CN115959931A - Method for preparing organic fertilizer by deep fermentation of livestock and poultry manure and straws - Google Patents
Method for preparing organic fertilizer by deep fermentation of livestock and poultry manure and straws Download PDFInfo
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W30/00—Technologies for solid waste management
- Y02W30/40—Bio-organic fraction processing; Production of fertilisers from the organic fraction of waste or refuse
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a method for preparing an organic fertilizer by deeply fermenting livestock and poultry manure and straws, which comprises the following steps: mixing the livestock and poultry manure with straws according to the weight ratio of 1:3, uniformly mixing, and adjusting the carbon-nitrogen ratio to be 25:1, simultaneously inoculating a microbial agent, wherein the inoculation amount is 3% by weight, and the water content is adjusted to 60%; obtaining a pre-fermented material; adding the pre-fermented material into an intelligent closed high-temperature aerobic fermentation tank, and stirring and fermenting for 5-16 days at the fermentation temperature of 60-70 ℃ under the condition of providing oxygen by a blower after the fermentation is started; and granulating the fermented finished product to obtain the organic fertilizer. The invention adopts the intelligent closed high-temperature aerobic fermentation tank to carry out closed high-temperature fermentation, and the fermentation mode is in-tank fermentation without pollution; and the adopted bacterial strain of the microbial agent is derived from high-temperature dominant flora in the natural fermentation process of straw compost, and can effectively promote the fermentation production process of the organic fertilizer taking straws and livestock and poultry manure as main raw materials through long-term screening and domestication, thereby improving the production efficiency and the fertilizer efficiency of the organic fertilizer.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to a method for preparing an organic fertilizer by deeply fermenting livestock and poultry manure and straws.
Background
The organic fertilizer contains rich organic nutrients such as humic acid, amino acid and the like, and also contains inorganic nutrients such as nitrogen, phosphorus, potassium and the like, and plays an important role in the agricultural development process of China. The organic fertilizer generally refers to a fertilizer prepared by microbial fermentation and complete decomposition of organic solid wastes, such as livestock and poultry manure, straws, cakes, agricultural and sideline products and solid wastes generated by food processing. In actual operation, most of the organic fertilizers are prepared by taking livestock and poultry manure and straws as main materials. Because the breeding industry in China is huge in scale, the production amount of livestock and poultry manure is also very incredible every year in China; the yield of crop straws in China is high, and the traditional crop straw treatment mode mainly comprises incineration, which causes serious air pollution. At present, organic fertilizers are prepared from materials such as livestock and poultry manure and straws, and become an effective way for recycling the livestock and poultry manure and the straws.
Straw is a general name of stem leaf (ear) parts of mature crops, the straw is rich in nitrogen, phosphorus, potassium, calcium, magnesium, organic matters and the like, and is a multipurpose renewable biological resource, livestock manure mainly refers to a kind of rural solid waste generated in livestock and poultry breeding industry, and comprises pig manure, cow manure, sheep manure, chicken manure, duck manure and the like.
Disclosure of Invention
An object of the present invention is to solve at least the above problems and/or disadvantages and to provide at least the advantages described hereinafter.
To achieve these objects and other advantages in accordance with the present invention, there is provided a method for preparing an organic fertilizer by deep fermentation of livestock and poultry manure and straw, comprising the steps of:
step one, mixing the livestock and poultry manure and the straws in a weight ratio of 1:3, uniformly mixing, and adjusting the carbon-nitrogen ratio to be 25:1, simultaneously inoculating a microbial agent, wherein the inoculation amount is 3% by weight, and the water content is adjusted to 60%; obtaining a pre-fermented material;
secondly, adding the pre-fermented material into an intelligent closed high-temperature aerobic fermentation tank, and stirring and fermenting for 5-16 days at the fermentation temperature of 60-70 ℃ under the condition of providing oxygen by a blower after fermentation starts; and granulating the fermented finished product to obtain the organic fertilizer.
Preferably, the livestock and poultry manure is one or more of pig manure, cattle manure, chicken manure and duck manure.
Preferably, the bacteria in the microbial agent are one or more of bacillus lentus, bacillus and bacillus brevis.
