CN105177083A - Method for improving enzymolysis efficiency of lignocellulose biomass - Google Patents
Method for improving enzymolysis efficiency of lignocellulose biomass Download PDFInfo
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Abstract
The invention discloses a method for improving the enzymolysis efficiency of lignocellulose biomass. The lignocellulose biomass is pretreated by coupling surfactant with ion liquid. The method specifically comprises the following steps that firstly, lignocellulose biomass raw materials are dried, smashed and ground to obtain powder; secondly, the ion liquid for treating the lignocellulose biomass is heated for 20-60 min at 100-150 DEG C, and constant stirring is conducted; thirdly, the powder obtained in the first step is weighed, the ion liquid treated in the second step and the surfactant are added, and stirring is conducted for 0.5-4 h under the condition of 70-130 DEG C; fourthly, deionized water is added, centrifugation is conducted for 15 min at the high speed of 7,000 r/min, then supernatant liquid is separated out and stored, and substratum sediment is taken, washed with the deionized water repeatedly and dried for 18-30 h at 60 DEG C for enzymolysis after the deionized liquid is removed. The pretreatment technology can remarkably improve the enzymolysis efficiency of the lignocellulose biomass.
Description
Technical field
The invention belongs to biomass processes technical field.More specifically, a kind of method improving lignocellulose biomass enzymolysis efficiency is related to.
Background technology
In recent years, along with the minimizing increasingly of the non-renewable fossil resources total amount such as oil, coal, the importance of ligno-cellulosic materials is day by day remarkable.Therefore, take lignocellulose as the study hotspot that raw material prepares that ethanol is biomass energyization utilization, this technology utilizes the lignocellulosic material such as agricultural stalk, municipal wastes to carry out processing to obtain the clean fuels such as ethanol.But because Mierocrystalline cellulose itself is the chain-like macromolecule compound be made up of a large amount of glucosyl group, its complex structure and there is mass crystallization district, add the package action of xylogen and hemicellulose, it is made to be difficult to water-soluble and general organic solvent, traditional pretreatment system has copper ammon solution system, dithiocarbonic anhydride/sodium hydroxide system steam explosion, high temperature pyrolysis, acid-base method, biological degradation etc., although treatment effect is good, because its consumption is large, be difficult to reclaim, and seriously polluted.Therefore be badly in need of finding a kind of green and effective treatment technology.
In recent years, ionic liquid, with the advantage of its uniqueness, becomes the excellent selection of biomass pre-treatment system.Compared with traditional chemical solvents, ionic liquid has non-volatility, thermostability is high with chemical stability, solvability good, performance is adjustable, be easy to features such as being separated, easily reclaim and recycle.
But carrying out and going deep into along with research, the drawback of its system also demonstrates gradually, the ion liquid dissolving effect that such as cost is low is bad, and the ionic liquid cost that solute effect is good is more high, and can cause Mierocrystalline cellulose recrystallization after ionic liquid process cellulose materials in washing process.
Summary of the invention
The technical problem to be solved in the present invention is the defect and the deficiency that overcome existing biomass energy treatment technology, a kind of tensio-active agent coupled ion liquid preconditioning technique is provided, tensio-active agent is utilized to have the effect reducing surface tension and surface free energy, in the process of ionic liquid process lignocellulose, add tensio-active agent and can reduce ionic liquid surface tension, the drawback appeared in unitary system can be overcome, thus improve ionic liquid to the dissolving power of ligno-cellulosic materials, improve enzymolysis sugar yield, reduce ionic liquid consumption, obtain a kind of green and efficient preprocessing lignocellulose MATERIALS METHODS, cost can be reduced and reduce environmental pollution.
The object of this invention is to provide a kind of method improving lignocellulose biomass enzymolysis efficiency.
Another object of the present invention is to provide aforesaid method and is improving the application in lignocellulose biomass enzymolysis efficiency, is especially improving the application in lignocellulose enzymolysis efficiency.
