CN109504672A - A kind of acidophilus leaching microbacteria embedding body and preparation method thereof - Google Patents
A kind of acidophilus leaching microbacteria embedding body and preparation method thereof Download PDFInfo
- Publication number
- CN109504672A CN109504672A CN201910042170.9A CN201910042170A CN109504672A CN 109504672 A CN109504672 A CN 109504672A CN 201910042170 A CN201910042170 A CN 201910042170A CN 109504672 A CN109504672 A CN 109504672A
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- suspension
- microorganism
- embedding
- leaching
- filler
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- 238000001179 sorption measurement Methods 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 229910001220 stainless steel Inorganic materials 0.000 description 1
- 239000010935 stainless steel Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 229910052682 stishovite Inorganic materials 0.000 description 1
- 159000000008 strontium salts Chemical class 0.000 description 1
- 239000011593 sulfur Substances 0.000 description 1
- 229910052717 sulfur Inorganic materials 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 229910052905 tridymite Inorganic materials 0.000 description 1
- 229920002554 vinyl polymer Polymers 0.000 description 1
- 239000002351 wastewater Substances 0.000 description 1
- 238000004065 wastewater treatment Methods 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
Classifications
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- 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
- C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
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- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Biomedical Technology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明属于微生物固定化技术领域，具体是涉及到一种嗜酸浸矿微生物包埋体及其制备方法，所述嗜酸浸矿微生物包埋体是在聚氨酯多孔亲水填料载体表层上，通过交联形成的网状结构，包括如下组成：聚氨酯多孔亲水填料、包埋剂、碳酸盐、嗜酸浸矿微生物悬液、交联剂和稳定剂，所述碳酸盐为碳酸钠、碳酸氢钠、碳酸钾、碳酸氢钾中的一种或多种，所述稳定剂为含多价金属离子的微生物培养液，所述嗜酸浸矿微生物悬液的pH小于3.0。所述的包埋体制备流程是让遇水膨胀的亲水填料依次浸入含包埋液和碳酸盐的混悬液、微生物悬液、交联液和稳定液。本发明解决了常规包埋体水气通透性差，且难以兼顾培养过程中高活性和高细胞浓度的微生物溶液连续产出的问题。
The invention belongs to the technical field of microbial immobilization, and in particular relates to an acidophilic leaching microorganism embedded body and a preparation method thereof. The network structure formed by cross-linking comprises the following components: polyurethane porous hydrophilic filler, embedding agent, carbonate, acidophilic leaching microorganism suspension, cross-linking agent and stabilizer, wherein the carbonate is sodium carbonate, One or more of sodium bicarbonate, potassium carbonate and potassium bicarbonate, the stabilizer is a microbial culture solution containing polyvalent metal ions, and the pH of the acidophilic ore-leaching microbial suspension is less than 3.0. The preparation process of the embedded body is that the hydrophilic filler swelled by water is immersed in the suspension containing the embedding liquid and carbonate, the microbial suspension, the cross-linking liquid and the stabilizing liquid in sequence. The invention solves the problems that the conventional embedding body has poor water vapor permeability and it is difficult to take into account the continuous production of the microorganism solution with high activity and high cell concentration in the culture process.
Description
Technical field
The invention belongs to a kind of immobilized microorganism technology fields, are specifically related to a kind of acidophilus leaching microbacteria embedding
Body and preparation method thereof.
Background technique
Biological metallurgy is under the catalysed oxidn of related microorganisms that valuable metal in mineral is molten in the form of ion
Solution is into leachate and is recycled, or harmful element in mineral is dissolved and removed, in conjunction with related process sides such as hydrometallurgys
The technology that method is formed.Biological metallurgy is found the history of last 100 years to the immersion Zn of element in mineral, but its base
Plinth theoretical research is more weak, is never developed, and until the middle and later periods in 20th century, which obtains certain hair finally
Exhibition.The huge advantage of biological metallurgy grows rapidly this technology, and biological metallurgy has obtained extensively in mining and metallurgy industry at present
Using after the biological metallurgy of copper, gold, uranium realizes industrialized production, nickel, cobalt, zinc, manganese also gradually realize industrial applications.
Biological metallurgy microculture condition is usually acidic environment, pH value about 1.0~2.0, and they are relied primarily on
Ferrous ion, elemental sulfur, sulphide ore of redox state etc. obtain energy and growth and breeding.If biological metallurgy microorganism directly into
Row batch culture can get compared with high microorganism yield, but every batch of incubation all successively have passed through lag phase → logarithmic phase → steady
Periodically → decline phase, cultivation cycle is longer, and logarithmic phase and stationary phase are difficult to define, microbial activity it is difficult to ensure that, reduce
High-activity microorganism yield；According to continuous culture, although the culture efficiency of microorganism can be improved, most of leaching mine
Microorganism is autotrophic microbe, and growth cycle is relatively long, when continuous feeding and discharging process speed control not at that time, microbial bacteria
Body will be largely lost, and it is unbalance to eventually lead to continuous cultivating system.
