CN103509784A - Screening method of mineralized microorganisms for self-repairing of concrete cracks - Google Patents
Screening method of mineralized microorganisms for self-repairing of concrete cracks Download PDFInfo
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Abstract
The invention discloses a screening method of mineralized microorganisms for self-repairing of concrete cracks. The method comprises the following steps: bacterial strains are activated and cultivated to prepare cell suspension; the cell suspension is added in alkali culture medium for cultivation after ultraviolet mutagenesis; the obtained culture solution is diluted into different concentrations and is cultivated on different flats; a plurality of single colonies are randomly picked to respectively store in holes of a microwell plate for inverted cultivation, wherein solid culture medium is placed in the microwell plate; the colonies in the microwell plate are inoculated in corresponding holes of a deep hole plate for stationary cultivation, wherein fermentation culture medium is placed in the deep hole plate; the deep hole plate is put in a deep hole plate centrifuger to separate out supernate as samples to be detected; and the calcium ion concentration and the calcium mineralization rate of the samples are detected to screen out excellent strains. The screening method provided by the invention has the characteristics of microquantity, miniaturization and parallelization, can synchronously process a plurality of samples, saves the manpower, material resources and time, and greatly improves the screening efficiency.
Description
[technical field]
The present invention relates to Microbial Breeding, relate in particular to the screening method of a kind of mineralising microorganism for distress in concrete selfreparing.
[background technology]
The cracking of concrete structure not only endangers the integrity of material of construction, causes bearing capacity and weather resistance to reduce, and may cause serious accident and loss of life and personal injury.At present, the inspection of distress in concrete and the main dependence of reparation manual operation, this method not only effect is limited but also costly, by contrast, concrete selfreparing is a kind of restorative procedure that has more sustainability and relatively low cost, becomes in recent years the study hotspot of field of civil engineering.Concrete selfreparing is divided into again physics selfreparing, chemical selfreparing and microorganism selfreparing.Wherein microorganism selfreparing refers to that the mineralising microorganism of mixing in concrete induces mineralising sedimentation to produce calcium carbonate to reach the object in automatic reparation crack after crack forms.With respect to traditional repairing and reinforcement material, microbial mineralization deposition material reaction conditions is gentle, negative effect is weak and consistency is good, can not cause irreversible reaction to structure and environment, and the eubiosis is had to active effect.
The bacterial strain with tosca activity has almost been contained each microbe groups, if be used for some kind of the microbial host bacillus of concrete selfreparing, wherein, anerobe produces carbonic acid gas as Pasteur genus bacillus mainly utilizes urase effect decomposing urea, and aerobic bacteria produces carbonic acid gas as Coriolis genus bacillus, false bacillus firmus etc. mainly utilize respiration, carbonic acid gas is converted into rapidly carbonate under high alkali environment, after reaching finite concentration, react formation calcium carbonate with calcium ion, crystal is grown up gradually, until stop up crack.Yet the effect of these bacterial strains in self-repair concrete practical application is barely satisfactory, its major cause is the dual deficiency of microorganism to the adaptive faculty of high alkali environment in concrete and tosca activity.
Efficient mineralising bacterial classification is the basis of concrete microorganism selfreparing.On the basis of setting up the easy activity rating of bacterial strain accurately method, from nature screening or to existing bacterial strain, carry out the important channel that genetic material transformation is acquisition excellent species always.
Tradition mineralising microbe to screen method is generally usingd 250ml triangular flask as fermenting container, every bottled nutrient solution 50ml, and each sample needs 3-4ml nitrite ion; Traditional method is inoculated sampling dilution with single passage pipettor and is waited operation; Traditional method is usingd spectrophotometer as detecting instrument, and each sample needs 50 μ L left and right fermented liquids, 3000 μ L colour developing application liquid, and the detection of every 1 sample needs 10s left and right; Therefore, conventional screening method workload is large, efficiency is low and with high costs.
[summary of the invention]
The technical problem to be solved in the present invention is to provide the screening method of the mineralising microorganism for distress in concrete selfreparing that a kind of workload is little, efficiency is high and with low cost.
