Biological polypeptide modified activated carbon soil conditioner and preparation method thereof

Abstract

The invention relates to the technical field of biomass waste reutilization and soil improvement, in particular to a biological polypeptide modified activated carbon soil conditioner and a preparation method thereof. The fertilizer comprises the following components in parts by weight: 5-7 parts of low-value animal waste protein, 5-7 parts of activated carbon, 6-8 parts of a landscaping substrate, 4-8 parts of vermiculite, 2-4 parts of a compound microbial agent and 4-8 parts of chitosan, low-value animal waste protein is prepared into functional polypeptide through a biotransformation technology, the functional polypeptide has the functions of adsorbing cations and chelating elements, then the functional polypeptide is used for modifying activated carbon, the functional polypeptide is organically mixed with other components according to a certain proportion to prepare the soil conditioner, and the functional polypeptide can be used as a fertilizer synergist in garden soil conservation and has a good application prospect. When the soil conditioner is used for treating the soil, nitrogen, phosphorus, potassium and trace elements in the soil can be enriched, the effectiveness of nutrient elements in the soil is improved, and the fixation of cadmium is reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of the reuse of biomass waste and soil improvement, and specifically relates to a bio-polypeptide modified activated carbon soil conditioner and a preparation method thereof. Background Art

[0002] Polypeptides can be used for plant protection as antibacterial and immune inducers, plant growth regulators, insecticides, herbicides, etc. Bio-polypeptides can increase the resistance of plants to drought and high temperature, and can improve the photosynthesis and growth rate of plants. In rice cultivation, the use of bio-polypeptides can increase rice yield, reduce soil salinization, etc., and make the plants stronger through promoting root growth and development, etc. Another study shows that the use of bio-polypeptides can significantly improve the resistance of crops to disasters such as drought, wind, and waterlogging, and shorten the crop growth cycle. In strawberry cultivation, bio-polypeptide fertilizers can significantly increase the yield and quality, increasing the fruit yield by 6.5% and the soluble sugar content by 7.1%. In vegetable cultivation, the use of bio-polypeptide fertilizers can increase the yield and quality, increasing the total yield by 2.8% and the VC content by 12.6%.

[0003] Activated carbon is a carbon-negative material, which can achieve soil carbon sequestration by improving the microbial carbon utilization efficiency and enhancing physical and chemical protection; in addition, activated carbon can reduce the emission of soil N2O by affecting the microbial community structure and nitrogen-related functional genes; it can also increase the soil pH value, conductivity and cation exchange capacity, and affect soil enzyme activity and bacterial community, etc. When combined with bio-polypeptides, it can efficiently adsorb pollutants, regulate the soil micro-ecology and promote crop growth, and is suitable for the remediation of heavy metal pollution, salinization and barren soil.

[0004] Traditional soil conditioners often use materials such as biochar, clay or microbial agents, but there are the following problems:

[0005] 1. Limited adsorption efficiency: Ordinary activated carbon lacks selectivity for the adsorption of heavy metals, and is easily saturated, relying only on physical adsorption, lacking the ability to promote soil nutrient cycling and microbial activity.

[0006] 2. Single function: Most conditioners only target a single problem (such as heavy metal pollution or water retention), lacking multi-functional synergistic effects. After adsorbing pollutants, they cannot be degraded or transformed, and are prone to secondary release.

[0007] 3. Insufficient stability: Microbial agents are easily inactivated in the soil, and the slow-release effect is poor.

[0008] 4. Poor synergy with plants: They do not integrate plant growth regulating components and are difficult to improve the stress resistance of crops.

[0009] In order to solve the above problems, the present invention is determined to provide a multifunctional soil conditioner that can efficiently adsorb heavy metals, enhance soil water retention, and promote microbial activity. Summary of the Invention

[0010] In order to solve the above problems, the present invention provides a bio-peptide modified activated carbon soil conditioner, which comprises, by weight: 5-7 parts of low-value animal waste protein, 5-7 parts of activated carbon, 6-8 parts of landscaping matrix, 4-8 parts of vermiculite, 2-4 parts of composite microbial agent, and 4-8 parts of chitosan; the activated carbon is a waste material from cystine production;

[0011] The low-value animal waste protein is one of blood, feathers, offal, chicken or duck meat;

[0012] The garden greening matrix is prepared by co-fermentation of garden waste and organic solid waste, wherein the organic solid waste is selected from any one or more of kitchen waste, livestock manure, riverbed sludge, and municipal sludge; and the garden waste is selected from any one or more of fallen branches and leaves, pruned branches and leaves, dead branches, and weeds. The co-fermentation process comprises: naturally drying the garden waste and the organic solid waste, crushing them to a diameter of 40-60 mm, and mixing them in a mass ratio of 0.8-1.2:1; then adjusting the fermentation temperature to 20-50° C., stirring once every 24-30 hours, and fermenting for 7-10 days. When the moisture content of the material drops to ≤30%, the garden greening matrix is obtained.

[0013] The composite microbial agent is obtained by mixing Bacillus lysinicola and Pseudomonas putida in a weight ratio of 1:2-3;

[0014] Description: Vermiculite's granular structure prevents soil hardening and promotes downward root extension, making it particularly suitable for seedlings with fragile root systems. It also improves soil drainage, preventing root rot caused by waterlogging. Chitosan can induce plants to produce disease-resistant substances such as chitinase, inhibiting fungal infections such as gray mold and powdery mildew, as well as bacterial infections. Its film-forming properties also physically block pests from feeding.

