CN112251357A - Method for screening livestock and poultry manure fermentation microbial inoculum - Google Patents
Method for screening livestock and poultry manure fermentation microbial inoculum Download PDFInfo
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Abstract
A method for screening a livestock and poultry manure fermentation microbial inoculum comprises the following steps of: 1. selecting fallen leaves to cover a soil surface area rich in humus, poking the surfaces of the fallen leaves, digging small pits with the depth of 25-35cm, the length of 55-65cm and the width of 25-35cm in the area, filling the pits into a plastic basin, pouring fresh pig manure into the plastic basin, sealing the pit mouth by using a plastic film, scattering dead branches or fallen leaves at the position of the plastic film, and enriching for 6-8 days; 2. after the step 1, weighing 8-12g of pig manure mixed liquid, adding 85-95mL of sterile water, and oscillating for 25-35 min; 3. after the step 2, diluting the uniformly oscillated pig manure suspension in different gradients to obtain pig manure solutions diluted in different gradients; 4. and 3, sucking 200 mu L of pig manure solution with each dilution concentration, respectively coating the pig manure solution on a Gao's first solid culture medium, a beef extract peptone agar culture medium and a Sha's agar culture medium, placing the pig manure solution in a constant-temperature incubator at 25-30 ℃ for culturing for 60-72h, and finally selecting colonies with different forms and excellent growth vigor for separation and purification to form single colonies.
Description
Technical Field
The invention relates to the technical field of microbial fermentation, in particular to a method for screening a livestock and poultry manure fermentation microbial inoculum.
Background
China is a big livestock breeding country and the country with the largest output of livestock manure in the world, and mainly takes pig manure, cow manure and chicken manure. Along with the improvement of the living standard of people, the demand of people for pork and meat products thereof is continuously improved, so the breeding scale is also continuously enlarged, and the yield of pig manure is also increased. However, most of the current large-scale pig farms are not provided with corresponding manure treatment facilities, and the serious environmental pollution problem is caused by improper treatment of the manure of the livestock.
At present, the livestock manure treatment methods used include chemical treatment methods, physical treatment methods, microbial fermentation methods and the like. Although the chemical treatment method has high efficiency of deodorization and decomposition of the pig manure, the equipment operation and maintenance cost is high, and farmers cannot bear the chemical treatment method; the physical treatment method has the defects of high early investment cost, low treatment efficiency, easy secondary pollution and the like.
The fermentation bed microbial inoculum of the current-stage biological fermentation method mainly adopts imported microbial inoculum products represented by Nippon Rockdong, so that the operation cost is high and the method is difficult for farmers to bear; the current domestic independently cultured microbial inoculum is low in commercialization degree, and generally shows the defects of incomplete deodorization, slow decomposition of pig manure and the like, so that the treatment capacity of a fermentation bed is low, the treatment period is long, and zero pollution discharge is difficult to realize.
Disclosure of Invention
The invention aims to solve the problems in the prior art and provides screening and application of pig manure fermented aspergillus fumigatus.
The invention adopts the technical scheme for solving the technical problems that: the method for screening the livestock and poultry manure fermentation microbial inoculum mainly comprises strain obtaining and strain screening,
the steps for obtaining the strain are as follows:
after the step 3 and the step 2, carrying out different gradient dilution on the uniformly oscillated pig manure suspension to obtain pig manure solutions with different gradient dilutions;
and 4, after the step 3, sucking 200 mu L of pig manure solution with each dilution concentration, respectively coating the pig manure solution on a Gao's first solid culture medium, a beef extract peptone agar culture medium and a Sha's agar culture medium, placing the pig manure solution in a constant-temperature incubator at 25-30 ℃ for culturing for 60-72h, and finally selecting colonies with different forms and excellent growth vigor for separation and purification to form single colonies.
The strain screening method comprises the following steps:
and 6, after the incubator is cultured in the step 5, performing accumulative determination for 2-3 weeks, comparing the strains with the minimum accumulative release amount, and taking the strains as raw materials of the fermentation inoculant.
For further improvement, the strain was subjected to a differential gradient dilution of 10 in step 3-2、10-3、10-4、10-5And 10-6Any one or more of them.
