CN109082391B - Controlled-release agricultural microbial inoculum - Google Patents
Controlled-release agricultural microbial inoculum Download PDFInfo
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- CN109082391B CN109082391B CN201810899582.XA CN201810899582A CN109082391B CN 109082391 B CN109082391 B CN 109082391B CN 201810899582 A CN201810899582 A CN 201810899582A CN 109082391 B CN109082391 B CN 109082391B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/30—Microbial fungi; Substances produced thereby or obtained therefrom
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
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- Plant Pathology (AREA)
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Abstract
The invention provides a controlled-release agricultural microbial inoculum which comprises microorganisms, a binder, dopamine or salt thereof and high polymer molecules. According to the invention, dopamine is attached to the surfaces of agricultural microbial inoculum particles, and then polymer molecules with environmental sensitivity are coated on the surfaces of the dopamine, so that the release amount of live bacteria coated in the dopamine can be automatically adjusted according to the change of environmental conditions, and the dopamine is a novel intelligent microbial inoculum capable of improving the utilization efficiency of the microbial inoculum. The controlled-release agricultural microbial inoculum can effectively release live bacteria, thereby inhibiting the activity of harmful microorganisms, improving the disease resistance and stress resistance of crops and sustainably improving the soil fertility condition. The controlled-release agricultural microbial inoculum has simple preparation process and environmental protection, does not use organic solvent, and is suitable for industrial mass production and application.
Description
Technical Field
The invention belongs to the technical field of development of agricultural microbial inoculum, and particularly relates to a controlled-release agricultural microbial inoculum.
Background
The application of the microbial agent can obviously increase the amount of microorganisms in soil, the increased activities of the microorganisms can promote the enhancement of the activity of soil enzymes and decompose and release insoluble mineral nutrients in the soil, and meanwhile, the microorganisms can secrete plant hormones so as to promote the growth of crops.
The use of the microbial agent greatly reduces the using amount of chemical fertilizers and pollution, and can enhance the disease resistance and stress resistance of plants through the interaction between microorganisms or plants, which has important significance for improving the quality and yield of crops and prolonging the harvest period and natural preservation period of agricultural products. The microbial agent is safe and convenient to use, low in price and free of environmental pollution, and is a new preparation for replacing chemical fertilizers in the future.
Disclosure of Invention
The invention provides a controlled-release agricultural microbial inoculum, which is a novel intelligent microbial inoculum capable of improving the utilization efficiency of the microbial inoculum and can automatically adjust the release amount of live bacteria wrapped in the dopamine according to the change of environmental conditions by attaching the dopamine on the surface of agricultural microbial inoculum particles and then coating the dopamine on the surface of the dopamine by polymer molecules with environmental sensitivity.
An object of the present invention is to provide a microbial agent comprising a microorganism, a binder, dopamine or a salt thereof, and a high polymer molecule.
Specifically, the microbial inoculum further comprises at least one of the following 1) to 4):
1) the binder comprises nutrients that provide the microorganisms with their metabolism or reproduction; specifically, the binder comprises starch and/or water;
2) the microorganisms include those that are beneficial for plant growth; specifically, the microorganism comprises bacillus subtilis, yeast and/or lactobacillus; more specifically, when the microorganism comprises bacillus subtilis, yeast and lactic acid bacteria, the mass ratio of the bacillus subtilis, the yeast and the lactic acid bacteria is 1: 1; specifically, the plant includes a crop;
3) the high polymer molecules comprise temperature sensitive high polymer molecules; specifically, the high polymer molecules include PNIPAm polymers;
4) the dopamine or the salt thereof covers the surfaces of the adhesive and the microorganism, and the high polymer molecules cover the surfaces of the dopamine or the salt thereof; specifically, the binder includes nutrients that can provide the microorganisms with their metabolism or reproduction; more specifically, the binder comprises starch and/or water; specifically, the microorganism comprises bacillus subtilis, yeast and/or lactobacillus; more specifically, when the binder includes starch and water, and when the microorganism includes bacillus subtilis, yeast and lactic acid bacteria, the mass ratio of the bacillus subtilis, the yeast, the lactic acid bacteria, the starch and the water is 2: 1.
