Low-temperature-resistant and high-temperature-resistant mixed bacterial agent for degrading cellulose and application thereof

Abstract

The invention provides a low-temperature-resistant and high-temperature-resistant mixed bacterial agent for degrading cellulose and application of the low-temperature-resistant and high-temperature-resistant mixed bacterial agent and belongs to the technical field of microbial strains. The mixed bacterial agent for degrading the cellulose, provided by the invention, comprises nine strains which are respectively named as MX-1: bacillus thickens, MX-4: bacillus cereus, CX-2: bacillus albicans, HX-1: bacillus pumilus, FX-5: bacillus altitudinis, HX-9: bacillus subtilis, FX-4: serratia nematophila, DX-5: Exiguobacterium and HX-6: streptomonas arabinowii. The mixed microbial agent provided by the invention can quickly start the sheep manure composting temperature in a low-temperature environment, improve and prolong the high-temperature period temperature and time, shorten the composting period, reduce the content of cellulose components, and effectively improve the composting efficiency and quality.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial strains, and particularly relates to a low-temperature and high-temperature resistant mixed bacterium agent for degrading cellulose and its application. Background Art

[0002] The aquaculture industry in China is gradually moving towards intensification and large-scale development. The annual cumulative discharge of livestock and poultry manure reaches up to 9.5 billion tons. The problem of manure treatment has become a bottleneck in the development of the livestock industry. The fertilizer utilization and resource utilization of livestock and poultry manure are one of the effective methods to solve the current harmless treatment of manure. However, the technology in some farms is still not perfect. According to investigations, the resource utilization rate of manure is only 15%, which has an adverse impact on the air and the surrounding environment. Therefore, how to carry out the harmless treatment of manure and improve the utilization rate is an important issue restricting the sustainable development of the aquaculture industry.

[0003] Aerobic composting is an effective method for the treatment and resource utilization of livestock and poultry manure. However, it is prone to difficult degradation or incomplete degradation due to the influence of temperature and composition. Cellulose is a kind of organic macromolecule substance that is difficult to degrade and is an important reason restricting the incomplete degradation of compost fermentation under natural conditions. Currently, the main methods for treating cellulose are physical, chemical treatments, and biodegradation methods. Among them, the physical and chemical methods have harsh reaction conditions and high equipment costs. In contrast, the biodegradation method has economy and high efficiency and has become the focus of current research. Existing research has confirmed that adding exogenous microorganisms to compost using the biodegradation method can improve the degradation efficiency. For example, after adding cellulose-degrading bacteria during the composting process by Shan Jianrong et al., the highest temperature can reach 62.5 °C, and the cellulose degradation rate is improved (Shan Jianrong, Quan Xin, Zhu Yongzhe, et al. Screening and optimization of enzyme production conditions of a low-temperature cellulose-degrading bacterium [J]. Chinese Journal of Ecology. 2021, 40(04): 1128-1136.); Wu Xiaosha et al. inoculated high-temperature-resistant cellulose-degrading bacteria in cow dung and straw compost, which can quickly initiate compost fermentation, rapidly increase the temperature, have a long high-temperature duration, and high cellulose degradation efficiency (Wu Xiaosha, Li Zaixing, Huang Yali, et al. Screening and identification of high-temperature lignocellulose-degrading bacteria and their application in composting [J]. Journal of Anhui Agricultural Sciences. 2021, 49(20): 68-71.). However, in winter in northern China, it is cold and the low-temperature period is long, and normal compost fermentation cannot be carried out. Many scholars have screened single cellulose-degrading bacteria, but due to the special structure of cellulose, a single strain cannot complete the degradation function, only providing good bacterial species resources for the resource utilization of cellulose substances. To solve this problem, screening cellulose-decomposing bacteria that are resistant to low temperature and high temperature and making them into a composite microbial bacterium agent is an important part of realizing the harmlessness of compost.

[0004] Termites exist in nature as decomposers, with a large number and wide distribution. During the process of evolution, intestinal microorganisms and the body have a mutualistic symbiosis. The parasitism of microorganisms in the intestine is beneficial for the termite body to absorb nutrients, carry out biological metabolism and physiological development. According to the different types of food ingested by termites, the diversity of intestinal microorganisms is also different. At present, the research on cellulose-degrading bacteria in the termite intestine is increasing, but for dung beetles, which are also decomposers, there are few reports on this research. Although cellulose-degrading bacteria have been successfully isolated from soil, marine environments and animal digestive tracts, there are also few studies on isolating and screening cellulose-degrading bacteria from horse manure. In this invention, samples of the intestines of termites and dung beetles, as well as the initial stage and high-temperature stage of horse manure compost fermentation, are taken to attempt to screen cellulose-degrading bacteria that are resistant to low temperature and high temperature, so as to contribute to the further promotion of the sustainable development of the breeding industry. Summary of the Invention

[0005] The purpose of the present invention is to provide a mixed bacterial agent for degrading cellulose, which can quickly start the temperature of sheep manure compost in a low-temperature environment, increase and extend the temperature and time of the high-temperature period, shorten the composting cycle, and reduce the content of cellulose components.

