CN105585742A - A preparing method of a polymer montmorillonite bacterium-carrying gel sphere - Google Patents

Abstract

A polymer montmorillonite composite hydrogel sphere and a preparing method thereof are disclosed. The sphere is prepared from 1-4% by weight of sodium alginate, 0.5-3% by weight of gelatin, 0.02-0.1 g/mL of montmorillonite and a proper amount of water. An auxiliary material comprises a mixed solution containing glutaraldehyde with a concentration of 0.4-0.7% and calcium chloride with a concentration of 3-6%. The preparing method includes preparing a sodium alginate mother liquor with a concentration of 5.0%, preparing a gelatin mother liquor with a concentration of 30.0%, adding a proper amount of the montmorillonite and a proper amount of distilled water, fully mixing, injecting the above-mentioned liquid mixture to 30 mL of the mixed solution containing the glutaraldehyde with a concentration of 0.4-0.7% and the calcium chloride with a concentration of 3-6% through an injector (with a syringe needle model number being 22-27 G) with the height being 5 cm so that smooth microspheres are immediately formed, allowing the immobilized spheres to stand in the solution for 4-10 h, pouring the mixed solution away, and washing the spheres with a proper amount of distilled water twice. An embedded vector prepared through the method is characterized by good cell embedding effects, good mass transfer performance, high ion exchange capability, high molecule adsorbability, the simple and feasible method, and the like.

Description

A kind of preparation method of macromolecule loaded Montmorillonite bacterium gel ball

Technical field

The present invention relates to a kind of preparation method of macromolecule hydrogel ball, belong to biochemical industry and environment-friendly function Material Field.

Background technology

The application of immobilized cell is extremely extensive, and this technology is own through being widely used in multiple fields such as industry, medical diagnosis, chemical analysis, environmental protection, energy development and correlation theory research at present. A desirable immobilized microorganism material will possess following condition substantially: (1) intensive microorganism, maintains the biomass concentration in reactor; (2) carrier has higher mechanical strength, and resistance to mass tranfer is little; (3) carrier is renewable, and reusable; (4) carrier is cheap, and process for fixation expense is low; (5) the secure bond intensity of carrier and microorganism is high, the impact of ability waterpower fluctuation; (6) carrier is nontoxic to microorganism, can Resistance to microbes.

Conventional immobilization material comprises the polytypes such as inorganic carrier, organic polymer carrier, complex carrier. Inorganic carrier have mechanical strength large, to microorganism nontoxicity, be difficult for by microorganism decomposition, acid and alkali-resistance, the characteristic such as cost is low, the life-span is long. Montmorillonite belongs to natural inorganic carrier, for smectite family monoclinic system, TOT type, its unit cell is to be made up of two-layer Si-O tetrahedron folder one deck Al-O octahedron, the factors such as crystal lattice, broken key, octahedra lamella dissociate all can cause montmorillonite to have electronegativity, make montmorillonite be easy to the organic cation of adsorption band positive charge. In addition, montmorillonite also has larger specific area, good characterization of adsorption, good nano-space structure, and this makes it have more superiority as the application of the aspects such as fixed cell carrier. But when inorganic carrier immobilized cell, mainly, by suction-operated and charge effect (model Dehua power, hydrogen bond and electrostatic interaction), Cell binding is insecure, easily comes off.

Natural polymer gel carrier in organic polymer carrier generally has the advantages such as good biocompatibility, preparation technology is simple, mass-transfer performance is good. Common natural polymer gel has agar, SN-263, calcium alginate, chitin etc. In these several natural carriers, agar strength is the poorest, and SN-263 price is more expensive, and chitin is insoluble to general reagent. By contrast, that calcium alginate has is cheap, preparation easily, the advantage such as mass-transfer performance is good, be most widely used. But sodium alginate intensity is lower, is under anaerobic easily decomposed by microorganism, and the life-span is shorter. Because organic and inorganic carrier material respectively have its pluses and minuses, and two class materials are complementary in many aspects. Therefore, this two classes material can be combined, composition composite carrier, to improve the performance of material.

