CN101348771B

CN101348771B - Imazethapyr pesticide residue degrading bacterial and inocula produced therefrom

Info

Publication number: CN101348771B
Application number: CN2007101332659A
Authority: CN
Inventors: 李顺鹏; 潘继杰; 黄星; 洪青
Original assignee: Nanjing Agricultural University
Current assignee: Nanjing Agricultural University
Priority date: 2007-09-26
Filing date: 2007-09-26
Publication date: 2012-05-23
Anticipated expiration: 2027-09-26
Also published as: CN101348771A

Abstract

The invention provides a degrading microbial inoculum for removing imazethapyr pesticide residue. The strain used is Gram-negative bacteria IM-4 which is identified to be Pseudomonas sp. The IM-4 strain is inoculated into a seed tank according to the inoculum size and cultured to the logarithmic phase; seed solution is inoculated into a productive tank for culture, and a culture medium used by the productive tank is the same as that used by the seed tank; and the air flow is 1 to between 0.6 and 1.2, the stirring speed is between 180 and 240 turns per minute, the culture temperature is between 30 and 35 DEG C, the culture time of the whole technological flow is between 36 and 48 hours, and then the microbial inoculum is obtained after the culture solution is fermented. Application of the degrading bacterium product can reduce the residue amount of imazethapyr in the soil by more than 65 percent and reduce the residue amount of the imazethapyr in 5 parts per thousand of an LB liquid medium by more than 85 percent. Therefore, the problem that the imazethapyr pesticide residue exceeds standard during the agricultural production process can be solved and nontoxic nuisanceless green agricultural products can be produced.

Description

The microbial inoculum of a kind of Imazethapyr pesticide residual degrading bacteria and production thereof

One, technical field

The microbial inoculum of a kind of Imazethapyr pesticide residual degrading bacteria of the present invention and production thereof belongs to biological high-tech field, is to utilize method of microorganism degraded chemical pesticide residual, is applicable to the production and the processing of green non-polluted farm product in the modern agriculture production.

Two, technical background

The use of chemical pesticide has promoted modern agricultural development widely; For the loss that causes because of reasons such as crop smotherings has in a large number been retrieved in agriculture prodn; And greatly improved the efficiency and the mechanization degree of agriculture prodn, in the modernization of agriculture prodn, played immeasurable effect.But agricultural chemicals all is some toxic and harmful substances simultaneously, and they have not only killed the deleterious biology to the mankind after getting into ecotope, also the ecosystem has been caused serious harm simultaneously.A large amount of uses of agricultural chemicals cause that pesticide residue exceed standard in the agricultural-food, have had a strong impact on the foreign exchange earning of people health and agricultural-food.The enhancing of Along with people's growth in the living standard and environmental protection consciousness, the whole society also more and more pays close attention to direct harm and potential impact that pesticide residue are brought, and people press for the green agricultural product of the cleaning of non agricultural chemical residuum pollution.But agricultural form and demographic situation from present China; Use to stop agricultural chemicals that to reduce farm output be impracticable as the nuisanceless production model of cost, agricultural chemicals also will continue in agriculture prodn, to bring into play enormous function as the effective means of control crop smothering.How solving this double-barreled question in the agriculture prodn just becomes the research focus of each subject.Laboratory and real application research show that all utilizing mikrobe to degrade eliminates pesticide residue " storehouse " capacity in the soil, thereby reduce the pesticide residue in the agricultural-food such as grain, vegetables, tealeaves, and the quality and the economic worth that improve agricultural-food are feasible.This seminar has had the patent of invention of degradation bacteria (open day: on March 19th, 1997), this patent was strain bacillus J5, a degrading methamidophos effectively for ZL96106317.3, publication number: CN1145408A in the prior art.

Three, summary of the invention

Technical problem the objective of the invention is to develop out a kind of novel bacterium for degradating residual agricultural chemical to practical problems and demand in the production practice, and the residual quantity of Imazethapyr is reduced, and production and use cost are lower.Use this degradation bacterial agent in the normal growth process of farm crop, normally to use chemical pesticide to carry out prevention and control of weeds and guarantee that the agricultural chemicals residual content meets the green food requirement in the agricultural-food.

Be main contents of the present invention below the technical scheme:

The present invention provides a kind of degradation bacterial agent of eliminating the Imazethapyr pesticide residue; Bacterial strain uses therefor is Gram-negative bacterium IM-4; Be deposited at Chinese microorganism strain on June 18th, 2007 and preserve management committee common micro-organisms center; Culture presevation number is: CGMCC NO.2093, and through being accredited as Rhodopseudomonas (Pseudomonas sp.).Main biological characteristics is G ^-, thalline is a rod-short, and size is 2.2 * 0.75 μ m, and oxydase, catalase, nitrate reduction, Hydrocerol A utilization react positive; Lipase reaction V.P. reaction, M.R react, indole reaction is negative; Can not hydrolyzed starch.

The technology of using above-mentioned Imazethapyr weedicide pesticide residual degrading bacteria to produce microbial inoculum is: inclined-plane kind-shake bottle a kinds-seeding tank-production jar-product (pack formulation and be liquid bacterial agent or solid absorption microbial inoculum).