Preferably, the classification name of the Lysinibacillus elongatus is Lysinibacillus elongatus (Lysinibacillus macroides SWUST-1), which has been registered and preserved in China general microbiological culture Collection center on 12.05.2022, with the preservation number of CGMCC No. M24857, the preservation address: beijing, china; the classification name of the Bacillus is Bacillus (Bacillus sp.SWUST-3), which has been registered and preserved in China general microbiological culture Collection center (CGMCC) at 12.05.2022, with the preservation number of CGMCC No. M24858 and the preservation address: beijing, china; the classification name of the Brevibacillus brevis is Brevibacillus brevis (Brevibacillus sp.SWUST-2), which is registered and preserved in China general microbiological culture Collection center (CGMCC) at 12/05 in 2022, the preservation number is CGMCC No. M24859, and the preservation address is as follows: beijing in China.
Preferably, the DNA gene sequence of the lysine bacillus elongatus (lysine bacillus macrocides SWUST-1) is shown in SEQ ID NO. 1.
Preferably, the DNA gene sequence of the Bacillus (Bacillus sp.SWUST-3) is shown as SEQ ID NO. 2.
Preferably, the DNA gene sequence of the Bacillus brevis (Brevibacillus sp. SWUST-2) is shown as SEQ ID NO. 3.
Preferably, the lysine bacillus elongatus adopts a secondary seed solution of lysine bacillus elongatus, and the preparation method comprises the following steps: picking two rings of elongated lysine bacillus (lysine microorganisms SWUST-1) slant seeds by using an inoculating loop, and culturing the two rings of elongated lysine bacillus slant seeds in a 250mL triangular flask filled with 50mL nutrient broth culture medium at 37 ℃ at 150r/min for 18h to prepare a first-level seed solution of the elongated lysine bacillus; inoculating the first-stage seed liquid of the lysine bacillus into 250mL of optimized culture medium with the inoculation amount of 3% (v/v), culturing at 37 ℃ at 150r/min for 30h in a shaking way to prepare a second-stage seed liquid of the elongated lysine bacillus;
the bacillus adopts bacillus secondary seed liquid, and the preparation method comprises the following steps: inoculating a loop to pick slant seeds of Bacillus bicolor (Bacillus sp.SWUST-3) in a 250mL triangular flask filled with 50mL nutrient broth culture medium, and culturing at 37 ℃ at 150r/min for 18h to obtain a first-level seed solution of Bacillus; inoculating the first-stage seed solution of Bacillus in a nutrient broth culture medium of 250mL at an inoculation amount of 6% (v/v), culturing at 37 deg.C and 150r/min for 30h in a shake flask to obtain the second-stage seed solution of Bacillus
The brevibacillus brevis adopts a second-stage brevibacillus brevis seed solution, and the preparation method comprises the following steps: picking two-ring Brevibacillus sp.SWUST-2 slant seeds by using an inoculating ring, and culturing the two-ring Brevibacillus sp.SWUST-2 slant seeds in a 250mL triangular flask filled with 50mL nutrient broth culture medium at 37 ℃ at 150r/min for 18h to prepare a first-stage Brevibacillus seed solution; inoculating the first-stage seed solution of the brevibacillus brevis into 250mL of nutrient broth culture medium with the inoculation amount of 6% (v/v), performing shake-flask culture at 37 ℃ for 30h at 150r/min, and preparing the second-stage seed solution of the brevibacillus brevis.
Preferably, the composition of the optimized medium is: CMC-Na5.0g, (NH) 4 ) 2 SO 4 2.0g、K 2 HPO 4 1.0g、MgSO 4 ·7H 2 O 0.5g、NaCl 0.5g、FeSO 4 ·7H 2 0.1g of O, 1000mL of distilled water and 7.0-7.2 of pH value.
Preferably, when the bacteria in the microbial agent are a mixture of the elongated lysine bacillus, bacillus and brevibacillus, the elongated lysine bacillus secondary seed liquid, the bacillus secondary seed liquid and the brevibacillus secondary seed liquid are mixed according to the ratio of 1-1.5:2:2, combining in proportion to obtain a microbial compound inoculant; the total bacterial colony number of the composite bacterial liquid reaches 2.5 multiplied by 10 9 CFU/mL。
The invention at least comprises the following beneficial effects: the invention adopts an intelligent closed high-temperature aerobic fermentation tank to carry out closed high-temperature fermentation, the fermentation mode is in-tank fermentation without pollution, aerobic microorganisms are rapidly propagated under the condition that an air feeder provides oxygen, the temperature of materials is rapidly raised, the temperature can reach 60-70 ℃ in a high-temperature period in the same day, organic matters are decomposed in the period, the moisture is rapidly reduced, pathogenic bacteria and weed seeds are killed, and the harmless, stable and quantitative reduction treatment of the materials is realized; and the adopted bacterial strain of the microbial agent is derived from high-temperature dominant flora in the natural fermentation process of straw compost, and can effectively promote the fermentation production process of the organic fertilizer taking straws and livestock and poultry manure as main raw materials through long-term screening and domestication, improve the production efficiency and the fertilizer efficiency of the organic fertilizer, and simultaneously improve the harmless treatment effect of the straws and the manure.
Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.
Description of the drawings:
FIG. 1 is a stained hydrolysis loop during primary screening of the strain of the invention;
FIG. 2 is a glucose standard curve during the rescreening of the strains of the invention.
The specific implementation mode is as follows:
the present invention is further described in detail below with reference to the attached drawings so that those skilled in the art can implement the invention by referring to the description text.
It will be understood that terms such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.
Example 1:
a method for preparing an organic fertilizer by deep fermentation of livestock and poultry manure and straws comprises the following steps:
step one, mixing pig manure and straw according to a weight ratio of 1:3, uniformly mixing, and adjusting the carbon-nitrogen ratio to be 25:1, simultaneously inoculating a lysinibacillus longissimus microbial inoculum, wherein the inoculation amount is 3% by weight, and the water content is adjusted to 60%; obtaining a pre-fermented material;
step two, adding the pre-fermented material into an intelligent closed high-temperature aerobic fermentation tank, and stirring and fermenting for 12 days at the fermentation temperature of 65 ℃ under the condition of providing oxygen by a blower after fermentation starts; and granulating the fermented finished product to obtain the organic fertilizer.
The preparation method of the lysine bacillus slender bacterial agent comprises the following steps: picking two rings of elongated lysine bacillus inclined plane seeds by using an inoculating loop, and culturing the two rings of elongated lysine bacillus inclined plane seeds in a 250mL triangular flask filled with 50mL nutrient broth culture medium at 37 ℃ at 150r/min for 18h to prepare an elongated lysine bacillus primary seed solution; inoculating the first-order seed liquid of elongated lysine bacillus into 250mL of optimized culture medium (CMC-Na 5.0g, (NH) with the inoculation amount of 3% (v/v) 4 ) 2 SO 4 2.0g、K 2 HPO 4 1.0g、MgSO 4 ·7H 2 O 0.5g、NaCl0.5g、FeSO 4 ·7H 2 0.1g of O, 1000mL of distilled water, and a pH value of 7.0-7.2), at 37 ℃ and 150r/min, and performing shake flask culture for 30h to prepare a second-stage seed solution of the elongated lysine bacillus, namely the elongated lysine bacillus microbial inoculum; the Classification and designation of the elongated lysine bacillus is elongated lysine bacillus (Lysinibacillus macrocrystals SWUST-1), which has been registered and preserved in China general microbiological culture Collection center (CGMCC) at 12/05 in 2022 with the preservation number of CGMCC No. M24857; the DNA gene sequence of the elongated lysine bacillus (lysine bacteria macrocides SWUST-1) is shown in SEQ ID NO. 1.
