CN109762765B - Decomposed solid fermentation microbial inoculum and application thereof in agricultural wastes - Google Patents
Decomposed solid fermentation microbial inoculum and application thereof in agricultural wastes Download PDFInfo
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- 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
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- Fodder In General (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a decomposed solid fermentation microbial inoculum using agricultural organic wastes such as cow dung, straws (walnut pruned branches and cotton stalks) and the like as raw materials, which comprises saccharomyces cerevisiae (saccharomyces cerevisiae)Saccharomyces cerevisiae) Bacillus methylotrophicus (A), (B) and (C)Bacillus methylotrophicus) Bacillus denitrificans thermophilus (B), (B)Geobacillus thermodenitrificans) Bacillus simplex (I) ((II))Bacillus simplex) And Lactobacillus buchneri: (Lactobacillus parabuchneri) Five strains of bacteria. The active microbial inoculum provided by the invention utilizes the synergistic effect of the five bacteria in compost materials to quickly form dominant flora, fully play the roles of deodorization and promotion of the decomposition process. Meanwhile, the problems of low effective utilization rate of agricultural organic wastes, long decomposition time of naturally fermented compost and poor product quality are solved. The fertilizer treated by the decomposition and fermentation can improve the soil fertility of farmland, regulate the pH value of soil and have good ecological benefit and economic benefit.
Description
Technical Field
The invention belongs to the technical field of biological fermentation, and further relates to a decomposed solid fermentation microbial inoculum and the technical field of application thereof in utilization of agricultural wastes.
Background
In recent years, with the rapid development of the forest and fruit industry, the planting areas of walnuts, red dates, apricots, apples, grapes and the like are continuously expanded, a large amount of agricultural solid wastes such as pruning and the like are randomly piled up to occupy a large amount of cultivated land, the crop diseases are easily spread, and the random burning causes the air pollution problem. The annual output of various agricultural wastes in China exceeds 7 hundred million tons, and the agricultural solid waste decomposing and returning technology is a precious renewable resource, and has important effects on improving the organic matter content of soil, improving the physical properties of the soil, fertilizing the soil fertility, increasing the microbial activity of the soil, reducing soil-borne diseases, improving the yield-increasing potential of crops and improving the agricultural ecological environment.
In addition, the rapid development of the breeding industry is accompanied by the rapid increase of the yield of the livestock and poultry manure, the livestock and poultry manure contains a large amount of organic matters and is easy to nourish pathogenic microorganisms, if the livestock and poultry manure is not treated properly, serious harm can be caused to the environment, such as spreading and spreading of epidemic diseases and parasite eggs, breeding of mosquito ropes, deterioration of water quality, even blackening and smelliness, serious standard exceeding of heavy metals in the soil can be caused by direct discharge of excessive unbounded land, and the soil, air, surface groundwater and the like can be polluted. It is predicted that the discharge amount of the livestock and poultry feces in China will reach 43 hundred million tons by 2020. The production of livestock and poultry manure exceeds industrial and domestic solid pollutants, and the problem of environmental pollution caused by the livestock and poultry manure is more and more prominent.
At present, the rapid decomposition and field return of agricultural wastes become hot spots for the fertilizer utilization of the agricultural wastes at home and abroad, and the research and development of efficient rapid decomposition microbial inoculum of the agricultural wastes is the key point for the efficient utilization of the agricultural wastes. The fast decomposition is an organic fertilizer formed by fermentation after adding a high-efficiency microbial preparation, and compared with the traditional compost, the organic matter and nutrient content of the organic fertilizer is higher, the improvement on the physical and chemical properties of soil is more obvious, and the organic fertilizer has certain influence on the growth and yield of crops. Meanwhile, the decomposed microbial preparation contains a large amount of beneficial high-temperature high-humidity microbial populations, can produce a large amount of high-activity enzymes, and can have certain influence on soil enzymes and microbial systems.
Disclosure of Invention
Based on the fact that the rapid decomposition and field return of agricultural wastes become hot spots for the fertilizer utilization of agricultural wastes at home and abroad at present, the key point for the efficient utilization of the agricultural wastes is to develop and develop the efficient rapid decomposition microbial inoculum of the agricultural wastes. The invention provides a decomposed solid fermentation microbial inoculum and application thereof in agricultural wastes, the microbial inoculum is fast in temperature rise and long in holding time after inoculation, macromolecular organic matters such as cellulose and the like can be rapidly degraded, the activity of ova, grass seeds and pathogenic bacteria is killed, the decomposition speed is improved, and the application in agricultural waste treatment obtains remarkable technical effects.
The invention adopts the following technical means to solve the technical problems:
the decomposed solid fermentation microbial inoculum is prepared by five strains including Saccharomyces cerevisiae (Saccharomyces cerevisiae), Bacillus methylotrophicus (Bacillus methylotrophicus), Bacillus thermophilus (Geobacillus thermonitrificans), Bacillus simplex (Bacillus simplexex) and Lactobacillus buchneri (Lactobacillus paracoccus) according to a specific proportion and a preparation method thereof, and further applied to agricultural wastes, can rapidly degrade macromolecular organic matters such as cellulose and the like, kills the activities of ova, grass seeds and pathogenic bacteria, improves the decomposition speed, and obtains remarkable technical effects when applied to agricultural waste treatment.
