CN103013877A - Brevibacillus borstelensis strain having capability of degrading thioanisole and application thereof - Google Patents
Brevibacillus borstelensis strain having capability of degrading thioanisole and application thereof Download PDFInfo
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- CN103013877A CN103013877A CN2012105405645A CN201210540564A CN103013877A CN 103013877 A CN103013877 A CN 103013877A CN 2012105405645 A CN2012105405645 A CN 2012105405645A CN 201210540564 A CN201210540564 A CN 201210540564A CN 103013877 A CN103013877 A CN 103013877A
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Abstract
The invention belongs to the technical field of microorganisms, and discloses a Brevibacillus borstelensis strain having a capability of degrading thioanisole and application thereof. The Brevibacillus borstelensis strain having a capability of degrading thioanisole is named as Brevibacillus borstelensis GIGAN1 and was collected at China Center for Type Culture Collection in Wuhan University in Wuhan, Hubei province, China on November 8<th>, 2012; and the collection number is CCTCC NO:M 2012451. The capability of the strain, of degrading thioanisole in the environment, can be up to 96.2%; and the strain can be used for degrading thioanisole in the environment, thereby achieving the purpose of environment renovation.
Description
Technical field
The invention belongs to microbial technology field, particularly a kind of Potsdam bacillus brevis strain and application thereof with degraded thioanisole ability.
Background technology
Thioanisole (thioanisole, MPS) claims again thioanisole, how as industrial chemicals, is usually used in the agricultural drugs such as the medicals such as synthetic antibiotic, antiulcer agent and sterilant, sterilant.Because it has lower olfact, therefore belong to malodorous compound.The pollution of such material exerts an influence to surrounding enviroment inhabitation and work crowd's psychology and sense organ, makes the people produce unhappiness, irritated mood with detesting, the quality of reduction working efficiency and life; Simultaneously also can bring serious harm to healthy, such as harm respiratory system, Digestive tract, blood circulation and neural system etc., affect the Metabolic activity of body.Once detect the thioanisole (0.5 μ g/l) of higher concentration in the foul smell that had the reported in literature paper pulp wastewater to produce.Therefore, thioanisole is because its special physico-chemical property often is selected as the study hotspot that the foul smell target substrates becomes the degraded deodorization in recent years.The effective ways of elimination thioanisole foul smell commonly used are microbiological deterioration, and therefore screening high efficiency, low cost thioanisole bacterium for degrading is the research emphasis of its abatement of pollution.Although had a lot about efficient genus bacillus and the report in environment murder by poisoning organic matter degradation thereof, still more rare about the efficient research of genus bacillus aspect organic foul waste gas purification at present at present.Potsdam bacillus brevis is to find in Shengli Oil Field and be used for the degraded oil hydrocarbon compound to improve the bacterium of oil productive rate the earliest.It is shaft-like that somatic cells generally is moderate-length, misaligned, and gramstaining is negative.At the solid culture primary surface, thalline forms translucent light yellow bacterium colony, and bacterium colony is smooth, neat in edge.Also do not find at present report and the patent of bacillus brevis degraded thioanisole aspect, relevant Potsdam.
Summary of the invention
For the shortcoming and deficiency that overcomes prior art, primary and foremost purpose of the present invention is to provide a kind of Potsdam bacillus brevis strain with degraded thioanisole ability.
Another object of the present invention is to provide described application with Potsdam bacillus brevis strain of degraded thioanisole ability.
Purpose of the present invention is achieved through the following technical solutions: a kind of Potsdam bacillus brevis strain with degraded thioanisole ability, called after Potsdam bacillus brevis (Brevibacillus borstelensis) GIGAN1, be preserved in the Chinese Typical Representative culture collection center (CCTCC) that is positioned at Hubei China province Wuhan Wuhan University on November 8th, 2012, deposit number is CCTCC NO:M 2012451;
Described Potsdam bacillus brevis strain with degraded thioanisole ability has following physiology and morphology biochemical characteristic:
The morphological character of this Potsdam bacillus brevis thalline is as follows:
A. adopt conventional Bacterial Physiological biochemical identification method and electron microscope observation, the cell dyeing of Potsdam bacillus brevis strain of screening is Gram-positive, and is shaft-like.
