Biodegradation method of retired double-base gunpowder and application of degradation product

Abstract

The invention discloses a biodegradation method of retired double-base gunpowder and application of a degradation product. The method comprises the following steps: inoculating mould into a shallow tray fermentation culture medium to ferment double-base gunpowder powder, inoculating bacteria, transferring into a compost fermentation culture medium, and continuing composting fermentation to obtain a biodegradation product of the double-base gunpowder; the mould is selected from trichoderma simonii, trichoderma koningii, penicillium or trichoderma viride, and the bacteria are bacillus subtilis or bacillus methylotrophicus; the shallow tray fermentation culture medium is composed of clinker and a raw material, the clinker comprises bran and water, the raw material comprises double-base gunpowder and water, the mass ratio of the bran to the double-base gunpowder is 1: 1, the mass ratio of the bran to the water in the clinker is 1: 2, and the mass ratio of the double-base gunpowder to the water in the raw material is 1: 1; the compost fermentation culture medium is composed of bran, double-base gunpowder and water according to the mass ratio of 5: 5: 12, and the inoculum size of the mould and the inoculum size of the bacteria are both 2.5%.

Description

Technical Field

[0001] The invention relates to a biodegradation method for retired double-base propellant and application of the degraded product. Background Art

[0002] Gunpowder is a chemical that causes intense combustion such as sparks and flames, and is the energy source for weapons to launch projectiles. The oldest gunpowder is black powder, one of the four great inventions of ancient China. It is a mixture of potassium nitrate, sulfur, and charcoal, and is still widely used today. Modern gunpowder has many varieties and complex compositions, usually composed of binders, oxidizers, fuels, curing agents, antioxidants, stabilizers, burning rate regulators and other added components.

[0003] Explosives are substances that can burn violently in a very short time. They are substances that explode under the action of a certain amount of external energy and are also called high explosives. In general, the chemical and physical properties of explosives are stable, but no matter whether the environment is sealed or not, how much explosives are used, or even when there is no oxygen supply from the outside, as long as there is strong energy excitation, the explosives will perform stable detonation work on the outside. When explosives explode, they can release a large amount of heat energy and produce high-temperature and high-pressure gas, which can destroy, throw, and compress surrounding substances.

[0004] Gunpowder and explosives are the main non-metallic materials recovered from dismantled ammunition during the destruction of retired and scrapped ammunition. The treatment of gunpowder and explosives is the main task of the destruction of retired and scrapped ammunition. The main components of retired and scrapped double-base gunpowder are nitrocellulose, followed by nitroglycerin. In order to increase its high flammability, ginar, aluminum powder, graphite, lead tannate, diphenylamine, alcohol ether solvents and other ingredients are often added to the formula. Some components of retired and scrapped double-base gunpowder can inhibit the growth of biological cells. Therefore, the difficulty of biodegradation of retired and scrapped double-base gunpowder is greatly increased. Due to the particularity of double-base gunpowder materials, related research reports are rare. Summary of the invention

[0005] The present invention aims to overcome the deficiencies of the prior art and provide a biodegradation method for retired double-base propellant and uses of the degraded product.

[0006] In order to achieve the above-mentioned purpose, the technical scheme provided by the present invention is as follows: the method is characterized in that the mold is inoculated into a shallow tray fermentation medium to ferment the double-base gunpowder powder, and then after inoculating bacteria, the medium is transferred to a compost fermentation medium for continued composting and fermentation to obtain a biodegradable product of the double-base gunpowder; the mold is selected from Trichoderma simmons, Trichoderma koningii, Penicillium or Trichoderma viride, and the bacteria are Bacillus subtilis or methylotrophic Bacillus; the shallow tray fermentation medium is composed of clinker and raw material, the clinker includes bran and water, the raw material includes double-base gunpowder powder and water, the mass ratio of the bran to the double-base gunpowder powder is 1:1, the mass ratio of the bran to the water in the clinker is 1:2, and the mass ratio of the double-base gunpowder powder to the water in the raw material is 1:1; the compost fermentation medium is composed of bran, double-base gunpowder powder and water in a mass ratio of 5:5:12, and the inoculation amount of the mold and bacteria is 2.5%.

[0007] Preferably, the method specifically comprises the following steps: (1) Prepare fungal seed culture medium, bacterial seed culture medium, shallow plate fermentation culture medium and compost fermentation culture medium; prepare 1000 mL of the fungal seed culture medium: add 40.0 g of bran to 1000 mL of water and boil for 30 min, filter with gauze, and then adjust the volume to 1000 mL, then add 10.0 g of glucose, 1.0 g of dipotassium hydrogen phosphate and 0.5 g of magnesium sulfate, put into a 2000 mL conical flask, sterilize at 118°C for 25 min, and set aside; prepare 1000 mL of the bacterial seed culture medium: add 5.0 g of yeast extract, 10.0 g of glucose, 5.0 g of sodium chloride and 1000 mL of water into a 2000 mL conical flask, sterilize at 118°C for 25 min, and set aside; prepare 6.5 kg of the shallow plate fermentation culture medium: add 2.0 kg of water to 1.5 kg of bran, stir and sterilize to make mature material, add 1.5 kg of double-base gunpowder powder and add 1.5 g of water to make mature material. kg, stir and sterilize to make raw material, and then mix the cooked material and the raw material; 11kg of the compost fermentation medium is prepared: 2.5 kg of bran, 2.5 kg of double-base gunpowder powder, and 6.0 kg of water are mixed and stirred, and then placed in a 20 L plastic barrel for standby use; (2) Shake flask seed preparation: Inoculate the mold at a rate of 2.5% into the mold seed culture medium and culture it at 28°C and 150 r / min for 4 days until the logarithmic phase to obtain mold seeds; inoculate the bacteria at a rate of 2.5% into the bacterial seed culture medium and culture it at 37°C and 200 r / min for 1 day until the logarithmic phase to obtain bacterial seeds; (3) Preparation of fermentation seeds: Inoculate the mold seeds into a fermentation tank at an inoculation rate of 2.5%, and culture them at 28°C, 100 r / min, and 8 L / min ventilation for 2.5-3 days until the fermentation liquid is nearly light yellow, thereby obtaining mold fermentation seeds; inoculate the bacterial seeds into another fermentation tank at an inoculation rate of 2.5%, and culture them at 37°C, 200 r / min, and 8 L / min ventilation for 1 day until the fermentation liquid is turbid, thereby obtaining bacterial fermentation seeds; the pressure in the fermentation tank is 0.02-0.04 MPa; (4) Shallow tray fermentation and compost fermentation: Inoculate 500 mL of mold fermentation seeds into 6.5 kg of shallow tray fermentation medium and ferment naturally in an environment above 15°C for 5-6 days. When mycelium grows and a small amount of green spores appear, add 500 mL of bacterial fermentation seeds and then transfer the whole to 11 kg of compost fermentation medium and continue compost fermentation for 15-16 days to obtain the degraded product.

