CN102051395A - Method for preparing bacterial cellulose from corn stalks - Google Patents
Method for preparing bacterial cellulose from corn stalks Download PDFInfo
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- CN102051395A CN102051395A CN 201010580800 CN201010580800A CN102051395A CN 102051395 A CN102051395 A CN 102051395A CN 201010580800 CN201010580800 CN 201010580800 CN 201010580800 A CN201010580800 A CN 201010580800A CN 102051395 A CN102051395 A CN 102051395A
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Abstract
The invention relates to a method for preparing bacterial cellulose from corn stalks. The method comprises the following steps: pretreating the corn stalks in an ionic liquid at 90-120 DEG C for 15 minutes-21 hours, then adding deionized water, stirring for regeneration, and adding cellulase to carry out enzymolysis, thereby obtaining an enzymolysis liquid; and detoxifying the enzymolysis liquid to obtain a culture medium for culturing the bacterial cellulose, adding yeast extract and tryptone to obtain a culture medium, adding a seed liquid of bacterial cellulose producing bacteria, and standing to culture at 25-30 DEG C for 8-28 days, thereby obtaining the bacterial cellulose. According to the invention, the farm crop byproduct corn stalks, which are often discarded or incinerated, are utilized as a raw material, thus a cheap carbon source is developed, which reduces the environmental pollution and can be used for producing products with high added value, thereby creating high economic benefit for related industries.
Description
Technical field
The invention belongs to the preparation field of bacteria cellulose, particularly relate to a kind of method for preparing bacteria cellulose with cornstalk.
Background technology
(Bacterial cellulose BC) is a kind of Mierocrystalline cellulose by microorganisms producing to bacteria cellulose.Compare with vegetable fibre, bacteria cellulose has high-crystallinity, high retentiveness, high Young's modulus, high purity, characteristics such as outstanding biocompatibility, so bacteria cellulose has wide application in fields such as biomedical materials such as medical dressing, food, papermaking, weavings.But the high production cost of bacteria cellulose has limited its technical scale, therefore reduces the difficult problem that needs to be resolved hurrily that the bacteria cellulose cost is a current industrial production.Cost height that the principal element that the bacteria cellulose cost is high is a carbon source in the substratum, so this experiment discloses invention and utilizes agriculture byproduct---cornstalk is produced the method for bacteria cellulose.
Lignocellulose such as rice straw, cornstalk stalk is the byproduct of farm crop, and annual such crop material of China has 7.05 hundred million tons approximately.The agricultural crop straw composition is Mierocrystalline cellulose, hemicellulose and xylogen, and wherein content of cellulose is near 40%.Mierocrystalline cellulose can be hydrolyzed to glucose, and hemicellulose then is hydrolyzed to five-carbon sugar and hexose, and these hydrolysis sugars can be used to produce bacteria cellulose by bacillus aceticus.But because the special construction of stalk, xylogen is severely limited the cellulosic effect of enzymic hydrolysis to the reactionlessness that the coating effect and the crystalline cellulose dense structure of Mierocrystalline cellulose and hemicellulose causes.Therefore, must carry out pre-treatment to lignocellulosic material, slough xylogen or change the physical chemistry structure of raw material to a certain extent, for example reduce degree of crystallinity, reduce the polymerization degree, increase porosity and surface-area etc., to promote enzyme-to-substrate to be in contact with one another and biochemical reaction takes place, improve enzymolysis speed and get sugared rate.At present the pretreatment process of stalk has a lot, and commonplace have physical method, chemical process, a biological method, ionic liquid be used for pretreated straw report seldom.Ionic liquid also of no use is both at home and abroad at present handled cornstalk and is used for the cellulosic report of culturing bacterium.
Ionic liquid is a kind of novel green solvent, the boiling point height, and steam can be ignored, and can not pollute atmosphere, and is not flammable, has better chemical stability, and recyclable utilization is beneficial to environmental protection.Because therefore ion liquid these characteristics are used to dissolve stalk.At present, there is research to report with ionic liquid pre-treatment rice straw, timber, bagasse, cornstalk, the cotton stalk is handled the back enzymatic hydrolyzation and is obviously improved.Being used for lignocellulose dissolved ionic liquid has [EPy] Br, [AMIM] [HCO2] etc.(AMIM) Cl does not have play-by-play it is useful on the dissolving of stalk.This experiment adopt (AMIM) Cl and (BMIM) Cl and LiCl/DMAc handle stalk, select for use (AMIM) Cl synthesis temperature low, nontoxic, pretreatment temperature is low and the time short, therefore characteristics such as rate of recovery height select this kind solvent pre-treatment cornstalk for use.
