CN112624818B

CN112624818B - Excrement composting fermentation retting agent capable of killing insects and promoting growth and application thereof

Info

Publication number: CN112624818B
Application number: CN202011500400.0A
Authority: CN
Inventors: 李慧芬; 王金龙; 魏秉培; 张大伟; 刘宝同; 冯海霞; 樊连梅
Original assignee: Qingdao Shangde Biotechnology Co ltd
Current assignee: Qingdao Shangde Biotechnology Co ltd
Priority date: 2020-12-21
Filing date: 2020-12-21
Publication date: 2021-06-08
Anticipated expiration: 2040-12-21
Also published as: CN112624818A

Abstract

The invention discloses a feces stacking fermentation retting agent for disinsection and growth promotion and application thereof. The manure composting and decomposing agent comprises a low-temperature composting starter and a high-temperature composting and decomposing pesticide; the low-temperature fermentation starter comprises a strain content of 50-55 multiplied by 10⁸The bacterial powder and the bacterial content of the CFU/g filamentous bacillus are 45-50 multiplied by 10⁸CFU/g of plant bacillus endophyte powder and a carrier X; the high-temperature retting insecticide comprises the bacteria content of 150-160 multiplied by 10⁸CFU/g of Bacillus subtilis desert subspecies powder with bacterium content of 40-50 multiplied by 10⁸CFU/g of clostridium butyricum powder and a carrier Y. When the excrement composting fermentation retting agent is used for treating excrement, the low-temperature composting starter and the high-temperature composting insecticide are inoculated step by step, when the total inoculation amount is 0.30% -0.50%, the death rate of ascarid eggs can reach more than 95%, the total amount of free amino acid can be increased, and then the plant growth is promoted.

Description

Excrement composting fermentation retting agent capable of killing insects and promoting growth and application thereof

Technical Field

The invention belongs to the technical field of microorganisms, and particularly relates to a pesticidal and growth-promoting manure composting and retting agent and application thereof.

Background

Roundworms are the most common parasites in humans, with infection rates of over 70%, especially in children, being relatively high. If the roundworm eggs are swallowed, the egg shells are digested, and larvae escape from the intestines, so that infection of different degrees is easily caused, and main symptoms such as fever, inappetence, bulimia, umbilical cord pain and the like can be caused, and sometimes more serious complications can be caused. In the process of manure fertilization, if the roundworm eggs are not effectively removed, the roundworm can hatch in soil and plant roots to cause plant diseases and insect pests and even enter plant fruits, so that the food sanitation and safety of human beings are critical. Therefore, in the pollution-free treatment of the excrement, the death rate of roundworm eggs is the most important hygienic index, and the judgment of the compliance of the treatment of the livestock excrement is directly influenced. At present, the harmless treatment of the livestock and poultry manure needs to conform to the national standard GBT 36195 plus 2018 harmless treatment technical specification, and the death rate of the roundworm eggs is required to be more than 95 percent.

In the existing treatment method of roundworm eggs in excrement, the physical and chemical means are expensive, and the excrement of a farm cannot be treated in a large scale. The biological method is simple and environment-friendly, can generate a large amount of heat through the life activities of microorganisms, improves the temperature of a system, and can maintain 55 ℃ and more than 15 days, so that roundworms die at high temperature and reach the technical standard of harmless treatment of excrement. In addition, in the microbial fermentation process, fecal organic matters serving as substrates, such as protein, fat, starch, fiber and the like, are also degraded by various enzymes generated by the microorganisms, so that growth stimulating substances, such as amino acids, micromolecular carbohydrates and the like, which are easily absorbed and utilized by plants or play a growth promoting role in plant roots are formed, and a certain growth promoting effect is achieved. The biological insecticidal method has the characteristics of simplicity, convenience, low cost, high efficiency, high comprehensive benefit and the like.

Then, most of the fecal biological treatment agents are applied under aerobic conditions, after fermentation is carried out for 2-3 d, turning is needed, the oxygen amount of a system is increased, some agents have requirements on the external environment temperature, the temperature is more than 25-28 ℃, otherwise, many microbial inoculum factors cannot grow at low temperature and cannot start the fermentation process, and the fecal biological treatment agents are invalid in winter. Therefore, it is important to research a microbial agent suitable for fecal treatment and capable of comprehensively utilizing aerobic bacteria and anaerobic bacteria.

Disclosure of Invention

The invention provides a feces composting and fermenting agent for disinsection and growth promotion and application thereof. When the excrement stacking fermentation retting agent is used for retting excrement, the excrement is subjected to low-temperature starting, decomposition high-temperature maintaining, decomposition medium-temperature maintaining and after-ripening processes, so that ascarid eggs are effectively killed, the total amount of free amino acids is increased, and the humification of materials is promoted.

The purpose of the invention is realized by the following technical scheme:

the invention provides a feces composting and fermenting agent for disinsection and growth promotion, which comprises a low-temperature composting starter and a high-temperature composting and fermenting insecticide; the low-temperature fermentation starter comprises filamentous bacillus powder, plant endophyte bacillus powder and a carrier X; the high-temperature retting insecticide comprises bacillus subtilis subspecies desert bacterial powder, clostridium butyricum bacterial powder and a carrier Y.

Further, the mass ratio of the filamentous bacillus powder to the plant endophyte bacillus powder to the carrier X is 1: 20.

Furthermore, the mass ratio of the bacillus subtilis subspecies desert powder, the clostridium butyricum and the carrier Y is 1: 15.

Further, the filamentous bacillus powder is obtained by fermenting and drying filamentous bacillus GBW-F006 with the preservation number of CGMCC No.20918, and the bacterium content of the filamentous bacillus powder is 50-55 multiplied by 10⁸CFU/g; the plant Bacillus endophyte powder is obtained by fermenting and drying plant Bacillus endophyte GBW-F008 with the preservation number of CGMCC No.20919, and the bacterium content of the plant Bacillus endophyte powder is 45-50 multiplied by 10⁸CFU/g。

Further, the bacillus subtilis desert subspecies powder is obtained by fermenting and drying bacillus subtilis S2 with the preservation number of CGMCC No.19824, and the bacterium content of the bacillus subtilis desert subspecies powder is 150-160 multiplied by 10⁸CFU/g; the clostridium butyricum powder is obtained by fermenting and drying clostridium butyricum GBW-N1 with the preservation number of CGMCC No.14499, and the bacterium content of the clostridium butyricum powder is 40-50 multiplied by 10⁸CFU/g。

Further, the carrier X comprises at least one of plant ash, humic acid and zeolite powder; the carrier Y comprises at least one of diatomite, vermiculite and weathered coal.

The invention also provides application of the manure composting and retting agent in preparation of a microorganism composting and fermenting agent for promoting plant growth.

Further, the using method of the manure composting and retting agent comprises the following steps: inoculating the low-temperature fermentation starter into excrement and batch, fermenting for 2.5-4d, then inoculating the high-temperature retting insecticide, continuously fermenting for 3-4d, continuously fermenting for 3-5d at a high temperature of more than 70 ℃, continuously fermenting for 8-10d at a high temperature of more than 55 ℃, after-ripening and fermenting for 6-7d, drying until the water content is 27% -29%, crushing, subpackaging, and directly mixing into plant planting soil.

