CN102586342A - Method for lowering fermentation inhibitor from source - Google Patents
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- CN102586342A CN102586342A CN2012100147967A CN201210014796A CN102586342A CN 102586342 A CN102586342 A CN 102586342A CN 2012100147967 A CN2012100147967 A CN 2012100147967A CN 201210014796 A CN201210014796 A CN 201210014796A CN 102586342 A CN102586342 A CN 102586342A
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Abstract
The invention discloses a method for lowering fermentation inhibitor from a source. In the method, a steam explosion technology and an airflow classification technology are combined to be applied to the pretreatment of wood fiber raw material; selective pretreatment is carried out by a second-section steam explosion combing technology according to the characteristic of each tissue of the fiber raw material effectively, so that the fiber tissue can achieve a good pretreatment effect and the thin-wall tissue can be prevented from excessively degrading; therefore, while the enzymolysis rate of the fiber raw material is improved, the side reaction can be effectively prevented, i.e. the inhibitor content is lowered, an detoxication unit operation introduction is omitted, and the technology is simplified. In addition, in the technology, the separating and the selective treatment of the fiber cell and the thin-wall cell provide a new path for the layered multistage conversion and biomass power utilization of the wood fiber raw material. The second-section steam explosion combing technology is favorable for rehydration uniformity of the fiber raw material, the steam explosion condition of the second section of material is lowered, the raw material content is reduced, and the use ratio of the raw material is improved.
Description
Technical field
The present invention relates to a kind of method that reduces the fermentation inhibitor in lignocellulose source, particularly is the pretreatment process of core with the steam explosion technology.
Background technology
The structure of lignocellulose with form complicatedly, wherein, the cellulosic structure of mainly forming with glucone is fine and close, more difficult hydrolysis, and the semicellulose of forming with multiple assorted glycosyl, structure is more open, pre-treatment can be hydrolyzed into monose.That is studying at present has multiple lignocellulosic material pretreatment process, for example acid hydrolysis, steam explosion, the explosion of ammonia fiber and a wet oxidation etc.Various preprocessing process all produces multiple compound, and its kind and content is difference with the difference of cellulosic material character and pretreatment condition, mainly forms 3 quasi-microorganism amicines: 1) weak acid: acetate, formic acid, levulinic acid etc.Acetate takes off acetyl by semicellulose and generates, and formic acid and levulinic acid are that (HMF, degraded product 5-Hydroxymethylfurfural), while formic acid also can be by the furfural generations of under sour environment, degrading for 5 hydroxymethyl furfural; 2) furans aldehydes: mainly be furfural (Furfural) and HMF, respectively by the generation of under sour environment, dewatering of pentose and hexose; 3) phenolic cpd: mainly form by lignin degradation.
The steam explosion technology is considered to pretreated tool prospect of vegetable fibre and most economical technology efficiently.Raw material is with being steam heated to 180-235 ℃, and dimension is pressed certain hour, and when unexpected decompression was spurted, the volume surge received the effect of mechanical force, and its solid materials is destructurized.Several advantages of steam explosion can be summarized as follows: (1) can be applicable to various plant biomass, and pretreatment condition is regulated control easily.(2) semicellulose, xylogen and cellulose iii kind component can be separated in three different flow processs, are respectively soluble component, the molten component of alkali and alkali indissolvable component.(3) cellulosic enzymolysis transformation efficiency can reach theoretical maximum.(4) xylogen after the process VT still can be used in the conversion of other chemical productss.(5) sugar of semicellulose generation can be converted into liquid fuel by complete utilization.(6) fermentation inhibitor that produces in the quick-fried process of vapour can reduce through the control quick-fried condition of vapour greatly.This pretreatment process is applicable to hardwood, cork, agricultural wastes, like various plant biomass such as bagasse, wheat straw, straw, corn stalk and other non-cellulose raw materials, and plays a greater and greater role just in this respect.But the maximum shortcoming of steam explosion preconditioning technique is exactly still to produce some content few fermentation inhibitor of other preconditioning techniques relatively.
These inhibitions have become one of main bottleneck of lignocellulose biological processing to the inhibition of subsequent fermenting mikrobe.The measure that must take necessary reply inhibition is to reduce or eliminate its restraining effect.People have attempted the detoxification treatment before several different methods is fermented to ligno-cellulose hydrolysate, comprise that the combination of biology, physics and chemical process etc. and different measure uses.Biological method comprises and utilizes certain enzyme (for example laccase laccase) or mikrobe that hydrolyzed solution is carried out detoxification treatment.Laccase is one type of cupric oxydo-reductase, specificity the phenols substrate molecule is carried out one-electron oxidation, generates corresponding living radical, and active intermediate changes dimer, oligomer and superpolymer subsequently into.Oxidative polymerization has taken place in the small molecular weight phenolic cpd in this course, has generated the lower high-molecular weight compounds of toxicity, thereby has reduced the toxicity of aldehydes matter in the lignocellulose pretreatment fluid.Usefulness such as Mussatto can only utilize the yeast saccharomyces cerevisiae mutant strain of acetate growth to handle hydrolyzed solution, make acetic acid concentration wherein be reduced to 0.4g/L by 6.8g/L.Physical method has that vacuum-drying concentrates, boiling, charcoal absorption, IX absorption and SX etc.; Vacuum concentration and boiling can make the volatility inhibition reduce in a large number, and IX and SX can effectively reduce acetate, Furan Aldehydes and content of phenolic compounds; Chemical process comprises utilizes various alkali (NH4OH, NaOH, Ca (OH) 2 etc.) and superfluous lime method etc. that hydrolyzed solution is handled, and wherein NH4OH can effectively remove the furans aldehyde material, and superfluous lime is handled can effectively improve monose utilization ratio in the hydrolyzed solution.In addition, the washing pretreating raw material also is a kind of simple detoxification process.2 kinds or several different methods are united use can reach better detoxification efficiency.
