Process for repairing polluted soil by tylosin waste liquid liquid fertilizer

Abstract

The invention provides a process for repairing polluted soil by using a tylosin waste liquid fertilizer, and belongs to the technical field of fertilizer preparation. The invention discloses a method for repairing polluted soil by using a liquid fertilizer prepared from a tylosin waste liquid, which comprises the following steps: mixing a tylosin fermentation waste liquid with plant fiber straw powder, and carrying out pretreatment; carrying out steam explosion treatment on the pretreated material to obtain a pretreated material; adding auxiliary nutritional raw materials and returned materials into the pretreated material, and adding a compound microbial agent for decomposing and fermenting to obtain a fermented material. According to the process for preparing the liquid fertilizer from the tylosin fermentation waste liquid to repair the polluted soil, the residual quantity of the tylosin is effectively reduced, and the prepared liquid fertilizer has a remarkable soil repairing effect and can greatly improve the microbial diversity, the enzymatic activity, the organic matter content and the cation exchange capacity of the soil, so that the crop yield is improved.

Description

Technical Field

[0001] The invention relates to the technical field of fertilizer preparation, and in particular to a process for repairing polluted soil by using tylosin waste liquid liquid fertilizer. Background Art

[0002] Antibiotics are indispensable drugs in clinical medicine and animal husbandry, and their production scale is expanding year by year. Macrolide antibiotics represented by tylosin are widely used in poultry and livestock farming because of their significant inhibitory effect on Gram-positive bacteria such as streptococci and staphylococci. However, the problem of waste liquid generated during the production of antibiotics is becoming increasingly prominent. These waste liquids contain not only incompletely reacted raw materials and intermediates, but also residual antibiotics, metal ions and other pollutants.

[0003] At present, the following methods are mainly used for the treatment of tylosin waste liquid: The first is the physical and chemical method, such as activated carbon adsorption and advanced oxidation. Zhang et al. (2018) reported on the use of modified activated carbon to treat tylosin wastewater. Although this method can effectively reduce COD and antibiotic content, it is costly and the waste activated carbon itself becomes a secondary pollution source. The ozone-hydrogen peroxide combined oxidation method proposed by Wang et al. (2020) has a degradation efficiency of more than 90%, but the equipment investment is large, the energy consumption is high, and toxic intermediates may be produced.

[0004] The second is biological treatment. Liu et al. (2019) reported a method for degrading tylosin waste liquid using a composite bacterial community. This method is environmentally friendly and low-cost, but the treatment cycle is long, the environmental conditions are high, and the degradation efficiency of antibiotic residues is unstable. In addition, CN107047930A discloses a harmless treatment method for fermentation drug residues. Although this method can effectively treat solid residues, it fails to solve the problem of liquid waste treatment.

[0005] Traditional soil remediation methods have obvious limitations: physical remediation methods such as soil leaching and heat treatment are costly and may damage the soil structure; chemical remediation methods such as chemical fixation and redox, although effective quickly, are prone to introduce secondary pollution; although biological remediation methods are relatively environmentally friendly, they have long cycles and unstable effects, and are difficult to meet the needs of rapid remediation of large areas of contaminated soil. Summary of the invention

[0006] The invention provides a process for repairing contaminated soil by using tylosin waste liquid as liquid fertilizer, which solves the technical problems that the solid fungus residue treatment method of fermentation drugs in the prior art is difficult to treat liquid waste liquid and lacks a special repair function design for contaminated soil.

[0007] The invention discloses a method for preparing liquid fertilizer from tylosin waste liquid to repair contaminated soil, comprising the following steps: The tylosin fermentation waste liquid is mixed with plant fiber straw powder for pretreatment; Performing steam explosion treatment on the pretreated material to obtain pretreated material; Adding auxiliary nutrient raw materials and re-cooked materials to the pre-treated materials, and adding composite microbial agents for decomposition and fermentation to obtain fermented materials; The fermented material is aged and then crushed, and activated carbon is added for adsorption treatment; The adsorption treated material is mixed with water and a biosurfactant is added to prepare a slurry; The slurry is filtered, sodium alginate and chitosan are added to the filtrate, and then a trace element regulator and an organic chelating agent are added to adjust the pH value to obtain a liquid fertilizer; The residual amount of tylosin in the liquid fertilizer is ≤0.5 mg / kg; The solid-state fermentation cycle is 8-10 days; The aging time is 9-11 days.

[0008] Preferably, the plant fiber straw powder has a grinding degree of 5-15 mesh and a moisture content of 5% to 15%; The mass ratio of the tylosin fermentation waste liquid to the plant fiber straw powder is (0.8-1.2): (0.4-0.6); Preferably, the steam explosion treatment comprises: First-stage steam explosion: temperature is 150-170℃, pressure is 1.2-1.8MPa, and pressure stabilization time is 3-7min; Secondary steam explosion: temperature is 190-210℃, pressure is 2.0-2.5MPa, and pressure stabilization time is 8-12min.

[0009] Preferably, the auxiliary nutrient raw material is added in an amount of 0.2% to 0.4%, and is composed of urea, superphosphate and potassium sulfate in a mass ratio of 1:2:5; The added amount of the re-clinker is 5% to 7%.

[0010] Preferably, the composite microbial agent comprises: Yeast ACCC20060, added at 2-4‰; Aspergillus niger ACCC30936, the addition amount is 1.5-2.5‰; Compound Bacillus, the addition amount is 3-5‰, added when the material is naturally heated to 35-40℃; The composite bacillus consists of Bacillus megaterium ACCC 02991, Bacillus subtilis ACCC 19374 and Bacillus amyloliquefaciens CGMCC 1.1043 in a mass ratio of 1:2:1.

