CN110699274A - Preparation and application methods of compound microbial agent for soil improvement - Google Patents
Preparation and application methods of compound microbial agent for soil improvement Download PDFInfo
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Abstract
The invention provides a compound microbial agent for soil improvement, which contains a compound microbial agent prepared by mixed fermentation of paenibacillus polymyxa, saccharomyces cerevisiae, lactococcus lactis and the like, wherein the number of active spores of the paenibacillus polymyxa in the compound microbial agent is more than or equal to 3 hundred million/ml; the number of active spores of the saccharomyces cerevisiae is more than or equal to 1 hundred million/ml; the number of live spores of lactococcus lactis is more than or equal to 2 hundred million/ml. The compound microbial agent can improve soil, increase the sugar content of apples cultivated in apple trees, and improve quality, can well solve the problem of soil hardening in soil improvement, increases the number of viable bacteria of beneficial microorganisms in soil, and can improve the number of microbial populations to about 10 to 15 hundred million. The invention is a green and environment-friendly biological agent, can well avoid chemical residues and environmental pollution caused by chemical fertilizers and chemical pesticides, and has wide application prospect in new biological agents or biological fertilizers.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a preparation and application method of a compound microbial agent.
Background
The soil is the foundation on which human beings live, is the most basic and important production data of agriculture, is the hub of material and energy circulation in the agricultural industry chain, is a natural resource with regeneration effect in the nature, and is an important nutrient source of plants, particularly surface plough layer soil. In recent years, the soil degradation phenomenon in China is increasingly serious, wherein the soil degradation phenomenon is typical of the decrease of the tiltability of farmlands, the harder the farmlands are, the more barren the soil is, the serious the growth of crops is hindered, and the yield and the quality of agricultural products are further seriously decreased. The hardening is taken as a typical expression of soil degradation, the main original condition is that chemical fertilizers and chemical pesticides are used for a long time, the propagation and population quantity of beneficial microorganisms in the soil are seriously influenced and damaged, and finally the soil tiltability becomes worse and worse, and the crop quality is seriously reduced.
Aiming at the problems, the compound microbial agent and the application thereof can help to solve the soil problem, and the three beneficial microorganisms are utilized to be planted and propagated in the soil in a large amount and the metabolites of the three beneficial microorganisms, so that the compound microbial agent not only has the function of improving the soil, but also can improve the quality of crops and the sugar content of apples. The invention is a high-efficiency green environment-friendly compound microbial agent, and has wide theoretical and practical significance in the research and development aspects and the application aspects of new biological fertilizers.
Disclosure of Invention
In order to solve the defects of the prior art, the invention provides a compound microbial agent and a preparation method thereof by utilizing the relationship of mutual beneficial symbiosis, synbiosis and the like among strains.
The invention provides a compound microbial agent, which contains paenibacillus polymyxa, saccharomyces cerevisiae and lactococcus lactis.
The paenibacillus polymyxa is preserved in the China general microbiological culture Collection center of the Committee for culture Collection of microorganisms, and the preservation number is CGMCC No. 11586; the saccharomyces cerevisiae is preserved in the China general microbiological culture Collection center of the Committee for culture Collection of microorganisms with the preservation number of CGMCCNo.11238; the lactococcus lactis is preserved in China center for type culture Collection with the preservation number of CCTCC NO: M2016235
However, the present invention is not limited to this bacterium, and three kinds of the complex microbial agents of the present invention are not limited to the specifically-numbered paenibacillus polymyxa, saccharomyces cerevisiae, and lactococcus lactis, and any of the microorganisms known in the art to belong to these bacteria can be applied to the complex microbial agent of the present invention.
The number of live spores of each bacterium in the composite microbial agent is respectively as follows: more than or equal to 3 hundred million/ml of paenibacillus polymyxa, more than or equal to 1 hundred million/ml of saccharomyces cerevisiae and more than or equal to 2 hundred million/ml of lactococcus lactis.
