Bacillus amyloliquefaciens, microbial agent, preparation method and application and citrus waste composting method

Abstract

The invention relates to bacillus amyloliquefaciens which is classified and named as bacillus amyloliquefaciens LDB1, the preservation number of the bacillus amyloliquefaciens is CCTCC No: M 20231897, and the preservation date is October 16, 2023. The invention relates to application of bacillus amyloliquefaciens in citrus waste compost. The invention relates to a microbial agent, which comprises bacillus amyloliquefaciens LDB1, bacillus amyloliquefaciens, bacillus subtilis and bacillus velezensis. The invention relates to application of a microbial agent in citrus waste compost. The method has the beneficial effects that lignin peroxidase, laccase and manganese peroxidase can be produced, good lignin degradation capability is achieved, degradation of lignocellulose in citrus waste compost can be effectively promoted by matching with other strains, the degradation rates of hemicellulose, cellulose and lignin are 33.8%, 36.5% and 38.5% respectively, and the method is suitable for industrial production. The strain and the microbial inoculum thereof have important application value in the aspect of recycling of high-lignin fiber organic wastes.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a bacillus amyloliquefaciens, a microbial agent, a preparation method and application thereof, and a citrus waste composting method. Background Art

[0002] With the growth of global citrus production and area, citrus organic waste has also increased accordingly. Among them, the average annual pruning volume of citrus branches reaches 15 to 18 t / ha. Most of the pruned branches are directly burned or discarded, resulting in greenhouse gas emissions and resource losses. Therefore, a large amount of citrus waste resources urgently need to be utilized.

[0003] Citrus branches are rich in lignocellulosic compounds, and composting them is a feasible resource recovery technology. However, the complex structure and low hydrolysis rate of lignocellulose limit the humification process of composting. Inoculation of efficient lignin-degrading bacteria (especially microorganisms naturally present in the composting system) can effectively promote the degradation of lignocellulose and accelerate the maturity of composting. So far, there has been no screening and application of efficient lignin-degrading bacteria in citrus waste composting. Screening lignin-degrading bacteria suitable for composting high-lignin fiber organic waste is of great significance to the green and sustainable development of the forestry and fruit industry system. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide Bacillus amyloliquefaciens, a microbial agent, a preparation method and application, and a citrus waste composting method to improve the utilization rate of high-lignin fiber organic waste to overcome the deficiencies in the above-mentioned prior art.

[0005] The technical solution of the present invention to solve the above technical problems is as follows:

[0006] The amyloliquefaciens that can produce laccase, lignin peroxidase and manganese peroxidase is classified and named as Bacillus amyloliquefaciens LDB1, and was deposited in the China Center for Type Culture Collection on October 16, 2023 with the deposit number: CCTCC No: M 20231897.

[0007] Based on the above technical solution, the present invention also provides an application of Bacillus amyloliquefaciens in composting citrus waste.

[0008] Based on the above technical solution, the present invention also provides a microbial agent, including the above-mentioned Bacillus amyloliquefaciens.

[0009] Furthermore, the microbial agent comprises: Bacillus amyloliquefaciens LDB1, Bacillus amyloliquefaciens, Bacillus subtilis and Bacillus velezensis.

[0010] Furthermore, the total number of viable bacteria in the microbial inoculant is 1.0×10 9 CFU / mL.

[0011] Furthermore, the ratio of the number of live cells of Bacillus amyloliquefaciens LDB1, Bacillus amyloliquefaciens, Bacillus subtilis and Bacillus velez is 1 to 10:1:1:1.

[0012] Furthermore, Bacillus amyloliquefaciens LDB1, Bacillus amyloliquefaciens, Bacillus subtilis and Bacillus velez were all screened from citrus organic waste compost.

[0013] Based on the above technical solution, the present invention also provides a method for preparing a microbial agent, comprising the following steps:

[0014] Bacillus amyloliquefaciens LDB1, Bacillus amyloliquefaciens, Bacillus subtilis and Bacillus Velezii were inoculated into the Luria-Bertani medium after high temperature sterilization. When the viable bacterial count was ≥1.0×10 8 CFU / mL, the fermentation broths of Bacillus amyloliquefaciens LDB1, Bacillus amyloliquefaciens, Bacillus subtilis, and Bacillus velez were obtained respectively;

[0015] The four fermentation broths were mixed in proportion, and the carrier auxiliary material biochar was added to obtain a liquid microbial agent.

