Compound microbial agent for cow dung compost fermentation as well as preparation method and application of compound microbial agent

Abstract

The invention belongs to the field of leavening agents, and particularly relates to a compound microbial agent for cow dung compost fermentation and a preparation method and application thereof. The compound microbial agent is a mixed bacterial agent of bacillus subtilis (HBU33), streptomyces althiophosphaticus (HBU11) and aspergillus niger (Aspergillus niger) HBUN19, and the compound microbial agent is a mixed bacterial agent of bacillus subtilis (HBU33), streptomyces althiophosphaticus (HBU11) and Aspergillus niger (HBUN19). The compound microbial agent is applied to cow dung compost, and can effectively shorten the fermentation time of cow dung compost, improve the compost temperature, promote organic matter degradation and lignocellulose decomposition, accelerate the composting process of fermented compost and reduce the loss of nutrient substances, thereby ensuring the stability of the compost quality.

Description

Technical Field

[0001] The invention belongs to the field of fermentation agents, and in particular relates to a composite microbial agent for cow dung compost fermentation, and a preparation method and application thereof. Background Art

[0002] With the development of animal husbandry, the annual output of livestock and poultry manure in my country is increasing year by year. The large amount of livestock and poultry manure produced has caused a series of serious hazards to the air, water, farmland, residential environment, human health, etc. Therefore, the harmless treatment of livestock and poultry manure has become a problem that must be solved in animal husbandry.

[0003] Aerobic composting fermentation is an effective treatment method for recycling livestock and poultry manure waste. It has the advantages of low treatment cost, ability to kill pathogens, and effective degradation of organic pollutants. It is widely used in northern provinces such as Hebei and Inner Mongolia in my country. However, in the early stage of aerobic composting fermentation of cow dung, the internal environment of cow dung is not suitable for the cultivation and reproduction of microorganisms required for fermentation, which makes the survival rate of beneficial microorganisms in the compost substrate low in the early stage of composting fermentation, and the formation of beneficial colonies is slow, which leads to a long fermentation cycle and a low degree of humification. At the same time, cow dung contains cellulose, lignin, a large amount of heavy metals and other harmful substances. It is difficult to harmlessly treat harmful substances using conventional composting methods, which can easily lead to safety issues in the application of produced organic fertilizers. In addition, the use of conventional methods to compost cow dung also has problems such as the release of a large amount of greenhouse gases and the easy loss of nutrients. Summary of the invention

[0004] In view of the problems of long aerobic composting fermentation cycle, difficulty in decomposing, containing difficult-to-degrade harmful substances, low fermentation temperature, easy loss of nutrients, etc. in existing cow dung composting, the purpose of the present invention is to provide a composite microbial agent for cow dung composting fermentation and a preparation method and application thereof.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] The invention discloses a composite microbial agent for cow dung composting fermentation. The composite microbial agent is a mixed agent of Bacillus subtilis HBU33, Streptomyces althioticus HBU11 and Aspergillus niger HBUN19.

[0007] Further, the Bacillus subtilis HBU33 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC) on October 28, 2024, at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number: CGMCC No: 32383;

[0008] The Streptomyces isothioic acid HBU11 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC) on October 28, 2024, at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, with a deposit number of CGMCC No: 32381.

[0009] The Aspergillus niger HBUN19 was deposited in the General Microbiology Center (CGMCC) of the China Culture Collection Administration on October 28, 2024, at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number: CGMCC No: 41558.

[0010] Furthermore, in the composite microbial agent, the ratio of the effective viable bacteria count of the fermentation broth of Bacillus subtilis HBU29, the spore count of Streptomyces isothiocyanate HBU11, and the spore count of Aspergillus niger HBUN19 is 1:1:1, and the effective viable bacteria count of each strain is ≥1×108 CFU / mL.

[0011] The method for preparing the composite microbial agent for cow dung composting fermentation comprises the following steps:

[0012] (1) taking a frozen Bacillus subtilis HBU33 strain and inoculating it into a sterilized LB liquid medium, and culturing it at 37° C. under shaking conditions for 24 hours to obtain a seed bacterial solution; taking the seed bacterial solution at a volume ratio of 1% and inoculating it into a sterilized and cooled LB medium, and fermenting and culturing it in a shaker for 48 hours to obtain a fermentation liquid of Bacillus subtilis HBU33;

[0013] (2) taking frozen and stored Streptomyces disulfide HBU11 spores and Aspergillus niger HBUN19 spores and inoculating them into sterilized liquid culture medium respectively, and culturing them under shaking conditions at 30° C. for 12 h to obtain seed bacterial liquid; taking an appropriate amount of seed bacterial liquid and applying it on a solid plate or eggplant bottle, culturing them at 28-30° C. for 5-6 days, and then washing and collecting the Streptomyces disulfide HBU11 spores and Aspergillus niger HBUN19 spores;

[0014] (3) By microscopic counting, the fermentation broth of Bacillus subtilis HBU33, spores of Streptomyces isothioic acid HBU11, and spores of Aspergillus niger HBUN19 were combined in a ratio of 1:1:1:1 of effective viable bacteria or spore counts to obtain a composite microbial agent for cow dung composting fermentation.