Example 2:
a method for preparing an organic fertilizer by deeply fermenting livestock and poultry manure and straws comprises the following steps:
step one, mixing pig manure and straw according to a weight ratio of 1:3, uniformly mixing, and adjusting the carbon-nitrogen ratio to be 25:1, simultaneously inoculating a bacillus agent, wherein the inoculation amount is 3% by weight, and the water content is adjusted to 60%; obtaining a pre-fermented material;
step two, adding the pre-fermented material into an intelligent closed high-temperature aerobic fermentation tank, and stirring and fermenting for 12 days under the condition that an air blower provides oxygen at the fermentation temperature of 65 ℃ after fermentation starts; granulating the fermented finished product to obtain an organic fertilizer;
the preparation method of the bacillus agent comprises the following steps: picking two-ring bacillus slant seeds by using an inoculating loop, and culturing the two-ring bacillus slant seeds in a 250mL triangular flask filled with 50mL nutrient broth culture medium at 37 ℃ at 150r/min for 18h to prepare a bacillus primary seed solution; inoculating the first-stage bacillus seed solution into 250mL of nutrient broth culture medium with the inoculation amount of 6% (v/v), performing shake-flask culture at 37 ℃ for 30h at 150r/min to obtain a second-stage bacillus seed solution, namely a bacillus microbial inoculum; the classification name of the Bacillus is Bacillus (Bacillus sp.SWUST-3), which has been registered and preserved in China general microbiological culture Collection center (CGMCC) at 12.05.2022, with the preservation number of CGMCC No. M24858; the DNA gene sequence of the Bacillus (Bacillus sp.SWUST-3) is shown as SEQ ID NO. 2;
example 3:
a method for preparing an organic fertilizer by deep fermentation of livestock and poultry manure and straws comprises the following steps:
step one, mixing pig manure and straw according to a weight ratio of 1:3, uniformly mixing, and adjusting the carbon-nitrogen ratio to be 25:1, simultaneously inoculating a Bacillus brevis microbial inoculum, wherein the inoculation amount is 3 percent by weight, and the water content is adjusted to 60 percent; obtaining a pre-fermented material;
step two, adding the pre-fermented material into an intelligent closed high-temperature aerobic fermentation tank, and stirring and fermenting for 12 days at the fermentation temperature of 65 ℃ under the condition of providing oxygen by a blower after fermentation starts; granulating the fermented finished product to obtain an organic fertilizer;
the preparation method of the brevibacillus brevis microbial inoculum comprises the following steps: selecting two rings of brevibacillus brevis inclined plane seeds by an inoculating loop, and culturing for 18h in a 250mL triangular flask filled with 50mL nutrient broth culture medium at 37 ℃ at 150r/min to prepare a first-level brevibacillus brevis seed solution; inoculating the first-stage seed liquid of the brevibacillus brevis into 250mL of nutrient broth culture medium with the inoculation amount of 6% (v/v), culturing at 37 ℃ for 150r/min for 30h in a shaking way to prepare second-stage seed liquid of the brevibacillus brevis; namely a brevibacillus agent; the classification name of the Brevibacillus brevis is Brevibacillus brevis (Brevibacillus sp.SWUST-2), which is registered and preserved in China general microbiological culture Collection center (CGMCC) at 12/05 in 2022, with the preservation number of CGMCC No. M24859; the DNA gene sequence of the Bacillus brevis (Brevibacillus sp. SWUST-2) is shown in SEQ ID NO. 3.
Example 4:
a method for preparing an organic fertilizer by deeply fermenting livestock and poultry manure and straws comprises the following steps:
step one, mixing pig manure and straw according to a weight ratio of 1:3, uniformly mixing, and adjusting the carbon-nitrogen ratio to be 25:1, simultaneously inoculating a compound microbial agent, wherein the inoculation amount is 3% by weight, and the water content is adjusted to 60%; obtaining a pre-fermented material;
step two, adding the pre-fermented material into an intelligent closed high-temperature aerobic fermentation tank, and stirring and fermenting for 12 days at the fermentation temperature of 65 ℃ under the condition of providing oxygen by a blower after fermentation starts; granulating the fermented finished product to obtain an organic fertilizer;
the preparation method of the compound microbial agent comprises the following steps: respectively picking two rings of elongated lysine bacillus, bacillus and brevibacillus oblique seeds by using inoculating loops, culturing the elongated lysine bacillus, bacillus and brevibacillus oblique seeds in a 250mL triangular flask filled with 50mL nutrient broth culture medium at 37 ℃ at 150r/min for 18h to respectively prepare elongated lysine bacillus primary seed liquid, bacillus primary seed liquid and brevibacillus primary seed liquid; the first-order seed liquid of the lysine bacillus was inoculated into 250mL of an optimized medium (CMC-Na5.0 g, (NH) in an amount of 3% (v/v) 4 ) 2 SO 4 2.0g、K 2 HPO 4 1.0g、MgSO 4 ·7H 2 O 0.5g、NaCl 0.5g、FeSO 4 ·7H 2 0.1g of O, 1000mL of distilled water, 7.0-7.2 