A combination of bacteria for aerobic composting of chicken manure, an immobilized bacterial agent containing the combination of bacteria, and a preparation method and a use method of the bacterial agent

Abstract

The present invention relates to the technical field of solid waste treatment and resource utilization. The present invention provides a combination of bacteria for aerobic composting of chicken manure, an immobilized bacterial agent containing the combination of bacteria, and a preparation method and a use method of the bacterial agent. The combination of bacteria consists of Bacillus amyloliquefaciens and Bacillus aryabhattai, and the preservation names are GJD2021101 and GWD2022101, respectively, and the preservation numbers are CCTCC M2024689 and CCTCC M2024688, respectively. When the immobilized bacterial agent containing the combination of bacteria is applied to the aerobic composting of chicken manure, it can make the organic matter degradation more complete and reduce NH4 + concentration, reducing NH3 and N2O emissions, greatly improving the seed germination index, and achieving the effect of promoting compost maturity while controlling pollutant gas emissions.

Description

Technical Field

[0001] The invention relates to the technical field of solid waste treatment and resource utilization, and in particular to a combined bacteria for aerobic composting of chicken manure, an immobilized bacterial agent containing the combined bacteria, and a preparation method and a use method of the bacterial agent. Background Art

[0002] The effective treatment and resource utilization of livestock and poultry manure is an inevitable requirement for sustainable development, but there are still problems such as low comprehensive utilization rate and insufficient harmlessness rate. Aerobic composting is an important way to utilize livestock and poultry manure resources. It can not only effectively treat livestock and poultry manure, but its compost products can also increase the content of soil humus and water-soluble organic matter, which is an important measure to achieve green agricultural development.

[0003] Aerobic composting is the process of decomposing unstable organic matter into safe and stable humus substances through microbial fermentation and a series of physical and chemical reactions under certain water content, carbon-nitrogen ratio and ventilation conditions. In order to ensure the normal progress of composting, high-carbon source auxiliary materials such as straw, garden prunings and sawdust are added to compost with livestock and poultry manure. However, ammonia (NH 3 ) and nitrous oxide (N 2 O), etc., which not only causes the loss of nitrogen in the compost, reduces the maturity of the compost and the quality of the product, but also exacerbates the greenhouse effect. The loss of nitrogen in aerobic composting accounts for about 40%, with NH 3 About 10% of nitrogen (NH 3 -N) accounts for 80-95% of the total nitrogen, with N 2 Nitrogen lost in the form of O (N 2 ON) accounts for about 4% of the total nitrogen.

[0004] Chicken manure has the advantages of high water content, low carbon-nitrogen ratio, and sufficient nitrogen. Although it is an ideal raw material for aerobic composting, it is more likely to cause nitrogen loss. 3 -N loss was 24.2%, significantly higher than pig manure (18.2%), sheep manure (18.6%) and cow manure (15.7%); 2 ON loss is 1.0%, which is higher than 0.7% of sheep manure and 0.6% of cow manure (Yuan Jing, Liu Yan, Tang Ruolan, et al. Review of carbon and nitrogen losses and greenhouse gas emissions during livestock and poultry manure composting [J]. Journal of Agricultural Environment Science, 2021, 40(11): 2428-2438+2590.). In addition, chicken manure is a highly toxic manure (high concentration of ammonium ions (NH 4 +)), its composting effect is not stable enough, and the seed germination index (GI) is difficult to meet the basic requirement of greater than 70% stipulated in the national standard "Organic Fertilizer" (NY / T525-2021).

[0005] Adding microbial agents is an effective means to promote composting maturity, and compared with liquid agents, solid agents (immobilized agents) are more conducive to the preservation and storage of bacteria. There have been some studies on the use of microbial agents to regulate the composting process. For example, there is an immobilized agent and its preparation method and application (CN113736708A) to accelerate the degradation of organic matter. However, previous studies mainly focused on the rapid start-up of composting and the promotion of composting maturity, and few studies have been conducted on controlling NH while promoting maturity. 3 and N 2 The promotion of compost maturity by microbial agents is mainly reflected in the adjustment of the microbial community structure in the early stage of composting, increasing the relative abundance of Bacillus and other bacterial genera, accelerating the utilization of compost materials, and the large amount of heat generated by microbial life activities promotes the temperature rise of the pile, thereby increasing the activity of thermophilic bacteria in the high temperature period of composting, and accelerating the composting process as a whole. However, the microbial agents currently studied, while promoting compost maturity, actually increase NH 3 and N 2 The emission of O is not controlled while pursuing the composting effect. This will not only cause secondary pollution to the environment, but also lead to a large loss of nitrogen and reduce the quality of compost. Summary of the invention