Above-mentioned purpose of the present invention is achieved through the following technical solutions:
Improve a method for lignocellulose biomass enzymolysis efficiency, be after adopting tensio-active agent coupled ion liquid to carry out pre-treatment to lignocellulose biomass, then carry out enzymolysis.
Particularly, the method for above-mentioned raising lignocellulose biomass enzymolysis efficiency comprises the steps:
S1. lignocellulose biomass raw material drying, pulverizing, grinding obtain powder;
S2. ionic liquid used for treatment of lignocellulosic biomass is heated 20 ~ 60min at 100 ~ 150 DEG C, and constantly stir;
S3. take the powder of step S1, add the ionic liquid after step S2 process and tensio-active agent, under 70 ~ 130 DEG C of conditions, stir 0.5 ~ 4h; Wherein, the weight ratio of powder and ionic liquid is 1:10, and the weight of tensio-active agent is 0.1 ~ 2% of powder;
S4. add deionized water, to isolate supernatant liquid after 7000r/min high speed centrifugation 15min, store; Take off a layer throw out, repeatedly clean with deionized water, remove ionic liquid, at 60 DEG C of drying 18 ~ 30h, for enzymolysis.
Wherein, preferably, ionic liquid described in step S2 is chlorination-1-butyl-3-Methylimidazole ([BMIM] Cl) or 1-allyl group-3-Methylimidazole villaumite ([AMIM] Cl).
Preferably, step S2 heats 30min at 120 DEG C.
Preferably, tensio-active agent described in step S3 is sodium lauryl sulphate (SDS) or cetyl trimethylammonium bromide (CTAB).
Preferably, the weight of tensio-active agent described in step S3 is 1% of powder.
Preferably, the condition stirred described in step S3 is 110 DEG C and stirs 1h.
Preferably, described in step S3, the method for enzymolysis is as follows:
According to the ratio of 2.5w/v%, get in the sodium citrate buffer and 0.02% sodium azide solution that pretreated lignocellulose biomass adds 0.1MpH4.8, shake up, add 50FPU/g cellulase (NovozymeNS22086) and 40CBU/g glucuroide (NovozymeNS22118) again, under 50 ± 0.5 DEG C of constant temperatures, 150r/min carries out saccharification 12 ~ 72h, and 13500rpm is centrifugal after completion of the reaction obtains enzyme hydrolyzate.
In addition, the application of method in treatment of lignocellulosic biomass of above-mentioned raising lignocellulose biomass enzymolysis efficiency, also within protection scope of the present invention.Specifically improving the application in lignocellulose enzymolysis efficiency.
The present invention is found by large quantity research, after ionic liquid process cellulose materials in washing process, Mierocrystalline cellulose recrystallization can be caused, thus affect the effect of cellulase hydrolysis, add the tension force that tensio-active agent can reduce ionic liquid, be conducive to treating processes intermediate ion liquid to contact more fully with substrate, increase the dissolving power of ionic liquid, improve fiber transformation efficiency.Research display, tensio-active agent coupled ion liquid pre-treatment rice straw is compared with untreated rice straw, and enzymolysis reducing sugar output can improve 50% ~ 54%; Compared with independent ionic liquid process rice straw, enzymolysis reducing sugar output can improve 8% ~ 15%.
The present invention has following beneficial effect:
1, with the Measures compare such as traditional dithiocarbonic anhydride/sodium hydroxide system steam explosion, high temperature pyrolysis, acid-base method, the inventive method processing efficiency is high, reaction conditions is gentle, corrosion-free and less to environmental impact to equipment, can not produce pollution.
2, compared with the ionic liquid pretreatment of unitary system, overcome the various drawbacks of single ionic liquid pretreatment system, the present invention is by adding tensio-active agent, reduce the effect of surface tension and surface free energy, be conducive to treating processes intermediate ion liquid to contact more fully with substrate, increase the dissolving power of ionic liquid, improve fiber transformation efficiency, improve the enzymolysis efficiency of lignocellulosic material.
