CN100396772C - A monocrotophos pesticide residue degrading bacterium and its production bacterium agent - Google Patents

Abstract

The invention provides a degrading bacterium for eliminating pesticide residues such as monocrotophos and the bacterial agent produced thereof, the bacterial strain is a Gram staining negative bacterium m-1, which is identified as Paracoccus (Paracoccus.sp.) The main biological characteristics are G-, the bacteria are short rod-shaped, aerobic, immobile, salt-intolerant, positive for contact enzymes and oxidases, unable to utilize citrate, positive for nitrate reduction, and produce gas during denitrification. Direct application of degrading bacteria products can reduce the amount of pesticide residues in the soil by more than 95%, solve the problem of excessive pesticide residues in agricultural production, and produce non-toxic and pollution-free green agricultural products.

Description

A monocrotophos pesticide residue degrading bacterium and its production bacterium agent

1. Technical field

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

2. Technical background

The use of chemical pesticides has greatly promoted the development of modern agriculture, restored a large number of losses caused by diseases and insect pests for agricultural production, and greatly improved the labor efficiency and mechanization degree of agricultural production, playing a role in the modernization of agricultural production. immeasurable effect. But at the same time, pesticides are some toxic and harmful chemicals. 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 the health of the people 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 form of agricultural production 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 play a huge role in agricultural production as an effective means of controlling diseases and insect pests. effect. 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 and eliminate the "storage" capacity of pesticide residues in the 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, but the strain has a narrow degradation spectrum and can only degrade one pesticide, methamidophos, so its use is limited.

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 for practical problems and needs in production practice. The use of this bacterial agent can reduce the amount of monocrotophos pesticide residues by more than 95%.

Technical scheme is main content of the present invention below:

The invention provides a degrading bacterium that eliminates pesticide residues such as monocrotophos, and its bacterial strain is a Gram staining reaction negative bacterium m-1 (deposited in the General Microbiology Center of China Microbial Strain Preservation Management Committee on October 12, 2005) , the strain preservation number is CGMCC ^NO.1483 ), and it has been identified that it belongs to Paracoccus (Paracoccus.sp.). Enzyme, oxidase positive, can not use citrate, nitrate reduction positive, denitrification gas production.

The process of using the monocrotophos pesticide residue-degrading bacteria to produce the bacterial agent is as follows: inclined plane seed-shake flask seed-seed tank-production tank-product (the packaging dosage form is liquid bacterial agent or solid adsorption bacterial agent).

Detailed implementation steps of the present invention are:

1) Inoculate the test tube species of the monocrotophos pesticide residue-degrading bacteria m-1 in a broth medium shake flask, and culture it with shaking until the logarithmic phase;

2) Inoculate the above-mentioned cultured strain into a 500-liter seed tank according to the inoculation amount of 10% of the volume of the seed tank culture medium, and cultivate to the logarithmic growth phase. The medium formula used in the seed tank is: 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) The seed liquid is inserted into the production tank for cultivation according to the inoculum amount of 10% of the medium volume of the production tank, and the medium used in the production tank is the same as the medium of the seed tank.

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 48-60 Hour. After the fermentation is completed, the culture solution is the bacterial agent.

Beneficial effects The pesticide residue degrading bacteria 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 the national grain, oil and vegetable production and export bases or places with green food trademark signs. The invention has important significance for protecting ecological environment, protecting people's health and improving the value of agricultural products.

Degrading bacteria m-1 can reduce the residual amount of monocrotophos pesticide by more than 95% (as shown in table 1), and also degrade amide herbicides simultaneously, which greatly reduces the workload in the production and use process and reduces the production cost. and usage costs. Using the degrading bacteria agent can normally use chemical pesticides to prevent and control diseases and insect pests 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. And the bacterium is a gram-negative bacterium, and the survival time in the field will not affect the effect of pesticide use in the future.

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

4. Specific implementation

Examples of the present invention are described below.

Activate the original seed of the microbial monocrotophos pesticide residue-degrading bacteria m-1 on a petri dish, measure its degradation performance, and inoculate it on the inclined surface of a test tube for later use. The test tube seeds were inoculated into 1000ml shake flasks containing 200ml broth medium, cultured with constant temperature shaking until logarithmic phase, and prepared to inoculate seed tanks. The seed tank is 500 liters, the feeding volume is 400 liters, the medium formula is: 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 value 7.2-7.5. After the feeding is completed, 121 ° C high-pressure damp heat sterilization, after cooling to 33 ° C, inoculate the above-mentioned cultivated shake flask strains into 500 liters of seed tanks according to the inoculation amount of 10%, and cultivate to logarithm During the growth period, the stirring speed was 220 rpm, and the amount of sterile air was 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 48-60 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 peat adsorption packaging bag.

The product obtained by the above method is used for field pesticide residue degradation test demonstration. Above-mentioned bacterium is applied in monocrotophos pesticide residue soil by using method requirement, after 7 days, take the water sample that flows out from soil and measure the content of monocrotophos residue with gas chromatography and liquid chromatography (by Ministry of Agriculture Food Quality Supervision, Inspection and Testing Center (Jinan) measures), the results are shown in Table 1, and the content of monocrotophos in the contrast reaches more than 60mg/L, and monocrotophos is not detected in the processing of the inoculum agent, and the results show that degrading bacteria remove 95% of the soil The above monocrotophos residues have a good degradation effect.

Table 1. Field degradation test of monocrotophos pesticide residues by degrading bacteria M-1

Sample serial number sample name Sample traits Monocrotophos (mg/L) 2004-H-128 Water sample (control) liquid 63.4 2004-H-129 Water sample (control) liquid 61.5 2004-H-130 Water sample (control) liquid 65.4 2004-H-131 Water sample (bacteria treatment) liquid Not detected (<0.01) 2004-H-132 Water sample (bacteria treatment) liquid Not detected (<0.01) 2004-H-133 Water sample (bacteria treatment) liquid Not detected (<0.01)

Claims (2)

1. A monocrotophos pesticide residue degrading bacterium, is characterized in that, this microorganism is the bacterial strain m-1 of the genus Paracoccus (Paracoccus.sp) of gram-staining negative, on October 12th, 2005 deposits in Chinese microbial bacteria Species Preservation Management Committee General Microbiology Center, the strain preservation number is CGMCC NO.1483, the main biological characteristics are G, the bacteria are short rod-shaped, aerobic, non-moving, not salt-tolerant, positive for contact enzymes and oxidases, and cannot With citrate, nitrate reduction is positive, and denitrification has gas production. 2. A pesticide residue degrading bacterial agent produced by the monocrotophos pesticide residue degrading bacteria according to claim 1 is produced by the following method: 1) Inoculate the test tube m-1 of the monocrotophos pesticide residue-degrading bacteria test tube species m-1 described in claim 1 in an LB medium shake flask, and vibrate and cultivate to the logarithmic phase; 2) Inoculate the above-mentioned cultured bacteria into a 500-liter seed tank according to the inoculation amount of 10% of the seed tank culture medium, and cultivate to the logarithmic growth phase. The medium formula used in the seed tank is: 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) The seed liquid is inserted into the production tank for cultivation according to the inoculation amount of 10% of the production tank culture medium, and the culture medium used in the production tank is the same as the seed tank culture medium; 4) During the cultivation process of seed tanks and production tanks, the ventilation volume 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 48- After 60 hours, the number of bacterial cells reaches more than 1 billion/ml after the fermentation is completed. After the fermentation is completed, the culture solution is directly packed into liquid dosage forms in plastic barrels or bottles, or into solid bacterial preparations in peat-adsorbed packaging bags. dosage form.