CN103952353A - Bacillus aerophilus, microbial agent and applications of bacillus aerophilus and microbial agent - Google Patents
Bacillus aerophilus, microbial agent and applications of bacillus aerophilus and microbial agent Download PDFInfo
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- CN103952353A CN103952353A CN201410168127.4A CN201410168127A CN103952353A CN 103952353 A CN103952353 A CN 103952353A CN 201410168127 A CN201410168127 A CN 201410168127A CN 103952353 A CN103952353 A CN 103952353A
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Abstract
The invention provides bacillus aerophilus with collection number of CGMCC No.8840. The invention also provides a microbial agent. The microbial agent comprises culture media and a thallus, wherein the thallus comprises bacillus aerophilus with collection number of CGMCC No.8840. The invention also provides applications of bacillus aerophilus and the microbial agent to degrading petroleum hydrocarbon. The marine petroleum pollution control efficiency can be more effectively improved by adopting bacillus aerophilus and the microbial agent.
Description
Technical field
The present invention relates to biological technical field, particularly, relate to a kind of aerosporus, a kind of microbiobacterial agent and their application.
Background technology
Along with the offshore production of oil and the development of transportation, the problem of petroleum pollution in ocean is on the rise.Drilling unit topples, the accident of oil pipeline explosion or oil tanker often occurs, as the offshore pollution that in July, 2010, oil pipeline blast in Dalian caused, and in April, 2010 BP of British Petroleum Company p.l.c. offshore drilling platform Gulfian pollution causing etc. of collapsing, according to American National ocean and air management office, estimate, about 5000 barrels of bottom, Gulfian oil well leakage of oil every day, by on June 1st, 2010, the oil that leaks into the Gulfian reached 1,700 ten thousand gallons to 2,700 ten thousand gallons.Capital causes bulk petroleum to leak in ocean environment, if do not taked scientific and effective counter-measure to be removed, all can cause marine ecology disaster difficult to the appraisal.
Facts have proved, once petroleum pollution in ocean occurs, if adopt an effective measure, loss will be much smaller.Up to now, the processing of sea surface oil stain is roughly divided into three kinds of physical treatment process, method of chemical treatment and biological treatments.The equipment that physical treatment process mainly adopts comprises: oil boom, oil recovery ship, oil skimmer vessel, oil suction hurdle, oil spill dispersant flusher, Floating oil bag, light oil reservoir, oily trawlnet etc.; Method of chemical treatment is to spray various chemical agents, as dispersion agent, washing agent, washing composition and other tensio-active agents etc., adopts and the oil slick on sea can be dispersed into minimum particle in this way, makes its emulsification, dispersion in seawater, dissolves or be deposited to seabed; Biological treatment is the ability that has oxidation and decompose oil according to certain micro-organisms, removes marine oil.
Wherein, biological treatment has the advantages such as Environmental compatibility is good, but the efficiency of biological treatment is subject to the restriction of the petroleum hydrocarbon degradation speed of microorganism, and the degradation rate of microorganism that at present can decomposing petroleum hydrocarbon is still lower.
Summary of the invention
In order to overcome the lower defect of speed of existing microorganism strains decomposing petroleum hydrocarbon, the invention provides a kind of aerosporus, the deposit number of this aerosporus (Bacillus aerophilus) is CGMCC NO.8840.
The present invention also provides a kind of microbiobacterial agent, and this microbiobacterial agent comprises substratum and thalline, and described thalline comprises that deposit number is the aerosporus of CGMCC NO.8840.
The present invention also provides aerosporus as above and the application of microbiobacterial agent as above in decomposing petroleum hydrocarbon.
The present invention also provides aerosporus as above and the application of microbiobacterial agent as above in degrading crude oil.
By technique scheme, microorganism strains of the present invention can be with higher speed decomposing petroleum hydrocarbon, thereby has improved the efficiency of administering petroleum pollution in ocean.
Other features and advantages of the present invention partly in detail are described the embodiment subsequently.
Biomaterial preservation
Aerosporus of the present invention (Bacillus aerophilus) is the pure growth of the present inventor's separation from the remote mountains lake bed deposits mud sample of Yantai, Shandong collection, its deposit number is CGMCC NO.8840, preservation date is on February 21st, 2014, depositary institution is China Committee for Culture Collection of Microorganisms's common micro-organisms center, address is positioned at No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, and Classification And Nomenclature is aerosporus (Bacillus aerophilus).
Embodiment
Below the specific embodiment of the present invention is elaborated.Should be understood that, embodiment described herein only, for description and interpretation the present invention, is not limited to the present invention.