[0015] Furthermore, the low-value animal waste protein is pretreated by:

[0016] (1) adding a composite alkaline protease to low-value animal waste protein at a mass ratio of 100:1-2, hydrolyzing the protein at 45-55° C. and a pH of 7.8-8.2 for 3.5-4.5 hours to obtain a protein hydrolyzate, adding Fe 3 O 4 nanoparticles to the protein hydrolyzate at a mass ratio of 1:0.5-0.7 and mixing the mixture to obtain a first polypeptide solution;

[0017] (2) Add a 0.5 - 1.2% MnCl₂ solution to the first polypeptide solution according to a mass ratio of 100:8 - 12, and stir and react at 50 - 60 °C for 1.5 - 2.5 h to obtain a second polypeptide solution;

[0018] (3) Add sodium alginate accounting for 1.5 - 2.5 wt% of the second polypeptide solution to the second polypeptide solution and stir for 8 - 12 min, then add chitosan accounting for 0.5 - 1.5 wt% of the second polypeptide solution, adjust the pH to 5.5 - 6.0, mix well, filter, concentrate under reduced pressure at 50 - 55 °C until the volume is reduced to 1 / 3 of the original solution, dehydrate under vacuum at 60 °C until the water content ≤ 15%, then cool to 55 °C and dry under vacuum until the water content is 8%, and finally perform directional magnetization treatment at a magnetic field strength of 0.55 - 0.65 T for 25 - 35 min;

[0019] Note: Treatment with compound alkaline protease for 4 h can increase the yield of small peptide segments (<3 kDa) from 18% by the boiling method to 65%, and tryptophan is retained intact. The addition of Fe₃O₄ nanoparticles can provide magnetic carriers and metal binding sites, increasing the heavy metal adsorption capacity by 40%; treatment with MnCl₂ solution can form Mn - polypeptide coordination bonds, stabilize the α - helix structure, and increase the thermal denaturation temperature by 15 °C; within the pH range of 5.5 - 6.0, peptide bond cleavage under acidic conditions (the hydrolysis rate increases by 30% when pH < 5) or deamidation reaction under alkaline conditions (the loss of glutamine reaches 40% when pH > 7) can be prevented. Mn 2+ has the strongest binding ability to the histidine residues of polypeptides in a weakly acidic environment, and the chelating capacity increases by 25%. Chitosan has a high degree of protonation when pH < 6.5, and the electrostatic interaction with sodium alginate is more sufficient. When the concentration of MnCl₂ solution exceeds 1.5%, excessive cross - linking of polypeptides will occur, resulting in a decrease in biological effectiveness.

[0020] Further, the preparation method of the compound alkaline protease is as follows:

[0021] Transfer the Bacillus subtilis strain to a Czapek slant medium, culture it at 37 - 40 °C and pH 7.0 for 12 - 14 h, then add Tris - HCl to adjust the pH to 8.5, continue to culture for 38 - 42 h, then place the cultured strain spores 30 - 35 cm away from the ultraviolet lamp and irradiate them under the condition of 28 - 32 W for 15 - 30 s. Then put the strain spores into a conical flask containing nutrient solution at a concentration of 0.005 - 0.008 g / L, and oscillate at a temperature of 39 - 41 °C and an oscillation speed of 120 - 140 r / min for 15 - 20 h, and dilute it to a concentration of 10 7 -10 9A bacterial suspension of [[ID=]], synchronously adding a catalyst accounting for 0.3 wt% of the bacterial suspension, and continuing to oscillate at 39 °C and 150 rpm for 6 - 8 h is sufficient;

[0022] The nutrient solution is prepared by mixing glucose, ammonium sulfate, calcium chloride, potassium dihydrogen phosphate, sodium chloride, zinc sulfate, and water according to a mass ratio of 12 - 15:0.1 - 0.2:0.002 - 0.004:0.3 - 0.5:0.06 - 0.08:0.005 - 0.008:1000.

[0023] Note: The composite protease (subtilisin, metalloprotease) secreted by Bacillus subtilis acts synergistically to efficiently break the hydrophobic bonds and peptide bonds in animal proteins, releasing active polypeptides containing carboxyl / amino groups. These polypeptides are more likely to adsorb on the surface of activated carbon through electrostatic interactions or hydrogen bonds; ultraviolet mutagenesis can enhance enzyme diversity, and the polypeptide chain lengths generated during the hydrolysis of animal proteins are more widely distributed, covering the modification requirements of different pore sizes of activated carbon; the optimized nutrient solution can avoid carbon catabolite repression, promote protease secretion, and increase the proportion of polypeptides <1 kDa in the hydrolysis product by 20% - 30%. These polypeptides are more likely to penetrate the micropores of activated carbon and enhance the modification depth; the activated carbon loaded with polypeptides after modification can promote the proliferation of soil microorganisms, accelerate the decomposition of organic matter, and improve the improvement effect; the organic acids in the fermentation products can neutralize alkaline soil, reducing the conductivity by 0.5 - 1.2 mS / cm. The sulfhydryl and carboxyl groups in the polypeptides can chelate Cd 2 + / Pb 2 +, increasing the passivation efficiency by 25% - 35%, thereby enhancing the performance of the soil conditioner.

[0024] Furthermore, the catalyst is composed of dopamine hydrochloride and phosphate buffer solution according to a weight ratio of 1:0.4 - 0.6;

[0025] Note: Adding dopamine hydrochloride as a catalyst can form a polydopamine coating, enabling the enzyme loading to reach 28 mg / g, and the magnetic responsiveness is close to that of the enzyme modified by the TA / PEI system. The thermal stability of the enzyme is improved: after mixing PBS and dopamine hydrochloride, the pH of the reaction system can be stabilized at 7.8 - 8.2, which not only avoids the oxidation and inactivation of dopamine under strong acidic conditions but also fits the optimal pH of Bacillus subtilis protease, which is 8.0 - 9.0; this pH range reduces the risk of autolysis of alkaline protease and extends its half-life by about 30%.

[0026] The present invention also provides a preparation method of a bio-polypeptide modified activated carbon soil conditioner, including the following steps:

[0027] S1. Crush and grind the low-value animal waste protein into mud, stir and emulsify it for 3 - 3.5 h under the conditions of a solid content of 10%, a temperature of 33 - 37 °C, and a rotation speed of 120 r / min, then sterilize it at 95 - 105 °C for 0.5 - 1 h, carry out biological fermentation and then ripen it in a storage tank for 4 - 5 days, remove impurities such as large-particle solids, suspended substances, and grease in the emulsion by sand filtration, and then add organic acid to adjust the pH value of the emulsion to 5.5 - 6.5, and fill it to obtain the biological polypeptide nutrient;

[0028] S2. At 55 - 65 °C, immerse the activated carbon in the biological polypeptide nutrient prepared in S1 for 3.5 - 4.5 h, add landscaping substrate, vermiculite, compound microbial inoculant, and chitosan to mix and granulate, and the particle size is 5 - 7 mm;

[0029] S3. Dry it in a forced-air drying oven at 55 - 65 °C for 22 - 26 h to obtain the biological polypeptide-modified activated carbon soil conditioner, and apply it to plant maintenance;

[0030] Note: The biological polypeptide-modified activated carbon soil conditioner obtained by the above method of the present invention has a 30 - 50% increase in the adsorption efficiency of mercury and cadmium, a 20% increase in soil water holding capacity, a microbial inoculant survival rate > 80% (stored at room temperature for 2 years), and can significantly reduce soil-borne diseases.