[0015] Furthermore, the liquid culture medium inoculated with the Aspergillus niger HBUN19 spores is a PDA liquid culture medium, and its seed liquid is coated on a PDA solid plate or an eggplant bottle.

[0016] Furthermore, the liquid culture medium inoculated with the spores of Streptomyces is Gao's No. 1 liquid culture medium, and its seed liquid is coated on Gao's No. 1 solid plate or eggplant bottle.

[0017] The invention also provides application of the composite microbial agent for cow dung composting fermentation in cow dung composting fermentation.

[0018] Specifically, the inoculation amount of the composite microbial agent for cow dung compost fermentation is 1% of the total mass of the cow dung compost, the fermentation temperature is 30-35° C., and the fermentation time is 25-30 days.

[0019] The beneficial effects of the present invention are:

[0020] The present invention obtains a composite microbial agent for cow dung compost fermentation by screening a fermentation bed mixed bedding (30% sawdust, 60% distiller's grains and 10% wheat straw). The composite microbial agent is applied to cow dung composting, which can effectively shorten the fermentation time of cow dung composting, increase the composting temperature, promote the degradation of organic matter and the decomposition of lignocellulose, accelerate the fermentation and composting maturity process, reduce the loss of nutrients, and thus ensure the stability of the compost quality. The cow dung organic fertilizer product obtained by fermenting the composite microbial agent of the present invention significantly improves the soil structure, improves the soil fertility, and promotes the good growth of corn crops and effectively increases the yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The temperature changes during composting after inoculation with different bacterial agents.

[0022] Figure 2 The changes in moisture content during composting after inoculation with different bacterial agents.

[0023] Figure 3 Changes in pH (Figure a) and EC value (Figure b) during composting after inoculation with different bacterial agents.

[0024] Figure 4 The changes in GI during composting after inoculation with different bacterial agents.

[0025] Figure 5 The changes of lignocellulose in the compost pile during the composting process after inoculation with different bacterial agents, including: Figure (a): hemicellulose; Figure (b): cellulose; Figure (c): lignin.

[0026] Figure 6 Effects of different fertilization treatments on maize plant height during the growth period.

[0027] Figure 7 Effects of different fertilization treatments on corn stem diameter.

[0028] Figure 8 Effects of different fertilization treatments on maize SPAD. DETAILED DESCRIPTION

[0029] The present invention will be further described below in conjunction with embodiments, but the present invention is not limited thereto.

[0030] Example 1

[0031] Screening of compound microbial agents

[0032] 1. Enrichment, separation and purification of functional bacteria

[0033] The unsterilized mixture (30% sawdust, 60% distiller's grains and 10% wheat straw) and cow manure were mixed evenly and added to a large test tube with a diameter of 30.36 mm and a length of 200 mm, and then placed in a 37°C incubator for fermentation for 30 days. Water was added every 7 days to keep the moisture content at 50%. The fermented mixture was used as the test material for strain screening to screen strains that efficiently remove ammonia and hydrogen sulfide and efficiently degrade lignin and cellulose.

[0034] Take 10 g of the fermented litter and put it in 90 mL of sterile water, shake it for 30 min, let it stand for 30 s, take 10 mL of the supernatant and add it into 100 mL of liquid NH3 selective medium (glucose 5 g, (NH4)2SO4 2.0 g, NaCl 2 g, FeSO4·7H2O0.4 g, MgSO4·7H2O 0.5 g, K2HPO4 1.0 g), H2S selective medium (KH2PO4 1.2 g, K2HPO4 1.2 g, NH4Cl0.4 g, MgCl2·6H2O 0.2 g, FeCl3 0.01 g, Na2S2O3·5H2O 8.0 g), sodium carboxymethyl cellulose medium (MgSO4 0.5 g, (NH4)2SO4 2 g, K2HPO4 1 g, NaCl 0.5 g, sodium carboxymethyl cellulose 5 g) and alkaline lignin medium (MgSO4 0.5g, (NH4)2SO4 2g, K2HPO4 1g, NaCl 0.5g, alkaline lignin 5g), 37℃, 180rpm culture for 7 days, continuous enrichment three times. Dilute the enriched bacterial solution appropriately, spread it on the corresponding solid selection plate, and culture at 37℃. Pick a single colony with good growth for streak purification.

[0035] 2. Initial screening of functional strains

[0036] 200 pure cultures were isolated and purified from four selective culture plates. These strains were initially screened to obtain 49 strains, including 24 bacteria, 15 actinomycetes and 10 fungi.

