CN103642873A - Method for producing microbial flocculant by using Paenibacillus mucilaginosus - Google Patents
Method for producing microbial flocculant by using Paenibacillus mucilaginosus Download PDFInfo
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Abstract
The invention belongs to preparation of biological polysaccharide, and particularly refers to a method for producing microbial flocculant by using Paenibacillus mucilaginosus. The method comprises the following steps of: A, performing bacterial domestication on the Paenibacillus mucilaginosus ACCC10013 by means of thermal shock; B, aerobically culturing the Paenibacillus mucilaginosus ACCC10013 domesticated in the step A in a sterile fermental cultivation medium containing carbon source, nitrogen source and growth factors, thus obtaining a fermentation broth containing microbial flocculant; C, separating the fermentation broth containing microbial flocculant, prepared in the step B, through a macroporous adsorption resin to produce the microbial flocculant. The method provided by the invention solves the problems, such as high cost and low purity of flocculant, of the existing technology, and has the advantages of low technological cost of preparation process, high yield, high product purity, high activity, and the like.
Description
Technical field
The invention belongs to the preparation of microbial polysaccharide, refer to especially a kind of method that adopts colloid series bacillus to produce microbial flocculant.
Background technology
Microbial flocculant is to utilize modern biotechnology, through techniques such as the fermented extracted of microorganism are refining, from microorganism or its secretory product, prepare and there is coherent meta-bolites, be a kind of nontoxic biological polymeric compound, mainly contain glycoprotein, polysaccharide, protein, Mierocrystalline cellulose and DNA and have thalline of flocculation activity etc.It is wide that microbial flocculant has flocculation scope, the high safe and harmless feature such as pollution-free of flocculation activity, and the kind of bacterium for producing flocculant is many, and growth is fast, is easy to realize suitability for industrialized production.Flocculation agent generally can be divided into inorganic flocculating agent, organic synthesis polymeric flocculant and natural organic high-molecular flocculant at present.Inorganic flocculating agent and organic synthesis polymeric flocculant are due to its good flocculating effect and be widely used compared with low use cost.Yet these flocculation agents exist larger insecurity and potential secondary pollution problem.As the aluminum ion in inorganic flocculating agent easily causes senile dementia, Ferric Salt Flocculants has corrosive nature to equipment and easily forms the compound of some difficult flocculation sediment; Some organic synthesis flocculation agent as polyacrylamide etc., is difficult for degraded, and monomer is carcinogenic, in some field, has limited or has banned use of.Therefore, develop that a kind of safety non-toxic, flocculation activity are high, the novel flocculant of non-secondary pollution to the production technique of product improve, the mankind's health and environment protection all have very important realistic meaning.Utilizing microbial flocculant to replace traditional flocculation agent is an inexorable trend of water treatment development.
Colloid series bacillus (Paenibacillus mucilaginosus) ACCC10013 produces a large amount of pod membranes in process of growth, the main component of pod membrane is polysaccharide, prove by experiment, this polysaccharide in fermentation liquid can be used as flocculation agent and is advantageously applied to the industries such as Treatment of Industrial Water, food.And the flocculating effect of this flocculation agent is good, and cost is low.
At present, the production method of traditional microbial flocculant is screening bacterium for producing flocculant, optimization of fermentation conditions, utilize the method for alcohol precipitation to purify, the method for traditional alcohol precipitation need be set up alcohol recovery device, carries out alcohol distillation and flows back to receipts, cost is higher, and has pollution, is not suitable for suitability for industrialized production.
Utilizing macroporous adsorbent resin to decolour with deproteinated to polysaccharide crude extract is a kind of method that new development is in recent years got up.The plurality of advantages such as macroporous adsorbent resin is the novel high molecular polymer of a class, and it has physics, chemical stability is high, adsorption selectivity is high, be not subject to the impact of inorganic salts existence, desorption condition is gentle, life cycle is long, it is easy to regenerate, cost is low.Utilize the selective adsorption feature of macroporous resin by separation of polysaccharides out.Utilize the macroporous adsorbent resin Deproteinated great advantage of decolouring to be: its operational condition is gentle, can not destroy the structure and activity of polysaccharide, and the impurity such as the pigment in Polysaccharide removing crude extract and albumen simultaneously, meet test requirements document.Adopt macroporous adsorbent resin to carry out aftertreatment to microbial flocculant and have no relevant report.