Further, the manure comprises chicken manure, pig manure and cow manure.

Further, the dosage of the batch is 30-40% of the mass of the excrement.

Further, the total inoculation amount of the manure composting and retting agent is 0.20-0.50%.

Preferably, the total inoculation amount of the manure composting and retting agent is 0.30-0.40%.

Further, the inoculation amount ratio of the low-temperature fermentation starter to the high-temperature retting insecticide is 1: 1-3: 5.

Further, the inoculation amount of the low-temperature fermentation starter is 0.10-0.25%, and the inoculation amount of the high-temperature retting insecticide is 0.10-0.25%.

Optimally, the inoculation amount of the low-temperature fermentation starter is 0.15-0.20%, and the inoculation amount of the high-temperature retting insecticide is 0.15-0.20%.

Furthermore, the excrement composting and fermenting agent can improve the excrement composting temperature, the death rate of roundworm eggs and the total amount of free amino acid.

Further, the manure composting and retting agent can improve the yield and fruit diameter of plants.

Further, the plants include cucumber and cherry.

Compared with the prior art, the invention has the following advantages and technical effects:

1. the excrement composting fermentation retting agent for disinsection and growth promotion comprehensively uses multiple bacilli and has a multi-bacterium composite effect.

2. The starter for fermenting the bacteria in the low-temperature reactor A comprises filamentous bacillus GBW-F006 which can tolerate low temperature and realize quick start, and plant bacillus GBW-F008 which can generate enzymes such as xylanase, lipase, cellulase, amylase, protease, phosphatase and the like, so that a system can continuously degrade a substrate, release nutrients such as micromolecular sugar, amino acid, quick-acting phosphorus, quick-acting potassium and the like, provide nutrients for the propagation of various bacteria in the starter, and finally become advantages.

3. The fermentation B is a high-temperature retting insecticide and comprises a bacillus subtilis subspecies desert S2 and a clostridium butyricum GBW-N1, the bacillus subtilis subspecies desert S2 has strong enzyme production and bacteriostasis characteristics, can rapidly grow and ferment in organic materials, generates a large amount of heat energy through aerobic reaction, rapidly promotes the temperature of a system to be 71-76 ℃, and the clostridium butyricum GBW-N1 grows due to gradual rarefied oxygen of the system to generate a large amount of organic acids represented by butyric acid, wherein the organic acids play an important role in plant growth, plant fruit tartaric acid formation, release of certain binding state mineral elements in soil and the like.

4. The preparation method of the composite microbial inoculum is simple and reasonable, does not need to turn over and throw, is convenient to use, and has good industrial implementation prospect.

Drawings

FIG. 1: bacterial colony, microscopic thallus and spore of filamentous bacillus GBW-F006;

FIG. 2: bacterial colony, microscopic thallus and spore of the plant Bacillus subtilis GBW-F008;

FIG. 3: bacterial colony, microscopic bacteria and spores of bacillus subtilis S2;

FIG. 4: bacterial colony, microscopic thallus and spore of clostridium butyricum GBW-N1;

FIG. 5: the two inoculated leavening agent bags of the manure composting and decomposition agent are used for carrying out step-by-step composting and decomposition on manure;

FIG. 6: influence of different manure composting and decomposition agent dosage on the composting temperature of the chicken manure;

FIG. 7: influence of different fecal composting retting agent dosage on death rate of chicken fecal ascaris egg;

the following drawings: 8: influence of different manure composting and decomposition agent dosage on the total amount of free amino acid in the chicken manure;

FIG. 9: influence of the inactivation and activity manure composting and decomposition agent on the pig manure composting temperature;

FIG. 10: influence of the inactivation and active fecal stacking fermentation retting agent on death rate of pig fecal roundworm eggs;

FIG. 11: influence of the inactivation and activity manure composting decomposition agent on the total amount of free amino acid in pig manure.

Detailed Description

The technical solution of the present invention will be further described in detail with reference to the following specific examples.

In the following examples, unless otherwise specified, the experimental methods used were all conventional methods, and materials, reagents and the like used were all available from biological or chemical reagents companies.

Example 1

Separation, screening and identification of filamentous bacillus GBW-F006

1. Isolation and screening of Bacillus filamentarius GBW-F006

Plant rhizosphere soil is taken from a greenhouse of multi-year-old vegetables such as Hebei cangzhou, Handan, Henan Pu Yang, Anyang, Shandong Qingdao, Haiyang, Qingzhou and Shouguang, is refrigerated and stored at 4 ℃ in a medical test sample bag and is separated in time, and the plant rhizosphere soil relates to 56 crops such as hot peppers, pumpkin, cauliflower, muskmelons, tomatoes, cucumbers and the like. Taking 2g of a soil sample, putting the soil sample into 18mL of sterile water, extracting the soil sample for 30min at 30 ℃ and 200rpm/min in a shaking way, taking sample liquid in 75 ℃ water bath for 20min, then carrying out gradient dilution, coating 100 mu l of each gradient dilution on an NA flat plate, carrying out inverted culture on the coated flat plate at 37 ℃ for 48h, picking out single bacterial colonies with different forms by using a sterilized bamboo stick, transferring the single bacterial colonies onto the NA inclined plane, carrying out microscopic examination after 48h of culture at 37 ℃ to determine spore production, and preserving the strain at 4 ℃ for later use.

Inoculating 67 strains obtained after low-temperature acclimatization into modified Nutrient Broth (NB) culture medium (beef extract 5g, peptone 10g, sodium chloride 5g, (NH)₄)₂SO₄ 2g，FeSO₄·7H₂O 0.03g，MgSO₄·7H₂0.05g of O, pH 7.2-7.4 and 1L of distilled water), culturing at 15 ℃ and 200rpm/min with shaking for 24h, and measuring the light absorption value of the culture solution at OD600 nm. 60 of the 67 strains are cultured at 15 ℃ for 24 hours, the absorbance value of OD600nm is less than 1.0, only 7 strains are cultured at 15 ℃ for 24 hours, and the absorbance value of OD600nm is more than 1.0, wherein the OD value of a culture solution at 15 ℃ of the strain GBW-F006 is the highest, so that the strain has good low temperature resistance, and the strain is separated from 11-year muskmelon rhizosphere soil of a family Xiahui plum, south Sharpu plum and village.

The colony, microscopic thallus and spore of the strain GBW-F006 on the NA culture medium are shown in figure 1. The bacterial colony of GBW-F006 on the NA culture medium is milky white, circular, 2-6mm in diameter, smooth and non-wrinkled in surface, non-transparent, neat in edge and non-halo as shown in figure 1 a; GBW-F006 10 × 100 fold oil microscopic examination result at 24h is shown in FIG. 1b, and the thallus is G⁺The spore end is cylindrical and elliptical, and the diameter of the spore end is 1-2 μm.

2. 16S rRNA sequencing of Bacillus filamentarius GBW-F006

And (2) amplifying by using a 16S rDNA universal primer by using the DNA of the strain GBW-F006 as a template, carrying out sequence determination on an amplified fragment, and carrying out Blast comparison analysis on the 16S rDNA sequencing result of the obtained strain GBW-F006 and a sequence in NCBI GenBank to show that the homologous similarity of the strain GBW-F006 and Bacillus filamentosus is 99.72%, thereby determining that the strain is Bacillus filamentosus.

