CN101392246A - Method for immobilizing white rot fungi by using bacteria cellulose film as vector - Google Patents
Method for immobilizing white rot fungi by using bacteria cellulose film as vector Download PDFInfo
- Publication number
- CN101392246A CN101392246A CN 200810051379 CN200810051379A CN101392246A CN 101392246 A CN101392246 A CN 101392246A CN 200810051379 CN200810051379 CN 200810051379 CN 200810051379 A CN200810051379 A CN 200810051379A CN 101392246 A CN101392246 A CN 101392246A
- Authority
- CN
- China
- Prior art keywords
- white
- rot fungi
- film
- medium
- bacteria cellulose
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
Abstract
The invention discloses a method for immobilizing white-rot fungi by utilizing a bacterial cellulose membrane as a carrier, which includes the following steps: the separation of the white-rot fungi and the preparation of spore solution of the white-rot fungi; the preparation of the bacterial cellulose membrane; the modification treatment of the bacterial cellulose membrane and the preparation of immobilized cells. After the bacterial cellulose membrane is processed by epoxy chloropropane, absolute ethyl alcohol, perchloric acid and the like, the modified bacterial cellulose membrane obtained is adopted as a carrier and reacts with a 25-percent glutaric dialdehyde solution and mixed with the spore solution of the white-rot fungi after the treatment of repetitive washing and the like, and then the solution is sealed and stays overnight at the temperature of 4 DEG C, and repetitively washed to obtain a bacterial cellulose membrane immobilized white-rot fungi. Three continuous batches of treatment tests for organics and chroma of xylitol production wastewater show that the treatment effects of immobilized white-rot fungi are not found to be obviously reduced and the immobilized white-rot fungi has obvious stability.
Description
Technical field
The invention belongs to using microbe and cell engineering field, relate to the method for a kind of fixation support preparation and utilize the method for this carrier immobilized cell.Be particularly related to a kind of method of utilizing the bacteria cellulose film as vector immobilizing white rot fungi.
Background technology
White-rot fungi (White rot fungi) is the saprophytic filamentous fungus of a class, because the outer lignin-degrading enzymes of its excretory born of the same parents has non-specific and need not the special performance of substrate for induction, make it have the degradation capability of wide spectrum, have important use at the bio-pulping of paper industry and aspects such as pulp bio-bleaching, water pollution control and soil remediation and be worth many structure differences, high toxicity, polymer hardly degraded organic substance.
Though white-rot fungi is widely used in the various environmental pollutant of degraded, but it is in suspension culture degraded system, also there are problems, as: processing efficiency has much room for improvement, the enzyme system poor stability, resistance, toxin immunity, anti-high load capacity are poor, and operating parameters such as temperature, pH and Pollutant levels are had relatively high expectations, sludge output is big, solid-liquid separation difficulty etc.And studies show that both at home and abroad in a large number, immobilized cell technology is introduced white-rot fungi degradation process to environmental pollutants can well overcome above problem.
The method that immobilizing white rot fungi is commonly used has entrapping method, absorption method etc., and fixation support commonly used has wood chip, polyurethane foam, gac, polyvinyl alcohol, alginate calcium etc.But above-mentioned carrier also exists some problems in actual application, as preparation process is complicated, immobilization efficiency is low, big to the influence of white rot fungi activity, operational stability is poor, easily environment is caused secondary pollution etc. after discarded.Bacteria cellulose film is a kind of by microorganism synthetic ultra micro pure cellulose film, by adjoining the glucopyranoside monomer with β-1, the straight-chain polysaccharide that 4 glycosidic links are formed by connecting, adjacent 6 carbon atoms that adjoin glucopyranoside are not in one plane, but be stable chair form three-dimensional arrangement, the β of several vicinities-1,4 dextran chain forms water insoluble under the effect of intramolecularly or intermolecular hydrogen bonding and high molecular polymer organic solvent.Have high degree of crystallinity, the superfine nano fibre network, high overlap ratio, biological fitness is strong, can be in the character that it is unique that nature is directly degraded etc.Have research that bacteria cellulose is used for the immobilization of plurality of enzymes as carrier at present, it has become the focus of domestic and international research as being a kind of novel nano-material.
Summary of the invention
The objective of the invention is: handle by bacteria cellulose film being carried out modification, make it become microbial immobilized excellent carrier, and then set up a kind of simple, effective means of immobilizing white rot fungi, it is more stable that white-rot fungi after the immobilization has activity, immunity from interference is stronger, characteristics such as can reuse.
