Compound fertilizer containing multiple microbial active substances as well as preparation method and application of compound fertilizer

Abstract

The invention relates to the field of compound fertilizers, in particular to a multi-microbial active substance compound fertilizer as well as a preparation method and application thereof. According to the scheme, through an innovative layered fermentation process and a multi-component synergistic effect, the microbial activity, the soil improvement and the crop yield increasing effect are remarkably improved. A layered inoculation strategy (bottom-layer aspergillus niger, middle-layer bacillus and surface-layer pseudomonas fluorescens) is adopted, and a breathable non-woven fabric isolation technology is combined, so that the defect of mutual inhibition of flora in traditional mixed fermentation is effectively avoided, and the survival rate of thalli is obviously improved compared with that of conventional mixed fermentation. And in combination with the function of a biochar-loaded carrier, a physical shelter is provided for thalli, and a slow-release system is formed through chemical bonding with humic acid. In addition, the introduction of diatomite is beneficial to improving the water retention rate and maintaining the yield increasing rate under the drought condition, and after the optimized nitrogen-phosphorus-potassium ratio and microbial metabolism are coordinated, the remarkable effect of increasing the yield of potatoes and cucumbers is achieved.

Description

Technical Field

[0001] The present invention relates to the field of compound fertilizers, in particular to a compound fertilizer containing multiple microbial active substances, a preparation method thereof and an application thereof. Background Art

[0002] With the increasing demand for sustainable agricultural development worldwide, the overuse of chemical fertilizers has led to increasingly prominent problems such as soil degradation, water pollution, and reduced agricultural product quality. Microbial active compound fertilizers, which combine nutrient supply, soil remediation, and disease control, have become a key development direction for green agriculture. Traditional microbial fertilizers often combine single-functional strains (such as nitrogen-fixing bacteria and phosphate-solubilizing bacteria) with chemical fertilizers, but these fertilizers have significant drawbacks. First, multi-strain combinations are susceptible to reduced activity due to nutrient competition or metabolic antagonism. For example, the conflicting pH requirements of nitrogen-fixing and phosphate-solubilizing bacteria often lead to synergistic failure. Second, microorganisms are susceptible to inactivation by environmental stresses (such as high temperature, drought, and ultraviolet light) during storage and application. Conventional encapsulation techniques (such as sodium alginate microcapsules) are costly and have unstable sustained-release effects. Third, the production process relies on commercial culture media and liquid fermentation, with raw material costs accounting for over 60%, resulting in a terminal price that is 2-3 times that of traditional chemical fertilizers, hindering their large-scale application.

[0003] Existing technologies also have shortcomings in adapting to regional environments. For example, in Yantai, Shandong, brown soils are commonly acidified (pH 5.5-6.5), lack organic matter, and suffer from salinization in coastal areas. Commercially available microbial fertilizers, however, mostly use generic bacterial strains, making them ill-suited to the fluctuating climate and soil characteristics of cold winters and wet summers. For example, the activity of standard Bacillus subtilis drops sharply at low temperatures (<10°C), making it unable to meet the winter needs of Yantai orchards. Conventional phosphate-solubilizing bacteria have a phosphate-solubilizing efficiency of less than 30% in acidic soils, leading to a mismatch between phosphorus release and crop demand.

[0004] While existing technologies have made some progress in improving the performance of microbial fertilizers through strain combination optimization and carrier modification, significant limitations remain. For example, liquid fermentation processes often rely on commercial culture media such as glucose and peptone (which account for over 60% of the cost), resulting in excessively high production costs and difficulty in large-scale application. Regarding carrier material selection, traditional bentonite and diatomaceous earth have low specific surface areas (typically <200 m² / g) and simple pore structures, resulting in a bacterial loading rate of less than 50%, and their slow-release performance is difficult to meet the needs of complex soil environments. Regarding regional adaptation, existing solutions often focus on a single environmental factor (such as the development of salt- or acid-resistant bacterial agents) and lack systematic design of multi-strain synergistic mechanisms, resulting in an imbalance between the cost of implementing the technology and its ecological benefits.

[0005] Specifically, although the liquid fermentation system can achieve high-density culture of bacteria, its high sterilization energy consumption and dependence on imported carbon sources for the culture medium make the price of the end product 2-3 times that of traditional chemical fertilizers; in carrier technology, conventional mineral materials are difficult to effectively fix functional bacterial communities due to insufficient surface active sites, and after field application, the bacteria are easily inactivated rapidly due to rain erosion or pH fluctuations; and at the regional adaptation level, most schemes only respond to specific stresses by adding a single stress-resistant bacterial species (such as saline-alkali land inoculants containing only halophilic bacteria), ignoring the functional complementarity of the bacterial community (such as phosphate solubility, nitrogen fixation, and disease resistance synergy) and the synergistic utilization value of organic waste, resulting in fragmented soil improvement effects.

[0006] Therefore, there is an urgent need to develop a low-cost, highly stable and precisely adapted multi-microbial compound fertilizer preparation technology for the regional environment. Through waste resource utilization (such as fruit branches and grape vines), functional complementary design of bacterial strains (combination of low-temperature-resistant, acid-resistant and salt-resistant bacterial communities) and process innovation (solid-state layered fermentation, biochar carrier coupling), we can break through the cost and efficiency bottlenecks of existing technologies and achieve a dual breakthrough in ecological and economic benefits.

