CN104651429B - A kind of method for irradiating pretreatment and the batch feeding realization high concentration of substrate enzyme hydrolysis of lignocellulosic - Google Patents
A kind of method for irradiating pretreatment and the batch feeding realization high concentration of substrate enzyme hydrolysis of lignocellulosic Download PDFInfo
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Abstract
The invention discloses a kind of methods for irradiating pretreatment and the batch feeding realization high concentration of substrate enzyme hydrolysis of lignocellulosic.It adds cellulase strategy using batch feeding and in batches using the lignocellulosic material of high dose x ray irradiation x pretreatment as substrate, the concentration of substrate in raising enzyme hydrolysis system, so as to obtain the hydrolyzate of high-concentration reduced sugar.Advantage of the invention is that x ray irradiation x preconditioning technique has many advantages, such as easy to operate, cleanliness without any pollution, can carry out at ambient temperature and pressure, without loss of material, significant effect, using batch feeding strategy, greatly improve the concentration of substrate in enzyme hydrolysis system, the concentration of substrate of processing is made to reach 36%, high-concentration reduced sugar is obtained while improving and digesting material concentration, the enzyme hydrolyzate middle and high concentration reduced sugar of acquisition reaches more than 200g/L, has reached domestic and international advanced level.The acquisition of lignocellulosic high-concentration reduced sugar enzyme hydrolyzate can improve later stage ethyl alcohol high gravity fermentation and reduce ethanol distillation energy consumption, so as to reduce the production cost of fibre fuel ethyl alcohol.
Description
Technical field
The invention belongs to lignocellulosic hydrolysis technology fields, and in particular to a kind of x ray irradiation x pre-processes wood fibre
The high concentration of substrate enzymatic hydrolysis process of element.
Background technology
With the exhaustion of fossil energy, reproducible biomass energy increasingly attracts people's attention.With wood fibre
Element is that the second generation alcohol fuel of raw material is considered as the important development direction of alcohol fuel.Lignocellulosic material is on the earth
One of most abundant regenerative resource.The global annual substantial amounts of agricultural crop straw such as big portion such as rape straw, cornstalk, rice straw
Divide and be dropped or burn, not only cause the wasting of resources, also result in environmental pollution.Lignocellulosic material production alcohol fuel tool
There is the features such as recyclability, nonstaining property, recycling can improve air quality, promote economic development from far-off regions, subtract
Few dependence to gasoline reduces greenhouse effects etc..But maximum problem is how to change the structure of lignocellulosic to increase at present
Add its reactivity worth that is saccharified.Various physics, process for chemically pretreating have certain limitation, in production application all there is
Processing cost is high, soda acid recycling is difficult, and environmental pollution is serious, has the problems such as inhibition to subsequent technique.Therefore seek a kind of high
Effect, effective preprocess method pollution-free, at low cost are extremely urgent.
Irradiation pretreatment is to utilize radio isotope Co60The energetic gamma rays released during decay act on wood fibre
Element destroys lignocellulosic physics and a kind of preprocess method of chemical constitution.Irradiation preprocessing process need not heat, entirely
Process can be carried out at room temperature, and the inhibition by-product generated is few.By high dosage irradiation treated wood fibre
Plain structure becomes loose, and hardness becomes smaller, and crystallinity declines, the microstructure of microcrystalline cellulose by smoothly becoming coarse, hemicellulose,
Cellulose and lignin molecule chain are largely cut off.Therefore, after high dosage irradiation pre-processes, the enzymolysis conversion of lignocellulosic
Rate significantly improves.X ray irradiation x preconditioning technique has easy to operate, cleanliness without any pollution, can carry out at ambient temperature and pressure, nothing
The advantages such as loss of material, significant effect.
