CN103923852A - Acidovorax sp. and application - Google Patents

Acidovorax sp. and application Download PDF

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Publication number
CN103923852A
CN103923852A CN201410083651.1A CN201410083651A CN103923852A CN 103923852 A CN103923852 A CN 103923852A CN 201410083651 A CN201410083651 A CN 201410083651A CN 103923852 A CN103923852 A CN 103923852A
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acidovorax
tsiklomitsin
shs
culture
tank
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CN103923852B (en
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马爱军
何任红
张小华
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Jiangsu Polytechnic College of Agriculture and Forestry
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Jiangsu Polytechnic College of Agriculture and Forestry
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Abstract

The invention discloses Acidovorax sp. SHS-01 which is collected in China General Microbiological Culture Collection Center. The collection number is CGMCC No.8388, and the collection date is Oct. 24th, 2013. The Acidovorax sp. provided by the invention has wide degradation spectrum, and can be used to reduce residual quantity of tetracycline in excrements of livestocks by more than 80%. Thus, compost maturity is promoted; the problem that effective composting is hard to carry out due to an inhibiting effect of tetracycline in excrements of livestocks to microorganisms is successfully solved; workload during production and use processes is greatly reduced; and production and use costs are decreased. The application of Acidovorax sp. is of great significance for protection of ecological environment and promotion of human health. In addition, the Acidovorax sp. SHS-01 has advantages of simple culture condition, low production and use cost, easy preservation and convenient usage, is easy for industrial production, and has a good development and application prospect.

