Microbial agent for decomposing cow dung and application thereof

Abstract

The invention discloses a microbial inoculum for decomposing cow dung and application of the microbial inoculum, and belongs to the technical field of biological fermentation. The microbial agent is prepared by the following steps: fermenting geobacillus stearothermophilus, trichoderma longibrachiatum, bacillus megatherium, bacillus amyloliquefaciens, bacillus subtilis and candida utilis to obtain a composite strain fermentation bacterial liquid, adding an adsorbent to obtain a mixed material, and then adding a protective agent. The protective agent relieves the condition that the viable count of thalli and the fermentation activity are reduced due to high-temperature stress, oxidative stress and dehydration stress in the fermentation process; the adsorbent enables thalli to be better protected in the drying and later preservation processes of the microbial inoculum, and the viable count in the finished microbial inoculum is ensured. According to the invention, six strains are combined for use, and the six strains realize better synergy in a mixed culture and fermentation manner, so that complementary advantages are achieved, and the composting fermentation effect is better.

Description

Technical Field

[0001] The invention relates to the technical field of biological fermentation, in particular to a bacterial agent for decomposing cow dung and application thereof. Background Art

[0002] Cow dung is rich in organic matter (14.5%), nitrogen (N) (0.30-0.45%), phosphorus (P2O5) (0.15-0.25%), and potassium (K2O) (0.10-0.15%). Through decomposition, these nutrients can be converted into a form that is more easily absorbed by plants and return to the soil, promoting ecological circulation and sustainable agricultural development. However, direct discharge of untreated cow dung can cause water pollution, soil acidification, and air odor. Therefore, decomposing cow dung can not only effectively reduce the number of harmful microorganisms and parasites and reduce environmental risks, but also promote nutrient circulation and improve soil structure.

[0003] However, compared with other livestock, cow dung has the lowest organic matter and nutrient content. At the same time, it has a fine texture, high fiber content, high water content, slow decomposition, low calorific value, and is a slow-acting fertilizer. In the process of microbial fermentation and decomposition of cow dung, the temperature limit often leads to incomplete decomposition, thereby reducing the fermentation efficiency; in addition, due to the low decomposition temperature, some pathogens in cow dung cannot be completely killed, which poses an ecological safety hazard. In the face of the above problems, in recent years, people have continuously explored new high-temperature fermentation strains and achieved corresponding achievements, laying the foundation for the efficient decomposition of livestock and poultry manure. However, since most of the microorganisms used to decompose livestock and poultry manure are mesophilic bacteria, even if strains with high temperature tolerance are screened out, the limitation of decomposition temperature cannot be completely solved. If the pathogens in cow dung are to be completely killed during the decomposition process, it will inevitably lead to the inactivation of some bacterial agents, thereby affecting the decomposition efficiency. Summary of the invention

[0004] The purpose of the present invention is to provide a bacterial agent for decomposing cow dung and its application, so as to solve the problems existing in the above-mentioned prior art. The bacterial agent product provided by the present invention is stable, has high composting fermentation efficiency and good effect.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The invention provides a bacterial agent for decomposing cow dung. The bacterial agent is prepared by fermenting Geobacillus stearothermophilus, Trichoderma longibrachiatum, Bacillus megaterium, Bacillus amyloliquefaciens, Bacillus subtilis and Candida utilis to obtain a composite bacterial fermentation liquid, adding an adsorbent to obtain a mixed material, and then adding a protective agent to obtain the mixed material.

[0007] The components of the adsorbent include light calcium carbonate, corn starch and bran;

[0008] The protective agent comprises mannitol, citric acid and glycerol.

[0009] Furthermore, in the adsorbent, the mass ratio of light calcium carbonate, corn starch and bran is 1:1:1.

[0010] Furthermore, the mass ratio of the adsorbent to the composite bacterial fermentation liquid is (1-2):1.

[0011] Furthermore, in the protective agent, the mass ratio of mannitol, citric acid and glycerol is 1:(1-2):(1-2).

