Bacillus licheniformis D1-2 and application thereof in ultrahigh-temperature compost of silkworm excrement

Abstract

The invention discloses bacillus licheniformis D1-2 and application thereof in ultrahigh-temperature compost of silkworm excrement, and belongs to the field of microorganisms, the bacillus licheniformis D1-2 is preserved in China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC No.31328. The invention further discloses a preparation method of the bacillus licheniformis D1-2. Bacillus licheniformis D1-2 is inoculated into silkworm excrement according to the inoculation amount of 6% for composting, the initial heating temperature of composting is 40 DEG C, stirring is conducted once every 3 days, the ventilation amount is 1.5 m < 3 > / min, and the composting time is 24 days or longer. The bacillus licheniformis D1-2 disclosed by the invention has very excellent high-temperature protease and amylase activity. The method is used for a silkworm excrement ultra-high-temperature composting technology, the composting time is shortened by 40%, the composting efficiency is remarkably improved, and technical support is provided for harmless treatment and resource utilization of silkworm excrement in industrial silkworm breeding.

Description

Technical Field

[0001] The invention belongs to the field of microorganisms, and particularly relates to a strain of Bacillus licheniformis D1-2 and application thereof in ultra-high temperature composting of silkworm excrement. Background Art

[0002] Silkworm breeding has always been an important part of China's agriculture. At present, with the advancement of agricultural modernization, traditional silkworm breeding is shifting towards an intensive and factory-based model. Factory-based silkworm breeding has overturned the traditional silkworm breeding industry, bringing significant improvements in production efficiency and increased economic benefits, but it has also brought new challenges, especially in the treatment of silkworm feces. Intensive and factory-based silkworm breeding means that a large number of silkworms are concentratedly raised in a limited space, which leads to a sharp increase in the production of silkworm feces. As the excrement of silkworms, silkworm feces, if not properly handled and directly discharged into the environment, can easily cause environmental problems such as eutrophication of water bodies, thereby destroying the balance of aquatic ecosystems and causing serious pollution to the environment. The harmless treatment of silkworm feces can not only effectively reduce environmental pollution, but also transform silkworm feces into valuable resources and realize resource utilization.

[0003] As an agricultural organic waste, silkworm excrement is rich in nutrients and organic matter and is suitable for use as high-quality organic fertilizer. Composting can not only effectively decompose the organic matter in it, but also release a large amount of nutrients such as nitrogen, phosphorus, and potassium. Its application as a fertilizer helps to improve soil structure and increase soil fertility. However, there is a great difference between the silkworm excrement produced by factory silkworm farming and the silkworm excrement produced by traditional silkworm farming. The composting treatment of silkworm excrement produced by factory silkworm farming still faces great challenges. At present, there is little research on the application of ultra-high temperature composting technology for silkworm excrement, so it is urgent to develop an ultra-high temperature composting technology for silkworm excrement.

[0004] As a new type of aerobic fermentation technology, ultra-high temperature composting has been proven in engineering practice to overcome many shortcomings of traditional high temperature composting. However, there are relatively few studies on ultra-high temperature composting, there are not many thermophilic microbial resources, and the composting process needs to be further improved. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a strain of Bacillus licheniformis D1-2 and application thereof in ultra-high temperature composting of silkworm excrement.

[0006] The technical solution of the present invention is: a strain of Bacillus licheniformis D1-2, which was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on July 16, 2024, with the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, and the deposit number is CGMCC No.31328.

[0007] A biological preparation containing the above-mentioned Bacillus licheniformis D1-2.

[0008] Application of the above-mentioned Bacillus licheniformis D1-2 or biological agent in silkworm excrement composting.

[0009] Furthermore, the application method is: Bacillus licheniformis D1-2 is inoculated into silkworm excrement at a rate of 6% for composting, the initial heating temperature of the compost is 40°C, it is stirred once every 3 days, and the ventilation volume is 1.5m 3 / min, composting time is more than 24 days.

[0010] Furthermore, the silkworm excrement matrix is ​​formed by mixing silkworm excrement and wood chips in a ratio of 1.6:1.

[0011] The strain D1-2 of the present invention has high protease activity and amylase activity. At 60°C, the protease activity is 40.05±2.54U / mL and the amylase activity is 174.01±15.45U / mL. The thermophilic bacterium D1-2 is identified as Bacillus licheniformis. The strain D1-2 has an optimum temperature of 50°C and can tolerate high temperatures above 70°C.

[0012] The verification test of the ultra-high temperature composting process conditions of silkworm excrement showed that the composting process can achieve silkworm excrement maturity in 24 days, and the germination index meets the national industry standard. The optimized composting process matures 16 days faster than the control group, shortening the composting cycle by 40%.