3. Strain preservation of filamentous Bacillus GBW-F006

And (3) performing strain preservation on the screened strain GBW-F006, wherein the preservation unit of the filamentous bacillus GBW-F006 is as follows: china general microbiological culture Collection center (CGMCC); address: western road No.1, north west city of township, beijing, institute of microbiology, china academy of sciences; the preservation date is as follows: 10 months and 20 days in 2020; the preservation number of the filamentous Bacillus filamniosus is as follows: CGMCC No. 20918.

4. Strain characteristics of Bacillus filamentous GBW-F006

The filamentous bacillus GBW-F006 can grow and reproduce at the temperature of 15-45 ℃, but the optimum growth temperature is 25-37 ℃, the bacillus can tolerate 9% of NaCl at most, the catalase and oxidase of the bacillus are positive, urease can be hydrolyzed, casein can be decomposed, and citrate can be used. GBW-F006 can also produce acid by using xylose, sucrose, inulin, glucose, sorbitol, salicin, D-trehalose, L-rhamnose, L-arabinose, alpha-galactosidase, beta-glucosidase, L-proline arylamine, etc. The filamentous bacillus GBW-F006 is separated from melon rhizosphere soil, can germinate continuously and slightly proliferate in the first 3d of the soil, and can inhibit various pathogenic fungi such as aerobic vibrio cholerae, anaerobic clostridium perfringens, fusarium equiseti, fusarium stratiotes, fusarium moniliforme, fusarium oxysporum, geotrichum candidum and bipolaris tritici, wherein the inhibition rate on the fusarium equiseti is up to 92.9%.

Second, separation, screening and identification of Bacillus plantarum GBW-F008

1. Isolation and screening of Bacillus plantarum GBW-F008

The root samples of 56 crops such as loofah, melon, pepper, colorful pepper, tomato, cucumber and the like are taken from a multi-year vegetable greenhouse of Hebei Gallery, Shandong Jinan, Taian, Laiyang, Zibo, chatting and Shouguang, are filled in a medical test sample bag, are refrigerated and stored at 4 ℃ and are separated in time. Sampling 2g of a sample, placing the sample into 18mL of sterile water, extracting the sample for 30min at 30 ℃ and 200rpm/min by shaking, carrying out water bath on the sample solution at 75 ℃ for 20min, then carrying out gradient dilution, coating 100 mu l of each gradient dilution on an NA flat plate, carrying out inverted culture on the coated flat plate at 37 ℃ for 48h, picking out single bacterial colonies with different forms by using a sterilized bamboo stick, transferring the single bacterial colonies onto an NA inclined plane, carrying out microscopic examination after 48h of culture at 37 ℃ to determine spore production, screening 72 spore-producing bacteria, and preserving the strains at 4 ℃ for later use.

1ml of improved LB culture medium containing tryptophan Try inducer is filled in each hole of a sterilized 48-hole culture plate, the culture plate is cooled after sterilization, then 72 screened spore bacteria are respectively inoculated in a 48-hole plate, shaking culture is carried out at 37 ℃ and 200rpm/min for 24h, centrifugation is carried out at 8000rpm for 5min, 200 mu l of sterile supernatant is taken and transferred into a 96-hole plate, according to the principle of Salkowski colorimetry, the spore bacteria fermentation liquor can generate indoleacetic acid IAA under the induction of tryptophan, the IAA reacts with Salkowski color developing agent to generate red, and the deeper the color indicates that the IAA content is higher. 72 strains of spore bacteria are screened, and after qualitative comparison of IAA production capacity, the red reaction of the strain GBW-F008 is deepest, which indicates that the IAA production capacity of the strain GBW-F008 is strongest in the 72 strains, and the strain is derived from a 11-year tomato shed in the village, the small town, the high new district, Tai' an and is separated from the roots of tomatoes.

The colony, microscopic thallus and spore of the bacterial strain GBW-F008 on the NA culture medium are shown in figure 2, and the colony of the GBW-F008 on the NA culture medium is white, opaque, round, neat in edge, moist and glossy in surface and 1-5mm in diameter as shown in figure 2 a; GBW-F008 showed 10X 100 fold oil microscopic examination result at 24h in FIG. 2b, the microscopic examination of the cells was G +, the cells were cylindrical and 2-3 μm in diameter, the proximal end of the spore appeared in an oblong shape, and the peritrichous flagellum was formed.

2. Molecular identification of Bacillus plantarum GBW-F008

The DNA of the strain GBW-F008 is used as a template, a 16S rDNA universal primer is used for amplification and the sequence of the DNA is determined, the 16S rDNA sequencing result of the obtained strain GBW-F008 and the sequence in GenBank are subjected to Blast comparison analysis, and the result shows that the homology of the strain GBW-F008 and Bacillus endo hygicus is the highest, so that the strain GBW-F008 is determined to be the Bacillus endophytic.

3. Strain preservation of Bacillus plantarum GBW-F008

And (3) performing strain preservation on the screened strain GBW-F008, wherein the preservation unit of the Bacillus plantarum GBW-F008: china general microbiological culture Collection center (CGMCC); address: western road No.1, north west city of township, beijing, institute of microbiology, china academy of sciences; the preservation date is as follows: 10 months and 20 days in 2020; the preservation number of the Bacillus endophyticus is as follows: CGMCC No. 20919.

4. Strain characteristics of Bacillus plantarum GBW-F008

The endophytic bacillus GBW-F008 can grow and propagate at the temperature of 20-42 ℃, but the most suitable growth temperature is 37 ℃, the endophytic bacillus GBW-F008 can normally grow in an improved NB culture medium with the pH of 4.5-9.6, the most suitable growth pH range is 6.5-7.5, the endophytic bacillus GBW-F008 can bear 10% of NaCl at most, urease can be generated, the reactions of catalase and oxidase are positive, and citrate can be used; GBW-F008 may also produce acid with xylose, inulin, glucose, D-ribose, cyclodextrin, gulose, D-trehalose, and other carbohydrates, as well as alpha-galactosidase, beta-glucosidase, leucine arylamine, etc. The plant bacillus GBW-F008 can produce amylase, xylanase, cellulase, lipase, protease and other enzymes, the hydrolysis ratio of the xylanase reaches 3.97, the plant bacillus GBW-F008 has the capabilities of dissolving phosphorus and potassium and the like, and the ratio of dissolving organic phosphorus reaches 2.31.

Thirdly, separating, screening and identifying Bacillus subtilis subspecies desert S2

1. Separation, screening and purification of Bacillus subtilis subspecies desert S2

10g of aquaculture soil sludge in Takaguchi region is taken, the aquaculture soil is shaken in 90mL of sterile water at 30 ℃ and 150r/min for 30min by a shaking table, 5 percent of soil suspension is taken and inoculated into a conical flask containing a basic culture medium, and the soil is cultured for 24h by the shaking table at constant temperature of 30 ℃ and 150 rpm. And (3) selecting a ring of fermentation liquid, streaking and separating on the surface of the TSA culture medium, and carrying out inverted culture in a constant-temperature incubator at 30 ℃ for multiple times until a single colony is separated.