[0006] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0007] The present invention provides a mixed bacterial agent for degrading cellulose. The mixed bacterial agent includes 9 strains, which are respectively named MX-1: Bacillus firmus, MX-4: Bacillus cereus, CX-2: Bacillus albus, HX-1: Bacillus pumilus, FX-5: Bacillus altitudinis, HX-9: Bacillus subtilis, FX-4: Serratia nematodiphila, DX-5: Exiguobacterium, HX-6: Luteimonas arabica.

[0008] The present invention also provides a preparation method of a solid bacterial agent of the mixed bacterial agent. After mixing the 9 strains, they are added to crushed corncobs according to an inoculation amount of 1%, and after drying, 500 g of glucose is added to obtain the solid bacterial agent.

[0009] Preferably, the bacterial concentrations of the 9 strains are respectively: MX-1: 6-8×10-7CFU / mL, MX-4: 8-9×10-10CFU / mL, FX-4: 5-7×10-6CFU / mL, FX-5: 3-4×10-7CFU / mL, CX-2: 5-6×10-6, DX-5: 7-8×10-6CFU / mL, HX-1: 5-6×10-8, HX-6: 7-8×10-7, HX-9: 7-8×10-6. Among them, the low-temperature bacteria are: FX-4, CX-2, DX-5, HX-1; the high-temperature bacteria are: MX-1, MX-4, FX-5, HX-6, HX-9; the mixing ratio is low-temperature bacteria: high-temperature bacteria = 2-4:1.

[0010] Preferably, the mesh number of the crushed corncob is 18 - 22 meshes.

[0011] Preferably, the added weight of glucose is 1 / 10 - 1 / 15 of the weight of the corncob.

[0012] The present invention also provides a solid bacterial agent obtained by the preparation method described above.

[0013] The present invention also provides an application of the mixed bacterial agent or the solid bacterial agent described above in the composting of sheep manure in the low - temperature season.

[0014] The present invention also provides a method for composting sheep manure using the mixed bacterial agent or the solid bacterial agent. Mix the mixed bacterial agent or the solid bacterial agent into the sheep manure pile body, and ferment at 13 - 16 °C for 20 - 23 days. During this period, after the composting starts and reaches the high - temperature period and lasts for 3 - 4 days, turn the pile twice.

[0015] Preferably, the water content of the sheep manure pile body before the start of composting is about 50 - 70%.

[0016] The present invention takes samples from the intestines of termites and dung beetles, and the initial stage and high - temperature stage of horse manure composting, screens out cellulose - degrading bacteria that are resistant to low temperature and high temperature, and successfully constructs a solid microbial fermentation bacterial agent for degrading cellulose that is resistant to low temperature and high temperature. Adding it to sheep manure for composting at 15 °C can enable the pile body to quickly reach the starting temperature, shorten the composting cycle, extend the high - temperature period, improve the composting efficiency and the quality of organic fertilizer, and significantly reduce the cellulose content in the pile body. Description of the Drawings

[0017] Figure 1 It is the standard glucose curve in Example 1;

[0018] Figure 2 It is the detection result of CMCase activity under the temperature conditions of 15 °C and 55 °C in Example 1;

[0019] Figure 3 It is the detection result of FPA activity under the temperature conditions of 15 °C and 55 °C in Example 1;

[0020] Figure 4 It is the LPS activity under the temperature conditions of 15 °C and 55 °C in Example 1;

[0021] Figure 5 It is the phylogenetic tree obtained in Example 1;

[0022] Figure 6 It is the temperature change result in Example 1;

[0023] Figure 7 It is the pH change result in Example 1;

[0024] Figure 8 Results of moisture content changes in Example 1

[0025] Figure 9 Results of GI changes in Example 1

[0026] Figure 10 Results of C / N content changes in Example 1

[0027] Figure 11 Results of organic matter content changes in Example 1

[0028] Figure 12 Results of total K content changes in Example 1

[0029] Figure 13 Results of total P content changes during the composting process in Example 1

[0030] Figure 14 Results of cellulose content changes in Example 1

[0031] Figure 15 Alpha index rarefaction curve in Example 1

[0032] Figure 16 Species composition at the phylum level in Example 1

[0033] Figure 17 Species composition at the genus level in Example 1 Specific implementation manners

[0034] The technical solutions provided by the present invention will be described in detail below in conjunction with examples, but they should not be construed as limiting the protection scope of the present invention.