In order to solve the problems of the technologies described above, the technical solution used in the present invention is that the screening method of a kind of mineralising microorganism for distress in concrete selfreparing, comprises the following steps:
101) bacterial strain is activated and cultivated, make cell suspending liquid;
102) cell suspending liquid is carried out adding in alkaline medium and cultivating after ultraviolet mutagenesis;
103) after being diluted to different concentration, cultivates respectively on different flat boards the nutrient solution obtaining in step 102; The random a plurality of single bacterium colonies of picking are stored in respectively in the hole of the microwell plate that solid medium is housed and are inverted and cultivate;
104) by the standing cultivation in hole corresponding to the deep-well plates that fermention medium is housed of the colony inoculation in microwell plate;
105) deep-well plates is positioned in deep-well plates whizzer and isolates supernatant liquor as testing sample;
106) calcium ion concn in working sample and calcium mineralization rate, filter out strain excellent.
Above-described screening method, step 101 comprises activation that bacterial strain is rule on agar plate, cultivate 20-28 hour for 28 ℃-32 ℃, get single bacterium colony point and be inoculated in new agar plate, after 28 ℃-32 ℃ cultivation 10-14h, with the sodium carbonate buffer of 90-110mM, wash lower lawn and make cell suspension.
Above-described screening method, step 102 comprises the rotor of cell suspending liquid and magnetic stirring apparatus is placed in to culture dish, and culture dish is placed on magnetic stirring apparatus, starts magnetic stirring apparatus, with after ultra violet lamp 10-20min, cell suspension is joined and in alkaline medium, cultivates 5-6h; Step 103 comprises the concentration of nutrient solution by certain gradient dilution, the diluent of quantitative different concns is coated to alkaline agar, each gradient is coated with a plurality of flat boards, cultivate 2-4 days for 28 ℃-32 ℃, the a plurality of single bacterium colonies of picking, be stored in the hole of the microwell plate that alkali solid substratum is housed 28 ℃-32 ℃, inversion cultivation 24-48h.
Above-described screening method, step 104 comprises the bacterium colony in microwell plate is equipped with in hole corresponding to the deep-well plates I of fermention medium, in 28 ℃ of-32 ℃ of standing cultivation 36-60h with corresponding being inoculated in of Multi-channel liquid transfer device; Fermented liquid in deep-well plates I is inoculated into accordingly and is equipped with in hole corresponding to the deep-well plates II of fermention medium, in 28 ℃ of-32 ℃ of standing cultivation 6-8 days; Deep-well plates II is positioned over to the centrifugal supernatant liquor obtaining in deep-well plates whizzer and is testing sample.
Above-described screening method, step 105 comprises with trace dilution method dilutes 3-5 doubly by testing sample, adds calcium ion colour developing application liquid in transparent microwell plate, adds testing sample to mix, lucifuge reaction 5-15min; After reaction finishes, adopt microplate reader to measure the absorbancy of color reaction liquid, according to typical curve and testing sample extension rate, calculate calcium ion concn and calcium mineralization rate in testing sample.
Above-described screening method, component and the preparation method of alkaline medium are as follows, yeast powder 5g/L, Tryptones 10g/L, sodium carbonate 10g/L; By above concentration weighing yeast powder and Tryptones, be dissolved in 900ml water, weigh sodium carbonate and be dissolved in 100ml water, after two kinds of solution sterilizations, mix; Component and the preparation method of alkali solid substratum are as follows: yeast powder 5g/L, Tryptones 10g/L, sodium carbonate 10g/L, agar powder 15g; By above concentration, weighing yeast powder, Tryptones and agar powder is dissolved in 900ml water; Weigh sodium carbonate and be dissolved in 100ml water, after two kinds of solution sterilizations, mix.
