CN111849804A - High-temperature-resistant composite microbial agent for degrading livestock and poultry manure and application thereof - Google Patents
High-temperature-resistant composite microbial agent for degrading livestock and poultry manure and application thereof Download PDFInfo
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
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- C05F17/00—Preparation of fertilisers characterised by biological or biochemical treatment steps, e.g. composting or fermentation
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Abstract
The invention discloses a high-temperature-resistant composite microbial agent for degrading livestock and poultry manure and application thereof, aims to further improve the composting effect of the livestock and poultry breeding manure, develops a composite microbial agent containing Bacillus sp bacteria NJAU-ND8 and Bacillus stearothermophilus, and provides composite microbial resources and technical support for the livestock and poultry breeding manure. The application of the composite microbial inoculum is that the composite microbial inoculum accelerates the degradation of livestock and poultry breeding feces. The composite microbial inoculum is used for degrading the chicken manure, the degradation rate of the chicken manure reaches 24.12 percent after half a month, and the degradation effect is better than that of single bacterium and blank treatment; the pig manure is degraded, and the degradation rate of the pig manure reaches 22.41 percent after half a month, which is better than that of single-bacterium and blank treatment; the sheep manure is degraded, and the degradation rate of the sheep manure reaches 34.12% after half a month, which is better than that of single bacterium and blank treatment.
Description
Technical Field
The invention relates to the field of agricultural microorganisms, in particular to a high-temperature-resistant composite microbial agent 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 object of the invention is: the invention aims to further improve the composting efficiency of livestock and poultry breeding waste, develops and develops a composite microbial inoculum, provides flora resources and technical support for livestock and poultry breeding waste composting industry, and provides a high-temperature-resistant composite microbial inoculum for degrading livestock and poultry manure and application thereof.
The technical scheme is as follows:
the technical scheme provided by the invention can be realized by the following steps: a microbial composition comprises Bacillus NJAU-ND8 and Geobacillus stearothermophilus B5.
As further description of the microbial composition, the microbial composition is applied to preparation of the composite microbial inoculum for efficiently degrading livestock and poultry manure.
The high-temperature-resistant livestock and poultry manure degradation composite microbial inoculum is prepared by mixing bacillus NJAU-ND8 and geobacillus stearothermophilus B5 in an equal volume ratio, wherein the bacillus NJAU-ND8 and geobacillus stearothermophilus B5 have the same concentration, and the concentration is not lower than 10 9CFU/ml。
As a further description of the complex microbial inoculum, the physiological characteristics of the complex microbial inoculum are as follows:
(1) the effective viable count of the strain is high, the liquid inoculation/inoculation body is adopted, and the concentration is 109CFU/ml is above;
(2) the strain is high temperature resistant and can grow at a high temperature of 55 ℃;
(3) the strain is salt-resistant and can grow in a culture medium containing 15% of salt;
(4) the bacillus NJAU-ND8 and the geobacillus stearothermophilus B5 both belong to bacillus bacteria, are harmless to crops and have no pathogenicity to human and animals;
(5) the activity of exo-beta-1, 4-glucanase reaches 12.548U, the activity of endo-beta-1, 4-glucanase reaches 13.027U, the activity of beta-glucosidase reaches 24.209U, the activity of neutral xylanase reaches 541.782U, the activity of filter paper enzyme reaches 0.605U, the activity of neutral protease reaches 328.180U, the activity of beta-amylase reaches 0.181U, and the activity of urease reaches 2.795U.
A preparation method of a high-temperature-resistant composite microbial inoculum for degrading livestock and poultry manure comprises the following steps:
(1) respectively inoculating activated strains of bacillus NJAU-ND8 and Geobacillus stearothermophilus B5 into an LB liquid culture medium;
(2) the liquid medium was incubated at 55 ℃ at 170r/min for 24 hours:
(3) after the cultivation is finished, adjusting OD value with sterile water to make the concentration of each bacterial liquid identical to 10 9CFU/ml；
(4) And finally, uniformly mixing according to the volume ratio of 1:1 to form the composite microbial inoculum.