Above-mentioned purpose realizes by the following technical solutions:
A kind of method of utilizing the bacteria cellulose film as vector immobilizing white rot fungi, form by following process and step:
A kind of method of utilizing the bacteria cellulose film as vector immobilizing white rot fungi, form by following process and step:
(1) separation of white-rot fungi White rot fungi and the preparation of spore liquid
A. the separation of white-rot fungi: get in the 100mL liquid nutrient medium after rotten wooden 2g adds sterilization, place 30 ℃ of constant temperature culture of biochemical incubator, observe the upgrowth situation of bacterium in the substratum at any time; Each ingredients constitute liquid nutrient medium quality usage percentage is respectively in the described liquid nutrient medium: KH
2PO
40.02%, MgSO
47H
2O 0.005%, CaCl
20.001%, glucose 1%, NH
3Cl 0.02%, H
2O
20.05%, sodium tartrate 0.028%, sodium succinate 0.054%, the potato extracting solution is 0.05% of a liquid nutrient medium volume, wherein the potato extracting solution is the 200g potato is boiled 30 minutes in 1L distilled water after, fall insolubles with 16 layers of filtered through gauze and make (as follows), sterilized 30 minutes for 121 ℃; After 48 hours, get this nutrient solution 10mL and repeat enrichment culture, after 3 enrichment culture, get final rich water and on the film solid media flat board, be coated with cultivation; Each ingredients constitute solid medium quality usage percentage is respectively in the described solid medium: glucose 2%, KH
2PO
40.3%, MgSO
47H
2O 0.15%, VITMAIN B1 0.0005%, and it is 80%, 121 ℃ of sterilization 30 minutes that agar 1.2%, potato extracting solution account for solid medium volume (referring to not add the preceding volume of agar, as follows) usage percentage; After 24 hours, the bacterial classification of picking white suede shape separations of ruling on flat board, after waiting white hypha to occur, the separation of ruling again of the single bacterium colony of picking, until repeating to obtain pure single strain, the inclined-plane preservation, standby;
B. the preparation of white-rot fungi spore liquid: preserve a small amount of mycelia of inclined-plane picking from the white-rot fungi that above-mentioned steps A. obtains, method of scoring is inoculated on the solid medium, the composition of described solid medium is identical with solid medium in the steps A, cultivate after 5 days for 30 ℃, the white-rot fungi spore increases in a large number, wash out conidium on the planar surface gently with sterilized water, and under aseptic condition with 16 metafiltration cloth filtering bacterium pieces, obtaining the oyster white spore suspension is the white-rot fungi spore liquid, and 4 ℃ of refrigerators are preserved standby;
(2) preparation of bacteria cellulose film
A. the separation of acetobacter xylinum Acetobacter xylinum: be inoculated in the 50mL enrichment medium after 100 times of the vinegar unstrained spirits dilutions, each ingredients constitute enrichment medium quality usage percentage is respectively in the enrichment medium: glucose 5%; Yeast extract paste 0.5%; Sodium acetate 0.2%; CaCO
31%; Regulate pH to 5.0 with acetate; 121 ℃ of down sterilizations after 30 minutes add that to account for enrichment medium volumetric usage per-cent be that 2% dehydrated alcohol and quality usage percentage are 0.5% nystatin, cultivate 5 days under 28 ℃ of temperature again, and liquid level length has oyster white colloid mycoderm person positive; Throw off the gel-film of enrichment culture primary surface, this film carefully is attached on first isolation medium flat board; Each ingredients constitute isolation medium quality usage percentage is respectively in the described isolation medium: glucose 5%; Peptone 0.5%; Yeast extract paste 0.5%; Na
2HPO
412H
2O 0.2%; KH
2PO
40.1%; MgSO
47H
2O 0.025%; Citric acid 0.2%; Agar 2%; PH5.8; Sterilized 30 minutes for 121 ℃; Take out again and be attached to second flat board, repeat so again 6 times; Flat board places 28 ℃ to cultivate the single bacterium colony access of picking inclined-plane after 48 hours, separates obtaining the canescence pure strain again through continuous three line;
B. the generation of bacteria cellulose film: the acetobacter xylinum list colony inoculation that will screen from the inclined-plane picking of steps A is in the 50mL fermention medium, and the per-cent that each amounts of components accounts for fermention medium quality consumption in the described fermention medium is respectively: glucose 5%; Peptone 0.5%; Yeast extract paste 0.5%; Na
2HPO
412H
2O 0.2%; KH
2PO
40.1%; MgSO
47H
2O 0.025%; Citric acid 0.2%; PH5.8; In 121 ℃ of sterilizations 30 minutes, 28 ℃ left standstill cultivation after 5 days, generate oyster white or faint yellow colloid mycoderm at liquid level; Film is taken out, wash repeatedly, remove striping surface medium and impurity with distilled water; Film is soaked in the NaOH solution of 0.1mol/L, 100 ℃ are boiled 20min again, remove thalline and residual substratum in the mycoderm, and film is creamy white translucent; Acetate with the weight percentage of distilled water and 0.5% washes repeatedly to neutrality then, is dried to constant weight in 80 ℃ again, obtains bacteria cellulose film;
(3) modification of bacteria cellulose film is handled
In 500mL band plug Erlenmeyer flask, add epoxy chloropropane 20～30mL respectively, dehydrated alcohol 5～10mL, perchloric acid 1～2mL, distilled water 3～6mL is with dilution with toluene to 100～200mL, after shaking up, add the bacteria cellulose film that 2～4g above-mentioned steps (2) obtains, in 60 ℃ of waters bath with thermostatic control, heated 4～6 hours, after question response finishes, take out the film washing with acetone, to remove small organic molecule residual on the film, place under 80 ℃ of conditions and dry; Place another 500mL Erlenmeyer flask to add quadrol 6～12mL the film of oven dry, distilled water 100～200mL, behind the shake well, in 80 ℃ of waters bath with thermostatic control, heated 4～6 hours, take out film then and be washed till neutrality with distilled water, use the salt acid elution of 1% weight percentage again after, still be washed till neutrality with distilled water, after the acetone rinse, natural air drying obtains the modified bacteria cellulose film;
(4) preparation of immobilized cell
In the 250mL Erlenmeyer flask, add the modified bacteria cellulose membrane carrier that 1～2g above-mentioned steps (3) obtains, add 20～30mL 0.03mol/L again, the phosphoric acid buffer of pH=7.0; And the glutaraldehyde solution of 25% weight percentage of 10～15mL, in 40 ℃ of water-baths, reacted 1～2 hour, take out film and wash with the phosphate buffer solution of 100mL distilled water and 100mL 0.03mol/LpH=7.0 successively, then natural air drying; The bacteria cellulose carrier that the above-mentioned glutaraldehyde of adding was handled in another 250mL Erlenmeyer flask, the phosphate buffer solution of 15～20mL0.03mol/L pH=7.0, the standby white-rot fungi spore liquid that 10～20mL above-mentioned steps (1) obtains, good seal, placed 12 hours down in 4 ℃, take out film successively with the 0.03mol/L phosphate buffer solution that contains 0.5mol/L NaCl of 4 ℃ of 250mL, its pH=7.0, and the washing of the phosphate buffer solution of the 0.03mol/L pH=7.0 of 4 ℃ of 50mL, obtain milky bacteria cellulose film immobilizing white rot fungi.