of pH value), 37 ℃ and 150r/min, and performing shake flask culture for 30h to prepare a second-stage seed solution of the elongated lysine bacillus; inoculating the first-level seed solution of the bacillus and the first-level seed solution of the brevibacillus into 250mL of nutrient broth culture medium with the inoculation amount of 6% (v/v), performing shake flask culture at 37 ℃ for 30h at 150r/min to respectively prepare a second-level seed solution of the bacillus and a second-level seed solution of the brevibacillus; mixing the long and thin bacillus lysinate secondary seed solution, the bacillus secondary seed solution and the bacillus brevis secondary seed solution according to the proportion of 1.5:2:2, combining in proportion to obtain a microbial compound inoculant; the total bacterial colony number of the composite bacterial liquid reaches 2.5 multiplied by 10 9 CFU/mL; the Classification and designation of the elongated lysine bacillus is elongated lysine bacillus (Lysinibacillus macrocrystals SWUST-1), which has been registered and preserved in China general microbiological culture Collection center (CGMCC) at 12/05 in 2022 with the preservation number of CGMCC No. M24857; the classification name of the Bacillus is Bacillus (Bacillus sp.SWUST-3), which has been registered and preserved in China general microbiological culture Collection center (CGMCC) at 12.05.2022, with the preservation number of CGMCC No. M24858; the classification name of the Brevibacillus brevis is Brevibacillus brevis (Brevibacillus sp. SWUST-2), which has been registered and preserved in China general microbiological culture Collection center in 12 th.05.2022, with the preservation number of CGMCC No. M24859. The DNA gene sequence of the lysine bacillus longituba (Lysinibacillus macrodes SWUST-1)The sequence is shown as SEQ ID NO. 1. The DNA gene sequence of the Bacillus (Bacillus sp.SWUST-3) is shown as SEQ ID NO. 2. The DNA gene sequence of the Bacillus brevis (Brevibacillus sp. SWUST-2) is shown in SEQ ID NO. 3.
Comparative example 1:
mixing pig manure and straw according to a weight ratio of 1:3, uniformly mixing, and adjusting the carbon-nitrogen ratio to be 25:1, simultaneously inoculating a slender lysine bacillus agent, adjusting the inoculation amount to be 3% by weight and the water content to be 60%, regularly monitoring the temperature and the water content, and keeping the temperature at 60 ℃; adding water to keep the water content of the compost to be about 60%, turning the compost when the temperature is obviously reduced, turning the compost for 3 times totally, and decomposing for 10 days after the temperature is not obviously increased any more to obtain the organic fertilizer; the properties are shown in table 1;
the preparation method of the lysinibacillus elongatus microbial inoculum is as in example 1.
Comparative example 2:
mixing pig manure and straw according to a weight ratio of 1:3, uniformly mixing, and adjusting the carbon-nitrogen ratio to be 25:1, simultaneously inoculating a bacillus agent, adjusting the water content to 60% according to the inoculation amount of 3% by weight, regularly monitoring the temperature and the water content, and keeping the temperature at 60 ℃; adding water to keep the water content of the compost about 60%, turning the compost when the temperature is obviously reduced, turning the compost for 3 times in total, and decomposing for 10 days after the temperature is not obviously increased any more to obtain the organic fertilizer; the properties are shown in table 1;
the preparation method of the bacillus agent is shown as an example 2.
Comparative example 3:
mixing pig manure and straw according to a weight ratio of 1:3, uniformly mixing, and adjusting the carbon-nitrogen ratio to be 25:1, simultaneously inoculating a brevibacillus brevis microbial inoculum, adjusting the water content to 60% according to the inoculation amount of 3% by weight, regularly monitoring the temperature and the water content, and keeping the temperature at 60 ℃; adding water to keep the water content of the compost about 60%, turning the compost when the temperature is obviously reduced, turning the compost for 3 times in total, and decomposing for 10 days after the temperature is not obviously increased any more to obtain the organic fertilizer; the properties are shown in table 1;
the preparation method of the brevibacillus brevis microbial inoculum is shown as an example 3.
Comparative example 4:
mixing pig manure and straw according to a weight ratio of 1:3, uniformly mixing, and adjusting the carbon-nitrogen ratio to be 25:1, simultaneously inoculating a compound microbial agent, adjusting the water content to 60% according to the inoculation amount of 3% by weight, periodically monitoring the temperature and the water content, and keeping the temperature at 60 ℃; adding water to keep the water content of the compost about 60%, turning the compost when the temperature is obviously reduced, turning the compost for 3 times in total, and decomposing for 10 days after the temperature is not obviously increased any more to obtain the organic fertilizer; the properties are shown in table 1; the preparation method of the complex microbial agent is shown as example 4.