[0012] Furthermore, the amount of the protective agent added is 10%-15% of the mass of the mixture.

[0013] Furthermore, after adding the protective agent, a drying step is also included.

[0014] Furthermore, the drying temperature is 34-45°C.

[0015] Furthermore, the water content of the bacterial agent is 10%-20%.

[0016] The invention also provides application of the bacterial agent in decomposing cow dung.

[0017] The invention also provides application of the bacterial agent in improving the decomposition efficiency of cow dung.

[0018] The present invention discloses the following technical effects:

[0019] In the bacterial agent provided by the present invention, six strains are used in combination in a mixed culture and fermentation manner, so that the six bacteria achieve better synergy and complement each other's advantages, thereby achieving better composting and fermentation effects. The preparation process of the bacterial agent adopts a mixed culture method, which is simpler, less prone to infection by miscellaneous bacteria, and simplifies the process.

[0020] The characteristic of Geobacillus stearothermophilus that it can produce high-temperature amylase ensures that the biological enzyme will not become ineffective due to excessive temperature during the composting heating process. At the same time, mannitol and citric acid are added to the protective agent during the preparation of the microbial agent. Mannitol, as a filler, has the function of regulating osmotic pressure and can prevent the effective ingredients from volatilizing with water vapor during the fermentation heating process, thereby alleviating the decrease in the number of live bacteria and fermentation activity caused by high temperature stress, oxidative stress and dehydration stress during the fermentation process. The combination of mannitol and citric acid, on the one hand, improves the survival rate of the bacteria during the fermentation process, and on the other hand, gives full play to the antioxidant effect of citric acid, making the obtained microbial agent product more stable.

[0021] The present invention uses light calcium carbonate, corn starch and bran as adsorbents in the process of preparing the microbial agent, so that the bacterial body is better protected during the drying and later storage of the microbial agent, thereby ensuring the number of live bacteria in the finished microbial agent. DETAILED DESCRIPTION

[0022] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0023] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0024] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0025] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0026] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0027] The microbial strains used in the embodiments of the present invention are as follows:

[0028] Geobacillus stearothermophilus, strain number CICC22988; Trichoderma longibrachiatum, strain number CICC 40144; Bacillus megaterium, strain number CICC 20611, all come from China Industrial Microbiological Culture Collection Center.

[0029] Bacillus amyloliquefaciens, strain number CGMCC 1.1414; Bacillus subtilis, strain number CGMCC 1.14985; Candida utilis, strain number CGMCC 2.2878, all come from China General Microbiological Culture Collection Center.

[0030] The above strains can be purchased from the above depository institutions.

[0031] Example 1

[0032] A bacterial agent for decomposing cow dung, the preparation method of which comprises the following steps:

[0033] (1) Bacteria activation:

[0034] Bacillus stearothermophilus, Trichoderma longibrachiatum, Bacillus amyloliquefaciens, Bacillus subtilis, Bacillus megaterium and Candida utilis were cultured and activated in an activation medium (sucrose 15 g / L, peptone 8 g / L, beef extract 8 g / L, dipotassium hydrogen phosphate 2 g / L, sodium acetate 5 g / L, Tween 80 1 g / L, magnesium sulfate 0.45 g / L, manganese sulfate 0.15 g / L, pH 6.5) and cultured at 35°C for 24 hours to obtain an activated bacterial solution of the composite strain.

[0035] (2) Expansion culture of bacterial liquid:

[0036] The activated bacterial solution of the composite strain obtained in step (1) was expanded and cultured in a fermenter at an inoculation rate of 2% for 24 h until the effective viable count reached 1×10 9 cfu / mL, and obtain the composite bacterial fermentation liquid.

[0037] (3) Preparation of adsorbent:

[0038] Mix light calcium carbonate, corn starch and bran in equal proportions, grind into powder, pass through a 30-mesh sieve, sterilize and dry to make an adsorbent for use.

