Compound microbial agent for reducing greenhouse gas emission in film composting process and application of compound microbial agent

Abstract

The invention belongs to the technical field of compost nitrogen conservation and synergistic emission reduction, and particularly discloses a compound microbial agent for reducing greenhouse gas emission in the membrane composting process and application of the compound microbial agent, and the compound microbial agent is prepared from fermentation liquor of actinomycetes (Leifsonia shikonensis N37, bacillus (Oceanobacillus sp) M11 and bacillus subtilis M8 according to the mass ratio of 1: 8: 1. Compared with the prior art, the compound microbial agent disclosed by the invention has the advantages that the emission of NH3 in a film composting process can be reduced, nitrogen fixation is promoted, and the nitrogen fertilizer benefit of a product is improved; the emission of greenhouse gases in the film composting process is reduced, and the environmental benefits of film composting are improved; therefore, the fertilizer efficiency of the film compost product is improved.

Description

Technical Field

[0001] The invention relates to the technical field of composting nitrogen preservation and coordinated emission reduction, in particular to a composite microbial agent for reducing greenhouse gas emissions in a membrane composting process and application thereof. Background Art

[0002] Composting is one of the methods of waste resource utilization, which can convert waste into organic fertilizer. The loss of nitrogen in traditional aerobic composting is mainly ammonia volatilization. In addition, greenhouse gases such as CO2, CH4, and N2O are also produced, causing environmental pollution and reducing the efficiency of nitrogen fertilizer products. Inoculating bacterial agents during the composting process can reduce the loss of nitrogen during the composting process and promote the maturity of compost products. Nano-membrane aerobic composting technology can effectively alleviate odor emissions during the composting process. In order to respond to the national carbon emission reduction policy, reduce ammonia emissions during membrane composting, and improve the fertilizer efficiency of membrane composting products, it is necessary to develop corresponding bacterial agents to regulate the membrane composting process, reduce greenhouse gas emissions, and improve the economic value of membrane composting products. Summary of the invention

[0003] In order to solve the above technical problems, the present invention provides a composite microbial agent for reducing greenhouse gas emissions in a membrane composting process and its application.

[0004] To achieve the above object, the present invention is implemented according to the following technical solutions:

[0005] One of the purposes of the present invention is to provide a composite microbial agent for reducing greenhouse gas emissions in a membrane composting process, the composite microbial agent being composed of fermentation broth of actinomycetes (Leifsonia shinshuensis) N37, Bacillus (Oceanobacillus sp) M11, and Bacillus subtilis (Bacillus subtilis) M8;

[0006] Among them, the actinomycetes (Leifsonia shinshuensis) N37 was deposited in the Guangdong Provincial Microbiological Culture Collection Center on July 8, 2024, with a preservation number of GDMCC No: 64848; the preservation number of the Bacillus (Oceanobacillus sp.) M11 is GDMCC No: 64847; the preservation number of the Bacillus subtilis (Bacillus subtilis) M8 is GDMCC No: 64849.

[0007] Preferably, the mass ratio of the fermentation broth of actinomycetes (Leifsonia shinshuensis) N37, Bacillus (Oceanobacillus sp) M11, and Bacillus subtilis (Bacillus subtilis) M8 in the composite microbial agent is 1:8:1.

[0008] Further, the fermentation broth of actinomycetes (Leifsonia shinshuensis) N37, Bacillus (Oceanobacillus sp) M11, and Bacillus subtilis (Bacillus subtilis) M8 is prepared by inoculating actinomycetes (Leifsonia shinshuensis) N37 / Bacillus (Oceanobacillus sp) M11 / Bacillus subtilis (Bacillus subtilis) M8 into LB broth medium at an inoculum amount of 1%, and culturing statically for 48 hours or culturing on a shaking table until CFU>10 8 Prepared.

