CN103194504A - Application of sulfonated lignin in wood fibre material enzyme hydrolysis saccharification - Google Patents
Application of sulfonated lignin in wood fibre material enzyme hydrolysis saccharification Download PDFInfo
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Abstract
The present invention discloses application of sulfonated lignin in wood fibre material enzyme hydrolysis saccharification. According to the invention, the sulfonated lignin is added in the enzyme hydrolysis process. The efficiency of enzyme hydrolysis saccharification can be improved, the retention time of the enzyme hydrolysis is shortened, also the amount of enzyme can be reduced, the enzyme activity is improved, and the enzyme hydrolysis cost of production of biological energy source from the wood fibre material is reduced.
Description
Technical field
The invention belongs to technical field of enzyme engineering, be specifically related to the application of a kind of sulfonated lignin in the performing enzyme hydrolysis on wood fiber raw material saccharification.
Background technology
Exhausted development and the progress that is seriously restricting world civilization day by day of fossil energy, seeking alternative renewable resource becomes the research emphasis of countries in the world.Since the seventies in last century, bioenergy obtains various countries investigator's favor as a kind of reproducible potential energy source.Simultaneously, the application of bioenergy can also reduce the greenhouse gases CO that brings by in traditional fossil energy use
2And a small amount of sulfur-bearing, nitrogenous obnoxious flavour, alleviate Greenhouse effect effectively.Characteristics such as s-generation biofuel, it is big that lignocellulose raw material possesses output, and cost is low make it possess brighter and clearer development prospect.
Utilize lignocellulose raw material to prepare bio-ethanol and mainly comprise pre-treatment, enzymic hydrolysis, fermentation and four steps of distillation.Wherein pre-treatment and enzymic hydrolysis are the committed steps that determines bio-ethanol production cost and production efficiency.
The lignocellulose raw material main chemical compositions is Mierocrystalline cellulose, hemicellulose and xylogen.In plant tissue, itself has crystalline texture Mierocrystalline cellulose, and hemicellulose is combined with the covalent linkage form with xylogen, and surrounds Mierocrystalline cellulose tightly, formed a kind of firm natural cover for defense, made catalyzer and general microorganism be difficult to contact and make with Mierocrystalline cellulose its degraded.Can break connection between Mierocrystalline cellulose, hemicellulose and the xylogen by pre-treatment, destroy cellulosic crystalline texture, separate or remove part hemicellulose and xylogen, increase the porosity of lignocellulose raw material, improve enzyme to cellulosic contact gear ratio surface-area and accessibility.
As one of three big moietys of plant cell wall, xylogen is considered to suppress the major obstacle of plant fiber material bio-transformation always.Change into by enzymic hydrolysis in the liquid fuel process in biomass, the ineffective adsorption of xylogen and enzyme and xylogen contact for enzyme and cause with Mierocrystalline cellulose passage obstruction is considered to influence the major cause that lignocellulose raw material changes into liquid fuel efficient.Because the complicacy of plant fiber material structure, behavior and the interaction of xylogen and enzyme of research xylogen in biotransformation is very difficult.The investigator is by adding various xylogen in the reaction substrate, or content of lignin and the xylogen in the reaction substrate is carried out modification study xylogen to the influence of enzymic hydrolysis in the minimizing reaction substrate.Many experiments show that the content of lignin that reduces in the reaction substrate is conducive to improve enzymic hydrolysis transformation efficiency sugared in the substrate.Therefore, the various pretreatment processs that are conducive to remove the raw material wood element are widely adopted for example alkaline process pre-treatment.
In recent years, the efficient utilization of enzyme and enzyme reclaim and more and more get more and more people's extensive concerning, because the expensive very big commercialization process that is restricting lignocellulose raw material production alcohol fuel of enzyme.Therefore, the auxiliary agent that can improve enzymic hydrolysis efficient is badly in need of and will be developed.But, in the enzymic hydrolysis process, add auxiliary agent and can increase extra-expense undoubtedly, so low-cost, the source is abundant, easily obtain, easily preparation, additive that efficient is high just more have application prospect.The enzymic hydrolysis additive need meet the following conditions: the high reactivity temperature that can act on enzyme; Can not have a negative impact to follow-up fermentation; Can be conducive to improve the enzymic hydrolysis efficient of substrate; Can be conducive to reduce the usage quantity of enzyme; Can be conducive to shorten the enzymic hydrolysis time.Study more enzymic hydrolysis auxiliary agent tensio-active agent, bovine serum albumin arranged, have polyvinyl alcohol (PEG), anthraquinone of ethylene oxide structure etc.
Sulfonated lignin is the xylogen through sulfonation modifying, has solubility property and surfactivity preferably, can be water-soluble, now obtaining widespread use than conglomerate, as cement water reducing agent, binding agent, pesticide dispersing agent, dye dispersant etc.