B. the morphological character of bacterium colony is: after the LB solid medium was cultivated 24h, colonial morphology was circular, milky white, and opaque, colony diameter is 1～2mm;
C. main physiological and biochemical property such as table 1:
Table 1
The 16S rDNA sequence of described Potsdam bacillus brevis strain with degraded thioanisole ability is shown in SEQ ID:No.1.
Described Potsdam bacillus brevis strain with degraded thioanisole ability can be applied to repairing environment, in its degradable environment, such as the thioanisole in atmosphere, water body and the soil.
The present invention has following advantage and beneficial effect with respect to prior art:
(1) Potsdam bacillus brevis that the bed mud screening obtains having degraded thioanisole ability is gushed in the present invention first from river, GuangZhou, Guangdong Province city.
(2) Potsdam of the present invention bacillus brevis has the ability of efficient degradation thioanisole, is the 240h that degrades under the 2mg/L condition at concentration of substrate, and degradation rate can reach 96.2%.
Description of drawings
Fig. 1 is the aspect graph of Potsdam bacillus brevis GIGAN1 of the present invention under electron microscope.
Embodiment
The present invention is described in further detail below in conjunction with embodiment and accompanying drawing, but embodiments of the present invention are not limited to this.
Potsdam bacillus brevis strain with degraded thioanisole ability of the present invention gush the bed mud from river, GuangZhou, Guangdong Province city separate, purifying and getting.Its separation purification method is as follows: used domestication substratum is that minimal medium (contains following composition: K in every liter of minimal medium
2HPO
43H
2O 1.2g, KH
2PO
41.2g, NH
4Cl 0.4g, MgSO
47H
2O 0.2g, FeSO
47H
2O 0.01g, CaCl
22H
2O 0.2g, MnSO
44H
2O 0.2g, CuSO
42H
2O 0.01g, ZnSO
47H
2O 0.2g, CoCl
26H
2O 0.09g, Na
2MoO
42H
2O 0.12g, H
3BO
30.006g).At first 3g mud is added the minimal medium that contains thioanisole 10mg/L, 37 ℃ of domestications are after 7 days, move into the minimal medium that contains thioanisole 20mg/L with the inoculum size of volume percent 5% and then tame a week, the concentration that progressively improves thioanisole is by that analogy tamed, and the working concentration of thioanisole is respectively 30mg/L, 40mg/L, 50mg/L.The rear inoculum size access beef extract-peptone liquid nutrient medium with volume percent 5% of domestication end (extractum carnis 3.0g/L, peptone 10.0g/L, NaCl 5.0g/L, pH 7.4～7.6) and cultivate 12h, get (the dilution 10 of 0.1ml bacteria suspension
-1～10
-7) be coated on the solid agar plate (K take thioanisole as sole carbon source
2HPO
43H
2O 1.2g/L, KH
2PO
41.2g/L, NH
4Cl 0.4g/L, MgSO
47H
2O 0.2g/L, FeSO
47H
2O 0.01g/L, CaCl
22H
2O 0.2g/L, MnSO
44H
2O 0.2g/L, CuSO
42H
2O 0.01g/L, ZnSO
47H
2O 0.2g/L, CoCl
26H
2O0.09g/L, Na
2MoO
42H
2O 0.12g/L, H
3BO
30.006g/L, agar powder 1.80g/L, thioanisole 10mg/L) until the single pure bacterium colony of dull and stereotyped upper appearance is pure bacterium.Select the bacterium colony of fast growth, regular edges.
Identify that with selecting the bacterium colony that obtains qualification result is as follows:
(1) morphological character of thalline:
A. adopt conventional Bacterial Physiological biochemical identification method and electron microscope observation, the cell dyeing of Potsdam bacillus brevis that screens is Gram-positive; At observed under electron microscope, its form is shaft-like, and the thalline size is 0.5～0.8 * 6～8 μ m, peritrichous, as shown in Figure 1;
B. the morphological character of bacterium colony: after the LB solid medium was cultivated 24h, colonial morphology be circle, oyster white, and opaque, colony diameter is 1～2mm
C. main physiological and biochemical property as table 2(according to " the elegant pearl in common bacteria system identification handbook east, Cai Miaoying work):
Table 2
The above results shows that the bacterium that the present invention screens is very similar to the physio-biochemical characteristics of Potsdam Brevibacillus.