In recent years, also occur some Immobilized culture technologies in the incubation of leaching microbacteria, the technology mainly by
Enzyme immobilization technology is derived, and is improved and is improved free microorganism system performance and grow up.Currently, in uranium
Large-scale industrial application has been obtained in the biological metallurgy technique of mine.Before entering biology leaching uranium, microorganism is fixed in inside
There is culture in the culture tank of 3 D elastic filler, allows microorganism to be fixed on filler, reach ferric ion in microculture
Regenerated purpose in slot.But the biofilm time of the method incubation is long, only iron oxidizing microorganisms of the part with adsorption capacity
Can in filler surface growth and breeding, microbial diversity in During Process of Long-term Operation it is difficult to ensure that, in washing away for air-flow and water flow
It acts on lower microbes to be inevitably largely lost, to seriously affect its popularization and application in production.Therefore, it is soaking
Needed in a hurry in mine microbial cultivation process it is a kind of can guarantee system iron oxidation activity and microorganism be largely lost
Immobilized culture technology.
Embedding is microbial immobilized common method, and there are two main classes for carrier material, and one kind is that natural polymer is more
Carbohydrate, such as sodium alginate, agar have many advantages, such as to be easy to form, and immobilization density small to Ecotoxicology is high, but such
Carrier Resistance to microbes ability is poor, and mechanical strength is lower, and service life is short；Another kind of is the high-molecular compound of synthesis, such as
Polyvinyl alcohol, polyacrylamide etc., strong, high mechanical strength, good chemical stability with Resistance to microbes performance, but
In embedding process, the formation condition of polymer supported volume grid is more violent, and high concentration crosslinking medicament is larger to microbe damage,
And it is poor to shape various structural controllability.Patent of invention " microbe carrier and preparation method thereof " (CN201010100571.4)
A kind of microbe carrier and preparation method thereof is provided, is a kind of microbe carrier with inierpeneirating network structure, not only has
Stable structure, and can guarantee the activity and diffusivity of institute's bearing microorganism and/or enzyme, but the method is only suitable for wastewater treatment
Process, microorganism are secured entirely on carrier inside, and microorganism is easy failure after expanding in carrier, needs to regularly replace.Invention
Patent " a kind of fixing means of microorganism " (CN201810707082.1) is big in order to solve the microbe damage in embedding process,
Carrier mechanical strength is lower, and microorganism is mixed with the type agent of helping and be fixed on nanosponges, although the method exists by service life short problem
The contact of microorganism with nutrients or pollutant will not be blocked while immobilized microorganism, but microorganism has firmly been trapped in sea
In silk floss, it is unfavorable for its growth and breeding, moisture vapor permeability is poor, is not suitable for forming efficient cultivating system.Equally, in biological metallurgy
Field, patent " referred to as the biomining microorganism of the work of BioSigma bioleaching seed (BBS) and alginates and iron ion
Capsule and its application that these microorganisms are inoculated with during bioleaching " (CN201380052933.9) is by leaching microbacteria packet
It is embedded in sodium alginate micro ball, avoids the loss of leaching microbacteria, but the microballoon surface layer that the method obtains is passed with internal microorganism
Matter efficiency difference is larger, and especially microorganism is difficult to obtain sufficient nutriment inside microballoon.In addition, leaching microbacteria culture
In system, autunezite easy to form leads to microballoon case-hardening, under the washing away of water flow and air-flow, removes layer by layer, is formed big
Measure solid waste.
Organic synthesis polymer immobilization method is also a kind of relatively conventional microorganism immobilization method, mechanical strength
It is high, chemical stability is good, Resistance to microbes is strong, to cytotoxic and it is cheap the advantages that be concerned, however, PVA
Investment has some defects, and the cross-link intensity such as PVA- boric acid is lower, the diffusion of embedding bacterium is hindered, after crosslinking
Bacterial activity loses easy adhesion of larger and immobilization particle etc..In order to increase the cross-link intensity of PVA- boric acid, improve immobilization
The hardness of grain and the bioactivity for improving embedding bacterium, polyvinyl alcohol immobilized new method embedding bacterium composite colloid particle is in poison
Property detection field in application, Xu Ying is superfine, Environmental Chemistry, and volume 34, the 10th phase, in October, 2015, it is different to have studied doping
The mode of additive is to study the influence to its performance.Calcium carbonate (CaCO is mainly investigated3), active carbon (AC), silica
(SiO2) and sodium alginate (SA).It is investigated from the result of research, the intensity of PVA doping AC or SA colloidal solid, which is substantially better than, mixes
Miscellaneous CaCO3Or SiO2Particle, main principle is the permeability for increasing sphere, and improves its cross-link intensity.Together
Sample, alginates are a kind of excellent immobilization materials, have that nontoxic, gel is simple, fixing condition is mild, fixed biomass
Greatly, the advantages that activity is high.But calcium-alginate-immobilized durability is poor, and with the extension of runing time, mechanical strength can gradually drop
It is low, so as to cause the disintegration of embedding body.In sewage treatment field, sodium alginate is usually further fixed on to wear-resistant filler
On come reduce fluid to embedding body damage, but be fixed on MBR filler or 3 D elastic filler outer layer there are still water flow rush
The problem of brush demoulding.Currently, most of embedding body is still with the macromolecules material such as such as alginate, polyvinyl alcohol, polyethylene glycol
Material is substrate, and embedding excessively tightly influences aqueous vapor transparent performance, and aerobic microbiological activity is affected, and embed it is excessively loose then
It is easy to cause microballoon (ball) to rupture, embedding effect is not achieved.In addition, it is contemplated that the cytotrophy inside embedding bead often supplies
The metabolite of deficiency, accumulation is excessive, the deterioration of gelled pill internal environment is bound to cause, so that micro- in the gelled pill of major diameter
Biological (cell) mortality increases, and therefore, Microsphere Size is the smaller the better, or even requires to be reduced to 250 μm hereinafter, this microballoon
Preparation difficulty it is big, it more difficult to adapt to extreme acidophilic microorganism growing environment.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of for the embedding body of acidophilus leaching microbacteria and its preparation side
Method solves microorganism and is easy to be lost in culture of continuous cultivation, and existing immobilization technology be difficult to take into account again high activity with
The problem of continuous output of high cell concentration microbial solution.
