CN111944726A - Soil microbial population regulator and preparation method and application thereof - Google Patents
Soil microbial population regulator and preparation method and application thereof Download PDFInfo
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Classifications
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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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- 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
Abstract
The invention belongs to the technical field of soil microbial regulation, and particularly relates to a soil microbial population regulator and a preparation method and application thereof. The invention adopts specific culture medium to culture the saccharomyces cerevisiae and the rhodotorula glutinis, simple crude protein is obtained by separating and purifying the obtained yeast co-culture solution, then pepsin is added for enzymolysis to generate yeast polypeptide, and the yeast polypeptide is compounded with the modified kaolin to obtain the soil microorganism regulator, which can stimulate the growth of actinomycetes, nitrogen-fixing flora, ammonia oxidation, nitrobacteria, phosphorus-dissolving bacteria and lactic acid flora, and has the functions of inhibiting the propagation of pathogenic bacteria and balancing the number of soil microorganism flora.
Description
Technical Field
The invention belongs to the technical field of soil microbial regulation, and particularly relates to a soil microbial population regulator and a preparation method and application thereof.
Background
Agriculture is an important prop industry in China, wherein planting is the most important component, in order to improve the growth and yield of crops, the traditional agriculture adopts a farming mode of using a large amount of pesticides and chemical fertilizers, the yield of the crops is increased, the environment is polluted, and a large amount of nutrient elements which are not absorbed by the crops enter the nature to accelerate the eutrophication of water bodies. The extensive agricultural development mode not only causes the waste of a large amount of nitrogen and phosphorus resources, but also causes the problems of water eutrophication, soil hardening and the like. The alteration and deterioration of soil structure and the imbalance of soil indigenous microbial flora are the main causes of the decrease of soil fertility and the reduction of plant growth. Soil is the root and guarantee of agricultural production, and under the double pressure of environmental protection consciousness and market competition at present, the planting industry must select a green, environment-friendly and energy-saving production mode. Among these, adjusting the coordination of soil microbiota to promote soil productivity is one of the preferred ways that can be employed.
The soil microbial flora activation needs to firstly improve the diversity of the soil microbial flora, and secondly increase the proportion of beneficial microbial flora, such as the relative abundance of actinomycetes, azotobacter, ammonia oxidation, nitrifying bacteria, phosphorus dissolving bacteria, lactic acid bacteria and the like, and balance the proportion of the microbial flora. The soil microbial flora is activated, so that the soil structure can be improved, the productivity degradation degree of the soil is slowed down, the influence of the fertilizer on the soil structure in the using process is reduced as far as possible, and the activation of carbon, nitrogen and phosphorus elements in the soil is accelerated, so that the growth of plants is stimulated. After a large amount of chemicals such as fertilizers and herbicides are used, the soil microbial population is easy to be unbalanced (beneficial microorganisms and indigenous microorganisms are greatly reduced); singularizing (decreasing relative abundance) nitrogen, deregulating the phosphorus cycle (blocking or reversing the pathway), etc., and by activating the soil microbiota, it is expected to solve the above problems.
Disclosure of Invention
The invention aims to provide a soil microbial community regulator, a preparation method and application thereof, and aims to solve the problems of easy unbalance, simplification and the like of the existing soil microbial community.
