CN101024819A - DU-E4LP seudomonas putida and use in degradating parathionmethyl - Google Patents
DU-E4LP seudomonas putida and use in degradating parathionmethyl Download PDFInfo
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- CN101024819A CN101024819A CN 200610008274 CN200610008274A CN101024819A CN 101024819 A CN101024819 A CN 101024819A CN 200610008274 CN200610008274 CN 200610008274 CN 200610008274 A CN200610008274 A CN 200610008274A CN 101024819 A CN101024819 A CN 101024819A
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Abstract
The invention supplies a methyl parathion degradation fungus DM-1 that the preservation NO is CGMCC No.1620. The classification designation is Bacillus subtilis. It is separated and selected from soil polluted by agricultural chemical. The fungus has good degradation effect to 50-500ppm methyl parathion at 30 degree centigrade.
Description
Technical field
The invention belongs to the microbiological deterioration field, specifically relate to a kind of parathion-methyl degradation bacteria, and the application in the degraded parathion-methyl.
Background technology
Parathion-methyl (Methyl Parathion is called for short MP) is a kind of organic phosphorous insecticide of high poison, and its chemical molecular formula is C
8H
10NO
5PS, chemical name are O, O-dimethyl O-(right-nitrophenyl) phosphorothionate.Because this sterilant has efficiently, the economic dispatch characteristics, it is widely used in agriculture production and the pest control.But simultaneously,, had a strong impact on the quality of people's healthy and agricultural exports, brought the series of negative effect because its residual hazard will cause the severe contamination of environment and food, and produce a large amount of sterilant refuses.How to handle residual parathion-methyl effectively, avoid environment and human infringement are become people's problem demanding prompt solution.
Use traditional physico-chemical process to handle residual parathion-methyl, difficulty is big, cost is higher, and processing efficiency is low, and causes secondary pollution easily.So generally select the microbiological deterioration technology of comparison environmental protection at present for use, to metabolism modes such as agricultural chemicals mineralising, hydrolysis and common metabolism, handle residual parathion-methyl by microorganism.As at document 1: the separation of parathion-methyl degradation bacteria DLL-1, evaluation and degradation property research, use and environmental organism journal [J], 1999,5 (supplementary issues): disclosed among the 147-150, as its sole carbon source or nitrogen, phosphorus source, use parathion-methyl degradation bacteria DLL-1 in agricultural chemicals with its hydrolysis or mineralising.This method is to the degraded of parathion-methyl the time, and the intermediate product of generation is a p-NP, and p-NP is one to have moderately toxic material, can bring potential to threaten to human beings'health.
Summary of the invention
The object of the present invention is to provide a kind of from be subjected to for a long time that screening and separating goes out the pesticide contaminated soil, have broad spectrum degrading organic phosphor compounds, can efficiently transform the parathion-methyl degradation bacteria of parathion-methyl, and the application in the degraded parathion-methyl in metabolic mode altogether.
The objective of the invention is to realize by the following technical solutions:
The invention provides a kind of parathion-methyl degradation bacteria DM-1, this parathion-methyl degradation bacteria is being preserved in " China Committee for Culture Collection of Microorganisms common micro-organisms center " (No. 13, North No.1 Row, Zhongguancun, Haidian District, Beijing City on February 17th, 2006, Institute of Microorganism, Academia Sinica), its preserving number is CGMCC No.1620, its classification called after subtilis Bacillus subtilis..
Parathion-methyl degradation bacteria provided by the invention, be from the soil that is subjected to pesticidal contamination for a long time, to separate and screening, concrete steps are as follows: the suspension that the pesticidal contamination soil sample of gathering is mixed with 20wt% with the inorganic salt nutrient solution, receive in the inorganic salt nutrient solution that contains the 50mg/L parathion-methyl shaking culture then with the inoculum size of 5wt%; Go down to posterity and cultivated for 6 generations, the concentration that improves parathion-methyl in the inorganic salt nutrient solution gradually is to 600ppm; Take out 1mL bacterium liquid from last nutrient solution, after the dilution, coat on the inorganic salt solid medium that contains the 500ppm parathion-methyl, 30 ℃ of thermostat containers are cultivated; Choose growth fast (transparent circle is arranged on the flat board), the bacterium colony rule obtains pure bacterial strain behind the stable bacterium colony that goes down to posterity, purifying 2～3 times; With isolated 5 strain bacterium as sieve again bacterial strain in the LB substratum enlarged culturing to identical OD value 1.0, inoculum size with 10% inserts and contains in the minimal medium of 100ppm parathion-methyl, in 30 ℃, the 180rpm shaking table is cultivated 48h, use the gas Chromatographic Determination degradation rate, finishing screen is chosen the highest strain bacterium of degradation rate, changes the beef extract-peptone inclined-plane over to and preserves.
