CN104830722A - Bacteria WY-4B having petroleum agglomeration function and uses thereof, and bacteria vector - Google Patents
Bacteria WY-4B having petroleum agglomeration function and uses thereof, and bacteria vector Download PDFInfo
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- CN104830722A CN104830722A CN201510220666.2A CN201510220666A CN104830722A CN 104830722 A CN104830722 A CN 104830722A CN 201510220666 A CN201510220666 A CN 201510220666A CN 104830722 A CN104830722 A CN 104830722A
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- 108090000913 Nitrate Reductases Proteins 0.000 claims description 2
- 102000004316 Oxidoreductases Human genes 0.000 claims description 2
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- 229920002472 Starch Polymers 0.000 claims description 2
- 241001148470 aerobic bacillus Species 0.000 claims description 2
- XHCADAYNFIFUHF-TVKJYDDYSA-N esculin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC(C(=C1)O)=CC2=C1OC(=O)C=C2 XHCADAYNFIFUHF-TVKJYDDYSA-N 0.000 claims description 2
- 238000000855 fermentation Methods 0.000 claims description 2
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- 230000035479 physiological effects, processes and functions Effects 0.000 claims description 2
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- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 claims description 2
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to bacteria WY-4B having a petroleum agglomeration function and uses thereof, and a bacteria vector, and belongs to the technical field of microorganisms. According to the present invention, a strain of the bacteria WY-4B having the petroleum agglomeration function is provided, and the uses of the bacteria WY-4B having the petroleum agglomeration function is petroleum pollution control in an agglomeration and/or degradation manner, and can be achieved in a floatable bacteria stick manner; the bacteria WY-4B having the petroleum agglomeration function is firstly screened from marine indigenous microorganisms; and the developed floatable bacteria stick has characteristics of low manufacturing cost, good use effect, and strong application value.
Description
Technical field
The invention belongs to microbial technology field, be specifically related to bacterium WY-4B (the Cobetia amphilecti with cohesion oil function, CGMCC No.10355) and utilize the application in this bacterial strain Marine Environmental Governance field, the application in the biological treating field of the petroleum pollution caused after especially relating to petroleum pollution.
Background technology
In recent years, due to the sharply increase of Crude Oil at Sea transport, offshore production platform and oil port quantity, and the accident of various Oil spills also occurs again and again, and petroleum pollution in ocean is increasingly sharpened.Because petroleum pollution in ocean will cause species diversity sharply to decline, especially contained in oil multiple aromatic hydrocarbons toxic compounds, stable, lasting in the environment and there is biological accumulation and scale effect because of it, and some compound has stronger carinogenicity, mutagenicity and teratogenecity especially.So it will be fatal to the destruction of ecotope particularly species diversity.Meanwhile, petroleum pollution is by the use value reducing beach environment, the self-purification capacity destroying coastal facility, affect the hydrometeorological condition in partial area and reduction ocean.Meanwhile, the recovery of these contaminated environment and recovering again of biotic population, need very long and difficult process.
Because ocean is different from terrestrial environment, especially float on the oil on sea, spread in sea in thin layer rapidly, rate of propagation is exceedingly fast, and pollution oceanic area can be made to increase sharply.But it is comparatively difficult for the improvement of the petroleum pollution floating on sea.In 716 events as Talien New Port petroleum pipe line in 2010 blast, adopt early stage physical method to collect and reveal oil, only alleviate petroleum pollution, and add new chemical course and impel the measures such as oil sedimentation, then cause secondary petroleum pollution.Therefore, the reconstruction that enforcement physico-chemical process carries out ecotope may realize hardly, and biological restoration pollution of the sea becomes the approach of unique feasible.But relative to land, marine plant is relatively deficient, the ecotope reparation of Marine Pollution also mainly relies on the Degradation of marine microorganism, especially depends on the various oceans original inhabitants' oil degradation microorganism having adapted to ocean environment.Oil degradation microorganism can utilize oil as its food source, fundamentally can solve the problem of petroleum pollution.But the report at present about characterization of oil-degrading marine micro-organisms is few, especially there is the report being condensed by oil and carry out the oil degradation microorganism of degradation function less.
