CN101781668A - Method for producing bacterial cellulose with wheat straws/spruces - Google Patents
Method for producing bacterial cellulose with wheat straws/spruces Download PDFInfo
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Abstract
本发明涉及一种利用麦秆/云杉生产细菌纤维素的方法，包括：(1)麦秆或云杉磨碎，再用稀硫酸或盐酸浸泡，在温度90℃～240℃下反应，接着通过抽滤将渣和酸水解液分开，收集水解液备用；(2)水解液的脱毒；(3)取上述的脱毒水解液作为培养基碳源，加氮源，配成培养基，接入醋酸杆菌或葡萄糖氧化杆菌细菌在25-30℃，160-250转/分钟摇床中培养或在25-30℃培养箱内静置培养，得到细菌纤维素。本发明制备的培养基碳源质量好，价格低，适合于工业化生产。
The invention relates to a method for producing bacterial cellulose by using wheat straw/spruce, comprising: (1) grinding wheat straw or spruce, soaking in dilute sulfuric acid or hydrochloric acid, reacting at a temperature of 90°C to 240°C, and then Separate the slag and the acid hydrolyzate by suction filtration, collect the hydrolyzate for subsequent use; (2) detoxify the hydrolyzate; (3) take the above-mentioned detoxified hydrolyzate as the carbon source of the medium, add nitrogen source, and make a culture medium, The bacterial cellulose is obtained by inserting bacteria of Acetobacter or Glucobacterium Oxidum and culturing them in a shaker at 25-30°C at 160-250 rpm or statically in an incubator at 25-30°C. The medium carbon source prepared by the invention has good quality and low price, and is suitable for industrialized production.
Description
Technical field
The invention belongs to the preparation field of bacteria cellulose, particularly relate to a kind of method of utilizing straw/dragon spruce to produce bacteria cellulose.
Background technology
Bacteria cellulose (Bacterial Cellulose, abbreviation BC) is called micro organism cellulose (Microbial Cellulose) again, it is a kind of biomaterial that broad prospect of application is arranged, compare with other higher plant Mierocrystalline cellulose of occurring in nature, it has the character of many uniquenesses, comprise high purity, high-crystallinity, high-polymerization degree, high retentiveness, high-tensile, Johnson ﹠ Johnson's thing adaptability etc.Therefore, this cellulose materials has great application prospect in fields such as artificial skin and blood vessel, medical dressing, tackiness agent, stereo set tympanum, papermaking, weaving, composite membranes.Yet bacteria cellulose culture medium cost height, problem such as cellulose output and productive rate are low but are its suitability for industrialized production and the bottleneck applied.
The production of bacteria cellulose relies on microbial fermentation, and the conventional fermentation raw material that initial people produce bacteria cellulose is the depleted Sucus Cocois, but the Sucus Cocois source is limited, can't satisfy the production needs.People had developed various substratum and had prepared bacteria cellulose with culturing micro-organisms afterwards.These substratum comprise carbon source, nitrogenous source and inorganic salt in forming, and carbon source is the chief component of substratum, large usage quantity, and the consumption of nitrogenous source is less relatively.Glucose, N.F,USP MANNITOL etc. are carbon sources commonly used in the bacteria cellulose preparation, all are commercialization reagent, use these commercialization carbon sources and make that the cost of bacteria cellulose is higher.Therefore develop wide material sources, low-price carbon source plays an important role to reducing the bacteria cellulose production cost.
Biomass such as agricultural crop straw are the renewable resourcess of a kind of enormous amount on the earth.China's biomass resource is abundant, and the annual biomass total amount that produces has more than 50 hundred million tons (dry weight), and therefore, the recycling of these biomass is that a systems engineering also is to demand the problem developed at present urgently.
The main chemical compositions of straw is made up of hemicelluloses such as Mierocrystalline cellulose, xylan and xylogen, is main agricultural crop straw in northern China.Spruce is the cold-resistant stronger needle arbor of shade tolerance, and the unit surface year increment is big, and the material quality better is a kind of important material of construction and pulpwood.On the Northern Hemisphere, dragon spruce is a kind of seeds of extensive existence.In China, dragon spruce mainly is distributed in the southwest, and accumulation is abundant.Polysaccharide content reaches 60-80% in the chemical constitution of dragon spruce, wherein mainly is Mierocrystalline cellulose, and other contains a small amount of mannosans.Dragon spruce is a kind of renewable resources, if the polysaccharide in the dragon spruce can be converted into soluble sugar, is that carbon source prepares bacteria cellulose with it, and not only the production for bacteria cellulose provides new way, and can improve the utility value of dragon spruce.
Summary of the invention
Technical problem to be solved by this invention provides a kind of method of utilizing straw/dragon spruce to produce bacteria cellulose, and the culture medium carbon source quality of this method preparation is good, and price is low, is suitable for industrial production.