Further perfecting, the main components of the Gao's No. one solid culture medium in the strain obtaining step 4 are 20g of soluble starch, 0.5g of NaCl, KNO31g, 0.5g of K2HPO 4.3H 2O 0.5, 0.5g of MgSO 4.7H 2O 0.5.5 g, 0.01g of FeSO 4.7H 2O 0.01, 20g of agar and 1L of purified water.
Further perfecting, the main components of the beef extract peptone agar culture medium in the strain obtaining step 4 are 40g of glucose, 10g of tryptic digest of casein and equal amount of gastric digest of animal tissue, 0.1g of chloramphenicol, 15g of agar and 1L of purified water.
Further perfecting, the main components of the saxarsia agar culture medium in the strain obtaining step 4 are 3g of beef extract, 10g of peptone, 5g of NaCl, 20g of agar and 1L of purified water.
Further perfecting, wherein the degradation rate formula is ammonia nitrogen degradation rate (%) (initial ammonia nitrogen concentration (-residual ammonia nitrogen concentration) ÷ initial ammonia nitrogen concentration multiplied by 100%.
And further perfecting, the preparation method of the pig manure mixed liquor in the strain screening step 3 comprises the steps of taking a plurality of fresh pig manure, adjusting the water content to 55-65%, uniformly mixing, filtering, selecting a plurality of conical bottles, and filling 150ml of pig manure solution into each conical bottle for sealed preservation.
Further perfected, the odor and malodor degree is graded as any one of no odor-Ms 0, barely sensed odor-Ms 1, weak odor-Ms 2, obvious odor-Ms 3, strong odor-Ms 4 and intolerable odor-Ms 5.
Further perfecting, the absorption liquid in the strain screening step 5 is boric acid solution with the mass concentration of 2%.
Further perfecting, and performing enzyme activity determination on the strains screened in the strain screening step 6, wherein the steps are as follows:
(1) the protease activity in the enzyme activity determination is determined by adopting an ultraviolet spectrophotometry method, acidic, neutral and alkaline buffer solutions are respectively prepared, and the activities of the acidic, neutral and alkaline proteases are determined.
(2) The activity of the alpha-amylase in the enzyme activity determination is determined by adopting a spectrophotometry method, and the enzyme activity is obtained by referring to a comparison table about absorbance and testing the activity of the alpha-amylase in a general test method (QB/T1803-1993) of an industrial enzyme preparation.
(3) The activity of the saccharifying enzyme in the enzyme activity determination is determined by adopting a titration method of a sodium thiosulfate standard solution.
(4) The lipase activity in the enzyme activity determination is determined by an acid-base titration method.
(5) The activity of the cellulase in the enzyme activity determination is determined according to a DNS method.
(6) The universal primer is used for amplifying a 16S rDNA segment of the strain, and BLAST homologous retrieval is carried out on the segment after sequencing and the existing sequence in the NCBI database, so that the strain is subjected to species identification on a molecular level.
Further perfecting, and the molecular identification result shows that the strain in the strain screening step 6 is aspergillus fumigatus.
The invention has the beneficial effects that: the method selects fallen leaves of a Baiyunshan forest park in the city of Lishui to cover a soil surface area containing humus, carries out microbial culture on the soil, obtains a bacterial colony with better two test results through comparative tests of different strains on ammonia nitrogen degradation rate and deodorization effect, shows the bacterial colony as aspergillus fumigatus through the identification result of extraction and culture molecular level, can be used for fermenting and retaining the advantages of high efficiency, cleanness, safety, convenient maintenance, low operation cost and the like in biological action, has lower commercialization degree than the current domestic independently cultured microbial inoculum, generally shows the defects of incomplete deodorization, slow decomposition of pig manure and the like, has better performance, and cultures a native microbial inoculum with good reaction prospect.
Drawings
FIG. 1 is a bar graph of ammonia nitrogen degradation rate of 13 strains (bacterium X) obtained by enrichment screening;
FIG. 2 is a bar graph of ammonia nitrogen degradation rate of 18 strains (fungus Z) obtained by enrichment screening;
FIG. 3 is a bar graph of ammonia nitrogen degradation rate of 18 strains (actinomycetes F) obtained by enrichment screening;
FIG. 4 is a bar graph of cumulative ammonia release of 7 strains selected by ammonia nitrogen degradation rate;
FIG. 5 is a line graph showing the cumulative ammonia release at 21 days for strain Z10;
FIG. 6 is a line graph showing the cumulative amount of released hydrogen sulfide of Z10 strain at 21 days;
FIG. 7 is a bar graph of seed germination rate and germination index;
FIG. 8 is a graph of seed germination;
FIG. 9 is a biochemical identification chart of strain Z10;
FIG. 10 is a schematic view of an enrichment region;
fig. 11 is product versus decomposition temperature.