Meanwhile, when oil leaks to the water surface, because component density a lot of in oil is lower than water-mass density, therefore, most oil floats on sea, and springs up and tidal fluctuations along with wave, and constantly diffusion spreads.Therefore, once there is Oil spills, oil meeting rapid spread, causes pollution in wide area.Because ocean environment is different from terrestrial environment, especially sea environment, climate impact is comparatively large, and environmental change is complicated.During generation Oil spills (especially oil port harbour often occurs), oil rate of propagation is exceedingly fast, and interception difficulty, namely allows to interception, also cannot carry out subsequent disposal.Up to the present, also sea oil effectively do not tackled, reclaim and the report of degradation technique, be not more loaded with the report of the microbe carrier of cohesion and degraded oil.
Summary of the invention
For solving the technical barrier of above-mentioned sea petroleum pollution process, first object of the present invention is to provide the bacterium that a strain has cohesion and degraded oil function simultaneously, contriver reaches out for efficient ocean original inhabitants' petroleum microorganism and studies, finally obtain Hai Kebeite Salmonella bacterial strain WY-4B (Cobetia amphilecti, CGMCC No.10355).Contriver requires to protect its purposes while this bacterial strain claimed.3rd object of the present invention is to provide one and has float function, simultaneously for the above-mentioned bacterium with cohesion and degraded oil function provides the obsolete fungus stick carrier in place of living away from home.
Technical scheme of the present invention is as follows:
Have the bacterium WY-4B (Cobetia amphilecti, CGMCCNo.10355) of cohesion oil function, its 16S rRNA sequence is as shown in SEQ ID No.1.
The described bacterium WY-4B with cohesion oil function is that contriver obtains from occurring in nature separation.Bacterial strain WY-4B is separated and obtains from the marine bottom sediment that Talien New Port petroleum pollution marine site gathers, and picker's name Liu length is built, picker's contact method 0411-87656046.
The described bacterium WY-4B with cohesion oil function submits preservation to, and concrete 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: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica;
Preservation date: on 01 13rd, 2015;
Deposit number: CGMCC No.10355;
The described Morphology and physiology biochemical character with the bacterium WY-4B of cohesion oil function is: colonial morphology is: cultivate after 7 days on LB substratum, in milk yellow, surface is gloss slightly, smooth, and colony edge is smooth.Gram-negative aerobic bacteria, oxidase negative, catalase positive, non-pigment produces, and thalline is shaft-like, and extremely raw or side raw 2-3 root flagellum, can move.All can grow within the scope of NaCl concentration 0-20%, the suitableeest NaCl concentration 3%, the temperature range 4-30 DEG C of growth, most be temperature range 15-20 DEG C, pH scope 4.5-10.5, optimal pH 6.5-8.5, not gelatin hydrolysate, can not utilize starch, chitin, Vitamin C2, xanthine, xanthoglobulin, tween 80.Do not produce H
2s, nitrate reductase is negative, produces indoles, can utilize glucose fermentation.
The described bacterium WY-4B purposes with cohesion oil function is to condense and/or the mode of degraded oil carries out the improvement of petroleum pollution.
Further, the described bacterium WY-4B with cohesion oil function is agglomerated together for the oil that will float on sea, then it is progressively degraded, clears up simultaneously.
Preferably, this purposes realizes with the form of floatability bacterium rod.
Described bacterium rod specifically obtains like this:
To pluck rear discarded edible mushrooms rod as carrier, with LB liquid medium activated spawn WY-4B, (final bacteria suspension concentration is for 10
9cfu/ml) as inoculum, utilize pressure pump, by edible mushrooms water injection needle, the bacteria suspension of strain W Y-4B is injected obsolete fungus stick, postvaccinal bacterium rod is cascaded, carry out circle around petroleum pollution district and even contain, prevent spreading of sea oil.