[0031] Further, in S2, the activated carbon is pretreated, and the pretreatment method is: place the activated carbon in 2.8 - 3.2 mol / L HNO3 according to a ratio of 10 g:20 mL, and carry out ultrasonic treatment for 25 - 35 min under the conditions of a power of 290 - 310 W and a temperature of 38 - 42 °C, then wash it with water until the activated carbon is neutral and then dry it, and finally grind it to 80 - 100 mesh;

[0032] Note: Under the action of ultrasonic waves, HNO3 can oxidize the surface of the activated carbon to generate oxygen-containing groups such as carboxyl and hydroxyl groups, significantly enhance the binding strength between the activated carbon and polypeptides or metal oxides, and improve its adsorption capacity for heavy metal ions and organic pollutants. The oxidation treatment enhances the hydrophilicity of the activated carbon surface, and the water contact angle is reduced by 50 - 60%, which is more conducive to the uniformity of subsequent polypeptide loading.

[0033] Further, during the ripening process in the storage tank and the crushing and grinding process, nitrogen gas atmosphere is introduced synchronously at a rate of 0.5 - 0.7 m 3 / h to protect, and the system pressure is maintained at 0.05 - 0.15 MPa;

[0034] Note: Nitrogen protection during the ripening process in the storage tank can avoid the loss of sulfhydryl groups caused by oxidation. Experiments show that this treatment can make the retention rate of active sulfhydryl groups reach 91%; nitrogen filling is required during crushing to avoid the increase in free radical content.

[0035] Further, the method of biological fermentation is as follows: inoculate yeast into the emulsified and sterilized raw materials at a mass percentage of 3-5%, then under the conditions of a temperature of 37-38°C and a relative humidity of 75-80%, pulse aerate until the dissolved oxygen content is 10-15%, ferment for 48-54 h, and terminate when the pH ≤ 4.2. Add 2% glucose, 1% yeast extract powder, and 0.1% MnCl2 every 12 h, and inactivate when the viable count of yeast drops to 25% of the initial amount;

[0036] Note: Microorganisms grow and reproduce in the raw materials, decompose organic substances such as proteins, and produce bio-polypeptides; the fermentation temperature of 37-38°C promotes yeast to secrete more exoproteases, increasing the degree of protein hydrolysis to 35-40%, generating more small-molecule active peptides. The MnCl2 added every 12 h serves as a cofactor, which can activate the manganese superoxide dismutase of yeast, reduce oxidative damage, and retain the thiol active sites of polypeptides; the glutathione analog in the fermented polypeptide can form a stable complex with Cd 2 +, reducing the available Cd by 60-70%. The addition of MnCl2 promotes the formation of manganese-containing peptide complexes, and the ion exchange efficiency with Cd 2 + is increased by 30%; Pulse aeration can avoid excessive oxidation and damage to the peptide chain structure; when the viable count is 25%, it indicates that the nutrients are exhausted and metabolites accumulate. Inactivation at this time can avoid the autolysis of bacteria and the release of proteases to destroy the structure of the generated polypeptides, preventing further pH decline caused by over-fermentation.

[0037] Compared with the existing technologies, the beneficial effects of the present invention are as follows:

[0038] (1) Low-value animal waste proteins are rich in proteins, but traditional treatment methods are prone to cause waste of resources and environmental pollution. The present invention prepares them into functional polypeptides through biological transformation technology, which has the functions of adsorbing cations and chelating elements. It can be used as a fertilizer synergist in garden soil maintenance, thereby improving fertilizer utilization efficiency. Treating the soil with it can enrich nitrogen, phosphorus, potassium, and trace elements in the soil, improve the effectiveness of soil nutrient elements, and reduce the fixation of phosphorus; using waste activated carbon (such as cystine production waste) as a carrier can reduce costs. The polypeptide can chelate heavy metals and promote the microbial degradation of organic pollutants, increasing the PAHs degradation rate by 40%.

[0039] (2) The present invention enzymatically hydrolyzes low-value animal waste proteins - Mn 2+The crosslinking-magnetization embedding synergy can efficiently decompose collagen into small-molecule polypeptides by using compound alkaline protease, improving its water solubility and plant absorption rate; the MnCl2 solution promotes the formation of Mn-oxygen coordination bonds between polypeptide chains, enhancing the structural stability and extending the slow-release time of polypeptides in the soil by 2-3 times; sodium alginate and chitosan form polyelectrolyte microcapsules through electrostatic interaction, with an encapsulation rate > 90%, protecting polypeptides from premature degradation by soil microorganisms and intelligently releasing polypeptides in response to acidic root exudates in the soil; the magnetization treatment aligns the Fe3O4-polypeptide complex along the magnetic field lines, forming a porous structure and further increasing the soil air permeability and water retention capacity.

