CN108384777B - Preparation method of porous microbial preparation for soil remediation - Google Patents
Preparation method of porous microbial preparation for soil remediation Download PDFInfo
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- CN108384777B CN108384777B CN201810487084.4A CN201810487084A CN108384777B CN 108384777 B CN108384777 B CN 108384777B CN 201810487084 A CN201810487084 A CN 201810487084A CN 108384777 B CN108384777 B CN 108384777B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09C—RECLAMATION OF CONTAMINATED SOIL
- B09C1/00—Reclamation of contaminated soil
- B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
Abstract
The invention discloses a preparation method of a porous microbial preparation for soil remediation, which comprises the steps of 1) uniformly mixing arthrobacter L HM7719 fermentation liquor and rhodopseudomonas palustris fermentation liquor, adding ACC deaminase, and uniformly mixing to obtain a composite microbial liquid, 2) mixing diatomite, sodium alginate, calcium carbonate and calcium chloride, adding the composite microbial liquid prepared in the step 1), uniformly mixing to obtain a dispersion liquid, 3) dropwise adding the dispersion liquid prepared in the step 2) into a magnesium sulfate solution to generate spherical particle precipitates, centrifugally separating the spherical particle precipitates, slowly dropwise adding a dilute hydrochloric acid solution into the spherical particle precipitates until no bubbles are generated, washing, centrifuging and drying.
Description
Technical Field
The invention belongs to the technical field of soil remediation, and particularly relates to a preparation method of a porous microbial preparation for soil remediation.
Background
Soil is a resource on which human beings rely for survival, and is an important place for material and energy circulation in an ecological system. Soil environment and ecological safety are important guarantees for sustainable development. With the rapid development of industry, the discharge of a large amount of industrial wastes causes heavy metal and organic matter contents in large-area land to seriously exceed standards, and has great influence on ecological environment, agricultural safety and sustainable development. The bioremediation of soil mainly comprises: (1) repairing the soil by utilizing the metabolic capability of microorganisms originally existing in the soil; (2) adding specific microorganisms with high pollutant decomposing efficiency to repair the soil. Since the composition of the pollutants in the soil polluted by industrial wastes is complex, the degradation capability of the pollutants by microorganisms originally existing in the soil is not high. The pollutants are transmitted through drinking water and food chains to endanger the health of residents, and the heavy metal pollution event is in a high-incidence situation. At present, few methods are available for the practical remediation of soil pollution. Due to pollution, the nutritional function, purification function, buffering function and organic matter supporting function of soil are losing, and remediation and treatment are urgently needed.
Disclosure of Invention
The purpose of the invention is as follows: the invention aims to provide a method for preparing a porous microbial preparation for soil remediation, which is simple to operate and suitable for industrial production, and the prepared porous microbial preparation for soil remediation can efficiently degrade organic pollutants in soil, reduce the content of heavy metals in the soil and promote plant growth.
The technical scheme is as follows: the invention provides a preparation method of a porous microbial preparation for soil remediation, which comprises the following steps:
1) uniformly mixing Arthrobacter (Arthrobacter sp.) L HM7719 fermentation liquor and Rhodopseudomonas palustris (Rhodopseudomonas palustris) fermentation liquor according to the mass ratio of 1: 1.2-1.5, adding ACC deaminase, and uniformly mixing to obtain a compound microbial solution;
2) mixing diatomite, sodium alginate, calcium carbonate and calcium chloride in water according to the mass ratio of 1: 1-2: 0.2-0.3, uniformly dispersing by using ultrasonic waves, adding the composite microbial bacteria liquid prepared in the step 1), and uniformly mixing to obtain a dispersion liquid;
3) dropwise adding the dispersion liquid prepared in the step 2) into a magnesium sulfate solution with the concentration of 0.05-0.1 mol/L to generate a spherical particle precipitate, centrifugally separating the spherical particle precipitate, slowly dropwise adding a dilute hydrochloric acid solution into the spherical particle precipitate until no bubbles are generated, washing the obtained solid substance with water, centrifuging, and drying to obtain the microbial preparation.
