CN114933983B - Microbial agent for reducing emission of livestock manure compost ammonia gas and greenhouse gas in cooperation, and preparation and application thereof - Google Patents
Microbial agent for reducing emission of livestock manure compost ammonia gas and greenhouse gas in cooperation, and preparation and application thereof Download PDFInfo
- Publication number
- CN114933983B CN114933983B CN202210417125.9A CN202210417125A CN114933983B CN 114933983 B CN114933983 B CN 114933983B CN 202210417125 A CN202210417125 A CN 202210417125A CN 114933983 B CN114933983 B CN 114933983B
- Authority
- CN
- China
- Prior art keywords
- microbial agent
- fermentation
- composting
- culture medium
- percent
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F11/00—Other organic fertilisers
- C05F11/08—Organic fertilisers containing added bacterial cultures, mycelia or the like
-
- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F17/00—Preparation of fertilisers characterised by biological or biochemical treatment steps, e.g. composting or fermentation
- C05F17/20—Preparation of fertilisers characterised by biological or biochemical treatment steps, e.g. composting or fermentation using specific microorganisms or substances, e.g. enzymes, for activating or stimulating the treatment
-
- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F17/00—Preparation of fertilisers characterised by biological or biochemical treatment steps, e.g. composting or fermentation
- C05F17/50—Treatments combining two or more different biological or biochemical treatments, e.g. anaerobic and aerobic treatment or vermicomposting and aerobic treatment
-
- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F3/00—Fertilisers from human or animal excrements, e.g. manure
-
- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05G—MIXTURES OF FERTILISERS COVERED INDIVIDUALLY BY DIFFERENT SUBCLASSES OF CLASS C05; MIXTURES OF ONE OR MORE FERTILISERS WITH MATERIALS NOT HAVING A SPECIFIC FERTILISING ACTIVITY, e.g. PESTICIDES, SOIL-CONDITIONERS, WETTING AGENTS; FERTILISERS CHARACTERISED BY THEIR FORM
- C05G3/00—Mixtures of one or more fertilisers with additives not having a specially fertilising activity
- C05G3/80—Soil conditioners
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
-
- 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
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Microbiology (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Molecular Biology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Mycology (AREA)
- Botany (AREA)
- Soil Sciences (AREA)
- Pest Control & Pesticides (AREA)
- Fertilizers (AREA)
- Treatment Of Sludge (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention belongs to the technical field of solid organic waste disposal and resource utilization, and particularly relates to a microbial agent for reducing emission of livestock manure compost ammonia gas and greenhouse gas in a synergistic manner, and a preparation method and application thereof. The compound microbial agent is applied to livestock manure composting fermentation, and can realize the synergistic emission reduction of ammonia and greenhouse gases in the composting process. Compared with the control group, the ammonia release amount of the treatment group added with the compound microbial agent is reduced by 57.5-66.2%, the methane release amount is reduced by 58.8-80.9%, and the nitrous oxide release amount is reduced by 56.7-64.1%. Meanwhile, compared with the control group, the total nitrogen content is improved by more than 27.48%, which is beneficial to improving the quality of the compost. Therefore, the composite microbial agent provided by the invention can not only remarkably reduce the emission of ammonia gas and greenhouse gas in the composting process, but also effectively shorten the composting time, improve the composting quality, meet the requirements of industrial large-scale production, and facilitate large-area popularization and use.
Description
Technical Field
The invention belongs to the technical field of solid organic waste disposal and resource utilization, and particularly relates to a microbial agent for reducing emission of livestock and poultry manure compost ammonia gas and greenhouse gas in a synergistic manner, and a preparation method and application thereof.
Background
With the rapid development of livestock and poultry breeding industry, a large amount of livestock and poultry breeding wastes are accumulated, so that serious environmental pollution is caused, and the sanitation and epidemic prevention and the human health of a farm are also affected. The livestock manure is rich in a large amount of organic matters, rich nitrogen, phosphorus and potassium, and trace elements such as copper, magnesium, sulfur, iron, zinc and the like, and is a high-quality raw material for manufacturing organic fertilizers. The organic fertilizer prepared by composting livestock manure provides 3050.8 ten thousand tons of nitrogen per year, can not only improve crop yield, but also promote the formation of soil aggregate structure, make soil soft, increase the air permeability of soil, improve the physicochemical properties of soil, promote the activity of soil microorganisms, accelerate the material circulation in the soil and the formation of humus, and is a valuable resource for sustainable development of agriculture.
Composting is one of effective ways for realizing harmless treatment and recycling of livestock manure, but ammonia (NH) caused in composting process 3 ) Volatilizing and greenhouse gases (nitrous oxide N) 2 O, methane CH 4 ) The emission not only pollutes the environment and affects the health of human bodies, but also reduces the nutrient content in the fertilizer. Greenhouse gases have a high global warming potential (global warming potentials, GWP), reported by IPCC as CH on the 100 year scale 4 、N 2 The global warming potentials of O are respectively CO 2 25 and 298 times. The prior study shows that NH is caused in the composting process of pig manure 3 And N 2 The emission of O causes a serious loss of nitrogen, in which NH 3 The loss of nitrogen caused by emission can reach 16.1 to 22.6 percent, N 2 The nitrogen loss caused by O emission can reach 0.1% -4.2%. In addition, the emission of ammonia in a large quantity not only seriously affects the production performance of animals and the health of raising personnel, but also can cause the formation of PM2.5 and acid rain, and further causes soil acidification, water surface eutrophication and groundwater pollution.
Therefore, in order to realize harmless treatment and recycling of the livestock manure, reduce the emission of ammonia and greenhouse gases in the composting process of the livestock manure, reduce the loss of nitrogen and improve the composting quality, a microbial agent for synergistically reducing the emission of the ammonia and the greenhouse gases in the composting process of the livestock manure is developed, and the microbial agent has great significance.
Disclosure of Invention
The invention aims to provide a microbial agent capable of rapidly decomposing livestock and poultry manure and realizing the emission reduction of ammonia and greenhouse gases in the composting process of the livestock and poultry manure, and a preparation method and application thereof.
The technical scheme of the invention is realized by the following steps:
a microbial agent for synergistically reducing emission of ammonia and greenhouse gases in livestock manure compost, which comprises the following components: bacillus subtilis (Bacillus subtilis) F2CGMCC No. 21117 and Saccharomyces cerevisiae (Saccharomyces cerevisiae) J8CGMCC No.21716.
The microbial agent is prepared by respectively fermenting and mixing bacillus subtilis CGMCC No. 21117 and Saccharomyces cerevisiae CGMCC No.21716.
The microbial agent has the bacterial count of 4.0X10 in the products after the fermentation of bacillus subtilis CGMCC No. 21117 and Saccharomyces cerevisiae CGMCC No.21716 9 ～6.0×10 9 Between cfu/g, the total number of viable bacteria is preferably not less than 50.0X10 8 cfu/g。
The preparation method of the microbial agent comprises the following steps:
1) Preparation of bacillus subtilis CGMCC No.21717 ferment
Firstly, bacillus subtilis is subjected to inclined plane strain activation and shake flask seed culture; culturing in a fermentation tank, and finally carrying out solid adsorption to obtain a powder product;
2) Preparation of Saccharomyces cerevisiae CGMCC No.21716 ferment
Firstly, activating saccharomyces cerevisiae through inclined plane strains and culturing shaking bottle seeds; and then culturing in a fermentation tank: finally, carrying out solid adsorption to obtain a powder product;
3) Microbial agent compounding
And (3) fully and uniformly mixing the bacillus subtilis fermented product powder product and the saccharomyces cerevisiae fermented product powder product.