The invention also discloses a preparation method of the decomposed solid fermentation microbial inoculum, which comprises the following steps:
(1) activating strains of five strains of Saccharomyces cerevisiae (Saccharomyces cerevisiae), Bacillus methylotrophicus (Bacillus methylotrophicus), Bacillus thermophilus (Geobacillus thermonitrificans), Bacillus simplex (Bacillus simplicix) and Lactobacillus buchneri (Lactobacillus parabuchner) stored in an environment of 0-4 ℃, and respectively carrying out test tube-shake flask culture to obtain primary seed liquid of each strain;
(2) continuously carrying out fermentation tank expanded culture on the seed liquid, and respectively fermenting seed liquid of Saccharomyces cerevisiae (Saccharomyces cerevisiae), Bacillus methylotrophicus (Bacillus methylotrophicus), Bacillus thermophilus (Geobacillus thermonitrificans), Bacillus simplex (Bacillus simplexex) and Lactobacillus buchneri (Lactobacillus parachui) to obtain zymocyte liquid; respectively detecting light absorption value and flora number of the fermented liquid by microscopy direct counting method and ultraviolet spectrophotometry, determining growth condition of flora, and detecting effective viable bacteria number of 1 × 109Stopping culturing at cfu/ml;
(3) after the fermentation of the single strains is finished, the fermentation method comprises the following steps of mixing Saccharomyces cerevisiae (Saccharomyces cerevisiae), Bacillus methylotrophicus (Bacillus methylotrophicus), Bacillus thermophilus (Geobacillus thermophilus), Bacillus simplex (Bacillus simplex) and Lactobacillus buchneri (Lactobacillus parabuchneri) according to the weight ratio of 1: 2: 2: 1: 1 to prepare a decomposed liquid composite fermentation microbial inoculum;
(4) and (3) mixing the decomposed liquid composite fermentation inoculum obtained in the step (3) according to the volume ratio of 1: 2, mixing the mixture with light calcium carbonate, uniformly stirring the mixture and drying the mixture until the water content is below 15 percent;
(5) and (3) adding a protective agent potassium sorbate into the composite microbial inoculum obtained in the step (4) according to the mass concentration of 1-1.5 per mill, and uniformly stirring to obtain the decomposed solid fermentation microbial inoculum.
The invention provides a decomposed solid fermentation microbial inoculum obtained by the preparation process.
In the invention, the strain culture medium and the culture method adopted by Saccharomyces cerevisiae (Saccharomyces cerevisiae) and Lactobacillus buchneri (Lactobacillus parabruchneri), and the formula of the FC2 culture medium is as follows: 2% sucrose; 0.5% yeast extract; 0.3% peptone; 0.2% disodium hydrogen phosphate; the inoculation amount is 1% -2%, and the cultivation is carried out for 24-36h at 180-.
In the invention, the strain culture medium and the culture method adopted by Bacillus methylotrophicus and Bacillus thermophilus are optimized and adopted by improved YM1 culture medium, and the formula of the culture medium is as follows: 0.5% corn steep liquor; 0.2% disodium hydrogen phosphate; 1% sucrose; the inoculation amount is 1-2%, the speed is 150-200 rpm, and the culture is carried out for 24-36 h.
In the invention, a strain culture medium and a culture method adopted by simple Bacillus (Bacillus simplex) are as follows: LBG culture medium is preferably adopted, and the formula of the LBG culture medium is as follows: 0.5% glucose, 1% peptone, 0.5% yeast extract, 1% sodium chloride; the inoculation amount is 1-2%, the speed is 150-200 rpm, and the culture is carried out for 24-36 h.
In the invention, five strains of Saccharomyces cerevisiae (Saccharomyces cerevisiae), Bacillus methylotrophicus (Bacillus methylotrophicus), Bacillus thermophilus (Geobacillus thermophilus), Bacillus simplex (Bacillus simplex) and Lactobacillus buchneri (Lactobacillus parabuchneri) which are adopted are harmless and safe strains allowed to be used by the agricultural department, are all purchased from the China general microbiological culture collection center (CGMCC), and can be obtained by common technicians through public channels.
Meanwhile, the invention provides the application of the decomposed solid fermentation microbial inoculum in agricultural wastes.
Further, the invention provides application of the decomposed solid fermentation microbial inoculum in agricultural wastes, wherein the agricultural wastes preferentially adopt 80% of fresh cow dung and 20% of straws in percentage by weight.
The invention provides application of a decomposed solid fermentation microbial inoculum in agricultural wastes, wherein 20% of straws are walnut straws or cotton straws preferentially.
