CN106086115A - A kind of biological ion liquid combined pretreatment method of lignocellulose - Google Patents
A kind of biological ion liquid combined pretreatment method of lignocellulose Download PDFInfo
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- CN106086115A CN106086115A CN201610770559.1A CN201610770559A CN106086115A CN 106086115 A CN106086115 A CN 106086115A CN 201610770559 A CN201610770559 A CN 201610770559A CN 106086115 A CN106086115 A CN 106086115A
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- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
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- C12P2201/00—Pretreatment of cellulosic or lignocellulosic material for subsequent enzymatic treatment or hydrolysis
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Abstract
The present invention relates to a kind of method utilizing antibacterial and ionic liquid combined pretreatment raising lignocellulose saccharification efficiency.First with a strain efficient lignin-degrading bacteria Sphingobacterium sp.LD 1 (preserving number is CGMCC No.10920), lignocellulosic material is carried out a biological disposal upon, remove partial lignin, hemicellulose, and destroy the structure that lignocellulose is fine and close to a certain extent；Further utilize ionic liquid at a certain temperature to lignocellulose pretreatment certain time.The present invention can reduce lignin, hemi-cellulose components proportion, hence it is evident that reduces the degree of crystallinity of lignocellulose, the accessible surface of increased fiber element enzyme, improves the saccharification efficiency of lignocellulose.The present invention have less demanding to equipment resistance to compression and corrosion resistance, the advantage such as be simple to operate and friendly to environment.
Description
Technical field
The invention belongs to lignocellulosic material preconditioning technique field, be specifically a kind of antibacterial
The method that Sphingobacterium sp.LD-1 improves enzymolysis efficiency with ionic liquid combined pretreatment lignocellulosic material.
Background technology
Along with global economy, the development of culture, people are increasing for the demand of regenerative resource, and wooden fibre
Dimension element is natural reproducible biomass resource the abundantest on the earth.In recent years, utilize lignocellulose to produce alcohol fuel to be subject to
Having arrived the extensive concern of domestic and international experts and scholars, the energy technology of development lignocellulosic materials for fuel ethanol, for solving to work as
Modern energy shortage, environmental pollution, crisis in food etc. have the most great meaning.
Lignocellulose constitutes the cell wall of plant, and it is mainly by cellulose, hemicellulose and lignin three part group
Become.Cellulose is the linear polymeric polymer connect by β-Isosorbide-5-Nitrae glycosidic bond by D-Glucose, by crystallization phase and non-knot
Crystalline phase is staggered to form, and crystalline configuration is fine and close, hinders the decomposition of cellulose.Hemicellulose be by several dissimilar pentoses and
The heteromultimer that hexose is constituted, it is combined in the surface of cellulose microfibers, and is connected with each other.Lignin is by phenylpropyl alcohol
Alkane and derivant thereof are the macromolecule aromatic compound that basic composition unit is formed, and Main Function is by forming intertexture net
Carry out sclereid wall, for secondary wall main component, during hydrocellulose, play barrier action.The mesh of preprocessing process
Destroy lignin, the hemicellulose parcel to cellulose exactly, reduce lignocellulose degree of crystallinity, increase enzyme can and table
Face, improves enzymolysis efficiency.
Ionic liquid refers to present liquid, the complete salt being made up of zwitterion, also in room temperature or under room temperature
It is referred to as low temperature molten salt.As a kind of novel green solvent, ionic liquid has extremely low vapour pressure, nonflammable, thermally-stabilised
Property is good, can design and go back reusable edible, is widely used in fields such as organic synthesis, catalysis, electrochemistry.Rogers in 2002
Et al. find ionic liquid at room temperature can dissolve cellulose, since then, ionic liquid at room temperature draws in lignocellulose pretreatment direction
Play the great attention of academia and business circles.Major part ionic liquid pretreatment lignocellulose is all to utilize ionic liquid pair
The dissolubility of lignocellulose high temperature, low solid under the conditions of be stirred vigorously lignocellulose dissolved, add anti-solvent and make fibre
Dimension element heavily analyses regeneration, to destroy lignocellulose structure, to reduce its degree of crystallinity to improve enzymatic saccharification rate.This technique is to equipment
Require harsh, cost is high.
The biological pretreatment being also widely used in (including fungus, antibacterial, actinomycetes etc.) lignocellulose producing and ethanol, especially
It it is white rot fungi.Biological method treatment conditions are gentle, do not cause environmental pollution, low cost, but pretreatment time length, efficiency are low,
Sugar composition has the shortcomings such as loss to hinder its development.