The composite microbial agent is prepared by respectively fermenting paenibacillus polymyxa, saccharomyces cerevisiae and lactococcus lactis to obtain secondary fermentation liquid, inoculating the secondary fermentation liquid of the three bacteria into a tertiary fermentation culture medium according to the volume ratio of 3:2:2 for fermentation, and finishing fermentation when the number of live spores of the paenibacillus polymyxa in the fermentation liquid is more than or equal to 3 hundred million/ml, the number of live spores of the saccharomyces cerevisiae is more than or equal to 1 hundred million/ml and the number of live spores of the lactococcus lactis is more than or equal to 2 hundred million/ml.
The invention provides a preparation method for preparing the compound microbial agent, which is characterized by comprising the following steps:
respectively carrying out primary seed fermentation and secondary seed fermentation on paenibacillus polymyxa, saccharomyces cerevisiae and lactococcus lactis to obtain secondary fermentation liquor;
respectively inoculating secondary fermentation liquor of paenibacillus polymyxa, saccharomyces cerevisiae and lactococcus lactis into a three-stage fermentation culture medium according to the volume ratio of 3%, 2% and 2% for fermentation, wherein when the number of live spores of each bacterium in the fermentation liquor is respectively as follows: and (3) finishing fermentation when the paenibacillus polymyxa is more than or equal to 3 hundred million/ml, the saccharomyces cerevisiae is more than or equal to 1 hundred million/ml and the lactococcus lactis is more than or equal to 2 hundred million/ml to prepare the compound microbial agent.
In the method, the first-stage and second-stage seed culture media adopted in fermentation of the paenibacillus polymyxa in the step (1) comprise, by weight, 3% -6% of brown sugar, 2% -4% of cane molasses, 0.8% -2.0% of peptone, 0.5% -1.5% of yeast extract, 0.3% -0.8% of NaCl0.3%, K2HPO40.1% -0.4%, 0.02% -0.09% of magnesium sulfate, 40.08% -0.16% of MnSO40, the balance being water, pH6.8-7.2, the first-stage and second-stage seed culture media adopted in fermentation of ② lactococcus lactis comprise, by weight, 2% -3.5% of sucrose, 1% -1.5% of soybean flour, 0.8% -2.0% of corn steep liquor dry powder, 0.1% -0.6% of hydrogen citrate, 0.01% -0.09% of ferrous sulfate, K2HPO40.03% -0.12%, 25. H2O0.01% -0.03%, 0.3875% -0.5% of diammonium citrate, 0.6%, the balance being water, 3.5% -3.5% of yeast extract, 3.5% of wheat malt, the balance being water, and the balance being water;
the fermentation method of the paenibacillus polymyxa in the step (1) comprises the following steps: inoculating the strain preserved on the slant of the paenibacillus polymyxa into 200mL of first-level seed culture medium, and performing shake flask fermentation culture at the fermentation temperature of 23-30 ℃, the rotation speed of 180r/min and the fermentation time of 24-36h to obtain first-level seed liquid. Transferring the primary seed solution into a secondary seed culture medium of a 50L fermentation tank, wherein the inoculation amount is 3%, the liquid loading amount is 20L, the fermentation temperature is 23-30 ℃, the stirring speed is 150r/min, and culturing for 36-48h to obtain a secondary seed solution for later use.
The fermentation method of the lactococcus lactis in the step (1) comprises the following steps: directly inoculating the strain preserved by the activated lactococcus lactis slant to a liquid seed culture medium, and culturing at 23-30 ℃ and 160r/min for 24-36h to obtain a first-stage seed solution; inoculating the shake flask seed solution into a 50L fermentation tank according to the inoculation amount of 2%, and fermenting at 23-30 deg.C for 36-48h at 120r/min for 20L to obtain secondary seed solution.
The fermentation method of the saccharomyces cerevisiae in the step (1) comprises the following steps: inoculating the slant-preserved strain of Saccharomyces cerevisiae to 300mL of first-stage seed culture medium, and performing shake flask fermentation culture at 23-30 deg.C, rotation speed of 180r/min for 24-36h to obtain first-stage seed solution. Transferring the primary seed solution into a secondary seed culture medium of a 50L fermentation tank, wherein the inoculation amount is 2%, the liquid loading amount is 20L, the fermentation temperature is 23-30 ℃, the stirring speed is 180r/min, and culturing for 36-48h to obtain a secondary seed solution for later use.