[0039] (4) Mixing:

[0040] The composite bacterial fermentation liquid prepared in step (2) and the adsorbent prepared in step (3) are mixed and stirred at a mass ratio of 1:2, so that the bacterial liquid is uniformly adsorbed on the adsorbent to obtain a mixed material.

[0041] (5) Add protective agent:

[0042] Mannitol, citric acid and glycerol are mixed in a mass ratio of 1:1:2 to prepare a protective agent, 10 wt% of the protective agent is added to the mixture obtained in step (4), stirred evenly, and placed in a low-temperature drying oven at 35-45°C to dry to a moisture content of 15%, thereby obtaining a bacterial agent for decomposing cow dung.

[0043] Example 2

[0044] A bacterial agent for decomposing cow dung, the preparation method of which comprises the following steps:

[0045] (1) Bacteria activation:

[0046] Bacillus stearothermophilus, Trichoderma longibrachiatum, Bacillus amyloliquefaciens, Bacillus subtilis, Bacillus megaterium and Candida utilis were cultured and activated in an activation medium (sucrose 15 g / L, peptone 8 g / L, beef extract 8 g / L, dipotassium hydrogen phosphate 2 g / L, sodium acetate 5 g / L, Tween 80 1 g / L, magnesium sulfate 0.45 g / L, manganese sulfate 0.15 g / L, pH 6.5) and cultured at 35°C for 24 hours to obtain an activated bacterial solution of the composite strain.

[0047] (2) Expansion culture of bacterial liquid:

[0048] The activated bacterial solution of the composite strain obtained in step (1) was expanded and cultured in a fermenter at an inoculation rate of 1.5% for 36 h until the effective viable count reached 1×10 10 cfu / mL, and obtain the composite bacterial fermentation liquid.

[0049] (3) Preparation of adsorbent:

[0050] Mix light calcium carbonate, corn starch and bran in equal proportions, grind into powder, pass through a 30-mesh sieve, sterilize and dry to make an adsorbent for use.

[0051] (4) Mixing:

[0052] The composite bacterial fermentation liquid prepared in step (2) and the adsorbent prepared in step (3) are mixed and stirred at a mass ratio of 1:1.5, so that the bacterial liquid is uniformly adsorbed on the adsorbent to obtain a mixed material.

[0053] (5) Add protective agent:

[0054] Mannitol, citric acid and glycerol are mixed in a mass ratio of 1:2:1.5 to prepare a protective agent, 15 wt% of the protective agent is added to the mixture obtained in step (4), stirred evenly, and placed in a low-temperature drying oven at 35-45° C. to dry to a moisture content of 10%, thereby obtaining a bacterial agent for decomposing cow dung.

[0055] Example 3

[0056] A bacterial agent for decomposing cow dung, the preparation method of which comprises the following steps:

[0057] (1) Bacteria activation:

[0058] Bacillus stearothermophilus, Trichoderma longibrachiatum, Bacillus amyloliquefaciens, Bacillus subtilis, Bacillus megaterium and Candida utilis were cultured and activated in an activation medium (sucrose 15 g / L, peptone 8 g / L, beef extract 8 g / L, dipotassium hydrogen phosphate 2 g / L, sodium acetate 5 g / L, Tween 80 1 g / L, magnesium sulfate 0.45 g / L, manganese sulfate 0.15 g / L, pH 6.5) and cultured at 35°C for 24 hours to obtain an activated bacterial solution of the composite strain.

[0059] (2) Expansion culture of bacterial liquid:

[0060] The activated bacterial solution of the composite strain obtained in step (1) was expanded and cultured in a fermenter at an inoculation rate of 1% for 30 h until the effective viable count reached 1×10 8 cfu / mL, and obtain the composite bacterial fermentation liquid.

[0061] (3) Preparation of adsorbent:

[0062] Mix light calcium carbonate, corn starch and bran in equal proportions, grind into powder, pass through a 30-mesh sieve, sterilize and dry to make an adsorbent for use.