[0006] The object of the present invention is to provide a combination of bacteria for aerobic composting of chicken manure, an immobilized bacterial agent containing the combination of bacteria, and a preparation method and a use method of the bacterial agent.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a combination of bacteria for aerobic composting of chicken manure, wherein the combination of bacteria consists of Bacillus amyloliquefaciens and Bacillus aggradiensis;

[0009] The deposited name of the Bacillus amyloliquefaciens is GJD2021101, the Latin name is Bacillusamyloliquefaciens, the deposit date is April 15, 2024, the depository is China Center for Type Culture Collection, and the deposit number is CCTCC M2024689;

[0010] The preservation name of the Bacillus aryabhattai is GWD2022101, the Latin name is Bacillus aryabhattai, the preservation date is April 15, 2024, the preservation unit is China Center for Type Culture Collection, and the preservation number is CCTCCM2024688.

[0011] The present invention also provides an application of the combined bacteria in preparing an immobilized bacterial agent.

[0012] The present invention also provides an immobilized bacterial agent containing the combined bacteria, wherein the total effective viable count of Bacillus amyloliquefaciens and Bacillus aggregatii in the immobilized bacterial agent is 1×10 8 ~5×10 8 CFU / g.

[0013] The present invention also provides a method for preparing the immobilized bacterial agent, comprising the following steps:

[0014] (1) inoculating Bacillus amyloliquefaciens and Bacillus arginii into liquid culture medium for cultivation respectively, thereby obtaining a liquid culture medium of Bacillus amyloliquefaciens and a liquid culture medium of Bacillus arginii;

[0015] (2) mixing the liquid inoculum of Bacillus amyloliquefaciens, trehalose and attapulgite uniformly, and then drying the mixture to obtain a dried mixed material A containing Bacillus amyloliquefaciens;

[0016] (3) uniformly mixing the liquid inoculum of Bacillus argyi with trehalose and attapulgite, and then drying the mixture to obtain a dried mixed material B containing Bacillus argyi;

[0017] (4) Mixing the mixed material A and the mixed material B to obtain an immobilized bacterial agent.

[0018] Preferably, the liquid culture medium in step (1) is cultured for 6 to 10 hours, and the liquid culture medium is LB liquid culture medium.

[0019] Preferably, the mass ratio of trehalose, attapulgite and liquid Bacillus amyloliquefaciens in step (2) is 1:6-10:23-43, and the mass ratio of trehalose, attapulgite and liquid Bacillus amyloliquefaciens in step (3) is 1:6-10:23-43.

[0020] Preferably, the drying temperatures in step (2) and step (3) are independently 40 to 50° C., and the drying times are independently 8 to 12 hours.

[0021] Preferably, in step (4), the mass ratio of the mixed material A to the mixed material B is 1:0.5-2.

[0022] The invention also provides an application of the immobilized bacterial agent in aerobic composting of chicken manure.

[0023] The present invention also provides a method for using the immobilized bacterial agent, comprising the following steps:

[0024] (1) mixing chicken manure and straw in a mass ratio of 7-10:1-2 to obtain a mixed composting raw material;

[0025] (2) The immobilized bacterial agent and the mixed composting raw materials are uniformly mixed in a mass ratio of 0.5 to 2:100 for aerobic composting.

[0026] Beneficial effects of the present invention:

[0027] The present invention provides a combination of bacteria for aerobic composting of chicken manure, an immobilized bacterial agent containing the combination of bacteria, and a preparation method and a use method of the bacterial agent. The combination of bacteria can be applied to the preparation of a bacterial agent for aerobic composting of chicken manure, and the immobilized bacterial agent containing the combination of bacteria can effectively reduce NH during the composting process while promoting the maturity of the compost. 3 and N 2 The immobilized bacterial agent of the present invention uses the physical porous material attapulgite as a carrier, and maintains NH 3 The relative abundance of transformed microorganisms can achieve rapid temperature rise and extend the high temperature period of aerobic composting of chicken manure, fully degrade organic matter, make the compost fully decomposed, control the emission of pollutant gases during the composting process, and improve the quality of compost, which has a relatively broad application prospect.

