CN1916157A - Filtering transformant of trichoderma koningii capable of degrading pollution of oxide, and method for immobilization preparation - Google Patents
Filtering transformant of trichoderma koningii capable of degrading pollution of oxide, and method for immobilization preparation Download PDFInfo
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- CN1916157A CN1916157A CN 200610030903 CN200610030903A CN1916157A CN 1916157 A CN1916157 A CN 1916157A CN 200610030903 CN200610030903 CN 200610030903 CN 200610030903 A CN200610030903 A CN 200610030903A CN 1916157 A CN1916157 A CN 1916157A
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Abstract
This invention relates to a method for screening and immobilization preparing Trichoderma transformant for degrading cyanide pollutant. The method comprises: (1) constructing Trichoderma mutant by combined restriction enzyme-mediated gene technique; (2) screening Trichoderma transformant with high degradable activity by activity comparison analysis of primary strain and its transformant cyanide degrading enzyme; (3) using sodium alginate as the carrier to embed transformant cell after determining sodium alginate and CaCl2 concentrations, hypha embedding amount, immobilization time, and degradation temperature and time to prepare immobilization cell globule to obtain Trichoderma transformant. The obtained Trichoderma transformant can be used to repair cyanogens-containing wastewater, has higher cyanide degradation rate than free cells, and can be conveniently recovered and recycled.
Description
Technical field
The present invention relates to a kind of screening and immobilized preparation of transformant of trichoderma koningii capable of degrading pollution of oxide, from the trichoderma transformant that the gene integration technology (REMI) of utilizing the restriction enzyme mediation obtains, screen and have the active trichoderma transformant of high cyanide degradation, will be applied to treatment of cyanide waste water behind its cell fixation.
Background technology
Because the modern industry development is to industry ecological protection pay attention to day by day, the waste of therefore effectively handling industrial discharge is needs of setting up green industry.Prussiate is to one of the highest chemical pollutant of environmental organism toxicity as the main component in the frequent waste that discharges of industrial and mining enterprises, and can longer-term enrichment in water body and soil.The prussiate toxic mechanism has three kinds at least: (1) forms mixture with divalence or tervalent metal, and (2) generate stable cyanogen or carbonitrile derivatives in enzymolysis process, and (3) form cyanalcohol with ketone groups.
At present handling the most commonly used for the cyanide pollution thing is exactly chemical method, but it costs an arm and a leg, and the sludge yield height is usually introduced other harmful reagent in treating processes, simultaneously very low for the degradation efficiency of some metal cyanides.Therefore, be badly in need of a kind of low cost, high efficiency method is handled by the waste water of cyanide pollution and soil, biological degradation then is one of the most promising method.
Up to now, have been found that a lot of microorganisms all have the ability of degraded prussiate, wood is mould to be one of them.If microbial host is by producing rhodanese and cyanogen hydratase degraded free cyanide.Yet in living microorganism processing prussiate process, find, it will be subjected to the influence of many-sided factor to the cyanide degradation activity, as with sewage or soil in the competition effects of other microorganisms, growth is subjected to the inhibition of certain chemical substance of environment (ion), anti-adversity ability difference etc.The mould antagonism fungi that conduct always for a long time has biological and ecological methods to prevent plant disease, pests, and erosion to be worth as soil inhabitant of wood, its fast growth, it is short to seize space time, competitive power is strong, the chlamydospore of its generation simultaneously is to high ambient temperature, high humidity, therefore the resistance height of adverse circumstances such as arid is a kind of of great practical value, can take into account the multi-functional cyanide degradation microorganism of control corps diseases.Yet, naturally the Trichoderma strains for degrading activity of screening is generally lower at present, and the workload of screening is big, is difficult to obtain the equal ideal degradation bacteria strains of each side characteristic, therefore need utilize the bio-technology improvement wild strain, filter out than the higher bacterial strain of wild strain cyanide degradation enzyme activity.