Summary of the invention
Technical problem to be solved by this invention provides the application of sulfonated lignin in the performing enzyme hydrolysis on wood fiber raw material saccharification.
In order to solve the problems of the technologies described above, the technical solution used in the present invention is as follows:
The application of sulfonated lignin in the performing enzyme hydrolysis on wood fiber raw material saccharification.
Wherein, described sulfonated lignin can be available from market or prepare voluntarily.Preparation then can be prepared by the material modification that contains xylogen voluntarily, and the described material that contains xylogen is plant fiber material, pulping waste liquor or enzymolysis residue.
Slurrying is an operation before the papermaking, is lignocellulose raw material is made fibrous screened stock by the method for physics or chemistry, is used for papermaking.The xylogen that removes from lignocellulose raw material is arranged in the pulping waste liquor.
The enzymolysis residue refers to that lignocellulose raw material is through the solid part that contains xylogen remaining behind the cellulase hydrolysis.
The concrete preparation method that sulfonated lignin prepares voluntarily for example, can be with the sulfate pulp-making waste liquid after oil slick reclaims, carry out Acid precipitation and obtain the vitriol xylogen, get the vitriol xylogen and be dissolved in water, after alkali regulator solution pH value to 11, centrifugation is except alkali insolubless such as sub-cloud silts, clear liquid 10wt% sulfuric acid adjust pH to 9, centrifugation discards supernatant liquid, throw out uses distilled water through repeatedly washing, to neutral.After the vacuum-drying of gained throw out, as the raw material of subsequent reactions.Take by weighing 1g xylogen sample, be made into 25% concentration, add S-WAT 1.3mmol, change in the 125mL stainless steel reaction jar, reacted 3 hours down at 160 ℃, obtain sulfonated lignin (reference: Yang Yiqin, Li Zhongzheng, the research that modified wood vitriol xylogen prepares dye dispersant, chemistry of forest product and industry, 23 (4), 31-36).
The method for preparing sulfonated lignin is not limited to above-mentioned a kind of, can prepare sulfonated lignin as long as be connected to sulfonic group at the side chain of lignin molecule or phenyl ring, reaction formula is as follows, middle one is reactant, first all is the product sulfonated lignin with the 3rd, because pharmaceutical chemicals consumption (S-WAT/formaldehyde), temperature of reaction, the difference of time can obtain the product of different sulfonation degree (sulfonation degree has characterized lignin molecule and connected sulfonic probability, i.e. sulfonic content in every gram xylogen) different molecular weight (200～10000 do not wait).
R=H?or?Phenylpropane
Wherein, described lignocellulose raw material is needlebush, leaf wood or grass.
The concrete application method of sulfonated lignin in the performing enzyme hydrolysis on wood fiber raw material saccharification is, sulfonated lignin is added in acetic acid-sodium-acetate buffer, add again in the pretreated enzymolysis substrate lignocellulose raw material, after mixing, add enzyme liquid, carry out the enzymic hydrolysis saccharification react, after reaction finished, centrifugal collection upper strata contained sugared clear liquid.
Wherein, the adding quality of described sulfonated lignin is 1～50% of pretreated lignocellulose raw material over dry quality.Higher consumption is feasible in theory, but higher addition improves contribution not quite to enzymolysis efficiency, and increases production cost, and preferable range is 10～30%.Concrete optimum addition is decided according to the kind of enzymic hydrolysis substrate, suggestion softwood 30%, and pourous wood 20%, Gramineae plants 10%.
Wherein, described acetic acid-sodium-acetate buffer pH4.0～5.0.
Wherein, described pretreated enzymolysis substrate lignocellulose raw material, pretreatment process is conventional pretreatment process, for example the dilute acid pretreatment in the acid system; Green liquor pre-treatment in the alkaline process; The yellow soda ash pre-treatment; Sulphite pre-treatment etc.
Wherein, acetic acid-the sodium-acetate buffer of sulfonated lignin is with after the enzymolysis substrate mixes, described enzymolysis concentration of substrate scope is 1～10wt%, higher or lower concentration is feasible in theory, but too high or too low concentration can cause increase or the operational inconvenience of production cost, advise that softwood enzymolysis concentration of substrate preferable range is 2～5%, pourous wood and grass can suitably be improved enzymolysis substrate concentration of substrate to 5%～10%.
Wherein, described enzyme is cellulase, zytase and beta fibers disaccharidase three's prozyme; The enzyme of three kinds of unit of enzyme volumes is lived than being cellulase: zytase: beta fibers disaccharidase=1FPU:1.2FXU:1CBU.
Wherein,
FPU is the filter paper enzyme activity unit of cellulase, and a FPU unit representation is substrate with Whatman1 filter paper, and cellulase is at pH4.8, and 50 ° of C of temperature produce the used enzyme amount of 1 μ mol glucose in 1min.