Summary of the invention
The object of the present invention is to provide a kind of method that adopts colloid series bacillus to produce microbial flocculant, the cost of the method is lower and yield is high, prepared microbial flocculant purity and active high.
Overall technology design of the present invention is:
Adopt colloid series bacillus to produce the method for microbial flocculant, comprise following processing step:
A, employing thermal shocking method are carried out strain domestication to colloid series bacillus (Paenibacillus mucilaginosus) ACCC10013;
B, contain carbon source, nitrogenous source and somatomedin without bacteria fermentation culture medium in the colloid series bacillus ACCC10013 after domestication in aerated culture steps A, obtain the fermented liquid that contains microbial flocculant;
C, employing macroporous adsorbent resin carry out separation to the fermented liquid that contains microbial flocculant of preparing in step B, make microbial flocculant.
Concrete technology contents of the present invention also has:
For ease of the production of microbial flocculant, improve the yield of product and improve purity and the activity of product, preferred technical scheme is that described steps A comprises following processing step:
A1, under aseptic condition, the bacterial classification spore in test tube slant is added to sterilized water, vibration is washed lower spore and is made spore suspension;
A2, the sterile chamber that spore suspension is housed is placed in to boiling water, homogeneous heating, after 1-10 minute takes out sterile chamber and cooling rapidly.
Test tube slant in described steps A 1 adopts high yield spore inclined-plane.
Fermention medium in described step B is comprised of the component of following quality percentage composition:
Starch 5%-20%, sucrose 5%-10%, ammonium sulfate 0.5%-2%, dipotassium hydrogen phosphate 1%-2%, iron trichloride 0.01%-0.5%, tween-80 0.001%-0.01%, yeast extract paste 0.1%-3%, surplus is tap water, pH=7.5.
Culture condition in described step B is:
Inoculum size is bacterial classification: without the mass ratio=4%-8% of bacteria fermentation culture medium.
Fluctuation temperature culture repeatedly in described step B refers in cultivation and proceeds to 15-16 hour for 1 hour, is warming up to 35 ℃-37 ℃ and cultivates 10 minutes, then be cooled to 30 ℃-32 ℃ cultivations 10 minutes, Fluctuation temperature culture process reciprocation cycle 1 hour.
Macroporous resin in described step C is selected S-8 macroporous adsorbent resin.
Adopting macroporous adsorbent resin to carry out the preferred technical scheme of aftertreatment to the fermented liquid that contains microbial flocculant is that described step C comprises following processing step:
The solid-liquid separation of C1, fermented liquid: the fermented liquid that contains microbial flocculant of preparing in step B is carried out to solid-liquid separation;
The pre-treatment of C2, resin: select macroporous adsorbent resin, by mass percent, be first that 5%HCl solution soaking was neutral with distilled water flushing to pH after 4 hours, by mass percent, be after after 5%NaOH solution soaking with distilled water flushing, to pH, to be neutral again, then with the ethanolic soln that mass percent is 95%, soak full resin, finally with the abundant drip washing of distilled water, wash ethanolic soln off;
The absorption of C3, resin
C3-1: pretreated resin is packed in resin column with wet method, with the abundant drip washing of deionized water, then with pH, being 5-7, the concentration phosphate buffered saline buffer that is 0.005-0.02mol/L carries out drip washing with the flow velocity of 2BV/h-5BV/h, makes resin chromatography column reach equilibrium state;
C3-2: the pH of the filtrate of preparing in regulating step C1, makes it equate with the phosphate buffered saline buffer in step C3-1;
C3-3: by the filtrate that mixes up pH in step C3-2 with the flow velocity of 1BV/h-4BV/h by reaching the resin chromatography column of equilibrium state in step C3-1, after resin chromatography column absorption 10BV filtrate, stop liquid feeding;
The wash-out of C4, microbial flocculant is collected
C4-1: the resin chromatography column of the phosphate buffer 1 BV that is 0.005-0.02mol/L by pH=5-7, concentration after with absorption filtrate in the flow velocity drip washing step C3-3 of 1BV/h-4BV/h;
C4-2: with the flocculation agent on the flow velocity wash-out resin chromatography column of 1BV/h-4BV/h, start to collect elutriant when effluent liquid has flocculation agent to occur, until flocculation agent is after testing by whole wash-outs with phosphoric acid salt+sodium-chlor damping fluid of pH=5-7.