A kind of method of utilizing wheat straws for producing bacterium cellulose of the present invention comprises:
(1) dilute acid hydrolysis of straw
Straw grinds with the plant pulverizer earlier, again with dilute sulphuric acid or hydrochloric acid (0.3%～7%, w/v) in reactor with 1: 6-1: 30 straw and the solid-liquid of diluted acid are than soaked overnight (12-24h), under 100 ℃～121 ℃ conditions of temperature, reacted 30～80 minutes then, then straw residue and acid hydrolysis liquid are separated by suction filtration, collect hydrolyzed solution, 4 ℃ of-8 ℃ of refrigerator cold-storages are standby;
(2) detoxification of hydrolyzed solution
Owing to contain certain toxic substance in the hydrolyzed solution, with hydrolyzed solution during as culture medium carbon source bacillus aceticus (Acetobacter aceti) or glucose oxidation and bacillus (Gluconobacter Oxydans) can not grow and synthetic bacteria cellulose, so the essential detoxification of hydrolyzed solution;
With NaOH, Ca (OH)
2(or lime) and ammoniacal liquor (NH
4OH) etc. alkali carries out detoxification to hydrolyzed solution, changes detoxification condition such as pH value, time, and in conjunction with gac or in conjunction with 10% laccase (enzyme is lived and is 2.75U/mL) hydrolyzed solution is carried out detoxification with the raising detoxification efficiency respectively;
Method 1:NaOH is transferred to 4.5-5.5 with hydrolyzed solution pH value, filtering-depositing, and be fine-tuning to pH4.5-5.5;
Method 2:NaOH is transferred to 4.5-5.5 with hydrolyzed solution pH value, adds charcoal absorption, and the reaction after-filtration falls gac and finely tunes the pH value again to 4.5-5.5;
Method 3:NaOH is transferred to 9.5-11 with hydrolyzed solution pH value, adds charcoal absorption, and the reaction after-filtration falls gac and readjusts the pH value to 4.5-5.5;
Method 4:NaOH is transferred to 9.5-11 with hydrolyzed solution pH value, reacts under 25-60 ℃ of warm water bath condition and spends the night, and filters also again adjust pH to 4.5-5.5;
Method 5:NaOH is transferred to 9.5-11 with hydrolyzed solution pH value, reacts under 25-60 ℃ of warm water bath condition and spends the night, and filters also again adjust pH and adds charcoal absorption then to 4.5-5.5, and the reaction after-filtration falls gac and finely tunes the pH value again to 4.5-5.5;
Method 6:NaOH is transferred to 4.5-5.5 with hydrolyzed solution pH value, adds 10% laccase (enzyme is lived and is 2.75U/mL) and react 12h-24h under 25-60 ℃ of warm water bath condition, filters out throw out and finely tunes the pH value again to 4.5-5.5;
Method 7:Ca (OH)
2Hydrolyzed solution pH value is transferred to 4.5-5.5, filtering-depositing, and be fine-tuning to pH4.5-5.5;
Method 8:Ca (OH)
2Hydrolyzed solution pH value is transferred to 4.5-5.5, adds charcoal absorption, the reaction after-filtration falls gac and finely tunes the pH value again to 4.5-5.5;
Method 9:Ca (OH)
2Hydrolyzed solution pH value is transferred to 9.5-11, adds charcoal absorption, the reaction after-filtration falls gac and readjusts the pH value to 4.5-5.5;
Method 10:Ca (OH)
2Hydrolyzed solution pH value is transferred to 9.5-11, under 25-60 ℃ of warm water bath condition, reacts and spend the night, filter also again adjust pH to 4.5-5.5;
Method 11:Ca (OH)
2Hydrolyzed solution pH value is transferred to 9.5-11, reacts under 25-60 ℃ of warm water bath condition and spend the night, filter also again adjust pH and add charcoal absorption then to 4.5-5.5, the reaction after-filtration falls gac and finely tunes the pH value again to 4.5-5.5;
Method 12:Ca (OH)
2Hydrolyzed solution pH value is transferred to 4.5-5.5, adds 10% laccase (enzyme is lived and is 2.75U/mL) and under 25-60 ℃ of warm water bath condition, react 12h-24h, filter out throw out and finely tune the pH value again to 4.5-5.5;
Method 13:25%-30% ammoniacal liquor is transferred to 9.5-11 with hydrolyzed solution pH value, under 25-60 ℃ of warm water bath condition, react and spend the night, filter and again adjust pH add charcoal absorption then to 4.5-5.5, the reaction after-filtration falls gac and also finely tunes the pH value again to 4.5-5.5;
Method 14:25%-30% ammoniacal liquor is transferred to 4.5-5.5 with hydrolyzed solution pH value, adds 10% laccase (enzyme is lived and is 2.75U/mL) and react 12h-24h under 25-60 ℃ of warm water bath condition, filters out throw out and finely tunes the pH value again to 4.5-5.5.
(3) preparation of bacteria cellulose
Get above-mentioned detoxification hydrolyzed solution as culture medium carbon source, add the nitrogenous source of 0.1-2%, 121 ℃ the sterilization 15-20min after as substratum, (USS biological sample preservation center ATCC provides: Acetobacter aceti subsp.xylinus (Gluconacetobacter xylinus) ATCC 23770 to insert bacillus aceticus with the inoculum size of 5%-10%, Acetobacteraceti subsp.xylinus (Gluconacetobacter xylinus) ATCC 53263, Gluconacetobacter xylinusATCC 53264, Gluconacetobacter xylinus ATCC 53524, Gluconacetobacter xylinus ATCC53582, Gluconacetobacter xylinus ATCC 53749, Gluconacetobacter xylinus ATCC 53750, Gluconacetobacter xylinus ATCC 700178, Gluconacetobacter hansenii ATCC 10821, Gluconacetobacter hansenii ATCC 23769) or glucose oxidation and bacillus bacteriums such as (Gluconobacter oxydans ATCC11894) at 25-30 ℃, cultivate or static cultivation in 25-30 ℃ of incubator in the 160-250 rev/min of shaking table, (6-25 days) all can access comparatively ideal bacteria cellulose through after a while.
Straw in the described step (1) is milled to 40 orders with the plant pulverizer earlier.
Gac in the described step (2) is that the gac of 1 quality %-6 quality % stirs 5-10min under room temperature.
Nitrogenous source in the described step (3) is one or more in the ammonium salts such as yeast extract, peptone, Tryptones, extractum carnis, ammonium sulfate.
The carbon source that described step (3) is used for culturing bacterium is the straw hydrolyzed solution for preparing through detoxification, reducing sugar amount (with glucose meter) by 7-30g/L is mixed with fermention medium with hydrolyzed solution, contain 0.1-1% yeast extract, 0.1-0.5% peptone, the medium pH value is 5.0.
Inoculum size in the described step (3) is 6%.
Wherein method 11, Ca (OH)
2The effect for preparing bacteria cellulose in conjunction with process of active carbon is best, can generate comparatively ideal bacteria cellulose film in the time about 11 days, and bacteria cellulose output is the highest, can reach 15.4mg/mL.When the hydrolyzed solution of this detoxification and other conventional carbon source relatively the time, under identical carbon source concentration and culture condition, the bacteria cellulose output of this detoxification hydrolyzed solution preparation still is higher than the conventional carbon source preparation, and the cellulose output when being carbon source with pure N.F,USP MANNITOL, sucrose or glucose exceeds 50.3%, 65.0% and 69.9% respectively.