FXU is 1, the enzyme of 4-beta-xylanase unit alive, a FXU unit refers to inscribe 1, the 4-beta-xylanase is at pH4.8, discharges the amount of the reducing sugar that is equivalent to 7.8 μ mol wood sugars under 50 ℃ of the temperature in 1min from the Arabic glycosyl polyxylose of azo wheat (Azo-wheat arabinoxylan).
CBU is the enzyme of the cellobiase unit that lives, and a CBU unit refers to the cellobiose to be substrate, and 1,4-beta fibers disaccharidase is at pH4.8,50 times 1min hydrolysis of temperature, 1 μ mol1, the enzyme amount that 4-beta fibers disaccharides glycosidic bond is required.
Wherein, the beta fibers disaccharidase is suitable excessive in the prozyme, to prevent that the accumulation because of cellobiose reduces the efficient that cellulose hydrolysis is glucose in the enzymic hydrolysis process.
Wherein, the prozyme consumption is the prozyme of 5～40FPU/ (g Mierocrystalline cellulose), and enzyme work is represented with Mierocrystalline cellulose filter paper enzyme activity FPU.
Wherein, the combinative enzyme hydrolysis temperature is 40～50 ° of C, and pH is 4.0～5.0, and hydrolysis time is 12～120h.Cross the activity that low or too high enzymic hydrolysis temperature and pH value can reduce enzyme liquid.The too short enzymic hydrolysis time will influence enzymic hydrolysis efficient, and the prolongation enzymic hydrolysis time is conducive to improve enzymic hydrolysis efficient in theory, but can cause the increase of investment in fixed assets and working cost.The combinative enzyme hydrolysis optimum condition is that temperature is 45～50 ° of C, and pH is 4.5～5.0, and hydrolysis time is 24～72h.
Wherein, enzymic hydrolysis saccharification react temperature is 40～50 ° of preferred 50 ° of C of C(), the reaction times is 12～120h, carries out the enzymic hydrolysis saccharification react under the agitation condition, the preferred 180r/min of mixing speed 150～200r/min().Temperature control preferably adopts air heating or heating in water bath.
Beneficial effect: the present invention presents following characteristic after sulfonated lignin is added the enzymic hydrolysis saccharification system of lignocellulose raw material as additive:
(1) can effectively improve enzymolysis efficiency, not change under enzyme dosage and the enzymic hydrolysis time prerequisite, lignocellulose raw material total reducing sugar transformation efficiency can improve 20%～50% after the pre-treatment;
(2) can effectively shorten the enzymic hydrolysis time, behind the enzyme digestion reaction 6h, enzymic hydrolysis total reducing sugar transformation efficiency can reach the effect of no any additives enzymolysis 48h;
(3) can effectively reduce enzyme dosage, tallerly when the enzymolysis total reducing sugar transformation efficiency when enzyme dosage is 20FPU/g is more additive-free than 40FPU/g go out 15%;
(4) can effectively improve the activity of enzyme, enzyme dosage increases to 40FPU/g by 20FPU/g, and enzymolysis total reducing sugar transformation efficiency improves 20%, and when not having sulfonated lignin and adding, increment has only 7.4%.
(5) this additive can not produce any disadvantageous effect to follow-up fermentation.
The present invention can effectively improve the total reducing sugar transformation efficiency of enzymolysis substrate by add sulfonated lignin in the enzymic hydrolysis process.Wherein softwood total reducing sugar transformation efficiency can improve more than 70%, and pourous wood and grass total reducing sugar transformation efficiency can be brought up to more than 80%.
Sulfonated lignin of the present invention can derive from any material that contains xylogen, as reproducible plant fiber material, pulping waste liquor or enzymolysis residue etc., xylogen is handled and can be obtained through suitable modification.Cost is low, and the source is wide, easily obtains, easily preparation.After enzymatic hydrolysis reaction finished, this additive can also efficient reuse in subsequent process, can effectively reduce the technical operation cost.If continue to build based on former pulp mill existing installation, adopt original pulping equipment to carry out the pretreatment stage of bio-ethanol preparation, to lignin in the pre-treatment black liquor simply process the back as auxiliary agent efficient for follow-up enzyme hydrolysis process, will accelerate the commercialization process of bio-ethanol preparation.
Description of drawings
Fig. 1 is that the interpolation of sulfonated lignin is to the influence of green liquor pre-treatment needlebush enzymolysis sugar transformation efficiency.
Fig. 2 is that the interpolation of sulfonated lignin is to the influence of green liquor pre-treatment needlebush enzymolysis time.
Fig. 3 is that the interpolation of sulfonated lignin is to the influence of green liquor pre-treatment needlebush enzyme dosage.
Fig. 4 is that the interpolation of sulfonated lignin is to the influence (to raw material sugar) of green liquor pre-treatment leaf wood enzymolysis sugar transformation efficiency.