[0028] Collection Instructions

[0029] Bacillus amyloliquefaciens, deposited name GJD2021101, Latin name Bacillusamyloliquefaciens, deposit date: April 15, 2024, deposited by: China Center for Type Culture Collection, address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, deposit number: CCTCC M2024689;

[0030] Bacillus aryabhattai, the deposited name is GWD2022101, the Latin name is Bacillus aryabhattai, the deposit date is April 15, 2024, the depository unit is China Center for Type Culture Collection, the address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, the deposit number is CCTCC M2024688. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a flow chart for strain screening, performance testing and strain identification.

[0032] Figure 2 The present invention is the preparation process of the immobilized bacterial agent.

[0033] Figure 3 These are the results of enzyme activity determination of various types of degradation bacteria of Bacillus amyloliquefaciens and Bacillus arginina, wherein A in the figure represents the cellulase activity of Bacillus amyloliquefaciens and Bacillus arginina; B in the figure represents the amylase activity of Bacillus amyloliquefaciens and Bacillus arginina; C in the figure represents the protease activity of Bacillus amyloliquefaciens and Bacillus arginina; and D in the figure represents the lipase activity of Bacillus amyloliquefaciens and Bacillus arginina.

[0034] Figure 4 This figure shows the effects of inoculation with immobilized bacteria and non-inoculation with immobilized bacteria on the seed germination index and temperature during chicken manure composting. Figure A represents the change of seed germination index with composting time; Figure B represents the change of temperature with composting time.

[0035] Figure 5 Figure 3 shows the effects of inoculation with immobilized bacteria and non-inoculation with immobilized bacteria on ammonium ions, organic matter content, ammonia emissions and nitrous oxide emissions during chicken manure composting. Figure A represents the change of ammonium ion content with composting time; Figure B represents the change of organic matter content with composting time; Figure C represents the change of cumulative ammonia emissions with composting time; Figure D represents the change of cumulative nitrous oxide emissions with composting time.

[0036] Figure 6 This is a comparison of the cumulative ammonia emissions, nitrous oxide emissions, organic matter degradation rate and ammonium ion content at the end of the 35th day of chicken manure composting between the treatments inoculated with immobilized bacteria and those not inoculated with immobilized bacteria. A in the figure represents the cumulative ammonia emissions at the end of composting; B in the figure represents the cumulative nitrous oxide emissions at the end of composting; C in the figure represents the degradation rate of organic matter at the end of composting; D in the figure represents the ammonium ion content at the end of composting DETAILED DESCRIPTION

[0037] The present invention provides a combination of bacteria for aerobic composting of chicken manure, wherein the combination of bacteria consists of Bacillus amyloliquefaciens and Bacillus aggradiensis.

[0038] The deposited name of the Bacillus amyloliquefaciens is GJD2021101, the Latin name is Bacillusamyloliquefaciens, the deposit date is April 15, 2024, the depository is China Center for Type Culture Collection, and the deposit number is CCTCC M2024689;

[0039] The preservation name of the Bacillus aryabhattai is GWD2022101, the Latin name is Bacillus aryabhattai, the preservation date is April 15, 2024, the preservation unit is China Center for Type Culture Collection, and the preservation number is CCTCCM2024688.

[0040] The present invention also provides an application of the combined bacteria in preparing an immobilized bacterial agent.

[0041] The present invention also provides an immobilized bacterial agent containing the combined bacteria, wherein the total effective viable count of Bacillus amyloliquefaciens and Bacillus aggregatii in the immobilized bacterial agent is 1×10 8 ~5×10 8 CFU / g.

[0042] In the present invention, the total effective viable count of Bacillus amyloliquefaciens and Bacillus arnoldii in the immobilized bacterial agent is preferably 2×10 8 ~4×10 8 CFU / g, more preferably 3×10 8 CFU / g.

[0043] The present invention also provides a method for preparing the immobilized bacterial agent, comprising the following steps:

[0044] (1) inoculating Bacillus amyloliquefaciens and Bacillus arginii into liquid culture medium for cultivation respectively, thereby obtaining a liquid culture medium of Bacillus amyloliquefaciens and a liquid culture medium of Bacillus arginii;

[0045] (2) mixing the liquid inoculum of Bacillus amyloliquefaciens, trehalose and attapulgite uniformly, and then drying the mixture to obtain a dried mixed material A containing Bacillus amyloliquefaciens;

[0046] (3) uniformly mixing the liquid inoculum of Bacillus argyi with trehalose and attapulgite, and then drying the mixture to obtain a dried mixed material B containing Bacillus argyi;

[0047] (4) Mixing the mixed material A and the mixed material B to obtain an immobilized bacterial agent.