[0035] Example 1

[0036] 1.1 Materials

[0037] 1.1.1 Sample sources

[0038] Adult worker termites of Odontotermes formosanus, Macrotermes barneyi, and Coptotermes formosanus were purchased from Maoming Jingfeng Pest Control Service Co., Ltd.; dung beetles were purchased from Qushou Natural Interesting Insects and Plants Museum; fresh horse manure was collected from the Bajun Horse Farm in Qingyuan District, Baoding City; fresh sheep manure was collected from farmers.

[0039] 1.2 Methods

[0040] 1.2.1 Screening of cellulose-degrading bacteria resistant to low temperature and high temperature

[0041] In the intestinal suspensions of termites and dung beetles and in the samples during the initial and high-temperature periods of horse manure fermentation, preliminary screening, isolation and purification, and selective cultivation were carried out according to the following methods:

[0042] ① Preparation of suspension: Select healthy worker termites and dung beetles and place them in a petri dish. After disinfection and washing, separate the heads and intestinal parts of the termites and dung beetles. Take 0.2 g of intestinal tissue and mix it with 1.8 ml of normal saline in a centrifuge tube, grind it thoroughly to obtain an intestinal suspension, mix it with normal saline at a volume ratio of 1:9, and perform 10-fold serial dilutions to 10 -1 -10 -8 ; For the fresh horse manure compost fermentation, at the high-temperature stage on the first day of fermentation, collect samples at the four corners and the middle of the compost pile using the five-point sampling method; Mix 10 g of horse manure sample with 90 ml of sterile water in a conical flask, shake well at 37 °C for 30 min, let it stand for 10 min after taking out, take the upper layer liquid and mix it with sterile water at a volume ratio of 1:9, and perform 10-fold serial dilutions to 10 -1 -10 -8 .

[0043] ② Primary culture: Select 200 μL each of the intestinal suspension and the suspension after horse manure compost fermentation at a 10 -3 -10 -8 gradient, and evenly spread the two samples on LB solid plates respectively. After incubating at 37 °C for 24 h, pick single colonies and perform streak culture using the streak plate method.

[0044] ③ Isolation and purification: Purify the single colonies on the plate 3 times until there is no growth of contaminants in the petri dish. Pick single colonies and perform proliferation culture in LB liquid medium at 37 °C and 180 rpm for 12 h, and then perform streak observation to ensure that the strain is single and free of contaminants.

[0045] ④ Screening: Dip a sterile cotton swab into each single colony and spot inoculate it on the CMC-Na medium, and incubate at 37 °C for 24 h. Use the CR staining method: Pour the prepared CR solution into the CMC-Na medium to cover the colonies, stain for 15 min, wash with 1 mol / L NaCl solution until decolorization produces a clear zone, and measure the radius (D) of the clear zone and the radius (d) of the colony.

[0046] Place the screened strains with cellulose degradation function at 15 °C, 25 °C, 37 °C, 45 °C, and 55 °C respectively for cultivation, and observe the growth status.

[0047] 1.2.2 Antagonism experiment

[0048] Dip an inoculation loop into a small amount of colonies respectively and perform cross streak culture on LB plates.

[0049] Results

[0050] When the ratio of the radius of the transparent zone to the radius of the colony > 2, it indicates that the microorganism has certain enzyme activity. Nine strains with cellulose degradation function were obtained through steps such as primary culture, isolation and purification, and selective culture, and were named FX-4, FX-5, MX-1, MX-4, FX-5, CX-2, HX-1, HX-9, and DX-5 respectively. (The same as the strains obtained in "Screening and Identification of Strains with Strong Decomposition Ability") It was found through experiments that the diameter ratios of the nine strains were all ≥ 2, and the D / d value of HX-1 was the largest at 6.0 cm, with the strongest cellulose decomposition ability and much higher than that of other strains. And there was no antagonistic phenomenon among the nine strains.

[0051] Strain DX-5 grew well at 15 °C; strains FX-4, FX-5, and HX-9 grew normally at 25 °C; strains MX-1, MX-4, FX-4, FX-5, CX-2, and HX-1 grew well at 45 °C, and strain HX-9 grew well at 55 °C.

[0052] 1.2.3 Determination of the enzyme activity of strains at different temperatures

[0053] ① Drawing of the glucose standard curve Prepare a 1 mg / mL standard glucose solution with anhydrous glucose. Prepare 7 stoppered test tubes, and pipette 0, 0.2, 0.4, 0.6, 0.8, 1.0, and 1.2 mL of the standard glucose solution into each tube, and make up the volume to 2.0 mL with water. Add 1.5 mL of DNS reagent, heat in boiling water for 5 min, quickly cool with running water, and then make up the volume to 25 mL and mix well to measure the OD540nm.

