CN102757920B - Bacillus pumilus and application thereof - Google Patents
Bacillus pumilus and application thereof Download PDFInfo
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- CN102757920B CN102757920B CN 201210264208 CN201210264208A CN102757920B CN 102757920 B CN102757920 B CN 102757920B CN 201210264208 CN201210264208 CN 201210264208 CN 201210264208 A CN201210264208 A CN 201210264208A CN 102757920 B CN102757920 B CN 102757920B
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- bacillus pumilus
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- crude oil
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Abstract
The invention discloses a bacillus pumilus 4070 whose preservation number is CGMCC No. 6073. In addition, the invention further discloses an application of the bacillus pumilus in biodegradation of petroleum contaminants. According to the invention, as the bacillus pumilus comes from soil, bacillus pumilus strains very fast becomes a dominant population after entering the soil and occupies a certain ecological niche, so as to effectively promote the biodegradation of petroleum hydrocarbons, optimize a soil microbial system, improve physical characteristics of the soil, enhance biological activity of the soil and recover hardened and degraded soils. The bacillus pumilus disclosed by the invention is a predominant strain screened with crude oil as a unique carbon source; the property of the crude oil can be changed by the strain in the growing and breeding process, so that components of the crude oil are changed, an effect of emusifying, wetting and dispersing the crude oil is achieved and the petroleum contaminants in the soil can be degraded within a short time.
Description
Technical field
The invention belongs to technical field of bioengineering, be specifically related to a kind of bacillus pumilus and application thereof.
Background technology
The microorganism recovery technique of oil-polluted soils, obtained abroad in recent ten years large development, but the petroleum hydrocarbon class pollutant degradation speed is slow, cause governance process longer perdurability, be an outstanding difficult point of this technology always, therefore seek the study hotspot that high efficient petroleum degrading bacteria is current microbial remedy technology for contaminated soil by petroleum.
Summary of the invention
Technical problem to be solved by this invention is for above-mentioned the deficiencies in the prior art, and a kind of bacillus pumilus that can be used for the degraded oil pollutent is provided.This bacterial strain is the dominant strain that screens take crude oil as sole carbon source, and bacterial strain can change the character of crude oil in the growth and breeding process, and the component of crude oil is changed, and plays emulsification, wetting and disperse the effect of crude oil.
For solving the problems of the technologies described above, the technical solution used in the present invention is: a kind of bacillus pumilus, it is characterized in that, described bacillus pumilus is Bacillus pumilus 4070, deposit number CGMCC No.6073, preservation day is on May 3rd, 2012, and depositary institution is China Committee for Culture Collection of Microorganisms's common micro-organisms center.
1, this bacterial strain has following character:
Morphological specificity: bacterium colony is flat larger, white, and surperficial little wrinkle, diameter 3mm～4mm, thalline are straight or little curved bacillus.
Physiological and biochemical property: gram positive bacterium.