Detailed Description
The invention will be further described with reference to the accompanying drawings in which:
with reference to the accompanying drawings:
the present invention will now be described with reference to specific embodiments and examples, but the present invention is not limited to the following.
Example 1
Strain NH3 degradation separation
(1) Sample preparation: the Baiyunshan of Lishui city is enriched with the strain separated from the pig manure for 7 days.
(2) The components of the culture medium: 50.0g of cane sugar, 10.0mL of ammonia water, 2.0g of monopotassium phosphate, 0.5g of magnesium sulfate heptahydrate, 0.1g of ferrous sulfate, 5.0mL of 1% zinc sulfate, 2.0g of sodium chloride and 1L of purified water.
(3) Primary screening: and inoculating the separated single colony into an ammonia selective medium agar plate or a shake flask, and primarily selecting strains which can grow by using ammonia water as a unique nitrogen source, wherein the strains using ammonia gas grow bacteria or are turbid and have obvious difference with non-growing bacteria and blank groups. Specific results are shown in table 1.
TABLE 1 utilization of ammonia by different strains
Note: the culture medium with ammonia as the only nitrogen source is adopted for culture, so that the strains which can use the ammonia as the nitrogen source are preliminarily screened, the strains which can use the ammonia all grow bacteria or are turbid in the preliminary screening culture medium (the growth condition is shown by plus), and the obvious difference exists between the strains which do not grow bacteria and a blank group (the growth condition is shown by minus).
In Table 1, 43 strains of deodorant strains capable of using ammonia gas as a nitrogen source were obtained, among which 18 strains of fungi, 13 strains of bacteria, and 18 strains of actinomycetes. Wherein the separation efficiency of the three strains of fungi, bacteria and actinomycetes is respectively 27.2%, 19.7% and 27.2%.
(4) Accurate screening of strains
After inoculating the screened strain with the inoculation amount of 10%, placing the strain at 30 ℃ for shake cultivation at 150r/min for 7d, and then calculating the degradation rate of ammonia nitrogen according to the following formula: ammonia nitrogen degradation rate (%) (initial ammonia nitrogen concentration-residual ammonia nitrogen concentration) ÷ initial ammonia nitrogen concentration x 100%. The specific results are shown in fig. 1, fig. 2 and fig. 3.
The ammonia selective medium was used to co-screen 49 strains, among which 13 strains of bacteria (X), 18 strains of fungi (Z) and 18 strains of actinomycetes (F). The 49 strains of bacteria show different degrees of degradation capability to ammonia nitrogen in the culture medium, and the ammonia nitrogen degradation rate is 33.8% -74.1%, as shown in figure 1, figure 2 and figure 3. Wherein the ammonia nitrogen degradation rate of 7 strains of X5, X12, Z6, Z13, Z23, F6 and F18 is over 70 percent and respectively is 72.0 percent, 74.1 percent, 70.9 percent, 72.4 percent, 71.2 percent, 70.7 percent and 74.0 percent. Because the ammonia selective medium takes ammonia water as a unique nitrogen source, the 7 strains theoretically have the functions of digesting ammonia nitrogen and reducing NH3 release in the fermentation process of pig manure.
Example 2
Screening by olfactory discrimination
(1) Sample preparation: the Baiyunshan of Lishui city is enriched with the strain separated from the pig manure for 7 days.
(2) Pig manure solution: taking a plurality of fresh pig manures, adjusting the water content to about 60%, uniformly mixing, filtering, filling 150mL of pig manures solution into each conical flask, and sealing.
(3) Screening: inoculating the separated strain according to the inoculation amount of 5 percent, naturally fermenting the control group without inoculating the strain, and putting the control group into a shaking table for culturing. The temperature of the shaking table is set to be 30 ℃, and the rotating speed is 120 r/min. Decomposing the pig manure by the strain, and grading according to the odor degree of the gas after 15 days. The specific results are shown in Table 2.
