CN112481130A - Method for extracting and preparing efficient composite composting microbial inoculum from cow dung - Google Patents
Method for extracting and preparing efficient composite composting microbial inoculum from cow dung 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
- C12N1/02—Separating microorganisms from their culture media
-
- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F17/00—Preparation of fertilisers characterised by biological or biochemical treatment steps, e.g. composting or fermentation
- C05F17/20—Preparation of fertilisers characterised by biological or biochemical treatment steps, e.g. composting or fermentation using specific microorganisms or substances, e.g. enzymes, for activating or stimulating the treatment
-
- 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
-
- 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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W30/00—Technologies for solid waste management
- Y02W30/40—Bio-organic fraction processing; Production of fertilisers from the organic fraction of waste or refuse
Abstract
The invention relates to a method for extracting and preparing a high-efficiency composite composting microbial inoculum from cow dung, which comprises the following steps: the method comprises the following steps of (1) separating and purifying strains, step (2) preparing a liquid compound microbial inoculum, step (3) preparing a fermentation raw material of a solid compound microbial inoculum, step (4) inoculating and culturing, step (5) fermenting, and step (6) preparing the solid compound microbial inoculum.
Description
Technical Field
The invention relates to a preparation method of a microbial inoculum, in particular to a method for extracting and preparing a high-efficiency composite composting microbial inoculum from cow dung, and belongs to the technical field of composting.
Background
High-temperature aerobic composting is one of the effective methods for treating kitchen waste. Composting is the process of biostabilizing organic solid waste under artificially controlled conditions. The kitchen waste has high organic matter content, comprehensive nutrient elements and low C/N ratio, is a good nutrient substance for microorganisms and is very suitable for being used as a composting raw material. The method has the advantages of simple treatment method and more retained nitrogen in the finished product. The disadvantages are large field required, long treatment period and difficult killing of some germs. How to improve the efficiency of treating kitchen garbage and how to make compost products harmless is a problem to be solved at present. Microbial inoculum is added in the composting process, so that the quantity of microorganisms in the compost can be increased, the temperature of the compost can be increased in the metabolism process of the microorganisms, the harmless effect of the compost is achieved, and the decomposition of organic matters in the kitchen garbage can be accelerated, so that the composting efficiency is promoted.
The invention patent CN105154373A provides a cow dung high-temperature decomposition agent and a preparation method thereof, and discloses a cow dung high-temperature decomposition agent which is prepared by fermenting and drying cow dung and a composite microbial inoculum; the cow dung particles of the cow dung high-temperature decomposition agent are loaded with mixed bacteria of bacillus subtilis, bacillus licheniformis, in-situ composting bacillus licheniformis, thermomyces lanuginosus and thermobifida fusca. However, the zymophyte strain is relatively single and uncertain in source, and whether cow dung is used as a carrier for stable propagation needs to be studied, so that the composting process is unstable and the cost is increased. The invention patent CN102795907A provides a method for producing organic compound fertilizer by cow dung, the steps of the invention are simple, the manufacture is convenient, but the decomposition is not carried out after the fermentation period, the index of the decomposition degree of the product can not meet the requirement, and the cow dung has long natural fermentation period and incomplete decomposition. The invention provides a preparation method of a cow dung organic fertilizer, which takes cow dung as a raw material, and maintains nutrient substances such as protein, microorganisms and the like in the cow dung to the maximum extent through a reasonable preparation method, so that pathogenic bacteria and harmful substances are killed to the maximum extent, the prepared cow dung organic fertilizer has high fertilizer efficiency, obvious yield increasing effect on crops and low cost. However, the method has relatively complex operation flow, uses three regulators of a disinfectant, a leavening agent and a culture agent, and has high cost and low decomposition degree. Therefore, a new solution to solve the above technical problems is urgently needed.
Disclosure of Invention
The invention provides a method for re-extracting and preparing high-efficiency composite composting microbial inoculum from cow dung aiming at the problems in the prior art, the technical scheme comprises a preparation method of a liquid composite microbial inoculum and a preparation method of a solid composite microbial inoculum, covers various functional bacteria in an aerobic composting process, has simple preparation process and ecological environmental protection, greatly improves composting efficiency and economic benefit, and has good application prospect.