2, the screening method of this bacterial strain is:
Step 1, enrichment: will take 10.0g with sterile manner from the soil sample of Xi'an Weiyang District Chang An University Weishui Campus Shaanxi Province's underground water and red clover rhizosphere reparation district, the 10cm left and right collection of ecotope Engineering Research Center testing ground soil petroleum pollution 10000mg/kg reparation district, in access 100.0mL oil/inorganic salt liquid substratum, be placed in 28 ± 1 ℃, 130r/min constant-temperature table enrichment culture 7d; Then draw the 5mL nutrient solution and again transfer in fresh oil/inorganic salt liquid substratum, then carry out 3 enrichments, domestication, each 7d; Consisting of of described oil/inorganic salt liquid substratum: NaNO
30.15%, (NH
4) SO
40.15%, K
2HPO
40.1%, MgSO
47H
2O0.05%, KCl0.05%, FeSO
47H
2O0.001%, CaCl
20.0002%, distilled water 1000mL, crude oil (extend oil dehydrating crude oil) 0.5% more than is mass percent, Medium's PH Value 7.0～7.5, the 105KPa 20min～25min that sterilizes;
Step 2, screening and purifying: the enrichment culture liquid after enrichment culture in step 1 is pressed the decimal dilution method dilution, get 10
-4, 10
-5, 10
-63 each 0.1mL of dilution nutrient solution coat on oil/inorganic salt solid plate substratum, be placed in and cultivate 48h in 37 ± 1 ℃ of constant incubators, choose the single bacterium colony of advantage of different colours and form, the separation of repeatedly ruling on oil/inorganic salt solid plate substratum, obtain the consistent purifying bacterial strain of form with purifying, the purifying bacterial strain is stored on the beef extract-peptone solid medium further screens, activate, obtain bacterial strain, stand-by; Consisting of of described oil/inorganic salt solid plate substratum: NaNO
30.15%, (NH
4) SO
40.15%, K
2HPO
40.1%, MgSO
47H
2O0.05%, KCl0.05%, FeSO
47H
2O0.001%, CaCl
20.0002%, agar 2%, distilled water 1000mL, crude oil (extend oil dehydrating crude oil) 0.5% more than is mass percent, Medium's PH Value 7.0～7.5, the 105KPa 20min～25min that sterilizes;
step 3, multiple sieve: the bacterial strain that in step 2, activation is good is inoculated in respectively in the inorganic salt liquid substratum that 100mL contains 1wt% n-dodecane and 1wt% hexanaphthene, 28 ± 1 ℃, the 130r/min shaking table is cultivated, after the obvious muddiness of nutrient solution, getting the lmL nutrient solution is inoculated in the inorganic salt solid medium that contains 1wt% n-dodecane and 1wt% hexanaphthene and cultivates, and then the inoculation of cultivating in containing the inorganic salt liquid substratum of 1wt% n-dodecane and 1wt% hexanaphthene, is cultivated 100mL, so repeat 3 times, preserve and finally still can make the inorganic salt liquid substratum that contains n-dodecane and hexanaphthene become muddy bacterial strain.
3, the authentication method of this bacterial strain is:
Step 1, extract total DNA:
101, get incubated overnight bacterial strain (at the most 2 * 10
9Individual), the centrifugal 1min of 10000r/min collects thalline, adds 180 μ L bacteriolyze solution (20mg/mL N,O-Diacetylmuramidase, 20mMTris-HCl pH8,2.5mMEDTA, 1%TritonX-100), resuspended bacterium liquid, 37 ℃ of water-bath 30min～60min; Then add 20 μ L Proteinase K solution (10mg/mL) in resuspended bacterium liquid, the mixing that fully vibrates, 56 ℃ of water-bath 30min are to the complete cracking of cell, then add 200 μ L solution B D, the mixing that fully vibrates, 70 ℃ of water-bath 10min, add 200 μ L dehydrated alcohols, mixing fully vibrates again;
102, adsorption column is put into collection tube, the bacterium liquid with pipettor after with vibration mixing in 101 all adds in adsorption column, standing 2min, and the centrifugal 3min of 12000r/min outwells filtrate; Add 500 μ L solution PW in adsorption column, the centrifugal 1min of 10000r/min outwells filtrate; Add 500 μ L rinsing solution Wash buffer in adsorption column, the centrifugal 1min of 10000r/min outwells filtrate; Adsorption column is put into collection tube, in the centrifugal 2min of 12000r/min, remove residual rinsing liquid Wash buffer;
103, take out adsorption column, put into the centrifuge tube of a new 1.5mL, add the elutriant Elution buffer of 50 μ L preheatings (60 ℃), standing 3min, the centrifugal 1min of 10000r/min room temperature collects DNA solution; Adopting concentration is that 2% agarose detects the total DNA that extracts, and clip size is in the 13kb left and right;
The pcr amplification of step 2,16SrDNA: the total DNA that extracts in the step 1 carries out pcr amplification, the PCR reaction system as template: DNA profiling 10pmol, upstream primer (10 μ M) 1 μ l, downstream primer (10 μ M) 1 μ l; DNTP (10Mm) 1 μ l; 10 * Taq reaction Buffer5 μ l; Taq (5U/ μ l) 0.25 μ l; Add water to 50 μ l; PCR reaction conditions: 98 ℃ of 5min of denaturation; The 95 ℃ of 35s that circulate, 55 ℃ of 35s, 72 ℃ of 30s, 35 circulations, last 72 ℃ are extended 8min, 4 ℃ of preservations; The primer sequence of described pcr amplification is:
Upstream primer (SEQ ID NO.1): 5'-CAGAGTTTGATCCTGGCT-3';
Downstream primer (SEQ ID NO.2): 5'-AGGAGGTGATCCAGCCGCA-3';
Step 3, bacterial strain 16SrDNA sequential analysis and comparison: to the product of pcr amplification in step 2 (clip size 1.4kb left and right) check order (being assisted to complete by Sangon Biotech (Shanghai) Co., Ltd.), the 16SrDNA sequence of the bacillus pumilus (Bacillus pumilus) of having delivered in the 16SrDNA portion gene sequence that obtains (SEQ ID NO.3) and GenBank is carried out homology relatively, and similarity reaches 100%.