[0008] The degradation product obtained by the above method can be used to prepare rapeseed growth promoter.

[0009] Nitrocellulose is rich in carbon and nitrogen. The main chain is linked by glucose β-1,4 glycosidic bonds, and the nitrogen element is the nitro group and the hydroxyl group of the glucose aglycone dehydrated to form a nitrate ester bond. Biological degradation of nitrocellulose can hydrolyze both glycosidic bonds and nitrate ester bonds. The hydrolysis products may be organic matter or nitrite or nitrate. After nitrocellulose is degraded by microorganisms, it may be absorbed and utilized by plants as nutrients in the form of small molecules. Solid-state fermentation can achieve the degradation of nitrocellulose. Solid fermentation is a commonly used fermentation method for organic composting. It is characterized by simplicity and ruggedness, low cost, good composting effect, and has promotion value in actual production.

[0010] As a high explosive, nitrocellulose has great risks in research and experiments due to its harsh storage and transportation conditions, and there are few studies and reports on it at home and abroad. Hot warfare, physical disassembly, and detonation are still the mainstream methods for the conventional destruction of retired and scrapped ammunition. After more than 40 years of peaceful construction, my country has accumulated some retired and scrapped ammunition that does not meet the needs of the times. Therefore, it is of practical significance to find a safe and environmentally friendly new way to degrade retired and scrapped ammunition. In theory, nitrocellulose is rich in nitrogen and carbon, and its organic matter can be used as a fertilizer, which points the way for the resource utilization of nitrocellulose.

[0011] The results of the cellulase activity and esterase activity of filter paper of nitrocellulose fermented by molds and the reducing sugar and nitrite ion of the degradation products showed that the mold hydrolyzed the β-1,4 glycosidic bonds and nitrate ester bonds of nitrocellulose, but the cellulase activity was higher than the esterase activity, and its molecular mechanism of degradation needs further study. The process of mixed fermentation of nitrocellulose by molds and bacteria is similar to that of organic fertilizer composting. After 21 days of degradation, the residual rate of nitrocellulose was 8.3%, which was more complete than the degradation by only inoculating molds. The content of water-soluble organic matter reached 12.62%, which is higher than the indicator (30%) of the organic fertilizer industry standard NY / T525-2021 in the agricultural industry.

[0012] Nitrocellulose is rich in carbon, hydrogen, oxygen, nitrogen and other elements, and is rich in organic matter after biodegradation. Therefore, the degradation products of nitrocellulose can theoretically be completely recycled into organic fertilizer nutrients. However, in addition to the main component nitrocellulose, double-base gunpowder also contains a small amount of alcohol ether solvents, diphenylamine, camphor, graphite and other components. Studies have shown that the toxicity of diphenylamine is equivalent to that of aniline, and the addition of 50-100 mg / kg diphenylamine to the soil has a significant inhibitory effect on the urease and total bacteria in the soil in the early stage. Therefore, investigating the impact of double-base gunpowder degradation products on soil and crop growth is an important indicator for evaluating the biodegradation of double-base gunpowder into fertilizer.

[0013] Studies have shown that returning crop residues to the field can increase crop yields and soil fertility. The present invention preliminarily explored a feasible way to realize resource utilization and fertilizer of nitrocellulose through partial biological degradation by conducting a study on the effects of double-base gunpowder powder degradation products on rapeseed seedling growth, soil pH, and soil microorganisms. The experimental results show that double-base gunpowder powder degradation products can promote a significant increase in plant height and weight in the rapeseed seedling stage, while the control group (CK) and the normal fertilizer group (NOR) grew slower, far beyond expectations. From March 11 to April 9, 2024, the temperature in Changsha was low in the early stage (see Appendix 1), and there was more rain, including 13 days of light rain, 4 days of moderate to heavy rain, 2 days of heavy rain, 1 day of cloudy weather, and 10 days of sunny weather, so the rapeseed seedlings grew slowly in the early stage. In addition, the lack of preparation in the early stage and the placement of potted plants in the open air caused the potted soil to be somewhat compacted. Although loosening measures were taken during the period, it still had a certain adverse effect on the growth of rapeseed. In the middle and late stages, potted rapeseed is placed in a light-transmitting rain shelter to avoid the interference of rain during the seedling stage. Since there is no significant difference between the control group and the normal fertilizer group, it shows that the fertility of the soil and fertilizer selected this time is not guaranteed. By adding double-base gunpowder powder degradation products, the volume of potted soil increased significantly, thereby increasing soil porosity, reducing bulk density, and improving soil structure. Anning Dominic Kwadwo's study found that the straw return treatment increased the carbon pool management index of the 0-20 cm soil layer (28.52%), and the total organic carbon and carbon pool management index in the soil were significantly positively correlated with the volatile organic matter components in the soil.

