CN113528352B - Trichoderma atroviride and application thereof in degrading chlorpyrifos - Google Patents
Trichoderma atroviride and application thereof in degrading chlorpyrifos Download PDFInfo
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- CN113528352B CN113528352B CN202110684510.5A CN202110684510A CN113528352B CN 113528352 B CN113528352 B CN 113528352B CN 202110684510 A CN202110684510 A CN 202110684510A CN 113528352 B CN113528352 B CN 113528352B
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09C—RECLAMATION OF CONTAMINATED SOIL
- B09C1/00—Reclamation of contaminated soil
- B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
- B09C1/105—Reclamation of contaminated soil microbiologically, biologically or by using enzymes using fungi or plants
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
- C02F3/347—Use of yeasts or fungi
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/30—Organic compounds
- C02F2101/306—Pesticides
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/30—Organic compounds
- C02F2101/40—Organic compounds containing sulfur
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2103/00—Nature of the water, waste water, sewage or sludge to be treated
- C02F2103/20—Nature of the water, waste water, sewage or sludge to be treated from animal husbandry
Abstract
The technical scheme of the invention discloses Trichoderma atroviride and application thereof in degrading chlorpyrifos, wherein the Trichoderma atroviride is Trichoderma atroviride (SG 3403), which is preserved in China general microbiological culture Collection center (CGMCC) No. 1 Hospital 3 of Shangyang district, beijing, in 8-28 days of 2012, and the preservation number is CGMCC No.6479. The trichoderma atroviride provided by the technical scheme of the invention can degrade chlorpyrifos in a water body, provides important trichoderma resources and application methods for biodegradation or bioremediation of chlorpyrifos in a water body around a farmland or a water body of a fishpond, and has important significance for developing a green aquaculture technology and protecting the water area environment of the farmland.
Description
Technical Field
The invention belongs to the technical field of microbial fermentation, and particularly relates to trichoderma atroviride and application thereof in chlorpyrifos degradation.
Background
The pesticide is widely applied to modern agricultural production, wherein the organophosphorus pesticide is one of the most widely used pesticides in the global scope, and plays an important role in preventing and treating crop diseases and insect pests. However, the organophosphorus pesticide has strong toxicity, and is easy to remain in crops, field soil and water after long-term use in a large amount, thereby causing the problems of food safety and environmental pollution.
Chlorpyrifos (CP) is a highly effective broad-spectrum organophosphorus insecticide, which is used in the prevention and control of various crop pests in large quantities. The most advantage of the chlorpyrifos is that the chlorpyrifos has higher control effect on underground pests of crops, and currently, no ideal pesticide capable of replacing the chlorpyrifos exists internationally. On the other hand, the pesticide has no high toxicity of pesticides such as methamidophos, omethoate and the like, has low production cost, and is still widely applied to agricultural production till now. However, the chlorpyrifos is a medium-toxicity organophosphorus pesticide in the genus of chlorpyrifos, has high acute toxicity, affects the central nervous system, the cardiovascular system and the respiratory system, can cause skin and eye allergy, and can cause residual quantity accumulation in the environment due to long-term unreasonable use, thereby causing great harm to the ecological environment.
The degradation of chemical pesticides is mainly carried out by physical methods, chemical methods and microbiological methods. The physical method mainly includes adsorption, illumination, ultrasonic wave and other methods, the chemical method mainly includes oxidant oxidation, enzyme preparation degradation and other methods, and the microbiological method mainly utilizes bacteria and fungi degradation method. The microbial degradation of pesticide residue has the advantages of low cost, no toxicity, no secondary pollution and the like, and is a bioremediation method with the most wide application prospect.
Disclosure of Invention
The technical problem to be solved by the technical scheme of the invention is to provide trichoderma atroviride for degrading chlorpyrifos in a water body.
In order to solve the technical problems, the technical scheme of the invention provides Trichoderma atroviride, which is Trichoderma atroviride (SG 3403) and has been preserved in China general microbiological culture Collection center of No. 1 Hospital No. 3 of West Lu No. 3 of the sunward area in Beijing at 8-28 days in 2012 with the preservation number of CGMCC No.6479.
