Complex microbial inoculant as well as preparation method and application thereof

Abstract

The invention provides a complex microbial inoculant as well as a preparation method and application thereof, and relates to the technical field of complex microbial inoculant preparation. The compound bacterial agent is prepared from bacillus pumilus JMB004, bacillus safeensis strain BXC22 and bacillus sp. EB35, and the compound bacterial agent is prepared from bacillus pumilus strain JMB004, bacillus safeensis strain BXC22 and bacillus sp. EB35. The three strains are obtained through autonomous screening and prepared into the complex microbial inoculant, dry powder can be stored at normal temperature, the storage life is prolonged to one year, and the composite microbial inoculant is convenient to carry. The complex microbial inoculant is only composed of three strains of bacteria, and the components are simple; the low-temperature composting agent is applied to composting treatment under a low-temperature condition, and is relatively high in temperature rise rate, relatively short in fermentation time and relatively high in decomposition degree.

Description

Technical Field

[0001] The present invention belongs to the technical field of composite bacterial agent preparation, and in particular relates to a composite bacterial agent, a preparation method and an application thereof. Background Art

[0002] Every year, China produces a large amount of waste such as livestock and poultry manure and crop straw. Agricultural solid waste such as crop straw and livestock and poultry manure is an important renewable resource. Aerobic composting is an important way to harmlessly treat and recycle agricultural waste such as crop straw and livestock and poultry manure. The composting process is a dynamic process achieved by the joint action of multiple microbial communities with very rapid microbial community structure succession. Cellulose substances are the main components of crop straw and livestock and poultry manure, and their degradation mainly depends on cellulose-degrading bacteria. Bacteria are easy to mass-produce because of their wide sources, fast reproduction speed, many types of enzymes secreted, and their ability to survive in various extreme environments.

[0003] Due to the limited number and variety of microorganisms in natural composting, the complex organic components in the compost cannot be effectively degraded, resulting in a slow composting process. The traditional natural composting method is greatly affected by the low ambient temperature in our area, which makes it difficult to start composting fermentation, so additional microbial agents need to be added to promote composting temperature rise.

[0004] The influence of environmental temperature on composting is great. In some special areas of my country or in autumn and winter, the low temperature time will last for 4 to 6 months, which will affect the rapid heating and effective fermentation of solid waste, and thus fail to effectively kill various pathogens in solid waste. Solid waste can only effectively kill pathogens under high temperature-50℃ or above fermentation conditions; low temperature also slows down the biological metabolism and chemical reactions in the composting process, prolongs the fermentation cycle, and thus leads to the reduction of the quality of organic fertilizer and certain restrictions on the use of composting technology. Studies have shown that when the temperature of the environment and the pile is below 20℃, the metabolic activities of most microorganisms in the pile are significantly slowed down or even stopped. When composting is carried out at an ambient temperature below 20℃, it is a broad low-temperature composting. At this time, the composting process is likely to be slowed down by the influence of ambient temperature. However, there are few studies on low-temperature cellulose degradation functional bacterial agents suitable for northern regions such as Inner Mongolia in my country.

[0005] One of the effective means to increase the composting temperature is to adopt the method of inoculating exogenous microorganisms. However, most of the current composting fermentation agents can only be effective when the temperature is above 20°C. Therefore, under low temperature conditions, it is very important to study low-temperature starting composite bacterial agents suitable for cattle and sheep manure and straw composting in northern regions such as Inner Mongolia, which can promote the rapid degradation and temperature rise of composting materials. Summary of the invention

[0006] The main purpose of the present invention is to provide a composite bacterial agent, a preparation method and an application. To achieve the above purpose, the present invention obtains three strains of bacteria through independent screening: Bacillus pumilus strain JMB004, Bacillus safensis strain BXC22 and Bacillus sp.EB35, and prepares a composite fermentation bacterial agent with simple components, containing only three strains of bacteria, and is applied to low-temperature composting treatment, with a faster heating rate, a shorter fermentation time and a higher degree of maturity.

[0007] The invention provides a composite bacterial agent, which consists of Bacillus pumilus strain JMB004, Bacillus safensis strain BXC22 and Bacillus sp. EB35.

[0008] Preferably, the volume ratio of Bacillus pumilus strain JMB004:Bacillus safensis strain BXC22:Bacillus sp. EB35 is 1-2:1-2:1-2.

[0009] More preferably, the volume ratio of Bacillus pumilus strain JMB004:Bacillus safensis strain BXC22:Bacillus sp. EB35 is 1:1:2 or 1:2:1.

[0010] The invention also provides a method for preparing the composite bacterial agent, which comprises mixing and fermenting Bacillus pumilus strain JMB004, Bacillus safensis strain BXC22 and Bacillus sp. EB35.

[0011] The present invention also provides a low-temperature composite bacterial agent dry powder, wherein the raw materials of the low-temperature composite bacterial agent dry powder include a microbial mixture and bran; the microbial mixture consists of Bacillus pumilus strain JMB004, Bacillus safensis strain BXC22 and Bacillus sp. EB35.