The preparation method of the microbial agent,
the slant strain activation culture medium in the step 1) comprises the following components in percentage by mass: 1% of peptone, 0.5% of NaCl, 0.5% of yeast extract, 1.5% -2.0% of agar, and the balance of water, wherein the pH value is 7.2-7.4;
the culture medium of the shake flask seeds in the step 1) is prepared by adding no agar into the slant culture medium in the step 1), and the specific operation is as follows: taking an activated fresh inclined plane strain as a loop, inoculating the activated fresh inclined plane strain into a shake flask seed liquid culture medium, wherein the loading amount is 100mL/500mL, the rotating speed is 180 rpm-200 rpm, and after culturing for 22-24 hours at the constant temperature of 35-37 ℃, carrying out water bath heat treatment for 10 minutes at the temperature of 80 ℃;
the culture medium in the fermentation tank is filled in an amount of 50-70% of the total volume, the inoculum size is 0.1-0.3% of the volume of the culture medium, the fermentation temperature is 30-37 ℃, the air is introduced at 180-200 rpm for stirring culture, the ratio of the aeration volume to the volume of the fermentation liquid is 0.9:1-1:1, and the tank pressure is 0.03-0.05 MPa; the fermentation end point is that the spore number is not less than 90% of the total bacterial number, and the viable bacterial number of the fermentation liquid is 2.5X10 10 ～3.0×10 10 cfu/mL; the fermentation tank culture medium comprises, by mass, 0.5-0.7% of glucose, 0.3-0.5% of starch, 0.8-1.2% of bean cake powder, 0.0062% of manganese sulfate, 0.5-0.8% of yeast powder, 0.3-0.5% of peptone, 0.01% of ferric chloride, 0.4% of potassium dihydrogen phosphate, 0.1% of calcium carbonate, 0.05% of magnesium sulfate, and the balance of water, wherein the pH value is 7.2-7.4;
the culture medium for activating the inclined plane strain in the step 2) is a PDA solid culture medium;
the culture medium of the seeds in the step 2) is prepared by adding no agar into the slant culture medium in the step 2), and the specific operation is as follows: taking an activated fresh inclined plane strain as a loop, inoculating the activated fresh inclined plane strain into a seed liquid culture medium, wherein the loading amount is 200 mL/500mL, the rotating speed is 180 rpm-200 rpm, and culturing at the constant temperature of 28-30 ℃ for 36-40 h, and stopping culturing.
The culture medium in the fermentation tank in the step 2) is filled in an amount of 50-70% of the total volume, the inoculum size accounts for 0.3-0.5% of the volume of the culture medium, the fermentation temperature is 28-30 ℃, the air is introduced at 150-180 rpm for stirring culture, the ratio of the aeration volume to the volume of the fermentation liquid is 1:1-1.1:1, the tank pressure is kept at 0.03-0.05 MPa, and the bacteria content of the fermentation liquid is 4.0x10 after the fermentation is completed for 46-48 hours 9 ～5.0×10 9 cfu/mL; the fermentation tank culture medium comprises the following components in percentage by mass: 3.5 to 4.0 percent of glucose, 1.5 to 2.0 percent of ammonium sulfate, 0.2 to 0.25 percent of yeast powder, 0.1 percent of monopotassium phosphate, 0.01 percent of sodium chloride, 0.05 percent of magnesium sulfate and the balance of water, and the pH value is 6.0 to 6.5.
The preparation method of the microbial agent,
step 1) finally carrying out solid adsorption: adsorbing the fermentation liquor with wheat bran according to the weight ratio of 1:2-1:3, dehumidifying and drying at low temperature after adsorption, and crushing, wherein the bacterial content of the powder product is 8.0x10 9 ～12.0×10 9 cfu/g.
Step 2) finally carrying out solid adsorption: adsorbing the fermentation liquid with wheat bran at a weight ratio of 1:1, performing low-temperature dehumidifying drying after adsorption, and pulverizing to obtain powder with a bacterial content of 3.0X10 9 ～4.0×10 9 cfu/g.
Step 3) according to 40-60% of bacillus subtilis ferment powder by weight percentage, 40-60% of saccharomyces cerevisiae ferment powder is fully and uniformly mixed; the microbial agent is 4.0X10 9 ～6.0×10 9 Between cfu/g, the total number of viable bacteria is preferably not less than 50.0X10 8 cfu/g。
The microbial agent is applied to the synergistic emission reduction of ammonia and greenhouse gases in livestock manure compost.
Further, the microbial agent is added during livestock manure composting.
The bacillus subtilis F2 and the saccharomyces cerevisiae J8 are preserved in the China general microbiological culture Collection center (CGMCC) at the 1 st month 25 of 2021, and the preservation numbers are CGMCC No. 21117 and CGMCC No.21716 respectively.
Classification and naming of bacillus subtilis F2: bacillus subtilis Bacillus subtilis
Preservation number: CGMCC No. 21017
Preservation time: 2021, 1 and 25 days
Preservation unit: CGMCC
Address: beijing city, chaoyang area, north Chenxi Lu No. 1 and 3
Saccharomyces cerevisiae J8 classification nomenclature: saccharomyces cerevisiae Saccharomyces cerevisiae
Preservation number: CGMCC No.21716.
Preservation time: 2021, 1 and 25 days
Preservation unit: CGMCC
Address: beijing city, chaoyang area, north Chenxi Lu No. 1 and 3
The invention has the beneficial effects that:
the bacillus subtilis and the saccharomyces cerevisiae are obtained by separating from decomposed livestock manure compost by the inventor. The bacillus subtilis has high temperature resistance, can secrete enzymes such as protease, amylase, cellulase and the like, has strong deodorizing capability and good ammonia reducing effect, and has the ammonia removing rate as high as 87.5-93.5% through an ammonia reducing simulation test; the saccharomyces cerevisiae has good deodorizing capability, particularly has good effect on removing hydrogen sulfide, and the removing rate of the hydrogen sulfide reaches 74.3-85.6% through a simulation test. In addition, the invention also discovers that the saccharomyces cerevisiae has better inhibition effect on the emission of methane and nitrous oxide in the composting process for the first time.
The compound microbial agent (bacillus subtilis and saccharomyces cerevisiae) is applied to livestock manure composting fermentation, so that ammonia and greenhouse gas are cooperatively reduced in the composting process. Compared with the control group, the ammonia release amount of the treatment group added with the compound microbial agent is reduced by 57.5-66.2%, the methane release amount is reduced by 58.8-80.9%, and the nitrous oxide release amount is reduced by 56.7-64.1%. The microbial agent is added in the composting process, so that the temperature of the piled body can be obviously increased, the composting decomposition is effectively accelerated, and the composting decomposition time is shortened. Meanwhile, the total nitrogen content of the composting treatment group added with the composite microbial agent is increased by more than 27.48% compared with that of the control group, and the composting treatment group is favorable for improving the composting quality. Therefore, the composite microbial agent provided by the invention can not only remarkably reduce the emission of ammonia gas and greenhouse gas in the composting process, but also effectively shorten the composting time, improve the composting quality, meet the requirements of industrial large-scale production, and facilitate large-area popularization and use.