The trichoderma atroviride can be applied to degrading chlorpyrifos.
Optionally, the tolerance concentration of trichoderma atroviride to chlorpyrifos is 40mg/L-50mg/L, and the spore production capacity of trichoderma atroviride is more than 60%.
Optionally, said dark greenwood is degraded when said chlorpyrifos is degradedThe mold produces paraoxonase, and the catalytic efficiency of the paraoxonase to the chlorpyrifos is 5.0 multiplied by 10 6 kcat/Kms -1 M -1 -6.05×10 6 kcat/Kms -1 M -1 The enzyme activity to the chlorpyrifos is 2.85U/mL-3.60U/mL.
The invention also provides a method for degrading chlorpyrifos by using trichoderma atroviride, which comprises the following steps: inoculating Trichoderma atroviride SG3403 strain on a potato glucose agar culture medium, and activating at 25-28 ℃; propagating spores on a potato dextrose agar medium and washing the spores to make a spore suspension; inoculating the spore suspension into a potato glucose medium to ensure that the concentration of the spores is 10 5 CFU/mL～10 6 CFU/mL; carrying out shake culture on the spores for 4 to 5 days at the rotating speed of 180 to 200rpm and the temperature of 25 to 28 ℃, and carrying out vacuum filtration, sterile water washing and mycelium pressing twice; inoculating 0.4-0.5 g of the squeezed mycelium into 100-110 mL of anhydrous water, adding chlorpyrifos, culturing for 120-125 hours at 25-28 ℃ and 180-200 rpm, keeping out of the sun, collecting 1.0-1.5 mL of liquid every 24-30 hours, carrying out solid phase microextraction-gas chromatography-mass spectrometry, and analyzing the hydrolysis dynamics of the chlorpyrifos.
Optionally, the method for preparing the potato dextrose agar medium comprises:
weighing 190-200 g of potato, peeling, cutting into small pieces, adding water, boiling for 30-35 min, filtering with four layers of gauze, adding 15-20 g of anhydrous glucose and 15-20 g of agar powder, cooling to a constant volume of 1L, and sterilizing at 120-121 ℃ for 15-30min.
Optionally, the spore suspension is inoculated in the potato glucose medium in an inoculation amount of 3% -5%.
Optionally, the method for preparing the potato glucose medium comprises: weighing 190-200 g of potato, peeling, cutting into small pieces, adding water, boiling for 30-35 min, filtering with four layers of gauze, adding 15-20 g of anhydrous glucose, cooling to constant volume of 1L, and sterilizing at 120-121 ℃ for 15-30min.
Optionally, when the trichoderma aureoviride is used for degrading chlorpyrifos, the mass concentration ratio of the trichoderma aureoviride to the chlorpyrifos is (3.7-4.0): (0.3-0.35).
According to the technical scheme, the chlorpyrifos-tolerant trichoderma atroviride SG3403 is obtained through screening, the culture of the trichoderma atroviride SG3403 has the paraoxonase activity for degrading chlorpyrifos, can be applied to remediation of organophosphorus pesticide chlorpyrifos pollution in farmland soil and natural water bodies, and the trichoderma atroviride SG3403 has strong environmental adaptability and high chlorpyrifos degrading activity and can be used for remediation of organophosphorus pesticide pollution in different ecological environments.
The method for degrading the chlorpyrifos by using the trichoderma atroviride in the technical scheme of the invention has the advantages that the degradation rate of the chlorpyrifos in the water of the fish pond can reach 36 percent within 48 hours, and the trichoderma resource and the application method are important for biodegradation or bioremediation of the chlorpyrifos in the water areas around the farmland or the water body of the fish pond, and have important significance for developing a green aquaculture technology and protecting the water area environment of the farmland.