In order to achieve the purpose of the invention, the technical scheme adopted by the invention is as follows:
in a first aspect, the present invention provides a method for preparing a soil microbiota regulator, comprising the steps of:
(1) preparing yeast polypeptide: inoculating Saccharomyces cerevisiae into S0 culture medium, culturing until ammonia nitrogen removal rate reaches 60%, total nitrogen removal rate reaches 50%, total phosphorus removal rate reaches 50%, and OD value is greater than 1 or cell density is greater than or equal to 1.5 × 108When the protein content of the saccharomyces cerevisiae is increased by more than 180% -200%, the thalli are centrifugally filtered to obtain the saccharomyces cerevisiae with accumulated polypeptide;
inoculating Rhodotorula glutinis in S0 culture medium, culturing until ammonia nitrogen removal rate reaches 70%, total nitrogen removal rate reaches 60%, total phosphorus removal rate reaches 50%, and OD value is greater than 1 or cell density is greater than or equal to 1.5 × 108When the protein content of the yeast is increased to be more than 180% -200%, centrifugally filtering the thalli to obtain the rhodotorula glutinis accumulated with the polypeptide;
putting the saccharomyces cerevisiae accumulated by the polypeptide and the rhodotorula glutinis accumulated by the polypeptide into an S0 culture medium for symbiotic culture to obtain a yeast co-culture solution;
centrifuging and washing the yeast co-culture solution to remove the culture medium to obtain a yeast mixture;
mixing the yeast mixture with water, heating to 47 ℃, adding sodium hydroxide until the concentration is 3% -4%, performing ultrasonic-alkali liquid wall breaking treatment, adjusting the pH to be neutral, performing centrifugal treatment, obtaining supernatant as yeast supernatant, and performing centrifugal and drying treatment on the obtained precipitate to obtain a yeast modified material;
mixing the yeast supernatant with 95% ethanol at 4 ℃, standing for 5h, and centrifuging to obtain a precipitate as yeast crude protein;
carrying out mixed enzymolysis treatment on the yeast crude protein and pepsin to obtain the yeast polypeptide;
(2) preparing modified kaolin: mixing metakaolin, calcium oxide, sodium silicate and the yeast modified material according to the mass ratio of (80-85): 6-8): 2-4): 5-10, then adding 10% of water for bonding, drying, calcining and sieving to obtain the modified kaolin;
(3) preparing a soil microbial community regulator: mixing the yeast polypeptide obtained in the step (1) with water according to the mass ratio of 1:10 to prepare a solution, adding the modified kaolin obtained in the step (2), mixing and adsorbing, wherein the mass ratio of the modified kaolin to the solution is 1:25, and obtaining the soil microbial population regulator;
wherein the S0 culture medium comprises 3g/L glucose and Na2HPO428.73mg/L、NH4Cl30mg/L、K2HPO4100mg/L、KCl50mg/L、MgSO47H2O40mg/L, pH 7.0;
the S1 culture medium is obtained by mixing millet, brown rice and quinoa according to the mass ratio of 5 (2-3) to (2-3) and sieving to obtain a first mixture, and mixing the first mixture with the S0 culture medium, wherein the mass ratio of the first mixture to the S0 culture medium is 1: 9;
the S2 culture medium is obtained by mixing rice, soybeans and corns according to the mass ratio of (4-5) to (2-3) to (2-4) and then sieving to obtain a second mixture, and mixing the second mixture with the S0 culture medium, wherein the mass ratio of the second mixture to the S0 culture medium is 1: 8.
In a preferred embodiment of the present invention, Saccharomyces cerevisiae is inoculated into S1 culture medium for culture, or Rhodotorula glutinis is inoculated into S2 culture medium for culture, the culture conditions are DO2-3mg/L, temperature is 25 deg.C, and rotation speed is 150 rpm.
In a preferred embodiment of the present invention, in the step of centrifuging and filtering the bacterial cells, the rotation speed of the centrifugation is 1000 rpm.
In a preferred embodiment of the present invention, the polypeptide-accumulating saccharomyces cerevisiae and the polypeptide-accumulating rhodotorula glutinis are placed in a S0 culture medium for symbiotic culture, and the ratio of the added amount of the polypeptide-accumulating saccharomyces cerevisiae to the added amount of the polypeptide-accumulating rhodotorula glutinis is 1: 2.
In a preferred embodiment of the present invention, the step of symbiotically culturing the polypeptide-accumulating Saccharomyces cerevisiae and the polypeptide-accumulating Rhodotorula glutinis in S0 medium under the condition of culturing at 25 deg.C and 150rpm for 1 d.
