CN101063121A - Micro-organism immobilization method using bacterial filament ball as carrier - Google Patents
Micro-organism immobilization method using bacterial filament ball as carrier Download PDFInfo
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- CN101063121A CN101063121A CN 200710072148 CN200710072148A CN101063121A CN 101063121 A CN101063121 A CN 101063121A CN 200710072148 CN200710072148 CN 200710072148 CN 200710072148 A CN200710072148 A CN 200710072148A CN 101063121 A CN101063121 A CN 101063121A
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- triangular flask
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- mycelium pellet
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Abstract
The invention discloses a microbe immobilized method with hypha ball as carrier, which comprises the following steps: 1, proceeding enriched culture; 2, preparing spore suspension; 3, seeding at the same time; reaching the goal of culturing aerobic bacteria and aspergillus bacteria sporinite at the same time. This invention saves washing , preparing bacteria suspending liquid and shock absorbing course, which shortens times, decreases steps and increases efficiency.
Description
Technical field
The present invention relates to a kind of microorganism immobilization method.
Background technology
Microbial immobilized technology is meant and by certain technique means (as utilizing solid support material, embedding substance or rationally controlling hydraulics etc.) microorganism is remained in the reactor microorganism.Mycelium pellet is the spheroid that is had certain physical strength and size by the autoflocculation phenomenon generation of mould, it is except self having certain absorption and degradation efficiency, recently also be used as a kind of novel biomass carrier gradually, fix other functional microorganisms and be applied in the biological sewage treatment.But, at present people adopt absorption method immobilized microorganism on mycelium pellet, this method complex operation step, consuming time, the fixation of microbe amount is limited, the inhomogeneous activity of immobilized microorganism and the mass transfer effect of whole spheroid of just certainly will influencing like this of the inner microbial profile of mixed bacterium pompon that form.And the simple fungal hyphae ball of cultivation becomes to need after the size to adopt absorption method to need earlier, again with its taking-up, wash stand-by repeatedly with sterilized water, and then enrichment culture treats fixation of bacteria, waits it to enter after the logarithmic phase, through centrifugal collection bacterium, bacterium is washed 2-3 time in sterilized water then, and then be made into bacterial suspension with sterilized water, and the simple fungal hyphae ball that will wash is again put into the bacterial suspension concussion absorption 10h for preparing and is reached saturatedly, and whole process is the shortest also to need 60h consuming time.
Summary of the invention
The present invention is existing with the problem of mycelium pellet as the microorganism immobilization method operation steps of carrier time length complicated and consuming time in order to solve, and proposes a kind of with the microorganism immobilization method of mycelium pellet as carrier.Realize by following steps as the microorganism immobilization method of carrier with mycelium pellet: one, enrichment culture: get a ring and treat that immobilized aerobic bacteria is inoculated in the triangular flask that fills 80～110mL liquid enrichment medium, places the shaking table enrichment culture standby after 24～30 hours triangular flask then under 30 ℃, 120～140 rev/mins condition; Two, preparation spore suspension: inclined-plane solid medium surperficial of aspergillus tubigensis spore arranged with aseptic normal saline flushing length and gently scrape the inclined-plane with transfering loop, washed fully until thalline, in the normal saline flushing media surface, catch the physiological saline that contains the aspergillus tubigensis spore that flows down in the flushing process with test tube, the light shaking test tube makes the spore in the suspension be homogeneously dispersed state then; Three, inoculation simultaneously: the spore suspension 2～5ml that takes out in the step 2 is standby, take out inoculum 20～50ml that enrichment is good in the step 1 again, then the spore suspension of 2～5ml and the good inoculum of enrichment of 20～50ml are inoculated in the triangular flask that fills 200～350ml balling-up substratum simultaneously, under 30 ℃, 120～140 rev/mins condition, place shaking table to cultivate 48～54 hours triangular flask then, promptly reach bacterium and be fixed on purpose on the aspergillus tubigensis mycelium pellet.The present invention cultivates simultaneously to aspergillus tubigensis thalline spore and bacterium, has saved the process of flushing, preparation bacterial suspension and concussion absorption, and the immobilization time only about 48h, has not only shortened the time but also reduced step, has also improved efficient greatly.