[0016] Furthermore, the Luria-Bertani medium is a medium sterilized at 121°C for 15 minutes, and the ingredients include: 10 g / L tryptone, 10 g / L NaCl, 5 g / L yeast extract, and 1000 mL water. Based on the above technical solution, the present invention also provides an application of a microbial agent in composting citrus waste.

[0017] Based on the above technical solution, the present invention also provides a citrus waste composting method, comprising the above microbial agent, and the steps are as follows:

[0018] Mix the microbial agent and compost raw materials in a ratio of 1 to 5:100 (v / m) and compost. Measure the temperature of the compost several times at 11 a.m. every day. If the temperature is higher than 55°C, turn the compost once a day.

[0019] If the temperature is below 50℃, turn the pile every 3-4 days;

[0020] When the temperature in the later stage of composting is lower than 30℃, stop turning the pile and the composting is finished.

[0021] Furthermore, the pile temperature was measured three times at 11:00 a.m. every day.

[0022] Compared with the prior art, the present invention has the following beneficial technical effects:

[0023] 1) Bacillus amyloliquefaciens LDB1 was inoculated onto an LB-agar medium plate, cultured at 35°C for 24 hours, and then transferred to LB liquid medium. After 64 hours of culture, the lignin peroxidase, manganese peroxidase and laccase activities of Bacillus amyloliquefaciens LDB1 were the highest, reaching 175.62±27.09U / mL, 1.59±0.11U / mL and 5.83±0.39U / mL, respectively, indicating that Bacillus amyloliquefaciens LDB1 has a strong lignin degradation ability and can coexist well with other Bacillus bacteria, and has extremely broad application prospects and practical value;

[0024] 2) The microbial agent composed of Bacillus amyloliquefaciens LDB1, Bacillus amyloliquefaciens, Bacillus subtilis and Bacillus velezensis can be used for mixed composting of citrus orchard organic waste (pruning branches, waste fruits), which can better realize the resource utilization of the above mixed wastes, and has obvious advantages in composting, and can effectively promote the degradation of lignocellulose in citrus waste composting. At the end of composting, the degradation rates of hemicellulose, cellulose and lignin in the control experiment were 30.9%, 32.0% and 23.2%, respectively, and the degradation rates of hemicellulose, cellulose and lignin at the end of composting after inoculation of microbial agent were 33.8%, 36.5% and 38.5%, respectively. The bacteria and the agent have important application value in the resource utilization of high lignin fiber organic waste (such as forestry and fruit industry waste);

[0025] 3) Microbial agents have obvious advantages in composting, which can effectively promote the synthesis and utilization of humus precursors and the humification process of composting. The increase in humic acid content in composting increased from 10.9% in the control experiment to 18.1%;

[0026] 4) Microbial agents have obvious advantages in composting, which can effectively shorten the heating time of the pile, shortening the time required for the compost to reach a high temperature of 55°C from 6 days in the control experiment to 5 days, and entering the maturity stage 6 days earlier, which can accelerate the compost heating, prolong the high temperature duration, promote the degradation of lignocellulose to increase the humus content, and effectively shorten the composting cycle;

[0027] 5) Microbial agents have obvious advantages in composting. The thermophilic period is extended by 3 days, the maximum composting temperature is increased from 66.7℃ in the control experiment to 68.3℃, and the seed germination index is increased by 7.4%, which improves the quality and safety of compost. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 They are Bacillus amyloliquefaciens LDB1, Bacillus subtilis, Bacillus velez and Bacillus amyloliquefaciens isolated from citrus waste compost;

[0029] Figure 2 It is a data chart of enzyme activity test of Bacillus amyloliquefaciens LDB1;

[0030] Figure 3 Provide a chart of compost temperature and moisture changes;

[0031] Figure 4 This is a graph showing changes in seed germination index;

[0032] Figure 5 It is a graph showing the changes in organic matter and lignocellulose content;

[0033] Figure 6 This is a chart showing changes in humus content. DETAILED DESCRIPTION

[0034] The principles and features of the present invention are described below in conjunction with the accompanying drawings. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0035] Example 1

[0036] The amyloliquefaciens that can produce laccase, lignin peroxidase and manganese peroxidase is classified and named as Bacillus amyloliquefaciens LDB1, and was deposited on October 16, 2023 with the deposit number: CCTCC No: M 20231897, and is deposited in: China Center for Type Culture Collection, and the deposit address is: Wuhan University, Wuhan, China.