[0054] As Figure 1 shown, the glucose standard curve equation and the correlation coefficient are respectively: y = 0.4896x + 0.0283, R 2 = 0.9975. The OD540nm values corresponding to each concentration are shown in Table 1. The linear relationship of this graph is good, which can provide accurate calculation data and reference for the determination of CMCase activity and FPA activity.

[0055] Table 1 OD540nm values corresponding to different glucose contents

[0056]

[0057]

[0058] ② Preparation of crude enzyme solution: 9 strains of bacteria were amplified and cultured at 37 °C and 180 r / min for 12 h. They were inoculated into 200 mL of fermentation medium (fermentation medium for enzyme production: 10.0 g / L tryptone, 10.0 g / L yeast extract, 10.0 g / L CMC-Na, 5.0 g / L NaCl, 1.0 g / L KH2PO4, 0.2 g / L MgSO4·7H2O, 1000 mL of pure water) at an inoculation amount of 2%, and cultured at 15 °C and 55 °C at 180 r / min for 5 d. 1 mL of the culture solution was centrifuged at 8000 r / min for 10 min at 4 °C, and the supernatant was the crude enzyme solution. Three parallel experiments were performed for each strain.

[0059] ③ Determination of CMCase activity: The 3,5-dinitrosalicylic acid method (DNS) was used to determine the activity of cellulase according to the amount of reducing sugar produced in the crude enzyme solution. A number of stoppered test tubes were numbered (1 was the blank control, and tubes 2, 3, and 4 were the parallel experiments of the corresponding strains). 1.5 mL of sodium citrate buffer was added and mixed evenly with 1 g of CMC-Na. After preheating in a 50 °C water bath for 5 min, 0.5 mL of the crude enzyme solution inactivated at high temperature for 10 min was added to tube 1, and 0.5 mL of the non-inactivated crude enzyme solution was added to the other tubes. They were incubated in a 50 °C water bath for 30 min, and immediately 1.5 mL of DNS reagent was added and shaken well. The test tube stopper was covered and heated in a boiling water bath for 10 min, quickly cooled with running water and made up to 25 mL, and mixed well to measure OD540nm. The results are as Figure 2 shown.

[0060] ④ Determination of FPA activity: The DNS method was used to determine the activity of filter paper enzyme according to the amount of reducing sugar produced in the crude enzyme solution. A number of stoppered test tubes were numbered for the corresponding strains (1 was the blank control, and tubes 2, 3, and 4 were the parallel experiments of the corresponding strains). 1.5 mL of sodium citrate buffer was added. Whatman No. 1 filter paper was cut into strips of 6 cm × 1 cm, and de-starch treatment was carried out (soaked in acetic acid for 48 h, washed with NaHCO3 to remove acetic acid and then dried), immersed in the bottom of the test tube, preheated in a 50 °C water bath for 5 min, and the remaining steps were the same as the above determination of CMCase activity. The results are as Figure 3 shown.

[0061] Definition of enzyme activity: Under the reaction conditions of 50 °C, the amount of cellulase required to catalyze the substrate to produce 1 μmol of glucose in 1 min, expressed as 1 U / mL.

[0062] The enzyme activity was calculated by the following formula:

[0063] Enzyme activity = OD540nm × (1 / k) × N × 1000 / T

[0064] k is the slope of the standard glucose curve; N is the dilution factor (this value is 1 if not diluted); T is the reaction time.

[0065] ⑤ The lipase activity was determined by sending the centrifuged crude enzyme solution to Qingdao Standard Testing Co., Ltd. The results are as Figure 4 shown.

[0066] Results

[0067] From Figure 2 , 3 , and 4, it can be seen that at 15°C, the highest CMCase activity and LPS activity of HX-9 are 18.49 U / mL and 275.92 U / L respectively, and the highest FPA activity of MX-4 is 33.48 U / mL; at 55°C, the highest CMCase activity, FPA activity, and LPS activity of HX-1, HX-6, CX-2, and FX-4 are 17.89 U / mL, 19.26 U / mL, and 227.3 U / L respectively.

[0068] 1.2.4 Molecular biological identification

[0069] The screened strains were subjected to PCR amplification using the universal bacterial primers 27F (5'-GAGTTTGATCMTGGCTCAG-3'SEQ ID NO.1) and 1492R (5'-ACGGCTACCTTGTTACGACTT-3'SEQ ID NO.2). The PCR reaction system (total volume 20 μL): 2×Rapid Taq Master Mix 10 μL, 0.5 μL of each upstream and downstream primer, 1 μL of DNA template, 8.9 μL of ddH2O. Reaction conditions: 95°C for 5 min; 95°C for 15 s, 56°C for 20 s, 72°C for 30 s, cycle 35 times; 72°C for 3 min of extension.