For ease of suitability for industrialized production, preferred and comparatively common technical scheme is, also comprises the preparation of seed liquor between described steps A, B, and wherein seed culture medium is comprised of the component of following quality percentage composition:
Starch 0.5-5g, dipotassium hydrogen phosphate 0.5-5g, peptone 0.5-5g, yeast extract paste 0.5-5g, ammonium sulfate 0.05-0.5g, magnesium sulfate 0.05-0.5g, distilled water 1000ml, agar 18g;
The preparation process of seed liquor is as follows: a packing gram formula bottle after seed culture medium preparation is carried out to sterilizing; After bacterial classification after domestication is activated, be seeded to the seed culture medium after sterilizing, at temperature 28-32 ℃, cultivate 36-60 hour.
Actication of culture in the preparation of described seed liquor after domestication is to carry out at normal temperatures, and the time is 4 hours.
The substantive distinguishing features that the present invention is obtained and significant technical progress are:
1, the flocculation agent composition that adopts zymotechnique to obtain in method of the present invention is more single, main component is slightly acidic polysaccharide, and active group is mainly hydroxyl, selects suitable macroporous adsorbent resin, carry out selective adsorption, can reach separation and purification preferably and concentrated effect, process recovery ratio is high, has greatly shortened the process time, reduced process costs, reduced the pollution to environment, the flocculation agent purity of gained is high, can be for grocery trade.
2, prepared flocculation agent purity and active high, flocculation agent output reaches as high as 8.1g/ml, and the yield of flocculation agent can reach more than 90%, and flocculation activity can reach more than 95%, the purity of flocculation agent at least reaches 97%(and can reach 99%).
Embodiment
Below in conjunction with embodiment, the present invention is described further; but not as a limitation of the invention; the content that protection scope of the present invention is recorded with claim is as the criterion, and any equivalence techniques means of making according to specification sheets are replaced, and all do not depart from protection scope of the present invention.
Embodiment 1
Adopt colloid series bacillus to produce the method for microbial flocculant, comprise following processing step:
A, employing thermal shocking method are carried out strain domestication to colloid series bacillus (Paenibacillus mucilaginosus) ACCC10013;
B, contain carbon source, nitrogenous source and somatomedin without bacteria fermentation culture medium in the colloid series bacillus ACCC10013 after domestication in aerated culture steps A, obtain the fermented liquid that contains microbial flocculant;
C, employing macroporous adsorbent resin carry out separation to the fermented liquid that contains microbial flocculant of preparing in step B, make microbial flocculant.
Described steps A comprises following processing step:
A1, under aseptic condition, the bacterial classification spore in test tube slant is added to sterilized water, vibration is washed lower spore and is made spore suspension;
A2, the sterile chamber that spore suspension is housed is placed in to boiling water, homogeneous heating, after 1-10 minute takes out sterile chamber and cooling rapidly.
Test tube slant in described steps A 1 adopts high yield spore inclined-plane.
Fermention medium in described step B is comprised of the component of following quality percentage composition:
Starch 5%, sucrose 5%, ammonium sulfate 0.5%, dipotassium hydrogen phosphate 1%, iron trichloride 0.01%, tween-80 0.001%, yeast extract paste 0.1%, surplus is tap water, pH=7.5.
Culture condition in described step B is:
Inoculum size is bacterial classification: without the mass ratio=4%-8% of bacteria fermentation culture medium.
Fluctuation temperature culture repeatedly in described step B refers in cultivation and proceeds to 15-16 hour for 1 hour, is warming up to 35 ℃-37 ℃ and cultivates 10 minutes, then be cooled to 30 ℃-32 ℃ cultivations 10 minutes, Fluctuation temperature culture process reciprocation cycle 1 hour.
Macroporous resin in described step C is selected S-8 macroporous adsorbent resin.