[0048] In the present invention, the culturing time of the liquid culture medium in step (1) is preferably 6 to 10 hours, more preferably 7 to 9 hours, and even more preferably 8 hours, and the liquid culture medium is preferably LB liquid culture medium.

[0049] In the present invention, the mass ratio of trehalose, attapulgite and Bacillus amyloliquefaciens liquid inoculant in step (2) is preferably 1:6-10:23-43, more preferably 1:8-9:28-38, and further preferably 1:8:33; the mass ratio of trehalose, attapulgite and Bacillus amyloliquefaciens liquid inoculant in step (3) is preferably 1:6-10:23-43, more preferably 1:8-9:28-38, and further preferably 1:8:33.

[0050] In the present invention, the temperature of the drying treatment in step (2) and step (3) is independently preferably 40-50°C, and further independently preferably 42-48°C, and the drying time is independently preferably 8-12h, and further independently preferably 9-11h.

[0051] In the present invention, the mass ratio of the mixed material A to the mixed material B in step (4) is preferably 1:0.5-2, more preferably 1:0.8-1.6, and even more preferably 1:1.2.

[0052] The invention also provides an application of the immobilized bacterial agent in aerobic composting of chicken manure.

[0053] The present invention also provides a method for using the immobilized bacterial agent, comprising the following steps:

[0054] (1) mixing chicken manure and straw in a mass ratio of 7-10:1-2 to obtain a mixed composting raw material;

[0055] (2) The immobilized bacterial agent and the mixed composting raw materials are uniformly mixed in a mass ratio of 0.5 to 2:100 for aerobic composting.

[0056] In the present invention, the mass ratio of chicken manure to straw in step (1) is preferably a mass ratio in terms of wet weight.

[0057] In the present invention, the mass ratio of chicken manure to straw in step (1) is preferably 8-9:1-2, and more preferably 85:15.

[0058] In the present invention, the mass ratio of the immobilized bacterial agent to the mixed composting raw material in step (2) is preferably a mass ratio in terms of wet weight.

[0059] In the present invention, the mass ratio of the immobilized bacterial agent to the mixed composting raw material in step (2) is preferably 0.8-1.6:100, more preferably 1-1.4:100, and even more preferably 1.2:100.

[0060] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0061] Example 1 Bacteria culture

[0062] a. Source of strains: collected from the high-temperature compost in the laboratory of the new teaching building of China Agricultural University. After the high-temperature compost was mixed evenly, the compost sample was placed in a ziplock bag and stored at 4°C.

[0063] b. Culture medium preparation:

[0064] Cellulose-degrading bacteria enrichment medium: peptone 1g, yeast powder 1g, NaCl 0.5g, MgSO 4 7H 2 O 0.5g, KH 2 PO 4 7H 2 O 0.5 g, FeSO 4 7H 2 O 0.025g, MnSO 4 ·4H 2 O 0.025 g, CMC-Na 10 g, H 2 O 1000mL, 1mol / LNaOH adjust the pH to 7.0.

[0065] Cellulose degrading bacteria screening plate culture medium: CMC 10.0g, yeast powder (paste) 1.0g, (NH 4 ) 2 SO 4 2.5 g, K 2 HPO 4 ·3H 2 O 0.25 g, NaCl 0.1 g, MgSO 4 7H 2 O 0.125 g, FeSO 4 7H 2 O 0.025g, MnSO 4 ·4H 2 O0.025g, H 2 O 1000ml, agar 18g / L, 1mol / LNaOH to adjust pH to 7.0.

[0066] LB plate medium: peptone 10g, yeast powder 5g, NaCl 10g, H 2 O 1000ml, agar 18g / L, adjust pH to 7.0 with 1mol / LNaOH.

[0067] c. Enrichment of degradation bacteria: Take 1g of the sample obtained in step a and put it in a 100ml conical flask, add 50ml of distilled water and shake it at 30℃, 150r / min for 30min. After standing for 15min, take 10ml of the supernatant and transfer it to 100ml of cellulose-degrading bacteria enrichment liquid culture medium on a sterile workbench, shake it continuously at 35℃, 150r / min, and observe the disintegration of the filter paper. When the filter paper under the liquid surface turns yellow and disintegrates, take 10ml of the culture medium and transfer it to fresh cellulose-degrading bacteria enrichment medium and continue shaking culture. Repeat the operation for 3 generations.