[0011] Preferably, the fermented material is crushed to 35-45 mesh; The activated carbon is powdered activated carbon, the addition amount is 1.3% to 1.7%, the specific surface area is ≥900m² / g, and the iodine adsorption value is ≥800mg / g; The adsorption treatment time is 5.5-6.5 hours.

[0012] Preferably, the mass ratio of the treated material to water is 1:(2.5-3.5); The biosurfactant is saponin, and the addition amount is 0.4% to 0.6%; The rotation speed of the high-speed stirring is 2800-3200 r / min, and the stirring time is 25-35 min.

[0013] Preferably, the filtrate is filtered using a 80-120 mesh screen; The addition amount of sodium alginate is 2.3% to 2.7%; The chitosan is added in an amount of 1.3% to 1.7%, has a molecular weight of 80-120 kDa, and a deacetylation degree of ≥85%; The rotation speed of the low-speed stirring is 100-140r / min.

[0014] Preferably, the trace element regulator contains at least one of boron, manganese, zinc and copper, with a total content of 0.2% to 0.4%; The organic chelating agent is sodium humate, and the addition amount is 0.6% to 1.0%; The pH value was adjusted to 6.5-7.0.

[0015] The present invention also provides an application of the liquid fertilizer prepared by the above method in repairing contaminated soil.

[0016] Compared with the prior art, the present invention has at least the following beneficial effects: The invention realizes the resource utilization of tylosin fermentation waste liquid, solves the problem of waste liquid treatment, and provides a highly efficient soil remediation product, which has significant environmental and social benefits.

[0017] 1. The present invention can effectively degrade and remove tylosin through a combined process of steam explosion treatment, microbial fermentation and activated carbon adsorption, so that the residual amount of tylosin in the prepared liquid fertilizer is reduced to below 0.1 mg / kg, reaching the safety use standard.

[0018] 2. The composite Bacillus fermentation technology adopted in the present invention can significantly improve the diversity of soil microorganisms. Experiments have shown that the average increase in the soil microbial diversity index can reach 30% to 40%.

[0019] 3. The liquid fertilizer prepared by the present invention can effectively restore the damaged soil biochemical function, so that the recovery rate of key enzyme activities such as urease, sucrase, catalase and dehydrogenase reaches 85% to 93%, which is close to the level of uncontaminated soil.

[0020] 4. The synergistic effect of sodium alginate and chitosan in the present invention can significantly improve soil structure and fertility, and increase the soil organic matter content by 15% to 20%.

[0021] 5. The present invention effectively increases the cation exchange capacity of the soil by adding trace element regulators and organic chelating agents, with an increase rate of 20% to 25%, thereby improving the soil's fertilizer retention and buffering capacity, and is beneficial to the storage and release of nutrients.

[0022] 6. The liquid fertilizer of the present invention can comprehensively improve the physical and chemical properties and biological activity of contaminated soil, increase crop yield by 15% to 18%, and has obvious economic benefits. DETAILED DESCRIPTION

[0023] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0024] The mass ratio of urea, superphosphate and potassium sulfate in the auxiliary nutrient raw materials used in this embodiment is 1:2:5; the composite bacillus used is composed of Bacillus megaterium ACCC 02991, Bacillus subtilis ACCC 19374 and Bacillus amyloliquefaciens CGMCC 1.1043 in a mass ratio of 1:2:1; the trace element regulator used is derived from Stanley Agricultural Group Co., Ltd., containing boron, manganese, zinc and copper; the powdered activated carbon used is derived from Fujian Yuanshi Carbon Co., Ltd.; the powdered activated carbon used is derived from Nanjing Jiali Carbon Co., Ltd., with an iodine adsorption value of 900 mg / g and a specific surface area of ​​950 m2 / g; the saponin biosurfactant used is derived from Shanghai Yuanye Biotechnology Co., Ltd., the product name is Bupleurum saponin B4, CAS is 58558-09-1; the chitosan used is derived from Shanghai Aladdin Biochemical Technology, with a molecular weight of 100 kDa, a deacetylation degree of 90%, and a product number of 226541; the remnant used is a liquid fertilizer produced in the previous batch.

[0025] Example 1 1 kg of tylosin fermentation waste liquid was mixed with 0.5 kg of plant fiber straw powder, the plant fiber straw powder had a pulverization degree of 10 mesh and a moisture content of 10%. The moisture content of the mixed material was controlled at 50% and the density was controlled at 0.8 g / ml.

[0026] The mixture was subjected to steam explosion treatment, with the first-level steam explosion temperature being 160°C, the pressure being 1.5MPa, and the pressure stabilization time being 5min; the second-level steam explosion temperature being 200°C, the pressure being 2.2MPa, and the pressure stabilization time being 10min, to obtain a pretreated material.

[0027] 0.3% of auxiliary nutrient raw materials and 6% of remnant material were added to the pretreated material, and then a composite microbial agent was added, including: yeast ACCC 20060 added in an amount of 3‰, Aspergillus niger ACCC 30936 added in an amount of 2‰, and when the material was naturally heated to 37°C, 4‰ of composite Bacillus was added.