In a preferred embodiment of the present invention, in the step of removing the culture medium from the yeast co-culture solution by centrifugation and washing, the centrifugation is performed under 10000rpm for 10 min.
As a preferred technical scheme of the invention, the yeast mixture and water are mixed and heated to 47 ℃, sodium hydroxide is added until the concentration is 3% -4%, the ultrasonic-alkali liquid wall breaking treatment is carried out, then the pH is adjusted to be neutral, the supernatant is obtained through centrifugation, the obtained precipitate is the yeast supernatant, and in the step of obtaining the yeast modified material through centrifugation and drying, the time of the ultrasonic-alkali liquid wall breaking treatment is 20-25 min; the centrifugation conditions of the centrifugation treatment are centrifugation at 2000rom for 20 min; the centrifugation rotating speed of the obtained sediment after centrifugation is 10000 rpm; the temperature of the drying treatment was 80 ℃.
In a preferred embodiment of the present invention, in step (2), the calcination is performed at 500 ℃ for 2 hours.
In a second aspect, the present invention provides a soil microbiota regulator, which is prepared by the above-described preparation method.
In a third aspect, the present invention provides the use of a soil microbial modulator for increasing the number of microbial populations in soil.
Compared with the prior art, the invention has the following beneficial effects:
firstly, the yeast can secrete different proteins in the growth process, the saccharomyces cerevisiae and the rhodotorula glutinis are cultured by adopting a specific culture medium, the obtained yeast co-culture solution is separated and purified to obtain simple crude protein, then pepsin is added for enzymolysis to generate yeast polypeptide, the growth of actinomycetes, azotobacter flora, ammonia oxidation bacteria, nitrobacteria, phosphorus dissolving bacteria and lactic acid bacteria can be stimulated, and the effects of inhibiting the propagation of pathogenic bacteria and balancing the number of soil microbiota are achieved.
In addition, the yeast polypeptide is adsorbed on the modified kaolin for storage, so that the use in agriculture is facilitated, the effects of local improvement and nutrient slow release of soil can be realized by adsorbing the yeast polypeptide and nitrogen and phosphorus nutrients through the slow release function of the kaolin, and the stability of the yeast polypeptide can be maintained.
Thirdly, the invention adopts specific culture media to culture the saccharomyces cerevisiae and the rhodotorula glutinis, and the specific culture media utilize the crushed grains as raw materials to provide nutrition for the saccharomyces cerevisiae and the rhodotorula glutinis, thus having the advantage of green production. Meanwhile, considering the problem that grains are aged after being stored for a period of time, the aged grains can be utilized by preparing the crushed grains into the culture medium and further processed into products required by agricultural production, so that the obtained soil microorganism regulator is a green product and green production is realized.
Finally, on the whole, the specific culture medium made of grains is adopted to culture the saccharomyces cerevisiae and the rhodotorula glutinis, so that the soil microbial regulator is obtained, the steps and parameters adopted from the beginning of fermentation production are that the nutrition of the biological fertilizer is kept as much as possible, then the effect of gradually releasing nutrient substances is realized through the modified kaolin, the obtained soil microbial regulator comprehensively utilizes the polypeptide produced in the fermentation process of the grains and the yeast, and the polypeptide is matched with nutrient elements, so that the microenvironment of a soil root system can be improved, and the quantity, the type, the proportion and the balance of soil microbial flora can be effectively regulated.
Drawings
FIG. 1 is a comparison of soil microorganisms before and after the application of a soil microorganism-regulating agent in example 1 of the present invention;
FIG. 2 is a comparison of the activity of soil enzymes before and after the application of a soil microorganism-regulating agent in example 1 of the present invention;
FIG. 3 is a comparison of the activity of soil enzymes before and after the application of a soil microorganism-regulating agent in example 1 of the present invention;
FIG. 4 is a comparison of soil microorganisms before and after the application of a soil microorganism-regulating agent in example 2 of the present invention;
FIG. 5 is a comparison of the number of soil microorganisms before and after the application of the soil microorganism-controlling agent in example 2 of the present invention;
FIG. 6 is a comparison of the activity of soil enzymes before and after the application of a soil microorganism-regulating agent in example 2 of the present invention;
FIG. 7 is a comparison of the activity of soil enzymes before and after the application of a soil microorganism-controlling agent in example 2 of the present invention.