Described inorganic salt nutrient solution is prepared in following ratio: in 1000ml water, and 0.5g NaCl, 0.2gMgSO
4, 0.5g (NH
4)
2SO
4, 1.5g K
2HPO
4, 0.5g KH
2PO
4, 0.05g CaCl
2, 0.02g FeSO
4, 0.002g yeast powder, 7.0,121 ℃ of autoclaving 20min of pH value;
Described inorganic salt solid medium is the agar that above-mentioned inorganic salt nutrient solution is added 1.5～2% (w/v) that account for inorganic salt nutrient solution gross weight.
Described LB substratum and beef extract-peptone and document 2: microbiology experiment [M], Beijing: Higher Education Publishing House, 1988, identical among the 75-78.
The testing conditions of used gas-chromatography is: HP5890II type gas chromatograph, electron capture detector; Chromatographic column: low-pole capillary column HP-1[100% polydimethylsiloxane, Polydimethylsiloxane 25m * 0.32mm (I.D.) * 0.25 μ m]; Service temperature: 300 ℃ of detector temperatures, 300 ℃ of injector temperatures, 210 ℃ of column temperatures; Post is pressed 6.9psi; Post flow 0.748ml/min; Minimum detectable activity: 0.05 μ g/ml; Standard specimen concentration 0.5ppm; The about 7.0min of parathion-methyl appearance time.
Peak area, standard specimen peak area and sample size calculate the content of parathion-methyl in the nutrient solution per sample.Calculation formula is:
In the formula: A
X--treat the test sample peak area; A
0--the standard peak area;
A--standard concentration;
V
0--the standard sample size; V
X--unknown sample sample size.
The degradation rate calculation formula is:
(Biolog.Inc. USA) identifies this bacterial strain to adopt microorganism computer classification identification systems.The bacterial strain of purifying is cultivated 16～24h on the Biolog substratum; Adopt the main method to prepare bacteria suspension, adjust turbidity to 52%T with GN/GP-IF; Bacteria suspension is inoculated in the GN-Microplate identification plate with every hole 150 μ l, and identification plate is cultivated 16～24h in 30 ℃; With Reader scanning identification plate, measure the light absorption value in each hole, the result of mensuration and database comparison provide qualification result automatically by software.Through this bacterial strain of Biolog technical evaluation is subtilis (Bacillussubtilis), called after parathion-methyl degradation bacteria DM-1.Qualification result sees Table 1.
Table 1, parathion-methyl degradation bacteria strain identification result
| NAME | PROB | SIM | DIST | |
|
| 1 | Bacillus |
100 | 0.68 | 4.84 | GP-ROD SB |
| 2 | Bacillus |
0 | 0.00 | 7.70 | GP-ROD SB |
| 3 | Bacillus |
0 | 0.00 | 10.05 | GP-ROD SB |
The morphological specificity and the growth characteristic of parathion-methyl degradation bacteria DM-1 bacterial strain provided by the invention are: gram positive bacterium, cell are shaft-like, long 1.2～1.5 μ m, and wide 0.8 μ m can form gemma, and strict aerobic growth belongs to bacillus.Colonial morphology rule on nutrient agar medium, surface irregularity, edge are spination.The vibration aerated culture produces mycoderm, not chromogenesis in the liquid medium within.Bacterial strain is grown in the 7%NaCl substratum, and optimum growth temperature is 30 ℃, and optimal pH is 7.