[48] . The double-base gunpowder powder degradation products in this experiment may be related to the decomposition of organic matter characterized in the degradation products. The degradation of its cellulose main chain is similar to the degradation of straw cellulose, which significantly reduces the soil pH value of the CK group (pH 7.1) to pH 6.39-6.49 in the degradation product treatment group. At the same time, the activity of acid phosphatase is significantly increased. The nitrocellulose used as experimental materials is rich in 11.8%-12.1% nitrogen. The soil urease activity of the double-base gunpowder powder degradation product treatment group is significantly increased by more than 5-10 times that of the control group. It can be seen that the double-base gunpowder powder degradation product can significantly improve soil fertility and nitrogen nutrients. Based on this, this experimental design focuses on soil enzymes (urease and acid sulfatase) related to fertility nutrients and acidic environment, and no data related to protective enzymes such as alkaline phosphatase and catalase have been obtained. Interestingly, the relative chlorophyll content of the double-base gunpowder powder degradation product treatment group was lower than that of the control group and the normal fertilizer group. However, due to the significant increase in the number of leaves and leaf area index of the degradation product treatment group, the total rapeseed green leaf area was still significantly increased.

[0014] Soil rhizosphere microorganisms play an important role in maintaining soil activity and fertility. Yang Jiangyi's research also found that in addition to increasing garlic yield, the application of organic fertilizers will also significantly increase the abundance ratio of bacteria / fungi in the rhizosphere microorganisms, achieving a change from soil fungi-dominated to bacteria-dominated. In order to investigate the effects of double-base gunpowder powder degradation products on soil microorganisms, the experiment analyzed the changes in bacterial community structure at five levels: phylum, class, order, family and genus. The results showed that double-base gunpowder powder degradation products have a regulatory effect on the structure of microorganisms in the soil, including Myxococcota, Bdellovibrio, Polyangia, Pseudomonadales, Comamonadaceae, Pseudomonadaceae, and Pseudomonas. Pseudomonas ), Cellulovibrio ( Cellvibrio ) was significantly positively correlated with plant height, dry weight, fresh weight and leaf area index. Among them, Vibrio celluli is widely present in the soil and can secrete cellulase and chitinase, which may play an important role in the decomposition of rice straw. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The effect of continuous fermentation of mixed bacteria in shallow trays on the degradation of nitrocellulose; Figure 2 The effect of double-base gunpowder powder degradation products on the growth indexes of rapeseed seedlings; in the figure: (A) plant height; (B) dry weight; (C) fresh weight; (D) number of leaves; (E) leaf area index; (F) relative chlorophyll content SPDA. The same letters indicate no significant difference between groups (p > 0.05, the same below) Figure 3The morphological observation of the effect of double-base gunpowder degradation products on rapeseed growth; Figure 4 The effect of double-base gunpowder powder degradation products on the pH and enzyme activities of potted soil; in the figure: (A) pH; (B) urease; (C) acid phosphatase; Figure 5 The effect of double-base gunpowder powder degradation products on the α diversity of potted soil microorganisms; In the figure: (A) ACE index; (B) Chao1 index; (C) Shannon index; (D) Simpson index; Figure 6 The effect of double-base gunpowder powder degradation products on soil microbial β diversity; Figure: (A) Venn diagram (Venn); (B) principal component analysis (PCA); (C) non-metric multidimensional scaling analysis (NMDS); (D) principal coordinate analysis (PCoA); Figure 7 The effect of double-base gunpowder powder degradation products on the community structure of soil microorganisms at the phylum level; In the figure: (A, B) phylum level; (C) Myxomycetes; (D) Acidobacteria; (E) Bdellovibrio; Figure 8 The effect of double-base gunpowder powder degradation products on the community structure of soil microorganisms at the class level; In the figure: (A, B) class level; (C) γ-Proteobacteria; (D) Polyangia; (E) α-Proteobacteria; Fig. 9 The effect of double-base gunpowder powder degradation products on the community structure of soil microorganisms at the order level; In the figure: (A, B) order level; (C) Pseudomonadales; (D) Sphingomonas; (E) Rhizobiales; Fig.10 The effect of double-base gunpowder powder degradation products on the community structure of soil microorganisms at the family level; In the figure: (A, B) family level; (C) Comamonadaceae; (D) Pseudomonadaceae; (E) Sphingomonasaceae; Fig.11 The effect of double-base gunpowder powder degradation products on the community structure of soil microorganisms at the genus level; In the figure: (A, B) genus level; (C) Vibrio; (D) Pseudomonas; (E) Sphingomonas; Fig.12 The effect of double-base gunpowder powder degradation products on soil microbial LEfSe analysis; Figure: (A) LDA graph; (B) LEfSe graph; Fig.13 Correlation analysis between rapeseed growth index, soil enzyme activity and microbial flora; in the figure: red indicates positive correlation, blue indicates negative correlation, and * indicates p < 0.05. DETAILED DESCRIPTION Example 1

[0016] (1) Bacterial species: Fungi: Trichoderma simmonii, Trichoderma koningii, Penicillium or Trichoderma viride; Bacteria: Bacillus subtilis or methylotrophic Bacillus. In this embodiment, the fungi are preferably Trichoderma koningii, and the bacteria are preferably Bacillus subtilis.

[0017] (2) Reagents: Wheat bran and anhydrous ethanol are both commercially available. Controlled substances such as concentrated sulfuric acid and acetone are purchased through the school's asset department and are purchased and used in accordance with the hazardous chemicals regulations.

[0018] (3) Preparation of mold seed culture medium: Add 40.0 g bran to 1000 mL water and boil for 30 min. Filter with gauze and then make the volume to 1000 mL. Then add 10.0 g glucose, 1.0 g potassium dihydrogen phosphate and 0.5 g magnesium sulfate. Pour into a 2000 mL conical flask and sterilize at 118°C for 25 min. Set aside.

[0019] (4) Bacterial seed culture medium: Place 5.0 g of yeast extract, 10.0 g of glucose, 5.0 g of sodium chloride, and 1000 mL of water into a 2000 mL conical flask, sterilize at 118°C for 25 min, and set aside.

[0020] (5) Shallow plate fermentation medium: Add 2.0 kg of water to 1.5 kg of bran, stir and sterilize to make a clinker. Add 1.5 kg of water to 1.5 kg of double-base gunpowder powder, stir and sterilize to make a raw meal. Then mix the clinker and the raw meal.