TABLE 2 olfactory screening
| Numbering | Grade | Numbering | Grade | Numbering | Grade | Numbering | Grade | Numbering | Grade | | Grade |
| X1 | |||||||||||
| 2 | |
2 | |
3 | |
1 | |
3 | |
3 | |
| |
4 | |
2 | |
3 | |
3 | |
3 | |
2 |
| |
2 | |
4 | |
2 | |
3 | |
4 | |
3 |
| |
2 | |
3 | |
4 | |
4 | |
4 | |
3 |
| |
2 | |
4 | |
2 | |
3 | |
3 | |
2 |
| |
3 | |
2 | |
4 | |
2 | |
2 | |
4 |
| |
2 | |
3 | |
2 | |
4 | |
4 | |
3 |
| |
2 | |
4 | |
2 | |
4 | |
2 | |
2 |
| |
3 | |
3 | |
3 | |
2 | |
5 | |
3 |
| |
2 | |
3 | |
3 | |
4 | |
2 | |
4 |
| |
4 | |
3 | |
2 | |
2 | |
4 | |
2 |
Note: strains with odor ratings of 0, 1, 2 were considered as effective strains.
Table 2 shows that 23 strains in total are considered as effective strains among 66 strains, and account for 34.8% of all strains.
Strains 7 were co-selected according to example 1 and example 2, and were: x5, X12, Z6, Z13, Z23, F6 and F18.
Example 3
Evaluation of 7 high-efficiency ammonia nitrogen degrading bacteria
(1) Sample preparation: the obtained 7 strains were subjected to degradation separation screening and olfactory discrimination screening by the strain NH 3.
(2) Boric acid absorption liquid: 10g of boric acid is weighed and dissolved in distilled water, and the volume is adjusted to 500 mL.
(3) Indicator (b): 0.10g of methyl red and 0.05g of methylene blue are dissolved in 150mL of a 95% ethanol solution.
(4) Titration solution: 2.7mL of 98% concentrated sulfuric acid was diluted to 1 liter with distilled water.
(5) Respectively inoculating 7 strains of bacteria into a big beaker filled with 400g of fresh pig manure, putting a small beaker filled with 20ml of boric acid absorption liquid with the concentration of 2% on the surface of the pig manure for absorbing the released NH3, sealing the cup opening by using a preservative film, and culturing in a constant-temperature incubator at 50 ℃. The cumulative ammonia release was measured for 2 weeks. Specific results are shown in table 3 and fig. 4.
Table 37 Strain 15d Ammonia cumulative Release amount
The data in table 3 show that the Z13 strain has the lowest ammonia release during 15 days of pig manure composting, which is 54.6% less than the blank control.
Example 4
Evaluation of deodorizing Effect of Z13 Strain
(1) Sample preparation: z13 Strain
(2) Ammonia gas determination reagent
1) Boric acid absorption liquid: 10g of boric acid is weighed and dissolved in distilled water, and the volume is adjusted to 500 mL.
2) Indicator (b): 0.10g of methyl red and 0.05g of methylene blue are dissolved in 150mL of a 95% ethanol solution.
3) Titration solution: 2.7mL of 98% concentrated sulfuric acid was diluted to 1 liter with distilled water.
(3) Hydrogen sulfide determination reagent
1) Stock solution: adding 30mL of distilled water into 50mL of concentrated sulfuric acid (98%), cooling, and adding 6g of p-aminodimethylaniline hydrochloride
2) FeCl3 solution: 100g of FeCl3 was dissolved in 100mL of distilled water and filtered if any precipitate was present.
3) Use solution: 2.5mL of stock solution was diluted to 100mL with 50% sulfuric acid.
4) Mixing color developing agents: the solution was mixed in a ratio of 1mL of the solution to 1 drop (0.04mL) of FeCl3 solution.
(4) The evaluation method comprises the following steps: the screened bacterial strain is inoculated into a big beaker filled with 400g of fresh pig manure, a small beaker filled with 20mL of boric acid absorption liquid with the concentration of 2% is placed on the surface of the pig manure for absorbing the released NH3, the cup opening is sealed by a preservative film and is placed in a constant temperature incubator at 50 ℃ for culture, the NH3 release amount is measured according to the Kay titration method, and the measurement result is shown in Table 4 and figure 5. The H2S release was determined in substantially the same manner as NH3 release, except that 20ml of zinc amine complex salt solution was added to a small beaker for absorption of released H2S, and H2S release was measured by zinc amine complex salt absorption colorimetry, the results of which are shown in table 5 and fig. 6. The control group was inoculated with no bacteria in pig manure, and the NH3 release was determined in the same manner as in H2S release.