Specifically, the microbial inoculum is prepared by the following method:
mixing and granulating microorganisms and a binder, stirring the mixture in Tris-HCl buffer solution with the pH value of 8-8.5 for more than 2 hours at room temperature together with dopamine hydrochloride, performing suction filtration, placing the product in a reaction container, adding ultrapure water, and introducing N2Adding monomer, cross-linking agent and catalyst of high-polymer molecule into the solution, and introducing N continuously in dark2Stirring at room temperature, transferring the reaction container to an ice bath, stirring, adding an initiator, transferring to a closed mold after stirring, polymerizing for 24 hours at room temperature, and crushing a product to obtain the high-performance high-temperature-resistant high-performance high-;
specifically, in the method, by mass, 1-10 parts of microorganisms, 1-10 parts of a binder, 1-10 parts of dopamine hydrochloride, 10-500 parts of monomers of high polymer molecules and 0.8-200 parts of a cross-linking agent are used;
more specifically, in the method, by mass, 7.5 parts of microorganisms, 2.5 parts of a binder, 1 part of dopamine hydrochloride, 12-60 parts of monomers of high polymer molecules and 1-20 parts of a cross-linking agent are used;
specifically, the binder comprises starch and/or water; specifically, the microorganism comprises bacillus subtilis, yeast and/or lactobacillus; more specifically, when the binder includes starch and water, and when the microorganism includes bacillus subtilis, yeast and lactic acid bacteria, the mass ratio of the bacillus subtilis, the yeast, the lactic acid bacteria, the starch and the water is 2: 1.
Still another object of the present invention is to provide a method for preparing a microbial inoculum, which comprises: mixing and granulating microorganisms and a binder, stirring the mixture in Tris-HCl buffer solution with the pH value of 8-8.5 for more than 2 hours at room temperature together with dopamine hydrochloride, performing suction filtration, placing the product in a reaction container, adding ultrapure water, and introducing N2Adding monomer, cross-linking agent and catalyst of high-polymer molecule into the solution, and introducing N continuously in dark2Stirring at room temperature, transferring the reaction container to an ice bath, stirring, adding an initiator, transferring to a closed mold after stirring, polymerizing for 24 hours at room temperature, and crushing the product to obtain the high-performance low-temperature-resistant high-performance low-temperature-resistant high-.
Specifically, the method further comprises at least one of the following 1) to 6):
1) the binder comprises nutrients that provide the microorganisms with their metabolism or reproduction; specifically, the binder comprises starch and/or water;
2) the microorganisms include those that are beneficial for plant growth; specifically, the microorganism comprises bacillus subtilis, yeast and/or lactobacillus; more specifically, when the microorganism comprises bacillus subtilis, yeast and lactic acid bacteria, the mass ratio of the bacillus subtilis, the yeast and the lactic acid bacteria is 1: 1; specifically, the plant includes a crop;
3) the high polymer molecules comprise temperature sensitive high polymer molecules; specifically, the high polymer molecules include PNIPAm polymers;
4) according to the mass, in the method, 1-10 parts of microorganism, 1-10 parts of binder, 1-10 parts of dopamine hydrochloride, 10-500 parts of monomer of high polymer molecule and 0.8-200 parts of cross-linking agent are used;
specifically, in the method, by mass, 7.5 parts of microorganisms, 2.5 parts of a binder, 1 part of dopamine hydrochloride, 12-60 parts of monomers of high polymer molecules and 1-20 parts of a cross-linking agent are used;
more specifically, the binder comprises starch and/or water; specifically, the microorganism comprises bacillus subtilis, yeast and/or lactobacillus; more specifically, when the binder comprises starch and water, and when the microorganism comprises bacillus subtilis, yeast and lactic acid bacteria, the mass ratio of the bacillus subtilis, the yeast, the lactic acid bacteria, the starch and the water is 2: 1;
5) the granulation comprises adding microorganism and binder into high-speed stirring granulator, granulating, boiling and drying at 30-37 deg.C until water content is 3-5%, and sieving with 20 mesh sieve;
6) mixing and granulating microorganisms and a binder, stirring the mixture in Tris-HCl buffer solution with the pH value of 8-8.5 for more than 2 hours at room temperature together with dopamine hydrochloride, performing suction filtration, placing the product in a reaction container, adding ultrapure water, and introducing N2Adding monomer NIPAM, cross-linking agent and catalyst into the solution, and adding 50ml of ultrapure water into each 1-5g of the product、NIPAM 11.3g/100g H2O, cross-linking agent clay 0.8-4g/100gH2O, catalyst TEMED 8. mu.L/100 gH2O; keep out of light and continue to lead to N2Stirring for 15min at room temperature, transferring the reaction container to an ice bath, stirring for 5min, adding an initiator KPS (KPS with the dosage of 2g/100g H)2O, stirring for 30s, transferring the mixture into a closed mold, and polymerizing for 24h at room temperature.