[0037] 3. Functional strain rescreening and species identification

[0038] All 49 strains obtained in the initial screening were further screened, and after the determination of ammonia, hydrogen sulfide, cellulose and lignin degradation rates, 10 strains with better ammonia and hydrogen sulfide degradation effects and 10 strains with better cellulose and lignin degradation effects were obtained; then 3 strains with the best effects of removing ammonia, hydrogen sulfide and degrading cellulose and lignin were selected for species identification and subsequent cow dung composting experiments.

[0039] By using 16SrDNA bacterial species identification and ITS fungal species identification methods, it was determined that the three strains were Bacillus subtilis HBU33, Streptomyces althioticus HBU11 and Aspergillus niger HBUN19. The composite microbial agent of the present invention is prepared from these three strains.

[0040] The Bacillus subtilis HBU33 was deposited in the General Microbiology Center of China Culture Collection Administration Committee (CGMCC) on October 28, 2024, at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with a deposit number of CGMCC No. 32383; the Streptomyces isothioic acid HBU11 was deposited in the General Microbiology Center of China Culture Collection Administration Committee (CGMCC) on October 28, 2024, at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with a deposit number of CGMCC No. 32381; the Aspergillus niger HBUN19 was deposited in the General Microbiology Center of China Culture Collection Administration Committee (CGMCC) on October 28, 2024, at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with a deposit number of CGMCC No. 41558.

[0041] Example 2

[0042] The preparation of the composite microbial agent of the present invention comprises the following steps:

[0043] (1) Take the frozen strain HBU33 and inoculate it into the sterilized LB liquid medium (yeast powder 5.0g, peptone 10.0g, NaCl 10.0g, distilled water 1000mL), shake and culture it at 37°C and 180rpm for 24h to obtain the seed bacterial solution. Take the seed bacterial solution at a volume ratio of 1% and inoculate it into the sterilized and cooled LB medium, ferment and culture it in a shaker for 48h at 37°C and 180rpm to obtain the fermentation liquid;

[0044] (2) The frozen spores of strain HBU11 were inoculated into sterilized Gao's No. 1 liquid culture medium (soluble starch 20 g, KNO3 1 g, K2HPO4 0.5 g, MgSO4·7H2O 0.5 g, NaCl 0.5 g, FeSO4·7H2O 0.01 g, distilled water 1000 mL, pH = 7.4-7.6), and cultured at 30°C and 180 rpm for 12 h to obtain the seed culture solution. Take an appropriate amount of bacterial liquid and spread it on Gao's No. 1 solid (soluble starch 20g, KNO3 1g, K2HPO4 0.5g, MgSO4·7H2O 0.5g, NaCl0.5g, FeSO4·7H2O0.01g, agar 20g, distilled water 1000mL, pH=7.4-7.6) plate or eggplant bottle, culture at 30℃ for 5-6 days, then wash and collect spores;

[0045] (3) Take the frozen HBUN19 strain spores and inoculate them into the sterilized PDA liquid medium (200 g potato, 20 g glucose, 1000 mL distilled water), shake and culture at 30°C and 180 rpm for 12 h to obtain the seed bacterial solution. Take an appropriate amount of the bacterial solution and spread it on a PDA solid (200 g potato, 20 g glucose, 20 g agar, 1000 mL distilled water) plate or eggplant bottle, culture at 28°C for 5-6 days, then wash and collect the spores;

[0046] (4) Through microscopic counting, the number of bacteria or spores was combined in a ratio of 1:1:1 to obtain a liquid composite bacterial agent, which was stored at 4°C for future use. There was no antagonism between the strains, and the effective viable bacterial count of each strain was determined to be 1×10 8 CFU / mL and above, which meets the standards of my country's agricultural microbial agents.

[0047] Example 3

[0048] The composite microbial agent for cow dung composting fermentation of the present invention is used in cow dung composting fermentation.

[0049] 1. Experiment on promoting cow dung composting with composite microbial agents

[0050] The test materials are cow dung and corn stalks after solid-liquid separation in a breeding farm. Cow dung was taken from a certain animal husbandry farm, and fresh dung samples after solid-liquid separation were collected and piled up in a centralized manner. Corn stalks were taken from the corn field in the plantation of the breeding farm.

[0051] The microbial agent purchased on the market is produced by Shandong Hengtai Biotechnology Co., Ltd. and is a commonly sold microbial agent (mainly containing Bacillus subtilis).

[0052] Three treatments were set up in this experiment, namely, adding commercially available microbial agents (treatment A), adding the composite microbial agent of Example 2 (treatment B) and natural composting (CK). The amount of composting in the three treatments was 1000 kg of cow dung + 200 kg of straw. Except for the natural fermentation composting (CK), the amount of microbial agents added in the other two treatments was 1% (according to the ratio of the mass of the composite microbial agent to the mass of the compost).