In order to achieve the purpose, the technical scheme of the invention is as follows, and the method for extracting and preparing the high-efficiency composite composting microbial inoculum from the cow dung comprises the following steps:
step (1), strain separation and purification: the mass ratio of the fresh cow dung to the auxiliary material mixture is 2-5: 1, uniformly mixing and then naturally fermenting; when the temperature of the first fermentation reaches 60-65 ℃, adding the fermentation product into a mixture of fresh cow dung and auxiliary materials according to the proportion of 1/10-1/4 to start second natural fermentation; and repeating the steps until the fermentation temperature of the fifth compost reaches 60-65 ℃, separating and purifying microorganisms in the materials obtained by the fifth fermentation by taking the materials obtained by the fifth fermentation as separation sources, and screening high-efficiency functional strains through enzyme production characteristics and carbon source utilization tests.
Step (2) preparation of liquid complex microbial inoculum: inoculating each strain to corresponding liquid culture medium, performing strain seed liquid amplification culture, measuring viable bacteria content of each seed liquid by plate counting method, and adjusting each seed liquid to 1.0 × 10 with sterile water8cfu/ml~2.0×108cfu/ml, according to viable count 1:1, mixing the seed solutions to prepare the liquid composite microbial inoculum.
Preparing a solid compound microbial inoculum fermentation raw material in the step (3): the solid composite microbial inoculum raw material is crushed to about 1cm in advance and then is dried, so that the water content is reduced to below 10-15%.
Step (4), inoculating and culturing: and uniformly spraying the liquid compound microbial inoculum and 2 strains of specific functional bacteria solution on the solid compound microbial inoculum fermentation raw material, so that the water content of the solid compound microbial inoculum fermentation raw material reaches 65-80%.
And (5) fermenting: and adding the mixed solid-state composite microbial inoculum fermentation raw materials into the mixture for aerobic fermentation, wherein the fermentation time is 30-40 days, and the fermentation temperature reaches 60-65 ℃.
Step (6) preparation of solid complex microbial inoculum: and (3) placing the fermented product in a constant-temperature oven, starting an air blower, drying at the constant temperature of 35 ℃ for 10 hours, drying, and then filling into a sterile sealed bag to obtain the solid-state composite microbial inoculum. In this scheme, contain abundant nutrient substance in the cow dung, can provide carrier and nutrient substance for the activity of microorganism, contain the useful microorganism of multiple intestinal in the cow dung again simultaneously, use cow dung as the isolated source, the microorganism that the separation obtained adds compost raw materials as the microbial inoculum, the adaptability of microorganism is strong, can expand the reproduction rapidly, change the microbial community structure of heap body, play a role in the heap body, make heap body temperature rise rapidly, and can keep for a long time in the high temperature period, fully inactivate harmful flora and weed seed etc. wherein, make the compost product reach innoxiousization. Therefore, the invention provides a method for preparing an efficient compound composting microbial inoculum by utilizing cow dung compost.
Further, the auxiliary material mixture comprises 1-2 parts of fallen leaves, 1-2 parts of corn straws, 0-1 part of coconut coir and 0-1 part of sawdust. The auxiliary material is used as dry matter to adjust the humidity and the carbon-nitrogen ratio of the cow dung, so that the mixed material is more suitable for the growth of microorganisms.
Further, the auxiliary material mixture is pretreated to ensure that the particle size is less than 1cm and the water content is less than 30%. The small granularity is to mix the auxiliary materials and the cow dung uniformly, and because the water content of the fresh cow dung is higher, the addition of the auxiliary materials needs to adjust the humidity of the materials to enable microorganisms to grow more suitably, and therefore the water content of the auxiliary materials needs to be controlled below 30%.
Further: the liquid composite microbial inoculum strain seed liquid amplification culture conditions are that the rotating speed of a shaking table is set to be 160-200 r/min, bacteria are cultured for 20-24 h at 37 ℃, fungi are cultured for 48-72 h at 30 ℃, and actinomycetes are cultured for 3-5 days at 28 ℃. The culture time can ensure that the microorganisms grow to a logarithmic phase, and the microorganisms have the strongest activity and the largest quantity.