TABLE 1
The screening and identification process of the elongated lysine bacillus, the bacillus and the brevibacillus comprises the following steps:
1. preliminary screening of strains (high temperature enrichment and high temperature culture)
Taking 10g of a manure sample in the composting process of the poultry manure, and putting the manure sample into a 250mL conical flask filled with 100mL of sterile normal saline under the aseptic condition; activating and enriching for 24h in a constant-temperature shaking incubator at the temperature of 50 ℃ and at the speed of 150 r/min;
diluting the activated bacteria liquid with sterile water in sequence until the bacteria liquid is released to 10 -5 、10 -6 、10 -7 Three gradients. 100 μ L of the diluted solution was added dropwise to a selection medium (sodium carboxymethylcellulose CMC-Na5.0g, ammonium sulfate (NH) 4 ) 2 SO 4 2.0g of dipotassium hydrogen phosphate K 2 HPO 4 1.0g, magnesium sulfate heptahydrate MgSO 4 ·7H 2 0.5g of O, 0.5g of sodium chloride NaCl, and ferrous sulfate heptahydrate FeSO 4 ·7H 2 0.1g of O, 20g of agar powder and 7.0-7.2 of pH. After dissolving the above substances, the volume was adjusted to 1000mL with distilled water. Sterilizing at 121 ℃ and 0.1Mpa for 20 min), uniformly coating, drying and fixing the bacterial liquid, inversely placing the plate into a constant-temperature incubator, and culturing at 50 ℃ for 72h.
Picking large and obvious single colony, repeatedly streaking in a flat plate for separation and purification, and observing by a gram-staining electron microscope until no mixed bacteria exist. And inoculating the separated and purified colonies to a screening culture medium plate for culturing for 72 hours at the temperature of 30 ℃. After bacterial colonies grow out, the bacterial colonies are stained by Congo red for 0.5h, then are soaked by 1mol/L sterile normal saline for 0.5h, and the enzyme production capacity of the bacterial strains is evaluated according to the diameter of a transparent hydrolysis ring generated by the bacterial colonies. Generally, the larger the hydrolysis loop, the stronger the enzyme-producing ability of the strain (see FIG. 1). The obtained strain was treated with 0.7mL of bacterial suspension: 0.3mL of glycerol is mixed evenly and stored in a refrigerator at minus 80 ℃ for standby.
2. Rescreening of strains (carboxymethyl cellulase activity assay)
Inoculating the cellulose-degrading bacteria obtained from the primary screening to a fermentation medium (sodium carboxymethylcellulose CMC-Na 10.0g, ammonium sulfate (NH) 4 ) 2 SO 4 2.0g of potassium hydrogen phosphate K 2 HPO 4 1.0g, magnesium sulfate heptahydrate MgSO 4 ·7H 2 0.5g of O, 0.5g of sodium chloride NaCl, and ferrous sulfate heptahydrate FeSO 4 ·7H 2 0.1g of O, 20g of agar powder and 7.0-7.2 of pH. After dissolving the above substances, the volume was adjusted to 1000mL with distilled water. Sterilizing at 121 ℃ and 0.1Mpa for 20 min), and culturing at 30 ℃ in a constant-temperature shaking incubator at 150r/min for 72h. Measuring the activity of carboxymethyl cellulase (CMCase), selecting a strain with higher enzyme activity as a target strain, and specifically comprising the following steps:
1. drawing of glucose standard curve
7 cleaned and dried colorimetric tubes of 10mL are respectively added with glucose and citric acid mixed solution with the total volume of 2mL 0, 0.04, 0.08, 0.12, 0.16, 0.20 and 0.40 mg/mL. Then, 1.5mL of DNS reagent was added, the mixture was subjected to boiling water bath for 10min, and after the mixture was washed and cooled with running water, 1.5mL of distilled water was added to adjust the volume to 5mL, and the absorbance at different glucose concentrations was measured by zeroing the mixture in a cuvette with a glucose concentration of 0 at a wavelength of 540nm (FIG. 2).
2. Preparation of crude enzyme solution
10mL of fermentation liquor is taken and placed in a cleaned and dried 50mL centrifuge tube, and is centrifuged for 5min at 5000r/min under the condition of 4 ℃, and supernatant fluid is taken to be crude enzyme liquid.