Accompanying drawing explanation
Fig. 1 is that the rice straw SEM after different pretreatments process schemes; Wherein, each group processing mode is respectively: a is Untreatedricestraw, b be 1%SDS, c be 1%CTAB, d be BMIMCl, e be BMIMCl+1%SDS, f is BMIMCl+1%CTAB.
Fig. 2 is the impact of different pretreatments on rice straw hydrolysis result.
Fig. 3 is that [BMIM] Cl and tensio-active agent adding proportion are on the impact of rice straw hydrolysis result.
Fig. 4 is tensio-active agent coupled ion liquid ([AMIM] Cl) impact on rice straw hydrolysis result.
Embodiment
Further illustrate the present invention below in conjunction with Figure of description and specific embodiment, but embodiment does not limit in any form to the present invention.Unless stated otherwise, the present invention adopts reagent, method and apparatus are the art conventional reagent, method and apparatus.
Unless stated otherwise, agents useful for same of the present invention and material are commercial.
embodiment 1
1, the collection of lignocellulose sample
The lignocellulosic material of this experiment is rice straw.Rice straw carries out air-dry process after collecting, and pulverizes after pulverizing, is the sample of this experiment.Rice straw is stored in the plastics bag of sealing, in drying place room temperature preservation.
2, tensio-active agent coupled ion liquid pre-treatment rice straw powder
(1) before pre-treatment rice straw powder, ionic liquid (chlorination-1-butyl-3-Methylimidazole, [BMIM] Cl) heated at 120 DEG C 30min and constantly stir, object removes the moisture in ionic liquid.
(2) 0.5g rice straw powder is taken in the bottle of band screw plug, add the tensio-active agent (sodium lauryl sulphate (SDS) or cetyl trimethylammonium bromide (CTAB)) of 5g ionic liquid and 1%, be placed on magnetic stirring apparatus, stir 0.5 ~ 4h under 70 ~ 130 DEG C of conditions after, add 45mL deionized water, mixed solution is placed in the centrifuge tube of 50mL, to be separated after 7000r/min high speed centrifugation 15min.Shift out supernatant liquid, store.
(3) take off a layer throw out, repeatedly clean with deionized water, remove ionic liquid, at 60 DEG C of drying at least 18h, taking-up is weighed, and preserves sample to carry out next step proteolysis assay.
3, the lignocellulose sample enzymatic saccharification before and after pre-treatment
Get 2.5%(w/v) rice straw powdered sample is placed in 50mL Erlenmeyer flask respectively before and after pre-treatment, add sodium citrate buffer and the 100 μ L0.02% sodium azide solutions of 0.1MpH4.8, shake up, then add 50FPU/g cellulase (NovozymeNS22086) and 40CBU/g glucuroide (NovozymeNS22118), be placed in 50 ± 0.5 DEG C of water bath with thermostatic control vibrators, rotating speed is saccharification 0,3,6 under 150r/min, 12,24,48,72h.React complete, 13500rpm centrifugal enzyme hydrolyzate, sampling DNS assay method analysis-reduction sugar content.
4, the mensuration of cellulase activity
(1) standard method adopting international theory and applied chemistry association (IUPAC) to recommend measures, and filter paper enzyme activity international unit equals per minute in enzymatic reaction and generates enzyme amount needed for 1 μm of ol glucose (representing with reducing sugar).The whatmanNo.1 filter paper bar (1 × 6cm) being rolled into tubular is put in test tube, add 0.5mL and dilute the cellulase solution after 50 times and 1.5mL citrate buffer solution (pH4.8) in 50 DEG C of water-bath 20min, reaction terminates the reducing sugar content of rear DNS method mensuration gained.
(2) glucose standard curve conversion enzyme activity is utilized to react reducing sugar amount.An enzyme activity unit U is defined as under this analysis condition, and per minute every milliliter disengages 1 μm of ole glucose ferment amount (μm ole/min.mL).