Further, the liquid complex microbial inoculum comprises 11 functional strains separated from cow dung compost: 3 medium temperature strains, 2 high temperature strains, 2 cellulose degrading bacteria, 2 lignin degrading bacteria and 2 starch degrading bacteria; contains most functional strains required by the composting process, can promote the composting to be harmless, increases the degradation rate, and improves the decomposition degree and the product quality of the compost product.
Further, the solid-state composite microbial inoculum is a mixture of fallen leaves and corn straws as a raw material, and the mixing mass ratio is 1: 1. The fallen leaves are thin and can be fully mixed with the microbial inoculum, the water absorption and the air permeability of the corn straws are good, and the humidity and the porosity of the materials can be adjusted.
Further, the solid complex microbial inoculum comprises 11 functional strains obtained from the liquid complex microbial inoculum and also comprises 2 fungi separated from the high-temperature period and the rotten period of the fallen leaf compost. The main components of the fallen leaves are cellulose and lignin, fungi separated from fallen leaf compost have strong capability of decomposing cellulose and lignin, the substances which are most difficult to decompose in the compost are macromolecular substances such as cellulose, lignin and the like, and the two fungi can promote the degradation process of the compost
Furthermore, the solid and liquid composite microbial inoculum can be applied to various organic waste composting systems, so that the composting time can be shortened by 20-30%, and the composting efficiency is improved.
The method for extracting and preparing the high-efficiency composite composting microbial inoculum from the cow dung has the following advantages:
1) the composite microbial inoculum is indigenous bacteria separated in the processes of cow dung composting and fallen leaf composting, and has strong adaptability and high activity; various functional bacteria covering the aerobic composting process: the medium-temperature strains, the high-temperature strains, the cellulose degrading bacteria, the lignin degrading bacteria and the starch degrading bacteria are enriched to prepare the microbial inoculum for the composting process, and the lag phase of microorganisms is shortened, so that the composting process rapidly enters the high-temperature phase, the composting process is accelerated, and the composting decomposition efficiency can be improved by 20%.
2) The medium-temperature strain and the high-temperature strain can quickly heat the compost, can continuously activate in the high-temperature period of the compost, prolong the high-temperature period of the compost, improve the inactivation rate of harmful bacteria in compost raw materials, and ensure that the compost is harmless.
3) Fungi produce a variety of biologically active enzyme systems, such as: cellulase, chitinase, xylanase and the like are added into the composite microbial inoculum to accelerate the degradation of composting raw materials, can be applied to various organic waste composting systems, and can shorten the composting time by 20-30%.
4) The preparation method of the composite microbial inoculum obtains two composite microbial inoculants for high-efficiency composting, namely a liquid composite microbial inoculum and a solid composite microbial inoculum.
5) The number of viable bacteria of the functional bacteria such as mesophilic strain, thermophilic strain, cellulose degradation bacteria, lignin degradation bacteria and starch degradation bacteria is 108cfu/mL, thereby ensuring that the compost composite microbial inoculum is 109cfu/mL (g) of a high-density microbial inoculum.
6) The two fungi separated from the fallen leaf compost have strong capability of decomposing cellulose and lignin, and promote the degradation of the refractory macromolecular substances in the composting process, thereby improving the compost maturity and the quality of compost products.
Therefore, the invention not only can change waste into valuable, reduce resource waste and environmental pollution, but also can promote the comprehensive development of livestock and poultry breeding and farmland planting in China, improve ecological effect and economic benefit, and has good application prospect.
The specific implementation mode is as follows:
in order to enhance the understanding and appreciation of the present invention, the technical solution is described in detail with reference to the following detailed description.
Example 1: a method for extracting and preparing a high-efficiency composite composting microbial inoculum from cow dung comprises the following steps:
(1) and (3) strain seed liquid amplification culture: preparing a culture medium, sterilizing and cooling in a high-pressure steam sterilization pot, inoculating each strain to a corresponding liquid culture medium, performing seed shake flask culture, setting the rotating speed of a shaking table to be 160r/min, culturing bacteria for 20-24 h at 30-37 ℃, culturing fungi for 48-72 h at 28-32 ℃, and culturing actinomycetes for 3-5 days at 28-30 ℃.