[0021] (6) Compost fermentation medium: Mix 2.5 kg of bran, 2.5 kg of double-base gunpowder powder, and 6.0 kg of water, stir and mix thoroughly, then place in a 20 L plastic bucket for later use.

[0022] (7) Preparation of double-base gunpowder powder: Dry the large-particle 1# double-base gunpowder stored in water naturally in the shade, take 500 g, seal it in a ziplock bag, and place it in a -80℃ refrigerator to completely prefreeze. Quickly transfer it to a stainless steel sealed pulverizer, turn on the pulverizer switch, pulverize at high speed for 0.5 min, pause for 5.0 min, and repeat this cycle 8-10 times. The powder is sieved through 80 mesh. During pulverization, use an infrared temperature detector to measure the pulverizer shell temperature from a distance to ensure that the pulverizer temperature does not exceed 50℃. The operator who controls the pulverizer switch should keep a safe distance of more than 10 m from the pulverizer to ensure the safety of the pulverization operation.

[0023] According to the seed culture medium formula, 200 mL of culture medium was prepared. After sterilization and cooling, the fungus was inoculated at an inoculum rate of 2.5%. Cultured at 28°C and 150 r / min for 4 days until the logarithmic phase to obtain fungus seeds.

[0024] According to the formula of bacterial seed culture medium, 200 mL of culture medium was prepared. After sterilization and cooling, 2.5% of the bacterial inoculum was inoculated and cultured at 37°C and 200 r / min for 1 day until the logarithmic phase to obtain bacterial seeds.

[0025] (1) Sterilization fermentation operating conditions: fermentation tank speed 100 r / min, temperature 28°C, ventilation volume 8 L / min.

[0026] (2) Bacterial fermentation operating conditions: fermenter speed 200 r / min, temperature 37°C, ventilation volume 8 L / min.

[0027] (3) Air disinfection: Check and clean the air filter pipe and fermenter of the 10 L fermenter; check and ensure that the temperature electrode, pH electrode, stirrer, spotlight, and valves are working properly. Remove the air filter of the fermenter, disinfect the fermenter and air pipe at 121℃ for 40 minutes, and cool the fermenter to room temperature.

[0028] (4) Sterilization of the tank: Prepare 8 L of culture medium material according to the fermentation medium formula, add it from the feed port, and fill it with tap water to the predetermined volume. Start the sterilization program, pass steam from the jacket, heat the fermenter to 90°C, and stop stirring; pass steam from the fermenter discharge pipe and air intake pipe respectively, while keeping the sampling valve, tail gas valve, and transfer exhaust valve open to discharge steam. When the tank temperature rises to 121°C, keep it warm for 25 minutes; then start the cooling program, after it drops to 100°C, pass sterile air, maintain a positive pressure of 0.02-0.03MPa, and cool to room temperature.

[0029] (5) Inoculation: Close the air inlet valve and inoculate 100 mL of shake bottle seeds at the inoculation port using the fire ring method with an inoculation volume of 2.5%.

[0030] (6) Fermentation start: Set the fermentation tank speed to 100 r / min, the temperature to 28℃, and the ventilation volume to 8 L / min. Maintain the pressure of the fermentation tank at 0.02-0.04MPa and start the fermentation. After 2.5-3 days of cultivation, when the fermentation liquid is nearly light yellow, it is mature mold fermentation seeds.

[0031] For bacterial fermentation, the fermentation tank was set at a speed of 200 r / min, a temperature of 37°C, and a ventilation volume of 8 L / min. The fermentation tank was kept at a pressure of 0.02-0.04 MPa and fermentation was started. After culturing for 1 day, when the fermentation liquid became turbid, it was considered mature bacterial fermentation seeds.

[0032] (7) Fermentation process detection: After fermentation starts, observe three times a day, record pH value, ventilation volume, temperature, and rotation speed to determine whether the fermentation is normal.

[0033] Inoculate 500 mL of mold fermentation seeds into 6.5 kg of shallow tray fermentation medium, and ferment naturally for 5-6 days in an environment above 15 ℃. When mycelium grows and a small amount of green spores appear, add 500 mL of bacterial fermentation seeds, and then transfer the whole to 11 kg of compost fermentation medium to continue composting and fermentation for 15-16 days. Measure the fermentation koji temperature at 12:00 noon every day, and measure the nitrocellulose content regularly. Measure the organic matter content at the end of fermentation.

[0034] Based on the mold fermentation, the effect of adding bacteria and continuing the fermentation on the degradation of nitrocellulose was investigated. Figure 1 .from Figure 1 It can be seen that the double-base gunpowder powder was continuously degraded by mold, and the residual rate of nitrocellulose was 13.7% at 21 days, and more than 70% of the nitrocellulose in the double-base gunpowder powder was degraded. The residue of the double-base gunpowder powder after degradation was completely humified, with a brown-black appearance, without any light green nitrocellulose, and the degraded material was sticky and loose, suitable for soil improvement. After 5 days of mold fermentation, 500 mL of bacterial fermentation seeds were added, and the fermentation was continued. The residual rate of nitrocellulose in the double-base gunpowder powder was 6.3%, and the degradation rate of nitrocellulose reached 93.4%. The degradation rate of shallow plate mixed bacteria fermentation was 23.4% higher than that of single mold degradation. The residue of the double-base gunpowder powder after degradation was completely humified, with the content of water-soluble organic matter accounting for 12.62% of the dry matter, and the appearance was brown-black and sticky. After drying, it has the potential of biological organic fertilizer. Example 2

[0035] (1) Preparation of potting soil: Place the light yellow clay soil from the slope to dry naturally in the shade (water content is about 8%), remove the larger stones, crush them with a mace, and pass them through a 5-mesh sieve to obtain soil particles with good granularity and loose texture (see attached). Figure 2 , attached Figure 3 ). Weigh 3 kg of soil per pot, a total of 40 pots for rapeseed pot design.