Description

A kind of acidovorax facilis and application thereof
Technical field
The invention belongs to environment and biological technical field, relate in particular to a kind of acidovorax facilis (Acidovorax sp.) SHS-01 and the application in tsiklomitsin degraded thereof.
Background technology
Microbiotic has kill microorganisms, suppresses microbial reproduction or growth, the effects such as disease, thereby a large amount of feeding antibiotics is used as fodder additives prevention and treats bacterially, is referred to as antimicrobial growth promotor.Microbiotic, in livestock and poultry body, only has small part to generate non-activity product through metabolic reactions such as the base that kicks the beam, cracking and the general acidifyings of glucose, and major part still excretes with fecaluria with original shape, so, in feces of livestock and poultry, generally contain antibiotics material.
Tsiklomitsin is in current livestock and poultry cultivation, to use one of the most general microbiotic, is a kind of Broad spectrum antibiotics.Correlative study demonstration, the tsiklomitsin concentration in ight soil can reach 200mg/kg.Before ight soil is used, it is processed to reduce antibiotic content, by the environmental risk that effectively reduces tsiklomitsin and may bring.Feces of livestock and poultry is carried out to compost treatment, and the advantage such as have that cost is low, efficiency is high, non-secondary pollution, ecological recovery are good, is significant for the compost of locating containing tsiklomitsin animal dung so filter out tsiklomitsin degradation bacteria.
Summary of the invention
Goal of the invention: in order to overcome the deficiencies in the prior art, the first object of the present invention is to provide a kind of acidovorax facilis tsiklomitsin to Degradation.
The second object of the present invention is to provide the application of a kind of acidovorax facilis in tsiklomitsin degraded.
Technical scheme: in order to solve above-mentioned the first object, the technical solution adopted in the present invention is: a kind of acidovorax facilis, its Classification And Nomenclature is acidovorax facilis (Acidovorax sp.) SHS-01, be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center (being called for short CGMCC), depositary institution address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode is 100101, deposit number is: CGMCC No.8388, preservation date is on October 24th, 2013.
In order to solve the second above-mentioned object, the technical solution adopted in the present invention is: the application of described acidovorax facilis in tsiklomitsin degraded.
Have particular application as:
(1) the test tube kind of the residual acidovorax facilis SHS-01 of tsiklomitsin that degrades is inoculated in LB substratum, shaking culture is to logarithmic phase;
(2) the cultured bacterial classification of step (1) is inoculated to the seeding tank into 500L by 10% inoculum size, the air flow of sterile air is 1:0.6-1:1.2, and stirring velocity is 180-240r/min, culture temperature is 30-35 ℃, incubation time is 60h, is cultured to logarithmic phase, obtains seed liquor;
(3) the resulting seed liquor of step (2) is produced to tank by 10% inoculum size access and carry out fermentation culture, produce tank used medium identical with seed tank culture base, the air flow of sterile air is 1:0.6-1:1.2, stirring velocity is 180-240r/min, culture temperature is 30-35 ℃, incubation time is 60h, and the nutrient solution after having fermented is the microbial inoculum being applied in tsiklomitsin degraded.
Further, in described step (1), the formula of LB substratum is: 1000mL distilled water, yeast powder 5.0g, peptone 10.0g, sodium-chlor 10.0g, pH7.0.
Further, in described step (2), the formula of seeding tank used medium is: glucose 0.8%, (NH 4) 2sO 41%, K 2hPO 40.2%, MgSO 40.05%, NaCl0.01%, CaCO 30.3%, yeast extract paste 0.02%, pH value 7.2-7.5.
The physiologically active feature of acidovorax facilis described in the present invention (Acidovorax sp.) SHS-01 is as follows:
Described acidovorax facilis (Acidovorax sp.) SHS-01 Main Biological is G-, and logarithmic phase thalline is rod-short, this bacterium energy liquefy gelatin, and hydrolyzation of glucose, fructose, lactose and maltose, can not be hydrolyzed N.F,USP MANNITOL, ribose, wood sugar and sucrose.
Beneficial effect: compared with prior art, advantage of the present invention is:
(1) a kind of acidovorax facilis disclosed by the invention (Acidovorax sp.) SHS-01 has wider degraded spectrum, can make the residual quantity of tsiklomitsin in feces of livestock and poultry reduce more than 80%, greatly reduce the workload in production and use procedure, reduced production and use cost.It is low that prepared microbial inoculum has production and application cost, easy to use, and the advantage that removal effect is good is adapted at using in livestock excrement composting process.The present invention is for preserving the ecological environment, and the healthy of to protect mankind has great importance.Thereby using this microbial inoculum can be the obvious degree of becoming thoroughly decomposed that promotes compost that reduces of tsiklomitsin content in feces of livestock and poultry.The present invention has successfully solved and has contained in feces of livestock and poultry owing to containing tsiklomitsin, microorganisms restraining effect being difficult to carry out the problem of effective compost.
(2) acidovorax facilis (Acidovorax sp.) SHS-01 culture condition is simple, production and application cost is low, easily preserves, easy to use, is easy to suitability for industrialized production, has good development prospect.
Accompanying drawing explanation
Fig. 1 is bacterial strain SHS-01 thalline violet staining photo of the present invention (1000x);
