CN114196663B - Soil-activated microbial inoculum immobilized microsphere and preparation method thereof - Google Patents
Soil-activated microbial inoculum immobilized microsphere and preparation method thereof Download PDFInfo
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Classifications
-
- 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
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/04—Enzymes or microbial cells immobilised on or in an organic carrier entrapped within the carrier, e.g. gel or hollow fibres
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K17/00—Soil-conditioning materials or soil-stabilising materials
- C09K17/40—Soil-conditioning materials or soil-stabilising materials containing mixtures of inorganic and organic compounds
-
- 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
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
- C12N11/12—Cellulose or derivatives thereof
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2101/00—Agricultural use
Abstract
The invention discloses a soil-activated microbial agent immobilized microsphere, which relates to the technical field of microbial immobilization and is mainly prepared from an activated microbial agent coating agent and a crosslinking auxiliary agent, wherein the mass ratio of the coating agent to the crosslinking auxiliary agent is 1:1; the excitation microbial inoculum coating agent is mainly prepared from the following raw materials in parts by weight: 10-15 parts of sodium alginate, 0-5 parts of sodium carboxymethylcellulose, 0-20 parts of a building agent, 2.5-4 parts of an excited microbial inoculum and 972-982 parts of water; the cross-linking auxiliary agent is prepared from 10-40 parts of calcium chloride and 960-990 parts of water, the building agent is kaolin or attapulgite, and the sodium carboxymethyl cellulose and the building agent are 0 part at the same time. The invention also provides a preparation method of the immobilized microsphere. The invention has the beneficial effects that: the materials used in the invention are natural, green, environment-friendly, safe and free from side effects, and can be stored for more than 180 days in a dry environment without degradation, and the activity of the microbial inoculum can be maintained to be more than 90%.
Description
Technical Field
The invention relates to the technical field of microorganism immobilization, in particular to a soil-activated microbial agent immobilized microsphere and a preparation method thereof.
Background
The production of crops is closely related to climate factors such as heat, illumination, precipitation and the like and soil factors, wherein the soil factors are one of the important factors which are manually controllable. Research has shown that soil factors conducive to crop production include abundant nutrients, loose soil structure, abundant soil biotics, etc., where the soil biotics include soil animals and soil microorganisms. By applying the microbial preparation to the field, some scholars and researchers optimize the biological richness of the soil, improve the soil structure and nutrient supply, promote crop growth and increase yield.
However, most of the traditional microbial agents are liquid products, are large and heavy, are not convenient for long-time storage and long-distance transportation, and further provide challenges for large-scale field application of the microbial agents. At present, solid microbial preparation products which are widely popularized and used are not seen in the market. A new lightweight, shelf-stable solid microbial formulation product is in need of development.
The patent application with publication number of CN 112646801A discloses a preparation method of microorganism immobilized microspheres, which uses a microorganism immobilization technology, takes biochar as a carrier, adopts an adsorption-crosslinking-embedding method to prepare a microbial inoculum, but after the immobilized micrococcus agent is stored for six months, the effective viable count is reduced from 425 hundred million cfu/mL to only more than 1 hundred million cfu/g, and the effective viable count is kept lower for a long time.
Disclosure of Invention
The technical problem to be solved by the invention is that the microbial immobilized microsphere in the prior art has lower effective viable count after long-time preservation, and provides a soil-activated microbial agent immobilized microsphere and a preparation method thereof.
The invention solves the technical problems by the following technical means:
the soil-activated microbial inoculum immobilized microsphere is mainly prepared from an activated microbial inoculum coating agent and a crosslinking auxiliary agent, wherein the mass ratio of the activated microbial inoculum coating agent to the crosslinking auxiliary agent is 1:1; the excitation microbial inoculum coating agent is mainly prepared from the following raw materials in parts by weight: 10-15 parts of sodium alginate, 0-5 parts of sodium carboxymethylcellulose, 0-10 parts of a building agent, 2.5-4 parts of an excited microbial inoculum and 972-982 parts of water; the sodium carboxymethyl cellulose and the building agent are 0 part at different time;
the cross-linking auxiliary agent is prepared from 10-40 parts of calcium chloride and 960-990 parts of water, and the building agent is kaolin or attapulgite.