Detailed Description
In order to make the objects, technical solutions and technical effects of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are clearly and completely described, and the embodiments described below are a part of the embodiments of the present invention, but not all of the embodiments. All other embodiments obtained by a person skilled in the art without making any inventive step are within the scope of protection of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions; the reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
Example 1
This example provides a method for preparing a soil microbiota regulator, comprising the steps of:
1. pretreatment and culture medium preparation of grain raw materials
Firstly, grains are pretreated, and the formula is as follows:
m1: wherein the cereal raw materials comprise millet, brown rice and quinoa in parts by weight, and 50g of millet, 20g of brown rice and 30g of wheat are added in each 100g of five-cereal raw materials. Mixing, pulverizing, and sieving with 100 mesh sieve.
M2: wherein the cereal raw materials comprise 50g of rice, 30g of soybean and 20g of corn in parts by weight per 100g of five-cereal raw materials. Mixing, pulverizing, and sieving with 100 mesh sieve.
S0 liquid medium: glucose 3g/L, Na2HPO428.73mg/L、NH4Cl30mg/L、K2HPO4100mg/L、KCl50mg/L、MgSO47H2O40mg/L, and adjusted the pH to 7.0 with 1mM NaOH.
S1 liquid medium: mixing M1 according to the weight ratio of 1:9 adding S0, mixing thoroughly to obtain suspension (M1+ S0) as S1 liquid culture medium.
S2 liquid medium: mixing M2 according to the weight ratio of 1:8, adding S0, and fully mixing to prepare a suspension, namely an S2 liquid culture medium (M2+ S0).
2. Culturing yeast and obtaining protein and polypeptide:
saccharomyces cerevisiae and Rhodotorula glutinis were obtained by purchase.
2.1, expanding culture of the saccharomyces cerevisiae:
selecting sterilized inoculating loop from brewing yeast, placing into 1000ml YM liquid culture medium, and aerobically culturing at 25 deg.C with shaking table rotation speed of 150rpm for 2 days, 2-3 mg/L. Then, the cells were centrifuged at 10000rpm to obtain cells. Then transferred into 1000ml of liquid medium S0 for culture. Centrifuging the thalli every 3-4 days, and performing centrifugation according to the weight ratio of 1:1 replacing S0 nutrient solution. Measuring the removal rates of ammonia nitrogen, total nitrogen and total phosphorus every other day from the third time, wherein when the removal rates respectively reach 70%, 50% and 50%, or the cell density is 1.5 x 108The cells were centrifuged/filtered at 1000rpm and replaced with a new volume of S1 liquid medium before the second stage.
And (3) carrying out amplification culture on the Rhodotorula glutinis: selecting sterilized inoculating loop from Rhodotorula glutinis strain, placing into 1000ml YM liquid culture medium, performing aerobic culture at 25 deg.C shaking table rotation speed of 150rpm for 2 days, and controlling dissolved oxygen at 2-3mg/L above. Then, the cells were centrifuged at 10000rpm to obtain cells. Then transferred into 1000ml of liquid medium S0 for culture. Centrifuging the thalli every 3-4 days, and performing centrifugation according to the weight ratio of 1:1 replacing S0 nutrient solution. And measuring the removal rates of ammonia nitrogen, total nitrogen and total phosphorus every other day from the third time, centrifuging/filtering the thallus at 1000rpm after the removal rates respectively reach 70%, 60% and 50% and the cell density is 1.5 x 108/ml, replacing a new equal volume of S2 liquid culture medium, and then entering the second stage.
