CN101525584A - Degradation bacteria for removing fomesafen pesticide residue and microbial inoculum prepared by same - Google Patents

Abstract

The invention provides a degradation microbial inoculum for removing fomesafen pesticide residue, which belongs to the high-technical field of biology. Gram-positive bacteria ZB-1 are used as a bacterial strain which is a bacillus (Bacillus sp.) by identification. The bacillus has the main biological characteristic of G<+>, a thalli is in the shape of a brevibacterium, and the size of the thalli is 0.5-1.0*1.5-5.0 microns, the gonite is round, and the bacillus is positive by catalase reaction, gelatin hydrolization and phenylalanine deaminase; the bacillus is negative by V-P reaction, M.R reaction, nitrate reaction, starch hydrolization and tyrosine hydrolization. The culture fluid which is obtained after the ZB-1 strain is fermented is the microbial inoculum. By using the degradation bacteria product, the pesticide residue of fomesafen in the earth is reduced by more than 67 percent, and the overproof problem of the fomesafen pesticide residue can be solved in agricultural production.

Description

A fomesafen pesticide residue degrading bacterium and its production bacterium

1. Technical field

The invention provides a fomesafen pesticide residue-degrading bacterium and a bacterial agent produced therefrom, which belong to the high-tech field of biology, use microorganisms to degrade chemical pesticide residues, and are suitable for the production of green and pollution-free agricultural products in modern agricultural production.

2. Background technology

The use of chemical pesticides has greatly promoted the development of modern agriculture, restored a large number of losses caused by plant diseases, insects, and weed damage for agricultural production, and greatly improved the labor efficiency and mechanization degree of agricultural production. played an invaluable role in the modernization process. But at the same time, pesticides are some toxic and harmful chemical substances. After they enter the ecological environment, they not only kill the organisms that are harmful to humans, but also cause serious harm to the ecological system. Extensive use of pesticides has caused pesticide residues in agricultural products to exceed the standard, seriously affecting people's health and the export of agricultural products. With the improvement of people's living standards and the enhancement of environmental protection awareness, the whole society is paying more and more attention to the direct harm and potential impact of pesticide residues. People urgently need clean and green agricultural products without pesticide residue pollution. However, judging from the current agricultural production form and population status in our country, the pollution-free production model at the cost of stopping the use of pesticides and reducing agricultural output is not feasible. Pesticides will continue to be used as an effective means of controlling plant diseases, insects, and weeds. important role in agricultural production. How to solve this dilemma in agricultural production has become a research hotspot in various disciplines. Both laboratory and practical application studies have shown that it is feasible to use microorganisms to degrade pesticide residues in soil, thereby reducing pesticide residues in agricultural products such as grain, vegetables, and tea, and improving the quality and economic value of agricultural products. In the prior art, our research group already has an invention patent for degrading bacteria (ZL96106317.3, publication number: CN1145408A, publication date: March 19, 1997), which is a strain of Bacillus J5, which can effectively degrade methylamine Phosphorus pesticide residues.

3. Contents of the invention

Technical Problems The purpose of the present invention is to develop a new type of pesticide residue degrading bacterial agent aimed at the practical problems and needs in production practice. Using this bacterial agent can reduce the residual amount of fomesafen by more than 67%, and the production and The use cost is lower. Using the degrading bacteria agent can normally use chemical pesticides to prevent and control weeds in the normal growth process of crops, so as to ensure that the pesticide residue content in agricultural products meets the requirements of green food.

Technical scheme Below is main content of the present invention:

The invention provides a fomesafen pesticide residue degrading bacterium ZB-1, which is a Gram-positive bacterium, and was preserved in the General Microbiology Center of China Microbial Strain Preservation Management Committee on November 3, 2008. The strain preservation number is : CGMCC No.2782, identified as Bacillus sp. The main biological characteristics are G ⁺ , the bacteria are short rod-shaped, the size is 0.5-1.0×1.5-5.0μm, the spores are round, and the contact enzyme reaction, gelatin hydrolysis, and phenylalanine deaminase are positive; VP reaction, MR reaction, nitrate reaction, starch hydrolysis, and tyrosine hydrolysis were negative.

