A small molecule peptide amino acid mixed nutrient solution for promoting vegetation restoration of mine wasteland, a preparation method and application thereof

Abstract

The application discloses a kind of small molecule peptide amino acid mixed nutrient solution for mine wasteland vegetation recovery and its preparation method and application, belong to environmental remediation technical field.Nutrient solution with high content small molecule peptide and amino acid obtained by specific enzymolysis of pig blood as core, compound molasses, alginic acid, mineral source potassium fulvic acid and EM fungicide are formed.The nutrient solution is suitable for the acid, high heavy metal pollution mine wasteland which has completed basic improvement and pioneer vegetation planting.The application method is: first irrigation after vegetation planting 5~7 days, repeat irrigation 3~6 times every 7~14 days.The application significantly improves the plant height and biomass of pioneer plants such as Sesbania cannabina and Sulfur Chrysanthemum under extreme adversity (such as the plant height of Sulfur Chrysanthemum increases by 38.62%) by easily absorbed organic nutrients and active substances, and cooperatively plays the functions of buffering acid and alkali, chelating heavy metals, improving rhizosphere microenvironment, etc., rapidly forms vegetation cover, fixes topsoil and accelerates ecological restoration.The raw materials are environmentally friendly, low in cost and easy to operate, and have good synergy with basic improvement measures, and are suitable for ecological restoration engineering of difficult sites such as metal mine dump and tailings pond.

Description

Technical Field

[0001] This invention relates to the field of environmental remediation and ecological engineering technology, specifically to a plant growth promoter and its application method, and more particularly to a small molecule peptide-amino acid mixed nutrient solution for rapid vegetation restoration in acidic, heavily heavy metal-polluted mine wastelands, its preparation method and application. Background Technology

[0002] Waste dumps and tailings ponds formed after metal mining operations have extremely harsh soil habitats, typically characterized by strong acidity (pH below 4.0), high salinity (EC values ​​exceeding 1 mS / cm), and severely excessive levels of various heavy metals (such as Fe, Cu, Zn, Pb, and Cr), accompanied by severe soil infertility and compaction. These adverse factors severely inhibit seed germination, seedling establishment, and growth, resulting in extremely slow or even non-existent natural ecological restoration processes in these sites. This leads to long-term soil erosion and heavy metal diffusion risks, posing a continuous threat to the surrounding ecological environment.

[0003] Currently, the ecological restoration of such extremely degraded habitats mainly adopts a comprehensive technical approach of "matrix improvement + vegetation reconstruction". Matrix improvement typically involves measures such as applying alkaline substances like lime to neutralize acidity, adding organic matter or topsoil to improve soil structure, and inoculating with heavy metal-tolerant microorganisms, aiming to create preliminary and basic living conditions for plants. Vegetation reconstruction focuses on selecting pioneer plant species with strong stress resistance. However, extensive engineering practice shows that even on initially improved substrates, due to residual acid stress, salt stress, and the combined toxic effects of heavy metals, the early growth of established pioneer plants is generally slow and fragile, and vegetation cover is difficult to form effectively in the short term. This directly leads to a long restoration cycle, high costs, and enormous pressure on later management, making it difficult to maintain stable restoration results.

[0004] In existing technologies, several fertilizers or nutrient solutions have been proposed for mine remediation or soil contaminated with heavy metals. For example, Chinese patent document CN102515946A discloses a nutrient solution to improve the germination rate and growth of plants in abandoned mining areas. Its main components are inorganic salts such as potassium nitrate, calcium chloride, and magnesium sulfate, with added plant growth regulators such as gibberellin and rooting powder. This technology focuses on providing inorganic nutrients and hormone stimulation, but under the combined stress of strong acidity, high salt, and high heavy metals, the absorption and utilization of inorganic nutrients by plants is severely hindered, and long-term use may exacerbate soil salinization. Another Chinese patent document, CN112934947B, discloses a method for preparing a conditioner using hydrothermal carbonization products of agricultural waste. Its nutrient solution mainly consists of conventional fertilizers such as potassium dihydrogen phosphate and urea. In addition, there are patented technologies (such as CN120842010A) that involve using microbial agents and mineral-derived potassium humate to remediate contaminated soil, or (such as CN120901074A) that utilize modified amino acids and humic acid to chelate heavy metals. These technologies either focus on physicochemical passivation, or on microbial remediation, or use conventional or modified organic materials, and cannot provide plants with easily absorbed organic nutrients under complex stress conditions to overcome early growth inhibition.