Drawings
FIG. 1 shows the colony morphology and corresponding conidiophore morphology of Trichoderma atroviride SG3403 according to an embodiment of the present invention;
FIG. 2 is the ITS sequence of Trichoderma atroviride SG3403 according to an embodiment of the invention;
FIG. 3 is the tef1 sequence of Trichoderma atroviride SG3403 according to an embodiment of the invention;
FIG. 4 is a phylogenetic tree of Trichoderma atroviride SG3403 according to an embodiment of the invention;
FIG. 5 shows the resistance of Trichoderma atroviride SG3403 to the degradation of chlorpyrifos;
FIG. 6 shows the degradation products of Trichoderma atroviride SG3403 to chlorpyrifos in pure water according to an embodiment of the invention;
FIG. 7 shows the efficiency of Trichoderma atroviride SG3403 degrading chlorpyrifos in aquarium water according to an embodiment of the invention.
Detailed Description
In order to make those skilled in the art better understand the technical solutions in the present application, the present invention will be further described below with reference to the following embodiments, and it is obvious that the described embodiments are only a part of the embodiments of the present application, and not all embodiments. All other embodiments obtained by a person of ordinary skill in the art based on the embodiments in the present application without making any creative effort shall fall within the protection scope of the present application.
The embodiment of the invention provides Trichoderma atroviride, which is Trichoderma atroviride (SG 3403) and has been preserved in China general microbiological culture collection management committee general microbiological center of No. 3 of No. 1 Hopki West Lu-1 Kyowa sunward, beijing city at 8 and 28 days 2012, and the preservation number is CGMCC No.6479.
Referring to FIG. 1, the Trichoderma atroviride SG3403 grows rapidly on potato dextrose agar medium, and the colony radius is 5.5 cm-8 cm on MA medium after culturing at 20 ℃ for 3 days (panel A). After 72 hours of incubation at 25 ℃ the colony radius on PDA medium was 42.8mm to 60.5mm (panel B). There were no sterile fimbriae. No diffusible pigment was produced, a relatively compact disc-like structure in the center of the colony was the main sporulation area of conidia, and coconut smell was observed on PDA. Conidiophores are regularly branched in a zigzag manner. Although relatively common in-register branches, the typical branching pattern of conidiophores is unilateral branching; the branch angle is approximately 90. The length of the bottle stalk is 6.0-9.7 mu m, the length-width ratio is 1.8-3.5, and the bottle stalk is straight or bent and sometimes hooked; 2-4 are arranged in vortex form, usually single birth. Conidia are green, subsphaeroidal to oval, smooth, (3.0-3.8) × (2.8-3.5 smooth). The ITS sequence and the tef1 sequence of trichoderma atroviride SG3403 are the main characteristic basis for identifying the strain, and the ITS sequence and the tef1 sequence are respectively shown in figures 2 and 3 and T.hamatum GJS 05-334, hyperomyces pernicious sus, T.asperellum CBS 433.97, T.atroviride CBS 142.95, T.atroviride CBS 119499, T.pubescens GJS 01-207 and T.virude strain GJS 92-14 in the sequence table. A phylogenetic tree of the strains constructed based on the ITS sequences is shown in FIG. 4.
The trichoderma atroviride SG3403 provided by the embodiment of the invention has obvious tolerance and surface adsorption capacity to chlorpyrifos, and can generate paraoxonase for hydrolyzing P-O bonds of the chlorpyrifos, and the trichoderma atroviride SG3403 is a functional strain capable of adsorbing and degrading the chlorpyrifos.
The tolerant concentration of the trichoderma atroviride to the chlorpyrifos is 40mg/L-50mg/L, and the spore production capacity of the trichoderma atroviride is more than 60%. When the chlorpyrifos is degraded, the trichoderma atroviride generates paraoxonase, and the catalytic efficiency of the paraoxonase on the chlorpyrifos is 5.0 multiplied by 10 6 kcat/Kms -1 M -1 -6.05×10 6 kcat/Kms -1 M -1 The enzyme activity to the chlorpyrifos is 2.85U/mL-3.60U/mL.