[0036] (2) Potted experiment design for rapeseed seedlings: The design was carried out according to Table 4-2, with a total of 5 groups, including the control group CTR, the normal fertilizer group NOR, the low-dose group of double-base gunpowder degradation products NCL, the medium-dose group of double-base gunpowder degradation products NCL, and the high-dose group of double-base gunpowder degradation products NCL. The soil and additives were weighed separately for each pot, and the soil and additives were mixed and poured into the pot. The first watering was done thoroughly, and each group had 8 parallels (Table 1 n = 8). 10-15 rapeseed seeds were sown in each pot. After germination, 7 rapeseed seedlings were retained for further potted experiments. The potted pots were placed in an outdoor rain shelter. The rain shelter was built with a "herringbone" galvanized iron frame. The "herringbone" was 1.5 meters high at the top, 1.2 meters high around, 2.5 meters wide, and 15 meters long. It was covered with transparent high-quality plastic film, ventilated around and surrounded by bird-proof nylon nets, and sunlight could directly enter the shed. Take pictures regularly every 10 days to record the weather temperature and barometer. No pesticides or pest control were applied during the seedling stage. After 30 days of seedling stage, the agronomic traits of rapeseed seedlings, soil enzymes and soil rhizosphere microorganisms (bacteria) were tested. No pest and disease protection was taken during the whole potting process.

[0037] Table 1 Experimental design Group Name Nutrients per pot (0 d) Rapeseed seedling stage (0-30 days) CTR 3 kg soil Normally spray 10 mL of MS nutrient solution and appropriate amount of tap water every 2 days NOR 3 kg soil + 50 g peat fertilizer Normally spray 10 mL of MS nutrient solution and appropriate amount of tap water every 2 days NCD-L Soil 3 kg + NC degradation product 50 g Normally spray 10 mL of MS nutrient solution and appropriate amount of tap water every 2 days NCD-M Soil 3 kg + NC degradation product 75 g Normally spray 10 mL of MS nutrient solution and appropriate amount of tap water every 2 days NCD-H Soil 3 kg + NC degradation product 100 g Normally spray 10 mL of MS nutrient solution and appropriate amount of tap water every 2 days (3) Testing of rapeseed seedling growth indicators: After 30 days of the experiment, the plant height, fresh weight, dry weight, number of leaves, leaf area index, relative chlorophyll content (SPDA), and soil pH of the potted rapeseed seedlings were tested.

[0038] Calculation of leaf number: randomly select 3 rapeseed seedlings from each pot of 7, count the number of all true leaves excluding cotyledons and incompletely expanded buds and leaves, and take the average value.

[0039] Calculation of leaf area index: referring to the determination method of Rong Feirong et al., the middle leaves of 3 rapeseed seedlings were randomly selected from each pot, and the SPDA values ​​of the middle part of the leaf and 1 cm above and below the middle were measured by a handheld chlorophyll meter, and the average value was taken.

[0040] The detection of leaf area index refers to the determination of Guan Chunyun: total leaf area per pot = total number of plants per pot × leaf area per plant; leaf area index per pot = total leaf area per pot / area of ​​each pot.

[0041] (4) Collection of potted soil samples: Soil samples were collected from three points in the flower pot using the "triangle" method. The sampling point was located at the midpoint between the center of the flower pot and the edge of the flower pot. A stainless steel round shovel was used to vertically rotate 5 cm downward to remove the soil block (φ=1.5 cm) and collect the soil sample 5 cm from the soil. The wet weight of the soil sample at each sampling point was about 2 g. The samples were mixed into one sample and marked. The soil samples were used for microbial flora analysis, pH determination, and soil enzyme activity analysis.

[0042] (5) Analysis of soil microbial communities The soil from each potted plant was sampled using the “triangle” method and mixed into one sample, which was then labeled and sent to Beijing Biomarker Biotechnology Co., Ltd. via cold chain for 16S rRNA bacterial flora analysis.

[0043] (6) Determination of soil pH: Take about 2 g of soil sample, soak and mix it with 10 mL of distilled water, and measure the soil pH with a Mettler pH electrode after 30 min.

[0044] (7) Determination of soil enzyme activity: Urease and acid phosphatase activities were detected according to the kit instructions of Beijing Solebow Biotechnology Co., Ltd.

[0045] Soil urease S-UE: Establishment of standard curve: Establish a standard curve based on the concentration of the standard tube (x, μg / mL) and the absorbance ΔA standard (y, ΔA standard). According to the standard curve, substitute the ΔA determination (y, ΔA determination) into the formula to calculate the sample concentration (x, μg / mL). Definition of soil urease S-UE activity calculation unit: 1 μg NH produced per gram of soil sample per day 3 -N is defined as one unit of enzyme activity. S-UE activity (U / g soil sample) = x × 10 × V total ÷ W ÷ T = 60 × x 10: dilution multiple; T: reaction time, 1d; V total: total volume of reaction system, 0.3 mL; W: sample mass, 0.05g.

[0046] Soil acid phosphatase (S-ACP): Activity unit definition: 1 nmol phenol released per gram of soil per day at 37°C is 1 enzyme activity unit. S-ACP (U / g soil sample) = [C standard solution × (A test tube-A blank tube) ÷ (A standard tube-A blank tube)] × V total × 1000 ÷ W ÷ T = 725 × (A test tube-A blank tube) ÷ (A standard tube-A blank tube) ÷ W C standard solution: 0.5 μmol / mL; V total: total volume of catalytic system, 1.45mL; W: soil sample mass, g; T: catalytic reaction time, 24 h = 1 d; 1000: unit conversion factor, 1μmol = 1000 nmol.

[0047] (8) Data processing and analysis SPSS 22 was used to perform Levene's test and Student's test for data variance, and the variance homogeneity test was met. Duncan's method was used for multiple comparisons. Origin 2022 Pro was used to draw the fresh weight, dry weight, plant height, number of leaves, leaf area index, relative chlorophyll content SPDA, soil pH, soil urease and acid phosphatase content, and different levels of microbial content in the soil of rapeseed seedlings. * p <0.05 indicated significant differences.