In addition, use different alkali lye to regulate after hydrolyzed solution pH to the 9.5-11 reaction again in conjunction with process of active carbon (method 5, method 11, method 13) than regulating hydrolyzed solution pH to 4.5-5.5 with different alkali lye again in conjunction with the method (method 6 of laccase, method 12, method 14) good to straw hydrolyzed solution detoxification efficiency, what wherein detoxification efficiency was best is with Ca (OH)
2Regulating hydrolyzed solution pH value, secondly is NaOH and ammoniacal liquor.
The straw main chemical compositions is made up of Mierocrystalline cellulose, hemicellulose and xylogen, but the straw complex structure.Mierocrystalline cellulose, hemicellulose are not only wrapped up by xylogen, and hemicellulose part covalency and xylogen combination, and Mierocrystalline cellulose then has the high-sequential crystalline structure, so the utilization of straw needs pre-treatment.Have only through pre-treatment, could remove xylogen to cellulosic parcel, thereby Mierocrystalline cellulose is come out, be beneficial to acid hydrolysis or enzymic hydrolysis and follow-up fermenting process.In hydrolytic process, though glucose is arranged, wood sugar, mixing sugar such as pectinose produce, but because reaction conditions is violent, also can generate many inhibitions to the toxic effect of organism of fermentation, the inhibitor in the hydrolyzed solution mainly contains: furfural, hydroxymethylfurfural, acetate, phenolic compound, syringic acid, hydroxy-benzoic acid, Vanillin and other toxic compounds.Carry out in the fermentation process so state hydrolyzed solution in the use, need be to the hydrolyzed solution detoxification, to reduce the influence that these toxic compounds are cultivated microbial fermentation.
A kind of method of utilizing dragon spruce to produce bacteria cellulose of the present invention comprises:
(1) dilute acid hydrolysis of dragon spruce
With air-dry 20 orders-80 order that is crushed to of dragon spruce, use 0.5%～8% (w/v) sulfuric acid or salt acid soak again, the solid-to-liquid ratio of dragon spruce and sulfuric acid or hydrochloric acid is 1: 5～1: 20, reacts 50～90min down at 100 ℃～240 ℃ then, reaction finishes the back suction filtration, collects hydrolyzed solution;
(2) detoxification of hydrolyzed solution
In the dragon spruce acid hydrolysis process, except producing monose and oligose, also produce some by products, metabolism has certain restraining effect to these by products to microbial growth, needs hydrolyzed solution is handled before utilizing hydrolyzed solution to produce bacteria cellulose.
With NaOH, Ca (OH)
2(or lime), ammoniacal liquor (NH
4OH) etc. alkali is regulated hydrolyzed solution pH value, in conjunction with gac or laccase hydrolyzed solution is handled.
Method 1: transfer hydrolyzed solution pH value to 4～6 with NaOH, centrifugal or remove by filter precipitation;
Method 2: transfer hydrolyzed solution pH value to 4～6 with NaOH, add gac and react 5min～60min, centrifugal or remove by filter precipitation;
Method 3: transfer hydrolyzed solution pH value to 9～11 with NaOH, add gac reaction 5min～60min, centrifugal or remove by filter precipitation, accent hydrolyzed solution pH value to 4.5～6;
Method 4: transfer hydrolyzed solution pH value to 9～11 with NaOH, in 20～60 ℃ of water-baths reaction 12～24 hours down, centrifugal or remove by filter precipitation, accent hydrolyzed solution pH value to 4～6;
Method 5: transfer hydrolyzed solution pH value to 9～11 with NaOH,, transfer hydrolyzed solution pH value to 4.5～6, add gac then and react 5min～30min in 20～60 ℃ of water-baths reaction 12～24 hours down, centrifugal or remove by filter precipitation;
Method 6: transfer hydrolyzed solution pH value to 4.5～6 with NaOH, centrifugal or remove by filter precipitation, add the laccase that 10% enzyme lives to 2.75U/mL and reacted 12～24 hours down in 30～60 ℃ of water-baths;
Method 7: with Ca (OH)
2Transfer hydrolyzed solution pH value to 4.5～6, centrifugal or remove by filter precipitation;
Method 8: with Ca (OH)
2Transfer hydrolyzed solution pH value to 4.5～6, add gac and react 5min～60min, centrifugal or remove by filter precipitation;
Method 9: with Ca (OH)
2Transfer hydrolyzed solution pH value to 9～11, add gac reaction 5min～60min, centrifugal or remove by filter precipitation, accent hydrolyzed solution pH value to 4～6;
Method 10: with Ca (OH)
2Transfer hydrolyzed solution pH value to 9～11, in 20～60 ℃ of water-baths reaction 12～24 hours down, centrifugal or remove by filter precipitation, accent hydrolyzed solution pH value to 4～6;
Method 11: with Ca (OH)
2Transfer hydrolyzed solution pH value to 9～11,, transfer hydrolyzed solution pH value to 4～6, add gac then and react 5min～60min in 20～60 ℃ of water-baths reaction 12～24 hours down, centrifugal or remove by filter precipitation;
Method 12: with Ca (OH)
2Transfer hydrolyzed solution pH value to 4～6, centrifugal or remove by filter precipitation, add laccase and reacted 12～24 hours down in 30～60 ℃ of water-baths;
Method 13: transfer hydrolyzed solution pH value to 9～11 with ammoniacal liquor,, transfer hydrolyzed solution pH value to 4～6, add gac then and react 5min～60min in 20～60 ℃ of water-baths reaction 12～24 hours down, centrifugal or remove by filter precipitation;
Method 14: transfer hydrolyzed solution pH value to 4～6 with ammoniacal liquor, centrifugal or remove by filter precipitation, add the laccase that 10% enzyme lives to 2.75U/mL and reacted 12～24 hours down in 30～60 ℃ of water-baths;
(3) get above-mentioned detoxification hydrolyzed solution as culture medium carbon source, add 0.1～2% nitrogenous source, 121 ℃ the sterilization 15-20min after as substratum; Inoculum size with 5%～10% inserts bacillus aceticus, and (USS biological sample preservation center ATCC provides: Acetobacter aceti subsp.xylinus (Gluconacetobacter xylinus) ATCC 23770, Acetobacteraceti subsp.xylinus (Gluconacetobacter xylinus) ATCC 53263, Gluconacetobacter xylinusATCC 53264, Gluconacetobacter xylinus ATCC 53524, Gluconacetobacter xylinus ATCC53582, Gluconacetobacter xylinus ATCC 53749, Gluconacetobacter xylinus ATCC 53750, Gluconacetobacter xylinus ATCC 700178, Gluconacetobacter hansenii ATCC 10821, Gluconacetobacter hansenii ATCC 23769) or glucose oxidation and bacillus bacteriums such as (Gluconobacter oxydans ATCC11894), at 25～30 ℃, leave standstill under 160～250rpm shaking culture or 25～30 ℃ and cultivated 6-25 days, obtain bacteria cellulose.