It is to be formed on carrier that a kind of acidophilus leaching microbacteria embedding body of the invention, which is in polyurethane cellular hydrophilic filler,
Reticular structure, including form as follows: polyurethane cellular hydrophilic filler, carbonate, acidophilus leaching microbacteria suspension, is handed over embedding medium
Join agent and stabilizer, packing volume expands after water is met in the polyurethane cellular hydrophilic filler, and the carbonate is sodium carbonate, carbonic acid
One of hydrogen sodium, potassium carbonate, saleratus are a variety of, and the stabilizer is the microbial culture medium of polyvalent metal ion, institute
The pH of acidophilus leaching microbacteria suspension is stated less than 3.0.
The polyurethane cellular hydrophilic filler is the elastomer modified containing hydrophilic high mol gel, preferably water-swellable,
The polyurethane cellular material that cubical expansivity is 110~200%, the polyurethane cellular hydrophilic filler preferably have following property
Matter: specific surface area is greater than 4000m2/m3, 0.8~2.2mm of aperture, porosity 98%, 1.1~1.3g/cm of density3.It can be made
Specification is 10~20 ± 1mm square or sphere.
The embedding medium includes the materials such as sodium alginate, polyvinyl alcohol, polyethylene glycol, and embedding medium of the invention is preferably sea
The mixture of mosanom and one of polyvinyl alcohol, polyethylene glycol, polyoxyethylene, hydroxypropyl methylcellulose or a variety of compositions；It is mixed
It closes in object, the weight percentage of sodium alginate is more than or equal to 50%.Relative to sodium alginate, polyvinyl alcohol, polyethylene glycol etc.
Single component embed body, mixed type, which embeds body, has more preferably stability, tough elasticity, water-retaining property and mechanical performance.
The crosslinking agent is the mixed solution of calcium chloride and boric acid, and calcium chloride concentration is 0.25~0.5mol/L, and boric acid is dense
Degree is 0.25~0.5mol/L.
The stabilizer is the microbial nutrient solution containing multivalent metal salt, and multivalent metal salt is preferably magnesium salts, calcium salt, molysite
Deng nutrient solution is preferably the mixed solution of ammonium sulfate, magnesium sulfate, dipotassium hydrogen phosphate, potassium chloride, calcium nitrate and ferrous sulfate, more
It is preferred that following component: 10~20g/L of green vitriol, 0.25~0.5g/L of magnesium sulfate, 1.5~3g/L of ammonium sulfate, phosphoric acid
Hydrogen 0.25~0.5g/L of dipotassium, 0.05~0.1g/L of potassium chloride, 0.005~0.01g/L of calcium nitrate, pH value 1.5~2.0.Stablize
Agent main function: filler expands completely in stabilizers, stablizes to the size under subsequent operating state；The acidity of stabilizer
Environment can sufficiently neutralize the carbonate in removing embedding suspension；Balance embeds the osmotic pressure in internal portion, avoids rupturing；Allow packet
It buries agent and polyvalent metal forms more stable cross-linked structure；Stabilizing solution principal component is microbial nutrient solution, mitigate embedding medium or
Crosslinking agent maintains microbial activity to the adverse effect of microorganism.
The present invention also provides a kind of preparation methods of acidophilus leaching microbacteria embedding body, include the following steps, by polyurethane
Porous hydrophilic filler immerses in suspension, and the suspension is the embedding medium mucus of dispersion and the mixture of carbonate, after taking-up,
Raffinate extra in hole is excluded, the filler for being attached with suspension is obtained；Then it is successively immersed to acidophilus leaching microbacteria to hang
It in liquid and crosslinking agent, is transferred in stabilizer, impregnates after draining residual solution, take out, drain raffinate and obtain the micro- life of thermophilic leaching-ore
Object embeds body.The mode for excluding raffinate extra in hole has centrifugation, extruding etc., drains residual solution or drains the side of raffinate
Formula has gravity to take off liquid etc..
In the suspension, the concentration expressed in percentage by weight of embedding medium is 1~5%, and the concentration of carbonate is 0.05~0.1mol/
L。
Acidophilus leaching microbacteria suspension is suspended in sulfuric acid solution for acidophilus leaching microbacteria and obtains, and the thermophilic leaching-ore is micro-
The concentration of biology is more than or equal to 109A/mL, the pH value of sulfuric acid solution are 1.5~2.0.
Preferably, polyurethane cellular hydrophilic filler is completely immersed in the suspension not solidified completely and is infiltrated, infiltrating time
For 30~60s, centrifugal dewatering excludes raffinate extra in hole, is centrifuged 100 × g, and the time is 3~5min.
After the filler for being attached with suspension is immersed acidophilus leaching microbacteria suspension, taken out when filler does not expand completely,
Infiltrating time is 30~60s；Alternatively, filler is immersed in the time in crosslinking agent as 1~5min；Alternatively, filler is immersed in surely
Determining the time in agent is 0.5~1h.
The preparation method of the suspension is to mix embedding medium with distilled water, and heating is completely dissolved, and is cooled to 50 DEG C
Hereinafter, being added immediately carbonate powder, it is stirred well to uniform state, embedding liquid A is obtained, keeps the temperature 30~45 DEG C.