In the separation of the white-rot fungi that top step (1) is mentioned and the screening, the rotten wood of used 2g is got and is picked up from China Haikou City, Hainan Province.
The evaluation of white-rot fungi: the steps A in the preparation of the isolation identification of above-mentioned steps (1) white-rot fungi and spore liquid. in the isolation identification of white-rot fungi, preserve inclined-plane picking list bacterium colony from white-rot fungi, insert and produce in the enzyme substratum, each ingredients constitute product enzyme substratum quality usage percentage is respectively in the described product enzyme substratum: glucose 1%, KH
2PO
40.2%, CaCl
20.01%, NH
3Cl 0.2%, MgSO
47H
2O 0.05%, FeSO
40.01%, NaCl 0.0001%, MnSO
40.00005%, VITMAIN B1 0.0005%, pH6.0-7.0 sterilized 30 minutes for 121 ℃; In cultivating 12 days in the 150rpm shaking table under 30 ℃, the centrifugal removal thalline of fermented liquid is got supernatant liquor and is the detection that crude enzyme liquid is used for white-rot fungi feature enzyme activity, and the result is as shown in table 1 below:
The mensuration of laccase and manganese peroxidase enzyme activity all adopts guaiacol method, with reference to " white-rot fungi biology and biotechnology ".An enzyme activity unit is defined as per minute and causes the needed enzyme liquid measure of one 0.01 unit change of wavelength 465nm place absorbancy.
By above feature enzyme activity detected result, identify that this bacterial strain is a white-rot fungi.
Steps A in the preparation of above-mentioned steps (2) bacteria cellulose film. in the isolation identification of acetobacter xylinum (Acetobacter xylinum), obtain pure strain through continuous three line separation, the visual inspection bacterium colony is canescence, can form the hide-like mycoderm, the microscopic examination bacterial classification is shaft-like, and its physiological and biochemical index detected result is as shown in table 2 below:
By above appearance features and detected result, identify that this bacterial strain is an acetobacter xylinum, with reference to " uncle's Jie Shi Bacteria Identification handbook (the 8th edition).
The present invention compared with prior art exists obvious improvement and positive effect:
1. with bacteria cellulose film as carrier immobilized white-rot fungi, on bacteria cellulose is used, provide new thinking and approach.
2. carrier purity height, biologically stable is strong.
3. mass-transfer performance is good, and is affected by environment little, and operational stability is good.
4. nontoxic, good biocompatibility.
5. tension stress is strong, good toughness.
6. easily degraded after discarded, and become the fertilizer of plant-growth by environmental microorganism.
Be the immobilization effect of checking bacteria cellulose film of the present invention to white-rot fungi, use two kinds of carriers respectively, be bacteria cellulose film and wood chip immobilizing white rot fungi, and 3 batches of Xylitol factory effluents (former water CODcr is 991mg/L, and colourity counts 0.801 with 340nm place light absorption value) have been handled respectively; Treatment step is: respectively add 1g different carriers immobilizing white rot fungi in two 250mL triangular flasks, 50mL Xylitol factory effluent, 30 ℃ of constant temperature shaking table 150rpm handled 7 days, measured treatment effect, discarded processed waste water, keep immobilized bacterium, add 50mL Xylitol factory effluent again in this triangular flask, 30 ℃ of constant temperature shaking table 150rpm handled 7 days, measured treatment effect, repeat so again 1 time, promptly with handling 3 batches of Xylitol factory effluents continuously with 1 batch of immobilizing white rot fungi; Treatment effect is as shown in table 3:
Table 3 different carriers immobilizing white rot fungi is to Xylitol production wastewater treatment effect
As can be seen from Table 3, the immobilized white-rot fungi of bacteria cellulose film does not occur obviously descending to 3 batches of Xylitol production wastewater treatment effects, significantly better than the immobilized white-rot fungi of wood chip, the immobilized white-rot fungi of bacteria cellulose film is suitable to the treatment effect of Xylitol factory effluent with not immobilized white-rot fungi simultaneously, the proof bacteria cellulose film is to the immobilization efficiency height of white-rot fungi, little to the bacterium activity influence, operational stability is good.
Above-mentioned wood chip to the immobilization step of white-rot fungi is: get wood chip 1～2g, the H with 2%
2O
2Behind the solution soaking 30min, be washed till neutrality, put into 20～40mL basic medium and soaked 24 hours with distilled water; Each ingredients constitute basic medium usage percentage is respectively in the described basic medium: glucose 1%, KH
2PO
40.04%, K
2HPO
40.16%, MgSO
47H
2O 0.02%, NH
3NO
30.05%, FeCl
36H
2O 0.005%, CaCl
2Sterilized 15 minutes for 0.0025%, 121 ℃; The elimination immersion liquid is with 121 ℃ of sterilizations of the wood chip 15min after soaking; Add the standby white-rot fungi spore liquid that 10～20mL above-mentioned steps (1) obtains, good seal was placed 7 days in 30 ℃ of constant incubators, promptly got the wood chip immobilizing white rot fungi.