As further description of the complex microbial inoculum, the complex microbial inoculum is applied to degradation of livestock and poultry feces, and the specific application is as follows: 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.
As a further description of the microbial composition, the bacillus NJAU-ND8 is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms, the preservation address is the institute of microbiology of China academy of sciences No. 3, Xilu No.1, Beijing, Toyobo, North-oriented south, the preservation date is 11 and 12 days in 2018, and the preservation number is CGMCC NO. 16737.
As a further description of the microbial composition, the Geobacillus stearothermophilus B5 is preserved in the China general microbiological culture Collection center of the China Committee for culture Collection of microorganisms, the preservation address is the institute of microbiology of China academy of sciences, No. 3, Xilu No.1, Beijing, Chaoyang, the preservation date is 11 months and 20 days in 2017, and the preservation number is CGMCC NO. 14935.
The invention has the beneficial effects that 1, the invention provides a microbial compound microbial inoculum which is used for efficiently converting livestock and poultry breeding wastes into organic fertilizers. The microbial compound bacterial agent contains 2 single bacteria for efficiently degrading livestock and poultry manure, and consists of bacillus NJAU-ND8 and geobacillus stearothermophilus B5. 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 rapidly 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.
2. The composite microbial agent is used for degrading the chicken manure, the degradation rate of the chicken manure reaches 24.12% after half a month, the degradation rate of the chicken manure of the bacillus stearothermophilus B5 is 17.66%, the degradation rate of the chicken manure of the bacillus NJAU-ND8 is only 11.77%, the degradation rate of the chicken manure of blank treatment is only 2.11%, and the degradation effect of the composite microbial agent on the chicken manure is better than that of single bacterium and blank treatment.
3. The composite microbial inoculum is used for degrading pig manure, the degradation rate of the pig manure reaches 22.41 percent after half a month, the degradation rate of the pig manure of Geobacillus stearothermophilus B5 is 11.37 percent, the degradation rate of the pig manure of Bacillus NJAU-ND8 is only 9.78 percent, the degradation rate of the pig manure subjected to blank treatment is only 3.58 percent, and the degradation effect of the composite microbial inoculum on the pig manure is better than that of single bacterium and blank treatment.
4. The composite microbial agent is used for degrading sheep manure, the degradation rate of the sheep manure reaches 34.12% after half a month, the degradation rate of the sheep manure of bacillus stearothermophilus B5 is 27.53%, the degradation rate of the sheep manure of bacillus NJAU-ND8 is only 21.38%, the degradation rate of the sheep manure of blank treatment is only 7.09%, and the degradation effect of the composite microbial agent on the sheep manure is better than that of single bacteria and blank treatment.
Drawings
FIG. 1 is a graph of 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 the degradation of pig manure;
figure 3 is a graph of the effect of different flora combinations on the degradation of sheep manure.
Detailed Description
(I) obtaining of Single and Complex bacteria
The Geobacillus stearothermophilus B5 and the Bacillus NJAU-ND8 strain preserved in a glycerol tube at the temperature of-80 ℃ are streaked and activated on an LB solid culture medium flat plate, and are cultured at the temperature of 55 DEG CThe box was incubated for hours. Single colonies of Geobacillus stearothermophilus B5 and Bacillus NJAU-ND8 are picked and cultured in a 3ml liquid test tube at 55 ℃ at 170r/min for 10 hours in a shaking way, and the bacterial liquid is used as seed liquid. The seed solution was transferred to LB liquid shake flasks at an inoculum size of 1% (v/v) and incubated at 55 ℃ at 170r/min for 24 hours. After the cultivation is finished, adjusting OD value with sterile water to make the concentration of each bacterial liquid be identical and be 10 9CFU/ml, and then uniformly mixing according to the volume ratio of 1:1 to form the composite microbial inoculum.
The preparation method of the LB culture solution is as follows, taking 1L culture medium as an example: 10g of egg white, 5g of yeast powder, 10g of NaCl10g and 20g of agar, metering the volume to 1000ml, naturally adjusting the pH value, and sterilizing at 121 ℃ for 20 min.