The reparation that live body degraded microorganism directly applies to contaminate environment is subjected to a lot of condition restriction, therefore generally needs just can better bring into play degradation effect through after the immobilization.Waste Water Treatment just began the seventies in 20th century to utilize immobilized cell to set up efficiently, and this technology has reactor organisms concentration height, and processing power is big, and sludge yield is few, and bacterial classification is high-purity efficient, adapts to advantages such as water quality and pH variation.Yet, utilize the immobilization technology fixation of microbe, need to solve problems such as the erosion of various ion pair immobilized cells in the waste water and fixation support structure be easily destroyed, for this reason, need research how to improve immobilized cell physical strength and stability, and the original high degrading activity of being maintained fixed cell.
Summary of the invention
The objective of the invention is at the deficiencies in the prior art, a kind of screening and immobilized preparation of transformant of trichoderma koningii capable of degrading pollution of oxide is provided, the trichoderma transformant that the obtains prussiate in the water body of effectively to degrade.
For realizing this purpose, the present invention at first utilizes the mediated gene of restriction endonuclease integration technology to make up trichoderma harzianum mutant strain, by active comparative analysis to cyanide degradation enzyme system between starting strain and transformant thereof, filter out trichoderma transformant, then sodium alginate and CaCl in having determined the immobilization process to cyaniding object height degrading activity
2On the basis of factors such as concentration, mycelia embedding amount, immobilization time, degradation temperature and time, use sodium alginate, preparation immobilized cell bead as carrier embedding transformant cell.Detect the immobilization trichoderma transformant at last to the cyanide wastewater recovery dynatron effect.
Method of the present invention specifically comprises the steps:
1, the structure of trichoderma transformant
Employing plasmid 30-40 μ g, concentration are that restriction enzyme HindIII10 μ l, the sorbyl alcohol damping fluid (1M pH7.5) of 10U/ μ l is STC 200 μ l, are mixed with enzyme-plasmid mixed solution, and is stand-by.With concentration is 10
6-10
8Behind the Trichoderma protoplastis suspension 100 μ l of/ml and the enzyme-plasmid mixed solution mixing that is mixed with; slowly adding concentration is PEG for the 60%2ml polyoxyethylene glycol; the STC that adds the 5ml precooling; centrifugal 15 minutes of 4 ℃ of following 2000rpm; abandon supernatant; add 3ml liquid regeneration culture medium; mixing; pour in the sterile petri dish; add the solid regenerated substratum that 15-20ml has dissolved well and has been cooled to 45-55 ℃, place after 6 hours down, pour the water agar 10-15ml that contains 300 μ g/ml Totomycin into for 20-25 ℃; be tiled on the regeneration culture medium, transformant occur after 2-3 days.The transformant cultivation is carried out postsearch screening at the PDA substratum that contains 250 μ g/ml Totomycin, to transformant that can normal growth carry out 5-7 for the PDA culture medium culturing after, again transformant is cultivated on the PDA substratum that contains 250 μ g/ml Totomycin, this moment still can normal growth be exactly the stable transformant that obtains by the REMI technology, they are inoculated into to be placed on the PDA inclined-plane in 4 ℃ of refrigerators preserve.
Wherein, being formulated as of described liquid regeneration culture medium: get ammonium sulfate 2.8g respectively, urea 600mg, vitriolate of tartar 4g, Calcium Chloride Powder Anhydrous 600mg, glucose 40g, sucrose 100g, MgSO
410H
2O 200mg, FeSO
47H
2O 10mg, ZnSO
47H
2O 1.8mg, MnSO
4H
2O 3.2mg, CoCl
26H
2O 4mg joins in the 800ml water, regulates pH value to 5.0.Solid medium then adds the 18-20g agar powder on the basis of liquid medium within.