Embodiment
Embodiment 1
The isolation identification of white-rot fungi and the preparation of spore liquid:
A. the isolation identification of white-rot fungi: get in the 100mL liquid nutrient medium after rotten wooden 2g adds sterilization, place 30 ℃ of constant temperature culture of biochemical incubator, observe the upgrowth situation of bacterium in the substratum at any time; Each ingredients constitute liquid nutrient medium quality usage percentage is respectively in the described liquid nutrient medium: KH
2PO
40.02%, MgSO
47H
2O 0.005%, CaCl
20.001%, glucose 1%, NH
3Cl 0.02%, H
2O
20.05%, it is 0.05%, 121 ℃ of sterilization 30 minutes that sodium tartrate 0.028%, sodium succinate 0.054%, potato extracting solution account for liquid nutrient medium volumetric usage per-cent; After 48 hours, get this nutrient solution 10mL and repeat enrichment culture, after 3 enrichment culture, get final rich water and on the film solid media flat board, be coated with cultivation; Each ingredients constitute solid medium quality usage percentage is respectively in the described solid medium: glucose 2%, KH
2PO
40.3%, MgSO
47H
2O 0.15%, VITMAIN B1 0.0005%, and it is 80%, 121 ℃ of sterilization 30 minutes that agar 1.2%, potato extracting solution account for solid medium volumetric usage per-cent; After 24 hours, the bacterial classification of picking white suede shape separations of ruling on flat board, after waiting white hypha to occur, the separation of ruling again of the single bacterium colony of picking, until repeating to obtain single strain, through being accredited as white-rot fungi, the inclined-plane preservation, standby.
B. the preparation of white-rot fungi spore liquid: preserve a small amount of mycelia of inclined-plane picking from white-rot fungi, method of scoring is inoculated on the solid medium that above-mentioned steps A. mentions, cultivate after 5 days for 30 ℃, the white-rot fungi spore increases in a large number, wash out conidium on the planar surface gently with sterilized water, and under aseptic condition with 16 metafiltration cloth filtering bacterium pieces, gained oyster white spore suspension is the white-rot fungi spore liquid, 4 ℃ of refrigerators are preserved standby.
Embodiment 2
The preparation of bacteria cellulose film:
A. the isolation identification of acetobacter xylinum (Acetobacter xylinum): be inoculated in the 50mL enrichment medium after 100 times of the vinegar unstrained spirits dilutions, each ingredients constitute enrichment medium quality usage percentage is respectively in the enrichment medium: glucose 5%; Yeast extract paste 0.5%; Sodium acetate 0.2%; CaCO
31%; Regulate pH to 5.0 with acetate; 121 ℃ of down sterilizations after 30 minutes add that to account for enrichment medium volumetric usage per-cent be that 2% dehydrated alcohol and quality usage percentage are 0.5% nystatin, cultivate 5 days under 28 ℃ of temperature again, and liquid level length has oyster white colloid mycoderm person positive.Throw off the gel-film of enrichment culture primary surface, this film carefully is attached on first isolation medium flat board; Each ingredients constitute isolation medium quality usage percentage is respectively in the described isolation medium: glucose 5%; Peptone 0.5%; Yeast extract paste 0.5%; Na
2HPO
412H
2O 0.2%; KH
2PO
40.1%; MgSO
47H
2O0.025%; Citric acid 0.2%; Agar 2%; PH5.8; Sterilized 30 minutes for 121 ℃; Take out and be attached to second flat board, so repeat 6 times, promptly every film on average pastes 8 flat boards.Flat board is put 28 ℃ and is cultivated the single bacterium colony access of picking inclined-plane after 48 hours, separates through continuous three line, through being accredited as the acetobacter xylinum bacterial classification again.
B. the generation of bacteria cellulose film: the acetobacter xylinum list colony inoculation that screens from above-mentioned steps A. inclined-plane picking is in the 50mL fermention medium, and the per-cent that each amounts of components accounts for fermention medium quality consumption in the described fermention medium is respectively: glucose 5%; Peptone 0.5%; Yeast extract paste 0.5%; Na
2HPO
412H
2O 0.2%; KH
2PO
40.1%; MgSO
47H
2O 0.025%; Citric acid 0.2%; PH5.8; In 121 ℃ of sterilizations 30 minutes, 28 ℃ left standstill cultivation after 5 days, generate oyster white or faint yellow colloid mycoderm at liquid level.Film is taken out, wash repeatedly, remove striping surface medium and impurity with distilled water.Film is soaked in the NaOH solution of 0.1mol/L, 100 ℃ are boiled 20min again, remove thalline and residual substratum in the mycoderm, and film is creamy white translucent.Acetate with distilled water and 0.5wt% washes repeatedly to neutrality then.80 ℃ are dried to constant weight, promptly get bacteria cellulose film.
Embodiment 3:
The modification of bacteria cellulose film is handled:
In 500mL band plug Erlenmeyer flask, add epoxy chloropropane 20mL respectively, dehydrated alcohol 5mL, perchloric acid 1mL, distilled water 3mL, with dilution with toluene to 100mL, after shaking up, add the 2g bacteria cellulose film that embodiment 2 obtains, heating is 4 hours in 60 ℃ of waters bath with thermostatic control, after question response finishes, takes out the film washing with acetone, to remove small organic molecule residual on the film, place under 80 ℃ of conditions and dry.The film of oven dry is placed another 500mL Erlenmeyer flask, add quadrol 6mL, distilled water 100mL, behind the shake well, heating is 4 hours in 80 ℃ of waters bath with thermostatic control, take out film then and be washed till neutrality with distilled water, after using 1wt% salt acid elution again, still be washed till neutrality, after the acetone rinse with distilled water, natural air drying promptly gets the modified bacteria cellulose film.
