CN114716030B - Spherical hydrogel and preparation method and application thereof - Google Patents
Spherical hydrogel and preparation method and application thereof Download PDFInfo
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- CN114716030B CN114716030B CN202210467972.6A CN202210467972A CN114716030B CN 114716030 B CN114716030 B CN 114716030B CN 202210467972 A CN202210467972 A CN 202210467972A CN 114716030 B CN114716030 B CN 114716030B
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Abstract
The invention belongs to the field of bioremediation. The spherical hydrogel disclosed by the invention has a core-shell structure, wherein the core-shell structure comprises a spherical inner core and a hydrogel shell; wherein, the spherical inner core comprises genetically engineered bacteria; the gene engineering bacteria are vibrio natriegens with introduced gene sequences phsA, phsB and phsC; the genetically engineered bacteria are attached to alginate; the hydrogel shell is prepared from a hydrogel precursor and MES buffer solution. The invention also relates to a preparation method of the spherical hydrogel and application of the spherical hydrogel in removing heavy metals in a water body. The invention has high-efficiency heavy metal removal effect and impact resistance to wastewater complex environment, can avoid leakage and solves the biosafety problem.
Description
Technical Field
The invention belongs to the field of bioremediation, and particularly relates to a spherical hydrogel and a preparation method and application thereof.
Background
With the development of productivity, the living demand of human beings is continuously increased, and various high pollution industries are rapidly developed. A large amount of industrial wastewater carries a large amount of pollutants into a water environment, and great threats are formed to the ecological environment and human health. In japan over the last century, water deficiency caused by mercury and pain caused by cadmium have given warnings to humans, and heavy metal contamination by industry has severely threatened human health and survival. In the face of increasing heavy metal wastewater pollution, an in-situ remediation technology needs to be developed urgently to effectively solve the pollution source threatening human health. At present, common methods for remedying heavy metals in wastewater mainly comprise physical, chemical and biological remediation methods. The physical repairing method refers to a method for separating heavy metal ions by adsorption and concentration under the condition of not changing the chemical forms of the heavy metal ions, and comprises an adsorption method, a solvent extraction method, an evaporation and solidification method, an ion exchange method, a membrane separation method and the like. The chemical repairing method refers to a method for removing heavy metal ions through chemical reaction, and comprises a chemical precipitation method, a chemical reduction method, an electrochemical method, a high-molecular heavy metal trapping agent method and the like. The bioremediation method is mainly a method for removing heavy metals by means of flocculation, absorption, accumulation, enrichment and the like of microorganisms or plants. The bioremediation method is an important purification means, has the advantages of simple equipment, no secondary pollution, wide and cheap material sources, economy, high efficiency and the like, is a heavy metal wastewater treatment method with great development potential, and has wide application prospect.
Among bioremediation methods, studies on the remediation of heavy metal pollutants in wastewater by using bacteria have been widely reported. However, the bacterial remediation process often requires screening of natural heavy metal tolerant strains, and the problems of long time consumption for domestication and screening, unsatisfactory effect and the like limit the wide application of the strains. The genetic engineering bacteria can avoid the defects of natural bacterial strain application, and the functional genes are introduced to construct the target bacterial strain through the genetic engineering technology, so that the long-term screening process can be avoided, the manpower and material resources are saved, and the tolerance capability and the removal effect of the bacteria on heavy metals can be greatly improved. However, the wastewater environment is complex, the flora diversity is wide, the application of the genetic engineering bacteria to the remediation of the heavy metals in the wastewater requires consideration of the adaptability of the genetic engineering bacteria to the complex wastewater environment, the potential biological safety problem is also important, and the method is limited in the in-situ remediation of the heavy metals in the wastewater. Aiming at the problems of adaptability of genetically engineered bacteria to complex environments and escape of genes carried by the genetically engineered bacteria and the complex environments, a specific restraint system needs to be developed to avoid leakage and threaten environmental safety, and in-situ bioremediation of heavy metals in wastewater is really realized. The development of a gene engineering bacterium immobilization method is a main strategy for overcoming potential biological safety problems of the gene engineering bacterium.
