CN110592066A - Charcoal-loaded nano zero-valent iron coupled phosphate solubilizing bacterium immobilized pellet and preparation method and application thereof - Google Patents
Charcoal-loaded nano zero-valent iron coupled phosphate solubilizing bacterium immobilized pellet and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a charcoal-loaded nano zero-valent iron coupled phosphate-solubilizing bacterium immobilized pellet as well as a preparation method and application thereof. The immobilized pellet prepared by the invention fully exerts the reduction and adsorption effects of the charcoal-loaded nano zero-valent iron on lead, simultaneously improves the resistance of phosphate-learning bacteria on the pressure of external heavy metals, and greatly strengthens the passivation of lead by the synergistic effect of the phosphate-learning bacteria and the external heavy metal. The immobilized pellet prepared by the method has the advantages of strong mechanical property, good mass transfer performance, outstanding phosphorus dissolving capacity, obvious lead passivation effect and the like, can be used for stabilizing soil lead, and has good application prospect.
Description
Technical Field
The invention belongs to the field of heavy metal pollution remediation, and particularly relates to a biochar-loaded nano zero-valent iron coupled phosphate solubilizing bacterium immobilized pellet as well as a preparation method and application thereof.
Background
Heavy metal pollution of farmland soil has great harm to environmental ecosystem and human health, heavy metal can not be biodegraded and is easy to accumulate in organisms and enter human bodies along with food chains, and thus human health and ecosystem balance are harmed. On the other hand, the low utilization rate of the phosphate fertilizer applied to farmland soil in season leads to the accumulation of a large amount of insoluble phosphorus in the soil, and the existence state of soil phosphorus can influence the migration and transformation of heavy metals. Therefore, the method has important significance in effectively stabilizing the heavy metals in the farmland soil, and the development of the high-efficiency, economic and environment-friendly heavy metal remediation technology is a problem to be solved urgently. At present, the traditional heavy metal restoration methods comprise a leaching method, a chemical precipitation method, an adsorption method and the like, but the methods have the problems of low efficiency, high cost, secondary pollution and the like when being specifically applied, the expected effect is difficult to achieve, and the microbial restoration is a very promising and ecologically benign method.
The phosphate solubilizing microorganism is used as an important component of soil microorganism, and can convert insoluble phosphorus (such as iron phosphorus, calcium phosphorus, medium-stability organic phosphorus, high-stability organic phosphorus and the like) in soil into phosphorus capable of being absorbed and utilized by plants by relying on metabolites (organic acid and phosphatase) of the microorganism or through synergistic action with other organisms, so that the utilization rate of the phosphorus in the soil can be greatly improved, the nutrient state of the phosphorus in the plants is improved, the growth of the plants is promoted, the form of heavy metal can be changed, and the remediation efficiency is improved. However, high concentrations of heavy metals inhibit the activity of phosphate solubilizing bacteria, especially bacteria in the free state, which can more easily limit the bioremediation process. .
Disclosure of Invention
The invention aims to solve the technical problems of high cost and low treatment efficiency in the prior art, develops a preparation method of a high-efficiency, ecological and environment-friendly biochar-loaded nano zero-valent iron coupled phosphate-solubilizing bacterium immobilized pellet, and applies the method to soil remediation.
The carrier-loaded nano metal coupling phosphate solubilizing bacterium immobilized pellet provided by the invention consists of a porous sphere and a load loaded on the pore wall of the porous sphere;
the material for forming the porous sphere is porous gel;
the load comprises a reinforced material and phosphate solubilizing bacteria mixed in the reinforced material;
in the carrier-loaded nano metal coupled phosphate solubilizing bacterium immobilized pellet, the porous gel is a product obtained by cross-linking polymerization of at least one of polyvinyl alcohol and sodium alginate;
the reinforced material is carbon material loaded with nano metal;
the phosphate solubilizing bacteria are selected from at least one of non-decarboxylating lechler bacteria, bacillus thuringiensis and pseudomonas putida;
in particular, the method comprises the following steps of,
the metal is iron;
the valence state of the nano metal is selected from at least one of zero valence, divalent state and trivalent state;
the nano metal is specifically nano zero-valent iron;
the carbon material is biochar or activated carbon;
the particle size of the carbon material is specifically less than or equal to 0.177 mm.
Specifically, the mass ratio of the carbon material to the nano metal is 3-5: 1;
the volume ratio of the mass of the reinforced material to the bacteria liquid of the phosphate solubilizing bacteria is 1: 50-62.5; specifically, the OD of the bacterial liquid of the phosphate solubilizing bacteria600＝0.6-0.8；
The diameter of the carrier-loaded nano metal coupling phosphate solubilizing bacteria immobilized pellet is 4mm-8 mm; in particular 6 mm.
The invention provides a preparation method of a biochar-loaded nano zero-valent iron coupled phosphate solubilizing bacteria immobilized pellet, which is characterized in that biochar-loaded nano zero-valent iron and phosphate solubilizing bacteria are embedded in a pellet formed by crosslinking polyvinyl alcohol, sodium alginate and a calcium chloride saturated boric acid solution, and the prepared immobilized pellet has the advantages of high mechanical strength, good mass transfer efficiency, quick treatment effect and the like.
The method for preparing the carrier-loaded nano metal coupled phosphate solubilizing bacterium immobilized pellet comprises the following steps:
1) adding the reinforced material and the seed solution of the phosphate solubilizing bacteria into a gel to obtain a thallus embedding solution;
2) and adding the thallus embedding liquid into a crosslinking liquid for crosslinking reaction, and obtaining the carrier-loaded nano metal coupling phosphate solubilizing bacteria immobilized pellet after the reaction is finished.
In step 1) of the above method, the gelling agent is at least one selected from an aqueous solution of polyvinyl alcohol and an aqueous solution of sodium alginate;
in the aqueous solution of the polyvinyl alcohol, the molecular weight of the polyvinyl alcohol is 75000-85000; the dosage ratio of the polyvinyl alcohol to the water is 8-10 g: 100 mL; specifically, 9 g: 100 mL;
in the sodium alginate aqueous solution, the dosage ratio of sodium alginate to water is 0.8-1 g: 100 mL.
In the step 2), the crosslinking solution is a saturated boric acid solution and 3% CaCl2Aqueous solution or 3% calcium chloride in saturated aqueous boric acid;
in the adding step, the speed of adding the thallus embedding liquid into the cross-linking liquid is 0.5-1 drop/s;
in the step of the crosslinking reaction, the stirring speed is 100-150 rpm; the temperature is room temperature; the time is 12-24 h.
The raw materials in the step 1) and the step 2) are sterilized before use.
In the above preparation method, all operations are performed under aseptic conditions.
In addition, the application of the carrier-loaded nano metal coupled phosphate solubilizing bacterium immobilized pellet in the remediation of heavy metal contaminated soil also belongs to the protection scope of the invention. Specifically, the heavy metal is lead, cadmium or zinc;
the dosage of the carrier-loaded nano metal coupled phosphate-solubilizing bacterium immobilized pellet is 2-10% of the weight of the heavy metal contaminated soil; specifically 5%.
The invention utilizes the biochar to stabilize the nano zero-valent iron, and then the nano zero-valent iron and the phosphate solubilizing bacteria are fixed in a grid structure formed by polyvinyl alcohol-sodium alginate to form immobilized beads. The biochar provides a favorable inhabitation environment for the growth of phosphate solubilizing bacteria, the reduction condition provided by the nano zero-valent iron is favorable for the release of soil phosphorus, an iron source is provided for the growth of the phosphate solubilizing bacteria in the oxidation process, and meanwhile, the nano particles are fixed in the pellets, so that the stability of the nano zero-valent iron is realized, and the secondary pollution caused by migration is avoided. Meanwhile, the phosphate solubilizing bacteria are fixed in the carrier, so that the phosphate solubilizing bacteria can obtain substances and energy sources from the outside of the carrier and continuously release soluble phosphorus, and can resist severe environment.
Compared with the prior art, the invention has the advantages that:
1) polyvinyl alcohol and sodium alginate are common immobilized carriers, the biochar has wide sources and low cost, the biochar-loaded nano zero-valent iron has large specific surface area and high reaction rate, and the material for producing the pellets has low price and low cost.
2) The immobilized phosphate solubilizing bacteria are used, so that insoluble phosphorus in soil can be activated, the investment of phosphorus-containing substances is reduced, secondary pollution is avoided, and heavy metals in soil can be passivated.
3) The biochar-loaded nano zero-valent iron coupled phosphate solubilizing bacterium immobilized pellet prepared by the method has high-efficiency phosphate solubilizing capability and certain heavy metal resistance, and can treat heavy metal pollution of farmland soil.
4) The nanotechnology is combined with the microbial remediation technology, so that the defects (such as slow single microbial remediation) caused by the traditional single technology are overcome, the environment-friendly and efficient remediation of the heavy metal pollution of the soil can be realized, and the method has a good application prospect.
Drawings
Fig. 1 is a schematic view and a schematic particle size of a biochar-loaded nano zero-valent iron coupled phosphate solubilizing bacterium immobilized pellet prepared in embodiment 1 of the present invention.
FIG. 2 is a graph showing the phosphate-solubilizing ability and lead-removing effect of immobilized beads and free bacteria in example 3 of the present invention.
FIG. 3 is a graph showing the effect of the addition of immobilized beads under lead pressure on lead removal in example 4 of the present invention.
Detailed Description
The present invention will be further illustrated with reference to the following specific examples, but the present invention is not limited to the following examples. The method is a conventional method unless otherwise specified. The starting materials are commercially available from the open literature unless otherwise specified.
The strain MRP-1 used in the following examples has been deposited in China general microbiological culture Collection center (CGMCC, address: No. 3 Siro 1 Kyoh-West Luo-Yang district, Beijing) at 22.08.2017, has a deposition number of CGMCC No.14561, and is classified and named as non-decarboxylating bacterium (Leclercia adecaboxylata);
the used strain MRP-2 is preserved in China general microbiological culture Collection center (CGMCC for short, the address is No. 3 Siro No.1 of Beijing city Kogyo No. 3 of the south China Committee for culture Collection of microorganisms) in 2018, 02 and 02 months, the preservation number is CGMCC NO.15337, and the strain is classified and named as Pseudomonas putida (Pseudomonas putida);
the strain MRP-3 has been preserved in China general microbiological culture Collection center (CGMCC, address: Siro No.1 Hospital No. 3 of Beijing market open-facing area north Chen) in 2018, 02/3, the preservation number is CGMCC NO.15338, and the strain is classified and named as Bacillus thuringiensis
The preparation method of the biochar-loaded nano zero-valent iron used in the following examples is various conventional methods, and can be prepared according to the following methods: treating biochar (the particle size is less than or equal to 0.177mm) with 200mL of 1mol/L hydrochloric acid solution for 12h, carrying out suction filtration, and then washing with distilled water until the pH value of a washing solution is 6.5-7.0; finally, the biochar is placed in an oven at 80 ℃ for drying for 24 h. By using a liquid phase reduction method, the method comprises the following steps of: the mass ratio of iron is 3: 1, preparing the charcoal-loaded nano zero-valent iron
Example 1 preparation of charcoal-loaded nanoscale zero-valent iron-coupled phosphate solubilizing bacterium immobilized pellet
1) 4.5g of polyvinyl alcohol was added to 50mL of deionized water, heated in a water bath at 40 ℃ overnight, then warmed to 80 ℃ until completely dissolved, and sterilized in an autoclave at 121 ℃ and 110Kpa for 30 min. When the temperature is reduced to 40 ℃, 0.5g of sodium alginate subjected to ultraviolet sterilization is added into the mixture, and the mixture is stirred uniformly until the sodium alginate is completely dissolved.
2) Enrichment culture is carried out on non-decarboxylating leclerian (Leclercia adecaboxylinata), and the components of a liquid culture medium comprise 3.0g of beef extract, 5.0g of sodium chloride, 10g of peptone and 1000mL of distilled water. And (3) filling 50mL of beef extract peptone liquid culture medium in a 150mL triangular conical flask, and sterilizing. A ring of non-decarboxylating lechi bacteria stored in a slant culture medium is selected and inoculated in a beef extract peptone liquid culture medium, and the culture is carried out at 120rpm and 37 ℃ for 24h to obtain the phosphate solubilizing bacteria seed liquid.
0.45g of ultraviolet sterilized biochar loaded with nano zero-valent iron and 25mL of OD600Adding 0.6-0.8 parts of phosphate solubilizing bacteria seed liquid slowly into the solution 1), and stirring and mixing uniformly.
3) The mixed solution is dripped into a saturated boric acid aqueous solution of 3 percent calcium chloride with pH of 6.8-7.0 by a peristaltic pump at the speed of 1 drop/s for crosslinking at room temperature for 24 hours, and stirring is carried out while reacting, wherein the stirring speed is 150 revolutions per minute.
4) And then cleaning the pellets to be neutral by using a saturated sodium chloride solution, and storing the pellets in a refrigerator at 4 ℃ for later use to obtain the biochar-loaded nano zero-valent iron coupled phosphate-solubilizing bacteria immobilized pellets.
The charcoal-loaded nanoscale zero-valent iron-coupled phosphate-solubilizing bacteria immobilized pellet obtained in example 1 is off-white in appearance, and the diameter of the pellet is measured by a vernier caliper, as shown in fig. 1, and is about 6 mm.
Example 2 and example 1 Performance determination of charcoal-loaded nanoscale zero-valent iron-coupled phosphate solubilizing bacterium immobilized pellet
1) Mechanical strength: the intensity coefficient of the pellets was represented by the ratio of intact pellets to the total number of original pellets after placing 60 pellets in a 250mL Erlenmeyer flask, adding 100mL deionized water, and shaking on a constant temperature shaker for 12 hours.
2) Expansibility: the diameter of 30 pellets was measured with a vernier caliper and then placed in a 250mL conical flask, 100mL of deionized water was added, and the flask was refrigerated at 4 ℃ for 6 hours, and then the diameter was measured as follows: the ratio of the sum of the diameters of the pellets measured after expansion to the sum of the diameters of the original pellets indicates the expansion properties of the pellets.
3) Mass transfer performance: the mass transfer performance of the immobilized beads was characterized by absorbance. Washing the pellet with distilled water, drying the surface water with filter paper, weighing 0.5g of the pellet in a clean beaker, adding 100 times diluted red ink into the beaker until the pellet is immersed, and measuring the absorbance at 406nm for 10 min.
TABLE 1 Performance parameters of charcoal-loaded nanoscale zero-valent iron-coupled phosphate solubilizing bacteria immobilized pellets obtained in example 1
As can be seen from Table 1, the charcoal-supported nano zero-valent iron coupled phosphate solubilizing bacterium immobilized pellet provided by the invention has good mechanical properties, expansion properties and mass transfer properties.
Determination of phosphate solubilizing ability and heavy metal removal ability of biochar-supported nano zero-valent iron coupled phosphate solubilizing bacteria immobilized pellet obtained in example 3 and example 1
(1) Determination of phosphate solubilizing ability
1) A ring of non-decarboxylating lechi (Leclercia adecarboxylata) thallus stored in a slant culture medium is selected and inoculated into a beef extract peptone liquid culture medium, and the beef extract peptone liquid culture medium is cultured at 120rpm and 37 ℃ for 24 hours to obtain a seed liquid.
2) The immobilized pellet soaked in normal saline is dried by using sterile cotton, inoculated in the same beef extract peptone liquid culture medium at 120rpm and 37 ℃ for 24h for activation.
3) Respectively inoculating the activated free bacteria and the immobilized pellets into sterilized Ca according to the inoculation amount of 2 percent3(PO4)2And (3) culturing in a liquid culture medium with a control in an incubator at 37 ℃ and 120rpm for 24h, centrifuging at 4500rpm for 10min, filtering the supernatant with a 0.45um filter membrane, and measuring the phosphate solubilizing capacity by a molybdenum blue colorimetric method.
Wherein the tricalcium phosphate liquid culture medium comprises the following components: 10.0g of glucose, 0.3g of sodium chloride, 0.3g of magnesium sulfate, 0.03g of manganese sulfate, 0.5g of ammonium sulfate, 0.3g of potassium chloride, 0.03g of ferric sulfate, 5.0g of calcium phosphate, 1000mL of distilled water and pH of 7.2-7.4.
In FIG. 2, a is the phosphate solubilizing ability of the charcoal-supported nano zero-valent iron coupled phosphate solubilizing bacteria immobilized pellet obtained in example 1. As can be seen from the figure, after the immobilized pellet is cultured in tricalcium phosphate culture medium for 24 hours, the soluble phosphorus content is higher than that of blank and free bacteria, and reaches 195.27 mg/L.
(2) Determination of the ability to remove heavy metals
1) A ring of non-decarboxylating lechi (Leclercia adecarboxylata) thallus stored in a slant culture medium is selected and inoculated into a beef extract peptone liquid culture medium, and the beef extract peptone liquid culture medium is cultured at 120rpm and 37 ℃ for 24 hours to obtain a seed liquid.
2) The immobilized pellet soaked in normal saline is dried by using sterile cotton, inoculated in the same beef extract peptone liquid culture medium at 120rpm and 37 ℃ for 24h for activation.
3) Inoculating activated free bacteria and immobilized pellet with 2% inoculation amount into sterilized liquid beef extract peptone culture medium containing 1mmol/L lead, setting control, culturing in incubator at 37 deg.C at 120rpm for 24 hr, centrifuging at 4500rpm for 10min, filtering the supernatant with 0.45um filter membrane, and measuring Pb in the supernatant by flame atomic absorption method2+And (4) concentration.
Wherein the method for configuring the liquid beef extract peptone medium containing 1mmol/L of lead comprises the following steps: using a lead nitrate stock solution with the concentration of 500mmol/L, sucking 0.1mL to 50mL beef extract peptone culture medium to prepare the culture condition with the heavy metal concentration of 1 mmol/L.
In FIG. 2, b is the ability of the charcoal-loaded nano-sized zero-valent iron coupled phosphate-solubilizing bacteria immobilized beads obtained in example 1 to remove heavy metal lead. As can be seen from the figure, the removal rate of the immobilized beads to the heavy metal lead reaches 69%, which is improved by 42% compared with that of the free bacteria, and the immobilized beads have better heavy metal removal efficiency.
Example 4 influence of the amount of the biochar-loaded nano zero-valent iron coupled phosphate solubilizing bacteria immobilized pellet obtained in example 1 on lead removal under heavy metal pressure
1) The immobilized pellet soaked in normal saline is dried by using sterile cotton, inoculated in the same beef extract peptone liquid culture medium at 120rpm and 37 ℃ for 24h for activation.
2) Inoculating the activated immobilized beads into sterilized liquid beef extract peptone medium containing 1mmol/L lead in an amount of 0.5%, 1%, 2.5%, 5%, respectively, setting control, culturing at 120rpm and 37 deg.C for 24 hr, centrifuging at 4500rpm for 10min, filtering the supernatant with 0.45 μm filter membrane, and measuring Pb in the supernatant by flame atomic absorption2+And (4) concentration.
Wherein the preparation method of the beef extract peptone liquid culture medium containing 1mmol/L of lead comprises the following steps: using a lead nitrate stock solution with the concentration of 500mmol/L, sucking 0.1mL to 50mL beef extract peptone culture medium to prepare a culture condition with the lead ion concentration of 1 mmol/L.
FIG. 3 is a graph showing the relationship between the amount of immobilized beads of charcoal-loaded nanoscale zero-valent iron-coupled phosphate solubilizing bacteria and the lead removal rate in example 1. As can be seen from the figure, the removal rate of lead ions increases with the addition amount of the immobilized beads, and when the addition amount of the immobilized beads is 5% (2.5g), the removal rate of lead ions can reach more than 80%, and the removal of lead ions can be basically satisfied.
Claims (9)
1. A carrier-loaded nano-metal coupled phosphate solubilizing bacterium immobilized pellet comprises a porous sphere and a load loaded on the pore wall of the porous sphere;
the material for forming the porous sphere is porous gel;
the load comprises a reinforced material and phosphate solubilizing bacteria mixed in the reinforced material.
2. The carrier-supported nano-metal coupled phosphate solubilizing bacterium immobilized bead as claimed in claim 1, wherein: the porous gel is a product obtained by cross-linking and polymerizing at least one of polyvinyl alcohol and sodium alginate serving as raw materials;
the reinforced material is carbon material loaded with nano metal;
the phosphate solubilizing bacteria are selected from at least one of non-decarboxylating lechler bacteria, bacillus thuringiensis and pseudomonas putida;
in particular, the method comprises the following steps of,
the metal is iron;
the valence state of the nano metal is selected from at least one of zero valence, divalent state and trivalent state;
the nano metal is specifically nano zero-valent iron;
the carbon material is biochar or activated carbon;
the particle size of the carbon material is specifically less than or equal to 0.177 mm.
3. The carrier-supported nano-metal coupled phosphate solubilizing bacterium immobilized bead as claimed in claim 2, wherein: the mass ratio of the carbon material to the nano metal is 3-5: 1;
the volume ratio of the mass of the reinforced material to the bacteria liquid of the phosphate solubilizing bacteria is 1: 50-62.5; specifically, the OD of the bacterial liquid of the phosphate solubilizing bacteria600＝0.6-0.8；
The diameter of the carrier-loaded nano metal coupling phosphate solubilizing bacteria immobilized pellet is 4mm-8 mm; in particular 6 mm.
4. A method for preparing the supported nanometal-coupled phosphate-solubilizing bacterium immobilized bead as defined in any one of claims 1 to 3, comprising:
1) adding the reinforced material and the seed solution of the phosphate solubilizing bacteria into a gel to obtain a thallus embedding solution;
2) and adding the thallus embedding liquid into a crosslinking liquid for crosslinking reaction, and obtaining the carrier-loaded nano metal coupling phosphate solubilizing bacteria immobilized pellet after the reaction is finished.
5. The method of claim 4, wherein: in the step 1), the gelling agent is at least one of an aqueous solution of polyvinyl alcohol and an aqueous solution of sodium alginate;
in the aqueous solution of the polyvinyl alcohol, the molecular weight of the polyvinyl alcohol is 75000-85000; the dosage ratio of the polyvinyl alcohol to the water is 8-10 g: 100 mL; specifically, 9 g: 100 mL;
in the sodium alginate aqueous solution, the dosage ratio of sodium alginate to water is 0.8-1 g: 100 mL.
6. The method according to claim 4 or 5, characterized in that: in the step 2), the crosslinking solution is a saturated boric acid solution and 3% CaCl2Aqueous solution or 3% calcium chloride in saturated aqueous boric acid;
in the adding step, the speed of adding the thallus embedding liquid into the cross-linking liquid is 0.5-1 drop/s;
in the step of the crosslinking reaction, the stirring speed is 100-150 rpm; the temperature is room temperature; the time is 12-24 h.
7. The method according to any one of claims 4-6, wherein: the raw materials in the step 1) and the step 2) are sterilized before use.
8. The use of the immobilized beads of the carrier-supported nanometal-coupled phosphate-solubilizing bacteria of any one of claims 1 to 3 in the remediation of heavy metal contaminated soil.
9. Use according to claim 8, characterized in that: the heavy metal is lead, cadmium or zinc;
the dosage of the carrier-loaded nano metal coupled phosphate-solubilizing bacterium immobilized pellet is 2-10% of the weight of the heavy metal contaminated soil; specifically 5%.
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