By taking macromolecule embedding medium polyvinyl alcohol and sodium alginate as an example, the solution temperature of polyvinyl alcohol is 75~80 DEG C, will
Polyvinyl alcohol is dissolved in water, and concentration expressed in percentage by weight is 1~5%, when being cooled to 50 DEG C or less, polyvinyl alcohol water solution is still
Completely solidification, be added immediately 0.05~0.1mol/L powdered sodium carbonate, stir, obtain embedding suspension, and
30~45 DEG C of heat preservations.After carrier infiltration embedding suspension, liquid ot-yet-hardened to be embedded is added in acid solution, preferably pH immediately
Value is 1.5~2.0 dilution heat of sulfuric acid.Powdered sodium carbonate generates carbon dioxide in dilution heat of sulfuric acid, is formed in embedding liquid a large amount of
Micropore, and assemble formed large-sized carbon dioxide bubble also its can be full of in the inside of embedding medium, increase and increase packet
The internal voids for burying agent create the microenvironment of a preferable growth and breeding, and the structure in this gap to microorganism
Retention and fixation to microorganism bring great benefit.
Acidophilus leaching microbacteria of the present invention include iron-oxidizing bacteria Leptospirillum ferrooxidans,
Leptospirillum ferriphilum、Leptospirillum ferrodiazotrophum、Ferrimicrobium
acidiphilum、Acidimicrobium ferrooxidans、Ferroplasma acidiphilum、Ferroplasma
acidarmanus、Ferroplasma thermophilum、Acidiplasma cupricumulans、Acidiplasma
Aeolicum etc., sulfur oxidizing bacterium Acidithiobacillus thiooxidans, Acidithiobacillus caldus,
Acidithiobacillus albertensis, Thiomonas cuprina, Metallosphaera cuprina etc., iron sulphur
Oxidation bacteria Acidithiobacillus ferrooxidans, Acidithiobacillus ferrivorans, Acidianus
brierleyi、Acidianus manzaensis、Sulfobacillus acidophilus、Sulfobacillus
Thermosulfidooxidans etc., or the mixed culture of the above leaching microbacteria.Its cell concentration is preferably greater than or equal to
109A/mL.Microbial concentration method includes that supercentrifugal process and ceramic membrane concentration method or two kinds of combination, high speed centrifugation are wanted
Revolving speed is asked to reach 10000~12000 × g, ceramic membrane aperture≤0.1 μm is concentrated in ceramic membrane.
The invention has the advantages that the present invention using polyurethane cellular hydrophilic filler as matrix, passes sequentially through embedding liquid, it is dense
Leaching microbacteria embedding body is prepared in the immersion treatment of contracting bacterium solution, crosslinked fluid and stabilizing solution.Most of leaching microbacteria it is big
Small is 100~2000nm, and the surface layer embedding body gel aperture of filling carrier is distributed between 50~5000nm, and the aperture of filler
About 1 ± 0.2mm, retention of most of leaching microbacteria by embedding gel and filler, microorganism maintenance dose is big, not easily runs off.
And Institute of Micro-biology needs nutriment that can directly contact through filler and gel layer with microorganism, filler gap is also microorganism
Suitable microenvironment is provided, microorganism growth is promoted.The method of preparation embedding body, it is micro- dexterously to incorporate leaching mine in the present invention
The characteristic of biological culture system and embedding medium, time of contact of the microorganism directly with all kinds of high concentrations embedding medicament in embedding process
It is relatively short, embedding suspension and crosslinked fluid can be effectively controlled to the active negative effect of acidophilic microorganism.This method is specific
Carbonate particle in embedding suspension is utilized and encounters acid microorganism suspension formation bubble theory, embeds micropore for microorganism
Formation creates necessary condition, and the air pocket that minute bubbles pool forms intercommunicating pore；The polyurethane cellular of water-swellable is hydrophilic
After filler infiltration to microorganism suspension, since the viscosity of microorganism suspension is lower, filler expansion rate can be increased, and be had swollen
The filler of swollen property, which can effectively avoid, blocks filler gap in microorganism suspension cross-linking process, and is conducive to be formed on the inner wall of gap
Surface layer embedding；Carbon dioxide bubble and the filler expansion of convergence are all conducive to microorganism suspension extra in support voids and are quickly discharged
(bleeding) avoids the loss of high concentration microorganism suspension.The embedding body that this method obtains, the microenvironment aqueous vapor of microorganism growth
Permeability is more excellent than traditional investment, can significantly improve the oxygen of the cell total amount and ferrous ion in leaching microbacteria cultivating system
Change efficiency, is suitable for industrialized leaching microbacteria culture of continuous cultivation.