2.2 two yeasts are cultured in the polypeptide production stage:
in the second stage, the saccharomyces cerevisiae is cultured in an S1 liquid culture medium under the conditions of accumulated yeast protein: DO2-3mg/L, temperature 25 ℃, shaking culture for 3 days under the condition of 150rpm of shaking table rotation speed, measuring the removal rate of ammonia nitrogen, total nitrogen and total phosphorus every other day, when the removal rate respectively reaches 60%, 50% and more than 50%. Replacing the equal volume S1 liquid culture medium, determining the protein content from the second stage, completing the culture when the protein content is increased by more than 180-200%, centrifuging/filtering and collecting for later use, wherein the thalli can be stored for 24 hours at 25 ℃.
In the second stage, rhodotorula glutinis is cultured in an S2 liquid culture medium under the conditions of yeast protein accumulation: DO2-3mg/L, temperature 25 ℃, shaking culture for 3 days under the condition of 150rpm of shaking table rotation speed, measuring the removal rate of ammonia nitrogen, total nitrogen and total phosphorus every other day, when the removal rate respectively reaches 60%, 50% and more than 50%. Replacing the equal volume S2 liquid culture medium, determining the protein content from the second stage, completing the culture when the protein content is increased by more than 180-200%, centrifuging/filtering and collecting for later use, wherein the thalli can be stored for 24 hours at 25 ℃.
2.3 obtaining of protein polypeptide and precipitate of yeast:
the two cultured yeasts were redissolved in S0 liquid medium at a ratio of 1:2, and carrying out symbiotic culture at 25 ℃ and 150r/min for 1 day in the same manner.
Then the yeast co-culture solution was centrifuged at 10000rpm for 15min, the medium was removed, the centrifuged yeast mixture was washed three times with distilled water, 15mL of distilled water was added to 1.5g of the yeast mixture, and heated to 47 ℃; then adding NaOH powder slowly into the yeast mixture solution until the concentration of NaOH reaches 3-4%, and continuously stirring to prevent overheating. Breaking cell wall with ultrasonic-alkali solution at 47 deg.C for 20-25min, and stopping at intermediate interval for 30 s; adjusting pH to 7, centrifuging at 2000rpm for 20min, collecting supernatant, cooling at 4 deg.C, and making into polypeptide. The precipitate is the residue of yeast extraction, mainly structural polysaccharides, fibers and cell residues. The remainder was centrifuged at 10000rpm and dried in an oven at a temperature of 80 ℃ or less, and used as a yeast-modified material.
Adding 3-4 times volume of iced 95% ethanol into the yeast supernatant in an environment of 4 ℃, continuously stirring for 1min, standing for 5h, centrifuging at 2000rpm for 20min, removing the supernatant, slightly washing off surface alcohol from the obtained precipitate with a small amount of water, and collecting the precipitate to obtain the yeast crude protein. Adding pepsin in a mass ratio of 0.5-1% into each 25g of crude protein for enzymolysis, wherein the molecular weight of the obtained peptide is distributed between dipeptide and decapeptide, and most of the obtained peptide is concentrated in tripeptide to heptapeptide and a small amount of amino acid below 10%, so as to obtain the yeast polypeptide.
3. Preparation and synthesis of soil microbial community regulator:
in order to keep the activity of the yeast polypeptide and facilitate the use, a special preparation method is designed. The soil microbial community regulator consists of two parts, namely A yeast polypeptide and B modified kaolin. The principle is that the main components and the adsorbent of the soil microbial community regulator are formed by utilizing the modified kaolin; and then mixing the adsorbent with the yeast polypeptide component, and adsorbing the yeast polypeptide on the adsorbent to obtain the soil microbial community regulator.
3.1 preparation of modified Kaolin:
the modified kaolin is prepared by two steps, the first step is as follows: obtaining metakaolin, and the second step: obtaining the biological modified kaolin. The metakaolin can be directly purchased or calcined at 800 ℃ for 2 hours to obtain the metakaolin.