[0068] d. Purification of degradation bacteria: dilute the enriched culture solution and spread it on LB medium for culture, pick a single colony and streak it, and purify it for 3 generations to obtain pure bacteria.

[0069] e. Identification of bacterial species: Gram staining and molecular biology 16S rDNA sequencing were performed on the obtained bacterial species. It was identified that both strains were Gram-positive bacteria, belonging to the genus Bacillus amyloliquefaciens and the genus Bacillus agglomerans, respectively.

[0070] f. The obtained strains are preserved. The deposited name of the Bacillus amyloliquefaciens is GJD2021101, which is Bacillus amyloliquefaciens in Latin. The deposit date is April 15, 2024. The depositor is China Center for Type Culture Collection. The deposit number is CCTCC M2024689. The deposited name of the Bacillus aryabhattai is GWD2022101, which is Bacillus aryabhattai in Latin. The deposit date is April 15, 2024. The depositor is China Center for Type Culture Collection. The deposit number is CCTCC M2024688.

[0071] Example 2 Bacterial performance determination

[0072] The activities of amylase, lipase, protease and cellulase of Bacillus amyloliquefaciens and Bacillus amyloliquefaciens were determined respectively. The determination methods are shown in Table 1. The determination results are shown in Table 1. Figure 3 shown.

[0073] The results showed that the Bacillus amyloliquefaciens and Bacillus aggrad had certain degradation capabilities for various organic matters. Compared with the two, the cellulase activity, lipase activity and protease activity of Bacillus amyloliquefaciens were better than those of Bacillus aggradfaciens, while the lipase activity of Bacillus aggradfaciens was better than that of Bacillus amyloliquefaciens.

[0074] Table 1 Enzyme activity determination method

[0075] Enzyme type to be tested Determination method Amylase 3,5-Dinitrosalicylic acid reduction method Lipase GB / T23535-2009 Acid-base titration Protease Folin-phenol method Cellulase DNS colorimetry

[0076] Table 2 Enzyme activities of Bacillus amyloliquefaciens and Bacillus arnoldii

[0077]

[0078] Example 3 Preparation of immobilized bacterial agent

[0079] The Bacillus amyloliquefaciens and Bacillus arnoldii obtained in Example 1 were used to prepare an immobilized bacterial agent, and the specific steps were as follows:

[0080] (1) Preparation of carrier: sterilize the attapulgite at 121° C. for 25 min to obtain a sterile attapulgite;

[0081] (2) Preparation of culture medium: 2 parts of peptone, 2 parts of sodium chloride, 1 part of yeast extract and 100 parts of water were mixed and sterilized at 121°C for 25 min to obtain a sterile culture medium (LB liquid medium);

[0082] (3) Cultivation of liquid bacterial agent: Bacillus amyloliquefaciens and Bacillus arnoldii were inoculated into LB liquid culture medium respectively, and cultured in a constant temperature shaker at 35°C for 8 h to obtain liquid bacteria A and B;

[0083] (4) Bacterial cell fixation: The liquid bacterial solutions A and B obtained in step (3) are mixed evenly in a ratio of 1 part trehalose, 8 parts sterile attapulgite obtained in step (1), and 33 parts liquid bacterial agent to obtain bacterial cells C and D;

[0084] (5) Preparation of solid bacterial agent: Dry the bacterial cells C and D obtained in step (4) in an oven at 45°C for 10 h;

[0085] (6) Grind the C and D dried in step (5) and mix them in a ratio of 1 part C to 1 part D to obtain an immobilized bacterial agent.

[0086] The immobilized bacterial agent prepared in this example was used as the experimental group.

[0087] Example 4 Method for aerobic composting using immobilized bacterial agents

[0088] The specific steps are as follows:

[0089] (1) chicken manure and corn stalks are mixed evenly in a ratio of 85:15 (wet weight) to obtain material 1;

[0090] (2) Material 1 and the prepared immobilized bacterial agent were mixed evenly in a ratio of 100:1.2 (wet weight). After mixing, they were placed in an aerobic composting fermentation tank and the ventilation rate was set to 0.25 L min. -1 kg -1 DM;

[0091] (3) The compost is turned over every 7 days. After 35 days, the composting is finished and the compost product is obtained.

[0092] Example 5 Determination of the effect of aerobic composting of chicken manure with immobilized bacterial agents

[0093] According to the aerobic composting method described in Example 4, the actual application effect of the immobilized bacterial agent was determined. The treatment without inoculation of the immobilized bacterial agent was used as the blank control (CK). The control group and the experimental group were tested twice, respectively. The obtained data were combined for subsequent composting effect analysis.