The microbial preparation prepared by the method is composed of particles with the particle size distribution of 1-500 microns, the particles are of a porous structure, and the pore size of the particles is mainly distributed in the range of 50-100 nm. The prepared spherical particles contain ingredients with strong adsorption capacity such as diatomite, sodium alginate, calcium sulfate and the like, and most of the ingredients of the composite microbial liquid in the dispersion liquid can be adsorbed into the spherical particles.
In the step 2), the mass ratio of the total mass of the added diatomite, the sodium alginate, the calcium carbonate and the calcium chloride to the mass of the added composite microbial liquid is 0.2-0.3: 1.
The preparation method of the Arthrobacter (Arthrobacter sp.) L HM7719 fermentation liquid comprises the steps of culturing Arthrobacter (Arthrobacter sp.) L HM7719 to logarithmic phase, inoculating strains cultured to logarithmic phase to L B culture medium according to the inoculation amount of 2% -5% of the volume of the culture medium, shaking a shaking table at 120-180 rpm at 25-37 ℃, and culturing for 40-48 hours to obtain the Arthrobacter (Arthrobacter sp.) L HM7719 fermentation liquid.
The Arthrobacter (Arthrobacter sp.) L HM7719 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC NO.8181, and is disclosed in the prior patent with the publication number of CN103555620A and the application number of 201310505522.2.
The Rhodopseudomonas palustris (Rhodopseudomonas palustris) can be a common Rhodopseudomonas palustris (Rhodopseudomonas palustris) which can be prepared according to a conventional method, for example, the commercially available Rhodopseudomonas palustris (Rhodopseudomonas palustris) is inoculated on a seed culture medium to be cultured to obtain a seed culture, and the seed culture is inoculated on a fermentation medium in a fermentation tank to be fermented.
The Rhodopseudomonas palustris (Rhodopseudomonas palustris) is preferably Rhodopseudomonas palustris2-8 (Rhodopseudomonas palustris 2-8). Specifically, the fermentation liquid can be prepared by the following method:
culturing Rhodopseudomonas palustris2-8 (Rhodopseudomonas palustris2-8) to logarithmic phase, then inoculating the strain cultured to logarithmic phase into a culture medium A according to the inoculum size of 2-5% of the volume of the culture medium, and culturing for 48-96 hours at 28-30 ℃ under the light anaerobic condition to obtain the Rhodopseudomonas palustris (Rhodopseudomonas palustris) fermentation liquor. The culture medium A comprises water and the following components in percentage by mass: 0.1% of ammonium chloride, 0.05% of sodium hydrogen phosphate, 0.02% of magnesium sulfate or magnesium chloride, 0.2% of sodium chloride, 0.1-0.2% of yeast extract or peptone, and the pH value of the culture medium A is 6.8-7.1.
The Rhodopseudomonas palustris2-8 (Rhodopseudomonas palustris2-8) is preserved in China Center for Type Culture Collection (CCTCC), and the preservation number is CCTCC NO: m205147, the bacterial species is disclosed in the patent documents with the publication number CN100575480C and the application number 200710089906.5.
The content of viable bacteria in Arthrobacter (Arthrobacter sp.) L HM7719 fermentation liquid is 5 × 108～5×1010cfu/g, viable bacteria content of Rhodopseudomonas palustris (Rhodopseudomonas palustris) fermentation broth of 5 × 109～5×1011cfu/g, and the concentration of ACC deaminase is 25-35U/mg.
The enzyme activity of the ACC deaminase is defined as that the enzyme amount of decomposing ACC (1-aminocyclopropanecarboxylic acid) into 1.0 mu mol α -ketobutyric acid per minute is defined as one enzyme activity unit at the pH of 8.5 and 30 ℃ in the presence of the ACC deaminase, and the ACC deaminase concentration is 25-35U/mg, namely that each mg of the ACC deaminase used for soil remediation contains 25-35 enzyme activity units of ACC deaminase.
The calcium carbonate added in the step 2) consists of calcium carbonate particles with the particle size of 200-300 meshes; the content of magnesium sulfate in the magnesium sulfate solution in the step 3) is based on the fact that the dispersion liquid in the step 2) can be fully precipitated.
The concentration of the dilute hydrochloric acid solution added in the step 3) is preferably 0.01-0.05 mol/L.