Straw in the described step (1) is milled to 40 orders with the plant pulverizer earlier.
Described step (2) gac is that the gac of 1 quality %-6 quality % stirs 5-10min under room temperature.
Nitrogenous source in the described step (3) is one or more in the ammonium salts such as yeast extract, peptone, Tryptones, extractum carnis, ammonium sulfate.
The substratum of described step (3) adopts the detoxification hydrolyzed solution as culture medium carbon source, reducing sugar amount (with glucose meter) by 7-30g/L is mixed with fermention medium with hydrolyzed solution, contain 0.1-1% yeast extract, 0.1-0.5% peptone, the medium pH value is 5.0.
Dragon spruce is after acid hydrolysis and handling, the sugared concentration of gained hydrolyzed solution is adjusted into 2.5g/L, add 0.5% yeast extract and 0.3% Tryptones and be mixed with substratum, inoculum size with 6% inserts bacillus aceticus, 30 ℃ of static cultivations 11 days, the output of gained bacteria cellulose is 11.9g/L, than under the similarity condition with N.F,USP MANNITOL, sucrose, glucose be you can well imagine the carbon source time-division high by 20.7%, 53.9% and 56.4%.
In hydrolytic process, though dragon spruce is the same with straw glucose arranged, mixing sugar such as seminose sugar produce, because reaction conditions is violent, also can generate many to the deleterious inhibition of organism of fermentation.Carry out in the fermentation process so state hydrolyzed solution in the use, need be to the hydrolyzed solution detoxification, to reduce the influence that these toxic compounds are cultivated microbial fermentation.
Beneficial effect
(1) the present invention is simple, and is with low cost, and raw material sources are extensive, is suitable for suitability for industrialized production;
(2) the present invention utilizes this inexpensive raw material in the straw, carry out dilute acid hydrolysis and detoxification, produce and a kind ofly can be used for culturing bacterium and prepare cellulosic culture medium carbon source, produce this emerging biomaterial of bacteria cellulose for large-scale industrialization new thinking and approach is provided; The straw wide material sources, cheap, preparation and the poison-removing method thereof of therefore producing the culture medium carbon source of bacteria cellulose have very high actual application value, and be with the obvious advantage; After tested, the culture medium carbon source that the present invention produced also can be used for the cultivation of other industrial microorganism, is a kind of carbon source of high quality and at a reasonable price;
(3) dragon spruce is a kind of renewable resources, is used for the fermentative preparation bacterial fibers and has not only improved its economic worth, and provide new way for the suitability for industrialized production of bacteria cellulose.
Description of drawings
The result of the bacteria cellulose that the straw hydrolyzed solution carbon source of Fig. 1 different methods preparation is produced.
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
Straw grinds with the plant pulverizer earlier, use dilute sulphuric acid (3% again, w/v) in reactor with the solid-liquid of 1: 6 straw and diluted acid than soaked overnight (12-24h), then 121 ℃ of reactions of temperature 60 minutes, then straw residue and acid solution suction filtration are separated, collect hydrolyzed solution, 4 ℃ of refrigerator cold-storages are standby.
Add NaOH hydrolysis clear liquid pH value is transferred to about 10, fall precipitation with filter paper filtering and obtain handling posthydrolysis liquid, more little adjust pH to 10.0.Seal with film then, place 30 ℃ of water-bath reaction 12-24 to spend the night, with diluted acid hydrolyzed solution pH value is transferred to 5.0 at last.Add 2% (mass percent) gac then, stir (5-10min under the room temperature condition) back and fall gac, obtain detoxification hydrolysis clear liquid, use the little adjust pH to 5.5 of dilute sulphuric acid again with filter paper filtering.Hydrolyzed solution after the detoxification is through surveying sugar, and as culture medium carbon source, yeast extract and the 0.1%-0.5% Tryptones of adding 0.1%-1% more therein are made into the cultivation that straw hydrolyzed solution substratum is used for microorganism.
Embodiment 2
Straw grinds with the plant pulverizer earlier, use dilute hydrochloric acid (1% again, w/v) in reactor with the solid-liquid of 1: 10 straw and diluted acid than soaked overnight (12-24h), then 121 ℃ of reactions of temperature 75 minutes, then straw residue and acid solution suction filtration are separated, collect hydrolyzed solution, 4 ℃ of refrigerator cold-storages are standby.
Add Ca (OH)
2Hydrolysis clear liquid pH value is transferred to about 10, is fallen precipitation with filter paper filtering and obtain handling posthydrolysis liquid, more little adjust pH to 10.0.Seal with film then, place 40 ℃ of water-bath reaction 12-24 to spend the night, with diluted acid hydrolyzed solution pH value is transferred to 5.0 at last.Add 2% (mass percent) gac then, stir (5-10min under the room temperature condition) back and fall gac, obtain detoxification hydrolysis clear liquid, use the little adjust pH to 5.5 of dilute sulphuric acid again with filter paper filtering.Hydrolyzed solution after the detoxification is through surveying sugar, and as culture medium carbon source, yeast extract and the 0.1%-0.5% Tryptones of adding 0.1%-1% more therein are made into the cultivation that straw hydrolyzed solution substratum is used for microorganism.