[0053] 1.1 Effect of the composite microbial agent obtained in Example 2 on composting temperature

[0054] The temperature of the pile is measured once at 9:00 and 17:00 every day. The average value of all sampling points is taken as the pile temperature of the day and the room temperature is measured. Figure 1 shown.

[0055] like Figure 1 As shown, the addition of the composite microbial agent of the present invention has a significant effect on the temperature change during the composting fermentation process. Compared with the A treatment and the CK group, the high temperature period of the pile body in the B treatment compost body arrived 5 days and 10 days earlier, and was maintained for more than 20 days, and the maximum temperature of the pile body could reach 74°C, which was 9°C and 15°C higher than that of the A treatment and the CK group; compared with the CK treatment, the temperature change of the pile body in the B treatment was more obvious and rapid during the high temperature period accompanied by the turning of the pile. Similarly, in the cooling and decomposition stages, the temperature drop of the B treatment was faster than that of the A treatment and the CK, indicating that the addition of the composite agent quickly started the initial temperature of the compost, increased the highest temperature of the compost, and prolonged the high temperature time.

[0056] 1.2 Effect of the composite microbial agent obtained in Example 2 on the moisture content of compost

[0057] The results were determined by 105℃ drying method. Figure 2 shown.

[0058] During the composting process, the moisture content will decrease as the fermentation progresses. Figure 2 It can be seen that the moisture content of the pile under the three groups of composting treatments has significant differences (P<0.05). On the 4th day of composting, the moisture content of the piles treated with CK, A, and B decreased, and the decrease in the B treatment was the most obvious, with a decrease of 11.8% and 7.4% compared with CK and A. On the 10th day of composting, the moisture content of the three groups of treatments also decreased significantly; on the 17th day of composting, both A and B treatments significantly reduced the moisture content of the pile, which was 13.5% and 8.4% lower than that of CK. On the 25th day of composting, that is, during the cooling period of composting, the moisture content of the piles treated with A and B was significantly lower than that of CK, with a reduction range of 5.6% to 8.9%. At the end of composting, the moisture content of the A treatment decreased by 50%, and the B treatment finally decreased by 52%. It can be seen that compared with the control group and the A treatment, adding the composite microbial agent of Example 2 to the cow dung composting process can promote the decrease in moisture content.

[0059] 1.3 Effect of the composite microbial agent obtained in Example 2 on the pH of compost

[0060] The pH was measured using a pH meter. Figure 3 Shown

[0061] The results of the pH test during composting are as follows: Figure 3 As shown in Figure 2, there was no significant difference in pH among the three composting treatments (P>0.05) ( Figure 3 a) An important indicator of composting is the concentration of soluble salts, the value of which mainly reflects the plant safety of the compost product. Figure 3 b It can be seen that the EC values ​​of the piles under the three composting treatments were significantly different (P<0.05). The change in conductivity showed an overall trend of first increasing and then decreasing during the composting fermentation process. On the 10th day of composting, the EC values ​​of the piles treated with CK, A, and B all increased. Compared with CK, the EC values ​​of A and B treatments showed significant differences (P<0.05). After the composting was completed, the conductivity of the three treatments reached the minimum value during the cooling period, among which the EC value of B treatment was the smallest, at 1.65μS / cm. This shows that the plant safety of the compost product is higher than that of the control.

[0062] 1.4 Effect of the composite microbial agent obtained in Example 2 on the physical and chemical indicators of compost

[0063] The physicochemical indices of different composting treatments showed significant differences (P<0.05, the same below; Table 1). On the 4th day of composting, the total nitrogen content of treatment B was significantly higher than that of treatments CK and A. On the 12th, 24th and 32nd days of composting, the total nitrogen content of treatment B was also significantly different from that of CK and A (P<0.05). The total nitrogen content showed a trend of first decreasing and then increasing during the fermentation process, and the change amplitude of treatment B was the largest among the three groups of treatments. There were significant differences in the total phosphorus content of the compost piles of the three groups of treatments (P<0.05). In the early stage of composting, the total phosphorus content of the piles of CK treatment was significantly higher than that of treatments A and B. As the fermentation progressed, there was no significant difference in the total phosphorus content of the piles of the three groups of treatments during the cooling period, while in the mature period, the total phosphorus content of the piles of treatment B showed significant differences from those of CK and A (P<0.05). The organic carbon in the pile is the main place for microorganisms to absorb nutrients and energy for life activities, and its content can better reflect the ability of microorganisms to degrade cellulose and lignin in materials. The results showed (Table 1) that on the 24th day of composting, the organic carbon content of treatment B showed a significant change compared with CK (P<0.05). After the end of composting, the organic carbon contents of CK, A and B were 34.88%, 36.6% and 37.67%, respectively. The organic carbon contents of the three groups of treatments decreased by 16.29%, 15.67% and 19.28% on the 4th to 32nd day of composting, respectively, indicating that the addition of the microbial agent of the present invention is conducive to the degradation of total organic carbon. Appropriate C / N is conducive to the reproduction and circulation of microorganisms in the compost pile, and has an important influence on the composting process. On the 4th day of composting, the C / N ratio of treatment B was significantly different from that of CK and A (P<0.05).