Drawings
FIG. 1 is a graph showing the temperature change of each treatment group with the composting time in example 5;
FIG. 2 is a graph showing the variation of ammonia release from each treatment group with the composting time in example 5;
FIG. 3 is a graph showing the methane release rate of each treatment group as a function of composting time in example 5;
FIG. 4 shows the variation of nitrous oxide release for each treatment group over the duration of composting in example 5.
Detailed Description
The following embodiments are intended to further illustrate the invention, not to limit it.
Example 1: screening of efficient dominant microorganism strains
1. Primary screening of strains
Thoroughly mixing thoroughly decomposed compost samples and enrichment culture medium (fresh pig manure) according to a certain proportion (weight ratio), adding 20% of sawdust and rice husk, regulating the water content of the materials to be about 60%, placing in a clean plastic bucket, standing at room temperature for culture, stirring for 1 time every 3 days, and determining the odor change and weight reduction rate change conditions in the culture process until the manure is basically odorless and the weight reduction is not obvious, continuing shaking culture until the concentration of the enrichment culture medium is gradually increased to 95%, and culturing each round until the weight reduction is not obvious. Taking 10g of domesticated samples with good effect, repeatedly smearing proper dilution on various separation plates, culturing at 30 ℃ for 2-3 days to obtain 32 strains, selecting 5 strains with large bacterial count and large bacterial colony as target strains, respectively named as F1, F2, F3, J7 and J8, purifying the strains and preserving on an inclined plane.
2. Deodorization bacterial strain rescreening
The dominant strains F1, F2 and F3 are inoculated into beef extract peptone liquid culture medium, J7 and J8 are inoculated into PDA liquid culture medium, after the culture is carried out to the logarithmic phase, the thalli are collected by centrifugation, and then a little sterile water is added into the thalli to prepare bacterial suspension.
Ammonia reduction experiment: taking 18mL of bacterial suspension in a 2L large beaker, and adding 2mL of 500mg/L ammonia water; then, a 50mL small beaker containing 20mL of 0.005N sulfuric acid absorbent was placed in the large beaker, and the beaker was covered with a double-layered plastic film and sealed.
Hydrogen sulfide reduction experiment: 10mL of the bacterial suspension is taken in a 2L large beaker, 10mL of 10mg/L hydrogen sulfide solution is added, then a 50mL small beaker filled with 20mL cadmium hydroxide absorption solution is put in the large beaker, and a double-layer plastic film is covered for sealing.
The above treatments were each repeated 3 times with equal amounts of sterile water as control and incubated in incubator at 30 ℃. After 24h, the small beaker was removed and the concentration of ammonia or hydrogen sulfide was measured. The determination of ammonia adopts a Nashi reagent colorimetric method, and the determination of hydrogen sulfide adopts a methylene blue colorimetric method.
Under laboratory conditions, F2 and J8 are found to have good deodorizing effect, wherein the F2 removal rate for ammonia is 87.5% -93.5% and the F2 removal rate for hydrogen sulfide is 35.2% -40.3%; the removal rate of J8 to ammonia is 62.3-68.6%, and the removal rate of J8 to hydrogen sulfide is 74.3-85.6%.
3. Identification of strains
3.1 F2 identification
Morphology identification: the fungus drops are off-white and are in irregular shapes, the edges are irregular, and the surfaces are rough; gram positive; the thallus is in a rod shape, the spores are elliptical, and the strain is neutral.
Physiological and biochemical test: gelatin liquefaction, casein hydrolysis, starch hydrolysis and positive nitrate reduction reaction; glucose, mannitol and lactose may be utilized.
16S rDNA sequence analysis: the genomic DNA of strain F2 was used as a template, and PCR amplification was performed using a bacterial 16S rDNA gene universal primer to obtain a 16S rDNA sequence of about 1.4kb in length, the 16S rDNA sequence of which is shown below.
ACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAA CCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTCTGAACCGCATGGTTCAGACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGC GGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGC AGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGCCGTTCAAA TAGGGCGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCG CAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAA TGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGA CGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACG CATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCT TACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCC CGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTAT GACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGT GAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAG
The sequence is subjected to homology comparison with reported sequences in NCBI GenBank database by BLAST, the result shows that the homology of F2 and bacillus subtilis reaches more than 99%, and the F2 is identified as bacillus subtilis (Bacillus subtilis) by combining morphological characteristics and physiological biochemical tests.
3.2 J8 identification
Morphological characteristics: the colony is white, takes the shape of spherical protrusions and has smooth surface; has a typical yeast cell morphology under a microscope.
And (3) performing ITS sequence analysis, namely performing PCR amplification by taking genomic DNA of the strain J8 as a template and ITS1 and ITS4 as primers to obtain an ITS sequence with the length of about 0.5kb, wherein the sequence is shown as follows.
GATTTGAGGTCAACTTTAAGAACATTGTTCGCCTAGACGCTCTCTTCTTATCGATAACGT TCCAATACGCTCAGTATAAAAAAGATTAGCCGCAGTTGGTAAAACCTAAAACGACCGTACTTGCATTATACCTCAAGCACGCAGAGAAACCTCTCTTTGGAAAAAAAAAACATCCA ATGAAAAGGCCAGCAATTTCAAGTTAACTCCAAAGAGTATCACTCACTACCAAACAGA ATGTTTGAGAAGGAAATGACGCTCAAACAGGCATGCCCCCTGGAATACCAAGGGGCGCAATGTGCGTTCAAAGATTCGATGATTCACGGAATTCTGCAATTCACATTACGTATCGCA TTTCGCTGCGTTCTTCATCGATGCGAGAACCAAGAGATCCGTTGTTGAAAGTTTTTAATATTTTAAAATTTCCAGTTACGAAAATTCTTGTTTTTGACAAAAATTTAATGAATAGATAA AATTGTTTGTGTTTGTTAACCTCTGGGCCCCG
Homology comparison of the sequence with reported sequences in NCBI GenBank database shows that J8 and Saccharomyces cerevisiae strain ATCC 18824 (KC 881067.1) are up to 99.5% similar, and J8 is identified as Saccharomyces cerevisiae (Saccharomyces cerevisiae) by combining morphological characteristics.
Example 2: preparation of microbial agent
1. Preparation of bacillus subtilis CGMCC No.21717 ferment
Activating inclined plane strains: the inclined plane strain is preserved in a refrigerator, inoculated in a fresh test tube inclined plane culture medium in a sterile operation, and cultivated overnight at a constant temperature of 37 ℃ to prepare the fresh test tube inclined plane strain. Slant culture medium: 1% of peptone, 0.5% of NaCl, 0.5% of yeast extract, 1.8% of agar and the balance of water, and the pH value is 7.3.