[0009] The second object of the present invention is to provide an application of a composite microbial agent for reducing greenhouse gas emissions in a membrane composting process in the preparation of membrane composting, comprising the following steps:

[0010] S1. Mix mushroom residue and laying hen manure in a mass ratio of 2:5, mix the composite microbial agent and the pile evenly, adjust the moisture of the pile to 60%, and cover the surface of the pile with a polymer semipermeable membrane; wherein the addition amount of the composite microbial agent is 2% of the wet weight of the pile;

[0011] S2. A ventilation duct is laid at the bottom of the pile, and then the pile is covered on the ventilation duct without turning the pile. The static composting mode is used for 30 days to obtain a finished membrane composting product.

[0012] Furthermore, the pile is intermittently ventilated through the ventilation duct, that is, ventilation for 10 minutes, rest for 50 minutes, and the ventilation rate is 0.6m 3 / h.

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] The composite microbial agent of the present invention can reduce the emission of NH3 during membrane composting, promote nitrogen fixation, and improve the efficiency of nitrogen fertilizer products; reduce the emission of greenhouse gases during membrane composting, and improve the environmental benefits of membrane composting; promote the maturity of membrane composting products, and improve the fertilizer efficiency of membrane composting products. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is a temperature change diagram of the compost in Example 2.

[0016] Figure 2 This is a diagram showing changes in humus in the compost in Example 2.

[0017] Figure 3 This is a graph showing the changes in ammonium nitrogen in the compost in Example 2.

[0018] Figure 4 This is a graph showing the changes in nitrate nitrogen in the compost in Example 2.

[0019] Figure 5 The germination index of cucumber seeds cultured in compost at different days in Example 2.

[0020] Figure 6 This is the CO2 emission pattern of the compost in Example 2.

[0021] Figure 7 This is the CH4 emission pattern of the compost in Example 2.

[0022] Figure 8 This is the N2O emission pattern of the compost in Example 2.

[0023] Fig. 9 This is the emission pattern of ammonia during the composting process in Example 2.

[0024] Fig.10 This is the growth-promoting effect of bacterial fertilizer on peanut seedlings. DETAILED DESCRIPTION

[0025] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments. The specific embodiments described herein are only used to explain the present invention and are not used to limit the invention.

[0026] Example 1. Preparation of composite microbial agent.

[0027] 1) Screening of Actinomycete (Leifsonia shinshuensis) N37

[0028] ① Preparation of solid culture medium: Weigh 8.64 g of nitrogen-free culture medium (N11764-250G nitrogen-free culture medium purchased from Beijing Puxitang Biotechnology Co., Ltd.) (43.2 g / L) into a 250 mL conical flask, add 200 mL of deionized water, stir until dissolved, sterilize in an autoclave at 121°C for 15 min, and pour into sterile culture dishes after sterilization, pouring 20 mL of nitrogen-free culture medium into each dish.

[0029] ② Preparation of supernatant: Weigh 2 g of soybean soil sample (field soil sample collected at longitude 113.85 and latitude 29.70 in 2022) into a 50 mL centrifuge tube, add 20 mL of PBS, shake and mix, place in a shaker (37 ° C, 120r) for 30 min, let stand for 10 min, and take 5 mL of supernatant.

[0030] ③ Plate streaking: Use a sterile inoculating loop to pick up the supernatant, and use the four-zone streak method to streak on the nitrogen-free culture medium, and then place it in a 37°C constant temperature incubator for 48 hours.

[0031] ④Purification and propagation: Select a single colony that can grow on a nitrogen-free medium, use an inoculation loop to pick up a single bacterium, and propagate it in a sterilized LB broth medium (purchased from Guangzhou Huankai Biotechnology Co., Ltd., with the formula: 10 g / L peptone, 5 g / L sodium chloride, 1 g / L glucose, 5 g / L yeast extract powder, and a final pH of 7.0±0.2) for 24 h. Divide the cultured bacterial solution into two portions and transfer them to sterile centrifuge tubes. Add an equal volume of 50% glycerol to one portion, and use the other portion for DNA extraction. Store the DNA at -20°C and the bacterial solution at -80°C.

[0032] ⑤PCR amplification: The amplification system is 25ul, add 12.5ul enzyme, 0.5ul upstream universal primer (27F), 0.5ul downstream universal primer (1492R), add 1ul DNA, add 10.5ul sterile water. The PCR program is: pre-denaturation 95℃, 5min; denaturation 95℃, 30s, annealing 56℃, 10s, extension 72℃, 10s, 34 cycles; final extension 72℃, 10min; storage at 4℃.