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

[0014] The Bacillus licheniformis D1-2 of the present invention has very excellent high-temperature protease and amylase activities and can be used in the ultra-high temperature composting technology of silkworm excrement. After optimization, the composting time is shortened by 40%, which significantly improves the composting efficiency and provides technical support for the harmless disposal and resource utilization of silkworm excrement in industrial silkworm breeding.

[0015] Collection information:

[0016] Bacillus licheniformis D1-2 was deposited at the General Microbiology Center of China Culture Collection Administration on July 16, 2024. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, and the deposit number is CGMCC No. 31328. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Colony of strain D1-2, Gram staining results and scanning electron microscope 30K magnification photos;

[0018] Figure 2 Phylogenetic tree of strain D1-2;

[0019] Figure 3 Growth of strain D1-2 at different temperatures;

[0020] Figure 4 Temperature changes of silkworm excrement high temperature composting;

[0021] Figure 5 Changes in moisture content of silkworm feces high temperature compost;

[0022] Figure 6 pH changes of silkworm feces during high temperature composting;

[0023] Figure 7 Changes in organic matter content in high-temperature composting of silkworm feces;

[0024] Figure 8 Changes in total nitrogen content of silkworm feces high temperature composting;

[0025] Fig. 9 Changes in total phosphorus content in silkworm excrement equipment compost;

[0026] Fig.10 Changes in total potassium content of silkworm feces high temperature compost;

[0027] Fig.11 Changes in seed germination index of silkworm feces high temperature compost;

[0028] Fig.12 Changes in humus during high-temperature composting of silkworm feces. DETAILED DESCRIPTION

[0029] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from commercial channels unless otherwise specified.

[0030] Main culture medium

[0031] LB medium: peptone 10 g, sodium chloride 10 g, yeast extract 5 g, distilled water 1000 mL, pH 7.0 ± 0.2.

[0032] LB solid medium: peptone 10 g, sodium chloride 10 g, yeast extract 5 g, agar 20 g, distilled water 1000 mL, pH 7.0 ± 0.2.

[0033] Protein hydrolysis medium: tryptone 17 g, glucose 2.5 g, sodium chloride 5 g, potassium dihydrogen phosphate 2.5 g, distilled water 1000 mL.

[0034] Protein plate culture medium: 10g skim milk powder, 20g agar. When preparing, first dissolve skim milk powder in 100mL Wahaha pure water as liquid A, and dissolve agar in 900mL Wahaha pure water as liquid B, and sterilize them separately. After sterilization, mix well in a sterile workbench and pour into a plate.

[0035] Starch hydrolysis medium: 5g beef extract, 10g peptone, 5g sodium chloride, 10g starch, 1000mL distilled water. When preparing, add 800mL water to dissolve beef extract, peptone and sodium chloride, then add starch and adjust to paste with 100mL water, and mix to volume.

[0036] Starch hydrolysis solid medium: 5g beef extract, 10g peptone, 5g sodium chloride, 10g starch, 20g agar, 1000mL distilled water. When preparing, add 800mL water to dissolve beef extract, peptone and sodium chloride, then add starch and adjust to paste with 100mL water, and mix to volume.

[0037] Example 1 Isolation, Screening and Identification of Thermophilic Microorganisms

[0038] 1. Sample source

[0039] The compost samples for separating and screening thermophilic microorganisms in this experiment were from an organic fertilizer production enterprise in Hangzhou, Zhejiang. During the high temperature period, multiple sampling was carried out at different vertical depths of 1 / 2 and 1 / 3, and the samples were placed in sealed bags, marked with information such as sampling time, compost temperature, and compost raw materials, and stored in a 4°C refrigerator for separation.

[0040] 2. Isolation and enzyme activity identification of thermophilic protein (starch) hydrolyzing bacteria

[0041] The compost sample was added to the protein (starch) hydrolysis medium at a ratio of 1:100 (m / v), and the degradation strains were enriched at 60°C. The obtained strains were further screened using protease and amylase screening medium. According to the colony morphology of the strains, 7 thermophilic bacteria capable of decomposing protein and starch were preliminarily obtained, and they were named A-2, 2-2, D1-2, CX-6, D-5-3, D-5-4, and D-6-1, respectively, as shown in Table 1.

[0042] Table 1 Colony morphology characteristics of thermophilic bacteria

[0043]

[0044]

[0045] By observing whether the strain produces a hydrolysis circle on the protein plate hydrolysis medium, the activity of the strain on protein hydrolysis can be preliminarily judged. It can be observed that 4 thermophilic bacteria can produce hydrolysis circles on the protein plate, namely A-2, 2-2, D1-2, and CX-6. The diameter D of the hydrolysis circle and the diameter d of the colony are measured, and their ratio is calculated. The ability of thermophilic strains to degrade protein is preliminarily judged according to the size of the ratio. As shown in Table 2, D1-2 has the highest ratio of 3.59±0.14, followed by A-2 with a ratio of 3.14±0.14, 2-2 with a ratio of 2.38±0.21, and CX-6 with the lowest ratio of only 2.23±0.08.