[0028] After 9 days of solid-state fermentation, the fermented material was aged for 10 days, then crushed to 40 mesh, and 1.5% powdered activated carbon was added for adsorption treatment for 6 hours. The adsorbed material was mixed with water at a mass ratio of 1:3, 0.5% saponin biosurfactant was added, and stirred at a high speed of 3000r / min for 30 minutes to prepare a slurry.

[0029] The slurry was filtered using a 100-mesh sieve, 2.5% sodium alginate and 1.5% chitosan were added to the filtrate, the mixture was stirred at a low speed of 120 r / min, 0.3% trace element regulator and 0.8% sodium humate were added as an organic chelating agent, the pH value was adjusted to 6.8, and liquid fertilizer was obtained.

[0030] The residual amount of tylosin in the prepared liquid fertilizer was 0.03 mg / kg.

[0031] Example 2 0.9 kg of tylosin fermentation waste liquid was mixed with 0.45 kg of plant fiber straw powder, the plant fiber straw powder had a pulverization degree of 7 mesh and a moisture content of 8%. The moisture content of the mixed material was controlled at 48% and the density was controlled at 0.75 g / ml.

[0032] The mixture was subjected to steam explosion treatment, with the first-stage steam explosion temperature being 155°C, the pressure being 1.3MPa, and the pressure stabilization time being 4min; the second-stage steam explosion temperature being 195°C, the pressure being 2.1MPa, and the pressure stabilization time being 9min, to obtain a pretreated material.

[0033] 0.25% of auxiliary nutrient raw materials and 5.5% of remnant material were added to the pretreated material, and then a composite microbial agent was added, including: yeast ACCC 20060 added in an amount of 2.5‰, Aspergillus niger ACCC 30936 added in an amount of 1.8‰, and when the material was naturally heated to 36°C, 3.5‰ of composite Bacillus was added.

[0034] After 8 days of solid-state fermentation, the fermented material was aged for 9 days and then crushed to 38 meshes, and 1.4% powdered activated carbon was added for adsorption treatment for 6 hours.

[0035] The adsorbed material was mixed with water at a mass ratio of 1:2.8, 0.45% of saponin biosurfactant was added, and the mixture was stirred at a high speed of 2900 r / min for 28 min to prepare a slurry.

[0036] The slurry was filtered using a 90-mesh screen, 2.4% sodium alginate and 1.4% chitosan were added to the filtrate, the mixture was stirred at a low speed of 110 r / min, 0.25% trace element regulator and 0.7% sodium humate were added as an organic chelating agent, the pH value was adjusted to 6.7, and liquid fertilizer was obtained.

[0037] The residual amount of tylosin in the prepared liquid fertilizer was 0.05 mg / kg.

[0038] Example 3 1.1 kg of tylosin fermentation waste liquid was mixed with 0.55 kg of plant fiber straw powder, wherein the plant fiber straw powder had a pulverization degree of 12 meshes and a moisture content of 12%.

[0039] The moisture content of the mixed material was controlled at 52%, and the density was controlled at 0.85 g / ml. The mixture was subjected to steam explosion treatment, with the first-stage steam explosion temperature being 165°C, the pressure being 1.7 MPa, and the pressure stabilization time being 6 min; the second-stage steam explosion temperature being 205°C, the pressure being 2.4 MPa, and the pressure stabilization time being 11 min, to obtain the pretreated material.

[0040] 0.35% of auxiliary nutrient raw materials and 6.5% of remnant material were added to the pretreated material, and then a composite microbial agent was added, including: yeast ACCC 20060 added in an amount of 3.5‰, Aspergillus niger ACCC 30936 added in an amount of 2.2‰, and when the material was naturally heated to 38°C, 4.5‰ of composite Bacillus was added.

[0041] After 10 days of solid-state fermentation, the fermented material was aged for 11 days, then crushed to 42 mesh, and 1.6% powdered activated carbon was added for adsorption treatment for 6.2 hours. The adsorbed material was mixed with water at a mass ratio of 1:3.2, 0.55% saponin biosurfactant was added, and high-speed stirring was performed at a speed of 3100r / min for 32 minutes to prepare a slurry.

[0042] The slurry was filtered using a 110-mesh screen, 2.6% sodium alginate and 1.6% chitosan were added to the filtrate, and the mixture was stirred at a low speed of 130 r / min. 0.35% trace element regulator and 0.9% sodium humate were added as an organic chelating agent, and the pH value was adjusted to 6.9 to obtain liquid fertilizer.

[0043] The residual amount of tylosin in the prepared liquid fertilizer was 0.05 mg / kg.

[0044] Example 4 0.8 kg of tylosin fermentation waste liquid was mixed with 0.4 kg of plant fiber straw powder, the plant fiber straw powder had a crushing degree of 5 mesh and a moisture content of 5%. The moisture content of the mixed material was controlled at 45% and the density was controlled at 0.7 g / ml.

[0045] The mixture was subjected to steam explosion treatment, with the first-level steam explosion temperature being 150°C, the pressure being 1.2MPa, and the pressure stabilization time being 3min; the second-level steam explosion temperature being 190°C, the pressure being 2.0MPa, and the pressure stabilization time being 8min, to obtain a pretreated material.

[0046] Add 0.2% of auxiliary nutrient raw materials and 5% of remnant material to the pretreated material, and then add composite microbial agents, including: yeast ACCC 20060 added in an amount of 2‰, Aspergillus niger ACCC 30936 added in an amount of 1.5‰, and when the material is naturally heated to 35°C, add composite Bacillus 3‰.

