CN103013858A - Ancylobacter Tet-1 and application thereof in microbial degradation of tetramethrin - Google Patents
Ancylobacter Tet-1 and application thereof in microbial degradation of tetramethrin Download PDFInfo
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- CN103013858A CN103013858A CN2012104769165A CN201210476916A CN103013858A CN 103013858 A CN103013858 A CN 103013858A CN 2012104769165 A CN2012104769165 A CN 2012104769165A CN 201210476916 A CN201210476916 A CN 201210476916A CN 103013858 A CN103013858 A CN 103013858A
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Abstract
The invention provides tetramethrin degrading bacteria-ancylobacter Tet-1 and application thereof in microbial degradation of tetramethrin. The strain is collected in the China Center for Type Culture Collection (CCTCC) in Wuhan University, Wuhan, China; the collection date is September 27th, 2012, and the collection number is CCTCC No: M2012387. The ancylobacter Tet-1 provided by the invention can be used for degrading tetramethrin in water body through a direct addition manner, and can safely, efficiently and quickly degrade residual tetramethrin in the water body; and the bacteria containing the strain are easy to prepare and convenient to use, and have low cost and good application prospect.
Description
(1) technical field
The present invention relates to a strain Tetramethrin degradation bacteria---shank Pseudomonas (Ancylobacter sp.) Tet-1, and the application in the microbiological deterioration Tetramethrin.
(2) background technology
In the past few decades, pyrethroid pesticide relies on the advantage of numerous uniquenesses such as it is efficient, low toxicity, low resistance, broad spectrum, and ranking among becomes the modern topmost member in the agricultural chemicals that uses, and accounts for the share in sterilant market, the world 30%.Along with the restriction that organic phosphorous insecticide is used, the consumption of pyrethroid insecticides can be increasing.Yet a lot of reports show that too high the remaining on the cash crop such as vegetables, tealeaves of pyrethroid pesticide meeting caused a series of food-safety problem.
Tetramethrin is a kind of of pyrethroid, belongs to powerful synthetic pesticide, and the sanitary insect pests such as mosquito, fly are had quick down effect, but the poor performance that causes death has anabiosis, therefore medicament many and other good disinsection effect the use that is mixed.Tetramethrin is widely used in the sterilant with its hypotoxic advantage, and this medicine is that world health organisation recommendations is used for one of sanitarian main sterilant.Because its Stability Analysis of Structures is difficult to degraded after using, and causes residuing in environment, Tetramethrin residual in the environment can enter water body by effects such as river rising in Ningxia and flowing into central Shaanxi, earth's surface streams, thus the harm hydrobiont.
The chemical structure of Tetramethrin and chemical property are all more stable, if remove with the method for physics, chemistry, processing cost is higher, and harmful byproduct is more, and microorganism has the effect of uniqueness to the metabolism of Tetramethrin.With the method for microbial metabolism degraded Tetramethrin, not only cost is low but also reduced and can effectively reduce secondary pollution, therefore has broad application prospects.And microorganism is that a class kind is many, breeding is fast, strong adaptability, organism that metabolic capacity is strong.Therefore, if the microorganism that the energy screening and separating goes out the Tetramethrin of effectively degrading, to reducing the content of Tetramethrin in environment, protection of the environment has profound significance.
(3) summary of the invention
The object of the invention provides the new Tetramethrin degradation bacteria of a strain---shank Pseudomonas (Ancylobacter sp.) Tet-1, and the application in the microbiological deterioration Tetramethrin.
The technical solution used in the present invention is:
Shank Pseudomonas (Ancylobacter sp.) Tet-1 is preserved in Chinese Typical Representative culture collection center, the address: China, and Wuhan, Wuhan University, preservation date is on September 27th, 2012, deposit number is CCTCC No:M 2012387.
