CN103642873B - Colloid series bacillus is adopted to produce the method for microbial flocculant - Google Patents
Colloid series bacillus is adopted to produce the method for microbial flocculant Download PDFInfo
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- CN103642873B CN103642873B CN201310651488.XA CN201310651488A CN103642873B CN 103642873 B CN103642873 B CN 103642873B CN 201310651488 A CN201310651488 A CN 201310651488A CN 103642873 B CN103642873 B CN 103642873B
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Abstract
The invention belongs to the preparation of microbial polysaccharide, refer to a kind of method adopting colloid series bacillus to produce microbial flocculant especially.Thermal shocking method is adopted to carry out strain domestication to colloid series bacillus ACCC10013, containing carbon source, nitrogenous source and somatomedin without aerated culture steps A in bacteria fermentation culture medium in domestication after colloid series bacillus ACCC10013, obtain the fermented liquid containing microbial flocculant, adopt macroporous adsorbent resin to be separated the fermented liquid containing microbial flocculant prepared in step B, make microbial flocculant.The invention solves the problems such as cost purity that is high, flocculation agent is low that prior art exists, have that preparation process process costs is low, yield is high, product purity and active advantages of higher.
Description
Technical field
The invention belongs to the preparation of microbial polysaccharide, refer to a kind of method adopting colloid series bacillus to produce microbial flocculant especially.
Background technology
Microbial flocculant utilizes modern biotechnology, prepare from microorganism or its secretory product through techniques such as the fermented extracted of microorganism are refining 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 the thalline etc. of flocculation activity.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.Current flocculation agent generally can be divided into inorganic flocculating agent, organic synthesis polymeric flocculant and natural organic high-molecular flocculant.Inorganic flocculating agent and organic synthesis polymeric flocculant are widely used due to its good flocculating effect and lower use cost.But these flocculation agents also 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 is as polyacrylamide etc., and not easily degrade, monomer is carcinogenic, has limited or prohibit the use in some field.Therefore, develop a kind of safety non-toxic, flocculation activity novel flocculant that is high, non-secondary pollution improves the production technique of product, the health of the mankind and environment protection all have very important realistic meaning.Microbial flocculant is utilized to replace traditional flocculation agent to be the inexorable trend that water treatment develops.
Colloid series bacillus (Paenibacillus mucilaginosus) ACCC10013 produces a large amount of pod membrane in process of growth, the main component of pod membrane is polysaccharide, prove by experiment, this polysaccharide in fermentation liquid can be advantageously applied to the industry such as Treatment of Industrial Water, food as flocculation agent.And the flocculating effect of this flocculation agent is good, 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 set up alcohol recovery device, carries out alcohol distillation and flows back to receipts, cost is higher, and there is pollution, is not suitable for suitability for industrialized production.
Macroporous adsorbent resin is utilized to decolour to polysaccharide crude extract and deproteinated is a kind of method that new development is in recent years got up.Macroporous adsorbent resin is the novel high molecular polymer of a class, and it has physics, chemical stability is high, adsorption selectivity is high, the impact that do not exist by inorganic salts, desorption condition is gentle, life cycle is long, regenerate the plurality of advantages such as easy, cost is low.Utilize the selective adsorption feature of macroporous resin by separation of polysaccharides out.Macroporous adsorbent resin Deproteinated great advantage of decolouring is utilized to be: its operational condition is gentle, can not destroy the structure and activity of polysaccharide, and can the impurity such as pigment simultaneously in Polysaccharide removing crude extract and albumen, 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 colloid series bacillus that adopts to produce the method for 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 carry out strain domestication to colloid series bacillus (Paenibacillus mucilaginosus) ACCC10013;
B, containing carbon source, nitrogenous source and somatomedin without aerated culture steps A in bacteria fermentation culture medium in colloid series bacillus ACCC10013 after domestication, obtain containing the fermented liquid of microbial flocculant;
C, employing macroporous adsorbent resin are separated the fermented liquid containing microbial flocculant prepared 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, described steps A comprises following processing step:
A1, aseptically the bacterial classification spore in test tube slant is added sterilized water, vibration is washed lower spore and is made spore suspension;
A2, the sterile chamber that spore suspension is housed is placed in boiling water, homogeneous heating, sterile chamber is taken out and cooling rapidly after 1-10 minute.
