CN101985642A - Method for preparing bacterial cellulose by using straw - Google Patents
Method for preparing bacterial cellulose by using straw Download PDFInfo
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- CN101985642A CN101985642A CN 201010580814 CN201010580814A CN101985642A CN 101985642 A CN101985642 A CN 101985642A CN 201010580814 CN201010580814 CN 201010580814 CN 201010580814 A CN201010580814 A CN 201010580814A CN 101985642 A CN101985642 A CN 101985642A
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Abstract
The invention relates to a method for preparing bacterial cellulose by using straw, which comprises the following steps of: preprocessing the straw for 15min to 21h in ionized water at the temperature of 90 to 120 DEG C, adding de-ionized water, stirring for regeneration, adding cellulose for enzymolysis and obtaining enzymolytic liquid; and performing detoxification treatment on the enzymolytic liquid, adding yeast extract and pancpin into the processed enzymolytic liquid to prepare a culture medium for culturing the bacterial cellulose, adding seed liquid of a bacterial cellulose production strain, standing the mixed solution for 8 to 28 days at the temperature of 25 to 30 DEG C and obtaining the bacterial cellulose. In the invention, usually discarded or burned straw which is the side product of the crop is used as a raw material and a cheap carbon source is developed, so that environment pollution is reduced, high value-added products can be produced and good economical benefit of relative industries can be created.
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 rice straw.
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---rice straw is produced the method for bacteria cellulose.
Lignocellulose stalks such as rice straw are the byproducts 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 rice straw 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 can be used to dissolve stalk.At present, there is research to report, handles the back enzymatic hydrolyzation and obviously improve with ionic liquid pre-treatment rice straw, timber, bagasse, cotton stalk.Be used for lignocellulose dissolved ionic liquid [EPy] Br and [AMIM] [HCO2] etc. are arranged.(AMIM) Cl does not see play-by-play it is used for the dissolving of stalk.(AMIM) Cl is adopted in this experiment and (BMIM) Cl and LiCl/DMAc handle stalk, select for use (AMIM) Cl to be because (AMIM) the Cl synthesis temperature is low, nontoxic, pretreatment temperature is low and the time short, therefore characteristics such as rate of recovery height select this kind solvent pre-treatment rice straw for use.
Summary of the invention
Technical problem to be solved by this invention provides a kind of method of utilizing rice straw to prepare bacteria cellulose, the farm crop by product of this method to abandon usually or to burn---rice straw 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 rice straw to prepare bacteria cellulose of the present invention comprises:
(1) ionic liquid pretreatment of rice straw, regeneration and enzyme digestion reaction
The ionic liquid pretreatment of rice straw: after rice straw 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/rice straw 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 do not degrade rice straw and enzymolysis solution, 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 pH to 9.5-11.0, detoxification reaction 12h-24h in 25 ℃ of-60 ℃ of water-baths filters and finely tunes pH to 4.5-5.0 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 rice straw in the described step (1) is pulverized number, and the concentration in ionic liquid is 3% (w/w), and rice straw is handled 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 (Achromobacter sp.), 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 rice straw, can improve enzymatic hydrolyzation, and preparation contains the enzymolysis solution of high concentration sugar, and the reason that the rice straw 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 rice straw 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 rice straw; The X-ray diffraction analysis finds that the degree of crystallinity of the rice straw 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 the rice straw surface after ionic liquid pretreatment, and the projection of some rice straw tissue surfaces is punched, and hole and crack occur, is convenient to the intrusion of enzyme, has improved the accessibility of enzyme.These all are the reasons that rice straw enzymolysis yield improves after the explanation pre-treatment.
The bacteria cellulose film output height (7.90g/L) that the detoxification enzymolysis solution is cultivated, and be carbon source with glucose, 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 rice straw 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 rice straw enzymolysis solution is cultivated the output height of the bacteria cellulose film dry weight that obtains, and is carbon source with glucose, and buffered soln is that the absolute pulling force of cellulose membrane cultivated of solvent is the highest.
Beneficial effect
The present invention is by the ionic liquid pretreatment rice straw, 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; Rice straw generally all is to abandon or burn as the byproduct of farm crop in addition, 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 rice straw is to the influence of pretreating effect in Fig. 1 ionic liquid.
The order number of Fig. 2 rice straw powder is to the influence of ionic liquid pretreatment effect, and A is the enzymic hydrolysis result of not pre-treatment rice straw, and B is the enzymic hydrolysis result of the rice straw of ionic liquid pretreatment.
Fig. 3 pretreatment time and temperature are to the influence of pretreating effect.
Enzymolysis graphic representation under the optimum pretreatment condition of Fig. 4.
The infrared spectrum characterization of the flocculent substance that Fig. 5 rice straw regeneration back forms.
