Northeast agriculture and forestry grassland soil remediation composite biological agent using corn straw as raw material, preparation method and application thereof

Abstract

The invention relates to a compound biological agent for soil remediation of northeast agriculture and forestry grassland by taking corn straw as a raw material as well as a preparation method and application of the compound biological agent. The compound biological agent is prepared from bacillus subtilis, paenibacillus mucilaginosus, saccharomycetes, Korean comamonas and an auxiliary agent. The corn straw is used as a main raw material, the composite biological agent is used for biological composting fermentation under the low-temperature condition in northeast, the prepared product has the unique characteristic of secreting polyglutamic acid and potassium under the low-temperature condition, the cost is low, the seed emergence rate is increased after application, the resistance of seedlings is enhanced, and the yield of the seedlings is increased. The soil physical and chemical properties can be effectively improved, the stress effect is reduced, effective nutrients in soil absorbed by crops are increased, and the crop yield is increased.

Description

Technical Field

[0001] The invention relates to a composite biological microbial agent for repairing agricultural, forestry and grassland soil in Northeast China using corn straw as raw material, a preparation method and application thereof, and belongs to the field of microbiology. Background Art

[0002] As a major agricultural country, my country boasts high crop yields, and consequently, a significant production of crop straw, ranking first in the world. Crop straw has become a vast renewable resource, with promising prospects for its comprehensive utilization. However, this resource is difficult to fully utilize, with much of it being burned. This not only wastes resources but also causes significant environmental pollution. Studies have shown that burning large areas of straw on the soil surface reduces the number and diversity of soil microorganisms, leading to a decrease in microbial community diversity. Straw burning also disrupts the soil structure, causing significant evaporation of surface water and a loss of soil water retention. This practice also severely pollutes the environment, wastes valuable biological resources, and disrupts soil structure, significantly reducing soil organic matter and nutrient content. In traditional agriculture, straw was primarily used for heating and animal feed. However, with the transition from traditional to modern agriculture, adjustments to rural feed and energy structures, and rapid economic development, the use of crop straw has undergone significant changes, resulting in a significant surplus of crop straw.

[0003] With the continuous increase in population and the increasing scarcity of arable land and water resources, the utilization of diverse soil types within the agroforestry-grassland system in Northeast China is a pressing issue. For soil beneath shelterbelts, trenching and root removal are often employed, increasing the area of ​​farmland occupied. This has severely curbed farmers' enthusiasm for establishing and maintaining shelterbelts, leading to some degradation of shelterbelts in many areas. Deep tillage, physical and chemical methods, and agricultural plant improvement methods are commonly used to improve soil quality and promote plant growth to a certain extent. However, these methods are costly and prone to secondary pollution. Soil remediation microbial agents are a relatively new type of soil remediation product that can more effectively and comprehensively utilize barrier soils. Many experts and scholars recognize the effectiveness of microbial remediation. They offer advantages such as low cost and zero secondary pollution.

[0004] Returning straw to the field is an effective measure to increase straw utilization and improve soil fertility, and it is also a major way to utilize straw as a resource. However, in northern China, due to temperature and other factors, straw decomposes slowly after entering the soil, failing to achieve the desired effect. To ensure rapid straw decomposition and improve soil fertility without affecting next-season farming, the application of functional composite microbial agents to rapidly and efficiently decompose straw at low temperatures and, using straw as the primary carrier, to prepare composite functional bioproducts for use in barrier soil remediation offers advantages and potential in terms of energy conservation and cost reduction, and holds great development potential.

[0005] In conclusion, a composite biological agent for soil remediation in Northeast China's agricultural, forestry and grassland areas using corn straw as raw material, its preparation method and its application have not been reported. Summary of the Invention

[0006] The purpose of the present invention is to solve the above problems and provide a composite biological agent for soil remediation in Northeast China's agricultural, forestry and grasslands using corn straw as raw material, a preparation method and application thereof.