[0033] ⑥Identification: The PCR product was sent to Guangzhou Shenggong Biotechnology Co., Ltd. for sequencing. The sequence was:

[0034]

[0035] 2) Screening of Oceanobacillus sp. M11

[0036] ①1① Weigh 4.2g LB agar medium (21g / L) into a 250mL conical flask, wherein the LB agar medium was purchased from Guangzhou Huankai Biotechnology Co., Ltd., and the LB agar medium formula is: peptone 10g / L, sodium chloride 5g / L, glucose 1g / L, yeast extract powder 5g / L, agar 15g / L, final pH 7.0±0.2; add 200mL deionized water, stir until dissolved, add 0.02g aniline blue solid, shake until the aniline blue solid is dissolved, sterilize at 121℃ in an autoclave for 15min, and pour into sterile culture dishes after sterilization, pour 20mL of culture medium into each dish to obtain aniline blue culture medium.

[0037] ② Weigh 4.2 g LB agar medium (21 g / L) into a 250 mL conical flask, wherein the LB agar medium was purchased from Guangzhou Huankai Biotechnology Co., Ltd., and the LB agar medium formula was: peptone 10 g / L, sodium chloride 5 g / L, glucose 1 g / L, yeast extract powder 5 g / L, agar 15 g / L, and the final pH was 7.0 ± 0.2; add 200 mL of deionized water, stir until dissolved, add 20 ul of guaiacol, shake well, sterilize at 121 ° C in an autoclave for 15 min, and pour into a sterile culture dish after sterilization. Pour 20 mL of culture medium into each dish to obtain guaiacol culture medium.

[0038] ③ Preparation of supernatant: Weigh 2 g of early, middle and late composting samples (membrane composting test samples collected from Runtian Composting Plant in Xinxing County, Yunfu City, Guangdong Province in 2022) into a 50 mL centrifuge tube, add 20 mL of PBS, shake and mix, place on a shaker (37°C, 120r) for 30 min, let stand for 10 min, and take 5 mL of supernatant.

[0039] ④ Dilute the supernatant to 10 -5 , 10 -6 , 10 -7 Multiples, aspirate 100ul of the diluted liquid, place it in aniline blue medium and guaiacol medium, spread it evenly with a spreading rod, and then place it in a 37℃ constant temperature incubator for 2h upright and then inverted for 48h.

[0040] ⑤ Select single colonies that produce transparent circles around the colonies on aniline blue culture medium and colonies that produce red color circles on guaiacol culture medium, use an inoculation loop to pick up single bacteria, and culture them in LB broth for 24 hours. Divide the cultured bacterial solution into two portions and transfer them to sterile centrifuge tubes. Add an equal volume of 50% glycerol to one portion, and use the other portion to extract DNA. Store the DNA at -20℃ and the bacterial solution at -80℃.

[0041] ⑥PCR amplification: The amplification system is 25ul, add 12.5ul enzyme, 0.5ul upstream universal primer (27F), 0.5ul downstream universal primer (1492R), add 1ul DNA, add 10.5ul sterile water. The PCR program is: pre-denaturation 95℃, 5min; denaturation 95℃, 30s, annealing 56℃, 10s, extension 72℃, 10s, 34 cycles; final extension 72℃, 10min; storage at 4℃.

[0042] ⑦ Identification: The PCR product was sent to Guangzhou Shenggong Biotechnology Co., Ltd. for sequencing. The sequence is:

[0043]

[0044] 3) Screening of Bacillus subtilis M8

[0045] ① Weigh 4.2 g LB agar medium (21 g / L) into a 250 mL conical flask, wherein the LB agar medium was purchased from Guangzhou Huankai Biotechnology Co., Ltd., and the LB agar medium formula was: 10 g / L peptone, 5 g / L sodium chloride, 1 g / L glucose, 5 g / L yeast extract powder, 15 g / L agar, and a final pH of 7.0 ± 0.2; add 200 mL of deionized water, stir until dissolved, add 0.02 g of aniline blue solid, shake until the aniline blue solid is dissolved, sterilize at 121 ° C in an autoclave for 15 min, and pour into a sterile culture dish after sterilization, pour 20 mL of culture medium into each dish to obtain aniline blue medium.