The stereoscan photograph of rice straw before and after Fig. 6 ionic liquid is handled, left side figure are before handling, and right figure is after handling.
The x-ray diffraction pattern of rice straw before and after Fig. 7 ionic liquid is handled.
The output of the bacteria cellulose film that Fig. 8 detoxification enzymolysis solution is cultivated.
The bacteria cellulose that Fig. 9 enzymolysis solution is cultivated and the infrared spectra spectrogram of Microcrystalline Cellulose.
The x-ray diffraction pattern of the bacteria cellulose that Figure 10 enzymolysis solution is cultivated.
The tension intensity of the bacteria cellulose film that Figure 11 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 rice straw
100 ℃, under the 500rpm magnetic agitation rotating speed, 150mg 40-60 purpose rice straw is placed ionic liquid (AMIM) Cl of 1.5g, 3g, 5g, pre-treatment 2h respectively.
(2) regeneration of rice straw
Add 5 times of deionized water regeneration in the rice straw ionic liquid mixed solution after above processing to the ionic liquid volume, stir 30min, with mixture centrifugal 10min under the 4000rpm rotating speed, precipitation is flushing repeatedly, until supernatant liquor is water white transparency, and collecting precipitation is standby.
(3) enzymolysis of regeneration rice straw
The 150mg precipitation is transferred in the 50ml triangular flask fully, add 30ml, the acetate buffer solution of 50mmol pH 4.8 adds the cellulase (11000U/g) of 90mg again, is put in the shaking water bath pot, 50 ℃, carry out enzyme digestion reaction under the 100rpm, draw reaction solution at regular intervals, centrifugal 5-20min separates do not degrade rice straw 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.
According to bibliographical information rice straw cellulose 36.20%, hemicellulose 18.06%.Main hydrolysis is hemicellulose and Mierocrystalline cellulose during enzymolysis, 54.26% of this two portions ingredients constitute rice straw weight.
Experimental result is seen Fig. 1.The result shows that the add-on of rice straw in ionic liquid is few more, and pre-treatment is just abundant more, and then the reducing sugar yield of back enzymic hydrolysis is just high more.
Embodiment 2
(1) pre-treatment of rice straw
90 ℃, under the 500rpm magnetic agitation rotating speed, place 50ml to fill the triangular flask of ionic liquid (AMIM) Cl of 5g, pre-treatment 10h the rice straw of 150mg different meshes (40-60 order, 60-80 order, 80 orders above, mix order number).
(2) regeneration of rice straw
Add 5 times of deionized waters to the ionic liquid volume in the rice straw ionic liquid mixed solution after above processing in Erlenmeyer flask, stir 30min, pour mixture into the B suction filtration, filter residue washes repeatedly, until filtrate is water white transparency, 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 rice straw.
(4) enzymolysis of regeneration rice straw
The filter residue of 150mg and the rice straw powder of untreated different meshes are transferred to the 50ml triangular flask fully, add 30ml, the acetate buffer solution of 50mmol pH 4.8, the cellulase (11U/mg) that adds 90mg again, be put in the shaking water bath pot, 50 ℃, carry out enzyme digestion reaction under the 100rpm, (be respectively 2,6,10,24h) draws reaction solution at regular intervals, centrifugal 5-20min separates do not degrade rice straw 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. 2.
Test-results shows that the order number of rice straw has certain influence to pretreating effect, and wherein 40-60 purpose pretreating effect is best, and the reducing sugar yield of back enzymic hydrolysis is the highest.The enzymic hydrolysis result of rice straw of being untreated is that the above reducing sugar yield of 80 orders is the highest.
Embodiment 3
(1) pre-treatment of rice straw
150mg 40-60 purpose rice straw is placed ionic liquid (AMIM) Cl of 5g respectively under 90 ℃, 100 ℃, 110 ℃ and 120 ℃ of four temperature, under the 500rpm rotating speed magnetic agitation, handle 6,10,12,15 and 18h under 90 ℃ of temperature, processing 1,2,3,4 and 5h under 100 ℃, handle down 0.5,1,1.5 and 2h for 110 ℃, handle 15,30,45,60 and 75min down for 120 ℃.
(2) regeneration of rice straw
Add 5 times in the rice straw ionic liquid mixed solution after above processing to the deionized water of ionic liquid volume, stir 30min, with mixture centrifugal 10min under the 4000rpm rotating speed, precipitation is flushing repeatedly, is water white transparency until supernatant liquor, and collecting precipitation is standby.
(3) enzymolysis of regeneration rice straw
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 do not degrade rice straw 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. 3.
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 best pretreating effect under each temperature (enzymolysis reducing sugar yield) is approaching.Wherein, handle 1h for 110 ℃, the reducing sugar yield can reach 41.7%.