TABLE 4Z13 cumulative ammonia release in 21 days (unit: mg/kg)
| Group of | | |
|
|
15d | 18d | 21d | ||
| Z13 | 17.20 | 22.60 | 31.90 | 40.70 | 49.60 | 63.10 | 80.71 | ||
| Control | 18.80 | 42.30 | 63.30 | 79.20 | 95.30 | 112.30 | 131.57 |
TABLE 5Z13 Hydrogen sulfide release rate cumulative over 21 days (unit: mg/kg)
| Group of | | |
|
|
15d | 18d | 21d | ||
| Z13 | 0.46 | 0.58 | 1.12 | 1.32 | 1.48 | 1.84 | 2.22 | ||
| Control | 2.21 | 3.12 | 3.33 | 3.46 | 3.79 | 4.07 | 4.45 |
During the 21-day fermentation, both NH3 and H2S released from the control group at each measurement point were higher than that of the Z10 group, the NH3 and H2S released from the control group were 131.57mg/kg and 4.45mg/kg, respectively, and the NH3 and H2S released from Z13 were 80.71mg/kg and 2.22mg/kg, respectively, which were reduced by 38.66% and 50.03%.
Example 5
Seed germination experiment
(1) Sample preparation: z13 Strain
(2) The content is as follows: weighing 10g of inoculated and fermented pig manure, adding 90ml of distilled water, mixing and stirring, placing the pig manure on a shaking table at 200r/min, shaking for 30min, filtering by using a piece of warp cloth, centrifuging for 10min at a rotating speed of 3000r/min, weighing 15ml of supernatant, pouring the supernatant into a culture dish filled with filter paper, and uniformly placing 20 cauliflower seeds. The pig manure fermentation group without inoculation is used as a negative control, and 15ml of distilled water is directly dripped on the filter paper for the positive control. After the three groups of samples are cultured in the dark at room temperature for 2d, the germination rate and the root length of the seeds are measured, and the germination index GI of the seeds is calculated, and the specific results are shown in Table 6 and FIG. 7.
(3) The GI calculation formula is as follows: GI (%) x 100% (treatment average germination rate × treatment average root length)/(control average germination rate × control average root length).
TABLE 6 germination index of seeds
The germination rate and the germination index of the seeds are the most reliable indexes for evaluating the rotten degree and the phytotoxicity of the pig manure at present. As can be seen from Table 5, the germination percentage and germination index of the negative control group are respectively 13.33% and 3.54%, which indicates that under the condition of fermentation without inoculation, the 21-day fermentation period is far less than the requirements of maturity and harmlessness, and the phytotoxicity is high, so that the germination percentage of the seeds is greatly inhibited, and the germination percentage is expressed as the root length of the germination; the germination rate and the germination index of the Z13 group reach 83.33 percent and 84.89 percent, and the pig manure is considered to be basically non-toxic to seeds because the germination rate exceeds 50 percent, so that the inoculation of the Z13 bacteria plays a remarkable role in promoting the decomposition and harmlessness of the pig manure. The higher germination index of the Z13 group further proves that organic matters in the pig manure are converted into nutrient substances which can be absorbed and utilized by seeds, so that the growth of buds is effectively promoted.
Example 6
Biochemical characterization of Z13
To further reveal the biochemical mechanism of the Z13 strain to degrade pig manure, the activities of protease, amylase, glucoamylase and cellulase were measured, and the results are shown in Table 7 and FIG. 9
TABLE 7 Biochemical characterization of the Z13 Strain
Example 7
16S rDNA molecular identification
The Z13 strain was compared by BLAST search for higher homology to strains listed in the NCBI data, and the homologous strain names and GenBank accession numbers are shown in table 8.
TABLE 8 GenBank accession numbers for homologous strains
The molecular characterization showed that the strain Z13 was Aspergillus fumigatus (Aspergillus fumigatus).