[0070] DNA extraction, PCR amplification were performed on 9 strains. The sequencing results were analyzed for sequence similarity comparison through the BLAST program on the NCBI website. The standard strains with high similarity to the 9 strains were used to analyze the homology and similarity of the target strains by MEGA7.0, and a phylogenetic tree was constructed, as Figure 5As shown in the figure. The results show that: Strain MX-1 and Bacillus sp. in Firmicutes, MX-4 and Bacillus cereus, CX-2 and Bacillus proteolyticus belong to the same branch with a homology of 95%; Strain HX-1 and Bacillus pumilus, FX-5 and Bacillus altitudinis, HX-9 and Bacillus subtilis belong to the same branch with a homology of 100%; Strain FX-4 and Serratia nematodiphila, DX-5 and Exiguobacterium aestuarii, HX-6 and Streptomonospora arabica do not belong to the same branch, but their homology with the standard strain is also 100%. Among them, Streptomonospora arabica is an actinomycete.

[0071] The physiological and biochemical characteristics of 9 strains of bacteria were identified, and the results are as follows:

[0072] MX-1: Aerobic bacteria, the most suitable growth temperature is 10 - 45°C. The colonies are round or irregular in shape on the LB plate, and the edges may be smooth or wavy, G+.

[0073] MX-4: Facultative anaerobic bacteria, the most suitable growth temperature is 28 - 35°C. The colonies are milky white, large, and irregular in shape on the LB plate, G+.

[0074] FX-4: Facultative anaerobic bacteria, can grow within the temperature range of 10 - 36°C. The catalase reaction is strongly positive, can ferment D-glucose and other sugars to produce acid, and some can also produce gas; the pigment produced shows red in an acidic environment and yellow under alkaline conditions.

[0075] FX-5: Aerobic bacteria, the cells are rod-shaped, irregular or curved, non-motile, the colonies are round, smooth on the surface, light blue; pH 7.0 is beneficial for spore formation during cultivation at 30°C; it has strong acid resistance and can grow in an acidic pH environment.

[0076] CX-2: Aerobic bacteria, the colonies are usually light yellow or white on the LB plate; the colony morphology is approximately round, the edges are irregular, the surface is dry, rough and slightly wrinkled, G+.

[0077] DX-5: Anaerobic bacteria, capable of surviving in an environment with a pH value of 6.5 - 11.5. As the culture time progresses, it will gradually transform into coccus; G+, with an appropriate growth temperature of 37°C, not capable of producing spores, and not tolerant of acidic environments.

[0078] HX-1: Aerobic bacteria, with uniform coloring, no capsule, peritrichous flagella, and capable of movement; G+; can grow between 10 - 50°C, with the optimal growth temperature being 30 - 40°C, and can grow within the range of 10 - 50°C; the optimal pH is 7.0 - 7.5, can grow within the pH range of 4 - 9, and has a relatively strong adaptability to neutral and slightly alkaline environments.

[0079] HX-6: Aerobic bacteria, with a growth temperature range of 16 - 45°C, a growth pH range of 5 - 9 (optimal 7), G+, spores are oval or rod-shaped, and the spore surface has folds.

[0080] HX-9: Aerobic bacteria, with relatively fast growth and reproduction rates, the colony surface is rough and opaque, dirty white or slightly yellow; thermophilic and acid-tolerant, capable of growing under high-temperature conditions and tolerating low temperatures; can secrete various extracellular enzymes; can utilize different types of carbon sources and nitrogen sources for growth, and has good adaptability.

[0081] The biochemical characteristics are shown in Table 1:

[0082] Biochemical characteristics of 9 strains of bacteria

[0083]

[0084]

[0085] After identification, the results are as follows: MX-1: Bacillus firmus, MX-4: Bacillus cereus, CX-2: Bacillus albus, HX-1: Bacillus pumilus, FX-5: Bacillus altitudinis, HX-9: Bacillus subtilis, FX-4: Serratia nematodiphila, DX-5: Exiguobacterium, HX-6: Luteimonas arabica.