[0046] ② Weigh 4.2 g LB agar medium (21 g / L) into a 250 mL conical flask, wherein the LB agar medium was purchased from Guangzhou Huankai Biotechnology Co., Ltd., and the LB agar medium formula was: peptone 10 g / L, sodium chloride 5 g / L, glucose 1 g / L, yeast extract powder 5 g / L, agar 15 g / L, and the final pH was 7.0 ± 0.2; add 200 mL of deionized water, stir until dissolved, add 20 ul of guaiacol, shake well, sterilize at 121 ° C in an autoclave for 15 min, and pour into a sterile culture dish after sterilization. Pour 20 mL of culture medium into each dish to obtain guaiacol culture medium.

[0047] ③ Preparation of supernatant: Weigh 2g of early, middle and late composting samples (membrane composting test samples collected from Runtian Composting Plant in Xinxing County, Yunfu City, Guangdong Province in 2022) into a 50mL centrifuge tube, add 20mL PBS, shake and mix, place on a shaker (37℃, 120r) for 30min, let stand for 10min, and take 5mL of supernatant.

[0048] ④ Dilute the supernatant to 10 -5 , 10 -6 , 10 -7 Multiples, aspirate 100ul of the diluted liquid, place it in aniline blue medium and guaiacol medium, spread it evenly with a spreading rod, and then place it in a 37℃ constant temperature incubator for 2h upright and then inverted for 48h.

[0049] ⑤ Select single colonies that produce transparent circles around the colonies on aniline blue culture medium and colonies that produce red color circles on guaiacol culture medium, use an inoculation loop to pick up single bacteria, and culture them in LB broth for 24 hours. Divide the cultured bacterial solution into two portions and transfer them to sterile centrifuge tubes. Add an equal volume of 50% glycerol to one portion, and use the other portion to extract DNA. Store the DNA at -20℃ and the bacterial solution at -80℃.

[0050] ⑥PCR amplification: The amplification system is 25ul, add 12.5ul enzyme, 0.5ul upstream universal primer (27F), 0.5ul downstream universal primer (1492R), add 1ul DNA, add 10.5ul sterile water. The PCR program is: pre-denaturation 95℃, 5min; denaturation 95℃, 30s, annealing 56℃, 10s, extension 72℃, 10s, 34 cycles; final extension 72℃, 10min; storage at 4℃.

[0051] ① Identification: The PCR product was sent to Guangzhou Shenggong Biotechnology Co., Ltd. for sequencing. The sequence was:

[0052]

[0053] Inoculate LB broth with 1% of Leifsonia shinshuensis N37, Oceanobacillus sp. M11, and Bacillus subtilis M8, respectively, and culture them statically for 48 h or on a shaking platform until CFU>10 8 The fermentation broths of three strains were prepared, and the fermentation broths of actinomycetes (Leifsonia shinshuensis) N37, Bacillus (Oceanobacillus sp) M11 and Bacillus subtilis (Bacillus subtilis) M8 were mixed in a mass ratio of 1:8:1 to obtain a composite microbial agent.

[0054] Example 2: Verification of composite microbial agents in reducing greenhouse gas emissions during membrane composting

[0055] Mix mushroom residue and laying hen manure in a mass ratio of 2:5, then mix the composite microbial agent and the pile, adjust the moisture of the pile to 60%, add 2% of the wet weight of the pile, and cover the surface of the pile with a polymer semipermeable membrane, recorded as the PB4 group; at the same time, set up a control (CK) group: mix mushroom residue and laying hen manure in a mass ratio of 2:5, adjust the moisture of the pile to 60%, and cover the surface of the pile with a polymer semipermeable membrane. Lay a ventilation duct at the bottom of the pile, and then cover the pile on the ventilation duct. The pile is intermittently ventilated through the ventilation duct, that is, ventilate for 10 minutes, and intermittently for 50 minutes, with a ventilation rate of 0.6m 3 / h; without turning the pile, the static composting mode was adopted for a total of 30 days to obtain the finished membrane compost.