[0040] (3) The compound alkaline protease of the present invention uses Bacillus subtilis as a raw material, and utilizes the synergistic action of subtilisin and metalloprotease secreted by itself to efficiently break the hydrophobic bonds and peptide bonds in animal proteins, releasing active polypeptides containing carboxyl / amino groups, so that these polypeptides are adsorbed on the surface of activated carbon through electrostatic interaction or hydrogen bonds. Compared with boiling hydrolysis, the secondary structure of the denatured protein can be exposed to active groups, and the yield of small peptide segments can be increased from 18% to 65%, and tryptophan is retained intact. Description of the Drawings

[0041] Figure 1 It is a comparison chart of the reduction ratio of available Cd in the soil after adding the bio-polypeptide modified activated carbon soil conditioner obtained in Examples 1-19 and Control Groups 1-4 of the present invention;

[0042] Figure 2 It is a comparison chart of the soil conductivity after adding the bio-polypeptide modified activated carbon soil conditioner obtained in Examples 1-19 and Control Groups 1-4 of the present invention;

[0043] Figure 3 It is a comparison chart of the yield of pakchoi after adding the bio-polypeptide modified activated carbon soil conditioner obtained in Examples 1-19 and Control Groups 1-4 of the present invention;

[0044] Figure 4 It is a comparison chart of the reduction ratio of available Cd in the soil after adding the bio-polypeptide modified activated carbon soil conditioner obtained in Examples 1, 20-32 and Control Groups 5-6 of the present invention;

[0045] Figure 5 It is a comparison chart of the soil conductivity after adding the bio-polypeptide modified activated carbon soil conditioner obtained in Examples 1, 20-32 and Control Groups 5-6 of the present invention;

[0046] Figure 6It is a comparison chart of the yields of pakchoi after adding the bio-polypeptide modified activated carbon soil conditioner obtained in Example 1, Examples 20 - 32 and Control Groups 5 - 6 of the present invention. Detailed implementation mode

[0047] To further elaborate on the methods adopted and the effects achieved by the present invention, the technical solutions of the present invention will be clearly and completely described below in combination with experiments.

[0048] Example 1: A bio-polypeptide modified activated carbon soil conditioner, by weight, includes: 6 parts of low-value animal waste protein, 6 parts of activated carbon, 7 parts of landscaping substrate, 6 parts of vermiculite, 3 parts of compound microbial inoculum, and 6 parts of chitosan;

[0049] The low-value animal waste protein is feathers;

[0050] The landscaping substrate is prepared by co-fermentation technology using garden waste and organic solid waste. Among them, the organic solid waste is selected from kitchen waste; the garden waste is selected from fallen leaves and branches. The process of the co-fermentation technology is: naturally air-dry and crush the garden waste and organic solid waste to a diameter of 50 mm, and mix them in a mass ratio of 1:1; then adjust the fermentation temperature to 35 °C, stir once every 27 h, ferment for 8.5 days, and when the moisture content of the material drops to 30%, the landscaping substrate is obtained;

[0051] The compound microbial inoculum is composed of: Pseudomonas putida mixed in a weight ratio of 1:2.5;

[0052] Based on the above preparation method of a bio-polypeptide modified activated carbon soil conditioner, it includes the following steps:

[0053] S1. Crush and grind the low-value animal waste protein into mud, stir and emulsify it for 3 h under the conditions of a solid content of 10%, a temperature of 33 - 37 °C, and a rotation speed of 120 r / min, then sterilize it at 100 °C for 0.8 h, carry out biological fermentation and then ripen it in a storage tank for 4.5 days. Use sand filtration to remove large-particle solids, suspended substances, oils and fats and other impurities in the emulsion, and then add organic acid to adjust the pH value of the emulsion to 6, and fill it to obtain bio-polypeptide nutrient;

[0054] The biological fermentation method comprises the following steps: inoculating yeast into emulsified and sterilized raw materials at a mass ratio of 4%, then subjecting the raw materials to pulse aeration until the dissolved oxygen content reaches 13% at a temperature of 37.5° C. and a relative humidity of 78%, and fermenting for 51 hours, terminating when the pH reaches 4.2. Supplementing the fermentation with 2% glucose, 1% yeast extract powder, and 0.1% MnCl2 every 12 hours, the yeast is inactivated when the number of viable cells decreases to 25% of the initial amount; and controlling the ventilation pipe to emit air pulses, wherein the air pulses have a duration of 1.2 minutes, an interval of 11 minutes, and an emission speed of 200 cm / s.

[0055] During the storage tank ripening process and the crushing and grinding process, the 3 A nitrogen atmosphere was introduced at a rate of / h to maintain the system pressure at 0.1MPa;

[0056] S2. At 60°C, the activated carbon was immersed in the biological polypeptide nutrient prepared in S1 for 4 hours, and then the landscaping matrix, vermiculite, composite microbial agent and chitosan were added to form granules, and the granule size was 6 mm.

[0057] S3, drying in a forced air drying oven at 60°C for 24 hours to obtain a bio-peptide modified activated carbon soil conditioner, which was then applied to plant maintenance;

[0058] In S2, the activated carbon was pretreated by placing the activated carbon in 3.0 mol / L HNO3 at a ratio of 10 g:20 mL, and ultrasonically treating it at a power of 300 W and a temperature of 40°C for 30 min. The activated carbon was then washed with water until it was neutral and then dried, and finally ground into 80-100 mesh.

[0059] Example 2: Different from Example 1, a bio-peptide modified activated carbon soil conditioner includes, by weight, 5 parts of low-value animal waste protein, 5 parts of activated carbon, 6 parts of landscaping matrix, 4 parts of vermiculite, 2 parts of composite microbial agent, and 4 parts of chitosan.

[0060] Example 3: Different from Example 1, a bio-peptide modified activated carbon soil conditioner includes, by weight, 7 parts of low-value animal waste protein, 7 parts of activated carbon, 8 parts of landscaping matrix, 8 parts of vermiculite, 4 parts of composite microbial agents, and 8 parts of chitosan.

[0061] Example 4: Different from Example 1, the process of the co-fermentation technology is as follows: the garden garbage and organic solid waste are naturally dried and crushed to a diameter of 40 mm, and then mixed in a mass ratio of 4:5; then the fermentation temperature is adjusted to 20°C, stirred once every 24 hours, and fermented for 7 days. When the moisture content of the material drops to 30%, the garden greening matrix is obtained.

[0062] Example 5: Different from Example 1, the process of the co-fermentation technology is as follows: The garden waste and organic solid waste are naturally dried and then crushed to a diameter of 60 mm, and then mixed according to a mass ratio of 6:5; then the fermentation temperature is adjusted to 50 °C, stirred every 30 h, and fermented for 10 days. When the moisture content of the material drops to 30%, the landscaping substrate is obtained.

[0063] Example 6: Different from Example 1, the composite microbial inoculant is obtained by mixing Lysinibacillus sphaericus and Pseudomonas putida in a weight ratio of 1:2.

[0064] Example 7: Different from Example 1, the composite microbial inoculant is obtained by mixing Lysinibacillus sphaericus and Pseudomonas putida in a weight ratio of 1:3.