[0094] (1) Determination of total dissolved carbon (TDC), dissolved organic carbon (DOC) and total dissolved nitrogen (TDN) contents in aerobic composting of chicken manure

[0095] The TDC, DOC and TDN contents of the composts of the control group and the experimental group were measured at the beginning of composting (D0), the 7th day of composting (D7), the 21st day of composting (D21) and the end of composting (D35). The results are shown in the table below.

[0096] Table 3 Determination of total dissolved carbon (TDC) during composting

[0097]

[0098] Table 4 Determination of dissolved organic carbon (DOC) during composting

[0099]

[0100] Table 5 Determination of total dissolved nitrogen (TDN) during composting

[0101]

[0102] During aerobic composting, the contents of total dissolved carbon, dissolved organic carbon, and total dissolved nitrogen can provide information about the decomposition and transformation of organic matter during the composting process. Total dissolved carbon can reflect the decomposition and oxidation process of organic compounds during the composting process. With the change of total dissolved carbon, we can understand the rate of conversion of organic matter into carbon dioxide and the degree of degradation of organic matter in the compost. Dissolved organic carbon is the carbon content of organic matter in water, which can reflect the decomposition and utilization of organic matter during the composting process, as well as the content of organic carbon available for plants in the soil after the composting is completed. Total dissolved nitrogen can provide information about the transformation of nitrogen during the composting process, including processes such as ammoniation and nitrification, and can also help evaluate the loss or retention of nitrogen in the composting system.

[0103] From the above results, it can be seen that for total dissolved carbon, the total dissolved carbon content of the experimental group increased during the treatment process, while that of the control group decreased, indicating that the application of immobilized bacteria promoted the decomposition and transformation of organic matter, and increased the total dissolved carbon content in the compost; for dissolved organic carbon, similar to the results of total dissolved carbon, the dissolved organic carbon content of the experimental group increased, while that of the control group decreased, indicating that immobilized bacteria contributed to the degradation and utilization of organic carbon; for total dissolved nitrogen, the total dissolved nitrogen content of the control group was 9.78 mg / L at the beginning and 6.14 mg / L at the end, while that of the experimental group was 7.78 mg / L at the beginning and 6.41 mg / L at the end. In terms of total dissolved nitrogen, the application of immobilized bacteria reduced the nitrogen loss during chicken manure composting.

[0104] Based on the above analysis, the inoculation of immobilized bacteria can effectively promote the decomposition and transformation of organic matter in aerobic composting, play a positive role in carbon transformation, increase the carbon content in the compost, further promote the formation of dissolved organic carbon, and reduce nitrogen loss during the composting process.

[0105] (2) Determination of temperature of aerobic composting of chicken manure

[0106] During aerobic composting, the compost temperature was measured daily using an automatic thermometer. Figure 4 As shown in B.

[0107] The results showed that in the experimental group inoculated with immobilized bacteria, the compost temperature was maintained above 60℃ from the first day (72.70℃) to the sixteenth day (65.70℃) of composting; in the control group, the compost temperature was maintained above 60℃ from the first day (63.80℃) to the fourth day (62.30℃), and the temperature decreased from the fifth day (59.00℃) to the seventh day (56.50℃), then rose back to above 60℃ and maintained until the thirteenth day (62.30℃), the temperature was 59.60℃ on the fourteenth day, and the temperatures on the fifteenth and sixteenth days were 63.90℃ and 61.50℃ respectively. After that, the compost temperature showed a clear downward trend and then tended to be stable. Comparison of composting temperatures between the experimental group and the control group showed that in the process of chicken manure composting, inoculation of immobilized bacterial agents could significantly increase the temperature of aerobic composting of chicken manure, prolong the duration of the high temperature period (>60°C), and reduce the temperature variation range during the high temperature period.

[0108] (3) Determination of seed germination index of aerobic composting of chicken manure

[0109] The experimental object selected for the determination of seed germination index (GI value) is radish seeds, and the specific variety is Jinghong No. 1.

[0110] Under the same external environmental conditions, the seed germination index of the blank control (CK) without inoculation of immobilized bacteria and the experimental group during aerobic composting of chicken manure were measured. The results are as follows Figure 4 As shown in A.