[0056] 1) Use TP101 metal probe thermometer to measure the changes of the temperature of the pile and the temperature of the composting environment. Measure the core temperature of the pile at 9 am and 5 pm every day, and take the average of the morning and evening temperatures as the temperature of the pile on that day. At the same time, record the morning and evening ambient temperatures and take the average of the temperatures as the temperature on that day. Figure 1As shown in the figure, the 0-2 days were the warming period, the 2-25 days were the high temperature period, and the 25-30 days were the cooling period. The highest temperature in the PB4 group occurred on the 2nd day, with a maximum temperature of 63.9°C. The highest temperature in the CK group occurred on the 2nd day, with a maximum temperature of 64.4°C. However, there was no significant difference in the maximum temperature between the two groups (P < 0.05). The duration of the high temperature period was 24 days for both groups, but during the high temperature period, the temperature in the PB4 group was higher than that in the CK group for 16 days, indicating that the addition of bacterial agents was more conducive to the maintenance of the temperature during the high temperature period. The high temperature period (> 50°C) in both groups lasted for more than 7 days, which met the standards for harmless treatment of livestock and poultry (GB36195-2018). During the cooling period, the temperature in the PB4 treatment group dropped faster, indicating that the addition of bacterial agents was conducive to the faster entry of the pile into the mature stage, which was conducive to shortening the composting time, and could improve the production efficiency of film composting products in production.

[0057] 2) Humus mainly includes humic acid and fulvic acid, which are complex and stable macromolecular organic compounds formed by the decomposition and transformation of organic matter during composting. The content of humus has a positive effect on regulating soil fertility, soil microbial structure and community, and crop growth. The determination of humus is carried out by sodium pyrophosphate sodium hydroxide extraction-high temperature external heating potassium dichromate oxidation capacity method. The total humus changes with the progress of composting. Figure 2 As shown in the figure, the humus content showed a downward trend in the early stage of composting, and increased on the 14th day. The highest humus content appeared in the composting sample on the 21st day. The highest humus value in the PB4 group was 22.5%, and the highest value in the CK group was 21.1%. The results showed that the addition of bacterial agents promoted the formation of humus and improved the maturity of the pile.

[0058] 3) As an important component of nitrogen in organic fertilizer, ammonium nitrogen has an important impact on the supply of soil nitrogen and the growth and development of plants after the organic fertilizer is returned to the field. Increasing the ammonium nitrogen content in organic fertilizer can reduce the amount of organic fertilizer used per unit area while meeting the demand for nitrogen fertilizer for crops, improve the utilization efficiency of organic fertilizer, and improve production benefits. Figure 3 The overall change of ammonium nitrogen during the composting process showed an upward trend. The ammonium nitrogen content decreased slightly from 7 to 21 days. At the end of composting, the ammonium nitrogen content in the CK group was 12.016 mg / g, and the ammonium nitrogen content in the PB4 group was 13.536 mg / g. The ammonium nitrogen content increased by 12.65%, indicating that the application of bacterial agents increased the ammonium nitrogen content of the membrane composting product, which is beneficial to the sequestration of nitrogen in the product.

[0059] Determination method: take 0.5g of sample, add 5mL of deionized water, vortex for 10min, centrifuge at 13000r / min for 5min, take 50μL of supernatant, add 10mL of 0.01mol / L dilute sulfuric acid, add 0.5mL of potassium sodium tartrate solution, add 0.5mL of nanoreagent, shake well, color for 10min, and measure the absorbance of the solution at 420nm.

[0060] Preparation of standard curve: Weigh 0.7855g of ammonium chloride and dissolve it in a 250mL volumetric flask to obtain a standard ammonium chloride solution with a concentration of 1mg / L. Pipette 0, 0.1, 0.3, 0.5, 1, 1.5, and 2mL of standard ammonium chloride solution, respectively, with ammonium nitrogen contents of 0, 2, 6, 10, 20, 30, and 40ug. Make up to 10mL with 0.01mol / L dilute sulfuric acid, add 0.5mL of potassium sodium tartrate solution, add 0.5mL of nanostructured reagent, shake well, develop color for 10min, and measure the absorbance of the solution at 420nm.

