CN110591941A - Bacillus subtilis with efficient degradation effect on organic phosphorus and preparation method thereof - Google Patents
Bacillus subtilis with efficient degradation effect on organic phosphorus and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a bacillus subtilis with high-efficiency degradation function on organic phosphorus and a preparation method thereof, the bacillus subtilis is bred by ion beam irradiation mutagenesis of bacillus with organic phosphorus degradation capability, is preserved in the Ministry of Gansu province of China center for industrial microorganism preservation and management, and has the preservation date as follows: and 6, 20 days in 2018, the preservation number is as follows: GSMSC 30266. The preparation process of the bacillus subtilis comprises the following steps: culturing and selecting a bacillus with strong degradation capability from bacillus with organic phosphorus degradation capability, and carrying out bacterial liquid treatment on the bacillus12C6+And (3) performing ion beam injection mutagenesis to obtain a mutant strain, and culturing, primarily screening and re-screening the mutant strain to obtain the required bacillus subtilis. The obtained bacillus subtilis can be used for degrading organophosphorus pesticide, has the advantages of high degradation speed and high efficiency, and particularly has good degradation capability on phoxim.
Description
Technical Field
The invention relates to the field of agricultural biotechnology, in particular to bacillus subtilis with efficient degradation effect on organic phosphorus and a preparation method thereof.
Background
The organophosphorus pesticide is a pesticide product widely produced and used at home and abroad, and is one of the important measures for ensuring high and stable agricultural yield at present. However, organophosphorus pesticides have mutagenicity and teratogenicity, and diseases of nervous system and immune system of mammals are mostly related to organophosphorus pesticides, and the diseases comprise mad cow disease, gulf war syndrome, Parkinson syndrome and the like. The organophosphorus pesticide is accumulated and migrated in soil, so that pesticide residue in agricultural products exceeds the standard, thereby causing the problem of food safety and restricting the export of agricultural products in China.
Microbial degradation of pesticides refers to the process of changing the chemical and physical properties of pesticides by changing the structure of the pesticides under the action of microorganisms by degrading the pesticides from macromolecular compounds to small molecular compounds and finally to H2O and CO2And the harmless degradation to the environment is realized. Compared with the conventional chemical (strong alkali hydrolysis and the like) and physical (light degradation and the like) degradation methods, the method has low efficiency, and the hydrolysis product is also an environmental pollutant, the method for degrading the organophosphorus pesticide by using the microorganisms has the advantages of low cost, small environmental influence, capability of reducing the pollutant concentration to the maximum extent and the like.
Disclosure of Invention
The invention aims to provide a bacillus subtilis with high-efficiency degradation function on organic phosphorus and a preparation method thereof.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
the bacillus subtilis is bred after ion beam irradiation mutagenesis of bacillus with the capability of degrading organic phosphorus, is rod-shaped, has the cell size of 1-1.5 multiplied by 1.5-4 microns, is not encapsulated and has spores; the bacillus subtilis is determined by 16S rDNA gene sequence analysis and homology comparison.
Preferably, the bacillus subtilis is preserved in the Ministry of Gansu of China center for the preservation and management of industrial microorganisms, and the preservation date is as follows: and 6, 20 days in 2018, the preservation number is as follows: GSMSC 30266.