[0111] Secondly, set up an additional comparison group, specifically:

[0112] Comparative group 1: using only the sterile attapulgite obtained by the treatment of step (1) of Example 3, aerobic composting of chicken manure was carried out according to the method described in Example 4, and the material 1 and the sterile attapulgite were evenly mixed in a ratio of 100:1.2 (wet weight);

[0113] Comparative Group 2: The bacterial agent was prepared by referring to the preparation method of the immobilized bacterial agent described in Example 3, except that step (4) the bacterial cell fixation step and step (5) were omitted, and aerobic composting of chicken manure was carried out according to the method described in Example 4.

[0114] The experiment was repeated twice for control group 1 and control group 2. The comparison results of seed germination index during aerobic composting of chicken manure among CK group, control group 1, control group 2 and experimental group are shown in Table 6:

[0115] Table 6 Comparison of seed germination index during aerobic composting of chicken manure

[0116]

[0117] The seed germination index (GI value) can comprehensively reflect the phytotoxicity of compost and is considered to be the most sensitive and reliable indicator for evaluating compost maturity, which can intuitively reflect the maturity of compost products.

[0118] The results show that during the chicken manure composting process, compared with the uninoculated treatment (CK), the GI value of the experimental group was 100.8% at the end of composting, which was about 2.7 times higher than that of the non-inoculated treatment (CK), while the GI value of the CK group could not even reach the standard (>70%). When sterile attapulgite and two liquid bacterial agents were added without immobilization treatment, the GI value of the control group 2 at the end of composting (57.92%) was lower than that of the control group 1 (69.54%) that only added sterile attapulgite. The GI value of the experimental group inoculated with the immobilized bacterial agent was significantly higher than that of the control groups 1 and 2 at the end of composting. The above shows that inoculation with immobilized bacterial agents can significantly improve the maturity of chicken manure compost products, decompose and reduce toxic substances produced during composting, and reduce toxic effects on plants.

[0119] (4) Determination of pollutant gas emissions from aerobic composting of chicken manure

[0120] The boric acid absorption method and gas chromatography were used to analyze the NH 3 and N 2 The emission of O is measured daily, and the daily cumulative emission results are as follows: Figure 5 C and Figure 5 D. At the end of composting (35 days), the total emissions were recorded. The comparison results between the CK group and the experimental group are shown in Figure 6 A and Figure 6 As shown in B.

[0121] Due to the high nitrogen content, chicken manure will produce a large amount of NH 3 Although the introduction of exogenous microbial agents can promote compost maturity, it may increase NH 3 The results of the present invention show that inoculation of immobilized bacteria can significantly reduce the emission of harmful gases during composting. At the end of composting, NH 3 and N 2 The cumulative emissions of O were 17.71 g kg -1 DM and 1.24 g kg -1 DM, CK group NH 3 and N 2 The cumulative emissions of O were 27.43 g·kg -1 DM and 1.49 g kg -1 DM, compared with the uninoculated CK group, the inoculation of immobilized bacteria reduced NH by 35.46% 3 Emissions and 16.57% of N 2 O emissions, reducing secondary pollution during the aerobic composting of chicken manure.

[0122] (5) Determination of organic matter degradation rate and ammonium ion content in aerobic composting of chicken manure

[0123] a. Determination of organic matter degradation rate:

[0124] At the beginning of aerobic composting of chicken manure (D0), the 7th day of composting (D7), the 21st day of composting (D21) and the end of composting (D35), the organic matter content (VS) of the compost was measured. The results are as follows: Figure 5 B, and as shown in Table 7; at the end of composting (D35), the organic matter degradation rates of the control group and the experimental group were as follows Figure 6 As shown in C.

[0125] Table 7 Comparison of organic matter content (%) during aerobic composting of chicken manure

[0126] Composting time CK (uninoculated) Experimental group (inoculation treatment) D0 83.0±0.1 80.9±0.2 D7 85.4±0.2 79.9±0.2 D21 71.1±0.1 66.0±0.6 D35 71.9±0.1 66.1±0.8

[0127] b. Determination of ammonium ions

[0128] Every 7 days, the chicken manure was aerobically composted for ammonium ions (NH 4 + ) content was determined, and the results were as follows Figure 5 A and Figure 6As shown in D.

[0129] At the end of composting, CK group NH 4 + Content: 7.76 g kg -1 DM, organic matter degradation rate was 13.35%, NH 4 + Content: 5.75g·kg -1 Compared with the control group, the organic matter degradation rate increased by 36.69% after inoculation of the immobilized bacteria, and at the same time, the NH 4 + During the composting process, the immobilized bacteria can promote the degradation of organic matter, accelerate the decomposition process of organic matter, increase the degradation rate of organic matter, and also promote the conversion of nitrogen and reduce NH 4 + content.