After three kinds of bacteria are inoculated into a three-stage fermentation culture medium in the step (2) of the preparation method of the compound microbial agent, the fermentation method comprises the following steps: controlling the fermentation temperature at 23-30 deg.C, performing aerobic fermentation for 24-36h to make Paenibacillus polymyxa become dominant flora and rapidly proliferate, reducing pH to 4.0-5.2, performing anaerobic fermentation at constant pressure, controlling the temperature at 23-30 deg.C, performing anaerobic fermentation for 24-36h to make Saccharomyces cerevisiae and lactococcus lactis become dominant flora and rapidly proliferate.
The invention has the following beneficial effects:
(1) the compound microbial agent provided by the invention has the advantages that the three beneficial microbial bacteria are propagated in the soil in a large amount, the number of the beneficial viable bacteria in the soil is increased, and the three beneficial microbial bacteria have the soil improvement function through metabolites of the three beneficial microbial bacteria, so that the sugar content of the apples can be increased, and the quality is improved.
(2) The method for preparing the compound microbial agent not only solves the problems of high energy consumption, complex process and the like of single-bacterium fermentation production, but also can improve the biological activity of each strain, expand the function of the single microbial agent, and solve the problems of strain degeneration and the like.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.
The paenibacillus polymyxa used in the following examples is preserved in China general microbiological culture Collection center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC No. 11586;
the saccharomyces cerevisiae used in the following examples is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms with the preservation number of CGMCCNo.11238;
the lactococcus lactis used in the following examples was deposited in the China center for type culture Collection with the accession number of CCTCC NO: M2016235.
Example 1
Preparing a compound microbial agent:
the invention provides a preparation method for preparing the compound microbial agent, which comprises the following steps:
respectively carrying out primary seed fermentation and secondary seed fermentation on paenibacillus polymyxa, saccharomyces cerevisiae and lactococcus lactis to obtain secondary fermentation liquor;
respectively inoculating secondary fermentation liquor of paenibacillus polymyxa, saccharomyces cerevisiae and lactococcus lactis into a three-stage fermentation culture medium according to the volume ratio of 3%, 2% and 2% for fermentation, wherein when the number of live spores of each bacterium in the fermentation liquor is respectively as follows: and (3) finishing fermentation when the paenibacillus polymyxa is more than or equal to 3 hundred million/ml, the saccharomyces cerevisiae is more than or equal to 1 hundred million/ml and the lactococcus lactis is more than or equal to 2 hundred million/ml to prepare the compound microbial agent.
In the method, the first-stage and second-stage seed culture medium adopted in the fermentation of the paenibacillus polymyxa in the step (1) comprises the following components in percentage by weight: 4% of brown sugar, 2.6% of cane molasses, 1.5% of peptone, 1.0% of yeast extract, 0.4% of NaCl0, K2HPO40.2%, 0.05% of magnesium sulfate, 40.12% of MnSO40 and the balance of water, wherein the pH value is 6.9;
the primary and secondary seed culture medium components adopted during the lactococcus lactis fermentation are as follows according to the weight percentage: 2.5 percent of cane sugar, 1.0 percent of soybean meal, 1.2 percent of corn steep liquor dry powder, 0.3 percent of diammonium hydrogen citrate, 0.08 percent of ferrous sulfate, K2HPO40.06 percent, MgSO 4.7 7H2O0.02 percent, MnSO 4.4 4H2O0.03 percent and the balance of water, wherein the pH value is 6.9;
the primary and secondary seed culture medium components adopted in the fermentation of the saccharomyces cerevisiae comprise the following components in percentage by weight: 1.5 percent of cane molasses, 1.0 percent of yeast powder, 1.0 percent of malt powder, 2.5 percent of peptone and the balance of water, wherein the pH value is 7.1;
the fermentation method of the paenibacillus polymyxa in the step (1) comprises the following steps: inoculating the strain preserved on the slant of the paenibacillus polymyxa in 200mL of first-level seed culture medium, and performing shake flask fermentation culture at the fermentation temperature of 28 ℃, the rotation speed of 180r/min and the fermentation time of 30h to obtain first-level seed liquid. Transferring the primary seed solution into a secondary seed culture medium of a 50L fermentation tank, wherein the inoculation amount is 3%, the liquid loading amount is 20L, the fermentation temperature is 28 ℃, the stirring speed is 150r/min, and culturing for 48h to obtain a secondary seed solution for later use.