(II) capability of single bacterium and compound bacterium to produce lignocellulose degrading enzyme, starch and protein hydrolase
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 a 3, 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 a 3, 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) Beta-amylase (beta-AL) viability assay
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 XV 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
And determining NH3-N generated by urea hydrolysis by urease by adopting an indophenol blue colorimetric method.
UE (ug/min/ml) (. DELTA.A-0.0373) ÷ 0.0915 × 10 × V anti-total ÷ V-like ÷ T ═ Δ A-0.0373) × 27.32
Wherein: 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; Δ A0: the difference of the light absorption values of the total amylase determination group and the control group; c, standard substance: 0.25 mu mol/ml standard tyrosine solution
According to the determination, the following results are obtained: the enzyme activities of different thermophilic bacterial strains, single bacteria and combined bacteria at 55 ℃ are shown in table 1. In all treatments, the enzyme activity of the combined bacteria (B5+ NJAU-ND8) is generally higher than that of all single bacteria combined into the composite bacteria. Compared with the single bacterium B5, the beta-GC, CX, FPA, NEX, beta-AL, NP and UE of the combined bacterium are all remarkably improved. Compared with single bacterium NJAU-ND8, the beta-GC, C1, CX, FPA, NEX, beta-AL, NP and UE of the combined bacterium are all obviously improved. The results show that compared with single bacteria B5 and NJAU-ND8, the composite bacteria containing B5+ NJAU-ND8 has better hydrolysis capability on starch, neutral protein, lignin, cellulose and urea.
TABLE 1 enzyme activities of different thermophilic strains of single and combined bacteria at 55 deg.C
Injecting the combined bacteria which are formed by mixing equal-volume equal concentrations of B5 and NJAU-ND 8; beta-GC represents beta-glucosidase, C1 represents exo-beta-1, 4 glucanase, Cx represents endo-beta-1, 4 glucanase, FPA represents filter paper enzyme, NEX represents neutral xylanase, beta-AL represents beta-amylase, NP represents neutral protease, and UE represents urease.
Example 1: degradation effect of single bacterium and compound bacterium 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. In all treatments, the degradation rate of chicken manure of the combined bacteria (NJAU-ND8+ B5) is the highest and is 24.12%, the degradation rate of chicken manure of B5 is 17.66%, the degradation rate of chicken manure of NJAU-ND8 is 11.77%, and the degradation rate of chicken manure of blank treatment is the lowest and is only 2.11%. Compared with blank treatment, single bacterium B5 treatment and single bacterium NJAU-ND8 treatment, the degradation effect of the combined bacteria (NJAU-N20+ NJAU-N30) on the chicken manure is remarkably improved by 22.01%, 6.46% and 12.35% respectively.
According to the indoor chicken manure degradation result, the degradation effect of the composite bacteria containing NJAU-ND8+ B5 on the chicken manure is optimal.
Example 2: degradation effect of single bacterium and compound bacterium on pig manure
The chicken manure was changed to pig manure, and the remaining operation steps were the same as in example 1.
Results and analysis
The effect of different flora combinations on the degradation of pig manure is shown in figure 2. In all treatments, the degradation rate of the combined bacteria (NJAU-ND8+ B5) is the highest and is 22.41%, the degradation rate of the single bacteria B5 is 11.37%, the degradation rate of the single bacteria NJAU-ND8 is 9.77%, and the degradation rate of the blank treatment is the lowest and is 3.57%. Compared with blank treatment, single bacterium B5 treatment and single bacterium NJAU-ND8 treatment, the degradation effect of the combined bacterium (NJAU-ND8+ B5) on the pig manure is remarkably improved by 18.84%, 11.05% and 12.64% respectively.
According to the indoor pig manure degradation result, the degradation effect of the composite flora containing NJAU-ND8+ B5 on the pig manure is optimal.
Example 3: degradation effect of single bacterium and composite bacterium on sheep manure
The chicken manure was changed to sheep manure, and the remaining operation steps were the same as in case 1.