Inoculating Trichoderma atroviride SG3403 strain on a potato glucose agar (PDA) culture medium, and activating at 25-28 deg.C; propagating spores on a potato dextrose agar culture medium and washing the spores to prepare a spore suspension; inoculating the spore suspension into potato glucose medium (PD) to make the concentration of the spores be 10 5 CFU/mL～10 6 CFU/mL; and carrying out shake culture on the spores for 4-5 days at the rotating speed of 180-200 rpm and the temperature of 25-28 ℃, and carrying out vacuum filtration, sterile water washing and mycelium pressing twice to obtain a mycelium culture.
The method for preparing the potato dextrose agar culture medium comprises the following steps: weighing 190-200 g of potato, peeling, cutting into small pieces, adding water, boiling for 30-35 min, filtering with four layers of gauze, adding 15-20 g of anhydrous glucose and 15-20 g of agar powder, cooling to a constant volume of 1L, and sterilizing at 120-121 ℃ for 15-30min.
Inoculating the spore suspension into the potato glucose medium in an inoculation amount of 3-5%. The preparation method of the potato glucose culture medium comprises the following steps: weighing 190-200 g of potato, peeling, cutting into small pieces, adding water, boiling for 30-35 min, filtering with four layers of gauze, adding 15-20 g of anhydrous glucose, cooling to constant volume of 1L, and sterilizing at 120-121 ℃ for 15-30min.
The embodiment of the invention also provides a method for degrading chlorpyrifos by trichoderma atroviride, which comprises the following steps:
step 1: inoculating Trichoderma atroviride SG3403 strain on a potato glucose agar culture medium, and activating at 25-28 ℃;
step 2: propagating spores on a potato dextrose agar culture medium and washing the spores to prepare a spore suspension;
and step 3: inoculating the spore suspension into a potato glucose medium to ensure that the concentration of the spores is 10 5 CFU/mL～10 6 CFU/mL；
And 4, step 4: performing shake culture on the spores for 4 to 5 days at the rotating speed of 180 to 200rpm and the temperature of 25 to 28 ℃, and performing vacuum filtration, sterile water washing and mycelium drying twice;
and 5: inoculating 0.4-0.5 g of the squeezed mycelium into 100-110 mL of anhydrous water, adding chlorpyrifos, culturing for 120-125 hours at 25-28 ℃ and 180-200 rpm, keeping out of the sun, collecting 1.0-1.5 mL of liquid every 24-30 hours, carrying out solid phase microextraction-gas chromatography-mass spectrometry, and analyzing the hydrolysis dynamics of the chlorpyrifos.
The preparation method of the potato dextrose agar medium and the potato dextrose medium is as described above. When the trichoderma aureoviride is used for degrading chlorpyrifos, the mass concentration ratio of the trichoderma aureoviride to the chlorpyrifos is (3.7-4.0): (0.3-0.35).
The method for detecting the degradation activity of trichoderma atroviride comprises the following steps:
preparation of modified Burk medium: caSO 4 ·2H 2 O 100mg，MgSO 4 ·7H 2 O 200mg，(NH 4 ) 2 SO 4 1g，MnSO 4 10mg，FeSO 4 ·7H 2 O 5mg，Na 2 MoO 4 ·2H 2 Adjusting the pH value of an improved Burk culture medium to 6.0 by using 5mol/L KOH, subpackaging the Burk culture medium with the chlorpyrifos as the only phosphorus source in a volume of 200mL/250mL triangular flask, and carrying out high-pressure moist heat sterilization at 115 ℃ for 20min, wherein the anhydrous glucose is 10 g;
the mycelium preparation (2 g of wet hyphae) was transferred to 200mL of modified Burk medium for culture, and chlorpyrifos was added to give a final concentration of 50mg/L in the modified Burk medium, and the culture was carried out at 28 ℃ and 180rpm, and the residual chlorpyrifos content was detected by gas chromatography GC/FPD. The experimental result shows that the degradation efficiency in 48 hours reaches more than 40 percent.
In some embodiments, the contaminated pond water from chlorpyrifos is treated with trichoderma atroviride. Trichoderma atroviride SG3403 degrades chlorpyrifos in fishpond water containing 50mg/L-100mg/L chlorpyrifos, the degradation rate reaches 36% -60% after 48 hours, and the residual of the chlorpyrifos in natural water can be effectively degraded.