Investment is that microorganism is limited in the confined space such as small grid or microcapsules of gel, can allow matrix infiltration and product diffuse out simultaneously. Investment is little on microbial activity impact, granule strength is high, is the process for fixation of current most study. Cross-linking method is by microorganism and the reagent reacting with two or more functional groups, makes microbial cells be interconnected to network structure, and reaches the object of fixation of microbe. In embedding process, allow natural organic high-molecular occur crosslinked, can make high-molecular gel more stable, firm. Although the gel-type vehicle of fixation of microbial cell can prevent the leakage of cell, be also the barrier of substrate and oxygen molecule diffusion simultaneously. Diffusion blocking can cause the decline of apparent reaction rates. Sometimes, even can change embedding and be fixed on due to mass transfer limitations the metabolism behavior of cell in gel particle. Therefore, in immobilized cell dynamics research process, should consider the dispersal behavior of material in gel. Meanwhile, the integrality of particle is subject to the impact of its mechanical strength, collision mutually, friction and shearing force, and these factors all can cause breakage of particles. Therefore, the mechanical strength of embedded particles and durability are also whether immobilized microorganism operation is successfully crucial.

Summary of the invention

Embedding carrier should possess microorganism nontoxic, and easily, mass-transfer performance is good in preparation, the features such as low price, and sodium alginate has easy to operate as carrier, and the activity of imbedded microbe is high. Gelatin is a kind of conventional protein gelling agent, and dissolubility is good and cheap, can well improve the molding effect of sodium alginate. Utilize micelle that sodium alginate prepared with gelatin coimmobilization can provide larger immobilization space compared with single application sodium alginate, be conducive to the diffusion of material. Its interlamination region of smectite structure characteristics determined has good ion-exchange performance and Molecular Adsorption feature. Sodium alginate, gelatin are prepared to gelled pill together with montmorillonite, can solve that the dispersion suspension that montmorillonite has in water is strong, Separation of Solid and Liquid speed is slow, flocculate costs and poor dehydration results, the shortcoming such as not easily separated.

Take appropriate sodium alginate be placed in boiling water stir it is fully dissolved, be cooled to room temperature, the sodium alginate mother liquor that compound concentration is 5.0%; Take appropriate gelatin be placed in boiling water stir it is fully dissolved, be cooled to room temperature, the gelatin mother liquor that compound concentration is 30.0%; Get the gelatin mother liquor, the sodium alginate mother liquor that prepare in right amount, add appropriate montmorillonite and distilled water to mix, making sodium alginate concentration is 1-4%, and gelatin concentration is 0.5-3%, and montmorillonite concentration is 0.02-0.1g/mL, and mixed liquor volume is 5-20mL. Syringe needle by syringe (syringe needle model 22-27G) highly injects 30mL by above-mentioned mixed liquor taking 5cm and contains concentration as the glutaraldehyde of 0.4-0.7% and concentration are as the calcium chloride mixed solution of 3-6%, forms immediately smooth microsphere. This immobilized spherule is left standstill in above-mentioned solution to 3-10h, outwell mixed solution, with appropriate distilled water washing bead twice, for subsequent use.

In the above-mentioned bead making, add after 30mL distilled water, then add the methylene blue solution that 500 μ L concentration are 0.2%, 30 DEG C, after 130rpm screen vibration 40min, take out solution, measure its absorbance under 620nm wavelength with visible spectrophotometer. Absorbance is less, illustrates that its mass-transfer performance is better. The consumption of montmorillonite is chosen to be: 0.02,0.04,0.06,0.08,0.1g/mL (now gelatin concentration is fixed as 3%, sodium alginate concentration fixture 1%), and OD₆₂₀Drop to 0.071 from 0.684. Use Folin phenol method to measure the protein content in supernatant, when montmorillonite concentration is 0.1g/mL, in supernatant, protein content is minimum. Gelatin concentration is chosen to be: 3%, 5%, 7%, 9%, 11% (now montmorillonite concentration is fixed as 0.08g/mL, sodium alginate concentration fixture 1%), OD₆₂₀Drop to 0.061 from 1.215; Use Folin phenol method to measure the protein content in supernatant, when gelatin concentration is 3%, in supernatant, protein content is minimum. When sodium alginate concentration is chosen to be: 1.0%, 1.5%, 2.0%, 2.5%, 3.0% (now gelatin concentration is fixed as 3%, montmorillonite concentration fixture 0.08g/mL), OD₆₂₀Drop to 0.096 from 0.196. Use Folin phenol method to measure the protein content in supernatant, when sodium alginate concentration is 3%, in supernatant, protein content is minimum.