Fig. 2 is the shaking flask Degrading experiment result schematic diagram of bacterial strain SHS-01 of the present invention to tsiklomitsin;
Fig. 3 is that the present invention uses bacterial strain SHS-01 to carry out composting test to tsiklomitsin degraded situation schematic diagram.
Embodiment
Below in conjunction with experimental example, elaborate the application of acidovorax facilis of the present invention (Acidovorax sp.) SHS-01.
The screening of acidovorax facilis (Acidovorax sp.) SHS-01, separation and evaluation:
(1) getting the active sludge 3.0mL obtaining from produce the purification tank for liquid waste of certain insecticide factory's production plant of tsiklomitsin, to add tsiklomitsin concentration be that during the 100mL inorganic salt liquid of 100mg/L is cultivated, its formula is: 1000mL distilled water, 1.50g K 2hPO 4, 0.50g KH 2pO 4, 0.20g MgSO 47H 2o, 1.00g NaCl, 1.00g (NH 4) 2sO 4, pH7.0, in 160r/min, shaking culture under 30 ℃ of conditions, was transferred in fresh minimal medium by 3% inoculum size every 5 days, transferred continuously 5 times;
(2) get enrichment bacterium liquid 1.0mL obtained above, add in 9.0mL sterilized water, be made into 10 -1pregnant solution, then draw that 1.0mL prepares 10 -1pregnant solution add in 9.0mL sterilized water, fully mix and be made into 10 -2pregnant solution, by that analogy, pregnant solution is carried out to gradient dilution.Drawing the concentration that the diluent 0.1mL of each gradient coats containing tsiklomitsin is that (culture medium prescription is 100mg/L inorganic salt solid medium: 1000mL distilled water, 1.50g K 2hPO 4, 0.50gKH 2pO 4, 0.20g MgSO 47H 2o, 1.00g NaCl, 1.00g (NH 4) 2sO 4, 20.00g agar, pH7.0) upper, cultivate 7 days for 30 ℃;
After (3) 7 days from above-mentioned inorganic salt solid medium picking list bacterium colony, in the LB of 3.0mL liquid nutrient medium, cultivate after 24 hours, LB liquid culture based formulas is: 1000mL distilled water, NaCl10.00g, peptone 10.00g, yeast powder 5.00g, pH7.0 centrifugal 2min under the condition of 8000r/min, remove supernatant liquor, add the sterilized water of 3.0mL to shake up, centrifugal 2min under the condition of 8000r/min still, according to said method use after twice of aseptic washing, add resuspended this bacterium of 3.0mL sterilized water.Draw this bacterium liquid of 1.0mL and add in the inorganic salt liquid substratum that 100mL tsiklomitsin concentration is 100mg/l, in 160r/min, under 30 ℃ of conditions, shaking culture, after 7 days, is surveyed its degradation effect with high performance liquid chromatography.The strain bacterial strain that degradation efficiency is higher is preserved, carried out subsequent experimental;
(4) degradation bacteria strains is inoculated on LB solid medium, its formula is: 1000mL distilled water, yeast powder 5.0g, peptone 10.0g, sodium-chlor 10.0g, pH7.0; After 30 ℃ of constant temperature culture 48h, observe colonial morphology, with observation by light microscope thalli morphology (1000X).The Physiology and biochemistry of bacterial strain is identified and is carried out according to < < common bacteria system identification handbook > >.Be accredited as SHS-01 and belong to (Acidovorax sp.); Called after: SHS-01.As shown in Figure 1, Main Biological is G-to its violet staining photo, and logarithmic phase thalline is rod-short, this bacterium energy liquefy gelatin, and hydrolyzation of glucose, fructose, lactose and maltose, can not be hydrolyzed N.F,USP MANNITOL, ribose, wood sugar and sucrose.
Acidovorax facilis (Acidovorax sp.) SHS-01 is carried out to shake flask test, checks its degradation effect to tsiklomitsin:
In containing the minimal medium of 100mg/L tsiklomitsin, its formula is: 1000mL distilled water, 1.50g K 2hPO 4, 0.50g KH 2pO 4, 0.20g MgSO 47H 2o, 1.00g NaCl, 1.00g (NH 4) 2sO 4, pH7.0, the inoculum size inoculation SHS-01 by 3%, in 30 ℃ of shaking culture, 48 hours sampling and measuring.Compare with blank, as shown in Figure 2, the absorption peak of processing sample obviously declines, and tsiklomitsin is degraded more than 90%.
The application of acidovorax facilis (Acidovorax sp.) SHS-01 in tsiklomitsin degraded:
Embodiment 1:
(1) original seed of acidovorax facilis SHS-01 is activated on culture dish, and measure degradation property, be inoculated on test tube slant standby.Test tube kind is inoculated in the 1000ml shaking flask containing 200ml LB substratum, and constant-temperature shaking culture is to logarithmic phase; Wherein the formula of LB substratum is: 1000mL distilled water, yeast powder 5.0g, peptone 10.0g, sodium-chlor 10.0g, pH7.0;
(2) prepare inoculation seeding tank, 500 liters of seeding tanks, 400 liters of charging capacitys, wherein culture medium prescription is: glucose 0.8%, (NH 4) 2sO 41%, K 2hPO 40.2%, MgSO 40.05%, NaCl0.01%, CaCO 30.3%, yeast extract paste 0.02%, pH value 7.2.121 ℃ of high pressure moist heat sterilizations after feeding intake, are cooled to after 30 ℃, cultivate 60h, above-mentioned cultured shaking flask bacterial classification is inoculated into 500 liters of seeding tanks by 10% inoculum size, be cultured to logarithmic phase, stirring velocity is 180r/min, and sterile air intake is 1:0.6;
(3) seed liquor that arrives logarithmic phase is produced to tank by 10% inoculum size access and cultivate, produce tank used medium composition identical with seed tank culture base.Produce 5 tons of tankages, 4.5 tons of charging capacitys.Production tank 1.1kg/cm after feeding intake 2pressure under, 121 ℃ of high pressure moist heat sterilizations, are cooled to after sterilizing below 35 ℃, logical sterile air keeps sterile state standby.Postvaccinal production tank temperature is controlled at 30 ℃, and in the culturing process of production tank, the air flow of sterile air is 1:0.6, and stirring velocity is 180r/min, cultivates 60h.After fermentation ends thalline quantity reach 1,000,000,000/more than mL, be the microbial inoculum being applied in tsiklomitsin degraded.The rear nutrient solution that fermented goes out tank and directly by plastic barrel or packing bottle, is distributed into liquid dosage form or adopts adsorption by peat to be distributed into solid fungicide formulation with packing bag.