The preparation method of the bacillus subtilis with the efficient degradation effect on organic phosphorus comprises the following steps:
step one, screening of starting strains: obtaining a plurality of bacilli with the capability of degrading organic phosphorus from Gansu center of China Industrial microorganism culture Collection, respectively coating the bacilli on Monkina organic phosphorus solid culture medium plates, culturing for 48h at 30 ℃, observing the size of a transparent ring around a bacterial colony, and selecting a strain LA with a larger transparent ring as a starting strain;
step two,12C6+Ion beam implantation mutagenesis: culturing the bacillus original strain screened in the step one in a liquid culture medium to logarithmic phase, sucking 1.5ml of bacterial liquid into a disposable culture dish, and mounting the culture dish on an irradiation disc;12C6+the ion beam injection LET is 60KeV, and the irradiation doses are respectively 50Gy, 80Gy, 120Gy, 150Gy, 210Gy and 270 Gy; calculating the survival rate of the bacterial strains in the bacterial liquid under each dosage, and drawing a survival rate curve;
step three, primary screening of mutant strains: comparing different irradiation doses12C6+Diluting the bacterial liquid subjected to ion beam mutagenesis treatment to 10% concentration-5、10-6、10-7Three gradients are adopted, 0.1mL of bacterial liquids with different concentrations are respectively sucked and added to a Monkina organophosphorus solid culture medium plate to be uniformly coated, inverted and cultured for 72 hours; simultaneously adding the strain LA which is not subjected to ion beam irradiation mutagenesis into the Monkina organophosphorus solid culture medium to be used as a control group, and selecting a transparent ring larger than the control groupThe bacterial colony is purely cultured and then is preserved for rescreening;
step four, re-screening mutant strains: taking lecithin as an organic phosphorus source, transferring the strains primarily screened in the third step into an inorganic salt liquid culture medium, culturing at the temperature of 30 ℃ and at the rotating speed of 200r/min for 72h, centrifuging, adding a proper amount of phosphorus-free activated carbon into 10mL of fermentation liquor to decolorize, at the rotating speed of 12000r/min, centrifuging at the temperature of 4 ℃ for 20min, taking 5mL of supernatant, measuring the available phosphorus in each bottle of mutant strains by adopting a molybdenum-antimony-scandium colorimetric method, measuring the wavelength of a spectrophotometer at 680nm, and selecting 1 strain of bacillus mutant strain LA-1 capable of efficiently degrading the organic phosphorus by taking the amount of the organic phosphorus converted into the available phosphorus as the basis of re-screening;
step five, identification of the strain: and D, identifying the bacillus mutant strain LA-1 screened out in the step four:
(1) morphological feature identification: observed under a microscope, the bacillus mutant strain LA-1 has straight rod-shaped cells with the cell size of 1-1.5 multiplied by 1.5-4 microns, is arranged in a chain shape, has no capsule, has spores and can move; the growth is rapid on a beef extract peptone culture medium, the surface of a bacterial colony is rough, opaque and light yellow, and the surface of the bacterial colony is wrinkled;
(2) 16S rDNA sequence-based identification: taking the genome DNA of the bacillus mutant strain LA-1 as a template, taking 27F and 1492R as primers to perform PCR amplification on 16S rDNA, performing gel electrophoresis on the obtained PCR amplification product, and sending the product to Shanghai biological engineering company Limited for sequencing to obtain a 16S rDNA sequence; homology comparison is carried out on the obtained 16S rDNA sequence in Genbank, and the 16S rDNA homology of the strain and the bacillus subtilis reaches 100 percent, which indicates that the bacillus mutant strain LA-1 belongs to the bacillus subtilis.
Preferably, in the step one, the content of each component in the Monkina organophosphorus solid medium is as follows: glucose 10g, (NH)4)2SO4 0.5g，NaCl 3g，KCl 0.3g，FeSO47H2O 0.03g，MnSO4H2O 0.03g，MgSO47H2O 0.3g，CaCO35g, 0.4g of yeast extract, 1000mL of distilled water and the pH value of the Monkina organic phosphorus solid culture medium is 7.0-7.5.
Preferably, the preparation method of the Monkina organophosphorus solid medium comprises the following steps:
(1) sterilizing the shell of fresh egg, cutting two ends of egg with dissecting knife, removing egg white, flowing yolk into sterilized conical flask, adding sterile water equal to yolk, and shaking to obtain yolk liquid diluent;
(2) adding 2g of agar powder into 100mL of Monkina culture medium, packaging, sterilizing, immediately adding egg yolk diluent after the sterilized Monkina culture medium is cooled to 50 ℃, adding 1mL of egg yolk diluent into 100mL of Monkina culture medium as an organic phosphorus source, uniformly mixing, and subpackaging in a culture dish to obtain the Monkina organic phosphorus solid culture medium.
Preferably, the content of each component in the inorganic salt culture medium in the step four is as follows: NaCl 0.2g, MgSO4 0.5g,K2SO4 0.5g,CaCO3 0.2g,FeSO47H2O0.001 g and distilled water 1000 mL.