The present invention obtains a series of bacterial strains from the active sludge of insecticide factory, after separation and purification, obtain can effectively the degrade bacterial strain of Tetramethrin of a strain, called after Tet-1.The 16S rDNA sequence (the Genbank number of logging in is JX878618) that bacterial strain Tet-1 records is carried out the BLAST compare of analysis in GenBank and RDP database, it is accredited as Ancylobacter belongs to (Ancylobacter sp.).Experimental result shows: Tet-1 can grow take Tetramethrin as sole carbon source, and behind the cultivation 7d, the Tet-1 bacterial strain is 54.0% to the degradation rate of the Tetramethrin of 50mg/L.There is no at present the bibliographical information that utilizes the strains for degrading Tetramethrin that separates in the mud, and the present invention can efficient degradation the solution Tetramethrin, utilize this bacterial strain solution Tetramethrin of effectively degrading, for its application in biological restoration provides certain theoretical basis.
Screening and the evaluation of shank Pseudomonas Tet-1 of the present invention (CCTCC No:M 2012387) bacterial strain:
1) substratum
Inorganic salt nutrient solution final concentration consists of: contain NaCl 1g, K in every liter of nutrient solution
2HPO
41.5g, KH
2PO
40.5g, (NH4)
2SO
41.5g, MgSO
40.1g 1ml trace element solution, solvent are water, natural pH value, high pressure steam sterilization (121 ℃, make after 20min), wherein contain MnSO in every liter of trace element solution
4H
2O 0.13g, ZnCl
20.23g, CuSO
4H
2O 0.03g, CoCl
26H
2O 0.42g, Na
2MoO
42H
2O 0.15g, AlCl
36H
2O 0.05g, solvent are water.
Enrichment culture liquid: in the inorganic salt nutrient solution, add Tetramethrin, so that the final concentration of Tetramethrin is 100 mg/L.
The LB liquid nutrient medium: contain yeast powder 10g in every liter of nutrient solution, peptone 5.0g, sodium-chlor 10.0g, solvent are water, natural pH value, high pressure steam sterilization (121 ℃ make after 20min).
The LB solid medium: contain yeast powder 10g in every liter of cultivation, peptone 5.0g, sodium-chlor 10.0g, agar 15.0g, solvent are water, natural pH value, high pressure steam sterilization (121 ℃ make after 20min).
2) strains separation purifying
Mud sample picks up from Hangzhou insecticide factory, get 5 g mud samples and place the 250ml Erlenmeyer flask, add 100ml enrichment culture liquid, (30 ℃ in 150rpm) 1 week, are got the turbid liquid in 5ml upper strata in fresh enrichment culture liquid 100 ml to dark shaking culture, continue (30 ℃ of dark shaking culture, 150rpm) 1 week repeats aforesaid operations process 3 times, and each inoculum of cultivating all is taken from the nutrient solution of cultivating gained last time.
3) identification of strains
The bacterial strain of above-mentioned acquisition is carried out morphological specificity and molecular biology identification, and the electromicroscopic photograph of this bacterial strain as shown in Figure 1.The main biological property of this bacterial strain is: the gramstaining reaction negative, and thalline is shaft-like, and bending is arranged, and atrichia, diameter are about 0.7～1.2 μ m, and bacterium colony is level and smooth, is white in color..The optimum growth conditions of this bacterial strain is pH value 7.0,30 ℃ of temperature.This bacterial strain is accredited as Ancylobacter through 16S rDNA sequential analysis and belongs to, so called after is shank Pseudomonas Tet-1(Ancylobacter sp. Tet-1).
The invention still further relates to the application of described shank Pseudomonas Tet-1 in the microbiological deterioration Tetramethrin.
Concrete, described degraded is carried out under 20 ~ 40 ℃, pH 4.0 ~ 10.0, dark condition, generally needs shaking culture.Preferably, described degraded is carried out under 30 ℃, pH 7.0,150rpm, dark condition.