2, the silicon particle of transformant is preserved
After mensuration to the mould cyanide degradation enzyme of wood, the active high trichoderma transformant of the degraded prussiate that obtains is placed PDA (potato-glucose-agar) culture medium culturing under 28 ℃ of conditions, the dull and stereotyped cultivation 5-7 days makes green spores be covered with whole culture dish.Get spiral mouth Glass tubing, put into the 1/4-1/2 of silicon particle to the Glass tubing volume, after dry sterilization was handled, cooling was used preceding ice bath 15-30 minute.The skimmed milk 0.5-2 milliliter of getting 5% sterilization adds in the culture dish, and level is rocked, and makes spore suspension.Draw the spore suspension of 0.5-1 milliliter with aseptic transfer pipet or rifle, be added to the silicon particle surface in the chilled Glass tubing, after closing the lid, shake rapidly, make silicon particle moistening and be stained with spore, put back to ice bath immediately.After 10-20 minute, tighten lid and put into 4 ℃ or-20 ℃ of refrigerators and preserve.
3, the cultivation of mycelia
Get the silicon particle of an above-mentioned infiltration spore suspension, be positioned on the substratum of PDA, cultivate after 5-7 days, green spores produces, picking length has the mycelia of spore to mix with the 5ml-6ml sterilized water, and the spore amount of picking makes mixing solutions be green and gets final product, and filters with three layers of cleansing tissue and obtains filtrate; Get 3ml filtrate and put into 50-100ml PDB (yeast extract 15g adds the water constant volume to 1000ml for potato 200g, glucose 20g) liquid nutrient medium, in shaking table, 28 ℃ of following 100-140rpm cultivated 60-70 hour down.With cultured mycelia, three layers of lens wiping paper filter, and with NaCl or the sterilized water of 0.7M substratum in the mycelia are rinsed well, obtain green wet mycelium.
4, the preparation of immobilized spherule
Place the ratio of 100ml distilled water in 2g-3g sodium alginate, 1g diatomite, sodium alginate, diatomite are placed distilled water, heated and stirred dissolving and moist heat sterilization are mixed with sodium alginate soln.
The preparation mass concentration is the CaCl of 2-3%
2Solution is loaded on moist heat sterilization in the bottle.In the ratio of putting into wet mycelia 5 grams in every 200ml sodium alginate soln, the sodium alginate soln that wet mycelia is put into after the sterilization stirs, and uses the 20-50ml syringe to draw the mycelia mixed solution, with syringe needle it is squeezed into the above-mentioned CaCl of 200ml-400ml
2Stir in the solution and at the uniform velocity, form the bead of φ 3-5mm, bead is at CaCl afterwards
24 ℃ were descended fixedly 2-3 hour in the solution, filtered being fixed bead.With sterilized mass concentration is the flushing of 0.9% NaCl solution, the unconjugated calcium ion of flush away.It is that the glutaraldehyde solution of 0.1-0.3% carried out crosslinked 30 seconds-2 minutes that continuation is put into volumetric concentration with bead.Behind aseptic water washing, the immobilized spherule that obtains preparing is the immobilization trichoderma transformant, 4 ℃ of preservations.
The immobilization trichoderma transformant that adopts the inventive method to obtain is joined in the basic ion substratum that contains prussiate 52ppm and 250ppm, the CN of 500ppm
-At 60 hours, 12 days, after 22 days, degradation rate reached more than 95% respectively, and the residual concentration of prussiate all is lower than national examination criteria.
The immobilized spherule that utilizes the present invention the to prepare prussiate of degrading, the degradation speed of its degradation speed specific ionization cell want fast, and convenient the recovery, and the bead of recovery is with 3% CaCl
2After fixing 2 hours, can be used for the cyanide degradation experiment of next batch again.
Embodiment
Below in conjunction with specific embodiment technical scheme of the present invention is further described.