[0007] Based on the above-mentioned technical pain points, the present invention innovatively proposes to use apple tree branch biochar and grape vines as core raw materials to construct a layered solid-state fermentation system, thereby simultaneously realizing the resource utilization of agricultural waste and the low-cost expansion of functional bacterial communities; by screening low-temperature-resistant, acid-resistant and salt-resistant functional bacterial communities (such as Pseudomonas fluorescens and halophilic Bacillus), combined with carrier sustained release and stress resistance enhancement technology, the survival rate and functional expression of the bacterial community in extreme environments are significantly improved. Summary of the Invention

[0008] In view of the deficiencies in the prior art, the present invention provides a multi-microbial active substance compound fertilizer and a preparation method and application thereof.

[0009] A multi-microbial active substance compound fertilizer, comprising the following components: Biochar loaded with bacteria; Basic fertilizer; Humic acid; diatomite.

[0010] Preferably, the multi-microbial active substance compound fertilizer is composed of the following components by mass percentage: 25-35% biochar loaded with bacteria; 55-65% basic fertilizer; 7-9% humic acid; 1-3% diatomaceous earth; The mass ratio of nitrogen, phosphorus and potassium in the basic fertilizer is 15: (9-10): 15.

[0011] The method for preparing the biochar loaded with bacteria comprises the following steps: (1) apple tree branches, grapevines, and wheat straw are mixed in a ratio of 5:3:(1-2) and crushed into 2-5 mm particles to obtain a fermentation substrate; the fermentation substrate and a 1 wt% calcium hydroxide aqueous solution are mixed in a mass ratio of 1:(0.5-1), and the mixture is allowed to stand for 2-3 days to obtain an alkali-treated fermentation substrate; (2) mixing the alkali-treated fermentation substrate obtained in step (1) and decomposed cow dung in a mass ratio of (90-99): (1-10) for fermentation, maintaining the fermentation temperature at 55-60°C and the humidity at 45-55%, turning the compost 1-3 times / day for 2-3 days, and obtaining a decomposed fermentation substrate; (3) First, a 14-16 cm thick layer of the decomposed fermentation substrate obtained in step (2) is laid on the bottom layer of the fermentation chamber and inoculated with Aspergillus niger. After 23-25 ​​hours, a 7-9 cm thick layer of the decomposed fermentation substrate obtained in step (2) is laid on the bottom layer as a middle layer and Bacillus subtilis and Bacillus gelatinosa are inoculated on the middle layer. After another 23-25 ​​hours, a 4-6 cm thick layer of the decomposed fermentation substrate obtained in step (2) is laid on the middle layer as a surface layer and Pseudomonas fluorescens is inoculated on the surface layer. The fermentation is continued for another 23-25 ​​hours, and the total fermentation time is 69-75 hours to obtain a fermentation product. The layers are separated by breathable non-woven fabrics to avoid mutual inhibition between strains and to allow metabolites to diffuse across the layers. (4) placing the apple tree branches in an oxygen-free environment, heating them to 490-510°C at a rate of 9-11°C / min, carbonizing them at a constant temperature for 0.5-1.5h, and then cooling them to room temperature at a rate of 9-11°C / min to obtain apple charcoal; (5) The fermentation product obtained in step (3) and the apple charcoal obtained in step (4) are mixed in a mass ratio of 1:1, trehalose and bentonite are added, and the mixture is stirred at a speed of 25-35 r / min for 1-3 h. After stirring, a pulse air flow dryer is used to reduce the moisture content to 8 wt%-10 wt% to obtain the biochar loaded with the bacteria.

[0012] In step (3), the inoculation amount of Aspergillus niger is 5wt%-8wt% of the dry weight of the bottom layer of the fermentation substrate; the inoculation amount of Bacillus subtilis is 3wt%-5wt% of the dry weight of the middle layer of the fermentation substrate; the inoculation amount of Bacillus gelatinosa is 2-4wt% of the dry weight of the middle layer of the fermentation substrate; and the inoculation amount of Pseudomonas fluorescens is 2wt%-3wt% of the dry weight of the surface layer of the fermentation substrate.

[0013] In step (3), the temperature in the fermentation chamber is controlled at 27-29°C, the humidity is controlled at 58-62%, the ventilation rate is 0.7-0.9 m³ / (h·kg) for the first 23-25 ​​hours, no ventilation is used for the middle 23-24 hours, and intermittent ventilation is used for 5-8 minutes every 25-30 minutes for the last 23-24 hours, with a ventilation rate of (0.1-0.3 m³ / (h·kg)).

[0014] The amount of trehalose added in step (5) is 4-6 wt% of the fermentation product obtained in step (3); the amount of bentonite added is 1-3 wt% of the fermentation product obtained in step (3).

[0015] The inlet temperature of the pulse air flow dryer in step (5) is 38-42°C and the outlet temperature is 30-35°C.

[0016] The present invention also provides a method for preparing a multi-microbial active substance compound fertilizer, comprising the following steps: mixing biochar loaded with bacteria, basic fertilizer, humic acid, and diatomaceous earth in the mass percentage ratio of (25-35): (55-65): (7-9): (1-3) to obtain the multi-microbial active substance compound fertilizer.

[0017] Beneficial effects of the present invention: This solution significantly improves microbial activity, soil improvement, and crop yields through an innovative layered fermentation process and the synergistic effects of multiple components. The stratified inoculation strategy (Aspergillus niger in the bottom layer, Bacillus spp. in the middle layer, and Pseudomonas fluorescens in the surface layer) combined with breathable non-woven fabric isolation technology effectively avoids the drawbacks of bacterial mutual inhibition in traditional mixed fermentation, significantly improving bacterial survival rates compared to conventional mixed fermentation. This technological breakthrough not only promotes organic matter decomposition through the efficient enzyme production of Aspergillus niger and the phosphate solubilization function of Bacillus spp., but also utilizes the iron carriers of Pseudomonas fluorescens to enhance plant disease resistance, forming a multifunctional linkage in the bacterial metabolic network. Furthermore, the composite protective agent composed of trehalose and bentonite reduces bacterial damage and enzyme protein inactivation during the drying process through the vitrification effect and physical adsorption, thereby improving enzyme activity stability and resolving the key technical bottleneck of activity loss during microbial fertilizer processing.

