CN101857849A - Bacillus thuringiensis strain separating, purifying and domesticating method and use - Google Patents
Bacillus thuringiensis strain separating, purifying and domesticating method and use Download PDFInfo
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Abstract
The invention discloses a bacterial strain, which is separated from oxadiazon herbicide production waste water treatment pool and is appraised as Bacillus thuringiensis strain ODPY. The bacterial strain can be used in the biochemical control of high-salinity waste water, particularly the biochemical control of waste water containing aromatic compound persistent organic pollutants such as chloroform and methylbenzene.
Description
One, technical field
The present invention relates to a kind of extracting method and purposes that is present in the microorganism in the mud, specifically separation and purification and acclimation method and the purposes of the bacillus thuringiensis in the strain Cun Zai Yu Evil humulone purification tank for liquid waste mud.
Two, background technology
According to relevant department's investigation, China's water consumption in 2008 is 8,000 hundred million tons, and wherein process water accounts for 25.4% of national total water amount, and the per capita water resources of China only has 2410 tons.Along with the development of producing, pressure that shortage of water resources brings and restriction are more and more serious.Have data to show, in the enterprise of family, the trade effluent annual emissions is steadily increasing surplus in the of national 70,000, reached 25,000,000,000 tons, accounted for 51% of waste water total release, about 6,200,000 tons of COD quantity discharged, pesticide industry is heavy polluter in chemical industry, also is to administer to pollute difficulty maximum, the maximum industry of investment.
Pesticide wastewater mainly comes from generation water in the building-up reactions and equipment and mill floor wash-down water, product purification washing water.Its factory effluent often has following characteristics: 1. organic content is very high, and toxicity is big, and the COD concentration of synthetic wastewater is generally 6000-90000mg/L, have in addition up to hundreds of thousands of mg/L; 2. pollutant component is complicated, contains various pesticide intermediates, phosphorus, sulfide and salt in the agricultural chemicals waste water.Most agricultural chemicals have germicidal action, can suppress the microbial metabolism activity, make the biosystem disorder; 3. the material of bio-refractory is more, and some pesticide molecule is aromatics or halogenated aryl hydrocarbon and organosulfur phosphorus compound, and these all belong to recalcitrant substance; 4. the wastewater flow rate of product discharging is very big, and the restriction of production management and operant level in addition makes water quality, the water yield of waste water often change and fluctuate, thereby brings difficulty to wastewater treatment.The discharging of agricultural chemicals organic waste water, directly cause total phosphorus, ammonia, nitrogen etc. to exceed standard, and then make body eutrophication, a large amount of breedings of phycophyta, the discharge of wastewater of compounds such as some high toxic agricultural chemicals and phenol, cyanogen has caused great harm to each kind of plant in the water body in addition, simultaneously underground water and surface water is polluted, the havoc environment influences human survival.
At present, the treatment technology of agricultural chemicals waste water is summarized and can be divided into biochemical process, physico-chemical process and chemical method etc.
The principle of agricultural chemicals waste water biochemical treatment is that a part of organism that the effects such as redox by microorganism self will absorb obtains microorganism growth, movable required energy when being decomposed into simple inorganics, another part organism then transfers the required trophoplasm of organism to, form new protoplasma or cellular material, make microorganism growth, breeding.Soluble organic in the waste water can directly be absorbed by microorganism by cell walls, and the insoluble organism that solid-state or colloidal invests cell walls then absorbs after the enzyme decomposition for microorganism secretion.
The used microorganism of biochemical treatment has two kinds of aerobic and anaerobism.Aerobic microbiological can be decomposed into toxic organic compound stable, odorless material such as carbonic acid gas, water, nitrate, hydrochloride, phosphoric acid salt in the presence of oxygen.Anaerobion could be decomposed into toxic organic compound materials such as methane, carbonic acid gas, ammonia, hydrogen sulfide under oxygen free condition.The key of biochemical treatment is to guarantee the condition of microorganism growth and breeding.Should keep enough dissolved oxygen amounts in the waste water; Potential of hydrogen should be controlled at pH9-11, and temperature is controlled at 15-50 ℃, is good with 18-40 ℃; Waste water should be controlled a certain proportion of carbon, nitrogen, phosphorus content and a certain amount of nutriment such as sanitary sewage, sulphur ammonium etc., to keep the essential nutriment of microorganism growth breeding.Biochemical treatment process and equipment also should adapt to the needs of microorganism growth, breeding.