[0046] Table 2 Protein hydrolysis transparent zone / colony diameter ratio

[0047]

[0048] The four thermophilic proteolytic bacteria 2-2, D1-2, A-2, and CX-6 screened out were tested for their protease activities by Folin-phenol method. The results showed that the protease activity of thermophilic bacteria D1-2 was the highest at 40.05±2.54U / mL, the protease activity of thermophilic bacteria A-2 was 30.90±7.37U / mL, followed by the protease activity of thermophilic bacteria 2-2 at 13.73±3.31U / mL, and the lowest was the thermophilic bacteria CX-6 at 5.11±1.14U / mL. Therefore, thermophilic bacteria D1-2 has a higher protease activity at high temperature and is suitable for use as a high-temperature composting agent for artificial silkworm excrement.

[0049] By spraying the strains with Lugol's iodine solution to observe whether there are transparent circles, it was observed that five strains had transparent circles, which were named D1-2, D-6-1, CX-6, D-5-3, and D-5-4. The diameter of the hydrolysis zone (D) and the colony diameter (d) were measured and their ratio was calculated (Table 3). According to the size of the ratio, the ability of thermophilic strains to degrade starch was preliminarily judged. It was found that the ratio of D1-2 was 4.43, the ratio of D-5-3 was 3.76, the ratio of D-6-1 was 3.43, the ratio of D-5-4 was 3.14, and the ratio of CX-6 was the smallest, only 2.90.

[0050] Table 3 Starch hydrolysis transparent circle / colony diameter ratio

[0051]

[0052]

[0053] The five thermophilic starch hydrolyzing bacteria D-5-3, D1-2, CX-6, D-6-1, and D-5-4 screened out were respectively tested for their amylase activity by DNS method. The results showed that the thermophilic bacteria D1-2 had the highest amylase activity, reaching 174.01±15.45U / ml. The amylase activity of thermophilic bacteria D-5-3 was 96.23±6.62U / ml, the amylase activity of D-6-1 was 69.13±14.79U / ml, and the amylase activity of thermophilic bacteria CX-6 was 53.14±5.41U / ml. The lowest amylase activity was D-5-4, which was only 40.13±3.54U / ml. The amylase activity of the re-screening was basically consistent with the results of the initial screening. Therefore, the thermophilic bacteria D1-2 had a higher amylase activity at high temperature and was suitable as a high-temperature composting and decomposing bacteria agent for artificial silkworm feces.

[0054] The above multiple comparative analysis showed that the protease activity and amylase activity of thermophilic bacteria D1-2 were both high, so thermophilic bacteria D1-2 was selected as the dominant strain.

[0055] 3. Morphological characteristics of strain D1-2

[0056] The colony of strain D1-2 is milky yellow and irregular in shape, with a raised center and jagged edges, and is opaque. It is a Gram-positive bacterium. The three-dimensional image of thermophilic bacteria D1-2 was observed using a scanning electron microscope at 30K times. The strain is long rod-shaped, with wrinkles on the surface of the bacteria and no flagella ( Figure 1 ).

[0057] 4. Molecular identification of strain D1-2

[0058] The genomic DNA of strain D1-2 was extracted using a bacterial DNA extraction kit, and then PCR amplification of the 16S rDNA fragment was performed. Gel electrophoresis showed that the fragment size was about 1500 bp. The purified PCR amplification product was sent to Shanghai Sangon Biotech Co., Ltd. for sequencing, and then the nucleic acid sequence (SEQ ID No.1) was compared with the NCBI database by BLAST homology. The results showed that the similarity between strain D1-2 and Bacillus licheniformis reached 100%. Based on the sequencing data, a phylogenetic tree was constructed using MEGA.11 ( Figure 2 ). Therefore, the strain D1-2 was named: Bacillus licheniformis D1-2.

[0059] 5. Effects of different temperatures on the growth of strain D1-2

[0060] The activated strain D1-2 was inoculated into LB medium at a rate of 0.5%, and the growth of the strain after culturing in an incubator at 20°C, 30°C, 40°C, 50°C, 60°C, and 70°C for 24 hours was as follows: Figure 3As shown in the figure, strain D1-2 basically does not grow below 20℃, but can grow between 30-70℃. With the increase of culture temperature, the growth speeds up, and the best growth is at 50℃. 600 It reached 1.43, and the growth rate slowed down after exceeding 50°C. It can be seen that the optimal growth temperature of strain D1-2 is 50°C, and it can tolerate high temperatures above 70°C.

[0061] 6. Deposit of strain D1-2

[0062] Bacillus licheniformis D1-2 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on July 16, 2024. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, and the deposit number is CGMCC No.31328.