The preparation of immobilized cell:
In the 250mL Erlenmeyer flask, add the above-mentioned modified bacteria cellulose membrane carrier that obtains of 1g, 20mL phosphoric acid buffer (0.03mol/L, pH=7.0), and 25% glutaraldehyde solution of 10mL, 40 ℃ of water-baths 1 hour, take out film and use 100mL distilled water and 100mL 0.03mol/L phosphate buffer solution (pH=7.0) washing, natural air drying successively.In another 250mL Erlenmeyer flask, add the bacteria cellulose carrier that above-mentioned glutaraldehyde was handled, 15mL 0.03mol/L phosphate buffer solution (pH=7.0), the white-rot fungi spore liquid 10mL that example 1 is standby, good seal, placed 12 hours down in 4 ℃, take out film and use the 0.03mol/L phosphate buffer solution (pH=7.0 of 4 ℃ of 250mL successively, contain 0.5mol/L NaCl) and 0.03mol/L phosphate buffer solution (pH=7.0) washing of 4 ℃ of 50mL, milky bacteria cellulose film immobilizing white rot fungi promptly got.
Embodiment 4:
The modification of bacteria cellulose film is handled:
In 500mL band plug Erlenmeyer flask, add epoxy chloropropane 25mL respectively, dehydrated alcohol 8mL, perchloric acid 1.5mL, distilled water 5mL, with dilution with toluene to 150mL, after shaking up, add the 3g bacteria cellulose film that above-mentioned example 2 obtains, heating is 5 hours in 60 ℃ of waters bath with thermostatic control, after question response finishes, takes out the film washing with acetone, to remove small organic molecule residual on the film, place under 80 ℃ of conditions and dry.The film of oven dry is placed another 500mL Erlenmeyer flask, add quadrol 9mL, distilled water 150mL, behind the shake well, heating is 5 hours in 80 ℃ of waters bath with thermostatic control, take out film then and be washed till neutrality with distilled water, after using 1% salt acid elution again, still be washed till neutrality, after the acetone rinse with distilled water, natural air drying promptly gets the modified bacteria cellulose film.
The preparation of immobilized cell:
In the 250mL Erlenmeyer flask, add the above-mentioned modified bacteria cellulose membrane carrier that obtains of 1.5g, 25mL phosphoric acid buffer (0.03mol/L, pH=7.0), and 25% glutaraldehyde solution of 13mL, 40 ℃ of water-baths 1.5 hours, take out film and use 100mL distilled water and 100mL 0.03mol/L phosphate buffer solution (pH=7.0) washing, natural air drying successively.In another 250mL Erlenmeyer flask, add the bacteria cellulose carrier that above-mentioned glutaraldehyde was handled, 18mL 0.03mol/L phosphate buffer solution (pH=7.0), the white-rot fungi spore liquid 15mL that example 1 is standby, good seal, placed 12 hours down in 4 ℃, take out film and use the 0.03mol/L phosphate buffer solution (pH=7.0 of 4 ℃ of 250mL successively, contain 0.5mol/L NaCl) and 0.03mol/L phosphate buffer solution (pH=7.0) washing of 4 ℃ of 50mL, milky bacteria cellulose film immobilizing white rot fungi promptly got.
Embodiment 5:
The modification of bacteria cellulose film is handled:
In 500mL band plug Erlenmeyer flask, add epoxy chloropropane 30mL respectively, dehydrated alcohol 10mL, perchloric acid 2mL, distilled water 6mL, with dilution with toluene to 200mL, after shaking up, add the 4g bacteria cellulose film that example 2 obtains, heating is 6 hours in 60 ℃ of waters bath with thermostatic control, after question response finishes, takes out the film washing with acetone, to remove small organic molecule residual on the film, place under 80 ℃ of conditions and dry.The film of oven dry is placed another 500mL Erlenmeyer flask, add quadrol 12mL, distilled water 200mL, behind the shake well, heating is 6 hours in 80 ℃ of waters bath with thermostatic control, take out film then and be washed till neutrality with distilled water, after using 1% salt acid elution again, still be washed till neutrality, after the acetone rinse with distilled water, natural air drying promptly gets the modified bacteria cellulose film.
The preparation of immobilized cell:
In the 250mL Erlenmeyer flask, add the above-mentioned modified bacteria cellulose membrane carrier that obtains of 2g, 30mL phosphoric acid buffer (0.03mol/L, pH=7.0), and 25% glutaraldehyde solution of 15mL, 40 ℃ of water-baths 2 hours, take out film and use 100mL distilled water and 100mL 0.03mol/L phosphate buffer solution (pH=7.0) washing, natural air drying successively.In another 250mL Erlenmeyer flask, add the bacteria cellulose carrier that above-mentioned glutaraldehyde was handled, 20mL 0.03mol/L phosphate buffer solution (pH=7.0), the white-rot fungi spore liquid 20mL that example 1 is standby, good seal, placed 12 hours down in 4 ℃, take out film and use the 0.03mol/L phosphate buffer solution (pH=7.0 of 4 ℃ of 250mL successively, contain 0.5mol/L NaCl) and 0.03mol/L phosphate buffer solution (pH=7.0) washing of 4 ℃ of 50mL, milky bacteria cellulose film immobilizing white rot fungi promptly got.