Adopting macroporous adsorbent resin to carry out the preferred technical scheme of aftertreatment to the fermented liquid that contains microbial flocculant is that described step C comprises following processing step:
The solid-liquid separation of C1, fermented liquid: the fermented liquid that contains microbial flocculant of preparing in step B is carried out to solid-liquid separation;
The pre-treatment of C2, resin: select macroporous adsorbent resin, by mass percent, be first that 5%HCl solution soaking was neutral with distilled water flushing to pH after 4 hours, by mass percent, be after after 5%NaOH solution soaking with distilled water flushing, to pH, to be neutral again, then with the ethanolic soln that mass percent is 95%, soak full resin, finally with the abundant drip washing of distilled water, wash ethanolic soln off;
The absorption of C3, resin
C3-1: pretreated resin is packed in resin column with wet method, with the abundant drip washing of deionized water, then carry out drip washing with pH is 5, concentration is 0.005mol/L phosphate buffered saline buffer with the flow velocity of 2BV/h, make resin chromatography column reach equilibrium state;
C3-2: the pH of the filtrate of preparing in regulating step C1, makes it equate with the phosphate buffered saline buffer in step C3-1;
C3-3: by the filtrate that mixes up pH in step C3-2 with the flow velocity of 1BV/h by reaching the resin chromatography column of equilibrium state in step C3-1, after resin chromatography column absorption 10BV filtrate, stop liquid feeding;
The wash-out of C4, microbial flocculant is collected
C4-1: the resin chromatography column of the phosphate buffer 1 BV that is 0.005mol/L by pH=5, concentration after with absorption filtrate in the flow velocity drip washing step C3-3 of 1BV/h;
C4-2: with the flocculation agent on the flow velocity wash-out resin chromatography column of 1BV/h, start to collect elutriant when effluent liquid has flocculation agent to occur, until flocculation agent is after testing by whole wash-outs with phosphoric acid salt+sodium-chlor damping fluid of pH=5.
Between described steps A, B, also comprise the preparation of seed liquor, wherein seed culture medium is comprised of the component of following quality percentage composition:
Starch 0.5g, dipotassium hydrogen phosphate 0.5g, peptone 0.5g, yeast extract paste 0.5g, ammonium sulfate 0.05g, magnesium sulfate 0.05g, distilled water 1000ml, agar 18g.
The preparation process of seed liquor is as follows: a packing gram formula bottle after seed culture medium preparation is carried out to sterilizing; Bacterial classification after domestication is seeded to the seed culture medium after sterilizing after activating, at 28 ℃ of temperature, cultivates 36 hours.
Actication of culture in the preparation of described seed liquor after domestication is to carry out at normal temperatures, and the time is 4 hours.
Adopt with the following method the activity of flocculation agent measured:
In 100ml graduated cylinder, add 0.5g kaolin suspension liquid, 5ml1%(wt%) CaCl
2solution and 2ml liquid to be measured, first rapid stirring 1 minute, then stir slowly 5 minutes, latter standing 10 minutes, by its absorbancy of 721 spectrophotometric determinations, wavelength was selected 550nm.To make CaCl
2solution in contrast, is determined flocculating rate by formula below.
Flocculating rate (%)=(A-B)/A * 100%
A is the optical density value at contrast supernatant liquor 550nm place; B is the optical density value at sample supernatant liquor 550nm place.
In embodiment 1, flocculation agent output is 8.1g/ml, and the yield of flocculation agent is 92%, and flocculation activity is 95%, and the purity of flocculation agent is 97%.
Embodiment 2
The difference of the present embodiment and embodiment 1 is:
Fermention medium in described step B is comprised of the component of following quality percentage composition:
Starch 20%, sucrose 10%, ammonium sulfate 2%, dipotassium hydrogen phosphate 2%, iron trichloride 0.5%, tween-80 0.01%, yeast extract paste 3%, surplus is tap water, pH=7.5.
Culture condition in described step B is:
Inoculum size is bacterial classification: without the mass ratio=4%-8% of bacteria fermentation culture medium.
Between described steps A, B, also comprise the preparation of seed liquor, wherein seed culture medium is comprised of the component of following quality percentage composition:
Starch 5g, dipotassium hydrogen phosphate 5g, peptone 5g, yeast extract paste 5g, ammonium sulfate 0.5g, magnesium sulfate 0.5g, distilled water 1000ml, agar 18g.
Macroporous resin in described step C is selected S-8 macroporous adsorbent resin.