[0005] On the other hand, the technology of preparing small molecule peptides from pig blood through enzymatic hydrolysis has been applied in the fields of feed and health products (e.g., CN105859874A, ZL201010130366.2), and in recent years, its application in the agricultural field has also begun to be explored, for example, as a component of cold-resistant (CN120887758A) or growth-promoting (CN120717842A) polypeptide fertilizers. However, these applications are mainly aimed at general farmland or cash crops, and their formulations and application methods have not been designed and optimized for the special and extreme habitat of acidic, heavily heavy metal-contaminated mine waste sites. Directly using pig blood peptides that have not been specifically optimized and compounded for mine remediation will have limited effects and may not be able to cope with complex soil toxins and abiotic stresses.

[0006] Therefore, there is an urgent need in this field to develop a specialized growth promoter and its application method that can seamlessly integrate with existing "substrate improvement-vegetation planting" technologies and specifically address the problem of early growth retardation in plants under extreme site conditions. This growth promoter should provide easily absorbed organic nutrients while possessing multiple functions such as mitigating acid damage, chelating heavy metals, and improving the rhizosphere microenvironment, thereby accelerating vegetation cover formation, shortening the restoration cycle, and improving the overall benefits and success rate of restoration projects. Summary of the Invention

[0007] To address the shortcomings of existing technologies, the present invention aims to provide a small-molecule peptide and amino acid mixed nutrient solution that can effectively promote vegetation restoration in acidic, heavy metal-contaminated mine wastelands, along with its preparation method, application method, and applications. Compared to existing technologies, the core innovation of this nutrient solution lies in using high-content small-molecule peptides and amino acids prepared from pig blood through a specific enzymatic hydrolysis process as the core organic nitrogen source and active substances, and scientifically combining them with molasses, alginic acid, mineral-derived potassium humate, and EM (Effective Microorganisms) to form a synergistic system. This system not only provides easily absorbed organic nutrients in the critical early stages of plant growth, rapidly promoting biomass accumulation, but also buffers soil pH, chelates and fixes heavy metal ions, and improves the rhizosphere microbial community, thereby significantly enhancing the establishment success rate and growth rate of pioneer plants under extreme adversity.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides a small molecule peptide-amino acid mixed nutrient solution for promoting vegetation restoration in abandoned mining areas, which is prepared by the following method: Enzymatic hydrolysis: Using fresh pig blood as raw material, add 0.2%~0.6% of a complex protease by weight of the pig blood, and hydrolyze for 4-6 hours under the conditions of pH 7.0-7.5 and temperature 50-55℃. The complex protease is composed of papain and alkaline protease in a weight ratio of 6:4. Inactivation and separation: The enzyme hydrolysate is heated to 85-90℃ and maintained for 10-15 minutes to inactivate the enzyme, and then solid-liquid separation is performed to obtain a clear enzyme hydrolysate; Purification: The clarified enzymatic hydrolysate is sequentially passed through ultrafiltration membranes with molecular weight cutoffs of 10 kDa and 1 kDa for fractionation and desalting, and the filtrate with a molecular weight of less than 1 kDa is collected. Concentration: The filtrate obtained in step C is concentrated under vacuum at a temperature below 60°C to obtain a small molecule peptide and amino acid concentrate with a solid content of 30%-40%, wherein the small molecule peptide content is ≥60%, the amino acid content is ≥40%, and the average molecular weight is less than 1000 Da. Compound preparation: Add 5% molasses, 2% alginic acid, 5% potassium humate and 2% EM bacterial agent with an effective live bacteria count of not less than 10 billion CFU / mL to the small molecule peptide amino acid concentrate obtained in step D by weight percentage, mix evenly to obtain the small molecule peptide amino acid mixed nutrient solution.

