CN112410246B - Microbial composition for degrading livestock and poultry manure and application thereof - Google Patents
Microbial composition for degrading livestock and poultry manure and application thereof Download PDFInfo
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Abstract
The invention provides a microbial composition for degrading livestock and poultry manure and application thereof. The microorganism combination consists of Thermomyces lanuginosus NJAU-F4-11 with the collection number of CGMCC NO.18134, Bacillus NJAU-N45 with the collection number of CGMCC NO.18019, Bacillus NJAU-ND8 with the collection number of CGMCC NO.16737 and Geobacillus stearothermophilus B5 with the collection number of CGMCC NO. 14935. The secretase of the microorganisms in the process of degrading the livestock and poultry manure has complementary functions and obvious synergistic effect, so that the livestock and poultry manure is quickly and fully degraded under the high-temperature condition, the production efficiency of converting the culture manure into the organic fertilizer is improved, and the economic benefit and the ecological environmental benefit of an enterprise are improved.
Description
Description of the cases
The invention is a divisional application of a Chinese invention patent application with the application date of 2019-9-17 and the application number of 2019108790148, and the invention name of the invention is 'a compound bacterial agent for degrading livestock and poultry excrement and application thereof'.
Technical Field
The invention belongs to the field of agricultural microorganisms, and discloses a microbial composition for degrading livestock and poultry manure and application thereof.
Background
At present, the annual production amount of livestock and poultry feces in China is about 38 hundred million tons. Large-scale livestock and poultry manure is a huge organic matter and nutrient resource, but if the treatment is not good, serious negative effects can be brought to the environment and the life of residents. 2010, the first national pollution source census bulletin shows that the chemical oxygen demand discharged by livestock and poultry breeding industry reaches 1268.26 ten thousand tons, which accounts for 96 percent of the total agricultural source discharge amount; the total nitrogen and phosphorus emissions were 102.48 and 16.04 million tons, accounting for 38 and 56% of the total agricultural source emissions, respectively. At present, livestock and poultry manure treatment and resource utilization mainly have three modes, namely energy utilization, fertilizer utilization and industrial treatment, wherein the energy utilization and the fertilizer utilization are main directions. In recent years, domestic livestock and poultry breeding waste treatment and resource utilization work has achieved certain effects, but the comprehensive realization of energy regeneration and fertilizer utilization of livestock and poultry manure still faces a lot of practical difficulties. At present, the aerobic fermentation of the livestock and poultry manure to prepare the organic fertilizer is one of the main ways of recycling the livestock and poultry manure, but the components of the livestock and poultry manure are complex and contain components such as protein, fat, organic acid, cellulose, hemicellulose, inorganic salt and the like, and the degradation of the livestock and poultry manure by a single strain or simply combined composite flora is insufficient and slow at present. Therefore, the development of the microbial complex bacteria with the function of efficiently degrading the livestock and poultry breeding wastes is the focus of the current research.
The complex microorganism is a community composed of two or more microorganisms. Compared with a single microbial agent, the compound microbial agent has more advantages, can adapt to complex environments, and has stronger and more complete efficacy. The composite bacteria are used in practical production, so that the degradation of livestock and poultry breeding waste can be promoted, and the economic benefit and the ecological environmental benefit of enterprises are improved.
Disclosure of Invention
The invention aims to further improve the composting efficiency of livestock and poultry breeding waste, develop and develop a compound microbial inoculum and provide flora resources and technical support for livestock and poultry breeding waste fertilizer industry.
The purpose of the invention can be realized by the following technical scheme:
a microorganism combination is characterized by consisting of Thermomyces lanuginosus NJAU-F4-11 with the preservation number of CGMCC NO.18134, Bacillus (Bacillus sp.) NJAU-N45 with the preservation number of CGMCC NO.18019, Bacillus (Bacillus sp.) NJAU-ND8 with the preservation number of CGMCC NO.16737 and Geobacillus stearothermophilus B5 with the preservation number of CGMCC NO. 14935.