[0007] In order to achieve the above-mentioned purpose of the present invention, the present invention adopts the following technical solutions: A composite biological agent for soil remediation in Northeast China's agricultural, forestry and grassland areas using corn straw as raw material, comprising Bacillus subtilis (ZY138), Paenibacillus subtilis (HZ117), yeast (H4), Comamonas koreaensis (L42) and adjuvants; the Bacillus subtilis is deposited at the General Microbiology Center of the China Culture Collection of Microorganisms, with a deposit number of CGMCC No. 32822 and a deposit date of November 27, 2024; the Paenibacillus subtilis is deposited at the General Microbiology Center of the China Culture Collection of Microorganisms, with a deposit number of CGMCC No. 32821 and a deposit date of November 27, 2024; the yeast is deposited at the General Microbiology Center of the China Culture Collection of Microorganisms, with a deposit number of CGMCC No. 18002 and a deposit date of June 19, 2019; and the Comamonas koreaensis is deposited at the CGMCC No. No.26059, the deposit date is November 7, 2022.

[0008] The composite bacterial agent consists of 2-4 parts of Bacillus subtilis, 1-3 parts of Paenibacillus subtilis, 1-3 parts of yeast, 1-3 parts of Comamonas koreana and 1-2 parts of auxiliary agents in parts by weight.

[0009] More preferably, the composite bacterial agent consists of 3 parts of Bacillus subtilis, 2 parts of Paenibacillus subtilis, 2 parts of yeast, 2 parts of Comamonas koreana and 1 part of auxiliary agent, calculated by weight.

[0010] The CFU ratio of Bacillus subtilis, Paenibacillus subtilis, yeast, and Comamonas koreana is (4-5): (3-4): (2-3): (1-2).

[0011] The auxiliary agent is at least one of diatomaceous earth, kaolin, bentonite, mica powder, calcium carbonate and starch.

[0012] Preferably, the total number of Bacillus subtilis is 1.5 billion CFU / g; Preferably, the total number of Paenibacillus mucilaginosus is 500 million CFU / g; Preferably, the total number of Comamonas koreacens is 500 million CFU / g; Preferably, the total number of yeast is 500 million CFU / g.

[0013] The preparation method of the composite biological agent comprises the following steps: (1) Bacillus subtilis (ZY136) No. 32822 and Paenibacillus mucilaginosus (HZ117) No. 32821 were inoculated into their respective fermentation media for fermentation culture; (2) The fermentation medium of Bacillus subtilis (ZY136) in step (1) is composed of NH ４ Cl 0.7~1%, K2HPO ４ 0.1~0.2%, CaCl2 0.05~0.07%, MgSO40.05~0.07%, MnSO40.005~0.01%, FeCl30.002~0.005%, citric acid 1.35~1.5%, L-glutamic acid 1~3%, and the rest is distilled water; pH of the culture medium = 4.8~7.2; (3) The fermentation medium of Bacillus subtilis (HZ117) in step (1) is composed of 0.5-15% sucrose, Na ２ HPO ４ 0.2~0.4%, MgSO40.05~0.1%, CaCO3 0.01~0.05%, FeCl30.005~0.01%, potassium feldspar 0.5~1.5%, and the rest is distilled water; the pH of the culture medium is 4.8~7.2; (4) Yeast H4 and Korean Comamonas L42 were placed in a culture medium at a strain ratio of 1:1, and cultured at a pH of 4.8-7.2 and a temperature of 5-15°C for 12-28 hours; (5) The composition of the culture medium in step (4) is, by weight percentage, soluble starch 2-3%, peptone 0.5-1%, (NH4)2SO4 0.5-1%, K2HPO4 0.07-0.1%, KH2PO4 0.03-0.5%, MgSO4·7H2O 0.01-0.05%, and the balance is water; (6) According to weight, the composite microbial agent is composed of 3 parts of Bacillus subtilis, 2 parts of Paenibacillus subtilis, 2 parts of yeast, 2 parts of Comamonas koreana and 1 part of auxiliary agent; finally, the amount of live bacteria in the composite microbial agent is 10 6 -10 9 cfu / g.