Above-described screening method, component and the preparation method of fermention medium are as follows: Pfansteihl sodium 5-10g/L, SODIUMNITRATE 1-2g/L, yeast powder 0.5-2g/L, potassium primary phosphate 0.01-0.02g/L, magnesium chloride 0.05--0.1g/L, CAPS11-22.13g/L, sodium-chlor 5-30g/L, sodium sulfate 3-4g/L, Repone K 0.677g/L, Potassium Bromide 0.05-0.15g/L, boric acid 0.02-0.03g/L, Sodium Fluoride 0.002-0.003g/L, calcium chloride 1-1.3g/L, strontium chloride 0.01-0.02g/L, deionized water 1L; According to above concentration, weighing calcium chloride, strontium chloride and magnesium chloride is dissolved in 180-220ml water; Weigh CAPS and be dissolved in 130-170ml water, with sodium hydroxide solution, the pH value of CAPS solution is adjusted to 9.6-10.5; Other components dissolved are in 630-670ml water; After above-mentioned 3 kinds of solution 115-121 ℃ sterilizing 15-30min, mix.
Above-described screening method, calcium atom spectroscopic analysis standard concentrated solution is drawn to each quantitative concentration ionic calcium soln with deionized water preparation gradient concentration ionic calcium soln with hyperchannel liquid-transfering gun to add in transparent microwell plate, each concentration adds 3 holes, with hyperchannel liquid-transfering gun, to being added with in the hole of gradient concentration ionic calcium soln, add quantitative calcium ion colour developing application liquid to mix, lucifuge reaction 5-15min; After reaction finishes, adopt microplate reader to measure the absorbancy at 575nm of color reaction liquid, take calcium ion concn as X coordinate, absorbance value is Y coordinate, obtains typical curve.
Above-described screening method, calcium ion colour developing application liquid is by developer and damping fluid equal-volume mixed preparing, wherein, the compound method of developer is as follows: in 500mg8-hydroxyquinoline, be dissolved in the concentrated hydrochloric acid of 5000 μ L, after adding o-cresolphthalein complexon 25mg to dissolve completely, add again 1mL Triton X-100 and mix, then add deionized water to 500ml; Damping fluid is the AMP aqueous solution of concentration 89.14g/L.
Above-described screening method, described bacterial strain is DSM8715.
The present invention has the feature of micro-ization, miniaturization and parallelization for the screening method of the mineralising microorganism of distress in concrete selfreparing, can process a large amount of samples simultaneously, saves human and material resources and time, significantly improves screening efficiency.
[accompanying drawing explanation]
Below in conjunction with the drawings and specific embodiments, the present invention is further detailed explanation.
Fig. 1 is the efficient mineralising microorganism high-pass screening process figure of the embodiment of the present invention 1.
Fig. 2 is the calcium ion color reaction product absorption curves figure of the embodiment of the present invention 296 well plate method.
Fig. 3 is the canonical plotting that the OCPC method of the embodiment of the present invention 296 well plate method detects calcium ion.
Fig. 4 is the calcium carbonate output figure of Bacillus pseudofirmus DSM8715 on solid medium under the different carbon source culture condition of the embodiment of the present invention 2
Fig. 5 is the active comparison diagram of the mineralising of the embodiment of the present invention efficient mineralising mutant strain of 1 strain DSM 8715 and starting strain.
[embodiment]
By the following examples the present invention is carried out to more specific detail, in embodiment, mentioned content is not limitation of the invention.
Embodiment 1
The method of the high flux screening of the mutagenesis of Bacillus pseudofirmus DSM8715 and efficient mineralising mutant strain is as follows:
By the strain DSM 8715(ATCC700159-DSM8715 of 4 ℃ of preservations) line activation on ASWM4 agar plate, cultivate 1 day for 30 ℃, with transfering loop, get single bacterium colony point and be connected to new ASWM4 agar plate and then with aseptic glass stick, fill whole flat board.
After 30 ℃ of cultivation 12h, with 100mM sodium carbonate solution, wash lower lawn and make cell suspension.By YK-Heber type bateria chamber counting cells concentration, cell suspension is diluted to 10 with 100mM sodium carbonate
8cells/mL.
The rotor of getting 7mL cell suspending liquid and magnetic stirring apparatus diameter 3cm is placed in the culture dish of diameter 9cm, culture dish is upper as for the magnetic stirring apparatus in super clean bench, adjusting rotary speed, padded magnetic stirring apparatus makes its daylight opening DLO from the about 20cm of ultraviolet lamp, open culture dish lid and ultraviolet lamp, after uv irradiating 16min, cell suspension is added to alkaline LB culture medium culturing 5-6h.