[0012] Preferably, in the low-temperature composite bacterial agent dry powder, the volume ratio of Bacillus pumilus strain JMB004, Bacillus safensis strain BXC22 and Bacillus sp. EB35 is 1-2:1-2:1-2.

[0013] More preferably, the volume ratio of Bacillus pumilus strain JMB004:Bacillus safensis strain BXC22:Bacillus sp. EB35 is 1:1:2 or 1:2:1.

[0014] The present invention also provides the application of the composite bacterial agent or the low-temperature composite bacterial agent dry powder.

[0015] Preferably, the application is to use the composite bacterial agent or the low-temperature composite bacterial agent dry powder to perform low-temperature composting.

[0016] Preferably, the temperature range of the low-temperature composting is 7°C to 16°C.

[0017] Preferably, the compost raw materials include fresh cattle and sheep manure and rice straw.

[0018] Preferably, the fresh weight ratio of the fresh cattle and sheep manure to the rice straw is 2:1.

[0019] Preferably, the mass ratio of the composite bacterial agent to the compost raw material is 0.4%, and the mass ratio of the low-temperature composite bacterial agent dry powder to the compost raw material is 0.4%.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The microbial mixture in the composite microbial agent provided by the present invention is composed of Bacillus pumilus, Bacillus safensis strain JMB004 and Bacillus sp. EB35. The composite microbial agent has simple components and only three strains of bacteria; when low-temperature composting is performed, the heating rate is fast, the fermentation time is short, and the degree of maturity is high.

[0022] The low-temperature composite bacterial agent dry powder provided by the present invention can be stored at room temperature, with a shelf life extended to 1 year, compared with liquid bacterial agents that are stored at a low temperature of 4°C, have a short shelf life and are inconvenient to carry, and can be conveniently carried to a composting base to carry out relevant experiments.

[0023] The present invention provides the use of the composite bacterial agent or the low-temperature composite bacterial agent dry powder in low-temperature composting. The composite bacterial agent or the low-temperature composite bacterial agent dry powder is applied to low-temperature composting, and the heating rate is fast, the fermentation time is short, and the degree of maturity is high. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the antagonism result among the three strains described in Example 1. DETAILED DESCRIPTION

[0025] The present invention provides a composite bacterial agent, which is composed of Bacillus pumilus strain JMB004, Bacillus safensis strain BXC22 and Bacillus sp. EB35. Preferably, the volume ratio of Bacillus pumilus strain JMB004: Bacillus safensis strain BXC22: Bacillus sp. EB35 is 1-2:1-2:1-2. More preferably, the volume ratio of Bacillus pumilus strain JMB004: Bacillus safensis strain BXC22: Bacillus sp. EB35 is 1:1:2 or 1:2:1. The references of Bacillus pumilus strain JMB004, Bacillus safensis strain BXC22 and Bacillus sp. EB35 of the present invention are Yue Linfang, Cheng Lixin, Li Yunhua, et al. Screening of room temperature and low temperature cellulose degrading bacteria and their enzyme activity characteristics [J]. Animal Husbandry and Feed Science, 2023, 44(02): 9-16; Bacillus pumilus strain JMB004, Bacillus safensis strain BXC22 and Bacillus sp. EB35 correspond to 30T-8-2, 30LF-5 and 30T-10 in Table 1 in the literature, respectively.

[0026] The present invention also provides a preparation method of the composite bacterial agent, which comprises the following steps of: mixing and fermenting the Bacillus pumilus strain JMB004, the Bacillus safensis strain BXC22, and the Bacillus sp. EB35.

[0027] The present invention also provides a low-temperature composite bacterial agent dry powder, the raw materials of which include a microbial mixture and bran; the microbial mixture consists of Bacillus pumilus strain JMB004, Bacillus safensis strain BXC22 and Bacillus sp. EB35. As an implementable embodiment, the volume ratio of Bacillus pumilus strain JMB004, Bacillus safensis strain BXC22 and Bacillus sp. EB35 is 1-2:1-2:1-2. Preferably, the volume ratio of Bacillus pumilus strain JMB004: Bacillus safensis strain BXC22: Bacillus sp. EB35 is 1:1:2 or 1:2:1. As an implementable embodiment, the preparation method of the low-temperature composite bacterial agent dry powder comprises the following steps: mixing the suspension of the microbial mixture with bran and fermenting to obtain a semi-solid composite wet powder, and after testing the effective live bacteria count in the wet powder to be qualified, drying to obtain a low-temperature composite bacterial agent dry powder. Preferably, the mixing ratio of the suspension of the microbial mixture and the bran is 1:1 in volume mass ratio; preferably, the fermentation temperature is 18°C, and the fermentation time is 5 to 7 days. Preferably, the effective live bacteria counts of Bacillus pumilus strain JMB004, Bacillus safensis strain BXC22 and Bacillus sp. EB35 in the obtained semi-solid composite wet powder are all greater than 10 9 CFU / g is qualified. As an implementable method, the obtained semi-solid composite wet powder is dried by a low-temperature blast drying oven method, the thickness of the dried material is controlled to be 2 to 2.5 cm, the drying temperature is controlled to be 30°C, and the moisture content is less than 14%, so as to prepare a low-temperature composite bacterial agent dry powder. The low-temperature composite bacterial agent dry powder prepared by the preparation method of the present invention has a moisture content of less than 14%; if stored under normal temperature and dry conditions, the dry powder can extend the shelf life to 1 year; it is convenient to carry to the composting base to carry out relevant experiments.