Summary of the invention
Technical problem to be solved by this invention provides a kind of method of utilizing cornstalk to prepare bacteria cellulose, the farm crop by product of this method to abandon usually or to burn---cornstalk is a raw material, be developed as a kind of carbon source of cheapness, not only reduced environmental pollution, and can be used to produce high value added product, for related industries has been created good economic benefit.
A kind of method of utilizing cornstalk to prepare bacteria cellulose of the present invention comprises:
(1) ionic liquid pretreatment of cornstalk, regeneration and enzyme digestion reaction
The ion liquid pre-treatment of cornstalk: after cornstalk pulverized, join in the ionic liquid, handle 15min-21h in 90 ℃-120 ℃, leave standstill cooling with the ratio of 1%-15% (w/w);
Ionic liquid/cornstalk mixed solution is regenerated: the deionized water of adding and ionic liquid 5-10 times volume, stir regeneration, and centrifugal then or filtration, precipitation or the filter residue that obtains with deionized water wash is extremely colourless until supernatant or filtrate again;
Simultaneously, the filtrate that the centrifugal supernatant liquor that obtains or filtration are obtained places matrass, temperature 100-170 ℃ of following air distillation or temperature 50-150 ℃ and-0.09～-vacuum degree condition of 0.1Mpa under underpressure distillation, remove water with the ionic liquid blend, be distilled to till the not water outlet the remaining ionic liquid that is recovery in the matrass; Wherein, the used cooling fluid of prolong is the cooling fluid below 10 ℃ in the still-process, connects in the preceding system of vacuum pump to install a cold-trap additional, and cold-trap places the vacuum flask that fills the cooled with liquid nitrogen agent;
Enzymolysis: adding buffered soln to solid-to-liquid ratio in above-mentioned precipitation or filter residue is 0.005g: 1ml～1g: 10ml, adding cellulase to final concentration again is 5-600U/mL, regulate pH to 4.0-5.5, under 40-60 ℃ of condition, react 1-60h, centrifugal or filtering separation is degrading maize stalk and enzymolysis solution not, with enzymolysis solution in 4 ℃ of refrigerations;
(2) enzymolysis solution prepares bacteria cellulose
With above-mentioned enzymolysis solution directly as the cellulosic substratum of culturing bacterium, add the yeast extract paste of relative enzymolysis solution 0.1-1% (w/v) and the Tryptones of 0.1-0.5% (w/v), be mixed with substratum, add the 6%-10% bacteria cellulose again and produce the seed liquor of bacterium, 25-30 ℃ leaves standstill cultivation and obtained bacteria cellulose in 8-28 days;
Or the enzymolysis solution that step (1) is made is with Ca (OH)
2Transfer enzymolysis solution pH to 9.5-11.0, detoxification reaction 12h-24h in 25 ℃ of-60 ℃ of water-baths filters and finely tunes pH4.5-5.0 then, adds stirring reaction 5-10min under the gac room temperature, filters the enzymolysis solution that obtains detoxification treatment; With the enzymolysis solution of detoxification treatment as the cellulosic substratum of culturing bacterium, add the yeast extract paste of relative enzymolysis solution 0.1-1% (w/v) and the Tryptones of 0.1-0.5% (w/v), be mixed with substratum, add the 6%-10% bacteria cellulose again and produce the seed liquor of bacterium, 25-30 ℃ leaves standstill cultivation and obtained bacteria cellulose in 8-28 days.
Ionic liquid in the described step (1) is (AMIM) Cl, (BMIM) Cl or LiCl/DMAc.
It is more than 40 orders that cornstalk in the described step (1) is pulverized number, and concentration is 3% (w/w) in ionic liquid, handles 1h for 110 ℃ in temperature.
Centrifugal condition in the described step (1) is speed 4000-10000rpm, time 5-20min.
The add-on of the gac in the described step (2) is the 1%-6% with respect to the enzymolysis solution quality.
It is acetic acid Pseudomonas (Acetobacter sp.) that bacteria cellulose in the described step (2) is produced bacterial strain, gluconobacter suboxydans belongs to (Gluconobacter sp.), glyconic acid genus acetobacter (Gluconacetobacter sp.), rhizobium (Rhizobium sp.), Sarcina (Sarcina sp.), Rhodopseudomonas (Pseudomounas sp.), achromobacter (Achromobactersp.), Alcaligenes (Alcaligenes sp.), aerobacter (Aerobacter sp.), Azotobacter (Azotobacter sp.), Agrobacterium (Agrobacterium sp.), pseudomonas cepacia (Seudomonas cepacia), campylobacter jejuni (Campylobacter jejuni) or tea fungus (kombucha); Wherein preferred strain is acetobacter xylinum (Acetobacter xylinum) or tea fungus.