The beneficial effects are that: the materials used in the invention are natural, green, environment-friendly, safe and free from side effects, and can be stored for more than 180 days in a dry environment without degradation, and the activity of the microbial inoculum can be maintained to be more than 90%.
The sodium alginate is crosslinked under the action of calcium ions to form calcium alginate gel, and in the phosphorus-containing environment in the field, the high affinity of phosphate radical and the calcium ions can lead to the extraction of the calcium ions in the gel, thereby leading to the degradation of the gel. If the environment is wet, the process can be facilitated, the immobilized microspheres can be rapidly and naturally degraded in the field environment, the soil-activated microbial inoculum is released, and the soil potential is activated.
If the weight fraction of calcium chloride is lower than the above value, the crosslinking reaction is slow and takes a long time, and the components in the coating agent, particularly bacteria, are scattered into the crosslinking auxiliary agent, the effect is deteriorated, and if the weight fraction of calcium chloride is higher than the above value, the consumption of calcium chloride is large and the cost is increased.
When sodium carboxymethyl cellulose is added, the sodium carboxymethyl cellulose is taken as a carboxymethyl derivative product of cellulose, so that the activity of degrading cellulose by the microbial inoculum is maintained, and the sodium carboxymethyl cellulose can be taken as a thickening agent to better promote the formation of microspheres. If sodium carboxymethylcellulose or a building agent is not added, the formed microspheres collapse into a film shape after being dried, but cannot be formed into spheres. If the amount of the auxiliary agent exceeds the above value, the gel voids are excessively filled, and the space for storing the cells is compressed, resulting in a decrease in the effectiveness of the product.
The present invention uses attapulgite or kaolin as a component for adsorbing bacteria and supporting a gel structure, can increase the reaction rate of forming microspheres, and the microspheres of the present invention can maintain a particle shape without flattening to form a film.
Preferably, the starter bacteria comprises Pseudomonas azotoformans CGMCC 1.7377 (Pseudomonas azotoformans) and Escherichia coli CGMCC 1.12873 (Escherichia coli), and the effective Colony Forming Unit (CFU) ratio of the two is 1:1.
The beneficial effects are that: the microbial inoculum used in the invention is a bacterial biological material with a cellulose degradation function, and the sodium carboxymethyl cellulose used in the invention has the function of stimulating the cellulose degradation capability of the used excitation microbial inoculum with the cellulose degradation function.
Pseudomonas azotoformans CGMCC 1.7377 can degrade cellulose naturally, and Escherichia coli CGMCC 1.12873 is modified by cellulase genes. Under normal nutritional conditions, cellulose is not the preferred carbon source for bacteria, but when cellulose alone is not present as a conventional carbon source (e.g., sucrose, glucose, etc.), cellulose-degrading bacteria specifically secrete enzymes that degrade cellulose, such as cellulases, etc., which is clearly advantageous for product application to the field.
The preparation method of the soil-activated microbial inoculum immobilized microsphere comprises the following steps:
(1) Excitation microbial inoculum thallus: inoculating the excited microbial inoculum into a liquid culture medium, performing shake culture for 20-22h at 28-30deg.C or 30-37deg.C or at 180-230rpm, and centrifuging to obtain excited microbial inoculum;
(2) Mixing sodium carboxymethylcellulose or a building agent with sodium alginate and water, and sterilizing to obtain a sterile excitation microbial inoculum coating solution;
(3) Mixing the excited microbial inoculum thallus in the step (1) with the excited microbial inoculum coating solution in the step (2) to obtain an excited microbial inoculum coating agent, then dripping the excited microbial inoculum coating agent into a crosslinking auxiliary agent, standing at normal temperature for crosslinking, and obtaining immobilized hydrogel microspheres after crosslinking;
(4) And (3) air-drying the immobilized hydrogel microspheres obtained in the step (3) under a sterile condition to obtain the soil-activated microbial inoculum immobilized microspheres.
The beneficial effects are that: the invention has simple preparation, simple and convenient operation and low cost, and can be produced in large scale.
According to the invention, the treatment of adding sodium carboxymethylcellulose has the advantages that the crosslinking speed is higher when the crosslinking auxiliary agent is dripped, the formed microspheres are more round and full, and the product is improved.