[0037] Screening of Bacillus amyloliquefaciens LDB1

[0038] The citrus shreds, leaves and discarded fruits were mixed in a ratio of (7:1:2) with an initial carbon-nitrogen ratio of 25 and a moisture content of 65%;

[0039] Collect mixed samples when the compost temperature reaches 50℃;

[0040] Take 10g of sample, add 90mL of sterile water, shake for 1h, and let stand for 15min;

[0041] The supernatant was heated to 10 -4 ~10 -6 Gradient dilution, pipette the dilution liquid onto the alkaline lignin solid medium, spread it and place it in an incubator for inverted culture. When clear colonies grow on the medium, pick out single colonies with different morphologies, and culture them under the same conditions until a purified strain is obtained;

[0042] Add 0.1g of aniline blue to 1L of nutrient agar medium, inoculate the purified strain into nutrient agar-aniline blue medium for rescreening, select bacteria that make the medium fade, and preliminarily judge the enzyme production ability of the strain based on the size of the fading circle, and finally obtain the target strain, such as Figure 1 shown.

[0043] Identification of Bacillus amyloliquefaciens LDB1

[0044] 16S rDNA sequencing was used for taxonomic molecular identification, and the sequencing results were subjected to Blast homology analysis on NCBI. The base sequence was most closely related to Bacillus amyloliquefaciens with a similarity of 99.7%. The strain was identified as Bacillus amyloliquefaciens and was named: Bacillus amyloliquefaciens LDB1.

[0045] Bacillus amyloliquefaciens LDB1 can survive in an environment of 55°C to 60°C and can be used in biological and environmental fields.

[0046] Bacillus amyloliquefaciens LDB1 was inoculated on LB-agar medium plate, cultured at 35℃ for 24h, and then transferred to LB liquid medium. After 64h of culture, the lignin peroxidase, manganese peroxidase and laccase activities of Bacillus amyloliquefaciens LDB1 were the highest, reaching 175.62±27.09U / mL, 1.59±0.11U / mL and 5.83±0.39U / mL, respectively. Figure 2 As shown, it has good lignin degradation ability and application prospects.

[0047] Lignin peroxidase activity determination: 1.6 mL sodium tartrate buffer (pH 3.0), 0.3 mL resveratrol and 1 mL enzyme solution were added to 3.0 mL reaction system, preheated in a 30 °C water bath for 3 min, and 0.1 mL H 2 O 2 , and the absorbance change within 1 min was measured at 310 nm.

[0048] Manganese peroxidase activity assay: 2.4 mL of acetic acid-sodium acetate buffer (pH 4.5), 0.1 mL of 2,6-DMP and 0.4 mL of enzyme solution were added to 3.0 mL of reaction system, preheated in a 30 °C water bath for 3 min, and 0.1 mL of H 2 O 2 , and the absorbance change within 1 min was measured at 469 nm.

[0049] Laccase activity determination: add 0.2 mL ABTS solution and 2.7 mL citric acid-sodium citrate buffer (pH 4.5) to 3 mL reaction system, preheat in a 30°C water bath for 3 min, add 0.1 mL enzyme solution, and measure the absorbance change at 420 nm within 1 min.

[0050] For the testing method, please refer to my country's organic fertilizer industry standard (NY 525-2021);

[0051] Seed germination index determination: The method for determining the seed germination index in organic fertilizers (NY 525-2021) was used;

[0052] Determination of lignocellulose content: The Vansoest washing method was used to determine the contents of hemicellulose, cellulose, lignin and ash in turn;

[0053] Determination of humus content: The humus content of the sample was measured by the potassium dichromate volumetric method-external heating method in organic fertilizer (NY 525-2021).

[0054] Example 2

[0055] An application of Bacillus amyloliquefaciens in composting citrus waste as described in Example 1, namely, application of Bacillus amyloliquefaciens LDB1 in composting citrus waste.

[0056] Example 3

[0057] The microbial agent includes the Bacillus amyloliquefaciens as described in Example 1, namely Bacillus amyloliquefaciens LDB1.