Comprehensive Physiology and biochemistry is identified, 16SrDNA sequential analysis qualification result, identifies that bacterial strain of the present invention is bacillus pumilus.
The present invention also provides the application of above-mentioned bacillus pumilus in the degraded oil pollutent.
The present invention compared with prior art has the following advantages:
1, bacillus pumilus of the present invention comes from soil, therefore become very soon dominant microflora after this bacterial strain enters soil, occupy certain ecological niche, effectively promote the degraded of petroleum hydrocarbon, optimize the soil microorganisms system, improve the physical behavior of soil, strengthen the biological activity of soil, repair the soil that hardens and degenerate.
2, bacillus pumilus of the present invention is the dominant strain that screens take crude oil as sole carbon source, and bacterial strain can change the character of crude oil in the growth and breeding process, and the component of crude oil is changed, and plays emulsification, wetting and disperse the effect of crude oil.
3, the bacillus pumilus of the present invention petroleum pollution in soil of can degrading at short notice.
Below by embodiment, technical scheme of the present invention is described in further detail.
Embodiment
The screening of bacterial strain:
Step 1, enrichment: will take 10.0g with sterile manner from the soil sample of Xi'an Weiyang District Chang An University Weishui Campus Shaanxi Province's underground water and red clover rhizosphere reparation district, the 10cm left and right collection of ecotope Engineering Research Center testing ground soil petroleum pollution 10000mg/kg reparation district, in access 100.0mL oil/inorganic salt liquid substratum, be placed in 28 ± 1 ℃, 130r/min constant-temperature table enrichment culture 7d; Then draw the 5mL nutrient solution and again transfer in fresh oil/inorganic salt liquid substratum, then carry out 3 enrichments, domestication, each 7d; Consisting of of described oil/inorganic salt liquid substratum: NaNO
30.15%, (NH
4) SO
40.15%, K
2HPO
40.1%, MgSO
47H
2O0.05%, KCl0.05%, FeSO
47H
2O0.001%, CaCl
20.0002%, distilled water 1000mL, crude oil (extend oil dehydrating crude oil) 0.5% more than is mass percent, Medium's PH Value 7.0～7.5, the 105KPa 20min～25min that sterilizes;
Step 2, screening and purifying: the enrichment culture liquid after enrichment culture in step 1 is pressed the decimal dilution method dilution, get 10
-4, 10
-5, 10
-63 each 0.1mL of dilution nutrient solution coat on oil/inorganic salt solid plate substratum, be placed in and cultivate 48h in 37 ± 1 ℃ of constant incubators, choose the single bacterium colony of advantage of different colours and form, the separation of repeatedly ruling on oil/inorganic salt solid plate substratum, obtain the consistent purifying bacterial strain of form with purifying, the purifying bacterial strain is stored on the beef extract-peptone solid medium further screens, activate, obtain bacterial strain, stand-by; Consisting of of described oil/inorganic salt solid plate substratum: NaNO
30.15%, (NH
4) SO
40.15%, K
2HPO
40.1%, MgSO
47H
2O0.05%, KCl0.05%, FeSO
47H
2O0.001%, CaCl
20.0002%, agar 2%, distilled water 1000mL, crude oil (extend oil dehydrating crude oil) 0.5% more than is mass percent, Medium's PH Value 7.0～7.5, the 105KPa 20min～25min that sterilizes;
