CN106520752A - Method for preparing biological carbon immobilized microorganism - Google Patents
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- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
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- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
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- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
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Abstract
The invention belongs to the technical field of environmental engineering, and discloses a method for preparing a biological carbon immobilized microorganism. The preparation method comprises the following steps: uniformly mixing a polyvinyl alcohol solution with a sodium alginate solution to form a mixed gel solution; mixing a biological carbon adsorbed microorganism bacterial suspension with the mixed gel solution to obtain a mixed bacterial liquid; dropping into a calcium chloride (CaCl2) solution with saturated boric acid for crosslinking, and transferring into an anhydrous sodium sulfate solution for crosslinking to prepare immobilized beads; and washing with normal saline for 3 times for later use. The prepared immobilized beads have the advantages of large specific surface area, high opening ratio, high moisture content and high microorganism activity.
Description
Technical field
The invention belongs to field of environment engineering technology, and in particular to a kind of charcoal immobilized microorganism preparation method.
Background technology
Immobilized microorganism technique is the new biotechnology grown up on the basis of enzyme immobilization technology, it
Microorganism concn can be increased substantially, making microorganism not easily run off and utilize time length, mithridatism and tolerance substantially increases,
And solid-liquid separation is easily, secondary pollution is few.
The immobilized method of microbial cell mainly includes absorption method, carrier combined techniqueses, cross-linking method and investment etc., single
Process for fixation cut both ways, the absorption-embedded-cross-linked combined type process for fixation for combining can be kept away to a certain extent
Exempt from the generation of these problems.
Polyvinyl alcohol as a kind of carrier of new immobilized microorganism technology, with chemical stability is good, antibiont
The advantages of capacity of decomposition height, high mechanical strength, no biotoxicity and excellent honest and clean price, become the focus of domestic and international research.With poly-
It is polyvinyl alcohol-borate method that vinyl alcohol prepares the method for immobilization particle most simple economy, but this method has two big disadvantages
End：One is the cohesion caused due to low cross-linking, hinders the mass transfer performances of globule；Another is boric acid to the toxic work of microorganism
With.Boric acid impact to cytoactive can effectively be reduced as cross-linking agent by the use of sodium sulfate, compared with traditional method due to
Boric acid crosslinking time short cell loss of activity is less, and the stability of carrier is more preferable.
Biomass carbon be it is a kind of containing carbon rich, finegrained porous mass, it is hot under anaerobic by biomass
Decompose what is produced, with specific surface area is big, adsorptivity strong, the advantages of pore structure is flourishing.Biomass carbon is used to adsorb fixation
Change microorganism and can effectively improve specific surface area, porosity and mass-transfer performance of carrier etc..
The content of the invention
It is an object of the invention to provide a kind of absorption-embedded-cross-linked combined type process for fixation for combining, is to micro-
Biological activity affects less process for fixation.
In addition, when the method for present invention offer is with biomass carbon as adsorbent, studying biomass carbon prepared by different temperatures
Impact to fixation support.
The present invention to achieve these goals, employs the following technical solutions realization：
A kind of charcoal immobilized microorganism preparation method, comprises the steps：
（1）, microorganism culture and the measure of growth curve, bacterial strain is placed in minimal medium and is grown 2 days and per hour
The growth curve of bacterial strain is surveyed, the point of interface of logarithmic (log) phase and stable phase is chosen, that is, the bacterium solution that grown 22h is tested；
（2）, biomass carbon added and cultivated the bacterium solution of 22h and is vibrated on shaking table；
（3）, by polyvinyl alcohol and sodium alginate each personal distilled water immersion overnight, polyvinyl alcohol water-bath dissolving after be down to room temperature with
Sodium alginate mixes, and forms mixed gel liquid and stands；
（4）, by step（2）With step（3）In solution mixing stand；
（5）, mixed liquor first instilled into the CaCl of saturation boric acid using dripping mode2Solution crosslinking, transfers to anhydrous sodium sulfate
Solution crosslinking, is obtained immobilization globule, standby with normal saline flushing 3 times.
Preferably, step（2）In, biomass carbon is prepared at being respectively 300 DEG C, 500 DEG C and 700 DEG C.
Preferably, step（1）In, the bacterial strain in microbial inoculum is as parent by pseudomonass and Sphingol single-cell
The fusant bacterial strain F14 obtained using Protoplast Fusion Technique, inoculum concentration is 20%.There is no spy to the species of bacterial strain in the present invention
Other restriction, other bacterial strains can equally adopt this method immobilization.