In view of the above, there is a need to develop an effective method for in-situ bioremediation of heavy metals in wastewater, so as to achieve in-situ remediation of wastewater and effectively solve the biosafety problem.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention develops a spherical hydrogel. The genetic engineering bacteria used by the invention have high-efficiency heavy metal removal capacity; the hydrogel encapsulation system combines the biocompatible multilayer hard shell and the alginate core, can realize multilayer and stable protection, can improve the adaptability of the functional microbial inoculum to the complex environment of wastewater, can avoid environmental leakage, and solves the biological safety problem.
The invention aims to provide a spherical hydrogel which has a core-shell structure, wherein the core-shell structure comprises a spherical inner core and a hydrogel shell;
wherein the content of the first and second substances,
the spherical inner core comprises genetically engineered bacteria;
the gene engineering bacteria are vibrio natriegens introduced with heavy metal removal genes phsA, phsB and phsC;
the gene nucleotide sequence of the phsA is shown as SEQ ID NO. 1;
the phsB gene nucleotide sequence is shown as SEQ ID NO. 2;
the gene nucleotide sequence of phsC is shown in SEQ ID NO. 3.
Further, the mass ratio of the spherical inner core to the hydrogel shell is as follows: (1-10):1.
Furthermore, the spherical inner core also comprises alginate, and the genetically engineered bacteria are attached to the alginate.
Another object of the present invention is to provide a method for preparing the spherical hydrogel, comprising the steps of:
s1, culturing genetically engineered bacteria, centrifuging and resuspending to obtain a bacterial suspension, mixing the bacterial suspension with an alginate solution, dripping the bacterial suspension into a calcium chloride solution, and reacting to obtain the spherical inner core;
s2, adding a cross-linking agent into a hydrogel precursor, wrapping the spherical inner core with the hydrogel precursor to obtain an intermediate product, adding the intermediate product into a MES buffer solution containing 1-ethyl-3-carbodiimide, N-hydroxysuccinimide and adipic dihydrazide, and reacting to obtain the spherical hydrogel;
wherein the content of the first and second substances,
the MES buffer solution comprises 0.05-0.2M 2-morpholine ethanesulfonic acid and 0.2-0.6M sodium chloride, and has pH of 5-7.
Further, in step S1, the concentration of the bacterial suspension is 10 6 -10 9 one/mL.
Further, in step S2, the hydrogel precursor includes the following components in percentage by mass: 1-2wt% of sodium alginate, 15-30wt% of acrylamide, 0.01-0.05wt% of ammonium persulfate and 0.01-0.015wt% of N, N' -methylene-bisacrylamide.
Further, the hydrogel shell is prepared from a hydrogel precursor.
Further, in the step S1, the reaction time is 10-30min.
Further, in the step S2, the reaction time is 1-3h.
The invention also aims to provide the application of the spherical hydrogel in removing heavy metals in water.
The invention has the following beneficial effects:
1. according to the invention, the genetic engineering bacteria are utilized to carry out bioremediation on heavy metals in the wastewater, so that the long-term screening process of microorganisms is shortened, and the removal effect of bacteria on the heavy metals is greatly improved;
2. according to the invention, a hydrogel packaging system is utilized to package genetically engineered bacteria for repairing heavy metals in wastewater, so that the impact resistance of functional bacteria to a complex wastewater environment is improved;
3. the hydrogel-encapsulated genetically engineered bacteria are used for treating heavy metals in wastewater in situ, so that the potential gene safety problem of the genetically engineered bacteria applied to the environment is solved, the safety is improved, and the in-situ remediation of the wastewater is really realized.
Drawings
FIG. 1 is a graph showing the physical size of the spherical hydrogel prepared in step S2 of example 1.
FIG. 2 is a graph showing the heavy metal removal results of the spherical hydrogel in test example 1.
Fig. 3 shows the removal rate of heavy metals from the spherical hydrogel in test example 1.
Detailed Description
In order to more clearly illustrate the technical solution of the present invention, the following examples are given, but the present invention is not limited thereto.
The experimental methods used in the following examples are all conventional methods unless otherwise specified; reagents, materials and the like used in the following examples are commercially available unless otherwise specified.