The invention provides a kind of aerosporus, the deposit number of this aerosporus (Bacillus aerophilus) is CGMCC NO.8840.
The present invention also provides a kind of microbiobacterial agent, and this microbiobacterial agent comprises substratum and thalline, and described thalline comprises that deposit number is the aerosporus of CGMCC NO.8840.
Wherein, the amount of the thalline containing in described microbiobacterial agent can in very large range change, and under preferable case, every gram of contained total viable count of described microbiobacterial agent can be (2-300) * 10
8cFU.
According to the present invention, the kind of described substratum can in very large range change, can be the various substratum of cultivating aerosporus that can be used in, for example, can be for the conventional substratum such as beef-protein medium, broth culture, LB substratum be as seed culture medium, for the preservation of bacterial classification; And the substratum of fermentation is generally used for production, the kind of these substratum is also conventionally known to one of skill in the art.Above-mentioned substratum can be commercially available or prepare according to the record of " microbiological culture media handbook " (Microbiology Culture Media Manual).For example, seed culture medium can be beef-protein medium, its extractum carnis that contains 1-3g/L, the peptone of 5-15g/L, the agar of the sodium-chlor of 3-8g/L and 15-20g/L.For example fermention medium can contain: the starch of 40-150g/L, the sodium-chlor of 0.5-8g/L, the calcium carbonate of 0.5-5g/L, the iron protochloride of the potassium primary phosphate of 2-10g/L and 1-10g/L.
Wherein, above-mentioned various substratum can carry out after sterilizing according to conventional sterilising method standby, for example sterilizing 10-30 minute under the condition of 115-125 ℃ and 1.5-2 standard atmospheric pressure.
Wherein, in order further to improve the effect of described microbiobacterial agent decomposing petroleum hydrocarbon, preferably, described microbiobacterial agent also contains nutritive salt; Described nutritive salt comprises NaBr, H
3bO
3, SrCl
2, NaF, Seignette salt and sodium malate.More preferably, the NaBr that contains 50-100mg in every kilogram of microbiobacterial agent, the H of 10-40mg
3bO
3, 10-40mg SrCl
2, the NaF of 1-5mg is, the sodium malate of the Seignette salt of 0.5-5g and 10-100mg.
Wherein, the preparation method of described microbiobacterial agent can comprise: aerosporus is inoculated in substratum and is cultivated, and the total viable count that makes every gram of microbiobacterial agent obtaining is (2-300) * 10
8cFU.The temperature of cultivating can be 28-32 ℃.
Wherein, in order further to improve the effect of described microbiobacterial agent decomposing petroleum hydrocarbon, preferably, the preparation method of described microbiobacterial agent can also comprise: the material obtaining after cultivating in fermention medium is mixed with nutritive salt; Described nutritive salt comprises NaBr, H
3bO
3, SrCl
2, NaF, Seignette salt and sodium malate.More preferably, the consumption of nutritive salt makes the NaBr that contains 50-100mg in every kilogram of described microbiobacterial agent, the H of 10-40mg
3bO
3, 10-40mg SrCl
2, the NaF of 1-5mg is, the sodium malate of the Seignette salt of 0.5-5g and 10-100mg.
The present invention also provides aerosporus as above and the application of microbiobacterial agent as above in decomposing petroleum hydrocarbon.
Wherein, described petroleum hydrocarbon can comprise alkane and/or aromatic hydrocarbons; The carbonatoms of described alkane can be 11-35, and the carbonatoms of described aromatic hydrocarbons can be 10-20.
The present invention also provides aerosporus as above and the application of microbiobacterial agent as above in degrading crude oil.
Further describe by the following examples the present invention:
Embodiment 1
The present embodiment is for illustrating the cultivation of aerosporus of the present invention and the preparation of microbiobacterial agent.
The aerosporus (Bacillus aerophilus) that is CGMCC NO.8840 by deposit number is inoculated in LB substratum (containing the Tryptones of 10g/L, the NaCl of the yeast extract of 5g/L, 10g/L and the water of surplus), is cultured to nectar degree OD
600value, for 0.6-0.8, obtains kind of a daughter bacteria liquid.
Above-mentioned kind of daughter bacteria liquid of 100mL is added to (starch that fermention medium contains 80g/L, the sodium-chlor of 3g/L, the calcium carbonate of 3g/L, the iron protochloride of the potassium primary phosphate of 5g/L and 5g/L) in 100L fermention medium, at 30 ℃, cultivate, in culturing process, sample and observe by ascites method, until the viable count of the aerosporus in every gram of nutrient solution is 10
9cFU, resulting nutrient solution is the microbiobacterial agent of the present embodiment.