Shake flask seed culture: taking an activated fresh inclined plane strain, inoculating the activated fresh inclined plane strain into a shake flask seed liquid culture medium in a sterile operation mode, wherein the loading amount is 100mL/500mL, the rotating speed is 200rpm, and performing constant-temperature culture for 24 hours at 37 ℃, and performing water bath heat treatment for 10 minutes at 80 ℃. The culture medium of the shake flask seeds is a slant culture medium without adding agar.
Culturing in a fermentation tank: the culture medium in the fermentation tank is filled in an amount of 60% of the total volume, the inoculation amount accounts for 0.1% of the volume of the culture medium, the fermentation temperature is 37 ℃, the air is introduced at 200rpm for stirring culture, the ratio of the aeration volume to the volume of the fermentation liquid is 1:1, and the tank pressure is 0.04MPa; the fermentation end point is that the spore number is not less than 90% of the total bacterial number, and the viable bacterial number of the fermentation liquid is 2.5X10 10 cfu/mL or so; the mass percentage of each component of the fermentation tank culture medium is 0.6 percent of glucose, 0.4 percent of starch and 1.0 percent of bean cake powderManganese sulfate 0.0062%, yeast powder 0.6%, peptone 0.4%, ferric chloride 0.01%, potassium dihydrogen phosphate 0.4%, calcium carbonate 0.1%, magnesium sulfate 0.05%, and water in balance, pH 7.3;
solid adsorption: adsorbing the fermentation liquor with wheat bran according to the weight ratio of 1:2, performing low-temperature dehumidifying and drying after adsorption, and crushing, wherein the bacterial content of the powder product is 1.0x10 10 cfu/g or so;
2. preparation of Saccharomyces cerevisiae CGMCC No.21716 ferment
Firstly, activating saccharomyces cerevisiae through inclined plane strains and culturing shaking bottle seeds;
the culture medium for activating the inclined plane strain is PDA culture medium;
taking an activated fresh inclined plane strain as a loop, inoculating the activated fresh inclined plane strain into a shake flask seed liquid culture medium, culturing at a constant temperature of 30 ℃ for 40 hours at a rotating speed of 200rpm and a loading amount of 200 mL/500mL, and stopping culturing. The culture medium of the shake flask seeds is PDA culture medium without agar.
And then culturing in a fermentation tank: the culture medium in the fermenter is 60% of the total volume, the inoculation amount is 0.4% of the culture medium volume, the fermentation temperature is 30 ℃, the air is introduced at 180rpm for stirring culture, the ratio of the aeration volume to the volume of the fermentation broth is 1.1:1, the tank pressure is kept at 0.04MPa, and the bacteria content of the fermentation broth after 48 hours fermentation is 4.5X10 9 cfu/mL or so; the fermentation tank culture medium comprises the following components in percentage by mass: glucose 4.0%, ammonium sulfate 2.0%, yeast powder 0.2%, potassium dihydrogen phosphate 0.1%, sodium chloride 0.01%, magnesium sulfate 0.05%, and water in balance, with pH6.5;
and finally, solid adsorption is carried out: adsorbing the fermentation liquid with wheat bran at a weight ratio of 1:1, performing low-temperature dehumidifying drying after adsorption, and pulverizing to obtain powder with a bacterial content of 3.5X10 9 cfu/g or so;
3. microbial agent compounding
The preparation method comprises the steps of mixing 60% of bacillus subtilis ferment powder, 40% of saccharomyces cerevisiae ferment powder and the like according to weight percentage. The total number of viable bacteria of the microbial agent is 6.0x10 9 cfu/g.
Example 3: preparation of microbial agent
1. Preparation of bacillus subtilis CGMCC No.21717 ferment
Activating inclined plane strains: the inclined plane strain is preserved in a refrigerator, inoculated in a fresh test tube inclined plane culture medium in a sterile operation, and cultivated overnight at a constant temperature of 37 ℃ to prepare the fresh test tube inclined plane strain. Slant culture medium: 1% of peptone, 0.5% of NaCl, 0.5% of yeast extract, 1.5% of agar and the balance of water, and the pH value is 7.2.
Shake flask seed culture: taking an activated fresh inclined plane strain, inoculating the activated fresh inclined plane strain into a shake flask seed liquid culture medium in a sterile operation mode, wherein the loading amount is 100mL/500mL, the rotating speed is 180rpm, and performing constant-temperature culture at 35 ℃ for 22 hours, and performing water bath heat treatment at 80 ℃ for 10 minutes. The culture medium of the shake flask seeds is a slant culture medium without adding agar.
Culturing in a fermentation tank: the culture medium in the fermentation tank is 70% of the total volume, the inoculation amount accounts for 0.2% of the volume of the culture medium, the fermentation temperature is 30 ℃, the air is introduced at 180rpm for stirring culture, the ratio of the aeration volume to the volume of the fermentation liquid is 0.9:1, and the tank pressure is 0.03MPa; the fermentation end point is that the spore number is not less than 90% of the total bacterial number, and the viable bacterial number of the fermentation liquid is 2.8x10 10 cfu/mL or so; the fermentation tank culture medium comprises, by mass, 0.5% of glucose, 0.3% of starch, 0.8% of bean cake powder, 0.0062% of manganese sulfate, 0.5% of yeast powder, 0.3% of peptone, 0.01% of ferric chloride, 0.4% of potassium dihydrogen phosphate, 0.1% of calcium carbonate, 0.05% of magnesium sulfate, and the balance of water, wherein the pH value is 7.2;
solid adsorption: adsorbing the fermentation liquid with wheat bran at a weight ratio of 1:3, performing low-temperature dehumidifying drying after adsorption, and pulverizing to obtain powder with a bacterial content of 8.0X10 9 cfu/g or so;
2. preparation of Saccharomyces cerevisiae CGMCC No.21716 ferment
Firstly, activating saccharomyces cerevisiae through inclined plane strains and culturing shaking bottle seeds;
the culture medium for activating the inclined plane strain is PDA culture medium;
taking an activated fresh inclined plane strain as a loop, inoculating the activated fresh inclined plane strain into a shake flask seed liquid culture medium, and culturing at the constant temperature of 28 ℃ for 40 hours at the rotating speed of 180rpm with the loading of 200 mL/500mL, and stopping culturing. The culture medium of the shake flask seeds is PDA culture medium without agar.
And then culturing in a fermentation tank: the culture medium in the fermenter is 70% of the total volume, the inoculation amount is 0.5% of the culture medium volume, the fermentation temperature is 28 ℃, the air is introduced at 150rpm for stirring culture, the ratio of the aeration volume to the volume of the fermentation broth is 1:1, the tank pressure is kept at 0.05MPa, and the bacteria content of the fermentation broth is 5.0X10 after 48 hours of fermentation is completed 9 cfu/mL or so; the fermentation tank culture medium comprises the following components in percentage by mass: glucose 4.0%, ammonium sulfate 2.0%, yeast powder 0.25%, potassium dihydrogen phosphate 0.1%, sodium chloride 0.01%, magnesium sulfate 0.05%, and water in balance, with pH6.0;
and finally, solid adsorption is carried out: adsorbing the fermentation liquid with wheat bran at a weight ratio of 1:1, performing low-temperature dehumidifying drying after adsorption, and pulverizing to obtain powder with bacterial content of 4.0X10 9 cfu/g or so;
3. microbial agent compounding
40% of bacillus subtilis ferment powder and 60% of saccharomyces cerevisiae ferment powder are mixed uniformly. The total number of viable bacteria of the microbial agent is 40.0x10 8 cfu/g.