Fig. 5 is that the interpolation of sulfonated lignin is to the influence (to slurry sugar) of green liquor pre-treatment leaf wood enzymolysis sugar transformation efficiency.
Fig. 6 is that the interpolation of sulfonated lignin is to the influence (to raw material sugar) of green liquor pre-treatment grass enzymolysis sugar transformation efficiency.
Fig. 7 is that the interpolation of sulfonated lignin is to the influence (to slurry sugar) of green liquor pre-treatment grass enzymolysis sugar transformation efficiency.
Fig. 8 is that the interpolation of sulfonated lignin is to the influence of unprocessed needlebush wood powder enzymolysis sugar transformation efficiency.
Fig. 9 is that the interpolation of sulfonated lignin is to the influence of unprocessed leaf wood wood powder enzymolysis sugar transformation efficiency.
Figure 10 is that the interpolation of sulfonated lignin is to the influence of unprocessed grass wood powder enzymolysis sugar transformation efficiency.
Embodiment
According to elaborating of following embodiment, the technician in this area and other fields can better understand the present invention.The described content of embodiment only is used for explanation the present invention, and should also can not limit the present invention described in detail in the claims.
The employed sulfonated lignin of following examples is a kind of sodium lignosulfonate of low sulfonation degree available from the Reax85A of Westvaco company.
Embodiment 1: the pre-treatment and the enzymatic hydrolysis process that with the needlebush are raw material.
Raw material:
The needlebush of experiment usefulness originates from Fujian, is provided by the paper mill, Fujian.Wood chip is about 1.5cm, wide about 2.5cm, thick about 0.3cm.Air-dry back balance moisture is standby.
By cellulase, zytase and beta fibers disaccharidase etc. mixes with mixed enzyme solution in enzymic hydrolysis.The enzyme of three kinds of unit of enzyme volumes is lived than being cellulase: zytase: beta fibers disaccharidase=1FPU:1.2FXU:1CBU.The enzyme work of mixed enzyme solution is counted 20FPU/ml with the cellulase filter paper enzyme activity.
Green liquor pretreatment:
Green liquor derives from the chemical recovery system of kraft process, mainly is made up of yellow soda ash and sodium sulphite.In homemade 10 * 1L oil bath pan, every jar takes by weighing the air-dried wood chip that is equivalent to over dry 150g, solid-to-liquid ratio is 1:4(g/mL) the adding cooking liquor, alkali charge is 20%, and sulphidity is 25%, after wood chip and cooking liquor mix, behind 80 ° of C turning down preimpregnation 30min, speed with 2 ° of C/min is warming up to 170 ℃, and after the boiling H factor reached 800, pre-treatment was finished.With digester take out be cooled to room temperature with cold water after, spend the night with wood chip after hot water injection's pre-treatment, with pharmaceutical chemicals residual in the wood chip and xylogen flush away and centrifuge dehydration, equilibrium water content and analyze yield in sealed vessel.All the other pre-treatment of chips are discongested pulping with paste roller mills (KRK, mill diameter 300mm, rotating speed 3000r/min), through centrifuge dehydration, tear up that to be placed in the sealed plastic bag equilibrium water content standby.
Enzyme hydrolysis process:
As in the 150mL Erlenmeyer flask, sulfonated lignin adds acetic acid-sodium-acetate buffer by a certain percentage with the pre-treatment of chips behind the mill (having discongested pulping), and the addition of sulfonated lignin is respectively 0,0.01,0.02,0.05,0.1,0.2,0.3,0.4,0.5g/g over dry substrate, concentration of substrate is 5wt%, and the pH value is 4.8, and enzyme dosage is for being respectively 20FPU/g and 40FPU/g.Erlenmeyer flask is placed constant temperature oscillator DHZ-2102, at 180r/min, oscillation treatment 6-96h under 50 ℃ of conditions.Hydrolysis finishes back centrifugal 15min under 5000r/min, obtains upper strata enzymic hydrolysis liquid glucose.
Experimental result:
The chemical ingredients of needlebush sees Table 1 before and after the pre-treatment.After the pre-treatment of needlebush process green liquor, the yield of slurry is 73.1%, and the decline of yield mainly is because the part of xylogen removes and carbohydrate degraded to a certain degree.Pretreatment fluid pH value is 9.4, is generally 13～14 much smaller than the pH(of kraft cooking).Low pH can effectively avoid alkaline hydrolysis and the secondary peeling reaction of carbohydrate in the raw material, keeps higher pre-treatment yield, and simultaneously suitable swollen fiber is conducive to the raising of follow-up enzymic hydrolysis sugar transformation efficiency.