The purified single strains were inoculated into a medium containing 100mL of seed medium (peptone 5g, yeast powder 1g, K)₂HPO₄ 1g，MgSO₄·7H₂0.5g of O, 5g of soluble starch, 1L of distilled water and pH of 7.4-7.6) in a 250mL triangular flask, carrying out shaking culture at 30 ℃ for 24h at a constant temperature of 150r/min, and determining the flocculation activity of the obtained culture solution.

The determination method of the flocculation activity comprises the following steps: 0.5g of kaolin, 80mL of distilled water and 2mL of 10g/L CaCl are added into a 100mL colorimetric cylinder₂And 2mL of culture solution, then adding distilled water to 100mL of scale mark, quickly shaking for 10 times respectively, standing for 30s, slowly shaking for 20 times, standing for 5min, and taking supernatant to determine absorbance at 550nm of a spectrophotometer. A blank control was set, and the control group was a kaolin suspension without added culture medium to determine the degree of flocculation of the culture medium. Flocculation rate E (%) [ (A-B)/A]X 100%, wherein A is the absorbance at 550nm of the control supernatantLuminosity; b is the absorbance of the sample supernatant at 550 nm. After flocculation comparison of different strain culture solutions, the strain with the best flocculation effect is selected, the flocculation rate of the strain can reach 71.1 percent, and the strain is named as S2.

The strain S2 is cultured on TSA culture medium at 36 deg.C for 16h, its colony is shown in FIG. 3a, it is light yellow, round, dry in surface, translucent, irregular and radial in edge, 2.5-3.5mm in diameter, and its thallus is shown in FIG. 3b, it is rod-shaped, 0.6-1.0 μm × 1.3-3.3 μm, single or paired arrangement, spore column shape, mesogenesis, cyst is not expanded, and it is gram-positive bacterium.

2. 16S rRNA sequence determination of Bacillus subtilis subspecies desert S2

And (3) amplifying the 16S rRNA universal primer by using the DNA of the strain S2 as a template, and performing sequence determination on the amplified fragment. The 16S rDNA sequencing result of the strain S2 is compared and analyzed with a sequence in GenBank, and the result shows that the homology of the strain S2 and Bacillus subtilis subsp.

3. Bacillus subtilis strain preservation of desert subspecies S2

And (3) performing strain preservation on the screened bacillus subtilis S2, wherein the preservation unit of the bacillus subtilis S2 is as follows: china general microbiological culture Collection center (CGMCC); address: western road No.1, north west city of township, beijing, institute of microbiology, china academy of sciences; the preservation date is as follows: year 2020, 15/05; the preservation number of the Bacillus subtilis is as follows: CGMCC No. 19824.

4. Strain characteristics of Bacillus subtilis subspecies deserticola S2

The bacillus subtilis desert subspecies S2 has wide salinity, can grow in the salinity range of 0-50 per mill, and has the optimal growth salinity of 0-20 per mill; the growth can be carried out normally at the temperature of 10-40 ℃, and the optimal growth temperature is 20-30 ℃; the beta-xylosidase, phenylalanine arylamine enzyme, tyrosine arylamine enzyme, alpha-galactosidase, beta-galactosidase, alanine-phenylalanine-proline arylamine enzyme, alanine arylamine enzyme, leucine arylamine enzyme, L-pyrrolidone arylamine enzyme, beta-N-acetylglucosaminidase and the like of the bacillus subtilis subspecies deserticola S2 are positive in reaction, and D-glucose, D-mannose, inulin, red tetrazole, cyclodextrin, glycogen, maltotriose, D-ribose, D-mannitol, D-trehalose, gulose, inositol and the like can be used for hydrolyzing the esculin, and the esculin can grow in 6.5 percent of sodium chloride. The bacillus subtilis desert subspecies S2 has high indoleacetic acid yield and good flocculation capacity, can effectively inhibit the number of vibrio, euglenophyta and dinoflagellate cells, increase the number of chlorella and diatom algae cells, and can effectively remove vibrio aquatic organisms and inhibit vibrio harveyi and mermaid photobacterium.

Screening, separating and identifying clostridium butyricum GBW-GBW-N1

1. Separation, screening and purification of clostridium butyricum GBW-GBW-N1

And (3) coating the bacterial suspension cultured by the collected sample on a tryptone-sulfite-cycloserine agar (TSC) culture medium after gradient dilution, separating and purifying for multiple times to obtain a single bacterial colony, and storing the single bacterial colony named as GBW-N1.

The bacterial colony of the bacterial strain GBW-N1 cultured on the TSC solid medium for 24 hours is shown in figure 4, and the bacterial colony is as shown in figure 4a, which is round, black, smooth in surface, slightly convex in the middle and irregular in edge; FIG. 4b shows that the thallus of the bacterial strain GBW-GBW-N1 cultured for 48h on the TSC liquid medium is rod-shaped, 1.1-1.2 Mum multiplied by 2.1-3.6 Mum, single arranged, gram positive, fusiform spore, mesogenic and cyst-expanded.

2. Molecular characterization of Clostridium butyricum GBW-N1

The DNA of the strain GBW-N1 is used as a template, 16S rRNA universal primers are used for amplification, sequence determination is carried out on amplified fragments, the 16S rDNA sequencing result of the obtained strain GBW-N1 is compared with the sequence in GenBank for analysis, and the result shows that the strain GBW-GBW-N11 has the highest homology with Clostridium butyricum, so that the strain GBW-N1 is determined to be Clostridium butyricum.

3. Strain deposit of Clostridium butyricum GBW-N1

And (3) performing strain preservation on the screened strain GBW-N1, wherein the preservation unit of the clostridium butyricum GBW-N1 is as follows: china general microbiological culture Collection center (CGMCC); address: western road No.1, north west city of township, beijing, institute of microbiology, china academy of sciences; the preservation date is as follows: year 2017, month 08, day 07; the preservation number of the Clostridium butyricum is CGMCC No. 14499.

4. Strain characteristics of Clostridium butyricum GBW-N1

The clostridium butyricum GBW-N1 can grow in the pH value range of 6-8, and the optimal growth pH value is 7.1-7.5; the growth can be normally carried out within the range of dissolved oxygen content of 1-5 mg/L, and the optimal growth dissolved oxygen concentration is 2-4 mg/L; can produce urease, beta-glucosidase and glycerol, and can degrade or decompose power-nitrate, simon citrate and semi-solid agar; can normally grow and propagate at the temperature of 30-45 ℃, and the optimal growth temperature is 35-38 ℃. The clostridium butyricum GBW-N1 is an anaerobic bacterium, can germinate and proliferate in an anaerobic environment, and has probiotic functions of enzyme production, acid production, bacteriostasis and the like.

Example 2

1. Preparation method of feces composting and decomposing agent for disinsection and growth promotion

The excrement composting fermentation retting agent for disinsection and growth promotion comprises a low-temperature composting starter (a fermentation agent A bag) and a high-temperature composting insecticide (a fermentation agent B bag).