The attapulgite or kaolin used in the invention is used as a component for adsorbing bacteria and supporting a gel structure, so that the corresponding treatment can form microspheres at the moment of dripping the crosslinking auxiliary agent, the reaction efficiency is greatly improved, the formed microspheres can still keep the particle shape after being dried and are not flat to form a film, and meanwhile, the microspheres are quickly sunk into the bottom of the crosslinking auxiliary agent due to higher density, so that the smooth crosslinking is ensured.
The materials used in the invention are natural, green, environment-friendly, safe and free from side effects, can be stored for more than 180 days in a dry environment without degradation, and can maintain the viability of the microbial inoculum.
The sodium alginate is crosslinked under the action of calcium ions to form calcium alginate gel, and in the phosphorus-containing environment in the field, the high affinity of phosphate radical and the calcium ions can lead to the extraction of the calcium ions in the gel, thereby leading to the degradation of the gel. If the environment is wet, the process can be facilitated, the immobilized microspheres can be rapidly and naturally degraded in the field environment, the soil-activated microbial inoculum is released, and the soil potential is activated.
If the weight fraction of calcium chloride is lower than the above value, the crosslinking reaction is slow and takes a long time, and the components in the coating agent, particularly bacteria, are scattered into the crosslinking auxiliary agent, the effect is deteriorated, and if the weight fraction of calcium chloride is higher than the above value, the consumption of calcium chloride is large and the cost is increased.
When sodium carboxymethyl cellulose is added, the sodium carboxymethyl cellulose is taken as a carboxymethyl derivative product of cellulose, so that the activity of degrading cellulose by the microbial inoculum is maintained, and the sodium carboxymethyl cellulose can be taken as a thickening agent to better promote the formation of microspheres. If sodium carboxymethylcellulose or a building agent is not added, the formed microspheres collapse into a film shape after being dried, but cannot be formed into spheres. If the amount of the auxiliary agent exceeds the above value, the gel voids are excessively filled, and the space for storing the cells is compressed, resulting in a decrease in the effectiveness of the product.
The coated microbial inoculum has higher requirements on temperature, acid-base property, biological cleanliness and the like in the preparation process, and the invention eliminates the interference from external biological factors through a sterilization step.
Different culture temperatures are set according to different stimulative bacteria.
Preferably, the liquid medium in the step (1) is a YEP medium.
Preferably, the YEP culture medium is prepared from the following raw materials in parts by weight: 10 parts of peptone, 10 parts of yeast extract, 5 parts of beef extract powder and 975 parts of water.
Preferably, in the step (1), the centrifugation is performed at 5000rpm at 4℃for 10min.
Preferably, the sterilization conditions in the step (2) are as follows: treating with steam at 121deg.C and 103.4kPa for 20-30min.
The beneficial effects are that: according to the invention, the dissolution and uniform distribution of sodium alginate and carboxymethylcellulose sodium in water can be better promoted through a high-temperature sterilization step.
Preferably, the stimulative bacteria and the stimulative bacteria coating solution in the step (3) are stirred for 10-60min at a speed of 200-1500 rpm.
Preferably, in the step (3), when the trigger microbial inoculum coating agent is added to the crosslinking auxiliary agent, the dripping position is 1-10 cm away from the liquid level of the crosslinking auxiliary agent.
Preferably, in the step (4), the air is dried naturally in a normal temperature, sterile, ventilated and dried environment.
The beneficial effects are that: not only provides a gentle dry environment, but also requires biological cleanliness.
Preferably, the starter bacteria is Pseudomonas azotoformans CGMCC 1.7377 (Pseudomonas azotoformans), and the culture temperature in the step (1) is 28-30 ℃.
Preferably, the starter culture is Escherichia coli CGMCC 1.12873 (Escherichia coli), and the culture temperature in the step (1) is 30-37 ℃.
The invention has the advantages that: the materials used in the invention are natural, green, environment-friendly, safe and free from side effects, and can be stored for more than 180 days in a dry environment without degradation, and the activity of the microbial inoculum can be maintained to be more than 90%.
The sodium alginate is crosslinked under the action of calcium ions to form calcium alginate gel, and in the phosphorus-containing environment in the field, the high affinity of phosphate radical and the calcium ions can lead to the extraction of the calcium ions in the gel, thereby leading to the degradation of the gel. If the environment is wet, the process can be facilitated, the immobilized microspheres can be rapidly and naturally degraded in the field environment, the soil-activated microbial inoculum is released, and the soil potential is activated.
If the weight fraction of calcium chloride is lower than the above value, the crosslinking reaction is slow and takes a long time, and the components in the coating agent, particularly bacteria, are scattered into the crosslinking auxiliary agent, the effect is deteriorated, and if the weight fraction of calcium chloride is higher than the above value, the consumption of calcium chloride is large and the cost is increased.
The sodium carboxymethyl cellulose serving as a carboxymethyl derivative product of cellulose can better promote the formation of microspheres. If sodium carboxymethylcellulose and a building agent are not added, the formed microspheres collapse into a film shape after being dried, but cannot be formed into spheres. If the amount of the auxiliary agent exceeds the above value, the gel voids are excessively filled, and the space for storing the cells is compressed, resulting in a decrease in the effectiveness of the product.
The attapulgite or kaolin used in the invention is used as a component for adsorbing bacteria and supporting a gel structure, so that the corresponding treatment can form microspheres at the moment of dripping the crosslinking auxiliary agent, the reaction efficiency is greatly improved, the formed microspheres can still keep the particle shape after being dried and are not flat to form a film, and meanwhile, the microspheres are quickly sunk into the bottom of the crosslinking auxiliary agent due to higher density, so that the smooth crosslinking is ensured.
The microbial inoculum used in the invention is a bacterial biological material with a cellulose degradation function, and the sodium carboxymethyl cellulose used in the invention has the function of stimulating the cellulose degradation capability of the used excitation microbial inoculum with the cellulose degradation function.
Pseudomonas azotoformans CGMCC 1.7377 can degrade cellulose naturally, and Escherichia coli CGMCC 1.12873 is modified by cellulase genes. Under normal nutritional conditions, cellulose is not the preferred carbon source for bacteria, but when cellulose alone is not present as a conventional carbon source (e.g., sucrose, glucose, etc.), cellulose-degrading bacteria specifically secrete enzymes that degrade cellulose, such as cellulases, etc., which is clearly advantageous for product application to the field.
Drawings
FIG. 1 is an image of soil-activated microbial agent immobilized hydrogel microspheres of comparative example 1, example 1-example 4 of the present invention; wherein A) comparative example 1; b) Example 1; c) Example 2; d) Example 3; e) Example 4. The short and long scale lengths were 5mm and 10mm, respectively.
FIG. 2 is an image of the soil-activated microbial agent immobilized microspheres of the present invention of comparative example 1, example 1-example 4 dried by natural air drying; wherein a) comparative example 1; b) Example 1; c) Example 2; d) Example 3; e) Example 4. The short and long scale lengths were 5mm and 10mm, respectively.
FIG. 3 is an electron microscopic image of the soil-activated microbial agent immobilized hydrogel microspheres of example 1 and example 2 after freeze-drying. Wherein a) microsphere surface electron microscopy image (125×); b) Microsphere surface electron microscope image (10000×); c) Microsphere surface electron microscope image (3000×); d) Electron microscope image of microsphere internal structure (137×); a-c are example 1 and d is example 2;
FIG. 4 is an image of comparative example 1, example 1-example 4 when crosslinked; wherein a) comparative example 1; b) Example 1; c) Example 2; d) Example 3; e) Example 4. The cross-linking time of three pictures from left to right for each row was 1 minute, 3 minutes and 10 minutes, respectively.
FIG. 5 is a graph showing the comparative effect of the immobilized microspheres of the soil-activated microbial agent on the microbial viability of the soil-activated microbial agent in example 3 of the present invention, wherein the control group is the microbial viability of the non-immobilized aqueous suspension of the soil-activated microbial agent.
The Pseudomonas azotoformans CGMCC 1.7377 (Pseudomonas azotoformans) and the Escherichia coli CGMCC 1.12873 (Escherichia coli) are public materials, and are purchased from the China general microbiological culture Collection center.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions in the embodiments of the present invention will be clearly and completely described in the following in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The test materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Those of skill in the art, without any particular mention of the techniques or conditions, may follow the techniques or conditions described in the literature in this field or follow the product specifications.
Example 1
The soil-activated microbial inoculum immobilized microsphere is formed by crosslinking and immobilizing a soil-activated microbial inoculum coating agent and a crosslinking auxiliary agent, wherein the soil-activated microbial inoculum coating agent comprises the following raw materials in parts by weight: 15 parts of sodium alginate, 5 parts of sodium carboxymethylcellulose, 3 parts of microbial inoculum, 977 parts of water and 1000 parts of crosslinking auxiliary agent; the crosslinking auxiliary agent comprises the following raw materials in parts by weight: 20 parts of calcium chloride and 980 parts of water.
The preparation method of the soil-activated microbial inoculum immobilized microsphere specifically comprises the following steps:
(1) Taking a sterilized universal YEP liquid culture medium, taking strains which are in a logarithmic phase and have a volume of one thousandth of the culture solution, respectively inoculating the strains into the culture solution, and performing shake culture to excite the microbial inoculum for 20 hours by using a constant-temperature shake incubator with the temperature of 30 ℃ and the rotating speed of 220 rpm;
(2) In order to determine the influence of the product on the viability of the thalli, the quantity of the thalli in the liquid culture medium in the step (1) is measured by adopting a method of cell counting under a microscope after dilution and diluting a coated flat plate, and the thalli is centrifugated at the temperature of 4 ℃ and the rotating speed of 5000rpm, so that the thalli of the soil excitation microbial inoculum is obtained through enrichment; wherein the thalli comprise escherichia coli CGMCC 1.12873 thalli and pseudomonas azotoformans CGMCC 1.7377 thalli, and the effective Colony Forming Unit (CFU) ratio of the two thalli is 1:1;
(3) Mixing sodium alginate, sodium carboxymethylcellulose, attapulgite, kaolin and water according to the mass ratio, and performing high-temperature steam treatment at 103.4kPa and 121 ℃ for 20 minutes to fully dissolve, mix and sterilize the component materials to obtain a sterile excitation microbial inoculum coating solution;
(4) Mixing the soil-activated microbial inoculum thallus obtained in the step (2) with the sterile activated microbial inoculum coating solution obtained in the step (3) according to the mass ratio to obtain an activated microbial inoculum coating agent;
the culture effect of bacteria is slightly different due to the difference of nutrient conditions, temperature, rotating speed, time and other factors of the culture. In the embodiment of the invention, the dosage of the thallus is 10≡10CFU thallus contained in every 1000 parts of coating agent, and practical experience shows that the fresh weight of the 10≡10CFU thallus is generally 2.5-4.0g. In production, the number of the thalli is limited in parts by weight, so that the method has the advantages of operability and simplicity.
(5) Dropwise adding the excited microbial inoculum coating agent obtained in the step (4) into a crosslinking auxiliary agent, standing and crosslinking for 2 hours at normal temperature to obtain soil excited microbial inoculum immobilized hydraulic microspheres, wherein the soil excited microbial inoculum immobilized hydraulic microspheres are shown as a B in fig. 1;
(6) And (3) naturally air-drying the soil-activated microbial agent immobilized hydraulic microspheres obtained in the step (5) in a normal-temperature, sterile, ventilated and dry environment to obtain dry soil-activated microbial agent immobilized microspheres, as shown in a figure 2 b.
Example 2
The soil-activated microbial inoculum immobilized microsphere is formed by crosslinking and immobilizing a soil-activated microbial inoculum coating agent and a crosslinking auxiliary agent, wherein the soil-activated microbial inoculum coating agent comprises the following raw materials in parts by weight: 15 parts of sodium alginate, 5 parts of sodium carboxymethylcellulose, 5 parts of attapulgite, 3 parts of a microbial inoculum, 972 parts of water and 1000 parts of a crosslinking auxiliary agent; the crosslinking auxiliary agent comprises the following raw materials in parts by weight: 20 parts of calcium chloride and 980 parts of water.
The preparation method of the soil-activated microbial inoculum immobilized microsphere specifically comprises the following steps:
(1) Taking a sterilized universal YEP liquid culture medium, taking strains which are in a logarithmic phase and have a volume of one thousandth of the culture solution, respectively inoculating the strains into the culture solution, and performing shake culture to excite the microbial inoculum for 20 hours by using a constant-temperature shake incubator with the temperature of 30 ℃ and the rotating speed of 200 rpm;
(2) In order to determine the influence of the product on the viability of the thalli, the method of adopting cell counting under a microscope after dilution and diluting a coated flat plate is adopted to measure the quantity of the thalli in the liquid culture medium in the step (1), and the thalli is centrifugated at the temperature of 4 ℃ and the rotating speed of 5000rpm, so that the thalli of the soil excitation microbial inoculum is obtained through enrichment; wherein the thalli comprise escherichia coli CGMCC 1.12873 thalli and pseudomonas azotoformans CGMCC 1.7377 thalli, and the effective Colony Forming Unit (CFU) ratio of the two thalli is 1:1;
(3) Mixing sodium alginate, sodium carboxymethylcellulose, attapulgite, kaolin and water according to the mass ratio, and treating with high-temperature steam of 103.4kPa and 121 ℃ for 30 minutes to fully dissolve, mix and sterilize the component materials to obtain a sterile excitation microbial inoculum coating solution;
(4) Mixing the soil-activated microbial inoculum thallus obtained in the step (2) with the sterile activated microbial inoculum coating solution obtained in the step (3) according to the mass ratio to obtain an activated microbial inoculum coating agent;
the culture effect of bacteria is slightly different due to the difference of nutrient conditions, temperature, rotating speed, time and other factors of the culture. In the embodiment of the invention, the dosage of the thallus is 10≡10CFU thallus contained in every 1000 parts of coating agent, and practical experience shows that the fresh weight of the 10≡10CFU thallus is generally 2.5-4.0g. In production, the number of the thalli is limited in parts by weight, so that the method has the advantages of operability and simplicity.
(5) Dropwise adding the excited microbial inoculum coating agent obtained in the step (4) into a crosslinking auxiliary agent, standing and crosslinking for 4 hours at normal temperature to obtain soil excited microbial inoculum immobilized hydraulic microspheres, wherein the soil excited microbial inoculum immobilized hydraulic microspheres are shown as C in figure 1;
(6) And (3) naturally air-drying the soil-activated microbial agent immobilized hydraulic microspheres obtained in the step (5) in a normal-temperature, sterile, ventilated and dry environment to obtain dry soil-activated microbial agent immobilized microspheres, as shown in a figure 2 c.
Example 3
This embodiment differs from embodiment 1 in that: the soil-activated microbial inoculant coating agent comprises the following raw materials in parts by weight: 15 parts of sodium alginate, 10 parts of kaolin, 3 parts of microbial inoculum, 972 parts of water, 1000 parts of cross-linking auxiliary agent, and the rest raw materials and steps are the same as those of the embodiment 1, and the obtained soil excitation microbial inoculum immobilized hydraulic microspheres and immobilized microspheres are respectively shown in a figure 1D and a figure 2D.
Example 4
This embodiment differs from embodiment 1 in that: the soil-activated microbial inoculant coating agent comprises the following raw materials in parts by weight: 10 parts of sodium alginate, 5 parts of sodium carboxymethylcellulose, 10 parts of kaolin, 3 parts of microbial inoculum, 972 parts of water, 1000 parts of cross-linking auxiliary agent, and the rest raw materials and steps are the same as those of example 1, and the obtained soil-activated microbial inoculum immobilized hydrogel microspheres and immobilized microspheres are respectively shown in the figure 1E and the figure 2E.
Example 5
This embodiment differs from embodiment 1 in that: the soil-activated microbial inoculant coating agent comprises the following raw materials in parts by weight: 15 parts of sodium alginate, 5 parts of kaolin, 3 parts of microbial inoculum, 977 parts of water, 1000 parts of cross-linking auxiliary agent and the rest raw materials and steps are the same as those of the example 1.
Comparative example 1
This comparative example differs from example 1 in that: the soil-activated microbial inoculant coating agent comprises the following raw materials in parts by weight: 15 parts of sodium alginate, 3 parts of microbial inoculum, 982 parts of water, 1000 parts of cross-linking auxiliary agent, wherein the cross-linking auxiliary agent comprises the following raw materials in parts by weight: 20 parts of calcium chloride and 980 parts of water.
The soil-activated microbial agent immobilized hydrogel microspheres and the immobilized microspheres obtained in comparative example 1 are shown in fig. 1 a and fig. 2 a, respectively.
FIG. 3 is an electron microscopic image of the soil-activated microbial agent immobilized hydrogel microspheres of example 1 and example 2 after freeze-drying.
From the protruding portion indicated by the red arrow in fig. 3 (c), a bright film structure is easily observed, and the film structure is an organic film formed by calcium alginate and sodium carboxymethyl cellulose, it can be inferred that the microsphere surface is covered by a layer of organic film structure, so that the influence of external general chemical substances on the microbial inoculum can be effectively isolated. From FIG. 3 (d), it can be seen that a large number of network structures and voids exist inside the microspheres, the network structures support the morphology of the microspheres, and the void structures become spaces for accommodating cells. Due to the nature of the sodium alginate and the sodium carboxymethyl cellulose material, the surface layer membrane structure of the microsphere is hydrophilic and breathable, and the lower basic metabolism requirement of the microbial inoculum can be maintained.
The coating rate of the microbial inoculum in all examples was measured and counted by the following method: (1) firstly, cell count detection is carried out on the concentration of thalli in the culture solution, and the 10≡10CFU thalli number (N) 0 ) After centrifugal collection, adding the mixture into an excited microbial inoculum coating solution to obtain a coating agent containing specific bacterial number; (2) dropwise adding a coating agent into the crosslinking auxiliary agent to obtain soil-activated microbial agent immobilized hydrogel microspheres; (3) sampling the cross-linking auxiliary agent after the reaction, detecting the concentration of uncoated thallus in the cross-linking auxiliary agent by adopting a cell technology method, and multiplying the concentration by the volume of the cross-linking auxiliary agent to obtain the number (N u ) The method comprises the steps of carrying out a first treatment on the surface of the (4) The coating ratio was calculated according to the following formula: coating ratio (%) = (N) 0 -N u )/N 0 *100。
The number of uncoated bacteria in the cross-linking aid is detected, and the result shows that the coating rate of the microbial inoculum in all the examples reaches more than 99%.
Fig. 4 is an image of example 1-example 4, comparative example 1, when crosslinked, with three pictures of each row having crosslinking times of 1 minute, 3 minutes, and 10 minutes, respectively, from left to right. It can be seen that the microbeads formed in examples 1, 2 and 4, to which sodium carboxymethylcellulose was added, were less prone to tailing, and that the microbeads formed in examples 2-4, to which attapulgite or kaolin was added, were more rapid.
FIG. 5 is a graph showing the effect of the immobilized microspheres of the soil-activated microbial agent on maintaining the microbial activity of the soil-activated microbial agent in example 3A line wherein the viability of the microbial agent in the microsphere is calculated and calculated according to the following method: (1) after the microspheres are dried, weighing microspheres with a certain mass (the actual weighing mass is 0.1000 g+/-0.0005 g, and after the microspheres are dried, weighing and split charging the microspheres into sterile Ep tubes for storage, and marking the actual weight), putting the microspheres into 20mL of sterile 0.2M phosphate buffer solution, and oscillating for 3 hours at 30 ℃ and 200rpm to fully crack the microspheres and release thalli; (2) sucking part of the solution from the phosphate buffer solution, diluting, coating the solution on a flat plate, counting and calculating to obtain the number N of viable bacteria in the microsphere c The method comprises the steps of carrying out a first treatment on the surface of the (3) After the microspheres are stored for a period of time, counting and calculating the number N of the residual viable bacteria in the microspheres by the same method as the step (1) and the step (2) t The method comprises the steps of carrying out a first treatment on the surface of the (4) The microbial inoculum activity was calculated according to the following formula: microbial inoculum viability (%) =n t /N c *100。
As can be seen from fig. 5, the microbial agent activity condition in the soil-activated microbial agent immobilized microsphere is detected after 180 days of storage, and the result shows that the microbial agent activity can still be maintained to be more than 90%, the microbial agent effectiveness is effectively maintained, and meanwhile, the storage and the transportation are convenient. Example 3 of the present invention in examples 2 to 4 works best.