Embodiment 1:
1, transformant makes up and obtains
Employing plasmid 40 μ g, concentration are that restriction enzyme HindIII10 μ l, the sorbyl alcohol damping fluid (1M pH7.5) of 10U/ μ l is STC 200 μ l, are mixed with enzyme-plasmid mixed solution, and is stand-by.With concentration is 10
6The Trichoderma protoplastis suspension 100 μ l of/ml and preceding prepared enzyme-plasmid mixed solution mixing of finishing after; slowly adding concentration is PEG for the 60%2ml polyoxyethylene glycol; the STC that adds the 5ml precooling; centrifugal 15 minutes of 4 ℃ of following 2000rpm; abandon supernatant; add 3ml liquid regeneration culture medium; mixing; pour in the 9cm sterile petri dish, add to have dissolved and good be cooled to 45 ℃, the solid regenerated substratum of 20ml; place after 6 hours down for 25 ℃; pour the water agar 10ml that contains 300 μ g/ml Totomycin into, be tiled on the regeneration culture medium, transformant occurs after 2 days.The transformant cultivation is carried out postsearch screening at the PDA substratum that contains 250 μ g/ml Totomycin, after transformant that can normal growth carried out 7 generation PDA culture medium culturing, again transformant is cultivated on the PDA substratum that contains 250 μ g/ml Totomycin, this moment still can normal growth be exactly the stable transformant that obtains by the REMI technology, they are inoculated into to be placed on the PDA inclined-plane in 4 ℃ of refrigerators preserve.
Wherein, being formulated as of described liquid regeneration culture medium: get ammonium sulfate 2.8g respectively, urea 600mg, vitriolate of tartar 4g, Calcium Chloride Powder Anhydrous 600mg, glucose 40g, sucrose 100g, MgSO
410H
2O 200mg, FeSO
47H
2O 10mg, ZnSO
47H
2O 1.8mg, MnSO
4H
2O 3.2mg, CoCl
26H
2O 4mg joins in the 800ml water, regulates pH value to 5.0; Solid medium then adds the 18-20g agar powder on the basis of liquid medium within.
2, TkA8 transformant screening and silicon particle are preserved:
The transformant that the REMI technology is obtained is degraded after the screening of prussiate enzyme activity, has obtained T30 transformant TkA8.Make green spores cover with whole ware in 28 ℃ of cultivations on the PDA after 6 days TkA8.In the preparation work before, silicon particle is filled 1/3 of spiral mouth Glass tubing, prepare 5% skim-milk 50ml, and moist heat sterilization.Before beginning silicon particle bacterial classification is preserved, earlier with silicon particle pipe ice bath 30 minutes.Take out cultured TkA8, in culture dish, put into the 5% aseptic skim-milk of 1ml, rock culture dish, make spore suspension, the spore suspension of drawing 0.5ml is gone in the silicon particle pipe in ice bath, takes out acutely rapidly and shakes, to guarantee that all being stained with spore on all silicon particles gets final product, again it is put back in the ice bath, take out after 15 minutes, put into-20 ℃ of refrigerators and preserve.
3, the cultivation of TkA8 transformant mycelia:
Take out one of TkA8 transformant silicon particle, be placed on the culture dish central authorities of good PDA, cultivate in 28 ℃ of incubators after 7 days, the inoculation hook is chosen the long mycelia that spore is arranged on 1/3 ware, mix with the 5ml sterilized water, obtain spore suspension after the filtration, get 3ml in 50ml PDB (potato 200g, glucose 20g, yeast extract 15g adds the water constant volume to 1000ml) in the liquid nutrient medium, 28 ℃, rotating speed 140rpm cultivated 70 hours.Afterwards, mycelia obtains the substratum in the sterile solution flush away mycelia of the NaCl of usefulness 0.7M by having put three layers of cleansing tissue Bs filtration.
4, the preparation of immobilized cell bead:
Before the immobilized cell preparation, take by weighing 6g sodium alginate and 2g diatomite earlier and be dissolved in the 200ml water, want heated and stirred in the dissolution process, the preparation mass concentration is 3%CaCl
2Solution 400ml, the equal moist heat sterilization of used solution.Dose volume concentration is that the 0.3%400ml glutaraldehyde solution is stand-by in addition.Sodium alginate-diatomite the 200ml of sterilization is poured in the beaker of 400ml, take by weighing 10g TkA8 wet mycelium (wet mycelium: sodium alginate-diatomite=1: 20), put into rotor and stir, be evenly distributed until mycelia in wherein.Get asepsis injector, draw sodium alginate-diatomite-mycelia mixture, use No. 12 syringe needles that the mycelia mixture is injected into 3%CaCl
2In the solution, constantly stir CaCl simultaneously
2Solution, the instantaneous formation of bead.After the mycelia sodium alginate mixture of 200ml all is prepared into bead, allow them at CaCl
2In the solution, fix 2 hours under 4 ℃ of conditions, filter being fixed bead, remove unnecessary calcium ion with the flushing of 0.9%NaCl solution after, with bead in 4 ℃ 0.3% glutaraldehyde solution crosslinked 1 minute, filter with sterilized water clean.Obtain stand-by immobilized spherule.Free mycelia is about 1: 18 with the ratio that is embedded into the alginate calcium bead of mycelia.
