CN1800353A - Bacterium for degrading phoxim pesticide residue and produced bacterium formulation - Google Patents

Abstract

The invention provides a degradation bacillus which can dispel the phoxime pesticide residue and its prodegradant. The used strain is Gram's staining reaction hysteroptosis X-12 which is Ochrobactrum.sp. The main biology characteristic is G-; the thaliana is the rod type whose size is (1.58-2.61)mu m*(0.79-1.23)mu m; it has flagellation around it and facultative anaerobes; the hydrogen peroxidase, oxidative enzyme, oxidation of ethanol and V.P. reaction is positive; the ketole reaction is negative; it can not be hydrolysed starched and oxide glucose to generate the acid so that the coagulate dydimic acid cow's milk; the Genbank landing number of the strain 16S rDNA is DQ093374.

Description

The microbial inoculum of a kind of degrading phoxim pesticide residue bacterium and production thereof

One, technical field

The microbial inoculum of a kind of degrading phoxim pesticide residue bacterium 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 reasons such as a large amount of due to illness insect pests cause has been retrieved in agriculture production, and greatly improved the efficiency and the mechanization degree of agriculture production, in the modernization of agriculture production, played immeasurable effect.But agricultural chemicals all is some toxic and harmful substances simultaneously, after they enter ecotope, has not only killed the deleterious biology to the mankind, 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.Along with the raising of people's living standard and the enhancing of 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 to bring into play enormous function as the effective means of control disease and pest in agriculture production.How solving this double-barreled question in the agriculture production just becomes the research focus of each subject.Laboratory and real application research all show pesticide residue " storehouse " capacity that utilizes microorganism to degrade to eliminate 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 (ZL96106317.3 of degradation bacteria in the prior art, publication number: CN1145408A, open day: on March 19th, 1997), this patent is a strain bacillus J5, degrading methamidophos effectively, but this strains for degrading spectrum is narrow, only can a kind of agricultural chemicals of degrading methamidophos, be restricted thereby use.

Three, summary of the invention

Technical problem the objective of the invention is at practical problems in the production practice and demand, develops out a kind of novel organic pesticide residue degrading microbial inoculum, uses this microbial inoculum can make the residual quantity of phoxim pesticide reduce more than 90%.

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

The invention provides a kind of degradation bacteria of eliminating phoxim pesticide residue, its bacterial strain is that a strain gramstaining reaction negative bacterium X-12 (was deposited at Chinese microorganism strain and preserves management committee common micro-organisms center on October 12nd, 2005, culture presevation number is CGMCC NO.1481), through identifying that belonging to the main biological characteristics of pale Bacillaceae (Ochrobactrum.sp.) is G-, thalline is shaft-like, the about 1.58 μ m of size * 0.79 μ m, peritrichous, facultative lonely; Catalase, oxydase, oxidation of ethanol and V.P. reaction are positive; The indole reaction feminine gender; Can not hydrolyzed starch, oxidizing glucose produces acid, makes the litmus milk acid cure solid, and the Genbank number of landing of this bacterial strain 16S rDNA is DQ093374.

The technology of using above-mentioned organophosphorus pesticide degradation 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) the test tube kind of the degradation bacteria X-12 of phoxim pesticide residue 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 the inoculum size of seed tank culture base 10%, be cultured to logarithmic phase.The used culture medium prescription of seeding tank is: glucose 0.8%, (NH ₄) ₂SO ₄1%, 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 inoculum size access of production jar substratum 10%, 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 48-60 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.

It is low, easy to use that the bacterium for degradating residual agricultural chemical that beneficial effect 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.

Use this degradation bacterial agent in the normal growth process of farm crop, normally to use chemical pesticide to carry out the prevention and control of plant diseases, pest control 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 production pesticide residue problem that exceeds standard, and has both given full play to the efficiently fast effect of chemical pesticide in plant pest management, can produce non-toxic and non-pollution green agricultural product again.

Four, embodiment

Below narrate embodiments of the invention.

The original seed of degrading phoxim pesticide residue bacterium X-12 of the present invention is activated on culture dish, and measure degradation property, be inoculated on the test tube slant standby.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 is: glucose 0.8%, (NH ₄) ₂SO ₄1%, 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 standby.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 48-60 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.

Product with the aforesaid method gained carries out field pesticides residue degrading experiment and demonstration.Leek sprayed phoxim pesticide after, above-mentioned microbial inoculum is imposed in the leek ground by the using method requirement, gather in the crops leek after 15～20 days, with the residual content of Volaton in gas-chromatography or the liquid chromatography for measuring leek (measuring) by Ministry of Agriculture food quality supervision verification test center (Jinan), from result's (table 1), find out, after degradation bacteria was handled, Volaton content did not detect substantially in the leek, far below the residual mean value 2.30mg/kg of the Volaton in the control group leek.

The degraded test-results of Volaton in the table 1 strain X-12 pair leek

	Numbering	1	2	3	On average
						Volaton content (mg/kg)	The control group treatment group	2.64 do not detect (＜0.02)	1.70 do not detect (＜0.02)	2.56 do not detect (＜0.02)	2.30 ± 0.52 does not detect (＜0.02)

Claims (2)

1. degrading phoxim pesticide residue bacterium, it is characterized in that, this microorganism is the strain X-12 of the pale Bacillaceae (Ochrobactrum.sp) of Gram-negative, be deposited at Chinese microorganism strain on October 12nd, 2005 and preserve management committee common micro-organisms center, culture presevation number is CGMCC NO.1481, and main biological characteristics is G ^-, thalline is shaft-like, the about 1.58 μ m of size * 0.79 μ m, and peritrichous, facultative lonely; Catalase, oxydase, oxidation of ethanol and V.P. reaction are positive; The indole reaction feminine gender; Can not hydrolyzed starch, oxidizing glucose produces acid, makes the litmus milk acid cure solid, and the Genbank number of landing of this bacterial strain 16S rDNA is DQ093374.

2. bacterium for degradating residual agricultural chemical of producing with the described degrading phoxim pesticide residue bacterium of claim 1 is to produce by the following method:

1) the described degrading phoxim pesticide residue bacterium of claim 1 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 the inoculum size of seed tank culture base 10%, be cultured to logarithmic phase, the used culture medium prescription of seeding tank is: glucose 0.8%, (NH ₄) ₂SO ₄1%, 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 inoculum size access of production jar substratum 10%, 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 48-60 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.