The inventor screens high-temperature strains growing at 55 ℃, designs 32 combinations of floras through the rod breaking theory, processes the floras and the single bacteria by 38 in total, determines the lignin, cellulose, starch and protein degrading capability of the single bacteria or the combined bacteria, determines the mutual relation among the screened strains, and compounds the strains which have no antagonistic action and synergistically degrade the macromolecular organic matter capability to obtain excellent compound floras, namely the compound floras containing thermomyces lanuginosus NJAU-F-4-11, bacillus NJAU-N45, bacillus NJAU-ND8 and bacillus B5.
The microbial composition disclosed by the invention is applied to preparation of the livestock and poultry excrement degradation composite microbial inoculum.
A composite bacterial preparation for degrading the dung of livestock and fowls is prepared from the dung of livestock and fowls, and the composite bacterial preparation whose concentration is not less than 109Each/ml of said cotton-like substanceThe bacterial liquid of the thermomyces thermophilus NJAU-F4-11 and the concentration are not less than 109CFU/ml of the bacillus NJAU-N45, the bacillus NJAU-ND8 and the bacillus B5 in equal volume ratio; the concentration of each bacterial liquid is the same.
The microbial compound inoculant is characterized in that:
(1) the effective viable count of the strain is high, and the liquid inoculum is adopted, and the concentration is 109More than one fungus per ml or CFU per ml (bacteria);
(2) the material is high temperature resistant and can grow at a high temperature of 55 ℃;
(3) salt tolerance, capable of production in media containing 15% salt;
(4) the 3 strains of bacteria are identified as bacillus bacteria, the fungi are identified as hyphomycete fungi, are harmless to crops and are not pathogenic to human and animals;
(5) the activity of exo-beta-1, 4-glucanase reaches 13.662U, the activity of endo-beta-1, 4-glucanase reaches 18.408U, the activity of beta-glucosidase reaches 42.395U, the activity of neutral xylanase reaches 635.283U, the activity of filter paper enzyme reaches 0.665U, the activity of neutral protease reaches 672.070U, the activity of alpha-amylase reaches 0.135U, the activity of beta-amylase reaches 0.222U, and the activity of urease reaches 30.146U.
The preparation method of the livestock and poultry manure degradation complex microbial inoculum comprises the following steps: inoculating NJAU-F-4-11 fungus into PDA liquid culture medium, and culturing at 55 deg.C and 170rpm for 2 days; respectively inoculating strains of bacillus NJAU-N45, bacillus NJAU-ND8 and B5 into LB culture medium, and culturing at 55 ℃ and 170rpm for 2 days; after the culture, the cells were collected by centrifugation at 4 ℃ and washed, and the concentration of the cells was adjusted to 10 with distilled water9CFU/ml or above, and fungus concentration of 109More than one bacterium per ml, and the concentration of each bacterium liquid is the same; and then uniformly mixing according to the volume ratio of 1:1:1:1 to obtain the livestock and poultry manure degradation composite microbial inoculum.
Wherein, the bacteria count the concentration through OD value, the fungus counts the spore number with the blood counting chamber.
Weighing a certain amount of livestock and poultry manure, placing the livestock and poultry manure into a 250ml triangular flask, sucking each bacterial liquid with the same total amount, adding the bacterial liquids into the triangular flask, uniformly mixing, placing the mixture into an incubator at 55 ℃ for culture, drying a sample after 15 days, weighing, taking the livestock and poultry manure without inoculation as a reference, and calculating the degradation rate of the livestock and poultry manure by a weight loss method.
The invention relates to an application of a composite bacterial agent for degrading livestock and poultry feces.
The invention relates to an application of a composite microbial inoculum for degrading livestock and poultry manure in preparation of an organic fertilizer by taking the livestock and poultry manure and straws as raw materials.