[0064] Table 1 Changes in total nitrogen, total phosphorus, total potassium, organic carbon content and C / N during composting

[0065]

[0066]

[0067] Note: Different lowercase letters in the same column represent the differences among CK, A, and B treatments at the same composting period. Different uppercase letters in the same row represent the differences among the same treatment at different composting periods (P<0.05).

[0068] 1.5 Effect of the composite microbial agent obtained in Example 2 on the seed germination index GI

[0069] The GI determination was carried out according to the method in Wang Xue's article "Development and Application of New Bio-organic Fertilizer from Livestock and Poultry Manure". Figure 4 shown.

[0070] Seed germination index (GI value) is an important indicator used to detect whether compost products have phytotoxicity and evaluate the maturity of compost. The change of GI value during the composting process shows ( Figure 4 ), as the compost fermentation continued, the GI value showed an upward trend, among which the GI value of the B treatment showed the most obvious upward trend. There was no significant difference in the GI values ​​of the three groups of treatments on the 4th and 10th days of composting (P>0.05). From the 17th day, the GI values ​​of the composts treated with CK, A and B showed significant changes (P<0.05). In the first 17 days of composting, the GI of the CK and A treatments was less than 80%. After 25 days, the GI value gradually reached more than 80%, while the GI value of the B treatment was already more than 80% on the 17th day of composting (compared to the GI value of natural and commercially available bacterial composts, which can exceed 80% one week in advance), and the change in GI value showed that B was greater than CK and A (P<0.05). At the end of composting, the GI value of the B treatment was 120%. It shows that the growth-promoting ability of the compost extract is improved after inoculation of the composite microbial agent of the present invention.

[0071] 1.6 Effect of the composite microbial agent obtained in Example 2 on the degradation of lignocellulose

[0072] The degradation of lignocellulose during composting can reflect the degree and maturity of composting. Figure 5 ), during the whole process of composting fermentation, hemicellulose (Figure a), cellulose (Figure b) and lignin (Figure c) were effectively degraded, among which the hemicellulose degradation in the pile was the largest. The hemicellulose and cellulose contents of CK, A and B treatments showed significant differences on the 10th day of composting (P<0.05), and until the 35th day, the hemicellulose and cellulose degradation rates of B were significantly increased compared with the CK treatment. The lignin content of the three treatments showed differences on the 4th day of composting (P<0.05), and until the end of composting, the lignin degradation of B treatment was more significant. The degradation of hemicellulose and cellulose mainly occurred in the high temperature period of composting, and the degradation of lignin started from the composting temperature rise period until the end of composting.

[0073] 2. Determination of related indicators of organic fertilizer products obtained by fermentation of cow dung compost with the composite microbial agent obtained in Example 2

[0074] According to my country's NY / T525-2021 agricultural standard, various indicators of the organic fertilizer product obtained by fermenting the composite microbial agent obtained in Example 2 with the cow dung compost in this experiment were compared. By comparing the indicators such as the mass fraction of organic matter, total nutrients, moisture and mechanical impurities in the generated compost, as shown in Table 2, it can be seen that they all meet the national organic fertilizer standard requirements. The moisture content is slightly higher than the organic fertilizer standard requirements. The reason may be that it rained during composting, resulting in high air humidity, thereby increasing the moisture content of the compost pile.

[0075] Table 2 Measured values ​​of relevant indicators of organic fertilizer products and industrial standard values ​​of organic fertilizer (dry basis)

[0076] index Measured value NY525—2021 Organic matter (%) 34.88～37.67 ≥30 <![CDATA[N+P2O5+K2O(%)]]> 3.75～4.30 ≥4.0 Moisture (%) 31～38 ≤30 pH 7.2～8.5 5.5～8.5 GI (%) 103～120 ≥70 Mechanical debris (%) <0.2 ≤0.5

[0077] Note: The mass fractions of organic matter and total nutrients (N+P2O5+K2O) are calculated on a drying basis.

[0078] 3. Effect of adding organic fertilizer obtained by fermentation of the composite microbial agent obtained in Example 2 to cow dung compost on soil fertility

[0079] The test site is located in a corn planting base of a certain animal husbandry company. The terrain is flat, high in the west and low in the east, with a gentle slope that stretches for dozens of miles. The demonstration research area is in a typical warm temperate monsoon climate environment, with annual sunshine hours between 2400 and 3000 hours, annual frost-free period of 120 to 200 days, annual average temperature between 0 and 13℃, annual average precipitation of 200 to 800mm, abundant rainfall throughout the year, and rainfall mostly concentrated in summer.