Embodiment 3
Straw grinds with the plant pulverizer earlier, use dilute sulphuric acid (2% again, w/v) in reactor with the solid-liquid of 1: 15 straw and diluted acid than soaked overnight (12-24h), then 100 ℃ of reactions of temperature 80 minutes, then straw residue and acid solution suction filtration are separated, collect hydrolyzed solution, 4 ℃ of refrigerator cold-storages are standby.
Add 25% ammoniacal liquor hydrolysis clear liquid pH value is transferred to 10, fall precipitation with filter paper filtering and obtain handling posthydrolysis liquid, more little adjust pH to 10.Seal with film then, place 25 ℃ of water-bath reactions to spend the night, with diluted acid hydrolyzed solution pH value is transferred to 5.0 at last.Add 2% (mass percent) gac then, stir (5-10min under the room temperature condition) back and fall gac, obtain detoxification hydrolysis clear liquid, use the little adjust pH to 5.5 of dilute sulphuric acid again with filter paper filtering.Hydrolyzed solution after the detoxification is through surveying sugar, and as culture medium carbon source, yeast extract and the 0.1%-0.5% Tryptones of adding 0.1%-1% more therein are made into the cultivation that straw hydrolyzed solution substratum is used for microorganism.
Embodiment 4
Straw grinds with the plant pulverizer earlier, use dilute sulphuric acid (3% again, w/v) in reactor with the solid-liquid of 1: 15 straw and diluted acid than soaked overnight (12-24h), then 110 ℃ of reactions of temperature 60 minutes, then straw residue and acid solution suction filtration are separated, collect hydrolyzed solution, 6 ℃ of refrigerator cold-storages are standby.
Add NaOH hydrolysis clear liquid pH value is transferred to 5.0, add the work of 10% enzyme and be in 50 ℃ of water-baths, to react 12h by the laccase of 2.75U/mL, filter out gac and finely tune pH value to 5.5 again.Hydrolyzed solution after the detoxification is through surveying sugar, and as culture medium carbon source, yeast extract and the 0.1%-0.5% Tryptones of adding 0.1%-1% more therein are made into the cultivation that straw hydrolyzed solution substratum is used for microorganism.
Straw grinds with the plant pulverizer earlier, use dilute sulphuric acid (3% again, w/v) in reactor with the solid-liquid of 1: 30 straw and diluted acid than soaked overnight (12-24h), then 110 ℃ of reactions of temperature 60 minutes, then straw residue and acid solution suction filtration are separated, collect hydrolyzed solution, 6 ℃ of refrigerator cold-storages are standby.
Add Ca (OH)
2Hydrolysis clear liquid pH value is transferred to 5.0, adds the work of 10% enzyme and be in 50 ℃ of water-baths, to react 12h by the laccase of 2.75U/mL, filter out gac and finely tune pH value to 5.5 again.Hydrolyzed solution after the detoxification is through surveying sugar, and as culture medium carbon source, yeast extract and the 0.1%-0.5% Tryptones of adding 0.1%-1% more therein are made into the cultivation that straw hydrolyzed solution substratum is used for microorganism.
Straw grinds with the plant pulverizer earlier, use dilute hydrochloric acid (3% again, w/v) in reactor with the solid-liquid of 1: 30 straw and diluted acid than soaked overnight (12-24h), then 121 ℃ of reactions of temperature 60 minutes, then straw residue and acid solution suction filtration are separated, collect hydrolyzed solution, 4 ℃ of refrigerator cold-storages are standby.
Add 25% ammoniacal liquor hydrolysis clear liquid pH value is transferred to 5.0, add the work of 10% enzyme and be in 40 ℃ of water-baths, to react 12h by the laccase of 2.75U/mL, filter out gac and finely tune pH value to 5.5 again.Hydrolyzed solution after the detoxification is through surveying sugar, and as culture medium carbon source, yeast extract and the 0.1%-0.5% Tryptones of adding 0.1%-1% more therein are made into the cultivation that straw hydrolyzed solution substratum is used for microorganism.
Embodiment 7
Straw grinds with the plant pulverizer earlier, use dilute sulphuric acid (1% again, w/v) in reactor with the solid-liquid of 1: 12 straw and diluted acid than soaked overnight (12-24h), then 121 ℃ of reactions of temperature 30 minutes, then straw residue and acid solution suction filtration are separated, collect hydrolyzed solution, 4 ℃ of refrigerator cold-storages are standby.
Add Ca (OH)
2Hydrolysis clear liquid pH value is transferred to about 10, is fallen precipitation with filter paper filtering and obtain handling posthydrolysis liquid, more little adjust pH to 10.0.Seal with film then, place 40 ℃ of water-bath reactions to spend the night, with diluted acid hydrolyzed solution pH value is transferred to 5.0 at last.Add 2% (mass percent) gac then, stir (5-10min under the room temperature condition) back and fall gac, obtain detoxification hydrolysis clear liquid, use the little adjust pH to 5.5 of dilute sulphuric acid again with filter paper filtering.Hydrolyzed solution after the detoxification is through surveying sugar, and as culture medium carbon source, yeast extract and the 0.1%-0.5% Tryptones of adding 0.1%-1% more therein are made into the cultivation that straw hydrolyzed solution substratum is used for microorganism.
Embodiment 8
Use above-mentioned the whole bag of tricks to the detoxification of straw hydrolyzed solution, and adjusting hydrolyzed solution sugar concentration is 25g/L, prepare glucose, N.F,USP MANNITOL, the sucrose of same concentration simultaneously respectively, yeast extract and the 0.1%-0.5% Tryptones of adding 0.1%-1% more therein are made into 50mL straw hydrolyzed solution substratum, dextrose culture-medium, N.F,USP MANNITOL substratum, sucrose medium respectively.Bacillus aceticus or glucose oxidation and bacillus are inserted static cultivation 8-15 days in 30 ℃ of incubators of straw hydrolyzed solution substratum with the inoculum size of 6-10%, can obtain comparatively ideal bacteria cellulose product or abundanter bacteria cellulose film, experimental result is seen Fig. 1.