[0063] Example 2 Application of Bacillus licheniformis D1-2 in Ultra-high Temperature Composting of Silkworm Feces

[0064] 1. Experimental methods

[0065] Orthogonal experiment was used to optimize the technology of ultra-high temperature composting of silkworm excrement. Based on the four factors and three levels in Table 4, the orthogonal experiment was designed according to Table 5.

[0066] A total of 9 treatment groups were set up in the orthogonal experiment. In each group, 160 kg of silkworm feces and 100 kg of sawdust as auxiliary material were weighed and mixed evenly, and then moved into the composting experimental device for composting. Different proportions of Bacillus licheniformis D1-2 were added to each experimental group, and different aeration, preheating at different temperatures and stirring frequencies were used for composting according to the orthogonal experimental parameter table 5.

[0067] During the composting process, the physical and chemical properties, maturity and germination index of the compost samples were measured, including the daily temperature, moisture content, pH value, conductivity value, organic matter content of the compost materials and seed germination index. The best composting reaction parameter formula combination was obtained based on the seed germination index and the accumulated temperature during the composting process.

[0068] Table 4 Experimental factor levels

[0069]

[0070] Table 5 Orthogonal test parameters

[0071]

[0072]

[0073] Composting was carried out according to 9 groups of treatments designed according to orthogonal experiments. Each treatment cycle was 12 days. Sampling was carried out on the 0th day, the 4th day, the 8th day and the 12th day respectively. When sampling, the pile was divided into three layers according to the thickness of the pile. The samples were mixed evenly and about 50g was taken and packed into self-sealing bags for storage at -50℃. The remaining samples were air-dried and passed through a 20-mesh sieve for subsequent experiments.

[0074] 2. Detection method

[0075] (1) Temperature

[0076] In this experiment, a PT100 temperature probe was used to monitor the temperature in real time. The temperature data was saved and recorded at three time points every day, and a temperature curve was drawn.

[0077] (2) pH value

[0078] Take 10 g of sample, put it into a 250 mL conical flask, mix it with carbon-free water at a ratio of 1:10 (W / V), place it in a horizontal shaker and oscillate horizontally for 2 hours. After standing for 30 minutes, use a pH meter to measure the pH value of the supernatant.

[0079] (3) Determination of seed germination index GI

[0080] Weigh 10.00g of the sample (fresh sample) and place it in a 250ml conical flask. After converting the moisture content of the sample, add the corresponding mass of water according to the solid-liquid ratio (mass / volume) of 1:10. After tightening the bottle cap, fix it vertically on a reciprocating horizontal oscillator, adjust the frequency to 100r / min, and the amplitude is not less than 40mm. Oscillate and extract at 25℃ for 1h. After removing and standing for 0.5h, take the supernatant and filter it on a filter device pre-installed with filter paper. Collect the filtered extract and shake it for analysis. The filtrate is used on the same day, or stored in an environment of 0℃~4℃ for no more than 48h. Place 1 or 2 qualitative filter papers in a 9cm culture dish, evenly place 10 plump cucumber seeds of basically the same size on it, add 10mL of the sample extract, cover the culture dish cover, and culture in a light-proof incubator at (25±2℃) for 48h. Count the number of germinated seeds and measure the taproot length one by one with a vernier caliper. Use pure water as a control and perform a blank test.

[0081] (4) Determination of nutrient content of organic fertilizer

[0082] Sample preparation

[0083] Weigh 0.5g to 1.0g (accurate to 0.0001g) of the air-dried sample that has passed through a 1 mm sieve, place it at the bottom of a 250mL conical flask or a digestion tube of appropriate volume, rinse the sample adhering to the wall of the flask / tube with a small amount of water, add 5mL sulfuric acid (ρ=1.84g / mL) and 1.5mL 30% hydrogen peroxide, shake carefully, place a small curved neck funnel at the mouth of the flask, and leave it overnight. Slowly heat until the sulfuric acid smokes, remove it, add 15 drops of hydrogen peroxide after it cools slightly, shake the conical flask or digestion tube gently, heat for 10 minutes, remove it, add 5 to 10 drops of hydrogen peroxide after it cools slightly, and digest it in batches until the solution is colorless or light yellow clear liquid, continue heating for 10 minutes, and remove it to cool, carefully add water to 20mL to 30mL, shake the conical flask or digestion tube gently, rinse the small curved neck funnel with a small amount of water, and collect the washing liquid into the conical flask or digestion tube. Transfer the digestion solution into a 100 mL volumetric flask, cool to room temperature, and add water to the mark. Allow to stand to clarify or filter with phosphorus-free filter paper into a triangular flask for later use.