Adopting macroporous adsorbent resin to carry out the preferred technical scheme of aftertreatment to the fermented liquid that contains microbial flocculant is that described step C comprises following processing step:
The solid-liquid separation of C1, fermented liquid: the fermented liquid that contains microbial flocculant of preparing in step B is carried out to solid-liquid separation;
The pre-treatment of C2, resin: select macroporous adsorbent resin, by mass percent, be first that 5%HCl solution soaking was neutral with distilled water flushing to pH after 4 hours, by mass percent, be after after 5%NaOH solution soaking with distilled water flushing, to pH, to be neutral again, then with the ethanolic soln that mass percent is 95%, soak full resin, finally with the abundant drip washing of distilled water, wash ethanolic soln off;
The absorption of C3, resin
C3-1: pretreated resin is packed in resin column with wet method, with the abundant drip washing of deionized water, then carry out drip washing with pH is 7, concentration is 0.02mol/L phosphate buffered saline buffer with the flow velocity of 5BV/h, make resin chromatography column reach equilibrium state;
C3-2: the pH of the filtrate of preparing in regulating step C1, makes it equate with the phosphate buffered saline buffer in step C3-1;
C3-3: by the filtrate that mixes up pH in step C3-2 with the flow velocity of 4BV/h by reaching the resin chromatography column of equilibrium state in step C3-1, after resin chromatography column absorption 10BV filtrate, stop liquid feeding;
The wash-out of C4, microbial flocculant is collected
C4-1: the resin chromatography column of the phosphate buffer 1 BV that is 0.02mol/L by pH=5-7, concentration after with absorption filtrate in the flow velocity drip washing step C3-3 of 4BV/h;
C4-2: with the flocculation agent on the flow velocity wash-out resin chromatography column of 4BV/h, start to collect elutriant when effluent liquid has flocculation agent to occur, until flocculation agent is after testing by whole wash-outs with phosphoric acid salt+sodium-chlor damping fluid of pH=7.
In the present embodiment, flocculation agent output is 7.9g/ml, and the yield of flocculation agent is 90%, and flocculation activity is 96%, and the purity of flocculation agent is 98%.
All the other technology contents are with embodiment 1.
Embodiment 3
The difference of the present embodiment and embodiment 1 is:
Fermention medium in described step B is comprised of the component of following quality percentage composition:
Starch 10%, sucrose 8%, ammonium sulfate 1%, dipotassium hydrogen phosphate 1.5%, iron trichloride 0.2%, tween-80 0.005%, yeast extract paste 1%, surplus is tap water, pH=7.5.
Culture condition in described step B is:
Between described steps A, B, also comprise the preparation of seed liquor, wherein seed culture medium is comprised of the component of following quality percentage composition:
Starch 3g, dipotassium hydrogen phosphate 2g, peptone 3g, yeast extract paste 3g, ammonium sulfate 0.3g, magnesium sulfate 0.3g, distilled water 1000ml, agar 18g.
Macroporous resin in described step C is selected S-8 macroporous adsorbent resin.
Adopting macroporous adsorbent resin to carry out the preferred technical scheme of aftertreatment to the fermented liquid that contains microbial flocculant is that described step C comprises following processing step:
The solid-liquid separation of C1, fermented liquid: the fermented liquid that contains microbial flocculant of preparing in step B is carried out to solid-liquid separation;
The pre-treatment of C2, resin: select macroporous adsorbent resin, by mass percent, be first that 5%HCl solution soaking was neutral with distilled water flushing to pH after 4 hours, by mass percent, be after after 5%NaOH solution soaking with distilled water flushing, to pH, to be neutral again, then with the ethanolic soln that mass percent is 95%, soak full resin, finally with the abundant drip washing of distilled water, wash ethanolic soln off;
The absorption of C3, resin
C3-1: pretreated resin is packed in resin column with wet method, with the abundant drip washing of deionized water, then carry out drip washing with pH is 5.5, concentration is 0.01mol/L phosphate buffered saline buffer with the flow velocity of 3BV/h, make resin chromatography column reach equilibrium state;
C3-2: the pH of the filtrate of preparing in regulating step C1, makes it equate with the phosphate buffered saline buffer in step C3-1;
C3-3: by the filtrate that mixes up pH in step C3-2 with the flow velocity of 2BV/h by reaching the resin chromatography column of equilibrium state in step C3-1, after resin chromatography column absorption 10BV filtrate, stop liquid feeding;
The wash-out of C4, microbial flocculant is collected
C4-1: the resin chromatography column of the phosphate buffer 1 BV that is 0.01mol/L by pH=5.5, concentration after with absorption filtrate in the flow velocity drip washing step C3-3 of 2BV/h;
C4-2: with the flocculation agent on the flow velocity wash-out resin chromatography column of 2BV/h, start to collect elutriant when effluent liquid has flocculation agent to occur, until flocculation agent is after testing by whole wash-outs with phosphoric acid salt+sodium-chlor damping fluid of pH=5.5.