One of agricultural chemicals waste water very main characteristics be exactly the salt concentration height, this has also just restricted the efficient improvement of this type of waste water.Organic high-salinity wastewater is meant that the saliferous total amount (comprises Na
+, Ca
2+, SO
4 2-, Cl
-Deng) being not less than 1% waste water, it comes from the industry that industrial aspect comprises has: printing and dyeing, chemical industry, pharmacy, papermaking, agricultural chemicals etc.Organic high-salinity wastewater is except that the salinity height, and organic content is also very high, as not treated direct discharging, can bring great pollution and destruction to environment, and then threatens human life and health.
Three, summary of the invention
First purpose of the present invention provides the anti-high salt of a strain, efficient degradation contains aromatic compound persistent organic bacillus thuringiensis bacterial strains such as chloroform, toluene.
Second purpose of the present invention provides bacillus thuringiensis bacterial strain (Bacillus thuringiensis) ODPY separation and purification and cultural method.
The 3rd purpose of the present invention provides the application of bacillus thuringiensis in the biological treating of waste water.
Bacillus thuringiensis bacterial strain (Bacillus thuringiensis) ODPY belongs to Gram-positive bacillus, belongs to bacillus, its thalline is shaft-like, amphimicrobian, amphitrichous, gemma is arranged, the higher tolerance of salinity is arranged, growth rapidly on the beef extract-peptone solid medium.
Technical scheme of the present invention is as follows:
1, produces certainly in the mud of agricultural chemicals oxadiazon purification tank for liquid waste and isolate bacillus thuringiensis bacterial strain (Bacillusthuringiensis) ODPY
A, under aseptic condition, the mud of producing agricultural chemicals oxadiazon purification tank for liquid waste is added static 0.5～2h behind the water mixing, get supernatant liquid 1mL, physiological saline gradient dilution 10 with 0.8%
-1～10
-8, obtain 8 parts of dilution bacterium liquid;
B, on aseptic worktable, get 8 flat boards, be cooled to about 40～50 ℃ separation and purification solid medium 15～20mL to each dull and stereotyped injection after melting, wait to solidify;
C, get and diluted each 0.1～0.2mL of good bacterium liquid among the A and evenly be applied on 8 flat boards with the triangle spreading rod of the bacterium of going out respectively, and number consecutively;
D, place 28～32 ℃ of incubators to cultivate 18～60h above-mentioned 8 flat boards that prepared;
2, the purifying of bacillus thuringiensis (Bacillus thuringiensis) ODPY
E, after the D step cultivate to finish, the more single bacterium colony of picking form in each flat board, by numbering, be placed on the separation and purification solid medium that has solidified on other 8 flat boards successively and line, place incubator to cultivate 18～60h for 28～32 ℃ the flat board that streaks line;
Above-mentioned E step repeats twice at least, observes under electron microscope at last, guarantees for behind pure single bacterium colony, and the bacterial classification inoculation of purifying in slant medium, is kept in 4 ℃ of refrigerators.
3, the evaluation of bacillus thuringiensis (Bacillus thuringiensis) ODPY
By bacterium colony observation, dyeing, observation by light microscope, electron microscope observation and a series of biochemical identification thereof, follow that " uncle's Jie Shi Bacteria Identification handbook is compared to obtaining bacterial strain, identifies that this bacterial strain belongs to bacillus; Carry out 16S rDNA sequential analysis again, identify that this bacterial strain is bacillus thuringiensis (Bacillus thuringiensis) ODPY.
4, the culture condition of bacillus thuringiensis (Bacillus thuringiensis) ODPY is: adding glucose in the inorganic salt liquid substratum is carbon source, and yeast powder is a nitrogenous source, and the pH value is 8.5.
Described 0.8% physiological saline prescription: NaCl 0.8g, water 100mL
Described separation and purification solid culture based formulas: NaCl 2～7g, peptone 9～12g, extractum carnis 2～5g, agar 10～30g, water 1000mL, the pH value is 6.0～9.0.