(2) Preparing a liquid composite microbial inoculum: measuring viable bacteria content of each seed liquid by plate counting method, and adjusting each seed liquid to 5.0 × 10 with sterile water8cfu/ml, in an ultra-clean workbench, according to the viable count of 1:1 mixing the seed liquids to obtain a liquid stateA composite microbial inoculum.
Example 2:
(1) and (3) strain seed liquid amplification culture: preparing a culture medium, sterilizing and cooling in a high-pressure steam sterilization pot, inoculating each strain to a corresponding liquid culture medium and performing seed shake flask culture under the corresponding temperature condition, setting the rotating speed of a shaking table to be 160r/min, culturing bacteria for 20-24 h at 30-37 ℃, culturing fungi for 48-72 h at 28-32 ℃, and culturing actinomycetes for 3-5 days at 28-30 ℃.
(2) Preparing a liquid composite microbial inoculum: measuring viable bacteria content of each seed liquid by plate counting method, and adjusting each seed liquid to 2.0 × 10 with sterile water8cfu/ml, in an ultra-clean workbench, according to the viable count of 1:1, mixing the seed solutions to obtain the liquid composite microbial inoculum.
(3) Preparing solid fermentation raw materials: cutting corn straws into 3-5 cm crushed materials in advance, drying the crushed materials in the sun for use, and mixing fresh cow dung and the crushed corn straws according to a ratio of 4: 6, the mixture is uniformly mixed, so that the water content of the mixed fermentation raw materials is 65 percent.
(4) Inoculating and culturing: spraying the liquid composite microbial inoculum on the compost raw materials according to the addition of 1.0 percent, and then uniformly mixing.
(5) Fermentation: adding the mixed fermentation raw materials into a composting pool of 1.0m multiplied by 1.2m for aerobic fermentation for 10 days.
(6) And (3) drying: and (3) placing the fermented product in a constant-temperature oven, starting an air blower, drying at the constant temperature of 35 ℃ for 10 hours, drying, and then filling into a sterile sealed bag to obtain the solid-state composite microbial inoculum.
Example 3:
(1) and (3) strain seed liquid amplification culture: preparing a culture medium, sterilizing and cooling in a high-pressure steam sterilization pot, inoculating each strain to a corresponding liquid culture medium and performing seed shake flask culture under the corresponding temperature condition, setting the rotating speed of a shaking table to be 160r/min, culturing bacteria for 20-24 h at 30-37 ℃, culturing fungi for 48-72 h at 28-32 ℃, and culturing actinomycetes for 3-5 days at 28-30 ℃.
(2) Preparing a liquid composite microbial inoculum: determination of viable bacteria content of various seed solutions by using plate counting methodMetering, and adjusting the seed liquid to 2.0X 10 with sterile water8cfu/ml, in an ultra-clean workbench, according to the viable count of 1:1, mixing the seed solutions to obtain the liquid composite microbial inoculum.
(3) Preparing solid fermentation raw materials: cutting corn straws into 3-5 cm crushed materials in advance, drying the crushed materials in the sun for use, and mixing fresh cow dung and the crushed corn straws according to a ratio of 4: 6, the mixture is uniformly mixed, so that the water content of the mixed fermentation raw materials is 65 percent.
(4) Inoculating and culturing: spraying the liquid composite microbial inoculum on the compost raw materials according to the addition of 0.75 percent, and then uniformly mixing.
(5) Fermentation: adding the mixed fermentation raw materials into a composting pool of 1.0m multiplied by 1.2m for aerobic fermentation for 12 days.
(6) And (3) drying: and (3) placing the fermented product in a constant-temperature oven, starting an air blower, drying at the constant temperature of 35 ℃ for 10 hours, drying, and then filling into a sterile sealed bag to obtain the solid-state composite microbial inoculum.
It should be noted that the above-mentioned embodiments are not intended to limit the scope of the present invention, and all equivalent modifications and substitutions based on the above-mentioned technical solutions are within the scope of the present invention as defined in the claims.