Preferably, step（3）In, dissolving detailed process is to take appropriate polyvinyl alcohol and sodium alginate in conical flask,
It is separately added into distilled water immersion overnight, shakes on sodium alginate shaking table overnight, poly-vinyl alcohol solution is put into 90 DEG C of water-baths by next day
Vibrated in agitator, vibration 3h or so is until dissolving.Poly-vinyl alcohol solution is down to room temperature and is mixed with sodium alginate.
Preferably, polyvinyl alcohol concentration is 6-10%, and sodium alginate concentration is 0.5-1%, specifically chosen to be shown in Table 1.
The polyvinyl alcohol of 1. variable concentrations of table（PVA）And sodium alginate（SA）Prepare the Performance comparision of fixation support
As shown in Table 1, when selecting polyvinyl alcohol concentration to be 8%, when sodium alginate concentration is 1%, balling-up effect is best, mass-transfer performance
Meet the requirements with mechanical strength.
Preferably, step（3）In, the time of repose of mixed gel liquid is 30min, and now solution can reach enough mixing
Close.
Preferably, step（5）In, in the CaCl of saturation boric acid2In solution, CaCl2Mass fraction be 4%, crosslinking when
Between be 30min, can form immobilized spherule, and less is affected on microbial activity.Anhydrous sodium sulfate solution concentration is 1M（Sulfur
Sour na concn is too low to be unfavorable for crosslinking, and too high cross-linking process has crystal precipitation, therefore selects 1M）, the time of crosslinking is 5h（Hand over
Connection overlong time is had an impact to microbial activity）.The concentration of above crosslinking time and cross-linking agent is selected according to following combined factors
Consider to determine：（1）During preparation immobilization globule, the balling-up effect of immobilization globule, the cost for whether having adhesion, medicine are asked
Topic；（2）Prepare.
Preferably, step（5）In, dripping refer to the asepsis injector that specification is 10ml at room temperature by mixed liquor in
10cm height is vertically instilled in cross-linking agent solution.
The present invention preparation principle be：The present invention is a kind of set absorption-embedded-cross-linked combined type immobilization being integrated
Method.Microorganism adsorption is fixed to its surface by the strong adsorptivity having by biomass carbon and big specific surface area；And with poly-
Vinyl alcohol and sodium alginate are occlusion vehicle, drip to crosslinked fluid and carry out crosslinking and can improve balling-up effect, and microorganism can
Preferably it is fixed in gel lattice without revealing, by microorganism, in boric acid, crosslinking time is transferred to sodium sulfate for a period of time afterwards
Toxic action of the boric acid to microorganism can be reduced in solution.
In addition, continue at after the globule being tentatively cross-linked to form is preserved a period of time at 4 DEG C in crosslinked fluid be crosslinked, can be with
With reference to physics freeze-thaw method and the advantage of chemical crosslink technique, formation mechanical strength is good, percent opening is high, moisture content is big, microbial activity
High fixation support.
Compared to the crosslinking of polyvinyl alcohol-boric acid, the preparation method and resulting product of the present invention have the advantage that and
Beneficial effect：
1st, microorganism immobilization method of the present invention is easy to operate, requires low, safe preparation process to conditioning apparatus.
2nd, in the present invention, cross-linking agent affects relatively low to Ecotoxicology, makes the biological activity of microorganism higher.
3rd, adsorbing agent biological matter charcoal is added to cause the specific surface area of fixation support bigger in the present invention, mass-transfer performance is more
It is good.
The present invention is reasonable in design, and a diameter of 3-4mm of obtained immobilization globule, specific surface area are big, and percent opening is high, moisture content
Greatly, microbial activity is high.
Specific embodiment
With reference to embodiment, the present invention is described in further detail, but embodiments of the present invention not limited to this.
Embodiment 1
A kind of charcoal immobilized microorganism preparation method, comprises the steps：
（1）, weigh the biomass carbons of 300 DEG C of 0.8g preparation, add 20ml to cultivate the bacterium solution of 22h；Wherein, bacterial strain is by vacation
The fusant bacterial strain F14 that Zymomonas mobiliss and Sphingol single-cell are obtained using Protoplast Fusion Technique as parent, inoculum concentration is
20%；
（2）, weigh 8g polyvinyl alcohol, 1g sodium alginates and be respectively put in 100ml conical flasks, be separately added into 50 ml and 20ml distillations
Water soaked overnight, vibrates on sodium alginate soln shaking table；Next day poly-vinyl alcohol solution water-bath vibration 3h or so is dropped to being uniformly dissolved
Sodium alginate soln is added to room temperature, and with 5ml distilled water flushing inwalls；
（3）, will add biomass carbon bacterium solution and step（2）In solution be mixed to get mixed bacteria liquid, and rushed with 5ml distilled water
Inwall is washed, now polyvinyl alcohol concentration is 8%, and sodium alginate concentration is 1%；
（4）, weigh 5g boric acid in 100ml distilled water, treat boric acid solution saturation separate out, take supernatant add CaCl2So that CaCl2
The ultimate density of solution is 4%, prepares the CaCl of saturation boric acid2Crosslinker solution；
（5）, weigh the anhydrous sodium sulfate of 14.2g in 100ml distilled water, prepare the anhydrous slufuric acid sodium solution of 1M；
（6）, be the asepsis injector of 10ml with specification at room temperature by step（3）Mixed liquor vertically instills saturation in 10cm height
The CaCl of boric acid2In solution, and it is stirred continuously, is crosslinked 30min；
（7）, anhydrous sodium sulfate solution crosslinking 5h that immobilization globule is transferred to 1M immobilization globule is obtained, rushed with normal saline
Wash 3 times it is standby.