The Physiology and biochemistry character of parathion-methyl degradation bacteria DM-1 bacterial strain provided by the invention is: catalase and oxydase reaction are positive; Methyl red and V.P. test are positive; Indole test is negative; Nitrate reduction test is negative; Glucose fermentation produces acid, aerogenesis; Energy gelatin hydrolysate and casein; Can utilize Citrate trianion; Can not hydrolyzed starch and urea.
The metabolic characteristic of parathion-methyl degradation bacteria DM-1 bacterial strain provided by the invention is: the organophosphorus degrading bacteria metabolism parathion-methyl of having reported at present is to produce p-NP by hydrolysis reaction, and bacterial strain DM-1 of the present invention is converted into p-aminophenol by reduction and hydrolysis reaction with parathion-methyl.This bacterial strain can tolerate the parathion-methyl of 0～600ppm, and can rapidly it be transformed fully.What is more important, bacterial strain DM-1 has very wide substrate utilization scope, can transform other organophosphorus pesticides efficiently, as paraoxon, thiophos and fenitrothion 95 etc., therefore, this bacterial strain has great application potential in the biology in situ reparation of the soil of organophosphorus pesticide pollution and water body.
Degradation property to parathion-methyl degradation bacteria DM-1 provided by the invention is measured.
One, glucose concn is to the influence of strain growth and degradation rate
With institute's isolating parathion-methyl degradation bacteria DM-1 bacterial strain enlarged culturing OD value about 1.5 in LB substratum (ditto), wash with sterilized water centrifugal back, be mixed with bacterium suspension, inoculum size with 10% is linked in the inorganic salt nutrient solution (ditto) that contains parathion-methyl 200ppm, (concentration of glucose is respectively 0g/L to add the inorganic salt liquid nutrient solution of glucose of different concns respectively, 1g/L, 2g/L, 5g/L, 10g/L, 20g/L, the pH value is 7.0) in, and be contrast not connect bacterium, each sample establish two parallel.30 ℃, 180r/min shaking table shaking culture 32h.Get nutrient solution 10ml, use equal volume of ethyl acetate 3 times, merge No. three times extracting solution, anhydrous sodium sulfate drying, gas Chromatographic Determination (testing conditions is as previously mentioned) is carried out in dilution, and ultraviolet spectrophotometer is measured OD
600
The result as shown in Figure 1, as can be known, during no glucose (concentration of glucose is 0g/L), parathion-methyl is degraded hardly; Under the condition of glucose as additional carbon, DM-1 has degradation capability preferably to MP, can the efficient degradation parathion-methyl.When glucose concn was on the low side, the growth of bacterial strain was relatively poor, and is also not good to the degradation effect of agricultural chemicals; Glucose concn is that the DM-1 bacterial strain all has good degradation effect in 2～20g/L scope.
Infer that thus DM-1 is that it needs environment that elementary carbon source is provided by the MP that degrades of metabolism altogether in degradation process.By last figure as can be known, whether glucose exists the degraded to MP to play decisive role, and its concentration height also has certain influence to degradation rate.The present invention shows that the common metabolic degradation process of toxic compounds is a special power consumption process, and the defeated people of energy is necessary in the external environment.Some poisonous difficult degradation compound of occurring in nature can not directly be degraded by microorganism, but adds a small amount of substrate that is easily utilized by microorganism such as glucose etc., can promote or start the degraded of difficult degradation compound, and it is led people's circulation.
Two, initial pH value is to the growth and the degraded influence of bacterial strain
With institute's isolating parathion-methyl degradation bacteria DM-1 bacterial strain in LB substratum (ditto) enlarged culturing to OD value about 1.5, wash with sterilized water centrifugal back, be mixed with bacterium suspension, insert with 10% inoculum size and contain in the inorganic salt nutrient solution (ditto) of 200ppm parathion-methyl, the initial pH value of nutrient solution is adjusted to 6.0 respectively, 6.5,7.0,8.0,9.0, and be contrast not connect bacterium, each sample establish two parallel.30 ℃, the 180r/min shaking table is cultivated 32h.Get nutrient solution 10ml, use equal volume of ethyl acetate 3 times, merge No. three times extracting solution, anhydrous sodium sulfate drying, gas Chromatographic Determination (testing conditions is as previously mentioned) is carried out in dilution, and ultraviolet spectrophotometer is measured OD
600
The result as shown in Figure 2, as can be known, in the scope of pH value 6.0～9.0, DM-1 strain growth and degradation effect are all better, wherein for the pH value is that 7.0 o'clock degradation rate is the highest, the increment maximum.