(1) The starter for low-temperature fermentation (starter A bag) contains bacteria with a content of 50-55 × 10⁸CFU/g filamentous bacillus GBW-F006 strain powder with the strain content of 45-50 multiplied by 10⁸The preparation method of the CFU/g Bacillus plantarum GBW-F008 bacterial powder and the carrier X comprises the following steps:

preparing the powder of the filamentous bacillus GBW-F006: inoculating an activated strain of filamentous bacillus GBW-F006 into a primary seed tank, maintaining the temperature in the tank at 36-38 ℃, the pressure in the tank at 0.05Mpa and the rotation speed in the tank at 170-180 rpm/min, culturing for 12-16 h, and transferring to a secondary fermentation tank after a logarithmic growth phase. Maintaining the fermentation temperature in the secondary fermentation tank at 35-37 ℃, the pressure in the secondary fermentation tank at 0.12Mpa, and the rotation speed in the secondary fermentation tank at 195-200 rpm/min, culturing for 46-48 h, and stopping the secondary fermentation tank when the OD600nm is not increased any more, and the spore rate is more than 98% and the spore maturity is more than 95% as determined by microscopic examinationThe amount of the fermentation bacteria is 350 multiplied by 10⁸CFU/ml～375×10⁸CFU/ml. Adding 5% corncob powder into the secondary fermentation liquid of the filamentous bacillus GBW-F006, and spray drying to obtain the bacterial powder, wherein the bacterial quantity of the semi-finished bacterial powder of the filamentous bacillus GBW-F006 is 1000 multiplied by 10⁸ CFU/g～1100×10⁸CFU/g。

The formula of the first-level seed tank is as follows: 5-6 g of peptone, 1-1.2 g of yeast powder and K₂HPO₄ 1～15g， MgSO4·7H₂0.5-0.8 g of O, 5-6 g of soluble starch, 7.4-7.6 of pH and 1L of distilled water. The formula of the culture solution of the secondary fermentation tank is as follows: 1-1.5 g of corn steep liquor dry powder, 2-2.5 g of cottonseed protein powder, 0.8-1 g of peptone, 0.5-0.8 g of yeast extract and KH₂PO₄ 0.1～0.3g，MgSO4·7H₂O 0.05～0.08g，MnSO₄·H₂0.03-0.05 g of neutral protease 5 ten thousand U/g 0.1-0.2 g, pH 7.3-7.5, and 1L of distilled water. The spray drying adopts an LPG-6000 spray drying tower, the air inlet is 135-140 ℃, and the air outlet is 70-75 ℃.

Preparing the powder of the Bacillus plantarum GBW-F008: inoculating an activated strain of Bacillus subtilis GBW-F008 in a first-stage seeding tank, maintaining the temperature in the tank at 35-37 ℃, the pressure in the tank at 0.05Mpa and the rotating speed in the tank at 165-175 rpm/min, culturing for 13-15 h, and transferring to a second-stage fermentation tank after a logarithmic growth phase. Maintaining the fermentation temperature in the secondary fermentation tank at 36-38 ℃, the pressure in the secondary fermentation tank at 0.13Mpa, and the rotation speed in the secondary fermentation tank at 180-190 rpm/min, culturing for 46-48 h, stopping the secondary fermentation tank when the OD600nm is not increased any more, and the spore rate is more than 98% and the spore maturity is more than 95% as determined by microscopic examination, wherein the general fermentation bacterial amount is 360 multiplied by 10⁸ CFU/ml～370×10⁸CFU/ml. Adding 6-8% of light calcium carbonate into the secondary fermentation liquid of the filamentous bacillus GBW-F006, and performing spray drying to obtain bacterial powder, wherein the bacterial quantity of the semi-finished bacterial powder of the plant bacillus GBW-F008 is 900 multiplied by 10⁸CFU/g～1000×10⁸CFU/g。

The formula of the first-level seed tank is as follows: 5-6 g of beef extract, 10-12 g of tryptone, 5-6 g of sodium chloride, (NH)₄₎₂SO₄ 2.5～3.5g，MnSO₄·H₂O 0.05～0.08g，MgSO₄·7H₂O 0.05-0.08 g, pH 7.4-7.6, and distilled water 1L. The formula of the culture solution of the secondary fermentation tank is as follows: 8-10 g of corn starch, 2-3 g of bean cake powder and K₂HPO₄ 2～3g， MgSO4·7H₂O 0.5～0.8g，MnSO₄·H₂O 0.5～0.8g，CaCO₃ 0.1～0.3g，FeCl₃·6H₂5-6 mg of O, 7.0-7.5 of pH and 1L of distilled water. The spray drying adopts an LPG-6000 spray drying tower, the air inlet is 126-135 ℃, and the air outlet is 65-70 ℃.

The mixing mass ratio of the semi-finished bacterial powder of the filamentous bacillus GBW-F006 and the plant bacillus GBW-F008 to the carrier X is as follows: semi-finished powder of filamentous bacillus GBW-F006: b, semi-finished product bacterial powder of B.plantarum GBW-F008: and mixing the carrier X at the ratio of 1:20 to obtain the low-temperature fermentation starter.

The carrier X is one or more of plant ash, humic acid and zeolite powder.

(2) The high-temperature retting pesticide (the fermentation agent B bag) comprises bacteria with the content of 150-160 multiplied by 10⁸CFU/g bacillus subtilis desert subspecies S2 strain powder with strain content of 40-50 multiplied by 10⁸CFU/g of Clostridium butyricum GBW-N1 bacterial powder and a carrier Y.

Preparing bacillus subtilis S2 strain powder: inoculating the activated strain of the bacillus subtilis S2 in the desert subspecies into a first-stage seed tank, maintaining the temperature in the tank at 37.2-37.6 ℃, the pressure in the tank at 0.06Mpa and the rotating speed in the tank at 200-210 rpm/min, culturing for 12-14 h, and transferring to a second-stage fermentation tank after logarithmic growth phase. Maintaining the fermentation temperature in the secondary fermentation tank at 36.5-37.5 ℃, the pressure in the secondary fermentation tank at 0.14Mpa, the rotating speed in the secondary fermentation tank at 210-215 rpm/min, culturing for 32-34 h, stopping the secondary fermentation tank when the OD600nm is not increased any more, and the spore rate is more than 99% and the spore maturity is more than 98% as determined by microscopic examination, wherein the general fermentation bacterial quantity is 420 multiplied by 10⁸ CFU/ml～450×10⁸CFU/ml. 5 to 8 percent of amylopectin is added into the secondary fermentation liquid of the bacillus subtilis subspecies desert S2, and the bacterial powder is obtained by spray drying, wherein the bacterial quantity of the semi-finished product bacterial powder is 2250 multiplied by 10⁸CFU/g ～2400×10⁸CFU/g。

The formula of the first-level seed tank is as follows: 6-8 g of cottonseed protein powder, 2-3 g of yeast extract and corn flour1.5-2 g, dipotassium hydrogen phosphate 2-2.5 g, MgSO4 & 7H₂0.5-0.8 g of O, 7.4-7.6 of pH and 1L of distilled water. The formula of the culture solution of the secondary fermentation tank is as follows: 2-3 g of peanut meal, 0.8-1.2 g of peptone, 0.5-0.8 g of yeast extract powder and KH₂PO₄0.8～1.2g，MgSO4·7H₂O 0.8～1.2g，MnSO₄·H₂0.5-0.8 g of O, 0.5-0.8 g of flavourzyme 10 ten thousand U/g, 7.6-7.8 of pH and 1L of distilled water. The spray drying adopts an LPG-6000 spray drying tower, the air inlet is 140-145 ℃, and the air outlet is 73-75 ℃.