[0018] Combined with the carrier function of biochar, it provides physical shelter for the bacteria and forms a slow-release system through chemical bonding with humic acid. Furthermore, the introduction of diatomaceous earth helps improve water retention, maintaining yield growth even under drought conditions. The optimized nitrogen, phosphorus, and potassium ratios, synergistic with microbial metabolism, significantly increase potato and cucumber yields.

[0019] The combined effect of these technological innovations gives this solution multi-dimensional advantages in soil improvement, crop yield increases, and environmental friendliness. Its "carrier-microbial community-process" synergistic mechanism not only overcomes the single-function limitations of traditional microbial fertilizers, but also, through the simultaneous optimization of microbial colonization efficiency, enzyme activity stability, and nutrient release rate, produces significant improvements beyond the combined effects of conventional technologies, fully demonstrating the unpredictability and creativity of the technology's results. DETAILED DESCRIPTION

[0020] Apple tree branches, grapevines, and wheat straw are local agricultural byproducts. The apple tree branches used in the embodiment are 1-2 year old branches after pruning of adult fruit trees, dried to a moisture content of 10%; the grapevines are vine residues produced by winter pruning of Cabernet Sauvignon grapevines; and the wheat is the remaining stems after harvest.

[0021] Humic acid, product number: 12418, Jiangsu Congzhong Chemical Co., Ltd.

[0022] Diatomaceous earth, product number: LI2346, Hubei Xinhongli Chemical Co., Ltd.

[0023] The decomposed cow dung is prepared by aerobic fermentation for 25-40 days, wherein the temperature is maintained at 55-65° C. for 3-5 days during the fermentation period and at 65-75° C. for 10-15 days during the high temperature period, the compost is turned over every 5-7 days, the fecal coliform count is 90 MPN / g, and the organic matter content is 50%.

[0024] Aspergillus niger (CGMCC No. 15234) was purchased from China General Microbiological Culture Collection Center.

[0025] Bacillus subtilis (ACCC 11025) was purchased from China Agricultural Microorganism Culture Collection Center.

[0026] Pseudomonas fluorescens (CCTCC M 2012345) was purchased from China Center for Type Culture Collection.

[0027] Breathable nonwoven fabric, average pore size: 10-30 μm.

[0028] Trehalose, product number: 10190, Xi'an Xinfengda Pharmaceutical Excipients Co., Ltd.

[0029] Bentonite, product number: 0002, Wuhan Profu Biotechnology Co., Ltd.

[0030] Example 1 A multi-microbial active substance compound fertilizer, comprising the following components by mass percentage: 30% biochar loaded with bacteria; 60% basic fertilizer; 8% humic acid; 2% diatomaceous earth; The mass ratio of nitrogen, phosphorus and potassium in the basic fertilizer is 15:10:15.

[0031] The method for preparing the biochar loaded with bacteria comprises the following steps: (1) Apple tree branches, grape vines, and wheat straw were mixed in a ratio of 5:3:2 and crushed into 3 mm particles to obtain a fermentation substrate; the fermentation substrate and a 1 wt% calcium hydroxide aqueous solution were mixed in a mass ratio of 1:0.6 and allowed to stand for 3 days to obtain an alkali-treated fermentation substrate; (2) mixing the alkali-treated fermentation substrate obtained in step (1) with decomposed cow dung in a mass ratio of 95:5 for fermentation, maintaining the fermentation temperature at 60°C and the humidity at 50%, turning the compost twice a day for 3 days, and obtaining a decomposed fermentation substrate; (3) First, a 15 cm thick layer of the decomposed fermentation substrate obtained in step (2) is laid on the bottom layer of the fermentation chamber and inoculated with Aspergillus niger. After 24 hours, an 8 cm thick layer of the decomposed fermentation substrate obtained in step (2) is laid on the bottom layer as a middle layer and Bacillus subtilis and Bacillus gelatinus are inoculated on the middle layer. After another 24 hours, a 5 cm thick layer of the decomposed fermentation substrate obtained in step (2) is laid on the middle layer as a surface layer and Pseudomonas fluorescens is inoculated on the surface layer. The fermentation is continued for another 24 hours, for a total of 72 hours, to obtain a fermentation product. The layers are separated by breathable non-woven fabrics to avoid mutual inhibition between strains and to allow metabolites to diffuse across the layers. (4) The apple tree branches were placed in an oxygen-free environment, heated to 500°C at a rate of 10°C / min, carbonized at a constant temperature for 1 hour, and then cooled to room temperature at a rate of 10°C / min to obtain apple charcoal; (5) The fermentation product obtained in step (3) and the apple charcoal obtained in step (4) were mixed in a mass ratio of 1:1, trehalose and bentonite were added, and the mixture was stirred at a speed of 30 r / min for 2 h. After the stirring was completed, a pulse air flow dryer was used to reduce the moisture content to 9 wt % to obtain the biochar loaded with the bacteria.

[0032] In step (3), the inoculation amount of Aspergillus niger is 6wt% of the dry weight of the bottom layer of the fermentation substrate; the inoculation amount of Bacillus subtilis is 4wt% of the dry weight of the middle layer of the fermentation substrate; the inoculation amount of Bacillus gelatinosa is 3wt% of the dry weight of the middle layer of the fermentation substrate; and the inoculation amount of Pseudomonas fluorescens is 3wt% of the dry weight of the surface layer of the fermentation substrate.