[0086] 1.2.5 Construction of high-temperature and low-temperature resistant cellulolytic bacterial agents

[0087] The growth curves were plotted, and after determining the time when each strain grew to 80% of the logarithmic phase (MX-1: 7×10 -7 CFU / mL, MX-4: 8.64×10 -10 CFU / mL, FX-4: 6×10 -6 CFU / mL, FX-5: 3.2×10 -7 CFU / mL, CX-2: 5.3×10 -6 , DX-5: 7.2×10 -6CFU / mL, HX-1: 5.2×10 -8 , HX-6: 7.6×10 -7 , HX-9: 7.8×10 -6 , with a mixing ratio of 3:1), 6 kg of 20-mesh corncobs were selected as the carrier of the microbial agent. After being washed and disinfected, they were added according to an inoculation amount of 1%. After culturing 200 mL of each bacterial liquid according to the ratio and mixing it with the corncobs, it was dried and then 500 g of glucose was added as a fermentation primer and mixed evenly with the microbial agent, and the preparation of the microbial agent was completed. The prepared solid microbial agent was yellowish-brown in color, extremely fine, and accompanied by the fishy smell of microorganisms.

[0088] 1.2.6 Evaluation of the fermentation effect of the solid microbial agent on sheep manure composting

[0089] Indoor sheep manure composting was carried out in March 2024, with the environmental temperature controlled at about 15 °C. Two groups of composting were set up. The control group was group C mixed with: corncobs + glucose, and the experimental group was group T mixed with: corncobs + bacterial liquid + glucose. The moisture content of the sheep manure pile was controlled at 60%. To ensure ventilation and no water accumulation at the bottom of the pile, several holes were drilled at the bottom of the plastic box. After the composting started to heat up and reached the high-temperature period and continued for 3 days with a cooling trend, it was timely turned over or supplemented with water until the composting ended. Sampling was carried out by the five-point sampling method at the initial stage (C1, T1), heating (C2, T2), high temperature (C3, T3), cooling (C4, T4), and maturity stage (C5, T5) of the composting, and then mixed and sealed. One part was stored at -20 °C for detecting physical and chemical indexes, and the other part was stored at -80 °C for microbial diversity analysis.

[0090] 1.2.7 Determination of physical and chemical indexes

[0091] During the composting period, the temperature at the sampling point was measured at 9:00 and 16:00 every day, and the average value was taken and the indoor temperature of the day was recorded; pH was measured using a pH meter; the moisture content was measured using the drying method; total organic C was determined with reference to the determination of total carbon content in plants TNAIA070-2021; total nitrogen determination referred to the Kjeldahl method; organic matter determination referred to the potassium dichromate heating method; total P referred to the molybdenum antimony resistance colorimetric method; total K referred to the flame photometry method; cellulose content was determined with reference to the method of Wang Xuekui (Wang Xuekui. Principles and Techniques of Plant Physiological and Biochemical Experiments. 2nd Edition [M]. Principles and Techniques of Plant Physiological and Biochemical Experiments. 2nd Edition, 2006.); the seed germination index (GI) was determined with reference to the method of Wang Xue (Wang Xue. Development and Application Research of New Biological Organic Fertilizers for Livestock and Poultry Manure [D]. University of Science and Technology Liaoning, 2012.). Each index was measured 3 times respectively.

[0092] Physical and chemical indexes

[0093] ① Temperature: From Figure 6It can be seen that the composting time of Group C is 26 days, and that of Group T is 22 days. The temperature change trends are basically the same, first increasing and then decreasing. During composting, the ambient temperature ranges from 15 to 20 °C. As composting progresses, the temperature of the compost pile gradually rises. The temperature of Group T rises to 50 °C one day earlier than that of Group C, and the average temperature reaches the highest 68 °C on the 7th day, which is higher than that of Group C. The high-temperature period of Group T lasts for 12 days in the range of 50 - 68 °C, with a 3-day longer duration compared to Group C. During the compost cooling and maturation stages, the temperatures of the two compost piles are maintained at 26 - 28 °C until the end of fermentation. The results show that Group T finishes composting 4 days earlier than Group C. This indicates that adding microbial inoculants can quickly initiate compost heating, increase the temperature during the high-temperature period, and extend the time.

[0094] ② pH: As Figure 7 It can be seen that during the entire composting process, the pH value shows a trend of first increasing and then decreasing. At the initial stage of composting, the pH values during the heating-up period and high-temperature period after adding microbial inoculants in Group T are significantly higher than those in Group C (P < 0.05), ranging from 8.44 to 8.92. During the high-temperature period, the pH remains at 8.7 - 9.0. During the cooling and maturation periods, the pH value decreases. At the end of composting, the pH values of Group C and Group T are 8.0 and 8.1 respectively, both showing a slightly alkaline nature, proving that adding microbial inoculants can accelerate the change of pH value.

[0095] ③ Moisture content: As Figure 8 It can be seen that the initial moisture contents are all above 60%. During the composting of the two groups, the moisture content gradually shows a decreasing trend as composting progresses. To ensure the quality of composting, warm water at about 40 °C is given to the compost pile in a timely manner after the temperature drops to ensure the extended time of the high-temperature period. At the end of composting, the moisture contents of Group C and Group T are reduced by 25% and 18% respectively compared to the initial stage. The test results show that adding microbial inoculants can promote water evaporation.