The present invention adopts the ionic liquid pretreatment cornstalk, can improve enzymatic hydrolyzation, and preparation contains the enzymolysis solution of high concentration sugar, and the reason that the cornstalk enzymolysis efficiency improves after the analysis pre-treatment; And then, and bacteria cellulose characterized the difference between the bacteria cellulose that the bacteria cellulose of inquiring into this method preparation at last and traditional method prepare with this enzymolysis solution fermentative preparation bacteria cellulose.
The enzymolysis raw material that adopts is the cornstalk after the regeneration, and directly enzymolysis is not gone through oven dry.The structure that has kept raw material to loosen after pre-treatment like this helps the contact and the catalytic hydrolysis of enzyme, and saves a step operation, saves the energy and manpower.
Infrared spectroscopy ionic liquid as can be known has cellulosic ability in the extracting cornstalk; The X-ray diffraction analysis finds that the degree of crystallinity of the cornstalk that the process ionic liquid was handled is lower than untreated, therefore also is beneficial to enzyme digestion reaction and carries out; The scanning electron microscope sem photo shows that the effect that the cornstalk surface does not have to puncture occurs.Explain the reason that the cornstalk enzymatic hydrolyzation improves after the pre-treatment from infrared and results X-ray diffraction two aspects.
The bacteria cellulose film output higher (2.01g/L) that the detoxification enzymolysis solution is cultivated, with glucose is carbon source, water is that the output of the bacteria cellulose film of solvent cultivation is 3.74g/L, is carbon source with glucose, and buffered soln is that the output of the bacteria cellulose film of solvent cultivation is 4.47g/L.Infared spectrum by comparison bacteria cellulose and the Microcrystalline Cellulose product that obtains of cornstalk enzymolysis solution cultivation as can be known is a pure cellulose.Is that carbon source is cultivated the bacteria cellulose that obtains than water and damping fluid for solvent glucose, and the output that the cornstalk enzymolysis solution is cultivated the bacteria cellulose film dry weight that obtains is not high.
Beneficial effect
The present invention is by the ionic liquid pretreatment cornstalk, improved enzymic hydrolysis efficient, the enzymolysis solution that obtains can be turned out bacteria cellulose through detoxification, for industrialization low cost production bacteria cellulose provides a feasible approach, helps this Mierocrystalline cellulose of commercial scale production; Cornstalk is as the byproduct of farm crop in addition, and one all is to abandon or burn, i.e. contaminate environment labor intensive again, come the culturing bacterium Mierocrystalline cellulose with it now, will be a kind of carbon source that is dirt cheap, both can increase the channel of an extra earning, can turn waste into wealth again to the peasant.
Description of drawings
The addition of cornstalk is to the influence of pretreating effect in Fig. 1 ionic liquid.
Fig. 2 pretreatment time and temperature are to the influence of pretreating effect.
Enzymolysis graphic representation under the optimum pretreatment condition of Fig. 3.
The infrared spectrum characterization of Fig. 4 cornstalk regeneration flocculent substance.
The stereoscan photograph of cornstalk before and after Fig. 5 ionic liquid is handled, left side figure are before handling, and right figure is after handling.
The x-ray diffraction pattern of cornstalk before and after Fig. 6 ionic liquid is handled.
The output of the bacteria cellulose film that Fig. 7 detoxification enzymolysis solution is cultivated.
The bacteria cellulose Infrared Characterization that Fig. 8 enzymolysis solution is cultivated.
The x-ray diffraction pattern of the bacteria cellulose that Fig. 9 enzymolysis solution is cultivated.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after the content of having read the present invention's instruction, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Embodiment 1
(1) pre-treatment of cornstalk
100 ℃, under the 500rpm magnetic agitation rotating speed, 150mg 40-60 purpose cornstalk is placed 1.5g respectively, 3g, among the ionic liquid of 5g (AMIM) Cl, pre-treatment 2h.
(2) regeneration of cornstalk
Add 5 times of deionized waters to the ionic liquid volume in the cornstalk ionic liquid mixed solution after above processing in Erlenmeyer flask, stir 30min, with the centrifugal 10min of mixture 4000r, precipitation is flushing repeatedly, is water white transparency until supernatant liquor, and collecting precipitation is standby.
(3) enzymolysis of regeneration cornstalk
The 150mg precipitation is transferred to the 50ml triangular flask fully, add 30ml, the acetate buffer solution of 50mmol pH 4.8, add the cellulase (11000U/g) of 90mg again, be put in the shaking water bath pot 50 ℃, carry out enzyme digestion reaction 12h under the 100rpm, centrifugal 5-20min separates not degrading maize stalk and enzymolysis solution under the 4000-10000rpm condition, gets enzymolysis solution and measures concentration of reduced sugar with the DNS method, calculates the reducing sugar yield.