[0033] In step (3), the temperature in the fermentation chamber is controlled at 28°C, the humidity is controlled at 60%, the ventilation rate in the first 24 hours is 0.8 m³ / (h·kg), there is no ventilation in the middle 24 hours, and intermittent ventilation is adopted for 8 minutes every 30 minutes in the last 24 hours, with a ventilation rate of (0.2 m³ / (h·kg)).

[0034] The amount of trehalose added in step (5) is 5wt% of the fermentation product obtained in step (3); the amount of bentonite added is 2wt% of the fermentation product obtained in step (3).

[0035] The inlet temperature of the pulse air flow dryer in step (5) is 40°C and the outlet temperature is 35°C.

[0036] A preparation method of a multi-microbial active substance compound fertilizer comprises the following steps: mixing biochar loaded with bacteria, basic fertilizer, humic acid, and diatomaceous earth in a mass percentage of 30:60:8:2 to obtain the multi-microbial active substance compound fertilizer.

[0037] Example 2 The difference from Example 1 is that the mass percentage of the biochar loaded with bacteria is 20%, and the mass percentage of the basic fertilizer is 80%.

[0038] A multi-microbial active substance compound fertilizer, comprising the following components by mass percentage: 20% biochar loaded with bacteria; 80% basic fertilizer; 8% humic acid; 2% diatomaceous earth; The mass ratio of nitrogen, phosphorus and potassium in the basic fertilizer is 15:10:15.

[0039] A preparation method of a multi-microbial active substance compound fertilizer comprises the following steps: mixing biochar loaded with bacteria, basic fertilizer, humic acid, and diatomaceous earth in a mass percentage of 30:60:8:2 to obtain the multi-microbial active substance compound fertilizer.

[0040] Example 3 The difference from Example 1 is that the mass percentage of the biochar loaded with bacteria is 40%, and the mass percentage of the basic fertilizer is 60%.

[0041] A multi-microbial active substance compound fertilizer, comprising the following components by mass percentage: 40% biochar loaded with bacteria; 50% basic fertilizer; 8% humic acid; 2% diatomaceous earth; The mass ratio of nitrogen, phosphorus and potassium in the basic fertilizer is 15:10:15.

[0042] A preparation method of a multi-microbial active substance compound fertilizer comprises the following steps: mixing biochar loaded with bacteria, basic fertilizer, humic acid, and diatomaceous earth in a mass percentage of 30:60:8:2 to obtain the multi-microbial active substance compound fertilizer.

[0043] Example 4 The difference from Example 1 is that step (2) does not adopt segmented ventilation, and the ventilation volume is 0.8 m³ / (h·kg).

[0044] The method for preparing the biochar loaded with bacteria comprises the following steps: (1) Apple tree branches, grape vines, and wheat straw were mixed in a ratio of 5:3:2 and crushed into 3 mm particles to obtain a fermentation substrate; the fermentation substrate and a 1 wt% calcium hydroxide aqueous solution were mixed in a mass ratio of 1:0.6 and allowed to stand for 3 days to obtain an alkali-treated fermentation substrate; (2) mixing the alkali-treated fermentation substrate obtained in step (1) with decomposed cow dung in a mass ratio of 95:5 for fermentation, maintaining the fermentation temperature at 60°C and the humidity at 50%, turning the compost twice a day for 3 days, and obtaining a decomposed fermentation substrate; (3) First, a 15 cm thick layer of the decomposed fermentation substrate obtained in step (2) is laid on the bottom layer of the fermentation chamber and inoculated with Aspergillus niger. After 24 hours, an 8 cm thick layer of the decomposed fermentation substrate obtained in step (2) is laid on the bottom layer as a middle layer and Bacillus subtilis and Bacillus gelatinus are inoculated on the middle layer. After another 24 hours, a 5 cm thick layer of the decomposed fermentation substrate obtained in step (2) is laid on the middle layer as a surface layer and Pseudomonas fluorescens is inoculated on the surface layer. The fermentation is continued for another 24 hours, for a total of 72 hours, to obtain a fermentation product. The layers are separated by breathable non-woven fabrics to avoid mutual inhibition between strains and to allow metabolites to diffuse across the layers. (4) The apple tree branches were placed in an oxygen-free environment, heated to 500°C at a rate of 10°C / min, carbonized at a constant temperature for 1 hour, and then cooled to room temperature at a rate of 10°C / min to obtain apple charcoal; (5) The fermentation product obtained in step (3) and the apple charcoal obtained in step (4) were mixed in a mass ratio of 1:1, trehalose and bentonite were added, and the mixture was stirred at a speed of 30 r / min for 2 h. After the stirring was completed, a pulse air flow dryer was used to reduce the moisture content to 9 wt % to obtain the biochar loaded with the bacteria.

[0045] In step (3), the inoculation amount of Aspergillus niger is 6wt% of the dry weight of the bottom layer of the fermentation substrate; the inoculation amount of Bacillus subtilis is 4wt% of the dry weight of the middle layer of the fermentation substrate; the inoculation amount of Bacillus gelatinosa is 3wt% of the dry weight of the middle layer of the fermentation substrate; and the inoculation amount of Pseudomonas fluorescens is 3wt% of the dry weight of the surface layer of the fermentation substrate.

[0046] In step (3), the temperature in the fermentation chamber is controlled at 28°C, the humidity is controlled at 60%, and the ventilation volume is 0.8 m³ / (h·kg).

[0047] The amount of trehalose added in step (5) is 5wt% of the fermentation product obtained in step (3); the amount of bentonite added is 2wt% of the fermentation product obtained in step (3).

[0048] The inlet temperature of the pulse air flow dryer in step (5) is 40°C and the outlet temperature is 35°C.