[0080] The test fertilizer was cow dung organic fertilizer fermented with the composite microbial agent obtained in Example 2, commercially available cow dung organic fertilizer A fermented with an agent, test fertilizer (urea (N) 46%, potassium sulfate (K2O) 50%, superphosphate (P2O5) 13%), the test crop was corn (provided by a certain animal husbandry company, the variety was Xianyu 1466), and the test soil was taken from a field test site (a certain animal husbandry ecological park), which was brown loam.

[0081] The field trial started in April 2021. The preliminary preparation work included tilling the soil, watering, fertilizing, building plots, etc. The trial set up 5 treatment levels:

[0082] Treatment 1 (CK): natural planting in the field, blank control;

[0083] Treatment 2 (CF): single fertilizer application, including 15 kg / mu of urea, 30 kg / mu of superphosphate, and 15 kg / mu of potassium sulfate;

[0084] Treatment 3 (AOF): Cow dung organic fertilizer fermented with the composite microbial agent obtained in Example 2 (conventional dosage, 1000 kg / mu);

[0085] Treatment 4 (BOF): 940 kg of organic fertilizer of cow dung fermented by the composite microbial agent obtained in Example 2 + chemical fertilizer (the conventional amount minus the nutrients contained in the organic fertilizer);

[0086] Treatment 5 (AOC): 940 kg of commercially available organic fertilizer fermented with bacterial agents + chemical fertilizer (conventional amount minus the nutrients contained in organic fertilizer);

[0087] Three plots were set up for each treatment, i.e., three replicates, and the area of ​​each plot was 9 m 2 ,The cells are randomly distributed and the layout is shown in Table 3 below.

[0088] Table 3 Field plot layout

[0089] CK BOF CF CF AOC AOF AOF CK AOC BOF AOC BOF AOF CF CK

[0090] The physical properties of the soil at the corn harvest stage under different fertilization treatments were measured. As shown in Table 4, the soil bulk densities at the corn harvest stage under the five fertilization treatments were significantly different (p<0.05), among which the soil bulk densities of the BOF and CK treatments were in sharp contrast, being 1.53 g / cm 3 and 1.69 g / cm 3 CK was 10.45% higher than BOF, indicating that the soil was very compact. The soil bulk density showed different trends with the change of fertilization methods. From the single application of chemical fertilizer or cow dung organic fertilizer to the combination of the two, the soil bulk density gradually decreased. The soil porosity at the corn harvest period under the five different fertilization treatments was significantly different (P<0.05). The soil saturated water content and field water holding capacity of BOF treatment were significantly different from those of CK (P<0.05), and its soil saturated water content and field water holding capacity increased by 25.46% and 63.68% compared with CK.

[0091] Table 4 Effects of different fertilization treatments on soil physical properties at harvest time

[0092]

[0093]

[0094] Note: Different lowercase letters indicate the differences among different fertilization treatments under the same index (P<0.05).

[0095] As shown in Table 5, there were significant differences in soil pH at the corn harvest period under different fertilization treatments (p<0.05). Among the fertilization treatments, CF had the lowest pH, which was 8.22, and CK had the highest, which was 8.57. However, the pH values ​​of various fertilization treatments were lower than those of CK, which indicated that all four fertilization methods could reduce the soil pH value, and the single application of chemical fertilizer had the most significant effect on reducing soil pH value. The soil conductivity at the harvest period showed significant differences under different fertilization treatments (p<0.05). The application of cow dung organic fertilizer alone or in combination with chemical fertilizer had a significant effect on the conductivity (p<0.05); the soil conductivity of the AOC treatment was the lowest, as low as 162.30us / cm, and the soil conductivity of the CF treatment was the highest, as high as 222.00us / cm. The application of chemical fertilizers will increase the conductivity of the soil, increase the potential risk of soil salinization, and is not conducive to the improvement of soil fertility. Therefore, it is necessary to pay attention to controlling the amount and method of fertilization in the subsequent application process. Under different fertilization treatments, soil organic matter in each group showed significant differences (p<0.05). The soil organic matter in CF, AOF, BOF, and AOC treatments was significantly different from that in CK treatment (p<0.05), increasing by 8.73%, 10.16%, 12.38%, and 8.65% respectively compared with CK. Except for BOF and AOC treatments, the other two groups of fertilization treatments had no significant effect on the total nitrogen, total phosphorus, and total potassium content of the soil (p>0.05), but the total nitrogen, total phosphorus, and total potassium content of the soil in each fertilization treatment were higher than those in CK.

[0096] Table 5 Effects of different fertilization treatments on soil chemical properties at harvest time

[0097]

[0098] Note: Different lowercase letters represent the differences among different fertilization treatments under the same index (P<0.05).