Not high concentration of reduced sugar after lignocellulosic enzymolysis is current cellulose fuel ethanol commercialization major technique bottle
One of neck.Under normal conditions, batch fermentation concentration of alcohol does not exceed 40g/L, and works as concentration of alcohol in zymotic fluid and be less than 4%
When, the energy consumption for the concentration of subsequent ethyl alcohol and rectifying will dramatically increase.The concentration of substrate for improving enzymatic hydrolysis system is that acquisition is highly concentrated
Spend reduced sugar direct method, and high concentration of substrate often lead to system stickiness increase, be unfavorable for system substance transfer and
Heat transfer makes enzymolysis efficiency low.Add substrate into system in batches and when enzyme can reduce reaction system viscosity, favorably
In the heat and mass transport of system, so as to obtain high-concentration reduced sugar.
The acquisition of lignocellulosic material high-concentration reduced sugar hydrolyzate is its high gravity fermentation of realization and reduces fiber second
The necessary condition of alcohol production cost.It is all that enzyme is carried out under relatively low concentration of substrate to irradiate pretreated lignocellulosic at present
Solution, it is impossible to obtain high concentration sugar, therefore the present invention utilizes batch feeding using the lignocellulosic of x ray irradiation x pretreatment as raw material
The method for adding cellulase in batches improves the concentration of substrate of irradiation preprocessing lignocellulose enzymatic hydrolysis system, so as to obtain height
The enzyme hydrolyzate of concentration reduced sugar.
The content of the invention
The object of the present invention is to provide a kind of easy to operate, cleanliness without any pollution, can carry out at ambient temperature and pressure, without material
Loss, the high concentration of substrate of raw material of effective x ray irradiation x pretreatment, the lignocellulolyticenzymes water of production high-concentration reduced sugar
The method of solution.
A kind of method for irradiating pretreatment and the batch feeding realization high concentration of substrate enzyme hydrolysis of lignocellulosic, with wooden fibre
Dimension element is pre-processed through x ray irradiation x for raw material, is added cellulase using batch feeding and in batches, is made raw material progress enzyme hydrolysis,
So as to obtain the enzyme hydrolyzate of high-concentration reduced sugar；
Batch feeding and cellulase step is added in batches be：
(1) Acetic acid-sodium acetate that lignocellulosic material is added to pH4.8 according to initial concentration for 18% buffers
In liquid, the cellulase of 45FPU/g substrates is added, in pH4.8, under the conditions of temperature is 50 DEG C and 130~160rpm of rotating speed, enzyme water
Solve 12h；
(2) lignocellulosic material is added so that the substrate mass concentration of processing reaches 24%, while increases by 45FPU/g
The substrate addition cellulase added, enzyme hydrolysis to 36h；
(3) lignocellulosic material is added so that the substrate mass concentration of processing reaches 30%, while increases by 45FPU/g
The substrate addition cellulase added, enzyme hydrolysis to 60h；
(4) lignocellulosic material is added so that the substrate mass concentration of processing reaches 36%, while increases by 45FPU/g
The substrate addition cellulase added continues hydrolysis to 144h, obtains hydrolyzate middle and high concentration reduced sugar and reach more than 200g/L.
The lignocellulosic material be main component for cellulose, hemicellulose and lignin agriculture and forestry organic waste material or
Fast-growing energy crop.Maize straw, rice straw, rape stalk, reed straw and one kind or several in Chinese silvergrass stalk can be selected
Kind.
The x ray irradiation x pretreatment is that lignocellulosic is dried or dried, and moisture≤10% places irradiation
In case, use60Co- gamma-rays carries out irradiation pretreatment, radiation dose rate 2kGy/h, and treatment dosage is 1000~1200kGy.It will
The lignocellulosic of x ray irradiation x pretreatment crushed 20 mesh sieves, obtained lignocellulosic material.