The method for producing the organic fertilizer by adopting the livestock and poultry manure degradation composite microbial inoculum comprises the following steps:
(1) mixing raw materials: cutting corn straws into pieces, mixing the cut corn straws with fresh sheep manure according to the carbon-nitrogen ratio of 25: 1, uniformly mixing, adjusting the initial water content to 65-70%, inoculating the livestock and poultry manure degradation complex microbial inoculum, wherein the inoculation amount is 7-10 ml/kg, uniformly mixing the pile materials after inoculation, and then building the pile materials into a strip pile shape, wherein the length and width of the pile base material are 1-1.2 meters, the height is 1.5-1.8 meters, and the length is not limited;
(2) composting and fermenting: after organic fertilizer fermentation base materials are piled in a fermentation shed in a strip pile type, manual pile turning fermentation is adopted, pile turning is started when the core temperature reaches above 70 ℃, pile turning is carried out for 1 time in 2 days, and fermentation is carried out for 20-22 days;
(3) after-ripening fermentation: and after compost fermentation is finished, after-ripening and stacking for 6-7 days, obtaining the organic fertilizer.
Advantageous effects
The invention provides a microbial compound microbial inoculum which is used for efficiently converting livestock and poultry breeding wastes into organic fertilizers. The microbial composite bacterial agent contains 4 kinds of single bacteria capable of degrading animal excrement effectively, and consists of 1 kind of fungi and 3 kinds of bacteria. The secretase of the microorganisms in the process of degrading the livestock and poultry manure has complementary functions and obvious synergistic effect, so that the livestock and poultry manure is quickly and fully degraded under the high-temperature condition, the production efficiency of converting the culture manure into the organic fertilizer is improved, and the economic benefit and the ecological environmental benefit of an enterprise are improved.
The composite bacterial agent is used for degrading the chicken manure, the degradation rate of the chicken manure is over 27 percent after half a month, the highest degradation rate of the combination of two bacteria is only 18 percent, and the highest degradation rate of a single bacterium is only 11.77 percent, so that the degradation effect of the composite bacteria reaches 2 times of that of the optimal single bacterium.
The composite microbial inoculum is used for degrading the pig manure, the degradation rate of the pig manure reaches 27.85% after half a month, the highest degradation rate of the combination of the two bacteria is only 23.4%, and the highest degradation rate of the single bacteria is only 20%, so that the degradation effect of the composite microbial inoculum on the pig manure is better than that of the combination of the two bacteria and the single bacteria.
The composite microbial inoculum is used for degrading sheep manure, the degradation rate of the sheep manure reaches 43.03% after half a month, the highest degradation rate of the combination of two bacteria is only 34.12%, and the highest degradation rate of single bacteria is only 27.66%, so that the degradation effect of the composite microbial inoculum on the sheep manure is better than that of the combination of two bacteria and single bacteria.
The screened bacterial strains and the composite flora are applied to industrial sheep manure decomposed compost of Jiangsu salt city Qianbao animal husbandry Limited company. The sheep manure and the combined bacteria are treated and stacked for 26 days, and the decomposition degree and the decomposition speed are obviously superior to those of single bacteria treatment and non-addition treatment.
Drawings
FIG. 1 shows the effect of different combinations of bacterial populations on the degradation of chicken manure;
FIG. 2 is a graph of the effect of different flora combinations on pig manure degradation;
FIG. 3 is the effect of different flora combinations on sheep manure degradation;
FIG. 4 shows the change of the remaining physicochemical properties of different treatments with the mixture of straw and sheep manure as the compost raw material;
biological material preservation information
NJAU-F4-11, classified name Thermomyces lanuginosus, and is preserved in China general microbiological culture Collection center, the preservation address is the microbial institute of China academy of sciences No. 3, North West Lu No.1 institute of North West Lu, Chao Yang district, Beijing, the preservation date is 2019, 6 months and 24 days, and the preservation number is CGMCC NO. 18134.
NJAU-N45, Bacillus sp.
NJAU-ND8, Bacillus sp, classified name, and is preserved in China general microbiological culture Collection center, the preservation address is the microbiological research institute of China academy of sciences, No. 3, Xilu No.1, North Chen, south China area, Beijing, the preservation date is 2018, 11 and 12 days, and the preservation number is CGMCC NO. 16737.