Example 1
The method for detecting the tolerance of trichoderma atroviride SG3403 to chlorpyrifos comprises the following steps: trichoderma atroviride (T. Atroviride) SG3403 is activated on PDA at 25 ℃, a bacterial cake with the diameter of 5mm is taken by a puncher and is transferred to a PDA culture medium plate containing 50mg/L chlorpyrifos, and the colony diameter is measured every 12 hours, so that the tolerance capacity of the Trichoderma atroviride SG3403 to the chlorpyrifos is detected. FIG. 5 shows the growth of Trichoderma atroviride SG3403 on PDA culture medium (control) containing chlorpyrifos (50 mg/L) and not containing chlorpyrifos, the colony diameter on the culture medium containing chlorpyrifos at 6 days after culture is 7.50cm, the colony diameter on the control culture medium not containing chlorpyrifos is 9.0cm, and the inhibition rate of chlorpyrifos on the growth of Trichoderma atroviride SG3403 colony is only 16.6%, therefore, the strain meets the requirement of drug resistance in application.
Example 2
The trichoderma atroviride SG3403 analyzes the activity and products of the chlorpyrifos degrading enzyme in pure water:
the Burk medium was dispensed into 200mL/250mL Erlenmeyer flasks and autoclaved at 115 ℃ for 20min. 2g of the mycelia (fresh weight) was transferred to 200mL of Burk's medium and cultured, and chlorpyrifos was added thereto so that the final concentration of chlorpyrifos was 50mg/L, and the culture was carried out at 28 ℃ and 180 rpm. Products of residual chlorpyrifos degradation for 48 hours are detected by gas chromatography GC/FPD, and mainly comprise 6-monochloro-2-pyridinol, 3,5, 6-trichloro-2-pyridinol, chlorpyrifos monooxide, 5, 6-dichloro-2-pyridinol and the like, as shown in figure 6. The activity of the extracted trichoderma atroviride paraoxonase protein on chlorpyrifos degradation in water is 5465.37U/mg.
Example 3
Degradation effect of trichoderma atroviride SG3403 on chlorpyrifos in fishpond water
1) An aquaculture pond water is a natural water body, the collection site is a Shunming culture farm (120.7 degrees E,30.8 degrees N) in Jiaxing city of Zhejiang, china, and 2 sampling points are randomly selected from three pond culture areas along opposite angles in the horizontal direction; in the vertical direction, the acquisition was performed at two depths of 0.2m and 0.7 m.
2) The culture conditions in the water environment refer to Burk culture medium, the culture container is 100mL brown penicillin bottles, each bottle contains 50mL culture medium, 50mg/L chlorpyrifos and 1% (W/V) Trichoderma atroviride culture are added, the culture is carried out at normal temperature (25 ℃), and the samples are respectively added on days 2 and 5. And identifying the extracellular degradation product of the chlorpyrifos in the trichoderma atroviride by adopting GC-MS or SPME-GC-MS. As shown in figure 3, the Trichoderma atroviride SG3403 can degrade chlorpyrifos in the water of the fish pond by 36% within 2 days and by more than 70% on the 5 th day. Compared with trichoderma reesei (t. Reesei), trichoderma atroviride SG3403 had an improvement in degradation efficiency of 27% on day 2 and 18% on day 7 (fig. 7). Although the test control strain also has a relatively degrading effect of Trichoderma harzianum (T.harzianum), the strain is not easy to be developed further due to its relatively poor spore-forming ability.
Although the present invention has been described with reference to the preferred embodiments, it is not intended to limit the present invention, and those skilled in the art can make modifications and variations of the present invention without departing from the spirit and scope of the present invention.