Experimental results show that, bacillus thuringiensis provided by the invention (Bacillus thuringiensis) ODPY, by biological degradation organic high-salinity wastewater effectively after the domestication, particularly contain aromatics persistent organic pollutant wastewaters such as chloroform, toluene, for the biological treating of such waste water provides function significant bacterial strain, have higher research and using value.
Four, embodiment
Embodiment 1: separation, the purifying of bacillus thuringiensis bacterial strain (Bacillus thuringiensis) ODPY, and concrete steps are:
A, under aseptic condition, the mud of producing agricultural chemicals oxadiazon purification tank for liquid waste is added static 0.5～2h behind the water mixing, get supernatant liquid 1mL, physiological saline gradient dilution 10 with 0.8%
-1～10
-8, obtain 8 parts of dilution bacterium liquid;
B, on aseptic worktable, get 8 flat boards, be cooled to about 40～50 ℃ beef extract-peptone solid medium 15～20mL to each dull and stereotyped injection after melting, wait to solidify;
C, get and diluted good bacterium liquid 0.1～0.2mL among the A and evenly be applied on 8 flat boards with the triangle spreading rod of the bacterium of going out respectively, and number consecutively;
D, place 30 ℃ incubator to cultivate 18～60h above-mentioned 8 flat boards that prepared;
E, from each flat board the more single bacterium colony of picking form, by numbering, be placed to successively on the beef extract-peptone solid medium that has solidified on other 8 flat boards and line, place 30 ℃ incubator to cultivate 18～60h the flat board that streaks line;
Repeatedly repeat above-mentioned E step, and under electron microscope, observe, guarantee to be pure single bacterium colony.
The physiological saline prescription of 0.8% in the above-mentioned A step is: NaCl 0.8g, water 100mL.
Beef extract-peptone solid culture based formulas in above-mentioned B, the E step is: NaCl 2-7g, peptone 9-12g, extractum carnis 2-5g, agar 10-30g, water 1000mL, the pH value is 5.0-9.0.
Embodiment 2: the strain identification of bacillus thuringiensis bacterial strain (Bacillus thuringiensis) ODPY
By the bacterial strain ODPY that obtains being carried out bacterium colony observation, dyeing, observation by light microscope, electron microscope observation, Physiology and biochemistry experiment.This bacterial strain colonial morphology is rounded, and the edge is spiculation, and color is creamy white, and is moistening glossy.The configuration of surface of bacterium colony is convex surface, and upper surface is level and smooth.Opticmicroscope is observed down, and the bacterium colony thalline is shaft-like, amphimicrobian, and amphitrichous has gemma; Gramstaining is positive, and can utilize multiple organic compound to grow for sole carbon source, according to " uncle Jie Shi Bacteria Identification handbook obtains preliminary evaluation, judges that this bacterial strain belongs to bacillus.
Again this bacterial strain is carried out the order-checking of 16S rDNA sequence, the calling sequence BLAST in ncbi database of institute is compared, the result shows, the corresponding name of planting of 16S rDNA gene order institute is the most close with bacillus thuringiensis (Bacillus thuringiensis), similarity reaches 99%, according to the bacterial system principle of classification, if the 16S rDNA homology of two bacterium is greater than 99%, be with a kind of bacterium on the gene level, therefore infer that this bacterial strain ODPY should belong to bacillus thuringiensis (Bacillus thuringiensis).According to 16S rDNA gene order, the genetic evolution relation of a few strain Bacillus that bacillus thuringiensis and sibship is nearest compares, and the result shows that the sibship of this bacterial strain ODPY and bacillus thuringiensis (Bacillus thuringiensis) is nearest.
In sum, identify that this bacterial strain is bacillus thuringiensis (Bacillus thuringiensis) ODPY.