In the present embodiment, flocculation agent output is 8.0g/ml, and the yield of flocculation agent is 93%, and flocculation activity is 98%, and the purity of flocculation agent is 99%.
All the other technology contents are with embodiment 1.
Embodiment 4
The difference of the present embodiment and embodiment 1 is:
Fermention medium in described step B is comprised of the component of following quality percentage composition:
Starch 18%, sucrose 6%, ammonium sulfate 1.5%, dipotassium hydrogen phosphate 1.8%, iron trichloride 0.05%, tween-80 0.003%, yeast extract paste 0.8%, surplus is tap water, pH=7.5.
Culture condition in described step B is:
Seed culture medium is comprised of the component of following quality percentage composition:
Starch 0.8g, dipotassium hydrogen phosphate 0.8g, peptone 1g, yeast extract paste 1g, ammonium sulfate 0.1g, magnesium sulfate 0.1g, distilled water 1000ml, agar 18g.
Macroporous resin in described step C is selected S-8 macroporous adsorbent resin.
Adopting macroporous adsorbent resin to carry out the preferred technical scheme of aftertreatment to the fermented liquid that contains microbial flocculant is that described step C comprises following processing step:
The solid-liquid separation of C1, fermented liquid: the fermented liquid that contains microbial flocculant of preparing in step B is carried out to solid-liquid separation;
The pre-treatment of C2, resin: select macroporous adsorbent resin, by mass percent, be first that 5%HCl solution soaking was neutral with distilled water flushing to pH after 4 hours, by mass percent, be after after 5%NaOH solution soaking with distilled water flushing, to pH, to be neutral again, then with the ethanolic soln that mass percent is 95%, soak full resin, finally with the abundant drip washing of distilled water, wash ethanolic soln off;
The absorption of C3, resin
C3-1: pretreated resin is packed in resin column with wet method, with the abundant drip washing of deionized water, then carry out drip washing with pH is 6.5, concentration is 0.01mol/L phosphate buffered saline buffer with the flow velocity of 3BV/h, make resin chromatography column reach equilibrium state;
C3-2: the pH of the filtrate of preparing in regulating step C1, makes it equate with the phosphate buffered saline buffer in step C3-1;
C3-3: by the filtrate that mixes up pH in step C3-2 with the flow velocity of 3BV/h by reaching the resin chromatography column of equilibrium state in step C3-1, after resin chromatography column absorption 10BV filtrate, stop liquid feeding;
The wash-out of C4, microbial flocculant is collected
C4-1: the resin chromatography column of the phosphate buffer 1 BV that is 0.01mol/L by pH=6.5, concentration after with absorption filtrate in the flow velocity drip washing step C3-3 of 3BV/h;
C4-2: with the flocculation agent on the flow velocity wash-out resin chromatography column of 3BV/h, start to collect elutriant when effluent liquid has flocculation agent to occur, until flocculation agent is after testing by whole wash-outs with phosphoric acid salt+sodium-chlor damping fluid of pH=6.5.
In the present embodiment, flocculation agent output is 7.8g/ml, and the yield of flocculation agent is 92%, and flocculation activity is 95%, and the purity of flocculation agent is 99%.
All the other technology contents are with embodiment 1.
Embodiment 5
The present embodiment is with the difference of embodiment 1:
Fermention medium in described step B is comprised of the component of following quality percentage composition:
Starch 7%, sucrose 8%, ammonium sulfate 0.8%, dipotassium hydrogen phosphate 1.4%, iron trichloride 0.4%, tween-80 0.008%, yeast extract paste 2%, surplus is tap water, pH=7.5.
Culture condition in described step B is:
Seed culture medium is comprised of the component of following quality percentage composition:
Starch 4g, dipotassium hydrogen phosphate 4g, peptone 3g, yeast extract paste 4g, ammonium sulfate 0.4g, magnesium sulfate 0.4g, distilled water 1000ml, agar 18g.
Macroporous resin in described step C is selected S-8 macroporous adsorbent resin.