[0049] Example 5 The difference from Example 1 is that step (2) adopts intermittent ventilation of 8 minutes every 30 minutes, and the ventilation volume is (0.2 m³ / (h·kg)).

[0050] The method for preparing the biochar loaded with bacteria comprises the following steps: (1) Apple tree branches, grape vines, and wheat straw were mixed in a ratio of 5:3:2 and crushed into 3 mm particles to obtain a fermentation substrate; the fermentation substrate and a 1 wt% calcium hydroxide aqueous solution were mixed in a mass ratio of 1:0.6 and allowed to stand for 3 days to obtain an alkali-treated fermentation substrate; (2) mixing the alkali-treated fermentation substrate obtained in step (1) with decomposed cow dung in a mass ratio of 95:5 for fermentation, maintaining the fermentation temperature at 60°C and the humidity at 50%, turning the compost twice a day for 3 days, and obtaining a decomposed fermentation substrate; (3) First, a 15 cm thick layer of the decomposed fermentation substrate obtained in step (2) is laid on the bottom layer of the fermentation chamber and inoculated with Aspergillus niger. After 24 hours, an 8 cm thick layer of the decomposed fermentation substrate obtained in step (2) is laid on the bottom layer as a middle layer and Bacillus subtilis and Bacillus gelatinus are inoculated on the middle layer. After another 24 hours, a 5 cm thick layer of the decomposed fermentation substrate obtained in step (2) is laid on the middle layer as a surface layer and Pseudomonas fluorescens is inoculated on the surface layer. The fermentation is continued for another 24 hours, for a total of 72 hours, to obtain a fermentation product. The layers are separated by breathable non-woven fabrics to avoid mutual inhibition between strains and to allow metabolites to diffuse across the layers. (4) The apple tree branches were placed in an oxygen-free environment, heated to 500°C at a rate of 10°C / min, carbonized at a constant temperature for 1 hour, and then cooled to room temperature at a rate of 10°C / min to obtain apple charcoal; (5) The fermentation product obtained in step (3) and the apple charcoal obtained in step (4) were mixed in a mass ratio of 1:1, trehalose and bentonite were added, and the mixture was stirred at a speed of 30 r / min for 2 h. After the stirring was completed, a pulse air flow dryer was used to reduce the moisture content to 9 wt % to obtain the biochar loaded with the bacteria.

[0051] In step (3), the inoculation amount of Aspergillus niger is 6wt% of the dry weight of the bottom layer of the fermentation substrate; the inoculation amount of Bacillus subtilis is 4wt% of the dry weight of the middle layer of the fermentation substrate; the inoculation amount of Bacillus gelatinosa is 3wt% of the dry weight of the middle layer of the fermentation substrate; and the inoculation amount of Pseudomonas fluorescens is 3wt% of the dry weight of the surface layer of the fermentation substrate.

[0052] In step (3), the temperature in the fermentation chamber is controlled at 28°C, the humidity is controlled at 60%, and intermittent ventilation is adopted with ventilation for 8 minutes every 30 minutes, and the ventilation volume is (0.2 m³ / (h·kg)).

[0053] The amount of trehalose added in step (5) is 5wt% of the fermentation product obtained in step (3); the amount of bentonite added is 2wt% of the fermentation product obtained in step (3).

[0054] The inlet temperature of the pulse air flow dryer in step (5) is 40°C and the outlet temperature is 35°C.

[0055] Comparative Example 1 The difference from Example 1 is that Aspergillus niger, Bacillus subtilis, Bacillus gelatinosa, and Pseudomonas fluorescens are inoculated at the same time and fermented for 72 hours.

[0056] The method for preparing the biochar loaded with bacteria comprises the following steps: (1) Apple tree branches, grape vines, and wheat straw were mixed in a ratio of 5:3:2 and crushed into 3 mm particles to obtain a fermentation substrate; the fermentation substrate and a 1 wt% calcium hydroxide aqueous solution were mixed in a mass ratio of 1:0.6 and allowed to stand for 3 days to obtain an alkali-treated fermentation substrate; (2) mixing the alkali-treated fermentation substrate obtained in step (1) with decomposed cow dung in a mass ratio of 95:5 for fermentation, maintaining the fermentation temperature at 60°C and the humidity at 50%, turning the compost twice a day for 3 days, and obtaining a decomposed fermentation substrate; (3) First, a 28 cm thick layer of the decomposed fermentation substrate obtained in step (2) is laid on the bottom layer of the fermentation chamber and inoculated with Aspergillus niger. At the same time, an 8 cm thick layer of the decomposed fermentation substrate obtained in step (2) is laid on the bottom layer as a middle layer and inoculated with Bacillus subtilis and Bacillus gelatinosa. A 5 cm thick layer of the decomposed fermentation substrate obtained in step (2) is laid on the middle layer as a surface layer and inoculated with Pseudomonas fluorescens. After fermentation for 72 hours, a fermentation product is obtained. The layers are separated by breathable non-woven fabrics to avoid mutual inhibition between strains and allow metabolites to diffuse across the layers. (4) The apple tree branches were placed in an oxygen-free environment, heated to 500°C at a rate of 10°C / min, carbonized at a constant temperature for 1 hour, and then cooled to room temperature at a rate of 10°C / min to obtain apple charcoal; (5) The fermentation product obtained in step (3) and the apple charcoal obtained in step (4) were mixed in a mass ratio of 1:1, trehalose and bentonite were added, and the mixture was stirred at a speed of 30 r / min for 2 h. After the stirring was completed, a pulse air flow dryer was used to reduce the moisture content to 9 wt % to obtain the biochar loaded with the bacteria.