The invention also aims to provide the microbial inoculum prepared by any method.
It is a further object of the present invention to provide the use of any of the agents of the present invention.
Specifically, the application comprises at least one of the following 1) to 4):
1) as a slow or controlled release product for bacteria that benefit plant growth;
2) as a product for beneficial plant growth;
3) as a product for regulating the ecological or nutritional status of soil microorganisms;
4) as a slow or controlled release product for fertilizers.
The plant includes a crop.
Specifically, when the high polymer molecules in the microbial inoculum comprise PNIPAm polymers, the slow release or controlled release is the slow release or controlled release of a temperature response mechanism; specifically, the release of the product is increased when the temperature is lower than the LCST of the PNIPAm polymer, and the release of the product is decreased when the temperature is higher than the LCST of the PNIPAm polymer.
It is a final object of the invention to provide a use of any of the methods of the invention.
Specifically, the application comprises at least one of the following 1) to 4):
1) use in the preparation of a product having a sustained or controlled release of bacteria that benefit plant growth or a product related thereto;
2) use in the preparation of a product having beneficial plant growth or a product related thereto;
3) the application in the preparation of products for regulating the ecological or nutritional status of soil microorganisms or related products thereof;
4) use in the manufacture of a product having a slow or controlled release fertiliser or a product related thereto.
The plant includes a crop.
Specifically, when the polymer molecule in the method comprises a PNIPAm polymer, the slow release or controlled release is a slow release or controlled release of a temperature response mechanism; specifically, the release of the product is increased when the temperature is lower than the LCST of the PNIPAm polymer, and the release of the product is decreased when the temperature is higher than the LCST of the PNIPAm polymer.
The invention has at least the following advantages:
(1) the controlled-release agricultural microbial inoculum provided by the invention can effectively release live bacteria, thereby improving the nutrient condition and nutrient supply condition of crops, inhibiting the activity of harmful microorganisms, improving the disease resistance and stress resistance of crops, and sustainably improving the soil fertility condition.
(2) The controlled-release agricultural microbial inoculum provided by the invention has the advantages of simple preparation process, environmental protection, no use of organic solvent and suitability for industrial mass production and application.
(3) In a specific embodiment, the invention utilizes the starch and the viable bacteria to mix and granulate, the obtained granules have poor water solubility, can keep an agglomerate state in water and are beneficial to coating dopamine or high polymer; in addition, the starch in the granules can be used as a nutrient substance, so that the quantity of live bacteria is increased, the starch degradation effect of the live bacteria is favorable for the disintegration of the granules to release the live bacteria, and an additional disintegrating agent is not required to be added in the granulation process.
(4) In a specific embodiment, the LCST of the temperature-sensitive PNIPAm polymer coated on the surfaces of agricultural microbial inoculum particles is about 32 degrees, and when the temperature is lower than the LCST, the molecular chain of the PNIPAm has stronger hydrophilicity and is represented as a stretched coil structure, so that living bacteria are easily released; when the temperature rises above the LCST, the PNIPAM brush is in a collapse state, so that the permeability of agricultural microbial inoculum particles of the multilayer polymer coating is reduced, the release rate is slowed down, the purpose of intelligently regulating and controlling the performance of the microbial inoculum according to the change of the environment is achieved, and the utilization efficiency of the microbial inoculum is improved.
(5) In a specific implementation mode, the temperature-sensitive PNIPAm polymer can be biodegraded, so that the problem of environmental pollution caused by large-scale application of fertilizers is avoided, the requirement of ecological green sustainable development is met, and the controlled-release agricultural microbial inoculum has huge actual and commercial application values.