[0061] Nassler's reagent: weigh 12g of sodium hydroxide (NaOH) and dissolve it in 60mL of water, and cool it; weigh 1.7g of mercuric chloride (HgCl2) and dissolve it in 30mL of water; weigh 3.5g of potassium iodide (KI) in 10mL of water, and slowly add the above mercuric chloride solution to the potassium iodide solution while stirring until the red precipitate formed no longer dissolves; while stirring, slowly add the sodium hydroxide solution cooled to room temperature to the mixed solution of mercuric chloride and potassium iodide, and then add the remaining mercuric chloride solution, mix well, and let it stand in the dark for 24h, pour out the supernatant, and store it in a brown bottle.

[0062] Potassium sodium tartrate solution: Weigh 50g of potassium sodium tartrate (KNaC4H6O6·4H2O) and dissolve it in 100mL of water. Heat to boil to drive off ammonia, and then cool and dilute to 100mL.

[0063] 4) Nitrate nitrogen is one of the nitrogen that can be directly absorbed and utilized by plants. During the composting process, nitrifying bacteria and denitrifying bacteria mainly affect its content. In order to improve the nitrogen fertilizer efficiency of compost products, it is also worth studying to increase the content of nitrate nitrogen in the products. Figure 4 As shown, in the early stage of composting, the nitrate nitrogen content increased. In the samples on the third day, the nitrate nitrogen content of the CK group was 3.92 mg / g, and the nitrate nitrogen content of the PB4 was 3.2 mg / g. This was mainly because in the early stage of composting, microorganisms multiplied in large numbers inside the pile, resulting in the formation of anaerobic zones locally, and nitrate nitrogen was generated by anaerobic nitrification. The nitrate nitrogen content decreased during the high temperature period. In the samples on the 21st day, the nitrate nitrogen content of the CK group and the PB4 group was 3.57 mg / g. At the end of composting, the nitrate nitrogen content of the PB4 group was 4.12 mg / g, and the nitrate nitrogen content of the CK group was 3.84 mg / g. The application of microbial agents increased the nitrate nitrogen content by 7.29%, which was beneficial to improving the nitrogen fertilizer efficiency of the product.

[0064] 5) The germination index is an indicator for judging the toxicity of compost products. High-quality organic fertilizers can promote seed rooting and germination, so the germination index can be used to indirectly reflect the maturity of compost products. Take 2g of compost samples from 0, 1, 3, 7, 14, 21, and 28 days, add 20mL of deionized water, vortex on a vortexer for 30 minutes, let stand for 30 minutes, lay a piece of filter paper at the bottom of the culture dish, put in 10 cucumber seeds (Yuexiu No. 3), repeat three times in each group, take 10mL of supernatant and add it to the culture dish, add 10mL of deionized water to the blank control group, and culture in the dark at 25°C for 48 hours, and then record the seed germination rate and seed root length. The germination index calculation formula is as follows:

[0065] GI (%) = (A1*A2) / (B1*B2);

[0066] Where: A1 is the germination rate of the experimental group; A2 is the average root length of the experimental group; B1 is the germination rate of the control group; B2 is the average root length of the control group.

[0067] The germination index of different days is as follows Figure 5 As shown, in the early stage of composting (0, 1, 4, 7), the germination index of the product was less than 80%, indicating that the product had a strong inhibitory effect on seed germination. The germination index on the 10th day was higher than 80%, indicating that the inhibitory effect of the product on seed germination was lifted. The germination index on the 21st day of composting was the highest, among which the germination index of the CK group was 114.62%, and that of the PB4 group was 138.34%. The germination index of the group with added microbial agent increased by 23.72% compared with that of the CK group on the 21st day. Although the germination index on the 28th day decreased, PB4 was still 126.48% higher than CK's 114.62%, an increase of 11.86%. It shows that the addition of microbial agent promoted the maturity of the pile, reduced the toxicity of the product, and promoted the rooting and germination of seeds.