This method sufficiently incorporates the advantage of bio-film colonization method and bioencapsulation method, its advantages further include: (1) embeds
Carbonate, the carbon dioxide gas generated when mixing with acid solution are added in agent, the process that bubble increases allows microorganism
Suspension further touches the embedding medium in polyurethane filler, and bubble can increase embedding body porosity, effectively avoid embedding medium
Three-phase mass transfer is obstructed problem caused by filling is fine and close；(2) compared to traditional biofilm, this programme is directly prepared in carrier
Microorganism is subjected to biofilm in the process, effectively shortens the biofilm time；(3) embedding compared to particle embedding method, in this programme
There is body multiple microorganism to retain structure, and one is 1mm porous structure possessed by polyurethane filler itself, not only mitigate aeration
Process can also maintain suspension microorganism in gap to be lost the violent scour of embedding structure；The other is surface layer embedding medium 50~
5000nm cross-linked structure, can high concentration effectively in holding system microorganism, realize High Density Cultivation；(4) the embedding body is
The embedding structure of microorganism is formed on the surface layer of porous carrier, oxygen, carbon dioxide and nutriment etc. are easily accessible filler sky
Gap content portion, directly contacts with microorganism, is easy to be utilized by microorganism, is conducive to the quick growth and breeding of microorganism, appropriately sized
Crosslinking hole be also conducive to microbial metabolic products and be proliferated thallus release；(5) a variety of leaching microbacteria Immobilized cultures, energy
Leaching microbacteria is effectively avoided to be easy to be lost during scale co-cultures, and then the failure of caused function of microbial population
Problem；(6) the embedding body pore water subtractive process in this programme can make to embed body volume-diminished, and microorganism is lockked in embedding body
In cross-linked structure, and carrier and embedding gel itself have water-retaining property, and remaining capillary water and combination water can also maintain microorganism
Activity be more suitable for permanent preservation and transport compared to liquid microbial inoculum.
There are three types of the influence factors of immobilized microorganism technology, are as follows: microorganism property, support and environmental properties
(influence research of the addition adsorbent to embedded immobilization microbial gelled pill performance, Yang Hui, Lanzhou Jiaotong University master opinion
Text).Embedding object in the present invention is extreme acidophilus leaching microbacteria, and growing environment pH value 1.0~3.0 needs to consider to fix
The material of change in acid condition erodible avoids fixed failure.And carrier often will affect substrate and oxygen in investment
The diffusion of gas, carrier, embedding medium, crosslinking agent etc. are to the fixation degree of microorganism, microenvironment and microorganism locating for microorganism
It all can significantly be influenced with exchanging for external environment.Polyvinyl alcohol-saturation boric acid cross-linking method is the current most common embedding
Method, in order to improve its effect, the direction made improvements has: the improvement of immobilization particle balling property, mechanical strength
It improves, reduces the loss of activity in immobilization process, adjust the specific gravity of immobilization particle, improve hydraulic performance etc..For example it is added
Sodium alginate, the aqueous solution of baric or strontium salt, silica sand or stainless steel washer, cotton, porous carrier or non-woven fabrics introduce sponge
Shape porous carrier, addition anthracite, active carbon, garnet etc..
The present invention inherently has very high mechanical strength using Hdyrophilic polyurethane filler as skeleton matrix.Firstly, embedding
The carbonate material such as powdered sodium carbonate that can generate gas is added in liquid, forms embedding suspension, at this time the property of embedding medium
Change there is no apparent.Then filler is immersed in embedding suspension, excludes raffinate extra in gap, then embedding is adhered to
Body immerses acid bacterium solution a moment, takes out immediately, the percarbonate particle in suspension will persistently generate two after touching acidic liquid
Carbon oxide gas, generates a large amount of micropores on embedding attachment, and the gel embedding liquid internal voids on surface layer increase.Gravity takes off liquid
Afterwards, microorganism attachment is immersed into a moment in crosslinked fluid at once again, and is taken out immediately, gravity takes off liquid and completes cross-linking process, obtains
It is crosslinked attachment.It after gravity takes off liquid, is dipped in stabilizing solution, arrives embedding body to crosslinked complete stability.Due to stabilization
Liquid main component is the microbial nutrient solution containing polyvalent metal ion, avoids negative effect of the cross-linking process to microorganism.
The present invention separates microbial inoculum and embedding agent solution, can mitigate high concentration embedding medium to microbial inoculum
Direct toxicity.Secondly, polyurethane filler is successively quickly immersed in embedding suspension and microorganism suspension, on the one hand make sodium carbonate
Reaction concentrate on embedding medium (such as sodium alginate, polyvinyl alcohol etc.) and filler, avoid meaningless waste of raw materials, carbon
The reaction environment of sour sodium, the generation position of carbon dioxide is more accurate in other words, on the other hand, keeps reaction controllable, avoids carbonic acid
The reaction of sodium and acid solution is too acutely or slight, reaches ideal effect.In addition, filler has been after having impregnated embedding suspension,
Rapid centrifugation takes off liquid and effectively prevents sticky embedding liquid obstruction support voids, conducive to the efficient progress of subsequent embedding process,
And improve the survival rate of embedding acidophilic microorganism.
The present invention using after crosslinking agent, putting it into the stabilizer for being suitable for the growth of acidophilus leaching microbacteria, is allowing load
Body expands completely, stablizes to the size of working condition, and stabilizer contains a large amount of nutritional ingredient, is supporting for microorganism growth
Divide and crosslinking agent reduces influence of the embedding medicament to microbial activity, and embed mechanics performance after stabilizer
It is obviously improved with anti-corrosion performance.
Detailed description of the invention
Fig. 1 is polyurethane cellular hydrophilic filler microstructure.
Fig. 2 is the encapsulating layer microstructure for embedding body skeleton matrix surface.
Fig. 3 is the form of one plant of leaching microbacteria Sulfobacillus sp..
Fig. 4 is the preparation flow of leaching microbacteria embedding body.
Fig. 5 is a kind of comparison diagram of the front and back of leaching microbacteria filler immobilization of the present invention.