[0057] In step (3), the inoculation amount of Aspergillus niger is 6wt% of the dry weight of the bottom layer of the fermentation substrate; the inoculation amount of Bacillus subtilis is 4wt% of the dry weight of the middle layer of the fermentation substrate; the inoculation amount of Bacillus gelatinosa is 3wt% of the dry weight of the middle layer of the fermentation substrate; and the inoculation amount of Pseudomonas fluorescens is 3wt% of the dry weight of the surface layer of the fermentation substrate.

[0058] In step (3), the temperature in the fermentation chamber is controlled at 28°C, the humidity is controlled at 60%, the ventilation rate in the first 24 hours is 0.8 m³ / (h·kg), there is no ventilation in the middle 24 hours, and intermittent ventilation is adopted for 8 minutes every 30 minutes in the last 24 hours, with a ventilation rate of (0.2 m³ / (h·kg)).

[0059] The amount of trehalose added in step (5) is 5wt% of the fermentation product obtained in step (3); the amount of bentonite added is 2wt% of the fermentation product obtained in step (3).

[0060] The inlet temperature of the pulse air flow dryer in step (5) is 40°C and the outlet temperature is 35°C.

[0061] Comparative Example 2 The difference from Example 1 is that trehalose and bentonite are not added.

[0062] The method for preparing the biochar loaded with bacteria comprises the following steps: (1) Apple tree branches, grape vines, and wheat straw were mixed in a ratio of 5:3:2 and crushed into 3 mm particles to obtain a fermentation substrate; the fermentation substrate and a 1 wt% calcium hydroxide aqueous solution were mixed in a mass ratio of 1:0.6 and allowed to stand for 3 days to obtain an alkali-treated fermentation substrate; (2) mixing the alkali-treated fermentation substrate obtained in step (1) with decomposed cow dung in a mass ratio of 95:5 for fermentation, maintaining the fermentation temperature at 60°C and the humidity at 50%, turning the compost twice a day for 3 days, and obtaining a decomposed fermentation substrate; (3) First, a 15 cm thick layer of the decomposed fermentation substrate obtained in step (2) is laid on the bottom layer of the fermentation chamber and inoculated with Aspergillus niger. After 24 hours, an 8 cm thick layer of the decomposed fermentation substrate obtained in step (2) is laid on the bottom layer as a middle layer and Bacillus subtilis and Bacillus gelatinus are inoculated on the middle layer. After another 24 hours, a 5 cm thick layer of the decomposed fermentation substrate obtained in step (2) is laid on the middle layer as a surface layer and Pseudomonas fluorescens is inoculated on the surface layer. The fermentation is continued for another 24 hours, for a total of 72 hours, to obtain a fermentation product. The layers are separated by breathable non-woven fabrics to avoid mutual inhibition between strains and to allow metabolites to diffuse across the layers. (4) The apple tree branches were placed in an oxygen-free environment, heated to 500°C at a rate of 10°C / min, carbonized at a constant temperature for 1 hour, and then cooled to room temperature at a rate of 10°C / min to obtain apple charcoal; (5) The fermentation product obtained in step (3) and the apple charcoal obtained in step (4) were mixed in a mass ratio of 1:1, and stirred at a speed of 30 r / min for 2 h. After stirring, a pulse air flow dryer was used to reduce the moisture content to 9 wt % to obtain the biochar loaded with the bacteria.

[0063] In step (3), the inoculation amount of Aspergillus niger is 6wt% of the dry weight of the bottom layer of the fermentation substrate; the inoculation amount of Bacillus subtilis is 4wt% of the dry weight of the middle layer of the fermentation substrate; the inoculation amount of Bacillus gelatinosa is 3wt% of the dry weight of the middle layer of the fermentation substrate; and the inoculation amount of Pseudomonas fluorescens is 3wt% of the dry weight of the surface layer of the fermentation substrate.

[0064] In step (3), the temperature in the fermentation chamber is controlled at 28°C, the humidity is controlled at 60%, the ventilation rate in the first 24 hours is 0.8 m³ / (h·kg), there is no ventilation in the middle 24 hours, and intermittent ventilation is adopted for 8 minutes every 30 minutes in the last 24 hours, with a ventilation rate of (0.2 m³ / (h·kg)).

[0065] The inlet temperature of the pulse air flow dryer in step (5) is 40°C and the outlet temperature is 35°C.

[0066] Comparative Example 3 The difference from Example 1 is that no biochar loaded with bacteria was added.

[0067] A multi-microbial active substance compound fertilizer, comprising the following components by mass percentage: 90% basic fertilizer; 8% humic acid; 2% diatomaceous earth; The mass ratio of nitrogen, phosphorus and potassium in the basic fertilizer is 15:10:15.

[0068] A preparation method of a multi-microbial active substance compound fertilizer comprises the following steps: mixing biochar loaded with bacteria, basic fertilizer, humic acid, and diatomaceous earth in a mass percentage of 30:60:8:2 to obtain the multi-microbial active substance compound fertilizer.

[0069] Comparative Example 4 The difference from Example 1 is that in step (2), the fermentation chamber is not ventilated.