[0084] Determination of Total Nitrogen in Organic Fertilizer

[0085] Add 10.0mL of boric acid-indicator mixture to a conical flask, place the conical flask on the ammonia receiving tray of the distillation instrument, and insert the condenser tube into the boric acid liquid surface. Aspirate 50.00mL of the unboiled liquid, add 200mL of water (depending on the amount of water replenishment of the distillation device) to the distillation flask. Connect the distillation tube to the distillation head of the ammonia determination instrument, add 15mL of 40% sodium hydroxide solution, and distill. When the distilled liquid reaches about 100mL, stop distillation. Titrate the distillate directly with sulfuric acid standard solution or hydrochloric acid standard solution, and the end point is when the color just changes from blue to purple-red. Record the volume of the consumed acid standard solution, and the calculation method refers to the organic fertilizer industry standard NY / T 525-2021.

[0086] Determination of Total Phosphorus in Organic Fertilizer

[0087] Take 0ml, 1.00ml, 2.50ml, 5.00ml, 7.50ml, 10.00ml, and 15.00ml of phosphorus standard solution and place them in 7 50ml volumetric flasks respectively, add water to about 30ml, add 2 drops of 2,4-dinitrophenol indicator, adjust to slightly yellow with 10% sodium hydroxide solution and low concentration sulfuric acid solution, add 10.0ml ammonium vanadate molybdate reagent, shake well, and dilute with water. This standard series of solutions contains 0μg, 50μg, 125μg, 250μg, 375μg, 500μg, and 750μg of phosphorus. After leaving it at room temperature for 20min, use a 1cm colorimetric dish to compare the color at a wavelength of 450nm in the spectrophotometer, adjust to zero with the 0μg standard solution, and read the absorbance. Draw a standard curve with the mass (μg) of phosphorus in the standard series of solutions as the horizontal axis and the corresponding absorbance as the vertical axis. Pipette a sample solution containing 50ug to 750μg of phosphorus into a 50mL volumetric flask, add water to 30mL, develop color and compare colors under the same conditions as the standard series solutions, adjust to zero with the blank test solution, and read the absorbance. Find the corresponding phosphorus mass (μg) on ​​the standard curve, and the calculation method refers to the fertilizer phosphorus determination industry standard NY / T 2541-2014.

[0088] Determination of Total Potassium in Organic Fertilizer

[0089] Accurately pipette 0mL, 2.50mL, 5.00mL, 10.00mL, 15.00mL, and 20.00mL of potassium standard solution into 6 100mL volumetric flasks, add water to the volume, and mix well. The mass concentrations of potassium in this standard series of solutions are 0μg / mL, 2.50μg / mL, 5.00ug / mL, 10.00ug / mL, 15.00μg / mL, and 20.00ug / mL, respectively. On the flame photometer with the selected working conditions, adjust the zero point and full scale (generally 80) of the instrument at the 0 point and the highest concentration point of the standard solution, and then measure the emission intensity value of each standard solution from low concentration to high concentration. Draw a standard curve with the mass concentration (μg / mL) of potassium in the standard series of solutions as the horizontal coordinate and the corresponding emission intensity as the vertical coordinate. Determination of sample solution The sample solution is directly (or after appropriate dilution) measured under the same conditions as the standard series solution, and the emission intensity of potassium is measured, and the corresponding potassium mass concentration (μg / mL) is found on the working curve. Note: The mass concentration of the standard series solution can be adjusted according to the sensitivity of different instruments. The calculation method refers to the determination of potassium fertilizer industry standard NY / T 2540-2014.

[0090] (5) Detection method of humus in compost samples:

[0091] 1) Preparation of sample solution

[0092] Weigh 5.00 g of air-dried sample that has passed through a 0.25 mm pore size sieve into a 250 mL conical flask, add 100 mL of sodium hydroxide-sodium pyrophosphate mixed extract, plug the flask tightly, and oscillate on a constant temperature oscillator at 25°C ± 2°C for 30 min ± 2 min. After a short rest, slightly rotate the conical flask and wash off the particles stuck to the bottle wall with the supernatant. Let it stand for about 24 hours (temperature is about 25°C), shake the solution thoroughly, filter or centrifuge it to make the filtrate clear, discard the residue, and collect the filtrate in a conical flask.

[0093] 2) Determination of humus content

[0094] Pipette 2.00mL~10.00mL (depending on the color of the filtrate) of the sample solution into a 150mL conical flask, neutralize it to pH 7.0 with 1mol / L sulfuric acid solution (can be tested with pH test paper), and filter through a 0.45μm membrane to obtain a sample that is humus.

[0095] 3) Determination of humic acid content

[0096] The pH value of the humus solution was adjusted to 1.0, and 6M HCl was added. The solution was stored at 4°C for 12 hours and centrifuged at 10,000 r / min for 15 minutes. The supernatant and precipitate obtained were fulvic acid and humic acid, respectively. Humic acid was dissolved with 0.05M NaHCO3. Finally, the TOC of HS, FA, and HA was analyzed using a Shimadzu organic carbon analyzer (Shimadzu, Japan).