[0028] The present invention also provides the application of the low-temperature composite bacterial agent in composting. As an implementable method, the ambient temperature range of the test is 7°C to 16°C. As an implementable method, the compost raw materials are fresh cattle and sheep manure and straw, the fresh weight ratio of the fresh cattle and sheep manure and straw is 2:1, the carbon-nitrogen ratio of the compost raw material is 26:1, the straw is crushed to a length of ≤5cm, and the mass ratio of the composite bacterial agent to the compost raw material is 0.4%. As an implementable method, before the composite bacterial agent is inoculated, the composite bacterial agent is first mixed with bran at a mass ratio of 1:10, stirred evenly, and sprinkled into the compost raw material; the moisture content of the material is adjusted to 55±5%, and the control standard is "holding a ball of material tightly with your hand, no liquid flows out, and the material does not disperse after letting go"; the pile is conical, with a pile height of 1.2m and a diameter of 2m. Mechanical turning and natural ventilation are used for compost turning. As an implementable embodiment, the compost management method is as follows: during the compost fermentation process, when the pile temperature reaches 40°C, the pile is turned; when the pile temperature continues to rise and reaches about 50°C, the pile is turned; the compost continues to ferment and reaches a high temperature of above 50°C and is maintained for 8 to 15 days, and then enters the post-maturity stage, the pile is turned once every 4 to 5 days, and the pile temperature gradually decreases until it approaches the ambient temperature, thereby obtaining fully mature fertilizer.

[0029] The invention is applied to low-temperature composting treatment, has a faster heating rate, a shorter fermentation time and a higher degree of maturity.

[0030] The references for the Bacillus pumilus strain JMB004, Bacillus safensis strain BXC22 and Bacillus sp. EB35 described in the present invention are Yue Linfang, Cheng Lixin, Li Yunhua, et al. Screening of room-temperature and low-temperature cellulose-degrading bacteria and their enzyme activity characteristics [J]. Animal Husbandry and Feed Science, 2023, 44(02): 9-16; they correspond to 30T-8-2, 30LF-5 and 30T-10 in Table 1 of the document, respectively.

[0031] The exoenzyme C1 described in the present invention is exo-β-glucanase, and its source is not particularly limited and can be obtained from commercial channels.

[0032] The pakchoi seeds of the present invention are purchased from Ershilitun Seed Breeding Farm in Machang Town, Qing County, Cangzhou City, Hebei Province.

[0033] In the data table of the present invention, Bacillus pumilus is Bacillus pumilus strain JMB004, Bacillus safensis is Bacillus safensis strain BXC22, and Bacillus sp. EB35 is Bacillus sp. EB35.

[0034] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0035] The experimental methods described in the following examples are conventional methods unless otherwise specified.

[0036] Unless otherwise specified, the reagents and materials mentioned above can be obtained from commercial sources.

[0037] Example 1

[0038] 1. Determination of enzyme activity of low-temperature strains:

[0039] (1) Preparation of crude enzyme solution

[0040] Three strains of bacteria selected independently: Bacillus pumilus strain JMB004, Bacillus safensis strain BXC22 and Bacillus sp. EB35 were inoculated at 2% in LB liquid medium and cultured at 18°C ​​and 160 r / min for 1 to 3 days until the number of viable cells reached 1×10 8 cfu / mL or above, centrifuge at 4000r / min for 10min, and the supernatant is used as the crude enzyme solution.

[0041] LB liquid medium: 5 g yeast extract, 10 g NaCl, 10 g peptone, 1000 mL water, pH 7.0.

[0042] (2) Enzyme activity assay

[0043] The activity of carboxymethylcellulose (endoenzyme CMCase) was determined using 2 mL 1% (W / V) carboxymethylcellulose-sodium (CMC-Na) buffer as the reaction substrate; the activity of filter paper enzyme (holoenzyme FPA) was determined using 2 mL citric acid-sodium hydrogen phosphate buffer soaked with 0.2 g (starch-free) filter paper strips; the exonuclease C1 was determined using 2 mL 1% (W / V) microcrystalline cellulose (MCC) buffer as the reaction substrate; and the β-glucosidase (β-Gase) was determined using 2 mL 1% salicin (W / V) buffer as the reaction substrate. 1 mL of the above fermentation crude enzyme solution was added to each of them, and the reaction was carried out at a constant temperature of 50°C for 30 minutes in a water bath. Then, 3 mL 3,5-dinitrosalicylic acid (DNS) was added to terminate the reaction. The mixture was boiled in a boiling water bath for 8-10 minutes, cooled, and ddH 2 O was fixed to 25 mL, and the absorbance was measured at 540 nm using a microplate reader. Enzyme activity definition: Under the reaction conditions of 50 ° C, the amount of enzyme required to catalyze the substrate to produce 1 μmol of glucose within 1 min is 1 U / mL. The enzyme activity of the isolated strain was measured, and the glucose content of the sample and the control was calculated by comparing with the standard glucose curve. The enzyme activity was calculated by the formula: U = K (m 1 -m 0 ) / 30, where U is the enzyme activity of the sample, and 1 unit of enzyme activity represents 1 mL of the original enzyme solution producing 1 μg of glucose in 1 min; K is the sample dilution multiple; m 1 is the amount of glucose in the sample; m 0 is the amount of control glucose; 30 is the reaction time between enzyme and substrate. CMCase, FPA, C1 and β-Gase were measured respectively, and the results are shown in Table 1:

[0044] Table 1 Results of enzyme activity determination of strains

[0045] Serial number strain CMCase (U / mL) β-Gase (U / mL) C1(U / mL) FPA (U / mL) 1 Bacillus safensis 30.71±0.49 29.27±0.36 35.35±2.32 31.18±1.59 2 Bacillus pumilus 26.82±3.40 29.80±0.83 35.16±2.64 35.55±1.33 3 Bacillus sp.EB35 48.15±0.35 46.10±0.94 57.96±0.68 46.81±1.81

[0046] 2. Preparation of low temperature leavening agent

[0047] (1) Antagonism between strains

[0048] The three activated strains were streaked on LB solid culture medium plates and cultured at 18°C ​​for 2-3 days. The growth of the two strains at the intersection was observed. If the two strains at the intersection grew relatively weakly or did not grow, it indicated that there was antagonism between the two strains. If the bacteria at the intersection grew well, it indicated that there was no antagonism between the two strains and they could be used for mixed culture. Figure 1 The results showed that no sterile zone was formed between the three strains and they could grow in contact with each other, indicating that there was no inhibitory effect between them.

[0049] (2) Composite bacterial agent combination

[0050] The three non-antagonistic strains in (1) were inoculated into LB liquid culture medium at an inoculum size of 2%, and cultured at 18°C ​​and 160 r / min for 1 to 3 days. Then, they were mixed at a certain volume ratio, and cultured at 18°C ​​and 160 r / min for 2 to 3 days to obtain a mixed bacterial suspension. The activity of various cellulases was determined using the same method as above to screen the optimal composite bacterial suspension.

[0051] Bacillus pumilus strain JMB004, Bacillus safensis strain BXC22 and Bacillus sp.EB35 were inoculated into LB liquid medium at a 2% inoculum volume, and cultured at 18°C ​​and 160 r / min for 2 days. The number of viable bacteria was greater than 10 8 CFU / mL, Bacillus pumilus strain JMB004, Bacillus safensis strain BXC22 and Bacillus sp.EB35 were mixed at a volume ratio of 1:1:1, 1:1:2, 2:1:1 and 1:2:1, respectively. The strain ratio settings are shown in Table 2. After the strains were mixed, the mixed bacterial suspension was prepared after shaking culture at 18°C ​​and 160r / min for 2 days. The number of viable bacteria was greater than 10 9 CFU / mL. The optimal compound bacterial agent was screened by enzyme activity assay.

[0052] Table 2 Strain ratio setting

[0053] Bacterial agent number Strain ratio Ⅰ Bacilluspumilus, Bacillussafensis and Bacillussp.EB35=1:1:1 Ⅱ Bacilluspumilus, Bacillussafensis and Bacillussp.EB35=1:1:2 Ⅲ Bacilluspumilus, Bacillussafensis and Bacillussp.EB35=2:1:1 Ⅳ Bacilluspumilus, Bacillussafensis and Bacillussp.EB35=1:2:1

[0054] Table 3 Determination of various enzyme activities of composite bacterial agents

[0055]

[0056] The activities of various enzymes in the composite agents are shown in Table 3. The results show that the enzyme activity of the screened composite agent II is the highest, followed by the composite agent IV. In the composite agent II, the CMCase of Bacillus pumilus strain JMB004, Bacillus safensis strain BXC22 and Bacillus sp. EB35 mixed in a ratio of 1:1:2 was 133.53±2.25U / mL, FPA was 124.01±5.20U / mL, C1 was 135.49±2.70U / mL and β-Gase was 115.59±4.08U / mL; the composite agent IV (Bacillus pumilus strain JMB004, Bacillus safensis strain BXC22 and Bacillus sp. sp.EB35 mixed ratio of 1:2:1) with CMCase of 123.75±3.68U / mL, FPA of 56.78±5.55U / mL, C1 of 69.73±5.51U / mL and β-Gase of 98.33±9.86U / mL.

[0057] Therefore, compound bacterial agents II and IV were applied to subsequent composting experiments to verify their effects.