On bacteria cellulose output, use Ca (OH)
2Being better than those in conjunction with the poison-removing method (method 11) of gac uses NaOH in conjunction with gac and the ammoniacal liquor poison-removing method in conjunction with gac.Through Ca (OH)
2Carbon source in conjunction with the preparation of the poison-removing method of gac, time about 8-15 days can generate comparatively ideal bacteria cellulose film, and through NaOH in conjunction with gac and ammoniacal liquor carbon source in conjunction with the poison-removing method of gac, though can form bacteria cellulose film, output does not have Ca (OH) yet
2Poison-removing method height in conjunction with gac.
As seen from Figure 1, Ca (OH)
2Bacteria cellulose output in conjunction with the poison-removing method (method 11) of gac is the highest, as Ca (OH)
2In conjunction with gac and other conventional carbon sources, for example (,) sucrose, when glucose and N.F,USP MANNITOL compare, Ca (OH)
2The substratum that is higher than conventional carbon source in conjunction with the bacteria cellulose output of the hydrolyzed solution of gac detoxification preparation.So under equal conditions, the bacteria cellulose output of substratum production that uses detoxification straw hydrolyzed solution preparation is a little more than with N.F,USP MANNITOL, sucrose or the glucose substratum as carbon source, because raw material straw wide material sources, cheap, therefore the preparation and the poison-removing method thereof of the culture medium carbon source of this production bacteria cellulose have very high actual application value, and be with the obvious advantage.
Embodiment 9
After the air-dry pulverizing of dragon spruce, the solid-to-liquid ratio with 1: 8 in reactor adds 3% (w/v) sulfuric acid, and reaction 20min under 180 ℃ reacts the end back and by suction filtration residue and hydrolyzed solution separated then, collects hydrolyzed solution.
Transfer hydrolyzed solution pH value to 10 with NaOH, in 30 ℃ of water-baths down reaction spend the night, transfer hydrolyzed solution pH value to 5, add gac then and react 60min, centrifugal or remove by filter precipitation;
Separating liquid with above-mentioned treated water is carbon source, adds 0.5% yeast extract and 0.3% peptone, and the sterilization back is as substratum.Insert bacterial classification with 6% inoculum size,, obtain bacteria cellulose 30 ℃, 160～250rpm shaking culture or static cultivation 12 days.
After the air-dry pulverizing of dragon spruce, the solid-to-liquid ratio with 1: 6 in reactor adds 6% (w/v) hydrochloric acid, and reaction 15min under 220 ℃ reacts the end back and by suction filtration residue and hydrolyzed solution separated then, collects hydrolyzed solution.
Transfer hydrolyzed solution pH value to 5.5 with NaOH, centrifugal or remove by filter precipitation, add laccase and reacted 24 hours down in 35 ℃ of ℃ of water-baths;
Separating liquid with above-mentioned treated water is carbon source, adds 0.3% yeast extract and 0.5% Tryptones, and the sterilization back is as substratum.Insert bacterial classification with 10% inoculum size,, obtain bacteria cellulose 30 ℃, 160～250rpm shaking culture or static cultivation 10 days.
After the air-dry pulverizing of dragon spruce, the solid-to-liquid ratio with 1: 10 in reactor adds 5% (w/v) sulfuric acid, and reaction 25min under 140 ℃ reacts the end back and by suction filtration residue and hydrolyzed solution separated then, collects hydrolyzed solution.
With Ca (OH)
2Transfer hydrolyzed solution pH value to 11, in 40 ℃ of water-baths down reaction spend the night, centrifugal or remove by filter precipitation, accent hydrolyzed solution pH value to 5.5;
Separating liquid with above-mentioned treated water is carbon source, adds 0.2% yeast extract and 0.6% peptone, and the sterilization back is as substratum.Insert bacterial classification with 6% inoculum size,, obtain bacteria cellulose 30 ℃, 160～250rpm shaking culture or static cultivation 15 days.
After the air-dry pulverizing of dragon spruce, the solid-to-liquid ratio with 1: 12 in reactor adds 3% (w/v) sulfuric acid, and reaction 45min under 130 ℃ reacts the end back and by suction filtration residue and hydrolyzed solution separated then, collects hydrolyzed solution.
Transfer hydrolyzed solution pH value to 10 with ammoniacal liquor, in 25 ℃ of water-baths down reaction spend the night, transfer hydrolyzed solution pH value to 5.5, add gac then and react 30min, centrifugal or remove by filter precipitation;
Separating liquid with above-mentioned treated water is carbon source, adds 1% peptone, and the sterilization back is as substratum.Insert bacterial classification with 7% inoculum size,, obtain bacteria cellulose 30 ℃, 160～250rpm shaking culture or static cultivation 12 days.
After the air-dry pulverizing of dragon spruce, the solid-to-liquid ratio with 1: 15 in reactor adds 2% (w/v) sulfuric acid, and reaction 60min under 120 ℃ reacts the end back and by suction filtration residue and hydrolyzed solution separated then, collects hydrolyzed solution.
With Ca (OH)
2Transfer hydrolyzed solution pH value to 5.0, centrifugal or remove by filter precipitation;
Separating liquid with above-mentioned treated water is carbon source, adds 0.5% beef extract and 0.3% peptone, and the sterilization back is as substratum.Insert bacterial classification with 10% inoculum size,, obtain bacteria cellulose 30 ℃, 160rpm shaking culture or static cultivation 10 days.
Embodiment 15
After the air-dry pulverizing of dragon spruce, the solid-to-liquid ratio with 1: 20 in reactor adds 1% (w/v) hydrochloric acid, and reaction 90min under 100 ℃ reacts the end back and by suction filtration residue and hydrolyzed solution separated then, collects hydrolyzed solution.