The process of using the fomesafen pesticide residue-degrading bacteria ZB-1 to produce the inoculum is as follows: test tube slant planting - shake flask planting - seed tank - production tank - product (the packaging dosage form is liquid inoculum or solid adsorption inoculum). Detailed implementation steps of the present invention are:

1. Inoculate the fomesafen pesticide residue-degrading bacterium ZB-1 test tube species in the broth culture medium shake flask, shake culture to the logarithmic phase;

2. the above-mentioned cultivated bacterial classification is inoculated into 500 liters of seed tanks by 10% inoculation amount of seed tank medium, cultivated to logarithmic growth phase, the used medium formula (mass percentage) of seed tanks: glucose 0.8%, NH ₄ NO ₃ 1%, K ₂ HPO ₄ 0.2%, MgSO ₄ 0.05%, NaCl 0.01%, CaCO ₃ 0.3%, yeast extract 0.02%, pH 7.2-7.5;

3. Put the seed liquid into the production tank for cultivation according to 10% of the inoculation amount of the production tank culture medium, and the culture medium used in the production tank is the same as the seed tank culture medium;

During the cultivation process of seed tanks and production tanks, the ventilation rate of sterile air is 1:0.6-1.2, the stirring speed is 180-240 rpm, the cultivation temperature is 30-35°C, and the cultivation time of the whole process is 36-48 Hour. After the fermentation is completed, the culture solution is the bacterial agent.

Beneficial Effects The pesticide residue degrading bacterial agent produced by the invention has the advantages of low production and use cost, convenient use and good removal effect, and is suitable for large-scale promotion and use in national grain, oil and vegetable production and export bases or areas with green food trademark marks. The invention has important significance for protecting ecological environment, protecting people's health and increasing the added value of agricultural products. Degrading bacteria ZB-1 can degrade more than 67% of fomesafen pesticide residues in the soil (Table 1). Using this degrading bacteria agent can normally use chemical pesticides for weed control in the normal growth process of crops to ensure that pesticides in agricultural products The residual content meets the requirements of green food.

The invention successfully solves the problem of excessive fomesafen pesticide residues in agricultural production, fully exerts the high-efficiency and rapid effect of chemical pesticides in weed control, and can produce non-toxic and pollution-free green agricultural products.

4. Specific implementation

The invention provides a degrading bacterial agent for eliminating fomesafen pesticide residues. The bacterial strain used is Gram-positive bacteria ZB-1, which is isolated from farmland soil where fomesafen pesticide has been used for a long time in Qiqihar City, Heilongjiang Province.

On November 3, 2008, it was preserved in the General Microorganism Center of China Microbiological Culture Collection Management Committee, the strain preservation number is: CGMCC No.2782, and it was identified as Bacillus sp. The main biological characteristics are G ⁺ , the bacteria are short rod-shaped, the size is 0.5-1.0×1.5-5.0μm, the spores are round, and the contact enzyme reaction, gelatin hydrolysis, and phenylalanine deaminase are positive; VP reaction, MR reaction, nitrate reaction, starch hydrolysis, and tyrosine hydrolysis were negative.

Degradation characteristic determination of degrading bacteria ZB-1 in inorganic salt yeast extract liquid medium: pick ZB-1 single colony in 3mlLB liquid medium (peptone 10 grams, yeast extract 5 grams, NaCl 10 grams, water 1000 milliliters, pH 7.0- In 7.2), shake culture at 30°C and 160rmp/min for 12h to obtain fresh bacterial liquid. In 100ml of 50mg/l fomesafen inorganic salt yeast extract liquid medium (NH ₄ NO ₃ 1.0 g, K ₂ HPO ₄ 1.5 g, KH ₂ PO ₄ 0.5 g, MgSO ₄ 0.2 g, NaCl 0.5 g, yeast Ointment 0.01 g, water 1000 ml, pH 7.0-7.2), insert 3 ml of centrifuged and suspended bacterial solution (bacterial solution concentration is 10 ⁷ cells/ml), cultivate in a shaker at 30°C (160rmp/min), timed Sampling was carried out to determine the content of fomesafen. The results are shown in Table 1. The degradation rate of fomesafen by the degrading bacteria ZB-1 in the inorganic salt yeast extract liquid medium reached 82.4%.

Determination of the degradation characteristics of fomesafen by the degrading bacteria ZB-1 in the soil: Weigh 1000g of soil, add fomesafen to make the drug concentration in the soil 10mg/kg, add ZB-1 bacterial liquid, mix well, and make the soil The concentration of ZB-1 in the medium was 10 ⁸ cells/gram of soil, and it was placed in a 30°C incubator under dark conditions for constant temperature cultivation, and samples were taken regularly to determine the residual amount of fomesafen in the soil. The results are shown in Table 1. Degradation in soil Bacteria ZB-1 degraded fomesafen up to 67.1%.