[0009] This invention provides a method for applying the aforementioned small molecule peptide-amino acid mixed nutrient solution. The key to this method lies in the precise timing and frequency of application. Specifically, the first irrigation is performed 5-7 days after the initial planting of pioneer vegetation in the acidic, high-heavy-metal-contaminated mine wasteland. Subsequent irrigations are repeated at 7-14 day intervals, for a total of 3-6 irrigations. A preferred method is to perform the first irrigation on the 5th day after planting, with 5 irrigations at 10-day intervals. Each application uses 20-30 kg of nutrient solution stock solution per 500 m², diluted 80-120 times with water before application and then evenly applied.

[0010] This invention provides a method for mine ecological restoration, comprising the following steps: S1. To carry out site leveling and basic improvement of abandoned mine sites with acidic and heavy metal pollution (such as adding lime, microbial agents and / or organic improvement substrates). S2. Plant stress-resistant pioneer plants on the site improved in step S1; S3. Using the application method described in the second aspect above, apply the small molecule peptide amino acid mixed nutrient solution described in the first aspect to the area where vegetation planting has been completed.

[0011] This invention provides the application of the aforementioned small molecule peptide-amino acid mixed nutrient solution in the preparation of formulations for promoting vegetation restoration in acidic, heavily heavy metal-contaminated mine wastelands. It is particularly suitable for extremely degraded habitats with initial soil pH ≤ 5.0, electrical conductivity ≥ 1.2 mS / cm, total iron content ≥ 60 mg / kg, total copper content ≥ 35 mg / kg, and total zinc content ≥ 90 mg / kg.

[0012] The beneficial effects of this invention include: Significantly promotes early plant growth and specifically addresses the bottlenecks of extreme site conditions: The nutrient solution provided by this invention is rich in small-molecule peptides, amino acids, and other organic nitrogen sources that are easily absorbed by plants, as well as active substances such as alginic acid and fulvic acid. Compared with existing technologies that mainly rely on inorganic salts or ordinary organic fertilizers, the organic nutrients provided by this invention have small molecular weights and high activity, enabling them to quickly penetrate plant cell membranes and be efficiently utilized even under adverse conditions. By precisely applying the solution at the critical early stage of plant growth (5-7 days after planting), it can effectively alleviate the inhibition of seedling growth caused by combined adverse stresses such as strong acid, high salt, and heavy metal toxicity. Experiments have shown that this method can significantly increase the plant height and biomass of various pioneer plants; for example, the plant height promotion effect on sulfur cosmos can reach 38.62%, thereby rapidly increasing vegetation cover and stabilizing the topsoil.

[0013] Synergistic Effect of Multiple Environmental Remediation Functions: This invention does not replace traditional substrate improvement, but rather serves as a reinforcement and promotion effect after "substrate improvement-vegetation planting." Building upon the initially improved site conditions, it further stimulates the growth potential of plants. The core components, small molecule peptides and amino acids, possess amphoteric properties, effectively buffering soil acidity and alkalinity. Their abundant carboxyl and amino functional groups form stable chelates with heavy metal ions such as Fe³⁺, Cu²⁺, and Zn²⁺ in the soil, reducing their bioavailability and mobility. The compounded mineral-derived potassium humate and alginic acid further enhance chelation, water retention, and soil loosening capabilities, while EM (Effective Microorganisms) helps establish a healthy rhizosphere microbial community, inhibiting pathogens and promoting nutrient cycling. This forms a multi-level, synergistic remediation model of "neutralization and detoxification - structural improvement - nutrient stimulation - microbial regulation," significantly improving overall remediation efficiency and stability.

[0014] The raw materials are environmentally friendly, turning waste into treasure, and offering significant cost advantages: the main raw material, pig blood, comes from slaughter by-products, which are abundant and inexpensive. Through bio-enzymatic hydrolysis technology, it is transformed into high-value-added small-molecule peptide products, achieving resource utilization of waste and aligning with the concept of a green circular economy. Compared to some solutions that use special marine raw materials or complex chemical modification processes, the raw materials of this invention are readily available, the process is relatively simple, and it is easier to scale up production and promote application in large-scale restoration projects.