Adopting macroporous adsorbent resin to carry out the preferred technical scheme of aftertreatment to the fermented liquid that contains microbial flocculant is that described step C comprises following processing step:
The solid-liquid separation of C1, fermented liquid: the fermented liquid that contains microbial flocculant of preparing in step B is carried out to solid-liquid separation;
The pre-treatment of C2, resin: select macroporous adsorbent resin, by mass percent, be first that 5%HCl solution soaking was neutral with distilled water flushing to pH after 4 hours, by mass percent, be after after 5%NaOH solution soaking with distilled water flushing, to pH, to be neutral again, then with the ethanolic soln that mass percent is 95%, soak full resin, finally with the abundant drip washing of distilled water, wash ethanolic soln off;
The absorption of C3, resin
C3-1: pretreated resin is packed in resin column with wet method, with the abundant drip washing of deionized water, then carry out drip washing with pH is 6.0, concentration is 0.015mol/L phosphate buffered saline buffer with the flow velocity of 4BV/h, make resin chromatography column reach equilibrium state;
C3-2: the pH of the filtrate of preparing in regulating step C1, makes it equate with the phosphate buffered saline buffer in step C3-1;
C3-3: by the filtrate that mixes up pH in step C3-2 with the flow velocity of 4BV/h by reaching the resin chromatography column of equilibrium state in step C3-1, after resin chromatography column absorption 10BV filtrate, stop liquid feeding;
The wash-out of C4, microbial flocculant is collected
C4-1: the resin chromatography column of the phosphate buffer 1 BV that is 0.015mol/L by pH=6.0, concentration after with absorption filtrate in the flow velocity drip washing step C3-3 of 4BV/h;
C4-2: with the flocculation agent on the flow velocity wash-out resin chromatography column of 4BV/h, start to collect elutriant when effluent liquid has flocculation agent to occur, until flocculation agent is after testing by whole wash-outs with phosphoric acid salt+sodium-chlor damping fluid of pH=6.0.
In the present embodiment, flocculation agent output is 7.6g/ml, and the yield of flocculation agent is 93%, and flocculation activity is 96%, and the purity of flocculation agent is 99%.
All the other technology contents are with embodiment 1.
Claims (10)
1. adopt colloid series bacillus to produce the method for microbial flocculant, it is characterized in that comprising following processing step:
A, employing thermal shocking method are carried out strain domestication to colloid series bacillus ACCC10013;
B, contain carbon source, nitrogenous source and somatomedin without bacteria fermentation culture medium in the colloid series bacillus ACCC10013 after domestication in aerated culture steps A, obtain the fermented liquid that contains microbial flocculant;
C, employing macroporous adsorbent resin carry out separation to the fermented liquid that contains microbial flocculant of preparing in step B, make microbial flocculant.
2. employing colloid series bacillus according to claim 1 is produced the method for microbial flocculant, it is characterized in that described steps A comprises following processing step:
A1, under aseptic condition, the bacterial classification spore in test tube slant is added to sterilized water, vibration is washed lower spore and is made spore suspension;
A2, the sterile chamber that spore suspension is housed is placed in to boiling water, homogeneous heating, after 1-10 minute takes out sterile chamber and cooling rapidly.
3. employing colloid series bacillus according to claim 2 is produced the method for microbial flocculant, it is characterized in that the test tube slant in described steps A 1 adopts high yield spore inclined-plane.
4. employing colloid series bacillus according to claim 1 is produced the method for microbial flocculant, and the fermention medium in the step B described in it is characterized in that is comprised of the component of following quality percentage composition:
Starch 5%-20%, sucrose 5%-10%, ammonium sulfate 0.5%-2%, dipotassium hydrogen phosphate 1%-2%, iron trichloride 0.01%-0.5%, tween-80 0.001%-0.01%, yeast extract paste 0.1%-3%, surplus is tap water, pH=7.5.
5. employing colloid series bacillus according to claim 1 is produced the method for microbial flocculant, it is characterized in that the culture condition in described step B is:
Inoculum size is bacterial classification: without the mass ratio=4%-8% of bacteria fermentation culture medium.
6. employing colloid series bacillus according to claim 5 is produced the method for microbial flocculant, Fluctuation temperature culture repeatedly in step B described in it is characterized in that refers in cultivation and proceeds to 15-16 hour for 1 hour, being warming up to 35 ℃-37 ℃ cultivates 10 minutes, be cooled to again 30 ℃-32 ℃ and cultivate 10 minutes, Fluctuation temperature culture process reciprocation cycle 1 hour.