[0096] ④ Seed germination index (GI): As Figure 9 It can be seen that the GI shows an upward trend. Initially, the GI values of Group C and Group T are 44% and 46% respectively. As composting progresses to the heating-up period and high-temperature period (in the abscissa, 1: initial stage of composting; 2: heating-up period; 3: high-temperature period; 4: cooling period; 5: maturation period), the GI value of Group T is extremely significantly higher than that of Group C (P < 0.01). After the end, the GI values of the two groups are 80% and 85% respectively, and it can be determined that the fermentation of this sheep manure has reached the harmless standard.

[0097] ⑤ C / N: As Figure 10It can be seen that the C / N shows a downward trend during the composting process. The initial C / N values of both groups are 2.16. As the composting progresses, the temperature gradually increases. During the high-temperature period, the T group is significantly lower than the C group (P < 0.01), and during the temperature-rising period and high-temperature period, the T group is significantly lower than the C group (P < 0.05). At the end of composting, the C / N values of the C group and the T group are 1.45 and 1.17 respectively. The results show that the addition of microbial inoculant makes the C / N decrease faster.

[0098] ⑥ Organic matter: such as Figure 11 It can be seen that the organic matter content shows a downward trend. In the initial stage of composting, the T group is not significantly different from the C group (P > 0.05). During the temperature-rising period, high-temperature period, and temperature-dropping period, the T group is significantly lower than the C group (P < 0.01). The initial organic matter contents of the C group and the T group are 50.68 g / kg and 49.82 g / kg respectively, and after composting, the contents drop to 29.25 g / kg and 28.77 g / kg respectively. The experiment shows that the addition of microbial inoculant can promote the decomposition of organic matter.

[0099] ⑦ Total K: such as Figure 12 It can be seen that the total K content shows an upward trend during the composting process. During the temperature-dropping period and maturity period, the T group is significantly higher than the C group (P < 0.05). The initial contents of the C group and the T group are 10.76 g / kg and 10.83 g / kg respectively, and the final contents are 15.88 g / kg and 17.06 g / kg respectively. It shows that the addition of microbial inoculant can increase the content of K element in the sheep manure compost.

[0100] ⑧ Total P: such as Figure 13 It can be seen that the total P content shows an upward trend during the composting process. During the temperature-dropping period and maturity period, the T group is significantly higher than the C group (P < 0.05). The initial contents of the C group and the T group are 19.71 g / kg and 20.84 g / kg respectively, and the final contents are 34.73 g / kg and 35.9 g / kg respectively. It shows that the addition of microbial inoculant can increase the content of P element in the sheep manure compost.

[0101] ⑨ Cellulose content: such as Figure 14 It can be seen that the cellulose content in sheep manure is about 183 g / kg in the initial stage of composting. As the composting fermentation progresses, the cellulose content gradually decreases. The cellulose degradation rate of the T group is significantly higher than that of the C group during the high-temperature period of composting (P < 0.05). Until the end of composting, the cellulose contents of the C group and the T group are 114.01 g / kg and 105.65 g / kg respectively. The experiment shows that the addition of microbial inoculant helps the degradation of cellulose in the compost.

[0102] 1.2.8 Species composition and dominant microorganisms 16S rDNA sequencing was carried out on the Illumina platform Miseq of Personalbio Co., Ltd. (Shanghai) to obtain the effective data of each sample and analyze the species composition and dominant flora.

[0103] 1.3 Data statistics and analysis

[0104] Use Excel 2010 for data entry and collation, use GraphPad Prism 8.0 to draw bar charts and line charts for experimental data, and use MEGA 7.0 to construct phylogenetic trees.

[0105] Sequencing results

[0106] As Figure 15 shown, from the rarefaction curves of Group C and Group T, the curves of C1 - C5 and T1 - T5 all tend to be flat, indicating that the sequencing volume of the sheep manure samples in this experiment has reached saturation, reflecting the diversity composition level of the microbial community.