Beneficial effects of the present invention：X ray irradiation x preconditioning technique have easy to operate, cleanliness without any pollution, can in room temperature and
It is carried out under normal pressure, without advantages such as loss of material, significant effects, treated, and lignocellulosic can directly carry out enzyme hydrolysis, without
Detoxification, it is simple for process, it is cost-effective.By x ray irradiation x pretreatment and batch feeding and cellulase coupling technique is added to wooden
Cellulose is degraded, and the viscosity of reaction system can be reduced by adding substrate and cellulase into system in batches, be conducive to body
The heat and mass transport of system can greatly improve the lignocellulosic concentration of substrate of enzyme digestion reaction system so that the substrate of processing is dense
Degree up to 36%, the enzyme hydrolyzate middle and high concentration reduced sugar of acquisition is more than 200g/L, has reached domestic and international advanced level.It is wooden
The acquisition of cellulose high-concentration reduced sugar enzyme hydrolyzate can improve later stage ethyl alcohol high gravity fermentation and reduce ethanol distillation energy consumption, from
And reduce the production cost of fibre fuel ethyl alcohol.
Description of the drawings
Fig. 1 is the influence that x ray irradiation x changes lignocellulosic biochemical component；
Fig. 2 is corn and the front and rear sugared yield of enzymolysis production of Chinese silvergrass stalk radiation treatment；
Fig. 3 is influence of the initial substrate concentration to enzyme hydrolysis production sugar；
Fig. 4 is influence of the cellulase additive amount to enzyme hydrolysis production sugar；
Fig. 5 is the reaction of 12h and 36h batch feedings 6% (concentration of substrate of processing up to 30%) Enzymatic hydrolysis；
Fig. 6 is 12h, 36, the reaction of 60h batch feedings 6% (concentration of substrate of processing up to 36%) Enzymatic hydrolysis.
Specific embodiment
Following embodiment is to further explanation of the invention, however, the present invention is not limited thereto.
Embodiment 1
Influence of the x ray irradiation x to lignocellulosic biochemical component and enzyme hydrolysis production sugar
1. influence of the x ray irradiation x to lignocellulosic biochemical component
Radiation treatment can directly interrupt lignocellulosic strand, reduce its degree of polymerization, realize the degradation of lignocellulosic.
Ligno-cellulosic materials are dried or dried, moisture≤10%, bundling is placed in irradiation box, is used under experimental conditions60Co-
Gamma-rays carries out irradiation pretreatment, radiation dose rate 2kGy/h, and treatment dosage is 400~1200kGy, and x ray irradiation x is located in advance
The ligno-cellulosic materials of reason crushed 20 mesh sieves, obtain lignocellulosic material.Such as the biochemical component changes of contents knot in Fig. 1
Shown in fruit, with the rise of irradiation dose, in straw and maize straw the constituent content of cellulose, hemicellulose and lignin by
It is decrescence small, illustrate that main biochemical component is degraded.After the processing of 1200kGy dosage, the fibre in straw and maize straw
The plain degradation rate of dimension is respectively 26.1% and 29.3%；And the degradation of hemicellulose is then more violent, hemicellulose degradation rate difference
For 77.8% and 78.1%, this is because the low polymerization degree attribute of hemicellulose is easier by caused by degrading；Lignin degradation
Rate is all higher than 10%, and the removing of lignin will promote the raising of the sugared yield of enzyme hydrolysis production.
2. influence of the x ray irradiation x to lignocellulosic enzyme hydrolysis production sugar
After x ray irradiation x is handled, ligno-cellulosic materials morphosis, molecular structure, supramolecular structure occur significantly
Change, cellulose hydrolyzation reaction efficiency can be greatly improved.Chinese silvergrass and maize straw batch enzymolysis produce sugared obtain before and after radiation treatment
Rate is as shown in Fig. 2, Chinese silvergrass and maize straw through the pretreatment of 1200kGy dosage x ray irradiation x, cellulose hydrolyzation production reduced sugar
Yield can respectively reach 398.1mg/g and 405.5mg/g, compared with without radiation treatment, can make enzymolysis production Reducing sugar point
7.1 and 6.5 times are not improved, illustrate that the sugared efficiency of lignocellulosic enzyme hydrolysis production can be greatly improved in x ray irradiation x pretreatment.
