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CN-122012109-A - Biological organic fertilizer with liquid peptidase for soil remediation and preparation process thereof

CN122012109ACN 122012109 ACN122012109 ACN 122012109ACN-122012109-A

Abstract

The invention discloses a biological organic fertilizer for soil restoration by liquid peptidase and a preparation process thereof, in particular to the technical field of agricultural environment restoration, the fertilizer comprises a liquid peptidase compound system, a multifunctional carrier matrix for loading the system and a synergistic microbial flora compounded with the multifunctional carrier matrix, wherein the carrier matrix comprises Fe 3 O 4 modified biochar, calcium alginate-chitosan microcapsules and modified attapulgite clay. The preparation process includes pre-activation of carrier, immobilization of enzyme complex, phased temperature control fermentation and magnetizing treatment. According to the invention, by constructing the enzyme-carrier-flora synergistic restoration micro-unit, synchronous and efficient removal and passivation of protein organic pollutants and heavy metals in soil are realized, the environmental stability and action durability of restoration factors are obviously improved, the soil aggregation structure can be effectively improved, and the microecological health is restored.

Inventors

  • Wang Baiwang
  • MIAO NING
  • WANG CHEN

Assignees

  • 北农阿木(河南)生物科技有限公司

Dates

Publication Date
20260512
Application Date
20260204

Claims (9)