step 3, multiple sieve: the bacterial strain that in step 2, activation is good is inoculated in respectively in the inorganic salt liquid substratum that 100mL contains 1wt% n-dodecane and 1wt% hexanaphthene, 28 ± 1, the 130r/min shaking table is cultivated, after the obvious muddiness of nutrient solution, get the lmL nutrient solution be inoculated into contain the 1wt% n-dodecane and and the inorganic salt solid medium of 1wt% hexanaphthene in cultivate, and then the inoculation of cultivating in containing the inorganic salt liquid substratum of 1wt% n-dodecane and 1wt% hexanaphthene, is cultivated 100mL, so repeat 3 times, preserve and finally still can make the inorganic salt liquid substratum that contains n-dodecane and hexanaphthene become muddy bacterial strain.
The evaluation of bacterial strain:
Step 1, extract total DNA:
101, get incubated overnight bacterial strain (at the most 2 * 10
9Individual), the centrifugal 1min of 10000r/min collects thalline, adds 180 μ L bacteriolyze solution (20mg/mL N,O-Diacetylmuramidase, 20mMTris-HCl pH8,2.5mMEDTA, 1%TritonX-100), resuspended bacterium liquid, 37 ℃ of water-bath 30min～60min; Then add 20 μ L Proteinase K solution (10mg/mL) in resuspended bacterium liquid, the mixing that fully vibrates, 56 ℃ of water-bath 30min are to the complete cracking of cell, then add 200 μ L solution B D, the mixing that fully vibrates, 70 ℃ of water-bath 10min, add 200 μ L dehydrated alcohols, mixing fully vibrates again;
102, adsorption column is put into collection tube, the bacterium liquid with pipettor after with vibration mixing in 101 all adds in adsorption column, standing 2min, and the centrifugal 3min of 12000r/min outwells filtrate; Add 500 μ L solution PW in adsorption column, the centrifugal 1min of 10000r/min outwells filtrate; Add 500 μ L rinsing solution Wash buffer in adsorption column, the centrifugal 1min of 10000r/min outwells filtrate; Adsorption column is put into collection tube, in the centrifugal 2min of 12000r/min, the rinsing liquid Wash buffer that leaves away residual;
103, take out adsorption column, put into the centrifuge tube of a new 1.5mL, add the elutriant Elution buffer of 50 μ L preheatings (60 ℃), standing 3min, the centrifugal 1min of 10000r/min room temperature collects DNA solution; Adopting concentration is that 2% agarose detects the total DNA that extracts, and clip size is in the 13kb left and right;
The pcr amplification of step 2,16SrDNA: the total DNA that extracts in the step 1 carries out pcr amplification, the PCR reaction system as template: DNA profiling 10pmol, upstream primer (10 μ M) 1 μ l, downstream primer (10 μ M) 1 μ l; DNTP (10Mm) 1 μ l; 10 * Taq reaction Buffer5 μ l; Taq (5U/ μ l) 0.25 μ l; Add water to 50 μ l; PCR reaction conditions: 98 ℃ of 5min of denaturation; The 95 ℃ of 35s that circulate, 55 ℃ of 35s, 72 ℃ of 30s, 35 circulations, last 72 ℃ are extended 8min, 4 ℃ of preservations; The primer sequence of described pcr amplification is:
Upstream primer (SEQ ID NO.1): 5'-CAGAGTTTGATCCTGGCT-3';
Downstream primer (SEQ ID NO.2): 5'-AGGAGGTGATCCAGCCGCA-3';
Step 3, bacterial strain 16SrDNA sequential analysis and comparison: to the product of pcr amplification in step 2 (clip size 1.4kb left and right) check order (being assisted to complete by Sangon Biotech (Shanghai) Co., Ltd.), the 16SrDNA sequence of the bacillus pumilus (Bacillus pumilus) of having delivered in the 16SrDNA portion gene sequence that obtains (SEQ ID NO.3) and GenBank is carried out homology relatively, and similarity reaches 100%.