The preparation method has the beneficial effects that the preparation method is simple, the porous microbial preparation for soil remediation can be efficiently prepared, the preparation method is suitable for work popularization and application, the prepared porous microbial preparation for soil remediation is composed of particles with the particle size distribution of 1-500 micrometers, the particles can be relatively uniformly dispersed in soil, the microbial preparation uses a composite material containing diatomite, calcium sulfate and sodium alginate as a carrier, the carrier has a good adsorption effect on heavy metal ions in the soil, in addition, the invention uses Arthrobacter (Arthrobacter sp) L HM7719 in the field of soil remediation, and long-time experimental screening shows that the Arthrobacter (Arthrobacter sp) L HM 9, Rhodopseudomonas palustris (Rhodopseudomonas palustris) and ACC deaminase can be matched to adjust the pH value of the soil, improve the soil hardening problem, degrade organic pollutants in the soil and promote plant growth.
Detailed Description
Example 1
The preparation method of the porous microbial preparation for soil remediation comprises the following steps:
1) preparation of Arthrobacter (Arthrobacter sp.) L HM7719 broth:
arthrobacter (Arthrobacter sp.) L HM7719 is placed in a triangular flask with seed culture foundation, seed culture is carried out to logarithmic phase under the conditions of pH7.2 and 30 ℃, then the strain cultured to logarithmic phase is inoculated into L B liquid culture medium according to the inoculation amount of 2% of the volume of the culture medium, shaking table is carried out at the temperature of pH 7.0 and 30 ℃ for 180rpm, and culture is carried out for 48 hours, thus obtaining the Arthrobacter (Arthrobacter sp.) L HM7719 fermentation liquor.
2) Preparation of Rhodopseudomonas palustris (Rhodopseudomonas palustris) fermentation broth:
inoculating activated Rhodopseudomonas palustris2-8 (Rhodopseudomonas palustris2-8) into a liquid culture medium A, carrying out anaerobic culture for 3 days under the conditions of illumination intensity of 2000lux and temperature of 28-30 ℃, then carrying out anaerobic culture and amplification for 2 times in the liquid culture medium A according to 3% of inoculation concentration under the conditions of illumination intensity of 20001ux and temperature of 28 ℃, and obtaining Rhodopseudomonas palustris2-8 fermentation liquor.
The liquid culture medium A comprises the following components in percentage by mass: 0.1% of ammonium chloride, 0.05% of sodium hydrogen phosphate, 0.02% of magnesium sulfate or magnesium chloride, 0.2% of sodium chloride, 0.1% of yeast extract and the balance of water, wherein the pH value of the liquid culture medium A is 7.0.
3) Preparing a compound microbial solution:
uniformly mixing Arthrobacter (Arthrobacter sp.) L HM7719 fermentation liquor obtained in the step 1) and Rhodopseudomonas palustris (Rhodopseudomonas palustris) fermentation liquor obtained in the step 2) according to the mass ratio of 1: 1.2, and adding ACC deaminase to obtain the composite microbial liquid, wherein the concentration of the ACC deaminase in the obtained composite microbial liquid is 30U/mg.
4) Mixing diatomite, sodium alginate, 200-300-mesh calcium carbonate particles and calcium chloride in water according to the mass ratio of 1: 1.5: 0.3, uniformly dispersing by ultrasonic, adding the composite microbial liquid prepared in the step 3), enabling the mass ratio of the total mass of the added diatomite, sodium alginate, calcium carbonate and calcium chloride to the mass of the added composite microbial liquid to be 0.2: 1, uniformly mixing to obtain a dispersion liquid, dropwise adding the dispersion liquid into a magnesium sulfate solution with the concentration of 0.1 mol/L by using a dropper to generate spherical particle precipitates, centrifugally separating the spherical particle precipitates, slowly dropwise adding a dilute hydrochloric acid solution with the concentration of 0.0 lmol/L into the separated spherical particle precipitates until no bubbles are generated, washing the obtained solid matter with water, centrifuging and drying to obtain the microbial preparation, wherein the prepared microbial preparation is composed of particles with the particle size distribution of 1-500 micrometers, the particles are of a porous structure, and the pore size of the particles is within the range of 50-100 nm.