Test tube slant in described steps A 1 adopts high yield spore inclined-plane.
Fermention medium in described step B is made up of the component of following mass percentage:
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 to and proceeds to 15-16 hour in cultivation for 1 hour, is warming up to 35 DEG C-37 DEG C and cultivates 10 minutes, then is cooled to 30 DEG C-32 DEG C and cultivates 10 minutes, Fluctuation temperature culture process reciprocation cycle 1 hour.
Macroporous resin in described step C selects S-8 macroporous adsorbent resin.
Adopting macroporous adsorbent resin to carry out the preferred technical scheme of aftertreatment to the fermented liquid containing microbial flocculant is that described step C comprises following processing step:
The solid-liquid separation of C1, fermented liquid: the fermented liquid containing microbial flocculant prepared in step B is carried out solid-liquid separation;
The pre-treatment of C2, resin: select macroporous adsorbent resin, be first 5%HCl solution soaking by mass percent be neutral with distilled water flushing to pH after 4 hours, again with mass percent be after 5%NaOH solution soaking after be neutral with distilled water flushing to pH, be then the full resin of ethanolic soln leaching of 95% by mass percent, finally wash ethanolic soln off with the abundant drip washing of distilled water;
The absorption of C3, resin
C3-1: pretreated resin is loaded in resin column in a wet process, with the abundant drip washing of deionized water, then with pH be 5-7, concentration is that the phosphate buffered saline buffer of 0.005-0.02mol/L carries out drip washing with the flow velocity of 2BV/h-5BV/h, makes resin chromatography post reach equilibrium state;
C3-2: the pH of the filtrate of preparing in regulating step C1, make it equal with the phosphate buffered saline buffer in step C3-1;
C3-3: by mix up pH in step C3-2 filtrate with the flow velocity of 1BV/h-4BV/h by reaching the resin chromatography post of equilibrium state in step C3-1, when stopping liquid feeding after resin chromatography post absorption 10BV filtrate;
The wash-out of C4, microbial flocculant is collected
C4-1: be that the phosphate buffer 1 BV of 0.005-0.02mol/L is with the resin chromatography post after absorption filtrate in the flow velocity rinsing step C3-3 of 1BV/h-4BV/h by pH=5-7, concentration;
C4-2: use the phosphoric acid salt+sodium chloride buffer of pH=5-7 with the flocculation agent on the flow velocity wash-out resin chromatography post of 1BV/h-4BV/h, start to collect elutriant, until flocculation agent is after testing by whole wash-out when effluent liquid has flocculation agent to occur.
For ease of suitability for industrialized production, preferably and comparatively common technical scheme also comprises the preparation of seed liquor between described steps A, B, wherein seed culture medium is made up of the component of following mass percentage:
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: after being prepared by seed culture medium, a packing gram formula bottle carries out sterilizing; Be seeded to the seed culture medium after sterilizing after being activated by bacterial classification after domestication, at temperature 28-32 DEG C, cultivate 36-60 hour.
Actication of culture in the preparation of described seed liquor after domestication carries out at normal temperatures, and the time is 4 hours.
Substantive distinguishing features acquired by the present invention and significant technical progress are:
1, the flocculation agent composition adopting 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, substantially reduces the process time, reduce process costs, decrease the pollution to environment, the flocculation agent purity of gained is high, may be used 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%, and 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 it is 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 equivalent technical elements of making according to specification sheets is replaced, and does not all 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 carry out strain domestication to colloid series bacillus (Paenibacillus mucilaginosus) ACCC10013;
B, containing carbon source, nitrogenous source and somatomedin without aerated culture steps A in bacteria fermentation culture medium in colloid series bacillus ACCC10013 after domestication, obtain containing the fermented liquid of microbial flocculant;
C, employing macroporous adsorbent resin are separated the fermented liquid containing microbial flocculant prepared in step B, make microbial flocculant.