B5, classified name of Geobacillus stearothermophilus, deposited in China general microbiological culture Collection center, with the preservation address of China academy of sciences microorganism institute 3, Xilu No.1, North Chen, of the Chaoyang district, Beijing, the preservation date of 2017, 11 months and 20 days, and the preservation number of CGMCC NO. 14935.
Detailed Description
8 strains of bacteria and fungi for efficiently degrading livestock and poultry excrement, which are separated and screened by Nanjing agriculture university resource and environmental science college, are selected, and comprise 4 strains of bacteria and 4 strains of fungi. The 4 kinds of fungi are NJAU-F3-2/NJAU-F4-11/NJAU-F2-13/NJAU-F3-5, and are further numbered as A, B, C and D respectively; the 4 kinds of bacteria are NJAU-N20/B5/NJAU-ND8/NJAU-ND45, and are further numbered as 1,2,3 and 4 respectively.
Inoculating fungi into PDA liquid culture medium, and culturing at 55 deg.C and 170rpm for 2 days; the bacteria were inoculated into LB medium and cultured at 55 ℃ and 170rpm for 2 days. After each strain grows well, centrifugally collecting the strain at 4 ℃, washing the strain, adjusting the concentration by using distilled water, adjusting the OD value of the strain by using sterile water, and counting the number of spores by using a blood counting chamber for fungus to ensure that the concentration of each strain liquid is 109The strain/ml (fungus) or CFU/ml (bacterium) is characterized in that the combination of 1 bacterium, 2 bacteria, 4 bacteria and 8 bacteria is set according to the rod breakage theory and random block design, and the total number of the combined bacteria is consistent with the total number of spores or colonies contained in the single bacteria treatment.
The single bacterium is combined with A, B, C, D,1,2,3 and 4; the two bacteria are combined with AB, A3, A4, AD, B1, B2, BC, C2, C3, CD, D1, D4,12,14,23 and 34; the combination of four bacteria comprises ABD1, AD12, ABCD, A123, AC23, ACD3, BD24, B134, B234, BC14, CD24 and C134; the combination of 8 bacteria was ABCD 1234.
EXAMPLE 1 ability of Single and Complex bacteria to produce lignocellulose degrading enzymes, starch and protein hydrolyzing enzymes
Both individual and combination strains were tested for enzyme activity associated with the degradation of lignin, cellulose, starch and proteins using enzyme activity kits (Suzhou Keming Biotechnology Co., Ltd.).
(1) Beta-glucosidase (beta-GC) activity assay
The beta-glucosidase decomposes p-nitrobenzene-beta-D-glucopyranoside to generate p-nitrobenzene, the p-nitrobenzene has a maximum absorption peak at 400nm, and the activity of the beta-glucosidase is calculated by measuring the rising rate of the light absorption value.
beta-GC (nmol/min/ml) (. DELTA.A +0.0027) ÷ 0.00543 XV anti-total ÷ V-like ÷ T ═ 61.39X (. DELTA.A +0.0027)
(2) Activity determination of exo-beta-1, 4-glucanase (C1)
The content of reducing sugar generated by degrading the microcrystalline cellulose catalyzed by exo-beta-1, 4-glucanase is measured by a3, 5-dinitrosalicylic acid method.
C1(ug/min/ml) 1000 × (. DELTA.A +0.0673) ÷ 6.4078 XV anti-total ÷ V-like ÷ T ═ 14.305 × (. DELTA.A +0.0673)
(3) Determination of endo-beta-1, 4-glucanase (Cx) Activity
The content of reducing sugar generated by degrading the endo-beta-1, 4-glucanase catalyzed by the carboxymethyl cellulose sodium is measured by a3, 5-dinitrosalicylic acid method.
Cx (ug/min/ml) 1000 × (. DELTA.A +0.0673) ÷ 6.4078 × V anti-total ÷ V-like ÷ T14.305 × (. DELTA.A +0.0673)
(4) Filter paper enzyme (FPA) activity assay
Reducing sugar generated by filter paper enzyme hydrolysis filter paper and 3, 5-dinitrosalicylic acid can generate a reddish brown amino compound, the maximum light absorption is realized at 540nm, the color depth of reaction liquid is in direct proportion to the amount of the reducing sugar in a certain range, and the activity of the filter paper enzyme can be measured and calculated.