[0047] After 8 days of solid-state fermentation, the fermented material was aged for 9 days, then crushed to 35 mesh, and 1.3% powdered activated carbon was added for adsorption treatment for 5.5 hours. The adsorbed material was mixed with water at a mass ratio of 1:2.5, 0.4% saponin biosurfactant was added, and stirred at a speed of 2800r / min for 25 minutes to prepare a slurry.

[0048] The slurry was filtered, and 2.3% sodium alginate and 1.3% chitosan were added to the filtrate using an 80-mesh screen, and the mixture was stirred at a low speed of 100 r / min. 0.2% trace element regulator and 0.6% sodium humate were added as an organic chelating agent, and the pH value was adjusted to 6.5 to obtain a liquid fertilizer.

[0049] The residual amount of tylosin in the prepared liquid fertilizer was 0.04 mg / kg.

[0050] Example 5 1.2 kg of tylosin fermentation waste liquid was mixed with 0.6 kg of plant fiber straw powder, the plant fiber straw powder had a crushing degree of 15 mesh and a moisture content of 15%. The moisture content of the mixed material was controlled at 55% and the density was controlled at 0.9 g / ml.

[0051] The mixture was subjected to steam explosion treatment, with the first-stage steam explosion temperature being 170°C, the pressure being 1.8MPa, and the pressure stabilization time being 7min; the second-stage steam explosion temperature being 210°C, the pressure being 2.5MPa, and the pressure stabilization time being 12min, to obtain a pretreated material.

[0052] 0.4% of auxiliary nutrient raw materials and 7% of remnant material were added to the pretreated material, and then a composite microbial agent was added, including: yeast ACCC 20060 added in an amount of 4‰, Aspergillus niger ACCC 30936 added in an amount of 2.5‰, and when the material was naturally heated to 40°C, 5‰ of composite Bacillus was added.

[0053] After 10 days of solid-state fermentation, the fermented material was aged for 11 days, then crushed to 45 mesh, and 1.7% powdered activated carbon was added for adsorption treatment for 6.5 hours. The adsorbed material was mixed with water at a mass ratio of 1:3.5, 0.6% saponin biosurfactant was added, and stirred at a high speed of 3200r / min for 35 minutes to prepare a slurry.

[0054] The slurry was filtered using a 120-mesh screen, 2.7% sodium alginate and 1.7% chitosan were added to the filtrate, the mixture was stirred at a low speed of 140 r / min, 0.4% trace element regulator and 1.0% sodium humate were added as an organic chelating agent, the pH value was adjusted to 7.0, and liquid fertilizer was obtained.

[0055] The residual amount of tylosin in the prepared liquid fertilizer was 0.07 mg / kg.

[0056] Comparative Example Comparative Example 1 1 kg of tylosin fermentation waste liquid was mixed with 0.5 kg of plant fiber straw powder, the plant fiber straw powder had a pulverization degree of 10 mesh and a moisture content of 10%. The moisture content of the mixed material was controlled at 50% and the density was controlled at 0.8 g / ml.

[0057] The mixture was directly used for the next step without steam explosion treatment. 0.3% of auxiliary nutrient raw materials and 6% of re-cooked material were added to the mixture, and then a composite microbial agent was added, including: yeast ACCC 20060 added in an amount of 3‰, Aspergillus niger ACCC 30936 added in an amount of 2‰, and when the material was naturally heated to 37°C, 4‰ of composite Bacillus was added.

[0058] After 9 days of solid-state fermentation, the fermented material was aged for 10 days and then crushed to 40 meshes, and 1.5% powdered activated carbon was added for adsorption treatment for 6 hours.

[0059] The adsorption treated material was mixed with water at a mass ratio of 1:3, 0.5% saponin biosurfactant was added, and the mixture was stirred at a high speed of 3000 r / min for 30 min to prepare a slurry.

[0060] The slurry was filtered using a 100-mesh sieve, 2.5% sodium alginate and 1.5% chitosan were added to the filtrate, the mixture was stirred at a low speed of 120 r / min, 0.3% trace element regulator and 0.8% sodium humate were added as an organic chelating agent, the pH value was adjusted to 6.8, and liquid fertilizer was obtained.

[0061] The residual amount of tylosin in the prepared liquid fertilizer was 1.8 mg / kg.

[0062] Comparative Example 2 1 kg of tylosin fermentation waste liquid was mixed with 0.5 kg of plant fiber straw powder, the plant fiber straw powder had a pulverization degree of 10 mesh and a moisture content of 10%. The moisture content of the mixed material was controlled at 50% and the density was controlled at 0.8 g / ml.

[0063] The mixture was subjected to steam explosion treatment, with the first-level steam explosion temperature being 160°C, the pressure being 1.5MPa, and the pressure stabilization time being 5min; the second-level steam explosion temperature being 200°C, the pressure being 2.2MPa, and the pressure stabilization time being 10min, to obtain a pretreated material.

[0064] 0.3% of auxiliary nutrient raw materials and 6% of remnant material were added to the pretreated material, and then a composite microbial agent was added, including: yeast ACCC 20060 added in an amount of 3‰, Aspergillus niger ACCC 30936 added in an amount of 2‰, and when the material was naturally heated to 37°C, 4‰ of composite Bacillus was added.

[0065] After 9 days of solid-state fermentation, the fermented material was aged for 10 days and then crushed to 40 mesh. No activated carbon was added for adsorption treatment. The material was mixed with water at a mass ratio of 1:3, 0.5% saponin biosurfactant was added, and high-speed stirring was performed at a speed of 3000 r / min for 30 minutes to prepare a slurry.