With 100mM sodium carbonate solution, inoculum concentration gradient is diluted to 10
-7, get 10
-4, 10
-5, 10
-6, 10
-7diluent 200 μ L coat alkaline LB agar plate, and 3 flat boards of each gradient coating, cultivate 2-4 days for 30 ℃.
Wherein, component and the preparation method of alkaline LB substratum are as follows, yeast powder 5g/L, Tryptones 10g/L, sodium carbonate 10g/L; By above concentration weighing yeast powder and Tryptones, be dissolved in 900ml water; Weighing sodium carbonate is dissolved in 100ml water; After two kinds of solution sterilizations, in aseptic Bechtop, mix and be sub-packed in culture vessel and can use in as triangular flask.
Random 1000 single bacterium colonies of picking, are stored in the hole of the 96 transparent microwell plates in hole that alkaline LB solid medium is housed, 30 ℃, inversion cultivation 24-48h; Number combination name of numbering, line number and the row of the transparent microwell plate in bacterial strain You Gaikong place in each hole.
Wherein, component and the preparation method of alkaline LB solid medium are as follows: yeast powder 5g/L, Tryptones 10g/L, sodium carbonate 10g/L, agar powder 15g; By above concentration, weighing yeast powder, Tryptones and agar powder is dissolved in 900ml water; Weighing sodium carbonate is dissolved in 100ml water; After two kinds of solution sterilizations, in aseptic Bechtop, mix while hot and be sub-packed in 96 orifice bore Zhong，Mei hole 300 μ L)
Bacterium colony in transparent microwell plate is equipped with in hole corresponding to the deep-well plates I of ASWM4 fermention medium with corresponding being inoculated in of Multi-channel liquid transfer device, covers 8 layers of gauze and at the foraminate plastic closure in corresponding aperture place, in 30 ℃ of standing cultivation 48h;
Fermented liquid in deep-well plates I is inoculated into accordingly and is equipped with in hole corresponding to the deep-well plates II of fermention medium, cover 8 layers of gauze and at the foraminate plastic closure in corresponding aperture place, in 30 ℃ of standing cultivation 168h; Deep-well plates II is positioned in deep-well plates whizzer to the centrifugal 10min of 3000 * g.Supernatant liquor in deep-well plates II hole is testing sample.
With trace dilution method, supernatant liquor is diluted to 3-5 doubly, in transparent microwell plate, add calcium ion colour developing application liquid, add testing sample to mix, with substratum, do blank, lucifuge reaction 5-15min.After reaction finishes, adopt microplate reader to measure the absorbancy of color reaction liquid, according to typical curve and fermented liquid extension rate, calculate calcium ion concn and calcium mineralization rate in supernatant liquor.
Calcium mineralization rate=(C
0-C
1)/C
0; C wherein
0refer to the initial calcium ion concn in substratum, initial calcium ion concn is the calcium ion concn in substratum, by the formula of substratum, is determined; C
1finger is through the residual calcium concn in 7 days cultivation secondary fermentation liquid, and the selection result has obtained 1 strain calcium mineralization rate and improved 11.1% mutant strain B388, and the calcium mineralization rate of bacterial strain the results are shown in Figure 5.
Embodiment 2:
The calcium carbonate output of Bacillus pseudofirmus DSM8715 on solid medium under different carbon source culture condition:
Prepare respectively minimum medium-sucrose B-Sugar, minimum medium-glucose B-Glucose, minimum medium-molasses B-Molasses, minimum medium-yeast powder B-yeast extract(ASWM4), minimum medium-solid medium Basic medium.Yeast powder composition in ASWM4 culture medium prescription is deleted and is basic medium substratum, with 1g/L sucrose, glucose or molasses, replace yeast powder respectively, be B-Sugar substratum, B-Glucose substratum and B-Molasses substratum.