Take by weighing 10 gram immobilized spherules in basic ion substratum, the weight of immobilized spherule is 1: 7.5 with the ratio of the volume of basic ion substratum, and the adding final concentration is 52 mcg/ml CN
-Potassium cyanide solution, 180 rev/mins of shaking culture were measured CN after 60 hours
-Concentration, degradation rate reaches 95%.
Immobilized spherule in the substratum completely of degrading is taken out, and sterilized water is put into 3%CaCl after cleaning
2In the solution, fix 2 hours for 4 ℃, the bead that obtains can continue to utilize the degraded cyanide-contained solution, degradation efficiency be prepared fresh bead 92%, and can recycle more than 6 times.
Embodiment 2:
1, transformant makes up and obtains
Adopting plasmid 30 μ g, the restriction enzyme HindIII10 μ l of 10U/ μ l, sorbyl alcohol damping fluid (1MpH7.5) is STC 200 μ l, is mixed with enzyme-plasmid mixed solution, stand-by.With concentration is 10
6The Trichoderma protoplastis suspension 100 μ l of/ml and preceding prepared enzyme-plasmid mixed solution mixing of finishing after; slowly adding concentration is PEG for the 60%2ml polyoxyethylene glycol; the STC that adds the 5ml precooling; centrifugal 15 minutes of 4 ℃ of following 2000rpm; abandon supernatant; add 3ml liquid regeneration culture medium; mixing; pour in the 9cm sterile petri dish, add to have dissolved and good be cooled to 55 ℃, the solid regenerated substratum of 15ml; place after 6 hours down for 25 ℃; pour the water agar 10ml that contains 300 μ g/ml Totomycin into, be tiled on the regeneration culture medium, transformant occurs after 3 days.The transformant cultivation is carried out postsearch screening at the PDA substratum that contains 250 μ g/ml Totomycin, after transformant that can normal growth carried out 5 generation PDA culture medium culturing, again transformant is cultivated on the PDA substratum that contains 250 μ g/ml Totomycin, this moment still can normal growth be exactly the stable transformant that obtains by the REMI technology, they are inoculated into to be placed on the PDA inclined-plane in 4 ℃ of refrigerators preserve.
2, TkB5 transformant screening and silicon particle are preserved:
The transformant that the REMI technology is obtained is degraded after the screening of prussiate enzyme activity, has obtained T30 transformant TkA8.Make green spores cover with whole ware in 28 ℃ of cultivations on the PDA after 6 days TkA8.In the preparation work before, silicon particle is filled 1/3 of spiral mouth Glass tubing, prepare 5% skim-milk 50ml, and moist heat sterilization.Before beginning silicon particle bacterial classification is preserved, earlier with silicon particle pipe ice bath 30 minutes.Take out cultured TkA8, in culture dish, put into the 5% aseptic skim-milk of 1ml, rock culture dish, make spore suspension, the spore suspension of drawing 0.5ml is gone in the silicon particle pipe in ice bath, takes out acutely rapidly and shakes, to guarantee that all being stained with spore on all silicon particles gets final product, again it is put back in the ice bath, take out after 15 minutes, put into-20 ℃ of refrigerators and preserve.