[0130] (6) Compost product indicators

[0131] The indicators of the compost products of the CK group and the experimental group at the end of composting (35 days) are summarized in Table 8. Compared with the uninoculated treatment, the experimental group inoculated with immobilized bacteria can increase the duration of the high temperature period, the degradation rate of compost organic matter and the GI value, and reduce NH 4 + concentration, reducing NH 3 and N 2 O emissions.

[0132] Table 8 Compost product indicators

[0133] Composting end indicator CK group (unvaccinated) Experimental group (inoculation treatment) Duration of high temperature period (>60℃) / day 12 16 Organic matter degradation rate / % 13.35 18.26 GI value / % 26.74 100.80 <![CDATA[NH 4 + Concentration / g·kg -1 DM]]> 7.76 5.75 <![CDATA[NH 3 Cumulative emissions / g·kg -1 DM]]> 27.43 17.71 <![CDATA[N 2 Cumulative emission of O / g kg -1 DM]]> 1.49 1.24

[0134] As can be seen from the above embodiments, the present invention provides a combination of bacteria for aerobic composting of chicken manure, an immobilized bacterial agent containing the combination of bacteria, and a preparation method and a use method of the bacterial agent. The combination of bacteria can be applied to aerobic composting of chicken manure, and the immobilized bacterial agent containing the combination of bacteria can accelerate the degradation of organic matter during the composting process. At the same time, due to the increase in the composting temperature and the duration of the high temperature period, the microbial activity is increased, and the degradation of NH 4 + Assimilation of NH 4 + The content of NH 3 The emission of NH 3 The assimilation of N 2O emission, because the inoculation treatment can degrade the materials more completely, and the toxic ion conversion is more thorough, so that the seed germination index is greatly improved, and the combined bacteria and the immobilized bacterial agent containing the combined bacteria have a relatively broad application prospect.

[0135] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing an immobilized bacterial agent, characterized in that: The steps include: (1) inoculating Bacillus amyloliquefaciens and Bacillus arginii into liquid culture medium for cultivation respectively, thereby obtaining a liquid culture medium of Bacillus amyloliquefaciens and a liquid culture medium of Bacillus arginii; (2) mixing the liquid inoculum of Bacillus amyloliquefaciens, trehalose and attapulgite uniformly, and then drying the mixture to obtain a dried mixed material A containing Bacillus amyloliquefaciens; (3) uniformly mixing the liquid inoculum of Bacillus argyi with trehalose and attapulgite, and then drying the mixture to obtain a dried mixed material B containing Bacillus argyi; (4) Mixing the mixed material A and the mixed material B to obtain an immobilized bacterial agent; The deposited name of the Bacillus amyloliquefaciens is GJD2021101, the Latin name is Bacillus amyloliquefaciens, the deposit date is April 15, 2024, the depository is China Center for Type Culture Collection, and the deposit number is CCTCCM2024689; The deposited name of the Bacillus aryabhattai is GWD2022101, the Latin name is Bacillus aryabhattai, the deposit date is April 15, 2024, the depository is China Center for Type Culture Collection, and the deposit number is CCTCC M2024688; The total effective viable count of Bacillus amyloliquefaciens and Bacillus aggregatii in the immobilized bacterial agent is 1×10 8 ~5×10 8 CFU / g.

2. The method for preparing the immobilized bacterial agent according to claim 1, characterized in that: The liquid culture medium in step (1) is cultured for 6 to 10 hours, and the liquid culture medium is LB liquid culture medium.

3. The method for preparing the immobilized bacterial agent according to claim 2, characterized in that: The mass ratio of trehalose, attapulgite and liquid Bacillus amyloliquefaciens in step (2) is 1:6-10:23-43, and the mass ratio of trehalose, attapulgite and liquid Bacillus amyloliquefaciens in step (3) is 1:6-10:23-43.

4. The method for preparing the immobilized bacterial agent according to claim 1, characterized in that: The temperature of the drying treatment in step (2) and step (3) is independently 40 to 50° C., and the drying time is independently 8 to 12 hours.

5. The method for preparing the immobilized bacterial agent according to claim 1, characterized in that: The mass ratio of the mixed material A to the mixed material B in step (4) is 1:0.5-2.

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