Transfer hydrolyzed solution pH value to 5.5 with ammoniacal liquor, centrifugal or remove by filter precipitation, add laccase and reacted 12 hours down in 50 ℃ of water-baths;
Separating liquid with above-mentioned treated water is carbon source, adds 0.5% yeast extract and 0.5% Tryptones, and the sterilization back is as substratum.Insert bacterial classification with 8% inoculum size,, obtain bacteria cellulose 30 ℃, 160rpm shaking culture or static cultivation 10 days.
After the air-dry pulverizing of dragon spruce, the solid-to-liquid ratio with 1: 7 in reactor adds 6% (w/v) hydrochloric acid, and reaction 40min under 120 ℃ reacts the end back and by suction filtration residue and hydrolyzed solution separated then, collects hydrolyzed solution.
With Ca (OH)
2Transfer hydrolyzed solution pH value to 5.0, add gac and react 60min, centrifugal or remove by filter precipitation;
Separating liquid with above-mentioned treated water is carbon source, adds 0.1% beef extract and 1.0% peptone, and the sterilization back is as substratum.Insert bacterial classification with 8% inoculum size,, obtain bacteria cellulose 30 ℃, 160～250rpm shaking culture or static cultivation 12 days.
Embodiment 17
After the air-dry pulverizing of dragon spruce, the solid-to-liquid ratio with 1: 9 in reactor adds 5% (w/v) hydrochloric acid, and reaction 20min under 150 ℃ reacts the end back and by suction filtration residue and hydrolyzed solution separated then, collects hydrolyzed solution.
Transfer hydrolyzed solution pH value to 11 with NaOH, add gac reaction 60min, centrifugal or remove by filter precipitation, accent hydrolyzed solution pH value to 5;
Separating liquid with above-mentioned treated water is carbon source, adds 0.2% yeast extract and 0.6% peptone, and the sterilization back is as substratum.Insert bacterial classification with 8% inoculum size,, obtain bacteria cellulose 30 ℃, 180rpm shaking culture or static cultivation 14 days.
Claims (5)
1. method of utilizing wheat straws for producing bacterium cellulose comprises:
(1) straw is milled to the 20-80 order with the plant pulverizer earlier, and dilute sulphuric acid or the hydrochloric acid with 0.3%～7%w/v soaks 12-24h in reactor again; Straw is 1 with the solid-liquid ratio of dilute sulphuric acid or hydrochloric acid: 6-1: 30, then 100 ℃～121 ℃ reactions 30～80 minutes, then straw residue and acid hydrolysis liquid are separated by suction filtration, and collect hydrolyzed solution, 4 ℃ of-8 ℃ of refrigerator cold-storages are standby;
(2) detoxification of hydrolyzed solution
Method 1:NaOH is transferred to 4.5-5.5 with hydrolyzed solution pH value, filtering-depositing, and be fine-tuning to pH4.5-5.5;
Method 2:NaOH is transferred to 4.5-5.5 with hydrolyzed solution pH value, adds charcoal absorption, and the reaction after-filtration falls gac and finely tunes the pH value again to 4.5-5.5;
Method 3:NaOH is transferred to 9.5-11 with hydrolyzed solution pH value, adds charcoal absorption, and the reaction after-filtration falls gac and readjusts the pH value to 4.5-5.5;
Method 4:NaOH is transferred to 9.5-11 with hydrolyzed solution pH value, reacts 12h-24h under 25-60 ℃ of warm water bath condition, filters also again adjust pH to 4.5-5.5;
Method 5:NaOH is transferred to 9.5-11 with hydrolyzed solution pH value, reacts 12h-24h under 25-60 ℃ of warm water bath condition, filters also again adjust pH and adds charcoal absorption then to 4.5-5.5, and the reaction after-filtration falls gac and finely tunes the pH value again to 4.5-5.5;
Method 6:NaOH is transferred to 4.5-5.5 with hydrolyzed solution pH value, adds 10% enzyme and lives to the laccase of 2.75U/mL reacts 12h-24h under 25-60 ℃ of warm water bath condition, filters out throw out and also finely tunes the pH value again to 4.5-5.5;
Method 7:Ca (OH)
2Hydrolyzed solution pH value is transferred to 4.5-5.5, filtering-depositing, and be fine-tuning to pH4.5-5.5;
Method 8:Ca (OH)
2Hydrolyzed solution pH value is transferred to 4.5-5.5, adds charcoal absorption, the reaction after-filtration falls gac and finely tunes the pH value again to 4.5-5.5;
Method 9:Ca (OH)
2Hydrolyzed solution pH value is transferred to 9.5-11, adds charcoal absorption, the reaction after-filtration falls gac and readjusts the pH value to 4.5-5.5;
Method 10:Ca (OH)
2Hydrolyzed solution pH value is transferred to 9.5-11, under 25-60 ℃ of warm water bath condition, reacts 12h-24h, filter also again adjust pH to 4.5-5.5;
Method 11:Ca (OH)
2Hydrolyzed solution pH value is transferred to 9.5-11, reacts 12h-24h under 25-60 ℃ of warm water bath condition, filter also again adjust pH and add charcoal absorption then to 4.5-5.5, the reaction after-filtration falls gac and finely tunes the pH value again to 4.5-5.5;
Method 12:Ca (OH)
2Hydrolyzed solution pH value is transferred to 4.5-5.5, adds 10% enzyme and live, filter out throw out and also finely tune the pH value again to 4.5-5.5 to the laccase of 2.75U/mL reacts 12h-24h under 25-60 ℃ of warm water bath condition;
Method 13:25%-30% ammoniacal liquor is transferred to 9.5-11 with hydrolyzed solution pH value, under 25-60 ℃ of warm water bath condition, react 12h-24h, filter and again adjust pH add charcoal absorption then to 4.5-5.5, the reaction after-filtration falls gac and also finely tunes the pH value again to 4.5-5.5;
Method 14:25%-30% ammoniacal liquor is transferred to 4.5-5.5 with hydrolyzed solution pH value, adds 10% enzyme and lives to the laccase of 2.75U/mL reacts 12h-24h under 25-60 ℃ of warm water bath condition, filters out throw out and also finely tunes the pH value again to 4.5-5.5;
(3) get above-mentioned detoxification hydrolyzed solution as culture medium carbon source, add 0.1～2% nitrogenous source, 121 ℃ the sterilization 15-20min after as substratum; Insert bacillus aceticus or glucose oxidation and bacillus with the inoculum size of 5%-10%,, cultivate in the 160-250 rev/min of shaking table or in 25-30 ℃ of incubator, leave standstill cultivation, through obtaining bacteria cellulose in 6-25 days at 25-30 ℃.