Preparation of clostridium butyricum GBW-N1 bacterial powder: inoculating an activated strain of clostridium butyricum GBW-N1 into a primary seed tank, maintaining the temperature in the tank at 37.5-38.2 ℃, the pressure in the tank at 0.05Mpa and the rotating speed in the tank at 30-45 rpm/min, culturing for 16-18 h, and transferring to a secondary fermentation tank after a logarithmic growth phase. Maintaining the fermentation temperature in the secondary fermentation tank at 36.5-37.5 ℃, the pressure in the secondary fermentation tank at 0.08Mpa, the rotating speed in the secondary fermentation tank at 50-55 rpm/min, culturing for 51-55 h, stopping the secondary fermentation tank when the OD600nm is not increased any more, and the spore rate is more than 95% and the spore maturity is more than 95% as determined by microscopic examination, wherein the general fermentation bacterial load is 120 multiplied by 10⁸CFU/ml～125×10⁸CFU/ml. Adding 5-8% of amylopectin into the secondary fermentation liquid of the bacillus subtilis subspecies desert S2, and spray drying to obtain bacterial powder, wherein the bacterial quantity of the semi-finished product bacterial powder is 600 multiplied by 10⁸CFU/g～750×10⁸CFU/g。

The formula of the first-level seed tank is as follows: 5-6 g of soytone, 5-6 g of yeast powder, 1-1.5 g of sodium metabisulfite, 0.5-1.5 g/L of ferric ammonium citrate, 7.4-7.6 of pH and 1L of distilled water. The formula of the culture solution of the secondary fermentation tank is as follows: 2-2.5 g of soybean meal powder, 0.5-0.8 g of disodium hydrogen phosphate, 0.25-0.35 g of dipotassium hydrogen phosphate, 0.05-0.08 g of manganese sulfate, 7.6-7.8 of pH and 1L of distilled water. The spray drying adopts an LPG-6000 spray drying tower, the air inlet is 85-90 ℃, and the air outlet is 50-55 ℃.

Uniformly mixing semi-finished bacterial powder of bacillus subtilis desert subspecies S2 and clostridium butyricum GBW-N1 with a carrier Y, wherein the mass ratio of the bacillus subtilis desert subspecies S2 to the clostridium butyricum GBW-N1 is as follows: semi-finished powder of bacillus subtilis S2 subspecies deserticola: semi-finished powder of clostridium butyricum GBW-N1: the carrier Y is 1:15, and the high-temperature retting pesticide is obtained after mixing.

The carrier Y is one or more of diatomite, vermiculite and weathered coal.

2. Application scheme of excrement composting fermentation retting agent capable of killing insects and promoting growth

The two fermentation agent bags of the manure composting and retting agent are used for the step-by-step composting and retting process of manure as shown in figure 5, and the specific application scheme steps are as follows:

1) feces: comprises conventional breeding manure such as chicken manure, pig manure, cow manure and the like;

2) adjusting indexes of a mixed carbon source and a substrate: miscellaneous wood chips: the water content is less than 8 percent, the pH is less than 7, and the C/N is more than 450: 1; wheat straw: the water content is less than 10%, the pH is less than 6.9, and the ratio of C to N is 60-70: 1; rice hull: the water content is less than 10%, the pH is less than 6, and the ratio of C to N is 70-80: 1; mushroom culture medium waste: the water content is less than 10%, the pH is less than 6, and the ratio of C to N is 30-35: 1; the basic indexes of the vegetable slag are that the water content is more than 70 percent, the pH value is less than 6, and the C/N is 8-15; adding 30-40% of batch mixture into 1 ton of feces before fermentation to form two or more than two of miscellaneous wood chips, wheat straws, rice hulls and mushroom culture medium waste, and uniformly distributing;

3) inoculating a starter A bag: a is a low-temperature fermentation starter, wherein the filamentous bacillus GBW-F006 can grow at a low temperature of 15 ℃, the bacillus plantarum GBW-F008 can grow at a low temperature of 20 ℃, the two can promote the system to enter a microbial fermentation state, continuously consume system oxygen, generate heat above 38ATP and promote the system to heat up, the filamentous bacillus GBW-F006 can continuously generate antibacterial substances, promote the reduction of bacteria such as fecal escherichia coli and the like in the system and gradually reduce the available nutrition of roundworm eggs, and the bacillus plantarum GBW-F008 continuously releases nutrients such as small molecular sugar, amino acid, available phosphorus, available potassium and the like through the produced xylanase, lipase, cellulase, amylase, protease, phosphatase and the like to provide nutrients for the propagation of various bacteria in the starter, so that the bacillus plantarum GBW-F008 finally becomes a dominant bacteria group, and the process takes about 2.5 d. The inoculation amount of the inoculated starter A bag is 0.1-0.25%.

4) And (3) system change: after the nutrients are prepared and started at low temperature, the water content of the system is reduced to 30-35%, the pH value is less than 6.8, and the ratio of C to N is 30-35: 1.

5) Inoculating a starter B package: b is a high-temperature retting pesticide, wherein the bacillus subtilis subspecies desert S2 has strong enzyme production and antibacterial properties, can quickly grow and ferment in organic materials, generates a large amount of heat energy through aerobic reaction, quickly promotes the system to be heated to 71-76 ℃, and grows due to gradual rarefied oxygen of the system by clostridium butyricum GBW-N1 to generate a large amount of organic acids represented by butyric acid, and the organic acids have important effects on plant growth, plant fruit acid formation, release of certain combined state mineral elements in soil and the like; in addition, the remaining filamentous bacillus, endophyte bacillus and the large amount of metabolites inoculated with the starter A package also play a continuous role in promoting nutrient release and boosting bacterial growth, and further promote the continuous temperature rise of the system and the death of roundworm eggs. This process starts at 3d and continues to 6 d. The inoculation amount of the inoculated starter B bag is 0.1-0.25%.

6) Maintaining the decomposing inoculant at high temperature: fermenting for 7-10 days, continuously maintaining at a high temperature of more than 70 ℃, and ensuring that the death rate of roundworm eggs is more than 95 percent;

7) medium temperature maintenance of the decomposing inoculant: fermenting for 11-20 days, continuously maintaining at a high temperature of more than 55 ℃, and ensuring that the death rate of roundworm eggs is more than 95 percent;

8) after-ripening: fermenting for 21d-27d, gradually entering a humification process, and gradually stabilizing the ratio of humic acid/caffeic acid (HA/FA);

9) drying: after fermenting for 28 days, drying the water to 27% -29% by adopting a rotary drum dryer;

10) crushing: crushing and sieving with a 8-mesh sieve;

11) index measurement: measuring moisture, organic matters, N, P, K, ascaris egg death rate, fecal colibacillus number and the like of the fermentation waste;

12) nutrient blending: and (5) blending nutrients according to the requirements of the finished product, and then subpackaging the finished product.