Sequence listing
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<120> Trichoderma atroviride and application thereof in degrading chlorpyrifos
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tgagcgtggt atcaccatcg acattgccct ctggaagttc gagactccca agtactatgt 420
caccgtcatt ggtatgtttt cgcttttcct cattgatact tggagaccaa gattctaacg 480
tgccgctctg tagacgctcc cggtcaccgt gatttcatca agaacatgat cactggtact 540
tcccaggctg actgcgctat cctgattatc gctgccggta ctggtgagtt cgaggctggt 600
atctccaagg atggccagac ccgtgagcac gctctgctcg cctacaccct gggtgtcaag 660
cagctcatcg ttgccatcaa caagatggac actgccaact gggccgaggc tcgttacctt 720
gagatcatca aggagacctc caacttcatc aagaaggtcg gcttcaaccc caagaccgtt 780
gccttcgtcc ccatctccgg cttcaacggc gacaacatgt tggctgcctc caccaactgc 840
ccctggtaca agggctggga gaaggagacc aaggctggca agtccaccgg caagaccctt 900
ctcgaggcca ttgacgccat tgagcccccc aagcgtccca cagacaagcc cctccgtctt 960
ccccttcagg atgtttacaa gatcggtggt attggaacag tccctgtcgg ccgtatcgag 1020
actggtatcc tcaagcccgg tatggtcgtt accttcgctc cctccaacgt caccactgaa 1080
gtcaagtccg ttgagatgca ccacgagcag ctcgtcgagg gtgtccccgg tgacaacgtt 1140
ggattcaacg tcaagaacgt ctccgtcaag gatatccgcc gtggtaacgt tgccggtgac 1200
tccaagaacg acccccccat gggtgccgct tctttcaacg cccaggtcat cgtcatgaac 1260
caccctggcc aggtcggtgc cggatacgtc c 1291
<210> 6
<211> 619
<212> DNA
<213> Trichoderma atroviride (Trichoderma atroviride)
<400> 6
gagaagttcg agaaggtaag ctcatttcat tgtttttctc attcattatg ggcacaattg 60
tgtccgacaa ttctgttctc agtcttgtca acactttttt ttccaccaag cattgcaccc 120
cgctttgcct acctacccct cctttggcac agcaaaattt ttctggctgc cttgtttggt 180
ttttagtggg gtgtcaaatt tttggcagca accccgctat tgccactgtc cctcacacat 240
tgcccaacaa tattctactt caatcaattt ctatttggtt cattgtgcta atcatacttc 300
aatcaatagg aagccgccga actcggcaag ggttccttca agtatgcgtg ggttcttgac 360
aagctcaagg ccgagcgtga gcgtggtatc accatcgaca ttgccctgtg gaagttcgag 420
actcccaagt actatgtcac cgtcattggt atgttttcag tccgactggt cggtattatc 480
ccaacatcat cattctaaca cgttacttta tagacgctcc cggtcaccgt gatttcatca 540
agaacatgat cactggtacc tcccaggccg attgcgctat cctcattatc gctgccggta 600
ctggtgagtt cgaggctgg 619
<210> 7
<211> 514
<212> DNA
<213> Trichoderma atroviride (Trichoderma atroviride)
<220>
<221> misc_feature
<222> (497)..(497)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (501)..(501)
<223> n is a, c, g, or t
<400> 7
tcgagaagtt cgagaaggta agctcatttc actgcttttt cactacgcgt tcctggtcca 60
atcgtgcccg acaattctgt tctcagtctt gtcaactttt ccctcgcagc atcacacccc 120
gctttgcctg cctctacccc tcattttgca cagcaaaaat tttctggcag tcttgtttgg 180
ctctgagtgg ggtgccaact tttgttggca gcgaccccgc tatcgccact gtccctcatc 240
catcgtccca acacattgtg ctcattcaat cgcatcgtct tttgcctcaa ttcctttgtg 300
attcattgtg ctgatcatgt ttcaaccaat aggaagccgc cgaactcggc aagggttctt 360
tcaagtatgc gtgggttctt gacaagctca aggccgagcg tgagcgtggt atcaccatcg 420
acattgccct ctggaagttc gagactccca gatactatgt caccgtcatt ggtatgtttt 480
tggttcccta aatgacngaa ngccatcatc attc 514
Claims (8)
1. A method for degrading chlorpyrifos by trichoderma aureoviride is characterized by comprising the following steps:
inoculating trichoderma atroviride SG3403 strain on a potato glucose agar culture medium, and activating at 25-28 ℃;
propagating spores on a potato dextrose agar medium and washing the spores to make a spore suspension;
inoculating the spore suspension into a potato glucose medium to ensure that the concentration of the spores is 10 5 CFU/mL~10 6 CFU/mL；