Comprehensive Physiology and biochemistry is identified, 16SrDNA sequential analysis qualification result, identifies that bacterial strain of the present invention is bacillus pumilus, and called after bacillus pumilus Bacillus pumilus4070, deposit number CGMCC No.6073.
The effect test of bacillus pumilus of the present invention:
1, the degradation capability test of bacillus pumilus to crude oil:
Bacillus pumilus of the present invention is inoculated in the inorganic salt liquid substratum that contains crude oil, in 28 ± 1 ℃, 130rpm fermentation culture 7 days, substratum forms: NaNO
30.15%, (NH
4) SO
40.15%, K
2HPO
40.1%, MgSO
47H
2O0.05%, KCl0.05%, FeSO
47H
2O0.001%, CaCl
20.0002%, crude oil 1%, distilled water 1000mL more than is mass percent, pH7.0～7.5, sterilization 20min～25min; Compare with the substratum of not inoculating bacillus pumilus, the color of fermented liquid is obviously deepened, and the crude oil in the fermented liquid of inoculation bacillus pumilus has the advantages that not hang bottle.This explanation, bacillus pumilus of the present invention are grown take crude oil as sole carbon source, have changed the character of crude oil, make the component of crude oil that variation occur.Crude oil after inoculation bacillus pumilus fermentation is examined under a microscope, found to have in crude oil bacterium liquid to exist, bacterium exists on water-oil interface, and take oil as carbon source, conforming produces oil substances, thereby plays the effect of emulsification, wetting and dispersion crude oil.
2, the Degrading experiment of bacillus pumilus to substratum PetroChina Company Limited.:
Bacillus pumilus of the present invention is inoculated in the inorganic salt liquid substratum that contains crude oil, in 28 ± 1 ℃, 130rpm fermentation culture 7 days, the inorganic salt liquid substratum that contains crude oil forms: NaNO
30.15%, (NH
4) SO
40.15%, K
2HPO
40.1%, MgSO
47H
2O0.05%, KCl0.05%g, FeSO
47H
2O0.001%, CaCl
20.0002%, crude oil 1%, distilled water 1000mL more than is mass percent, pH7.0～7.5, sterilization 20min～25min; Then getting respectively the 10mL fermented liquid is inoculated in the fresh inorganic salt liquid substratum that contains crude oil of 100mL (substratum forms the same), contains the inorganic salt liquid substratum of n-dodecane and contain in the inorganic salt liquid substratum of hexanaphthene, in 28 ± 1 ℃, 130rpm fermentation culture 7 days, the inorganic salt liquid substratum that contains n-dodecane forms: NaNO
30.15%, (NH
4) SO
40.15%, K
2HPO
40.1%, MgSO
47H
2O0.05%, KCl0.05%g, FeSO
47H
2O0.001%, CaCl
20.0002%, n-dodecane 1%, distilled water 1000mL more than is mass percent, pH7.0～7.5, sterilization 20min～25min; The inorganic salt liquid substratum that contains hexanaphthene forms: NaNO
30.15%, (NH
4) SO
40.15%, K
2HPO
40.1%, MgSO
47H
2O0.05%, KCl0.05%g, FeSO
47H
2O0.001%, CaCl
20.0002%, hexanaphthene 1%, distilled water 1000mL more than is mass percent, pH7.0～7.5, sterilization 20min～25min; Take nonvaccinated three kinds of liquid nutrient mediums as blank, in 28 ± 1 ℃, the 130rpm fermentation culture was taken a sample in 7 days, detected respectively the fermented liquid of inoculation bacillus pumilus and total petroleum hydrocarbon (TPH) degradation rate, straight-chain paraffin degradation rate and the naphthenic hydrocarbon degradation rate of blank, the results are shown in Table 1.