Embodiment 3: the optimization of bacillus thuringiensis bacterial strain (Bacillus thuringiensis) ODPY culture condition
One, the suitableeest growth pH value determines
Investigate the growing state of bacillus thuringiensis bacterial strain (Bacillus thuringiensis) ODPY in the environment of different initial pH value substratum, concrete operation method is: follow the unitary variant principle, fix other culture condition such as 30 ℃ of constant temperature shaking table temperature, rotating speed 150rpm, transfer the pH (prescription is with the separation and purification substratum) of beef extract-peptone liquid nutrient medium to make its initial pH value be respectively 7.0,7.5,8.0,8.5,9.0,9.5, take a sample behind the shaking culture 24h, measure bacterial concentration OD with absorbance method
600Test-results shows that the suitableeest growth pH value of bacillus thuringiensis bacterial strain (Bacillus thuringiensis) ODPY is 8.5.
Two, the optimization of carbon source
Investigate the utilize situation of bacillus thuringiensis bacterial strain (Bacillus thuringiensis) ODPY to several carbon sources such as glucose, sucrose, starch, concrete grammar is: follow the unitary variant principle, fixed nitrogen derived components corn steep liquor 3g/L and other components of inorganic salt liquid substratum, changing the carbon source composition is glucose, sucrose, starch (content is 150mg/L), inoculation bacillus thuringiensis bacterial strain (Bacillus thuringiensis) ODPY, 30 ℃ of constant temperature shaking table temperature, rotating speed 150rpm, lucifuge is cultivated 36h, measures OD
600Test-results shows that suitable carbon source is a glucose.
Three, the optimization of nitrogenous source
Investigate the utilize situation of bacillus thuringiensis bacterial strain (Bacillus thuringiensis) ODPY to corn steep liquor and yeast powder, concrete grammar is: follow the unitary variant principle, fixed carbon derived components glucose 150mg/L and other components of inorganic salt liquid substratum, changing the nitrogenous source composition is corn steep liquor or yeast powder, inoculation bacillus thuringiensis bacterial strain (Bacillus thuringiensis) ODPY, 30 ℃ of constant temperature shaking table temperature, rotating speed 150rpm, lucifuge is cultivated 36h, measures OD
600Test-results shows that suitable nitrogenous source is a yeast powder.
Four, the domestication of bacillus thuringiensis bacterial strain (Bacillus thuringiensis) ODPY
The bacterial strain of getting behind the purifying is inoculated in the minimal medium by the access amount of 5%～10% (W/V), tames with peak concentration pressure type method for domesticating, makes that the peak concentration of oxadiazon is 100mg/L.Domestication is cultivated in 25～35 ℃, 120～170rpm shaking table, is inoculated in new substratum every 5～10d by 5%～10% inoculum size, and the domestication time is 2～4 months altogether.
Described inorganic salt liquid culture medium prescription: K
2HPO
44～8g, KH
2PO
40.2～0.8g, MgSO
40.2～0.6g, (NH
4)
2SO
41～5g, MnSO
4H
2O 0.04～0.08g, FeSO
47H
2O 0.008～0.02g, CaCl
20.01～0.03g, ammonium sulfate 0.1g, oxadiazon 0.1g, water 1000mL.
Embodiment 4: the characteristic that bacillus thuringiensis bacterial strain (Bacillus thuringiensis) is effectively degraded to organic pollutant under the various salinity
Get agricultural chemicals oxadiazon factory effluent, investigate under the different salinity degraded situation organic component in the waste water.The concrete operations step is:
A, to fix other culture condition constant, get the known waste water of measuring certain salinity, be diluted to salinity successively and be respectively 40g, 60g, 80g, 100g, 120g/L, insert 3% beef-protein medium and 10% bacillus thuringiensis (Bacillusthuringiensis) ODPY inward, place the constant temperature shaking table lucifuge of 28～35 ℃ of temperature, rotating speed 150rpm to cultivate 90～120h.
B, on aseptic worktable, get five flat boards, be cooled to about 30～50 ℃ beef extract-peptone solid medium 15～20mL to each dull and stereotyped injection after melting respectively, wait to solidify;
C, survey COD and the corresponding gradient dilution bacterium of COD liquid every 18～25h sampling and carry out flat board and be coated with.
Used beef extract-peptone solid culture based formulas in the experiment: NaCl 2～7g, peptone 9～12g, extractum carnis 2～5g, agar 10～30g, water 1000mL, the pH value is 6.0～9.0.