3. Determination of enzyme Activity
The enzyme activity of the fermentation liquor is measured by adopting 3,5-dinitrosalicylic acid color development method, the crude enzyme liquid degrades cellulose to generate reducing sugar, the reducing sugar can reduce nitro of 3,5-dinitrosalicylic acid into amino to generate new amino compound, and color development reaction which makes the solution turn yellow is generated. Under the condition of a certain reducing sugar concentration, the color depth of the mixed solution is increased along with the increase of the reducing sugar concentration, namely the enzyme activity is in positive correlation with the chromogenic reaction degree.
CMCase viability assay: 4 sterilized and dried 10mL colorimetric tubes are taken and numbered, 1.5mL of citric acid buffer solution is added into the No. 0 tube to serve as a blank sample, and 1.5mL of 1.0% CMC-Na solution is added into the other three colorimetric tubes respectively. Respectively adding 0.5mL of crude enzyme solution into 4 colorimetric tubes, performing water bath at 50 ℃ for 30min, adding 1.5mL of DNS reagent into a boiling water bath for 10min after the water bath is finished, washing with tap water, rapidly cooling, and adding 1.5mL of distilled water. The absorbance was measured at a wavelength of 540nm (Table 2).
TABLE 2
| Strain numbering | CMCase/U |
| 1# | 25.573 |
| 2# | 12.345 |
| 3# | 26.692 |
| 4# | 20.694 |
| 5# | 27.742 |
| 6# | 17.807 |
| 7# | 10.869 |
| 8# | 16.937 |
| 9# | 18.165 |
| 10# | 11.609 |
3. Molecular characterization of strains
And selecting 1#, 3# and 5# strains with high enzyme activity for sequencing identification. The colony DNA is extracted by a kit method, 27F (GAGAGTTTGATCCTGGCTCAG) and 1492R (TACGGCTACCTTGTTACGAC) are respectively used as a forward primer and a reverse primer for PCR amplification, and the 3 strains have an amplification band at about 1500bp through electrophoresis detection. The sequence of the 1# strain is shown as SEQ ID NO. 3; the sequence of the 3# strain is shown in SEQ ID NO. 1; the sequence of the 5# strain is shown in SEQ ID NO. 2; splicing sequencing results by using ContigExpress, and removing parts with inaccurate two ends; the spliced sequences were aligned in NCBI database (blast. NCBI. Nlm. Nih. Gov), strain # 1: brevibacillus sp. (Brevibacillus sp.); 3# Strain lysine slender bacillus (Lysinibacillus macrioides); strain # 5 Bacillus (Bacillus sp.).
While embodiments of the invention have been described above, it is not limited to the applications set forth in the description and the embodiments, which are fully applicable to various fields of endeavor for which the invention may be embodied with additional modifications as would be readily apparent to those skilled in the art, and the invention is therefore not limited to the details given herein and to the examples shown and described without departing from the generic concept as defined by the claims and their equivalents.
Claims (10)
1. A method for preparing an organic fertilizer by deeply fermenting livestock and poultry manure and straws is characterized by comprising the following steps:
step one, mixing the livestock and poultry manure and the straws in a weight ratio of 1:3, uniformly mixing, and adjusting the carbon-nitrogen ratio to be 25:1, simultaneously inoculating a microbial agent, wherein the inoculation amount is 3% by weight, and the water content is adjusted to 60%; obtaining a pre-fermented material;
secondly, adding the pre-fermented material into an intelligent closed high-temperature aerobic fermentation tank, and stirring and fermenting for 5-16 days at the fermentation temperature of 60-70 ℃ under the condition of providing oxygen by a blower after fermentation starts; and granulating the fermented finished product to obtain the organic fertilizer.
2. The method for preparing organic fertilizer by deeply fermenting livestock and poultry manure and straw as claimed in claim 1, wherein the livestock and poultry manure is one or more of pig manure, cow manure, chicken manure and duck manure.
3. The method for preparing the organic fertilizer by the advanced fermentation of the livestock and poultry manure and the straws as claimed in claim 1, wherein the bacteria in the microbial agent are one or more of bacillus gracilis, bacillus subtilis and bacillus brevis.
4. The method for preparing the organic fertilizer by deeply fermenting the livestock and poultry manure and the straw as claimed in claim 3, wherein the Classification and naming of the lysine bacillus longitubus (lysine bacillus macrodes SWUST-1) is that the lysine bacillus longitubus has been registered and preserved in the China general microbiological culture Collection center (CGMCC) on 12.05.2022, with the preservation number of CGMCC No. M24857; the classification name of the Bacillus is Bacillus (Bacillus sp.SWUST-3), which has been registered and preserved in China general microbiological culture Collection center (CGMCC) at 12.05.2022, with the preservation number of CGMCC No. M24858; the classification name of the Brevibacillus brevis is Brevibacillus brevis (Brevibacillus sp. SWUST-2), which is registered and preserved in China general microbiological culture Collection center on 12/05 in 2022, with the preservation number of CGMCC No. M24859.