According to bibliographical information cornstalk cellulose 37.68%, hemicellulose 24.58%.Main hydrolysis is hemicellulose and Mierocrystalline cellulose during enzymolysis, 62.26% of this two portions ingredients constitute cornstalk weight.
Experimental result is seen Fig. 1.Test-results shows that the amount of cornstalk in ionic liquid is few more, and pre-treatment is abundant more, and then the reducing sugar yield of back enzymic hydrolysis is high more.
(1) pre-treatment of cornstalk
100 ℃, under the 500rpm magnetic agitation rotating speed, place 50ml to fill the triangular flask of ionic liquid (AMIM) Cl of 5g, pre-treatment 2h the cornstalk of 150mg different meshes (40-60 order, 60-80 order more than 80 orders, mix the order number).
(2) regeneration of cornstalk
Add 5 times of deionized water regeneration to the ionic liquid volume in the cornstalk ionic liquid mixed solution after above processing, stir 30min, pour mixture into the B suction filtration, filter residue washes repeatedly, is water white transparency until filtrate, collects filter residue and filtrate for later use.
(3) ion liquid distillation is reclaimed
The filtrate of collecting is placed matrass, 130 ℃ of temperature down and-0.09～-vacuum degree condition of 0.1Mpa under the water of underpressure distillation removal and ionic liquid blend, be distilled to till the not water outlet the remaining ionic liquid that is recovery in the matrass.The used cooling fluid of prolong is the cooling fluid below 10 ℃ in the distillation, connects in the preceding system of vacuum pump to install a cold-trap additional, and cold-trap places the vacuum flask that fills the cooled with liquid nitrogen agent.The ionic liquid rate of recovery is about 98%.It is respond well that the ionic liquid of this recovery is used to handle cornstalk.
(4) enzymolysis of regeneration cornstalk
The filter residue of 150mg and the cornstalk powder of untreated different meshes are transferred to the 50ml triangular flask fully, add 30ml, the acetate buffer solution of 50mmol pH 4.8, add the cellulase (11U/mg) of 90mg again, be put in the shaking water bath pot 50 ℃, carry out enzyme digestion reaction 12h under the 100rpm, centrifugal 5-20min separates not degrading maize stalk and enzymolysis solution under the 4000-10000rpm condition, gets enzymolysis solution and measures concentration of reduced sugar with the DNS method, calculates the reducing sugar yield.Experimental result sees Table 1.
The different meshes of table 1 cornstalk is to the influence of ion liquid pretreating effect
Test-results shows that the order number of cornstalk is different influential to pretreating effect, and wherein 40-60 purpose effects of pretreatment is best, and the reducing sugar yield of back enzymolysis is the highest, but the enzymolysis yield with mix being more or less the same of order number.In order to save operation, select stepless mixing order number for use.
(1) pre-treatment of cornstalk
With 150mg 40-60 purpose cornstalk at 90 ℃, 100 ℃, 110 ℃, place ionic liquid (AMIM) Cl of 5g under 120 ℃ of four temperature, under the 500rpm rotating speed magnetic agitation, handle 4,6,8,10 and 12h for 90 ℃; Handle 1,1.5,2,2.5 and 3h for 100 ℃; Handle 20,40,60,80 and 100min for 110 ℃; Handle 5,10,15,20,30min for 120 ℃.
(2) regeneration of cornstalk
The deionized water that adds 5 times of volumes stirs 30min in Erlenmeyer flask, and with the centrifugal 10min of mixture 4000rpm, precipitation is flushing repeatedly, is water white transparency until supernatant liquor, and collecting precipitation is standby.
(3) enzymolysis of regeneration cornstalk
The 150mg precipitation is transferred to the 50ml triangular flask fully, add 30ml, the acetate buffer solution of 50mmol pH 4.8, add the cellulase (11U/mg) of 90mg again, be put in the shaking water bath pot 50 ℃, carry out enzyme digestion reaction 6h under the 100rpm, reaction finishes back centrifugal 5-20min under the 4000-10000rpm condition and separates not degrading maize stalk and enzymolysis solution, gets enzymolysis solution and measures concentration of reduced sugar with the DNS method, calculates the reducing sugar yield.Experimental result is seen Fig. 2.
Test-results shows that temperature is low more, and the time that reaches optimum pretreating effect is long more, and temperature is high more, and the time that reaches optimum pretreating effect is short more.And the optimum pretreating effect under each temperature (enzymolysis reducing sugar yield) is approaching.Wherein handle 1h for 110 ℃, the reducing sugar yield can reach 49.9%.
(1) pre-treatment of cornstalk
At 110 ℃, ionic liquid (AMIM) Cl that is dissolved in 5g under the condition of 500rpm handles 1h with the cornstalk of 150mg mixing order number.