Example 8
Product comparison
The pig manure is from a certain family farm in Limony city of Lishui city of Zhejiang province and is aired for 1 day before use. Certain LB bacterial powder has the function of catalytic decomposition. Composting the pig manure, wherein the compost adopts a circular stack body and is piled into a circular stack shape, the initial mass of each stack body is 500kg, and the three stacks are combined: adding LB bacterial agent into the pile body 1, adding LB bacterial agent and aspergillus fumigatus bacterial agent into the pile body 2, adding LB bacterial agent into the pile body 3, wherein the inoculum size is 15% of the pile body, and fully and uniformly mixing the materials. The temperature of the heap was measured in situ using digital thermometers, and the heap temperature was recorded at 10:00, 14:00, 18:00 days during composting, with the average being the temperature of the heap for the day, while the ambient temperature (one measurement every 3 days) was recorded, and the results are shown in FIG. 11. Taking a proper amount of compost samples, extracting the compost samples by using distilled water, oscillating the compost samples for 4 hours, putting 10.00mL of supernatant into a culture dish, putting filter paper into the culture dish, uniformly putting 30 cauliflower seeds into the culture dish, culturing the cauliflower seeds for 48 hours under the dark condition at room temperature, measuring the germination index GI of the seeds, and using the distilled water as a control, wherein the results are shown in Table 9.
TABLE 9 comparative germination index for products
| Group of | Germination index/% of seeds |
| Stack 1 | 73 |
| |
79 |
| |
84 |
During composting, the microorganisms decompose the organic matter in the heap to release heat, and the heap 3 reaches 72 ℃ at 9d and is maintained above 55 ℃ for 15 d. It is generally considered that when the GI value reaches 80% to 85%, it indicates that the compost has become thoroughly decomposed, and after the composting is completed, the germination index of the seeds of the compost 1 is 73%, the germination index of the compost two is 79%, and the germination index of the compost 3 is 84%, indicating that the compost product has become thoroughly decomposed.
While the invention has been shown and described with reference to a preferred embodiment, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the appended claims.
Claims (10)
1. A method for screening a fermentation bacterial agent of livestock and poultry manure mainly comprises strain obtaining and strain screening,
the steps for obtaining the strain are as follows:
step 1, selecting fallen leaves to cover a soil surface area rich in humus, poking the surfaces of the fallen leaves, digging small pits with the depth of 25-35cm, the length of 55-65cm and the width of 25-35cm in the area, filling the small pits into a plastic basin, pouring fresh pig manure into the plastic basin, sealing the pit opening by using a plastic film, scattering dead branches or fallen leaves at the position of the plastic film, and enriching for 6-8 days to form pig manure mixed liquid;
step 2, after the step 1, weighing 8-12g of pig manure mixed liquor, adding 85-95mL of sterile water, and oscillating for 25-35min to obtain pig manure suspension;
after the step 3 and the step 2, carrying out different gradient dilution on the uniformly oscillated pig manure suspension to obtain pig manure solutions with different gradient dilutions;
and 4, after the step 3, sucking 200 mu L of pig manure solution with each dilution concentration, respectively coating the pig manure solution on a Gao's first solid culture medium, a beef extract peptone agar culture medium and a Sha's agar culture medium, placing the pig manure solution in a constant-temperature incubator at 25-30 ℃ for culturing for 60-72h, and finally selecting colonies with different forms and excellent growth vigor for separation and purification to form single colonies.
The strain screening method comprises the following steps:
step 1, inoculating the single colony obtained in step 4 in strain acquisition into an ammonia selective medium agar plate or a shake flask, wherein strains using ammonia gas grow bacteria or are turbid, and have obvious difference with strains not growing bacteria and blank groups, and selecting strains capable of growing by using ammonia water as a unique nitrogen source;
step 2, inoculating the strains screened in the step 1 by 10 percent of inoculation amount, placing the strains in an environment of 20-30 ℃, performing shake cultivation for 6-8 days at 150r/min, and calculating the ammonia nitrogen degradation rate according to an ammonia nitrogen degradation rate formula after the cultivation is finished;
step 3, inoculating the single bacterial colony obtained in the step 4 in the bacterial colony obtaining process into the pig manure mixed solution according to the inoculation amount of 5% to separate the bacterial strain, putting the obtained bacterial strain into a shaking table for culturing, setting the temperature of the shaking table to be 25-35 ℃, setting the rotating speed to be 110-;
step 4, screening out the same strains with better effects in the step 2 and the step 3;
step 5, inoculating the same bacterial strain obtained in the step 4 into a big beaker filled with 400g of fresh pig manure, and then putting an absorption liquid filled with 20mL on the surface of the pig manure for absorbing and releasing NH3Sealing the cup mouth with a preservative film, culturing in a constant temperature incubator at 50 ℃, and measuring the release amount according to a Kjeldahl method;
and 6, after the incubator is cultured in the step 5, performing accumulative determination for 2-3 weeks, comparing the strains with the minimum accumulative release amount, and taking the strains as raw materials of the fermentation inoculant.