[0066] The slurry was filtered using a 100-mesh sieve, 2.5% sodium alginate and 1.5% chitosan were added to the filtrate, the mixture was stirred at a low speed of 120 r / min, 0.3% trace element regulator and 0.8% sodium humate were added as an organic chelating agent, the pH value was adjusted to 6.8, and liquid fertilizer was obtained.

[0067] The residual amount of tylosin in the prepared liquid fertilizer was 1.2 mg / kg.

[0068] Comparative Example 3 1kg of tylosin fermentation waste liquid was mixed with 0.5kg of plant fiber straw powder, the powder had a crushing degree of 10 mesh and a moisture content of 10%. The moisture content of the mixed material was controlled at 50% and the density was controlled at 0.8g / ml.

[0069] The mixture was subjected to steam explosion treatment, with the first-level steam explosion temperature being 160°C, the pressure being 1.5MPa, and the pressure stabilization time being 5min; the second-level steam explosion temperature being 200°C, the pressure being 2.2MPa, and the pressure stabilization time being 10min, to obtain a pretreated material.

[0070] 0.3% of auxiliary nutrient raw materials and 6% of remnant were added to the pretreated material, and solid-state fermentation was carried out for 9 days. The fermented material was aged for 10 days, then crushed to 40 mesh, and 1.5% of powdered activated carbon was added for adsorption treatment for 6 hours. The adsorbed material was mixed with water at a mass ratio of 1:3, 0.5% of saponin biosurfactant was added, and high-speed stirring was carried out at a speed of 3000r / min for 30 minutes to make a slurry.

[0071] The slurry was filtered, and 2.5% sodium alginate and 1.5% chitosan were added to the filtrate using a 100-mesh screen, and the mixture was stirred at a low speed of 120 r / min. 0.3% trace element regulator and 0.8% sodium humate were added as an organic chelating agent, and the pH value was adjusted to 6.8 to obtain a liquid fertilizer.

[0072] The residual amount of tylosin in the prepared liquid fertilizer was 2.3 mg / kg.

[0073] Performance Testing 1. Determination method of tylosin residue Weigh 10g of liquid fertilizer sample into a 250mL conical flask, prepare a mixture of methanol and water in a volume ratio of 7:3 as the extraction solvent, add 100mL of extraction solvent to the sample, seal it and place it in an ultrasonic cleaner with a power of 300W and a frequency of 40kHz. The water bath temperature is controlled at 25℃ and the extraction is carried out for 30min. The extract is transferred to a 50mL centrifuge tube and centrifuged at 10000r / min at 4℃ for 15min. Take the supernatant and filter it into a brown sample bottle using a 0.22μm PTFE filter membrane.

[0074] Chromatographic analysis: Chromatographic column: Agilent ZORBAX SB-C18 column (250 mm × 4.6 mm, 5 μm); Mobile phase: acetonitrile: water = 35:65 (v / v), HPLC grade; Flow rate: 1.0 mL / min; Detection wavelength: 290nm; Column temperature: 30°C; Injection volume: 20 μL; Running time: 25min.

[0075] Preparation of standard curve: prepare 0.05, 0.1, 0.5, 1.0, 2.0, and 5.0 mg / L tylosin standard solutions and draw a standard curve.

[0076] Content calculation: Calculate the tylosin residue in the sample according to the standard curve, in mg / kg.

[0077] 2. Determination method of soil microbial diversity index Soil sampling: The five-point sampling method was used to collect 0-20 cm surface soil samples in each plot with an area of ​​10 m². After mixing, the samples were divided into quarters and placed in sterile sampling bags.

[0078] Sample pretreatment: Pass the soil sample through a 2 mm sieve to remove stones and plant debris, and store it in a 4 °C refrigerator for no more than 24 hours.

[0079] DNA extraction: 0.5 g of soil was weighed and total DNA was extracted using the FastDNA SPIN Kit for Soil (MP Biomedicals).

[0080] For specific operations, refer to the instructions of the kit: add phosphate buffer and MT buffer, homogenize at a speed of 6.5 m / s for 45 seconds, take the supernatant after centrifugation, add protein precipitation solution, centrifuge again, transfer the supernatant to a new tube, add binding matrix suspension, mix for 10 minutes, discard the supernatant after centrifugation, add SEWS-M solution to wash twice, and elute DNA with 100 μL of RNase-free DNase sterile water preheated at 55°C.

[0081] PCR amplification: Bacterial 16S rRNA V3-V4 region primers: 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3') Fungal ITS1 region primers: ITS1F (5'-CTTGGTCATTTAGAGGAAGTAA-3') and ITS2 (5'-GCTGCGTTCTTCATCGATGC-3') PCR reaction system (50 μL): template DNA 1 μL, forward and reverse primers 1 μL each, 2×Phanta Max MasterMix 25 μL, add ddH2O to 50 μL PCR amplification conditions: 95°C pre-denaturation for 5 min; 95°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 45 s, 30 cycles; 72°C final extension for 10 min Sequencing Processing: The PCR products were detected by agarose gel electrophoresis, and the target fragments were recovered by gel excision.

[0082] Paired-end sequencing was performed using the Illumina MiSeq PE300 platform.

[0083] Data filtering: remove adapter sequences, low-quality reads, and chimera sequences.

[0084] Data Analysis: Sequencing data were analyzed using QIIME2 software.

[0085] OTU clustering: 97% similarity threshold, using the UPARSE algorithm.