Get the immobilized spherule of preparing under above-mentioned different condition and be placed in 30mL distilled water, stir vibration with magnetic stirring apparatus with 30 DEG C, maximum (top) speed, measure the light absorption value of solution at 660nm place after 2h, light absorption value is less, illustrates that mechanical strength is larger. Through measuring its OD₆₆₀Be about 0.006-0.025, bead percentage of damage 0-5%.

Detailed description of the invention

Specific embodiment one

From refrigerator, take out e. coli jm109, get wherein 20 μ L and be linked in the LB culture medium of 100mL, 37 DEG C, 140rpm shaking table cultivation 48h. Get 80 milliliters of above-mentioned LB culture mediums, in centrifuge 4000r/min, centrifugal 15min, with twice of physiological saline washing thalline. A part for the cenobium that above-mentioned centrifugation is obtained joins in the liquid of 10mL containing sodium alginate, gelatin, montmorillonite, making sodium alginate final concentration is 4%, gelatin final concentration is 3%, montmorillonite final concentration is 0.1g/mL, mix rear with syringe injection 30mL glutaraldehyde, calcium chloride solution (glutaraldehyde concentration is 0.6%, and calcium chloride concentration is 6%), after crosslinked 2.5-3 hour, the supernatant that inclines, adds 30 ml physiological salines and continues aging. After 12h, change physiological saline, after 46-48h, measure the OD of the aqueous solution₆₂₀Be 0.037 (embedding bacterium is not 0.967), illustrate that bacterium dissolution rate is low. Get the bead of certain mass, add 30mL physiological saline, add 500 μ L0.2% methylene blues. After absorption 1h, survey OD₆₂₀, methylene blue clearance is 88%. Get after 0.5 gram of bead crushes and add in LB culture medium and cultivate, after 20h, survey its supernatant OD₆₂₀Be 0.616, illustrate and still have a large amount of viable bacterias to exist, coated plate is cultivated also has bacterium colony to exist.

Specific embodiment two

From refrigerator, take out e. coli jm109, get wherein 20 μ L and be linked in the LB culture medium of 100mL, 37 DEG C, 140rpm shaking table cultivation 48h. Get 80 milliliters of above-mentioned LB culture mediums, in centrifuge 4000r/min, centrifugal 15min, with twice of physiological saline washing thalline. The cenobium that above-mentioned centrifugation is obtained is got a part and is joined in the liquid of 10mL containing sodium alginate, gelatin, montmorillonite, and making sodium alginate final concentration is 1%, and gelatin final concentration is 0.5%, and montmorillonite final concentration is 0.02g/mL. Mix and inject 30 milliliters of glutaraldehydes, calcium chloride solution (wherein glutaraldehyde concentration is 0.6%, and calcium chloride concentration is 6%) with syringe afterwards, after crosslinked 2.5-3 hour, the supernatant that inclines, adds 30 ml physiological salines and continues aging. The gel obtaining is sheet. After 12h, change physiological saline, after 6h, measure the OD of the aqueous solution₆₂₀Be 0.109 (embedding bacterium is not 0.967), illustrate that most of bacterium throws away by embedding. Get after 0.15 gram of bead crushes and add in LB culture medium, after 20h, measure its supernatant OD₆₂₀Be 0.114, illustrate and still have viable bacteria to exist, coated plate is cultivated also has bacterium colony to exist.