Formula and the preparation method of ASWM4 fermention medium are as follows: Pfansteihl sodium 5-10g/L, SODIUMNITRATE 1-2g/L, yeast powder 0.5-2g/L, potassium primary phosphate 0.01-0.02g/L, magnesium chloride 0.05--0.1g/L, CAPS(3-encircles aminopropanesulfonic acid) 11-22.13g/L, sodium-chlor 5-30g/L, sodium sulfate 3-4g/L, Repone K 0.677g/L, Potassium Bromide 0.05-0.15g/L, boric acid 0.02-0.03g/L, Sodium Fluoride 0.002-0.003g/L, calcium chloride 1-1.3g/L, strontium chloride 0.01-0.02g/L, deionized water 1L.
Compound method: weigh calcium chloride, strontium chloride and magnesium chloride according to above concentration and be dissolved in 200ml water; Weigh CAPS and be dissolved in 150ml water, with 6M sodium hydroxide solution, the pH value of CAPS solution is adjusted to 9.6-10.5; Other compositions are dissolved in 650ml water; By after above 3 kinds of solution 115-121 ℃ high pressure moist heat sterilization 15-30min, in aseptic Bechtop, mix and with Multi-channel liquid transfer device divide be filled in 96 orifice plates standby.
In 24 orifice plates that are listed as to 4 row 6, in each hole, add 2mL substratum, every kind of substratum only adds row, be 4 holes of every kind of substratum, in 5 kinds of substratum Gong20Ge holes, be added with substratum, with the adjustable liquid-transfering gun of 8 passage, respectively inoculate 10 μ L strain DSM 8715 seed liquor, 3 holes in every kind of culture medium inoculated 1 row, another one hole is not inoculated, 30 ℃ of standing cultivations 7 days, take and not inoculate solid medium as contrast.
With dissecting needle, the solid medium in each hole is smash into fritter, with spoon, dig out, put into 50mL centrifuge tube, add 30mL deionized water, 100 ℃ of water-baths, until substratum melts, are put on vortex oscillation device and shake 2min, 100 ℃ of standing 10min of water-bath, the supernatant that inclines, then the deionized water that adds 30mL to boil, supernatant is removed in vibration hypsokinesis, so repeatedly, wash altogether 5 times and supernatant is removed in the hypsokinesis of vibrating, finally put into 80 ℃ of baking ovens dry 12 hours, evaporate the remaining calcium carbonate that is to moisture completely.
In centrifuge tube, add 2mL0.5M dilute hydrochloric acid respectively, screw vortex vibration 1min after lid, standing 12h.On whizzer, with the centrifugal 15min of 4500 * g, respectively get supernatant liquor 50-100 μ L, be transferred to each hole and be equipped with in the corresponding aperture of transparent microwell plate of 200 μ L0.5M dilute hydrochloric acid, with hyperchannel liquid-transfering gun pressure-vaccum, mix.
With the hyperchannel liquid-transfering gun that range is 10 microlitres, drawing 3.75 μ L adds in another new transparent microwell plate, the hyperchannel liquid-transfering gun that with range is 300 microlitres adds 300 μ L calcium ions colour developing application liquid to being added with in the hole of testing sample, not add the ASWM4 substratum of calcium chloride, do blank, lucifuge reaction 5-15min.After reaction finishes, adopt microplate reader to measure the absorbancy at 575nm of color reaction liquid, according to typical curve and extension rate, calculate in different holes calcium ion concn in supernatant liquor, and then volume calculates the calcium carbonate output in different holes more per sample.
The preparation of o-cresolphthalein complexon (OCPC) developer: take oxine 500mg and put in beaker, add concentrated hydrochloric acid 5000 μ L, make its dissolving and proceed in 500ml volumetric flask, add again o-cresolphthalein complexon 25mg, until completely dissolved, add 1mL Triton X-100 (TritonX-100) and mix, then add deionized water to 500ml, put 4 ℃ of preservations in polyethylene bottle.
The preparation of AMP ealkaline buffer: in the mid-deionized water 500mL of 1L volumetric flask, add AMP89.14g, add water to until completely dissolved 1L, put 4 ℃ of preservations in polyethylene bottle.