[0048] 2.1 Effects of double-base propellant powder degradation products on rapeseed growth indicators Through five groups of rapeseed pot experiments (8 parallels in each group, n = 8), the effects of different addition amounts of double-base gunpowder degradation products on the growth of rapeseed seedlings were investigated. Figure 2 ,from Figure 2 (AC) It can be seen that different concentrations of double-base gunpowder degradants affect the plant height, fresh weight, and dry weight of rapeseed. As the concentration of double-base gunpowder degradants increases, the overall growth of rapeseed does not change significantly, but it shows an upward trend, among which the NCD-H group has the best effect. Figure 2 A shows that compared with the CK group (average plant height 7.93 cm), the NOR group (9.27 cm) had no significant change, while the plant height of the NCD-L group, NCD-M group, and NCD-H group increased significantly ( p < 0.05), increased to 14.23 cm, 14.50 cm, and 15.30 cm, respectively. Figure 2 B shows that compared with the CK group (average plant weight 0.045 g), the dry weight of the NOR group (0.095 g) did not change significantly, while the dry weight of the NCD-L group, NCD-M group, and NCD-H group increased significantly ( p < 0.05), increased to 0.216 g, 0.244 g, and 0.248 g, respectively. Figure 2 C shows that compared with the CK group, the fresh weight of the NCD-L group, NCD-M group, and NCD-H group increased significantly ( p < 0.05). The fresh weight of the CK group was 0.48 g, and after treatment with the NCD-L group, NCD-M group, and NCD-H group, the fresh weight increased to 2.32 g, 2.70 g, and 2.98 g, respectively. The fresh weight of the NOR group was 0.90 g, which was not significantly different from that of the CK group.

[0049] In addition, the number of rapeseed leaves, leaf area index and relative chlorophyll content also changed after the soil was treated with different concentrations of double-base gunpowder degradation products. Figure 2 D shows that compared with the CK group, the number of leaves in the NCD-L group, NCD-M group, and NCD-H group increased significantly ( p < 0.05), increased by 16%, 18.67%, and 14.67%, respectively. There was no significant difference in the number of leaves between the NOR group and the CK group. Figure 2 E shows that compared with the CK group, the leaf area index of the NCD-L group, NCD-M group, and NCD-H group increased significantly ( p < 0.05), increased from 0.21 to 0.74, 0.98, and 0.97, respectively. The leaf area index of the NOR group was 0.37, which was not significantly different from that of the CK group. Figure 2 F shows that compared with the CK group, the relative chlorophyll content of the NCD-M group and the NCD-H group was significantly reduced ( p <0.05), there was no significant change in the NCD-L group compared with the CK group, but it showed an upward trend. The results showed that the appropriate concentration of double-base gunpowder degradants can increase the relative content of chlorophyll. In summary, different concentrations of double-base gunpowder degradants have a promoting effect on the growth of rapeseed.

[0050] from Figure 3 It can be seen intuitively from the rapeseed potted plants that the addition of double-base gunpowder powder degradation products has a very obvious effect on the plant height, number of leaves and total biomass of rapeseed in the seedling stage, which shows that it plays a promoting role in the growth of rapeseed.

[0051] The pH, urease and acid phosphatase activities in soil treated with double-base powder degradation products were as follows: Figure 4 The results showed that compared with the CK group (pH 7.1), the soil pH of the NOR group (pH 6.55), NCD-L group (pH 6.41), NCD-M group (pH 6.39), and NCD-H group (pH 6.49) decreased significantly ( p < 0.05, which may be due to the accumulation of organic matter and organic acid by the degradation of fertilizers and nitrocellulose, which produces a neutralization reaction and thus reduces the soil pH. Compared with the CK group (urease 45.45 U / g, phosphatase activity 4504.98 U / g), the urease activity of the NCD-L group (urease 245.91 U / g, phosphatase activity 7552.08 U / g), the NCD-M group (urease 260.05 U / g, phosphatase activity 7797.25 U / g), and the NCD-H group (urease 452.77 U / g, phosphatase activity 7525.81 U / g) increased significantly ( p < 0.05) Among them, the urease activity of the NCD-H group was the highest, at 452.77 U / g, which was nearly 10 times higher. The acid phosphatase activity of the NCD-L group, NCD-M group, and NCD-H group increased significantly ( p < 0.05), up to 7500 U / g or more, indicating that different concentrations of double-base gunpowder powder degradation products can improve the environmental conditions of rapeseed soil by adjusting soil pH and increasing soil enzyme activity, thereby promoting the growth of rapeseed.

[0052] (1) Analysis of α-diversity of soil microorganisms in rapeseed potted plants Figure 5 is α diversity analysis. Ace and Chao1 indexes can be used to measure species abundance. Simpson and Shannon indexes are used to measure species diversity. Figure 5AB showed that compared with the CK group, the Ace index and Chao1 index of the NOR group, NCD-L group, NCD-M group and NCD-H group increased significantly ( p <0.05), indicating that NCD can increase the species abundance of soil microorganisms, and the effect is similar to that of NOR. Figure 5 C shows that the Shannon index of the NCD-L group increased significantly compared with the CK group ( p <0.05), although the changes in the NOR group, NCD-M group, and NCD-H group were not significant, they all showed an upward trend. Figure 5 D is the Simpson index, and there is no significant difference among the five groups of data.