(1) pre-treatment of rice straw
At 110 ℃, ionic liquid (AMIM) Cl that is dissolved in 5g under the condition of 500rpm handles 1h with 150mg 40-60 purpose rice straw.
(2) regeneration of rice straw
Add 5 times in the rice straw ionic liquid mixed solution after above processing to the deionized water of ionic liquid volume, stir 30min, with mixture centrifugal 10min under the 4000rpm rotating speed, precipitation is flushing repeatedly, is water white transparency until supernatant liquor, and collecting precipitation is standby.
(3) enzymolysis of regeneration rice straw
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, draws reaction solution at regular intervals, and centrifugal 5-20min separates do not degrade rice straw 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. 4.The reducing sugar yield reaches 56.1% behind the enzymolysis 58h, and the rice straw enzymolysis reducing sugar yield of not handling through ionic liquid has only 29.8%.
(1) pre-treatment of rice straw
At 110 ℃, place ionic liquid (AMIM) Cl, (BMIM) Cl of 5g or LiCl/DMAc to handle 1h under the condition of 500rpm 150mg40-60 purpose rice straw, with the centrifugal 10min of rice straw ionic liquid mixed solution 4000rpm, it is standby to get supernatant liquor.
(2) regeneration of solute in the ionic liquid supernatant liquor
Add 5 times to the deionized water of supernatant liquor volume in the supernatant liquor of above acquisition, stir 30min, with mixture centrifugal 10min under the 4000rpm rotating speed, precipitation is flushing repeatedly, is water white transparency until supernatant liquor, and collection regeneration flocks is standby.
(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. 5.Be dissolved in the ionic liquid rice straw 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 rice straw, increase rice straw 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 rice straw
At 110 ℃, ionic liquid (BMIM) Cl that is dissolved in 5g under the condition of 500rpm handles 1h with 150mg 40-60 purpose rice straw.
(2) regeneration of rice straw
Add 5 times in the rice straw ionic liquid mixed solution after above processing to the deionized water of ionic liquid volume, stir 30min, with mixture centrifugal 10min under the 4000rpm rotating speed, precipitation is flushing repeatedly, is water white transparency until supernatant liquor, and collecting precipitation is standby.
(3) morphologic observation
The lyophilize of the rice straw of will regenerating precipitation is observed its form with model for the TM-1000 scanning electron microscope.Experimental result is seen Fig. 6.Photo shows that the white protruding of rice straw surface breaks, and increased the chance that enzyme infiltrates after ionic liquid is handled.
Embodiment 7
(1) pre-treatment of rice straw
At 110 ℃, place ionic liquid (AMIM) Cl of 5g to handle 1h under the condition of 500rpm 150mg 40-60 purpose rice straw.
(2) regeneration of rice straw
Add 5 times in the rice straw ionic liquid mixed solution after above processing to the deionized water of ionic liquid volume, stir 30min, with mixture centrifugal 10min under the 4000rpm rotating speed, precipitation is flushing repeatedly, is water white transparency until supernatant liquor, and collecting precipitation is standby.
(3) degree of crystallinity is measured
The rice straw lyophilize of will regenerating is the D/Max-2550PCX-ray diffraction determination with model.Experimental result is seen Fig. 7.The degree of crystallinity of handling back regeneration rice straw is 44.78%, and the degree of crystallinity of the rice straw that is untreated is 50.26%, and the degree of crystallinity of handling the back rice straw has descended 5.48%.The decline of degree of crystallinity helps the carrying out of enzyme digestion reaction.
(1) pre-treatment of rice straw
At 110 ℃, the ionic liquid that is dissolved in 150g under the condition of 500rpm is handled 1h with the rice straw of 5g mixing order number.
(2) regeneration of rice straw
Add 5 times in the rice straw ionic liquid mixed solution after above processing to the deionized water of ionic liquid volume, stir 30min, with mixture centrifugal 10min under the 4000rpm rotating speed, precipitation is flushing repeatedly, is water white transparency until supernatant liquor, and collecting precipitation is standby.
(3) enzymolysis of regeneration rice straw
The 150mg precipitation is transferred to the 250ml triangular flask fully, add 60ml, the acetate buffer solution of 50mmol pH 4.8, the cellulase (11U/mg) that adds 3g again, be put in the shaking water bath pot, 50 ℃, carry out enzyme digestion reaction 60h under the 100rpm, centrifugal 5-20min separates rice straw residue and enzymolysis solution under the 4000rpm condition, gets enzymolysis solution and measures concentration of reduced sugar with the DNS method.