Embodiment 2:
(1) original seed of acidovorax facilis SHS-01 is activated on culture dish, and measure degradation property, be inoculated on test tube slant standby.Test tube kind is inoculated in the 1000ml shaking flask containing 200ml LB substratum, and constant-temperature shaking culture is to logarithmic phase; Wherein the formula of LB substratum is: 1000mL distilled water, yeast powder 5.0g, peptone 10.0g, sodium-chlor 10.0g, pH7.0;
(2) prepare inoculation seeding tank, 500 liters of seeding tanks, 400 liters of charging capacitys, wherein culture medium prescription is: glucose 0.8%, (NH 4) 2sO 41%, K 2hPO 40.2%, MgSO 40.05%, NaCl0.01%, CaCO 30.3%, yeast extract paste 0.02%, pH value 7.5.121 ℃ of high pressure moist heat sterilizations after feeding intake, are cooled to after 35 ℃, cultivate 60h, above-mentioned cultured shaking flask bacterial classification is inoculated into 500 liters of seeding tanks by 10% inoculum size, be cultured to logarithmic phase, stirring velocity is 240r/min, and sterile air intake is 1:1.2;
(3) seed liquor that arrives logarithmic phase is produced to tank by 10% inoculum size access and cultivate, produce tank used medium composition identical with seed tank culture base.Produce 5 tons of tankages, 4.5 tons of charging capacitys.Production tank 1.1kg/cm after feeding intake 2pressure under, 121 ℃ of high pressure moist heat sterilizations, are cooled to after sterilizing below 35 ℃, logical sterile air keeps sterile state standby.Postvaccinal production tank temperature is controlled at 35 ℃, and in the culturing process of production tank, the air flow of sterile air is 1:1.2, and stirring velocity is 240r/min, cultivates 60h.After fermentation ends thalline quantity reach 1,000,000,000/more than mL, be the microbial inoculum being applied in tsiklomitsin degraded.The rear nutrient solution that fermented goes out tank and directly by plastic barrel or packing bottle, is distributed into liquid dosage form or adopts adsorption by peat to be distributed into solid fungicide formulation with packing bag.
Embodiment 3:
(1) original seed of acidovorax facilis SHS-01 is activated on culture dish, and measure degradation property, be inoculated on test tube slant standby.Test tube kind is inoculated in the 1000ml shaking flask containing 200ml LB substratum, and constant-temperature shaking culture is to logarithmic phase; Wherein the formula of LB substratum is: 1000mL distilled water, yeast powder 5.0g, peptone 10.0g, sodium-chlor 10.0g, pH7.0;
(2) prepare inoculation seeding tank, 500 liters of seeding tanks, 400 liters of charging capacitys, wherein culture medium prescription is: glucose 0.8%, (NH 4) 2sO 41%, K 2hPO 40.2%, MgSO 40.05%, NaCl0.01%, CaCO 30.3%, yeast extract paste 0.02%, pH value 7.4.121 ℃ of high pressure moist heat sterilizations after feeding intake, are cooled to after 33 ℃, cultivate 60h, above-mentioned cultured shaking flask bacterial classification is inoculated into 500 liters of seeding tanks by 10% inoculum size, be cultured to logarithmic phase, stirring velocity is 220r/min, and sterile air intake is 1:0.9;
(3) seed liquor that arrives logarithmic phase is produced to tank by 10% inoculum size access and cultivate, produce tank used medium composition identical with seed tank culture base.Produce 5 tons of tankages, 4.5 tons of charging capacitys.Production tank 1.1kg/cm after feeding intake 2pressure under, 121 ℃ of high pressure moist heat sterilizations, are cooled to after sterilizing below 35 ℃, logical sterile air keeps sterile state standby.Postvaccinal production tank temperature is controlled at 33 ℃, and in the culturing process of production tank, the air flow of sterile air is 1:0.9, and stirring velocity is 220r/min, cultivates 60h.After fermentation ends thalline quantity reach 1,000,000,000/more than mL, be the microbial inoculum being applied in tsiklomitsin degraded.The rear nutrient solution that fermented goes out tank and directly by plastic barrel or packing bottle, is distributed into liquid dosage form or adopts adsorption by peat to be distributed into solid fungicide formulation with packing bag.
Acidovorax facilis (Acidovorax sp.) SHS-01 is the application to tsiklomitsin degraded in composting test:
Compost adopts fresh cattle ight soil, and choosing proportioning raw materials is cow dung: straw section: straw powder: tap water is 17.6:0.36:0.23:0.45, and the C/N in this raw material is 25:1, and moisture control is 75%, and tsiklomitsin content is 50mg/kg.Microbial inoculum prepared by above-described embodiment 1-3 and being seeded in heap body through the inoculum size of sterilising treatment (121 ℃, the microbial inoculum that 30min) prepared by above-described embodiment 1-3 with 0.5%(mass ratio), starts to bank up after mixing.After 50 days composting process, measure respectively tsiklomitsin content in fertile heap, as shown in Figure 3, after the microbial inoculum 50d that sterilized is processed, the degradation rate of tsiklomitsin is reached more than 80%, and after microbial inoculum 50d after sterilising treatment to the degradation rate of tsiklomitsin 30% left and right only, result shows, a kind of acidovorax facilis of the present invention (Acidovorax sp.) SHS-01 can be directly used in livestock excrement composting tsiklomitsin is degraded, and has successfully solved and has contained in feces of livestock and poultry owing to containing tsiklomitsin, microorganisms restraining effect being difficult to carry out the problem of effective compost.