Beneficial effect of the present invention is:
1. the present invention filters out the bacterium WY-4B with cohesion oil function first from the indigenous microorganism of ocean;
2. the floatability bacterium rod of contriver's research and development, cheap for manufacturing cost, result of use is good, has very strong using value.
Accompanying drawing explanation
Fig. 1 bacterial strain WY-4B of the present invention is to oil cohesion and degradation effect schematic diagram;
Fig. 2 adds oil state of aggregation enlarged view after oil degradation bacteria strain WY-4B;
The colonial morphology figure of Fig. 3 bacterial strain WY-4B;
The scanning electron microscope diagram of Fig. 4 bacterial strain WY-4B;
The phylogenetic tree of Fig. 5 bacterial strain WY-4B;
Bacterium rod discarded after Fig. 6 edible mushroom collection;
Fig. 7 is the tool hole inoculating needle for the inoculation of bacterium rod;
The bacterium rod series connection schematic diagram of Fig. 8 bacterial strain WY-4B.
Embodiment
In the mode specifically implemented, technical scheme of the present invention is further described below, but the present invention is not limited to embodiment content in any form.
One, the preparation of bacterium rod floating carrier
Embodiment
To pluck rear discarded edible mushrooms rod as carrier, with LB liquid medium activated spawn WY-4B, (final bacteria suspension concentration is for 10
9cfu/ml) as inoculum, utilize pressure pump, by edible mushrooms water injection needle, the bacteria suspension of strain W Y-4B is injected obsolete fungus stick, postvaccinal bacterium rod is cascaded, carry out circle around petroleum pollution district and even contain, prevent spreading of sea oil.Containing effect preferably for reaching, multilayer can be contained by parallel for postvaccinal series connection bacterium rod.After floating oil and the bacterium rod being vaccinated with bacterial strain WY-4B, oil by the absorption of bacterium rod, will adhere to bacterium rod surface, along with the prolongation containing the time, the oil sticking to bacterium rod surface will present patch, the progressively aggegation of patch oil, invest bacterium rod side in spherical or particulate state, be convenient to collect and cleaning.
Two, bacterial strain WY-4B oil degradation performance measurement
(1) Erlenmeyer flask of 150ml adds ASM substratum 50ml/ bottle 121 DEG C of sterilizing 30min; ASM culture medium prescription:
NaCl:30g, NH
4nO
3: 1g, KH
2pO
4: 0.2245g, Na
2hPO
412H
2o:5g, natural sea-water 1000ml, micro solution: 10ml, pH 7.5.Sterilising temp 121 DEG C of 30min.
Micro solution is filled a prescription:
| CaCl 2 | FeCl.6H 2O | CuSO 4 | MnCl.4H 2O | ZnSO 4·7H 2O | Distilled water |
| 2mg | 50mg | 0.5mg | 0.5mg | 10mg | 1000ml |
(2) 250ul crude oil and micro solution 0.5ml is added by the substratum of bacterium of having gone out
(3) cell concentration 10 of bacterial strain is controlled
8cfu/ml, adds 1ml bacteria suspension respectively in Erlenmeyer flask.
(4) not add the blank ASM substratum (with the addition of 250ul crude oil) of bacterium for contrast.
(5) 15 DEG C of shaking tables cultivate 7d, rotating speed 150rmp/min, and experiment in triplicate.
(6) measuring method of petroleum degradation rate: the method adopting extraction and colorimetric.
A: by the interpolation bacterial strain of above-mentioned cultivation 7d and the nutrient solution that do not add in each Erlenmeyer flask of bacterial strain, after adding sherwood oil 20ml respectively, pour in separating funnel and fully mix, then leaves standstill 15min.B: the separating funnel lower switches after layering is opened, by aqueous phase separation out, leaves residual petroleum, carry out centrifugal treating, centrifugal speed 1000r/min, centrifugal 15min, then supernatant liquor is poured in 50ml volumetric flask.