Claims (1)
1, a kind of method of utilizing the bacteria cellulose film as vector immobilizing white rot fungi is
Form by following process and step:
(1) separation of white-rot fungi White rot fungi and the preparation of spore liquid
A. the separation of white-rot fungi: get in the 100mL liquid nutrient medium after rotten wooden 2g adds sterilization, place 30 ℃ of constant temperature culture of biochemical incubator, observe the upgrowth situation of bacterium in the substratum at any time; Each ingredients constitute liquid nutrient medium quality usage percentage is respectively in the described liquid nutrient medium: KH
2PO
40.02%, MgSO
47H
2O 0.005%, CaCl
20.001%, glucose 1%, NH
3Cl 0.02%, H
2O
20.05%, sodium tartrate 0.028%, sodium succinate 0.054%, potato extracting solution are 0.05% of liquid nutrient medium volume, wherein the potato extracting solution is the 200g potato is boiled 30 minutes in 1L distilled water after, to fall insolubles with 16 layers of filtered through gauze and make; Sterilized 30 minutes for 121 ℃; After 48 hours, get this nutrient solution 10mL and repeat enrichment culture, after 3 enrichment culture, get final rich water and on the film solid media flat board, be coated with cultivation; Each ingredients constitute solid medium quality usage percentage is respectively in the described solid medium: glucose 2%, KH
2PO
40.3%, MgSO
47H
2O 0.15%, VITMAIN B1 0.0005%, and it is 80% that agar 1.2%, potato extracting solution account for solid medium volumetric usage per-cent; Sterilized 30 minutes for 121 ℃; After 24 hours, the bacterial classification of picking white suede shape separations of ruling on flat board, after waiting white hypha to occur, the separation of ruling again of the single bacterium colony of picking, until repeating to obtain pure single strain, the inclined-plane preservation, standby;
B. the preparation of white-rot fungi spore liquid: preserve a small amount of mycelia of inclined-plane picking from the white-rot fungi that above-mentioned steps A. obtains, method of scoring is inoculated on the solid medium, the composition of described solid medium is identical with solid medium in the steps A, cultivate after 5 days for 30 ℃, the white-rot fungi spore increases in a large number, wash out conidium on the planar surface gently with sterilized water, and under aseptic condition with 16 metafiltration cloth filtering bacterium pieces, obtaining the oyster white spore suspension is the white-rot fungi spore liquid, and 4 ℃ of refrigerators are preserved standby;
(2) preparation of bacteria cellulose film
A. the separation of acetobacter xylinum Acetobacter xylinum: be inoculated in the 50mL enrichment medium after 100 times of the vinegar unstrained spirits dilutions, each ingredients constitute enrichment medium quality usage percentage is respectively in the enrichment medium: glucose 5%; Yeast extract paste 0.5%; Sodium acetate 0.2%; CaCO
31%; Regulate pH to 5.0 with acetate; 121 ℃ of down sterilizations after 30 minutes add that to account for enrichment medium volumetric usage per-cent be that 2% dehydrated alcohol and quality usage percentage are 0.5% nystatin, cultivate 5 days under 28 ℃ of temperature again, and liquid level length has oyster white colloid mycoderm person positive; Throw off the gel-film of enrichment culture primary surface, this film carefully is attached on first isolation medium flat board; Each ingredients constitute isolation medium quality usage percentage is respectively in the described isolation medium: glucose 5%; Peptone 0.5%; Yeast extract paste 0.5%; Na
2HPO
412H
2O 0.2%; KH
2PO
40.1%; MgSO
47H
2O 0.025%; Citric acid 0.2%; Agar 2%; PH5.8; Sterilized 30 minutes for 121 ℃; Take out again and be attached to second flat board, repeat so again 6 times; Flat board places 28 ℃ to cultivate the single bacterium colony access of picking inclined-plane after 48 hours, separates obtaining the canescence pure strain again through continuous three line;
B. the generation of bacteria cellulose film: the acetobacter xylinum list colony inoculation that screens from the inclined-plane picking of steps A is in the 50mL fermention medium, and the per-cent that each amounts of components accounts for fermention medium quality consumption in the described fermention medium is respectively: glucose 5%; Peptone 0.5%; Yeast extract paste 0.5%; Na
2HPO
412H
2O 0.2%; KH
2PO
40.1%; MgSO
47H
2O0.025%; Citric acid 0.2%; PH5.8; In 121 ℃ of sterilizations 30 minutes, 28 ℃ left standstill cultivation after 5 days, generate oyster white or faint yellow colloid mycoderm at liquid level; Film is taken out, wash repeatedly, remove striping surface medium and impurity with distilled water; Film is soaked in the NaOH solution of 0.1mol/L, 100 ℃ are boiled 20min again, remove thalline and residual substratum in the mycoderm, and film is creamy white translucent; Acetate with the weight percentage of distilled water and 0.5% washes repeatedly to neutrality then, is dried to constant weight in 80 ℃ again, obtains bacteria cellulose film;
(3) modification of bacteria cellulose film is handled
