positive, Indoles is produced, can be grown in the range of NaCl concentration 0-20%, most suitable NaCl concentration 3%, 4-30 DEG C of the temperature range of growth, 15-20 DEG C of optimum temperature range, pH scope 4.5-10.5, optimal pH 7.0-8.0, not gelatin hydrolysate, it is impossible to utilize starch, grape Sugar, lactose, galactolipin, maltose, sucrose, ribose, fructose and mannose can be utilized.
3. the psychrotropic bacteria BW02 with oil degradation function is carrying out sea in a manner of oil degradation as claimed in claim 1 The purposes of the improvement of bedstone oily pollution.
It is by the psychrotropic bacteria with oil degradation function described in claim 1 4. a kind of seabed settles oil degradation microbial inoculum BW02 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：
The fermentation material of psychrotropic bacteria BW02 with oil degradation function is the low temperature with oil degradation function that will have been activated Bacterium BW02 composts are with propagating culture medium according to 1:5-10 mass ratioes mix；The solid fermentation 3~7 days at 20~50 DEG C and obtain Fermentation material；
The described psychrotropic bacteria BW02 composts activated with oil degradation function be LB inclined-planes picking it is to be activated with Activated and obtained at 25-50 DEG C in the psychrotropic bacteria BW02 bacterial strains access activated liquid LB culture mediums of oil degradation function；From And cell concentration is 10 after being activated10Cfu/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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