Preferably, the PCR reaction system of 16S rDNA described in the fifth step is 50 μ L: 10 × PCR Buffer (Mg)2+)5 mu L of the solution; dNTP mix (2.5mM) 5. mu.L; rTaq DNA polymerase (0.5U/. mu.L) 1. mu.L, 27F (10. mu. mol/L) 1. mu.L, 1492R (10. mu. mol/L) 1. mu.L; 50ng of template genome DNA; ddH2O to 50 μ L; the reaction conditions of PCR are 95 ℃ for 5min, 94 ℃ for 45s, 60 ℃ for 45s and 72 ℃ for 90s, and 35 cycles; 10min at 72 ℃.
The ion beam irradiation treatment of the microbial strains has shown great advantages and advancement in providing new fermentation strains with high conversion rate for the food industry and the pharmaceutical industry and realizing industrialization, and initiates a new situation of microbial breeding application. Ion beam irradiation has a unique position of importance in life science research because it has advantages that conventional radiation sources do not have: the method has the advantages of large energy transmission linear density (LET), high Relative Biological Efficiency (RBE), small repairing effect after damage, good spatial resolution of energy deposition, small oxygen effect, small difference of cell sensitivity to heavy ion beams in different time phases and the like. With the continuous development of the research and application of microorganisms, the ion beam irradiation technology will promote the accelerated development of the microorganism industry.
The effect of degrading organophosphorus pesticides phoxim and omethoate by the bacillus subtilis LA-1 bred by ion beam irradiation mutagenesis is verified: adding phoxim (manufacturer: Anhui Kangda chemical industry Co., Ltd.) with 40% of effective constituent and omethoate (manufacturer: Chongqing pesticide chemical industry Co., Ltd.) with 40% of effective constituent into inorganic salt liquid culture medium respectively, making the addition amount of the inorganic salt liquid culture medium to be 800 mug/mL, carrying out shake culture for 72h under the conditions that the temperature is 30 ℃ and the rotating speed is 200r/min, and then determining the content of organophosphorus pesticide in the fermentation liquor by adopting a high performance liquid chromatography. After the bacillus subtilis LA-1 is cultured in an inorganic salt culture medium, the contents of organophosphorus pesticides phoxim and omethoate are respectively 52.4 mu g/mL and 243 mu g/mL, and the degradation rates of mutant strains LA-1 to phoxim and omethoate are respectively 93.5 percent and 69.6 percent.
The bacillus subtilis LA-1 bred by the method can be used for degrading organophosphorus pesticide, has the advantages of high degradation speed and high efficiency, and particularly has remarkable degradation capability on phoxim.
Drawings
FIG. 1 is a schematic representation of the effect of irradiation dose on the survival of mutant strains;
FIG. 2 is a diagram of the determination of the content of organophosphorus pesticide phoxim in an inorganic salt culture medium by high performance liquid chromatography;
FIG. 3 is a diagram of the determination of the content of the organophosphorus pesticide omethoate in the inorganic salt culture medium by high performance liquid chromatography.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings.
The bacillus subtilis is bred after ion beam irradiation mutagenesis of bacillus with the capability of degrading organic phosphorus, is rod-shaped, has the cell size of 1-1.5 multiplied by 1.5-4 microns, has no capsule and spores, and is determined to be the bacillus subtilis through 16S rDNA gene sequence analysis and homology comparison. The bacillus subtilis is preserved in the Gansu center of China center for Industrial microorganism preservation and management, and the preservation date is as follows: and 6, 20 days in 2018, the preservation number is as follows: GSMSC 30266.