[0097] 3. Results

[0098] (1) Temperature changes during silkworm excrement composting

[0099] In the initial stage of composting, there is still a large amount of decomposable organic matter in the pile, which provides a source of rapid proliferation for microbial life activities. The pile releases a large amount of heat due to the violent reaction, and the temperature of the pile rises rapidly in this stage. Figure 4 It can be seen that the temperature of various experimental groups under different composting technical conditions showed an upward trend. Among them, treatment three, treatment four, and treatment five had faster temperature rise, higher temperature during the high temperature period, and higher accumulated temperature. Among them, treatment three experimental group had the highest accumulated temperature.

[0100] like Figure 4 It can be seen that the heating time of treatments 1 and 2, which were groups with lower initial heating temperatures, was relatively slow, while the heating time of the other experimental groups with higher initial heating temperatures was relatively fast. After the temperature in the pile exceeded the initial heating temperature, the composting equipment stopped heating, and microorganisms could continue to generate heat by decomposing organic matter in the pile. Among the treatments, treatment 3 had the longest high temperature time, the highest compost temperature, and the highest accumulated temperature.

[0101] (2) Changes in moisture content during silkworm excrement composting

[0102] Moisture content is one of the key factors in composting. Appropriate moisture in the pile can ensure the growth and metabolism of microorganisms, provide reaction media for biochemical reactions, and is also an important indicator of the composting process. During the composting process, as the temperature of the pile rises, the water evaporates and the moisture content of the pile gradually decreases. Figure 5 It can be seen that the moisture content of the nine treatments showed a downward trend during the composting process, and the proportion of moisture content reduction was positively correlated with the composting temperature and aeration volume. The moisture content decreased faster in the high temperature stage and slower in the temperature rise stage. The moisture content of the nine different treatment groups decreased by 14%-56% after the end of composting, among which treatment 2 had the lowest moisture content and treatment 4 had the highest moisture content.

[0103] (3) Changes in pH during silkworm excrement composting

[0104] pH value affects the life activities of microorganisms and is also an important indicator of the quality of compost products. Figure 6 It can be seen that in the early stage of the composting process, the pH value decreased due to the decomposition of macromolecular organic matter to produce organic acids, while the pH of the piles in treatments 3, 5, and 7 gradually increased after 4 days. This may be due to the high ventilation frequency of these three treatments, which provided relatively sufficient oxygen for the piles. During the temperature rise stage, the activity of microorganisms in the piles was enhanced, and ammonia substances were produced after a large amount of easily degradable organic matter was degraded, causing the pH to rise. At the end of the composting of each experimental group, the pH of treatments 3, 5, and 7 were 6.5, 6.0, and 6.3, respectively.

[0105] (4) Changes in organic matter content during silkworm excrement composting

[0106] Organic matter is an important quality indicator of compost products. The organic matter in the initial material is a carbon and nitrogen source for microbial metabolism and is one of the important conditions for the normal growth and metabolism of microorganisms. During the composting process, due to the decomposition and utilization of microorganisms, the amount of organic matter in the compost material will gradually decrease. Figure 7 As shown, the organic matter content of different treatments gradually decreased, among which the organic matter content of treatment three experimental group changed the most.

[0107] (5) Changes in total nitrogen content in silkworm excrement compost

[0108] In the composting process, at the beginning, the nitrogen in the materials mainly exists in the form of organic nitrogen, such as protein, amino acids, etc., and the total nitrogen content is relatively high. As the composting process progresses, microorganisms begin to decompose organic materials and convert organic nitrogen into inorganic nitrogen compounds such as ammonia nitrogen, nitrite and nitrate. As the composting continues, the changes in nitrogen gradually stabilize. Figure 8As shown, the total nitrogen content of the nine different experimental treatments was 3.5% at the beginning, and the total nitrogen content of the different treatments at the end of composting was 3.05% ± 0.06, 3.13% ± 0.03, 2.26% ± 0.02, 2.75% ± 0.04, 2.64% ± 0.02, 3.05% ± 0.06, 3.08% ± 0.07, and 3.3% ± 0.05. Among all the treatments, the total nitrogen content of treatment three was the lowest, and the loss rate was the largest, reaching 37.14%. It may be that the microbial activity in treatment three was higher, and the decomposition rate of organic nitrogen was faster.

[0109] (6) Changes in total phosphorus content in silkworm excrement composting equipment

[0110] Changes in total phosphorus content in silkworm excrement compost Fig. 9 As shown. The initial content of total phosphorus in different treatments was 0.95%. As the composting progressed, the total phosphorus content of different treatments showed an increasing trend overall. At the end of composting, the total phosphorus contents of the nine groups of different treatments were 1.29% ± 0.02, 1.24% ± 0.01, 1.40% ± 0.01, 1.34% ± 0.01, 1.38% ± 0.02, 1.32% ± 0.01, 1.25% ± 0.02, 1.21% ± 0.01, and 1.33% ± 0.01. Compared with the initial stage of composting, the total phosphorus content of treatment three had the largest growth rate of 60.9%.