[0015] Standardized operation and stable, predictable results: This invention clearly defines the complete technical parameters from nutrient solution preparation to field application, particularly the start time, intervals, and cycles of application, establishing a standardized operating procedure. Examples show that application according to the method of this invention not only significantly promotes plant growth but also continuously improves the soil environment (e.g., shifting the soil pH from strongly acidic to a more suitable slightly acidic range for plant growth and reducing the content of bioavailable heavy metals). This predictable and stable effect is beneficial for engineering management and the evaluation of remediation effectiveness. Figure 1 This is a flowchart illustrating the process for preparing small molecule peptide amino acid nutrient solutions. Detailed Implementation

[0016] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with the accompanying drawings and embodiments. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0017] Example 1: Preparation of a mixed nutrient solution of small molecule peptides and amino acids

[0018] Raw material pretreatment: Collect fresh pig blood and immediately mix it with a 0.5% (w / v) sodium citrate anticoagulant solution, stirring evenly to prevent coagulation.

[0019] Enzymatic hydrolysis: Take 1000 kg of crushed pig blood, adjust the pH to 7.2 with NaOH solution, and preheat to 52℃. Add 0.4% of the weight of the pig blood of a complex protease (papain to alkaline protease weight ratio 6:4), and hydrolyze for 5 hours at a constant temperature of 52℃ with continuous stirring. During this period, the pH of the reaction system is maintained at 7.0-7.5 by automatically adding dilute NaOH solution.

[0020] Inactivation and solid-liquid separation: The enzyme hydrolysate was rapidly heated to 88°C and held for 12 minutes to completely inactivate the enzyme. The inactivated solution was then filtered through a plate and frame filter and centrifuged at high speed (8000 rpm, 20 minutes) to obtain a clear enzyme hydrolysate.

[0021] Membrane separation purification: The clarified enzymatic hydrolysate is pumped into an ultrafiltration system. It first passes through an ultrafiltration membrane with a molecular weight cutoff of 10 kDa to remove incompletely hydrolyzed large protein molecules and other impurities. The filtrate then passes through an ultrafiltration membrane with a molecular weight cutoff of 1 kDa, collecting the filtrate with a molecular weight less than 1 kDa. This process removes salts while yielding a component rich in small peptides and amino acids.

[0022] Vacuum concentration: The filtrate was concentrated under vacuum at 55°C until the solid content reached 35%, yielding a concentrated solution of small molecule peptides and amino acids. Analysis showed that the concentrated solution contained 65% small molecule peptides, 35% amino acids, and an average molecular weight of approximately 800 Da.

[0023] Compound finished product: Take 1000 kg of the above concentrated liquid as the base liquid, and add 50 kg of molasses, 20 kg of alginic acid, 50 kg of mineral potassium humate and 20 kg of EM bacteria (effective live bacteria count of 10 billion CFU / mL) in sequence. Stir and mix thoroughly to obtain the finished product of small molecule peptide amino acid mixed nutrient solution.

[0024] Example 2: Application of small molecule peptide-amino acid mixed nutrient solution in the remediation of acidic iron ore spoil heaps

[0025] Site Overview: The test site is located at the Yitunbulak spoil heap in Ruoqiang County. The soil at this site is highly acidic, highly saline, and severely contaminated with heavy metals. Specific physicochemical properties are shown in Tables 1 and 2.

[0026] Table 1. Basic properties of soil at the test site Mean ± Standard Error 3.53 ± 0.50 1.26 ± 0.05 3.69 ± 0.51 17.76 ± 7.32 Table 2 Heavy metal content in soil of the test site average value 107.30 40.85 126.7 18.61 23.81 Repair and Experimental Design: Basic treatment and vegetation restoration: The experimental plot was leveled and loose stones were removed. In-situ substrate improvement was then carried out, with the addition of lime, microbial agents, and organic substrate amendments. After improvement, white clover, sesbania, ryegrass, bermudagrass, bahiagrass, sulfur cosmos, and cosmos seeds were evenly sown, and shrubs such as Amorpha fruticosa were planted.