[0086] Diversity indices were calculated: Shannon index (reflecting community richness and evenness), ACE index and Chao1 index (reflecting community richness).

[0087] 3. Soil enzyme activity determination method Urease activity determination: weigh 5 g of air-dried soil sample into a 100 mL conical flask, add 5 mL of toluene, let stand for 15 min, add 10 mL of pH 7.0 phosphate buffer and 10 mL of 10% urea solution, incubate at 37 °C for 24 h, filter, take 5 mL of filtrate, add 4 mL of sodium phenol solution and 3 mL of sodium hypochlorite solution, color develop for 20 min, measure the absorbance at a wavelength of 630 nm with a spectrophotometer, and calculate the NH3-N content (mg / kg).

[0088] Determination of sucrase activity: weigh 5 g of fresh soil into a 100 mL conical flask, add 15 mL of 8% sucrose solution and 5 mL of toluene, incubate at 37 °C for 24 h, filter, take 2 mL of filtrate, add 2 mL of DNS reagent, heat in a boiling water bath for 5 min, cool and make up to 25 mL, measure the absorbance at a wavelength of 540 nm with a spectrophotometer, and calculate the reducing sugar content (mg / g).

[0089] Determination of catalase activity: weigh 2 g of fresh soil into a 100 mL conical flask, add 40 mL of distilled water and 5 mL of 0.3% H2O2 solution, shake for 20 min and filter, take 25 mL of the filtrate, add 2.5 mL of 3 mol / L H2SO4 to terminate the reaction, titrate with 0.1 mol / L KMnO4, and calculate the amount of H2O2 consumed (mL / g).

[0090] Determination of dehydrogenase activity: weigh 5 g of fresh soil into a 50 mL test tube, add 5 mL of 0.5% TTC solution and 2 mL of glucose solution, incubate at 37 °C for 24 h, add 10 mL of methanol to extract the generated TPF, measure the absorbance at a wavelength of 485 nm using a spectrophotometer, and calculate the TPF content (μg / g).

[0091] 4. Determination of soil organic matter content Sample preparation: The soil sample was air-dried, ground, and passed through a 2 mm sieve. About 20 g of the soil sample was taken and ground again to pass through a 0.25 mm sieve. 0.500 g of the soil sample that passed through the 0.25 mm sieve was weighed into a 250 mL conical flask.

[0092] Oxidation reaction: Accurately pipette 10.00mL of 0.8000mol / L potassium dichromate standard solution, quickly add 20mL of concentrated H2SO4 containing 5g silver sulfate / L, slowly shake well, cool to room temperature, and add 2-3 drops of o-phenanthroline indicator.

[0093] Titration process: Use 0.2000mol / L ammonium ferrous sulfate standard solution for titration. The end point is when the color changes from orange-yellow to brick-red.

[0094] A blank control test was also performed.

[0095] Content calculation: organic matter (g / kg) = [c(FeSO4)·V1-c(FeSO4)·V0]·0.003·1.724·1000 / (m·1-w) Wherein: c (FeSO4) is the concentration of ammonium ferrous sulfate standard solution (mol / L); V1 is the blank titration volume (mL); V0 is the sample titration volume (mL); m is the mass of the soil sample (g); w is the soil moisture content (%); 1.724 is the conversion factor; 0.003 is the mass of 1 mmol carbon (g).

[0096] Optimization of the determination method of soil cation exchange capacity Sample preparation: Take 2.000 g of air-dried soil sample, pass it through a 2 mm sieve, place it in a 100 mL centrifuge tube, add 30 mL of 1 mol / L ammonium acetate solution (pH 7.0), shake for 30 min, centrifuge at 4000 r / min for 5 min, pour off the supernatant, repeat the above operation 3 times, adding 30 mL of ammonium acetate each time.

[0097] Washing steps: add 30 mL of 95% ethanol, shake for 15 minutes, centrifuge at 4000 r / min for 5 minutes, discard the supernatant, and repeat washing twice to ensure that non-adsorbed ammonium ions are removed.

[0098] Replacement steps: Add 30mL of 1mol / L KCl solution, shake for 30min, centrifuge at 4000r / min for 5min, take the supernatant into a 100mL volumetric flask, repeat the extraction 4 times, combine the extracts, and make up to 100mL with 1mol / L KCl.

[0099] Determination steps: Take 25mL of the extract into a distillation flask, add 10mL of 10mol / L NaOH solution, and distill with steam. The receiving liquid is 25mL of 2% boric acid solution containing an indicator. Distill about 150mL and titrate with 0.01mol / L H2SO4 standard solution until it turns gray-purple.

[0100] Calculation formula: Cation exchange capacity (cmol / kg) = (V-V0) × c × 100 × 100 / (m × 25 × 1-w) Where: V is the sample titration volume (mL); V0 is the blank titration volume (mL); c is the concentration of H2SO4 standard solution (mol / L); m is the mass of the soil sample (g); w is the soil moisture content (%).

[0101] 5. Crop yield determination method Experimental plot design: A randomized block design was adopted, with 3 replicate plots for each treatment. The plot area was 10m², and a 0.5m protection zone was set between the plots. The irrigation, fertilization and management measures of each plot were kept consistent, and only the experimental factors were adjusted.

[0102] Fertilization treatment: Apply liquid fertilizer at a rate of 50 kg / mu, using the uniform spray method to ensure that the fertilizer is evenly distributed in the plot, and record parameters such as fertilization time, weather conditions, and soil moisture content.