Described steps A comprises following processing step:
A1, aseptically the bacterial classification spore in test tube slant is added sterilized water, vibration is washed lower spore and is made spore suspension;
A2, the sterile chamber that spore suspension is housed is placed in boiling water, homogeneous heating, sterile chamber is taken out and cooling rapidly after 1-10 minute.
Test tube slant in described steps A 1 adopts high yield spore inclined-plane.
Fermention medium in described step B is made up of the component of following mass percentage:
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 to and proceeds to 15-16 hour in cultivation for 1 hour, is warming up to 35 DEG C-37 DEG C and cultivates 10 minutes, then is cooled to 30 DEG C-32 DEG C and cultivates 10 minutes, Fluctuation temperature culture process reciprocation cycle 1 hour.
Macroporous resin in described step C selects S-8 macroporous adsorbent resin.
Adopting macroporous adsorbent resin to carry out the preferred technical scheme of aftertreatment to the fermented liquid containing microbial flocculant is that described step C comprises following processing step:
The solid-liquid separation of C1, fermented liquid: the fermented liquid containing microbial flocculant prepared in step B is carried out solid-liquid separation;
The pre-treatment of C2, resin: select macroporous adsorbent resin, be first 5%HCl solution soaking by mass percent be neutral with distilled water flushing to pH after 4 hours, again with mass percent be after 5%NaOH solution soaking after be neutral with distilled water flushing to pH, be then the full resin of ethanolic soln leaching of 95% by mass percent, finally wash ethanolic soln off with the abundant drip washing of distilled water;
The absorption of C3, resin
C3-1: pretreated resin is loaded in resin column in a wet process, with the abundant drip washing of deionized water, then with pH be 5, concentration is that the phosphate buffered saline buffer of 0.005mol/L carries out drip washing with the flow velocity of 2BV/h, makes resin chromatography post reach equilibrium state;
C3-2: the pH of the filtrate of preparing in regulating step C1, make it equal with the phosphate buffered saline buffer in step C3-1;
C3-3: by mix up pH in step C3-2 filtrate with the flow velocity of 1BV/h by reaching the resin chromatography post of equilibrium state in step C3-1, when stopping liquid feeding after resin chromatography post absorption 10BV filtrate;
The wash-out of C4, microbial flocculant is collected
C4-1: be that the phosphate buffer 1 BV of 0.005mol/L is with the resin chromatography post after absorption filtrate in the flow velocity rinsing step C3-3 of 1BV/h by pH=5, concentration;
C4-2: use the phosphoric acid salt+sodium chloride buffer of pH=5 with the flocculation agent on the flow velocity wash-out resin chromatography post of 1BV/h, start to collect elutriant, until flocculation agent is after testing by whole wash-out when effluent liquid has flocculation agent to occur.
Also comprise the preparation of seed liquor between described steps A, B, wherein seed culture medium is made up of the component of following mass percentage:
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: after being prepared by seed culture medium, a packing gram formula bottle carries out sterilizing; Be seeded to the seed culture medium after sterilizing after being activated by bacterial classification after domestication, cultivate 36 hours at temperature 28 DEG C.
Actication of culture in the preparation of described seed liquor after domestication carries out at normal temperatures, and the time is 4 hours.
Adopt and with the following method the activity of flocculation agent measured:
0.5g aqueous suspension ofkaolin is added, 5ml1%(wt% in 100ml graduated cylinder) CaCl
2solution and 2ml liquid to be measured, first rapid stirring 1 minute, then stir 5 minutes slowly, latter standing 10 minutes, by its absorbancy of 721 spectrophotometric determinations, wavelength selected 550nm.To make CaCl
2solution in contrast, by formula determination flocculating rate 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 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 made up of the component of following mass percentage:
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.
Also comprise the preparation of seed liquor between described steps A, B, wherein seed culture medium is made up of the component of following mass percentage:
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 selects S-8 macroporous adsorbent resin.