Claims (8)
1. A method for extracting and preparing a high-efficiency composite composting microbial inoculum from cow dung is characterized by comprising the following steps:
step (1), strain separation and purification: the mass ratio of the fresh cow dung to the auxiliary material mixture is 2-5: 1, uniformly mixing and then naturally fermenting; when the temperature of the first fermentation reaches 60-65 ℃, adding the fermentation product into a mixture of fresh cow dung and auxiliary materials according to the proportion of 1/10-1/4 to start second natural fermentation; repeating the above steps until the fermentation temperature of the fifth compost reaches 60-65 ℃, separating and purifying microorganisms in the fifth fermented materials as separation sources, and screening high-efficiency functional strains through enzyme production characteristics and carbon source utilization tests;
step (2) preparation of liquid complex microbial inoculum: inoculating each strain to corresponding liquid culture medium respectivelyPerforming amplification culture of strain seed solution, measuring viable bacteria content of each seed solution by plate counting method, and adjusting each seed solution to 1.0 × 10 with sterile water8cfu/ml~2.0×108cfu/ml, according to viable count 1:1, mixing the seed solutions to prepare a liquid composite microbial inoculum;
preparing a solid compound microbial inoculum fermentation raw material in the step (3): crushing a solid composite microbial inoculum raw material to about 1cm in advance, and then drying the crushed solid composite microbial inoculum raw material to reduce the water content to below 10-15%;
step (4), inoculating and culturing: uniformly spraying the liquid compound microbial inoculum and 2 strains of specific functional bacteria solution on a solid compound microbial inoculum fermentation raw material to enable the water content of the solid compound microbial inoculum fermentation raw material to reach 65-80%;
and (5) fermenting: adding the mixed solid-state composite microbial inoculum fermentation raw materials into the mixed solid-state composite microbial inoculum for aerobic fermentation, wherein the fermentation time is 30-40 days, and the fermentation temperature reaches 60-65 ℃;
step (6) preparation of solid complex microbial inoculum: and (3) placing the fermented product in a constant-temperature oven, starting an air blower, drying at the constant temperature of 35 ℃ for 10 hours, drying, and then filling into a sterile sealed bag to obtain the solid-state composite microbial inoculum.
2. The method for extracting and preparing a high-efficiency composite composting microbial inoculum from cow dung according to claim 1, wherein the auxiliary material mixture comprises 1-2 parts of fallen leaves, 1-2 parts of corn stalks, 0-1 part of coconut coir and 0-1 part of sawdust.
3. The method for extracting and preparing a high-efficiency composite composting microbial inoculum from cow dung as claimed in claim 2, wherein the auxiliary material mixture is pretreated to have a particle size of less than 1cm and a water content of less than 30%.
4. The method for extracting and preparing a high-efficiency composite composting microbial inoculum from cow dung according to claim 3, wherein the conditions for the seed liquid expanded culture of the liquid composite microbial inoculum strain are that the rotating speed of a shaking table is set to be 160-200 r/min, bacteria are cultured for 20-24 h at 30-37 ℃, fungi are cultured for 48-72 h at 28-32 ℃, and actinomycetes are cultured for 3-5 days at 28-30 ℃.
5. The method for extracting and preparing a high-efficiency composite compost microbial inoculum from cow dung as claimed in claim 3 or 4, wherein the liquid composite microbial inoculum comprises 11 functional strains separated from cow dung compost: 3 mesophilic strains (2 bacteria and 1 actinomycete), 2 thermophilic strains (1 bacteria and 1 fungus), 2 cellulose degrading strains (2 fungi), 2 lignin degrading strains (2 fungi) and 2 starch degrading strains (2 fungi), wherein the viable count of each strain in the composite microbial inoculum reaches 1.0 multiplied by 108cfu/ml~9.0×108cfu/ml。
6. The method for extracting and preparing the high-efficiency composite composting microbial inoculum from the cow dung as claimed in claim 5, wherein the solid composite microbial inoculum is a mixture of fallen leaves and corn stalks, and the mixing mass ratio is 1: 1.
7. The method for extracting and preparing a high-efficiency composite compost microbial inoculum from cow dung as claimed in claim 6, wherein the solid composite microbial inoculum comprises 11 functional strains obtained from a liquid composite microbial inoculum and also comprises 2 fungi separated from a high-temperature period and a rotten period of fallen leaf compost.
8. The method for extracting and preparing the efficient composite composting microbial inoculum from the cow dung as claimed in claim 6, wherein the solid and liquid composite microbial inoculum can be applied to various organic waste composting systems, so that the composting time is shortened by 20-30%.
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