[0065] Example 8: Different from Example 1, in S1, the low-value animal waste protein is crushed and ground into mud, stirred and emulsified at a solid content of 10%, a temperature of 33 °C, and a rotation speed of 120 r / min for 3 h, then sterilized at 95 °C for 0.5 h, post-ripened in a storage tank for 4 days after biological fermentation, the large-particle solid particles, suspended solids, and grease in the emulsion are removed by sand filtration, and then the pH value of the emulsion is adjusted to 5.5 with an organic acid, and filled to obtain the biological polypeptide nutrient.

[0066] Example 9: Different from Example 1, in S1, the low-value animal waste protein is crushed and ground into mud, stirred and emulsified at a solid content of 10%, a temperature of 37 °C, and a rotation speed of 120 r / min for 3.5 h, then sterilized at 105 °C for 1 h, post-ripened in a storage tank for 5 days after biological fermentation, the large-particle solid particles, suspended solids, and grease in the emulsion are removed by sand filtration, and then the pH value of the emulsion is adjusted to 6.5 with an organic acid, and filled to obtain the biological polypeptide nutrient.

[0067] Example 10: Different from Example 1, in S1, yeast is inoculated into the emulsified and sterilized raw material at a mass percentage of 3%, and then under the conditions of a temperature of 37 °C and a relative humidity of 75%, pulsed aeration is carried out until the dissolved oxygen content is 10%, and fermentation is carried out for 48 h and terminated when the pH is 4.2.

[0068] Example 11: Different from Example 1, in S1, yeast is inoculated into the emulsified and sterilized raw material at a mass percentage of 5%, and then under the conditions of a temperature of 38 °C and a relative humidity of 80%, pulsed aeration is carried out until the dissolved oxygen content is 15%, and fermentation is carried out for 54 h and terminated when the pH is 4.2.

[0069] Example 12: Different from Example 1, in S1, the duration of the air pulse is 1 min, the intermittent time is 10 min, and the emission speed of the air pulse is 100 cm / s.

[0070] Example 13: Different from Example 1, in S1, the duration of the air pulse is 1.5 minutes, the intermittent time is 13 minutes, and the emission speed of the air pulse is 300 cm / s.

[0071] Example 14: The difference from Example 1 is that in S1, the storage tank post-ripening process and the crushing and grinding process are both synchronized according to 0.5m 3 A nitrogen atmosphere was introduced at a rate of / h to maintain the system pressure at 0.05MPa.

[0072] Example 15: The difference from Example 1 is that in S1, the storage tank post-ripening process and the crushing and grinding process are both synchronized according to 0.7m 3 Nitrogen atmosphere was introduced at a rate of / h to maintain the system pressure at 0.15MPa.

[0073] Example 16: The difference from Example 1 is that, in S2, activated carbon is immersed in the biological polypeptide nutrient prepared in S1 at 55°C for 3.5 hours, and then a landscaping matrix, vermiculite, a composite microbial agent, and chitosan are added to form a mixed granule with a particle size of 5 mm;

[0074] S3. After drying in a forced air drying oven at 55° C. for 22 h, a bio-peptide-modified activated carbon soil conditioner was prepared and applied to plant maintenance.

[0075] Example 17: The difference from Example 1 is that, in S2, activated carbon is immersed in the biological polypeptide nutrient prepared in S1 at 65°C for 4.5 hours, and then a landscaping matrix, vermiculite, a composite microbial agent, and chitosan are added to the mixture and granulated to a particle size of 7 mm;

[0076] S3. After drying in a forced air drying oven at 65° C. for 26 h, a bio-peptide-modified activated carbon soil conditioner was prepared and applied to plant maintenance.

[0077] Example 18: Different from Example 1, the activated carbon was pretreated. The pretreatment method was as follows: the activated carbon was placed in 2.8 mol / L HNO3 at a ratio of 10 g:20 mL, and ultrasonically treated for 25 min at a power of 290 W and a temperature of 38°C. The activated carbon was then washed with water until it was neutral and then dried. Finally, it was ground into 80-100 mesh.

[0078] Example 19: Different from Example 1, the activated carbon was pretreated. The pretreatment method was as follows: the activated carbon was placed in 3.2 mol / L HNO3 at a ratio of 10 g:20 mL, and ultrasonically treated for 35 min at a power of 310 W and a temperature of 42°C. The activated carbon was then washed with water until it was neutral and then dried. Finally, it was ground into 80-100 mesh.

[0079] Example 20: Different from Example 1, the low-value animal waste protein is pretreated, and the pretreatment method is as follows:

[0080] (1) Add compound alkaline protease to the low-value animal waste protein according to a mass ratio of 100:1.5, and carry out hydrolysis treatment at 50 °C and pH 8.0 for 4 h to obtain a protein hydrolysate. Add Fe3O4 nanoparticles to the protein hydrolysate according to a mass ratio of 1:0.6 and mix well to obtain a first polypeptide solution;

[0081] (2) Add 0.8% MnCl2 solution to the first polypeptide solution according to a mass ratio of 100:10, and stir and react at 55 °C for 2 h to obtain a second polypeptide solution;

[0082] (3) Add sodium alginate accounting for 2.0 wt% of the second polypeptide solution to the second polypeptide solution and stir for 10 min, then add chitosan accounting for 1.0 wt% of the second polypeptide solution, adjust the pH to 5.8, mix well, filter, evaporate and concentrate under reduced pressure at 53 °C until the volume is reduced to 1 / 3 of the original solution, dehydrate under vacuum at 60 °C until the water content is 15%, then cool to 55 °C and dry under vacuum until the water content is 8%, and finally carry out directional magnetization treatment at a magnetic field strength of 0.6 T for 30 min;

[0083] The preparation method of the compound alkaline protease is as follows:

[0084] Transfer the Bacillus subtilis strain to a Czapek slant medium, culture it at 38 °C and pH 7.0 for 13 h, then add Tris-HCl to adjust the pH to 8.5 and continue to culture for 40 h. Then place the cultured strain spores 33 cm away from the ultraviolet lamp and irradiate them under the condition of 30 W for 22 s. Then put the strain spores into a conical flask containing nutrient solution according to a concentration of 0.006 g / L, and oscillate at a temperature of 40 °C and an oscillation speed of 130 r / min for 18 h, and dilute it into a bacterial suspension with a concentration of 10 8 cells / mL. Synchronously add a catalyst accounting for 0.3 wt% of the bacterial suspension. The catalyst is composed of dopamine hydrochloride and phosphate buffer in a weight ratio of 1:0.5, and continue to oscillate at 39 °C and 150 rpm for 6 - 8 h;

[0085] The nutrient solution is prepared by mixing glucose, ammonium sulfate, calcium chloride, potassium dihydrogen phosphate, sodium chloride, zinc sulfate, and water according to a mass ratio of 14:0.15:0.003:0.4:0.07:0.007:1000.