Results and analysis
The effect of different flora combinations on the degradation of sheep manure is shown in figure 3. In all treatments, the degradation rate of the combined bacteria (NJAU-ND8+ B5) is the highest and is 34.12%, the degradation rate of the single bacteria B5 is 27.66%, the degradation rate of the single bacteria NJAU-ND8 is 21.77%, and the degradation rate of the blank treatment is the lowest and is 7.09%. Compared with blank treatment, single bacterium B5 treatment and single bacterium NJAU-ND8 treatment, the combined bacterium (NJAU-ND8+ B5) has obvious improvement on the degradation effect of sheep manure, and the degradation effect is respectively improved by 27.03%, 6.46% and 12.35%.
According to the indoor sheep manure degradation result, the degradation effect of the composite flora containing NJAU-ND8+ B5 on sheep manure is optimal.
According to the embodiment 1, the embodiment 2 and the embodiment 3, the microbial inoculum containing the composite flora (NJAU-ND8+ 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.
The technical solutions described above only represent the preferred technical solutions of the present invention, and some possible modifications to some parts of the technical solutions by those skilled in the art all represent the principles of the present invention, and fall within the protection scope of the present invention.
Claims (8)
1. A microbial composition, which consists of Bacillus NJAU-ND8 and Geobacillus stearothermophilus B5.
2. The microbial composition according to claim 1, wherein the microbial composition is applied to preparation of a composite microbial agent for efficiently degrading livestock and poultry manure.
3. The high-temperature-resistant composite microbial agent for degrading livestock and poultry manure is characterized in that the composite microbial agent is formed by mixing bacillus NJAU-ND8 and geobacillus stearothermophilus B5 in an equal volume ratio, wherein the bacillus NJAU-ND8 and the geobacillus stearothermophilus B5 are the same in concentration, and the concentration is not lower than 10 9CFU/ml。
4. The high-temperature-resistant livestock and poultry manure degrading composite microbial inoculum according to claim 3, which is characterized by comprising the following physiological characteristics:
(1) the effective viable count of the strain is high, the liquid inoculation/inoculation body is adopted, and the concentration is 109CFU/ml is above;
(2) the strain is high temperature resistant and can grow at a high temperature of 55 ℃;
(3) the strain is salt-resistant and can grow in a culture medium containing 15% of salt;
(4) the bacillus NJAU-ND8 and the geobacillus stearothermophilus B5 both belong to bacillus bacteria, are harmless to crops and have no pathogenicity to human and animals;
(5) the activity of exo-beta-1, 4-glucanase reaches 12.548U, the activity of endo-beta-1, 4-glucanase reaches 13.027U, the activity of beta-glucosidase reaches 24.209U, the activity of neutral xylanase reaches 541.782U, the activity of filter paper enzyme reaches 0.605U, the activity of neutral protease reaches 328.180U, the activity of beta-amylase reaches 0.181U, and the activity of urease reaches 2.795U.
5. A preparation method of a high-temperature-resistant composite microbial inoculum for degrading livestock and poultry manure is characterized by comprising the following steps:
(1) respectively inoculating activated strains of bacillus NJAU-ND8 and Geobacillus stearothermophilus B5 into an LB liquid culture medium;
(2) The liquid medium was incubated at 55 ℃ at 170r/min for 24 hours:
(3) after the cultivation is finished, adjusting OD value with sterile water to make the concentration of each bacterial liquid identical to 109CFU/ml；
(4) And finally, uniformly mixing according to the volume ratio of 1:1 to form the composite microbial inoculum.
6. The high-temperature-resistant livestock and poultry manure degrading composite microbial inoculum according to claim 3, wherein the composite microbial inoculum is applied to livestock and poultry manure degradation.
7. The microbial composition of claim 1, wherein the bacillus NJAU-ND8 is deposited in the China general microbiological culture Collection center (CGMCC) with a collection number of CGMCC No. 16737.
8. The microbial composition of claim 1, wherein the Geobacillus stearothermophilus B5 is deposited in the China general microbiological culture Collection center (CGMCC) with the deposition number of CGMCC No. 14935.
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