The preparation method of the genetically engineered bacterium XG204 in the embodiment of the invention comprises the following steps:
engineered operon fragments (5136 bp) containing heavy metal removal genes phsA, phsB and phsC were prepared according to the literature "Sambrook J, fritsch E F, maniatis T]Cloning into a plasmid vector pTrc99A by the method disclosed in Cold spring plasmid Press,1989", to construct a recombinant vector; then, 100 mu L of vibrio natriegens and 200ng of the recombinant vector are mixed evenly and subjected to electric pulse for 1 time (1.7 kV/cm); after the recovery of the culture, ampicillin-resistant plates were applied (V2 salt (204 mmol/L NaCl,4.2mmol/L KCl,23.14mmol/L MgCl) was added to LB medium 2 ) Ampicillin concentration of 200 mug/mL) and culturing for 12h, and then screening out the genetic engineering bacterium XG204.
The basic medium in the examples of the present invention comprises the following components: 25g/L LB broth, 11.9g/L NaCl, 0.313g/L KCl, 2.2g/L MgCl 2 (ii) a The LB broth is: the BS 1302.
The N, N, N ', N' -tetramethylethylenediamine in the embodiment of the invention is: allatin T121820.
The components of the hydrogel precursor in the embodiment of the invention are as follows: 2wt% sodium alginate, 25wt% acrylamide, 0.03wt% ammonium persulfate and 0.01wt% N, N' -methylenebisacrylamide.
The MES buffer solution in the embodiment of the invention comprises the following components: 0.1M 2-morpholinoethanesulfonic acid, 0.2M sodium chloride, pH 6.
Example 1
A spherical hydrogel having a core-shell structure comprising a spherical inner core and a hydrogel shell;
wherein the content of the first and second substances,
the spherical inner core comprises genetically engineered bacteria;
the genetic engineering bacteria are vibrio natriegens introduced with heavy metal removal genes phsA, phsB and phsC;
the gene nucleotide sequence of the phsA is shown as SEQ ID NO. 1;
the phsB gene nucleotide sequence is shown as SEQ ID NO. 2;
the gene nucleotide sequence of phsC is shown in SEQ ID NO. 3.
The mass ratio of the spherical inner core to the hydrogel shell is as follows: 3:1.
The preparation method of the spherical hydrogel comprises the following steps:
s1, culturing the genetically engineered bacterium XG204 in a basal culture medium at 37.0 ℃ for 4h, centrifuging (4000rpm, 5min), and re-suspending to obtain the genetically engineered bacterium XG with the concentration of 10 8 Mixing the bacterial suspension with a 5.5wt% sodium alginate solution (bacterial suspension: sodium alginate solution =2, 1,v/v), then dripping into 15mL of a 5.2wt% calcium chloride solution, and reacting for 10min to obtain the spherical inner core;
s2, adding N, N, N ', N' -tetramethylethylenediamine into a hydrogel precursor (N, N, N ', N' -tetramethylethylenediamine: hydrogel precursor =0.15, 100 v/v), then wrapping the spherical core with the hydrogel precursor (hydrogel precursor: spherical core =3, 1,m/m) to obtain an intermediate product, then adding the intermediate product into a MES buffer solution containing 0.001% by mass of 1-ethyl-3-carbodiimide, 0.0004% by mass of N-hydroxysuccinimide, and 0.0008% by mass of adipic acid dihydrazide, and reacting for 3.5 hours to obtain the spherical hydrogel.
FIG. 1 is a graph showing the physical size of the spherical hydrogel prepared in step S2 of example 1.
Example 2
A spherical hydrogel having a core-shell structure comprising a spherical inner core and a hydrogel shell;
wherein the content of the first and second substances,
the spherical inner core comprises genetically engineered bacteria;
the gene engineering bacteria are vibrio natriegens introduced with heavy metal removal genes phsA, phsB and phsC;
the gene nucleotide sequence of the phsA is shown as SEQ ID NO. 1;
the phsB gene nucleotide sequence is shown as SEQ ID NO. 2;
the gene nucleotide sequence of phsC is shown in SEQ ID NO. 3.
The mass ratio of the spherical inner core to the hydrogel shell is as follows: 2:1.