[0086] Example 21: Different from Example 20, in (1), compound alkaline protease was added to low-value animal waste protein at a mass ratio of 100:1, and hydrolysis treatment was carried out at 45 °C and pH 7.8 for 3.5 h to obtain a protein hydrolysate. Fe3O4 nanoparticles were added to the protein hydrolysate at a mass ratio of 1:0.5 and mixed evenly to obtain a first polypeptide solution.

[0087] Example 22: Different from Example 20, in (1), compound alkaline protease was added to low-value animal waste protein at a mass ratio of 50:1, and hydrolysis treatment was carried out at 55 °C and pH 8.2 for 4.5 h to obtain a protein hydrolysate. Fe3O4 nanoparticles were added to the protein hydrolysate at a mass ratio of 1:0.7 and mixed evenly to obtain a first polypeptide solution.

[0088] Example 23: Different from Example 20, in (2), 0.5% MnCl2 solution was added to the first polypeptide solution at a mass ratio of 25:2, and stirring reaction was carried out at 50 °C for 1.5 h to obtain a second polypeptide solution.

[0089] Example 24: Different from Example 20, in (2), 1.2% MnCl2 solution was added to the first polypeptide solution at a mass ratio of 25:3, and stirring reaction was carried out at 60 °C for 2.5 h to obtain a second polypeptide solution.

[0090] Example 25: Different from Example 20, in (3), sodium alginate accounting for 1.5 wt% of the second polypeptide solution was added to the second polypeptide solution and stirred for 8 min, then chitosan accounting for 0.5 wt% of the second polypeptide solution was added, and the pH was adjusted to 5.5, followed by mixing treatment, filtration, concentration under reduced pressure at 50 °C until the volume was reduced to 1 / 3 of the original solution, vacuum dehydration at 60 °C until the water content was 15%, then cooling to 55 °C and vacuum drying until the water content was 8%, and finally directional magnetization treatment was carried out at a magnetic field strength of 0.55 T for 25 min.

[0091] Example 26: Different from Example 20, in (3), sodium alginate accounting for 2.5 wt% of the second polypeptide solution was added to the second polypeptide solution and stirred for 12 min, then chitosan accounting for 1.5 wt% of the second polypeptide solution was added, and the pH was adjusted to 6.0, followed by mixing treatment, filtration, concentration under reduced pressure at 55 °C until the volume was reduced to 1 / 3 of the original solution, vacuum dehydration at 60 °C until the water content was 15%, then cooling to 55 °C and vacuum drying until the water content was 8%, and finally directional magnetization treatment was carried out at a magnetic field strength of 0.65 T for 35 min.

[0092] Example 27: Different from Example 20, it was cultured for 12 h at 37 °C and pH 7.0, then Tris-HCl was added to adjust the pH to 8.5, and the culture was continued for 38 h. Then the cultured strain spores were placed 30 cm away from the ultraviolet lamp and irradiated for 15 s under the condition of 28 W. Then the strain spores were put into a conical flask containing nutrient solution at a concentration of 0.005 g / L and shaken for 15 h at a temperature of 39 °C and an oscillation speed of 120 r / min, and diluted to a concentration of 10 7 cells / mL of bacterial suspension.

[0093] Example 28: Different from Example 20, it was cultured for 14 h at 40 °C and pH 7.0, then Tris-HCl was added to adjust the pH to 8.5, and the culture was continued for 42 h. Then the cultured strain spores were placed 35 cm away from the ultraviolet lamp and irradiated for 30 s under the condition of 32 W. Then the strain spores were put into a conical flask containing nutrient solution at a concentration of 0.008 g / L and shaken for 20 h at a temperature of 41 °C and an oscillation speed of 140 r / min, and diluted to a concentration of 10 9 cells / mL of bacterial suspension.

[0094] Example 29: Different from Example 20, the catalyst is composed of dopamine hydrochloride and phosphate buffer solution in a weight ratio of 1:0.4.

[0095] Example 30: Different from Example 20, the catalyst is composed of dopamine hydrochloride and phosphate buffer solution in a weight ratio of 1:0.6.

[0096] Example 31: Different from Example 20, the nutrient solution is prepared by mixing glucose, ammonium sulfate, calcium chloride, potassium dihydrogen phosphate, sodium chloride, zinc sulfate, and water in a mass ratio of 12:0.1:0.002:0.3:0.06:0.005:1000.

[0097] Example 32: Different from Example 20, the nutrient solution is prepared by mixing glucose, ammonium sulfate, calcium chloride, potassium dihydrogen phosphate, sodium chloride, zinc sulfate, and water in a mass ratio of 15:0.2:0.004:0.5:0.08:0.008:1000.

[0098] Experimental Example: The description basis of this experimental example is the recording scheme in Example 1, aiming to clarify the actual application effect of the present invention. Application Test: A modifier (1.5 wt%) was applied to Cd-contaminated soil (Cd content 2.5 mg / kg), and after 60 days of planting pakchoi, the effect of the bio-polypeptide modified activated carbon soil modifier in Examples 1 - 32 and Control Groups 1 - was detected on the contaminated soil. Among them, the available Cd in the soil of Example 1 decreased by 72.3% (38.5% in Control Group 1); the yield of pakchoi increased by about 14%, and the Cd content in the leaves was lower than the national standard limit (0.2 mg / kg); the soil electrical conductivity (EC) decreased from 4.8 dS / m in Control Group 1 to 2.1 dS / m.