[0063] (4) Mixing:

[0064] The composite bacterial fermentation liquid prepared in step (2) and the adsorbent prepared in step (3) are mixed and stirred at a mass ratio of 1:1, so that the bacterial liquid is uniformly adsorbed on the adsorbent to obtain a mixed material.

[0065] (5) Add protective agent:

[0066] Mannitol, citric acid and glycerol are mixed in a mass ratio of 1:1.5:1 to prepare a protective agent, 10 wt% of the protective agent is added to the mixture obtained in step (4), stirred evenly, and placed in a low-temperature drying oven at 35-45° C. to dry to a moisture content of 20%, thereby obtaining a bacterial agent for decomposing cow dung.

[0067] Comparative Example 1

[0068] Compared with Example 1, the difference is that in step (1), the bacterial strain does not contain Bacillus stearothermophilus.

[0069] Comparative Example 2

[0070] Compared with Example 1, the difference is that in step (5), the protective agent does not contain mannitol.

[0071] Comparative Example 3

[0072] Compared with Example 1, the difference is that in step (5), the mannitol in the protective agent is replaced by an equal amount of potassium sorbate.

[0073] Comparative Example 4

[0074] Compared with Example 1, the difference is that in step (5), mannitol, citric acid and glycerol are mixed in a mass ratio of 1:1:2 to prepare a protective agent;

[0075] The mixed material obtained in step (4) is placed in a low-temperature drying oven and dried at 35-45° C. to a moisture content of 15%. Then, 10 wt % of a protective agent is added thereto and stirred evenly to obtain a bacterial agent for decomposing cow dung.

[0076] Effect verification

[0077] Experimental location: A large cattle farm in Yinchuan City, Ningxia Hui Autonomous Region, with an ambient temperature of 22℃±4℃.

[0078] 7 tons of cow dung, with a moisture content of 83.5%, divided into 7 piles, each with 1 ton, numbered 1, 2, 3, 4, 5, 6, 7;

[0079] A mixture of chopped straw, sawdust, rice husk and peat soil was added to each pile of cow dung, so that the C / N ratio of the seven piles of cow dung was 20:1 and the moisture content was 55-60%; then the bacterial agents prepared in Examples 1-3 and Comparative Examples 1-4 were added respectively according to a mass ratio of bacterial agent mass: total compost mass = 1.5:1000, first mixed with a small amount of cow dung, and then gradually mixed into the entire cow dung pile for composting and fermentation.

[0080] During the composting process, the temperature changes and the disappearance of odor were recorded. The experimental results are shown in Table 1:

[0081] Table 1 Fermentation experimental data of different bacterial agents decomposing cow dung

[0082] serial number Bacterial agents used Time to heat up to 65℃ (h) Time for odor to disappear (h) Composting is finished (d) No. 1 Example 1 40.2 55.3 11 No. 2 Example 2 41.1 57.5 12 No.3 Example 3 40.7 56.2 12 No. 4 Comparative Example 1 53.5 75.5 15 No. 5 Comparative Example 2 50.3 73.8 15 No. 6 Comparative Example 3 51.6 75.2 16 No.7 Comparative Example 4 47.8 67.1 14

[0083] The data in Table 1 show that the bacterial agents prepared in Examples 1 to 3 and Comparative Examples 1 to 4 have obvious differences in the effects of decomposing cow dung for composting. Compared with Comparative Examples 1 to 4, the bacterial agents in Examples 1 to 3 effectively shorten the heating time, odor disappearance time and composting completion time of cow dung composting.

[0084] The samples of No. 1 to No. 7 composts after fermentation were taken and the main physical and chemical indicators were tested. The results are shown in Table 2.