[0058] Furthermore, the microbial agent comprises: Bacillus amyloliquefaciens LDB1, Bacillus amyloliquefaciens, Bacillus subtilis and Bacillus velezensis, wherein the total number of viable bacteria of the microbial agent is 1.0×10 9 CFU / mL, while the ratio of the viable cell counts of Bacillus amyloliquefaciens LDB1, Bacillus amyloliquefaciens, Bacillus subtilis and Bacillus velez was 1-10:1:1:1.

[0059] Among them, Bacillus amyloliquefaciens, Bacillus subtilis and Bacillus velezensis are preferably: Bacillus amyloliquefaciens NR_041455, Bacillus subtilis NR_027552 and Bacillus velezensis NR_075005, respectively. The information of the above strains can be queried in the NCBI database.

[0060] The preparation method of the microbial agent comprises the following steps:

[0061] Bacillus amyloliquefaciens LDB1, Bacillus amyloliquefaciens, Bacillus subtilis and Bacillus Velezii were inoculated into the Luria-Bertani medium after high temperature sterilization. When the viable bacterial count was ≥1.0×10 8 CFU / mL, the fermentation broths of Bacillus amyloliquefaciens LDB1, Bacillus amyloliquefaciens, Bacillus subtilis, and Bacillus velez were obtained respectively;

[0062] The fermentation broth of Bacillus amyloliquefaciens LDB1, the fermentation broth of Bacillus amyloliquefaciens, the fermentation broth of Bacillus subtilis and the fermentation broth of Bacillus Velez are mixed in a ratio of 1 to 10:1:1:1, preferably in a ratio of 1:1:1:1, and the carrier auxiliary material biochar is added to obtain a total viable cell count of 1.0×10 9 CFU / mL of liquid microbial agent.

[0063] After adding biochar, the degradation rates of cellulose, hemicellulose, and lignin in the compost maturity period increased, and the humus content increased, indicating that biochar may provide a suitable living environment for composite microorganisms and enhance the ability of strains to degrade lignocellulose. In addition, microbial 16S rRNA sequencing analysis found that adding biochar can promote the succession of microbial communities at different stages of composting.

[0064] Luria-Bertani medium is a medium sterilized at 121°C for 15 minutes, and its ingredients include: tryptone 10g / L, NaCl 10g / L, yeast extract 5g / L, and water 1000mL.

[0065] Example 4

[0066] An application of the microbial agent as described in Example 3 in composting citrus waste.

[0067] Example 5

[0068] A citrus waste composting method, comprising the microbial agent as described in Example 3, comprises the following steps:

[0069] The citrus pruning branches and waste fruits are mixed in a ratio of 3:1, the pruning branches are crushed into a particle size of 1 cm to 2 cm, the waste fruits are crushed, the moisture content of the pile is 60%, and the C / N ratio is about 30, so as to obtain compost raw materials;

[0070] The liquid microbial agent comprising Bacillus amyloliquefaciens LDB1, Bacillus amyloliquefaciens, Bacillus subtilis and Bacillus Velezii is mixed with the compost raw material at a mass ratio of 1 to 5:100 (v / m), and the amount of the agent added is 1% to 5%, preferably 1:100 (v / m), and the amount of the agent added is 1%;

[0071] The mixed material is placed in a polypropylene compost barrel with a height of 65 cm and a diameter of 35 cm. A ventilation hole is set on the top and bottom of the compost barrel, and a stainless steel porous screen is placed 5 cm from the bottom of the barrel to achieve filtrate filtration and uniform oxygen supply;

[0072] Turn the pile every 2 days during the first 7 days of composting, and every 4-5 days thereafter;

[0073] Measure the temperature at the center of the pile at 11 a.m. every day, and measure the moisture content of the pile every 10 days or so. If it is lower than 30%, add water in time. If the temperature is higher than 55°C, turn the pile once a day.

[0074] If the temperature is below 50℃, turn the pile every 3-4 days;

[0075] When the temperature in the later stage of composting is lower than 30℃, stop turning the pile and the composting is finished.

[0076] The results showed that the compost temperature increased rapidly, and after 5 days the compost temperature was higher than 55°C, with the highest temperature being 68.3°C, and the thermophilic period lasted for 20 days. Figure 3 As shown;

[0077] At the end of composting, the seed germination index was 107.2%, the organic matter degradation rate was 24.3%, the hemicellulose, cellulose and lignin degradation rates were 33.8%, 36.5% and 38.5%, and the humus content increased by 18.1%. Figure 4 , Figure 5 as well as Figure 6 shown.