The modified kaolin comprises the components of metakaolin, calcium oxide, sodium silicate and yeast modified materials. The formula is prepared according to the mass ratio, wherein metakaolin accounts for 80%, calcium oxide accounts for 6%, sodium silicate accounts for 4%, and yeast modified material accounts for 10%. All the components are uniformly mixed and added into the mixture according to the mass ratio of powder to water of 10: 1, drying, calcining at 500 ℃ for 2h, cooling, and sieving by a 100-mesh sieve to obtain the modified kaolin.
3.2 preparation of soil microbial activator:
the mass ratio of the pure water to the yeast polypeptide is 10: 1 to prepare a solution, and then mixing the modified kaolin with the solution according to the mass ratio of 1:25 for 4 hours to obtain the soil microbial community regulator, and directly using the soil microbial community regulator without drying or drying the soil microbial community regulator at low temperature for later use.
Example 2
This example is essentially the same as example 1, except that the modified kaolin was formulated as: 80% of metakaolin, 5% of calcium oxide, 4% of sodium silicate and 11% of yeast modified material. All the components are uniformly mixed and added into the mixture according to the mass ratio of powder to water of 9: 1, drying, calcining at 500 ℃ for 2h, cooling, and sieving by a 100-mesh sieve to obtain the modified kaolin. The mass ratio of the pure water to the yeast polypeptide is 10: 1 to prepare a solution, and then mixing the modified kaolin with the solution according to the mass ratio of 1: shaking, mixing and adsorbing for 4h at the ratio of 25, centrifuging/filtering to obtain precipitate to obtain soil microbial population regulator, drying at the low temperature below 50 ℃ for later use, and standing for a long time within half a year.
Experimental example 1
During the planting of flowering cabbage in Guangzhou, the soil microbial population regulator obtained in example 1 was applied 1 time per week at a rate of 100g per square meter. The crops are 30 groups planted in the Chinese cabbage, the soil microbiota regulator group is not applied for comparison, the crops are used for 4 times within one month during the growth period, the fertilizer is used as usual, and the soil around the plants is respectively taken for analysis after the soil microbiota regulator is used for the first time and the fourth time. After being used, the change of soil microorganism and crops is measured,
in the soil environment, the actinomycetes play an important role in guaranteeing the soil health, can accelerate the transformation and circulation of nutrients such as nitrogen, phosphorus and potassium in the soil, can resist and inhibit the growth and the propagation of harmful organisms (soil-borne diseases) through nutrient resistance, and is an important indicator for evaluating the soil health and safety; the fungi are important components of soil microorganisms, can accelerate soil substance circulation, inhibit the occurrence of soil-borne diseases and the like, maintain the safety and health of soil, and are important indexes for evaluating the safety and fertility of the soil. The specific results are as follows:
in the two uses, the water content of the soil is not obviously changed. However, the total number of bacteria, fungi and actinomycetes were higher than those in the control group by using the soil microbial population regulator, and it was revealed that the soil microbial population regulator could increase the number of three major microbial populations, and among them, the effect was maintained better with the passage of time (fig. 1).
Then analyzing the soil enzyme activity, and respectively tracking carbon (sucrase), nitrogen (urease), phosphorus (alkaline phosphatase) and dehydrogenase (respiration), wherein the soil dehydrogenase is an intracellular enzyme, has no activity outside cells and is positively correlated with the soil microbial activity, and the activity can represent the soil microbial activity and functional diversity. The results show that: the soil microbial community regulator can improve the activity of four enzyme activities, wherein better effect than that of a control group can be maintained as time goes on, and the process of carbon, nitrogen, phosphorus and respiratory activity of soil is accelerated while the activity of microorganisms is promoted, so that the soil microbial community regulator has the effects of improving the activity of soil microorganisms and promoting plant growth (figures 2-3).