Embodiment 2
The optimization of batch enzyme hydrolysis (disposably adding in raw material and enzyme) primary condition
Influence lignocellulosic enzymolysis efficiency key factor include initial substrate concentration, enzyme additive amount, hydrolysis time and
Substrate structure characteristic.Corn stalk raw material of the x ray irradiation x dosage for 1200kGy processing is added in 0.05M Acetic acid-sodium acetates
In buffer solution (pH4.8), add 45FPU/g substrates cellulase (CTec2, Novozymes, Denmark),
In pH4.8, under the conditions of temperature is 50 DEG C and rotating speed 160rpm, enzyme hydrolysis 72h, concentration of reduced sugar is surveyed with DNS methods in enzyme hydrolyzate
It is fixed, optimize batch enzyme hydrolysis primary condition.The influence that initial substrate concentration is saccharified to feed supplement is primarily looked at, from figure 3, it can be seen that
When initial substrate concentration is 6%, 12%, 18%, in enzyme hydrolyzate concentration of reduced sugar respectively reach 51.2,82.3,96.0g/
L, concentration of reduced sugar increase with initial substrate concentration and are increased, and unobvious occur for substrate inhibition.When initial substrate concentration reaches
During to 24%, hydrolysis reaction is slack-off in reaction 12h compared with other initial substrate concentrations, and it is concentration of substrate mistake to trace it to its cause
Height, the viscosity of system increase, and resistance to mass tranfer increases, and enzyme-to-substrate contact difficulty is consequently increased, so as to influence enzyme hydrolysis degradation
Efficiency.It is thus determined that 18% is optimal initial concentration of substrate condition.Can also be drawn from Fig. 3, when initial substrate concentration for 6%,
12%th, 18% when, have very high enzyme hydrolysis efficiency in enzyme digestion reaction 12h, about more than 60% total reducing sugars, which generates, to exist
It reacts in 12h, as the reaction time promotes, base consumption is serious, and enzyme hydrolysis efficiency is gradually reduced after 48h.
The influence (initial substrate concentration 18%) that cellulase is saccharified to feed supplement is investigated, as shown in figure 4, comparing enzyme addition
30 and 40FPU/g substrates are measured, the concentration of reduced sugar of acquisition is all substantially lower with 50FPU/g substrates than additive amount 45；45 He of additive amount
The Factor of Enzymolysis Corn Stalk reaction efficiency curve of 50FPU/g is similar, but is examined in feed supplement saccharifying from the reaction time is shortened
Consider, enzyme hydrolysis efficiency and the sugared yield of production should be maintained at high-level state reacting interior for 24 hours, when additive amount is 45FPU/g substrates
When, the sugared yield of enzymatic hydrolysis reaction production is 50FPU/g substrate highers than additive amount, it follows that, 45FPU/g substrates are x ray irradiation x
The optimal enzyme additive amount of pretreated straw enzymatic hydrolysis reaction.
Embodiment 3
Batch feeding and cellulose hydrolyzation is added batch-wise
The acquisition of lignocellulosic material high-concentration reduced sugar hydrolyzate is its high gravity fermentation of realization and reduces fiber second
The necessary condition of alcohol production cost.In order to obtain high-concentration reduced sugar enzyme hydrolyzate, in the optimal conditions of batch enzyme hydrolysis experiment
Under, batch feeding saccharification strategy study is carried out to the maize straw of x ray irradiation x pretreatment.Maize straw is added in 12h and 36h
6% (wt), enzyme additive amount add 45FPU by every gram of maize straw substrate added, and the concentration of substrate of final process is 30%, enzyme
Hydrolysis 144h, as shown in figure 5, it is 152.1g/L to obtain final reducing sugar concentration, compared with the batch enzyme water of concentration of substrate 18%
Concentration of reduced sugar improves 58.4% in solution liquid.
To further improve concentration of reduced sugar in enzyme hydrolyzate, the basis of maize straw 6% (wt) is added in 12h and 36h
On, 6% maize straw is with the addition of in 60h, enzyme additive amount adds 45FPU by every gram of corn substrate added, final process
Concentration of substrate is 36%, enzymatic hydrolysis reaction 144h, as shown in fig. 6, the concentration of reduced sugar in enzyme hydrolyzate is obtained as 202.3g/L,
Wherein glucose content is 128.8g/L, Xylose Content 52.5g/L, and it is 56.1% to realize total reducing sugars yield.