Table 1. needlebush raw material and the composition analysis of green liquor pre-treatment disposed slurry
Green liquor pre-treatment disposed slurry is through the KRK defibrination, and the slurry that obtains disperseing carries out enzymolysis.Table 2 and table 3 are respectively the sulfonated lignin addition to the influence of enzymic hydrolysis yield and enzymic hydrolysis transformation efficiency.When not having sulfonated lignin to add, the yield of enzymolysis dextran, xylan, seminose and total reducing sugar is respectively 15.4%, 3.2%, 3.3% and 21.9%.In the enzymic hydrolysis system, add a certain proportion of sulfonated lignin in acetic acid-sodium-acetate buffer, enzymatic saccharification efficient obviously improves along with the increase of sulfonated lignin adding proportion, when the sulfonated lignin addition is 0.3g/g, enzymolysis sugar yield reaches maximum, at this moment, the yield of dextran, xylan, seminose and total reducing sugar is respectively 29.8%, 4.5%, 4.5% and 38.8%.Enzymolysis total reducing sugar yield amplification is up to 77%.
Table 2. sulfonated lignin addition is to the influence of enzymolysis sugar yield (to raw material)
Table 3. sulfonated lignin addition is to the influence of enzymolysis sugar transformation efficiency (to sugar in the raw material)
1Sugared content * 100% in enzymolysis sugar transformation efficiency=enzymolysis sugar yield/slurry
Corresponding enzymolysis sugar transformation efficiency is also deferred to above-mentioned rule, and along with the increase of sulfonated lignin addition obviously raises, when the sulfonated lignin addition was 0.3g/g, enzymolysis total reducing sugar transformation efficiency was 62%, 35.1% during far above no sulfonated lignin interpolation.
The interpolation of sulfonated lignin can not only obviously improve enzymolysis efficiency, can also shorten enzymolysis time.When the addition of sulfonated lignin was 0.3g/g, the total reducing sugar transformation efficiency of enzymolysis 24h correspondence was 70.4%, and the total reducing sugar transformation efficiency 47.7% when not having interpolation far above 96h is seen Fig. 2, and only needed enzymolysis 6h just can reach the non-additive effect of enzymolysis 48h.
Secondly, the interpolation of sulfonated lignin can also reduce enzyme dosage.Enzyme dosage is the 20FPU/g Mierocrystalline cellulose, and when the addition of sulfonated lignin was 0.3g/g, enzymolysis total reducing sugar transformation efficiency was 60.7%, was higher than the total reducing sugar transformation efficiency 45.4% that the 40FPU/g Mierocrystalline cellulose does not have interpolation, saw Fig. 3.
In addition, as shown in Figure 3, the activity that the interpolation of sulfonated lignin not only can inhibitory enzyme can effectively improve the activity of enzyme on the contrary.When not having sulfonated lignin to add, enzyme dosage is elevated to 40FPU/g from 20FPU/g, and enzymolysis efficiency has only improved 7.4%, and when the sulfonated lignin addition was 0.3g/g, enzyme dosage was elevated to 40FPU/g from 20FPU/g, and enzymolysis efficiency can improve 20%.
Embodiment 2: the pre-treatment and the enzymatic hydrolysis process that with the leaf wood are raw material.
Raw material:
The leaf wood of experiment usefulness originates from the northern Suzhou.Wood chip is about 1.5cm, wide about 2.5cm, thick about 0.2cm.Air-dry back balance moisture is standby.
By cellulase, zytase and beta fibers disaccharidase mix with mixed enzyme solution in enzymic hydrolysis.The enzyme of three kinds of unit of enzyme volumes is lived than being cellulase: zytase: beta fibers disaccharidase=1FPU:1.2FXU:1CBU.The enzyme work of mixed enzyme solution is counted 20FPU/ml with the cellulase filter paper enzyme activity.
Green liquor pretreatment:
Green liquor derives from the chemical recovery system of kraft process, mainly is made up of yellow soda ash and sodium sulphite.In homemade 10 * 1L oil bath pan, every jar takes by weighing the air-dried wood chip that is equivalent to over dry 150g, solid-to-liquid ratio is 1:4(g/mL) the adding cooking liquor, alkali charge is 20%, and sulphidity is 25%, after wood chip and cooking liquor mix, behind 80 ℃ of turning down preimpregnation 30min, speed with 2 ° of C/min is warming up to 160 ° of C, and after the boiling H factor reached 400, pre-treatment was finished.With digester take out be cooled to room temperature with cold water after, spend the night with wood chip after hot water injection's pre-treatment, with pharmaceutical chemicals residual in the wood chip and xylogen flush away and centrifuge dehydration, equilibrium water content and analyze yield in sealed vessel.All the other pre-treatment of chips are discongested pulping with paste roller mills (KRK, mill diameter 300mm, rotating speed 3000r/min), through centrifuge dehydration, tear up that to be placed in the sealed plastic bag equilibrium water content standby.