Detailed implementation step of the present invention is:

1. Imazethapyr pesticide residual degrading bacteria IM-4 test tube kind is inoculated in broth culture and shakes in the bottle, shaking culture is to logarithmic phase;

2. above-mentioned cultured bacterial classification is inoculated into 500 liters of seeding tanks by 10% inoculum size of seed tank culture base, be cultured to logarithmic phase, the culture medium prescription (mass percent) that seeding tank is used: 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 10% inoculum size access of production jar substratum, it is identical with the seed tank culture base to produce the used substratum of jar;

The air flow of sterile air is 1 in the culturing process of seeding tank and production jar: 0.6-1.2, and stirring velocity is 180-240 rev/min, and culture temperature is 30-35 ℃, and the whole process flow incubation time is 36-48 hour.Nutrient solution was microbial inoculum after fermentation was accomplished.

Beneficial effect

It is low, easy to use that the bacterium for degradating residual agricultural chemical that uses this invention to produce has the production use cost, and the advantage that removal effect is good is adapted at national grain oil & vegetable production export base or has the local big area of green food brand mark to promote the use of.The present invention protects people health for preserving the ecological environment, and near the value that improves agricultural-food has great importance.

Evidence, degradation bacteria IM-4 can make the residual quantity of Imazethapyr in the soil reduce more than 65%, can make the residual quantity of 5 ‰ LB liquid nutrient medium Imazethapyrs reduce more than 85%.Use this degradation bacterial agent in the normal growth process of farm crop, normally to use chemical pesticide to carry out prevention and control of weeds and guarantee that the agricultural chemicals residual content meets the green food requirement in the agricultural-food.And this bacterium is a Gram-negative bacteria, the effect that agricultural chemicals uses after the survival time in field suitably can not influence.

The present invention has successfully solved in the agriculture prodn Imazethapyr pesticide residue problem that exceeds standard, and has both given full play to the efficiently fast effect of chemical pesticide in prevention and control of weeds, can produce non-toxic and non-pollution green agricultural product again.

Four, embodiment

From the agricultural land soil that receives the Imazethapyr chronic pollution, be separated to Imazethapyr efficient degrading bacteria IM-4, through being accredited as Rhodopseudomonas (Pseudomonas sp.).Be preserved in Chinese microorganism strain on June 18th, 2007 and preserve management committee common micro-organisms center, culture presevation number is: CGMCC NO.2093.

Degradation bacteria IM-4 degradation characteristic is measured in the 5 ‰ LB liquid nutrient mediums: the single bacterium colony of picking IM-4 is in 5ml LB liquid nutrient medium, and 30 ℃, 165r/min shaking culture 24h obtain fresh bacterium liquid.In the 50mg/L of 100ml Imazethapyr 5 ‰ LB liquid nutrient mediums, the bacterium liquid after the centrifugal suspension of access 5ml is cultivated (170r/min) in 30 ℃ of shaking tables; Timing sampling is measured; Measure the content of Imazethapyr, the result sees table 1, and degradation bacteria IM-4 degraded Imazethapyr reaches 85.42% in the 5 ‰ LB liquid nutrient mediums.

Degraded in soil bacterium Imazethapyr degradation characteristic is measured: take by weighing 1000g soil, add Imazethapyr, make that to contain concentration in the soil be 10mg/kg; Add IM-4 bacterium liquid; Mixing places constant temperature culture under 30 ℃ of incubator dark conditions, and timing sampling is measured the residual quantity of Imazethapyr in soil; The result sees table 1, and degraded in soil bacterium IM-4 degraded Imazethapyr reaches 66.70%.

Table 1: bacterial strain IM-4 in 5 ‰ LB liquid nutrient medium sweetenings of the soil to the degradation characteristic of Imazethapyr

Substratum	Soil	5‰LB
			Degradation rate (%)	66.70	85.42

With original seed activation on petridish of the degradation bacteria IM-4 of weedicide pesticide residue of the present invention, and measure degradation property, be inoculated on the test tube slant subsequent use.The test tube kind is inoculated in and contains 200ml broth culture 1000ml and shake in the bottle, and constant-temperature shaking culture is prepared the inoculation seeding tank to logarithmic phase.500 liters of seeding tanks, 400 liters of charging capacitys, culture medium prescription (mass percent): 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.The back 121 ℃ of high pressure moist heat sterilizations that finish that feed intake, be cooled to 33 ℃ after, the above-mentioned cultured bottle bacterial classification that shakes is inoculated into 500 liters of seeding tanks by 10% inoculum size, be cultured to logarithmic phase, stirring velocity is 220 rev/mins, sterile air feeding amount is 1: 0.8.The seed liquor that arrives logarithmic phase is produced jar by 10% inoculum size access cultivate, it is identical with the seed tank culture base to produce the used medium component of jar.Produce 5 tons of tankages, 4.5 tons of charging capacitys.Production jar 1.1kg/cm after feeding intake ²Pressure under, 121 ℃ of high pressure moist heat sterilizations, below the sterilization postcooling to 35 ℃, logical sterile air keeps sterile state subsequent use.A postvaccinal production jar temperature is controlled at 30-35 ℃, and the air flow of sterile air is 1 in the culturing process of production jar: 0.8-1.0, and stirring velocity is 240 rev/mins, the whole process flow incubation time is 36-48 hour.After the fermentation ends thalline quantity reach 1,000,000,000/more than the ml.

Fermentation is accomplished the back nutrient solution and is gone out jar and directly be distributed into liquid dosage form or adopt peat to adsorb with plastic barrel or packing bottle and be distributed into the solid fungicide formulation with packing bag.The residual quantity that can make Imazethapyr in the soil of using of degradation bacteria product reduces more than 65%.

Claims

1. Imazethapyr pesticide residual degrading bacteria; Bacterial strain IM-4 for the Pseudomonas (Pseudomonassp.) of Gram-negative; Be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on June 18th, 2007, culture presevation number is: CGMCC NO.2093.