Be in the inorganic salt nutrient solution of 50 mg/L at the Tetramethrin final concentration, add the cell suspension that contains shank Pseudomonas Tet-1 and consist of reaction system, about 20 ~ 40 ℃, 4.0 ~ 10.0 times dark shaking culture 7 d of pH, can make the quality residual quantity of Tetramethrin in the reaction solution less than 50%; Shank Pseudomonas Tet-1 final concentration is 1 * 10 to the described cell suspension add-on that contains shank Pseudomonas Tet-1 in the reaction system in order to make
7~ 5 * 10
7Individual/ml.
In the practical application, described bacterial strain usually need to be through overactivation and enlarged culturing, and detailed process is as follows:
(1) slant activation: shank Pseudomonas Tet-1 is inoculated in slant medium, cultivated 5 ~ 7 days for 25 ~ 45 ℃, obtain the thalline inclined-plane; The final concentration of described slant medium consists of: yeast powder 10g/L, and peptone 5.0g/L, sodium-chlor 10.0g/L, agar 15.0g/L, solvent are water;
(2) seed culture: picking one transfering loop thalline is seeded to the inorganic salt nutrient solution from step (1) thalline inclined-plane, cultivates 5 ~ 7 days for 25 ~ 45 ℃, obtains seed liquor; Described inorganic salt nutrient solution final concentration consists of: contain NaCl 1g, K in every liter of nutrient solution
2HPO
41.5g, KH
2PO
40.5g, (NH4)
2SO
41.5g, MgSO
40.1g 1ml trace element solution, solvent are water, natural pH value, high pressure steam sterilization (121 ℃, make after 20min), wherein contain MnSO in every liter of trace element solution
4H
2O 0.13g, ZnCl
20.23g, CuSO
4H
2O 0.03g, CoCl
26H
2O 0.42g, Na
2MoO
42H
2O 0.15g, AlCl
36H
2O 0.05g, solvent are water;
(3) enlarged culturing: the seed liquor that step (2) is obtained is seeded in the LB liquid nutrient medium with the inoculum size of volumetric concentration 10 ~ 20%, 30 ℃, 150rpm vibration are supported to logarithmic phase, obtain bacterium liquid, bacterium liquid is centrifugal, abandon supernatant, precipitation is that 7.0 phosphoric acid buffer suspends with the pH value, obtains to contain the cell suspension of shank Pseudomonas Tet-1, and this cell suspension can add the degraded that is used for Tetramethrin in water body; Described LB liquid nutrient medium final concentration consists of: contain yeast powder 10g in every liter of cultivation, and peptone 5.0g, sodium-chlor 10.0g, solvent are water, natural pH value.
Thalli growth amount of the present invention adopts ultraviolet spectrophotometer to detect, and represents by measuring the absorbance of thalline (being the mycetocyte nutrient solution) at the 600nm place.
The present invention adopts reversed-phased high performace liquid chromatographic to detect the residual quantity of Tetramethrin in the inorganic salt nutrient solution.RPLC testing conditions: Grace Alltima C18 Column (4.6 mm * 250 mm, 5 μ m), moving phase is methyl alcohol: water=95: the 5(volume ratio), flow velocity is 0.9 mL/min, and column temperature is 25 ℃, sample size is 20 μ L.
Compared with prior art, beneficial effect of the present invention is mainly reflected in:
Shank Pseudomonas Tet-1 of the present invention can be applied to by the mode that directly adds the degraded of Tetramethrin in the water body, the residual Tetramethrin in the water body of safely, efficiently, fastly degrading, the microbial inoculum preparation technology who contains this bacterial strain is simple, with low cost, easy to use, have good application prospect.
(4) description of drawings
Fig. 1 is the Electronic Speculum figure of Tetramethrin degradation bacteria of the present invention;
Fig. 2 is the canonical plotting of Tetramethrin;
Fig. 3 is Tetramethrin degradation bacteria of the present invention under the pure culture condition being the degradation curve figure of Tetramethrin of 50mg/L and Tetramethrin degradation bacteria to concentration in Tetramethrin concentration is growth curve chart under the 50mg/L pure culture condition.