7. employing colloid series bacillus according to claim 1 is produced the method for microbial flocculant, it is characterized in that the macroporous resin in described step C is selected S-8 macroporous adsorbent resin.
8. according to the employing colloid series bacillus described in claim 1 or 7, produce the method for microbial flocculant, it is characterized in that described step C comprises following processing step:
The solid-liquid separation of C1, fermented liquid: the fermented liquid that contains microbial flocculant of preparing in step B is carried out to solid-liquid separation;
The pre-treatment of C2, resin: select macroporous adsorbent resin, by mass percent, be first that 5%HCl solution soaking was neutral with distilled water flushing to pH after 4 hours, by mass percent, be after after 5%NaOH solution soaking with distilled water flushing, to pH, to be neutral again, then with the ethanolic soln that mass percent is 95%, soak full resin, finally, with the abundant drip washing of distilled water, wash ethanolic soln off;
The absorption of C3, resin
C3-1: pretreated resin is packed in resin column with wet method, with the abundant drip washing of deionized water, then with pH, being 5-7, the concentration phosphate buffered saline buffer that is 0.005-0.02mol/L carries out drip washing with the flow velocity of 2BV/h-5BV/h, makes resin chromatography column reach equilibrium state;
C3-2: the pH of the filtrate of preparing in regulating step C1, makes it equate with the phosphate buffered saline buffer in step C3-1;
C3-3: by the filtrate that mixes up pH in step C3-2 with the flow velocity of 1BV/h-4BV/h by reaching the resin chromatography column of equilibrium state in step C3-1, after resin chromatography column absorption 10BV filtrate, stop liquid feeding;
The wash-out of C4, microbial flocculant is collected
C4-1: the resin chromatography column of the phosphate buffer 1 BV that is 0.005-0.02mol/L by pH=5-7, concentration after with absorption filtrate in the flow velocity drip washing step C3-3 of 1BV/h-4BV/h;
C4-2: with the flocculation agent on the flow velocity wash-out resin chromatography column of 1BV/h-4BV/h, start to collect elutriant when effluent liquid has flocculation agent to occur, until flocculation agent is after testing by whole wash-outs with phosphoric acid salt+sodium-chlor damping fluid of pH=5-7.
9. employing colloid series bacillus according to claim 1 is produced the method for microbial flocculant, it is characterized in that also comprising between described steps A, B the preparation of seed liquor, and wherein seed culture medium is comprised of the component of following quality percentage composition:
Starch 0.5-5g, dipotassium hydrogen phosphate 0.5-5g, peptone 0.5-5g, yeast extract paste 0.5-5g, ammonium sulfate 0.05-0.5g, magnesium sulfate 0.05-0.5g, distilled water 1000ml, agar 18g;
The preparation process of seed liquor is as follows: a packing gram formula bottle after seed culture medium preparation is carried out to sterilizing; After bacterial classification after domestication is activated, be seeded to the seed culture medium after sterilizing, at temperature 28-32 ℃, cultivate 36-60 hour.
10. employing colloid series bacillus according to claim 1 is produced the method for microbial flocculant, and the actication of culture in the preparation of the seed liquor described in it is characterized in that after domestication is to carry out at normal temperatures, and the time is 4 hours.
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Citations (2)
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|---|---|---|---|---|
| CN101475249A (en) * | 2009-01-19 | 2009-07-08 | 浙江理工大学 | Method for treating cultivation wastewater by using microbial flocculant and compound fertilizer obtained thereby |
| CN102952834A (en) * | 2012-10-31 | 2013-03-06 | 河北省微生物研究所 | Method for producing microbial polysaccharide fermentation broth by using Paenibacillus mucilaginosus |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101475249A (en) * | 2009-01-19 | 2009-07-08 | 浙江理工大学 | Method for treating cultivation wastewater by using microbial flocculant and compound fertilizer obtained thereby |
| CN102952834A (en) * | 2012-10-31 | 2013-03-06 | 河北省微生物研究所 | Method for producing microbial polysaccharide fermentation broth by using Paenibacillus mucilaginosus |
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| 万红贵 等: "大孔吸附树脂对芽孢杆菌胞外多糖静态吸附性能的研究", 《中国酿造》, no. 7, 31 December 2011 (2011-12-31), pages 130 - 133 * |
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