Description of drawings
Fig. 1 is the sem photograph of the mixed bacterium pompon internal structure of absorption method formation, wherein the top among Fig. 1 is 700 times of sem photographs of the mixed bacterium pompon internal structure of absorption method formation, and lower part is 3500 times of sem photographs of the mixed bacterium pompon internal structure of absorption method formation; Fig. 2 is 3000 times of sem photographs that do not carry out the internal structure of immobilized simple fungal hyphae ball; Fig. 3 is 3000 times of sem photographs that adopt the mixed bacterium pompon internal structure of method formation of the present invention.
Embodiment
Embodiment one: realize by following steps as the microorganism immobilization method of carrier with mycelium pellet in the present embodiment: one, enrichment culture: get a ring and treat that immobilized aerobic bacteria is inoculated in the triangular flask that fills 80～110mL liquid enrichment medium, places the shaking table enrichment culture standby after 24～30 hours triangular flask then under 30 ℃, 120～140 rev/mins condition; Two, preparation spore suspension: inclined-plane solid medium surperficial of aspergillus tubigensis spore arranged with aseptic normal saline flushing length and gently scrape the inclined-plane with transfering loop, washed fully until thalline, in the normal saline flushing media surface, catch the physiological saline that contains the aspergillus tubigensis spore that flows down in the flushing process with test tube, the light shaking test tube makes the spore in the suspension be homogeneously dispersed state then; Three, inoculation simultaneously: the spore suspension 2～5ml that takes out in the step 2 is standby, take out inoculum 20～50ml that enrichment is good in the step 1 again, then the spore suspension of 2～5ml and the good inoculum of enrichment of 20～50ml are inoculated in the triangular flask that fills 200～350ml balling-up substratum simultaneously, under 30 ℃, 120～140 rev/mins condition, place shaking table to cultivate 48～54 hours triangular flask then, promptly reach bacterium and be fixed on the intravital purpose of aspergillus tubigensis spore.
Embodiment two: the difference of present embodiment and embodiment one is that enrichment medium is to be mixed by 5g NaCl, 3g extractum carnis, 10g peptone and 1000mL distilled water in the step 1.Other step is identical with embodiment two.
Embodiment three: the difference of present embodiment and embodiment one is to get in the step 1 ring and treats that immobilized aerobic bacteria is inoculated in the triangular flask that fills 90～100mL liquid enrichment medium.Other step is identical with embodiment two.
Embodiment four: the difference of present embodiment and embodiment one is to place the shaking table enrichment culture standby after 26～28 hours triangular flask under 30 ℃, 125～130 rev/mins condition in the step 1.Other step is identical with embodiment two.
Embodiment five: the difference of present embodiment and embodiment one is that the balling-up substratum in the step 3 is by 30g sucrose, 0.5g NH
4Cl, 0.1g K
2HPO
3H
2O, 0.1g MgSO
47H
2O, 0.1g FeSO
47H
2O and 1000mL distilled water mix.Other step is identical with embodiment two.
Embodiment six: the difference of present embodiment and embodiment one is that the spore suspension 3～4ml that takes out in the step 2 in the step 3 is standby, take out inoculum 30～40ml that enrichment is good in the step 1 again, then the spore suspension of 3～4ml and the good inoculum of enrichment of 30～40ml are inoculated in the triangular flask that fills 240～300ml balling-up substratum simultaneously.Other step is identical with embodiment two.
Embodiment seven: the difference of present embodiment and embodiment one is to place shaking table to cultivate 50～52 hours triangular flask under 30 ℃, 125～130 rev/mins condition in the step 3.Other step is identical with embodiment two.