[0053] (2) Analysis of β-diversity of rhizosphere soil microorganisms Figure 6 A is the Venn diagram, from which we can see that the number of OTUs in the five groups is 57. The numbers of OTUs in the CK group, NOR group, NCD-L group, NCD-M group, and NCD-H group are 2358, 8244, 13126, 9286, and 11898, respectively. The numbers of OTUs in the NOR group, NCD-L group, NCD-M group, and NCD-H group are significantly more than that in the CK group, while the numbers of OTUs in the NCD-L group, NCD-M group, and NCD-H group are significantly more than that in the NOR group, indicating that NCD can increase the diversity of rhizosphere soil microorganisms in rapeseed, and the effect is better than that of NOR. Figure 6 (BD) showed the beta diversity of soil microorganisms, including principal component analysis (PCA), principal coordinate analysis (PCoA) and non-metric multidimensional scaling (NMDS). The contribution rates of the first principal component (PC1) and the second principal component (PC2) were 33.41% and 14.33%, respectively. The CK group and the NCD-L group, NCD-M group, and NCD-H group were significantly separated, indicating that there were differences in the soil microbial community structure between those not inoculated with double-base gunpowder powder degraders and those inoculated with double-base gunpowder powder degraders. The results of PCoA and NMDS were consistent with the results of PCA. These results indicate that double-base gunpowder powder degraders can affect the microbial community structure in the soil.

[0054] (1) Effects of double-base gunpowder degradation products on the phylum-level community structure of soil microorganisms in potted plants Figure 8 (AB) shows the top ten microbial phyla, with the dominant phyla being Proteobacteria, Bacteroidota, Myxococcota, and Actinobacteriota, which account for more than 50%. Figure 8C shows that compared with the CK group, the abundance of Myxococcota in the NOR group did not change significantly, while the abundance in the NCD-L, NCD-M and NCD-H groups increased significantly ( p <0.05), 10.73%, 10.67%, and 11.33% respectively. Figure 8 D shows that compared with the CK group, the abundance of Actinobacteriota in the NOR group, NCD-L group, NCD-M group, and NCD-H group was significantly reduced (p<0.05), from 8.23% to 6.11%, 3.93%, 3.50%, and 2.53%, respectively. Figure 8 E shows that compared with the CK group, the abundance of Bdellovibrio in the NOR group did not change significantly, while the abundance of Bdellovibrio in the NCD-L group, NCD-M group, and NCD-H group increased significantly ( p <0.05), were 2.72%, 2.93% and 2.66% respectively.

[0055] (2) Effects of double-base gunpowder degradation products on the community structure of soil microorganisms at the class level Figure 8 (AB) shows the top ten microorganisms at the class level, with the top three microorganisms being Gammaproteobacteria, Bacteroidia, and Alphaproteobacteria. Figure 8 C shows that compared with the CK group, the abundance of Gammaproteobacteria in the NOR group and the NCD-L group did not change significantly, while the abundance in the NCD-M group and the NCD-H group increased significantly ( p <0.05), increased from 22.41% to 35.28% and 37.38% respectively. Figure 8 D shows that compared with the CK group, the abundance of Polyangia in the NOR group did not change significantly, while the abundance in the NCD-L group, NCD-M group, and NCD-H group increased significantly ( p <0.05), 9.78%, 9.70%, and 9.21% respectively. Figure 8 E shows that compared with the CK group, the abundance of Alphaproteobacteria in the NCD-L group did not change significantly, while the abundance in the NOR group, NCD-M group, and NCD-H group decreased significantly ( p <0.05), were 8.61%, 7.99% and 7.63% respectively.

[0056] (3) Effects of double-base gunpowder degradation products on the order-level community structure of soil microorganisms The effects of double-base gunpowder degradation products on the order-level community structure of soil microorganisms are shown in Fig. 9 . Fig. 9 (AB) shows the top ten microorganisms at the order level, with the dominant bacteria belonging to Burkholderiales, Xanthomonadales, and Pseudomonadales. Fig. 9 C shows that compared with the CK group, the abundance of Pseudomonadales in the NOR group did not change significantly, while the abundance of Pseudomonadales in the NCD-L group, NCD-M group, and NCD-H group increased significantly ( p <0.05), with abundances of 7.67%, 12.10%, and 15.30%, respectively. Fig. 9 D shows that compared with the CK group, the abundance of Sphingomonadales in the NOR group, NCD-L group, NCD-M group, and NCD-H group was significantly reduced (p<0.05), from 4.43% to 2.69%, 2.56%, 1.95%, and 1.65%, respectively. Fig. 9 E shows that compared with the CK group, the abundance of Rhizobiales in the NOR group, NCD-L group, NCD-M group, and NCD-H group was significantly reduced ( p <0.05), from 3.50% to 2.32%, 2.37%, 2.21% and 2.34% respectively.

[0057] (4) Effects of double-base gunpowder powder degradation products on the community structure of soil microorganisms at the family level Effects of double-base gunpowder degradation products on the community structure of soil microorganisms at the family level Fig.10 . Fig.10 (AB) are the top ten microorganisms at the family level, Fig.10 C shows that compared with the CK group, the abundance of Comamonadaceae in the NOR group did not change significantly, while the abundance in the NCD-L, NCD-M and NCD-H groups increased significantly ( p <0.05), with abundances of 5.24%, 5.76%, and 6.45%, respectively. Fig.10 D shows that compared with the CK group, the abundance of Pseudomonadaceae in the NOR group and the NCD-L group did not change significantly, while the abundance in the NCD-M group and the NCD-H group increased significantly ( p <0.05), from 0.90% to 2.85% and 6.20% respectively. Fig.10E shows that compared with the CK group, the abundance of Sphingomonadaceae in the NOR group, NCD-L group, NCD-M group, and NCD-H group was significantly reduced ( p <0.05), from 4.43% to 2.69%, 2.56%, 1.95% and 1.65% respectively.

[0058] (5) Effects of double-base gunpowder degradation products on the community structure of soil microorganisms at the genus level The effects of double-base gunpowder degradation products on the community structure of soil microorganisms at the genus level are shown in Fig.11 . Fig.11 (AB) AB are the top ten microorganisms at the genus level, Fig.11 C shows that compared with the CK group, the NCD-L group, NCD-M group and NCD-H group had a higher number of Vibrio spp. Cellvibrio ) abundance increased significantly ( p <0.05), and the abundance increased from 0.21% to 3.52%, 7.30%, and 7.50%, respectively. Fig.11 D shows that compared with the CK group, the Pseudomonas spp. Pseudomonas ) abundance did not change significantly, but increased significantly in the NCD-M and NCD-H groups ( p <0.05), from 0.90% to 2.84% and 6.20% respectively. Fig.11 E shows that compared with the CK group, the NCD-L group, NCD-M group and NCD-H group had a higher number of Sphingomonas genus ( Sphingomonas ) abundance decreased significantly ( p <0.05), from 3.14% to 1.53%, 1.04% and 0.81% respectively.