By implementing the technical scheme of the invention, the following beneficial effects can be achieved:
(1) the decomposed solid fermentation microbial inoculum provided by the invention has the characteristics of strong adaptability synergy and certain high temperature resistance by utilizing five strains of Saccharomyces cerevisiae (Saccharomyces cerevisiae), Bacillus methylotrophicus (Bacillus methylotrophicus), Bacillus thermophilus (Geobacillus thermonitrificans), Bacillus simplex (Bacillus simplexex) and Lactobacillus buchneri (Lactobacillus paracuchneri) in the decomposing process of cow dung and straws, generates various bioactive enzymes to participate in the decomposing process, has the characteristics of novel strain combination, stable structure, strong enzyme activity, high bacterial count and the like, can quickly form dominant bacterial groups after entering compost materials, can grow and reproduce in a large quantity, releases cellulase, lipase, amylase, dehydrogenase, protease and other bioactive substances, and quickly decomposes lignocellulose, crude fiber, fat, starch, protein and the like in the compost materials into low-molecular organic matters. Meanwhile, growth promoting and disease resisting functions of Bacillus methylotrophicus and Bacillus simplex are utilized to promote the growth of plants and prevent diseases, so that the decomposed fermentation product has certain effects of promoting the growth of crops and preventing diseases; the thermophilic denitrified bacillus (Geobacillus thermosulfinitificans) can convert unavailable organic nitrogen into available inorganic nitrogen, so that the nitrogen utilization rate of decomposed products is improved; the acid production function of Lactobacillus buchneri and Saccharomyces cerevisiae can effectively adjust the pH value of compost materials, reduce alkalinity, degrade ammonia (amine) containing components in the materials, and control the emission of odor gas such as ammonia gas, thereby achieving the purposes of deodorizing, improving the quality of compost finished products and promoting the composting efficiency.
(2) The decomposed solid fermentation microbial inoculum provided by the invention is applied to agricultural wastes, the temperature rise is fast after the microbial inoculum is inoculated, the retention time is long, the activities of worm eggs, grass seeds and the like in the cow dung can be rapidly killed, and the decomposition speed of straws and cow dung is improved. The loose space structure of the light calcium carbonate greatly improves the microorganism adsorption rate, increases the combination degree with the bottom materials in the composting process, enables the microbial inoculum to be uniformly mixed in the composting materials, quickly forms dominant flora, fully plays the role of deodorization and promotes the decomposing process. The fertilizer treated by the decomposition and fermentation can be used as an agricultural organic fertilizer, the soil fertility of farmlands is improved, the soil pH value is adjusted, and good ecological benefit and economic benefit are achieved.
(3) The preparation process of the decomposed solid fermentation microbial inoculum provided by the invention is simple and suitable for industrial production, and a non-toxic protective agent is adopted in the preparation process, so that the safety of the microbial inoculum is ensured, and the preparation efficiency can be improved. Meanwhile, the prepared decomposed solid fermentation microbial inoculum consists of saccharomyces cerevisiae, bacillus methylotrophicus, bacillus denitrificans, simple bacillus and lactobacillus buchneri which have excellent environment adaptability and strong synergistic effect, the microbial inoculum is heated quickly after inoculation, the holding time is long, macromolecular organic matters such as cellulose and the like can be degraded quickly, the activity of ova, grass seeds and pathogenic bacteria is killed, the decomposition speed is improved, and the decomposed solid fermentation microbial inoculum is suitable for being used for treating agricultural wastes and shows remarkable technical effects.
Drawings
Fig. 1 is a graph showing temperature changes during the decomposition process.
Detailed Description
The present invention will be described below by way of examples, but the present invention is not limited to the following examples.
The materials of the invention are: saccharomyces cerevisiae (Saccharomyces cerevisiae), Bacillus methylotrophicus (Bacillus methylotrophicus), Bacillus thermophilus (Geobacillus thermonitrificans), Bacillus simplex (Bacillus simplex), Lactobacillus buchneri (Lactobacillus parabuchneri), potassium sorbate and light calcium carbonate can be purchased through public channels, and equipment and instruments adopted in the process are common equipment in the field.
All materials, reagents and equipment selected for use in the present invention are well known in the art, but do not limit the practice of the invention, and other reagents and equipment well known in the art may be suitable for use in the practice of the following embodiments of the invention.
The following examples further illustrate the present invention but are not to be construed as limiting 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.