[0107] As Figure 16 shown, the species composition and dominant microorganisms in the sheep manure compost samples at the phylum level of Group C and Group T. This figure only shows the top 10 with the highest abundances. The species composition mainly includes Actinobacteria, Bacilli, Gammaproteobacteria, Alphaproteobacteria, etc. The change rules of the diversity of various microbial communities are different in Group C and Group T, and there are also great differences in the abundance ratios. Actinobacteria is the dominant microorganism in each stage of the two groups, with a relatively similar proportion of 42.0%; Bacilli has a relatively high abundance ratio in the C2 - C3 and T2 - T3 stages, especially in the C2 and T2 heating periods, which are 39% and 63.81% respectively, and in the C3 and T3 high - temperature periods, they are 35.7% and 46.77% respectively; the abundance of Gammaproteobacteria gradually decreases with the decrease of temperature; Alphaproteobacteria is the dominant microorganism in the compost cooling period (C4, T4) and the maturity period (C5, T5), and the abundances of C4, T4, C5, and T5 are 12.75%, 17.13%, 15.47%, and 11.52% respectively; the abundances of the remaining microbial communities are relatively low in the overall compost, with ratios not exceeding 10%. The experimental results show that adding the compound microbial agent will change the growth and reproduction of bacteria, change the abundance ratios of each strain in different compost stages, and increase the number of dominant microorganisms.

[0108] As Figure 17As shown, the species composition and dominant microorganisms in the horizontal sheep manure compost samples of Group C and Group T. A total of 24 genera were detected, and only the top 10 with the highest abundances are shown in this figure. The species composition mainly includes: Corynebacterium, Saccharomonospora, Actinomadura, Halomonas, Planifilum, Bacillus, Luteimonas, Others, etc. Except for Others with the highest proportion, Corynebacterium is the dominant microorganism in the initial stage and temperature-rising stage of composting. The abundances of C1 and T1 are 25.33% and 27.07% respectively. At the temperature-rising stage, the abundances of C2 and T2 increase to 50.37% and 38.23%; Corynebacterium is the dominant microorganism in the temperature-dropping stage and maturity stage. The abundances of C4, T4, C5, and T5 are 15.73%, 20.55%, 17.58%, and 10.6% respectively; Planifilum is the dominant microorganism in the high-temperature stage. The abundance ratios of C3 and T3 are 9.92% and 15.13%. The proportions of the remaining Bacillus, Luteimonas, etc. are very small in each group and each stage, but it can still be seen that they are the dominant microorganisms in the temperature-rising stage and the initial stage of composting. The test results show that adding a compound microbial agent before composting will change the abundance ratios of various microorganisms in different periods of composting, and the number of dominant microorganisms increases.

[0109] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A mixed bacterial agent for degrading cellulose, characterized in that: The mixed bacterial agent includes 9 strains, which are named MX-1: Bacillus firmicola, MX-4: Bacillus cereus, CX-2: Bacillus albus, HX-1: Bacillus brevis, FX-5: Bacillus trexonema, HX-9: Bacillus subtilis, FX-4: Serratia nematophila, DX-5: Exiguobacterium, HX-6: Arabidopsis arabinophila; among them, the low-temperature bacteria are: FX-4, CX-2, DX-5, HX-1; the high-temperature bacteria are: MX-1, MX-4, FX-5, HX-6, HX-9.

2. A method for preparing a solid bacterial agent containing the mixed bacterial agent according to claim 1, characterized in that: The 9 strains are mixed and added to the crushed corn cobs at an inoculation rate of 1%, and then glucose is added after drying to obtain the solid bacterial agent.

3. The preparation method according to claim 2, characterized in that: The bacterial concentrations of the 9 strains were: MX-1: 6-8×10 -7 CFU / mL, MX-4: 8-9×10 -10 CFU / mL, FX-4: 5-7×10 -6 CFU / mL, FX-5: 3-4×10 -7 CFU / mL, CX-2: 5-6×10 -6 DX-5: 7-8×10 -6 CFU / mL, HX-1: 5-6×10 -8 、HX-6:7-8×10 -7 、HX-9:7-8×10 -6 Among them, the low-temperature bacteria are: FX-4, CX-2, DX-5, HX-1; the high-temperature bacteria are: MX-1, MX-4, FX-5, HX-6, HX-9; the mixing ratio is low-temperature bacteria: high-temperature bacteria = 2-4:

1.

4. The preparation method according to claim 3, characterized in that: The corn cob has a mesh size of 18-22 after being crushed.

5. The preparation method according to claim 3, characterized in that: The weight of the added glucose is 1 / 10-1 / 15 of the weight of the corn cob.

6. A solid bacterial agent obtained by the preparation method according to any one of claims 2 to 5.

7. Use of the mixed bacterial agent according to claim 1 or the solid bacterial agent according to claim 6 in sheep manure composting in low temperature seasons.

8. A method for composting sheep manure using the mixed bacterial agent according to claim 1 or the solid bacterial agent according to claim 6, characterized in that: The mixed bacterial agent or the solid bacterial agent is mixed into the sheep manure pile, and fermented at 13-16° C. for 20-23 days. During the fermentation, the pile is turned twice after the high temperature period is reached and lasts for 3-4 days after the composting starts.

9. The method according to claim 8, characterized in that The water content of the sheep manure pile before composting is about 50-70%.

Images