(2) adopt the DNA extraction method to extract bacteria total DNA.Adopt the 16S rDNA universal primer amplification of bacterium.
Extract total DNA method:
A) with microbionation in 37 ℃ of the freshly prepared sterilization of 5mL LB substratum, shaking table is cultured to logarithmic phase.Get 1.5mL bacterium liquid to the 1.5mL centrifuge tube, 8000rpm, the centrifugal 2min of room temperature collects thalline (if the bacterium amount is very little, can repeat step).
B) abandon supernatant liquor, stay bacterial sediment, control is done.Add 450 μ L extraction buffer (100mMTris-HCL (pH8.0), 100mM Na
2-EDTA (pH8.0), 100mM phosphate buffered saline buffer (pH8.0), 1.5mM NaCl, the CTAB of mass percent 1%) in centrifuge tube, vibration is abundant, the suspension thalline.
C) add 12 μ L100mg/mL N,O-Diacetylmuramidases, abundant mixing, level vibration 200rpm, 37 ℃, 30min.Add 8 μ L10mg/mL Proteinase Ks, abundant mixing, level vibration 200rpm, 37 ℃, 30min.
D) add 50 μ L 20%(w/w) SDS solution, mixing gently, 65 ℃ of dissolving 2h, per 15～20min is inverted up and down gently, and water-bath is placed on 5min protein precipitation in 4 ℃ of ice baths, 12000rpm, 5min, 4 ℃ are centrifugal, collect supernatant liquor in another sky centrifuge tube.
E) add isopyknic chloroform-primary isoamyl alcohol (24:1V/V), the about 1min of extracting, 12000rpm, 5min, 4 ℃ are centrifugal, carefully draw supernatant liquor.Add 300 μ L Virahols, mixing, precipitation at room temperature 1h or spend the night.
F) 12000rpm, 10min, 4 ℃ are centrifugal, collect thick DNA, use 200uL 70%(v/v) ice washing with alcohol precipitation 3min, 12000rpm, 10min, 4 ℃.Dry alcohol residue in the air, add 50 μ L TE damping fluid dissolving DNAs, be kept at-20 ℃.
The primer sequence that adopts increases to 16S rDNA gene:
27F：5’-AGAGTTTGATCCTGGCTCAG-3’
1492R：5’-GGTTACCTTGTTACGACTT-3’
Pcr amplification system and condition:
A) pcr amplification system (50 μ L system)
taq?buffer：5μl
NTPs(concentration 2.5 μ M): 4 μ l
Primer (concentration: 10 μ M): each 1 μ l of primer up and down
Taq enzyme (concentration 5U/ μ l): 0.25 μ l
Template: 1 μ l
Add the sterilization deionized water and supply 50 μ l systems.
B) PCR instrument condition is set: 94 ℃ of 4min of denaturation; 94 ℃ of 1min, 55 ℃ of 1min, 72 ℃ of 2min circulate 30 times; Extend 72 ℃ of 10min.