Example 1: preparation of decomposed solid fermentation microbial inoculum
The invention provides a preparation method of a decomposed solid fermentation microbial inoculum, which comprises the following steps:
(1) activating various slant strains stored in an environment of 0-4 ℃, namely Saccharomyces cerevisiae (Saccharomyces cerevisiae), Bacillus methylotrophicus (Bacillus methylotrophicus), Bacillus thermophilus (Geobacillus thermonitrificans), Bacillus simplex (Bacillus simplicix) and Lactobacillus buchneri (Lactobacillus parachui). Culturing the strain: saccharomyces cerevisiae (Saccharomyces cerevisiae) and Lactobacillus buchneri (Lactobacillus parabruchneri) were cultured in FC2 medium; the FC2 culture medium comprises the following components: 2% sucrose; 0.5% yeast extract; 0.3% peptone; 0.2% disodium hydrogen phosphate; the inoculation amount is 1% -2%, and the cultivation is carried out for 24-36h at 180-. FC2 culture medium, Bacillus methylotrophicus (Bacillus methylotrophicus) and Bacillus denitrificans (Geobacillus thermoentificans) adopt optimized YM1 culture medium, and the formula of the culture medium is as follows: 0.5% corn steep liquor; 0.2% disodium hydrogen phosphate; 1% sucrose; the inoculation amount is 1-2%, the speed is 150-200 rpm, and the culture is carried out for 24-36 h. The Bacillus simplex simpliciens adopts LBG culture medium, and the formula of the LBG culture medium is as follows: 0.5% glucose, 1% peptone, 0.5% yeast extract, 1% sodium chloride; the inoculation amount is 1-2%, the speed is 150-200 rpm, and the culture is carried out for 24-36 h. Performing test tube-shake flask culture on the five strains respectively to obtain first-level seed liquid of each strain;
(2) continuously carrying out fermentation tank amplification culture on the seed solution, and fermenting saccharomyces cerevisiae, bacillus methylotrophicus, bacillus denitrificans thermophilus, bacillus simplex and lactobacillus buchneri respectively to obtain a zymocyte solution; by microscopyRespectively detecting light absorption value and flora number of the fermented liquid by direct counting method and ultraviolet spectrophotometry, determining growth condition of flora, and detecting effective viable bacteria number of 1 × 109Stopping culturing when cfu/ml is about;
(3) after the fermentation of the single strains is finished, the fermentation method comprises the following steps of mixing Saccharomyces cerevisiae (Saccharomyces cerevisiae), Bacillus methylotrophicus (Bacillus methylotrophicus), Bacillus thermophilus (Geobacillus thermophilus), Bacillus simplex (Bacillus simplex) and Lactobacillus buchneri (Lactobacillus parabuchneri) according to the weight ratio of 1-3: 1-3: 1-3: 1-3: 1-3 to prepare a decomposed liquid composite fermentation microbial inoculum;
(4) and (4) mixing the complex microbial inoculum liquid obtained in the step (3) according to the volume ratio of 1: 2, mixing the mixture with light calcium carbonate, uniformly stirring the mixture and drying the mixture until the water content is below 15 percent;
(5) and (3) adding a protective agent potassium sorbate into the composite microbial inoculum obtained in the step (4) according to the mass concentration of 1-1.5 per mill, and uniformly stirring to obtain the decomposed solid fermentation microbial inoculum.
Example two: preparation of decomposed solid fermentation microbial inoculum
Activating each slant strain stored in an environment of 0-4 ℃, and performing test tube-shake flask culture on five strains respectively to obtain first-level seed liquid of each strain; continuously performing fermentation tank enlargement culture on the seed solution, detecting light absorption value and flora number of the fermentation solution by microscopy direct counting method and ultraviolet spectrophotometry, determining growth condition of flora, and detecting effective viable bacteria number of 1 × 109Stopping culturing when cfu/ml is about; after fermentation of each single strain is finished, mixing Saccharomyces cerevisiae (Saccharomyces cerevisiae), Bacillus methylotrophicus (Bacillus methylotrophicus), Bacillus thermophilus (Geobacillus thermophilus), Bacillus simplex (Bacillus simplex) and Lactobacillus buchneri (Lactobacillus parabuchner) according to a weight ratio of 1: 2: 2: 3: 3 to obtain a decomposed liquid composite fermentation microbial inoculum; and (3) mixing the obtained complex microbial inoculum liquid according to the volume ratio of 1: 2, mixing the mixture with light calcium carbonate, uniformly stirring the mixture and drying the mixture until the water content is below 15 percent; adding potassium sorbate with the mass concentration of 1-1.5 per mill for protectionUniformly stirring the mixture to obtain the decomposed solid fermentation inoculum.
Example three: preparation of decomposed solid fermentation microbial inoculum
Based on the second embodiment, each strain is activated and cultured to obtain the first-level seed liquid of each strain; the seed liquid is continuously subjected to fermentation tank amplification culture, and the number of detected effective viable bacteria is 1 multiplied by 109Stopping culturing when cfu/ml is about; after fermentation of each single strain is finished, mixing Saccharomyces cerevisiae (Saccharomyces cerevisiae), Bacillus methylotrophicus (Bacillus methylotrophicus), Bacillus thermophilus (Geobacillus thermophilus), Bacillus simplex (Bacillus simplex) and Lactobacillus buchneri (Lactobacillus parabuchner) according to a weight ratio of 1: 2: 2: 1: 1 to obtain a decomposed liquid composite fermentation microbial inoculum; and (3) mixing the obtained complex microbial inoculum liquid according to the volume ratio of 1: 2, mixing the mixture with light calcium carbonate, uniformly stirring the mixture and drying the mixture until the water content is below 15 percent; adding a potassium sorbate protective agent with the mass concentration of 1-1.5 per mill, and uniformly stirring to obtain the decomposed solid fermentation microbial inoculum.