Specific embodiment
Embodiment 1
A kind of microorganism fixing means, comprises the following steps that
Step 1, the sodium alginate for weighing 10g is added distilled water 500mL, is completely dissolved with microwave stove heating after mixing, temperature
When degree is cooled to 50 DEG C, the micro-nano potassium carbonate of 3g is added, average grain diameter 266nm is uniformly mixing to obtain embedding suspension A, protects
45 DEG C of temperature；
Step 2, by cultured 10L leptospirillum ferriphilum (Leptospirillum ferriphilum YSK) and in
Thermophilic sulphur oxidation of sulfureted bacillus (Sulfobacillus thermosulfidooxidans YN22) is spent, supercentrifuge is passed through
10000 × g is centrifuged 20 minutes, is collected bacterium mud with the dilute sulfuric acid suspension bacterium mud of the pH2.0 of 500L and is obtained 3.6 × 109A/mL's
Microorganism suspension B；
Step 3, the calcium chloride for weighing 20g, is dissolved with distilled water, is settled to 500mL, obtains crosslinked fluid C；
Step 4, ammonium sulfate 1.5g, magnesium sulfate 0.25g, dipotassium hydrogen phosphate 0.25g, potassium chloride 0.05g, calcium nitrate are weighed
0.005g adjusts pH value to 2.0, green vitriol 20g is added, is settled to after dissolution after the dissolution of 500mL distilled water
500L, then pH value 2.0 is adjusted with the concentrated sulfuric acid, obtain stabilizing solution D；
Step 5, taking cube polyurethane water absorbent gel filler that specification is 10mm, (specific surface area is greater than 4000m2/m3, hole
Rate 98%, 1.2 ± 0.75g/cm of density3, appearance is similar to spongiosa bread internal structure, and buying is in the white environmental protection science and technology of nation, Shandong Province
Co., Ltd, model PPC-1) it is placed in embedding suspension A and impregnates 60 seconds, 100 × g is centrifuged 5min, removes more in gap
Remaining embedding liquid；
Step 6, it then by step 5 treated filler immerses in microorganism suspension B 60 seconds, gravity takes off liquid and drains in gap
Solution, be immediately transferred in crosslinked fluid C, impregnate 5 minutes, gravity take off liquid drain the solution in gap；
Step 7, the filler after step 6 processing is placed in stabilizing solution and is impregnated 60 minutes, it is extra that the de- liquid of gravity drains
Solution obtains leaching microbacteria embedding body.
The embedding body is added in the aeration reactor of 5L with the dosage of 20%v/v, and 45 DEG C of temperature control, aeration intensity
0.1vvm, after 5 days precultures, the oxidation-reduction potential in system reaches 687mV；Continuously preparing high iron solution process
In, iron oxidation efficiency reaches as high as 2.06g Fe2+/ (Lh), can be in iron oxidation efficiency 2g Fe2+The premise of/(Lh) or more
Lower continuous operation 1 week or more, the high iron solution of cyclic regeneration can be used for uranium ore or discarded circuit board Bioleaching；Utilizing 9K
During culture medium continuously prepares bacterial suspension, under the conditions of 48 hours residence times, leakage fluid dram suspension microorganism quantity can reach 1
~2 × 108A/mL holds time 3 months or more.
Embodiment 2
A kind of microorganism fixing means, comprises the following steps that
Step 1, the polyvinyl alcohol of 20g and the sodium alginate of 30g are weighed respectively, and distilled water 1L is added after mixing, uses microwave
Stove heating is completely dissolved, and when temperature is cooled to 50 DEG C, the powdered sodium carbonate of 5g is added, powder diameter is stirred evenly less than 5 μm
To embedding suspension A, 30 DEG C are kept the temperature；
Step 2, by cultured 50L Thiobacillus ferrooxidans (Acidithiobacillus ferrooxidans ATCC
23270) 5L, is concentrated by the ceramic membrane reactor in 0.1 μm of aperture, concentration bacterium solution pass through again 10000 × g of supercentrifuge from
The heart 20 minutes, bacterium mud was collected with the dilute sulfuric acid suspension bacterium mud of the pH 2.0 of 1L and obtains 1.3 × 1010The microorganism suspension of a/mL
B；
Step 3, the calcium chloride of 30g and the boric acid of 25g are weighed respectively, are dissolved with distilled water, are settled to 1L, obtain crosslinked fluid
C；
Step 4, ammonium sulfate 1.5g, magnesium sulfate 0.25g, dipotassium hydrogen phosphate 0.25g, potassium chloride 0.05g, calcium nitrate are weighed
0.005g adjusts pH value to 2.0, green vitriol 20g is added, is settled to after dissolution after the dissolution of 500mL distilled water
1L, then pH value 2.0 is adjusted with the concentrated sulfuric acid, obtain stabilizing solution D；
Step 5, taking cube polyurethane water absorbent gel filler that specification is 20mm, (specific surface area is greater than 4000m2/m3, aperture 1
± 0.2mm, porosity 98%, 1.2 ± 0.75g/cm of density3, appearance is similar to towel gourd internal structure, and buying is in nation, Shandong Province
White Environmental Protection Technology Co., Ltd, model PPC-2) it is placed in embedding suspension A and impregnates 60 seconds, on low speed centrifuge, revolving speed
100 × g is centrifuged 5min, removes the extra embedding liquid in gap；
Step 6, it then by step 5 treated filler immerses in microorganism suspension B 30 seconds, gravity takes off liquid and drains in gap
Solution, be immediately transferred in crosslinked fluid C, impregnate 3 minutes, gravity take off liquid drain the solution in gap；
Step 7, the filler after step 6 processing is placed in stabilizing solution and is impregnated 30 minutes, it is extra that the de- liquid of gravity drains
Solution obtains leaching microbacteria embedding body.