Example 4: preparation of microbial agent
1. Preparation of bacillus subtilis CGMCC No.21717 ferment
Activating inclined plane strains: the inclined plane strain is preserved in a refrigerator, inoculated in a fresh test tube inclined plane culture medium in a sterile operation, and cultivated overnight at a constant temperature of 37 ℃ to prepare the fresh test tube inclined plane strain. Slant culture medium: 1% of peptone, 0.5% of NaCl, 0.5% of yeast extract, 2.0% of agar and the balance of water, and the pH value is 7.4.
Shake flask seed culture: taking an activated fresh inclined plane strain, inoculating the activated fresh inclined plane strain into a shake flask seed liquid culture medium in a sterile operation mode, wherein the loading amount is 100mL/500mL, the rotating speed is 200rpm, and performing constant-temperature culture for 24 hours at 35 ℃, and performing water bath heat treatment for 10 minutes at 80 ℃. The culture medium of the shake flask seeds is a slant culture medium without adding agar.
Culturing in a fermentation tank: the culture medium in the fermenter is filled in an amount of 50% of the total volume, the inoculation amount is 0.3% of the volume of the culture medium, the fermentation temperature is 35 ℃, the air is introduced at 200rpm for stirring culture, the aeration volume and the fermentation liquid are introducedThe product ratio is 1:1, and the tank pressure is 0.05MPa; the fermentation end point is that the spore number is not less than 90% of the total bacterial number, and the viable bacterial number of the fermentation liquid is 3.0X10 10 cfu/mL or so; the fermentation tank culture medium comprises, by mass, 0.7% of glucose, 0.5% of starch, 1.2% of bean cake powder, 0.0062% of manganese sulfate, 0.8% of yeast powder, 0.5% of peptone, 0.01% of ferric chloride, 0.4% of potassium dihydrogen phosphate, 0.1% of calcium carbonate, 0.05% of magnesium sulfate, the balance of water and pH 7.4;
solid adsorption: adsorbing the fermentation liquor with wheat bran according to the weight ratio of 1:2, performing low-temperature dehumidifying and drying after adsorption, and crushing, wherein the bacterial content of the powder product is 1.2 multiplied by 10 10 cfu/g or so;
2. preparation of Saccharomyces cerevisiae CGMCC No.21716 ferment
Firstly, activating saccharomyces cerevisiae through inclined plane strains and culturing shaking bottle seeds;
the culture medium for activating the inclined plane strain is PDA culture medium;
taking an activated fresh inclined plane strain as a loop, inoculating the activated fresh inclined plane strain into a shake flask seed liquid culture medium, and culturing at the constant temperature of 30 ℃ for 36 hours at the rotating speed of 200rpm with the loading of 200 mL/500mL, and stopping culturing. The culture medium of the shake flask seeds is PDA culture medium without agar.
And then culturing in a fermentation tank: the culture medium in the fermenter is 50% of the total volume, the inoculation amount is 0.3% of the culture medium volume, the fermentation temperature is 30 ℃, the air is introduced at 180rpm for stirring culture, the ratio of the aeration volume to the volume of the fermentation broth is 1.1:1, the tank pressure is kept at 0.03MPa, and the bacteria content of the fermentation broth after 46 hours fermentation is 4.0X10 9 cfu/mL or so; the fermentation tank culture medium comprises the following components in percentage by mass: glucose 3.5%, ammonium sulfate 1.5%, yeast powder 0.2%, monopotassium phosphate 0.1%, sodium chloride 0.01%, magnesium sulfate 0.05%, and water in balance, with pH6.3;
and finally, solid adsorption is carried out: adsorbing the fermentation liquid with wheat bran at a weight ratio of 1:1, performing low-temperature dehumidifying drying after adsorption, and pulverizing to obtain powder with a bacterial content of 3.0X10 9 cfu/g or so;
3. microbial agent compounding
The weight percentage of the bacillus subtilis ferment powder 50 percent, 50 percent of saccharomyces cerevisiae ferment powder and fully and uniformly mixing. The total number of viable bacteria of the microbial agent is 5.5X10 9 cfu/g.
Example 5: application of composite microbial agent in fresh pig manure compost
1. Livestock manure composting production
The fresh pig manure, rice bran and sawdust are added with water and uniformly mixed in a composting fermentation tank, so that the C/N ratio is between 20 and 25, the water content is about 55 to 65 percent, and the pH value is natural. The fully and uniformly mixed compost raw materials are equally divided into 4 groups of 3 parallel compost, and the compost quantity of each parallel test is about 5 tons. Test period 20d, subsequent treatment 10d. The test period is 20 days before, the test is mechanically turned once every 3 days, and the post-maturation treatment period is not turned.
2. Test group
The CK group is a control group, and no microbial agent is added in the compost; test group T1, 0.3% of the Saccharomyces cerevisiae fermentum powder prepared in example 3 was added to the compost, and the total number of viable bacteria was 4.0X10 9 cfu/g or so; test group T2, 0.3% of the Bacillus subtilis ferment powder prepared in example 3 was added to the compost, and the total number of viable bacteria was 8.0X10 9 cfu/g or so; test T3 group, 0.3% of the composite microbial agent prepared in example 3 (40% of bacillus subtilis ferment powder and 60% of Saccharomyces cerevisiae ferment powder) was added into the compost, and the total number of viable bacteria was 40.0X10 8 cfu/g. The main ingredients of the compost raw materials are shown in table 1.
TABLE 1 physicochemical Properties in compost raw Material
3. Experimental results
1) Temperature variation
The composting time was continued for 30d and the temperature change for each treatment group is shown in figure 1. The temperature change trend of the test group is similar to that of the control group, and three typical composting stages of heating, high temperature and cooling are carried out. However, the holding time at 50℃or higher was different, and the holding time at high temperature was 4d, 10d, 14d, and 15d for the control group CK and the test groups T1, T2, and T3, respectively. Compared with the control group, the test group added with the microbial agent has fast temperature rise and long lasting time Wen Wei, and obviously accelerates the composting process.
2) Ammonia gas release
As shown in FIG. 2, NH is treated during the high temperature period of composting 3 The release amount is larger. The highest peak of ammonia gas production during composting of all test groups appears at day 7, and the ammonia gas release amounts of T1, T2 and T3 are 35.7ppm, 28.9ppm and 26.5ppm respectively, and the highest peak of ammonia gas release amount of the control group appears at day 10 and is 78.3ppm. When composting is completed for 25 days, the ammonia release amount of T1, T2 and T3 is 0, and the ammonia release amount of the control group CK is 5.3ppm at the end of composting. In the whole composting process, compared with a control group, the test group added with the microbial inoculum effectively reduces the volatilization of ammonia, wherein the T2 and T3 test groups have good inhibition effect on the release of ammonia, and compared with the CK group, the maximum release amount of ammonia is respectively reduced by 63.1 percent and 66.2 percent. During composting, no hydrogen sulfide release could be detected in all treatment groups.