Adopting macroporous adsorbent resin to carry out the preferred technical scheme of aftertreatment to the fermented liquid containing microbial flocculant is that described step C comprises following processing step:
The solid-liquid separation of C1, fermented liquid: the fermented liquid containing microbial flocculant prepared in step B is carried out solid-liquid separation;
The pre-treatment of C2, resin: select macroporous adsorbent resin, be first 5%HCl solution soaking by mass percent be neutral with distilled water flushing to pH after 4 hours, again with mass percent be after 5%NaOH solution soaking after be neutral with distilled water flushing to pH, be then the full resin of ethanolic soln leaching of 95% by mass percent, finally wash ethanolic soln off with the abundant drip washing of distilled water;
The absorption of C3, resin
C3-1: pretreated resin is loaded in resin column in a wet process, with the abundant drip washing of deionized water, then with pH be 7, concentration is that the phosphate buffered saline buffer of 0.02mol/L carries out drip washing with the flow velocity of 5BV/h, makes resin chromatography post reach equilibrium state;
C3-2: the pH of the filtrate of preparing in regulating step C1, make it equal with the phosphate buffered saline buffer in step C3-1;
C3-3: by mix up pH in step C3-2 filtrate with the flow velocity of 4BV/h by reaching the resin chromatography post of equilibrium state in step C3-1, when stopping liquid feeding after resin chromatography post absorption 10BV filtrate;
The wash-out of C4, microbial flocculant is collected
C4-1: be that the phosphate buffer 1 BV of 0.02mol/L is with the resin chromatography post after absorption filtrate in the flow velocity rinsing step C3-3 of 4BV/h by pH=5-7, concentration;
C4-2: use the phosphoric acid salt+sodium chloride buffer of pH=7 with the flocculation agent on the flow velocity wash-out resin chromatography post of 4BV/h, start to collect elutriant, until flocculation agent is after testing by whole wash-out when effluent liquid has flocculation agent to occur.
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 made up of the component of following mass percentage:
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:
Also comprise the preparation of seed liquor between described steps A, B, wherein seed culture medium is made up of the component of following mass percentage:
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 selects S-8 macroporous adsorbent resin.
Adopting macroporous adsorbent resin to carry out the preferred technical scheme of aftertreatment to the fermented liquid containing microbial flocculant is that described step C comprises following processing step:
The solid-liquid separation of C1, fermented liquid: the fermented liquid containing microbial flocculant prepared in step B is carried out solid-liquid separation;
The pre-treatment of C2, resin: select macroporous adsorbent resin, be first 5%HCl solution soaking by mass percent be neutral with distilled water flushing to pH after 4 hours, again with mass percent be after 5%NaOH solution soaking after be neutral with distilled water flushing to pH, be then the full resin of ethanolic soln leaching of 95% by mass percent, finally wash ethanolic soln off with the abundant drip washing of distilled water;
The absorption of C3, resin
C3-1: pretreated resin is loaded in resin column in a wet process, with the abundant drip washing of deionized water, then with pH be 5.5, concentration is that the phosphate buffered saline buffer of 0.01mol/L carries out drip washing with the flow velocity of 3BV/h, makes resin chromatography post reach equilibrium state;
C3-2: the pH of the filtrate of preparing in regulating step C1, make it equal with the phosphate buffered saline buffer in step C3-1;
C3-3: by mix up pH in step C3-2 filtrate with the flow velocity of 2BV/h by reaching the resin chromatography post of equilibrium state in step C3-1, when stopping liquid feeding after resin chromatography post absorption 10BV filtrate;
The wash-out of C4, microbial flocculant is collected
C4-1: be that the phosphate buffer 1 BV of 0.01mol/L is with the resin chromatography post after absorption filtrate in the flow velocity rinsing step C3-3 of 2BV/h by pH=5.5, concentration;
C4-2: use the phosphoric acid salt+sodium chloride buffer of pH=5.5 with the flocculation agent on the flow velocity wash-out resin chromatography post of 2BV/h, start to collect elutriant, until flocculation agent is after testing by whole wash-out when effluent liquid has flocculation agent to occur.
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 made up of the component of following mass percentage:
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 made up of the component of following mass percentage:
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 selects S-8 macroporous adsorbent resin.