[0099] 1. Explore the influence of the composition and preparation method of the bio-polypeptide modified activated carbon soil modifier on its performance

[0100] Control Group 1: Different from Example 1, the addition of low-value animal waste protein was missing in the soil modifier.

[0101] Control Group 2: Different from Example 1, the addition of Lysinibacillus was missing in the compound microbial inoculum.

[0102] Control Group 3: Different from Example 1, there was no aeration treatment.

[0103] Control Group 4: Different from Example 1, the activated carbon was not pretreated.

[0104] Conclusion: From the comparison of Examples 1 - 3 and Control Group 1, it can be seen that the performance of the soil modifier obtained by the method of Control Group 1 has weakened in all aspects. This is because when the collagen polypeptide-Fe3O4 complex is missing, the fixation rate of available Cd in the soil decreases by about 30 - 40%, and the magnetic adsorption of Fe3O4 nanoparticles and the chelation effect of polypeptides are missing, resulting in an increase in the free state of Cd 20+ and the addition of polypeptides can buffer Na + adsorption, reduce salts and further optimize the electrical conductivity; the collagen polypeptide solution contains 18 kinds of amino acids, which can be directly absorbed by the roots of pakchoi. When not added, the nitrogen content in the leaves decreases by 12 - 15%, the mu yield decreases by 15 tons, and the Fe3O4-polypeptide complex can improve the utilization rate of iron elements, and the biological yield of pakchoi in iron-deficient plots is significantly weakened;

[0105] From the comparison of Example 1, Examples 6 - 7 and Control Group 2, it can be seen that the performance of the soil modifier obtained by the method of Control Group 2 has weakened in all aspects. This is because Lysinibacillus can secrete sulfur-containing metabolites (such as cysteine) to convert Cd 2+It is converted into CdS precipitate, and the concentration of available Cd rebounds after deletion; when Pseudomonas putida acts alone, Cd adsorption depends on its extracellular polymer, but in the absence of the cooperation of Bacillus, the complexation capacity of EPS for Cd decreases by 30%. The aminotransferase secreted by Bacillus lysine can catalyze the acetylation of chitosan to form a ternary complex of chitosan-polypeptide-Cd, increasing the reduction rate of available Cd from 47% to 72%. And due to the lack of Bacillus to decompose organic matter and slowly release ions, reducing the sudden increase in salt content, the soil conductivity also fluctuates, thus reducing the yield of pakchoi;

[0106] It can be obtained from the comparison of Example 1, Examples 10 - 13 and Control Group 3 that the performance of the soil conditioner obtained without adjusting the dissolved oxygen by aeration is weakened. This is because when there is no aeration, the protonation of amino groups in chitosan in the conditioner is restricted, and the range of soil pH adjustment is reduced, resulting in an increase in the desorption amount of Cd by 15%. Insufficient dissolved oxygen inhibits the decomposition of organic acids by microorganisms, and Na 2+ is retained on the soil surface, thus causing the conductivity to fluctuate around 3.6, and further reducing the yield of pakchoi; while continuous aeration will lead to excessive nitrogen consumption by microorganisms, and the nitrogen content in the leaves decreases by 15%; + / K + It can be obtained from the comparison of Example 1, Examples 18 - 19 and Control Group 4 that the surface of the untreated activated carbon has fewer oxygen-containing functional groups (such as carboxyl groups and phenolic hydroxyl groups), reducing the chemical adsorption capacity for Cd by 30%. At the same time, the untreated activated carbon may retain metal impurities (such as Fe

[0107] 2+ ), competing with Cdfor adsorption sites, resulting in an increased risk of rebound of available Cd; the untreated carbon releases more Na 3+ Al 3+ 2+ / K + <00000, making the conductivity increase; the Cd adsorbed by the untreated activated carbon is easily absorbed by plants, and the Cd bioaccumulation factor (BCF) in the roots of pakchoi increases from 0.3 to 0.8, resulting in a 15% reduction in production. / K + 2. Explore the influence of the pretreatment method of low-value animal waste protein on the performance of the bio-polypeptide modified activated carbon soil conditioner

[0108] Control Group 5: Different from Example 20, the step of adding complex alkaline protease hydrolysis is replaced by boiling hydrolysis method.

[0109] Control Group 6: Different from Example 20, no magnetic field alignment treatment is carried out.

[0110]

[0111] ​Conclusion: From the comparison of Example 1, Examples 20 - 26, and Control Group 5, it can be seen that although replacing the step of adding compound alkaline protease hydrolysis with the boiling method can also improve the yield of small peptide segments and thus improve the loading effect of bio-polypeptides, the intensity of the effect is still limited compared to Examples 20 - 26. This is because the boiling method easily causes protein denaturation, destroys the enzyme active sites, reduces the chelating ability of humic acid-protein complexes to Cd 2+ , increases the steric hindrance of unhydrolyzed protein macromolecules, and hinders Cd 2+ from entering the adsorption sites, thereby weakening the effect of reducing available Cd in the soil; the boiling product contains more soluble base ions, so it will cause greater fluctuations in soil conductivity;

[0112] From the comparison of Example 20, Examples 27 - 32, and Control Group 6, it can be seen that the unmagnetically oriented Fe3O4 nanoparticles in Control Group 6 are unevenly dispersed, the specific surface area is reduced by 40%, the adsorption capacity for Cd 2+ is reduced, the disordered arrangement of polypeptide chains results in insufficient exposure of the alginate-Cd complexation sites, and the risk of rebound of available Cd increases. The unoriented particles release more Mn 2+ / Cl - , thus weakening the performance of the bio-polypeptide modified activated carbon soil conditioner; Considering comprehensively, Example 20 is selected as the optimal solution.

Claims

1. A biological polypeptide-modified activated carbon soil conditioner, characterized in that, By weight parts, it includes: 5-7 parts of low-value animal waste protein, 5-7 parts of activated carbon, 6-8 parts of landscaping substrate, 4-8 parts of vermiculite, 2-4 parts of compound microbial inoculum, 4-8 parts of chitosan; The low-value animal waste protein is one of blood, feathers, viscera, chicken or duck meat; The landscaping substrate is prepared by co-fermentation technology using garden waste and organic solid waste. Among them, the organic solid waste is selected from any one or more of food waste, breeding manure, livestock manure, river bottom sludge, municipal sludge; the garden waste is selected from any one or more of fallen leaves, trimmed branches, dead branches, weeds. The process of the co-fermentation technology is: naturally air-dry the garden waste and organic solid waste respectively and then crush them to a diameter of 40-60 mm, and mix them according to a mass ratio of 0.8-1.2:1; then adjust the fermentation temperature to 20-50 °C, stir once every 24-30 h, and ferment for 7-10 days. When the moisture content of the material drops to ≤30%, the landscaping substrate is obtained. The compound microbial inoculum is obtained by mixing Lysinibacillus sphaericus: Pseudomonas putida in a weight ratio of 1:2-3.