FPA (U/ml) (. DELTA.A +0.0255) ÷ 0.2805 XV anti-total/V-like/T ═ 0.416X (. DELTA.A +0.0255)
(5) Determination of Activity of neutral xylanase (NEX)
NEX catalyzes xylan to be degraded into reducing oligosaccharide and monosaccharide in a neutral environment, the degrading oligosaccharide and monosaccharide further react with 3, 5-dinitrosalicylic acid under the condition of boiling water bath to generate color development, a characteristic absorption peak is formed at 540nm, the color depth of reaction liquid is in direct proportion to the amount of reducing sugar generated by enzymolysis, and the activity of NEX can be calculated by measuring the rate of increase of the light absorption value of the reaction liquid at 540 nm.
NEX(nmol/min/ml)＝(△A－0.00058)÷1.6904÷150÷T×106＝657×(△A－0.00058)
(6) Activity assay for alpha-amylase (alpha-AL) and beta-amylase (beta-AL)
Reducing sugar reduces 3, 5-dinitrosalicylic acid to generate a brownish red substance. Alpha-amylase is not acid tolerant and beta-amylase is not heat tolerant. According to the above characteristics, the activity of another amylase can be determined by inactivating one of them.
Alpha-amylase (mg/min/ml) (. DELTA.A +0.1778) ÷ 3.7215 XV anti-total ÷ V-like ÷ T ═ 0.1075 × (. DELTA.A +0.1778)
Total amylase (mg/min/ml) × 5 × (. DELTA.A +0.1778) ÷ 3.7215 × V anti-total ÷ V-like ÷ T ═ 0.5375 × (. DELTA.A)0+0.1778)
Beta-amylase (mg/min/ml) ═ Total amylase Activity-alpha-amylase Activity
(7) Determination of Neutral Protease (NP) Activity
Under neutral conditions, NP catalyzes casein to hydrolyze tyrosine; under alkaline conditions, tyrosine reduces phosphomolybdic acid compounds to generate tungsten blue, and a characteristic absorption peak is shown at 680 nm.
NP (nmol/min/ml) ═ C standard × Δ A ÷ Δ A standard × V inverse total ÷ V1 ÷ T ═ 125 × Δ A ÷ Δ A standard
(8) Urease (UE) viability assay
Method for measuring NH generated by urease hydrolysis of urea by indophenol blue colorimetry3-N。
UE (ug/min/ml) (. DELTA.A-0.0373) ÷ 0.0915 × 10 × V anti-total ÷ V-like ÷ T ═ Δ A-0.0373) × 27.32
Note: v, reverse total: the total volume of the reaction system; and V sample: adding the volume of the sample into the reaction system; v1 is added into the volume of the crude enzyme liquid in the reaction system; t: reaction time; a: measuring the difference of the absorbance values of the group and the control group; delta A0: the difference of the light absorption values of the total amylase determination group and the control group; c standardProduct (2) preparation: 0.25 mu mol/ml standard tyrosine solution
Results and analysis
The enzyme activities of different thermophilic bacterial strains, single bacteria and combined bacteria at 55 ℃ are shown in table 1. In all strain combinations, the higher the diversity of the combined strain, the stronger the degradation capability of lignin, cellulose, starch and protein, and the stronger the activity of the single bacterium enzyme of the composition. In all combined bacteria treatment, the enzyme activity of the B +2+3+4(NJAU-F4-11+ B5+ NJAU-ND8+ NJAU-N45) combination is generally higher than that of any two bacteria, other four bacteria and eight bacteria, and is higher than that of all single bacteria combined into compound bacteria. The results show that the composite bacteria containing NJAU-F4-11+ B5+ NJAU-ND8+ NJAU-N45 has the optimal effect on the hydrolysis capacity of starch, neutral protein, lignin, cellulose and urea in all strain combinations.