5. The method for preparing organic fertilizer by the advanced fermentation of the livestock and poultry manure and the straws as claimed in claim 4, wherein the DNA gene sequence of the elongated lysine bacillus (lysine bacillus macroides SWUST-1) is shown as SEQ ID NO. 1.
6. The method for preparing the organic fertilizer by the deep fermentation of the livestock and poultry manure and the straw as claimed in claim 4, wherein the DNA gene sequence of the Bacillus (Bacillus sp.SWUST-3) is shown as SEQ ID NO. 2.
7. The method for preparing organic fertilizer by deeply fermenting livestock and poultry manure and straws as claimed in claim 4, wherein the DNA gene sequence of the Bacillus brevis (Brevibacillus sp. SWUST-2) is shown as SEQ ID NO. 3.
8. The method for preparing organic fertilizer by deeply fermenting livestock and poultry manure and straw as claimed in claim 4,
the lysine bacillus slender rod adopts a lysine bacillus slender rod secondary seed solution, and the preparation method comprises the following steps: using an inoculating loop to pick two loops of inclined surface seeds of lysine bacillus longilineans (lysine bacillus macrodes SWUST-1) in a 250mL triangular flask filled with 50mL nutrient broth culture medium, and culturing at 37 ℃ and 150r/min for 18h to prepare a primary seed liquid of the lysine bacillus longilineans; inoculating the first-stage seed liquid of the lysine bacillus into 250mL of optimized culture medium with the inoculation amount of 3% (v/v), culturing at 37 ℃ at 150r/min for 30h in a shaking way to prepare a second-stage seed liquid of the elongated lysine bacillus;
the bacillus adopts bacillus secondary seed liquid, and the preparation method comprises the following steps: inoculating a loop to pick slant seeds of Bacillus bicyloides (Bacillus sp.SWUST-3) in a 250mL triangular flask filled with 50mL nutrient broth culture medium, and culturing at 37 ℃ at 150r/min for 18h to obtain a first-level seed solution of Bacillus; inoculating the first-stage seed solution of Bacillus in a nutrient broth culture medium of 250mL at an inoculation amount of 6% (v/v), culturing at 37 deg.C and 150r/min for 30h in a shake flask to obtain the second-stage seed solution of Bacillus
The brevibacillus brevis adopts a second-stage seed solution of brevibacillus brevis, and the preparation method comprises the following steps: selecting slant seeds of Brevibacillus bicolor (Brevibacillus sp. SWUST-2) by using an inoculating loop, and culturing for 18h in a 250mL triangular flask filled with 50mL nutrient broth culture medium at 37 ℃ at 150r/min to prepare a first-grade seed solution of Brevibacillus brevis; inoculating the first-stage seed liquid of the brevibacillus brevis into 250mL of nutrient broth culture medium with the inoculation amount of 6% (v/v), culturing at 37 ℃ at 150r/min for 30h in a shaking way, and preparing the second-stage seed liquid of the brevibacillus brevis.
9. The method for preparing the organic fertilizer by the advanced fermentation of the livestock and poultry manure and the straws as claimed in claim 8, wherein the optimized culture medium comprises the following components: CMC-Na5.0g, (NH) 4 ) 2 SO 4 2.0g、K 2 HPO 4 1.0g、MgSO 4 ·7H 2 O 0.5g、NaCl 0.5g、FeSO 4 ·7H 2 0.1g of O, 1000mL of distilled water and 7.0-7.2 of pH value.
10. The method for preparing the organic fertilizer by the advanced fermentation of the livestock and poultry manure and the straws as claimed in claim 8, wherein when the bacteria in the microbial agent are the mixture of the elongated lysine bacillus, the bacillus and the brevibacillus, the elongated lysine bacillus secondary seed solution, the bacillus secondary seed solution and the brevibacillus secondary seed solution are mixed according to the ratio of 1-1.5:2:2, combining in proportion to obtain a microbial compound inoculant; the total bacterial colony number of the composite bacterial liquid reaches 2.5 multiplied by 10 9 CFU/mL。
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