[0014] The specific application method is as follows: using field tests, applying composite microbial agents to corn straw composting tests, using the fermentation products to improve the soil on barrier soil, and measuring crop yields by monitoring soil bulk density, pH, organic matter and other physical and chemical indicators to determine its application effect in actual production.

[0015] The advantages of the present invention are: This invention utilizes a strain of organisms that efficiently secrete cellulase and ligninase at low temperatures for straw return to fields in Northeast China. By using a composite bio-inoculant to ferment and degrade corn straw at low temperatures, the resulting barrier soil is used in Northeast China's agriculture, forestry, and grassland systems. This effectively addresses the challenges of straw burning and returning to fields in the region, while also rapidly reversing the stress effects of barrier soil on crops.

[0016] 2. The composite biological agent product produced by the present invention has the unique characteristics of producing cellulase and ligninase under low temperature conditions, as well as secreting polyglutamic acid and potassium dissolving. It is also low in cost. After application, it can effectively improve the physical and chemical properties of the soil, increase the seed germination rate, and enhance the salt and alkali resistance of the seedlings. DETAILED DESCRIPTION

[0017] The present invention is further described in detail below with reference to the embodiments.

[0018] A composite bio-microbial agent for soil remediation in Northeast China's agricultural, forestry and grasslands using corn straw as raw material, composed of Bacillus subtilis Bacillus subtilis (ZY138) and Paenibacillus mucilaginosus Paenibacillus mucilaginosus (HZ117), yeast Saccharomyces (H4) and Korean Comamonas (L42) and auxiliary agents; the classification name of Bacillus subtilis is Bacillus subtilis, The taxonomic name of Paenibacillus mucilaginosus is Paenibacillus mucilaginosus, The classification name of yeast is Saccharomyces , the classification name of Comamonas in Korea is Comamonas koreensis; The depository institution of the Bacillus subtilis is the General Microbiology Center of the China Culture Collection Administration Committee for Microorganisms, the deposit number of Bacillus subtilis is CGMCC No.32822, and the deposit date is November 27, 2024; the depository institution of the Bacillus subtilis is the General Microbiology Center of the China Culture Collection Administration Committee for Microorganisms, the deposit number of the Bacillus subtilis is CGMCC No.32821, and the deposit date is November 27, 2024; the deposit number of the yeast is CGMCC No.18002, and the deposit date is June 19, 2019; the deposit number of the Korean Comamonas is CGMCC No.26059, and the deposit date is November 7, 2022.

[0019] The composite bacterial agent consists of 2-4 parts of Bacillus subtilis, 1-3 parts of Paenibacillus subtilis, 1-3 parts of yeast, 1-3 parts of Comamonas koreana and 1-2 parts of auxiliary agents in parts by weight.

[0020] More preferably, the composite bacterial agent consists of 3 parts of Bacillus subtilis, 2 parts of Paenibacillus subtilis, 2 parts of yeast, 2 parts of Comamonas koreana and 1 part of auxiliary agent, calculated by weight.

[0021] The CFU ratio of Bacillus subtilis, Paenibacillus subtilis, yeast, and Comamonas koreana is (4-5): (3-4): (2-3): (1-2).

[0022] The auxiliary agent is at least one of diatomaceous earth, kaolin, bentonite, mica powder, calcium carbonate and starch.

[0023] Preferably, the total number of Bacillus subtilis is 1.5 billion CFU / g; Preferably, the total number of Paenibacillus mucilaginosus is 500 million CFU / g; Preferably, the total number of Comamonas koreacens is 500 million CFU / g; Preferably, the total number of yeast is 500 million CFU / g.