Embodiment 2
The present embodiment is for illustrating the cultivation of aerosporus of the present invention and the preparation of microbiobacterial agent.
The aerosporus (Bacillus aerophilus) that is CGMCC NO.8840 by deposit number is inoculated in LB substratum (containing the Tryptones of 10g/L, the NaCl of the yeast extract of 5g/L, 10g/L and the water of surplus), is cultured to nectar degree OD
600value, for 0.6-0.8, obtains kind of a daughter bacteria liquid.
Above-mentioned kind of daughter bacteria liquid of 100mL is added to (starch that fermention medium contains 80g/L, the sodium-chlor of 3g/L, the calcium carbonate of 3g/L, the iron protochloride of the potassium primary phosphate of 5g/L and 5g/L) in 100L fermention medium, at 30 ℃, cultivate, in culturing process, sample and observe by ascites method, until the viable count of the aerosporus in every gram of nutrient solution is 10
9cFU, in every kg nutrient solution obtained above, adds the NaBr of 80mg, the H of 30mg
3bO
3, 20mg SrCl
2, the NaF of 3mg is, the sodium malate of the Seignette salt of 2g and 50mg, the material obtaining after mixing is the microbiobacterial agent of the present embodiment.
Embodiment 3
The present embodiment is for illustrating the cultivation of aerosporus of the present invention and the preparation of microbiobacterial agent.
The aerosporus (Bacillus aerophilus) that is CGMCC NO.8840 by deposit number is inoculated in LB substratum (containing the Tryptones of 10g/L, the NaCl of the yeast extract of 5g/L, 10g/L and the water of surplus), is cultured to nectar degree OD
600value, for 0.6-0.8, obtains kind of a daughter bacteria liquid.
Above-mentioned kind of daughter bacteria liquid of 100mL is added to (starch that fermention medium contains 40g/L, the sodium-chlor of 6g/L, the calcium carbonate of 1g/L, the iron protochloride of the potassium primary phosphate of 8g/L and 8g/L) in 100L fermention medium, at 30 ℃, cultivate, in culturing process, sample and observe by ascites method, until the viable count of the aerosporus in every gram of nutrient solution is 10
9cFU, in every kg nutrient solution obtained above, adds the NaBr of 50mg, the H of 10mg
3bO
3, 40mg SrCl
2, the NaF of 5mg is, the sodium malate of the Seignette salt of 5g and 100mg, the material obtaining after mixing is the microbiobacterial agent of the present embodiment.
Embodiment 4
The present embodiment is for illustrating the cultivation of aerosporus of the present invention and the preparation of microbiobacterial agent.
The aerosporus (Bacillus aerophilus) that is CGMCC NO.8840 by deposit number is inoculated in LB substratum (containing the Tryptones of 10g/L, the NaCl of the yeast extract of 5g/L, 10g/L and the water of surplus), is cultured to nectar degree OD
600value, for 0.6-0.8, obtains kind of a daughter bacteria liquid.
Above-mentioned kind of daughter bacteria liquid of 100mL is added to (starch that fermention medium contains 120g/L, the sodium-chlor of 1g/L, the calcium carbonate of 4g/L, the iron protochloride of the potassium primary phosphate of 3g/L and 2g/L) in 100L fermention medium, at 30 ℃, cultivate, in culturing process, sample and observe by ascites method, until the viable count of the aerosporus in every gram of nutrient solution is 10
9cFU, in every kg nutrient solution obtained above, adds the NaBr of 90mg, the H of 35mg
3bO
3, 15mg SrCl
2, the NaF of 2mg is, the sodium malate of the Seignette salt of 1g and 15mg, the material obtaining after mixing is the microbiobacterial agent of the present embodiment.
Comparative example 1
By the article number purchased from ATCC, be
49342
tMgenus bacillus (Bacillus sp.) be inoculated into LB substratum (containing the Tryptones of 10g/L, the NaCl of the yeast extract of 5g/L, 10g/L and the water of surplus) in, be cultured to nectar degree OD
600value, for 0.6-0.8, obtains kind of a daughter bacteria liquid.
Above-mentioned kind of daughter bacteria liquid of 100mL is added to (starch that fermention medium contains 80g/L, the sodium-chlor of 3g/L, the calcium carbonate of 3g/L, the iron protochloride of the potassium primary phosphate of 5g/L and 5g/L) in 100L fermention medium, at 30 ℃, cultivate, in culturing process, sample and observe by ascites method, until the viable count of the genus bacillus in every gram of nutrient solution is 10
9cFU, resulting nutrient solution is the microbiobacterial agent of this comparative example.