(2) regeneration of cornstalk
The deionized water that adds 5 times of volumes stirs 30min in Erlenmeyer flask, and with the centrifugal 10min of mixture 4000r, precipitation is flushing repeatedly, is water white transparency until supernatant liquor, and collecting precipitation is standby.
(3) enzymolysis of regeneration cornstalk
The 150mg precipitation is transferred to the 50ml triangular flask fully, add 30ml, the acetate buffer solution of 50mmol pH 4.8 adds the cellulase (11U/mg) of 90mg again, is put in the shaking water bath pot, 50 ℃, 100rpm carries out enzyme digestion reaction, and (be respectively 2,6,12,24,46,58h) draws reaction solution at regular intervals, and centrifugal 5-20min separates not degrading maize stalk and enzymolysis solution under the 4000-10000rpm condition, get enzymolysis solution and measure concentration of reduced sugar, calculate the reducing sugar yield with the DNS method.
Experimental result is seen Fig. 3.The reducing sugar yield reaches 65.7% behind the enzymolysis 58h, and the cornstalk enzymolysis reducing sugar yield of not handling through ionic liquid has only 22.2%.
(1) pre-treatment of cornstalk
At 110 ℃, ionic liquid (AMIM) Cl, (BMIM) Cl or the LiCl/DMAc that are dissolved in 5g under the condition of 500rpm handle 1h with the cornstalk of 150mg mixing order number, and with the centrifugal 10min of cornstalk ionic liquid mixed solution 4000rpm, it is standby to get supernatant liquor.
(2) regeneration of solute in the ion supernatant liquor
Add 5 times of deionized waters to the supernatant liquor volume in Erlenmeyer flask in the supernatant liquor of above acquisition, stir 30min, with the centrifugal 10min of mixture 4000rpm, precipitation is flushing repeatedly, is water white transparency until supernatant liquor, and it is standby to collect the regeneration flocks.
(3) Infrared Characterization
The flocks material lyophilize of will regenerating, with model be NEXUS-670 infrared-the Raman spectrometer sweep measuring.Experimental result is seen Fig. 4.Be dissolved in the ionic liquid cornstalk regeneration flocculent substance and learn it mainly is Mierocrystalline cellulose through infrared analysis, show that ionic liquid can go out the Mierocrystalline cellulose extracting in the cornstalk, increase cornstalk and organize the space, not only improved the accessibility of enzyme, and the Mierocrystalline cellulose of stripping facile hydrolysis more, thereby quicken the carrying out of enzyme digestion reaction.
(1) pre-treatment of cornstalk
At 110 ℃, ionic liquid (BMIM) Cl that is dissolved in 5g under the condition of 500rpm handles 1h with the cornstalk of 150mg mixing order number.
(2) regeneration of cornstalk
The deionized water that adds 5 times of volumes stirs 30min in Erlenmeyer flask, and with the centrifugal 10min of mixture 4000rpm, precipitation is flushing repeatedly, is water white transparency until supernatant liquor, and collecting precipitation is standby.
(3) morphologic observation
The lyophilize of the cornstalk of will regenerating precipitation is observed its form with model for the TM-1000 scanning electron microscope.Experimental result is seen Fig. 5.Photo shows that the effect that the cornstalk surface does not have to puncture occurs, but has produced some cracks after handling, and has increased the chance that enzyme infiltrates.
Embodiment 7
(1) pre-treatment of cornstalk
At 110 ℃, the ionic liquid that is dissolved in 5g under the condition of 500rpm is handled 1h with the cornstalk of 150mg mixing order number.
(2) regeneration of cornstalk
Add 5 times of deionized waters to the ionic liquid volume in the cornstalk ionic liquid mixed solution after above processing in Erlenmeyer flask, stir 30min, with the centrifugal 10min of mixture 4000rpm, precipitation is flushing repeatedly, is water white transparency until supernatant liquor, and collecting precipitation is standby.
(3) degree of crystallinity is measured
The cornstalk lyophilize of will regenerating is the D/Max-2550PCX-ray diffraction determination with model.Experimental result is seen Fig. 6.The degree of crystallinity of handling back regeneration cornstalk is 35.32%, and the degree of crystallinity of the cornstalk that is untreated is 44.83%, and the degree of crystallinity of handling the back cornstalk has descended 9.51%.The decline of degree of crystallinity helps the carrying out of enzyme digestion reaction.
(1) pre-treatment of cornstalk
At 110 ℃, the ionic liquid that is dissolved in 150g under the condition of 500rpm is handled 1h with the cornstalk of 5g mixing order number.
(2) regeneration of cornstalk
The deionized water that adds 5 times of volumes stirs 30min in Erlenmeyer flask, and with the centrifugal 10min of mixture 4000rpm, precipitation is flushing repeatedly, is water white transparency until supernatant liquor, and collecting precipitation is standby.