[0111] (7) Changes in total potassium content in silkworm excrement compost

[0112] Changes in total potassium content in silkworm excrement compost Fig.10 As shown. The initial content of total potassium in different treatments was 2.3%. As the composting progressed, the total potassium content of different treatments showed an increasing trend. At the end of composting, the total potassium content of the nine groups of different treatments was 2.63% ± 0.01, 2.46% ± 0.02, 2.80% ± 0.03, 2.43% ± 0.01, 2.55% ± 0.01, 2.44% ± 0.02, 2.57% ± 0.03, 2.50% ± 0.01, and 2.52% ± 0.01. Compared with the initial stage of composting, the total potassium content of treatment three increased most significantly.

[0113] (8) Changes in germination index in silkworm excrement compost

[0114] Seed germination index (GI) is an important parameter for judging the maturity of compost. Fig.11As shown in the figure, the germination index of silkworm excrement material was low at the beginning, indicating that it was highly toxic to plants. As the composting time prolonged, the germination index of the nine different treatments showed a gradual upward trend. At the end of 12 days of composting, the germination index of treatment three reached 37.9% ± 0.64, which was much higher than that of other treatments. The germination indexes of treatment one, treatment two, treatment four, treatment five, treatment six, treatment seven, treatment eight, and treatment nine were 17.04%, 12.47%, 18.67%, 23.49%, 23.00%, 11.01%, 10.68%, and 14.19%, respectively. However, due to the short composting time, the germination index of each treatment was still low overall.

[0115] (9) Changes in humus in silkworm excrement compost

[0116] Humus (HS) is a type of polymer composed of a series of molecules formed by organic materials under the action of microorganisms and enzymes. It is generally composed of one to multiple aromatic rings attached to a variety of active functional groups. The change pattern of humus content during composting is inconsistent and is related to the composting raw materials and composting process parameters. This study used silkworm feces and sawdust as the main raw materials to conduct nine groups of composting experiments with different treatments. The results are as follows Fig.12 As shown. The experiment shows that the HS content of treatment 1, treatment 3, treatment 5, and treatment 8 experimental groups is higher than that of the initial sample, while the HS content of the other experimental groups is lower than that of the initial sample. Wei Zimin et al.'s research shows that HS first rises and then falls during the composting process. In the early stage of composting, the humus synthesis capacity is extremely strong, and the amount of HS formed reaches a peak; then, because the decomposition rate is greater than the formation rate, while new HS is formed during the composting process, the original HS is also constantly mineralized, and the HS mass fraction shows a downward trend and tends to stabilize. The reason why the humus content in this experiment is higher than the initial sample may be that the composting time is short.

[0117] Different treatments have different effects on the humus of silkworm feces compost. The HA content of treatment one, treatment three, treatment five, and treatment nine experimental groups is higher than the initial sample, while the other experimental groups are lower than the initial sample. During the composting process, fulvic acid (FA) generally shows a downward trend. Compared with HA, FA has the opposite change pattern. The humification index (HI) is the ratio of humic acid to fulvic acid. It is an important indicator for evaluating the humification level of compost and can better reflect the changes in the properties of compost humus components. The higher the humic acid content, the better the humification degree. Fig.12 As shown in the experimental results, the humification coefficients of treatments three and five were significantly different from those of the other experimental treatment groups. The humification coefficients of treatments three and five were higher than those of the other treatment groups, indicating that the humification degree of treatment three experimental group was better.

[0118] Based on the 4-factor 3-level orthogonal test, the accumulated temperature during the 12-day composting and the seed germination index of the final composting material were used as evaluation indicators to conduct orthogonal experimental range analysis, and the effects of the initial heating temperature, stirring frequency, aeration volume, and inoculum volume on the composting process were compared and analyzed, and the best combination was obtained. The calculation results are shown in Table 6.

[0119] The increase in temperature during aerobic composting is an important indicator of the maturity of the compost. During aerobic composting, the composting process can be divided into four periods: heating stage, high temperature stage, cooling stage and maturity stage according to the change in the temperature of the compost body. The accumulated temperature during the composting process is used as an evaluation index. The range analysis of the relevant orthogonal test on the accumulated temperature during 12 days of composting is shown in Table 6. In this orthogonal test, the accumulated temperature of the treatment group 3 with the highest accumulated temperature during the composting process reached 828.83℃ after 12 days of composting. The larger the k value at different levels of each factor, the better the treatment effect. When the accumulated temperature during the composting process is used as the basis for judgment, the optimal process conditions for the high-temperature aerobic composting technology of silkworm feces are A2B3C3D3, that is, the initial heating temperature is 40℃, stirring once every 3 days, and 1.5m 3 / min ventilation volume, 6% bacterial inoculation volume. The magnitude of the range R value reflects the influence of various factors on the experimental results. The influence of various factors on the pile temperature is in the following order: aeration volume > inoculation volume > stirring frequency > initial heating temperature.