Example four: preparation of decomposed solid fermentation microbial inoculum
Based on the second embodiment, each strain is activated and cultured to obtain the first-level seed liquid of each strain; the seed liquid is continuously subjected to fermentation tank amplification culture, and the number of detected effective viable bacteria is 1 multiplied by 109Stopping culturing when cfu/ml is about; after the fermentation of each single strain is finished, the fermentation liquor is prepared by mixing Saccharomyces cerevisiae (Saccharomyces cerevisiae), Bacillus methylotrophicus (Bacillus methylotrophicus), Bacillus thermophilus (Geobacillus thermophilus), Bacillus simplex (Bacillus simplex) and Lactobacillus buchneri (Lactobacillus parabuchneri) according to the weight ratio of 2: 3: 3: 1: 1 to obtain a decomposed liquid composite fermentation microbial inoculum; and (3) mixing the obtained complex microbial inoculum liquid according to the volume ratio of 1: 2, mixing the mixture with light calcium carbonate, uniformly stirring the mixture and drying the mixture until the water content is below 15 percent; adding a potassium sorbate protective agent with the mass concentration of 1-1.5 per mill, and uniformly stirring to obtain the decomposed solid fermentation microbial inoculum.
Example five: preparation of decomposed solid fermentation microbial inoculum
Based onIn the second embodiment, each strain is activated and cultured to obtain the first-stage seed liquid of each strain; the seed liquid is continuously subjected to fermentation tank amplification culture, and the number of detected effective viable bacteria is 1 multiplied by 109Stopping culturing when cfu/ml is about; after the fermentation of each single strain is finished, mixing Saccharomyces cerevisiae (Saccharomyces cerevisiae), Bacillus methylotrophicus (Bacillus methylotrophicus), Bacillus thermophilus (Geobacillus thermophilus), Bacillus simplex (Bacillus simplex) and Lactobacillus buchneri (Lactobacillus parabuchner) according to the weight ratio of 3: 1: 1: 2: 2 to obtain a decomposed liquid composite fermentation microbial inoculum; and (3) mixing the obtained complex microbial inoculum liquid according to the volume ratio of 1: 2, mixing the mixture with light calcium carbonate, uniformly stirring the mixture and drying the mixture until the water content is below 15 percent; adding a potassium sorbate protective agent with the mass concentration of 1-1.5 per mill, and uniformly stirring to obtain the decomposed solid fermentation microbial inoculum.
Example six: preparation of decomposed solid fermentation microbial inoculum
Based on the second embodiment, each strain is activated and cultured to obtain the first-level seed liquid of each strain; the seed liquid is continuously subjected to fermentation tank amplification culture, and the number of detected effective viable bacteria is 1 multiplied by 109Stopping culturing when cfu/ml is about; after the fermentation of each single strain is finished, the fermentation liquor is prepared by mixing Saccharomyces cerevisiae (Saccharomyces cerevisiae), Bacillus methylotrophicus (Bacillus methylotrophicus), Bacillus thermophilus (Geobacillus thermophilus), Bacillus simplex (Bacillus simplex) and Lactobacillus buchneri (Lactobacillus parabuchneri) according to the weight ratio of 2: 2: 3: 3: 1 to obtain a decomposed liquid composite fermentation microbial inoculum; and (3) mixing the obtained complex microbial inoculum liquid according to the volume ratio of 1: 2, mixing the mixture with light calcium carbonate, uniformly stirring the mixture and drying the mixture until the water content is below 15 percent; adding a potassium sorbate protective agent with the mass concentration of 1-1.5 per mill, and uniformly stirring to obtain the decomposed solid fermentation microbial inoculum.
Example seven: optimization of preparation process of decomposed solid fermentation inoculant
The following different formulas are respectively adopted to prepare the components of the decomposed fermentation active microbial inoculum:
formula 1: saccharomyces cerevisiae (Saccharomyces cerevisiae), Bacillus methylotrophicus (Bacillus methylotrophicus), Bacillus thermophilus (Geobacillus thermoacidophilus), Bacillus simplex (Bacillus simplex) and Lactobacillus buchneri (Lactobacillus parachuticum) in a weight ratio of 1: 2: 2: 3: 3 to obtain the decomposed liquid composite fermentation inoculum.
And (2) formula: saccharomyces cerevisiae (Saccharomyces cerevisiae), Bacillus methylotrophicus (Bacillus methylotrophicus), Bacillus thermophilus (Geobacillus thermoacidophilus), Bacillus simplex (Bacillus simplex) and Lactobacillus buchneri (Lactobacillus parachuticum) in a weight ratio of 1: 2: 2: 1: 1 to prepare the decomposed liquid composite fermentation inoculum.