Three, concentration of substrate is to the influence of strain growth and degradation rate
With institute's isolating parathion-methyl degradation bacteria DM-1 bacterial strain in LB substratum (ditto) enlarged culturing to OD value about 1.5, wash with sterilized water centrifugal back, be mixed with bacterium suspension, the inoculum size with 10% inserts and contains 50ppm, 100ppm, 200ppm, 300ppm, 400ppm, 500ppm parathion-methyl, pH value are 7.0, glucose concn is in the inorganic salt nutrient solution (ditto) of 5g/L, and be contrast not connect bacterium, each sample establish two parallel.30 ℃, the 180r/min shaking table is cultivated 32h.Get nutrient solution 10ml, use equal volume of ethyl acetate 3 times, merge No. three times extracting solution, anhydrous sodium sulfate drying, gas Chromatographic Determination (testing conditions is as previously mentioned) is carried out in dilution, and ultraviolet spectrophotometer is measured OD
600
The result as shown in Figure 3, as can be known, strain growth and degradation effect were all better when substrate (parathion-methyl) concentration was 50～500ppm, degradation rate descended to some extent after concentration of substrate was higher than 300ppm, show that bacterial strain is subjected to the concentration affects of parathion-methyl to the degraded of parathion-methyl, the concentration of parathion-methyl surpasses finite concentration has restraining effect to the metabolism of bacterial strain.
Four, the DM-1 bacterial strain is to the degraded dynamic studies of MP
With institute's isolating parathion-methyl degradation bacteria DM-1 bacterial strain in LB substratum (ditto) enlarged culturing to OD value about 1.5, wash with sterilized water centrifugal back, be mixed with bacterium suspension, insert with 10% inoculum size and to contain that 200ppm parathion-methyl, pH value are 7.0, glucose concn is in the inorganic salt nutrient solution (ditto) of 5g/L, and be contrast not connect bacterium, each sample establish two parallel.30 ℃, the 180r/min shaking table is cultivated.Respectively at 3h, 6h, 12h, 20h, 28h, 38h get nutrient solution 10ml, with equal volume of ethyl acetate 3 times, merge No. three times extracting solution, anhydrous sodium sulfate drying, gas Chromatographic Determination (testing conditions is as previously mentioned) is carried out in dilution, ultraviolet spectrophotometer mensuration OD
600
The result as shown in Figure 4, as can be known, bacterial strain is through after the cultivation of 12h, the speed of growth begins to accelerate, and enters the growth platform phase behind the 28h, and after this slow decline arranged.The concentration of parathion-methyl reduces along with the growth of bacterial strain, in 0～28h degraded very fast, behind the 28h since pesticide concentration seldom, degradation rate descends to some extent.As seen the common metabolic capacity of bacterial strain is closely related with the increment of bacterial strain, along with OD
600The increase of value, the concentration of parathion-methyl reduces gradually, is almost transformed fully behind 28h.
Adopt gas chromatography/mass spectrometry coupling (GC-MS) that the product of bacterial strain DM-1 degraded parathion-methyl of the present invention is studied, the GC-MS testing conditions is: adopt the Agilent6890N-5973N-MSDGC-MS analyser, chromatographic column: DB-5MS[60m * 0.25mm (I.D.) * 0.25 μ m] capillary column.Operational condition---chromatogram column temperature: temperature programming, 60 ℃ of beginnings keep 1min, rise to 120 ℃ with 20 ℃/min, keep 2min, rise to 280 ℃ with 10 ℃/min, keep 2min; Injector temperature: 250 ℃; Carrier gas (He) flow 1.0mL/min; Sample size: 1 μ L; Ionization mode: EI; Ionizing energy: 70eV; Ion source temperature: 230 ℃; Quadrupole temperature: 150 ℃; Quality of scanning scope 30～280amu; Sweep velocity: 1.0s/scan.