3, the mycelial cultivation of TkB5 transformant:
Take out two of TkB5 transformant silicon particles, be placed in the culture dish of good PDA, cultivate in 28 ℃ of incubators after 5 days, the inoculation hook is chosen 1/2 ware mycelia, mix with the 5ml sterilized water, obtain spore suspension after the filtration, get 5 in 100ml PDB liquid nutrient medium, 28 ℃, rotating speed 140rpm, cultivate after 60 hours, mycelia obtains by having put three layers of cleansing tissue Bs filtration, with sterilized water residual substratum is rinsed well.
4, the preparation of immobilized spherule:
Before the immobilized spherule preparation, take by weighing the 3g sodium alginate earlier and 1 gram diatomite is dissolved in 100 ml waters, want heated and stirred in the dissolution process, 200 milliliters of preparation 3%CaCl2 solution, the equal moist heat sterilization of used solution.Prepare 0.3% glutaraldehyde 200ml solution for later use in addition.100 milliliters in the sodium alginate-diatomite of sterilization poured in 200 milliliters the beaker, take by weighing 5g TkB5 wet mycelium and be put in wherein, be stirred to mycelia and be evenly distributed.Get the asepsis injector of No. 12 syringe needles of tool, draw sodium alginate-diatomite-mycelia mixture, inject 3%CaCl
2In the solution, constantly stir the instantaneous formation of bead.200 milliliters mycelia beads are added CaCl
2Solution is fixed 3 hours under 4 ℃ of conditions, filters being fixed bead, after removing unnecessary calcium ion with 0.9%NaCl solution flushing, with bead in 4 ℃ 0.3% glutaraldehyde solution crosslinked 2 minutes, filter with sterilized water and clean, promptly obtain stand-by immobilized spherule.
Take by weighing immobilized spherule that 12.5g prepares and put into the basic medium of 100ml pH6.5, add 500ppm CN
-The potassium cyanide of final concentration, 28 ℃, 140 rev/mins, shaking culture.After 22 days, the concentration degradable of the KCN concentration in the substratum is to 0.2ppm.
Claims (1)
1, a kind of screening of transformant of trichoderma koningii capable of degrading pollution of oxide and immobilized preparation is characterized in that may further comprise the steps:
1) structure of trichoderma transformant: adopt restriction enzyme HindIII10 μ l, the STC sorbyl alcohol damping fluid 200 μ l of plasmid 30-40 μ g, 10U/ μ l, be mixed with enzyme-plasmid mixed solution; With concentration is 10
6-10
8Behind the Trichoderma protoplastis suspension 100 μ l of/ml and the enzyme-plasmid mixed solution mixing that is mixed with, slowly adding concentration is the 60%2ml polyoxyethylene glycol, the STC that adds the 5ml precooling, centrifugal 15 minutes of 4 ℃ of following 2000rpm, abandon supernatant, add 3ml liquid regeneration culture medium, mixing, pour in the sterile petri dish, add the solid regenerated substratum that 15-20ml has dissolved well and has been cooled to 45-55 ℃, place after 6 hours down, pour the water agar 10-15ml that contains 300 μ g/ml Totomycin into for 20-25 ℃, be tiled on the regeneration culture medium, transformant occur after 2-3 days; The transformant cultivation is carried out postsearch screening at the PDA substratum that contains 250 μ g/ml Totomycin, to the transformant of normal growth carry out 5-7 for the PDA culture medium culturing after, again transformant is cultivated on the PDA substratum that contains 250 μ g/ml Totomycin, stable transformant that still can normal growth is inoculated on the PDA inclined-plane, is placed in 4 ℃ of refrigerators and preserves; Wherein, being formulated as of described liquid regeneration culture medium: get ammonium sulfate 2.8g respectively, urea 600mg, vitriolate of tartar 4g, Calcium Chloride Powder Anhydrous 600mg, glucose 40g, sucrose 100g, MgSO
410H
2O 200mg, FeSO
47H
2O 10mg, ZnSO
4.7H
2O 18mg, MnSO
4.H
2O 32mg, CoCl
26H
2O 4mg joins in the 800ml water, regulates pH value to 5.0; Solid medium then adds the 18-20g agar powder on the basis of liquid medium within;