[0024] The preparation method of the composite biological agent comprises the following steps: (1) Bacillus subtilis (ZY136) No. 32822 and Paenibacillus mucilaginosus (HZ117) No. 32821 were inoculated into their respective fermentation media for fermentation culture; (2) The fermentation medium of Bacillus subtilis (ZY136) in step (1) is composed of NH ４ Cl 0.7~1%, K2HPO ４0.1~0.2%, CaCl2 0.05~0.07%, MgSO40.05~0.07%, MnSO40.005~0.01%, FeCl30.002~0.005%, citric acid 1.35~1.5%, L-glutamic acid 1~3%, and the rest is distilled water; pH of the culture medium = 4.8~7.2; (3) The fermentation medium of Bacillus subtilis (HZ117) in step (1) is composed of 0.5-15% sucrose, Na ２ HPO ４ 0.2~0.4%, MgSO40.05~0.1%, CaCO3 0.01~0.05%, FeCl30.005~0.01%, potassium feldspar 0.5~1.5%, and the rest is distilled water; the pH of the culture medium is 4.8~7.2; (4) Yeast H4 and Korean Comamonas L42 were placed in a culture medium at a strain ratio of 1:1, and cultured at a pH of 4.8-7.2 and a temperature of 5-15°C for 12-28 hours; (5) The composition of the culture medium in step (4) is, by weight percentage, soluble starch 2-3%, peptone 0.5-1%, (NH4)2SO4 0.5-1%, K2HPO4 0.07-0.1%, KH2PO4 0.03-0.5%, MgSO4·7H2O 0.01-0.05%, and the balance is water; (6) According to weight, it is composed of 3 parts of Bacillus subtilis, 2 parts of Bacillus subtilis, 2 parts of yeast, 2 parts of Comamonas koreana and 1 part of auxiliary agent; mix them evenly to prepare a composite biological agent; finally, the amount of live bacteria in the obtained composite biological agent is 10 6 -10 9 cfu / g.

[0025] The specific application method is as follows: using field tests, applying composite microbial agents to corn straw composting tests, using the fermentation products to improve the soil on barrier soil, and measuring crop yields by monitoring soil bulk density, pH, organic matter and other physical and chemical indicators to determine its application effect in actual production. Example

[0026] The composting process is as follows: Adjust the moisture content of one ton of crushed corn stalks to 50%. Pile the corn stalks parallel to the ground. Apply the prepared compound bio-inoculation agent to the surface every 20 cm, compact the pile, and maintain a 1.5-meter height. Cover the pile with agricultural plastic film. After 45 days of fermentation, the compound bio-organic fertilizer is obtained. Composting period: March 13, 2024 - April 26, 2024. Composting location: Shenyang.

[0027] Index inspection: Determine the straw compost temperature at different periods and the lignin, cellulose and hemicellulose content of the straw.

[0028] The results showed that, under an average spring temperature of 6°C, the straw compost treated with the compound bio-inoculation agent achieved an average temperature of 58.7°C, an 86.7% increase over the control, while the lignin, cellulose, and hemicellulose contents decreased by 20.6-66.7% compared to the control. These results demonstrate that the compound bio-inoculation agent can increase composting temperature and accelerate the decomposition of lignin, cellulose, and hemicellulose in straw. Example

[0029] The experimental site is a slightly salinized soil near the shelterbelt, and the soil type is brown soil.

[0030] Before the experiment, the topsoil was collected for testing. The basic physical and chemical properties of the soil are shown in Table 1.

[0031] Table 1. Basic physical and chemical properties of soil

[0032] The test crop was corn, and the variety was Zhengdan 958.

[0033] Experimental design: The experiment has three treatments with a plot area of ​​50 m²; the experiment is repeated three times and arranged in random blocks.

[0034] Treatment 1: Conventional fertilization + soil remediation compound bio-organic fertilizer (the product of corn straw fermented with a compound bacterial agent).

[0035] Treatment 2: conventional fertilization + equal amount of inactivated substrate.

[0036] Treatment 3: conventional fertilization.

[0037] Test method: Treatment 1 is to apply soil remediation compound biological organic fertilizer on the basis of conventional fertilization, with a dosage of 100 kg per 667m².

[0038] Treatment 2 was based on conventional fertilization, with the same amount of inactivated substrate as in treatment 1 applied to the bottom.