Detailed Description
As used in the following examples, the experimental procedures used were all conventional ones unless otherwise specified.
Materials and the like used in the following examples are commercially available unless otherwise specified.
The following examples and their detailed description are presented to illustrate and understand the present invention and are not to be construed as limiting the invention.
Example 1 preparation of temperature-responsive controlled-Release agricultural microbial inoculum
Preparation of agricultural microbial inoculum granules
Adding Bacillus subtilis, yeast, lactobacillus, starch and water at a mass ratio of 2:1 into a high-speed stirring granulator, granulating, boiling and drying at 30-37 deg.C until the water content is 3-5%, and sieving with a 20-mesh sieve to obtain agricultural microbial inoculum granule.
(II) agricultural microbial inoculum granule surface coating and attaching dopamine
Weighing 1.5g of Tris, placing the Tris in a beaker, adding about 900mL of deionized water, and adjusting the pH value of a Tris-HCl solution to 8-8.5 by using 6M HCl; adding 50g of the prepared agricultural microbial inoculum granules into the Tris-HCl solution, and fixing the volume of the solution to 1L; and adding 5g of dopamine hydrochloride, stirring at room temperature for more than 2h, and performing suction filtration to obtain agricultural microbial inoculum particles coated and attached with dopamine on the surfaces.
Coating of (III) temperature-sensitive high polymer molecular material
Taking 1-5g of the agricultural microbial inoculum particles with dopamine attached to the surfaces, placing the agricultural microbial inoculum particles into a 100ml reaction vessel, and adding 50ml of ultrapure water. General formula (N)2Removing dissolved oxygen in the solution for 0.5h, adding monomer NIPAM, crosslinking agent and catalyst into the solution, wherein the specific dosage is NIPAM 11.3g/100g H2O, cross-linking agent clay 0.8-4g/100g H2O, catalyst TEMED 8. mu.L/100 gH2O, continuously introducing N in dark under the condition of being coated by tinfoil2And rapidly stirring at room temperature for 15 min. Then the reaction vessel was transferred to an ice bath and stirred slowly for 5min, initiator KPS (KPS amount 2g/100g H) was added2O), stirring for 30s, quickly transferring the mixture into a closed mold, and polymerizing for 24h at room temperature. The product was washed centrifugally in ultrapure water to remove unreacted monomers and impurities. And finally, crushing the product to obtain agricultural microbial inoculum particles coated with temperature-sensitive polymer molecules on the surfaces, thereby obtaining the temperature-response controlled-release agricultural microbial inoculum.
Example 2 live bacterium release experiment of temperature-responsive controlled-release agricultural microbial inoculum
The temperature response controlled release agricultural microbial inoculum prepared by the method in the embodiment 1 is taken and averagely divided into 9 parts, the 18 parts are respectively placed in the environment with the temperature of 15 ℃, 25 ℃ and 35 ℃ for 10 days, 1 month and 3 months, and then the release amount of viable bacteria of the temperature response controlled release agricultural microbial inoculum is detected under different temperatures and different placing times.
The method for detecting the release amount of the viable bacteria by a flat plate detection method comprises the following steps:
1) preparing a solution according to the mass-to-volume ratio of the placed sample to the sterile water of 1: 10, and filtering the solution through a 3-micron filter membrane to filter agricultural microbial inoculum particles which do not release live bacteria and are coated with dopamine and agricultural microbial inoculum with temperature-sensitive polymer molecules coated on the surfaces;
2) collecting filtrate, adding glass beads, standing for 20min, and shaking on rotary shaker at 200r/min for 30min to obtain mother liquor bacterial suspension;
3) respectively sucking 5.0ml of the mother liquid bacterial suspension by using a sterile pipette, adding the mother liquid bacterial suspension into 45ml of sterile water, and performing serial dilution according to the volume ratio of 1: 10 to respectively obtain a series of diluted bacterial suspensions with different dilution times;
4) taking 3 continuous suitable dilutions for each sample, respectively sucking 0.1ml of bacterial suspension with different dilutions by using a sterile pipette, adding the bacterial suspension to a solid agar culture medium, and respectively and uniformly coating the bacterial suspension with different dilutions on the solid agar culture medium by using a sterile glass scraper; repeating each dilution for 3 times, while using sterile water as blank control, and culturing in 37 deg.C constant temperature incubator for 12-36 h;
5) colony count and calculation of viable count in sample: the method is carried out according to the method in the national standard GB 20287-2006.