TABLE 1 enzyme activities of different thermophilic strains of single and combined bacteria at 55 deg.C
Note that β -GC represents β -glucosidase, C1 represents exo- β -1,4 glucanase, Cx represents endo- β -1,4 glucanase, FPA represents filter paper enzyme, NEX represents neutral xylanase, α -AL represents α -amylase, β -AL represents β -amylase, NP represents neutral protease, and UE represents urease.
Example 2 degradation Effect of Single and Complex bacteria on Chicken manure
Weighing a certain amount of chicken manure, placing the chicken manure into a 250ml triangular flask, sucking each bacterial liquid with the same total amount, adding the bacterial liquids into the triangular flask, uniformly mixing, placing the mixture into a 55-degree incubator for culture, drying a sample after 15 days, weighing, and calculating the degradation rate of the chicken manure by a weight loss method by taking the chicken manure without inoculating strains as a reference.
Results and analysis
The effect of different flora combinations on the degradation of chicken manure is shown in figure 1. Among all the single-bacterium treatments, the degradation rate of the No. 2 bacterium (B5) to the chicken manure is the highest and is 17.66%. Among the combinations of the two bacteria, the combination of bacteria No. 2 and bacteria No. 3 (B5+ NJAU-ND8) showed the highest degradation rate of 24.12%. In the combination of the 4 bacteria, the degradation rate of the combination of the fungus B, the bacteria No. 2, the bacteria No. 3 and the bacteria No. 4(NJAU-F4-11+ NJAU-ND8+ NJAU-N45+ B5) is 37.05 percent at most. The optimal combination of 4 bacteria has a degradation rate on the chicken manure which is obviously higher than that of the chicken manure treated by two bacteria and single bacteria. In addition, the combined degradation rate of 8 bacteria was 35.32%, and the blank group was degraded by 2.11%.
According to the indoor chicken manure degradation result, the compound bacteria containing NJAU-F4-11+ NJAU-ND8+ NJAU-N45+ B5 has the optimal chicken manure degradation effect.
Example 3 degradation Effect of Single and Complex bacteria on pig manure
The chicken manure was changed to pig manure, and the remaining operation steps were the same as in example 2.
Results and analysis
The effect of different flora combinations on the degradation of pig manure is shown in figure 2. Among all the single-bacterium treatments, the degradation rate of the No. 2 bacterium (B5) to the pig manure is the highest and is 11.37%. In the combination of the two bacteria, the degradation rate of the combination of the fungus B and the bacteria No. 2 (NJAU-F4-11+ B5) is the highest and is 23.80 percent. In the combination of the 4 bacteria, the degradation rate of the combination of the fungus B, the bacteria No. 2, the bacteria No. 3 and the bacteria No. 4(NJAU-F4-11+ NJAU-ND8+ NJAU-N45+ B5) is 34.11 percent at most. The optimal combination of 4 bacteria has a degradation rate on the chicken manure which is obviously higher than that of the chicken manure treated by two bacteria and single bacteria. In addition, the combined degradation rate of 8 bacteria was 30.28%, and the blank group was degraded by 3.57%.
According to the indoor pig manure degradation result, the degradation effect of the composite flora containing NJAU-F4-11+ NJAU-ND8+ NJAU-N45+ B5 on the pig manure is optimal.
Example 4 degradation effect of Single and Complex bacteria on sheep manure
The chicken manure was changed to sheep manure, and the remaining operation steps were the same as in case 2.
EXAMPLES results and analysis
The effect of different flora combinations on the degradation of sheep manure is shown in figure 3. Of all the single-bacterium treatments, the degradation rate of the No. 2 bacterium (B5) to the sheep manure is the highest and is 27.66%. Among the combinations of the two bacteria, the combination of bacteria No. 2 and bacteria No. 3 (B5+ NJAU-ND8) showed the highest degradation rate of 34.12%. In the combination of the 4 bacteria, the degradation rate of the combination of the fungus B, the bacteria No. 2, the bacteria No. 3 and the bacteria No. 4(NJAU-F4-11+ NJAU-ND8+ NJAU-N45+ B5) is 43.03 percent at most. The optimal combination of 4 bacteria has a degradation rate on the chicken manure which is obviously higher than that of the chicken manure treated by two bacteria and single bacteria. In addition, the combined degradation rate of 8 bacteria was 41.32%, and the blank group was degraded by 7.09%.