(3) enzymolysis of regeneration cornstalk
The 150mg precipitation is transferred to the 250ml triangular flask fully, add 60ml, the acetate buffer solution of 50mmol pH 4.8, add the cellulase (11U/mg) of 3g again, be put in the shaking water bath pot 50 ℃, 100rpm carries out enzyme digestion reaction 60h, centrifugation cornstalk residue and enzymolysis solution under the 4000rpm condition are collected enzymolysis solution, survey its concentration of reduced sugar with DNS.
(4) detoxification of enzymolysis solution
Ca (OH)
2Transfer enzymolysis solution pH to 10.0, detoxification reaction 12h in 30 ℃ of water-baths filters and finely tunes pH5.0 then, add under the gac room temperature with respect to enzymolysis solution quality 1% to react 5-10min, and, filter and obtain enzymolysis solution;
(5) preparation of substratum preparation and bacteria cellulose
With above-mentioned detoxification enzymolysis solution as carbon source, the Tryptones that adds the yeast extract and 0.5% (w/v) of 0.3% (w/v) again, be mixed with substratum, 121 ℃ of sterilization 20min, inoculum size with 6% adds bacillus aceticus, gluconobacter suboxydans or tea fungus, 30 ℃ leave standstill to cultivate and can obtain bacteria cellulose film in 12 days, and wherein blank is for being solvent with water and hac buffer respectively, and the glucose of adding same concentrations is as carbon source.
(6) dry weight of film
The bacteria cellulose that cultivation obtains contains a large amount of impurity, in order to remove these impurity, at first adopts clear water to wash away partial impurities, then all is transferred to through the G of constant weight in advance
3In the glass hessian crucible, in 105 ℃ ± 0.1 baking oven, dry to constant weight, the balance calculating of weighing by analysis, the over dry that promptly obtains bacteria cellulose is heavy.Test-results is seen Fig. 7.The bacteria cellulose output that the detoxification enzymolysis solution is cultivated is than being that the bacteria cellulose output that obtains of carbon source (water and buffering solution are reagent) is low with glucose.
Embodiment 9
The processing of bacteria cellulose film: fermention medium leaves standstill to be cultivated after 18 days, take out bacteria cellulose film, distilled water flushing repeatedly after, place 1% NaOH solution, 80 ℃ of insulation 120min, remove remaining thalline and substratum, wash repeatedly to neutrality with deionized water, this moment, bacteria cellulose film became translucent oyster white.
Infrared Characterization: will handle standby bacteria cellulose film lyophilize, dried sample obtains the sample collection of illustrative plates through the KBr pressed disc method through the scanning of fourier infrared instrument.Experimental result is seen Fig. 8.The results of FT-IR shows that the bacteria cellulose that enzymolysis solution is cultivated is a pure cellulose.
The processing of bacteria cellulose film: fermention medium leaves standstill to be cultivated after 18 days, take out bacteria cellulose film, distilled water flushing repeatedly after, the NaOH solution of adding 1%, 80 ℃ of insulation 120min, remove remaining thalline and substratum, wash repeatedly to neutrality with deionized water, this moment, bacteria cellulose film became translucent oyster white.
X-ray diffraction characterizes: will handle standby bacteria cellulose lyophilize.Degree of crystallinity test condition: power: 40kV, 300mA, goes on foot wide: 0.02 ° by sweep limit 5-60 °.Experimental result is seen Fig. 9.X-ray diffraction obtains the bacteria cellulose degree of crystallinity that enzymolysis solution is cultivated, and is solvent than water and damping fluid respectively, and glucose is the bacteria cellulose degree of crystallinity height that carbon source is cultivated.
Embodiment 11
(1) pre-treatment of cornstalk
At 110 ℃, the ionic liquid that is dissolved in 150g under the condition of 500rpm is handled 1h with the cornstalk of 5g mixing order number.
(2) regeneration of cornstalk
The deionized water that adds 5 times of volumes stirs 30min in Erlenmeyer flask, and with the centrifugal 10min of mixture 4000rpm, precipitation is flushing repeatedly, is water white transparency until supernatant liquor, and collecting precipitation is standby.
(3) enzymolysis of regeneration cornstalk
The 150mg precipitation is transferred to the 250ml triangular flask fully, add 60ml, the acetate buffer solution of 50mmol pH 4.8, add the cellulase (11U/mg) of 3g again, be put in the shaking water bath pot 50 ℃, 100rpm carries out enzyme digestion reaction 60h, centrifugation cornstalk residue and enzymolysis solution under the 4000rpm condition are collected enzymolysis solution, survey its concentration of reduced sugar with DNS.