Claims (5)

1. an acidovorax facilis, its Classification And Nomenclature is acidovorax facilis (Acidovorax sp.) SHS-01, is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is: CGMCC No.8388, preservation date is on October 24th, 2013.
2. the application of acidovorax facilis claimed in claim 1 in tsiklomitsin degraded.
3. application according to claim 2, is characterized in that, adopts following Production Flow Chart to obtain being applied to the acidovorax facilis in tsiklomitsin degraded:
(1) the test tube kind of the residual acidovorax facilis SHS-01 of tsiklomitsin that degrades is inoculated in LB substratum, shaking culture is to logarithmic phase;
(2) the cultured bacterial classification of step (1) is inoculated to the seeding tank into 500L by 10% inoculum size, the air flow of sterile air is 1:0.6-1:1.2, and stirring velocity is 180-240r/min, culture temperature is 30-35 ℃, incubation time 60h, is cultured to logarithmic phase, obtains seed liquor;
(3) the resulting seed liquor of step (2) is produced to tank by 10% inoculum size access and carry out fermentation culture, produce tank used medium identical with seed tank culture base, the air flow of sterile air is 1:0.6-1:1.2, stirring velocity is 180-240r/min, culture temperature is 30-35 ℃, incubation time 60h, the nutrient solution after having fermented is the microbial inoculum being applied in tsiklomitsin degraded.
4. application according to claim 3, is characterized in that: in described step (1), the formula of LB substratum is: 1000mL distilled water, yeast powder 5.0g, peptone 10.0g, sodium-chlor 10.0g, pH7.0.
5. application according to claim 3, is characterized in that: in described step (2), the formula of seeding tank used medium is: glucose 0.8%, (NH 4) 2sO 41%, K 2hPO 40.2%, MgSO 40.05%, NaCl0.01%, CaCO 30.3%, yeast extract paste 0.02%, pH value 7.2-7.5.
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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103468598A (en) * 2012-11-07 2013-12-25 上海大学 Humic acid degrading strain, and screening method and application method thereof

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103468598A (en) * 2012-11-07 2013-12-25 上海大学 Humic acid degrading strain, and screening method and application method thereof

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
WATARU WATANABE等: "Bacterial Degradation and Reduction in the Estrogen activity of 4-nonylphenol", 《BIOCONTROL SCIENCE》 *
YOSHIYUKI OHTSUBO 等: "Complete Genome Sequence of Acidovorax sp. Strain KKS102, a Polychlorinated-Biphenyl Degrader", 《JOURNAL OF BACTERIOLOGY》 *
沈颖 等: "猪粪中四环素类抗生素残留物的生物降解", 《过程工程学报》 *

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