According to the indoor sheep manure degradation result, the sheep manure degradation effect is optimal by the composite flora containing NJAU-F4-11+ NJAU-ND8+ NJAU-N45+ B5.
Example 5 application of functional bacteria in straw-sheep manure mixed raw material compost
The screened optimal single bacterium B5 and NJAU-F4-11+ NJAU-ND8+ NJAU-N45+ B5 composite bacteria agent are inoculated to a mixed raw material of pure straw and sheep manure for composting in a fertilizer factory of Qianbao animal husbandry of Yangcheng of Jiangsu, and 3 treatments are designed in the test: the combined bacteria of the sheep manure and the straws, the single bacteria of the sheep manure and the straws and the straw are respectively marked as Y3, Y2 and Y1.
Test procedure
1. Preparing raw materials: cutting up corn straws in an outdoor storage yard, and mixing the corn straws with fresh sheep manure by using a forklift according to the C/N value of a stack body of 25: 1, and after mixing, the initial water content of the stockpile is about 68 percent.
2. Preparing materials: adding high-efficiency single bacteria and combined flora into compost according to the proportion of 1%, wherein the water content of the compost is about 68% and the difference between the water content and the water content is not large;
3. material distribution: transferring the mixture to a fermentation bin by using a forklift for bar type fermentation, collecting two samples after distributing, naturally drying one sample, and storing the other fresh sample in a refrigerator at the temperature of-80 ℃; (sampling method: adopting five-point sampling method, collecting equal amount of samples from four directions and middle position of compost, and mixing uniformly)
4. Turning: turning the pile once every 2 days, and collecting two samples after every 2 times of turning is finished;
5. recording: recording the temperature of the pile and the room temperature every morning and evening, wherein the temperature is recorded as 11:00 in the morning and 17:00 in the afternoon;
6. and (3) finishing composting: after 20 days of composting, collecting a sample for the last time, stopping turning the compost after 6 times of collecting the sample, and enabling the compost to enter an after-ripening fermentation stage;
7. after-ripening treatment: after the post-ripening and stacking for 6 days, the fermentation is finished after the 7 th sample collection.
8. And (3) detection: detecting compost samples according to NY525-2012 standards, and detecting indexes such as water content, pH and the like of fresh samples; the air-dried sample is used for detecting indexes such as organic matters and N, P, K;
results and analysis
The variation of nutrient content for different treatments with straw and sheep manure mixture as compost raw material is shown in table 2 and figure 4. Compared with the treatment without adding bacteria and with adding single bacteria, the temperature rise is fastest in the composting process by adding the composite bacteria heap Y3, and the highest temperature in the high-temperature period can reach 75.5 ℃; the water content of the added composite bacteria compost Y3 is reduced fastest in the composting process, the water content is 33.08% at the end of composting, and compared with the water content which is not subjected to bacteria addition treatment, the water content is reduced by 7.99% at the end of composting; the pH change trends of the three heaps are approximately the same, when composting is finished, the pH of the heaps treated by adding no bacteria and single bacteria is greater than 8.5, the pH of the heaps treated by adding composite bacteria Y3 is 8.41 and less than 8.5, and the three heaps meet the national standard NY 525-2012; the germination index of the Y3 compost is 75.8% at the end of composting, is greater than that of single-bacterium and non-bacterium treatment, the rotten effect is best, the content of organic matters is 45.17% at the end of composting, is greater than 45%, and meets the national standard NY 525-2012; compared with the treatment without adding bacteria and the treatment with adding single bacteria, the indexes of nitrogen, phosphorus and potassium in the fertilizer after the composting is finished by adding the composite bacteria heap Y3 are the highest, namely 1.64%, 1.54% and 3.24%, respectively, and the sum of the nitrogen, the phosphorus and the potassium is 6.42%, compared with the treatment without adding bacteria, the nitrogen, the potassium and the phosphorus are increased by 0.35% and are more than 5%, and the national standard NY525-2012 is met.