U=(W×N×5.56)/(V×t)=(W×1×5.56)/(0.05×20)
U: enzymic activity
W: glucose content (mg)
N: dilution ratio
V: enzyme liquid add-on (mL) in reaction solution
T: reaction times (min)
μm ole number of 5.56:1mg glucose
5, the mensuration of reducing sugar amount
(1) preparation (every 100mL) of DNS reagent: Seignette salt 30g is dissolved in 80mL hot water to be treated to dissolve completely, add NaOH1.6g more quantitatively to 100mL, add 1gDNS (3,5-Dinitrosalicylicacid) again, load in light tight bottle after dissolving.
(2) get 0.1mL, concentration known is 0.1mg/mL ~ 5mg/mL glucose solution, add in 0.3mL dinitrosalicylic acid (Dinitrosalicylicacid, DNS) reagent.Heat 5 minutes in the water of 100 DEG C, add 0.6mL distilled water afterwards, leave standstill to room temperature, after using point luminometer to measure its light absorption value under the light of wavelength for 575nm, the DNS typical curve of formation determination reducing sugar concentration.
(3) according to DNS typical curve, get 0.1mL determinand sample and add 0.3mLDNS reagent, adding 0.6mL distilled water after mixed solution is heated 5 minutes in the water of 100 DEG C puts to room temperature, utilize wavelength for 575nm rayed, and survey its light absorption value with a point luminometer, reducing sugar concentration can be conversed.
6, the comparison before and after rice straw powder pre-treating
(1) the present embodiment compares the effect of different treatment technology, and result shows, and the rice straw composition analysis after untreated and various different treatment technical finesse is as shown in table 1 below.
After individually adding 1%SDS, 1%CTAB, BMIMCl process rice straw, the decreasing ratio of xylogen is followed successively by 12.66%, 8.66%, 24.98%, and after 1%SDS coupling BMIMCl and 1%CTAB coupling BMIMCl pre-treatment rice straw, delignification rate can rise to 49.38% and 34.48% respectively.This also shows that tensio-active agent coupled ion liquid treatment rice straw more easily destroys the package action of xylogen, increases the touch opportunity of fiber and enzyme, thus improves enzymolysis efficiency.
Table 1 is untreated with the impact of different pretreatments technology on rice straw composition
Note: SDS is sodium lauryl sulphate; CTAB is cetyl trimethylammonium bromide; [BMIM] Cl is chlorination-1-butyl-3-Methylimidazole
(2) accompanying drawing 1 is that rice straw SEM after different pretreatments process schemes, and observe and find, untreated rice straw Mierocrystalline cellulose (a) structure is compacted very much, and surface is more smooth, does not have slight crack.The rice straw surface microscopic topographic that independent tensio-active agent (b, c) and independent ionic liquid (d) process loses original baculum shelf structure, and it is broken and with irregular cavity and groove to become cavity.And tensio-active agent coupled ion liquid treatment (e, f) larger destruction is suffered on rice straw surface, rice straw structure becomes more loose, do not see the baculum shelf structure that rice straw is original completely, present irregular particulate state or bulk, thus Mierocrystalline cellulose and enzyme accessibility are increased, improve hydrolysis result.
(3) accompanying drawing 2 is the impact of different pretreatments on rice straw hydrolysis result, as can be seen from Figure 2, all samples is obvious at the raising hydrolysis result of 0h to 24h, extend enzymolysis substantially to remain unchanged to 72h output of sugar, this is because when enzymolysis product runs up to a certain degree, inhibit the activity of enzyme conversely; Add surfactant SDS with the rice straw after CTAB process compared with untreated rice straw, cellulose conversion rate improves 53.72% and 49.82% respectively, this is mainly due in treating processes, the degraded of tensio-active agent to xylogen has certain promoter action, reduces the content of sample xylogen further.And in tensio-active agent coupled ion liquid pre-treatment rice straw process, being added with of tensio-active agent is beneficial to preprocessing process intermediate ion liquid and substrate contact, increase the dissolving power of ionic liquid, make treating processes more abundant, thus more easily destroy the package action of xylogen, also not easily greatly reduced by the composition of enzymolysis in rice straw after making process, improve enzymolysis output of sugar.