Its 16S rDNA gene, sequencing result is as follows:
GGTGGGCACCTCTAGCGGCTGGCTCCTTGCGGTTACCTCACCGACTTCGGGTGTTGCAAACTCCCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCGACTTCATGCAGGCGAGTTGCAGCCTGCAATCCGAACTGAGACTGGTTTTAAGAGATTGGCATACTCTCGCGAGCTAGCTTCCCGTTGTACCAGCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCGCCTTCCTCCGTCTTGTCGACGGCAGTCTCTCTAGAGTGCCCAACTGAATGCTGGCAACTAAAGATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCACCGCTGCCCCGAAGGGAAGCCCTATCTCTAGGACGGTCAGCGGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCACTCTTGCGAGCGTACTCCCCAGGCGGAGTGCTTATTGCGTTAGCTGCGGCACTGAGGGTATTGAAACCCCCAACACCTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCGCCTCAGCGTCAGTTACAGACCAGAAAGCCGCCTTCGCCACTGGTGTTCCTCCACATCTCTACGCATTTCACCGCTACACGTGGAATACCGCTTTCCTCTTCTGCACTCAAGCTACACAGTTTCCGATGCGAACCGGGGTTGAGCCCCGGGCTTTAACACCAGACTTACATAGCCGCCTGCGCGCGCTTTACGCCCAATAATTCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTCGTCAGGTACCGTCAAGGTACCGCCCTGTTCGAACGGTACTTGTTCGTCTCTGACAACAGAACTTTACAATCCGAAGACCTTCATCGTTCACGCGGCGTTGCTCCATCAGACTTTCGTCCATTGTGGAAAATTCCCTACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGGTCACCCTCTCAGGTCGGCTACGCATCGTCGCCTTGGTAGGCCGTTACCCCACCAACTAGCTAATGCGCCGCAGGCCCATCCGTAAGTGGTAGCTTGCGCCACCTTTCCGTCTCCTCTCATGCGAGAGAAGACCCTATCCGGTATTAGCATGAGTTTCCCCATGTTATCCCGAGCTTACGGGCAGGTTGCCTACGTGTTACTCACCCGTCCGCCGCTAGCCCCCGAAGGGACTCGCTCGACTGCATTAAGTCTGCGTATTTCCA；
For comparing, listed gene order among the 16S rDNA gene order of 1432bp and the Genbank finds that bacterial strain and Brevibacillus borstelensis sp.R-16402 have the highest homology to reach 100% with long.
Comprehensive above-mentioned physio-biochemical characteristics, 16S rRNA gene order result, the bacterium that the present invention screens should belong to Potsdam Brevibacillus, and be a novel species of this genus, called after Potsdam bacillus brevis (Brevibacillus borstelensis) GIGAN1, be preserved in the Chinese Typical Representative culture collection center (CCTCC) that is positioned at Wuhan, China Wuhan University on November 8th, 2012, deposit number is CCTCC NO:M2012451.
Embodiment 2
Potsdam bacillus brevis GIGAN1 that the present invention's screening obtains has preferably degradation capability to thioanisole:
Take by weighing K
2HPO
43H
2O 1.2g, KH
2PO
41.2g, NH
4Cl 0.4g, MgSO
47H
2O 0.2g, FeSO
47H
2O 0.01g, CaCl
22H
2O 0.2g, MnSO
44H
2O 0.2g, CuSO
42H
2O 0.01g, ZnSO
47H
2O 0.2g, CoCl
26H
2O 0.09g, Na
2MoO
42H2O 0.12g, H
3BO
30.0.06g, then be settled to 1L with deionized water, the 100ml/ bottle is divided in the shaking flask of 250ml, takes out behind the sterilization 30min under 121 ℃ of conditions, add thioanisole under aseptic condition, the concentration of thioanisole is followed successively by 2mg/L, 4mg/L, 8mg/L, 16mg/L, 24mg/L.During the GIGAN1 bacterial classification inoculation that inclined-plane (slant medium is beef-protein medium) preserved is cultivated to the beef-protein medium, at 30 ℃, activation thalline 15h in the shaking table of 220r/min, inoculum size access with volume percent 5% contains in the minimal medium of thioanisole, begin Degrading experiment under 30 ℃, the amount recycling Henry's law that adopts HP6890 to install thioanisole in hydrogen flame ionization detector (FID) the gas Chromatographic Determination headspace gas additional is measured its degradation rate in liquid phase.Gas-chromatography equipment HP-5MS capillary column (0.25mm * 0.25 μ m * 30m, AgilentTechnologies), testing conditions is: 230 ℃ of detector temperatures, 200 ℃ of injector temperatures, 50 ℃ of maintenances of column temperature 0 minute, kept 2 minutes after being increased to 150 ℃ with 15 ℃/min, be increased to 230 degree with 25 ℃/min.Adopt the airtight pin sample introduction of Agilent 500 μ L, sample size is 300 μ L, not shunt modes.The result is as shown in table 3:
The degradation rate of the thioanisole of the different starting point concentrations of table 3
| Thioanisole concentration | Degradation rate |
| 2mg/L | 96.2% |
| 4mg/L | 74.9% |
| 8mg/L | 68.4% |
| 16mg/L | 63.1% |
| 24mg/L | 55.7% |
As can be seen from Table 3, the present invention screening obtains Potsdam bacillus brevis GIGAN1 the degradation capability of thioanisole is reached as high as 96.2%.