Embodiment eight: realize by following steps as the microorganism immobilization method of carrier with mycelium pellet in the present embodiment: one, enrichment culture: get a ring and treat that immobilized aerobic bacteria is inoculated in the triangular flask that fills 100mL liquid enrichment medium, places the shaking table enrichment culture standby after 24 hours triangular flask then under 30 ℃, 140 rev/mins condition; Two, preparation spore suspension: inclined-plane solid medium surperficial of aspergillus tubigensis spore arranged with aseptic normal saline flushing length and gently scrape the inclined-plane with transfering loop, washed fully until thalline, in the normal saline flushing media surface, catch the physiological saline that contains the aspergillus tubigensis spore that flows down in the flushing process with test tube, the light shaking test tube makes the spore in the suspension be homogeneously dispersed state then; Three, inoculation simultaneously: the spore suspension 2ml that takes out in the step 2 is standby, take out the inoculum 20ml that enrichment is good in the step 1 again, then the spore suspension of 2ml and the good inoculum of enrichment of 20ml are inoculated in the triangular flask that fills 200ml balling-up substratum simultaneously, under 30 ℃, 140 rev/mins condition, triangular flask placed shaking table to cultivate then 48 hours, promptly reach bacterium and be fixed on purpose on the aspergillus tubigensis mycelium pellet.
Enrichment medium in the present embodiment is to be mixed by 5g NaCl, 3g extractum carnis, 10g peptone and 1000mL distilled water; The balling-up substratum is by 30g sucrose, 0.5g NH4Cl, 0.1gK
2HPO
3H
2O, 0.1g MgSO
47H
2O, 0.1g FeSO
47H
2O and 1000mL distilled water mix.
Adopt the method among absorption method and the present invention to carry out simultaneously cultivating as the bacterium immobilization of carrier with the aspergillus tubigensis mycelium pellet, after treating that two kinds of methods are all fixedly finished, get each one of the mixed bacterium pompon that two kinds of process for fixation form at random, cut back observation internal structure separately under electron microscope, compare with the internal structure of not carrying out immobilized simple fungal hyphae ball simultaneously, as seen from the figure: the top among Fig. 1 is 700 times of sem photographs of the mixed bacterium pompon internal structure of absorption method formation, lower part is 3500 times of sem photographs of the mixed bacterium pompon internal structure of absorption method formation, adopt the bacterium of the mixed bacterium pompon inside of adsorption of immobilization method formation only to anchor at the place that mycelia intersects to form platform among Fig. 1, and do not having just do not have bacterium to exist on the mycelia of intersecting; Fig. 3 is 3000 times of sem photographs that adopt the mixed bacterium pompon internal structure of the method formation among the present invention, no matter whether crosslinked mycelia is, the all very uniform aligned growth of bacterium is on each root fungus silk, and the also obvious mixed bacterium pompon of the bacterial number of immobilization growth on the unit surface more than Fig. 1, bacterium is evenly distributed among Fig. 3, seldom occur piling up and stratified phenomenon, so just do not influence the supply of the energy and the oxygen of mycelium pellet inside, can not influence the mass transfer of mycelium pellet to inner bacterium, the bacterium that is fixed so just can keep higher activity.Fig. 2 is 3000 times of sem photographs that do not carry out the internal structure of immobilized simple fungal hyphae ball.
Claims (7)
1, with the microorganism immobilization method of mycelium pellet as carrier, it is characterized in that realizing by following steps as the microorganism immobilization method of carrier: one, enrichment culture: get a ring and treat that immobilized aerobic bacteria is inoculated in the triangular flask that fills 80～110mL liquid enrichment medium, places the shaking table enrichment culture standby after 24～30 hours triangular flask then under 30 ℃, 120～140 rev/mins condition with mycelium pellet; Two, preparation spore suspension: inclined-plane solid medium surperficial of aspergillus tubigensis spore arranged with aseptic normal saline flushing length and gently scrape the inclined-plane with transfering loop, washed fully until thalline, in the normal saline flushing media surface, catch the physiological saline that contains the aspergillus tubigensis spore that flows down in the flushing process with test tube, the light shaking test tube makes the spore in the suspension be homogeneously dispersed state then; Three, inoculation simultaneously: the spore suspension 2～5ml that takes out in the step 2 is standby, take out inoculum 20～50ml that enrichment is good in the step 1 again, then the spore suspension of 2～5ml and the good inoculum of enrichment of 20～50ml are inoculated in the triangular flask that fills 200～350ml balling-up substratum simultaneously, under 30 ℃, 120～140 rev/mins condition, place shaking table to cultivate 48～54 hours triangular flask then, promptly reach bacterium and be fixed on purpose on the aspergillus tubigensis mycelium pellet.