2. method of utilizing dragon spruce to produce bacteria cellulose comprises:
(1) with the air-dry pulverizing of dragon spruce, use 0.5%～8%w/v sulfuric acid or salt acid soak again, the solid-to-liquid ratio of dragon spruce and sulfuric acid or hydrochloric acid is 1: 5～1: 20, reacts 50～90min down at 100 ℃～240 ℃ then, reaction finishes the back suction filtration, collects hydrolyzed solution;
(2) detoxification of hydrolyzed solution
Method 1: transfer hydrolyzed solution pH value to 4～6 with NaOH, centrifugal or remove by filter precipitation;
Method 2: transfer hydrolyzed solution pH value to 4～6 with NaOH, add gac and react 5min～60min, centrifugal or remove by filter precipitation;
Method 3: transfer hydrolyzed solution pH value to 9～11 with NaOH, add gac reaction 5min～60min, centrifugal or remove by filter precipitation, accent hydrolyzed solution pH value to 4.5～6;
Method 4: transfer hydrolyzed solution pH value to 9～11 with NaOH, in 20～60 ℃ of water-baths reaction 12～24 hours down, centrifugal or remove by filter precipitation, accent hydrolyzed solution pH value to 4～6;
Method 5: transfer hydrolyzed solution pH value to 9～11 with NaOH,, transfer hydrolyzed solution pH value to 4.5～6, add gac then and react 5min～30min in 20～60 ℃ of water-baths reaction 12～24 hours down, centrifugal or remove by filter precipitation;
Method 6: transfer hydrolyzed solution pH value to 4.5～6 with NaOH, centrifugal or remove by filter precipitation, add the laccase that 10% enzyme lives to 2.75U/mL and reacted 12～24 hours down in 30～60 ℃ of water-baths;
Method 7: with Ca (OH)
2Transfer hydrolyzed solution pH value to 4.5～6, centrifugal or remove by filter precipitation;
Method 8: with Ca (OH)
2Transfer hydrolyzed solution pH value to 4.5～6, add gac and react 5min～60min, centrifugal or remove by filter precipitation;
Method 9: with Ca (OH)
2Transfer hydrolyzed solution pH value to 9～11, add gac reaction 5min～60min, centrifugal or remove by filter precipitation, accent hydrolyzed solution pH value to 4～6;
Method 10: with Ca (OH)
2Transfer hydrolyzed solution pH value to 9～11, in 20～60 ℃ of water-baths reaction 12～24 hours down, centrifugal or remove by filter precipitation, accent hydrolyzed solution pH value to 4～6;
Method 11: with Ca (OH)
2Transfer hydrolyzed solution pH value to 9～11,, transfer hydrolyzed solution pH value to 4～6, add gac then and react 5min～60min in 20～60 ℃ of water-baths reaction 12～24 hours down, centrifugal or remove by filter precipitation;
Method 12: with Ca (OH)
2Transfer hydrolyzed solution pH value to 4～6, centrifugal or remove by filter precipitation, add laccase and reacted 12～24 hours down in 30～60 ℃ of water-baths;
Method 13: transfer hydrolyzed solution pH value to 9～11 with ammoniacal liquor,, transfer hydrolyzed solution pH value to 4～6, add gac then and react 5min～60min in 20～60 ℃ of water-baths reaction 12～24 hours down, centrifugal or remove by filter precipitation;
Method 14: transfer hydrolyzed solution pH value to 4～6 with ammoniacal liquor, centrifugal or remove by filter precipitation, add the laccase that 10% enzyme lives to 2.75U/mL and reacted 12～24 hours down in 30～60 ℃ of water-baths;
(3) get above-mentioned detoxification hydrolyzed solution as culture medium carbon source, add 0.1～2% nitrogenous source, 121 ℃ the sterilization 15-20min after as substratum; Insert bacillus aceticus or glucose oxidation and bacillus with 5%～10% inoculum size, under 25～30 ℃, 160～250rpm shaking culture or 25～30 ℃, leave standstill and cultivated 6-25 days, obtain bacteria cellulose.
3. a kind of method of utilizing straw or dragon spruce production bacteria cellulose according to claim 1 and 2, it is characterized in that: the gac that the middle gac of described step (2) is 1 quality %-6 quality % stirs 5-10min under room temperature.
4. a kind of method of utilizing straw or dragon spruce production bacteria cellulose according to claim 1 and 2, it is characterized in that: the nitrogenous source in the described step (3) is one or more in yeast extract, peptone, Tryptones, extractum carnis, the ammonium sulfate.
5. a kind of method of utilizing straw or dragon spruce production bacteria cellulose according to claim 1 and 2, it is characterized in that: the substratum of described step (3) adopts the detoxification hydrolyzed solution as culture medium carbon source, reducing sugar amount by 7-30g/L is mixed with fermention medium with hydrolyzed solution, contain 0.1-1% yeast extract, 0.1-0.5% peptone, the medium pH value is 5.0: wherein the reducing sugar amount is with glucose meter.
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| WO2016029431A1 (en) * | 2014-08-29 | 2016-03-03 | Shanghai Zhiyi Information Technology Ltd | Processing of plant material into bacterial feedstock |
| US9708580B2 (en) | 2012-05-15 | 2017-07-18 | Shanghai Zhiyi Information Technology Ltd. | Bacterial culture media and methods for their preparation and use |
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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 |
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| CN102242166B (en) * | 2011-06-28 | 2014-10-15 | 东华大学 | Preparation method of Bacterial Cellulose (BC) with carbon source of inulin |
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