  1. 1. A liquid peptidase used for soil remediation is characterized by comprising the following components: A liquid peptidase complex system; a multifunctional carrier matrix carrying a liquid peptidase complex system; a synergistic microbial flora compounded with a liquid peptidase compounding system and a multifunctional carrier matrix; wherein the multifunctional carrier matrix comprises biochar modified by Fe 3 O 4 nano particles, calcium alginate-chitosan microcapsules and modified attapulgite clay.
  2. 2. The bio-organic fertilizer according to claim 1, wherein the liquid peptidase complex system comprises: A thermostable liquid peptidase which is a commercial protease derived from Thermomyces lanuginosus or an engineered variant thereof; heavy metal chelating peptidase, which is a recombinant protease obtained by introducing a cysteine-rich metal binding domain into the Bacillus licheniformis protease sequence; the hydrophobic substrate penetration enhancing peptidase is a fusion protease obtained by fusion expression of protease and hydrophobic peptide fragment, and the host for fusion expression is bacillus subtilis WB600.
  3. 3. The bio-organic fertilizer according to claim 1, wherein the multifunctional carrier matrix is composed of the following components in parts by weight: 30-40 parts of decomposed straw biochar modified by Fe 3 O 4 nano particles; 20-25 parts of calcium alginate-chitosan microcapsules; 15-20 parts of modified attapulgite clay loaded with Fe 3 O 4 nano particles; 10-15 parts of humic acid-amino acid chelate; Wherein, the specific surface area of the decomposed straw biochar modified by Fe 3 O 4 nano particles is more than 400m2/g, the magnetic saturation strength is more than 35emu/g, the chelation rate of humic acid-amino acid chelate is more than or equal to 85%, and the enzyme encapsulation rate of calcium alginate-chitosan microcapsule is more than or equal to 80%.
  4. 4. The biological organic fertilizer according to claim 1, wherein the synergistic microbial flora is compounded by strains with complementary functions, and the synergistic microbial flora comprises the following components in terms of total colony forming units: 40-50% of an enzyme activity protective bacterial group consisting of strains selected from the genus pseudomonas capable of secreting extracellular polysaccharides to stabilize the liquid peptidase activity; 30-40% of heavy metal passivation bacteria group, which consists of heavy metal resistant strains which are selected from the genus greedy copper or rhizobium and carry heavy metal resistance and transformation gene clusters, wherein the Cd resistance concentration is more than or equal to 100mg/L, pb and the resistance concentration is more than or equal to 500 mg/L; 20-30% of a soil structure modifying bacterial group consisting of a strain selected from the group consisting of Streptomyces species capable of secreting extracellular polysaccharides and phytohormone analogues to promote soil aggregate formation; The strain is a strain deposited in public strain deposit center or a strain having a corresponding function isolated and identified from contaminated soil or plant rhizosphere by conventional screening methods.
  5. 5. A preparation process for preparing the liquid peptidase according to any one of claims 1-4 for soil remediation of a biological organic fertilizer, which is characterized by comprising the following steps: s1, pre-activating a carrier: s1.1, soaking the decomposed straw biochar in 0.1M FeCl 3 solution, performing ultrasonic treatment at 40kHz for 30min, and performing vacuum drying at 60 ℃; s1.2, heating the dried biochar to 450 ℃ at 5 ℃ per min in an N 2 atmosphere, and performing heat treatment for 2 hours to obtain a Fe 3 O 4 @biochar composite carrier; S1.3, mixing attapulgite clay and FeCl 3 solution according to a mass ratio of 1:3, stirring for 2 hours, drying, and calcining at 400 ℃ for 1.5 hours to obtain modified attapulgite clay; S1.4, mixing humic acid and amino acid according to a molar ratio of 1:1.2, reacting for 3 hours at the pH of 7 and 60 ℃, and spray drying to obtain humic acid-amino acid chelate; S2, constructing a multifunctional carrier matrix: S2.1, preparing a liquid peptidase compound system, namely mixing thermostable liquid peptidase, heavy metal chelate peptidase and hydrophobic substrate penetration enhancing peptidase according to an enzyme activity unit ratio of 2:1:1.5, and preparing an enzyme solution with a total protein concentration of 2.5mg/mL by using phosphate buffer solution with a pH value of 5.5; S2.2, mixing enzyme solution with 1% (w/v) chitosan acetic acid solution in equal volume, and stirring for 2 hours at 4 ℃ and 150rpm for electrostatic adsorption; S2.3, dropwise adding 2% (w/v) sodium alginate solution and 0.1M CaCl 2 solution into the mixed solution to form gel microcapsules with the particle size of 50-100 mu M; S2.4, mixing the Fe 3 O 4 @biochar composite carrier, the calcium alginate-chitosan microcapsule, the modified attapulgite clay and the humic acid-amino acid chelate according to the weight ratio of 30-40:20-25:15-20:10-15; s2.5, treating for 1h in a fluidized bed at 40 ℃ and a fluidization number of 1.5 to uniformly compound the components to obtain a multifunctional carrier matrix for carrying the enzyme complex; s3, fermenting by controlling the temperature in stages: S3.1, preparing a synergistic microbial flora, namely mixing an enzyme activity protection bacterial group, a heavy metal passivation bacterial group and a soil structure improvement bacterial group according to a colony forming unit ratio of 40-50 percent to 30-40 percent to 20-30 percent; S3.2, adding a multifunctional carrier matrix carrying an enzyme complex into a fermentation culture medium according to the concentration of 30g/L, and inoculating 10% (v/v) of synergistic microbial flora, wherein the fermentation culture medium comprises 8-12g/L of glucose, 4-6g/L of peptone, 2-4g/L of yeast powder, 1.5-2.5g/L of potassium dihydrogen phosphate, 0.4-0.6g/L of magnesium sulfate heptahydrate and 0.5-1.5mL/L of trace element solution; Wherein the specific components of the microelement solution are zinc sulfate heptahydrate 0.1g/L, manganese sulfate monohydrate 0.05g/L, copper sulfate pentahydrate 0.01g/L, cobalt chloride hexahydrate 0.005g/L and sodium molybdate crystal 0.005g/L; s3.3, performing aerobic fermentation in the first stage, namely culturing for 48 hours at 35-38 ℃ and 180rpm under the pH of 6.5-7; s3.4, performing second stage micro-aerobic fermentation, namely culturing for 48 hours at 30-32 ℃ and 50rpm under the condition that the concentration of dissolved oxygen is 2-3 mg/L; s3.5, curing at low temperature in the third stage, namely culturing for 48 hours under the standing condition of 15-18 ℃; s4, magnetization treatment and drying: s4.1, applying an alternating magnetic field with the frequency of 50Hz and the intensity of 10mT for 6 hours at the end of fermentation; s4.2, vacuum freeze-drying the fermentation product to constant weight at-45 ℃ under the condition of 0.1Pa to obtain the biological organic fertilizer.
  6. 6. The process according to claim 5, wherein in step S1.3, the concentration of FeCl 3 solution is 0.15M, and the calcination is performed under the protection of N 2 .
  7. 7. The preparation process according to claim 5, wherein in the step S2.4, the specific weight ratio of each component is 35 parts of Fe 3 O 4 @biochar composite carrier, 22 parts of calcium alginate-chitosan microcapsule, 18 parts of modified attapulgite clay and 12 parts of humic acid-amino acid chelate.
  8. 8. The preparation process according to claim 5, wherein in the step S3.1, the synergistic microorganism flora comprises 45% of enzyme activity protective bacteria group, 35% of heavy metal passivation bacteria group and 20% of soil structure improvement bacteria group.
  9. 9. The process of claim 5, wherein the step S3.4 comprises the step of fermenting the carrier with micro-organism to form a biomembrane complex on the surface of the carrier, the thickness of the membrane is 50-100 μm, the viable cell density of the biomembrane is more than or equal to 1X 10 8 CFU/cm2, and the extracellular polysaccharide content is more than or equal to 8g/L.