[0027] Experimental setup: Two treatment zones were set up: a control zone (only basic improvement and vegetation planting as described in step a above) and an experimental treatment zone (after step a, the nutrient solution of this invention was applied). An untreated original site was used as a blank control zone.

[0028] Nutrient solution application: The first application was initiated on the experimental treatment area 5 days after grass seeding. Each time, 25 kg of the nutrient solution stock solution prepared according to the method in Example 1 was taken and diluted in approximately 2.0 m³ of water (approximately 80 times dilution), and then evenly irrigated over a 500 m² area. This process was repeated every 10 days thereafter, for a total of 5 applications.

[0029] Effectiveness evaluation: Effects on plant growth: After application, the average plant height of representative plants in each region was measured. The results are shown in Table 3. The application of small molecule peptide nutrient solution significantly promoted the growth of most pioneer plants, especially sulfur cosmos.

[0030] Table 3 Effects of small molecule peptide nutrient solution on plant height Tian Jing 83.05 ± 20.4 103.1 ± 13.1 +24.14% sulfur cosmos 49.20 ± 10.8 68.20 ± 10.22 +38.62% Cosmos 44.06 ± 8.86 60.48 ± 8.07 +37.26% Amorpha fruticosa 45.52 ± 5.40 57.05 ± 3.85 +25.33% Zinnia 51.80 ± 10.95 62.81 ± 8.69 +21.25% locust 43.25 ± 10.28 51.20 ± 6.09 +18.38% Impact on soil properties: Post-application tests (Table 4) showed that the soil environment in the experimental treatment area was continuously improved. Notably, the soil pH in the experimental treatment area (6.60) was lower than that in the control area (8.19) which only underwent basic soil improvement. This is not a negative effect, but rather indicates that the nutrient solution of this invention has a buffering and regulating effect, avoiding excessive soil alkalization (pH>8) that may result from the use of lime alone, and stabilizing the soil pH in a neutral to slightly acidic range (6.0-7.5) more suitable for the growth of most plants. At the same time, the soil electrical conductivity (EC value) further decreased, and the available content of heavy metals such as total Fe and total Cu in the soil also decreased further compared with the control area, confirming the chelation and fixation effect of the nutrient solution of this invention on heavy metals.

[0031] Table 4. Main physicochemical properties of soil after application of small molecule peptide nutrient solution pH 3.496 ± 0.272 8.190 ± 0.164 6.597 ± 1.237 EC (mS / cm) 3.682 ± 1.560 0.3637 ± 0.117 0.2591 ± 0.079 NAG-pH 2.458 ± 0.326 6.398 ± 0.637 4.399 ± 0.816 <![CDATA[NAG (kg H2SO4 / t)]]> 29.80 ± 16.39 3.273 ± 1.562 12.860 ± 6.037 Zn (mg / kg) 138.6 ± 75.8 118.6 ± 31.7 107.3 ± 63.2 Cu (mg / kg) 50.37± 17.33 32.87± 6.99 23.81± 3.08 Pb (mg / kg) 19.71 ± 8.26 16.27 ± 3.149 13.67± 3.83 Fe (g / kg) 53.25 ± 7.62 29.89 ± 6.63 23.58 ± 5.62 Example 3: Discussion of Application Parameters

[0032] Under extreme site conditions similar to Example 2, the application plan can be flexibly adjusted within the parameter range provided by this invention, depending on the specific circumstances of the initial stage of vegetation restoration. Enhanced growth promotion program: If the seedlings are significantly weak in the early stages, the first application can be brought forward to the 5th day after planting, the irrigation interval can be shortened to 7 days, and the total number of applications can be increased to 6 to provide more intensive and continuous nutrient support.

[0033] Maintain growth promotion program: If the site conditions improve well in the early stage and the plants adapt and grow well, the first application time can be postponed to 7-10 days after planting, the watering interval can be extended to 10-14 days, and the total number of applications can be reduced to 3-4 times, providing assistance during the critical period.

[0034] The optimal selection of specific parameters can be determined by comprehensively considering the soil improvement effect, climatic conditions (such as rainfall), and the actual growth status of plants.