Adopting macroporous adsorbent resin to carry out the preferred technical scheme of aftertreatment to the fermented liquid containing microbial flocculant is that described step C comprises following processing step:
The solid-liquid separation of C1, fermented liquid: the fermented liquid containing microbial flocculant prepared in step B is carried out solid-liquid separation;
The pre-treatment of C2, resin: select macroporous adsorbent resin, be first 5%HCl solution soaking by mass percent be neutral with distilled water flushing to pH after 4 hours, again with mass percent be after 5%NaOH solution soaking after be neutral with distilled water flushing to pH, be then the full resin of ethanolic soln leaching of 95% by mass percent, finally wash ethanolic soln off with the abundant drip washing of distilled water;
The absorption of C3, resin
C3-1: pretreated resin is loaded in resin column in a wet process, with the abundant drip washing of deionized water, then with pH be 6.5, concentration is that the phosphate buffered saline buffer of 0.01mol/L carries out drip washing with the flow velocity of 3BV/h, makes resin chromatography post reach equilibrium state;
C3-2: the pH of the filtrate of preparing in regulating step C1, make it equal with the phosphate buffered saline buffer in step C3-1;
C3-3: by mix up pH in step C3-2 filtrate with the flow velocity of 3BV/h by reaching the resin chromatography post of equilibrium state in step C3-1, when stopping liquid feeding after resin chromatography post absorption 10BV filtrate;
The wash-out of C4, microbial flocculant is collected
C4-1: be that the phosphate buffer 1 BV of 0.01mol/L is with the resin chromatography post after absorption filtrate in the flow velocity rinsing step C3-3 of 3BV/h by pH=6.5, concentration;
C4-2: use the phosphoric acid salt+sodium chloride buffer of pH=6.5 with the flocculation agent on the flow velocity wash-out resin chromatography post of 3BV/h, start to collect elutriant, until flocculation agent is after testing by whole wash-out when effluent liquid has flocculation agent to occur.
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 made up of the component of following mass percentage:
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 made up of the component of following mass percentage:
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 selects S-8 macroporous adsorbent resin.
Adopting macroporous adsorbent resin to carry out the preferred technical scheme of aftertreatment to the fermented liquid containing microbial flocculant is that described step C comprises following processing step:
The solid-liquid separation of C1, fermented liquid: the fermented liquid containing microbial flocculant prepared in step B is carried out solid-liquid separation;
The pre-treatment of C2, resin: select macroporous adsorbent resin, be first 5%HCl solution soaking by mass percent be neutral with distilled water flushing to pH after 4 hours, again with mass percent be after 5%NaOH solution soaking after be neutral with distilled water flushing to pH, be then the full resin of ethanolic soln leaching of 95% by mass percent, finally wash ethanolic soln off with the abundant drip washing of distilled water;
The absorption of C3, resin
C3-1: pretreated resin is loaded in resin column in a wet process, with the abundant drip washing of deionized water, then with pH be 6.0, concentration is that the phosphate buffered saline buffer of 0.015mol/L carries out drip washing with the flow velocity of 4BV/h, makes resin chromatography post reach equilibrium state;
C3-2: the pH of the filtrate of preparing in regulating step C1, make it equal with the phosphate buffered saline buffer in step C3-1;
C3-3: by mix up pH in step C3-2 filtrate with the flow velocity of 4BV/h by reaching the resin chromatography post of equilibrium state in step C3-1, when stopping liquid feeding after resin chromatography post absorption 10BV filtrate;
The wash-out of C4, microbial flocculant is collected
C4-1: be that the phosphate buffer 1 BV of 0.015mol/L is with the resin chromatography post after absorption filtrate in the flow velocity rinsing step C3-3 of 4BV/h by pH=6.0, concentration;
C4-2: use the phosphoric acid salt+sodium chloride buffer of pH=6.0 with the flocculation agent on the flow velocity wash-out resin chromatography post of 4BV/h, start to collect elutriant, until flocculation agent is after testing by whole wash-out when effluent liquid has flocculation agent to occur.