The preparation method of the spherical hydrogel comprises the following steps:
s1, culturing the genetically engineered bacterium XG204 in a basal culture medium at 37.2 ℃ for 3.5h, centrifuging (4000rpm, 8min), and resuspending to obtain the genetically engineered bacterium XG with the concentration of 10 9 Mixing the bacterial suspension with a 6.0wt% sodium alginate solution (bacterial suspension: sodium alginate solution =2:1, v/v), then dripping the bacterial suspension into a 20mL 5.5wt% calcium chloride solution, and reacting for 20min to obtain the spherical inner core;
s2, adding N, N, N ', N' -tetramethylethylenediamine into a hydrogel precursor (N, N, N ', N' -tetramethylethylenediamine: hydrogel precursor =0.12, 100 v/v), then wrapping the spherical core with the hydrogel precursor (hydrogel precursor: spherical core =2, 1,m/m) to obtain an intermediate product, then adding the intermediate product into a MES buffer solution containing 0.0015% by mass of 1-ethyl-3-carbodiimide, 0.00035% by mass of N-hydroxysuccinimide, and 0.0009% by mass of adipic dihydrazide, and reacting for 3.5 hours to obtain the spherical hydrogel.
Comparative example 1
A spherical hydrogel, the comparative example is a non-core-shell structure, and the preparation method comprises the following steps:
culturing the genetically engineered bacterium XG204 in a basal medium at 37.0 ℃ for 4h, centrifuging (4000rpm, 5min), and re-suspending to obtain a concentrated solutionDegree of 10 8 one/mL of the bacterial suspension, and then the bacterial suspension was mixed with 5.5% wt sodium alginate solution (bacterial suspension: sodium alginate solution =2, 1,v/v), and then dropped into 15mL of 5.2% wt calcium chloride solution, and after reaction for 10min, the spherical hydrogel was obtained.
Comparative example 2
A spherical hydrogel, the preparation method of this comparative example differs from that of example 1 in that: in step S2, the spherical inner core is wrapped with a 2.5wt% sodium alginate solution, and the other preparation steps are the same as in example 1.
Test example 1
Heavy Metal removal Performance test
The test method comprises the following steps:
the spherical hydrogels prepared in examples 1 and 2 and comparative examples 1 and 2 were placed in a basal medium, respectively, and 0.1mM of an aqueous solution containing lead ions was added thereto, and after culturing at 37 ℃ for 48 hours, the lead ion concentration in the medium was measured.
And (3) testing results:
the test results are shown in table 1 and fig. 2 and 3.
Table 1 heavy metal removal performance test results
FIG. 2 is a graph showing the results of removing heavy metals from the spherical hydrogel in test example 1, in the left container, the spherical hydrogel prepared in example 1 was put into a lead ion solution of 0.1mM, and the inner core of the spherical hydrogel was blackened, which indicates that microorganisms interacted with lead ions to form precipitates, thereby removing lead ions; in the right container, the spherical hydrogel prepared in example 1 was placed in deionized water without lead ions, and the spherical hydrogel core was not significantly changed. Fig. 3 shows the removal rate of heavy metals from the spherical hydrogel in test example 1. As can be seen from the table 1, the figures 2 and 3, the spherical hydrogel disclosed by the invention has a high-efficiency heavy metal removal effect, the removal rate of lead ions can reach more than 80%, and the spherical hydrogel has good water body restoration capability, while the spherical hydrogel prepared by the comparative example has low stability, so that thalli cannot be fixed in the hydrogel, thallus leakage occurs, the removal effect of heavy metals is poor, and the hidden danger of secondary pollution to the environment exists.
Test example 2
Impact resistance test for wastewater complex environment
The test method comprises the following steps:
after placing the spherical hydrogels prepared in examples 1 and 2 and comparative examples 1 and 2 into LBV2 medium with pH =4 respectively and shaking-incubating for 4h, taking out the alginate inner core, washing the outer surface with LBV2 medium, placing into a mixture of phosphate buffer (PBS, 1 mL) and sodium citrate (55 mM) for shaking-dissolving, taking the dissolved solution for streak-culturing on LBV2 plate, counting, and calculating the cell survival rate according to the following formula:
and (3) testing results:
the test results are shown in table 2.