Embodiment 2
A kind of charcoal immobilized microorganism preparation method, comprises the steps：
（1）, weigh the biomass carbons of 500 DEG C of 0.8g preparation, add 20ml to cultivate the bacterium solution of 22h；
（2）, weigh 8g polyvinyl alcohol, 1g sodium alginates and be respectively put in 100ml conical flasks, be separately added into 50ml and 20ml distilled water
Soaked overnight, vibrates on sodium alginate soln shaking table；Next day poly-vinyl alcohol solution water-bath vibration 3h or so is down to being uniformly dissolved
Room temperature adds sodium alginate soln, and with 5ml distilled water flushing inwalls；
（3）, will add biomass carbon bacterium solution and step（2）In solution be mixed to get mixed bacteria liquid, and rushed with 5ml distilled water
Inwall is washed, now polyvinyl alcohol concentration is 8%, and sodium alginate concentration is 1%；
（4）, weigh 5g boric acid in 100ml distilled water, treat boric acid solution saturation separate out, take supernatant add CaCl2So that CaCl2
The ultimate density of solution is 4%, prepares the CaCl of saturation boric acid2Crosslinker solution；
（5）, weigh the anhydrous sodium sulfate of 14.2g in 100ml distilled water, prepare the anhydrous slufuric acid sodium solution of 1M；
（6）, be the asepsis injector of 10ml with specification at room temperature by step（3）Mixed liquor vertically instills saturation in 10cm height
The CaCl of boric acid2In solution, and it is stirred continuously, is crosslinked 30min；
（7）, anhydrous sodium sulfate solution crosslinking 5h that immobilization globule is transferred to 1M immobilization globule is obtained, rushed with normal saline
Wash 3 times it is standby.
Embodiment 3
A kind of charcoal immobilized microorganism preparation method, comprises the steps：
（1）, weigh the biomass carbons of 700 DEG C of 0.8g preparation, add 20ml to cultivate the bacterium solution of 2h；
（2）, weigh 8g polyvinyl alcohol, 1g sodium alginates and be respectively put in 100ml conical flasks, be separately added into 50ml and 20ml distilled water
Soaked overnight, vibrates on sodium alginate soln shaking table；Next day poly-vinyl alcohol solution water-bath vibration 3h or so is down to being uniformly dissolved
Room temperature adds sodium alginate soln, and with 5ml distilled water flushing inwalls；
（3）, will add biomass carbon bacterium solution and step（2）In solution be mixed to get mixed bacteria liquid, and rushed with 5ml distilled water
Inwall is washed, now polyvinyl alcohol concentration is 8%, and sodium alginate concentration is 1%；
（4）, weigh 5g boric acid in 100ml distilled water, treat boric acid solution saturation separate out, take supernatant add CaCl2So that CaCl2
The ultimate density of solution is 4%, prepares the CaCl of saturation boric acid2Crosslinker solution；
（5）, weigh the anhydrous sodium sulfate of 14.2g in 100ml distilled water, prepare the anhydrous slufuric acid sodium solution of 1M；
（6）, be the asepsis injector of 10ml with specification at room temperature by step（3）Mixed liquor vertically instills saturation in 10cm height
The CaCl of boric acid2In solution, and it is stirred continuously, is crosslinked 30min；
（7）, anhydrous sodium sulfate solution crosslinking 5h that immobilization globule is transferred to 1M immobilization globule is obtained, rushed with normal saline
Wash 3 times it is standby.