The fermentation method of the lactococcus lactis in the step (1) comprises the following steps: directly inoculating the strain preserved by the activated lactococcus lactis slant to a liquid seed culture medium, and culturing at 28 ℃ and 160r/min for 30h to obtain a first-stage seed solution; inoculating the shake flask seed solution into a 50L fermentation tank according to the inoculation amount of 2%, wherein the liquid loading amount is 20L, and fermenting at 28 ℃ and 120r/min for 48h to obtain a secondary seed solution for later use.
The fermentation method of the saccharomyces cerevisiae in the step (1) comprises the following steps: inoculating the saccharomyces cerevisiae slant preserved strain into 300mL of first-level seed culture medium, and performing shake flask fermentation culture at 28 ℃, the rotation speed of 180r/min and the fermentation time of 20h to obtain first-level seed liquid. Transferring the primary seed solution into a secondary seed culture medium of a 50L fermentation tank, wherein the inoculation amount is 2%, the liquid loading amount is 20L, the fermentation temperature is 28 ℃, the stirring speed is 180r/min, and culturing for 48h to obtain a secondary seed solution for later use.
After three kinds of bacteria are inoculated into a three-stage fermentation culture medium in the step (2) of the preparation method of the compound microbial agent, the fermentation method comprises the following steps: and controlling the fermentation temperature at 28 ℃, carrying out aerobic fermentation for 30h to enable the Paenibacillus polymyxa to become dominant flora and rapidly proliferate, reducing the pH to 4.0-5.2, carrying out anaerobic fermentation at constant pressure, controlling the temperature at 28 ℃, and carrying out anaerobic fermentation for 28h to enable the Saccharomyces cerevisiae and lactococcus lactis to become dominant flora and rapidly proliferate. And finally, finishing fermentation when the number of the live spores of the paenibacillus polymyxa in the fermentation liquor is more than or equal to 3 hundred million/ml, the number of the live spores of the saccharomyces cerevisiae is more than or equal to 1 hundred million/ml and the number of the live spores of the lactococcus lactis is more than or equal to 2 hundred million/ml, thus preparing the composite microbial agent.
Example 2:
the invention relates to an improvement application of a compound microbial agent to soil
This example was carried out by the following method: the method comprises the following steps:
(1) turning over the hardened soil by a shallow cultivator and smashing soil blocks by a rotary cultivator;
(2) spraying on the surface layer of soil, and diluting the compound microbial agent with water, wherein the compound microbial agent diluted with water comprises the following components in proportion of 1: 300 is mixed with water, sprayed on the surface of the soil and covered by 2-3 cm of broken soil, then the compound microbial agent is applied again, and finally a layer of 3-5 cm of broken soil is covered.
(3) After the biological fertilizer is used in soil, the biological fertilizer can well solve the soil hardening problem and increase and improve the microbial population of the soil. And the microbial population can be recovered to about 10 to 15 hundred million, and no chemical residue and no environmental pollution are generated in the process of improving the soil, so that the microbial agent is an environment-friendly microbial agent.
Example 3:
application of compound microbial agent in aspect of fruit trees
In this example, in order to improve the gala quality of apple, the test site: in the test field of apples in the Yangling area, the area of a small area of the test of the apples is 120 square meters, and 12 fruit trees are planted in the small area.
1. And (3) test treatment: processing one: the compound microbial agent prepared in the first embodiment is prepared by fully mixing the apple with water 50 days before harvesting (ratio: 1: 300), then treating the apple with an infiltration irrigation technology, fully mixing the apple with water 50L per square meter, then spraying the apple onto leaf surfaces once 30 days before harvesting, and then spraying the apple with the diluted apple liquid 30L per square meter; and (5) processing: changing the bacterial liquid in the first treatment into equivalent commercial chemical fertilizer; and (3) treatment III: and replacing the bacterial liquid in the first treatment with clean water with the same amount.