Example 2
The preparation method of the porous microbial preparation for soil remediation comprises the following steps:
1) preparation of Arthrobacter (Arthrobacter sp.) L HM7719 broth:
arthrobacter (Arthrobacter sp.) L HM7719 is placed in a triangular flask with seed culture foundation, seed culture is carried out to logarithmic phase under the condition of pH7.2 and 30 ℃, then the strain cultured to logarithmic phase is inoculated into L B liquid culture medium according to the inoculation amount of 5% of the volume of the culture medium, shaking table is carried out at the temperature of pH 7.0 and 35 ℃, 150rpm is shaken, and culture is carried out for 48 hours, so as to obtain the Arthrobacter (Arthrobacter sp.) L HM7719 fermentation liquor.
2) Preparation of Rhodopseudomonas palustris (Rhodopseudomonas palustris) fermentation broth
Inoculating activated Rhodopseudomonas palustris2-8 (Rhodopseudomonas palustris2-8) into a liquid culture medium A, carrying out anaerobic culture for 3 days under the conditions of illumination intensity of 20001ux and temperature of 28-30 ℃, then carrying out anaerobic culture and amplification for 2 times in the liquid culture medium A according to the inoculation concentration of 2 percent under the conditions of illumination intensity of 20001ux and temperature of 30 ℃, and obtaining the Rhodopseudomonas palustris2-8 fermentation liquor.
The liquid culture medium A comprises the following components in percentage by mass: 0.1% of ammonium chloride, 0.05% of sodium hydrogen phosphate, 0.02% of magnesium sulfate or magnesium chloride, 0.2% of sodium chloride, 0.2% of peptone and the balance of water, wherein the pH value of the liquid medium A is 7.0.
3) Preparing a compound microbial solution:
uniformly mixing Arthrobacter (Arthrobacter sp.) L HM7719 fermentation liquor obtained in the step 1) and Rhodopseudomonas palustris (Rhodopseudomonas palustris) fermentation liquor obtained in the step 2) according to the mass ratio of 1: 1.5, and adding ACC deaminase to obtain the microbial soil remediation agent, wherein the concentration of the ACC deaminase in the obtained microbial soil remediation agent is 35U/mg.
4) Mixing diatomite, sodium alginate, 200-300-mesh calcium carbonate particles and calcium chloride in water according to the mass ratio of 1: 2: 0.3: 0.2, uniformly dispersing by ultrasonic waves, adding the composite microbial liquid prepared in the step 3), enabling the mass ratio of the total mass of the added diatomite, sodium alginate, calcium carbonate and calcium chloride to be 0.3: 1, uniformly mixing to obtain a dispersion liquid, dropwise adding the dispersion liquid into a magnesium sulfate solution with the concentration of 0.1 mol/L by using a dropper to generate spherical particle precipitates, centrifugally separating the spherical particle precipitates, slowly dropwise adding a dilute hydrochloric acid solution with the concentration of 0.03 mol/L into the spherical particle precipitates obtained by separation until no bubbles are generated, washing the obtained solid matter with water, centrifuging and drying to obtain the microbial preparation, wherein the prepared microbial preparation is composed of particles with the particle size distribution of 1-500 micrometers, the particles are of a porous structure, and the pore size of the particles is within the range of 50-100 nm.
Example 3
The preparation method of the porous microbial preparation for soil remediation comprises the following steps:
1) preparation of Arthrobacter (Arthrobacter sp.) L HM7719 broth:
arthrobacter (Arthrobacter sp.) L HM7719 is placed in a triangular flask with seed culture foundation, seed culture is carried out to logarithmic phase under the condition of pH7.2 and 30 ℃, then the strain cultured to logarithmic phase is inoculated into L B liquid culture medium according to the inoculation amount of 3% of the volume of the culture medium, shaking table is carried out at the temperature of pH 7.0 and 28 ℃, and culture is carried out for 48 hours, thus obtaining the Arthrobacter (Arthrobacter sp.) L HM7719 fermentation liquor.