Performing shake culture on the spores for 4-5 days at the rotation speed of 180rpm-200rpm and the temperature of 25-28 ℃, and performing vacuum filtration, sterile water washing and mycelium pressing twice;
inoculating 0.4g to 0.5g of the press-dried mycelia into 100mL to 110mL of anhydrous water, adding chlorpyrifos, culturing for 120 hours to 125 hours at the temperature of 25 ℃ to 28 ℃ and under the conditions of 180rpm to 200rpm, keeping out of the sun, collecting 1.0mL to 1.5mL of liquid every 24 hours to 30 hours, carrying out solid phase microextraction-gas chromatography-mass spectrometry detection, and analyzing the hydrolysis dynamic of the chlorpyrifos;
the Trichoderma atroviride is Trichoderma atroviride SG3403, which has been deposited in China general microbiological culture Collection center of China Committee for culture Collection, china, no. 3 of West Lu No. 1 Hospital, beijing, chaozhou, chaoyang, on 28 days of 8 months in 2012, and has a collection number of CGMCC No.6479.
2. The method of using trichoderma atroviride for the degradation of chlorpyrifos, as claimed in claim 1, wherein the method of preparing said potato dextrose agar medium comprises: weighing 190g to 200g of potatoes, peeling the potatoes, cutting the potatoes into small pieces, adding water, boiling for 30min to 35min, filtering the small pieces with four layers of gauze, adding 15g to 20g of anhydrous glucose and 15g to 20g of agar powder, cooling to a constant volume of 1L, and sterilizing at 120-121 ℃ for 15min to 30min.
3. The method for degrading chlorpyrifos according to claim 1, wherein the spore suspension is inoculated in the potato dextrose medium in an inoculum size of 3-5%.
4. The method of using trichoderma atroviride for the degradation of chlorpyrifos, as claimed in claim 1, wherein the method of preparing the potato glucose medium comprises: weighing 190g to 200g of potatoes, peeling the potatoes, cutting the potatoes into small pieces, adding water, boiling for 30min to 35min, filtering the small pieces with four layers of gauze, adding 15g to 20g of anhydrous glucose, cooling to a constant volume of 1L, and sterilizing at 120-121 ℃ for 15min to 30min.
5. The method for degrading chlorpyrifos by trichoderma aureoviride according to claim 1, wherein when the trichoderma aureoviride is used for degrading chlorpyrifos, the mass concentration ratio of trichoderma aureoviride hyphae to chlorpyrifos is (3.7-4.0): (0.3 to 0.35).
6. The application of Trichoderma atroviride in degrading chlorpyrifos is characterized in that the Trichoderma atroviride is SG3403, which is preserved in China general microbiological culture collection management center of No. 3 Hopkins West Lu No. 1 Hopkin, ind. Tokyo area, kyoho, in 8-28 days 2012, and the preservation number is CGMCC No.6479.
7. The use of trichoderma aureoviride for degrading chlorpyrifos according to claim 6, wherein the trichoderma aureoviride is resistant to chlorpyrifos at a concentration of 40mg/L to 50mg/L and the trichoderma aureoviride has a sporulation capacity of more than 60%.
8. The use of trichoderma aureoviride for degrading chlorpyrifos according to claim 6, wherein said trichoderma aureoviride produces paraoxonase when said chlorpyrifos is degraded, and the catalytic efficiency of said paraoxonase on said chlorpyrifos is 5.0 x 10 6 kcat/Kms -1 M -1 -6.05×10 6 kcat/Kms -1 M -1 The enzyme activity to the chlorpyrifos is 2.85U/mL-3.60U/mL.
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