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 (8)
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 carry out strain domestication to colloid series bacillus ACCC10013;
B, containing carbon source, nitrogenous source and somatomedin without aerated culture steps A in bacteria fermentation culture medium in colloid series bacillus ACCC10013 after domestication, obtain containing the fermented liquid of microbial flocculant;
C, employing S-8 macroporous adsorbent resin are separated the fermented liquid containing microbial flocculant prepared in step B, make microbial flocculant;
Culture condition in described step B is:
Inoculum size is bacterial classification: without the mass ratio=4%-8% of bacteria fermentation culture medium.
2. employing colloid series bacillus according to claim 1 produces the method for microbial flocculant, it is characterized in that described steps A comprises following processing step:
A1, aseptically the bacterial classification spore in test tube slant is added sterilized water, vibration is washed lower spore and is made spore suspension;
A2, the sterile chamber that spore suspension is housed is placed in boiling water, homogeneous heating, sterile chamber is taken out and cooling rapidly after 1-10 minute.
3. employing colloid series bacillus according to claim 2 produces 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 produces the method for microbial flocculant, it is characterized in that the fermention medium in described step B is made up of the component of following mass percentage:
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 produces the method for microbial flocculant, the Fluctuation temperature culture repeatedly that it is characterized in that in described step B refers to and proceeds to 15-16 hour in cultivation for 1 hour, be warming up to 35 DEG C-37 DEG C to cultivate 10 minutes, be cooled to 30 DEG C-32 DEG C again to cultivate 10 minutes, Fluctuation temperature culture process reciprocation cycle 1 hour.
6. employing colloid series bacillus according to claim 1 produces 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 containing microbial flocculant prepared in step B is carried out solid-liquid separation;
The pre-treatment of C2, resin: select macroporous adsorbent resin, be first 5%HCl solution soaking by mass percent be neutral with distilled water flushing to pH after 4 hours, again with mass percent be after 5%NaOH solution soaking after be neutral with distilled water flushing to pH, it is then the full resin of ethanolic soln leaching of 95% by mass percent, finally with the abundant drip washing of distilled water, wash ethanolic soln off;
The absorption of C3, resin
C3-1: pretreated resin is loaded in resin column in a wet process, with the abundant drip washing of deionized water, then with pH be 5-7, concentration is that the phosphate buffered saline buffer of 0.005-0.02mol/L carries out drip washing with the flow velocity of 2BV/h-5BV/h, makes resin chromatography post reach equilibrium state;
C3-2: the pH of the filtrate of preparing in regulating step C1, make it equal with the phosphate buffered saline buffer in step C3-1;
C3-3: by mix up pH in step C3-2 filtrate with the flow velocity of 1BV/h-4BV/h by reaching the resin chromatography post of equilibrium state in step C3-1, when stopping liquid feeding after resin chromatography post absorption 10BV filtrate;
The wash-out of C4, microbial flocculant is collected
C4-1: be that the phosphate buffer 1 BV of 0.005-0.02mol/L is with the resin chromatography post after absorption filtrate in the flow velocity rinsing step C3-3 of 1BV/h-4BV/h by pH=5-7, concentration;
C4-2: use the phosphoric acid salt+sodium chloride buffer of pH=5-7 with the flocculation agent on the flow velocity wash-out resin chromatography post of 1BV/h-4BV/h, start to collect elutriant, until flocculation agent is after testing by whole wash-out when effluent liquid has flocculation agent to occur.
7. employing colloid series bacillus according to claim 1 produces the method for microbial flocculant, and it is characterized in that the preparation also comprising seed liquor between described steps A, B, wherein seed culture medium is made up of the component of following mass percentage:
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: after being prepared by seed culture medium, a packing gram formula bottle carries out sterilizing; Be seeded to the seed culture medium after sterilizing after being activated by bacterial classification after domestication, at temperature 28-32 DEG C, cultivate 36-60 hour.
8. employing colloid series bacillus according to claim 1 produces the method for microbial flocculant, and it is characterized in that the actication of culture in the preparation of described seed liquor after domestication carries out at normal temperatures, the time is 4 hours.
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