And (3) formula: saccharomyces cerevisiae (Saccharomyces cerevisiae), Bacillus methylotrophicus (Bacillus methylotrophicus), Bacillus thermophilus (Geobacillus thermoacidophilus), Bacillus simplex (Bacillus simplex) and Lactobacillus buchneri (Lactobacillus parachuticum) in a weight ratio of 2: 3: 3: 1: 1 to prepare the decomposed liquid composite fermentation inoculum.
And (4) formula: saccharomyces cerevisiae (Saccharomyces cerevisiae), Bacillus methylotrophicus (Bacillus methylotrophicus), Bacillus thermophilus (Geobacillus thermoacidophilus), Bacillus simplex (Bacillus simplex) and Lactobacillus buchneri (Lactobacillus parachuticum) in a weight ratio of 3: 1: 1: 2: 2 to obtain the decomposed liquid composite fermentation inoculum.
And (5) formula: saccharomyces cerevisiae (Saccharomyces cerevisiae), Bacillus methylotrophicus (Bacillus methylotrophicus), Bacillus thermophilus (Geobacillus thermoacidophilus), Bacillus simplex (Bacillus simplex) and Lactobacillus buchneri (Lactobacillus parachuticum) in a weight ratio of 2: 2: 3: 3: 1 to prepare the decomposed liquid composite fermentation inoculum.
The five different formulas are respectively prepared into the decomposed fermentation active microbial inoculum according to the following processes:
activating each slant strain stored in the environment of 0-4 ℃, and performing test tube-shake flask culture on five strainsCulturing to obtain first-stage seed liquid of each strain; continuously performing fermentation tank enlargement culture on the seed solution, detecting light absorption value and flora number of the fermentation solution by microscopy direct counting method and ultraviolet spectrophotometry, determining growth condition of flora, and detecting effective viable bacteria number of 1 × 109Stopping culturing when cfu/ml is about; after the fermentation of each single strain is finished, mixing the single strains according to the weight ratio to prepare a decomposed liquid composite fermentation microbial inoculum; and (3) mixing the obtained complex microbial inoculum liquid according to the volume ratio of 1: 2, mixing the mixture with light calcium carbonate, uniformly stirring the mixture and drying the mixture until the water content is below 15 percent; adding a potassium sorbate protective agent with the mass concentration of 1-1.5 per mill, and uniformly stirring to obtain the decomposed solid fermentation microbial inoculum. Mixing 80% of cow dung and 20% of crushed straws according to the weight ratio, stacking and decomposing outdoors, adding 1 per mill of decomposed solid fermentation microbial inoculum prepared in different formulas at the beginning to keep the water content of the materials at 60%, stacking and decomposing outdoors for four weeks to prepare a substrate obtained by treated agricultural wastes, taking 30 healthy tomato seeds, and placing under the growth condition of 25 ℃ to monitor the germination rate after 21 days.
1. Orthogonal optimization decomposed fermentation active microbial inoculum
Designing a single-factor experiment, respectively exploring the decomposed solid fermentation microbial inoculum prepared by different adding proportions of saccharomyces cerevisiae, bacillus methylotrophicus, bacillus denitrificans, bacillus simplex and lactobacillus buchneri, mixing 80% of cow dung and 20% of crushed straw according to the weight ratio, carrying out outdoor stacking decomposition, preparing a substrate obtained by treated agricultural wastes, and detecting the influence on the germination rate of tomato seeds. Five-factor three-level orthogonal experiments were performed on the basis of the single-factor experiments, see table 1.
TABLE 1 orthogonal test factors and levels
The results and analysis of the orthogonal optimization test are shown in Table 2.
TABLE 2 results and analysis of orthogonal experiments
As can be seen from Table 2, in the range of experimental design, Bacillus simplex (Bacillus simplex) has the greatest effect on the quality of the decomposing fermentative active agent, followed by Saccharomyces cerevisiae (Saccharomyces cerevisiae), Bacillus methylotrophicus (Bacillus methylotrophicus), Bacillus stearothermophilus (Geobacillus thermonitrificans) and Bacillus brucei (Lactobacillus parachui). The best raw material of the active microbial inoculum for decomposing fermentation is A1B2C2D1E1Namely Saccharomyces cerevisiae (Saccharomyces cerevisiae), Bacillus methylotrophicus (Bacillus methylotrophicus), Bacillus thermophilus (Geobacillus thermophilus), Bacillus simplex (Bacillus simplex) and Lactobacillus buchneri (Lactobacillus parabuchneri) in a weight ratio of 1: 2: 2: 1: 1, and preparing the decomposed solid fermentation inoculum according to the preparation process provided by the second embodiment to the sixth embodiment.