[0059] (6) Analysis of soil microbial selection and discrimination LEfSe analysis can be used to understand the different biomarkers in different groups. Fig.12 As shown in Figure 2, the main marker bacteria in the CK group were Firmicutes, Acidobacteriota, and Sphingomonas. Sphingomonas The main marker bacteria of the NOR group were Lysobacterium ( Lysobacter ) and Chloroflexi. The main marker bacteria of the NCD-L group were Sorangium ( Sorangium ) and Micrococcus ( MicrococcaceaeThe main marker bacteria of NCD-M group were Fibrobacteria and Bdellovibrionota. The main marker bacteria of NCD-H group were Myxococcota, Gammaproteobacteria, Pseudomonadales, Comamonadaceae and Fibrobacter. Cellvibrio ). The results showed that different concentrations of double-base powder degradation products had a regulatory effect on the structure of soil microorganisms.

[0060] In order to explore the relationship between rapeseed growth indicators and the activity of enzymes in the soil and soil microorganisms, a correlation analysis was conducted. Fig.13 The results showed that the Myxococcota, Bdellovibriota, Polyangia, Pseudomonadales, Comamonadaceae, Pseudomonadaceae, Pseudomonas Pseudomonas ), Cellulovibrio ( Cellvibrio ) was significantly positively correlated with plant height, dry weight, fresh weight, leaf area index, and Acidobacteria. Actinobacteriota was significantly negatively correlated with plant height, dry weight, fresh weight, leaf area index, and Bdellovibrio, Gammaproteobacteria, Pseudomonadales, and Fibrovibrion. Cellvibrio ) was significantly negatively correlated with SPDA.

Claims

1. A biodegradation method for retired double-base propellants, characterized in that: The method comprises the following steps: inoculating mold into a shallow tray fermentation medium to ferment double-base gunpowder powder, then inoculating bacteria and transferring the medium to a compost fermentation medium for further composting and fermentation to obtain a biodegradable product of the double-base gunpowder; the mold is selected from Trichoderma simmonsii, Trichoderma koningii, Penicillium or Trichoderma viride, and the bacteria are Bacillus subtilis or methylotrophic Bacillus; the shallow tray fermentation medium consists of a clinker and a raw material, the clinker comprises bran and water, the raw material comprises double-base gunpowder powder and water, the mass ratio of the bran to the double-base gunpowder powder is 1:1, the mass ratio of the bran to the water in the clinker is 1:2, and the mass ratio of the double-base gunpowder powder to the water in the raw material is 1:1; the compost fermentation medium consists of bran, double-base gunpowder powder and water in a mass ratio of 5:5:12, and the inoculation amount of the mold and bacteria is 2.5% each.

2. The biodegradation method of retired double-base gunpowder according to claim 1, characterized in that: The method specifically comprises the following steps: (1) Prepare fungal seed culture medium, bacterial seed culture medium, shallow plate fermentation culture medium and compost fermentation culture medium; prepare 1000 mL of the fungal seed culture medium: add 40.0 g of bran to 1000 mL of water and boil for 30 min, filter with gauze, and then adjust the volume to 1000 mL, then add 10.0 g of glucose, 1.0 g of dipotassium hydrogen phosphate and 0.5 g of magnesium sulfate, put into a 2000 mL conical flask, sterilize at 118°C for 25 min, and set aside; prepare 1000 mL of the bacterial seed culture medium: add 5.0 g of yeast extract, 10.0 g of glucose, 5.0 g of sodium chloride and 1000 mL of water into a 2000 mL conical flask, sterilize at 118°C for 25 min, and set aside; prepare 6.5 kg of the shallow plate fermentation culture medium: add 2.0 kg of water to 1.5 kg of bran, stir and sterilize to make mature material, add 1.5 kg of double-base gunpowder powder and add 1.5 g of water to make mature material. kg, stir and sterilize to make raw material, and then mix the cooked material and the raw material; 11kg of the compost fermentation medium is prepared: 2.5 kg of bran, 2.5 kg of double-base gunpowder powder, and 6.0 kg of water are mixed and stirred, and then placed in a 20 L plastic barrel for standby use; (2) Shake flask seed preparation: Inoculate the mold at a rate of 2.5% into the mold seed culture medium and culture it at 28°C and 150 r / min for 4 days until the logarithmic phase to obtain mold seeds; inoculate the bacteria at a rate of 2.5% into the bacterial seed culture medium and culture it at 37°C and 200 r / min for 1 day until the logarithmic phase to obtain bacterial seeds; (3) Preparation of fermentation seeds: Inoculate the mold seeds into a fermentation tank at an inoculation rate of 2.5%, and culture them at 28°C, 100 r / min, and 8 L / min ventilation for 2.5-3 days until the fermentation liquid is almost light yellow, thereby obtaining mold fermentation seeds; inoculate the bacterial seeds into another fermentation tank at an inoculation rate of 2.5%, and culture them at 37°C, 200 r / min, and 8 L / min ventilation for 1 day until the fermentation liquid is turbid, thereby obtaining bacterial fermentation seeds; the pressure in the fermentation tank is 0.02-0.04 MPa; (4) Shallow tray fermentation and compost fermentation: Inoculate 500 mL of mold fermentation seeds into 6.5 kg of shallow tray fermentation medium and ferment naturally in an environment above 15°C for 5-6 days. When mycelium grows and a small amount of green spores appear, add 500 mL of bacterial fermentation seeds and then transfer the whole to 11 kg of compost fermentation medium and continue compost fermentation for 15-16 days to obtain the degraded product.

3. Use of the degradation product obtained by the method according to claim 1 or 2 in the preparation of a rapeseed growth promoter.

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