2) Preparation of Rhodopseudomonas palustris (Rhodopseudomonas palustris) fermentation broth
Inoculating activated Rhodopseudomonas palustris2-8 (Rhodopseudomonas palustris2-8) into a liquid culture medium A, carrying out anaerobic culture for 3 days under the conditions of illumination intensity of 2000lux and temperature of 30 ℃, then carrying out anaerobic culture and amplification for 2 times in the liquid culture medium A according to the inoculation concentration of 5 percent under the conditions of illumination intensity of 20001 lux and temperature of 30 ℃ to obtain the Rhodopseudomonas palustris2-8 fermentation liquor.
The liquid culture medium A comprises the following components in percentage by mass: 0.1% of ammonium chloride, 0.05% of sodium hydrogen phosphate, 0.02% of magnesium sulfate or magnesium chloride, 0.2% of sodium chloride, 0.1% of peptone and the balance of water, wherein the pH value of the liquid medium A is 7.0.
3) Preparing a compound microbial solution:
uniformly mixing Arthrobacter (Arthrobacter sp.) L HM7719 fermentation liquor obtained in the step 1) and Rhodopseudomonas palustris (Rhodopseudomonas palustris) fermentation liquor obtained in the step 2) according to the mass ratio of 1: 1.4, and adding ACC deaminase to obtain the microbial soil remediation agent, wherein the concentration of the ACC deaminase in the obtained microbial soil remediation agent is 25U/mg.
4) Mixing diatomite, sodium alginate, 200-300-mesh calcium carbonate particles and calcium chloride in water according to the mass ratio of 1: 0.2, ultrasonically dispersing uniformly, adding the composite microbial liquid prepared in the step 3), enabling the mass ratio of the total mass of the added diatomite, sodium alginate, calcium carbonate and calcium chloride to the mass of the added composite microbial liquid to be 0.2: 1, uniformly mixing to obtain a dispersion liquid, dropwise adding the dispersion liquid into a magnesium sulfate solution with the concentration of 0.05 mol/L by using a dropper to generate spherical particle precipitates, centrifugally separating the spherical particle precipitates, slowly dropwise adding a dilute hydrochloric acid solution with the concentration of 0.05 mol/L into the spherical particle precipitates obtained by separation until no bubbles are generated, washing the obtained solid matter with water, centrifuging, and drying to obtain the microbial preparation, wherein the prepared microbial preparation is composed of particles with the particle size distribution of 1-500 micrometers, the particles are of a porous structure, and the pore diameter of the particles is within the range of 50-100 nm.
Comparative example 1
The preparation method of the porous microbial preparation for soil remediation comprises the following steps:
1) preparation of Arthrobacter (Arthrobacter sp.) L HM7719 broth:
arthrobacter (Arthrobacter sp.) L HM7719 is placed in a triangular flask with seed culture foundation, seed culture is carried out to logarithmic phase under the conditions of pH7.2 and 30 ℃, then the strain cultured to logarithmic phase is inoculated into L B liquid culture medium according to the inoculation amount of 2% of the volume of the culture medium, shaking table is carried out at the temperature of pH 7.0 and 30 ℃ for 180rpm, and culture is carried out for 48 hours, thus obtaining the Arthrobacter (Arthrobacter sp.) L HM7719 fermentation liquor.
2) Preparing a compound microbial solution:
adding ACC (adenosine triphosphate) into the Arthrobacter (Arthrobacter sp.) L HM7719 fermentation liquor prepared in the step 1) to prepare composite microbial liquid, wherein the concentration of the ACC deaminase in the obtained composite microbial liquid is 30U/mg.
3) Mixing diatomite, sodium alginate, 200-300-mesh calcium carbonate particles and calcium chloride in water according to the mass ratio of 1: 1.5: 0.3, uniformly dispersing by ultrasonic, adding the composite microbial liquid prepared in the step 3), enabling the mass ratio of the total mass of the added diatomite, sodium alginate, calcium carbonate and calcium chloride to the mass of the added composite microbial liquid to be 0.2: 1, uniformly mixing to obtain a dispersion liquid, dropwise adding the dispersion liquid into a magnesium sulfate solution with the concentration of 0.1 mol/L by using a dropper to generate spherical particle precipitates, centrifugally separating the spherical particle precipitates, slowly dropwise adding a dilute hydrochloric acid solution with the concentration of 0.01 mol/L into the separated spherical particle precipitates until no bubbles are generated, washing the obtained solid matter with water, centrifuging, and drying to obtain the microbial preparation, wherein the prepared microbial preparation is composed of particles with the particle size distribution of 1-500 micrometers, the particles are of a porous structure, and the pore size of the particles is within the range of 50-100 nm.