Summary of the invention
For solving above-mentioned technical problem, it is an object of the invention to utilize the side of a kind of bio combined ionic liquid pretreatment
Method improves the enzymatic saccharification efficiency of lignocellulose.
The present invention adopts the following technical scheme that
(1) lignocellulose is dried, pulverizes, and controlling particle diameter is 180 μm～425 μm；
(2) the LD-1 thalline being saved in LB inclined-plane is inoculated in LB fluid medium, at a temperature of 25 DEG C～40 DEG C, training
Support 16～24h, obtain the seed liquor of LD-1；
(3) the wherein said each composition proportion of LB fluid medium is: peptone 10g, yeast powder 5g, sodium chloride 10g, distillation
Water 1L；Described LB inclined-plane is the agar of addition 15～20g/l on the basis of above-mentioned formula；
(4) the LD-1 seed liquor obtained by previous step is centrifuged 1～5 minute under the conditions of 6000～12000rpm, discards
The supernatant, collects thalline；
(5) further the LD-1 thalline collected is pressed 5～15% (volume ratio) inoculum concentration, be inoculated in lignocellulose liquid
In culture medium, at a temperature of 25 DEG C～40 DEG C, natural pH, cultivates 3～7 days；
(6) the wherein said each composition proportion of lignocellulose fluid medium is: lignocellulose 10.0g,
K2HPO41.0g, (NH4)2SO42.0g, KH2PO41.0g, MgSO40.2g, CaCl20.1g, FeSO40.05g, MnSO40.02g, steams
Distilled water 1L；
(7), after the lignocellulose cleaning after LD-1 cultivates being neutrality to pH, dry to permanent in 50 DEG C～60 DEG C
Weight；
(8) it is under conditions of 1:2～1:5, both to be mixed at LD-1 preprocessing lignocellulose and ionic liquid mass ratio
Uniformly, at 90～130 DEG C, stand 2～6h, be then cooled to room temperature, add deionized water, filter, wash filtering residue, after drying
Obtain preprocessing lignocellulose；
(9) it is 10～30mg/mL to mix preprocessing lignocellulose with citrate buffer according to solid-to-liquid ratio, then presses
According to 10～40FPU/g substrates ratio add cellulase, under the conditions of 100～200rpm/min, 40～60 DEG C, enzymolysis 12～
48 hours.
Ionic liquid described in step (8) is 1-ethyl-3 Methylimidazole. acetate ([Emim] [Ac]).
Citrate buffer described in step (9) concentration be 0.1mol/L, pH be 4.8.
The advantage of the preprocess method that the present invention provides is:
(1) two-step method utilizing efficient lignin-degrading bacteria LD-1 biological treatment to be combined with ionic liquid, removes major part
Lignin component, reduces lignocellulose degree of crystallinity, the accessible surface of increased fiber element enzyme, improves the saccharifying effect of lignocellulose
Rate；
(2) use efficient lignin degradation antibacterial that lignocellulose is carried out pretreatment, significantly contract compared with fungus pretreatment
The short Biological Pretreatment cycle also saves content of cellulose greatly；
(3) react with the solid condition of height during ionic liquid pretreatment, use standing mode, it is not necessary to cellulose is entered
Row regeneration is heavily analysed, and simplifies technical process, and significantly reduces energy consumption.
Accompanying drawing illustrates:
Fig. 1: each change of component of lignocellulose after the present invention combines pretreatment
Fig. 2: lignocellulose enzymolysis effect analysis after the present invention combines pretreatment
Fig. 3: combine the lignocellulose pretreatment traversing of probe electronic microscope photos of pretreatment through the present invention
Detailed description of the invention:
The present invention is further illustrated below by embodiment.