Degraded product of the present invention is carried out mass spectroscopy, and its mass spectrum analyzes as can be known that this metabolite is respectively O as shown in Figure 5 and Figure 6, O-dimethyl O-(p-aminophenyl) phosphorothionate (amino methyl thiophos, C
8H
12NO
3PS), molecular weight is 233, and characteristic peak is 233,124,108; And p-aminophenol, molecular weight is 109, characteristic peak is 109,80,53.
When with the method for prior art parathion-methyl being degraded, generally obtaining meta-bolites is p-NP, and it is displaing yellow in neutrality or alkaline aqueous solution.And in the present invention, in degradation process, never see the xanchromatic phenomenon, and with the inorganic salt solution adjust pH 7.5 after cultivating, UV scanning OD
410(p-NP characteristic peak) do not have absorption peak to occur yet.As seen, DM-1 bacterium of the present invention is an approach that is different from conventional catabolism fully to the common pathways metabolism of parathion-methyl.
When using parathion-methyl degradation bacteria of the present invention that parathion-methyl is degraded, it is that 7 inorganic salt nutrient solution is inoculated parathion-methyl degradation bacteria of the present invention by 10% inoculum size that use contains 5g/L glucose pH value, at 30 ℃ of parathion-methyls to 50～500ppm good degradation effect is arranged.
Description of drawings
Fig. 1 is that glucose concn is to the degradation rate of parathion-methyl and the influence curve of strain growth;
Fig. 2 is that initial pH value is to the degradation rate of parathion-methyl and the influence curve of strain growth;
Fig. 3 is that concentration of substrate is to the degradation rate of parathion-methyl and the influence curve of strain growth;
Fig. 4 is the degradation curve of MP and the growth curve of DM-1;
Fig. 5 is the mass spectrum of the degraded product I amino methyl thiophos of DM-1 degraded parathion-methyl;
Fig. 6 is the mass spectrum of the degraded product II amino methyl thiophos of DM-1 degraded parathion-methyl.
Embodiment
From the soil that is subjected to pesticidal contamination for a long time that Tianjin insecticide factory gathers on every side, take a sample, the pesticidal contamination soil sample of gathering is mixed with the suspension of 20wt% with the inorganic salt nutrient solution, receive in the inorganic salt nutrient solution that contains the 50mg/L parathion-methyl shaking culture then with the inoculum size of 5wt%; Go down to posterity and cultivated for 6 generations, the concentration that improves parathion-methyl in the inorganic salt nutrient solution gradually is to 600ppm; Take out 1mL bacterium liquid from last nutrient solution, after the dilution, coat on the inorganic salt solid medium that contains the 500ppm parathion-methyl, 30 ℃ of thermostat containers are cultivated; Choose growth fast (transparent circle is arranged on the flat board), the bacterium colony rule obtains pure bacterial strain behind the stable bacterium colony that goes down to posterity, purifying 2～3 times; With isolated 5 strain bacterium as sieve again bacterial strain in the LB substratum enlarged culturing to identical OD value 1.0, inoculum size with 10% inserts and contains in the minimal medium of 100ppm parathion-methyl, in 30 ℃, the 180rpm shaking table is cultivated 48h, use the gas Chromatographic Determination degradation rate, finishing screen is chosen the highest strain bacterium of degradation rate, changes the beef extract-peptone inclined-plane over to and preserves.This parathion-methyl degradation bacteria is being preserved in " China Committee for Culture Collection of Microorganisms common micro-organisms center " (No. 13, North No.1 Row, Zhongguancun, Haidian District, Beijing City on February 17th, 2006, Institute of Microorganism, Academia Sinica), its preserving number is CGMCCNo.1620, its classification called after subtilis Bacillus subtilis..
Described inorganic salt nutrient solution is prepared in following ratio: in 1000ml water, and 0.5g NaCl, 0.2gMgSO
4, 0.5g (NH
4)
2SO
4, 1.5g K
2HPO
4, 0.5g KH
2PO
4, 0.05g CaCl
2, 0.02g FeSO
4, 0.002g yeast powder, 7.0,121 ℃ of autoclaving 20min of pH value;
Described inorganic salt solid medium is the agar that above-mentioned inorganic salt nutrient solution is added 1.5～2% (w/v) that account for inorganic salt nutrient solution gross weight.