The preparation of colour developing application liquid: before use, how many according to institute's expense, OCPC developer is mixed with AMP damping fluid equal-volume.After mixing, draw 300 μ L colour developing application liquid and add in transparent micropore plate hole, by microplate reader, detect its absorbance value to wavelength 575nm light, if OD575 below 0.3, represents that this solution is qualified, can use.
The making of typical curve: dilute calcium atom spectroscopic analysis standard concentrated solution (calcium atom spectroscopic analysis standard concentrated solution 1.00gCa, sigma company product) to 10g/L(250mM with 100mL volumetric flask constant volume) after packing, in-20 ℃, save backup.With deionized water, prepare gradient concentration ionic calcium soln: 0.25mM, 0.5mM, 1mM, 2mM, 3mM, 4mM, 5mM.With the hyperchannel liquid-transfering gun of 10 microlitres, drawing each concentration ionic calcium soln of 3.75 μ L adds in transparent microwell plate, each concentration adds 3 holes, with the hyperchannel liquid-transfering gun of 300 microlitres, to being added with in the hole of gradient concentration ionic calcium soln, add 300 μ L calcium ions colour developing application liquid to mix, with deionized water, do blank, lucifuge reaction 5-15min.After reaction finishes, adopt microplate reader to measure the absorbancy at 575nm of color reaction liquid, take calcium ion concn as X-coordinate, OD575 value is ordinate zou, drawing standard curve, and result is as Fig. 3.The binary fit equation of typical curve is: y=0.1009x-0.0006, R2=0.9992.
Under different carbon source culture condition, the residual calcium detected result of Bacillus pseudofirmus DSM8715 fermented liquid is as Fig. 4.
The above embodiment of the present invention be take microorganism culturing that microwell plate is service platform and the foundation of activity determination method, for realizing high-throughput in bacterial screening process, lays a good foundation.The bacterial strain screening method of microwell plate system has the feature of micro-ization, miniaturization and parallelization, therefore adopts the efficient mineralising microorganism of high-throughput Policy Filtering can process a large amount of samples simultaneously, saves human and material resources and time, significantly improves screening efficiency.
The above embodiment high-throughput screening method of the present invention is usingd 96 orifice plates as fermenting container ，Mei hole dress liquid 300 μ L, and each sample only needs 300 μ L nitrite ions, saves nutrient solution and nitrite ion, and cost significantly reduces;
The high-throughput screening method of the above embodiment of the present invention is with Multi-channel liquid transfer device (6 passages, 8 passages or 12 passage pipettors) inoculate operation such as sampling dilution etc., each operation can correspondingly complete inoculation, sampling or the dilution of 6-12 sample, the mass of realizing operation, has improved operation efficiency;
Above each sample of embodiment high-flux detection method of the present invention only needs 3 μ L left and right, and 96 sample detection times of every plate only need 30s, have greatly shortened detection required time, have improved detection efficiency.
Claims (10)
1. for a screening method for the mineralising microorganism of distress in concrete selfreparing, it is characterized in that, comprise the following steps:
101) bacterial strain is activated and cultivated, make cell suspending liquid;
102) cell suspending liquid is carried out adding in alkaline medium and cultivating after ultraviolet mutagenesis;
103) after being diluted to different concentration, cultivates respectively on different flat boards the nutrient solution obtaining in step 102; The random a plurality of single bacterium colonies of picking are stored in respectively in the hole of the microwell plate that solid medium is housed and are inverted and cultivate;
104) by the standing cultivation in hole corresponding to the deep-well plates that fermention medium is housed of the colony inoculation in microwell plate;
105) deep-well plates is positioned in deep-well plates whizzer and isolates supernatant liquor as testing sample;
106) calcium ion concn in working sample and calcium mineralization rate, filter out strain excellent.
2. screening method according to claim 1, it is characterized in that, step 101 comprises activation that bacterial strain is rule on agar plate, cultivate 20-28h for 28 ℃-32 ℃, get single bacterium colony point and be inoculated in new agar plate, after 28 ℃-32 ℃ cultivation 10-14h, with the sodium carbonate buffer of 90-110mM, wash lower lawn and make cell suspension.