The petroleum degradation rate of table 1 bacterial strain and function thereof
| The TPH degradation rate | The straight-chain paraffin degradation rate | The naphthenic hydrocarbon degradation rate | |
| 4070 bacterial strains | 37.4% | 41.6% | 26.9% |
| Blank | 9.8% | 13.8% | 11.1% |
As can be seen from Table 1, the degradation rate of total petroleum hydrocarbon improves 27.6% than blank under bacillus pumilus of the present invention exists, the degradation rate of straight-chain paraffin and naphthenic hydrocarbon all has a more substantial increase, and bacillus pumilus of the present invention can effectively degrade straight-chain paraffin and naphthenic hydrocarbon are described.
3, the degraded situation of bacillus pumilus to soil PetroChina Company Limited.
Bacillus pumilus of the present invention is inoculated in 37 ± 1 ℃ of activation 36h in the beef extract-peptone solid medium, with the bacterial classification after activation according to the 5%(mass percent) ratio adds in different dustiness soil, all add 5% wheat bran in soil, the ratio of C in soil: N: P is 100: 5: 1, and N, P source is respectively NH
4NO
3And K
2HPO
4, the dustiness of soil PetroChina Company Limited. is respectively 0.3%, 0.7% and 1.5%, and keeping soil moisture content is 20%～25%, take the soil that do not add bacterial classification as blank.Through the degraded of 21 days, TPH(total petroleum hydrocarbon) degradation rate result is as shown in table 2.
The petroleum degradation rate of table 2 soil petroleum pollution bacterial strain
| The petroleum pollution degree | 0.3% | 0.7% | 1.5% |
| 4070 bacterial strains | 43.5% | 39.5% | 37.4% |
| Blank | 15.6% | 12.2% | 10.7% |
Degraded by bacillus pumilus, TPH in 3 kinds of petroleum pollution degree Soil Under Conditions all has obvious reduction, more blankly respectively improved 27.9%, 27.3% and 26.7%, can find out that bacillus pumilus can reach degradation effect preferably at soil a middle or short term.
In sum, bacillus pumilus of the present invention comes from soil, therefore become very soon dominant microflora after this bacterial strain enters soil, occupy certain ecological niche, can degrade at short notice petroleum pollution in soil, effectively promote the degraded of petroleum hydrocarbon, optimize the soil microorganisms system, improve the physical behavior of soil.
The above; it is only preferred embodiment of the present invention; be not that the present invention is done any restriction, every any simple modification, change and equivalent structure of above embodiment being done according to the invention technical spirit changes, and all still belongs in the protection domain of technical solution of the present invention.
Claims (2)
1. a bacillus pumilus, is characterized in that, described bacillus pumilus is Bacillus pumilus 4070, deposit number CGMCC No.6073.
2. the application of the described bacillus pumilus of claim 1 in the degraded oil pollutent.
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| CN105132401A (en) * | 2015-07-28 | 2015-12-09 | 云南大学 | Pectinase for wood processing |
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| CN105316268B (en) * | 2015-12-08 | 2018-11-13 | 山东宝源生物科技股份有限公司 | One plant of bacillus pumilus for producing gibberellin and its application in degraded oil |
| CN106178381A (en) * | 2016-07-04 | 2016-12-07 | 四川行之智汇知识产权运营有限公司 | A kind of oil stain remover based on microorganism |
| CN106947725B (en) * | 2017-05-24 | 2020-05-22 | 北京本农科技发展有限公司 | Microbial agent and application thereof in saline-alkali soil improvement |
| CN110468082B (en) * | 2019-09-19 | 2020-05-19 | 四川农业大学 | Bacillus aryabhattai OCB-6 and application thereof |
| CN110713947A (en) * | 2019-10-30 | 2020-01-21 | 中国石油集团川庆钻探工程有限公司长庆井下技术作业公司 | Compound microbial agent for repairing petroleum pollution and preparation method thereof |
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Cited By (1)
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