Embodiment 4
The batch feeding of example 3 is used to the rice straw of x ray irradiation x dosage 1200kGy processing and adds cellulase
Method carries out enzyme hydrolysis experiment, and rice straw raw material initial mass volumetric concentration is added to Acetic acid-sodium acetate for 18% (wt)
In buffer solution (pH4.8), the cellulase of addition 45FPU/g (substrate), in pH4.8, temperature for 50 DEG C and rotating speed 130~
Under the conditions of 160rpm, after enzyme hydrolysis 12h, then rice straw raw material is added, making the concentration of substrate of processing, to reach 24% (dense from originating
Degree is started), while cellulase is added in by the amount of 45FPU/g (substrate added) cellulase, enzyme hydrolysis 36h adds rice
Stalk makes the concentration of substrate of processing reach 30% (being started from initial concentration), while fine by 45FPU/g (substrate added)
The amount of the plain enzyme of dimension adds in cellulase, and enzyme hydrolysis to 60h adds rice straw raw material, reaches the concentration of substrate of processing for the third time
It (is started to 36% from initial concentration), while cellulase is added by the amount of 45FPU/g (substrate added) cellulase, continued
144h is hydrolyzed, obtain enzyme hydrolyzate middle and high concentration reduced sugar reaches more than 200.5g/L, realizes that total reducing sugars yield is
55.7%.It is demonstrated experimentally that x ray irradiation x pretreatment and batch feeding and addition cellulase coupling technique to lignocellulosic into
Row degradation, can greatly improve the lignocellulosic concentration of substrate of enzymatic hydrolysis system, the concentration of substrate of processing up to 36%, acquisition
Enzyme hydrolyzate middle and high concentration reduced sugar reaches more than 200g/L, has reached domestic and international advanced level.Lignocellulosic high concentration reduces
The acquisition of carbohydrase hydrolyzate can improve later stage ethyl alcohol high gravity fermentation and reduce ethanol distillation energy consumption, this is for reducing fibre fuel
The production cost of ethyl alcohol has very important significance.
Claims (1)
1. a kind of method for irradiating pretreatment and the batch feeding realization high concentration of substrate enzyme hydrolysis of lignocellulosic, feature exist
In, the corn straw smashing that x ray irradiation x pre-processes is crossed into 20 mesh sieves, obtains lignocellulosic material, using batch feeding and point
It criticizes and adds cellulase, raw material is made to carry out enzyme hydrolysis, so as to obtain the enzyme hydrolyzate containing reduced sugar；
Batch feeding and cellulase step is added in batches be：
(1) the Acetic acid-sodium acetate buffer solution that lignocellulosic material is added to pH4.8 according to initial concentration for 18%
In, the cellulase of addition 45FPU/g substrates, in pH4.8, under the conditions of temperature is 50 DEG C and 130~160rpm of rotating speed, enzyme hydrolysis
12h；
(2) lignocellulosic material is added so that the substrate mass concentration of processing reaches 24%, while increased by 45FPU/g
Substrate adds cellulase, enzyme hydrolysis to 36h；
(3) lignocellulosic material is added so that the substrate mass concentration of processing reaches 30%, while increased by 45FPU/g
Substrate adds cellulase, enzyme hydrolysis to 60h；
(4) lignocellulosic material is added so that the substrate mass concentration of processing reaches 36%, while increased by 45FPU/g
Substrate adds cellulase, continues hydrolysis to 144h, obtains hydrolyzate middle and high concentration reduced sugar and reach more than 200g/L；
The x ray irradiation x pretreatment is that lignocellulosic is dried or dried, and moisture≤10% is placed in irradiation box,
With60Co- gamma-rays carries out irradiation pretreatment, radiation dose rate 2kGy/h, treatment dosage 1200kGy.
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| 辐照预处理对玉米秸秆酶解产糖率的影响;唐洪涛;《中国优秀硕士学位论文全文数据库》;20121215;第12页2.2.2.2部分、第15页2.3.2部分 * |
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