The above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims (9)
1. A soil-activated microbial inoculum immobilized microsphere is characterized in that: the microbial agent is mainly prepared from an excitation microbial agent coating agent and a crosslinking auxiliary agent, wherein the mass ratio of the coating agent to the crosslinking auxiliary agent is 1:1; the excitation microbial inoculum coating agent is mainly prepared from the following raw materials in parts by weight: 10-15 parts of sodium alginate, 0-5 parts of sodium carboxymethylcellulose, 0-10 parts of a building agent, 2.5-4 parts of an excited microbial inoculum and 972-982 parts of water; the sodium carboxymethyl cellulose and the building agent are 0 part at different time;
the cross-linking auxiliary agent is prepared from 10-40 parts of calcium chloride and 960-990 parts of water, and the building agent is kaolin or attapulgite;
the microbial inoculum is Pseudomonas azotoformans CGMCC 1.7377 (Pseudomonas azotoformans
azotoformans) and Escherichia coli CGMCC 1.12873 (Escherichia coli), the effective Colony Forming Units (CFU) ratio of the two is 1:1.
2. The method for preparing the soil-activated microbial agent immobilized microsphere according to claim 1, wherein the method comprises the following steps: the method comprises the following steps:
(1) Excitation microbial inoculum thallus: inoculating the excited microbial inoculum into a liquid culture medium, performing shake culture for 20-22h at 28-30deg.C or 30-37deg.C or at 180-230rpm, and centrifuging to obtain excited microbial inoculum;
(2) Mixing sodium carboxymethylcellulose or a building agent with sodium alginate and water, and sterilizing to obtain a sterile excitation microbial inoculum coating solution;
(3) Mixing the excited microbial inoculum thallus in the step (1) with the excited microbial inoculum coating solution in the step (2) to obtain an excited microbial inoculum coating agent, then dripping the excited microbial inoculum coating agent into a crosslinking auxiliary agent, standing at normal temperature for crosslinking, and obtaining immobilized hydrogel microspheres after crosslinking;
(4) And (3) air-drying the immobilized hydrogel microspheres obtained in the step (3) under a sterile condition to obtain the soil-activated microbial inoculum immobilized microspheres.
3. The method for preparing soil-activated microbial agent immobilized microspheres according to claim 2, wherein the method comprises the following steps: the liquid culture medium in the step (1) is YEP culture medium.
4. The method for preparing soil-activated microbial agent immobilized microspheres according to claim 3, wherein the method comprises the following steps: the YEP culture medium is prepared from the following raw materials in parts by weight: 10 parts of peptone, 10 parts of yeast extract, 5 parts of beef extract powder and 975 parts of water.
5. The method for preparing soil-activated microbial agent immobilized microspheres according to claim 2, wherein the method comprises the following steps: and (3) centrifuging in the step (1) at 4 ℃ and 5000rpm for 10min.
6. The method for preparing soil-activated microbial agent immobilized microspheres according to claim 2, wherein the method comprises the following steps: the sterilization conditions in the step (2) are as follows: treating with steam at 121deg.C and 103.4kPa for 20-30min.
7. The method for preparing soil-activated microbial agent immobilized microspheres according to claim 2, wherein the method comprises the following steps: and (3) stirring the bacteria-exciting agent thallus and the bacteria-exciting agent coating solution in the step (3) at a rotating speed of 200-1500rpm for 10-60min.
8. The method for preparing soil-activated microbial agent immobilized microspheres according to claim 2, wherein the method comprises the following steps: when the excited microbial inoculum coating agent is added to the cross-linking auxiliary agent in the step (3), the height of the dripping position from the liquid level of the cross-linking auxiliary agent is 1-10 cm.
9. The method for preparing soil-activated microbial agent immobilized microspheres according to claim 2, wherein the method comprises the following steps: and (3) naturally air-drying in the step (4) in a normal-temperature, sterile, ventilated and dry environment.
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