Comparative example 2
The preparation method of the porous microbial preparation for soil remediation comprises the following steps:
1) preparation of Rhodopseudomonas palustris (Rhodopseudomonas palustris) fermentation broth:
inoculating activated Rhodopseudomonas palustris2-8 (Rhodopseudomonas palustris2-8) into a liquid culture medium A, carrying out anaerobic culture for 3 days under the conditions of illumination intensity of 20001ux and temperature of 28-30 ℃, then carrying out anaerobic culture and amplification for 2 times in the liquid culture medium A according to 3% of inoculation concentration under the conditions of illumination intensity of 2000lux and temperature of 28 ℃, and obtaining Rhodopseudomonas palustris2-8 fermentation liquor.
The liquid culture medium A comprises the following components in percentage by mass: 0.1% of ammonium chloride, 0.05% of sodium hydrogen phosphate, 0.02% of magnesium sulfate or magnesium chloride, 0.2% of sodium chloride, 0.1% of yeast extract and the balance of water, wherein the pH value of the liquid culture medium A is 7.0.
2) Preparing a compound microbial solution:
adding ACC deaminase into the Rhodopseudomonas palustris (Rhodopseudomonas palustris) fermentation liquor prepared in the step 1) to prepare a compound microorganism bacterial liquor. The concentration of ACC deaminase in the obtained composite microbial liquid is 30U/mg.
3) Mixing diatomite, sodium alginate, 200-300-mesh calcium carbonate particles and calcium chloride in water according to the mass ratio of 1: 1.5: 0.3, uniformly dispersing by ultrasonic, adding the composite microbial liquid prepared in the step 3), enabling the mass ratio of the total mass of the added diatomite, sodium alginate, calcium carbonate and calcium chloride to the mass of the added composite microbial liquid to be 0.2: 1, uniformly mixing to obtain a dispersion liquid, dropwise adding the dispersion liquid into a magnesium sulfate solution with the concentration of 0.1 mol/L by using a dropper to generate spherical particle precipitates, centrifugally separating the spherical particle precipitates, slowly dropwise adding a dilute hydrochloric acid solution with the concentration of 0.01 mol/L into the separated spherical particle precipitates until no bubbles are generated, washing the obtained solid matter with water, centrifuging, and drying to obtain the microbial preparation, wherein the prepared microbial preparation is composed of particles with the particle size distribution of 1-500 micrometers, the particles are of a porous structure, and the pore size of the particles is within the range of 50-100 nm.
Comparative example 3
The preparation method of the porous microbial preparation for soil remediation comprises the following steps:
1) preparation of Arthrobacter (Arthrobacter sp.) L HM7719 broth:
arthrobacter (Arthrobacter sp.) L HM7719 is placed in a triangular flask with seed culture foundation, seed culture is carried out to logarithmic phase under the conditions of pH7.2 and 30 ℃, then the strain cultured to logarithmic phase is inoculated into L B liquid culture medium according to the inoculation amount of 2% of the volume of the culture medium, shaking table is carried out at the temperature of pH 7.0 and 30 ℃ for 180rpm, and culture is carried out for 48 hours, thus obtaining the Arthrobacter (Arthrobacter sp.) L HM7719 fermentation liquor.
2) Preparation of Rhodopseudomonas palustris (Rhodopseudomonas palustris) fermentation broth:
inoculating activated Rhodopseudomonas palustris2-8 (Rhodopseudomonas palustris2-8) into a liquid culture medium A, carrying out anaerobic culture for 3 days under the conditions of illumination intensity of 2000lux and temperature of 28-30 ℃, then carrying out anaerobic culture and amplification for 2 times in the liquid culture medium A according to 3% of inoculation concentration under the conditions of illumination intensity of 20001ux and temperature of 28 ℃, and obtaining Rhodopseudomonas palustris2-8 fermentation liquor.