[0103] Observation during the growth period: Record growth indicators such as plant height, stem diameter, leaf area index, etc. every week, observe and record the performance of key growth periods such as flowering period and filling period, and use the five-point sampling method in the field, selecting 10 plants for measurement each time.

[0104] Harvest at maturity: Harvest the entire plot at the physiological maturity of the crop, remove the edge row effect, and take the 6m center of the plot. 2 The yield was measured, the fresh weight was determined, and the dry weight was determined after natural drying to the standard moisture content.

[0105] Yield determination: 100 wheat plants were randomly sampled from each plot to measure plant height, ear length, number of grains per ear, and 1000-grain weight.

[0106] Thousand-grain weight determination: Randomly select 3 portions of seeds, each with 1000 seeds, and dry them at 105℃ for 4 hours to constant weight.

[0107] Number of grains per ear: 30 ears were randomly selected, the number of grains per ear was counted and the average value was taken.

[0108] Calculate the yield increase rate: (treatment yield - control yield) / control yield × 100%.

[0109] Test Results Table 1. Tylosin residue record sample Tylosin residue (mg / kg) Example 1 0.03 Example 2 0.05 Example 3 0.05 Example 4 0.04 Example 5 0.7 Comparative Example 1 1.8 Comparative Example 2 1.2 Comparative Example 3 2.3 Table 2 Changes in soil microbial diversity index after 60 days of application sample Shannon Index Growth ACE Index Growth Chao1 Index Growth Average increase Example 1 38.5% 36.2% 37.3% 37.3% Example 2 35.2% 34.5% 34.8% 34.8% Example 3 39.8% 38.6% 39.1% 39.2% Example 4 31.6% 30.5% 31.2% 31.1% Example 5 40.2% 39.5% 40.0% 39.9% Comparative Example 1 18.3% 17.5% 17.8% 17.9% Comparative Example 2 22.6% 21.9% 22.4% 22.3% Comparative Example 3 15.4% 14.7% 15.1% 15.1% Control (no fertilizer) 0 0 0 0 Table 3. Recovery rate of various soil enzyme activities relative to uncontaminated control soil after 60 days of application sample Urease Sucrase Catalase Dehydrogenase Average recovery rate Example 1 89.5% 87.2% 88.9% 87.5% 88.3% Example 2 87.3% 85.4% 86.8% 85.9% 86.4% Example 3 92.1% 90.6% 91.8% 90.9% 91.4% Example 4 85.5% 84.2% 85.7% 84.6% 85.0% Example 5 93.6% 91.8% 93.2% 92.5% 92.8% Comparative Example 1 62.4% 60.8% 61.5% 60.2% 61.2% Comparative Example 2 70.30% 68.7% 69.5% 68.2% 69.2% Comparative Example 3 58.1% 56.4% 57.2% 55.9% 56.9% Contamination control 43.2% 41.5% 42.8% 40.9% 42.1% Table 4. Increasing rate of soil organic matter content after 60 days of application Table 5. Increase rate of soil cation exchange capacity after 60 days of application sample Cation exchange capacity increase rate Example 1 23.5% Example 2 21.2% Example 3 24.7% Example 4 20.3% Example 5 25.2% Comparative Example 1 12.8% Comparative Example 2 15.4% Comparative Example 3 10.5% Contamination control 0.8% Table 6. Crop yield increase rate sample Production increase rate Example 1 17.3% Example 2 16.2% Example 3 17.9% Example 4 15.2% Example 5 18.0% Comparative Example 1 8.5% Comparative Example 2 10.2% Comparative Example 3 7.3% Contamination control 0% Data analysis As can be seen from Table 1, the tylosin residues in the products of Examples 1-5 of the present invention are all lower than 0.1 mg / kg, which meets the standard. In Comparative Examples 1-3, due to the lack of key process steps, the tylosin residues are significantly higher than those in the embodiments, and Comparative Example 3 is even as high as 2.30 mg / kg, which indicates that the combined process of steam explosion treatment, microbial fermentation and activated carbon adsorption adopted by the present invention can effectively degrade and remove tylosin, so that the waste liquid can be safely used to prepare liquid fertilizer.

[0110] As can be seen from Table 2, after applying the products of Examples 1-5, the average increases in soil microbial diversity index were 37.3%, 34.8%, 39.2%, 31.1% and 39.9%, respectively, all within the range of 30% to 40% as stated in Claim 11. The average increases in Comparative Examples 1-3 were only 17.9%, 22.3% and 15.1%, significantly lower than those in the Examples, indicating that the liquid fertilizer prepared by the present invention can effectively promote the recovery of soil microbial communities and improve diversity.

[0111] As can be seen from Table 3, the average recovery rates of soil enzyme activities in Examples 1-5 are 88.3%, 86.4%, 91.4%, 85.0% and 92.8%, respectively. Among them, the recovery effects of Examples 3 and 5 are the most significant, and the activities of four key enzymes, urease, sucrase, catalase and dehydrogenase, are all restored to more than 90%, close to the level of uncontaminated soil; the average recovery rates of Comparative Examples 1-3 are only 61.2%, 69.2% and 56.9%, which are significantly lower than those in the Examples, indicating that the liquid fertilizer of the present invention can effectively repair the damaged soil biochemical function and restore its self-purification ability.