Test-results shows, under each salinity condition, bacillus thuringiensis (Bacillus thuringiensis) ODPY all can grow and each COD value also has 50～83% ranges of decrease, degradation rate to each substrate reaches respectively: chloroform 82～94%, toluene 82～92%, other aromatics 80～95%.
Embodiment 5: bacillus thuringiensis bacterial strain (Bacillus thuringiensis) ODPY is to the degraded situation of agricultural chemicals oxadiazon factory effluent
Get the waste water that the agricultural chemicals oxadiazon produces, investigate under the different COD degraded situation organic component in the waste water.The concrete operations step is:
A, follow the unitary variant principle, fix other culture condition, get the known waste water of measuring certain C OD, be diluted to that COD is respectively 5000,7500,10000,12500,15000mg/L, insert 3% beef-protein medium and 10% bacillus thuringiensis (Bacillus thuringiensis) ODPY inward, place the constant temperature shaking table lucifuge of 30 ℃ of temperature, rotating speed 150rpm to cultivate 90～120h.
B, on aseptic worktable, get five flat boards, be cooled to about 30～50 ℃ beef extract-peptone solid medium 14～22mL to each dull and stereotyped injection after melting respectively, wait to solidify;
C, survey COD and the corresponding gradient dilution bacterium of each COD liquid every 24h sampling and carry out flat board and be coated with.
Used beef extract-peptone solid culture based formulas in the experiment: NaCl 2～7g, peptone 9～12g, extractum carnis 2～5g, agar 10～30g, water 1000mL, the pH value is 6.0～9.0.
Test-results shows, under each COD situation, bacillus thuringiensis (Bacillus thuringiensis) ODPY all can grow, and each COD all has 52～80% ranges of decrease, degradation rate to each substrate reaches respectively: chloroform 83～93%, toluene 82～94%, other aromatics 81～93%.
Claims (7)
1. separation and purification and the acclimation method of a bacillus thuringiensis strain (Bacillus thuringiensis) ODPY is characterized in that:
A, under aseptic condition, the mud of producing agricultural chemicals oxadiazon purification tank for liquid waste is added static 0.5～2h behind the water mixing, get supernatant liquid 1mL, physiological saline gradient dilution 10 with 0.8%
-1～10
-8, obtain 8 parts of dilution bacterium liquid;
B, on aseptic worktable, get 8 flat boards, be cooled to about 40～50 ℃ beef extract-peptone solid medium 15～20mL to each dull and stereotyped injection after melting, wait to solidify;
C, get and diluted each 0.1～0.2mL of good bacterium liquid among the A and evenly be applied on 8 flat boards with the triangle spreading rod of the bacterium of going out respectively, and number consecutively;
D, place 30 ℃ incubator to cultivate 18～60h above-mentioned 8 flat boards that prepared;
E, from each flat board the more single bacterium colony of picking form, by numbering, be placed on the beef extract-peptone solid medium that has solidified on other 8 flat boards successively and line, place 30 ℃ incubator to cultivate 18～60h the flat board that streaks line, this process repeats twice at least.
F, the access amount of the bacterial strain behind the purifying by 5%～10% (W/V) is inoculated in the minimal medium, peak concentration is 100mg/L.Domestication is cultivated in 25～35 ℃, 120～170rpm shaking table, is inoculated in new substratum every 5～10d by 5%～10% inoculum size, and the domestication time is no less than 2 months.
2. method according to claim 1 is characterized in that: described separation and purification solid culture based formulas: NaCl 2～7g, peptone 9～12g, extractum carnis 2～5g, agar 10～30g, water 1000mL, the pH value is 6.0～9.0.
3. method according to claim 1 is characterized in that: described inorganic salt liquid culture medium prescription: contain K in every 1000mL water
2HPO
44～8g, KH
2PO
40.2～0.8g, MgSO
40.2～0.6g, (NH
4)
28O
41～5g, MnSO
4H
2O0.04～0.08g, FeSO
47H
2O 0.008～0.02g, CaCl
20.01～0.03g, ammonium sulfate 0.1g, oxadiazon 0.1g.
4. the purposes of a bacillus thuringiensis (Bacillus thuringiensis) ODPY is characterized in that: the application in biochemistry improvement organic waste water.
5. the purposes of bacillus thuringiensis according to claim 1 (Bacillus thuringiensis) ODPY is characterized in that: the application in biochemistry improvement organic high-salinity wastewater.