Embodiment 1
(1) rice straw cleaned, dry, pulverize, control its particle diameter and be distributed between 180 μm～425 μm；
(2) the LD-1 thalline being saved in LB inclined-plane is inoculated in LB fluid medium, at a temperature of 30 DEG C, cultivates 18h,
Obtain the seed liquor of LD-1；
(3) the wherein said each composition proportion of LB fluid medium is: peptone 10g, yeast powder 5g, sodium chloride 10g, steams
Distilled water 1L, described LB inclined-plane is the agar adding 15g/l on the basis of above-mentioned formula；
(4) the LD-1 seed liquor obtained by previous step is centrifuged 2 minutes under the conditions of 10000rpm, discards the supernatant,
Collect thalline；
(5) further the LD-1 thalline collected is pressed 10% (volume ratio) inoculum concentration, be inoculated in the training of lignocellulose liquid
Support in base, at a temperature of 30 DEG C, natural pH, cultivates 3 days；
(6) the wherein said each composition proportion of lignocellulose fluid medium is: lignocellulose 10.0g,
K2HPO41.0g, (NH4)2SO42.0g, KH2PO41.0g, MgSO40.2g, CaCl20.1g,FeSO40.05g, MnSO40.02g, steams
Distilled water 1L；
(7), after the lignocellulose cleaning after LD-1 cultivates being neutrality to pH, dry to constant weight in 55 DEG C；
(8) under conditions of LD-1 preprocessing lignocellulose is 1:3 with [Emim] [Ac] mass ratio, both are mixed all
Even, at 130 DEG C, stand 2～6h, be then cooled to room temperature, add deionized water, filter, wash filtering residue, dried acquisition is pre-
Process lignocellulose；
(9) it is that 25mg/mL mixes with citrate buffer by preprocessing lignocellulose according to solid-to-liquid ratio, according still further to
The ratio of 12FPU/g substrate adds cellulase, under the conditions of 110～150rpm/min, 50 DEG C, enzymolysis 48 hours；
By the enforcement of the present embodiment, as shown in Figure 1, in the rice straw after treated by the present method, lignin is fine with half
Dimension cellulose content significantly reduces, and cellulose ratios promotes；Pretreated rice straw enzymolysis after 48 hours reducing sugar yield the highest
Reach 98% (such as accompanying drawing 2), and after the most pretreated rice straw enzymolysis, Reducing sugar is only 20.7%, through this embodiment
After pretreatment, rice straw enzymolysis efficiency rises to original 4.7 times；Shown in scanning electron microscope image (such as accompanying drawing 3), without
The rice straw (a, b) of pretreatment is in direct rod shape and bar footpath is relatively thick, and surfacing is smooth, and through the pretreated water of the present embodiment
Rice straw (c, d) rigid structure destroys the most completely, and surface erosion is serious, and a large amount of celluloses come out.
Embodiment 2
(1) rice straw cleaned, dry, pulverize, control its particle diameter and be distributed between 180 μm～425 μm；
(2) the LD-1 thalline being saved in LB inclined-plane is inoculated in LB fluid medium, at a temperature of 30 DEG C, cultivates 18h,
Obtain the seed liquor of LD-1；
(3) the wherein said each composition proportion of LB fluid medium is: peptone 10g, yeast powder 5g, sodium chloride 10g, distillation
Water 1L, described LB inclined-plane is the agar adding 15g/l on the basis of above-mentioned formula；
(4) the LD-1 seed liquor obtained by previous step is centrifuged 2 minutes under the conditions of 10000rpm, discards the supernatant,
Collect thalline；
(5) further the LD-1 thalline collected is pressed 10% (volume ratio) inoculum concentration, be inoculated in the training of lignocellulose liquid
Support in base, at a temperature of 30 DEG C, natural pH, cultivates 3 days；
(6) the wherein said each composition proportion of lignocellulose fluid medium is: lignocellulose 10.0g,
K2HPO41.0g, (NH4)2SO42.0g, KH2PO41.0g, MgSO40.2g, CaCl20.1g, FeSO40.05g, MnSO40.02g, steams
Distilled water 1L；
(7), after the lignocellulose cleaning after LD-1 cultivates being neutrality to pH, dry to constant weight in 55 DEG C；
(8) under conditions of LD-1 preprocessing lignocellulose and [Emim] [Ac] mass ratio are 1:2～1:5, both are mixed
Close uniformly, at 110 DEG C, stand 4～6h, be then cooled to room temperature, add deionized water, filter, wash filtering residue, obtain after drying
Obtain preprocessing lignocellulose；
(9) it is that 25mg/mL mixes with citrate buffer by preprocessing lignocellulose according to solid-to-liquid ratio, according still further to
The ratio of 12FPU/g substrate adds cellulase, under the conditions of 110～150rpm/min, 50 DEG C, enzymolysis 48 hours；
After the present embodiment pretreated rice straw enzymolysis, Reducing sugar is the highest rises to 86.9% from 20.7%,
Enzymolysis efficiency rises to original 4.1 times.