3) Methane emission
As shown in fig. 3, methane emissions occur primarily during the high temperature period of composting. The test groups T1, T2 and T3 reached a peak methane emission at 5d composting, 1.0g.kg respectively -1 .d -1 、2.5g.kg -1 .d -1 、0.9g.kg -1 .d -1 The method comprises the steps of carrying out a first treatment on the surface of the The methane emission reaches the peak value at the time of composting 7d of the control group CK, and the methane emission is 4.7g.kg -1 .d -1 Far higher than the test group to which the microbial agent was added. In the whole composting process, compared with a control group, the test group added with the microbial inoculum effectively reduces the emission of methane, wherein the T1 and T3 test groups have the best effect of inhibiting methane, and the maximum release amount of methane is respectively reduced by 78.7 percent and 80.9 percent compared with the control group CK.
4) Nitrous oxide emissions
As shown in fig. 4, the nitrous oxide emissions from all the test groups fluctuated during the first week, rising sharply within 10 days, peaking and declining sharply near the end of composting. The nitrous oxide emission from the control CK was gradually increased 10d before pre-composting and peaked at 10d, at 3.9g.kg -1 .d -1 . The emission peak values of T1, T2 and T3 are respectively 1.6g.kg -1 .d -1 、2.0g.kg -1 .d -1 、1.4g.kg -1 .d -1 Compared with the maximum release amount of CK oxidized nitrous oxide in the control group, the maximum release amount of CK oxidized nitrous oxide in the control group is respectively reduced by 58.9%, 48.7% and 64.1%.
5) Composting maturity index change before and after composting
As can be seen from Table 2, at the end of composting, the total nitrogen content of all the test groups T1, T2 and T3 is increased by 20.70%, 22.35% and 27.48%, respectively, wherein the total nitrogen content of the T3 is highest, and the total nitrogen content of the control group is reduced by 2.85%; the organic matter contents of all test groups and control groups are reduced to different degrees, and the organic matter contents of T1, T2, T3 and CK are 63.30%, 62.75%, 65.01% and 66.08% respectively, which are far higher than the national standard of organic fertilizers; when composting is finished, the carbon-nitrogen ratio of all test groups is lower than 20, the composting standard is met, and the carbon-nitrogen ratio of the control group is 22.55, so that the composting requirement is not met; as can be seen from table 3, as composting proceeds, the GI of each treatment group gradually increases, and by the time of composting 20d, the GI of each test group is greater than 100%, and the complete composting requirement is reached, wherein the GI value of the T3 test group reaches 94.5% at 15d, and the GI of the control group at the end of composting is only 67.8%, and the composting standard is not reached. From the data, the microbial agent is added into the compost, so that the composting of the compost can be accelerated, the quality of the compost can be improved, and particularly, the T3 test group added with the composite microbial agent has the best effect, the highest composting speed and high organic matter and total nitrogen content.
According to the data, the addition of the composite microbial agent in the livestock manure composting process is beneficial to reducing the emission of ammonia gas and greenhouse gas, and can accelerate composting and decomposition and improve the quality of the compost.
TABLE 2 carbon to nitrogen variation for different treatments before and after composting
TABLE 3 variation of seed germination index
Example 6: application of composite microbial agent in fresh cow dung compost
1. Livestock manure composting production
Mixing the fresh cow dung with the ingredients such as the conditioner according to the weight ratio of 5:1, or adjusting the C/N ratio according to the physical and chemical properties of the materials to ensure that the C/N ratio is between 20 and 25.
The fresh cow dung is dehydrated and crushed for use, and the conditioner is mushroom residue, crushed straw, sawdust and the like.
The product, cow dung and conditioner are mixed with water in a composting trough, the water content is about 55%, the pH value is natural, and the inoculum size is 0.3%. The composting amount of each group is about 1 ton. Test period 25d, post-maturation treatment 10d. The test is carried out by mechanically turning every 2 days 15 days before the test period, the test is carried out by mechanically turning every 5 days between 15 days and 25 days, and the post-ripening treatment period is not turned.
2. Test group
The test CK group is a control group, and is not inoculated with compost rotten microbial agents; test group T1, inoculating commercial composting microbial agent 1 (containing 70% Bacillus subtilis and 30% Lactobacillus plantarum) with bacterial content of 5.5X10 9 cfu/g or so; test group T2, inoculated with commercial compost-decomposing microbial inoculant 2 (containing 60% Bacillus amyloliquefaciens and 40% Saccharomyces cerevisiae) at a biomass of 6.0X10 9 cfu/g or so; test T3 group is inoculated with the composite microbial agent of the invention example 4, and the microbial agent comprises the following components in percentage by weight: 50% of bacillus subtilis ferment powder, 50% of saccharomyces cerevisiae ferment powder and 5.5 multiplied by 10 of bacteria content 9 cfu/g or so; the test T4 group is inoculated with the composite microbial agent of the embodiment 2, and the microbial agent comprises the following components in percentage by weight: 60% of bacillus subtilis ferment powder, 40% of saccharomyces cerevisiae ferment powder and 6.0x10% of bacteria content 9 cfu/g.
3. Experimental results
1) Ammonia gas release
The ammonia release amounts for each treatment group are shown in table 4. The highest peak of ammonia release amount during composting of the T3 and T4 test groups inoculated with the microbial inoculum of the invention appears on day 5, and the ammonia release amounts are 28.6ppm and 26.1ppm respectively. The highest peak of ammonia gas production of the T1 and T2 test groups inoculated with the commercial microbial agents appeared on day 7, and the ammonia gas release amounts were 40.3ppm and 37.2ppm, respectively. The highest peak of the control treated ammonia appeared at day 10 with a release of 67.3ppm. Compared with a control, the treatment of the microbial agent is effective in reducing the emission of ammonia, and the maximum release amounts of ammonia in test groups T1, T2, T3 and T4 are respectively reduced by 40.1%, 44.8%, 57.5% and 61.2% compared with the maximum release amounts of ammonia in CK groups, so that the release amount of ammonia in the microbial agent is obviously lower than that in the market microbial agent.
TABLE 4 Ammonia emission (ppm) during composting
2) Methane emission
Methane emissions during composting are shown in table 5, with methane emissions occurring primarily in the early stages of composting. At composting time 5d, methane emission reaches the peak value, and the emission amounts of CK, T1, T2, T3 and T4 are respectively 3.4g.kg -1 .d -1 、3.0g.kg -1 .d -1 、3.2g.kg -1 .d -1 、1.3g.kg -1 .d -1 、1.4g.kg -1 .d -1 . From the above data, the test groups T3 and T4 to which the microbial agents of the present invention were added had significantly lower methane emissions than the control group CK, and the test groups T1 and T2 to which the commercial microbial agents were inoculated. Compared with the maximum release amount of CK methane in a control group, the maximum release amount of the CK methane in a T3 test group and a T4 test group is respectively reduced by 61.8 percent and 58.8 percent, which proves that the microbial agent has better inhibiting effect on the methane emission in the composting process.