Table 1: Degradation characteristics of fomesafen by strain ZB-1 in inorganic salt yeast extract liquid medium and soil

culture medium Inorganic salt yeast paste the soil Degradation rate 82.4% 67.1%

The original seed of the pesticide residue degrading bacteria ZB-1 of the present invention is activated on a petri dish, and inoculated on the inclined surface of a test tube for later use.

Slant medium formula: 10 grams of peptone, 5 grams of yeast extract, 10 grams of NaCl, 1000 ml of water, 2 grams of agar, pH7.0-7.2.

Inoculate the slant strain of the test tube into a 1000ml shake flask containing 200ml of broth medium (3 grams of beef extract, 10 grams of peptone, 5 grams of NaCl, 1000 milliliters of water, pH 7.0-7.2), and cultivate it to the logarithmic phase with constant temperature oscillation. Prepare the seed pots for inoculation. 500 liters of seed tank, 400 liters of feeding capacity, medium formula (mass percentage): glucose 0.8%, NH ₄ NO ₃ 1%, K ₂ HPO ₄ 0.2%, MgSO ₄ 0.05%, NaCl 0.01%, CaCO ₃ 0.3%, Yeast paste 0.02%, pH 7.2-7.5. After the feeding is completed, sterilize under high pressure at 121°C, cool to 33°C, inoculate the above cultured shake flask strains into a 500-liter seed tank at an inoculum size of 10%, and cultivate until the logarithmic growth phase, with a stirring speed of 220 rpm /min, the sterile air intake rate is 1:0.8. The seed solution that has reached the logarithmic phase is inserted into the production tank for cultivation according to the inoculum amount of 10%, and the composition of the medium used in the production tank is the same as that of the seed tank medium. The capacity of the production tank is 5 tons, and the feeding volume is 4.5 tons. Under the pressure of 1.1kg/cm ² , the production tank after feeding is sterilized by high-pressure damp heat at 121°C, cooled to below 35°C after sterilization, and kept sterile by sterile air for standby. The temperature of the production tank after inoculation is controlled at 30-35°C, the ventilation volume of sterile air during the cultivation process of the production tank is 1:0.8-1.0, the stirring speed is 240 rpm, and the cultivation time of the whole process is 36-48 hours . After the fermentation, the number of bacteria reached more than 1 billion/ml. After the fermentation is completed, the culture solution is taken out of the tank and directly packed into a liquid dosage form with a plastic packaging barrel or a packaging bottle, or into a solid microbial dosage form with a packaging bag for peat adsorption.

Claims (2)

1. the degradation bacteria ZB-1 of a removing fomesafen pesticide residue, be gram-positive microorganism, through being accredited as genus bacillus (Bacillus sp.), be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on November 3rd, 2008, culture presevation number is: CGMCC No.2782.

2. bacterium for degradating residual agricultural chemical of producing with the described degradation bacteria for removing fomesafen pesticide residue ZB-1 of claim 1 produces by the following method:

1) the described degradation bacteria for removing fomesafen pesticide residue ZB-1 of claim 1 bacterial classification inoculation is shaken in the bottle in broth culture, shaking culture is to logarithmic phase;

2) above-mentioned cultured bacterial classification is inoculated into 500 liters of seeding tanks by volume ratio 10% inoculum size of seed tank culture base, be cultured to logarithmic phase, the used mass percent culture medium prescription of seeding tank is: glucose 0.8%, NH ₄NO ₃1%, K ₂HPO ₄0.2%, MgSO ₄0.05%, NaCl 0.01%, CaCO ₃0.3%, yeast extract paste 0.02%, pH value 7.2-7.5;

3) seed liquor is produced a jar cultivation by the volume 10% inoculum size access of production jar substratum, it is identical with the seed tank culture base to produce the used substratum of jar;

4) air flow of sterile air is 1 in the culturing process of seeding tank and production jar: 0.6-1.2, stirring velocity is 180-240 rev/min, culture temperature is 30-35 ℃, the whole process incubation time is 36-48 hour, after the fermentation ends thalline quantity reach 1,000,000,000/more than the ml, fermentation is finished the back nutrient solution and is gone out jar and directly be distributed into liquid dosage form with plastic barrel or packing bottle or adopt peat to adsorb and be distributed into the solid fungicide formulation with packing bag.