Fig. 4 is that Tetramethrin degradation bacteria of the present invention is the degradation curve figure of the Tetramethrin of 50mg/L to concentration under condition of different pH;
Fig. 5 is that Tetramethrin degradation bacteria of the present invention is the degradation curve figure of the Tetramethrin of 50mg/L to concentration under condition of different temperatures.
(5) embodiment
The present invention is described further below in conjunction with specific embodiment, but protection scope of the present invention is not limited in this:
Embodiment 1: the screening of bacterial strain and evaluation
1) substratum
Inorganic salt nutrient solution: NaCl 1g, K
2HPO
41.5g, KH
2PO
40.5g, (NH4)
2SO
41.5g, MgSO
40.1g, the 1ml trace element solution, distilled water complements to 1000ml, stirs after the mixing, natural pH value, high pressure steam sterilization (121 ℃, make after 20min), wherein contain MnSO in every liter of trace element solution
4H
2O 0.13g, ZnCl
20.23g, CuSO
4H
2O 0.03g, CoCl
26H
2O 0.42g, Na
2MoO
42H
2O 0.15g, AlCl
36H
2O 0.05g complements to 1000ml with distilled water.
Enrichment culture liquid: in the inorganic salt nutrient solution, add Tetramethrin solution, so that the concentration of Tetramethrin is 100 mg/L.
The LB nutrient solution: yeast powder 10g, peptone 5.0g, sodium-chlor 10.0g, distilled water complements to 1000ml, stirs after the mixing, natural pH value, high pressure steam sterilization (121 ℃ make after 20min).
The LB solid medium: yeast powder 10g, peptone 5.0g, sodium-chlor 10.0g, agar 15.0g, distilled water complements to 1000ml, stirs after the mixing, natural pH value, high pressure steam sterilization (121 ℃ make after 20min).
2) strains separation purifying
Mud sample picks up from Hangzhou insecticide factory, get 5 g mud samples and place the 250ml Erlenmeyer flask, add 100ml enrichment culture liquid, (30 ℃ in 150rpm) 1 week, are got the turbid liquid in 5ml upper strata in fresh enrichment culture liquid to dark shaking culture, continue (30 ℃ of dark shaking culture, 150rpm) 1 week repeats aforesaid operations process 3 times, and each inoculum of cultivating all is taken from the nutrient solution of cultivating gained last time.
Get last cultivation gained nutrient solution a little carry out gradient dilution, the nutrient solution 150 μ l that get after the dilution coat on the LB solid plate that contains the 50mg/L Tetramethrin, place constant incubator (30 ℃) to cultivate, after growing bacterium colony on the flat board, each bacterium colony of picking purifying repeatedly on the LB solid plate, until bacterium colony is single, each bacterium colony behind the purifying is connected to respectively (30 ℃ of shaking culture in the LB liquid tube, 150rpm) spend the night, be connected to after cultured bacterium liquid is centrifugal and cultivate 7 d in the enrichment culture liquid, detect the residual quantity of Tetramethrin in each enrichment culture liquid by high performance liquid chromatography (HPLC), at last screening obtains the bacterial strain of strain energy efficient degradation Tetramethrin, called after Tet-1(CCTCC No:M 2012387).
3) identification of strains
The bacterial strain of above-mentioned acquisition is carried out morphological specificity and molecular biology identification, and the electromicroscopic photograph of this bacterial strain as shown in Figure 1.The main biological property of this bacterial strain is: the gramstaining reaction negative, and thalline is shaft-like, and bending is arranged, and atrichia, diameter are about 0.7～1.2 μ m, and bacterium colony is level and smooth, is white in color.The optimum growth conditions of this bacterial strain is pH value 7.0,30 ℃ of temperature.This bacterial strain is accredited as Ancylobacter through 16S rDNA sequential analysis and belongs to.