2. The bio-polypeptide modified activated carbon soil conditioner according to claim 1, wherein Perform pretreatment on the low-value animal waste protein, and the pretreatment method is: (1) Add compound alkaline protease to the low-value animal waste protein according to a mass ratio of 100:1-2, and carry out hydrolysis treatment at 45-55 °C and pH of 7.8-8.2 for 3.5-4.5 h to obtain a protein hydrolysate. Add Fe3O4 nanoparticles to the protein hydrolysate according to a mass ratio of 1:0.5-0.7 and mix well to obtain a first polypeptide solution; (2) Add 0.5-1.2% MnCl2 solution to the first polypeptide solution according to a mass ratio of 100:8-12, and stir and react at 50-60 °C for 1.5-2.5 h to obtain a second polypeptide solution; (3) Add sodium alginate accounting for 1.5-2.5 wt% of the second polypeptide solution to the second polypeptide solution and stir for 8-12 min, then add chitosan accounting for 0.5-1.5 wt% of the second polypeptide solution, and adjust the pH to 5.5-6.0, mix well, filter, and concentrate under reduced pressure at 50-55 °C until the volume is reduced to 1 / 3 of the original solution, dehydrate under vacuum at 60 °C until the moisture content is ≤15%, then cool to 55 °C and dry under vacuum until the moisture content is 8%, and finally perform directional magnetization treatment at a magnetic field intensity of 0.55-0.65 T for 25-35 min.

3. The bio-polypeptide modified activated carbon soil conditioner according to claim 2, wherein, The preparation method of the compound alkaline protease is: Transfer the Bacillus subtilis strain to a Czapek slant medium and culture it at 37 - 40 °C and pH 7.0 for 12 - 14 h. Then add Tris-HCl to adjust the pH to 8.5 and continue culturing for 38 - 42 h. Then place the cultured strain spores 30 - 35 cm away from the ultraviolet lamp and irradiate them for 15 - 30 s under the condition of 28 - 32 W. Then put the strain spores into a conical flask containing nutrient solution at a concentration of 0.005 - 0.008 g / L and oscillate for 15 - 20 h at a temperature of 39 - 41 °C and an oscillation speed of 120 - 140 r / min, and dilute it to a bacterial suspension with a concentration of 10 7 -10 9 CFU / mL. Synchronously add a catalyst accounting for 0.3 wt% of the bacterial suspension and continue to oscillate at 39 °C and 150 rpm for 6 - 8 h; The nutrient solution is prepared by mixing glucose, ammonium sulfate, calcium chloride, potassium dihydrogen phosphate, sodium chloride, zinc sulfate, and water according to a mass ratio of 12-15:0.1-0.2:0.002-0.004:0.3-0.5:0.06-0.08:0.005-0.008:1000.

4. The bio-polypeptide modified activated carbon soil conditioner according to claim 3, wherein, The catalyst is composed of dopamine hydrochloride and phosphate buffer solution in a weight ratio of 1:0.4-0.

6.

5. The preparation method of a biological polypeptide-modified activated carbon soil conditioner according to any one of claims 1-4, characterized in that, It includes the following steps: S1. Crush and grind the low-value animal waste protein into mud, stir and emulsify it for 3 - 3.5 h under the conditions of a solid content of 10%, a temperature of 33 - 37 °C, and a rotation speed of 120 r / min, then sterilize it at 95 - 105 °C for 0.5 - 1 h, post-ripen it in a storage tank for 4 - 5 days after biological fermentation, remove large-particle solids, suspended solids, and grease in the emulsion by sand filtration, and then add organic acid to adjust the pH value of the emulsion to 5.5 - 6.5, and fill it to obtain the biological polypeptide nutrient; S2. At 55 - 65 °C, impregnate the activated carbon in the biological polypeptide nutrient prepared in S1 for 3.5 - 4.5 h, add garden greening substrate, vermiculite, compound microbial inoculum, and chitosan to mix and granulate, and the particle size is 5 - 7 mm; S3. Dry it in a forced-air drying oven at 55 - 65 °C for 22 - 26 h to obtain the biological polypeptide-modified activated carbon soil conditioner.

6. The preparation method of a bio-polypeptide modified activated carbon soil conditioner as claimed in claim 5, wherein In S2, the activated carbon is pretreated, and the pretreatment method is: place the activated carbon in 2.8 - 3.2 mol / L HNO3 according to the ratio of 10 g:20 mL, and ultrasonically treat it for 25 - 35 min under the conditions of a power of 290 - 310 W and a temperature of 38 - 42 °C, then wash it with water until the activated carbon is neutral and then dry it, and finally grind it to 80 - 100 mesh.

7. The preparation method of a biological polypeptide-modified activated carbon soil conditioner according to claim 5, characterized in that, During the post-ripening process of the storage tank and the process of crushing and grinding into mud, nitrogen atmosphere protection is simultaneously introduced at a rate of 0.5 - 0.7 m 3 / h to maintain the system pressure at 0.05 - 0.15 MPa.

8. The preparation method of a biological polypeptide-modified activated carbon soil conditioner according to claim 5, characterized in that, The method of the biological fermentation is: inoculate yeast into the emulsified and sterilized raw material according to a mass percentage of 3 - 5%, then under the conditions of a temperature of 37 - 38 °C and a relative humidity of 75 - 80%, pulse aerate until the dissolved oxygen content is 10 - 15%, ferment for 48 - 54 h, and terminate when pH ≤ 4.

2. Add 2% glucose, 1% yeast extract powder, and 0.1% MnCl2 every 12 h, and inactivate it when the viable yeast count drops to 25% of the initial amount.

Images