(4) preparation of substratum preparation and bacteria cellulose
With above-mentioned enzymolysis solution as carbon source, the Tryptones that adds the yeast extract and 0.5% (w/v) of 0.3% (w/v) again, be mixed with substratum, 121 ℃ of sterilization 20min, inoculum size with 10% adds bacillus aceticus, gluconobacter suboxydans or tea fungus, 30 ℃ leave standstill to cultivate and can obtain bacteria cellulose film in 28 days, and wherein blank is for being solvent with water and hac buffer respectively, and the glucose of adding same concentrations is as carbon source.
(5) dry weight of film
The bacteria cellulose that cultivation obtains contains a large amount of impurity, in order to remove these impurity, at first adopts clear water to wash away partial impurities, then all is transferred to through the G of constant weight in advance
3In the glass hessian crucible, in 105 ℃ ± 0.1 baking oven, dry to constant weight, the balance calculating of weighing by analysis, the over dry that promptly obtains bacteria cellulose is heavy.
Claims (6)
1. method of utilizing cornstalk to prepare bacteria cellulose comprises:
(1) ionic liquid pretreatment of cornstalk, regeneration and enzyme digestion reaction
The ionic liquid pretreatment of cornstalk: after cornstalk pulverized, join in the ionic liquid, handle 15min-21h in 90 ℃-120 ℃, leave standstill cooling with the ratio of 1%-15% (w/w);
Ionic liquid/cornstalk mixed solution is regenerated: the deionized water of adding and ionic liquid 5-10 times volume, stir regeneration, and centrifugal then or filtration, precipitation or the filter residue that obtains with deionized water wash is extremely colourless until supernatant or filtrate again;
Simultaneously, the filtrate that the centrifugal supernatant liquor that obtains or filtration are obtained temperature 100-170 ℃ of following air distillation or temperature 50-150 ℃ and-0.09～-vacuum degree condition of 0.1Mpa under underpressure distillation, remove water with the ionic liquid blend, be distilled to till the not water outlet, reclaim and promptly get ionic liquid;
Enzymolysis: adding buffered soln to solid-to-liquid ratio in above-mentioned precipitation or filter residue is 0.005g: 1ml～1g: 10ml, adding cellulase to final concentration again is 5-600U/mL, regulate pH to 4.0-5.5, under 40-60 ℃ of condition, react 1-60h, centrifugal or filtering separation is degrading maize stalk and enzymolysis solution not, with enzymolysis solution in 4 ℃ of refrigerations;
(2) enzymolysis solution prepares bacteria cellulose
With above-mentioned enzymolysis solution directly as the cellulosic substratum of culturing bacterium, add the yeast extract paste of relative enzymolysis solution 0.1-1% (w/v) and the Tryptones of 0.1-0.5% (w/v), be mixed with substratum, add the 6%-10% bacteria cellulose again and produce the seed liquor of bacterium, 25-30 ℃ leaves standstill cultivation and obtained bacteria cellulose in 8-28 days;
Or the enzymolysis solution that step (1) is made is with Ca (OH)
2Transfer enzymolysis solution pH to 9.5-11.0, detoxification reaction 12h-24h in 25 ℃ of-60 ℃ of water-baths filters and finely tunes pH4.5-5.0 then, adds stirring reaction 5-10min under the gac room temperature, filters the enzymolysis solution that obtains detoxification treatment; With the enzymolysis solution of detoxification treatment as the cellulosic substratum of culturing bacterium, add the yeast extract paste of relative enzymolysis solution 0.1-1% (w/v) and the Tryptones of 0.1-0.5% (w/v), be mixed with substratum, add the 6%-10% bacteria cellulose again and produce the seed liquor of bacterium, 25-30 ℃ leaves standstill cultivation and obtained bacteria cellulose in 8-28 days.
2. a kind of method of utilizing cornstalk to prepare bacteria cellulose according to claim 1 is characterized in that: the ionic liquid in the described step (1) is (AMIM) Cl, (BMIM) Cl or LiCl/DMAc.
3. a kind of method of utilizing cornstalk to prepare bacteria cellulose according to claim 1 is characterized in that: it is more than 40 orders that the cornstalk in the described step (1) is pulverized number, and concentration is 3% (w/w) in ionic liquid, handles 1h for 110 ℃ in temperature.
4. a kind of method of utilizing cornstalk to prepare bacteria cellulose according to claim 1 is characterized in that: the centrifugal condition in the described step (1) is speed 4000-10000rpm, time 5-20min.
5. a kind of method of utilizing cornstalk to prepare bacteria cellulose according to claim 1 is characterized in that: the add-on of the gac in the described step (2) is the 1%-6% with respect to the enzymolysis solution quality.