[0099] 4. Effect of organic fertilizer obtained by fermentation of cow dung compost with the composite microbial agent obtained in Example 2 on the growth and yield of corn crops

[0100] The experimental site and experimental design were the same as in 3.

[0101] Depend on Figure 6 It can be seen that different fertilization treatments have significant differences in plant height during the growth cycle of corn (P<0.05). At the seedling stage, the plant height of corn under the five fertilization treatments did not change significantly. At the jointing stage, the plant height of CF, AOF, BOF, and AOC treatments were significantly different from that of CK treatment (P<0.05), with an increase range of 14.84%-30.47%. Among them, BOF treatment had the best effect on promoting plant height, which increased by 30.47% compared with the control group.

[0102] The stem diameter of corn is an important indicator of its own and plays an important role in the transport of nutrients and water throughout the growth cycle. Figure 7 It can be seen that different fertilization treatments have significant differences in stem diameter during the growth cycle of corn (P<0.05). In the seedling stage, except for the CF treatment, the stem diameter of other fertilization treatments increased compared with the control group. Among them, the BOF treatment had the greatest effect on promoting plant height, which increased by 40.00% compared with the control group. In the jointing and tasseling stages, the stem diameter of the five fertilization treatments also showed significant differences (P<0.05). In the filling stage, the stem diameter of the CF treatment was significantly different from that of the other four treatments (P<0.05). The stem diameter of the CF treatment increased by 9.52%, 4.55%, 4.55%, and 9.09% compared with the CK, AOF, BOF, and AOC treatments. In the harvest stage, the stem diameter of the five fertilization treatments had different changes, specifically BOF>AOC>AOF>CF>CK.

[0103] Depend on Figure 8 It can be seen that under different fertilization treatments, as the growth time goes by, the chlorophyll content of rice stems and leaves under the five fertilization treatments has a similar trend in the entire growth cycle, all showing a slow increase. The SPAD values ​​of corn under the five fertilization treatments showed different differences in their entire growth cycle (p<0.05). In the seedling stage, the SPAD value of corn in the BOF treatment was the highest, and the CK group was the lowest. The SPAD values ​​of CF, AOF, BOF and AOC treatments were significantly different from those of CK (p<0.05); in the tasting stage, the SPAD values ​​of corn in the BOF and CF, AOF, CK groups were significantly different (p<0.05); the changes in the filling stage and the tasting stage were the same.

[0104] As shown in Table 6, the effects of different fertilization treatments on corn yield and its components were significantly different (P<0.05). Compared with the CK treatment, the corn ear diameter, ear length, number of grains per ear, 100-grain weight and yield of the other four fertilization treatments were significantly improved, indicating that the four fertilization methods can achieve the effect of increasing corn yield. The ear diameter under the BOF treatment was significantly different from that of the other groups, and the ear diameter of the AOC group and the AOF group also formed a significant difference, indicating that the combination of cow dung organic fertilizer and chemical fertilizer is conducive to the growth of corn ear diameter. The ear length, number of kernels per ear, 100-kernel weight and yield under different fertilization treatments showed significant differences. The ear length of CF, AOF, BOF and AOC treatments increased by 4.82%, 11.87%, 16.51% and 8.60% respectively compared with CK, and the number of kernels per ear increased by 10.60%, 12.00%, 12.40% and 10.40% respectively compared with CK; the 100-kernel weight buried at the four fertilization sites increased by 2.94%, 1.35%, 3.17% and 2.22% respectively compared with CK.

[0105] The degree of bald tip under different fertilization treatments showed significant differences (P<0.05), and CK had the most serious bald tip. Compared with CK, the degree of corn bald tip in CF, AOF, BOF and AOC treatments decreased by 18.58%, 17.67%, 36.47% and 36.09%, respectively. Corn bald tip is a common phenomenon in the planting and growth process, which is caused by various comprehensive factors and is an irreversible process. Severe bald tip can lead to nutritional deficiency, spikelet degeneration, and a significant reduction in yield during the growth period. In summary, the combination of homemade cow dung organic fertilizer and chemical fertilizer has the most significant agronomic effect on corn. The agronomic effects of the five different types of fertilization treatments are BOF>AOC>AOF>CF>CK.

[0106] Table 6 Effects of different fertilization treatments on corn yield and its components

[0107]

[0108] Note: Different lowercase letters indicate the differences among different fertilization treatments under the same index (P<0.05).

[0109] From the above results, it can be seen that adding the liquid composite bacterial agent prepared by the present invention to improve the fermentation effect of cow dung in cow dung compost can effectively shorten the composting time and improve the composting efficiency and organic fertilizer quality. The cow dung organic fertilizer product generated by composting has a significant effect in improving soil structure, increasing soil fertility, promoting good growth of crops, and effectively increasing production. The specific effects are as follows:

[0110] (1) During the composting process, the addition of composite microbial agents caused the high temperature period to appear 5 days and 2 days earlier than that of natural and commercial agents, and the duration of the high temperature period was prolonged, with the maximum temperature reaching 74°C.