The preparation method of the bacillus subtilis with the efficient degradation effect on organic phosphorus comprises the following steps:
step one, screening of starting strains: obtaining a plurality of bacilli with the capability of degrading organic phosphorus from Gansu center of China Industrial microorganism culture Collection, respectively coating the bacilli on a plurality of Monkina organic phosphorus solid culture medium plates, culturing for 48h at 30 ℃, observing the size of a transparent ring around a bacterial colony, and selecting a strain LA with a larger transparent ring as a starting strain;
step two,12C6+Ion beam implantation mutagenesis: culturing the bacillus original strain screened in the step one in a liquid culture medium to logarithmic phase, sucking 1.5ml of bacterial liquid into a disposable culture dish, and mounting the culture dish on an irradiation disc;12C6+the ion beam injection LET is 60KeV, and the irradiation doses are respectively 50Gy, 80Gy, 120Gy, 150Gy, 210Gy and 270 Gy; calculating the survival rate of the bacterial strains in the bacterial liquid under each dosage, and drawing a survival rate curve;
step three, primary screening of mutant strains: comparing different irradiation doses12C6+Diluting the bacterial liquid subjected to ion beam mutagenesis treatment to 10% concentration-5、10-6、10-7Three gradients are adopted, 0.1mL of bacterial liquids with different concentrations are respectively sucked and added to a Monkina organophosphorus solid culture medium plate to be uniformly coated, inverted and cultured for 72 hours; simultaneously adding the strain LA which is not subjected to ion beam irradiation mutagenesis into a Monkina organophosphorus solid culture medium to serve as a control group, selecting a bacterial colony of which the transparent ring is larger than the control group, and preserving and reserving for re-screening after pure culture;
step four, re-screening mutant strains: taking lecithin as an organic phosphorus source, transferring the strains primarily screened in the third step into an inorganic salt liquid culture medium, culturing at the temperature of 30 ℃ and at the rotating speed of 200r/min for 72h, centrifuging, adding a proper amount of phosphorus-free activated carbon into 10mL of fermentation liquor to decolorize, at the rotating speed of 12000r/min, centrifuging at the temperature of 4 ℃ for 20min, taking 5mL of supernatant, measuring the available phosphorus in each bottle of mutant strains by adopting a molybdenum-antimony-scandium colorimetric method, measuring the wavelength of a spectrophotometer at 680nm, and selecting 1 strain of bacillus mutant strain LA-1 capable of efficiently degrading the organic phosphorus by taking the amount of the organic phosphorus converted into the available phosphorus as the basis of re-screening;
step five, identification of the strain: and D, identifying the bacillus mutant strain LA-1 screened out in the step four:
(1) morphological feature identification: observed under a microscope, the bacillus mutant strain LA-1 has straight rod-shaped cells with the cell size of 1-1.5 multiplied by 1.5-4 microns, is arranged in a chain shape, has no capsule, has spores and can move; the growth is rapid on a beef extract peptone culture medium, the surface of a bacterial colony is rough, opaque and light yellow, and the surface of the bacterial colony is wrinkled;
(2) 16S rDNA sequence-based identification: taking the genome DNA of the bacillus mutant strain LA-1 as a template, taking 27F and 1492R as primers to perform PCR amplification on 16S rDNA, performing gel electrophoresis on the obtained PCR amplification product, and sending the product to Shanghai biological engineering company Limited for sequencing to obtain a 16S rDNA sequence; homology comparison is carried out on the obtained 16S rDNA sequence in Genbank, and the 16S rDNA homology of the strain and the bacillus subtilis reaches 100 percent, which indicates that the bacillus mutant strain LA-1 belongs to the bacillus subtilis.
The content of each component in the Monkina organophosphorus solid culture medium in the step one is as follows: glucose 10g, (NH)4)2SO40.5g，NaCl 3g，KCl 0.3g，FeSO47H2O 0.03g，MnSO4H2O 0.03g，MgSO47H2O 0.3g，CaCO35g, 0.4g of yeast extract, 1000mL of distilled water and the pH value of the Monkina organic phosphorus solid culture medium is 7.0-7.5.
The preparation method of the Monkina organophosphorus solid medium comprises the following steps:
(1) sterilizing the shell of fresh egg, cutting two ends of egg with dissecting knife, removing egg white, flowing yolk into sterilized conical flask, adding sterile water equal to yolk, and shaking to obtain yolk liquid diluent;
(2) adding 2g of agar powder into 100mL of Monkina culture medium, packaging, sterilizing, immediately adding egg yolk diluent after the sterilized Monkina culture medium is cooled to 50 ℃, adding 1mL of egg yolk diluent into 100mL of Monkina culture medium as an organic phosphorus source, uniformly mixing, and subpackaging in a culture dish to obtain the Monkina organic phosphorus solid culture medium.