Example 3

The test adopts white feather broiler manure produced in a certain chicken raising factory in Huiyang in Shouguang in 2020 and 5-7 months. The test chicken manure was mixed evenly and divided into 6 groups of 2 tons each, and 3 replicates were performed each. In order to investigate the influence of the inoculation amount of the pesticidal growth-promoting manure composting and retting agent on the manure treatment effect, the design of each group is as follows:

TABLE 1 different manure composting retting agent inoculum (%, m/m) groups

The composting fermentation retting test is 1 month in period, the influence of the inoculation amount on indexes of the treated excrement, such as the composting temperature of the excrement, the ascarid egg death rate, the total amount of free amino acid and the like, is mainly considered, the fermented products of each group are applied to the base fertilizer application of the cucumber, and the indexes of the emergence rate of cucumber seedlings, the per mu yield of the cucumber and the like after the equal amount of fermentation retting test is compared and applied by the same method. Uniformly measuring the average temperature of 5 different points below 50cm at the highest position of the fermentation pile at the fecal composting temperature; the ascarid egg death rate refers to the determination of the ascarid egg death rate in GB/T19524.2-2004 fertilizer; the determination of free amino acids in the GB/T30987-; the plant application effect refers to NY T1536 and 2007 field test technical procedures of microbial fertilizers and fertilizer efficiency evaluation guidelines.

FIGS. 6 to 8 show the effect of different amounts of composting agents on the indexes of chicken manure composting temperature, ascarid mortality, total free amino acids, etc. The test result shows that after 12d of fermentation, namely after the step inoculation of the low-temperature composting starter A bag and the high-temperature composting insecticide B bag, 4d of decomposition and high-temperature maintenance are carried out, and the second day of medium-temperature maintenance is carried out, the total inoculation amount of AB is 0.20-0.50% of the A2B2, A3B3, A4B4 and A5B5 groups, the feces composting temperature is 71.5-74.3 ℃, the roundworm egg death rate is 96.1-97.3%, the roundworm egg death rate is more than 95%, the national standard requirements can be met, the total inoculation amount of AB is 0.10% of the A1B1 group and the non-inoculation control group, the feces composting temperature is 50.2 ℃ and 58.7 ℃, the roundworm egg death rate is 85.1% and 72.8%, respectively, and the national standard requirements are not met. In the 12d fermentation, the A3B3, A4B4 and A5B5 groups with the total AB inoculation amount of 0.30-0.50% achieve the total free amino acid amount of 4.55-4.61%, which is more than 4.5%, while the A2B2 group with the total AB inoculation amount of 0.20%, the A1B1 group with the total free amino acid amount of 4.45% and the A1B1 group with the total AB inoculation amount of 0.10% and the non-inoculated control group respectively have the total free amino acid amount of 3.85-3.65% and are lower. After 12 days, the total amount of free amino acids is continuously increased, particularly when 28 days are adopted, the total amount of AB inoculation is 0.30-0.50%, the total amount of free amino acids is up to 7.24-7.38% of groups A3B3, A4B4 and A5B 5.

The production indexes of the chicken manure treated by different manure composting and retting agents after being applied to cucumber planting are shown in table 2, the A2B2, A3B3, A4B4 and A5B5 groups with the total AB inoculation amount of 0.20-0.50 percent have the germination rate of cucumber seeds of more than 93 percent and are obviously higher than the CK and A1B1 groups with the total AB inoculation amount of 0-0.10 percent. Aiming at the cucumber per mu yield index, the cucumber yield of the A2B2 group is obviously lower than that of the latter three groups (P is less than 0.05), the A3B3, A4B4 and A5B5 groups with the AB total inoculation amount of 0.30-0.50 percent, the cucumber per mu yield is more than 15700 kg/mu and is obviously higher than that of other groups (P is less than 0.05), and particularly, the yield is increased by 25.8-28.1 percent relative to the control group.

TABLE 2 influence of chicken manure treated with different manure composting and retting agent inoculum size on cucumber emergence rate and yield per mu

The test results show that the total inoculation quantity of AB is 0.20-0.50%, the composting temperature of excrement is higher than 71 ℃, the ascarid mortality index can reach the national standard, the total quantity of free amino acid is gradually increased along with the continuous degradation of substances such as protein, and when 28d of fermentation treatment is finished, the total quantity of three groups of free amino acid with the total inoculation quantity of AB of 0.30-0.50% reaches the highest, and the three groups can realize the per mu yield increase of 25.8-28.1% on cucumber application tests, and the economic benefit is obviously higher than that of other groups. From the aspects of application cost, economic benefit and the like, the total dosage of the A package low-temperature fermentation starter and the B package high-temperature retting insecticide is recommended to be 0.30-0.40 percent, namely 0.15-0.20 percent of A and 0.15-0.20 percent of B.

Example 4

The test is carried out by adopting Duroc pig manure produced in a certain pig farm in Linyi Junan, 8-9 months. The test pig manure was mixed evenly and divided into 5 groups of 2 tons each, and 3 replicates were performed each. In order to investigate the influence of the active group and the inactivation group of the insecticidal growth-promoting manure composting and retting agent on the manure treatment effect, the following groups are designed, wherein a control CK group is not inoculated with a fermenting agent; the total amount of the inactivated leaven in the M groups is 0.30 percent; n groups of seeds are inoculated with 0.30 percent of active leaven; group I inoculates 0.30% of starter culture in total, of which only 0.15% of low temperature fermentation starter is active and 0.15% of high temperature retting insecticide is inactivated; group J was inoculated with 0.30% total starter culture, of which only 0.15% of the high temperature retting insecticide was active and 0.15% of the low temperature fermentation starter was inactivated.

TABLE 3 different manure composting and retting agent groups

The composting fermentation retting test is 1 month in period, the influence of the inoculation amount on indexes such as the composting temperature of excrement after excrement treatment, the ascarid egg death rate, the total amount of free amino acid and the like is mainly considered, the fermented products of each group are applied to base fertilizer application before cherry trees are subjected to fruit setting, and indexes such as cherry yield per mu and cherry fruit diameter are compared after the same amount of fermentation retting test is applied by the same method. Uniformly measuring the average temperature of 5 different points below 50cm at the highest position of the fermentation pile at the fecal composting temperature; the ascarid egg death rate is determined by referring to the ascarid egg death rate in GB/T19524.2-2004 fertilizer. Free amino acids were determined in reference to the GB/T30987-. The plant application effect refers to NY T1536 and 2007 field test technical procedures of microbial fertilizers and fertilizer efficiency evaluation guidelines.