[0068] 6) In order to mitigate the trend of global warming and respond to the national carbon reduction policy, it is urgent to reduce the emission of greenhouse gases during composting. Greenhouse gases mainly include CO2, CH4 and N2O, among which CO2 emission is the main gas causing the greenhouse effect. At the same time, CO2 is also the main greenhouse gas produced during composting. In this study, at 5 pm every day, the static box method was used to collect the composting tail gas at 0 and 60 minutes, and the gas was collected using aluminum foil bags. The Agilent 7890B was used to measure the content of CO2, CH4 and N2O in the tail gas. The CO2 emission pattern is as follows: Figure 6 As shown in Figure 2, the CH4 emission pattern is as follows: Figure 7 As shown in the figure, the N2O emission pattern is as follows Figure 8 shown.

[0069] The emission of CO2 was mainly concentrated in the early stage of composting. The CO2 emission of CK group reached the maximum value of 8.45×10 4 The CO2 emission of the PB4 group reached a maximum of 8.35×10 4 mg / kg. Comprehensively considering the entire composting process, the cumulative CO2 emission of the CK group was 126.11×10 4 mg / kg, the cumulative CO2 emission of PB4 group was 114.2×10 4 mg / kg, the CO2 emission reduction rate of bacterial agent addition was 9.44%.

[0070] CH4 emissions are mainly concentrated in the high temperature period of composting, and there is an emission peak at the end of composting. Figure 7 The results showed that the CH4 emission of the PB4 group was significantly lower than that of the CK group. The highest daily emission of the CK group was 386.54 mg / kg on the 28th day, and the highest daily emission of the PB4 group was 200.97 mg / kg on the 28th day. The lowest value appeared on the 0th day, with 10.08 mg / kg in the CK group and 11.28 mg / kg in the PB4 group. In terms of cumulative emissions, the CK group was 4877.37 mg / kg and the PB4 was 2805.52 mg / kg. The addition of bacterial agents reduced CH4 emissions by 42.48%.

[0071] N2O emissions such as Figure 8 As shown, the emissions are mainly concentrated in the early and late stages of composting. The highest daily emission of the CK group is 196.15 mg / kg, and the highest daily emission of the PB4 group is 249.85 mg / kg. Taking the emissions during the entire composting process into consideration, the cumulative emissions of the CK group are 877.47 mg / kg, and the cumulative emissions of the PB4 group are 847.98 mg / kg. In summary, the addition of bacterial agents can reduce N2O emissions by 3.36%.

[0072] PB4 bacterial agent has a significant effect on reducing greenhouse gas emissions, among which the reduction rate for CH4 is the highest at 42.48%. However, CO2 emissions are four orders of magnitude greater than CH4 and N2O, and GHG (greenhouse gas) as a whole is greatly affected by CO2 emissions. Therefore, this bacterial agent has positive benefits for reducing greenhouse gas emissions in the membrane composting process and can reduce the pollution of composting to the environment.

[0073] 7) The emission pattern of ammonia during composting is as follows: Fig. 9 As shown, 0.5 mol / L dilute sulfuric acid was used as the absorption liquid for ammonia, and the static box method was used to absorb ammonia in the tail gas from 8 pm to 9 am. The detection method was the same as that for ammonium nitrogen. Ammonia emissions were mainly concentrated in the early stage of composting. As the composting progressed, ammonia emissions gradually decreased. The highest ammonia emissions occurred on the first day. The highest ammonia emissions in the CK group were 53.97 mg / m 2The maximum ammonia emission of PB4 group is 53.38mg / m 2 The cumulative NH3 emissions of each group were CK (235.88±10.71mg / m 2 )、PB4(204.46±4.71mg / m 2 ). The cumulative NH3 emission of the PB4 group was significantly lower than that of the CK group in the first 7 days (P < 0.05). At the end of composting, the cumulative NH3 emissions were CK (2882.79 ± 41.06 mg) and PB4 (2538.37 ± 26.38 mg). The PB4 group significantly reduced the cumulative ammonia emission compared with the CK group (P < 0.05), and the reduction rate was 11.97%.