C: pour 15ml sherwood oil again into by being separated the aqueous phase obtained in B, then carry out the 2nd extraction, leave standstill 15min, repeat B program.
D: last, gets 10ml sherwood oil cleaning separating funnel, and all supernatant liquors and separating funnel scavenging solution is all imported in above-mentioned 50ml volumetric flask.
E: by volumetric flask liquid capacity-fixed to 50ml, then as the solution to be measured of colorimetric estimation.
(7) spectrophotometric colo measures
The detection method of petroleum degradation rate: ultraviolet spectrophotometry (wavelength 225nm) measures degradation rate and defines by following formula:
C
0remaining oil in control sample, C
1it is remaining oil in working sample.
The petroleum degradation rate of bacterial strain WY-4B
Three, flocculating result measures
Cohesion measuring method is shown in the performance measurement of WY-4B oil degradation.After bacterial strain WY-4B is accessed ASM substratum, observe oil cohesion situation every day, after the 5th day, oil is condensed into larger one or more particulate state, and the contrast oil do not added in the culturing bottle of bacterial strain WY-4B still swims in nutrient solution surface, there is not conglomerate or particle phenomenon.
Annex:
Sequence table
SEQ ID No.1 (the 16S rRNA nucleotide sequence of bacterial strain WY-4B):
TGATTTTTACATGTGATCGCGGAACAAGTCGAGCGGAACGATCCTAGCTTGCTAGGAGGCGTCGAGCGGCGGACGGGTGAGTAATGCATGGGAATCTGCCCGATAGTGGGGGACAACCTGGGGAAACTCAGGCTAATACCGCATACGTCCTACGGGAGAAAGCAGGGGATCTTCGGACCTTGCGCTATCGGATGAGCCCATGTCGGATTAGCTTGTTGGTGAGGTAATGGCTCACCAAGGCGACGATCCGTAGCTGGTCTGAGAGGATGATCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGAAAGCCTGATCCAGCCATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTCAGCGAGGAAGAACGCCTTGGGATTAATACTCCCGAGGAAAGACATCACTCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCGCGTAGGTGGCTAAGTCAGCCAGGTGTGAAAGCCCCGGGCTCAACCTGGGAACGGCATCTGGAACTGCTTGGCTAGAGTGCAGGAGAGGAAGGTAGAATTCCCGGTGTAGCGGTGAAATGCGTAGAGATCGGGAGGAATACCAGTGGCGAAGGCGGCCTTCTGGACTGACACTGACACTGAGGTGCGAAAGCGTGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCAACTAGCCGTTGGGTCCCTTGAGGACTTAGTGGCGCAGCTAACGCAATAAGTTGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACCCTTGACATCCAGAGGACTTTCCAGAGATGGATTGGTGCCTTCGGGAACTCTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGTAACGAGCGCAACCCCTATCCTTATTTGCCAGCGAGTAATGTCGGGAACTCTAAGGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGGCCCTTACGGGTAGGGCTACACACGTGCTACAATGGCAAGTACAAAGGGTTGCAATACTGCGAAGTGGAGCCAATCCCATAAAGCTTGCCTCAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTGGATCAGAATGCCACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGACTGCACCAGAAGTGGTTAGCCTAACCTTCGGGAGGGCGATCACCATCGGTTGTCGTTCGCG
<110> Dalian Nationality College
<120> has bacterium WY-4B and its purposes and the bacteria carrier of cohesion oil function
<160> 1
<210> 1
<211> 1449
<212> rDNA
<213> Bacillus thermotolerans
<400> 1