Above-described embodiment is the better embodiment of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under spirit of the present invention and the principle, substitutes, combination, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (4)
1. Potsdam bacillus brevis strain with degraded thioanisole ability, it is characterized in that: name is called Potsdam bacillus brevis (Brevibacillus borstelensis) GIGAN1, be preserved in the Chinese Typical Representative culture collection center that is positioned at Hubei China province Wuhan Wuhan University on November 8th, 2012, deposit number is CCTCC NO:M 2012451.
2. Potsdam bacillus brevis strain with degraded thioanisole ability according to claim 1, it is characterized in that: its 16S rDNA sequence is shown in SEQ ID:No.1.
3. claim 1 or the 2 described application of Potsdam bacillus brevis strain in repairing environment with degraded thioanisole ability.
4. the application of Potsdam bacillus brevis strain in repairing environment with degraded thioanisole ability according to claim 3 is characterized in that: described Potsdam bacillus brevis strain with degraded thioanisole ability is used for the thioanisole of degraded environment;
Described environment comprises atmosphere, water body and soil.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103667104A (en) * | 2013-10-09 | 2014-03-26 | 中国科学院广州地球化学研究所 | Bacillus having ethanethiol degradation ability and application thereof |
| CN104328078A (en) * | 2014-11-20 | 2015-02-04 | 黑龙江省科学院微生物研究所 | Brevibacillus borstelensis for producing neutral protease |
| CN112195115A (en) * | 2020-08-13 | 2021-01-08 | 长江大学 | Brevibacillus borstelensis, preparation, method for producing surfactant and application |
| WO2021068416A1 (en) * | 2019-10-12 | 2021-04-15 | 湖南恒凯环保科技投资有限公司 | Brevibacillus nitrificans strain yj1 and application thereof |
| CN114164145A (en) * | 2021-11-23 | 2022-03-11 | 福建农林大学 | Brevibacillus borstelensis, neutral protease and application thereof |
| CN114456982A (en) * | 2022-03-03 | 2022-05-10 | 青岛蔚蓝赛德生物科技有限公司 | Bacillus brevis and application thereof in degradation or digestion of sludge |
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| CN103667104A (en) * | 2013-10-09 | 2014-03-26 | 中国科学院广州地球化学研究所 | Bacillus having ethanethiol degradation ability and application thereof |
| CN104328078A (en) * | 2014-11-20 | 2015-02-04 | 黑龙江省科学院微生物研究所 | Brevibacillus borstelensis for producing neutral protease |
| CN104328078B (en) * | 2014-11-20 | 2017-02-15 | 黑龙江省科学院微生物研究所 | Brevibacillus borstelensis for producing neutral protease |
| WO2021068416A1 (en) * | 2019-10-12 | 2021-04-15 | 湖南恒凯环保科技投资有限公司 | Brevibacillus nitrificans strain yj1 and application thereof |
| CN112195115A (en) * | 2020-08-13 | 2021-01-08 | 长江大学 | Brevibacillus borstelensis, preparation, method for producing surfactant and application |
| CN114164145A (en) * | 2021-11-23 | 2022-03-11 | 福建农林大学 | Brevibacillus borstelensis, neutral protease and application thereof |
| CN114164145B (en) * | 2021-11-23 | 2022-09-02 | 福建农林大学 | Brevibacillus borstelensis, neutral protease and application thereof |
| CN114456982A (en) * | 2022-03-03 | 2022-05-10 | 青岛蔚蓝赛德生物科技有限公司 | Bacillus brevis and application thereof in degradation or digestion of sludge |
| CN114456982B (en) * | 2022-03-03 | 2023-09-26 | 青岛蔚蓝赛德生物科技有限公司 | Brevibacillus brevis and application thereof in degrading or digesting sludge |
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