[0074] 8) Fig.10 This is the growth-promoting effect of bacterial fertilizer on peanut seedlings. Fig.10 It can be seen that the application of microbial agents has a significant growth-promoting effect on peanuts. On the 7th, 14th, 21st, 28th, 35th, and 70th days, the growth-promoting effect of the PB4 group on plants was 9.77%, 8.55%, 2.11%, 2.41%, 4.63%, and 7.30% respectively compared with CK. Combined with the total weight of the aboveground plants, the total weight of the underground roots, and the weight of the fruit, the PB4 group has a significant growth-promoting effect on the plants, indicating that the application of microbial agents improves the fertilizer efficiency of organic fertilizers, promotes the growth of peanut plants, and increases the yield.

[0075] Table 1 Effect of microbial agent application on plant growth

[0076]

[0077] In summary, the composite microbial agent of the present invention can reduce the emission of ammonia during membrane composting and reduce the impact of compost odor on the environment; reduce the emission of greenhouse gases during composting and contribute to carbon emission reduction during composting; promote compost maturity, increase the degradation rate of lignin, increase the content of humus in the compost, improve the maturity of compost products, increase the ammonium nitrogen content in the compost, promote the fixation of nitrogen in the compost, and increase the fertilizer efficiency.

[0078] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. All technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. A composite microbial agent for reducing greenhouse gas emissions in membrane composting, characterized in that: The composite microbial agent is composed of fermentation liquid of actinomycetes (Leifsonia shinshuensis) N37, Bacillus (Oceanobacillus sp) M11, and Bacillus subtilis (Bacillus subtilis) M8; Among them, the actinomycetes (Leifsonia shinshuensis) N37 was deposited in the Guangdong Provincial Microbiological Culture Collection Center on July 8, 2024, with a preservation number of GDMCC No: 64848; the preservation number of the Bacillus (Oceanobacillus sp.) M11 is GDMCC No: 64847; the preservation number of the Bacillus subtilis (Bacillus subtilis) M8 is GDMCC No: 64849.

2. The composite microbial agent for reducing greenhouse gas emissions in the membrane composting process according to claim 1, characterized in that: The mass ratio of the fermentation liquid of actinomycetes (Leifsonia shinshuensis) N37, Bacillus (Oceanobacillus sp) M11 and Bacillus subtilis (Bacillus subtilis) M8 in the composite microbial agent is 1:8:

1.

3. The composite microbial agent for reducing greenhouse gas emissions in the membrane composting process according to claim 2, characterized in that: The fermentation liquid of actinomycetes (Leifsonia shinshuensis) N37, Bacillus (Oceanobacillus sp) M11, and Bacillus subtilis (Bacillus subtilis) M8 is prepared by inoculating actinomycetes (Leifsonia shinshuensis) N37 / Bacillus (Oceanobacillus sp) M11 / Bacillus subtilis (Bacillus subtilis) M8 into LB broth medium at an inoculum amount of 1%, and culturing the culture medium statically for 48 hours or culturing the culture medium on a shaking platform until the CFU is greater than 10 8 Prepared.

4. Use of the composite microbial agent for reducing greenhouse gas emissions in membrane composting as claimed in any one of claims 1 to 3 in the preparation of membrane composting.

5. The use of the composite microbial agent for reducing greenhouse gas emissions in the membrane composting process according to claim 4 in the preparation of membrane composting, characterized in that: The following steps are involved: S1. Mix mushroom residue and laying hen manure in a mass ratio of 2:5, mix the composite microbial agent and the pile evenly, adjust the moisture of the pile to 60%, and cover the surface of the pile with a polymer semipermeable membrane; wherein the addition amount of the composite microbial agent is 2% of the wet weight of the pile; S2. A ventilation duct is laid at the bottom of the pile, and then the pile is covered on the ventilation duct without turning the pile. The static composting mode is used for 30 days to obtain a finished membrane composting product.

6. The use of the composite microbial agent for reducing greenhouse gas emissions in the membrane composting process according to claim 5 in preparing membrane composting, characterized in that: The pile is intermittently ventilated through the ventilation duct, that is, ventilation for 10 minutes, intermittent for 50 minutes, and the ventilation rate is 0.6m 3 / h.

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