Specific embodiment three

Saccharomycete bacterial classification 100 μ L are linked in the YPD culture medium of 100mL to 28 DEG C, 140rpm shaking table cultivation 48h. Get 80 milliliters of above-mentioned YPD culture mediums, in centrifuge 4000r/min, centrifugal 15min, uses physiological saline washed twice. The cenobium that above-mentioned centrifugation is obtained joins in the liquid of 10mL containing sodium alginate, gelatin, montmorillonite, making sodium alginate final concentration is 4%, gelatin final concentration is 3%, montmorillonite final concentration is 0.1g/mL, mix rear with 30 milliliters of glutaraldehydes of syringe injection, calcium chloride solution (wherein glutaraldehyde concentration is 0.6%, and calcium chloride concentration is 6%), after crosslinked 2.5-3 hour, the supernatant that inclines, adds 30 ml physiological salines and continues aging. After 12h, change physiological saline, after 46-48h, measure the OD of the aqueous solution₆₂₀Be 0.050 (embedding bacterium is not 0.800), illustrate that bacterium dissolution rate is low. Get respectively the bead of certain mass, add 30mL physiological saline. Subsequently, add 500 μ L0.2% methylene blues. After absorption 1h, survey OD₆₂₀, methylene blue clearance is 92%. Get after 0.5 gram of bead crushes and add in YPD culture medium, after 20h, measure supernatant light absorption value OD₆₂₀Be 0.560, illustrate and still have a large amount of viable bacterias to exist. Coated plate is cultivated also proves to have a large amount of viable bacterias to exist.

Specific embodiment four

Saccharomycete bacterial classification 100 μ L are linked in the YPD culture medium of 100mL to 37 DEG C, 140rpm shaking table cultivation 48h. Get 80 milliliters of above-mentioned YPD culture mediums, in centrifuge 4000r/min, centrifugal 15min, uses physiological saline washed twice. The cenobium that above-mentioned centrifugation is obtained joins in the liquid of 10mL containing sodium alginate, gelatin, montmorillonite, making sodium alginate final concentration is 1%, gelatin final concentration is 0.5%, montmorillonite final concentration is 0.02g/mL, mix rear with 30 milliliters of glutaraldehydes of syringe injection, calcium chloride solution (wherein glutaraldehyde concentration is 0.6%, and calcium chloride concentration is 6%), after crosslinked 2.5-3 hour, the supernatant that inclines, adds 30 ml physiological salines and continues aging. The gel obtaining is sheet. After 12h, change physiological saline, after 6h, measure the light absorption value OD of the aqueous solution₆₂₀Be 0.078 (embedding bacterium is not 0.800), illustrate that most of bacterium throws away by embedding. Get after 0.25 gram of bead crushes and add in YPD culture medium, after 20h, measure supernatant light absorption value OD₆₂₀Be 0.415, illustrate and still have viable bacteria to exist, coated plate is cultivated also proves to have a large amount of viable bacterias to exist.

Claims (6)

1. a macromolecule montmorillonite composite aquogel, is characterized in that,

Raw material by following weight portion is made: sodium alginate 1-4%; Gelatin 0.5-3%; Montmorillonite 0.02-0.1g/mL; Water is appropriate.

2. auxiliary material comprise: the calcium chloride mixed solution that the glutaraldehyde of 0.4-0.7% and concentration are 3-6%.

3. the modified montmorillonite used composite aquogel preparation method described in is as follows: take appropriate sodium alginate be placed in boiling water stir it is fully dissolved, be cooled to room temperature, the sodium alginate mother liquor that compound concentration is 5.0%; Take appropriate gelatin be placed in boiling water stir it is fully dissolved, be cooled to room temperature, the gelatin mother liquor that compound concentration is 30.0%; Get the gelatin mother liquor, the sodium alginate mother liquor that prepare in right amount, and add appropriate imvite and distilled water to mix, making sodium alginate concentration is 1-4%, and gelatin concentration is 0.5-3%, and montmorillonite concentration is 0.02-0.1g/mL, and making mixed liquor volume is 5-20ml.

4. the syringe needle by syringe (syringe needle model 22-27G) highly injects 30ml by above-mentioned mixed liquor taking 5cm and contains concentration as the glutaraldehyde of 0.4-0.7% and concentration are as the calcium chloride mixed solution of 3-6%, forms immediately smooth microsphere.

5. this immobilized spherule is left standstill in above-mentioned solution to 4-10h, outwell mixed solution, wash bead twice with appropriate distilled water.

6. subsequently this ball is put into distilled water or physiological saline saves backup.