Example eight: effect of decomposed solid fermentation bacterial agent in application of different agricultural wastes
Mixing fresh chicken manure or pig manure or cow manure or sheep manure with different proportions and walnut stalks or cotton stalks crushed to 1-2cm, stacking and decomposing outdoors, adding 1 per thousand of decomposed solid fermentation microbial inoculum prepared by the optimal raw material proportioning scheme obtained in the seventh embodiment at the beginning to keep the water content of the materials at 60%, stacking and decomposing outdoors for four weeks to prepare a substrate obtained by treated agricultural wastes, taking 30 healthy tomato seeds, and placing under the growth condition of 25 ℃ to monitor the germination rate after 21 days. As can be seen from Table 3, the germination rate of the microbial inoculum decomposed group added with 80% of cow dung is obviously higher than that of other groups, which shows that the combination of 80% of cow dung, 20% of walnut straw and cotton stalk and the composite microbial inoculum can effectively degrade toxic and harmful substances in the walnut straw and the cotton stalk and improve the straw decomposition degree.
Table 3: effect of decomposed solid fermentation bacterial agent in application of different agricultural wastes
Example nine: application of decomposed solid fermentation microbial inoculum to temperature change in decomposition process of cow dung and straw waste
Mixing 80% of fresh cow dung and 20% of crushed walnut stalks, composting outdoors, adding 1 per mill of the decomposed solid fermentation microbial inoculum prepared according to the optimal proportion in the seventh embodiment at the beginning, composting at 15 days, and monitoring temperature change in the composting process. Referring to the attached figure 1, the maximum temperature of the treatment with the added microbial inoculum can be raised to 72 ℃, and the maximum temperature of the control is 53.4 ℃, which shows that the complex microbial inoculum has obvious promotion effect on the decomposition process.
Example ten: influence of decomposed solid fermentation inoculant on tomato germination rate by applying walnut stalk decomposed products
Crushing the walnut stalks into chips of 1-2cm, adding 1 per mill of the decomposed solid fermentation microbial inoculum prepared by the optimal raw material proportioning scheme obtained in the seventh embodiment, uniformly stirring to keep the water content of the materials at 60%, and stacking the materials outdoors for decomposing for four weeks to obtain the substrate obtained by the treated agricultural wastes. 30 healthy tomato seeds were taken and placed at 25 ℃ for 21 days to monitor germination rate. As can be seen from Table 4, the germination rate of the decomposed group with the added microbial inoculum is obviously higher than that of the naturally decomposed group, which shows that the compound microbial inoculum can effectively degrade toxic and harmful substances in the walnut stalks and improve the decomposition degree of the stalks.
Table 4: tomato seed germination test
Example eleven: the nutrient component change of the decomposed product after the decomposed solid fermentation microbial inoculum is added to the cow dung to carry out the decomposed fermentation treatment
Mixing 80% of fresh cow dung and 20% of cotton stalks, keeping the water content of the raw materials at 50% -60%, adding 1 ‰ of the composite microbial inoculum prepared according to the optimal proportioning scheme obtained in the seventh embodiment, uniformly stirring, subpackaging with plastic bags, compacting, sealing, culturing at 35 ℃ for about 30 days, and detecting the change of nutritional components of the decomposed product after the cow dung is subjected to decomposition fermentation treatment by adding the composite microbial inoculum. The data in Table 5 show that key indexes such as organic matters, total nitrogen content and the like can be greatly improved by adding the decomposed solid fermentation inoculant.
Table 5: analysis result of main physical and chemical indexes of organic fertilizer
Note: the data in the table are the average values of three replicates, Y1 is the group added with the fungicide, and Y2 is the naturally decomposed control group.
Example twelve: growth promotion effect of decomposed product prepared from decomposed solid fermentation inoculant in tomato seedling culture
The use method of the compound microbial inoculum in the decomposition of the mixture of 80 percent of cow dung and 20 percent of cotton stalks comprises the following steps of drying the fermented material in the embodiment to the moisture content of below 15 percent for later use. In the tomato plug seedling test, the sample application of the compound microbial inoculum rotten product is used for the fertilizer effect test of the tomato in the seedling stage. Taking vermiculite as a substrate, adding water to a wet state, putting the soaked tomato seeds, and covering a layer of substrate. Culturing under room temperature illumination condition, growing 2 cotyledons on the seedling after one week, and applying the decomposed product into the matrix about 1cm away from the root of the seedling. Watering every day, controlling seedlings without fertilizing, managing the same watering, recording the growth situation after 30 days, observing the growth difference of the seedlings among treatments, and randomly sampling and selecting 10 seedlings for measurement in each treatment.
The data in Table 6 show that the average plant height of the strain treated by adding the microbial inoculum is 47.16% higher than that of the control group, and the plant height of the strain treated by not adding the microbial inoculum is 3.62% higher than that of the control group.