Described LB substratum and beef extract-peptone and document 2: microbiology experiment [M], Beijing: Higher Education Publishing House, 1988, identical among the 75-78.
The testing conditions of used gas-chromatography is: HP5890II type gas chromatograph, electron capture detector; Chromatographic column: low-pole capillary column HP-1[100% polydimethylsiloxane, Polydimethylsiloxane 25m * 0.32mm (I.D.) * 0.25 μ m]; Service temperature: 300 ℃ of detector temperatures, 300 ℃ of injector temperatures, 210 ℃ of column temperatures; Post is pressed 6.9psi; Post flow 0.748ml/min; Minimum detectable activity: 0.05 μ g/ml; The about 7.0min of standard specimen concentration 0.5ppm parathion-methyl appearance time.
Peak area, standard specimen peak area and sample size calculate the content of parathion-methyl in the nutrient solution per sample.Calculation formula is:
In the formula: A
X--treat the test sample peak area; A
0--the standard peak area;
A--standard concentration;
V
0--the standard sample size; V
X--unknown sample sample size.
The degradation rate calculation formula is:
The parathion-methyl degradation bacteria of embodiment 2, evaluation embodiment 1
(Biolog.Inc., USA) bacterial strain that embodiment 1 is obtained is identified to adopt microorganism computer classification identification systems.The bacterial strain of purifying is cultivated 16～24h on the Biolog substratum; Adopt the main method to prepare bacteria suspension, adjust turbidity to 52%T with GN/GP-IF; Bacteria suspension is inoculated in the GN-Microplate identification plate with every hole 150 μ l, and identification plate is cultivated 16～24h in 30 ℃; With Reader scanning identification plate, measure the light absorption value in each hole, the result of mensuration and database comparison provide qualification result automatically by software.Through this bacterial strain of Biolog technical evaluation is subtilis (Bacillus subtilis), called after parathion-methyl degradation bacteria DM-1.Qualification result sees Table 1.
Table 1, parathion-methyl degradation bacteria strain identification result
| NAME | PROB | SIM | DIST | |
|
| 1 | |
100 | 0.68 | 4.84 | GP- |
| 2 | |
0 | 0.00 | 7.70 | GP- |
| 3 | |
0 | 0.00 | 10.05 | GP-ROD SB |
With the isolating parathion-methyl degradation bacteria DM-1 bacterial strain of institute enlarged culturing OD value about 1.5 in LB substratum (ditto) among the embodiment 1, wash with sterilized water centrifugal back, be mixed with bacterium suspension, inoculum size with 10% is linked in the inorganic salt nutrient solution (ditto) that contains parathion-methyl 200ppm, (concentration of glucose is respectively 0g/L to add the inorganic salt liquid nutrient solution of glucose of different concns respectively, 1g/L, 2g/L, 5g/L, 10g/L, 20g/L, the pH value is 7.0) in, and be contrast not connect bacterium, each sample establish two parallel.30 ℃, 180r/min shaking table shaking culture 32h.Get nutrient solution 10ml, use equal volume of ethyl acetate 3 times, merge No. three times extracting solution, anhydrous sodium sulfate drying, gas Chromatographic Determination (testing conditions is as previously mentioned) is carried out in dilution, and ultraviolet spectrophotometer is measured OD
600
The result as shown in Figure 1, as can be known, during no glucose (concentration of glucose is 0g/L), parathion-methyl is degraded hardly; Under the condition of glucose as additional carbon, DM-1 has degradation capability preferably to MP, can the efficient degradation parathion-methyl.When glucose concn was on the low side, the growth of bacterial strain was relatively poor, and is also not good to the degradation effect of agricultural chemicals; Glucose concn is that the DM-1 bacterial strain all has good degradation effect in 2～20g/L scope.
Infer that thus DM-1 is that it needs environment that elementary carbon source is provided by the MP that degrades of metabolism altogether in degradation process.By last figure as can be known, whether glucose exists the degraded to MP to play decisive role, and its concentration height also has certain influence to degradation rate.The present invention shows that the common metabolic degradation process of toxic compounds is a special power consumption process, and the defeated people of energy is necessary in the external environment.Some poisonous difficult degradation compound of occurring in nature can not directly be degraded by microorganism, but adds a small amount of substrate that is easily utilized by microorganism such as glucose etc., can promote or start the degraded of difficult degradation compound, and it is led people's circulation.