6. a kind of method of utilizing cornstalk to prepare bacteria cellulose according to claim 1 is characterized in that: it is acetic acid Pseudomonas (Acetobacter sp.) that the bacteria cellulose in the described step (2) is produced bacterial strain, gluconobacter suboxydans belongs to (Gluconobacter sp.), glyconic acid genus acetobacter (Gluconacetobacter sp.), rhizobium (Rhizobium sp.), Sarcina (Sarcina sp.), Rhodopseudomonas (Pseudomounas sp.), achromobacter (Achromobactersp.), Alcaligenes (Alcaligenes sp.), aerobacter (Aerobacter sp.), Azotobacter (Azotobacter sp.), Agrobacterium (Agrobacterium sp.), pseudomonas cepacia (Seudomonas cepacia), campylobacter jejuni (Campylobacter jejuni) or tea fungus (kombucha); Wherein preferred strain is acetobacter xylinum (Acetobacter xylinum) or tea fungus.
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102994588A (en) * | 2012-12-26 | 2013-03-27 | 东华大学 | Cultural method for continuous thickening of bacterial cellulose |
| CN104031956A (en) * | 2014-06-05 | 2014-09-10 | 陕西科技大学 | Bacterial cellulose fermentation medium made from apple pomace and method for producing bacterial cellulose by utilizing medium |
| WO2016029431A1 (en) * | 2014-08-29 | 2016-03-03 | Shanghai Zhiyi Information Technology Ltd | Processing of plant material into bacterial feedstock |
| CN106047960A (en) * | 2016-07-22 | 2016-10-26 | 南京理工大学 | Optimizing method for ferment-producing bacterial cellulose by using gluconic acid as sole carbon source |
| CN106191162A (en) * | 2016-07-14 | 2016-12-07 | 华南理工大学 | A kind of Bacterial cellulose and environment-friendly preparation method thereof thereof and application |
| CN108315382A (en) * | 2018-04-27 | 2018-07-24 | 江苏省农业科学院 | A method of preparing bacteria cellulose using bean curd yellow pulp water |
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| CN101781667A (en) * | 2009-03-10 | 2010-07-21 | 东华大学 | Method for producing bacterial cellulose with wheat straws/maize straws |
| CN101787381A (en) * | 2009-09-14 | 2010-07-28 | 哈尔滨工业大学 | Method for preparing fermentable reducing sugar by adopting ionic liquids to treat cellulose biomass |
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Patent Citations (2)
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| CN101781667A (en) * | 2009-03-10 | 2010-07-21 | 东华大学 | Method for producing bacterial cellulose with wheat straws/maize straws |
| CN101787381A (en) * | 2009-09-14 | 2010-07-28 | 哈尔滨工业大学 | Method for preparing fermentable reducing sugar by adopting ionic liquids to treat cellulose biomass |
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| CN102994588A (en) * | 2012-12-26 | 2013-03-27 | 东华大学 | Cultural method for continuous thickening of bacterial cellulose |
| CN102994588B (en) * | 2012-12-26 | 2014-03-19 | 东华大学 | Cultural method for continuous thickening of bacterial cellulose |
| CN104031956A (en) * | 2014-06-05 | 2014-09-10 | 陕西科技大学 | Bacterial cellulose fermentation medium made from apple pomace and method for producing bacterial cellulose by utilizing medium |
| WO2016029431A1 (en) * | 2014-08-29 | 2016-03-03 | Shanghai Zhiyi Information Technology Ltd | Processing of plant material into bacterial feedstock |
| CN106191162A (en) * | 2016-07-14 | 2016-12-07 | 华南理工大学 | A kind of Bacterial cellulose and environment-friendly preparation method thereof thereof and application |
| CN106047960A (en) * | 2016-07-22 | 2016-10-26 | 南京理工大学 | Optimizing method for ferment-producing bacterial cellulose by using gluconic acid as sole carbon source |
| CN106047960B (en) * | 2016-07-22 | 2019-06-04 | 南京理工大学 | It is a kind of to make the optimization method that sole carbon source carries out fermented-producing bacteria cellulose with gluconic acid |
| CN108315382A (en) * | 2018-04-27 | 2018-07-24 | 江苏省农业科学院 | A method of preparing bacteria cellulose using bean curd yellow pulp water |
| CN109107551A (en) * | 2018-09-18 | 2019-01-01 | 浙江蓝德能源科技发展有限公司 | A kind of device and method using ionic liquid regenerated carbon |
| CN111057722A (en) * | 2019-12-24 | 2020-04-24 | 刘洋 | Method for fermenting corn straws by using black tea leavening agent |
| CN113481256A (en) * | 2021-08-10 | 2021-10-08 | 武汉纺织大学 | Method for producing bacterial cellulose by jasmine flower culture |
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Application publication date: 20110511 |