[0078] Comparative Example 1

[0079] The citrus pruning branches and waste fruits are mixed in a ratio of 3:1, the pruning branches are crushed into a particle size of 1 cm to 2 cm, the waste fruits are crushed, the moisture content of the pile is 60%, and the C / N ratio is about 30, so as to obtain compost raw materials;

[0080] No liquid microbial agent containing Bacillus amyloliquefaciens LDB1, Bacillus amyloliquefaciens, Bacillus subtilis and Bacillus velez is added;

[0081] The mixed material is placed in a polypropylene compost barrel with a height of 65 cm and a diameter of 35 cm. A ventilation hole is set on the top and bottom of the compost barrel, and a stainless steel porous screen is placed 5 cm from the bottom of the barrel to achieve filtrate filtration and uniform oxygen supply;

[0082] Turn the pile every 2 days during the first 7 days of composting, and every 4-5 days thereafter;

[0083] Measure the temperature of the pile center three times at 11 a.m. every day, and measure the moisture content of the pile once every 10 days or so. If it is lower than 30%, add water in time. If the temperature is higher than 55°C, turn the pile once a day.

[0084] If the temperature is below 50℃, turn the pile every 3-4 days;

[0085] When the temperature in the later stage of composting is lower than 30℃, stop turning the pile and the composting is finished.

[0086] The results showed that the temperature of the pile rose slowly. After 6 days, the compost temperature was higher than 55℃, with the highest temperature being 65.7℃. The thermophilic period lasted for 15 days. Figure 3 As shown;

[0087] At the end of composting, the seed germination index of the compost was 99.8%, the degradation rate of organic matter was 18.7%, the degradation rates of hemicellulose, cellulose and lignin were 30.9%, 32.0% and 23.2%, and the increase in humus content was 10.9%. Figure 4 , Figure 5 as well as Figure 6 shown.

[0088] Comparative Example 2

[0089] The citrus pruning branches and waste fruits are mixed in a ratio of 3:1, the pruning branches are crushed into a particle size of 1 cm to 2 cm, the waste fruits are crushed, the moisture content of the pile is 60%, and the C / N ratio is about 30, so as to obtain compost raw materials;

[0090] The exogenous microbial agent and the compost raw material were mixed at a mass ratio of 1:100, and the amount of the agent added was 1%. The exogenous microbial agent included Bacillus subtilis, Bacillus licheniformis, Trichoderma and Aspergillus niger, which were provided by the Microbial Engineering Center of Huazhong Agricultural University.

[0091] The mixed material is placed in a polypropylene compost barrel with a height of 65 cm and a diameter of 35 cm. A ventilation hole is set on the top and bottom of the compost barrel, and a stainless steel porous screen is placed 5 cm from the bottom of the barrel to achieve filtrate filtration and uniform oxygen supply;

[0092] Turn the pile every 2 days during the first 7 days of composting, and every 4-5 days thereafter;

[0093] Measure the temperature at the center of the pile at 11 a.m. every day, and measure the moisture content of the pile every 10 days or so. If it is lower than 30%, add water in time. If the temperature is higher than 55°C, turn the pile once a day.

[0094] If the temperature is below 50℃, turn the pile every 3-4 days;

[0095] When the temperature in the later stage of composting is lower than 30℃, stop turning the pile and the composting is finished.

[0096] The results showed that the temperature of the pile rose slowly. After 6 days, the compost temperature was higher than 55℃, with the highest temperature being 66.7℃. The thermophilic period lasted for 16 days. Figure 3 As shown;

[0097] At the end of composting, the seed germination index of the compost was 104.9%, the degradation rate of organic matter was 21.1%, the degradation rates of hemicellulose, cellulose and lignin were 31.7%, 32.8% and 29.0%, and the increase in humus content was 17.5%. Figure 4 , Figure 5 as well as Figure 6 shown.