The liquid culture medium A comprises the following components in percentage by mass: 0.1% of ammonium chloride, 0.05% of sodium hydrogen phosphate, 0.02% of magnesium sulfate or magnesium chloride, 0.2% of sodium chloride, 0.1% of yeast extract and the balance of water, wherein the pH value of the liquid culture medium A is 7.0.
3) Preparing a compound microbial solution:
uniformly mixing Arthrobacter (Arthrobacter sp.) L HM7719 fermentation liquor prepared in the step 1) and Rhodopseudomonas palustris (Rhodopseudomonas palustris) fermentation liquor prepared in the step 2) according to the mass ratio of 1: 1.2 to prepare the compound microbial liquid.
4) Mixing diatomite, sodium alginate, 200-300-mesh calcium carbonate particles and calcium chloride in water according to the mass ratio of 1: 1.5: 0.3, uniformly dispersing by ultrasonic, adding the composite microbial liquid prepared in the step 3), enabling the mass ratio of the total mass of the added diatomite, sodium alginate, calcium carbonate and calcium chloride to the mass of the added composite microbial liquid to be 0.2: 1, uniformly mixing to obtain a dispersion liquid, dropwise adding the dispersion liquid into a magnesium sulfate solution with the concentration of 0.1 mol/L by using a dropper to generate spherical particle precipitates, centrifugally separating the spherical particle precipitates, slowly dropwise adding a dilute hydrochloric acid solution with the concentration of 0.01 mol/L into the separated spherical particle precipitates until no bubbles are generated, washing the obtained solid matter with water, centrifuging, and drying to obtain the microbial preparation, wherein the prepared microbial preparation is composed of particles with the particle size distribution of 1-500 micrometers, the particles are of a porous structure, and the pore size of the particles is within the range of 50-100 nm.
Example 4
Testing the degradation capability of the porous microbial preparation for soil remediation on heavy metals in soil:
1) taking polluted soil around the waste slag pile of the smelting stone, air-drying, mashing, sieving with a 20-mesh sieve, fully and uniformly mixing, placing in an autoclave, sterilizing at 110 ℃ for 100 minutes, and cooling for later use.
2) Averagely dividing the sterilized soil obtained in the step 1) into 18 parts, respectively placing the 18 parts in a sterilized container, and dividing the 18 parts of sterilized soil into 6 groups of 3 parts. Taking the microbial preparations prepared in examples 1-3 and comparative examples 1-3, adding the microbial preparation prepared in each example/comparative example into one group of soil correspondingly, and uniformly mixing, wherein the mass of the microbial preparation added into each part of soil accounts for 0.5 percent of the mass of the soil.
3) The removal rate of exchangeable states of heavy metals in the treated soil was measured after two months, and the results are shown in table 1 by averaging 3 parts per group.
TABLE 1
The degradation capability of the porous microbial preparation for soil remediation on petroleum hydrocarbon in soil is tested:
1) the method comprises the steps of taking the contaminated soil of a gas station, crushing, screening, removing impurities such as plant roots and large stones in the contaminated soil, crushing large pieces of soil, and adjusting the pH value of the soil to be within the range of 6.5-7.5.
2) Averagely dividing the soil treated in the step 1) into 18 parts, respectively placing the 18 parts in a sterilized container, and dividing the 18 parts of sterilized soil into 6 groups of 3 parts. Taking the composite microbial soil remediation agent prepared in the examples 1-3 and the comparative examples 1-3, adding the composite microbial soil remediation agent prepared in each example/comparative example into one group of soil correspondingly, and uniformly mixing, wherein the mass of the composite microbial soil remediation agent added into the soil accounts for 0.5% of the mass of the soil.
3) After one month, the removal rate of VPH, EPA and TPH in the treated soil was measured. VPH is equivalent to gasoline and mainly comprises aliphatic hydrocarbon, aromatic hydrocarbon, olefin and naphthenic hydrocarbon of C5-C10; the EPH is equivalent to diesel oil, mainly comprises the sum of organic matters which can be extracted by dichloromethane between C10 and C40 and are not adsorbed by magnesium silicate; TPH is total petroleum hydrocarbon (sum of VPH and EPH). The results are shown in Table 2, averaged for each of the 3 parts.