2) silicon particle of transformant is preserved: the active high trichoderma transformant of the degraded prussiate that will obtain after degrading enzymatic activity is measured places the PDA culture medium culturing under 28 ℃ of conditions, the dull and stereotyped cultivation 5-7 days, make green spores be covered with whole culture dish, get spiral mouth Glass tubing, put into the 1/4-1/2 of silicon particle to the Glass tubing volume, after dry sterilization was handled, cooling was used preceding ice bath 15-30 minute; The skimmed milk 0.5-2 milliliter of getting 5% sterilization adds in the culture dish, and level is rocked, and makes spore suspension; Spore suspension with aseptic transfer pipet or rifle absorption 0.5-1 milliliter is added to the silicon particle surface in the chilled Glass tubing, after closing the lid, shake rapidly, make silicon particle moistening and be stained with spore, put back to ice bath immediately, after 10-20 minute, tighten lid and put into 4 ℃ or-20 ℃ of refrigerators and preserve;
3) cultivation of mycelia: the silicon particle of getting an above-mentioned infiltration spore suspension, be positioned on the substratum of PDA, cultivate and produce green spores after 5-7 days, picking length has the mycelia of spore to mix with the 5-6ml sterilized water to make mixing solutions to be green, filter then, get 3ml filtrate and put into 50-100ml PDB liquid nutrient medium, in shaking table, 28 ℃, 100-140rpm were cultivated 60-70 hour down; Cultured mycelia is filtered, and substratum in the mycelia is rinsed well, obtain green wet mycelium with NaCl or the sterilized water of 0.7M;
4) preparation of immobilized spherule: place the ratio of 100ml distilled water in 2g-3g sodium alginate, 1g diatomite, sodium alginate, diatomite are placed distilled water, heated and stirred dissolving and moist heat sterilization are mixed with sodium alginate soln; The preparation mass concentration is the CaCl of 2-3%
2Solution, be loaded on moist heat sterilization in the bottle, in the ratio of putting into wet mycelia 5 grams in every 200ml sodium alginate soln, the sodium alginate soln that wet mycelia is put into after the sterilization stirs, use the 20-50ml syringe to draw the mycelia mixed solution, it is squeezed into the above-mentioned CaCl of 200ml-400ml with syringe needle
2Stir in the solution and at the uniform velocity, form the bead of φ 3-5mm, bead is at CaCl afterwards
24 ℃ were descended fixedly 2-3 hour in the solution, filtered being fixed bead; After being the flushing of 0.9% NaCl solution with sterilized mass concentration, it is that the glutaraldehyde solution of 0.1-0.3% carried out crosslinked 30 seconds-2 minutes that continuation is put into volumetric concentration with bead, behind aseptic water washing, and the immobilized spherule that obtains preparing, be the immobilization trichoderma transformant, 4 ℃ of preservations.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102586118A (en) * | 2012-03-05 | 2012-07-18 | 杭州师范大学 | Method for separating and screening nitrogen fixation blue-green algae with polychlorinated biphenyl degradation function |
| CN111494866A (en) * | 2020-05-10 | 2020-08-07 | 浙江澄宇环保新材料股份有限公司 | Process for screening and repairing tailing slag |
-
2006
- 2006-09-07 CN CN 200610030903 patent/CN1916157A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102586118A (en) * | 2012-03-05 | 2012-07-18 | 杭州师范大学 | Method for separating and screening nitrogen fixation blue-green algae with polychlorinated biphenyl degradation function |
| CN102586118B (en) * | 2012-03-05 | 2016-04-20 | 杭州师范大学 | A kind of separating screening method with polychlorobiphenyl degradation function Azotica |
| CN111494866A (en) * | 2020-05-10 | 2020-08-07 | 浙江澄宇环保新材料股份有限公司 | Process for screening and repairing tailing slag |
| CN111494866B (en) * | 2020-05-10 | 2021-07-23 | 浙江澄宇环保新材料股份有限公司 | Process for screening and repairing tailing slag |
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