[0111] (2) During the composting process, the GI value of composting with the addition of composite microbial agents can exceed 80% one week earlier than that of natural and commercially available compost agents.

[0112] (3) The composite microbial agent increased the degradation rate of organic matter, the reduction rate of C / N ratio, the decomposition rate of lignocellulose and the degradation of soluble salts, which was beneficial to the dehydration of the pile and accelerated the fermentation and maturity of the compost. At the same time, it improved the utilization of nitrogen in the pile and ensured the stability of the compost quality.

[0113] (4) Adding cow dung organic fertilizer produced by compound microbial agents can increase the soil organic matter content to 14.16 g / kg, which is 12.38% and 8.65% for super natural compost and commercial compost fertilizer.

[0114] (5) Compared with corn grown with natural compost, the application of cow dung organic fertilizer can increase corn ear diameter by 13.01%, ear length by 16.51%, number of grains per ear by 12.40% and yield by 14.20%.

[0115] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A composite microbial agent for cow dung composting fermentation, characterized in that: The composite microbial agent is Bacillus subtilis ( Bacillus subtilis ) HBU33, Streptomyces isothioicola ( Streptomyces althioticus ) HBU11 and Aspergillus niger ( Aspergillus niger ) Mixed bacterial agent of HBUN19.

2. The composite microbial agent for sewage treatment according to claim 1, characterized in that: The Bacillus subtilis HBU33 was deposited in the General Microbiology Center of China Culture Collection Administration Committee (CGMCC) on October 28, 2024, at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with a deposit number of CGMCC No. 32383; the Streptomyces isothioic acid HBU11 was deposited in the General Microbiology Center of China Culture Collection Administration Committee (CGMCC) on October 28, 2024, at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with a deposit number of CGMCC No. 32381; the Aspergillus niger HBUN19 was deposited in the General Microbiology Center of China Culture Collection Administration Committee (CGMCC) on October 28, 2024, at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with a deposit number of CGMCC No. 41558.

3. The composite microbial agent for cow dung composting fermentation according to claim 1, characterized in that: In the composite microbial agent, the ratio of the effective viable bacteria count of the fermentation broth of Bacillus subtilis HBU29, the spore count of Streptomyces isothiocyanate HBU11, and the spore count of Aspergillus niger HBUN19 is 1:1:1, and the effective viable bacteria count of each species is ≥1×10 8 CFU / mL.

4. The method for preparing the composite microbial agent for cow dung composting fermentation according to claim 1, characterized in that: The following steps are involved: (1) Inoculate the frozen Bacillus subtilis HBU33 strain into a sterilized LB liquid medium, and culture it at 37°C under shaking conditions for 24 hours to obtain a seed culture solution; inoculate the seed culture solution into a sterilized and cooled LB medium at a volume ratio of 1%, and ferment and culture it in a shaker for 48 hours to obtain a fermentation liquid of Bacillus subtilis HBU33; (2) Take frozen and stored Streptomyces disulfide HBU11 spores and Aspergillus niger HBUN19 spores and inoculate them into sterilized liquid culture medium respectively, and culture them at 30°C with shaking for 12 hours to obtain seed culture liquid; take an appropriate amount of seed culture liquid and spread it on a solid plate or eggplant bottle, culture it at 28-30°C for 5-6 days, and then wash and collect the Streptomyces disulfide HBU11 spores and Aspergillus niger HBUN19 spores; (3) By microscopic counting, the fermentation broth of Bacillus subtilis HBU33, spores of Streptomyces isothiocyanate HBU11, and spores of Aspergillus niger HBUN19 were combined in a ratio of 1:1:1:1 of effective viable bacteria or spore counts to obtain a composite microbial agent for cow dung composting fermentation.

5. The preparation method according to claim 4, characterized in that: The liquid culture medium inoculated with the Aspergillus niger HBUN19 spores is a PDA liquid culture medium, and its seed liquid is coated on a PDA solid plate or an eggplant bottle.

6. The preparation method according to claim 4, characterized in that: The liquid culture medium inoculated with the spores of Streptomyces is Gao's No. 1 liquid culture medium, and the seed liquid is coated on Gao's No. 1 solid plate or eggplant bottle.

7. Use of the composite microbial agent for cow dung composting fermentation according to claim 1 in cow dung composting fermentation.

8. The use according to claim 7, characterized in that: The inoculation amount of the composite microbial agent for cow dung compost fermentation is 1% of the total mass of the cow dung compost, the fermentation temperature is 30-35° C., and the fermentation time is 25-30 days.

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