The inorganic salt culture medium comprises the following components in percentage by weight: NaCl 0.2g, MgSO4 0.5g,K2SO40.5g,CaCO3 0.2g,FeSO47H2O0.001 g and distilled water 1000 mL.
The PCR reaction system of the 16S rDNA in the step five is 50 mu L: 10 × PCR Buffer (Mg)2+)5 mu L of the solution; dNTP mix (2.5mM) 5. mu.L; rTaq DNA polymerase (0.5U/. mu.L) 1. mu.L, 27F (10. mu. mol/L) 1. mu.L, 1492R (10. mu. mol/L) 1. mu.L; 50ng of template genome DNA; ddH2O to 50 μ L; the reaction conditions of PCR are 94 ℃ for 5min, 94 ℃ for 45s, 60 ℃ for 45s and 72 ℃ for 90s, and 35 cycles; 10min at 72 ℃.
The bacillus subtilis LA-1 prepared by the method provided by the invention is used for testing the degradation effect of organophosphorus pesticide phoxim:
adding phoxim with 40% of active ingredient content into an inorganic salt culture medium to ensure that the addition amount of the phoxim in the culture medium is 800 mug/mL, inoculating bacillus subtilis LA-1 according to 1% of inoculation amount, placing the bacillus subtilis LA-1 at the temperature of 30 ℃ and the rotation speed of 200r/min for shaking culture for 72h, centrifuging at 12000r/min for 10min, taking supernatant fluid to perform high performance liquid chromatography to detect the residual quantity of the phoxim, the organophosphorus pesticide:
(1) preparation of standard working fluid
The standard substance of phoxim was precisely weighed and dissolved in methanol-water (7:3, V/V) to prepare a 1mg/L stock solution of the standard substance, and then diluted to standard working solutions having concentrations of 0.5. mu.g/mL, 1. mu.g/mL, 2. mu.g/mL, 5. mu.g/mL, 10. mu.g/mL, and 20. mu.g/mL.
(2) Sample processing
Absorbing 1mL of fermentation liquor of phosphate-solubilizing bacteria, placing the fermentation liquor in a test tube with a plug scale, adding 5mL of dichloromethane, violently shaking, standing and separating, dehydrating a lower-layer organic phase by using anhydrous sodium sulfate, adding 1mL of the dehydrated organic phase into a centrifuge tube, blow-drying by using nitrogen, metering the volume to 1mL by using a methanol solution, and filtering by using a 0.22-micrometer filter membrane to be detected.
(3) Chromatographic conditions
A chromatographic column: 1.0X 150mm, 3.5 μm SB-C18 column, mobile phase: methanol-water (7:3, V/V), flow rate: 0.4mL/min, sample size: 10 μ L, column temperature: 30 ℃, detection wavelength: 254 nm. The chromatogram of the organophosphorus pesticide phoxim is shown in figure 2, the phoxim content is 52.4 mu g/mL after the culture in the inorganic salt culture medium, and the degradation rate of the bacillus subtilis LA-1 is 93.5%.
The bacillus subtilis LA-1 is used for testing the degradation effect of the organophosphorus pesticide omethoate:
adding omethoate with the effective component content of 40% into an inorganic salt culture medium, leading the adding amount of the omethoate in the culture medium to be 800 mug/mL, inoculating bacillus subtilis LA-1 according to the inoculation amount of 1%, placing the mixture into shaking culture at the temperature of 30 ℃ and the rotating speed of 200r/min for 72h, centrifuging at 12000r/min for 10min, taking the supernatant, and carrying out high performance liquid chromatography to determine the residual quantity of the organophosphorus pesticide omethoate:
(1) preparation of standard working fluid
The omethoate standard substance is precisely weighed and dissolved in the formazan to prepare a standard substance stock solution with the concentration of 1mg/L, and then diluted into standard working solutions with the concentrations of 0.5g/mL, 1 mu g/mL, 2 mu g/mL, 5 mu g/mL, 10 mu g/mL and 20 mu g/mL.