[0035] The above embodiments fully demonstrate that the small molecule peptide amino acid mixed nutrient solution and its matching application method provided by the present invention can effectively promote the early growth of pioneer plants planted on abandoned mine sites with strong acidity and high heavy metal pollution, especially for pioneer plants such as legumes and asteraceae. It provides an efficient, reliable and highly operable solution for accelerating the ecological restoration process of such extremely difficult sites.

Claims

1. A small molecule peptide-amino acid mixed nutrient solution for promoting vegetation restoration in abandoned mining areas, characterized in that, It is prepared by the following method: Enzymatic hydrolysis: Using fresh pig blood as raw material, add 0.2%~0.6% of a complex protease by weight of the pig blood, and hydrolyze for 4-6 hours under the conditions of pH 7.0-7.5 and temperature 50-55℃. The complex protease is composed of papain and alkaline protease in a weight ratio of 6:

4. Inactivation and separation: The enzyme hydrolysate is heated to 85-90℃ and maintained for 10-15 minutes to inactivate the enzyme, and then solid-liquid separation is performed to obtain a clear enzyme hydrolysate; Purification: The clarified enzymatic hydrolysate is sequentially passed through ultrafiltration membranes with molecular weight cutoffs of 10 kDa and 1 kDa for fractionation and desalting, and the filtrate with a molecular weight of less than 1 kDa is collected. Concentration: The filtrate obtained in step C is concentrated under vacuum at a temperature below 60°C to obtain a small molecule peptide and amino acid concentrate with a solid content of 30%-40%, wherein the small molecule peptide content is ≥60%, the amino acid content is ≥40%, and the average molecular weight is less than 1000 Da. Compound preparation: Add 5% molasses, 2% alginic acid, 5% potassium humate and 2% EM bacterial agent with an effective live bacteria count of not less than 10 billion CFU / mL to the small molecule peptide amino acid concentrate obtained in step D by weight percentage, mix evenly to obtain the small molecule peptide amino acid mixed nutrient solution.

2. The small molecule peptide-amino acid mixed nutrient solution according to claim 1, characterized in that, The application method is as follows: after the pioneer vegetation is planted in the abandoned mining area, the first watering is carried out 5 to 7 days later, and then the watering is repeated at intervals of 7 to 14 days, for a total of 3 to 6 waterings.

3. The application method according to claim 2, characterized in that, The first irrigation was carried out on the 5th day after the vegetation was planted, with an interval of 10 days between irrigations, for a total of 5 irrigations.

4. The application method according to claim 2 or 3, characterized in that, The dosage of the small molecule peptide amino acid mixed nutrient solution for each application is 20-30 kg / 500 m². It should be diluted with water 80-120 times before application.

5. The application of the small molecule peptide amino acid mixed nutrient solution according to claim 1 in promoting vegetation restoration in abandoned mine sites with acidic and heavy metal pollution.

6. The application according to claim 5, characterized in that, The initial pH of the soil in the acidic, heavy metal-contaminated mine wasteland is ≤ 5.0, the soil electrical conductivity is ≥ 1.2 mS / cm, and the total iron content is ≥ 60 g / kg.

7. The application according to claim 6, characterized in that, The total copper content in the soil of the abandoned mine site is ≥ 35 mg / kg, and the total zinc content is ≥ 90 mg / kg.

8. The application according to any one of claims 5-7, characterized in that, The vegetation is pioneer plants, including at least one of legumes, cosmos, grasses, shrubs and trees; preferably, the pioneer plants include at least one of sesbania, sulfur cosmos, cosmos, purple locust, zinnia and black locust.

9. A method for ecological restoration of a mine, characterized in that, The steps are performed in the following order: S1. To carry out site leveling and basic improvement of mine waste sites that are acidic and heavily contaminated with heavy metals, wherein the basic improvement includes adding lime, microbial agents and / or organic improvement substrates; S2. Plant pioneer plants on the site improved in step S1; S3. Using the method described in any one of claims 2 to 4, apply the small molecule peptide amino acid mixed nutrient solution of claim 1 to the area where step S2 was completed.

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