2. The test method comprises the following steps: after picking, the coloring degree and sweetness of the apples under each test treatment are compared, the coloring result is shown in a table I, and the sweetness detection is shown in a table II.
TABLE one apple coloring test results under three treatments
As can be seen from Table 1, the coloring effect of the compound microbial agent prepared in example 1 on apples is obviously better than that of the second treatment and the third treatment, and the coloring ratio of more than 50% is respectively 70.1%, 51.1% and 46.8%, so that the compound microbial agent prepared in example 1 has an obvious promoting effect on apple coloring.
Sweetness test results of apples under two or three test treatments
| Process one | Treatment two | Treatment three | |
| Sweetness level | 8.5 | 7.6 | 6.8 |
The second table shows that the effect of the compound microbial agent on sweetness improvement of the apple is obviously better than that of the second treatment and the third treatment, and the sweetness is improved by 25% compared with that of the control, which shows that the compound microbial agent prepared by the embodiment of the invention has an obvious promotion effect on the sweetness of the apple.
Example 4:
the invention relates to an application of a composite microbial agent in improving the number of soil microorganisms.
Selecting a test field with the square meter of 15 as a test cell.
And (3) test treatment: processing one: the compound microbial agent prepared in the first embodiment is fully mixed with water (the ratio is 1: 300) and sprayed and applied to soil of a test cell, and the usage amount of the diluted bacterial liquid is 20L per square meter; and (5) processing: replacing the compound microbial agent in the first treatment with a commercially available soil conditioner; and (3) treatment III: and replacing the bacterial liquid in the first treatment with clear water.
The test method comprises the following steps: the application is carried out 3 times in total in each treatment group, each time is separated by one week, and after 30 days of treatment once after week, sampling investigation is carried out to count the total number of the soil microorganisms.
The results of the soil microorganism quantity survey are shown in the third table.
Number of soil microorganisms under three treatments
As can be seen from the third table, the compound microbial agent has an obvious promotion effect on the recovery of the number of microorganisms in soil, is more efficient than the improvement brought by the commercially available soil conditioner, and can increase the number of microorganisms to about 10-15 hundred million.
In conclusion, the compound microbial agent has obvious promotion effects on soil improvement and restoration and improvement of apple quality, belongs to an environment-friendly biological agent, does not pollute the environment, and can solve the soil problem caused by excessive use of pesticide and fertilizer in the past.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (8)
1. A compound microbial agent is characterized by containing Paenibacillus polymyxa, Saccharomyces cerevisiae and lactococcus lactis.
2. The complex microbial inoculant according to claim 1, wherein the number of viable spores of each respective bacteria is: more than or equal to 3 hundred million/ml of paenibacillus polymyxa, more than or equal to 1 hundred million/ml of saccharomyces cerevisiae and more than or equal to 2 hundred million/ml of lactococcus lactis.
3. The compound microbial agent as claimed in claim 1 or 2, wherein after the paenibacillus polymyxa, saccharomyces cerevisiae and lactococcus lactis are fermented respectively to obtain secondary fermentation broth, the secondary fermentation broth of the three bacteria is inoculated into a tertiary fermentation medium according to the volume ratio of 3:2:2 for fermentation, and the fermentation is finished when the number of live spores of the paenibacillus polymyxa is more than or equal to 3 hundred million/ml, the number of live spores of the saccharomyces cerevisiae is more than or equal to 1 hundred million/ml and the number of live spores of the lactococcus lactis is more than or equal to 2 hundred million/ml in the fermentation broth, so as to obtain the compound microbial agent.