In addition, result of study display of the present invention, the best pretreatment condition of different surfaces promoting agent coupled ion liquid treatment rice straw: temperature is 110 DEG C, time is 60min, SDS and CTAB addition is 1%, 72h enzymolysis produces reducing sugar amount and is respectively 23.09mg/100mg rice straw, 21.92mg/100mg rice straw, improves 53.72% and 49.82% respectively than untreated rice straw fiber transformation efficiency.
embodiment 2 [BMIM] Cl and tensio-active agent adding proportion are on the impact of rice straw hydrolysis result
The present embodiment is with tensio-active agent adding proportion for single factor test, and processing mode is with embodiment 1, and best pretreatment condition: temperature is 110 DEG C, the time is that 60min, SDS and CTAB addition is 1%, 72h enzymolysis.
Have studied on [BMIM] Cl and tensio-active agent adding proportion on the impact of rice straw hydrolysis result, result as shown in Figure 3.
As shown in Figure 3, cellulose conversion rate increases in the rear downward trend that first rises with tensio-active agent addition, and when tensio-active agent addition is 1% of substrate, reducing sugar output reaches maximum.Being added with of tensio-active agent is beneficial to removes hemicellulose in rice straw and xylogen, thus improves pretreating effect.Tensio-active agent adds too much, may form micelle in the solution, can have side effects, cause hydrolysis result to decline to the hydrophobic interaction of xylogen and surfactant hydrophobic group.
embodiment 3 tensio-active agent coupled ion liquid ([AMIM] Cl) impact on rice straw hydrolysis result
The present embodiment studies different ionic liquid coupled surface promoting agents to the impact of rice straw hydrolysis result, and replace ionic liquid [BMIM] Cl with ionic liquid ([AMIM] Cl), result as shown in Figure 4.
As can be seen from Figure 4, all samples is obvious at the raising hydrolysis result of 0h to 24h, extends enzymolysis and substantially remains unchanged to 70h output of sugar, this is because when enzymolysis product runs up to a certain degree, inhibit the activity of enzyme conversely; Add surfactant SDS with the rice straw after CTAB process compared with untreated rice straw, cellulose conversion rate significantly improves, this is mainly due in treating processes, and the degraded of tensio-active agent to xylogen has certain promoter action, reduces the content of sample xylogen further.And in tensio-active agent coupled ion liquid pre-treatment rice straw process, being added with of tensio-active agent is beneficial to preprocessing process intermediate ion liquid and substrate contact, increase the dissolving power of ionic liquid, make treating processes more abundant, thus more easily destroy the package action of xylogen, also not easily greatly reduced by the composition of enzymolysis in rice straw after making process, improve enzymolysis output of sugar.
Claims (10)
1. improve a method for lignocellulose biomass enzymolysis efficiency, it is characterized in that, be after adopting tensio-active agent coupled ion liquid to carry out pre-treatment to lignocellulose biomass, then carry out enzymolysis.
2. improve the method for lignocellulose biomass enzymolysis efficiency according to claim 1, it is characterized in that, comprise the steps:
S1. lignocellulose biomass raw material drying, pulverizing, grinding obtain powder;
S2. ionic liquid used for treatment of lignocellulosic biomass is heated 20 ~ 60min at 100 ~ 150 DEG C, and constantly stir;
S3. take the powder of step S1, add the ionic liquid after step S2 process and tensio-active agent, under 70 ~ 130 DEG C of conditions, stir 0.5 ~ 4h; Wherein, the weight ratio of powder and ionic liquid is 1:10, and the weight of tensio-active agent is 0.1 ~ 2% of powder;
S4. add deionized water, to isolate supernatant liquid after 7000r/min high speed centrifugation 15min, store; Take off a layer throw out, repeatedly clean with deionized water, remove ionic liquid, at 60 DEG C of drying 18 ~ 30h, for enzymolysis.