(4) enzymolysis solution detoxification
With Ca (OH)
2Transfer enzymolysis solution pH value to 10.0, detoxification reaction 12h in 30 ℃ of water-baths filters and finely tunes pH5.0 then, adds under the gac room temperature with respect to enzymolysis solution quality 1% and reacts 5-10min, filters and obtains 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. 8.
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 high with glucose, so the carbon source that obtains by this method of ionic liquid processing rice straw is a kind of high-quality carbon source.
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, 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.
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. 9.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; Sweep limit 5-60 °; Go on foot wide: 0.02 °.Experimental result is seen Figure 10.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
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.
The bacterial fibers pulling force characterizes: will handle standby bacteria cellulose film scissors is the strip of 3cm * 1cm, four in each sample is done parallel test, with universal material test machine test tension intensity, the distance of setting between two anchor clamps is 3cm, and speed is 100mm/min.The pulling force of the absolute pulling force of BC film=directly record.Test-results is seen Figure 11.The pulling force of the bacteria cellulose film that enzymolysis solution is cultivated is the low of carbon source than damping fluid for solvent glucose, is the pulling force height that carbon source is cultivated than water for solvent glucose.
(1) pre-treatment of rice straw
At 110 ℃, the ionic liquid that is dissolved in 150g under the condition of 500rpm is handled 1h with the rice straw of 5g mixing order number.
(2) regeneration of rice straw
Add 5 times in the rice straw ionic liquid mixed solution after above processing to the deionized water of ionic liquid volume, stir 30min, with mixture centrifugal 10min under the 4000rpm rotating speed, precipitation is flushing repeatedly, is water white transparency until supernatant liquor, and collecting precipitation is standby.
(3) enzymolysis of regeneration rice straw
The 150mg precipitation is transferred to the 250ml triangular flask fully, add 60ml, the acetate buffer solution of 50mmol pH 4.8, the cellulase (11U/mg) that adds 3g again, be put in the shaking water bath pot, 50 ℃, carry out enzyme digestion reaction 60h under the 100rpm, centrifugal 5-20min separates rice straw residue and enzymolysis solution under the 4000rpm condition, gets enzymolysis solution and measures concentration of reduced sugar with the DNS method.
(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 rice straw to prepare bacteria cellulose comprises:
(1) ionic liquid pretreatment of rice straw, regeneration and enzyme digestion reaction
The ionic liquid pretreatment of rice straw: after rice straw 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/rice straw 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 do not degrade rice straw and enzymolysis solution, 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 pH to 9.5-11.0, detoxification reaction 12h-24h in 25 ℃ of-60 ℃ of water-baths filters and finely tunes pH to 4.5-5.0 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 rice straw 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 rice straw to prepare bacteria cellulose according to claim 1 is characterized in that: it is more than 40 orders that the rice straw in the described step (1) is pulverized number, and the concentration in ionic liquid is 3% (w/w), and rice straw is handled 1h for 110 ℃ in temperature.
4. a kind of method of utilizing rice straw 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 rice straw 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 rice straw 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 (Achromobacter sp.), 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 (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102392062A (en) * | 2011-11-18 | 2012-03-28 | 东华大学 | Method for preparing bacterial cellulose by using decayed fruits as raw materials |
| WO2016029431A1 (en) * | 2014-08-29 | 2016-03-03 | Shanghai Zhiyi Information Technology Ltd | Processing of plant material into bacterial feedstock |
| CN105671091A (en) * | 2016-04-08 | 2016-06-15 | 中国农业科学院棉花研究所 | Method for pretreating cotton straw with ionic liquid [Bmim]Cl |
| 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 |
| CN107586801A (en) * | 2017-10-19 | 2018-01-16 | 南京理工大学 | A kind of method that bacteria cellulose is prepared using cotton stalk |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101298620A (en) * | 2007-05-10 | 2008-11-05 | 河南科技大学 | Method for preparing ethanol by one-step fermentation after ion liquid stage treatments from native cellulose material |
| 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)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101298620A (en) * | 2007-05-10 | 2008-11-05 | 河南科技大学 | Method for preparing ethanol by one-step fermentation after ion liquid stage treatments from native cellulose material |
| 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 |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102392062A (en) * | 2011-11-18 | 2012-03-28 | 东华大学 | Method for preparing bacterial cellulose by using decayed fruits as raw materials |
| WO2016029431A1 (en) * | 2014-08-29 | 2016-03-03 | Shanghai Zhiyi Information Technology Ltd | Processing of plant material into bacterial feedstock |
| CN105671091A (en) * | 2016-04-08 | 2016-06-15 | 中国农业科学院棉花研究所 | Method for pretreating cotton straw with ionic liquid [Bmim]Cl |
| 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 |
| CN107586801A (en) * | 2017-10-19 | 2018-01-16 | 南京理工大学 | A kind of method that bacteria cellulose is prepared using cotton stalk |
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