Embodiment 2: the preparation of mycetocyte suspension
(1) slant culture: shank Pseudomonas Tet-1 is inoculated in slant medium, cultivated 6 days for 30 ℃, obtain the thalline inclined-plane; The final concentration of described slant medium consists of: yeast powder 10g, peptone 5.0g, sodium-chlor 10.0g, agar 15.0g, water 1000ml;
(2) seed culture: picking one transfering loop thalline is seeded to the inorganic salt nutrient solution from step (1) thalline inclined-plane, cultivates 6 days for 30 ℃, obtains seed liquor; Described inorganic salt nutrient solution final concentration forms with embodiment 1;
(3) enlarged culturing: the seed liquor that step (2) is obtained is seeded in the LB liquid nutrient medium (100 mL) with the inoculum size of volumetric concentration 10 ~ 20%, 30 ℃, 150rpm shaking culture are to logarithmic phase, obtain bacterium liquid, with the centrifugal (6000rpm of bacterium liquid, 5min), abandon supernatant, precipitation is 7.0 phosphoric acid buffer suspension with the pH value, acquisition contains shank Pseudomonas Tet-1 cell suspension 100 mL, and wherein the shank Pseudomonas Tet-1 concentration in the cell suspension is 0.2 ~ 1 * 10
9Individual/ml; Described LB liquid nutrient medium final concentration forms with embodiment 1; PH is that the prescription of the phosphoric acid buffer of 7.0 0.2 mol/L is: get the SODIUM PHOSPHATE, MONOBASIC 39ml of 0.2 mol/L and the Sodium phosphate dibasic 61ml of 0.2mol/L, be settled to 1000ml with ultrapure water, behind the high pressure steam sterilization (121 ℃, 20min) and get final product.
Embodiment 3: the Tetramethrin degradation experiment
1) detection of cell concentration and Tetramethrin content in the inorganic salt nutrient solution:
The thalli growth amount adopts ultraviolet spectrophotometer to detect, and the absorbance of thalline at the 600nm place represents in the nutrient solution by measuring.
This experiment adopts reversed-phased high performace liquid chromatographic to detect the residual quantity of Tetramethrin in the inorganic salt nutrient solution.RPLC testing conditions: Grace Alltima C18 Column(4.6 mm * 250 mm, 5 μ m), moving phase is methyl alcohol: water=95: the 5(volume ratio), flow velocity is 0.9 mL/min, and column temperature is 25 ℃, and sample size is 20 μ L.
2) Tetramethrin degradation experiment:
Get 5 250ml Erlenmeyer flasks, all add 100ml inorganic salt nutrient solution, (121 ℃ of high pressure steam sterilizations, add Tetramethrin 20min), make Tetramethrin concentration be 50 mg/L, respectively get the mycetocyte suspension 5ml that embodiment 2 methods obtain, be inoculated in respectively in this inorganic salt nutrient solution, making shank Pseudomonas Tet-1 final concentration is 1 ~ 5 * 10
7Individual/ml, respectively at 20,25, (pH 7.0, and 150rpm), every day, timing sampling was measured residual amine chrysanthemum ester concentration, the results are shown in Figure 5 for 30,35,40 ℃ of cultivation shaking tables.
Get 6 250ml Erlenmeyer flasks, all add 100ml inorganic salt nutrient solution, (121 ℃ of high pressure steam sterilizations, add Tetramethrin 20min), make Tetramethrin concentration be 50 mg/L, respectively get the mycetocyte suspension 5ml that embodiment 2 methods obtain, be inoculated in respectively in this inorganic salt nutrient solution, making shank Pseudomonas Tet-1 final concentration is 1 ~ 5 * 10
7Individual/ml, respectively at pH 4.0,6.0,7.0,8.0,9.0,10.0 cultivate shaking table, and (30 ℃, 150rpm), every day, timing sampling was measured residual amine chrysanthemum ester concentration, the results are shown in Figure 4.