4. A method for preparing the complex microbial inoculant of any one of claims 1-3, comprising the steps of:
respectively carrying out primary seed fermentation and secondary seed fermentation on paenibacillus polymyxa, saccharomyces cerevisiae and lactococcus lactis to obtain secondary fermentation liquor;
and respectively inoculating the secondary fermentation liquid of the paenibacillus polymyxa, the saccharomyces cerevisiae and the lactococcus lactis into the tertiary fermentation culture medium according to the volume ratio of 3%, 2% and 2% of the tertiary fermentation culture medium for fermentation, and finishing fermentation when the number of the live spores of the paenibacillus polymyxa in the fermentation liquid is more than or equal to 3 hundred million/ml, the number of the live spores of the saccharomyces cerevisiae is more than or equal to 1 hundred million/ml and the number of the live spores of the lactococcus lactis is more than or equal to 2 hundred million/ml, so as to prepare the composite microbial agent.
5. The method as claimed in claim 4, wherein the first and second seed culture media used in the fermentation of ① Paenibacillus polymyxa in step (1) comprise, by weight, 3% -6% of brown sugar, 2% -4% of cane molasses, 0.8% -2.0% of peptone, 0.5% -1.5% of yeast extract, 0.3% -0.8% of NaCl0, K2HPO40.1% -0.4% of magnesium sulfate, 0.02% -0.09% of MnSO40.08% -0.16% of water, the balance being pH6.8-7.2, the first and second seed culture media used in the fermentation of lactococcus lactis ② comprise, by weight, 2% -3.5% of sucrose, 1% -1.5% of soybean meal, 0.8% -2.0% of corn steep liquor dry powder, 0.1% -0.6% of diammonium hydrogen citrate, 0.01% -0.09% of ferrous sulfate, K2HPO40.03% -0.12% of sucrose, 4.7% -0.03% of peptone, 0.63.63% -4.860.25% of MnSO, 0.1% -0.5% of yeast extract, the balance being water, the balance being 3% -3.5% of malt extract, 3.5% of yeast extract, the balance being water, 3.5% -3.5% of yeast extract.
6. The method according to any one of claims 4 to 5, wherein, after the three kinds of bacteria are inoculated into the tertiary fermentation medium in the step (2), the fermentation method is: controlling the fermentation temperature at 23-30 ℃, carrying out aerobic fermentation for 24-36h to enable the Paenibacillus polymyxa to become dominant flora and rapidly proliferate, reducing the pH to 4.0-5.2, carrying out constant-pressure anaerobic fermentation, controlling the temperature at 23-30 ℃, carrying out anaerobic fermentation for 24-36h to enable the Saccharomyces cerevisiae and lactococcus lactis to become dominant flora and rapidly proliferate, and finally enabling the number of live spores of the Paenibacillus polymyxa to be more than or equal to 3 hundred million/ml, the number of live spores of the Saccharomyces cerevisiae to be more than or equal to 1 hundred million/ml, and the number of live spores of the lactococcus lactis to be more than or equal to 2 hundred million/ml.
7. The compound microbial inoculant according to claim 1, wherein the compound microbial inoculant is mainly applied to soil improvement and apple sugar content increase to improve quality.
8. The complex microbial agent according to claim 1, wherein the complex microbial agent of the present invention is prepared by mixing the following components in a ratio of 1: 300, and then the mixture is fully mixed with water and is combined with an infiltrating irrigation technology and soil surface layer spraying treatment for fruit tree cultivation.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111470889A (en) * | 2020-05-28 | 2020-07-31 | 上海绿乐生物科技有限公司 | Special microbial agent for soil improvement and preparation method thereof |
| CN113200613A (en) * | 2021-04-22 | 2021-08-03 | 青海洁神环境科技股份有限公司 | Microbial nutrient solution for sewage treatment and preparation method and application thereof |
| CN113387754A (en) * | 2020-12-28 | 2021-09-14 | 菏泽金正大生态工程有限公司 | Biological fertilizer for apple cultivation and preparation method thereof |
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| CN113387754A (en) * | 2020-12-28 | 2021-09-14 | 菏泽金正大生态工程有限公司 | Biological fertilizer for apple cultivation and preparation method thereof |
| CN113200613A (en) * | 2021-04-22 | 2021-08-03 | 青海洁神环境科技股份有限公司 | Microbial nutrient solution for sewage treatment and preparation method and application thereof |
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