[0112] As can be seen from Table 4, after applying Examples 1-5, the increase rates of soil organic matter content were 18.5%, 16.8%, 19.3%, 15.2% and 20.1%, respectively; while the increase rates of Comparative Examples 1-3 were only 11.3%, 13.5% and 9.6%, all of which failed to meet the standard requirements. The significant increase in soil organic matter content indicates that the liquid fertilizer of the present invention can not only repair pollution, but also improve soil structure and enhance soil fertility.

[0113] As can be seen from Table 5, the increase rates of soil cation exchange capacity in Examples 1-5 are 23.5%, 21.2%, 24.7%, 20.3% and 25.2% respectively; the increase rates in Comparative Examples 1-3 are only 12.8%, 15.4% and 10.5%, which are much lower than the effects of the embodiments. The improvement of cation exchange capacity indicates that the liquid fertilizer of the present invention can significantly improve the soil's fertilizer retention and buffering capacity, which is beneficial to the storage and release of nutrients.

[0114] As can be seen from Table 6, the increase rates of crop yield in the plots treated in Examples 1-5 were 17.3%, 16.2%, 17.9%, 15.2% and 18.0%, respectively; while the increase rates of yield in Comparative Examples 1-3 were only 8.5%, 10.2% and 7.3%, which were significantly lower than the effects of the embodiments. The increase in crop yield directly reflects the comprehensive effect of the liquid fertilizer of the present invention in repairing contaminated soil while improving soil fertility, and has obvious economic benefits.

[0115] In summary, the present invention achieves an effective reduction in the amount of tylosin residues by preparing liquid fertilizer from tylosin fermentation waste liquid to repair contaminated soil, and the prepared liquid fertilizer has a significant soil repair effect, can greatly increase soil microbial diversity, enzyme activity, organic matter content and cation exchange capacity, and thus increase crop yields.

[0116] Although the embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that all equivalent changes and improvements made within the scope of the present invention should still fall within the scope of the patent coverage of the present invention.

Claims

1. A method for preparing liquid fertilizer from tylosin waste liquid to repair contaminated soil, characterized in that: The following steps are involved: The tylosin fermentation waste liquid is mixed with plant fiber straw powder for pretreatment; Performing steam explosion treatment on the pretreated material to obtain pretreated material; Adding auxiliary nutrient raw materials and re-cooked materials to the pre-treated materials, and adding composite microbial agents for decomposition and fermentation to obtain fermented materials; The fermented material is aged and then crushed, and activated carbon is added for adsorption treatment; The adsorption treated material is mixed with water and a biosurfactant is added to prepare a slurry; The slurry is filtered, sodium alginate and chitosan are added to the filtrate, and then a trace element regulator and an organic chelating agent are added to adjust the pH value to obtain a liquid fertilizer; The residual amount of tylosin in the liquid fertilizer is ≤0.5 mg / kg; The solid-state fermentation cycle is 8-10 days; The aging time is 9-11 days.

2. The method according to claim 1, characterized in that The plant fiber straw powder has a grinding degree of 5-15 meshes and a moisture content of 5% to 15%; The mass ratio of the tylosin fermentation waste liquid to the plant fiber straw powder is (0.8-1.2): (0.4-0.6).

3. The method according to claim 1, characterized in that The steam explosion treatment comprises: First-stage steam explosion: temperature is 150-170℃, pressure is 1.2-1.8MPa, and pressure stabilization time is 3-7min; Secondary steam explosion: temperature is 190-210℃, pressure is 2.0-2.5MPa, and pressure stabilization time is 8-12min.

4. The method according to claim 1, characterized in that: The auxiliary nutrient raw material is added in an amount of 0.2% to 0.4%, and is composed of urea, superphosphate and potassium sulfate in a mass ratio of 1:2:5; The added amount of the re-clinker is 5% to 7%.

5. The method according to claim 1, characterized in that The composite microbial agent comprises: Yeast ACCC20060, added at 2-4‰; Aspergillus niger ACCC30936, the addition amount is 1.5-2.5‰; Compound Bacillus, the addition amount is 3-5‰, added when the material is naturally heated to 35-40℃; The composite bacillus consists of Bacillus megaterium ACCC 02991, Bacillus subtilis ACCC 19374 and Bacillus amyloliquefaciens CGMCC 1.1043 in a mass ratio of 1:2:

1.

6. The method according to claim 1, characterized in that The fermented material is crushed into 35-45 mesh; The activated carbon is powdered activated carbon, the addition amount is 1.3% to 1.7%, the specific surface area is ≥900m² / g, and the iodine adsorption value is ≥800mg / g; The adsorption treatment time is 5.5-6.5 hours.

7. The method according to claim 1, characterized in that The mass ratio of the treated material to water is 1:(2.5-3.5); The biosurfactant is saponin, and the addition amount is 0.4% to 0.6%; The rotation speed of the high-speed stirring is 2800-3200 r / min, and the stirring time is 25-35 min.

8. The method according to claim 1, characterized in that: The filtrate is filtered using a 80-120 mesh screen; The addition amount of sodium alginate is 2.3% to 2.7%; The chitosan is added in an amount of 1.3% to 1.7%, has a molecular weight of 80-120 kDa, and a deacetylation degree of ≥85%; The rotation speed of the low-speed stirring is 100-140r / min.

9. The method according to claim 1, characterized in that: The trace element regulator contains at least one of boron, manganese, zinc and copper, with a total content of 0.2% to 0.4%; The organic chelating agent is sodium humate, and the addition amount is 0.6% to 1.0%; The pH was adjusted to 6.5-7.

0.

10. A process for remediating contaminated soil using the liquid fertilizer prepared by the method according to any one of claims 1 to 10.