(2) Sample processing
Sucking 5mL of fermentation liquid of bacillus subtilis LA-1 into a centrifuge tube, uniformly mixing with equal volume of ethyl acetate, violently shaking for 20min for extraction, standing for 30min until an extractant and a water phase are separated, sucking an upper organic phase, evaporating to dryness by using a rotary evaporator, adding equal volume of methanol-water (1:9, V/V) for dissolution, and filtering by using a filter membrane with the aperture of 0.22 mu m to be detected.
(3) Chromatographic conditions
A chromatographic column: 3.0X 150mm, 3.5 μm ZORBAX Eclipse Plus C18 column, mobile phase: methanol-water (9:1, V/V), flow rate: 1.0mL/min, sample size: 10 μ L, column temperature: 30 ℃, detection wavelength: 200 nm. The chromatogram of the organophosphorus pesticide omethoate is shown in figure 3, the omethoate content is 243 mug/mL after the culture in the inorganic salt culture medium, and the degradation rate of the bacillus subtilis LA-1 is 69.6%.
Experiments prove that the bacillus subtilis LA-1 bred by ion beam irradiation mutagenesis has better degradation effect on organophosphorus pesticides phoxim and omethoate, wherein the phoxim has higher degradation speed and higher degradation efficiency.
Claims (7)
1. A bacillus subtilis with high-efficiency degradation effect on organic phosphorus is characterized in that: the bacillus subtilis is prepared by cultivating bacillus with the capability of degrading organic phosphorus after ion beam radiation mutagenesis, has a rod shape, a cell size of 1-1.5 multiplied by 1.5-4 microns, no capsule and spores, and is determined to be the bacillus subtilis through 16S rDNA gene sequence analysis and homology comparison.
2. The bacillus subtilis having a high degrading effect on organic phosphorus according to claim 1, wherein: the bacillus subtilis is preserved in the Gansu center of China center for Industrial microorganism preservation and management, and the preservation date is as follows: and 6, 20 days in 2018, the preservation number is as follows: GSMSC 30266.
3. A method for preparing a bacillus subtilis with efficient degradation effect on organic phosphorus, wherein the bacillus subtilis is the bacillus subtilis as claimed in any one of claims 1 or 2, and is characterized in that: it comprises the following steps:
step one, screening of starting strains: obtaining a plurality of bacilli with the capability of degrading organic phosphorus from Gansu center of China Industrial microorganism culture Collection, respectively coating the bacilli on Monkina organic phosphorus solid culture medium plates, culturing for 48h at 30 ℃, observing the size of a transparent ring around a bacterial colony, and selecting a strain LA with a larger transparent ring as a starting strain;
step two,12C6+Ion beam implantation mutagenesis: culturing the bacillus original strain screened in the step one in a liquid culture medium to logarithmic phase, sucking 1.5ml of bacterial liquid into a disposable culture dish, and mounting the culture dish on an irradiation disc;12C6+the ion beam injection LET is 60KeV, and the irradiation doses are respectively 50Gy, 80Gy, 120Gy, 150Gy, 210Gy and 270 Gy; calculating the survival rate of the bacterial strains in the bacterial liquid under each dosage, and drawing a survival rate curve;
step three, primary screening of mutant strains: comparing different irradiation doses12C6+Diluting the bacterial liquid subjected to ion beam mutagenesis treatment to 10% concentration-5、10-6、10-7Three gradients are adopted, 0.1mL of bacterial liquids with different concentrations are respectively sucked and added to a Monkina organophosphorus solid culture medium plate to be uniformly coated, inverted and cultured for 72 hours; simultaneously adding the strain LA which is not subjected to ion beam irradiation mutagenesis into a Monkina organophosphorus solid culture medium to serve as a control group, selecting a bacterial colony of which the transparent ring is larger than the control group, and preserving and reserving for re-screening after pure culture;