The results are shown in Table 1. The results in table 1 show that the temperature-responsive controlled-release agricultural microbial inoculum prepared by the method in example 1 can adjust the release rate of the live bacteria wrapped by the microbial inoculum according to different temperatures, and when the temperature is lower than the LCST 32 ℃, the lower the temperature is, the greater the release amount of the live bacteria is; when the temperature is higher than LCST 32 ℃, the release amount of the living bacteria is reduced sharply. The experimental result shows that the temperature response controlled release agricultural microbial inoculum provided by the invention is very suitable for fertilizing early rice and winter wheat. In high-temperature summer, the temperature response controlled-release agricultural microbial inoculum provided by the invention can automatically store the wrapped live bacteria without loss, and the utilization efficiency of the microbial inoculum is improved.
In addition, the release amount of viable bacteria in the sample, presumably due to the metabolic activity of the bacteria and the consumption of nutrients, at the same temperature and at different standing times, should be greater than the amount shown in Table 1 if the sample is placed in the field, because the field environment is favorable for the metabolic reproduction of the bacteria.
TABLE 1
The above-mentioned embodiments only express the embodiments of the present invention, and the description is more specific and detailed, but not understood as the limitation of the patent scope of the present invention, but all the technical solutions obtained by using the equivalent substitution or the equivalent transformation should fall within the protection scope of the present invention.
Claims (2)
1. The microbial inoculum is characterized by comprising microorganisms, a binder, dopamine hydrochloride and high polymer molecules, wherein 1) the binder is starch and water; 2) the microorganisms are bacillus subtilis, saccharomycetes and lactic acid bacteria, and the mass ratio of the bacillus subtilis to the saccharomycetes to the lactic acid bacteria is 1:1: 1; 3) the high polymer molecule is PNIPAm polymer; 4) the dopamine hydrochloride is covered on the surfaces of the adhesive and the microorganism, the high polymer molecule is covered on the surface of the dopamine hydrochloride, and the microbial inoculum is prepared by the following method:
mixing and granulating microorganisms, starch and water, stirring the mixture in Tris-HCl buffer solution with the pH value of 8-8.5 for more than 2 hours at room temperature together with dopamine hydrochloride, performing suction filtration, putting the product in a reaction container, adding ultrapure water, and introducing N2Adding NIPAM monomer of high polymer molecule, cross-linking agent clay and TEMED catalyst into the solution, and introducing N continuously in dark2Stirring at room temperature, transferring the reaction container to an ice bath, stirring, adding an initiator KPS, stirring, transferring to a closed mold, polymerizing for 24 hours at room temperature, and crushing a product to obtain the compound;
according to the mass, in the method, 1-10 parts of microorganism, 1-10 parts of binder, 1-10 parts of dopamine hydrochloride, 78-500 parts of monomer NIPAM10 of high polymer molecule and 0.8-200 parts of cross-linking agent clay;
the mass ratio of the bacillus subtilis to the yeast to the lactobacillus to the starch to the water is 2:2:2:2: 1.
2. The use of the microbial inoculum according to claim 1, wherein the use comprises at least one of the following 1) to 2):
1) as a slow or controlled release product for bacteria that benefit plant growth;
2) As a product for regulating the ecological or nutritional status of soil microorganisms.
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| CN104789226A (en) * | 2015-03-13 | 2015-07-22 | 迪斯科化工集团股份有限公司 | Charcoal-based microbial soil conditioner and preparation method thereof |
| CN107556112A (en) * | 2017-09-25 | 2018-01-09 | 农德志 | Biological slow-released selenium-enriched organic fertilizer |
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| CN104789226A (en) * | 2015-03-13 | 2015-07-22 | 迪斯科化工集团股份有限公司 | Charcoal-based microbial soil conditioner and preparation method thereof |
| CN107556112A (en) * | 2017-09-25 | 2018-01-09 | 农德志 | Biological slow-released selenium-enriched organic fertilizer |
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