3. screening method according to claim 1, it is characterized in that, step 102 comprises the rotor of cell suspending liquid and magnetic stirring apparatus is placed in to culture dish, culture dish is placed on magnetic stirring apparatus, start magnetic stirring apparatus, with after ultra violet lamp 10-20min, cell suspension is joined and in alkaline medium, cultivates 5-6h; Step 103 comprises the concentration of nutrient solution by certain gradient dilution, the diluent of quantitative different concns is coated to alkaline agar, each gradient is coated with a plurality of flat boards, cultivate 2-4 days for 28 ℃-32 ℃, the a plurality of single bacterium colonies of picking, be stored in the hole of the microwell plate that alkali solid substratum is housed 28 ℃-32 ℃, inversion cultivation 24-48h.
4. screening method according to claim 1, is characterized in that, step 104 comprises the bacterium colony in microwell plate is equipped with in hole corresponding to the deep-well plates I of fermention medium, in 28 ℃ of-32 ℃ of standing cultivation 36-60h with corresponding being inoculated in of Multi-channel liquid transfer device; Fermented liquid in deep-well plates I is inoculated into accordingly and is equipped with in hole corresponding to the deep-well plates II of fermention medium, in 28 ℃ of-32 ℃ of standing cultivation 6-8 days; Deep-well plates II is positioned over to the centrifugal supernatant liquor obtaining in deep-well plates whizzer and is testing sample.
5. screening method according to claim 1, is characterized in that, step 105 comprises with trace dilution method dilutes 3-5 doubly by testing sample, adds calcium ion colour developing application liquid in transparent microwell plate, adds testing sample to mix, lucifuge reaction 5-15min; After reaction finishes, adopt microplate reader to measure the absorbancy of color reaction liquid, according to typical curve and testing sample extension rate, calculate calcium ion concn and calcium mineralization rate in testing sample.
6. screening method according to claim 3, is characterized in that, component and the preparation method of alkaline medium are as follows, yeast powder 5g/L, Tryptones 10g/L, sodium carbonate 10g/L; By above concentration weighing yeast powder and Tryptones, be dissolved in 900ml water, weigh sodium carbonate and be dissolved in 100ml water, after two kinds of solution sterilizations, mix; Component and the preparation method of alkali solid substratum are as follows: yeast powder 5g/L, Tryptones 10g/L, sodium carbonate 10g/L, agar powder 15g; By above concentration, weighing yeast powder, Tryptones and agar powder is dissolved in 900ml water; Weigh sodium carbonate and be dissolved in 100ml water, after two kinds of solution sterilizations, mix.
7. screening method according to claim 4, it is characterized in that, component and the preparation method of fermention medium are as follows, Pfansteihl sodium 5-10g/L, SODIUMNITRATE 1-2g/L, yeast powder 0.5-2g/L, potassium primary phosphate 0.01-0.02g/L, magnesium chloride 0.05--0.1g/L, CAPS11-22.13g/L, sodium-chlor 5-30g/L, sodium sulfate 3-4g/L, Repone K 0.677g/L, Potassium Bromide 0.05-0.15g/L, boric acid 0.02-0.03g/L, Sodium Fluoride 0.002-0.003g/L, calcium chloride 1-1.3g/L, strontium chloride 0.01-0.02g/L, deionized water 1L; According to above concentration, weighing calcium chloride, strontium chloride and magnesium chloride is dissolved in 180-220ml water; Weigh CAPS and be dissolved in 130-170ml water, with sodium hydroxide solution, the pH value of CAPS solution is adjusted to 9.6-10.5; Other components dissolved are in 630-670ml water; After above-mentioned 3 kinds of solution 115-121 ℃ sterilizing 15-30min, mix.
8. screening method according to claim 5, it is characterized in that, calcium atom spectroscopic analysis standard concentrated solution is drawn to each quantitative concentration ionic calcium soln with deionized water preparation gradient concentration ionic calcium soln with hyperchannel liquid-transfering gun to add in transparent microwell plate, each concentration adds 3 holes, with hyperchannel liquid-transfering gun, to being added with in the hole of gradient concentration ionic calcium soln, add quantitative calcium ion colour developing application liquid to mix, lucifuge reaction 5-15min; After reaction finishes, adopt microplate reader to measure the absorbancy at 575nm of color reaction liquid, take calcium ion concn as X coordinate, absorbance value is Y coordinate, obtains typical curve.