Table 6: influence of different treatment organic fertilizers on growth of tomato seedlings
| Numbering | Plant height | Number of leaves | Length of cotyledon | Width of cotyledon |
| N1 | 15.04 | 5.45 | 2.62 | 0.75 |
| N2 | 10.59 | 3.09 | 2.11 | 0.79 |
| CK | 10.22 | 2.09 | 2.08 | 0.69 |
Note: the data in the table above are average values of ten randomly extracted samples, N1 is a group to which a microbial inoculum is added, N2 is a natural rotten product without the microbial inoculum, and a control group is treated by applying tap water.
As described above, the present invention can be preferably implemented, and the above-mentioned embodiments only describe the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various changes and modifications of the technical solution of the present invention made by those skilled in the art without departing from the design spirit of the present invention shall fall within the protection scope defined by the present invention.
Claims (9)
1. A decomposed solid fermentation microbial inoculum is characterized in that: the decomposed solid fermentation inoculant comprises a composite decomposed active inoculant formed by combining five bacteria, namely Saccharomyces cerevisiae (Saccharomyces cerevisiae), Bacillus methylotrophicus (Bacillus methylotrophicus), Bacillus thermophilus (Geobacillus thermonitritificians), Bacillus simplex (Bacillus simplexex) and Lactobacillus buchneri (Lactobacillus parachui); wherein the effective viable count of each bacterium in the composite decomposed active microbial inoculum is 1 multiplied by 109cfu/mL, the ratio of each strain is 1: 2: 2: 1: 1, and mixing and combining the components in a ratio of 1.
2. A method for preparing the decomposed solid fermentation inoculum according to claim 1, comprising the steps of:
(1) activating each slant strain stored in an environment of 0-4 ℃, and respectively carrying out test tube-shake flask culture on five strains of saccharomyces cerevisiae, methylotrophic bacillus, thermophilic denitrified bacillus, simple bacillus and lactobacillus buchneri to obtain first-level seed liquid of each strain;
(2) continuously carrying out fermentation tank amplification culture on the seed solution, and fermenting saccharomyces cerevisiae, bacillus methylotrophicus, bacillus denitrificans thermophilus, bacillus simplex and lactobacillus buchneri respectively to obtain a zymocyte solution; respectively detecting light absorption value and flora number of the fermented liquid by microscopy direct counting method and ultraviolet spectrophotometry, determining growth condition of flora, and detecting effective viable bacteria number of 1 × 109Stopping culturing at cfu/mL;
(3) after the fermentation of the single strains is finished, saccharomyces cerevisiae, methylotrophic bacillus, thermophilic denitrified bacillus, simple bacillus and lactobacillus buchneri are mixed according to the ratio of 1: 2: 2: 1: 1 to prepare a decomposed liquid composite fermentation microbial inoculum;
(4) and (4) mixing the complex microbial inoculum liquid obtained in the step (3) according to the volume ratio of 1: 2, mixing the mixture with light calcium carbonate, uniformly stirring the mixture and drying the mixture until the water content is below 15 percent;
(5) and (3) adding a protective agent potassium sorbate into the composite microbial inoculum obtained in the step (4) according to the mass concentration of 1-1.5 per mill, and uniformly stirring to obtain the decomposed solid fermentation microbial inoculum.
3. The method for preparing the decomposed solid fermentation inoculant according to claim 2, wherein saccharomyces cerevisiae and lactobacillus buchneri adopt an optimized FC2 culture medium, and the formula of the culture medium is as follows: 2% sucrose; 0.5% yeast extract; 0.3% peptone; 0.2% disodium hydrogen phosphate; the inoculation amount is 1-2%, the speed is 150-200 rpm, and the culture is carried out for 24-36 h.
4. The method for preparing decomposed solid fermentation inoculant according to claim 2, wherein the bacillus methylotrophicus and the bacillus denitrificans use an optimized YM1 culture medium, and the formula of the culture medium is as follows: 0.5% corn steep liquor; 0.2% disodium hydrogen phosphate; 1% sucrose; the inoculation amount is 1-2%, the speed is 150-200 rpm, and the culture is carried out for 24-36 h.
5. The method for preparing the decomposed solid fermentation inoculant according to claim 2, wherein the simple bacillus adopts LBG culture medium, and the formula of the LBG culture medium is as follows: 0.5% glucose, 1% peptone, 0.5% yeast extract, 1% sodium chloride; the inoculation amount is 1-2%, the speed is 150-200 rpm, and the culture is carried out for 24-36 h.
6. The application of the decomposed solid fermentation inoculant prepared by the method for preparing the decomposed solid fermentation inoculant according to claim 2 in decomposing agricultural wastes.
7. The application of the decomposed solid fermentation inoculant in agricultural waste decomposition treatment according to claim 6, wherein the prepared decomposed solid fermentation inoculant can be applied to agricultural waste of 80% of cow dung and 20% of straws.
8. The application of the decomposed solid fermentation inoculant in agricultural waste decomposition treatment according to claim 7, wherein 20% of straws are cotton straws.
9. The application of the decomposed solid fermentation inoculant in agricultural waste decomposition treatment according to claim 7, wherein 20% of straws are walnut straws.
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