[0058] (3) Preparation of composite bacterial agent solid powder

[0059] The suspensions of the better combinations of composite bacterial agents II and IV were placed in a sterilized plastic basin with bran at a volume mass ratio of 1:1 (v / m), with a thickness of 2 cm. After stirring and moistening for 1 hour, the surface was covered with sterile gauze and cultured at 18°C ​​for 5-7 days. The semi-solid composite wet powder was obtained, and the effective live bacterial counts of all strains in the semi-solid composite wet powder were greater than 10 by the dilution coating method. 9 The obtained semi-solid composite wet powder is dried by low-temperature blast drying oven method, the thickness of the dried material is controlled to be 2-2.5 cm, the drying temperature is controlled to be 30° C., the moisture content is less than 14%, and the low-temperature composite bacterial agent dry powder is prepared.

[0060] Results: The low-temperature composite bacterial agent dry powder was obtained, with a moisture content of less than 14%, and was stored under dry conditions at room temperature; the dry powder could extend the shelf life to 1 year; and it was convenient to carry to the composting base for related experiments.

[0061] Example 2

[0062] Experimental location: Inner Mongolia Pangyou Biotechnology Co., Ltd.; ambient temperature is 7℃~16℃.

[0063] (1) Test bacterial agent

[0064] The addition of composite bacterial agent II—Bacillus pumilus strain JMB004, Bacillus safensis strain BXC22 and Bacillus sp. EB35=1:1:2 was applied in composting treatment.

[0065] (2) Pile establishment

[0066] The compost raw materials are fresh cow and sheep manure and rice straw, with a fresh weight ratio of 2:1, a carbon-nitrogen ratio of 26:1, rice straw crushed to a length of ≤5cm, and a mass ratio of compound bacteria to compost raw materials of 0.4%. Before the compound bacteria inoculation, the compound bacteria is first mixed with bran at a mass ratio of 1:10, stirred evenly, and sprinkled into the compost raw materials. The moisture content of the material is adjusted to 55±5%, and the control standard is "holding a ball of material tightly with your hand, no liquid flows out, and the material does not fall apart after letting go". The pile is conical, with a height of 1.2m and a diameter of 2m. Mechanical turning and natural ventilation are used for compost turning.

[0067] (3) Compost management

[0068] During the composting fermentation process, when the pile temperature reaches 40℃, turn the pile; when the pile temperature continues to rise and reaches about 50℃, turn the pile; the compost continues to ferment and reaches a high temperature of above 50℃ and maintains for 8 to 15 days, then enters the post-composting stage, turn the pile once every 4 to 5 days, and the pile temperature gradually decreases until it approaches the ambient temperature.

[0069] (4) Determination of relevant indicators during composting maturity

[0070] A. Compost Sample Collection

[0071] During the composting process, a thermometer was used to observe and record the daily ambient and central temperature of the compost. Samples were collected on the 0th, 7th, 14th, 21st, 28th, 35th and 42nd days of composting, with each sample weighing about 1200g. The collected samples were used to test the moisture content, pH value and germination index of compost seeds.

[0072] B. Composting temperature

[0073] Use a thermometer, insert it vertically into the pile from the top to the depth of 20-30cm, 50-60cm and 80-90cm respectively, measure and record the pile temperature and the ambient temperature of the day at 9:00, 13:00 and 17:00 every day. The daily pile temperature value is the average value of the three time points.

[0074] The results showed that on the 14th day, the ambient temperature was 10.2℃ and the pile temperature was 43.2℃, and then the pile temperature kept rising. On the 28th day, the ambient temperature was 12.4℃ and the pile temperature was 54.7℃, and then the pile temperature kept falling. On the 35th day, the ambient temperature was 10.4℃ and the pile temperature was 40.3℃. On the 42nd day, the pile temperature was 23.3℃, ​​close to the ambient temperature of 15.4℃. The pile temperature was maintained above 50℃ for 8 days, which met the requirements of the agricultural industry standard "Technical Specifications for Harmless Treatment of Livestock and Poultry Manure NY / T 1168-2006": The pile temperature was maintained above 50℃ for no less than 7 days.

[0075] C. Moisture content of compost

[0076] First, dry the aluminum box for the test to constant weight, with a mass of m 0 ; Weigh 20.0g of compost sample, mass m 1 ; Dry at 105℃ for 4-6h, cool to constant weight, mass is m 2 ;

[0077] Moisture content calculation: Moisture content = (m 0 +m 1 -m 2 / m 1 )×100%.

[0078] The results of compost moisture content measurement showed that the initial moisture content of the compost was 63.13%, after which the moisture content of the compost began to decrease at a rapid rate. On the 42nd day, the moisture content of the compost dropped to 27.15%.

[0079] D. pH value of compost

[0080] Take 10.0 g of compost sample, mix it with distilled water in a mass-to-volume ratio of 1:10, shake it at 160 r / min for 30 to 40 min at room temperature, filter it, and use the extract for pH determination.

[0081] The results showed that the pH value of the compost showed a trend of first increasing and then decreasing. At the end of the fermentation on the 42nd day, the pH value of the compost was 8.07, which was alkaline and met the requirements of the organic fertilizer agricultural industry standard NY / T525-2021: pH value 5.5-8.5.