Description

Biological organic fertilizer with liquid peptidase for soil remediation and preparation process thereof Technical Field The invention relates to the technical field of agricultural environment restoration, in particular to a biological organic fertilizer for soil restoration by liquid peptidase and a preparation process thereof. Background With the rapid development of industry and agriculture, the problem of soil pollution is increasingly serious, especially the concurrent combined pollution of organic pollutants and heavy metals becomes a difficult problem to be solved in the field of soil remediation, and the biological organic fertilizer has great potential in soil remediation due to the advantages of environmental friendliness, lower cost and the like. However, existing bio-organic fertilizers and repair techniques thereof still have obvious limitations: 1. The functional singleness is strong, commercial products or researches focus on degrading specific organic pollutants (such as pesticides and petroleum hydrocarbon) or passivating heavy metals, and a multifunctional integrated system capable of synchronously and efficiently treating the combined pollution of protein organic matters and heavy metals is lacking; 2. The core active ingredients (such as enzyme preparation and functional microorganisms) are easy to inactivate in a complex soil environment, have short half-life, have limited protection effect by a conventional physical adsorption or embedding method, and are difficult to maintain long-term restoration efficacy; 3. in the prior art, enzymes, microorganisms and carriers are simply and physically mixed, effective synergic and functional coupling is lacked among the components, the repair efficiency is improved to a limited extent, and in addition, most products cannot achieve pollution removal and restoration of the self-microecological structure and physical properties of soil, so that the repair effect is incomplete. Therefore, the biological organic fertilizer capable of repairing the composite polluted soil synergistically, efficiently and permanently and synchronously improving the ecological function of the soil and the preparation method thereof are developed, and the biological organic fertilizer has important practical significance and application value. Disclosure of Invention In order to overcome the defects in the prior art, the invention provides a biological organic fertilizer for soil remediation by liquid peptidase and a preparation process thereof, which are used for solving the problems in the background art. In order to achieve the aim, the invention provides the following technical scheme that the liquid peptidase is used for the biological organic fertilizer for soil remediation, and comprises the following components: A liquid peptidase complex system; a multifunctional carrier matrix carrying a liquid peptidase complex system; a synergistic microbial flora compounded with a liquid peptidase compounding system and a multifunctional carrier matrix; wherein the multifunctional carrier matrix comprises biochar modified by Fe 3O4 nano particles, calcium alginate-chitosan microcapsules and modified attapulgite clay. Preferably, the liquid peptidase complex system comprises: A thermostable liquid peptidase which is a commercial protease derived from Thermomyces lanuginosus or an engineered variant thereof; heavy metal chelating peptidase, which is a recombinant protease obtained by introducing a cysteine-rich metal binding domain into the Bacillus licheniformis protease sequence; the hydrophobic substrate penetration enhancing peptidase is a fusion protease obtained by fusion expression of protease and hydrophobic peptide fragment, and the host for fusion expression is bacillus subtilis WB600. Preferably, the multifunctional carrier matrix is composed of the following components in parts by weight: 30-40 parts of decomposed straw biochar modified by Fe 3O4 nano particles; 20-25 parts of calcium alginate-chitosan microcapsules; 15-20 parts of modified attapulgite clay loaded with Fe 3O4 nano particles; 10-15 parts of humic acid-amino acid chelate; Wherein, the specific surface area of the decomposed straw biochar modified by Fe 3O4 nano particles is more than 400m2/g, the magnetic saturation strength is more than 35emu/g, the chelation rate of humic acid-amino acid chelate is more than or equal to 85%, and the enzyme encapsulation rate of calcium alginate-chitosan microcapsule is more than or equal to 80%. Preferably, the synergistic microbial flora is compounded by strains with complementary functions, and the composition comprises, based on the total colony forming units: 40-50% of an enzyme activity protective bacterial group consisting of strains selected from the genus pseudomonas capable of secreting extracellular polysaccharides to stabilize the liquid peptidase activity; 30-40% of heavy metal passivation bacteria group, which consists of heavy metal resistant strains