Embodiment 4, initial pH value influence the growth and the degraded of parathion-methyl degradation bacteria DM-1 bacterial strain of the present invention
With 1 isolating parathion-methyl degradation bacteria DM-1 bacterial strain of embodiment in LB substratum (ditto) enlarged culturing to OD value about 1.5, wash with sterilized water centrifugal back, be mixed with bacterium suspension, insert with 10% inoculum size and contain in the inorganic salt nutrient solution (ditto) of 200ppm parathion-methyl, the initial pH value of nutrient solution is adjusted to 6.0 respectively, 6.5,7.0,8.0,9.0, and be contrast not connect bacterium, each sample establish two parallel.30 ℃, the 180r/min shaking table is cultivated 32h.Get nutrient solution 10ml, use equal volume of ethyl acetate 3 times, merge No. three times extracting solution, anhydrous sodium sulfate drying, gas Chromatographic Determination (testing conditions is as previously mentioned) is carried out in dilution, and ultraviolet spectrophotometer is measured OD
600
The result as shown in Figure 2, as can be known, in the scope of pH value 6.0～9.0, DM-1 strain growth and degradation effect are all better, wherein for the pH value is that 7.0 o'clock degradation rate is the highest, the increment maximum.
With 1 isolating parathion-methyl degradation bacteria DM-1 bacterial strain of embodiment in LB substratum (ditto) enlarged culturing to OD value about 1.5, wash with sterilized water centrifugal back, be mixed with bacterium suspension, the inoculum size with 10% inserts and contains 50ppm, 100ppm, 200ppm, 300ppm, 400ppm, 500ppm parathion-methyl, pH value are 7.0, glucose concn is in the inorganic salt nutrient solution (ditto) of 5g/L, and be contrast not connect bacterium, each sample establish two parallel.30 ℃, the 180r/min shaking table is cultivated 32h.Get nutrient solution 10ml, use equal volume of ethyl acetate 3 times, merge No. three times extracting solution, anhydrous sodium sulfate drying, gas Chromatographic Determination (testing conditions is as previously mentioned) is carried out in dilution, and ultraviolet spectrophotometer is measured OD
600
The result as shown in Figure 3, as can be known, strain growth and degradation effect were all better when substrate (parathion-methyl) concentration was 50～500ppm, degradation rate descended to some extent after concentration of substrate was higher than 300ppm, show that bacterial strain is subjected to the concentration affects of parathion-methyl to the degraded of parathion-methyl, the concentration of parathion-methyl surpasses finite concentration has restraining effect to the metabolism of bacterial strain.
With 1 isolating parathion-methyl degradation bacteria DM-1 bacterial strain of embodiment in LB substratum (ditto) enlarged culturing to OD value about 1.5, wash with sterilized water centrifugal back, be mixed with bacterium suspension, insert with 10% inoculum size and to contain that 200ppm parathion-methyl, pH value are 7.0, glucose concn is in the inorganic salt nutrient solution (ditto) of 5g/L, and be contrast not connect bacterium, each sample establish two parallel.30 ℃, the 180r/min shaking table is cultivated.Respectively at 3h, 6h, 12h, 20h, 28h, 38h get nutrient solution 10ml, with equal volume of ethyl acetate 3 times, merge No. three times extracting solution, anhydrous sodium sulfate drying, gas Chromatographic Determination (testing conditions is as previously mentioned) is carried out in dilution, ultraviolet spectrophotometer mensuration OD
600
The result as shown in Figure 4, as can be known, bacterial strain is through after the cultivation of 12h, the speed of growth begins to accelerate, and enters the growth platform phase behind the 28h, and after this slow decline arranged.The concentration of parathion-methyl reduces along with the growth of bacterial strain, in 0～28h degraded very fast, behind the 28h since pesticide concentration seldom, degradation rate descends to some extent.As seen the common metabolic capacity of bacterial strain is closely related with the increment of bacterial strain, along with OD
600The increase of value, the concentration of parathion-methyl reduces gradually, is almost transformed fully behind 28h.