Comparative example 2
By the article number purchased from purchased from ATCC, be
49342
tMgenus bacillus (Bacillus sp.) be inoculated into LB substratum (containing the Tryptones of 10g/L, the NaCl of the yeast extract of 5g/L, 10g/L and the water of surplus) in, be cultured to nectar degree OD
600value, for 0.6-0.8, obtains kind of a daughter bacteria liquid.
Above-mentioned kind of daughter bacteria liquid of 100mL is added to (starch that fermention medium contains 80g/L, the sodium-chlor of 3g/L, the calcium carbonate of 3g/L, the iron protochloride of the potassium primary phosphate of 5g/L and 5g/L) in 100L fermention medium, at 30 ℃, cultivate, in culturing process, sample and observe by ascites method, until the viable count of the genus bacillus in every gram of nutrient solution is 10
9cFU, in every kg nutrient solution obtained above, adds the NaBr of 80mg, the H of 30mg
3bO
3, 20mg SrCl
2, the NaF of 3mg is, the sodium malate of the Seignette salt of 2g and 50mg, the material obtaining after mixing is the microbiobacterial agent of this comparative example.
Test implementation example 1
In this test implementation example, in the effect of the decomposing petroleum hydrocarbon of the microbiobacterial agent of laboratory environment test implementation example 1-4 and comparative example 1-2.
Seashore silt (taking from Yantai, Shandong Laizhou Wan, dry and sterilizing), water, n-Hexadecane and phenanthrene are mixed by weight (1000:500:20:20), obtain simulating petroleum hydrocarbon contaminated pending sample.
Get each 20mL of microbiobacterial agent of embodiment 1-4 and comparative example 1-2, with the above-mentioned pending sample mix of 1kg, under 25 ℃ of lucifuges, cultivate after 30 or 60 days the sample after being processed respectively.Sample after processing with dichloromethane extraction, and the n-Hexadecane in the sample after processing with gas chromatography determination and luxuriant and rich with fragrance residual quantity, and calculate n-Hexadecane and luxuriant and rich with fragrance degradation rate, result is as shown in table 1.
Table 1
Visible according to the data of table 1, deposit number of the present invention be the aerosporus (Bacillus aerophilus) of CGMCC NO.8840 with respect to other genus bacillus, there is the ability of excellent decomposing petroleum hydrocarbon.
Test implementation example 2
In this test implementation example, in the effect of the degrading crude oil of the microbiobacterial agent of laboratory environment test implementation example 1-4 and comparative example 1-2.
Seashore silt (taking from Yantai, Shandong Laizhou Wan, dry and sterilizing), water and crude oil (purchased from Shandong Dongying Shengli Oil Field) are mixed by weight (1000:500:40), obtain the pending sample that Simulation of Crude Oil is polluted.
Get each 2L of microbiobacterial agent of embodiment 1-4 and comparative example 1-2, respectively with the above-mentioned pending sample mix of 100kg, place out of doors after 15 days (testing location is Yantai, Shandong Laizhou Wan, and the time is on September 10 to 25) sample after being processed.Sample after processing with 100mL dichloromethane extraction 100g, is extracted liquid.By gravimetric determination oil degradation rate, concrete operations comprise extraction liquid are transferred in flask, under the condition of 40 ℃, rotate evaporative removal methylene dichloride, weighing is with the flask gross weight of oil residues dirt, deduct the weight of flask own, obtain the weight of residual crude oil, by the weight of degrading crude oil, divided by the weight of processing front crude oil, obtain oil degradation rate.And to processing the alkane of carbonatoms 11-35 and the aromatic hydrocarbons of carbonatoms 10-20 in front crude oil and residual crude oil, carry out chromatographic peak sxemiquantitative by GC-MS(gas chromatography-mass spectrography), and calculate degradation rate, result is as shown in table 2.
Table 2
Visible according to the data of table 2, deposit number of the present invention is that the aerosporus (Bacillus aerophilus) of CGMCC NO.8840 is with respect to other genus bacillus, the ability with excellent degrading crude oil, and alkane degradation ability is better than Biodegradation of PAHs ability.
More than describe the preferred embodiment of the present invention in detail; but the present invention is not limited to the detail in above-mentioned embodiment, within the scope of technical conceive of the present invention; can carry out multiple simple variant to technical scheme of the present invention, these simple variant all belong to protection scope of the present invention.