According to the embodiment 1, the embodiment 2, the embodiment 3, the embodiment 4 and the embodiment 5, the microbial inoculum containing the compound flora (NJAU-F4-11+ NJAU-ND8+ NJAU-N45+ B5) has a good decomposition effect on livestock and poultry manure, can promote the degradation of livestock and poultry wastes, and improves the economic benefit and the ecological environmental benefit of enterprises.
TABLE 2 variation of nutrient content for different treatments with straw and sheep manure mixture as compost raw material
Claims (6)
1. A microorganism combination is characterized by consisting of Thermomyces lanuginosus NJAU-F4-11 with the collection number of CGMCC NO.18134, Bacillus (Bacillus sp.) NJAU-N45 with the collection number of CGMCC NO.18019, Bacillus (Bacillus sp.) NJAU-ND8 with the collection number of CGMCC NO.16737 and Geobacillus stearothermophilus B5 with the collection number of CGMCC NO. 14935.
2. The use of the microbial combination of claim 1 in the preparation of a complex microbial agent for degrading livestock and poultry manure.
3. Use of the combination of microorganisms according to claim 1 for the degradation of animal manure.
4. The use of the combination of microorganisms according to claim 1 for accelerating the composting fermentation of livestock and poultry manure.
5. The use of the microbial composition of claim 1 in the preparation of organic fertilizer from livestock and poultry manure and straw.
6. A method for producing organic fertilizer by using the microorganism combination as claimed in claim 1, which is characterized by comprising the following steps:
(1) preparing a complex microbial inoculum using the combination of microorganisms of claim 1: the compound microbial inoculum is prepared from the compound microbial inoculum with the concentration of not less than 109The bacterial liquid of Thermomyces lanuginosus NJAU-F4-11 with the preservation number of CGMCC NO.18134 and the concentration of the bacterial liquid is not less than 109CFU/ml, and is formed by mixing the bacteria liquids of Bacillus (Bacillus sp.) NJAU-N45 with the collection number of CGMCC NO.18019, Bacillus (Bacillus sp.) NJAU-ND8 with the collection number of CGMCC NO.16737 and Geobacillus stearothermophilus B5 with the collection number of CGMCC NO.14935 in the same volume ratio; wherein the concentration of each bacterial liquidThe same;
(2) mixing raw materials: cutting corn straws into pieces and mixing with fresh sheep manure according to the C/N value of a pile body of 25: 1, uniformly mixing, adjusting the initial water content to 65-70%, inoculating the composite microbial inoculum in the step (1), wherein the inoculation amount is 7-10 ml/kg, uniformly mixing the pile materials after inoculation, and then building the pile materials into a strip pile shape, wherein the length and width of the pile base material are 1-1.2 meters, the height is 1.5-1.8 meters, and the length is not limited;
(3) composting and fermenting: after organic fertilizer fermentation base materials are piled in a fermentation shed in a strip pile type, manual pile turning fermentation is adopted, pile turning is started when the core temperature reaches above 70 ℃, pile turning is carried out for 1 time in 2 days, and fermentation is carried out for 20-22 days;
(4) after-ripening fermentation: after compost fermentation is finished, after-ripening and stacking for 6-7 days to obtain an organic fertilizer;
the compound microbial inoculum is mainly prepared by the following method: inoculating NJAU-F-4-11 fungus into PDA liquid culture medium, and culturing at 55 deg.C and 170rpm for 2 days; respectively inoculating strains of bacillus NJAU-N45, bacillus NJAU-ND8 and B5 into LB culture medium, and culturing at 55 ℃ and 170rpm for 2 days; after the culture, the cells were collected by centrifugation at 4 ℃ and washed, and the concentration of the cells was adjusted to 10 with distilled water9CFU/ml or above, and fungus concentration of 109More than one bacterium per ml, and the concentration of each bacterium liquid is the same; and then uniformly mixing according to the volume ratio of 1:1:1:1 to obtain the composite microbial inoculum.
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