Experimental example 2
During the planting of tomato seedlings in Zephyranthus, the soil microbial population regulator obtained in example 2 was applied 1 time per week at a rate of 100g per square meter. The crops are 30 groups planted in the Chinese cabbage, the group without applying the soil microbial community regulator is used for comparison, the crops are used for 4 times in one month during the growth period, the fertilizer is used as usual, and the soil around the plants is respectively taken for analysis after the soil microbial community regulator is used for the first time and the fourth time. After being used, the change of soil microorganism and crops is measured,
the results are as follows: in the two uses, the water content and the pH value of the soil are not obviously changed. The soil microbial community regulator is shown to maintain relatively stable changes of soil moisture and pH without obviously changing the soil structure in 1 month of continuous use. However, the total number of bacteria, the total number of fungi and the total number of actinomycetes were higher than those of the control group by using the soil microbiota regulator, and it was revealed that the soil microbiota regulator could increase the number of three major microbial populations during the planting of tomato seedlings, and among them, the effect could be maintained better with the passage of time. The bacteria are the main force of carbon nitrogen phosphorus circulation, the increase of the quantity reflects the increase of the potential activity of the soil, the fungi and the actinomycetes have important effects on the decomposition of the difficultly degraded soil substances, and the soil microbial population regulator is obviously enhanced and the effect is maintained for more than 1 month (figures 4-5).
And then analyzing the soil enzyme activity, and respectively tracking carbon (sucrase), nitrogen (urease), phosphorus (alkaline phosphatase) and dehydrogenase (respiration), wherein the soil dehydrogenase is an intracellular enzyme, has no activity outside cells, is positively correlated with the soil microbial activity, and can represent the soil microbial activity and functional diversity. The results show that: the soil microbial flora regulator can improve the activity of four enzyme activities, wherein better effect than that of a control group can be maintained over time, and the soil microbial flora regulator also accelerates the process of carbon, nitrogen, phosphorus and respiratory activity of soil while promoting the activity of microorganisms, and has the effects of improving the activity of soil microorganisms and promoting plant growth (figures 6-7).
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (10)
1. A method for preparing a soil microbiota regulator, comprising the steps of:
(1) preparing yeast polypeptide: inoculating Saccharomyces cerevisiae into S0 culture medium, culturing until ammonia nitrogen removal rate reaches 60%, total nitrogen removal rate reaches 50%, total phosphorus removal rate reaches 50%, and OD value is greater than 1 or cell density is greater than or equal to 1.5 × 108When the protein content of the saccharomyces cerevisiae is increased by more than 180% -200%, the thalli are centrifugally filtered to obtain the saccharomyces cerevisiae with accumulated polypeptide;
inoculating Rhodotorula glutinis in S0 culture medium, culturing until ammonia nitrogen removal rate reaches 70%, total nitrogen removal rate reaches 60%, total phosphorus removal rate reaches 50%, and OD value is greater than 1 or cell density is greater than or equal to 1.5 × 108At each mL, the cells were centrifuged and filtered, and then inoculatedCulturing in S2 culture medium, and centrifuging and filtering thallus when the increase of yeast protein content is over 180% -200% to obtain Rhodotorula glutinis with accumulated polypeptide;
putting the saccharomyces cerevisiae accumulated by the polypeptide and the rhodotorula glutinis accumulated by the polypeptide into an S0 culture medium for symbiotic culture to obtain a yeast co-culture solution;
centrifuging and washing the yeast co-culture solution to remove the culture medium to obtain a yeast mixture;
mixing the yeast mixture with water, heating to 47 ℃, adding sodium hydroxide until the mass concentration is 3% -4%, performing ultrasonic-alkali liquid wall breaking treatment, adjusting the pH to be neutral, performing centrifugal treatment, obtaining supernatant as yeast supernatant, and performing centrifugal and drying treatment on the obtained precipitate to obtain a yeast modified material;