2, according to claim 1 with the microorganism immobilization method of mycelium pellet as carrier, it is characterized in that enrichment medium is to be mixed by 5g NaCl, 3g extractum carnis, 10g peptone and 1000mL distilled water in the step 1.
3, according to claim 1 with the microorganism immobilization method of mycelium pellet as carrier, it is characterized in that getting in the step 1 ring and treat that immobilized aerobic bacteria is inoculated in the triangular flask that fills 90～100mL liquid enrichment medium.
4, according to claim 1 with the microorganism immobilization method of mycelium pellet as carrier, it is characterized in that under 30 ℃, 125～130 rev/mins condition, placing the shaking table enrichment culture standby after 26～28 hours triangular flask in the step 1.
5, according to claim 1 with the microorganism immobilization method of mycelium pellet as carrier, it is characterized in that the balling-up substratum in the step 3 is by 30g sucrose, 0.5g NH
4Cl, 0.1g K
2HPO
3H
2O, 0.1g MgSO
47H
2O, 0.1g FeSO
47H
2O and 1000mL distilled water mix.
6, according to claim 1 with the microorganism immobilization method of mycelium pellet as carrier, it is characterized in that the spore suspension 3～4ml that takes out in the step 2 in the step 3 is standby, take out inoculum 30～40ml that enrichment is good in the step 1 again, then the spore suspension of 3～4ml and the good inoculum of enrichment of 30～40ml are inoculated in the triangular flask that fills 240～300ml balling-up substratum simultaneously.
7, according to claim 1 with the microorganism immobilization method of mycelium pellet as carrier, it is characterized in that under 30 ℃, 125～130 rev/mins condition, placing shaking table to cultivate 50～52 hours triangular flask in the step 3.
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| CN 200710072148 CN101063121A (en) | 2007-04-29 | 2007-04-29 | Micro-organism immobilization method using bacterial filament ball as carrier |
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| CN 200710072148 CN101063121A (en) | 2007-04-29 | 2007-04-29 | Micro-organism immobilization method using bacterial filament ball as carrier |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103255123A (en) * | 2013-04-27 | 2013-08-21 | 沈阳大学 | Method for mycelium pellet to form mixed mycelium pellet by adsorbing photosynthetic bacteria |
| CN103436520A (en) * | 2013-09-16 | 2013-12-11 | 江南大学 | Preparation method of novel immobilized enzyme with brewer yeast spore as carrier |
| CN107058282A (en) * | 2017-06-19 | 2017-08-18 | 曲阜师范大学 | A kind of method that denitrifying bacteria immobilization is carried out by carrier of Trichoderma viride |
| CN108707598A (en) * | 2018-05-29 | 2018-10-26 | 江南大学 | A method of using filamentous fungi as the intensified anti-nitrated bacterial nitrogen removal of carrier |
-
2007
- 2007-04-29 CN CN 200710072148 patent/CN101063121A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103255123A (en) * | 2013-04-27 | 2013-08-21 | 沈阳大学 | Method for mycelium pellet to form mixed mycelium pellet by adsorbing photosynthetic bacteria |
| CN103255123B (en) * | 2013-04-27 | 2015-04-22 | 沈阳大学 | Method for mycelium pellet to form mixed mycelium pellet by adsorbing photosynthetic bacteria |
| CN103436520A (en) * | 2013-09-16 | 2013-12-11 | 江南大学 | Preparation method of novel immobilized enzyme with brewer yeast spore as carrier |
| CN107058282A (en) * | 2017-06-19 | 2017-08-18 | 曲阜师范大学 | A kind of method that denitrifying bacteria immobilization is carried out by carrier of Trichoderma viride |
| CN107058282B (en) * | 2017-06-19 | 2020-11-24 | 曲阜师范大学 | Method for immobilizing denitrifying bacteria by taking trichoderma viride as carrier |
| CN108707598A (en) * | 2018-05-29 | 2018-10-26 | 江南大学 | A method of using filamentous fungi as the intensified anti-nitrated bacterial nitrogen removal of carrier |
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