[0082] E. Compost seed germination index

[0083] 20 seeds of Chinese cabbage were selected, and 10 mL of the water extract obtained with the above pH value was used in a culture dish covered with filter paper for the test group, and 10 mL of distilled water was used in a culture dish covered with filter paper for the control group. After culturing in an incubator at 25°C in the dark for 72 hours, the seed germination index and root length were measured;

[0084] Calculate the seed germination index (GI):

[0085] Seed germination index (GI) % = (germination rate of experimental group × root length of experimental group) / (germination rate of control group × root length of control group) × 100%.

[0086] The results showed that the seed germination index increased with the extension of compost fermentation time. At the beginning of composting, the seed germination index was 18.6%, and on the 28th day, the seed germination index was 50.5%; on the 42nd day, the seed germination index reached 75.5%, and the harmless treatment was completely completed, that is, the compost was fully decomposed.

[0087] F. Compost odor and color

[0088] The manual identification method is adopted: (more than 4 people) qualitatively determine the sensory properties of the fermentation pile.

[0089] Qualitative criteria for sensory properties:

[0090] The color of the compost was divided into yellow-brown, dark brown and dark brown; the smell was divided into odor, slight ammonia, slight odor and slight earthy smell; the particle size was divided into lumps, blocks and no blocks (loose). During the composting process, the apparent characteristics of the compost, such as color, odor and morphology, were recorded.

[0091] At the beginning of composting, the compost is yellow-brown and has a distinct odor. As the compost fermentation time increases, the color of the compost becomes darker, and the odor gradually increases and then slowly fades away. At the end of the fermentation, when the compost is 42 days old, the compost is dark brown, odorless and has a slight earthy smell. It is loose and has white mycelium. It does not attract mosquitoes and flies and has no insect eggs, indicating that the compost has been fully decomposed.

[0092] Manual identification method of compost odor and color and description of results References: Tian Wei. Study on material changes, microbial diversity and maturity evaluation during high-temperature composting of cow dung [D]. Changsha: Hunan University, 2012. Wu Qingshan. Screening of cellulose-degrading bacteria and its application in sheep dung composting fermentation [D]. Guizhou Normal University, 2018. Example 3

[0093] Composite bacterial agent IV (Bacillus pumilus, Bacillus safensis and Bacillus sp. EB35 = 1:2:1) was added to the compost and the operation method of Example 2 was repeated.

[0094] The results of compost temperature measurement showed that the ambient temperature was 10.2℃ and the pile temperature was 53.5℃ on the 14th day, and the pile temperature continued to rise. On the 28th day, the ambient temperature was 12.4℃ and the pile temperature was 63.6℃. After that, the pile temperature continued to decline. On the 35th day, the ambient temperature was 10.4℃ and the pile temperature was 45.4℃; on the 42nd day, the temperature was 21.6℃, close to the ambient temperature of 15.4℃. The pile continued to ferment at above 45℃ for about 22 days, and the pile temperature was maintained above 50℃ for 15 days. The compost reached above 50℃ on the 15th day and above 60℃ on the 28th day. Continuous high temperature above 50℃ can effectively kill pathogens and insect eggs in the pile.

[0095] The results of compost moisture content measurement showed that the initial moisture content of the compost was 64.35%, and the moisture content decreased rapidly. On the 42nd day, the moisture content of the compost dropped to 25.97%, which meets the requirements of the organic fertilizer agricultural industry standard NY / T 525-2021: The moisture content of the compost is ≤30%.

[0096] The pH value of the compost showed a trend of first increasing and then decreasing. On the 42nd day of fermentation, the pH value of the compost was 7.88.

[0097] The test results of the compost seed germination index show that the seed germination index increases with the extension of compost fermentation time. At the beginning of composting, the seed germination index was 19.9%, on the 28th day the compost seed germination index was 55.8%, and on the 42nd day the seed germination index reached 86.4%, which means that the compost was completely decomposed and met the requirements of the organic fertilizer agricultural industry standard (NY / T 525-2021): the seed germination index GI ≥ 70%.

[0098] The compost is yellow-brown in the early stage and has a distinct odor. As the compost fermentation time increases, the color of the pile becomes darker and the odor gradually increases and then slowly fades away. At the end of the fermentation, on the 42nd day of composting, the pile is dark brown, odorless and has a slight earthy smell. It is loose and has white mycelium. It does not attract mosquitoes and flies and has no insect eggs, indicating that the compost has been fully decomposed.

[0099] Example 4

[0100] Add a commercially available bacterial agent to compost.

[0101] The operation method of Example 2 was repeated.

[0102] The composting temperature results showed that on the 14th day, the ambient temperature was 10.2℃ and the compost body temperature was 47.4℃, and then the compost body temperature kept rising. On the 28th day, the ambient temperature was 12.4℃ and the compost body temperature was 56.2℃, and then the compost body temperature kept falling. On the 35th day, the ambient temperature was 10.4℃ and the compost body temperature was 39.2℃. On the 42nd day, the temperature was 22.2℃, close to the ambient temperature of 15.4℃. The compost body temperature was maintained above 50℃ for 8 days.

[0103] The results of compost moisture content determination showed that the initial moisture content of the compost was 60.06%, and the moisture content decreased rapidly, and on the 42nd day the moisture content of the compost dropped to 28.03%.