FIGS. 9 to 11 show the effects of the inactivated and activated feces stacking retting agent on the indexes of feces stacking retting temperature, death rate of ascaris suum ova, total amount of free amino acids, and the like. The test result shows that the manure composting temperature is separated when fermenting for 3d, wherein the temperature of the active N group inoculated with the active low-temperature fermentation starter and the active high-temperature retting insecticide reaches 58.3 ℃ at the highest, then the temperature of the active N group inoculated with the active low-temperature fermentation starter is 43.5 ℃, the temperature of other groups at 3d is 29.8-38.6 ℃, the fermentation temperature is gradually increased and gradually maintained along with the inoculation of the high-temperature retting insecticide at 6-12d, and the temperature of the active N group is 71-73 ℃ at the highest 76 ℃; and the group I only inoculated with the active low-temperature reactor fermentation starter has the stack temperature of only 51.5-54 ℃ because the high-temperature retting insecticide is in an inactivated state and lacks the subsequent temperature-raising insecticidal capacity after 6-12 days; group J inoculated with active high-temperature retting insecticide only, and the stack temperature of 6-12d is 61.6-63.5 ℃ due to lack of initial start and nutrient decomposition supply; the highest temperature of the control group and the inactivation group M is lower than 51.5 ℃, and the requirement of maintaining the high temperature above 55 ℃ is not met.

The 12 th day of fermentation of each group, namely the low-temperature fermentation starter and the high-temperature retting insecticide which undergo the step-by-step inoculation activity undergo 4 days of decomposition and high-temperature maintenance, the ascarid egg death rate of the active N groups is more than 95 percent and reaches the national standard requirement on the second day of medium-temperature maintenance, and the ascarid egg death rates of the CK and the inactivated groups are 73.6 to 75.1 percent respectively and do not reach the national standard requirement; the I group inoculated with the active low-temperature fermentation starter or the J group inoculated with the active high-temperature retting insecticide has stronger indexes than the inactivated group, but the death rate of roundworm eggs is 82.6-86.4% at 12 days and 85.1-93.8% at 28 days, and still does not meet the national standard requirements.

The total free amino acid amount of the active N group is gradually increased from 6d, the fermentation 28d reaches the highest value of 0.79%, the free amino acid amount is increased by 88.1% compared with the control group by 0.42%, the difference is obvious (P is less than 0.05), and the free amino acid amount of the inactivated M group is only increased by 2.3% compared with the control group without obvious difference (P is more than 0.05). In contrast, in the group I inoculated with the active low-temperature fermentation starter or the group J inoculated with the active high-temperature retting insecticide, although each index is stronger than that of the inactivated group, the total amount of free amino acid is respectively improved by 9.5-33.3 percent relative to the control group, and the amplification is obviously lower than that of the active N group (P is less than 0.05).

The results show that the excrement retting temperature can be increased only by inoculating the active low-temperature fermentation starter and the active high-temperature retting insecticide simultaneously, so that the up-to-standard and high ascarid egg death rate is finally realized, the total amount of free amino acid is greatly increased, and the up-to-standard and nutrient-value-added standard excrement treatment product is finally formed.

The production indexes of the processed pig manure applied to cherry tree planting under different manure composting and retting agent dosages are shown in table 4.

TABLE 4 influence of pig manure treated with inactivated and active manure composting retting agent on cherry yield and fruit diameter

The results show that the ascarid egg death rate of the three groups of the sterilized M group, the I group inoculated with the active low-temperature fermentation starter or the J group inoculated with the active high-temperature retting insecticide does not reach the standard, although the stacking temperature is increased to a certain degree compared with a control group, the content of free amino acid nutrients is increased, a certain positive result is achieved on the improvement of cherry production indexes, but the ascarid egg death rate does not reach the standard due to the partial inactivation of the components of the starter, and the potential threat exists on the farmland environment and the food safety; the pig manure treated by the active N groups has obvious insecticidal effect, the national standard requirement can be met after the 12 th fermentation of the ascarid egg death rate group, the safety and the reliability are realized, the total amount of free amino acid in the pig manure can be obviously improved, finally, in the cherry planting test, the increase of the cherry yield reaches 18.6%, the increase of the cherry fruit diameter reaches 15%, and the difference is obvious (P is less than 0.05).

Summary of the test: when the feces stacking fermentation retting agent with biological activity and insecticidal growth promotion is used for treating feces, a low-temperature stacking fermentation starter and a high-temperature retting insecticide are inoculated step by step, when the total inoculation amount of the two is 0.30-0.50%, the feces is maintained at a high temperature after being decomposed after being started at a low temperature, and the death rate of ascarid eggs is basically over 95% after 12 days of fermentation, so that the insecticidal effect is quickly realized to achieve the hygienic index. In addition, through the process of medium-temperature maintenance and after-ripening, the total amount of free amino acid is continuously increased, the material is further humified, and the method is beneficial to the synthesis of various life activities or stimulating substances of plants, and finally realizes the purpose of promoting the growth of the plants.

The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions.

Claims

1. The excrement composting and retting agent is characterized in that: the manure composting and decomposing agent comprises a low-temperature composting starter and a high-temperature composting and decomposing pesticide; the low-temperature fermentation starter comprises filamentous bacillus powder, plant endophyte bacillus powder and a carrier X; the high-temperature retting insecticide comprises bacillus subtilis subspecies desert bacterial powder, clostridium butyricum bacterial powder and a carrier Y;

the mass ratio of the filamentous bacillus powder to the plant endophyte bacillus powder to the carrier X is 1:1: 20;

the mass ratio of the bacillus subtilis subspecies desert powder to the clostridium butyricum to the carrier Y is 1:1: 15;

the filamentous bacillus powder adopts filamentous bacillus with the preservation number of CGMCC No.20918Bacillus filamentosus GBW-F006 is obtained by fermentation and drying, and the bacterium content is 50-55 multiplied by 10⁸CFU/g; the plant endospore bacillus powder adopts plant endospore bacillus with preservation number of CGMCC number 20919Bacillus endophyticusGBW-F008 is obtained by fermentation and drying, and the bacterium content of the GBW-F008 is 45-50 multiplied by 10⁸CFU/g；

The desert subspecies powder of the bacillus subtilis adopts the bacillus subtilis with the preservation number of CGMCC No.19824Bacillus subtilis S2 is obtained by fermentation and drying, and the bacterium content is 150-160 multiplied by 10⁸CFU/g; the clostridium butyricum powder adopts clostridium butyricum with the preservation number of CGMCC No.14499Clostridium butyricum GBW-N1 is obtained by fermentation and drying, and the bacterium content is 40-50 multiplied by 10⁸CFU/g；

The carrier X comprises at least one of plant ash, humic acid and zeolite powder; the carrier Y comprises at least one of diatomite, vermiculite and weathered coal.

2. Use of the manure composting retting agent of claim 1 for manure composting.

3. The use according to claim 2, characterized in that said manure composting agent is used in a method comprising: inoculating the low-temperature fermentation starter into excrement and batch, fermenting for 2.5-4d, then inoculating the high-temperature retting insecticide, continuously fermenting for 3-4d, continuously fermenting for 3-5d at a high temperature of more than 70 ℃, continuously fermenting for 8-10d at a high temperature of more than 55 ℃, after-ripening and fermenting for 6-7d, drying until the water content is 27% -29%, crushing, subpackaging, and directly mixing into plant planting soil; the total inoculation amount of the manure composting and decomposing agent is 0.20-0.50%; the inoculation amount ratio of the low-temperature fermentation starter to the high-temperature retting insecticide is 1: 1-3: 5.