TGATTTTTACATGTGATCGCGGAACAAGTCGAGCGGAACGATCCTAGCTTGCTAGGAGGCGTCGAGCGGCGGACGGGTGAGTAATGCATGGGAATCTGCCCGATAGTGGGGGACAACCTGGGGAAACTCAGGCTAATACCGCATACGTCCTACGGGAGAAAGCAGGGGATCTTCGGACCTTGCGCTATCGGATGAGCCCATGTCGGATTAGCTTGTTGGTGAGGTAATGGCTCACCAAGGCGACGATCCGTAGCTGGTCTGAGAGGATGATCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGAAAGCCTGATCCAGCCATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTCAGCGAGGAAGAACGCCTTGGGATTAATACTCCCGAGGAAAGACATCACTCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCGCGTAGGTGGCTAAGTCAGCCAGGTGTGAAAGCCCCGGGCTCAACCTGGGAACGGCATCTGGAACTGCTTGGCTAGAGTGCAGGAGAGGAAGGTAGAATTCCCGGTGTAGCGGTGAAATGCGTAGAGATCGGGAGGAATACCAGTGGCGAAGGCGGCCTTCTGGACTGACACTGACACTGAGGTGCGAAAGCGTGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCAACTAGCCGTTGGGTCCCTTGAGGACTTAGTGGCGCAGCTAACGCAATAAGTTGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACCCTTGACATCCAGAGGACTTTCCAGAGATGGATTGGTGCCTTCGGGAACTCTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGTAACGAGCGCAACCCCTATCCTTATTTGCCAGCGAGTAATGTCGGGAACTCTAAGGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGGCCCTTACGGGTAGGGCTACACACGTGCTACAATGGCAAGTACAAAGGGTTGCAATACTGCGAAGTGGAGCCAATCCCATAAAGCTTGCCTCAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTGGATCAGAATGCCACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGACTGCACCAGAAGTGGTTAGCCTAACCTTCGGGAGGGCGATCACCATCGGTTGTCGTTCGCG
Claims (4)
1. a strain has the bacterium WY-4B of cohesion oil function, and it is characterized in that, its deposit number is CGMCC No.10355, and its 16SrRNA sequence is as shown in SEQ ID No.1.
2. a strain as claimed in claim 1 has the bacterium WY-4B of cohesion oil function, Morphology and physiology biochemical character: colonial morphology is: cultivate on LB substratum after 7 days, in milk yellow, surface is gloss slightly, smooth, colony edge is smooth, Gram-negative aerobic bacteria, oxidase negative, catalase positive, non-pigment produces, thalline is shaft-like, extremely life or side raw 2-3 root flagellum, can move, all can grow within the scope of NaCl concentration 0-20%, the suitableeest NaCl concentration 3%, the temperature range 4-30 DEG C of growth, most be temperature range 15-20 DEG C, pH scope 4.5-10.5, optimal pH 6.5-8.5, not gelatin hydrolysate, starch can not be utilized, chitin, Vitamin C2, xanthine, xanthoglobulin, tween 80, do not produce H
2s, nitrate reductase is negative, produces indoles, can utilize glucose fermentation.
3. a strain as claimed in claim 1 bacterium WY-4B with cohesion oil function carries out the purposes of the improvement of petroleum pollution in the mode of cohesion and/or degraded oil.
4. a floatability bacterium rod, be that the bacterium WY-4B with cohesion oil function according to claim 1 is prepared into bacteria suspension, inject discarded edible fungus stick, postvaccinal bacterium rod is cascaded, carry out circle even to contain around petroleum pollution district, prevent spreading of sea oil.
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| CN108018234A (en) * | 2017-12-14 | 2018-05-11 | 华侨大学 | One plant of bacterial strain for producing algin catenase and its application |
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| JP2013188176A (en) * | 2012-03-14 | 2013-09-26 | Picaso Cosmetic Laboratory Ltd | New microorganism, variant thereof and method for producing polysaccharides by using the same |
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| CN108018234A (en) * | 2017-12-14 | 2018-05-11 | 华侨大学 | One plant of bacterial strain for producing algin catenase and its application |
| CN108018234B (en) * | 2017-12-14 | 2020-10-30 | 华侨大学 | Bacterial strain for producing alginate lyase and application thereof |
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| CN104830722B (en) | 2017-12-08 |
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