2. The method for screening a fermentation inoculum of livestock manure according to claim 1, which comprises: the strain is obtained in step 3 with different gradient dilution of 10-2、10-3、10-4、10-5And 10-6Any one or more of them.
3. The method for screening a fermentation inoculum of livestock manure according to claim 1, which comprises: the main components of the Gauss No. I solid culture medium in the strain obtaining step 4 are 20g of soluble starch, 0.5g of NaCl, KNO31g, K2HPO 4.3H 2O 0.5g, MgSO 4.7H 2O 0.5.5 g, FeSO 4.7H 2O 0.01.01 g, agar 20g and purified water 1L.
4. The method for screening a fermentation inoculum of livestock manure according to claim 1, which comprises: the main components of the beef extract peptone agar culture medium obtained in the step 4 are 40g of glucose, 10g of tryptic digest of casein and equal amount of gastric digest of animal tissue, 0.1g of chloramphenicol, 15g of agar and 1L of purified water.
5. The method for screening a fermentation inoculum of livestock manure according to claim 1, which comprises: the main components of the saxarsia agar culture medium obtained in the step 4 are 3g of beef extract, 10g of peptone, 5g of NaCl, 20g of agar and 1L of purified water.
6. The method for screening a fermentation inoculum of livestock manure according to claim 1, which comprises: the degradation rate formula is ammonia nitrogen degradation rate (%) (initial ammonia nitrogen concentration (-residual ammonia nitrogen concentration) ÷ initial ammonia nitrogen concentration multiplied by 100%.
7. The method for screening a fermentation inoculum of livestock manure according to claim 1, which comprises: the preparation method of the pig manure mixed liquid in the strain screening step 3 comprises the steps of taking a plurality of fresh pig manure, adjusting the water content to be 55-65%, uniformly mixing, filtering, selecting a plurality of conical bottles, and filling 150ml of pig manure solution into each conical bottle for sealed storage.
8. The method for screening a fermentation inoculum of livestock manure according to claim 1, which comprises: the odor and malodor degree is rated as any one of no odor-Ms 0, barely sensed odor-Ms 1, weak odor-Ms 2, obvious odor-Ms 3, strong odor-Ms 4 and unbearable odor-Ms 5.
9. The method for screening a fermentation inoculum of livestock manure according to claim 1, which comprises: the absorption liquid in the strain screening step 5 is boric acid solution with the mass concentration of 2%.
10. The method for screening a fermentation inoculum of livestock manure according to claim 1, which comprises: the strain in the strain screening step 6 is aspergillus fumigatus.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113430122A (en) * | 2021-06-23 | 2021-09-24 | 四川农业大学 | Complex microbial inoculant suitable for ex-situ fermentation bed and preparation method and application thereof |
| CN114657230A (en) * | 2022-02-23 | 2022-06-24 | 中国农业科学院北京畜牧兽医研究所 | Method for evaluating fermentation characteristics of fiber raw material by combining bionic digestion with in-vitro fermentation |
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| CN110982744A (en) * | 2019-12-17 | 2020-04-10 | 石家庄亚特生物科技有限公司 | Biological quick deodorant for municipal domestic waste, sludge and livestock and poultry manure |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113430122A (en) * | 2021-06-23 | 2021-09-24 | 四川农业大学 | Complex microbial inoculant suitable for ex-situ fermentation bed and preparation method and application thereof |
| CN114657230A (en) * | 2022-02-23 | 2022-06-24 | 中国农业科学院北京畜牧兽医研究所 | Method for evaluating fermentation characteristics of fiber raw material by combining bionic digestion with in-vitro fermentation |
| CN114657230B (en) * | 2022-02-23 | 2023-11-24 | 中国农业科学院北京畜牧兽医研究所 | Method for evaluating fermentation characteristics of fiber raw materials by combining bionic digestion and in-vitro fermentation |
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