mixing the yeast supernatant with 95% ethanol at 4 ℃, standing for 5h, and centrifuging to obtain a precipitate as yeast crude protein;
carrying out mixed enzymolysis treatment on the yeast crude protein and pepsin to obtain the yeast polypeptide;
(2) preparing modified kaolin: mixing metakaolin, calcium oxide, sodium silicate and the yeast modified material according to the mass ratio of (80-85): 6-8): 2-4): 5-10, then adding 10 wt% of water for bonding, drying, calcining and sieving to obtain the modified kaolin;
(3) preparing a soil microbial community regulator: mixing the yeast polypeptide obtained in the step (1) with water according to the mass ratio of 1:10 to prepare a solution, adding the modified kaolin obtained in the step (2), mixing and adsorbing, wherein the mass ratio of the modified kaolin to the solution is 1:25, and obtaining the soil microbial population regulator;
wherein the S0 culture medium comprises 3g/L glucose and Na2HPO428.73mg/L、NH4Cl 30mg/L、K2HPO4 100mg/L、KCl50mg/L、MgSO4 7H2O40mg/L and pH 7.0;
the S1 culture medium is obtained by mixing millet, brown rice and quinoa according to the mass ratio of 5 (2-3) to (2-3) and sieving to obtain a first mixture, and mixing the first mixture with the S0 culture medium, wherein the mass ratio of the first mixture to the S0 culture medium is 1: 9;
the S2 culture medium is obtained by mixing rice, soybeans and corns according to the mass ratio of (4-5) to (2-3) to (2-4) and then sieving to obtain a second mixture, and mixing the second mixture with the S0 culture medium, wherein the mass ratio of the second mixture to the S0 culture medium is 1: 8.
2. The method for preparing a soil microbiota regulator according to claim 1, wherein in the step of inoculating in S1 culture medium for culture and inoculating in S2 for culture, the culture conditions are as follows: DO2-3mg/L, temperature 25 ℃, rotation speed 150 rpm.
3. The method for producing a soil microorganism population regulator according to claim 1, wherein in the step of centrifuging the cells, the rotation speed of the centrifugation is 1000 rpm.
4. The method for preparing a soil microbial population regulator according to claim 1, wherein the step of symbiotically culturing said polypeptide-accumulating saccharomyces cerevisiae and said polypeptide-accumulating rhodotorula glutinis performed in S0 medium, and the ratio of the addition amount of said polypeptide-accumulating saccharomyces cerevisiae to that of said polypeptide-accumulating rhodotorula glutinis is 1: 2.
5. The method for preparing a soil microbial population regulator according to claim 1, wherein in the step of subjecting the polypeptide-accumulating Saccharomyces cerevisiae and the polypeptide-accumulating Rhodotorula glutinis to symbiotic culture in S0 medium, the symbiotic culture is performed under the condition of culturing at 150rpm for 1 day at 25 ℃.
6. The method for preparing a soil microbiota regulator according to claim 1, wherein in the step of removing the culture medium by centrifugation and washing of the yeast co-culture solution, the centrifugation is performed under conditions of centrifugation at 10000rpm for 10 min.
7. The method for preparing a soil microbial population regulator according to claim 1, wherein the step of mixing the yeast mixture with water, heating the mixture to 47 ℃, adding sodium hydroxide to a concentration of 3% -4%, performing ultrasonic-alkali liquid wall breaking treatment, then adjusting the pH to be neutral, performing centrifugation treatment, obtaining a supernatant as a yeast supernatant, centrifuging and drying the obtained precipitate to obtain the yeast modified material is carried out, wherein the time of the ultrasonic-alkali liquid wall breaking treatment is 20min-25 min; the centrifugation conditions of the centrifugation treatment are centrifugation at 2000rom for 20 min; the centrifugation rotating speed of the obtained sediment after centrifugation is 10000 rpm; the temperature of the drying treatment was 80 ℃.
8. The method for preparing a soil microbial population regulator according to claim 1, wherein in the step (2), the calcination is performed under the condition of calcination at 500 ℃ for 2 hours.
9. A soil microbiota regulator prepared by the method for preparing a soil microbiota regulator according to any one of claims 1 to 8.
10. Use of a soil microbiota regulator according to claim 9 for increasing the number of soil microbiota populations.
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