The embedding body is added in the aeration reactor of 10L with the dosage of 10%v/v, 30 DEG C of temperature control, pre- by 3 days
After culture, the oxidation-reduction potential in system reaches 721mV；During continuously preparing high iron solution, iron oxidation efficiency highest
Up to 3.91g Fe2+/ (Lh), can be in iron oxidation efficiency 2g Fe2+Continuous operation 2 weeks or more under the premise of/(Lh) or more,
The high iron solution of cyclic regeneration can be used for uranium ore or discarded circuit board Bioleaching；Bacterium is continuously being prepared using 9K culture medium
During suspension, the residence time 36 hours, leakage fluid dram suspension microorganism quantity can reach 3~6 × 108A/mL, holds time 3
A month or more.
The present invention relates to a kind of leaching microbacteria embedding body preparation method, the side that microbial bacteria is embedded with surface layer
Formula is fixed on inside elastic filler, reduces the loss of microbial bacteria.Filler and surface layer cross-linked network with gap structure
Nutriment will not be blocked to contact with microorganism, can improve the culture efficiency of microbial bacteria, filler long service life, and will not be right
Environment causes secondary pollution.
Embodiment 3
A kind of microorganism fixing means, comprises the following steps that
Step 1, the polyvinyl alcohol of 5g, the sodium alginate of 5g polyoxyethylene and 10g are weighed respectively, and distilled water is added after mixing
1L is completely dissolved with microwave stove heating, when temperature is cooled to 50 DEG C, the nano-calcium carbonate hydrogen sodium powder end of 5g is added, average grain diameter is about
280nm is uniformly mixing to obtain embedding suspension A, keeps the temperature 45 DEG C；
Step 2, by cultured 50L Sulfobacillus acidophilus (Sulfobacillus acidophilus TPY), lead to
The ceramic membrane reactor for crossing 0.1 μm of aperture is concentrated into 5L, and concentration bacterium solution passes through 10000 × g of supercentrifuge again and is centrifuged 20 minutes,
It collects bacterium mud and obtains 2.1 × 10 with the dilute sulfuric acid suspension bacterium mud of the pH 2.0 of 1L10The microorganism suspension B of a/mL；
Step 3, the calcium chloride of 30g and the boric acid of 20g are weighed respectively, are dissolved with distilled water, are settled to 1L, obtain crosslinked fluid
C；
Step 4, ammonium sulfate 1.5g, magnesium sulfate 0.25g, dipotassium hydrogen phosphate 0.25g, potassium chloride 0.05g, calcium nitrate are weighed
0.005g adjusts pH value to 2.0, green vitriol 20g is added, is settled to after dissolution after the dissolution of 500mL distilled water
1L, then pH value 2.0 is adjusted with the concentrated sulfuric acid, obtain stabilizing solution D；
Step 5, taking cube polyurethane water absorbent gel filler that specification is 20mm, (specific surface area is greater than 4000m2/m3, aperture 1
± 0.2mm, porosity 98%, 1.2 ± 0.75g/cm of density3, appearance is similar to towel gourd internal structure) and it is placed in embedding suspension
It is impregnated 60 seconds in A, on low speed centrifuge, 100 × g of revolving speed is centrifuged 5min, removes the extra embedding liquid in gap；
Step 6, it then by step 5 treated filler immerses in microorganism suspension B 30 seconds, gravity takes off liquid and drains in gap
Solution, be immediately transferred in crosslinked fluid C, impregnate 3 minutes, gravity take off liquid drain the solution in gap；
Step 7, the filler after step 6 processing is placed in stabilizing solution and is impregnated 30 minutes, it is extra that the de- liquid of gravity drains
Solution obtains leaching microbacteria embedding body.
The embedding body is added in the aeration reactor of 100L with the dosage of 10%v/v, and 45 DEG C of temperature control, by 3 days
After preculture, the oxidation-reduction potential in system reaches 709mV；During continuously preparing high iron solution, iron oxidation efficiency is most
High reachable 2.91g Fe2+/ (Lh), can be in iron oxidation efficiency 2g Fe2+Under the premise of/(Lh) or more continuous operation 2 weeks with
On, the high iron solution of cyclic regeneration can be used for uranium ore or discarded circuit board Bioleaching；It is continuously prepared using 9K culture medium
During bacterial suspension, the residence time 36 hours, leakage fluid dram suspension microorganism quantity can reach 2~4 × 108A/mL, when maintenance
Between 3 months or more.
Comparative example 1
This comparative example 1 is compared with embodiment 2, and difference is, the sodium carbonate in step 1 replaces with calcium carbonate, other steps
It is identical.
It is measured under equal conditions, after 3 days precultures, the oxidation-reduction potential in system is only 519mV；Continuous preparation is high
During ferrous solution, iron oxidation efficiency highest only 1.75g Fe2+/ (Lh) is continuously preparing bacterial suspension using 9K culture medium
In the process, residence time 36 hours, leakage fluid dram suspension microorganism quantity only 3.0~4.7 × 107A/mL.
It has been recognised by the inventors that foring calcium sulfate during calcium carbonate and acid reaction, calcium sulphate powders block skeleton matrix
In gap, influence mass transfer.