[0039] Treatment 3 was conventional fertilization: 40 kg / 667 m² of compound fertilizer (20-10-10) was applied at the time of corn sowing, and 20 kg / 667 m² of urea was applied at the corn tasseling stage.

[0040] Test requirements: During the test period, except for fertilization according to the test plan, no other fertilizers will be applied, and other management measures will remain the same; each time the crops are harvested, weighed, and the yield is calculated separately for each plot, and the growth and economic traits of the crops are observed and recorded.

[0041] Field management: On April 24, 2023, soil remediation compound biological organic fertilizer was applied, and the plots were divided and sown. On September 25, 2023, the economic characteristics of corn were investigated in each plot, and then the corn was harvested and the yield was measured.

[0042] Test results and analysis: 1. Effects of different treatments on soil physical and chemical properties.

[0043] Table 2 shows the interannual changes in soil physical and chemical properties at the experimental site. In Treatment 1, soil pH, salt content, and bulk density showed a downward trend, decreasing by 2.31%, 10.34%, and 8.63%, respectively, while soil organic matter increased by 9.25%. In summary, the application of the soil remediation compound bio-organic fertilizer significantly improved soil salinization, rapidly modified the soil topsoil, and fully utilized soil functions, providing strong support for soil ecosystem restoration and crop yield increases.

[0044] Table 2. Changes in soil physical and chemical indicators under different treatments

[0045] 2. Effects of different treatments on the nutritional growth and economic traits of corn.

[0046] Table 3 shows that the application of soil-remediation compound bio-organic fertilizer had a significant impact on the nutritional growth and economic traits of corn. It alleviated leaf senescence, increased fresh ear length, and reduced bald tip length. Plant height, ear length, and ear diameter increased by 3.9%, 8.5%, and 9.0%, respectively, compared to Treatment 3. This indicates that soil-remediation compound bio-organic fertilizer promoted the nutritional growth of corn and fostered favorable economic traits.

[0047] Table 3. Effects of different treatments on maize physiological traits

[0048] 3. Effects of different treatments on corn yield.

[0049] The 2023 yield results are shown in Table 4. As can be seen from Table 4, corn yield increased by 7.21% with soil-remediation compound bio-organic fertilizer application compared to conventional fertilization. To further verify the differences between soil-remediation compound bio-organic fertilizer application and substrate treatments and conventional fertilization, corn yield results were tested using SPSS 19.0 (Table 5) and the Ducan method for multiple comparisons (Table 6). The results of the variance analysis showed that soil-remediation compound bio-organic fertilizer application had a statistically significant effect on corn yield, with the differences reaching an extremely significant level. The results of the multiple analysis showed that the yield of Treatment 1 was significantly different from that of Treatments 2 and 3, while the yield difference between Treatments 2 and 3 was not significant.

[0050] Table 4. Corn production statistics

[0051] Table 5. Analysis of variance of corn plot yield

[0052] Table 6. Duncan multiple comparisons

[0053] Conclusion: Application of a soil-remediation compound bio-organic fertilizer using corn straw fermentation products as its primary raw material significantly impacted maize nutritional growth and economic traits, alleviating leaf senescence while increasing fresh ear length and reducing bald tip length. In 2023, bottom application of a soil-remediation agent significantly increased maize yield by 7.21% compared to conventional fertilization, demonstrating a significant income-increasing effect.

Claims

1. A composite biological agent for soil remediation in Northeast China's agricultural, forestry and grasslands using corn stalks as raw materials, characterized in that: The invention is composed of Bacillus subtilis (ZY138), Bacillus mucilaginosus (HZ117), yeast (H4), Comamonas koreaensis (L42) and auxiliary agents; the depository of the Bacillus subtilis is the General Microbiology Center of the China Culture Collection Administration Committee for Microorganisms, the depository number of Bacillus subtilis is CGMCC No.32822, and the depository date is November 27, 2024; the depository of the Bacillus mucilaginosus is the General Microbiology Center of the China Culture Collection Administration Committee for Microorganisms, the depository number of the Bacillus mucilaginosus is CGMCC No.32821, and the depository date is November 27, 2024; the depository number of the yeast is CGMCC No.18002, and the depository date is June 19, 2019; the depository number of the Comamonas koreaensis is CGMCC No.26059, and the depository date is November 7, 2022.