[0104] The pH value of the compost showed a trend of first increasing and then decreasing. On the 42nd day of fermentation, the pH value of the compost was 7.93, which was alkaline.

[0105] The test results of compost seed germination index showed that the seed germination index increased with the extension of compost fermentation time. At the beginning of composting, the seed germination index was 20.7%, on the 28th day, the seed germination index was 53.1%, and on the 42nd day, the seed germination index reached 79.3%, which means that the harmless treatment was completely completed, that is, the compost was fully decomposed.

[0106] At the beginning of composting, the compost is yellow-brown and has a distinct odor. As the compost fermentation time increases, the color of the compost becomes darker, and the odor gradually increases and then slowly fades away. At the end of the fermentation, when the compost is 42 days old, the compost is dark brown, odorless and has a slight earthy smell. It is loose and has white mycelium. It does not attract mosquitoes and flies and has no insect eggs, indicating that the compost has been fully decomposed.

[0107] Conclusion: Low-temperature fermentation agents Ⅱ and Ⅳ were applied to composting, and the temperature, pH value, moisture content and seed germination index of three groups of composts: compound bacterial agent group Ⅱ, compound bacterial agent group Ⅳ and a commercial bacterial agent group were measured, and the apparent characteristics of the compost such as color, odor and state were recorded. During the composting process, the temperature rise rate of compound bacterial agent group Ⅳ was faster than that of compound bacterial agent group Ⅱ and a commercial bacterial agent group; the fermentation end point was the 42nd day, and the moisture content of the compost dropped to 27.15%, 25.97% and 28.03% respectively; the pH values ​​of the composts of the three bacterial agent groups were 8.07, 7.88 and 7.93 respectively; the seed germination index of the bacterial agent groups reached 75.5%, 86.4% and 79.3% respectively, and all of them were completely harmless, that is, the compost was fully decomposed. Among them, the seed germination index of the composite bacterial agent group IV was the highest and the degree of maturity was also the highest; the compost temperature was maintained above 50°C for 15 days at the longest, and the compost reached above 50°C on the 15th day and above 60°C on the 28th day. Only the compost temperature of the composite bacterial agent group IV rose to above 60°C.

[0108] The commercially available microbial agent is Jinbaobei fertilizer fermentation agent, which is purchased from Beijing Huaxia Kangyuan Technology Co., Ltd. Jinbaobei fertilizer fermentation agent contains various beneficial bacteria: yeast, actinomycetes, etc.; organic matter carriers, active enzymes and trace elements.

[0109] Comparative Example 1

[0110] The control group was formed without adding any bacterial agent to the compost, and the operation method was the same as that in Example 2.

[0111] Experimental results:

[0112] 1. Composting temperature

[0113] The results showed that on the 14th day, the ambient temperature was 10.2℃, and the control group was 18.2℃; on the 28th day, the ambient temperature was 12.4℃, and the control group was 37.2℃; after that, the temperature of the pile continued to decrease, and on the 42nd day, the ambient temperature was 15.4℃, and the control group was 21.2℃. During the entire composting process, the temperature of the pile did not rise above 40℃ and 50℃.

[0114] 2. Moisture content of compost

[0115] The results showed that the moisture content of the compost was 61.59% at the beginning, and then the moisture content of the compost began to decrease. On the 42nd day, the moisture content of the compost dropped to 42.30%, which was higher than the agricultural industry standard.

[0116] 3. Compost pH

[0117] It showed a trend of first rising and then falling. On the 42nd day of fermentation, the pH value of the control group was 8.58, slightly higher than the agricultural industry standard.

[0118] 4. Compost seed germination index

[0119] The test results showed that the seed germination index increased with the extension of composting time. At the beginning of composting, the seed germination index was 21.9%, and the control group was 40.2% on the 28th day; the control group was 58.3% on the 42nd day, which was not mature.

[0120] 5. Compost smell and color

[0121] At the beginning of composting, the compost is yellow-brown with a distinct smell of manure. After 42 days of composting, the pile becomes dark brown with a slight odor, with lumps in some parts, attracting mosquitoes and flies, and a small amount of insect eggs are bred in the deep layers, and it is not fully decomposed.

[0122] Table 4 Composting temperature (℃)

[0123] 0d 3d 5d 7d 14d 21d 28d 35d 42d 1 16.1±0.07a 13.2±0.13c 10.3±0.2e 7.2±0.13g 10.2±0.07e 8.3±0.2f 12.4±0.27d 10.4±0.33e 15.4±0.13b 2 15.2±0.13g 15.1±0.2g 15.2±0.07g 17.3±0.27f 18.2±0.13e 20.1±0.07d 37.2±0.2a 28.3±0.13b 21.2±0.07c 3 14.3±0.2i 17.2±.07h 19.1±0.13g 25.1±0.2e 43.2±0.27c 50.2±0.13b 54.7±0.07a 40.3±0.27d 23.3±0.03f 4 14.5±0.07i 17.4±.13h 22.2±0.2f 35.2±.07e 53.5±0.33c 58.3±0.2b 63.6±0.13a 45.4±0.07d 21.6±0.2g 5 14.4±0.13h 18.1±0.2g 18.3±0.27g 28.4±.33e 47.4±0.13c 52.2±0.07b 56.2±0.2a 39.2±0.13d 22.2±0.07f