6. the purposes of bacillus thuringiensis according to claim 1 (Bacillus thuringiensis) ODPY is characterized in that: the application in agricultural chemicals oxadiazon production organic waste water.
7. the purposes of bacillus thuringiensis according to claim 1 (Bacillus thuringiensis) ODPY is characterized in that: administer the application that contains in chloroform, toluene, the aromatics persistent organic pollutant wastewater at biochemistry.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102191205A (en) * | 2011-04-17 | 2011-09-21 | 南京农业大学 | Bacterial strain B1 for converting insoluble phosphate into soluble phosphate |
| CN102598979A (en) * | 2012-03-02 | 2012-07-25 | 天津师范大学 | Method for using Bacillus thruingiensis fermentation liquor to adjust proline and malondialdehyde in Festuca arundinacea |
| CN103693804A (en) * | 2013-11-29 | 2014-04-02 | 西安近代化学研究所 | Method for treating energetic material synthetic wastewater |
| CN104261568A (en) * | 2014-08-29 | 2015-01-07 | 东华大学 | Method of degrading phosphate tri(2-chloroethyl) ester by adopting bacillus thuringiensis |
| CN104560791A (en) * | 2014-12-16 | 2015-04-29 | 华南农业大学 | Bacillus thuringiensis capable of degrading pesticide and application thereof |
| CN117660225A (en) * | 2023-11-17 | 2024-03-08 | 宁夏农林科学院植物保护研究所（宁夏植物病虫害防治重点实验室） | Bacillus thuringiensis LSYS-16 with disease prevention and insect killing effects and application thereof |
| CN117660225B (en) * | 2023-11-17 | 2024-05-14 | 宁夏农林科学院植物保护研究所（宁夏植物病虫害防治重点实验室） | Bacillus thuringiensis LSYS-16 with disease prevention and insect killing effects and application thereof |
-
2010
- 2010-05-21 CN CN201010182472A patent/CN101857849A/en active Pending
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102191205A (en) * | 2011-04-17 | 2011-09-21 | 南京农业大学 | Bacterial strain B1 for converting insoluble phosphate into soluble phosphate |
| CN102191205B (en) * | 2011-04-17 | 2012-10-03 | 南京农业大学 | Bacterial strain B1 for converting insoluble phosphate into soluble phosphate |
| CN102598979A (en) * | 2012-03-02 | 2012-07-25 | 天津师范大学 | Method for using Bacillus thruingiensis fermentation liquor to adjust proline and malondialdehyde in Festuca arundinacea |
| CN102598979B (en) * | 2012-03-02 | 2013-05-08 | 天津师范大学 | Method for using Bacillus thruingiensis fermentation liquor to adjust proline and malondialdehyde in Festuca arundinacea |
| CN103693804A (en) * | 2013-11-29 | 2014-04-02 | 西安近代化学研究所 | Method for treating energetic material synthetic wastewater |
| CN103693804B (en) * | 2013-11-29 | 2016-01-20 | 西安近代化学研究所 | A kind for the treatment of process of energetic material synthetic wastewater |
| CN104261568A (en) * | 2014-08-29 | 2015-01-07 | 东华大学 | Method of degrading phosphate tri(2-chloroethyl) ester by adopting bacillus thuringiensis |
| CN104560791A (en) * | 2014-12-16 | 2015-04-29 | 华南农业大学 | Bacillus thuringiensis capable of degrading pesticide and application thereof |
| CN104560791B (en) * | 2014-12-16 | 2017-05-10 | 华南农业大学 | Bacillus thuringiensis capable of degrading pesticide and application thereof |
| CN117660225A (en) * | 2023-11-17 | 2024-03-08 | 宁夏农林科学院植物保护研究所（宁夏植物病虫害防治重点实验室） | Bacillus thuringiensis LSYS-16 with disease prevention and insect killing effects and application thereof |
| CN117660225B (en) * | 2023-11-17 | 2024-05-14 | 宁夏农林科学院植物保护研究所（宁夏植物病虫害防治重点实验室） | Bacillus thuringiensis LSYS-16 with disease prevention and insect killing effects and application thereof |
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