Enzyme hydrolysis process:
With the pre-treatment of chips behind the mill (having discongested pulping) as in the 150mL Erlenmeyer flask, sulfonated lignin adds acetic acid-sodium-acetate buffer by a certain percentage, and addition is respectively 0,0.05,0.1,0.2,0.3,0.4,0.5g/g over dry substrate, concentration of substrate is that 5%, pH value is 4.8, and enzyme dosage is 40FPU/g.Erlenmeyer flask is placed constant temperature oscillator DHZ-2102, at 180r/min, oscillation treatment 48h under 50 ° of C conditions.Hydrolysis finishes back centrifugal 15min under 5000r/min, obtains upper strata enzymic hydrolysis liquid glucose.
Experimental result:
Leaf wood raw material total sugar content is, 64.5%, and wherein arabinan content has only 2.2%, degrade substantially through arabinan after the green liquor pre-treatment, so total reducing sugar refers to the summation of dextran and xylan behind the enzymolysis.Content of lignin is 25.2% in the pre-treatment disposed slurry, is 19.9% with respect to raw material, and the delignification rate reaches 25%, and the part of xylogen removes and will be conducive to the raising of follow-up enzymolysis efficiency.
Table 4. leaf wood raw material and the composition analysis of green liquor pre-treatment disposed slurry
Green liquor pre-treatment disposed slurry is through the KRK defibrination, and the slurry that obtains disperseing carries out enzymolysis.Table 5 and table 6 are respectively the sulfonated lignin addition to the influence of enzymic hydrolysis yield and enzymic hydrolysis transformation efficiency.When not having sulfonated lignin to add, the yield of enzymolysis dextran, xylan and total reducing sugar is respectively 30.3,8.7 and 39.0%.In enzymatic hydrolysis system, add a certain proportion of sulfonated lignin in acetic acid-sodium-acetate buffer, enzymatic saccharification efficient obviously improves along with the increase of sulfonated lignin adding proportion, when the sulfonated lignin addition is 0.2g/g, enzymolysis sugar yield reaches maximum, and the yield of dextran, xylan and total reducing sugar is respectively 41.9,10.4 and 52.3%.
Corresponding enzymolysis sugar transformation efficiency is also deferred to above-mentioned rule, raising along with the increase of sulfonated lignin addition, when the sulfonated lignin addition is 0.2g/g, is 81.2% to raw material total reducing sugar transformation efficiency, this moment, the transformation efficiency to total reducing sugar in the slurry reached 93.3%, as Fig. 5.
Table 5. sulfonated lignin addition is to the influence of enzymolysis sugar yield
Table 6. sulfonated lignin addition is to the influence of enzymolysis sugar transformation efficiency
Embodiment 3: the pre-treatment and the enzymatic hydrolysis process that with the grass are raw material.
Raw material:
Grass south reed of experiment usefulness originates from the Hunan.Raw material is cut to being about 3cm, and air-dry back balance moisture is standby.
By cellulase, zytase and beta fibers disaccharidase mix with mixed enzyme solution in enzymic hydrolysis.The enzyme of three kinds of unit of enzyme volumes is lived than being cellulase: zytase: beta fibers disaccharidase=1FPU:1.2FXU:1CBU.The enzyme work of mixed enzyme solution is counted 20FPU/ml with the cellulase filter paper enzyme activity.
Green liquor pretreatment:
Green liquor derives from the chemical recovery system of kraft process, mainly is made up of yellow soda ash and sodium sulphite.In homemade 10 * 1L oil bath pan, every jar takes by weighing the air-dried wood chip that is equivalent to over dry 100g, solid-to-liquid ratio is 1:6(g/mL) the adding cooking liquor, alkali charge is 12%, sulphidity is 20%, after wood chip and cooking liquor mix, behind 80 ℃ of turning down preimpregnation 30min, speed with 2 ° of C/min is warming up to 140 ° of C, and pre-treatment is finished behind the insulation 1h.With digester take out be cooled to room temperature with cold water after, with hot water injection's pre-treatment disposed slurry, with pharmaceutical chemicals residual in the wood chip and xylogen flush away and centrifuge dehydration, equilibrium water content and analyze yield in sealed vessel.All the other pre-treatment of chips are discongested pulping with paste roller mills (KRK, mill diameter 300mm, rotating speed 3000r/min), through centrifuge dehydration, tear up that to be placed in the sealed plastic bag equilibrium water content standby.
Enzyme hydrolysis process:
With the pre-treatment of chips behind the mill (having discongested pulping) as in the 150mL Erlenmeyer flask, sulfonated lignin adds acetic acid-sodium-acetate buffer by a certain percentage, and addition is respectively 0,0.05,0.1,0.2,0.3,0.4,0.5g/g over dry substrate, concentration of substrate is that 5%, pH value is 4.8, and enzyme dosage is for being respectively the 20FPU/g Mierocrystalline cellulose.Erlenmeyer flask is placed constant temperature oscillator DHZ-2102, at 180r/min, oscillation treatment 48h under 50 ℃ of conditions.Hydrolysis finishes back centrifugal 15min under 5000r/min, obtains upper strata enzymic hydrolysis liquid glucose.