TABLE 2
Claims (6)
1. A preparation method of a porous microbial preparation for soil remediation is characterized by comprising the following steps:
1) uniformly mixing Arthrobacter (Arthrobacter sp.) L HM7719 fermentation liquor and Rhodopseudomonas palustris (Rhodopseudomonas palustris) fermentation liquor according to the mass ratio of 1: 1.2-1.5 to obtain a composite microbial inoculum, adding ACC deaminase into the composite microbial inoculum, and uniformly mixing to obtain a composite microbial inoculum;
2) mixing diatomite, sodium alginate, calcium carbonate and calcium chloride in water according to the mass ratio of 1: 1-2: 0.2-0.3, uniformly dispersing by using ultrasonic waves, adding the composite microbial bacteria liquid prepared in the step 1), and uniformly mixing to obtain a dispersion liquid;
3) dropwise adding the dispersion liquid prepared in the step 2) into a magnesium sulfate solution with the concentration of 0.05-0.1 mol/L to generate spherical particle sediment, centrifugally separating the spherical particle sediment, slowly dropwise adding a dilute hydrochloric acid solution into the spherical particle sediment obtained by separation until no bubbles are generated, washing the obtained solid substance with water, centrifuging and drying to obtain the microbial preparation,
in the step 2), the mass ratio of the total mass of the added diatomite, sodium alginate, calcium carbonate and calcium chloride to the mass of the composite microbial liquid is 0.2-0.3: 1,
in the compound microorganism bacterial liquid, the live bacteria content of Arthrobacter (Arthrobacter sp.) L HM7719 fermentation liquid is 5 × 108～5×1010cfu/g, viable bacteria content of Rhodopseudomonas palustris (Rhodopseudomonas palustris) fermentation broth of 5 × 109～5×1011cfu/g, and the concentration of ACC deaminase is 25-35U/mg.
2. The method for preparing the porous microbial preparation for soil remediation according to claim 1, wherein the method for preparing the Arthrobacter (Arthrobacter sp.) L HM7719 fermentation broth comprises the steps of culturing Arthrobacter (Arthrobacter sp.) L HM7719 to logarithmic phase, inoculating the strain cultured to logarithmic phase into L B culture medium according to the inoculation amount of 2% -5% of the volume of the culture medium, shaking the table at 25-37 ℃ and 120-180 rpm, and culturing for 40-48 hours to obtain the Arthrobacter (Arthrobacter sp.) L HM7719 fermentation broth.
3. The method for producing a porous microbial preparation for soil remediation according to claim 1, wherein the Rhodopseudomonas palustris (Rhodopseudomonas palustris) is Rhodopseudomonas palustris2-8 (Rhodopseudomonas palustris 2-8).
4. The method of claim 1, wherein the Rhodopseudomonas palustris (Rhodopseudomonas palustris) fermentation broth is prepared by the method comprising: culturing Rhodopseudomonas palustris2-8 (Rhodopseudomonas palustris2-8) to logarithmic phase, then inoculating the strain cultured to logarithmic phase into a culture medium A according to the inoculum size of 2-5% of the volume of the culture medium, and culturing for 48-96 hours at 28-30 ℃ under the light anaerobic condition to obtain the Rhodopseudomonas palustris (Rhodopseudomonas palustris) fermentation liquor; the culture medium A comprises water and the following components in percentage by mass: 0.1% of ammonium chloride, 0.05% of sodium hydrogen phosphate, 0.02% of magnesium sulfate or magnesium chloride, 0.2% of sodium chloride, 0.1-0.2% of yeast extract or peptone, and the pH value of the culture medium A is 6.8-7.1.
5. The method for preparing a porous microbial preparation for soil remediation according to claim 1, wherein the calcium carbonate is composed of calcium carbonate particles having a particle size of 200 to 300 mesh.
6. The method for preparing a porous microbial preparation for soil remediation according to claim 1, wherein the concentration of the dilute hydrochloric acid solution is 0.01 to 0.05 mol/L.
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