Comparative example 2
This comparative example 2 is compared with embodiment 2, and difference is, step 5 and 6 replacements are as follows: taking specification is cube poly- ammonia of 20mm
(specific surface area is greater than 4000m to ester water absorbent gel filler2/m3, 1 ± 0.2mm of aperture, porosity 98%, 1.2 ± 0.75g/ of density
cm3, appearance is similar to towel gourd internal structure) and it is placed in the mixed solution of embedding suspension A and microorganism suspension B and impregnates 60 seconds
Clock squeezes the extra solution in removal gap, is immediately transferred in crosslinked fluid C, impregnates 3 minutes, and gravity takes off liquid and drains gap
In solution.Other steps and embodiment 2 are identical.
It is measured under equal conditions, after 3 days precultures, the oxidation-reduction potential in system is only 524mV；Continuous preparation high-speed rail
In solution processes, iron oxidation efficiency is up to 2.05g Fe2+/ (Lh) is continuously preparing bacterial suspension mistake using 9K culture medium
Cheng Zhong, the residence time 36 hours, leakage fluid dram suspension microorganism quantity only 5.2~10 × 107A/mL, after operation 1 week, culture is anti-
Trench bottom is answered the embedding medium largely to fall off occur.
Almost, effect embedding body is unsatisfactory, and possible reason is to compare for the effect and comparative example 1 of comparative example 2
In example 2, acidophilic microorganism shifts to an earlier date to be mixed with neutral embedding liquid, and microbial activity is decreased obviously, and is easy to appear a large amount of gas
Bubble, influences the impregnation process of carrier；It is exposed in acidic environment for a long time before embedding liquid is uncrosslinked and is easy to appear hydrolysis；Suspension A
It is difficult to control with the bubble size formed after microorganism suspension B mixing, air pocket affects carrier infiltration, and solution hardly enters load
Internal portion, wastes the effective ratio area of carrier, and deposit efficiency is greatly reduced.Carbon dioxide, which does not reach, as a result, is wrapping
It buries and generates more porous in body and while containing the purpose of microorganism.
Comparative example 3
This comparative example 3 is compared with embodiment 2, and difference is, removes step 7, is extended filler in step 6 and is immersed in crosslinking agent
Time in C is 1h, other steps are identical.
It is measured under equal conditions, after 3 days precultures, the oxidation-reduction potential in system is 527mV；Continuous preparation high-speed rail
In solution processes, iron oxidation efficiency is up to 0.87g Fe2+/ (Lh) is continuously preparing bacterial suspension mistake using 9K culture medium
Cheng Zhong, the residence time 36 hours, leakage fluid dram suspension microorganism quantity was 2.3~5.1 × 107A/mL.
The effect of comparative example 3 is better than comparative example 1,2, but is worse than embodiment 2, and possible reason is one side crosslinking agent
Optimal pH be 8~9 and acidophilus leaching microbacteria optimum pH value it is not identical, lead to that optimum efficiency, another party is not achieved
Face, stabilizer can improve the microenvironment of microorganism, can reduce influence of the cross-linking process to microbial activity.
Comparative example 4
This comparative example 4 is compared with embodiment 2, and difference is, is extended filler in step 6 and is immersed in the time in crosslinking agent C
For 1h, other steps are identical.
It is measured under equal conditions, after 3 days precultures, the oxidation-reduction potential in system is 633mV；Continuous preparation high-speed rail
In solution processes, iron oxidation efficiency is up to 0.89g Fe2+/ (Lh) is continuously preparing bacterial suspension mistake using 9K culture medium
Cheng Zhong, the residence time 36 hours, leakage fluid dram suspension microorganism quantity was 3.5~5.2 × 107A/mL.
It is found through experiments that, the effect of comparative example 4 and comparative example 3 is similar, illustrates the main mesh of stabilizer of the invention
Be not only be used as microbial culture medium, also have the function of raising its cross-linking effect.
Technical solution and its effect to above-described embodiment and comparative example compare, and the result is shown in described in table 1.
The technical solution and Contrast on effect table of the different embodiment and comparative examples of table 1
Wherein, the most unit of high-speed rail oxidation efficiency is g Fe2+/ (Lh), the unit of leakage fluid dram micro organism quantity is/
mL。
In above scheme, the embedding effect and microbial activity of embodiment 2 are most strong.
The above description is only an embodiment of the present invention, is not intended to limit the scope of the invention, the present invention relates to
The preparation method of microorganism embedding body be equally applicable to the microorganisms of the other fields such as waste water, air pollution control and embedded
Journey, it is all using equivalent structure or equivalent flow shift made by description of the invention and accompanying drawing content, directly or indirectly transport
Used in other related technical areas, it is included within the scope of the present invention.
Claims (10)
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| CN111470625B (en) * | 2020-05-06 | 2022-03-29 | 北京工业大学 | Preparation and application of anaerobic hydrolysis acidification composite bacteria embedded bioactive filler |
| CN112358029A (en) * | 2020-09-10 | 2021-02-12 | 安徽普氏生态环境工程有限公司 | Novel biological composite filler and preparation method and application thereof |
| CN113207710A (en) * | 2021-04-24 | 2021-08-06 | 山东宠言生物科技有限公司 | Probiotic cat litter capable of deodorizing and inhibiting bacteria and preparation method thereof |
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| CN114149072A (en) * | 2021-12-28 | 2022-03-08 | 华夏碧水环保科技有限公司 | A kind of preparation method and filler system of water treatment filler that can be loaded with microorganisms |
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| CN114480364B (en) * | 2022-01-07 | 2024-01-26 | 成都大学 | Microorganism capsule for recovering valuable metals and preparation method and application thereof |
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