[0085] Table 2 Main physical and chemical indicators of samples after composting

[0086] serial number pH Conductivity Organic matter (%) Total nitrogen (%) Total phosphorus (%) Total potassium (%) No. 1 5.7 10.57 67.1 6.89 2.31 3.62 No. 2 5.6 10.13 66.5 6.77 2.23 3.52 No.3 5.9 10.38 66.7 6.45 2.28 3.45 No. 4 8.2 7.34 43.4 3.5 1.38 2.18 No. 5 8.3 7.11 40.1 2.25 1.41 2.23 No. 6 6.6 8.16 53.4 4.84 1.65 2.56 No.7 6.5 8.89 54.5 5.01 1.77 2.84

[0087] It can be found from the data in Table 2 that adding the bacterial agents prepared in Examples 1 to 3 can significantly improve key indicators such as organic matter and total nitrogen content compared to Comparative Examples 1 to 4.

[0088] In order to verify the growth-promoting effect of cow dung decomposition on crops, tomato seedling experiments were carried out using the products obtained after composting No. 1 to No. 7.

[0089] In the tomato seedling tray experiment, compost products No. 1 to 7 were used for the fertilizer effect experiment of tomato seedlings. Vermiculite was used as the substrate, water was added until it was moist, and the soaked tomato seeds were placed in it, and then covered with a layer of substrate. Cultivated under room temperature and light conditions, after the seedlings grew two cotyledons after one week, the sample was applied to the substrate, about 1 cm away from the root of the seedlings. Watering was done every day, and the control seedlings were not fertilized, with the same watering management. After 30 days, their growth was recorded, and the growth differences of the seedlings between the treatments were observed. Ten seedlings were randomly sampled for each treatment for measurement. The results are shown in Table 3.

[0090] Table 3 Effects of different treatments of organic fertilizer on the growth of tomato seedlings

[0091] serial number Plant height (cm) Number of leaves Cotyledon length (cm) Cotyledon width (cm) No. 1 15.52 5 2.87 0.79 No. 2 15.35 6 2.63 0.77 No.3 15.51 5 2.79 0.78 No. 4 11.54 3 2.21 0.69 No. 5 10.77 2 2.13 0.67 No. 6 12.32 3 2.32 0.71 No.7 12.85 4 2.38 0.73 CK 10.15 2 2.05 0.65

[0092] From the data in Table 3, it can be found that the tomato seedlings treated with the bacterial agents of Examples 1 to 3 have obvious advantages in average plant height, leaf number and cotyledon size over the tomato seedlings treated with the bacterial agents of Comparative Examples 1 to 4.

[0093] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A bacterial agent for decomposing cow dung, characterized in that: The composite bacterial fermentation liquid is obtained by fermenting Geobacillus stearothermophilus, Trichoderma longibrachiatum, Bacillus megaterium, Bacillus amyloliquefaciens, Bacillus subtilis and Candida utilis, an adsorbent is added to obtain a mixed material, and a protective agent is added to obtain the mixed material; The components of the adsorbent include light calcium carbonate, corn starch and bran; The protective agent comprises mannitol, citric acid and glycerol.

2. The bacterial agent according to claim 1, characterized in that In the adsorbent, the mass ratio of light calcium carbonate, corn starch and bran is 1:1:

1.

3. The bacterial agent according to claim 2, characterized in that The mass ratio of the adsorbent to the composite bacterial fermentation liquid is (1-2):

1.

4. The bacterial agent according to claim 1, characterized in that In the protective agent, the mass ratio of mannitol, citric acid and glycerol is 1:(1-2):(1-2).

5. The bacterial agent according to claim 4, characterized in that The added amount of the protective agent is 10%-15% of the mass of the mixed material.

6. The bacterial agent according to claim 1, characterized in that After adding the protective agent, a drying step is also included.

7. The bacterial agent according to claim 6, characterized in that The drying temperature is 34-45°C.

8. The bacterial agent according to claim 6, characterized in that The water content of the bacterial agent is 10%-20%.

9. Use of the bacterial agent as described in any one of claims 1 to 8 in decomposing cow dung.

10. Use of the microbial agent according to any one of claims 1 to 8 in improving the decomposition efficiency of cow dung.