TABLE 2 impact resistance test for wastewater complex environments
| Survival rate | Example 1 | Example 2 | Comparative example 1 | Comparative example 2 |
| 0h | 81.4％ | 82.5％ | 86.2％ | 83.3％ |
| 4h | 72.5％ | 75.1％ | Leakage of thallus | Leakage of thallus |
As can be seen from Table 2, the survival rate of the genetically engineered bacteria encapsulated by the spherical hydrogel prepared by the invention can exceed 70% in a complex environment, and the genetically engineered bacteria have strong impact resistance.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Furthermore, it should be understood that although the present specification describes embodiments, not every embodiment includes only a single embodiment, and such description is for clarity purposes only, and it is to be understood that all embodiments may be combined as appropriate by one of ordinary skill in the art to form other embodiments as will be apparent to those of skill in the art from the description herein.
Claims (9)
1. The spherical hydrogel is characterized by having a core-shell structure, wherein the core-shell structure comprises a spherical inner core and a hydrogel shell;
wherein the content of the first and second substances,
the spherical inner core comprises genetically engineered bacteria;
the gene engineering bacteria are vibrio natriegens introduced with heavy metal removal genes phsA, phsB and phsC;
the gene nucleotide sequence of the phsA is shown as SEQ ID NO. 1;
the phsB gene nucleotide sequence is shown as SEQ ID NO. 2;
the gene nucleotide sequence of phsC is shown in SEQ ID NO. 3.
2. The spherical hydrogel according to claim 1, wherein the spherical inner core and the hydrogel shell are present in a mass ratio of: (1-10):1.
3. The spherical hydrogel of claim 1, further comprising alginate in the spherical inner core, wherein the genetically engineered bacteria are attached to the alginate.
4. A process for producing a spherical hydrogel according to any one of claims 1 to 3, which comprises the steps of:
s1, culturing genetically engineered bacteria, centrifuging and resuspending to obtain a bacterial suspension, mixing the bacterial suspension with an alginate solution, dripping the mixture into a calcium chloride solution, and reacting to obtain the spherical inner core;
s2, adding a cross-linking agent into a hydrogel precursor, wrapping the spherical inner core with the hydrogel precursor to obtain an intermediate product, adding the intermediate product into a MES buffer solution containing 1-ethyl-3-carbodiimide, N-hydroxysuccinimide and adipic dihydrazide, and reacting to obtain the spherical hydrogel;
wherein the content of the first and second substances,
the MES buffer solution comprises 0.05-0.2M 2-morpholine ethanesulfonic acid and 0.2-0.6M sodium chloride, and has pH of 5-7.
5. The method for producing a spherical hydrogel according to claim 4, wherein the concentration of the bacterial suspension in step S1 is 10 6 -10 9 one/mL.
6. The method for preparing the spherical hydrogel according to claim 4, wherein in step S2, the hydrogel precursor comprises the following components in percentage by mass: 1-2wt% of sodium alginate, 15-30wt% of acrylamide, 0.01-0.05wt% of ammonium persulfate and 0.01-0.015wt% of N, N' -methylene-bisacrylamide.
7. The method for producing the spherical hydrogel according to claim 4, wherein the reaction time in step S1 is 10 to 30min.
8. The method for producing the spherical hydrogel according to claim 4, wherein the reaction time in the step S2 is 1 to 3 hours.
9. Use of the spherical hydrogel according to any one of claims 1 to 3 for the removal of heavy metals from a water body.
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| CN101845447A (en) * | 2010-04-22 | 2010-09-29 | 国家海洋局第二海洋研究所 | Beta-agarase encoding gene and gene acquisition method |
| CN103045563A (en) * | 2012-11-28 | 2013-04-17 | 国家海洋局第一海洋研究所 | Low temperature beta-agarase and coding gene and application thereof |
| CN106318880A (en) * | 2015-06-15 | 2017-01-11 | 中国科学院微生物研究所 | Bacillus amyloliquefaciens and its bacterial depressant and use |
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| CN101845447A (en) * | 2010-04-22 | 2010-09-29 | 国家海洋局第二海洋研究所 | Beta-agarase encoding gene and gene acquisition method |
| CN103045563A (en) * | 2012-11-28 | 2013-04-17 | 国家海洋局第一海洋研究所 | Low temperature beta-agarase and coding gene and application thereof |
| CN106318880A (en) * | 2015-06-15 | 2017-01-11 | 中国科学院微生物研究所 | Bacillus amyloliquefaciens and its bacterial depressant and use |
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