Experimental result:
Grass raw material total sugar content is 65.8%, is 79.1% through total reducing sugar after the green liquor pre-treatment to slurry, is 59.2% to raw material, and the total reducing sugar degradation rate is 10%.Content of lignin is 13.2% in the pre-treatment disposed slurry, has only 9.9% with respect to raw material, and the delignification rate is up to 57%.This explanation green liquor pre-treatment has extremely excellent selectivity to the delignification of grass, removed most lignin in slurry having kept 90% high glycan sugar.Passage has been opened in removing to follow-up enzymolysis of xylogen, will be conducive to the raising of follow-up enzymolysis efficiency.
Table 7. grass raw material and the composition analysis of green liquor pre-treatment disposed slurry
Green liquor pre-treatment disposed slurry is through the KRK defibrination, and the slurry that obtains disperseing carries out enzymolysis.Table 8 and table 9 are respectively the sulfonated lignin addition to the influence of enzymic hydrolysis yield and enzymic hydrolysis transformation efficiency.When not having sulfonated lignin to add, the yield of enzymolysis dextran, xylan, pectinose and total reducing sugar is respectively 31.0,10.7,0.6 and 42.2%.In enzymatic hydrolysis system, add a certain proportion of sulfonated lignin in acetic acid-sodium-acetate buffer, enzymatic saccharification efficient obviously improves along with the increase of sulfonated lignin adding proportion, when the sulfonated lignin addition is 0.1g/g, enzymolysis sugar yield reaches maximum, and the yield of dextran, xylan, arabinan and total reducing sugar is respectively 40.3,14.9,0.9 and 56.2%.
Corresponding enzymolysis sugar transformation efficiency is also deferred to above-mentioned rule, raising along with the increase of sulfonated lignin addition, when the sulfonated lignin addition is 0.1g/g, is 85.4% to raw material total reducing sugar transformation efficiency, this moment, the transformation efficiency to total reducing sugar in the slurry reached 95.9%, as Fig. 7.That is to say that every gram substrate only need add 0.1 gram sulfonated lignin just can make the raising rate of enzymolysis efficiency reach 33%, this illustrates again that also this kind additive is a kind of enzymolysis of lignocellulose raw material efficiently auxiliary agent.
Table 8. sulfonated lignin addition is to the influence of enzymolysis sugar yield
Table 9. sulfonated lignin addition is to the influence of enzymolysis sugar transformation efficiency
Comparative Examples 4: be the direct enzymatic hydrolysis process of wood powder of raw material with the needlebush.
Raw material:
The needlebush of experiment usefulness originates from Fujian, is provided by the paper mill, Fujian.Wood chip is about 1.5cm, wide about 2.5cm, thick about 0.3cm.It is standby to wear into dressing sieve to 40～80 order back balance moisture with pulverizer.
By cellulase, zytase and beta fibers disaccharidase mix with mixed enzyme solution in enzymic hydrolysis.The enzyme of three kinds of unit of enzyme volumes is lived than being cellulase: zytase: beta fibers disaccharidase=1FPU:1.2FXU:1CBU.The enzyme work of mixed enzyme solution is counted 20FPU/ml with the cellulase filter paper enzyme activity.
Enzyme hydrolysis process:
As in the 150mL Erlenmeyer flask, sulfonated lignin adds acetic acid-sodium-acetate buffer by a certain percentage with needlebush 40～80 order wood powders, and addition is respectively 0,0.05,0.1,0.2,0.3,0.4g/g over dry substrate, concentration of substrate is that 5%, pH value is 4.8, and enzyme dosage is 40FPU/g.Erlenmeyer flask is placed constant temperature oscillator DHZ-2102, at 180r/min, oscillation treatment 48h under 50 ℃ of conditions.Hydrolysis finishes back centrifugal 15min under 5000r/min, obtains upper strata enzymic hydrolysis liquid glucose.
Experimental result:
Experiment shows that the interpolation of sulfonated lignin can not improve the enzymolysis efficiency of not pretreated needlebush wood powder.Along with the interpolation of sulfonated lignin, enzymolysis sugar yield and transformation efficiency are constant substantially, as table 10 and Fig. 8.
Table 10. sulfonated lignin addition is to the influence of needlebush wood powder enzymolysis sugar yield
Comparative Examples 5: be the direct enzymatic hydrolysis process of wood powder of raw material with the leaf wood.
Raw material:
The leaf wood wood of experiment usefulness originates from the northern Suzhou.Wood chip is about 1.5cm, wide about 2.5cm, thick about 0.2cm.It is standby to wear into dressing sieve to 40～80 order back balance moisture with pulverizer.
Experiment uses enzyme and enzyme hydrolysis process with Comparative Examples 4.