The GC-MS testing conditions is: adopt the Agilent6890N-5973N-MSDGC-MS analyser, chromatographic column: DB-5MS[60m * 0.25mm (I.D.) * 0.25 μ m] capillary column.Operational condition---chromatogram column temperature: temperature programming, 60 ℃ of beginnings keep 1min, rise to 120 ℃ with 20 ℃/min, keep 2min, rise to 280 ℃ with 10 ℃/min, keep 2min; Injector temperature: 250 ℃; Carrier gas (He) flow 1.0mL/min; Sample size: 1 μ L; Ionization mode: EI; Ionizing energy: 70eV; Ion source temperature: 230 ℃; Quadrupole temperature: 150 ℃; Quality of scanning scope 30～280amu; Sweep velocity: 1.0s/scan.
Degraded product of the present invention is carried out mass spectroscopy, and its mass spectrum analyzes as can be known that this metabolite is respectively O as shown in Figure 5 and Figure 6, O-dimethyl O-(p-aminophenyl) phosphorothionate (amino methyl thiophos, C
8H
12NO
3PS), molecular weight is 233, and characteristic peak is 233,124,108; And p-aminophenol, molecular weight is 109, characteristic peak is 109,80,53.
Claims (6)
1, a kind of parathion-methyl degradation bacteria DM-1, this parathion-methyl degradation bacteria is being preserved in " China Committee for Culture Collection of Microorganisms common micro-organisms center " on February 17th, 2006, its preserving number is CGMCCNo.1620, its classification called after subtilis Bacillus subtilis..
2, parathion-methyl degradation bacteria as claimed in claim 1, be from the soil that is subjected to pesticidal contamination for a long time, to separate and screening, concrete steps are as follows: the suspension that the pesticidal contamination soil sample of gathering is mixed with 20wt% with the inorganic salt nutrient solution, receive in the inorganic salt nutrient solution that contains the 50mg/L parathion-methyl shaking culture then with the inoculum size of 5wt%; Go down to posterity and cultivated for 6 generations, the concentration that improves parathion-methyl in the inorganic salt nutrient solution gradually is to 600ppm; Take out 1mL bacterium liquid from last nutrient solution, after the dilution, coat on the inorganic salt solid medium that contains the 500ppm parathion-methyl, 30 ℃ of thermostat containers are cultivated; It is fast to choose growth, and the bacterium colony rule obtains pure bacterial strain behind the stable bacterium colony that goes down to posterity, purifying 2～3 times; With isolated 5 strain bacterium as sieve again bacterial strain in the LB substratum enlarged culturing to identical OD value 1.0, inoculum size with 10% inserts and contains in the minimal medium of 100ppm parathion-methyl, in 30 ℃, the 180rpm shaking table is cultivated 48h, use the gas Chromatographic Determination degradation rate, finishing screen is chosen the highest strain bacterium of degradation rate, changes the beef extract-peptone inclined-plane over to and preserves.
3, parathion-methyl degradation bacteria as claimed in claim 2 is characterized in that: described inorganic salt nutrient solution is prepared in following ratio: in 1000ml water, and 0.5g NaCl, 0.2g MgSO
4, 0.5g (NH
4)
2SO
4, 1.5gK
2HPO
4, 0.5g KH
2PO
4, 0.05g CaCl
2, 0.02g FeSO
4, 0.002g yeast powder, 7.0,121 ℃ of autoclaving 20min of pH value.
4, parathion-methyl degradation bacteria as claimed in claim 2 is characterized in that: described inorganic salt solid medium is the agar that above-mentioned inorganic salt nutrient solution is added 1.5～2% (w/v) that account for inorganic salt nutrient solution gross weight.
5, the application of the described parathion-methyl degradation bacteria of a kind of claim 1 in the degraded parathion-methyl.
6, the application of parathion-methyl degradation bacteria as claimed in claim 5 in the degraded parathion-methyl, it is characterized in that: use that to contain 5g/L glucose pH value be that 7 inorganic salt nutrient solution is inoculated parathion-methyl degradation bacteria of the present invention by 10% inoculum size, degrade at 30 ℃ of parathion-methyls to 50～500ppm.
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