In 500mL band plug Erlenmeyer flask, add epoxy chloropropane 20～30mL respectively, dehydrated alcohol 5～10mL, perchloric acid 1～2mL, distilled water 3～6mL is with dilution with toluene to 100～200mL, after shaking up, add the bacteria cellulose film that 2～4g above-mentioned steps (2) obtains, in 60 ℃ of waters bath with thermostatic control, heated 4～6 hours, after question response finishes, take out the film washing with acetone, to remove small organic molecule residual on the film, place under 80 ℃ of conditions and dry; Place another 500mL Erlenmeyer flask to add quadrol 6～12mL the film of oven dry, distilled water 100～200mL, behind the shake well, in 80 ℃ of waters bath with thermostatic control, heated 4～6 hours, take out film then and be washed till neutrality with distilled water, use the salt acid elution of 1% weight percentage again after, still be washed till neutrality with distilled water, after the acetone rinse, natural air drying obtains the modified bacteria cellulose film;
(4) preparation of immobilized cell
In the 250mL Erlenmeyer flask, add the modified bacteria cellulose membrane carrier that 1～2g above-mentioned steps (3) obtains, add 20～30mL 0.03mol/L again, the phosphoric acid buffer of pH=7.0; And the glutaraldehyde solution of 25% weight percentage of 10～15mL, in 40 ℃ of water-baths, reacted 1～2 hour, take out film and wash with the phosphate buffer solution of 100mL distilled water and 100mL0.03mol/LpH=7.0 successively, then natural air drying; The bacteria cellulose carrier that the above-mentioned glutaraldehyde of adding was handled in another 250mL Erlenmeyer flask, the phosphate buffer solution of 15～20mL0.03mol/L pH=7.0, the standby white-rot fungi spore liquid that 10～20mL above-mentioned steps (1) obtains, good seal, placed 12 hours down in 4 ℃, take out film successively with 250mL4 ℃ the 0.03mol/L phosphate buffer solution that contains 0.5mol/L NaCl, its pH=7.0, and the washing of the phosphate buffer solution of 50mL4 ℃ 0.03mol/L pH=7.0, obtain milky bacteria cellulose film immobilizing white rot fungi.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200810051379 CN101392246A (en) | 2008-10-29 | 2008-10-29 | Method for immobilizing white rot fungi by using bacteria cellulose film as vector |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200810051379 CN101392246A (en) | 2008-10-29 | 2008-10-29 | Method for immobilizing white rot fungi by using bacteria cellulose film as vector |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101392246A true CN101392246A (en) | 2009-03-25 |
Family
ID=40492756
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200810051379 Pending CN101392246A (en) | 2008-10-29 | 2008-10-29 | Method for immobilizing white rot fungi by using bacteria cellulose film as vector |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101392246A (en) |
Cited By (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101967471A (en) * | 2009-11-10 | 2011-02-09 | 东华大学 | Method for preparing immobilized enzyme by taking bacterial cellulose bead/membrane as vector |
| CN101973640A (en) * | 2010-09-21 | 2011-02-16 | 东北电力大学 | Method for treating malachite green dye waste water |
| CN102010069A (en) * | 2010-09-21 | 2011-04-13 | 东北电力大学 | Method for treating methyl orange dye waste water |
| CN102226174A (en) * | 2011-04-21 | 2011-10-26 | 钟春燕 | Bacterial cellulose immobilized yeast and application thereof |
| CN102234670A (en) * | 2010-04-20 | 2011-11-09 | 中国科学院过程工程研究所 | Method for producing bacterial cellulose through solid state fermentation by using inert adsorption carrier |
| CN102276736A (en) * | 2011-07-10 | 2011-12-14 | 东华大学 | Selective amination method of bacterial cellulose |
| CN101591448B (en) * | 2009-06-22 | 2012-02-15 | 天津科技大学 | Method for preparing bacterial cellulose membrane with high rehydration |
| WO2012056110A3 (en) * | 2010-10-27 | 2012-06-14 | Upm-Kymmene Corporation | Cell culture material based on microbial cellulose |
| CN103266065A (en) * | 2013-05-15 | 2013-08-28 | 内蒙古农业大学 | Method for quickly culturing verticillium dahliae |
| CN105638247A (en) * | 2016-02-03 | 2016-06-08 | 程雪娇 | Method for breeding fungi by means of rotten wood |
| CN106350544A (en) * | 2016-08-24 | 2017-01-25 | 合肥国轩高科动力能源有限公司 | Environment-friendly preparation method of porous graphene |
| US9593304B2 (en) | 2012-09-25 | 2017-03-14 | Upm-Kymmene Corporation | Three-dimensional discontinuous entity for cell culturing |
| CN110029136A (en) * | 2018-01-11 | 2019-07-19 | 西安宁远清洁技术有限公司 | A kind of preparation method of biological cellulose membrane |
| CN115487340A (en) * | 2022-10-31 | 2022-12-20 | 振德医疗用品股份有限公司 | Bacterial cellulose dressing loaded with active oxygen and preparation method thereof |
| CN116554543A (en) * | 2023-05-26 | 2023-08-08 | 南京工业大学 | Immobilization medium and preparation method and application thereof |
-
2008
- 2008-10-29 CN CN 200810051379 patent/CN101392246A/en active Pending
Cited By (23)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101591448B (en) * | 2009-06-22 | 2012-02-15 | 天津科技大学 | Method for preparing bacterial cellulose membrane with high rehydration |
| CN101967471B (en) * | 2009-11-10 | 2012-06-13 | 东华大学 | Method for preparing immobilized enzyme by taking bacterial cellulose bead/membrane as vector |
| CN101967471A (en) * | 2009-11-10 | 2011-02-09 | 东华大学 | Method for preparing immobilized enzyme by taking bacterial cellulose bead/membrane as vector |