TABLE 5 methane emissions during composting (g.kg) -1 .d -1 )
3) Nitrous oxide emissions
As shown in Table 6, the nitrous oxide emissions of all treatment groups were low in the early stage of composting, rapidly increased in the middle of composting, peaked at 15 days, and rapidly decreased at the end of composting. Compared with the CK in the control group, the microbial agent in the market has no inhibition effect on the discharge of nitrous oxide in the composting process, and the microbial agent in the invention has better inhibition effect on nitrous oxide, and compared with the CK nitrous oxide in the control group, the maximum release amount of the T3 and T4 test groups is reduced by 63.3 percent and 56.7 percent respectively.
TABLE 6 nitrous oxide emissions during composting (g.kg) -1 .d -1 )
By combining the data, the composite microbial agent can be added in the fresh cow dung composting process, so that the emission of ammonia and greenhouse gases in the composting process can be reduced cooperatively.
Sequence listing
<110> Hunan province institute of microorganisms
<120> a microbial agent for reducing emission of livestock manure compost ammonia gas and greenhouse gas in a synergistic manner, and preparation and application thereof
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1373
<212> DNA
<213> Bacillus subtilis (Bacillus subtilis)
<400> 1
acagatggga gcttgctccc tgatgttagc ggcggacggg tgagtaacac gtgggtaacc 60
tgcctgtaag actgggataa ctccgggaaa ccggggctaa taccggatgg ttgtctgaac 120
cgcatggttc agacataaaa ggtggcttcg gctaccactt acagatggac ccgcggcgca 180
ttagctagtt ggtgaggtaa cggctcacca aggcgacgat gcgtagccga cctgagaggg 240
tgatcggcca cactgggact gagacacggc ccagactcct acgggaggca gcagtaggga 300
atcttccgca atggacgaaa gtctgacgga gcaacgccgc gtgagtgatg aaggttttcg 360
gatcgtaaag ctctgttgtt agggaagaac aagtgccgtt caaatagggc ggcaccttga 420
cggtacctaa ccagaaagcc acggctaact acgtgccagc agccgcggta atacgtaggt 480
ggcaagcgtt gtccggaatt attgggcgta aagggctcgc aggcggtttc ttaagtctga 540
tgtgaaagcc cccggctcaa ccggggaggg tcattggaaa ctggggaact tgagtgcaga 600
agaggagagt ggaattccac gtgtagcggt gaaatgcgta gagatgtgga ggaacaccag 660
tggcgaaggc gactctctgg tctgtaactg acgctgagga gcgaaagcgt ggggagcgaa 720
caggattaga taccctggta gtccacgccg taaacgatga gtgctaagtg ttagggggtt 780
tccgcccctt agtgctgcag ctaacgcatt aagcactccg cctggggagt acggtcgcaa 840
gactgaaact caaaggaatt gacgggggcc cgcacaagcg gtggagcatg tggtttaatt 900
cgaagcaacg cgaagaacct taccaggtct tgacatcctc tgacaatcct agagatagga 960
cgtccccttc gggggcagag tgacaggtgg tgcatggttg tcgtcagctc gtgtcgtgag 1020
atgttgggtt aagtcccgca acgagcgcaa cccttgatct tagttgccag cattcagttg 1080
ggcactctaa ggtgactgcc ggtgacaaac cggaggaagg tggggatgac gtcaaatcat 1140
catgcccctt atgacctggg ctacacacgt gctacaatgg acagaacaaa gggcagcgaa 1200
accgcgaggt taagccaatc ccacaaatct gttctcagtt cggatcgcag tctgcaactc 1260
gactgcgtga agctggaatc gctagtaatc gcggatcagc atgccgcggt gaatacgttc 1320
ccgggccttg tacacaccgc ccgtcacacc acgagagttt gtaacacccg aag 1373
<210> 2
<211> 502
<212> DNA
<213> Saccharomyces cerevisiae (Saccharomyces cerevisiae)
<400> 2
gatttgaggt caactttaag aacattgttc gcctagacgc tctcttctta tcgataacgt 60
tccaatacgc tcagtataaa aaagattagc cgcagttggt aaaacctaaa acgaccgtac 120
ttgcattata cctcaagcac gcagagaaac ctctctttgg aaaaaaaaaa catccaatga 180
aaaggccagc aatttcaagt taactccaaa gagtatcact cactaccaaa cagaatgttt 240
gagaaggaaa tgacgctcaa acaggcatgc cccctggaat accaaggggc gcaatgtgcg 300
ttcaaagatt cgatgattca cggaattctg caattcacat tacgtatcgc atttcgctgc 360
gttcttcatc gatgcgagaa ccaagagatc cgttgttgaa agtttttaat attttaaaat 420
ttccagttac gaaaattctt gtttttgaca aaaatttaat gaatagataa aattgtttgt 480
gtttgttaac ctctgggccc cg 502
Claims (10)
1. The microbial agent for the synergistic emission reduction of ammonia and greenhouse gas in livestock manure compost is characterized by comprising bacillus subtilis (Bacillus subtilis) F2CGMCC No. 21117 and Saccharomyces cerevisiae (Saccharomyces cerevisiae) J8CGMCC No.21716.
2. The microbial agent according to claim 1, wherein the microbial agent is prepared by respectively fermenting and mixing bacillus subtilis CGMCC No. 21117 and Saccharomyces cerevisiae CGMCC No.21716.
3. The microbial agent according to claim 1, wherein the total bacterial count of bacillus subtilis CGMCC No. 217117 and Saccharomyces cerevisiae CGMCC No.21716 is 4.0X10 9 ～6.0×10 9 cfu/g。
4. A method for producing a microbial agent according to any one of claims 1 to 3, comprising the steps of:
1) Preparation of bacillus subtilis CGMCC No.21717 ferment
Firstly, bacillus subtilis is subjected to inclined plane strain activation and shake flask seed culture; culturing in a fermentation tank, and finally carrying out solid adsorption to obtain a powder product;
2) Preparation of Saccharomyces cerevisiae CGMCC No.21716 ferment
Firstly, activating saccharomyces cerevisiae through inclined plane strains and culturing shaking bottle seeds; and then culturing in a fermentation tank: finally, carrying out solid adsorption to obtain a powder product;
3) Microbial agent compounding
And (3) fully and uniformly mixing the bacillus subtilis fermented product powder product and the saccharomyces cerevisiae fermented product powder product.
