CN108118018B - Bacillus aryabhattai W-5 and application thereof - Google Patents
Bacillus aryabhattai W-5 and application thereof Download PDFInfo
- Publication number
- CN108118018B CN108118018B CN201810136152.2A CN201810136152A CN108118018B CN 108118018 B CN108118018 B CN 108118018B CN 201810136152 A CN201810136152 A CN 201810136152A CN 108118018 B CN108118018 B CN 108118018B
- Authority
- CN
- China
- Prior art keywords
- bacillus aryabhattai
- atrazine
- strain
- soil
- aryabhattai
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Images
Classifications
-
- 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
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
-
- A—HUMAN NECESSITIES
- A62—LIFE-SAVING; FIRE-FIGHTING
- A62D—CHEMICAL MEANS FOR EXTINGUISHING FIRES OR FOR COMBATING OR PROTECTING AGAINST HARMFUL CHEMICAL AGENTS; CHEMICAL MATERIALS FOR USE IN BREATHING APPARATUS
- A62D3/00—Processes for making harmful chemical substances harmless or less harmful, by effecting a chemical change in the substances
- A62D3/02—Processes for making harmful chemical substances harmless or less harmful, by effecting a chemical change in the substances by biological methods, i.e. processes using enzymes or microorganisms
-
- 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
-
- A—HUMAN NECESSITIES
- A62—LIFE-SAVING; FIRE-FIGHTING
- A62D—CHEMICAL MEANS FOR EXTINGUISHING FIRES OR FOR COMBATING OR PROTECTING AGAINST HARMFUL CHEMICAL AGENTS; CHEMICAL MATERIALS FOR USE IN BREATHING APPARATUS
- A62D2101/00—Harmful chemical substances made harmless, or less harmful, by effecting chemical change
- A62D2101/04—Pesticides, e.g. insecticides, herbicides, fungicides or nematocides
-
- A—HUMAN NECESSITIES
- A62—LIFE-SAVING; FIRE-FIGHTING
- A62D—CHEMICAL MEANS FOR EXTINGUISHING FIRES OR FOR COMBATING OR PROTECTING AGAINST HARMFUL CHEMICAL AGENTS; CHEMICAL MATERIALS FOR USE IN BREATHING APPARATUS
- A62D2101/00—Harmful chemical substances made harmless, or less harmful, by effecting chemical change
- A62D2101/20—Organic substances
- A62D2101/26—Organic substances containing nitrogen or phosphorus
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biochemistry (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- General Engineering & Computer Science (AREA)
- Environmental & Geological Engineering (AREA)
- Emergency Management (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Business, Economics & Management (AREA)
- Toxicology (AREA)
- General Chemical & Material Sciences (AREA)
- Soil Sciences (AREA)
- Mycology (AREA)
- Medicinal Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Purification Treatments By Anaerobic Or Anaerobic And Aerobic Bacteria Or Animals (AREA)
Abstract
The invention relates to Bacillus aryabhattai W-5 and application thereof. The strain is preserved in Guangdong province microorganism culture collection center in 2018, 1 month and 29 days, and the preservation number is GDMCC No. 60320. The Bacillus aryabhattai W-5 provided by the invention is screened from surrounding soil (brick red soil) of pesticide factory Zhanjiang river biochemistry industry Co., Ltd in Zhanjiang river city, Yangxi city, Guangdong province, has a good degradation effect on herbicides, especially has a good degradation effect on atrazine in high-salinity saline-alkali soil such as coastal mudflat, inland saline-alkali soil and the like, and can be popularized and applied in a large scale.
Description
Technical Field
The invention belongs to the field of microorganisms, and particularly relates to bacillus aryabhattai W-5 and application thereof.
Background
Atrazine (Atrazine) is widely used as a triazine herbicide worldwide, and is a typical teratogenic, carcinogenic and mutagenic 'triple-causative' pollutant containing chlorine atoms. The atrazine has stable structure, is not easy to degrade, and has broad-spectrum toxicity and bioaccumulation. The atrazine is used in the beginning of 80 years in China, is mainly used as a herbicide for dry crops such as corn, sorghum, woodland, fruit tea garden and the like, and the dosage is increased by about 20 percent every year. The soil is a storage warehouse and a transfer station of atrazine in the environment, and the atrazine mainly takes aging, migration, degradation and other actions after entering the soil. The aging and migration behaviors only delay or transfer the damage of atrazine in soil, the potential risk still exists, and the degradation is the main way of reducing the damage. Atrazine pollution has directly affected world grain production safety and agricultural ecological health, and further affected human health.
The pesticide atrazine has low residual concentration, wide range, great difficulty and high cost by adopting the traditional physical and chemical remediation method and has secondary pollution. The microbial remediation technology has good treatment effect, high mineralization degree and no secondary pollution, and is widely concerned and researched. The microbial remediation technology is characterized in that a large number of high-efficiency degrading strains are added to form dominant flora in a certain time, so that the environmental purification and ecological effect recovery of a polluted area are realized. Microbial remediation technology has been applied to pesticide contaminated sites around the world, with a large number of atrazine degrading strains, such as: rhodococcus (Rhodococcus), Pseudomonas (Pseudomonas), Agrobacterium (Agrobacterium), Acinetobacter (Acinetobacter), etc. have been successfully used for remediation of soil atrazine contamination. However, microbial remediation also faces application limitations of low environmental adaptability of strains, specificity of degraded substrates, competition with indigenous microorganisms, and the like. According to the specific environmental conditions of the polluted site, the selection of proper high-quality strains is the key for determining whether the microbial remediation technology can be successfully applied.
The saline-alkali soil contains a large amount of soluble salt (0.6-3%), the pH value is 7.1-9.5, 2.8 hundred million acres of saline-alkali soil can be developed and utilized in China, and the method has extremely high scientific research and strategic significance. The development and utilization strength of saline-alkali soil is improved by China, and saline-alkali tolerant cash crops such as medlar, grapes and seawater rice are planted in a large scale. When the cash crops are planted, a large amount of fertilizer and pesticide is continuously applied to the soil. At present, the research on the atrazine degrading pesticide bacteria at home and abroad is steadily carried out, but the bacterial strains for effectively repairing the atrazine polluted farmland soil under the environment with relatively high salt content such as coastal mudflats, inland saline-alkali soil and the like are not reported.
In view of this, screening the atrazine degrading strain resources which can be popularized and applied to special environments such as coastal beaches, inland saline-alkali soil and the like is a very good development direction.
Disclosure of Invention
The invention aims to overcome the defect and deficiency that the strain in the prior art can not effectively restore atrazine pollution of farmland soil in an environment with relatively high salt content, and provides a strain of Bacillus aryabhattai (Bacillus aryabhattai) W-5. The Bacillus aryabhattai (Bacillus aryabhattai) W-5 provided by the invention has a good degradation effect on the pesticide atrazine in coastal beach or inland saline-alkali soil with high salt content.
The invention also aims to provide the application of the Bacillus aryabhattai W-5 in degrading herbicides.
The invention also aims to provide an atrazine degrading microbial inoculum.
In order to achieve the purpose, the invention adopts the following technical scheme:
a strain of Bacillus aryabhattai (Bacillus aryabhattai) W-5 is deposited in Guangdong province microorganism strain collection center in 2018, 1 month and 29 days, and the deposition number is GDMCC No. 60320.
Through a large number of experimental researches, the inventor screens out a strain capable of efficiently degrading atrazine from the surrounding soil (brick red soil) of the pesticide factory Zhanjiang river, Tuxi county, Zhanjiang city, spring river, biochemistry industry Co., Ltd. The strain is named as bacterium.W-5, is called W-5 for short, is primarily identified as Bacillus aryabhattai, is preserved in Guangdong province microorganism strain preservation center in 29 months 1 and 2018, is preserved in No. 59 building 5 of Michelia Tokyo 100 Dazhou, Guangzhou city, and has the preservation number of GDMCC No. 60320.
The 16S rDNA sequence of the strain is shown in SEQ ID NO: 1 is shown.
The Bacillus aryabhattai (Bacillus aryabhattai) W-5 provided by the invention has the following morphological characteristics of thallus.
Preferably, the Bacillus aryabhattai (Bacillus aryabhattai) W-5 is a gram-positive bacterium, having spores; the thallus is rod-shaped, has motility and obligate aerobe.
The Bacillus aryabhattai (Bacillus aryabhattai) W-5 provided by the invention has the following colony morphology characteristics.
Preferably, bacterial colonies formed after the bacterial strains are cultured on a beef extract peptone medium for 24 hours are round or irregular, and bacterial colonies formed after 48 hours are round, milky white, 2-3 mm in diameter, irregular in edge and flat and wet.
The molecular classification status of Bacillus aryabhattai W-5 provided by the invention is determined as follows.
And extracting total DNA of bacteria by a kit method. The 16S rDNA of the bacteria is amplified by adopting the universal primers F63 and R1087 of the bacteria and PCR, an obvious band appears near 1000bp, the PCR amplification product is recovered and then subjected to sequence determination, the obtained DNA sequence is input into GenBank, and all sequences in a database are compared and analyzed by a Blast program, and the result shows that the 16S rDNA sequence of the strain has higher homology with Bacillus aryabhattai in the GenBank. Combining the morphological characteristics and the result of 16S rDNA sequence, the strain belongs to Bacillus aryabhattai (Bacillus aryabhattai) and is named as bacterium.W-5.
The optimal culture conditions of the Bacillus aryabhattai (Bacillus aryabhattai) W-5 are as follows: the pH range is 5-10, the temperature range is 20-35 ℃, the salinity range is 0-6%, the ventilation volume has no obvious influence on the growth of the strain, glucose, fructose and sucrose are preferably used as carbon sources, and tryptone and yeast powder are preferably used as nitrogen sources.
The application of the Bacillus aryabhattai W-5 in degrading herbicides is also within the protection scope of the invention.
The Bacillus aryabhattai (Bacillus aryabhattai) W-5 provided by the invention can greatly degrade herbicides in soil, is inoculated into seed liquid (OD600nm is approximately equal to 2.00) of a W-5 strain according to the inoculation amount of 3%, and the residual concentrations of different pesticides are measured after shaking culture for 48 hours on a constant temperature shaking bed at 30 ℃ at 180r/min, wherein the degradation rate of atrazine is 97.75%, the degradation rate of pentachlorophenol is 83.15%, the degradation rate of simazine is 91.46%, and the degradation rate of dichlorodiphenyl trichloroethane is 62.44%.
Preferably, the herbicide is a halogen herbicide.
Preferably, the herbicide is atrazine.
Preferably, the salt content in the degraded soil is not higher than 6%.
Under the environment with relatively high salt content (soluble salt content is 0.6-3%) such as coastal mudflat, inland saline-alkali soil and the like, the Bacillus aryabhattai W-5 provided by the invention also has a good effect of degrading atrazine.
Preferably, the degraded soil is coastal mudflat or inland saline-alkali soil.
An agent for degrading atrazine comprises Bacillus aryabhattai W-5 and adjuvants.
The Bacillus aryabhattai (Bacillus aryabhattai) W-5 provided by the invention is prepared into a microbial inoculum, and has better effects of degrading atrazine and promoting plant growth.
Preferably, the cell density OD600nm of the Bacillus aryabhattai (Bacillus aryabhattai) W-5 in the microbial inoculum is 2.00.
Compared with the prior art, the invention has the following beneficial effects:
the Bacillus aryabhattai W-5 provided by the invention is screened from surrounding soil (brick red soil) of a pesticide factory in Yangtze river, Yangtze city, Yangtze river biochemistry industry Co., Ltd, Zhanjiang river, spring river, and biochemistry industry Co., Ltd, has a good degradation effect on herbicides, especially has a good degradation effect on atrazine in high-salinity saline-alkali soil such as coastal mudflats and inland saline-alkali soil, and can be popularized and applied in a large scale.
Drawings
FIG. 1 shows morphological characteristics of Bacillus aryabhattai (Bacillus aryabhattai) W-5 strain;
FIG. 2 is a graph showing the relationship between growth of Bacillus aryabhattai W-5 and atrazine degradation;
FIG. 3 is a graph showing the relationship between growth of Bacillus aryabhattai W-5 and atrazine degradation at different salinity (48 h);
FIG. 4 is a diagram showing the optimum growth conditions for Bacillus aryabhattai (Bacillus aryabhattai) W-5;
FIG. 5 is a graph showing the effect of Bacillus aryabhattai (Bacillus aryabhattai) W-5 in degrading pesticides;
FIG. 6 is a graph showing the effect of Bacillus aryabhattai (Bacillus aryabhattai) W-5 in permanent planting across saline-alkali soil to degrade atrazine;
FIG. 7 is a graph showing the effect of Bacillus aryabhattai (Bacillus aryabhattai) W-5 in field.
Detailed Description
The invention is further illustrated by the following examples. These examples are intended to illustrate the invention and are not intended to limit the scope of the invention. Experimental procedures without specific conditions noted in the examples below, generally according to conditions conventional in the art or as suggested by the manufacturer; the raw materials, reagents and the like used are, unless otherwise specified, those commercially available from the conventional markets and the like. Any insubstantial changes and substitutions made by those skilled in the art based on the present invention are intended to be covered by the claims.
EXAMPLE 1 enrichment, isolation and purification of the Strain, characterization
(1) Enrichment of degrading bacteria
Accurately weighing 10g of soil of each sampling point, putting the soil into a 250mL triangular flask (added with glass beads) filled with 90mL sterile water, oscillating for 30min by a shaking table to fully mix the sample with the water, and dispersing cells to form uniform bacterial suspension. Standing for 20-30s, sucking 2mL of supernatant into an atrazine-containing basic inorganic salt culture medium (with atrazine as the only nitrogen source) at 100mg/L, and shaking and culturing at 30 deg.C and constant temperature on a shaking bed at 180 r/min. And taking the culture every 5 days and inoculating the culture into a fresh culture medium, wherein the transfer amount is 10%, and the content of the atrazine in the culture medium is increased by 100mg/L each time. And (5) continuously performing acclimation culture until the final concentration of the atrazine acclimation is increased to 400mg/L, and finishing the enrichment culture. Blank control without adding bacterial liquid is available for each enrichment culture.
(2) Isolation and purification of degrading bacteria
Respectively diluting the enrichment culture with atrazine acclimation concentration of 400mg/L to 10-1、10-2、10-3、10-4、10-5、10-6、10-7The respective treatments were determined by preliminary experiments to be 10-4、10-5、10-6The diluted solution (100 μ L) was spread on a solid medium plate (400mg/L) to which corresponding atrazine concentration had been added, and the plate was placed on a 30-day plate℃Culturing in a constant temperature incubator. Screening high-efficiency atrazine pesticide degrading bacteria, observing transparent degrading rings day by day, and recording degrading bacteria colonies (D) and degrading ring diameters (D). Separating, purifying and culturing the strain with larger degradation ring for 3 times to obtain a purified strain, and transferring to a nutrient agar slant for storage.
(3) Characterization of properties
3.1 morphological characteristics of the cells
Gram-positive bacteria, with spores. The thallus is rod-shaped, has motility and obligate aerobe. The results of the experiment are shown in FIG. 1.
3.2 colony morphology characteristics
The bacterial colony formed after 24 hours of culture on the beef extract peptone medium is circular or irregular, and the bacterial colony is all circular after 48 hours, is milk white, has the diameter of about 2-3 mm, and is irregular in edge, flat and moist. The test results are shown in FIG. 1.
3.3 molecular biological Properties
And extracting total DNA of bacteria by a kit method. The 16S rDNA of the bacteria is amplified by adopting the universal primers F63 and R1087 of the bacteria and PCR, an obvious band appears near 1000bp, the PCR amplification product is recovered and then subjected to sequence determination, the obtained DNA sequence is input into GenBank, and all sequences in a database are compared and analyzed by a Blast program, and the result shows that the 16S rDNA sequence of the strain has higher homology with Bacillus aryabhattai in the GenBank. Combining the morphological characteristics and the result of 16S rDNA sequence, the strain belongs to Bacillus aryabhattai (Bacillus aryabhattai) and is named as bacterium.W-5.
The sequencing results were as follows:
CGAGCGAACTGATTAGAAGCTTGCTTCTATGACGTTAGCGGCGGACGGGTGAGTAACACGTGGGCAACCTGCCTGTAAGACTGGGATAACTTCGGGAAACCGAAGCTAATACCGGATAGGATCTTCTCCTTCATGGGAGATGATTGAAAGATGGTTTCGGCTATCACTTACAGATGGGCCCGCGGTGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGCATAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTAGGGAAGAACAAGTACGAGAGTAACTGCTCGTACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGAATTATTGGGCGTAAAGCGCGCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGAAAAGCGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGGCTTTTTGGTCTGTAACTGACGCTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAACTCTAGAGATAGAGCGTTCCCCTTCGGGGGACAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTTAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAAAGGGCTGCAAGACCGCGAGGTCAAGCCAATCCCATAAAACCATTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGGGAGTAAACCGTAAAGGAGCTA。
example 2 application of Bacillus aryabhattai (Bacillus aryabhattai) W-5 in degrading herbicides
(1) Experiment of effect of Bacillus aryabhattai (Bacillus aryabhattai) W-5 strain on degrading atrazine
Inoculating seed liquid (OD) of W-5 strain into basal salt culture medium with atrazine concentration of 400mg/L according to 3% inoculation amount600nmAbout.2.00) and shaking and culturing on a constant temperature shaking bed at 30 ℃ for 180 r/min. The strain growth (OD) was measured by sampling every 4h600nm) And the concentration of atrazine. The results show that: the W-5 strain can obviously degrade the atrazine, and the growth amount of the W-5 strain and the residual amount of the atrazine have obvious negative correlation. After 48 hours of the experiment, the atrazine degradation rate in the treatment of adding the W-5 strain reaches 97.75 percent. The specific experimental results are shown in FIG. 2.
(2) Growth of Bacillus aryabhattai (Bacillus aryabhattai) W-5 strain at different salinity and atrazine degradation effect experiment
Preparing 400mg/L atrazine inorganic salt culture medium, inoculating seed liquid (OD600nm is approximately equal to 2.00) of the W-5 strain according to the inoculation amount of 3 percent until the salinity is 0 percent, 2 percent, 4 percent, 6 percent, 8 percent and 10 percent of the culture medium, placing the culture medium in a constant temperature incubator at 30 ℃ and shaking-culturing for 48 hours at the rotating speed of 180r/min, and determining the growth condition (OD600nm) of the strain and the concentration of the atrazine. The results show that: the strain grows well in a substrate with the salt content of less than 6 percent, and when the salinity is more than 8 percent, the growth and degradation effects of the strain are poor. The specific experimental results are shown in FIG. 3.
(3) Experiment of optimal growth conditions of Bacillus aryabhattai (Bacillus aryabhattai) W-5 strain
Optimum pH: the pH of the medium was measured and adjusted to 4, 5, 6, 7, 8, 9, 10 with 10mol/L NaOH solution and 10mol/L HCl solution. After sterilization at 121 ℃ for 25min, the culture medium: the volume ratio of the bacterial suspension is 100: 1, adding the bacterial suspension into the newly-configured culture medium, repeating the steps for three times, placing the culture medium in a 30-DEG C constant-temperature incubator, performing shake culture at the rotating speed of 180r/min for 48 hours, and measuring the quantity of the bacteria by using an ultraviolet spectrophotometer under the condition that the wavelength lambda is 600.
Optimum temperature: preparing an atrazine inorganic salt culture medium, adjusting the pH value to 7.0, sterilizing at 121 ℃ for 25min, and mixing the components in the culture medium: the volume ratio of the bacterial suspension is 100: 1, adding the bacterial suspension into the newly-configured culture medium, placing the newly-configured culture medium into an incubator at 15, 20, 25, 30, 35 and 40 ℃ respectively, performing shake culture at the rotating speed of 180r/min for 48h, repeating three times, and measuring the quantity of bacteria by using an ultraviolet spectrophotometer at the wavelength lambda of 600.
Optimum salinity: preparing an atrazine inorganic salt culture medium, adjusting the pH value to 7.0, sterilizing at 121 ℃ for 25min, and mixing the components in the culture medium: the volume ratio of the bacterial suspension is 100: 1, adding the bacterial suspension into a culture medium with the salinity of 0%, 2%, 4%, 6%, 8% and 10%, repeating the steps for three times, placing the culture medium in a constant-temperature incubator at 30 ℃ and shaking and culturing the culture medium for 48 hours at the rotating speed of 180r/min, and measuring the quantity of the bacteria by using an ultraviolet spectrophotometer under the condition that the wavelength lambda is 600.
Optimum ventilation: the measurement was carried out using a 500ml Erlenmeyer flask. Preparing an atrazine inorganic salt culture medium, adjusting the pH value to 7.0, sterilizing at 121 ℃ for 25min, and mixing the components in the culture medium: the volume ratio of the bacterial suspension is 100: 1, 50, l00,150, 200, 250 and 300mL in a bottle, and placing the bottles in a 30 ℃ constant temperature incubator for shake culture at the rotating speed of 180r/min for 48h, and measuring the number of bacteria by using an ultraviolet spectrophotometer under the wavelength lambda of 600.
Optimum carbon source: adding glucose, lactose, fructose, sucrose, soluble starch and maltose with the final concentration of 1g/L into a carbon-free nitrogen-free inorganic salt culture medium respectively, adding glucose with the final concentration of 500mg/L as a carbon source, adjusting the pH value to 7.0, sterilizing at 121 ℃ for 25min, and then adding the culture medium: the volume ratio of the bacterial suspension is 100: 1, adding the bacterial suspension into the newly-configured culture medium, repeating the steps for three times, placing the culture medium in a 30-DEG C constant-temperature incubator, performing shake culture at the rotating speed of 180r/min for 48 hours, and measuring the quantity of the bacteria by using an ultraviolet spectrophotometer under the condition that the wavelength lambda is 600.
Most suitable nitrogen source: adding tryptone with the final concentration of 1g/L, yeast powder, urea, ammonium chloride and sodium nitrate into a carbon-free and nitrogen-free inorganic salt culture medium respectively, adding glucose with the final concentration of 500mg/L as a carbon source, adjusting the pH value to 7.0, sterilizing at 121 ℃ for 25min, and then adding the culture medium: the volume ratio of the bacterial suspension is 100: 1, adding the bacterial suspension into the newly-configured culture medium, repeating the steps for three times, placing the culture medium in a 30-DEG C constant-temperature incubator, performing shake culture at the rotating speed of 180r/min for 48 hours, and measuring the quantity of the bacteria by using an ultraviolet spectrophotometer under the condition that the wavelength lambda is 600.
The result shows that the proper pH range of the strain is 5-10, the temperature range is 20-35 ℃, the salinity range is 0-6%, the ventilation volume has no obvious influence on the growth of the strain, and glucose, fructose and sucrose are preferably used as carbon sources, and tryptone and yeast powder are preferably used as nitrogen sources. The results of all the above experiments are shown in detail in FIG. 4.
(4) Experimental chart of Bacillus aryabhattai (Bacillus aryabhattai) W-5 strain for degrading other pesticides
Common herbicides atrazine, pentachlorophenol, simazine, imazamox, clomazone, dichlorodiphenyl trichloroethane and nicosulfuron in the market are selected to prepare a basic salt culture medium with the concentration of 100mg/L, seed liquid (OD600nm is approximately equal to 2.00) of a W-5 strain is inoculated according to the inoculation amount of 3 percent, and the residual concentrations of different pesticides are measured after shaking culture is carried out for 48 hours on a constant temperature shaking bed at 30 ℃ for 180 r/min. The results show that: the strain has good degradation effect on atrazine, simazine, pentachlorophenol and dichlorodiphenyl trichloroethane pesticides, has no obvious degradation effect on imazamox, clomazone and nicosulfuron, and supposes that the W-5 strain is favorable for degrading halogen-containing herbicides. The specific experimental results are shown in FIG. 5.
(5) Soil colonization effect graph of Bacillus aryabhattai (Bacillus aryabhattai) W-5 strain
The solid culture medium with atrazine concentration of 400mg/L is placed on the outer ring of a culture dish, the inner ring is covered with soil (soil of the seaside tidal flat land of Zhanjiang city), and then the W-5 strain is added at the central part. Culturing in a constant temperature incubator at 30 ℃, and observing that the W-5 strain can negatively plant atrazine passing through soil and metabolizing outer rings. The results show that: after 5d of culture, the W-5 strain can be effectively planted in the coastal beach salinized soil to degrade outer ring atrazine (generate obvious transparent rings). The results of the experiment are shown in FIG. 6.
(6) Experiment of field effect of Bacillus aryabhattai (Bacillus aryabhattai) W-5 on promoting growth of plants on atrazine-polluted soil
Selecting a W-5 strain to carry out a field effect test in atrazine-polluted soil, and dividing the field effect test into 3 treatment strains in total, wherein the CK: no strain was added, W-5-2%: adding 2% (weight ratio) of inoculum size of strain seed liquid (OD600nm ≈ 2.00), W-5-4%: the seed solution of the strain was added in an inoculum size of 4%. The atrazine pollution concentration of the soil is 1.0mg/kg, the weight of the soil of each treatment is 3.0kg, and each treatment is repeated for 3 times. Water washing rice of Gramineae for germination, moistening with filter paper for preserving water, placing in 30 deg.C incubator, exposing after two days, and sowing in seedbed. When the seedlings are about 25cm high, the seedlings are transplanted into pots, and 6 seedlings are planted in each pot. After 45 days of planting, rice growth was measured at 7 and 11 days in 2017. The results show that: no strain control was added, 4 rice plants died, and 2 surviving rice plants were short, yellow and withered leaf surfaces. The bacterial liquid W-5 is inoculated in the inoculation amount of 2 percent, the rice is completely survived, but the plant is short. The seed liquid of the W-5 strain is inoculated with the inoculation amount of 4 percent, the rice grows healthily, the rice ears are full, and the rice growth is not influenced by the atrazine pollution of soil. The results of the experiment are shown in FIG. 7.
Sequence listing
<110> Guangdong ocean university
<120> Bacillus aryabhattai W-5 strain and application thereof
<160>1
<170>SIPOSequenceListing 1.0
<210>1
<211>1411
<212>DNA
<213> Bacillus aryabhattai (Bacillus aryabhattai)
<400>1
cgagcgaact gattagaagc ttgcttctat gacgttagcg gcggacgggt gagtaacacg 60
tgggcaacct gcctgtaaga ctgggataac ttcgggaaac cgaagctaat accggatagg 120
atcttctcct tcatgggaga tgattgaaag atggtttcgg ctatcactta cagatgggcc 180
cgcggtgcat tagctagttg gtgaggtaac ggctcaccaa ggcaacgatg catagccgac 240
ctgagagggt gatcggccac actgggactg agacacggcc cagactccta cgggaggcag 300
cagtagggaa tcttccgcaa tggacgaaag tctgacggag caacgccgcg tgagtgatga 360
aggctttcgg gtcgtaaaac tctgttgtta gggaagaaca agtacgagag taactgctcg 420
taccttgacg gtacctaacc agaaagccac ggctaactac gtgccagcag ccgcggtaat 480
acgtaggtgg caagcgttat ccggaattat tgggcgtaaa gcgcgcgcag gcggtttctt 540
aagtctgatg tgaaagccca cggctcaacc gtggagggtc attggaaact ggggaacttg 600
agtgcagaag agaaaagcgg aattccacgt gtagcggtga aatgcgtaga gatgtggagg 660
aacaccagtg gcgaaggcgg ctttttggtc tgtaactgac gctgaggcgc gaaagcgtgg 720
ggagcaaaca ggattagata ccctggtagt ccacgccgta aacgatgagt gctaagtgtt 780
agagggtttc cgccctttag tgctgcagct aacgcattaa gcactccgcc tggggagtac 840
ggtcgcaaga ctgaaactca aaggaattga cgggggcccg cacaagcggt ggagcatgtg 900
gtttaattcg aagcaacgcg aagaacctta ccaggtcttg acatcctctg acaactctag 960
agatagagcg ttccccttcg ggggacagag tgacaggtgg tgcatggttg tcgtcagctc 1020
gtgtcgtgag atgttgggtt aagtcccgca acgagcgcaa cccttgatct tagttgccag 1080
catttagttg ggcactctaa ggtgactgcc ggtgacaaac cggaggaagg tggggatgac 1140
gtcaaatcat catgcccctt atgacctggg ctacacacgt gctacaatgg atggtacaaa 1200
gggctgcaag accgcgaggt caagccaatc ccataaaacc attctcagtt cggattgtag 1260
gctgcaactc gcctacatga agctggaatc gctagtaatc gcggatcagc atgccgcggt 1320
gaatacgttc ccgggccttg tacacaccgc ccgtcacacc acgagagttt gtaacacccg 1380
aagtcggtgg gagtaaaccg taaaggagct a 1411
Claims (5)
1. A strain of Bacillus aryabhattai (Bacillus aryabhattai) W-5 is characterized in that the strain is preserved in Guangdong province collection of microorganisms in 2018, 1 month and 29 days, and the preservation number is GDMCC No. 60320.
2. The use of Bacillus aryabhattai W-5 in degrading herbicide as claimed in claim 1, wherein the herbicide is one or more of atrazine, pentachlorophenol, simazine or dichlorodiphenyl trichloroethane.
3. Use according to claim 2, wherein the salt content in the degraded soil is not higher than 6%.
4. An atrazine-degrading bacterial agent, comprising the Bacillus aryabhattai W-5 of claim 1 and an auxiliary material.
5. The microbial inoculum according to claim 4, wherein the cell density OD600nm of Bacillus aryabhattai W-5 is 2.00.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810136152.2A CN108118018B (en) | 2018-02-09 | 2018-02-09 | Bacillus aryabhattai W-5 and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810136152.2A CN108118018B (en) | 2018-02-09 | 2018-02-09 | Bacillus aryabhattai W-5 and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108118018A CN108118018A (en) | 2018-06-05 |
| CN108118018B true CN108118018B (en) | 2020-10-02 |
Family
ID=62234323
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810136152.2A Expired - Fee Related CN108118018B (en) | 2018-02-09 | 2018-02-09 | Bacillus aryabhattai W-5 and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108118018B (en) |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| TWI652344B (en) | 2018-06-22 | 2019-03-01 | 國立臺灣大學 | Bacillus aryabhattai mn1, culture method and microbial agent thereof, and use of the microbial agent |
| CN110452831B (en) * | 2019-07-04 | 2022-02-11 | 天津科技大学 | Kitchen waste degrading bacterium and application thereof |
| CN111534472B (en) * | 2020-06-11 | 2022-04-19 | 浙江工业大学 | Bacillus aryabhattai WZZ10 and application thereof in chiral resolution of 2-tetrahydrofurfuryl acid |
| CN114503992A (en) * | 2022-02-23 | 2022-05-17 | 江苏仁信作物保护技术有限公司 | Herbicide containing imazamox and imazapyr and preparation method thereof |
| CN114958802A (en) * | 2022-03-18 | 2022-08-30 | 上海市农业科学院 | Hydrolase from bacillus elongatus and method for repairing atrazine pollution of soil by using hydrolase |
| CN116004453B (en) * | 2022-12-15 | 2023-09-01 | 黑龙江八一农垦大学 | Atrazine degrading strain, fermentation liquor and application thereof |
| CN117701479B (en) * | 2024-02-06 | 2024-04-12 | 华南农业大学 | Bacterial strain capable of degrading benzonitrile herbicide, microbial agent and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2008137980A (en) * | 2006-12-05 | 2008-06-19 | Kumiai Chem Ind Co Ltd | Composition for agriculture and horticulture |
| CN103865857A (en) * | 2014-03-28 | 2014-06-18 | 山东潍坊润丰化工股份有限公司 | Halophilic bacteria fungicide as well as preparation thereof, biological treatment system containing fungicide and application of fungicide in treating triazine wastewater |
| CN104877950A (en) * | 2015-06-26 | 2015-09-02 | 黑龙江大学 | Bacillus pumilus capable of degrading atrazine efficiently |
-
2018
- 2018-02-09 CN CN201810136152.2A patent/CN108118018B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2008137980A (en) * | 2006-12-05 | 2008-06-19 | Kumiai Chem Ind Co Ltd | Composition for agriculture and horticulture |
| CN103865857A (en) * | 2014-03-28 | 2014-06-18 | 山东潍坊润丰化工股份有限公司 | Halophilic bacteria fungicide as well as preparation thereof, biological treatment system containing fungicide and application of fungicide in treating triazine wastewater |
| CN104877950A (en) * | 2015-06-26 | 2015-09-02 | 黑龙江大学 | Bacillus pumilus capable of degrading atrazine efficiently |
Non-Patent Citations (6)
| Title |
|---|
| Biodegradation of triazine herbicide metribuzin by the strain Bacillus sp. N1;Hao Zhang等;《J Environ Sci Health B》;20141231;第49卷(第2期);第79-86页 * |
| Isolation and Characterization of Atrazine Mineralizing Bacillus subtilis Strain HB-6;Wang Jinhua等;《PLOS ONE》;20140919;第9卷(第9期);e107270 * |
| 噻吩磺隆降解菌Bacillus subtilis LXL-7的分离与应用;李晓楼;《生物技术通报》;20160526;第32卷(第05期);第172-179页 * |
| 氯嘧磺隆高效降解菌的分离、鉴定及其降解特性;关靓等;《东北林业大学学报》;20090625;第37卷(第06期);第77-79页 * |
| 短小芽孢杆菌(Bacillus pumilis)对精喹禾灵的降解特性;周丽兴等;《农药》;20060925;第45卷(第09期);第627-629页 * |
| 莠去津高效降解菌的分离与鉴定;吕惠萍;《化工技术与开发》;20170215;第46卷(第02期);第22-24页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108118018A (en) | 2018-06-05 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108118018B (en) | Bacillus aryabhattai W-5 and application thereof | |
| CN111235076B (en) | Bacillus anethoides, microbial inoculum thereof and application thereof in heavy metal remediation field | |
| CN111423996B (en) | Complex microbial inoculant, preparation method thereof and application thereof in soil remediation field | |
| CN105255782B (en) | There is fiber bacterium and the purposes of reducing power to Cr VI | |
| CN109182178B (en) | Strain with chromium tolerance and Cr (VI) removal capacity and application thereof in-situ remediation of moderately and slightly chromium-polluted soil | |
| CN102533619B (en) | Preparation method of hydrochloric ether efficient aerobic degradation mixed bacteria and application thereof | |
| CN107136122B (en) | Biocontrol microbial inoculum for preventing and treating potato late blight | |
| CN106148232B (en) | The bacterium bacterial strain and its application of one plant of antagonism plant pathogenetic bacteria | |
| CN108192838B (en) | Bacillus amyloliquefaciens with dual functions of inorganic phosphorus degradation and disease prevention | |
| CN113913327B (en) | Saline-alkali-resistant growth-promoting bacterium YS-AT1 capable of secreting IAA and application thereof | |
| CN110643548A (en) | Microbacterium flavum for efficiently degrading aniline and application thereof | |
| CN113444660A (en) | Bacillus amyloliquefaciens strain LXBA.1 and application thereof | |
| CN107916238B (en) | Efficient chlorothalonil degrading bacterium and application thereof in greenhouse soil environment | |
| CN115011510A (en) | Salt-tolerant growth-promoting stenotrophomonas maltophilia, microbial agent thereof and application thereof | |
| Feng et al. | Characterization of Pseudomonas mendocina LR capable of removing nitrogen from various nitrogen-contaminated water samples when cultivated with Cyperus alternifolius L. | |
| CN109868242B (en) | Salt-tolerant acetoin-producing bacillus subtilis and application thereof | |
| CN109576177B (en) | Chinese micro-rod strain SM8 and application thereof in salt tolerance and growth promotion | |
| CN109136142B (en) | Streptomyces alboflavus, method for preparing biocontrol microbial inoculum by using same and application thereof | |
| CN109355232B (en) | Termite fungus IS7 and application thereof in plant growth promotion | |
| CN114934000B (en) | Salt-tolerant bacillus amyloliquefaciens and screening and application thereof | |
| CN114085788B (en) | Pseudomonas amazonensis strain and application thereof | |
| CN112501084B (en) | Rhizosphere probiotic Klebsiella ZH07 and application thereof | |
| CN114874953A (en) | Peanut rhizosphere biocontrol bacterium-Bacillus belgii SW-1 and application thereof | |
| CN114292775A (en) | Toluene degrading strain and application thereof | |
| CN108467842B (en) | Bacterial strain and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20201002 Termination date: 20220209 |