| CN102234670B (en) * | 2010-04-20 | 2014-06-04 | 中国科学院过程工程研究所 | Method for producing bacterial cellulose through solid state fermentation by using inert adsorption carrier |
| CN102234670A (en) * | 2010-04-20 | 2011-11-09 | 中国科学院过程工程研究所 | Method for producing bacterial cellulose through solid state fermentation by using inert adsorption carrier |
| CN101973640A (en) * | 2010-09-21 | 2011-02-16 | 东北电力大学 | Method for treating malachite green dye waste water |
| CN102010069A (en) * | 2010-09-21 | 2011-04-13 | 东北电力大学 | Method for treating methyl orange dye waste water |
| CN101973640B (en) * | 2010-09-21 | 2011-11-16 | 东北电力大学 | Method for treating malachite green dye waste water |
| US10612003B2 (en) | 2010-10-27 | 2020-04-07 | Upm-Kymmene Corporation | Plant derived cell culture material |
| WO2012056110A3 (en) * | 2010-10-27 | 2012-06-14 | Upm-Kymmene Corporation | Cell culture material based on microbial cellulose |
| US9631177B2 (en) | 2010-10-27 | 2017-04-25 | Upm-Kymmene Corporation | Drug delivery compositions |
| CN102226174A (en) * | 2011-04-21 | 2011-10-26 | 钟春燕 | Bacterial cellulose immobilized yeast and application thereof |
| CN102276736B (en) * | 2011-07-10 | 2013-02-20 | 东华大学 | Selective amination method of bacterial cellulose |
| CN102276736A (en) * | 2011-07-10 | 2011-12-14 | 东华大学 | Selective amination method of bacterial cellulose |
| US9593304B2 (en) | 2012-09-25 | 2017-03-14 | Upm-Kymmene Corporation | Three-dimensional discontinuous entity for cell culturing |
| CN103266065A (en) * | 2013-05-15 | 2013-08-28 | 内蒙古农业大学 | Method for quickly culturing verticillium dahliae |
| CN105638247A (en) * | 2016-02-03 | 2016-06-08 | 程雪娇 | Method for breeding fungi by means of rotten wood |
| CN106350544A (en) * | 2016-08-24 | 2017-01-25 | 合肥国轩高科动力能源有限公司 | Environment-friendly preparation method of porous graphene |
| CN106350544B (en) * | 2016-08-24 | 2020-01-17 | 合肥国轩高科动力能源有限公司 | Green preparation method of porous graphene |
| CN110029136A (en) * | 2018-01-11 | 2019-07-19 | 西安宁远清洁技术有限公司 | A kind of preparation method of biological cellulose membrane |
| CN115487340A (en) * | 2022-10-31 | 2022-12-20 | 振德医疗用品股份有限公司 | Bacterial cellulose dressing loaded with active oxygen and preparation method thereof |
| CN115487340B (en) * | 2022-10-31 | 2023-11-17 | 振德医疗用品股份有限公司 | Bacterial cellulose dressing loaded with active oxygen and preparation method thereof |
| CN116554543A (en) * | 2023-05-26 | 2023-08-08 | 南京工业大学 | Immobilization medium and preparation method and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101392246A (en) | Method for immobilizing white rot fungi by using bacteria cellulose film as vector | |
| Tabaii et al. | Comparison of bacterial cellulose production among different strains and fermented media | |
| CN101973640B (en) | Method for treating malachite green dye waste water | |
| Krishna et al. | Laccase production using Pleurotus ostreatus 1804 immobilized on PUF cubes in batch and packed bed reactors: influence of culture conditions | |
| Reda et al. | Production of bacterial pectinase (s) from agro-industrial wastes under solid state fermentation conditions | |
| CN101880632B (en) | Laccase and preparation method and special production strain thereof | |
| Prasongsuk et al. | Decolourization of pulp mill wastewater using thermotolerant white rot fungi | |
| CN103627654B (en) | A kind of starch degradation bacterium and the application in water treatment thereof | |
| CN101892184B (en) | Black pepper peel degumming strain and use thereof | |
| CN105925492B (en) | A kind of method of biodegrade deoxynivalenol enol | |
| CN101481671A (en) | Cultivation method and use of Bacillus foecalis alkaligenes from sea | |
| WO2015087143A1 (en) | Consortium of fungi immobilised on a laminar lignocellulose carrier for the treatment of wastewater and method for producing same | |
| CN102010069B (en) | Method for treating methyl orange dye waste water | |
| Villena et al. | Production of lignocellulolytic enzymes by Aspergillus niger biofilms at variable water activities | |
| CN101942764B (en) | Method for modifying polyester fabric biologically | |
| CN103173378A (en) | Method for fast screening microbial flocculant producing bacteria | |
| CN105969701B (en) | A kind of sphingolipid monad for the PVA that degrades | |
| CN105936879B (en) | Bacillus subtilis K13 and its cultural method and application | |
| CN1308435C (en) | Laccase, preparing method thereof and strain special for same | |
| CN104611270A (en) | Streptomyces strain and application thereof | |
| CN103990441B (en) | A kind of adsorbent for heavy metal preparation method based on modified bacteria cellulose | |
| CN102690773A (en) | Enterobacteria strain FY-07 and method thereof for producing bacterial cellulose by static liquid submerged fermentation | |
| KR20040086139A (en) | Biopulp for non-woody fiber plants and biopulping method thereof | |
| Widden et al. | Decomposition of cotton by Trichoderma species: influence of temperature, soil type, and nitrogen levels | |
| CN108913629A (en) | A kind of bacterium of cellulase-producing and the preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Open date: 20090325 |