[0120] During aerobic composting, the pile gradually ferments to reduce the biological toxicity of the pile. The seed germination index is an important biological indicator to characterize the maturity of the compost. Therefore, the seed germination index value can be used as one of the evaluation references. The seed germination index value of the pile on the last day of composting was used as the evaluation index to conduct an orthogonal test range analysis. The relevant orthogonal test range analysis of the seed germination index value is shown in Table 7. As shown in Table 7, in this orthogonal test, the germination index of the treatment group 3 on the last day of composting was the highest, and the germination index reached 37.93%. The larger the k value at different levels of each factor, the better the treatment effect. When the germination index on the last day of composting is used as the basis for judgment, the optimal process conditions for the high-temperature aerobic composting technology of silkworm feces are A2B3C3D3, that is, the initial heating temperature is 40°C, stirring once every 3 days, and 1.5m 3 / min ventilation volume, 6% bacterial inoculation volume. The magnitude of the range R value reflects the influence of various factors on the experimental results. The influence of various factors on the pile temperature is in the following order: aeration volume > inoculation volume > stirring frequency > initial heating temperature.

[0121] Table 6 Orthogonal test range analysis 1

[0122]

[0123]

[0124] Table 7 Orthogonal test range analysis II

[0125]

[0126] 4. Parameter optimization verification test

[0127] Through orthogonal experimental range analysis, the optimal process conditions for high-temperature aerobic composting of silkworm feces are A2B3C3D3, that is, initial heating temperature of 40℃, stirring once every 3 days, 1.5m 3 / min ventilation volume, 6% inoculation amount of bacterial agent. However, due to the short composting time in the condition optimization, the germination index of the nine treatments did not reach the organic fertilizer industry standard. In order to verify the optimal process conditions, the time for composting to complete decomposition. The optimal process conditions were used for composting verification in the fermentation box (experimental group), and the traditional ground box composting was used as the control group (according to 24kg of silkworm feces and 16kg of sawdust, weighed and mixed evenly to make the moisture content reach about 60%, and 2% of Bacillus licheniformis D1-2 was accessed, and ground box composting was used. The test time was 5-6 months, and the aerobic composting cycle was 40 days). In the control group, the temperature was measured in the upper, middle and lower layers of the pile every afternoon, and samples were regularly taken to determine the germination index of the compost material.

[0128] It can be seen from Table 8 that the highest temperature of the experimental group is 78.6°C, which is 9.1°C higher than that of the control group. In addition, the fermentation period of the experimental group material to meet the national industry standard (NY / T 525-2021) is only 24 days, which is 16 days faster than the control group, shortening the composting period by 40%.

[0129] Table 8 Control group experimental results

[0130]

[0131] Through orthogonal experimental design, the effects of initial temperature, stirring frequency, aeration volume, and inoculation volume on the maturity of ultra-high temperature composting of silkworm feces were explored, and the following conclusions were drawn:

[0132] (1) According to the orthogonal test results, the GI value and the accumulated temperature value were comprehensively analyzed as evaluation indicators for range analysis, and the optimal process conditions for high-temperature composting of silkworm feces were obtained as follows: A2B3C3D 3， That is, the initial heating temperature is 40°C, stirring once every 3 days, 1.5m 3 / min ventilation volume, 6% bacterial inoculation volume. The influence of each factor is in the following order: aeration volume > inoculation volume > stirring frequency > initial heating temperature.

[0133] (2) Under the optimal high-temperature composting process conditions for silkworm feces, verification experiments showed that the germination index of the compost could reach 85 after 24 days of composting, achieving complete decomposition of silkworm feces, which met national industry standards. The optimized composting process matured 16 days faster than the control group, shortening the composting cycle by 40%.

Claims

1. A strain of Bacillus licheniformis D1-2, deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, with the deposit number of CGMCC No.31328.

2. A biological preparation containing the Bacillus licheniformis D1-2 according to claim 1.

3. Use of the Bacillus licheniformis D1-2 according to claim 1 or the biological preparation according to claim 2 in silkworm excrement composting.

4. The use according to claim 3, characterized in that: The application method is: Bacillus licheniformis D1-2 is inoculated into the silkworm excrement matrix at a rate of 6% for composting, the initial heating temperature of the compost is 40°C, the compost is stirred once every 3 days, and the ventilation volume is 1.5m 3 / min, composting time is more than 24 days.

5. The use according to claim 4, characterized in that: The silkworm excrement matrix is ​​prepared by mixing silkworm excrement and wood chips in a ratio of 1.6:1.

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