Get the 250ml Erlenmeyer flask, add 100ml inorganic salt nutrient solution, (121 ℃ of high pressure steam sterilizations, add Tetramethrin 20min), making Tetramethrin concentration is 50 mg/L, get the mycetocyte suspension 5ml that embodiment 2 methods obtain, be inoculated in this inorganic salt nutrient solution, making shank Pseudomonas Tet-1 final concentration is 1 ~ 5 * 10
7Individual/ml, 1 experiment that does not contain this bacterial classification is set accordingly as blank, then together place dark shaking culture in the shaking table (30 ℃, pH7.0,150rpm).Be 0,1,2,3,4,5,6 at incubation time, timing sampling during 7d, detect the increment of thalline in the inorganic salt nutrient solution and the residual quantity of Tetramethrin, the results are shown in Figure 3.
Tetramethrin standard substance (concentration 100 mg/L) are dissolved with sterilized water, and at reversed-phase liquid chromatography testing standard curve, the Tetramethrin typical curve as shown in Figure 2, the typical curve equation is (y=13.63x+7.384, R2=0.999, y is peak area, x is Tetramethrin concentration).Bacterial strain of the present invention can find out that OD600 is increased to 0.45 from 0.02, illustrates that thalli growth is good to the growth curve of the degradation curve of the Tetramethrin of 50 mg/L concentration and thalline as shown in Figure 3.Observe Fig. 3, can find, cultivate 7 d after, Tetramethrin degradation bacteria of the present invention is 54 ± 0.9% to the degradation rate of the Tetramethrin of 50 mg/L, all do not add the percent hydrolysis of blank behind 7 d of bacterium all less than 5%.Fig. 4 and Fig. 5 show that the optimum degradation condition of this bacterial strain is pH value 7.0,30 ℃ of temperature.
Experimental result shows that this bacterial classification has preferably degradation capability to the Tetramethrin of concentration 50 mg/L, and this bacterial classification is novel Tetramethrin degradation bacteria, therefore, this bacterium has very large promoter action to degradation pathway and the degrading genes of research Tetramethrin, and the degraded of Tetramethrin in the environment is especially had certain positive effect to the concentrated reparation of Tetramethrin.
Claims (4)
1. shank Pseudomonas (Ancylobacter sp.) Tet-1 is preserved in Chinese Typical Representative culture collection center, the address: China, and Wuhan, Wuhan University, preservation date is on September 27th, 2012, deposit number is CCTCC No:M 2012387.
2. the application of shank Pseudomonas Tet-1 as claimed in claim 1 in the microbiological deterioration Tetramethrin.
3. application as claimed in claim 2 is characterized in that described degraded carries out under 20 ~ 40 ℃, pH 4.0 ~ 10.0, dark condition.
4. application as claimed in claim 3 is characterized in that described degraded carries out under 30 ℃, pH 7.0,150rpm, dark condition.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1420848A (en) * | 1999-07-12 | 2003-05-28 | 克莱沃有限公司 | Method of purifying water, suitable bacteria for said method and use thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1420848A (en) * | 1999-07-12 | 2003-05-28 | 克莱沃有限公司 | Method of purifying water, suitable bacteria for said method and use thereof |
Non-Patent Citations (2)
| Title |
|---|
| 翁林捷: "拟除虫菊酯类农药微生物修复回顾与展望", 《中国农学通报》, vol. 25, no. 16, 31 December 2009 (2009-12-31) * |
| 莫如友, 等: "胺菊酯降解菌的筛选鉴定及其降解特性", 《湖南科技大学学报（自然科学版）》, vol. 27, no. 03, 25 September 2012 (2012-09-25) * |
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Application publication date: 20130403 |