5. The method for preparing a microbial agent according to claim 4, wherein,
the culture medium in the fermentation tank is filled in an amount of 50-70% of the total volume, the inoculum size is 0.1-0.3% of the volume of the culture medium, the fermentation temperature is 30-37 ℃, the air is introduced at 180-200 rpm for stirring culture, the ratio of the aeration volume to the volume of the fermentation liquid is 0.9:1-1:1, and the tank pressure is 0.03-0.05 MPa; the fermentation end point is that the spore number is not less than 90% of the total bacterial number, and the viable bacterial number of the fermentation liquid is 2.5X10 10 ～3.0×10 10 cfu/mL; the fermentation tank culture medium comprises, by mass, 0.5-0.7% of glucose, 0.3-0.5% of starch, 0.8-1.2% of bean cake powder, 0.0062% of manganese sulfate, 0.5-0.8% of yeast powder, 0.3-0.5% of peptone, 0.01% of ferric chloride, 0.4% of potassium dihydrogen phosphate, 0.1% of calcium carbonate, 0.05% of magnesium sulfate, and the balance of water, wherein the pH value is 7.2-7.4;
the culture medium in the fermentation tank in the step 2) is filled in an amount of 50 to 70 percent of the total volume, the inoculation amount accounts for 0.3 to 0.5 percent of the volume of the culture medium, the fermentation temperature is 28 to 30 ℃, the air is introduced for 150 to 180rpm for stirring culture, and the aeration volume and the volume of fermentation liquor are increasedThe ratio is 1:1-1.1:1, the tank pressure is kept at 0.03-0.05 MPa, and the bacterial count of the fermentation liquor after the fermentation is completed for 46-48 hours is 4.0x10 9 ～5.0×10 9 cfu/mL; the fermentation tank culture medium comprises the following components in percentage by mass: 3.5 to 4.0 percent of glucose, 1.5 to 2.0 percent of ammonium sulfate, 0.2 to 0.25 percent of yeast powder, 0.1 percent of monopotassium phosphate, 0.01 percent of sodium chloride, 0.05 percent of magnesium sulfate and the balance of water, and the pH value is 6.0 to 6.5.
6. The method for preparing a microbial agent according to claim 4, wherein,
step 1) finally carrying out solid adsorption: the fermentation liquid is adsorbed by wheat bran according to the weight ratio of 1:2-1:3, and the powder product is subjected to low-temperature dehumidifying and drying after adsorption and crushing, and the bacterial content of the powder product is 8.0 multiplied by 10 9 ～12.0×10 9 cfu/g.
7. The method for preparing a microbial agent according to claim 4, wherein,
step 2) finally carrying out solid adsorption: adsorbing the fermentation liquid with wheat bran at a weight ratio of 1:1, performing low-temperature dehumidifying drying after adsorption, and pulverizing to obtain powder with a bacterial content of 3.0X10 9 ～4.0×10 9 cfu/g.
8. The method for preparing a microbial agent according to claim 4, wherein,
step 3) according to 40-60% of bacillus subtilis ferment powder by weight percent and 40-60% of saccharomyces cerevisiae ferment powder by weight percent, the sum of the two weight percent is 100%, and the two weight percent are fully and uniformly mixed; the total number of viable bacteria of the microbial agent is 4.0X10 9 ～6.0×10 9 cfu/g.
9. The use of the microbial agent according to any one of claims 1-3 or prepared by the method according to any one of claims 4-8, for the synergistic emission reduction of ammonia and greenhouse gases in the composting of livestock manure.
10. The use according to claim 9, wherein the microbial agent is added during composting of livestock manure.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210417125.9A CN114933983B (en) | 2022-04-20 | 2022-04-20 | Microbial agent for reducing emission of livestock manure compost ammonia gas and greenhouse gas in cooperation, and preparation and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210417125.9A CN114933983B (en) | 2022-04-20 | 2022-04-20 | Microbial agent for reducing emission of livestock manure compost ammonia gas and greenhouse gas in cooperation, and preparation and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114933983A CN114933983A (en) | 2022-08-23 |
| CN114933983B true CN114933983B (en) | 2023-06-09 |
Family
ID=82861833
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210417125.9A Active CN114933983B (en) | 2022-04-20 | 2022-04-20 | Microbial agent for reducing emission of livestock manure compost ammonia gas and greenhouse gas in cooperation, and preparation and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114933983B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115636698B (en) * | 2022-09-30 | 2024-03-15 | 新乡医学院 | Method for performing innocent treatment on livestock manure by utilizing saccharomycetes and application of method |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106244475A (en) * | 2016-08-19 | 2016-12-21 | 北京市农林科学院 | A kind of low temperature resistant compost fermentation microbial inoculum with deodorization functions and application thereof |
| KR20190081204A (en) * | 2017-12-29 | 2019-07-09 | 서남대학교 산학협력단 | Method for producing organic fertilizer using blood of animal by-product |
-
2022
- 2022-04-20 CN CN202210417125.9A patent/CN114933983B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN114933983A (en) | 2022-08-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107586745B (en) | Livestock manure compost deodorization nitrogen-retention strain, microbial inoculum and preparation method and application thereof | |
| CN101863692B (en) | Method for preparing bioorganic fertilizer by using city life sludge | |
| CN106047762B (en) | bacillus subtilis and application thereof in kitchen waste | |
| CN103053432A (en) | Fermenting bed bioactive padding and preparation and application methods thereof | |
| CN101973795A (en) | Sludge aerobic composting method by utilizing composting compound microbial inoculum | |
| CN103232944A (en) | Microorganism bacterium agent for straw and excrement mixed composting | |
| CN102834504A (en) | Mixed strain culture for the disposal of food waste, and food waste disposal method using same | |
| CN107176891A (en) | It is a kind of to promote the biological agent and its production technology of stalk fast degradation | |
| CN113969246B (en) | Nitrogen-preserving strain, composite microbial inoculum, and preparation method and application of composite microbial inoculum | |
| CN114907161B (en) | Organic fertilizer containing biocontrol microbial inoculum and preparation method thereof | |
| CN107245462A (en) | A kind of high temperature resistant deodorizing and nitrogen protecting fermentation organic fertilizer microbial inoculum and preparation method thereof | |
| CN114933983B (en) | Microbial agent for reducing emission of livestock manure compost ammonia gas and greenhouse gas in cooperation, and preparation and application thereof | |
| Chen et al. | Strategy to strengthen rural domestic waste composting at low temperature: choice of ventilation condition | |
| CN111621446A (en) | Organic material decomposition agent and preparation method thereof | |
| CN110591974A (en) | Preparation and use method of thermophilic microbial agent for leather sludge drying | |
| CN108277187B (en) | Microbial agent for efficient biological drying of municipal sludge | |
| CN112980748B (en) | Brevibacillus brevis, application and method for producing humic acid by converting lignite | |
| CN106399157A (en) | Microbial agent with facility agricultural soil remediation function, and preparation and application of microbial agent | |
| CN115838668B (en) | Preparation method of pig manure composite fermentation microbial inoculum, fermentation microbial inoculum prepared by using same, pig manure bio-organic fertilizer and preparation method and application thereof | |
| Luo et al. | Enhancing the carbon content of coal gangue for composting through sludge amendment: A feasibility study | |
| CN105586286A (en) | Method for producing heavy metal absorbent through microbiological treatment on lignite and related compound microbial agent | |
| CN114350579B (en) | Sludge aerobic composting composite microbial inoculum and preparation method and application thereof | |
| CN116023182A (en) | Method for improving composting degree of goose manure by comprehensively utilizing environment-friendly ferment and rice straw biochar | |
| CN107937299B (en) | Thermophilic Pb mineralization bacteria and method for passivating Pb in sludge high-temperature compost by using same | |
| CN117049910B (en) | Method for producing bio-organic fertilizer by treating municipal sludge with microbial agent |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |