CN114107090B - Biochemical composite material and preparation method and application thereof - Google Patents
Biochemical composite material and preparation method and application thereof Download PDFInfo
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- CN114107090B CN114107090B CN202111252716.7A CN202111252716A CN114107090B CN 114107090 B CN114107090 B CN 114107090B CN 202111252716 A CN202111252716 A CN 202111252716A CN 114107090 B CN114107090 B CN 114107090B
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- 239000002131 composite material Substances 0.000 title claims abstract description 65
- 238000002360 preparation method Methods 0.000 title claims abstract description 12
- 239000002689 soil Substances 0.000 claims abstract description 74
- 108010046334 Urease Proteins 0.000 claims abstract description 70
- 229910001385 heavy metal Inorganic materials 0.000 claims abstract description 40
- 230000001580 bacterial effect Effects 0.000 claims abstract description 30
- 239000013501 sustainable material Substances 0.000 claims abstract description 20
- 244000005700 microbiome Species 0.000 claims abstract description 14
- 238000000034 method Methods 0.000 claims description 37
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 32
- 239000004202 carbamide Substances 0.000 claims description 32
- 235000015097 nutrients Nutrition 0.000 claims description 12
- 239000000725 suspension Substances 0.000 claims description 12
- 239000000463 material Substances 0.000 claims description 10
- 230000003100 immobilizing effect Effects 0.000 claims description 9
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 8
- 235000012054 meals Nutrition 0.000 claims description 8
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 7
- 229910052793 cadmium Inorganic materials 0.000 claims description 7
- BDOSMKKIYDKNTQ-UHFFFAOYSA-N cadmium atom Chemical compound [Cd] BDOSMKKIYDKNTQ-UHFFFAOYSA-N 0.000 claims description 7
- 239000001110 calcium chloride Substances 0.000 claims description 7
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 7
- 238000002156 mixing Methods 0.000 claims description 5
- 230000008569 process Effects 0.000 claims description 5
- 229910052757 nitrogen Inorganic materials 0.000 claims description 4
- 238000012258 culturing Methods 0.000 claims description 2
- 230000000694 effects Effects 0.000 abstract description 21
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 abstract description 12
- 239000000843 powder Substances 0.000 abstract description 12
- 230000008021 deposition Effects 0.000 abstract description 8
- 230000009286 beneficial effect Effects 0.000 abstract description 6
- 229910000019 calcium carbonate Inorganic materials 0.000 abstract description 6
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 abstract description 5
- 229910052791 calcium Inorganic materials 0.000 abstract description 5
- 239000011575 calcium Substances 0.000 abstract description 5
- 238000004519 manufacturing process Methods 0.000 abstract description 5
- 230000007613 environmental effect Effects 0.000 abstract description 3
- 239000000109 continuous material Substances 0.000 abstract description 2
- 238000004321 preservation Methods 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 53
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 12
- 238000004458 analytical method Methods 0.000 description 10
- 239000003153 chemical reaction reagent Substances 0.000 description 10
- 241001118866 Bacillus sp. WA Species 0.000 description 9
- 238000002474 experimental method Methods 0.000 description 9
- 238000005067 remediation Methods 0.000 description 9
- 239000000523 sample Substances 0.000 description 9
- 239000001963 growth medium Substances 0.000 description 8
- 238000009631 Broth culture Methods 0.000 description 7
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 6
- 239000008367 deionised water Substances 0.000 description 6
- 229910021641 deionized water Inorganic materials 0.000 description 6
- 238000001514 detection method Methods 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 238000005033 Fourier transform infrared spectroscopy Methods 0.000 description 5
- 238000001556 precipitation Methods 0.000 description 5
- 230000001954 sterilising effect Effects 0.000 description 5
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 239000011159 matrix material Substances 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 229910052751 metal Inorganic materials 0.000 description 4
- 239000002184 metal Substances 0.000 description 4
- 238000001179 sorption measurement Methods 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 108050001049 Extracellular proteins Proteins 0.000 description 3
- 239000000969 carrier Substances 0.000 description 3
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- 238000005516 engineering process Methods 0.000 description 3
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- 239000008363 phosphate buffer Substances 0.000 description 3
- 230000003068 static effect Effects 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 240000008042 Zea mays Species 0.000 description 2
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 2
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 235000005822 corn Nutrition 0.000 description 2
- 238000011161 development Methods 0.000 description 2
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- 239000010793 electronic waste Substances 0.000 description 2
- 231100000584 environmental toxicity Toxicity 0.000 description 2
- 125000000524 functional group Chemical group 0.000 description 2
- 238000003384 imaging method Methods 0.000 description 2
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- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 2
- 238000005342 ion exchange Methods 0.000 description 2
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- 239000011259 mixed solution Substances 0.000 description 2
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- 239000011148 porous material Substances 0.000 description 2
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- 230000008439 repair process Effects 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 241000609240 Ambelania acida Species 0.000 description 1
- 102100036008 CD48 antigen Human genes 0.000 description 1
- 229910021532 Calcite Inorganic materials 0.000 description 1
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 101000716130 Homo sapiens CD48 antigen Proteins 0.000 description 1
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- 235000010627 Phaseolus vulgaris Nutrition 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000012271 agricultural production Methods 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 239000010905 bagasse Substances 0.000 description 1
- 231100000693 bioaccumulation Toxicity 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 230000033558 biomineral tissue development Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- YKYOUMDCQGMQQO-UHFFFAOYSA-L cadmium dichloride Chemical compound Cl[Cd]Cl YKYOUMDCQGMQQO-UHFFFAOYSA-L 0.000 description 1
- XIEPJMXMMWZAAV-UHFFFAOYSA-N cadmium nitrate Inorganic materials [Cd+2].[O-][N+]([O-])=O.[O-][N+]([O-])=O XIEPJMXMMWZAAV-UHFFFAOYSA-N 0.000 description 1
- 230000002308 calcification Effects 0.000 description 1
- 229910001424 calcium ion Inorganic materials 0.000 description 1
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- 239000012895 dilution Substances 0.000 description 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 229910000397 disodium phosphate Inorganic materials 0.000 description 1
- 235000019800 disodium phosphate Nutrition 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 239000002657 fibrous material Substances 0.000 description 1
- 238000000799 fluorescence microscopy Methods 0.000 description 1
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- 239000012535 impurity Substances 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 229910000403 monosodium phosphate Inorganic materials 0.000 description 1
- 235000019799 monosodium phosphate Nutrition 0.000 description 1
- 238000010899 nucleation Methods 0.000 description 1
- 239000006916 nutrient agar Substances 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- NMHMNPHRMNGLLB-UHFFFAOYSA-N phloretic acid Chemical compound OC(=O)CCC1=CC=C(O)C=C1 NMHMNPHRMNGLLB-UHFFFAOYSA-N 0.000 description 1
- 230000008635 plant growth Effects 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 238000004382 potting Methods 0.000 description 1
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- 239000012488 sample solution Substances 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 1
- 238000003900 soil pollution Methods 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
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Classifications
-
- 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
-
- 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
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- 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
-
- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
- C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
- C12Y305/01—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in linear amides (3.5.1)
- C12Y305/01005—Urease (3.5.1.5)
Abstract
The invention relates to the technical cross field of microorganisms and environmental engineering, and particularly discloses a biochemical composite material and a preparation method and application thereof. The biochemical composite material comprises urease producing strain and sustainable material carrier, wherein the urease producing strain is preserved in the microorganism strain preservation center of Guangdong province in 2021, 3 and 15 days, and the preservation number is GDMCC NO. 61559. The urease-producing strain which can promote the calcium carbonate deposition induced by microorganisms is screened out; the biochemical composite material has the advantages of high urease production capacity, optimal bacterial reproduction capacity and high resistance to heavy metal environment, and is beneficial to repairing heavy metal polluted soil; the invention also adopts corncob powder as a continuous material carrier, has low cost, and has extremely high promotion effect on enrichment of urease-producing strains in soil and calcium deposition induced by the strains as a carrier.
Description
Technical Field
The invention relates to the technical cross field of microorganisms and environmental engineering, in particular to a biochemical composite material and a preparation method and application thereof.
Background
Heavy metal soil pollution poses a threat to biodiversity, agricultural production, overall environment and human health. Various soil remediation techniques are currently employed in many countries to reduce soil metal concentrations, such as physicochemical remediation and phytoremediation, however, these techniques still suffer from a number of drawbacks. Microbial calcification is a special biomineralization method as a new research field of environmental rock and soil in recent years, and the core technology is MICP (Microbially Induced Calcite Precipitation, calcium carbonate precipitation is induced by microorganisms), urease for decomposing urea can be generated by metabolism of microorganisms in nature, and carbonate ions generated after urea decomposition are combined with free metal calcium ions in nature to generate a gel crystal process, so that heavy metal immobilization is achieved, and the rigidity and strength of soil texture are improved. At present, the traditional method for repairing the heavy metal contaminated soil has certain limitation, and the microbial repairing technology is nondestructive, green and economical and environment-friendly, so that the method is gradually developed into a soil repairing technology with great prospect.
In heavy metal contaminated soil, the MICP reaction largely fixes the heavy metal ions in an exchanged state into a carbonate precipitation form having internal stability. By this step, the biotoxicity and bioavailability of the heavy metals are reduced. It is therefore highly desirable and urgent to develop an efficient, low cost, sustainable ecological metal removal process. However, exogenous urease-producing microorganisms are limited in survival in this soil remediation mode due to competition with local microorganisms and unfavorable growth conditions in open soil. Furthermore, direct incorporation of exogenous inoculation microorganisms reduces their vertical transport and colonization ability in the soil. Thus, there is a need for a suitable seeding material to better utilize microbial induced calcium carbonate precipitation for soil remediation.
Biochemical Composite Materials (BCM) formed by combining the method for fixing soil heavy metals by using the microorganisms and biochar as carriers have also been proved to be beneficial to plant growth. Biochar, however, is more expensive as a thermally decomposed matrix than naturally available fibrous or porous materials. In this context, it is contemplated to introduce other cost-effective matrices or biological materials for immobilization and stabilization of urease-producing bacteria.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a biochemical composite material, a preparation method and application thereof. The urease-producing strain with high yield of urease is screened out, so that the biologically induced calcium carbonate deposition (MICP) can be promoted; the strain is added into the biochemical composite material, so that the biochemical composite material has high-efficiency calcium carbonate precipitation capability, is beneficial to the restoration of heavy metal contaminated soil, and has higher urease activity, urease yield and optimal bacterial reproduction capability.
The first object of the invention is to provide a urease-producing strain, the sequence of which is shown as SEQ ID NO. 1, wherein the urease-producing strain is Bacillus sp.WA which is deposited in the microorganism strain deposit center of Guangdong province on the 3 rd month 15 th year of 2021, and the deposit address is 59 th floor 5 of the 100 th university of Mitsui, guangzhou city, and the deposit number is GDMCC NO. 61559.
The Bacillus sp.WA is separated from soil collected from an electronic waste field, and is subjected to bacterial culture for 24 hours under the static culture condition of 25-35 ℃.
The invention provides a second object, and provides application of the urease-producing strain in preparing urease.
The invention provides a third purpose of the urease-producing strain in reducing urea content in heavy metal soil.
The Bacillus sp.WA can catalyze urea to hydrolyze into ammonia and carbonate in an environment of adding a nutrient source and urea, and further allows carbonate ions to combine with free heavy metal ions to form metal carbonate, so that the soil heavy metal immobilization is realized.
The fourth object is to provide a biochemical composite material comprising the urease producing strain and a sustainable material carrier.
The biochemical composite material has the advantages of high urease production capacity and high resistance to heavy metal environment. And the biochemical composite material can not generate extra substances with environmental toxicity, can not generate secondary pollution, and promotes sustainable development.
The yield of urease produced by adding the biochemical composite material consisting of Bacillus sp.WA and sustainable material carrier can reach 4U/mg-5U/mg.
As a preferred embodiment of the biochemical composite material according to the present invention, the sustainable material carrier includes at least one of corncob meal, okara, wood chips and bagasse, and more preferably, the sustainable material carrier is corncob meal.
Referring to fig. 5, when the sustainable material carrier is corncob meal, it is low in cost and has extremely high promotion effect on enrichment of urease-producing strains in soil and strain-induced calcium deposition as a carrier, without using high-cost biochar as a bacterial carrier.
The fifth object of the present invention is to provide a method for preparing the biochemical composite material, comprising the following steps:
s1, culturing the urease-producing strain in a nutrient medium containing a nitrogen source at the temperature of 30-40 ℃ and the pH value of 6.0-7.0 to obtain a bacterial suspension;
s2, mixing and centrifuging the bacterial suspension prepared in the step S1 and a sustainable material carrier to obtain the biochemical composite material.
More preferably, the urease producing strain in step S1 is under aerobic and static culture conditions.
As a preferred embodiment of the method for preparing a biochemical composite according to the present invention, the ratio of the volume of the bacterial suspension to the mass of the sustainable material carrier is (10-20): 1, more preferably the ratio of the volume of the bacterial suspension to the mass of the sustainable material carrier is 10:1.
When the ratio of the volume of the bacterial suspension to the mass of the sustainable material carrier is in the above range, the adsorption fixing ability of the sustainable material carrier to bacteria is better, and when the ratio of the volume of the bacterial suspension to the mass of the sustainable material carrier is 10:1, the adsorption fixing ability of the sustainable material carrier to bacteria is optimal, and fig. 4 provides a photograph of the biochemical composite material observed by a scanning electron microscope at the mass ratio.
As a preferred embodiment of the preparation method of the biochemical composite material, in the step S2, the bacterial suspension prepared in the step S1 and the sustainable material carrier are mixed for 8-16 hours at the temperature of 30-40 ℃ and the rotating speed of 120-160 rpm/min.
As a preferred embodiment of the method for preparing a biochemical composite material according to the present invention, the nitrogen source includes urea, and the concentration of the urea is 2% -3%.
As a preferred embodiment of the method for preparing a biochemical composite material according to the present invention, the nutrient medium comprises calcium chloride, and the concentration of the calcium chloride is less than 25ppm.
The sixth object is to provide a method for immobilizing heavy metals in soil, which adopts the urease-producing strain or the biochemical composite material to immobilize heavy metals in soil.
When the urease-producing strain or the biochemical composite material is used for fixing the heavy metals in soil, a large amount of chemical fixing agents are not needed to be added, so that the secondary pollution caused by chemical agents is reduced.
As a preferred embodiment of the method for immobilizing heavy metals in soil according to the present invention, the above biochemical composite material is added to the soil contaminated with heavy metals for 2 to 4 weeks.
As a preferred embodiment of the method for immobilizing heavy metals in soil according to the present invention, the concentration of the biochemical composite material is 10% (v: w) g material/kg soil to 20% (v: w) g material/kg soil.
As a preferred embodiment of the method for immobilizing heavy metals in soil according to the present invention, the heavy metals include cadmium, and each kilogram of soil includes 5mg to 12mg of cadmium.
The seventh object of the invention is to provide the application of the biochemical composite material in repairing heavy metal contaminated soil.
The biochemical composite material disclosed by the invention is applied to repairing heavy metal polluted soil, and a large amount of chemical fixing agents are not required to be added, so that the repairing cost can be reduced.
When the biochemical composite material is added into heavy metal contaminated soil, the highest functional group on the surface of a soil sample is found, which is favorable for the adsorption of the strain, the high density of the strain and the improvement of the efficiency of the biological calcium deposition induced by the strain. According to the soil remediation experiment for 28 days, the ion exchange state heavy metal in the soil added with the biochemical composite material is reduced by half and reduced to below national standard (0.5 mg/kg), and the carbonate binding state content in a stable form is increased by two times, so that the problem of heavy metal pollution of the soil is effectively solved.
Compared with the prior art, the invention has the following beneficial effects:
1) The invention screens a urease-producing strain of high-yield urease, which can promote microorganism-induced calcium carbonate deposition (MICP);
2) The biochemical composite material has the advantages of high urease production capacity and high resistance to heavy metal environment; the biochemical composite material does not generate extra substances with environmental toxicity, does not generate secondary pollution, and promotes sustainable development;
3) The corn cob powder is used as a continuous material carrier, the cost is low, and the corn cob powder is used as a carrier to have extremely high promotion effect on enrichment of urease-producing strains in soil and calcium deposition induced by the strains, so that high-cost biochar is not required to be used as a bacterial carrier, and the production cost is saved;
4) In the soil restoration process, the tested soil added with the biochemical composite material has the highest urease activity, the highest yield and the highest bacterial reproduction capacity.
Drawings
FIG. 1 is a bacterial screening process of example 1;
FIG. 2 is a graph showing the results of analysis of urease activity of urease-producing strains;
FIG. 3 is an electron scanning microscope (SEM) imaging of the biochemical composite prepared in example 2;
FIG. 4 is an electron scanning microscope (SEM) image of the biochemical composite prepared in example 2;
FIG. 5 is a Fourier Transform Infrared (FTIR) spectrum of a biochemical composite prepared from two materials, corncob meal and okara, as bacterial carriers;
FIG. 6 is a Fourier transform Infrared Spectroscopy (FTIR) spectrum of the biochemical composite prepared in example 2;
FIG. 7 is a graph of urease activity of the biochemical composite material prepared in example 2;
FIG. 8 is a graph showing cell densities of soil samples to which the biochemical composite material, the urease-producing strain, the corncob meal, and the urea nutrient solution prepared in example 2 were added, respectively (a is a soil sample to which the biochemical composite material was added, b is a soil sample to which the urease-producing strain was added, c is a soil sample to which the corncob meal was added, and d is a soil sample to which the urea nutrient solution was added);
FIG. 9 is a graph showing the analysis of heavy metal distribution between the biochemical composite material prepared in example 2 and a control sample under the same experiment after the biochemical composite material is placed in cadmium-containing soil;
fig. 10 is a flowchart of the soil remediation experiment in test example one.
Detailed Description
For a better description of the objects, technical solutions and advantages of the present invention, the present invention will be further described with reference to the accompanying drawings and specific embodiments.
In the following examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are commercially available.
EXAMPLE 1 enrichment, purification and analysis of urease-producing Strain
1. Enrichment of urease-producing strains
1.1 urease-producing strain of the present invention was isolated from soil collected from some electronic waste site in Guiyu town, guangdong province, and was subjected to bacterial culture at 25℃to 35℃for 24 hours under static culture conditions. After removing large particle impurities through a 2mm pore size screen surface, 5 g portions of each are placed in a clean 50mL conical flask, 25mL of 85% sodium chloride (NaCl) solution is added, and 50mg/L cadmium chloride (CdCl) is prepared for the solution 2 ) And 4% urea;
1.2 the solution prepared in step 1.1 was placed in a constant temperature shaking incubator, the set temperature of which was 37℃and the set rotational speed was 120rpm, and the incubation time was 7 days.
2. Purification of urease-producing strains
2.1 taking the solution treated in the step 1.2, and diluting each part of the solution into 10 of the original solution concentration -2 、10 -3 、10 -4 ；
Taking 0.5mL of the solution sample with each concentration, and coating the solution sample on a urea solid culture medium containing 40% of urea;
putting the culture medium into a constant temperature incubator at 30 ℃ and observing colony propagation conditions every day;
2.2, taking the culture medium with a relatively vigorous colony morphology in the step 2.1, and selecting a colony with a good morphology and active urease reaction with a culture medium matrix, wherein the colony is specifically embodied as a purple color ring which is outwards diffused around the colony;
inoculating the solid urea culture medium containing 40% of urea in the step 2.1 by a sterile inoculating loop through a flat line drawing method;
2.3, taking the culture medium with a relatively vigorous colony morphology in the step 2.2, selecting 3-5 colonies which have good morphology and active urease reaction with a culture medium matrix, inoculating the colonies into 5mL of broth culture solution (LB), and placing the broth culture solution into a constant-temperature incubator; the temperature of the constant temperature incubator is set to be 37 ℃; the incubator time range was 24 hours.
3. Urease activity assay of urease-producing strains
3.1 taking the broth treated in step 2.3, 200. Mu.L of the broth was taken separately in clean 250mL Erlenmeyer flasks using a pipette, three replicates were taken per flask of broth, and in each Erlenmeyer flask, were mixed:
pinhole sterilized sodium hydroxide (NaOH) solution (1 m,30 μl);
step one, pinhole sterilizing urea solution (40%, 5 mL);
in the last step, pinhole sterilizing calcium chloride (CaCl) 2 ) Solution (25 mM,10 mL);
in the previous step, broth (LB) (85 mL) was sterilized in the presence of a pinhole.
3.2, placing the conical flask in the step 3.1 into a constant temperature shaking incubator; the temperature of the constant-temperature shaking incubator is set to be 37 ℃; the rotation speed of the constant temperature shaking incubator was set at 160rpm.
3.3, putting 1mL of each solution into a centrifuge for centrifugation to obtain a supernatant containing extracellular proteins; the rotational speed of the centrifuge is 10000rpm; the centrifugal time of the centrifugal machine is 10min; the centrifuge temperature was set at 4 ℃.
3.4 preparation of ammonium ion solution as a standard solution for urease Activity analysis, the concentration of ammonium ion solution was 0mM,0.5mM,1mM,2mM,3mM,4mM,5mM.
3.5 preparing phosphate buffer solution as blank control solution for urease activity analysis, wherein the specific preparation method comprises the following steps:
27.6 g of pure sodium dihydrogen phosphate (NaH) was taken 2 PO 4 ) Dissolving the powder in 1L of deionized water, and marking the powder as solution A; 28.4 g of pure sodium hydrogen phosphate (Na) 2 HPO 4 ) Dissolving the powder in 1L of deionized water, and marking as a solution B; mixing 5.3mL of the A solution and 94.7mL of the B solution with 100mL of deionized water to obtain 200mL of mixed solution; 100mL of the mixed solution was taken and 900mL of deionized water was added to dilute 1L of phosphate buffer.
3.6 preparation of Nahner reagent (Nessler's reagent) and 200mM urea solution as reagents for urease activity analysis, the specific preparation method is:
mixing 15mL of Nahner reagent solution with 60mL of deionized water for dilution to obtain Nahner reagent for detection; 0.6 g of urea was dissolved in 100mL of the phosphate buffer prepared in step 3.5 to obtain a urea solution for detection.
3.7, taking with a pipette: the conical flask in the step 3.2 contains urea culture solution of purified urease-producing strains, the supernatant containing extracellular proteins in the step 3.3, ammonium ion solution in the step 3.4 and phosphate buffer solution in the step 3.5, and the samples are placed in a 96-well plate, and Nahner reagent and urea solution are added as reagents for urease activity analysis, and the specific method is as follows:
adding 10 mu L of urea culture solution containing purified urease-producing strain, and marking as A;
adding 10 μl of the supernatant containing extracellular proteins, labeled B;
adding ammonium ion solution, wherein the volume of each concentration is 50 mu L, and the mark is C;
10 mu L of phosphate buffer is added and marked as D;
after adding the four solutions to be tested, 150 mu L of Navier reagent and 40 mu L of 200mM urea solution are added into the ABD solution;
150. Mu.L of Nahner reagent was added to the solution C.
3.8, placing the 96-well plate in the step 3.7 into an enzyme-labeled instrument to measure the absorbance of the sample solution at the wavelength of 480nm, wherein the specific method comprises the following steps:
setting the spectroscopic wavelength to 480nm; setting the detection temperature to be 30 ℃; setting the detection interval to be 5 minutes;
in step 3.2, the urea culture solution containing the purified urease-producing strain was subjected to the test of steps 3.7 to 3.8 in total at 0, 12, 24, 48, 72, 96, 120, and 144 hours after the start of the culture.
3.9 from the urea culture solution containing the purified urease-producing strain in step 3.2, 200. Mu.L of each was placed in a 96-well plate with a pipette, and the optical density OD600 was measured in a spectrophotometer to detect the bacterial reproduction efficiency, the specific implementation method was as follows:
setting the spectroscopic wavelength to 600nm; setting the detection temperature to be 30 ℃; the detection interval was set to 5 minutes.
Analysis results:
referring to FIG. 1, the bacterial strains screened were positive on urease identification medium.
As a result of the urease activity analysis, referring to FIG. 2, the urease activity of the strain E after 24 hours was optimal, and thus, a subsequent study was conducted as a candidate bacterium.
Therefore, the strain obtained by screening according to the invention is named as Bacillus sp.WA which is deposited in the microorganism strain collection of Guangdong province at the 3 rd month 15 th year of 2021, and the deposited address is building 5 of the 100 th university of Mitsui, guangzhou, and the deposited number is GDMCC NO:61559.
Example 2, biochemical composite Material and method for producing the same
1) According to the urease activity and bacterial optical density data obtained in the step 3.8, bacillus sp.WA with high urease activity and high optical density is reasonably selected as one of raw materials of the biochemical composite material, inoculated into a culture dish taking nutrient agar as a matrix, and stored in a refrigerator at 4 ℃;
2) The urea nutrient solution (NBU) is prepared as a forming medium of the biochemical composite material, and the specific method comprises the following steps:
2.6 grams of nutrient broth powder was weighed and dissolved in 170mL deionized water;
next, 20mL of 250mM pinhole sterilized calcium chloride (CaCl) was added 2 ) A solution;
then 10mL of 40% pinhole sterilized urea solution is added;
step one, adding 60 mu L of pinhole sterilizing sodium hydroxide (NaOH) solution;
after the above steps are completed, 200mL of urea nutrient solution (NBU) is obtained;
3) Grinding appropriate amount of corncob into powder in a wall breaking machine, and sterilizing under ultraviolet lamp; the sterilization time is 7 days;
4) Inoculating the Bacillus sp.WA culture medium stored in the step 1) into 5mL broth culture solution by using a sterile inoculating loop, and placing the broth culture solution in a constant-temperature shaking incubator for culture, wherein the specific method comprises the following steps of:
setting the culture temperature to be 37 ℃; setting the rotation speed of the oscillator to 160rpm; the culture time is 24 hours;
5) Taking the broth culture solution containing Bacillus sp.WA in the step 4), taking 200 mu L by a pipette and inoculating the broth culture solution into 200mL of urea nutrient solution prepared in the step 2), and placing the broth culture solution in a constant-temperature shaking incubator for culture, wherein the specific method comprises the following steps of:
setting the culture temperature to be 37 ℃; setting the rotation speed of the oscillator to 160rpm; the culture time is 24 hours;
6) Weighing 20 g of corncob powder in the step 3), mixing with the urea nutrient solution containing Bacillus sp.WA in the step 5), and placing into a constant-temperature shaking incubator for culture, wherein the specific method comprises the following steps:
setting the culture temperature to be 37 ℃; setting the rotation speed of the oscillator to 160rpm; the culture time is 12 hours, and the biochemical composite material is obtained. Electron scanning microscope (SEM) imaging of the biochemical composite is shown in fig. 3 and 4.
The results of the repair coefficients of biochemical composites prepared using different bacterial vectors are shown in table 1.
TABLE 1
Repair coefficient (%) = ((Final Cd concentration-Initial Cd concentration)/Initial Cd concentration) ×100%.
According to the invention, two materials, namely corncob powder and bean dregs, are selected as bacterial carriers (refer to figure 5), and only the corncob powder has good bacterial attachment and enrichment effects. Therefore, for the subsequent soil remediation experiments, a biochemical composite material using corncob meal as a carrier was selected (example 2).
Test example one, soil remediation experiment
The biochemical composite material prepared in the example 2 is applied to the soil containing cadmium element for experiments and a blank control group is arranged, and the specific method is as follows:
taking 8 empty plastic barrels, and respectively putting 1.5 kg of common potting soil and 15 mg of cadmium nitrate powder into the 8 barrels; the 8 barrels are labeled BCM1, BCM2, B1, B2, CP1, CP2, C1, C2, respectively, wherein the corresponding added materials are:
TABLE 2
Soil samples at days 0, 7, 14, 21, and 28 after the addition of the above materials were analyzed by FTIR and fluorescence microscopy, respectively, and the results are shown in fig. 6 and 8.
As shown in FIG. 6, the highest functional groups (the lowest line) on the surface of the soil sample added with the biochemical composite material in example 2 are beneficial to the adsorption of urease-producing strain and further beneficial to the improvement of the biological calcium deposition efficiency induced by the urease-producing strain.
As shown in FIG. 7, the urease activity in the soil samples at days 7, 14, 21 and 28 after the addition of the biochemical composite material prepared in example 2 was higher than that of the other soil samples. The urease activity in the soil sample at day 28 after the biochemical composite material prepared in example 2 was taken can be as high as 4U/mg to 5U/mg.
As shown in FIG. 8, the biochemical composite material prepared in example 2 had the highest bacterial density and the highest bioaccumulation ability.
FIG. 9 is a graph showing soil heavy metal distribution analysis according to the experimental method specified in Tessier (1979) standard method. The result shows that after the soil remediation experiment for 28 days, the cadmium in the ion exchange state in the soil added with the biochemical composite material is reduced by half and is reduced to below national standard (0.5 mg/kg), and the content of the carbonate bonding state in a stable form is increased by two times, so that the problem of heavy metal pollution of the soil is effectively solved.
As shown in FIG. 10, the test soil to which the biochemical composite material prepared in example 2 was added exhibited the highest urease activity and yield and optimal bacterial reproduction, whereas the urease activity and yield and optimal bacterial reproduction of the test soil of the other control group and the blank group were not as good as those of the test soil to which the biochemical composite material prepared in example 2 was added.
Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or substituted equally without departing from the spirit and scope of the technical solution of the present invention.
SEQUENCE LISTING
<110> Guangdong Israel academy of engineering
<120> a biochemical composite material, and preparation method and application thereof
<130> 2021.10.22
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1490
<212> DNA
<213> Synthesis
<400> 1
ggctcaggat gaacgctggc ggcgtgccta atacatgcaa gtcgagcgaa cttttgggag 60
cttgctccca aaagttagcg gcggacgggt gagtaacacg tgggcaacct gcctataaga 120
ctgggataac ttcgggaaac cggagctaat accggataac atatgagacc acatggtctc 180
atattaaaag atggctttta gctatcactt atagatgggc ccgcggcgca ttagctagtt 240
ggtgaggtaa tggctcacca aggcgacgat gcgtagccga cctgagaggg tgatcggcca 300
cactgggact gagacacggc ccagactcct acgggaggca gcagtaggga atcttccgca 360
atggacgaaa gtctgacgga gcaacgccgc gtgagcgaag aaggtcttcg gatcgtaaag 420
ctctgttgtt agggaagaac aagtaccgtt caaatagggc ggtaccttga cggtacctaa 480
ccagaaagcc acggctaact acgtgccagc agccgcggta atacgtaggt ggcaagcgtt 540
atccggaatt attgggcgta aagcgcgcgc aggcggtcct ttaagtctga tgtgaaagcc 600
cacggctcaa ccgtggaggg tcattggaaa ctgggggact tgagtgcaga agaggagagc 660
ggaattccac gtgtagcggt gaaatgcgta gagatgtgga ggaacaccag tggcgaaggc 720
ggctctctgg tctgtaactg acgctgaggc gcgaaagcgt ggggagcgaa caggattaga 780
taccctggta gtccacgccg taaacgatga gtgctaagtg ttagagggtt tccgcccttt 840
agtgctgcag caaacgcatt aagcactccg cctggggagt acggtcgcaa gactgaaact 900
caaaggaatt gacgggggcc cgcacaagcg gtggagcatg tggtttaatt cgaagcaacg 960
cgaagaacct taccaggtct tgacatcccg tggcaactcc taaaatagag ctttcccctt 1020
cgggggacac ggtgacaggt ggtgcatggt tgtcgtcagc tcgtgtcgtg agatgttggg 1080
ttaagtcccg caacgagcgc aacccttgat cttagttgcc agcattaagt tgggcactct 1140
aaggtgactg ccggtgacaa accggaggaa ggtggggatg acgtcaaatc atcatgcccc 1200
ttatgacctg ggctacacac gtgctacaat ggatggtaca aagggcagcg aaaccgcgag 1260
gttaagccaa tcccataaaa ccattctcag ttcggattgc aggctgcaac tcgcctgcat 1320
gaagccggaa tcgctagtaa tcgcggatca gcatgccgcg gtgaatacgt tcccgggcct 1380
tgtacacacc gcccgtcaca ccacgagagt ttgtaacacc cgaagtcggt ggggtaaccg 1440
taaggagcca gccgcctaag gtgggacaga tgattggggt gaagtcgtaa 1490
Claims (15)
1. A urease-producing strain is characterized in that the urease-producing strain is bacillusBacillus sp.WAThe bacillusBacillus sp.WAHas been deposited with the Guangdong province microorganism strain collection at 2021, 3 and 15, with the deposit number GDMCC NO. 61559.
2. Use of a urease-producing strain according to claim 1 for the preparation of urease.
3. Use of a urease-producing strain according to claim 1 for reducing urea content in heavy metal soil.
4. A biochemical composite material comprising the urease producing strain of claim 1 and a sustainable material carrier, the sustainable material carrier being corncob meal.
5. The method for preparing a biochemical composite material according to claim 4, comprising the steps of:
s1, culturing the urease-producing strain in a nutrient medium containing a nitrogen source at the temperature of 30-40 ℃ and the pH value of 6.0-7.0 to obtain a bacterial suspension;
s2, mixing and centrifuging the bacterial suspension prepared in the step S1 and a sustainable material carrier to obtain the biochemical composite material.
6. The process according to claim 5, wherein the ratio of the volume of the bacterial suspension to the mass of the sustainable material carrier is from (10 to 20): 1.
7. The method of claim 6, wherein the ratio of the volume of the bacterial suspension to the mass of the sustainable material carrier is 10:1.
8. The method according to claim 5, wherein in the step S2, the bacterial suspension prepared in the step S1 is mixed with the sustainable material carrier at a temperature of 30 to 40 ℃ at a rotation speed of 120 to 160rpm/min for 8 to 16 hours.
9. The method of claim 5, wherein the nitrogen source comprises urea and the concentration of urea is 2% to 3%.
10. The method of claim 5, wherein the nutrient medium comprises calcium chloride at a concentration of less than 25ppm.
11. A method for immobilizing heavy metals in soil, wherein the urease-producing strain according to claim 1 or the biochemical composite material according to claim 4 is used for immobilizing heavy metals in soil.
12. The method of immobilizing heavy metals in soil according to claim 11, wherein the biochemical composite material according to claim 4 is added to the soil contaminated with heavy metals for 2 to 4 weeks.
13. The method for immobilizing heavy metals in soil according to claim 11 or 12, wherein the concentration of the biochemical composite material is 10% (v: w) g material/kg soil to 20% (v: w) g material/kg soil.
14. The method of immobilizing heavy metals in soil according to claim 11, wherein the heavy metals comprise cadmium and each kilogram of soil comprises 5mg to 12mg cadmium.
15. The use of the biochemical composite material according to claim 4 for repairing heavy metal contaminated soil.
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| CN103289919A (en) * | 2012-04-23 | 2013-09-11 | 清华大学 | Urease-producing microorganisms and method for solidifying heavy metals in subgrade by using same |
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| CN103289919A (en) * | 2012-04-23 | 2013-09-11 | 清华大学 | Urease-producing microorganisms and method for solidifying heavy metals in subgrade by using same |
| KR20170006330A (en) * | 2015-07-07 | 2017-01-18 | 한국지질자원연구원 | Sporosarcina sp. having a capability of producing urease and the Use thereof |
| CN113278558A (en) * | 2021-05-26 | 2021-08-20 | 南宁市拜欧生物工程有限责任公司 | Bacillus subtilis for high yield of urease and application method thereof |
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