Embodiment 4
A kind of charcoal immobilized microorganism preparation method, comprises the steps：
（1）, weigh the biomass carbons of 300 DEG C of 0.8g preparation, add 20ml to cultivate the bacterium solution of 22h；
（2）, weigh 8g polyvinyl alcohol, 1g sodium alginates and be respectively put in 100ml conical flasks, be separately added into 50ml and 20ml distilled water
Soaked overnight, vibrates on sodium alginate soln shaking table；Next day poly-vinyl alcohol solution water-bath vibration 3h or so is down to being uniformly dissolved
Room temperature adds sodium alginate soln, and with 5ml distilled water flushing inwalls；
（3）, will add biomass carbon bacterium solution and step（2）In solution be mixed to get mixed bacteria liquid, and rushed with 5ml distilled water
Inwall is washed, now polyvinyl alcohol concentration is 8%, and sodium alginate concentration is 1%；
（4）, weigh 5g boric acid in 100ml distilled water, treat boric acid solution saturation separate out, take supernatant add CaCl2So that CaCl2
The ultimate density of solution is 4%, prepares the CaCl of saturation boric acid2Crosslinker solution；
（5）, weigh the anhydrous sodium sulfate of 14.2g in 100ml distilled water, prepare the anhydrous slufuric acid sodium solution of 1M；
（6）, be the asepsis injector of 10ml with specification at room temperature by step（3）Mixed liquor vertically instills saturation in 10cm height
The CaCl of boric acid2In solution, and it is stirred continuously, is crosslinked 30min；
（7）, immobilization globule placed into 4 DEG C of Refrigerator store 20h, transfer to 1M anhydrous sodium sulfate solution crosslinking 5h be obtained it is solid
Surely change globule, it is standby with normal saline flushing 3 times.
Performance comparision after the biomass carbon immobilization prepared under 2 different temperatures of table
It should be noted last that, above example is only unrestricted to illustrate technical scheme, although with reference to this
Inventive embodiments have been described in detail, it will be understood by those within the art that, technical scheme is carried out
Modification or equivalent, without departure from the spirit and scope of technical scheme, which all should cover claim protection
In scope.
Claims (10)
1. a kind of charcoal immobilized microorganism preparation method, it is characterised in that：Comprise the steps：
（1）, configuration poly-vinyl alcohol solution and sodium alginate soln, polyvinyl alcohol soaked overnight, shook on sodium alginate shaking table
Polyvinyl alcohol water-bath dissolving is down to room temperature and is mixed with sodium alginate by night, next day, forms mixed gel liquid and stands；
（2）, will add biomass carbon microbial inoculum and mixed gel liquid mixing, obtain mixed bacteria liquid；
（3）, configuration saturation boric acid solution, wait have boric acid separate out after, take supernatant add calcium chloride, obtain final product crosslinked fluid saturation boric acid
CaCl2Solution；
（4）, mixed bacteria liquid is first instilled the CaCl of saturation boric acid2Solution crosslinking, transfers to anhydrous sodium sulfate solution crosslinking；
（5）, immobilization globule is obtained, it is standby with normal saline flushing.
2. charcoal immobilized microorganism preparation method according to claim 1, it is characterised in that：Step（1）In, gather
Ethylene determining alcohol is 6-10%, and sodium alginate concentration is 0.5-1%.
3. charcoal immobilized microorganism preparation method according to claim 2, it is characterised in that：Step（1）In, gather
Ethylene determining alcohol is 8%, and sodium alginate concentration is 1%.
4. charcoal immobilized microorganism preparation method according to claim 1, it is characterised in that：Step（2）In, it is raw
Material charcoal is to prepare at 300 DEG C, 500 DEG C or 700 DEG C.
5. charcoal immobilized microorganism preparation method according to claim 1, it is characterised in that：Step（2）In, it is micro-
Bacterial strain in biological bacterium solution is obtained using Protoplast Fusion Technique as parent by pseudomonass and Sphingol single-cell
Fusant bacterial strain F14.
6. charcoal immobilized microorganism preparation method according to claim 1, it is characterised in that：Step（4）In, will
Mixed bacteria liquid first instills the CaCl of saturation boric acid using dripping mode2Solution crosslinking, transfers to anhydrous sodium sulfate solution crosslinking；
Wherein, dripping is referred to.
7. charcoal immobilized microorganism preparation method according to claim 1, it is characterised in that：Step（4）In, chlorine
The mass fraction for changing calcium is 4%, and crosslinking time is 30min.
8. charcoal immobilized microorganism preparation method according to claim 7, it is characterised in that：Step（4）In, sulfur
Sour na concn is 1M, and crosslinking time is 5h.
9. charcoal immobilized microorganism preparation method according to claim 8, it is characterised in that：Step（4）In, will
Mixed bacteria liquid first instills the CaCl of saturation boric acid2Then immobilization globule is preserved 20h in 4 DEG C, then is turned by solution crosslinking 30min
The anhydrous sodium sulfate solution crosslinking 5h of 1M is moved to, immobilization globule is obtained.
10. a kind of charcoal immobilized microorganism globule, it is characterised in that：Prepared by claim 1-9 methods described.
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