[0124] Note: 1 is the ambient temperature; 2 is the control group; 3 is the compound bacterial agent group II; 4 is the compound bacterial agent group IV; 5 is the commercial bacterial agent group

[0125] Table 5 Compost moisture content (%)

[0126]

[0127]

[0128] Table 6 Compost pH

[0129] 0d 7d 14d 21d 28d 35d 42d Blank Group <![CDATA[ 8 .09±0.07c]]> <![CDATA[ 8 .14±0.09c]]> <![CDATA[ 8 .29±0.09bc]]> <![CDATA[ 8 .48±0.16ab]]> <![CDATA[ 8 .67±0.05a]]> <![CDATA[ 8 .63±0.14a]]> <h2 style=";text-align:left;direction:ltr"><![CDATA[ <h2 style=";text-align:left;direction:ltr"> 8 <h2 style=";text-align:left;direction:ltr"> .58±0.13ab]]><h2 style=";text-align:left;direction:ltr"> Compound bacterial agent group II <![CDATA[ 8 .13±0.13c]]> <![CDATA[ 8 .21±0.2c]]> <![CDATA[ 8 .32±0.15bc]]> <![CDATA[ 8 .68±0.15ab]]> <![CDATA[ 8 .76±0.18a]]> <![CDATA[ 8 .13±0.11c]]> <![CDATA[ 8 .07±0.06c]]> Compound bacterial agent group Ⅳ <![CDATA[ 8 .01±0.17cd]]> <![CDATA[ 8 .38±0.21bc]]> <h2 style=";text-align:left;direction:ltr"><![CDATA[ <h2 style=";text-align:left;direction:ltr"> 8 <h2 style=";text-align:left;direction:ltr"> .65±0.2ab]]><h2 style=";text-align:left;direction:ltr"> <![CDATA[ 8 .87±0.1a]]> <h2 style=";text-align:left;direction:ltr"><![CDATA[ <h2 style=";text-align:left;direction:ltr"> 8 <h2 style=";text-align:left;direction:ltr"> .97±0.03a]]><h2 style=";text-align:left;direction:ltr"> <![CDATA[ 8 .39±0.13bc]]> <![CDATA[ 7 .88±0.15d]]> Commercially available bacterial agent group <![CDATA[ 8 .09±0.27d]]> <![CDATA[ 8 .28±0.17cd]]> <h2 style=";text-align:left;direction:ltr"><![CDATA[ <h2 style=";text-align:left;direction:ltr"> 8 <h2 style=";text-align:left;direction:ltr"> .39±0.26abc]]><h2 style=";text-align:left;direction:ltr"> <![CDATA[ 8 .73±0.11ab]]> <![CDATA[ 8 .79±0.11a]]> <h2 style=";text-align:left;direction:ltr"><![CDATA[ <h2 style=";text-align:left;direction:ltr"> 8 <h2 style=";text-align:left;direction:ltr"> .35±0.13abc]]><h2 style=";text-align:left;direction:ltr"> <![CDATA[ 7 .93±0.03d]]>

[0130] Table 7 Compost GI value (%)

[0131]

[0132] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A composite bacterial agent, characterized in that: The composite bacterial agent consists of Bacillus pumilus strain JMB004, Bacillus safensis strain BXC22 and Bacillus sp. EB35.

2. The composite bacterial agent according to claim 1, characterized in that: The volume ratio of Bacillus pumilus strain JMB004: Bacillus safensis strain BXC22: Bacillus sp. EB35 is 1-2:1-2:1-2.

3. The method for preparing the composite bacterial agent according to claim 1 or 2, characterized in that: Bacillus pumilus strain JMB004, Bacillus safensis strain BXC22 and Bacillus sp. EB35 were mixed and fermented.

4. A low-temperature composite bacterial agent dry powder, characterized in that: The raw materials of the low-temperature composite bacterial agent dry powder include a microbial mixture and bran; the microbial mixture consists of Bacillus pumilus strain JMB004, Bacillus safensis strain BXC22 and Bacillus sp. EB35.

5. Use of the composite bacterial agent as claimed in claim 1 or 2 or the low-temperature composite bacterial agent dry powder as claimed in claim 4.

6. The use according to claim 5, characterized in that: The application is to use the composite bacterial agent or the low-temperature composite bacterial agent dry powder to carry out low-temperature composting.

7. The use according to claim 6, characterized in that: The temperature range of the low-temperature composting is 7°C to 16°C.

8. The use according to any one of claims 5 to 7, characterized in that: The compost raw materials include fresh cattle and sheep manure and rice straw.

9. The use according to claim 8, characterized in that: The fresh weight ratio of the fresh cattle and sheep manure to the rice straw is 2:

1.

10. The use according to claim 8, characterized in that: The mass ratio of the composite bacterial agent to the compost raw material is 0.4%, and the mass ratio of the low-temperature composite bacterial agent dry powder to the compost raw material is 0.4%.

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