It should be noted that in addition, each concrete technical characterictic described in above-mentioned embodiment, in reconcilable situation, can combine by any suitable mode, for fear of unnecessary repetition, the present invention is to the explanation no longer separately of various possible array modes.
In addition, between various embodiment of the present invention, also can carry out arbitrary combination, as long as it is without prejudice to thought of the present invention, it should be considered as content disclosed in this invention equally.
Claims (10)
1. an aerosporus, is characterized in that: the deposit number of this aerosporus (Bacillus aerophilus) is CGMCC NO.8840.
2. a microbiobacterial agent, this microbiobacterial agent comprises substratum and thalline, it is characterized in that: described thalline comprises that deposit number is the aerosporus of CGMCC NO.8840.
3. microbiobacterial agent according to claim 2, is characterized in that: every gram of contained total viable count of described microbiobacterial agent is (2-300) * 10
8cFU.
4. according to the microbiobacterial agent described in claim 2 or 3, it is characterized in that: described substratum is at least one in beef-protein medium, broth culture and LB substratum.
5. according to the microbiobacterial agent described in claim 2 or 3, it is characterized in that: the starch that described substratum contains 40-150g/L, the sodium-chlor of 0.5-8g/L, the calcium carbonate of 0.5-5g/L, the iron protochloride of the potassium primary phosphate of 2-10g/L and 1-10g/L.
6. according to the microbiobacterial agent described in claim 2 or 3, it is characterized in that: described microbiobacterial agent also contains nutritive salt; Described nutritive salt comprises NaBr, H
3bO
3, SrCl
2, NaF, Seignette salt and sodium malate.
7. microbiobacterial agent according to claim 6, is characterized in that, contains the NaBr of 50-100mg, the H of 10-40mg in every kilogram of microbiobacterial agent
3bO
3, 10-40mg SrCl
2, the NaF of 1-5mg is, the sodium malate of the Seignette salt of 0.5-5g and 10-100mg.
8. the application of the microbiobacterial agent described in any one in decomposing petroleum hydrocarbon in aerosporus claimed in claim 1 and claim 2-7.
9. application according to claim 8, is characterized in that: described petroleum hydrocarbon comprises alkane and/or aromatic hydrocarbons; The carbonatoms of described alkane is 11-35, and the carbonatoms of described aromatic hydrocarbons is 10-20.
10. the application of the microbiobacterial agent described in any one in degrading crude oil in aerosporus claimed in claim 1 and claim 2-7.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105039200A (en) * | 2015-06-10 | 2015-11-11 | 哈尔滨工业大学（威海） | Deep-sea bacillus aerophilus capable of inhibiting aflatoxin |
| CN107446840A (en) * | 2017-07-10 | 2017-12-08 | 江苏南资环保股份有限公司 | One plant of 2 pyridazinone degradation bacteria of 6 hydroxyl of phenyl 3 and its application |
| CN116355808A (en) * | 2023-04-18 | 2023-06-30 | 黑龙江八一农垦大学 | Bacillus aerophilus and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102851235A (en) * | 2012-06-28 | 2013-01-02 | 北京世纪金道石油技术开发有限公司 | Ureibacillus sp. and bacterial agents and application thereof |
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2014
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102851235A (en) * | 2012-06-28 | 2013-01-02 | 北京世纪金道石油技术开发有限公司 | Ureibacillus sp. and bacterial agents and application thereof |
Non-Patent Citations (1)
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| 刘陈立等: "海洋石油降解微生物的分离鉴定", 《海洋学报》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105039200A (en) * | 2015-06-10 | 2015-11-11 | 哈尔滨工业大学（威海） | Deep-sea bacillus aerophilus capable of inhibiting aflatoxin |
| CN107446840A (en) * | 2017-07-10 | 2017-12-08 | 江苏南资环保股份有限公司 | One plant of 2 pyridazinone degradation bacteria of 6 hydroxyl of phenyl 3 and its application |
| CN107446840B (en) * | 2017-07-10 | 2020-07-10 | 江苏南资环保科技有限公司 | 2-phenyl-6-hydroxy-3-pyridazinone degrading bacterium and application thereof |
| CN116355808A (en) * | 2023-04-18 | 2023-06-30 | 黑龙江八一农垦大学 | Bacillus aerophilus and application thereof |
| CN116355808B (en) * | 2023-04-18 | 2024-02-20 | 黑龙江八一农垦大学 | Bacillus aerophilus and application thereof |
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|---|---|
| CN103952353B (en) | 2016-03-30 |
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