2. The biological agent according to claim 1, characterized in that The composite bacterial agent consists of 2-4 parts of Bacillus subtilis, 1-3 parts of Paenibacillus subtilis, 1-3 parts of yeast, 1-3 parts of Comamonas koreana and 1-2 parts of auxiliary agents in parts by weight.

3. The biological agent according to claim 1, characterized in that More preferably, the composite bacterial agent consists of 3 parts of Bacillus subtilis, 2 parts of Paenibacillus subtilis, 2 parts of yeast, 2 parts of Comamonas koreana and 1 part of auxiliary agent, calculated by weight.

4. The biological agent according to claim 1, characterized in that The CFU ratio of Bacillus subtilis, Paenibacillus subtilis, yeast, and Comamonas koreana is (4-5): (3-4): (2-3): (1-2).

5. The biological agent according to claim 1, characterized in that The auxiliary agent is at least one of diatomaceous earth, kaolin, bentonite, mica powder, calcium carbonate and starch.

6. The biological agent according to claim 1, characterized in that Preferably, the total number of Bacillus subtilis is 1.5 billion CFU / g; Preferably, the total number of Paenibacillus mucilaginosus is 500 million CFU / g; Preferably, the total number of Comamonas koreacens is 500 million CFU / g; Preferably, the total number of yeast is 500 million CFU / g.

7. The biological agent according to claim 1, characterized in that The preparation method of the composite biological agent comprises the following steps: (1) Bacillus subtilis (ZY136) No. 32822 and Paenibacillus mucilaginosus (HZ117) No. 32821 were inoculated into their respective fermentation media for fermentation culture; (2) The fermentation medium of Bacillus subtilis (ZY136) in step (1) comprises 0.7-1% NH4Cl, 0.1-0.2% K2HPO4, 0.05-0.07% CaCl2, 0.05-0.07% MgSO4, 0.005-0.01% MnSO4, 0.002-0.005% FeCl3, 1.35-1.5% citric acid, 1-3% L-glutamic acid, and the remainder distilled water; the pH of the medium is 4.8-7.2; (3) The fermentation medium of Paenibacillus subtilis (HZ117) in step (1) comprises 0.5-15% sucrose, 0.2-0.4% Na2HPO4, 0.05-0.1% MgSO4, 0.01-0.05% CaCO3, 0.005-0.01% FeCl3, 0.5-1.5% potassium feldspar, and the remainder is distilled water; the pH of the medium is 4.8-7.2; (4) Yeast H4 and Comamonas koreana L42 were placed in a culture medium at a strain ratio of 1:1, and cultured at a pH of 4.8-7.2 and a temperature of 5-15°C for 12-28 hours; (5) The composition of the culture medium in step (4) is, by weight percentage, soluble starch 2-3%, peptone 0.5-1%, (NH4)2SO4 0.5-1%, K2HPO4 0.07-0.1%, KH2PO4 0.03-0.5%, MgSO4·7H2O 0.01-0.05%, and the balance is water; (6) According to weight, the composite microbial agent is composed of 3 parts of Bacillus subtilis, 2 parts of Paenibacillus subtilis, 2 parts of yeast, 2 parts of Comamonas koreana and 1 part of auxiliary agent; finally, the amount of live bacteria in the composite microbial agent is 10 6 -10 9 cfu / g.

8. The biological agent according to claim 6, characterized in that The specific application method is as follows: using field tests, applying composite microbial agents to corn straw composting tests, using the fermentation products to improve the soil on barrier soil, and measuring crop yields by monitoring soil bulk density, pH, organic matter and other physical and chemical indicators to determine its application effect in actual production.

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