[0070] The method for preparing the biochar loaded with bacteria comprises the following steps: (1) Apple tree branches, grape vines, and wheat straw were mixed in a ratio of 5:3:2 and crushed into 3 mm particles to obtain a fermentation substrate; the fermentation substrate and a 1 wt% calcium hydroxide aqueous solution were mixed in a mass ratio of 1:0.6 and allowed to stand for 3 days to obtain an alkali-treated fermentation substrate; (2) mixing the alkali-treated fermentation substrate obtained in step (1) with decomposed cow dung in a mass ratio of 95:5 for fermentation, maintaining the fermentation temperature at 60°C and the humidity at 50%, turning the compost twice a day for 3 days, and obtaining a decomposed fermentation substrate; (3) First, a 15 cm thick layer of the decomposed fermentation substrate obtained in step (2) is laid on the bottom layer of the fermentation chamber and inoculated with Aspergillus niger. After 24 hours, an 8 cm thick layer of the decomposed fermentation substrate obtained in step (2) is laid on the bottom layer as a middle layer and Bacillus subtilis and Bacillus gelatinus are inoculated on the middle layer. After another 24 hours, a 5 cm thick layer of the decomposed fermentation substrate obtained in step (2) is laid on the middle layer as a surface layer and Pseudomonas fluorescens is inoculated on the surface layer. The fermentation is continued for another 24 hours, for a total of 72 hours, to obtain a fermentation product. The layers are separated by breathable non-woven fabrics to avoid mutual inhibition between strains and to allow metabolites to diffuse across the layers. (4) The apple tree branches were placed in an oxygen-free environment, heated to 500°C at a rate of 10°C / min, carbonized at a constant temperature for 1 hour, and then cooled to room temperature at a rate of 10°C / min to obtain apple charcoal; (5) The fermentation product obtained in step (3) and the apple charcoal obtained in step (4) were mixed in a mass ratio of 1:1, trehalose and bentonite were added, and the mixture was stirred at a speed of 30 r / min for 2 h. After the stirring was completed, a pulse air flow dryer was used to reduce the moisture content to 9 wt % to obtain the biochar loaded with the bacteria.

[0071] In step (3), the inoculation amount of Aspergillus niger is 6wt% of the dry weight of the bottom layer of the fermentation substrate; the inoculation amount of Bacillus subtilis is 4wt% of the dry weight of the middle layer of the fermentation substrate; the inoculation amount of Bacillus gelatinosa is 3wt% of the dry weight of the middle layer of the fermentation substrate; and the inoculation amount of Pseudomonas fluorescens is 3wt% of the dry weight of the surface layer of the fermentation substrate.

[0072] In step (3), the temperature in the fermentation chamber is controlled at 28° C., the humidity is controlled at 60%, and there is no ventilation.

[0073] The amount of trehalose added in step (5) is 5wt% of the fermentation product obtained in step (3); the amount of bentonite added is 2wt% of the fermentation product obtained in step (3).

[0074] The inlet temperature of the pulse air flow dryer in step (5) is 40°C and the outlet temperature is 35°C.

[0075] Test Example 1 Bacterial survival rate (CFU / g) test; Sample treatment: Take 0.5g of the biochar loaded with bacteria prepared in Examples 1-6 and Comparative Examples 1-2, add 4.5mL of sterile saline, vortex and shake for 10min; gradient dilution to 10 -6 (Aspergillus niger), 10 -7 (Bacillus), 10 -8 (Pseudomonas fluorescens); Culture medium selection: Aspergillus niger: PDA medium (containing 0.1% streptomycin to inhibit bacteria); Bacillus subtilis / Bacillus gelatinosa: LB medium; Pseudomonas fluorescens: KB medium (containing 0.05% cycloheximide to inhibit fungi).

[0076] Culture counting: After coating, culture at 28°C for 48 hours (bacteria) or 72 hours (fungi), and record the number of colonies; Calculation formula: CFU / g = number of colonies × dilution factor × 20 (corrected for dilution volume).

[0077] The results are shown in Table 1.

[0078] Table 1: Test results of bacterial survival rate (CFU / g)

[0079] Test Example 2 Cellulase / xylanase activity (U / g) test; Enzyme extraction: 1 g of the bacterial-loaded biochar prepared in each example or comparative example was added to 10 mL of pH 5.0 acetate buffer, extracted at 4°C for 2 h, and centrifuged (8000 rpm, 10 min) to obtain the supernatant.

[0080] Reaction system: Cellulase: 1% sodium carboxymethyl cellulose (CMC-Na) substrate, reaction at 50°C for 30 min, reducing sugar determination by DNS method; Xylanase: 1% birch xylan substrate, reaction at 50℃ for 10 min, and xylose production was measured by DNS method.

[0081] Activity definition: 1U = the amount of enzyme required to generate 1 μmol of reducing sugar per minute.

[0082] The results are shown in Table 2.

[0083] Table 2: Cellulase / xylanase activity (U / g) test results

[0084] Test Example 3 Crop yield increase test: Field experiment design: Three crops, cucumber (fruiting vegetable), spinach (leafy vegetable), and potato (root vegetable), were selected; a randomized block design with three replicates per group was used, and the multi-microbial active substance compound fertilizers prepared in Examples 1-6 and Comparative Examples 1-3 were applied, with Comparative Example 3 serving as the reference group; the fertilizer application rate was 150 kg / mu, and the yield, single fruit weight, and disease incidence were recorded.

[0085] Data analysis: Yield increase rate = (yield of treatment group – yield of control example 3) / yield of conventional fertilizer group × 100%; Duncan multiple comparison (p < 0.05) was used to determine significance.

[0086] Table 3: Crop yield increase test results

[0087] From the test results of Table 1, Table 2 and Table 3, it can be seen that Example 1 has the best strain survival rate, strain enzyme production ability and yield increase effect; the difference between Example 2 and Example 1 is that the mass percentage of biochar loaded with bacteria is 20%, and the mass percentage of basic fertilizer is 80%. The difference between Example 3 and Example 1 is that the mass percentage of biochar loaded with bacteria is 40%, and the mass percentage of basic fertilizer is 60%. Its strain survival rate, enzyme activity and yield increase rate are all lower than those of Example 1, which indicates that the optimal amount of biochar loaded with bacteria is 30wt%. The difference between Example 4 and Example 1 is that the segmented ventilation strategy is not adopted, and excessive oxidation accelerates carbon source consumption, resulting in a decrease in cellulase activity and an imbalance in bacterial metabolism; the difference between Example 5 and Example 1 is that the high-oxygen ventilation stage in the first 24 hours is not adjusted, and CO2 accumulation inhibits the activity of aerobic bacteria, resulting in a decrease in the survival rate of Aspergillus niger, and temperature fluctuations aggravate bacterial stress; the difference between Comparative Example 1 and Example 1 is that the bacterial community is not isolated by stratification, and the survival rate and enzyme activity drop sharply due to the mutual inhibition effect of bacterial species; the difference between Comparative Example 2 and Example 1 is that trehalose and bentonite are not added, and the bacterial body is dehydrated due to drying damage, and the yield increase rate decreases simultaneously.