Experimental result:
Experiment shows that the interpolation of sulfonated lignin can not improve the enzymolysis efficiency of not pretreated leaf wood wood powder.Along with the interpolation of sulfonated lignin, enzymolysis sugar yield and transformation efficiency are constant substantially, as table 11 and Fig. 9.
Table 11. sulfonated lignin addition is to the influence of leaf wood wood powder enzymolysis sugar yield
Comparative Examples 6: be the direct enzymatic hydrolysis process of wood powder of raw material with the grass.
Raw material:
Grass south reed of experiment usefulness originates from the Hunan.Raw material is cut to being about 3cm, and it is standby to wear into dressing sieve to 40～80 order back balance moisture with pulverizer.
Experiment uses enzyme and enzyme hydrolysis process with Comparative Examples 4.
Experimental result:
Table 12. sulfonated lignin addition is to the influence of grass wood powder enzymolysis sugar yield
Experiment shows that the interpolation of sulfonated lignin can not improve the enzymolysis efficiency of not pretreated grass wood powder.Along with the interpolation of sulfonated lignin, enzymolysis sugar yield and transformation efficiency are constant substantially, as table 12 and Figure 10.
Claims (10)
1. the application of sulfonated lignin in the performing enzyme hydrolysis on wood fiber raw material saccharification.
2. application according to claim 1 is characterized in that, described sulfonated lignin is available from market or prepare voluntarily.
3. application according to claim 2 is characterized in that, described sulfonated lignin is prepared by the material modification that contains xylogen.
4. application according to claim 3 is characterized in that, the described material that contains xylogen is plant fiber material, pulping waste liquor or enzymolysis residue.
5. application according to claim 1 is characterized in that, described lignocellulose raw material is needlebush, leaf wood or grass.
6. application according to claim 1 is characterized in that, sulfonated lignin is added in acetic acid-sodium-acetate buffer, adds in the pretreated enzymolysis substrate lignocellulose raw material again, after mixing, adds enzyme liquid, carries out the enzymic hydrolysis saccharification react.
7. application according to claim 6 is characterized in that, the adding quality of described sulfonated lignin is 1～50% of pretreated lignocellulose raw material quality.
8. application according to claim 6 is characterized in that, described enzymolysis concentration of substrate scope is 1～10wt%
9. application according to claim 6 is characterized in that, described enzyme is the mixture of cellulase, zytase and beta fibers disaccharidase; The enzyme of three kinds of unit of enzyme volumes is lived than being cellulase: zytase: beta fibers disaccharidase=1FPU:1.2FXU:1CBU.
10. application according to claim 6 is characterized in that, enzymic hydrolysis saccharification react temperature is 40～50 ° of C, and the reaction times is 12～120h, carries out enzymic hydrolysis saccharification react, mixing speed 150～200r/min under the agitation condition.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103497974A (en) * | 2013-09-02 | 2014-01-08 | 华南理工大学 | Method for improving cellulose enzymatic hydrolysis and saccharification yield |
| CN109022497A (en) * | 2018-09-13 | 2018-12-18 | 山东科技大学 | A method of improving lignocellulosic enzymatic hydrolysis and fermentation producing and ethanol yield |
| CN111676257A (en) * | 2020-06-16 | 2020-09-18 | 天津科技大学 | Method for improving efficiency of producing fermentable sugar by high-concentration enzymatic hydrolysis of broad-leaved wood cellulose |
| CN113215209A (en) * | 2021-06-11 | 2021-08-06 | 南京林业大学 | Method for promoting hydrolysis of lignocellulose |
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2013
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Non-Patent Citations (1)
| Title |
|---|
| ZJ WANG: "Lignosulfonate and elevated pH can enhance enzymatic saccharification of lignocelluloses", 《LIGNOSULFONATE AND ELEVATED PH CAN ENHANCE ENZYMATIC SACCHARIFICATION OF LIGNOCELLULOSES》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103497974A (en) * | 2013-09-02 | 2014-01-08 | 华南理工大学 | Method for improving cellulose enzymatic hydrolysis and saccharification yield |
| CN109022497A (en) * | 2018-09-13 | 2018-12-18 | 山东科技大学 | A method of improving lignocellulosic enzymatic hydrolysis and fermentation producing and ethanol yield |
| CN109022497B (en) * | 2018-09-13 | 2021-08-27 | 山东科技大学 | Method for improving yield of ethanol produced by lignocellulose enzymolysis and fermentation |
| CN111676257A (en) * | 2020-06-16 | 2020-09-18 | 天津科技大学 | Method for improving efficiency of producing fermentable sugar by high-concentration enzymatic hydrolysis of broad-leaved wood cellulose |
| CN113215209A (en) * | 2021-06-11 | 2021-08-06 | 南京林业大学 | Method for promoting hydrolysis of lignocellulose |
| CN113215209B (en) * | 2021-06-11 | 2023-11-03 | 南京林业大学 | Method for promoting lignocellulose hydrolysis |
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