step four, re-screening mutant strains: taking lecithin as an organic phosphorus source, transferring the strains primarily screened in the third step into an inorganic salt liquid culture medium, culturing at the temperature of 30 ℃ and at the rotating speed of 200r/min for 72h, centrifuging, adding a proper amount of phosphorus-free activated carbon into 10mL of fermentation liquor to decolorize, at the rotating speed of 12000r/min, centrifuging at the temperature of 4 ℃ for 20min, taking 5mL of supernatant, measuring the available phosphorus in each bottle of mutant strains by adopting a molybdenum-antimony-scandium colorimetric method, measuring the wavelength of a spectrophotometer at 680nm, and selecting 1 strain of bacillus mutant strain LA-1 capable of efficiently degrading the organic phosphorus by taking the amount of the organic phosphorus converted into the available phosphorus as the basis of re-screening;
step five, identification of the strain: and D, identifying the bacillus mutant strain LA-1 screened out in the step four:
(1) morphological feature identification: observed under a microscope, the bacillus mutant strain LA-1 has straight rod-shaped cells with the cell size of 1-1.5 multiplied by 1.5-4 microns, is arranged in a chain shape, has no capsule, has spores and can move; the growth is rapid on a beef extract peptone culture medium, the surface of a bacterial colony is rough, opaque and light yellow, and the surface of the bacterial colony is wrinkled;
(2) 16S rDNA sequence-based identification: taking the genome DNA of the bacillus mutant strain LA-1 as a template, taking 27F and 1492R as primers to perform PCR amplification on 16S rDNA, performing gel electrophoresis on the obtained PCR amplification product, and sending the product to Shanghai biological engineering company Limited for sequencing to obtain a 16S rDNA sequence; homology comparison is carried out on the obtained 16S rDNA sequence in Genbank, and the 16S rDNA homology of the strain and the bacillus subtilis reaches 100 percent, which indicates that the bacillus mutant strain LA-1 belongs to the bacillus subtilis.
4. The method for preparing Bacillus subtilis having an efficient degradation effect on organophosphorus according to claim 3, wherein: the content of each component in the Monkina organophosphorus solid culture medium in the step one is as follows: glucose 10g, (NH)4)2SO4 0.5g，NaCl 3g，KCl 0.3g，FeSO47H2O 0.03g，MnSO4H2O 0.03g，MgSO47H2O 0.3g，CaCO35g, 0.4g of yeast extract, 1000mL of distilled water and the pH value of the Monkina organic phosphorus solid culture medium is 7.0-7.5.
5. The method for preparing Bacillus subtilis having a high degrading effect on organophosphorus according to claim 3 or 4, wherein: the preparation method of the Monkina organophosphorus solid medium comprises the following steps:
(1) sterilizing the shell of fresh egg, cutting two ends of egg with dissecting knife, removing egg white, flowing yolk into sterilized conical flask, adding sterile water equal to yolk, and shaking to obtain yolk liquid diluent;
(2) adding 2g of agar powder into 100mL of Monkina culture medium, packaging, sterilizing, immediately adding egg yolk diluent after the sterilized Monkina culture medium is cooled to 50 ℃, adding 1mL of egg yolk diluent into 100mL of Monkina culture medium as an organic phosphorus source, uniformly mixing, and subpackaging in a culture dish to obtain the Monkina organic phosphorus solid culture medium.
6. The method for preparing Bacillus subtilis having an efficient degradation effect on organophosphorus according to claim 5, wherein: the inorganic salt culture medium in the step four comprises the following components in percentage by weight: NaCl 0.2g, MgSO4 0.5g,K2SO40.5g,CaCO3 0.2g,FeSO47H2O0.001 g and distilled water 1000 mL.
7. The organic phosphorus reducing agent of claim 6 with high efficiencyThe preparation method of the bacillus subtilis is characterized by comprising the following steps: the PCR reaction system of the 16S rDNA in the step five is 50 mu L: 10 × PCR Buffer (Mg)2+)5 mu L of the solution; dNTP mix (2.5mM) 5. mu.L; rTaq DNA polymerase (0.5U/. mu.L) 1. mu.L, 27F (10. mu. mol/L) 1. mu.L, 1492R (10. mu. mol/L) 1. mu.L; 50ng of template genome DNA; ddH2O to 50 μ L; the reaction conditions of PCR are 94 ℃ for 5min, 94 ℃ for 45s, 60 ℃ for 45s and 72 ℃ for 90s, and 35 cycles; 10min at 72 ℃.
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