[0098] Comparative Example 3

[0099] The citrus pruning branches and waste fruits are mixed in a ratio of 3:1, the pruning branches are crushed into a particle size of 1 cm to 2 cm, the waste fruits are crushed, the moisture content of the pile is 60%, and the C / N ratio is about 30, so as to obtain compost raw materials;

[0100] A liquid microbial agent containing Bacillus amyloliquefaciens LDB1, Bacillus amyloliquefaciens, Bacillus subtilis and Bacillus Velez is mixed with a compost raw material at a mass ratio of 1:100, with an agent addition amount of 1%, and rice straw biochar accounting for 5% of the mass ratio of the compost raw material is added as an auxiliary material; the mixture is loaded into a polypropylene compost barrel with a height of 65 cm and a diameter of 35 cm, a ventilation hole is arranged on the upper and lower parts of the compost barrel, and a stainless steel porous screen is placed 5 cm at the bottom of the barrel to achieve filtrate filtration and uniform oxygen supply;

[0101] Turn the pile every 2 days during the first 7 days of composting, and every 4-5 days thereafter;

[0102] Measure the temperature at the center of the pile at 11 a.m. every day, and measure the moisture content of the pile every 10 days or so. If it is lower than 30%, add water in time. If the temperature is higher than 55°C, turn the pile once a day.

[0103] If the temperature is below 50℃, turn the pile every 3-4 days;

[0104] When the temperature in the later stage of composting is lower than 30℃, stop turning the pile and the composting is finished.

[0105] The results showed that the temperature of the pile rose slowly. After 5 days, the compost temperature was higher than 55℃, with the highest temperature being 69.2℃. The thermophilic period lasted for 20 days. Figure 3 As shown;

[0106] At the end of composting, the seed germination index of the compost was 104.5%, the degradation rate of organic matter was 29.3%, the degradation rates of hemicellulose, cellulose and lignin were 39.1%, 37.4% and 42.5% respectively, and the increase of humus content was 18.6%. Figure 4 , Figure 5 as well as Figure 6 shown.

[0107] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A Bacillus amyloliquefaciens that can produce laccase, lignin peroxidase and manganese peroxidase, characterized in that: Its classification name is: Bacillus amyloliquefaciens LDB1, and it was deposited on October 16, 2023 with the preservation number: CCTCC No: M 20231897 in: China Center for Type Culture Collection.

2. Use of the Bacillus amyloliquefaciens as claimed in claim 1 in composting citrus waste.

3. A microbial agent, characterized in that: The invention comprises the Bacillus amyloliquefaciens as claimed in claim 1.

4. The microbial agent according to claim 3, characterized in that: The strains include Bacillus amyloliquefaciens LDB1, Bacillus amyloliquefaciens, Bacillus subtilis and Bacillus velezensis.

5. The microbial agent according to claim 3 or 4, characterized in that: The total number of viable bacteria in the microbial agent is 1.0×10 9 CFU / mL.

6. The microbial agent according to claim 3, 4 or 5, characterized in that: The ratio of the viable cell counts of Bacillus amyloliquefaciens LDB1, Bacillus amyloliquefaciens, Bacillus subtilis and Bacillus velez is 1 to 10:1:1:

1.

7. A method for preparing the microbial agent according to any one of claims 3 to 6, characterized in that: The steps include: Bacillus amyloliquefaciens LDB1, Bacillus amyloliquefaciens, Bacillus subtilis and Bacillus Velezii were inoculated into the Luria-Bertani medium after high temperature sterilization. When the viable bacterial count was ≥1.0×10 8 CFU / mL, the fermentation broths of Bacillus amyloliquefaciens LDB1, Bacillus amyloliquefaciens, Bacillus subtilis, and Bacillus velez were obtained respectively; The four fermentation broths were mixed in proportion, and the carrier auxiliary material biochar was added to obtain a liquid microbial agent.

8. The preparation method according to claim 7, characterized in that: The Luria-Bertani medium is a medium sterilized at 121° C. for 15 minutes, and its ingredients include: 10 g / L tryptone, 10 g / L NaCl, 5 g / L yeast extract, and 1000 mL water.

9. Use of the microbial agent according to any one of claims 3 to 6 in composting citrus waste.

10. A citrus waste composting method, characterized in that: The method comprises the microbial agent according to any one of claims 3 to 6, wherein the steps are as follows: Mix the microbial agent and compost raw materials in a ratio of 1 to 5:100 (v / m) and compost. Measure the temperature of the compost several times at 11 a.m. every day. If the temperature is higher than 55°C, turn the compost once a day. If the temperature is below 50℃, turn the pile every 3-4 days; When the temperature in the later stage of composting is lower than 30℃, stop turning the pile and the composting is finished.

Images