3. improve the method for lignocellulose biomass enzymolysis efficiency according to claim 2, it is characterized in that, ionic liquid described in step S2 is chlorination-1-butyl-3-Methylimidazole or 1-allyl group-3-Methylimidazole villaumite.
4. improve the method for lignocellulose biomass enzymolysis efficiency according to claim 2, it is characterized in that, step S2 heats 30min at 120 DEG C.
5. improve the method for lignocellulose biomass enzymolysis efficiency according to claim 2, it is characterized in that, tensio-active agent described in step S3 is sodium lauryl sulphate or cetyl trimethylammonium bromide.
6. improve the method for lignocellulose biomass enzymolysis efficiency according to claim 2, it is characterized in that, the weight of tensio-active agent described in step S3 is 1% of powder.
7. improve the method for lignocellulose biomass enzymolysis efficiency according to claim 2, it is characterized in that, the condition stirred described in step S3 is 110 DEG C and stirs 1h.
8. improve the method for lignocellulose biomass enzymolysis efficiency according to claim 2, it is characterized in that, described in step S3, the method for enzymolysis is as follows:
According to the ratio of 2.5w/v%, get in the sodium citrate buffer and 0.02% sodium azide solution that pretreated lignocellulose biomass adds 0.1MpH4.8, shake up, add 50FPU/g cellulase and 40CBU/g glucuroide again, under 50 ± 0.5 DEG C of constant temperatures, 150r/min carries out saccharification 12 ~ 72h, and 13500rpm is centrifugal after completion of the reaction obtains enzyme hydrolyzate.
9. described in claim 1, improve the application of method in treatment of lignocellulosic biomass of lignocellulose biomass enzymolysis efficiency.
10. applying according to claim 9, it is characterized in that, is improving the application in lignocellulose enzymolysis efficiency.
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| CN105838741A (en) * | 2016-03-11 | 2016-08-10 | 青岛农业大学 | Method for fermentation production of isoprenoid compound via peanut shell degradation sugar |
| CN106086109A (en) * | 2016-06-07 | 2016-11-09 | 江南大学 | A kind of method that ionic liquid mixed system preprocessing lignocellulose improves its enzymolysis efficiency |
| CN107361243A (en) * | 2017-07-20 | 2017-11-21 | 通化师范学院 | A kind of feed addictive for promoting cold water fish growth and application thereof |
| CN109517862A (en) * | 2018-12-04 | 2019-03-26 | 齐鲁工业大学 | A method of biological enzyme formulation lignocellulose degradation is cooperateed with using glyoxaline ion liquid |
| CN110066840A (en) * | 2019-03-08 | 2019-07-30 | 华南农业大学 | A kind of method that hydro-thermal-microwave coupled ion liquid two-step method lignocellulosic material improves enzymolysis efficiency |
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| CN105838741A (en) * | 2016-03-11 | 2016-08-10 | 青岛农业大学 | Method for fermentation production of isoprenoid compound via peanut shell degradation sugar |
| CN106086109A (en) * | 2016-06-07 | 2016-11-09 | 江南大学 | A kind of method that ionic liquid mixed system preprocessing lignocellulose improves its enzymolysis efficiency |
| CN107361243A (en) * | 2017-07-20 | 2017-11-21 | 通化师范学院 | A kind of feed addictive for promoting cold water fish growth and application thereof |
| CN109517862A (en) * | 2018-12-04 | 2019-03-26 | 齐鲁工业大学 | A method of biological enzyme formulation lignocellulose degradation is cooperateed with using glyoxaline ion liquid |
| CN110066840A (en) * | 2019-03-08 | 2019-07-30 | 华南农业大学 | A kind of method that hydro-thermal-microwave coupled ion liquid two-step method lignocellulosic material improves enzymolysis efficiency |
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