Embodiment 3
(1) rice straw cleaned, dry, pulverize, control its particle diameter and be distributed between 180 μm～425 μm；
(2) the LD-1 thalline being saved in LB inclined-plane is inoculated in LB fluid medium, at a temperature of 30 DEG C, cultivates 18h,
Obtain the seed liquor of LD-1；
(3) the wherein said each composition proportion of LB fluid medium is: peptone 10g, yeast powder 5g, sodium chloride 10g, distillation
Water 1L, described LB inclined-plane is the agar adding 15g/l on the basis of above-mentioned formula；
(4) the LD-1 seed liquor obtained by previous step is centrifuged 2 minutes under the conditions of 10000rpm, discards the supernatant,
Collect thalline；
(5) further the LD-1 thalline collected is pressed 10% (volume ratio) inoculum concentration, be inoculated in the training of lignocellulose liquid
Support in base, at a temperature of 30 DEG C, natural pH, cultivates 3 days；
(6) the wherein said each composition proportion of lignocellulose fluid medium is: lignocellulose 10.0g,
K2HPO41.0g, (NH4)2SO42.0g, KH2PO41.0g, MgSO40.2g, CaCl20.1g, FeSO40.05g, MnSO40.02g, steams
Distilled water 1L；
(7), after the lignocellulose cleaning after LD-1 cultivates being neutrality to pH, dry to constant weight in 55 DEG C；
(8) under conditions of LD-1 preprocessing lignocellulose and [Emim] [Ac] mass ratio are 1:2～1:5, both are mixed
Close uniformly, at 130 DEG C, stand 2h, be then cooled to room temperature, add deionized water, filter, wash filtering residue, obtain pre-after drying
Process lignocellulose；
(9) it is that 25mg/mL mixes with citrate buffer by preprocessing lignocellulose according to solid-to-liquid ratio, according still further to
The ratio of 12FPU/g substrate adds cellulase, under the conditions of 110～150rpm/min, 50 DEG C, enzymolysis 48 hours；
After the present embodiment pretreated rice straw enzymolysis, Reducing sugar is the highest rises to from 20.7%
91.08%, enzymolysis efficiency rises to original 4.4 times.
Note: the antibacterial Sphingobacterium sp.LD-1 that this patent relates to is preserved in China on May 28th, 2015
Microbiological Culture Collection administration committee common micro-organisms center (address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3), protects
Hide numbering: CGMCC NO.10920, Classification And Nomenclature is Sphingobacterium Sphingobacterium sp.
Claims (4)
1. the biology of a wood fibre-ionic liquid combined pretreatment method, it is characterised in that: include following step:
(1) antibacterialSphingobacteriumSp. LD-1 pretreatment: willSphingobacteriumSp. LD-1 thalline presses 5 ~ 15%
(volume ratio) inoculum concentration, is inoculated in lignocellulose fluid medium, in 25 ~ 40 DEG C, under the conditions of natural pH, cultivate 3 ~ 7 days,
Gained lignocellulose ultra-pure water cleans 3 times, dries to constant weight；
(2) ionic liquid pretreatment: be that 1:2 ~ 1:5 is mixed in mass ratio by step (1) gained lignocellulose and [Emim] [Ac]
Close uniformly, after 90 DEG C ~ 130 DEG C isoperibols stand 2 ~ 6 h, be cooled to room temperature, add deionized water, filter, wash filtering residue, dry
Preprocessing lignocellulose is obtained after dry.
The combination preprocess method of raising lignocellulose saccharification result the most according to claim 1, is characterized by step
(1) in, lignocellulose is through antibacterialSphingobacteriumSp. LD-1 25 ~ 40 DEG C, under the conditions of natural pH, cultivate 3 ~ 7
My god.
The combination preprocess method of raising lignocellulose saccharification result the most according to claim 1, is characterized by step
(2) ionic liquid described in is 1-ethyl-3 Methylimidazole. acetate ([Emim] [Ac]).
The combination preprocess method of raising lignocellulose saccharification result the most according to claim 1, is characterized by step
(2) in, lignocellulose is mixed homogeneously for 1:2 ~ 1:5 in mass ratio with [Emim] [Ac], and 90 DEG C ~ 130 DEG C isoperibols stand 2 ~
6 h。
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Cited By (1)
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| CN107177646A (en) * | 2017-06-16 | 2017-09-19 | 中南大学 | A kind of method of utilization lignin-degrading bacteria reinforcing abandoned biomass acid system pretreatment |
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| CN107177646A (en) * | 2017-06-16 | 2017-09-19 | 中南大学 | A kind of method of utilization lignin-degrading bacteria reinforcing abandoned biomass acid system pretreatment |
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