9. screening method according to claim 8, it is characterized in that, calcium ion colour developing application liquid is by developer and damping fluid equal-volume mixed preparing, wherein, the compound method of developer is as follows: in 500mg8-hydroxyquinoline, be dissolved in the concentrated hydrochloric acid of 5000 μ L, after adding o-cresolphthalein complexon 25mg to dissolve completely, then add 1mL Triton X-100 and mix, then add deionized water to 500ml; Damping fluid is the AMP aqueous solution of concentration 89.14g/L.
10. according to the screening method described in arbitrary claim in claim 1 to 9, it is characterized in that, described bacterial strain is DSM8715.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104403959B (en) * | 2014-09-18 | 2017-07-18 | 深圳大学 | One plant of efficient calcium mineralising Alkaliphilic bacillus and its application |
| CN107601941A (en) * | 2017-11-01 | 2018-01-19 | 深圳大学 | A kind of microbial augmentation regeneration aggregate and preparation method thereof |
| CN107975385A (en) * | 2017-10-16 | 2018-05-01 | 山东科技大学 | A kind of coal mine roadway anchoring whitewashing microorganism self-healing system and its construction method |
| CN113121145A (en) * | 2021-04-15 | 2021-07-16 | 同济大学 | Concrete crack self-repairing material based on microbial collaborative mineralization and application |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102906257A (en) * | 2010-03-11 | 2013-01-30 | 埃欧金生物制品公司 | Modified family 5 cellulases and uses thereof |
| CN103289979A (en) * | 2012-10-29 | 2013-09-11 | 北京仁峰科技有限公司 | Method for preparing alkaline protease by fermentation method |
-
2013
- 2013-09-18 CN CN201310430855.3A patent/CN103509784A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102906257A (en) * | 2010-03-11 | 2013-01-30 | 埃欧金生物制品公司 | Modified family 5 cellulases and uses thereof |
| CN103289979A (en) * | 2012-10-29 | 2013-09-11 | 北京仁峰科技有限公司 | Method for preparing alkaline protease by fermentation method |
Non-Patent Citations (9)
| Title |
|---|
| HENK M. JONKERS & ERIK SCHLANGEN: "Development of a bacteria-based self healing concrete", 《TAILOR MADE CONCRETE STRUCTRUES》 * |
| 吴松刚: "《高等师范院校新世纪教材 微生物工程》", 31 December 2004 * |
| 徐晶: "基于微生物矿化沉积的混凝土裂缝修复研究进展", 《浙江大学学报（工学版）》 * |
| 田志茗等: "当量溶液分光光度法同时测定微量钙和镁", 《光谱实验室》 * |
| 谭俊: "基于多尺度创新原理的工业微生物高通量筛选平台构建及应用研究", 《中国博士学位论文全文数据库 工程科技I辑》 * |
| 贾永峰等: "原生质体紫外诱变高通量选育盐霉素高产菌", 《中国医药工业杂志》 * |
| 钱春香等: "自修复混凝土中微生物矿化方解石的形成机理", 《硅酸盐学报》 * |
| 陈伟等: "羧甲基壳聚糖对钙离子的络合", 《福建医科大学学报》 * |
| 陈坚 等: "《发酵工程原理与技术》", 31 December 2012 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104403959B (en) * | 2014-09-18 | 2017-07-18 | 深圳大学 | One plant of efficient calcium mineralising Alkaliphilic bacillus and its application |
| CN107975385A (en) * | 2017-10-16 | 2018-05-01 | 山东科技大学 | A kind of coal mine roadway anchoring whitewashing microorganism self-healing system and its construction method |
| CN107601941A (en) * | 2017-11-01 | 2018-01-19 | 深圳大学 | A kind of microbial augmentation regeneration aggregate and preparation method thereof |
| CN113121145A (en) * | 2021-04-15 | 2021-07-16 | 同济大学 | Concrete crack self-repairing material based on microbial collaborative mineralization and application |
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