[0088] In summary, Example 1 avoided mutual inhibition through layered inoculation, optimized metabolism through segmented ventilation, and synergistically enhanced efficiency between the composite carrier and the protective agent, and was significantly superior to other schemes in terms of bacterial colonization, enzyme activity release, and fertilizer efficiency stability.

Claims

1. A multi-microbial active substance compound fertilizer, characterized in that: By mass percentage, it is composed of the following components: 25-35% biochar loaded with bacteria; 55-65% basic fertilizer; 7-9% humic acid; 1-3% diatomaceous earth; The method for preparing the biochar loaded with bacteria comprises the following steps: (1) mixing apple tree branches, grape vines, and wheat straw in a ratio of 5:3:(1-2) and crushing them into 2-5 mm particles to obtain a fermentation substrate; mixing the fermentation substrate and a 1 wt% calcium hydroxide aqueous solution in a mass ratio of 1:(0.5-1), and letting it stand for 2-3 days to obtain an alkali-treated fermentation substrate; (2) mixing the alkali-treated fermentation substrate obtained in step (1) and decomposed cow dung in a mass ratio of (90-99):(1-10) for fermentation, maintaining the fermentation temperature at 55-60° C. and the humidity at 45-55%, turning the compost 1-3 times / day for 2-3 days, and obtaining a decomposed fermentation substrate; (3) First, a 14-16 cm thick layer of the decomposed fermentation substrate obtained in step (2) is laid on the bottom layer of the fermentation chamber and inoculated with Aspergillus niger. After 23-25 ​​hours, a 7-9 cm thick layer of the decomposed fermentation substrate obtained in step (2) is laid on the bottom layer as a middle layer and Bacillus subtilis and Bacillus gelatinus are inoculated on the middle layer. After another 23-25 ​​hours, a 4-6 cm thick layer of the decomposed fermentation substrate obtained in step (2) is laid on the middle layer as a surface layer and Pseudomonas fluorescens is inoculated on the surface layer. The fermentation is continued for another 23-25 ​​hours, and the total fermentation time is 69-75 hours to obtain a fermentation product. The layers are separated by breathable non-woven fabrics to avoid mutual inhibition of bacterial species and allow metabolites to diffuse across the layers. (4) placing the apple tree branches in an anaerobic environment, heating the temperature to 490-510°C at a rate of 9-11°C / min, carbonizing at a constant temperature for 0.5-1.5h, and then cooling the temperature to room temperature at a rate of 9-11°C / min to obtain apple charcoal; (5) The fermentation product obtained in step (3) and the apple charcoal obtained in step (4) are mixed in a mass ratio of 1:1, trehalose and bentonite are added, and the mixture is stirred at a speed of 25-35 r / min for 1-3 h. After stirring, a pulse air flow dryer is used to reduce the moisture content to 8wt%-10wt% to obtain the biochar loaded with the bacteria.

2. The multi-microbial active substance compound fertilizer according to claim 1, characterized in that: The mass ratio of nitrogen, phosphorus and potassium in the basic fertilizer is 15:(9-10):

15.

3. The multi-microbial active substance compound fertilizer according to claim 1, characterized in that: In step (3), the inoculation amount of Aspergillus niger is 5wt%-8wt% of the dry weight of the bottom layer of the fermentation substrate; the inoculation amount of Bacillus subtilis is 3wt%-5wt% of the dry weight of the middle layer of the fermentation substrate; the inoculation amount of Bacillus gelatinous is 2-4wt% of the dry weight of the middle layer of the fermentation substrate; and the inoculation amount of Pseudomonas fluorescens is 2wt%-3wt% of the dry weight of the surface layer of the fermentation substrate.

4. The multi-microbial active substance compound fertilizer according to claim 1, characterized in that: In step (3), the temperature in the fermentation bin is controlled at 27-29° C., the humidity is controlled at 58-62%, the ventilation volume in the first 23-25 ​​hours is 0.7-0.9 m³ / (h·kg), there is no ventilation in the middle 23-24 hours, and intermittent ventilation is adopted for the last 23-24 hours, with ventilation for 5-8 minutes every 25-30 minutes, and the ventilation volume is (0.1-0.3 m³ / (h·kg)).

5. The multi-microbial active substance compound fertilizer according to claim 1, characterized in that: The amount of trehalose added in step (5) is 4-6wt% of the fermentation product obtained in step (3); the amount of bentonite added is 1-3wt% of the fermentation product obtained in step (3).

6. The multi-microbial active substance compound fertilizer according to claim 1, characterized in that: The inlet temperature of the pulse air flow dryer in step (5) is 38-42°C and the outlet temperature is 30-35°C.

7. A method for preparing a composite fertilizer containing multiple microbial active substances as claimed in any one of claims 1 to 6, characterized in that: The method comprises the following steps: mixing the biochar loaded with bacteria, basic fertilizer, humic acid and diatomaceous earth in the mass percentages of (25-35): (55-65): (7-9): (1-3) to obtain the composite fertilizer containing multiple microbial active substances.

Images