But the detoxification step has increased the fermentation costs of bio-based product undoubtedly, makes technological process more complicated, also causes the loss of a part of fermentable sugar simultaneously, so must reduce the detoxification link before the fermentation during the fermentation as far as possible.Feasible method has the high resistant strain of seed selection, improves himself intrinsic tolerance, perhaps optimizes the generation of hydrolysis process control inhibition from the source, reduces its content.Lignocellulosic material has complicated, inhomogenous multilevel hierarchy.See that from chemical ingredients the chemical ingredients of lignocellulose raw material comprises Mierocrystalline cellulose, semicellulose and xylogen.See from the cell composition, comprise fibrous fibrocyte and heteroproteose cell (comprising conduit, parenchyma cell, epidermic cell etc.).Fibrocyte is a cell main, the most basic in the plant fiber material, is the sustentacular tissue of plant material, and fibrocyte generally has flourishing secondary wall (being that thickness is bigger).Another kind of important cell is a parenchyma cell in the plant material, in plant-growth, plays a part to store nutrition the characteristics that the parenchyma cell chamber is big, wall thin, length is short.In view of the structure and the modal difference of 2 types of cells, can know that these 2 types of desired pretreatment conditions of cell are different.Fibrocyte, the cell walls degree of lignification is high, and compact structure receives in the thermal process its mass-and heat-transfer resistance big, and is difficult for being torn; Parenchyma cell, wall is thin and the chamber is big, promptly is beneficial to mass-and heat-transfer, is beneficial to the water vapor flash distillation again its physics is torn.In the different material, content, the structure of these two types of cells have nothing in common with each other, and this is just from having determined the difference of the treatment condition that it is required in essence.Therefore, can be to different tissues cell optimization of treatment conditions respectively, thus reach best separately hydrolysis effect and side reaction extent control in minimum.
Two sections quick-fried carding technologies of vapour of the present invention's exploitation; Two sections quick-fried technologies of vapour that are different from traditional indication; Here be meant that to adopt the quick-fried condition of relatively mild vapour to carry out first section vapour quick-fried; With first section stock grading, obtain parenchyma and fibrous tissue through airflow classification device, it is quick-fried again fibrous tissue to be carried out second section vapour under appropriate condition.Two sections quick-fried carding technologies of vapour effectively optionally carry out pre-treatment to the feature request of its each tissue; Can guarantee that fibrous tissue reaches pretreating effect preferably, can avoid parenchymatous excessive degradation again, thereby when improving the enzymatic hydrolyzation of fibrous material; Can effectively control the generation of side reaction again; Promptly reduce inhibitor contents, saved the introducing of detoxification unit operation, simplified technology.In addition; In this technology; The isolation and selection property pre-treatment of fibrocyte and parenchyma cell, for the layered multi-stage of lignocellulose raw material transform and the living weight right with new way is provided, and two sections quick-fried carding technologies of vapour also help the rehydration of fibrous material even; Reduce the quick-fried condition of vapour of second section material and reduced raw material content, improved utilization ratio of raw materials.
Summary of the invention
The objective of the invention is has become one of main bottleneck of lignocellulose biological processing to producing fermentation inhibitor in the present lignocellulose preprocessing process; And the normal detoxification operation that adopts has not only increased the fermentation costs of bio-based product; Make technological process more complicated; And cause the problems such as loss of a part of fermentable sugar, developed two sections novel quick-fried carding technologies of vapour.Two sections quick-fried carding technologies of vapour here; Two sections quick-fried technologies of vapour that are different from traditional indication; Be meant that to adopt the quick-fried condition of relatively mild vapour to carry out first section vapour quick-fried; With first section stock grading, obtain parenchyma and fibrous tissue through airflow classification device, it is quick-fried again fibrous tissue to be carried out second section vapour under appropriate condition.
Technical scheme of the present invention is following:
The novel two sections quick-fried carding technologies of vapour that reduce the fermentation inhibitor content in lignocellulose source from the source provided by the invention, it may further comprise the steps: be 0.5～1.5MP at pressure 1)
A, the time is that the quick-fried condition of the vapour of 1～10min carries out stalk is carried out first section steam explosion processing; 2) through airflow classification device first section gas-explosive material carried out classification, obtain parenchyma and fibrous tissue; 3) fibrous tissue that branch is combed is 0.5～1.5MPa at pressure, and the time is to carry out second section steam explosion under the condition of 1～10min to handle; 4) material quick-fried to first section vapour respectively, after dividing comb and second section vapour quick-fried carries out enzymolysis and fermentation; 5) measure respectively stalk, first section gas-explosive material, divide the transformation efficiency of water ratio, the inhibitor contents in the water lotion, enzymatic hydrolyzation and the tunning of parenchyma and fibrous tissue and second section gas-explosive material behind the comb.
Among the present invention, enzymatic hydrolysis condition is that the solid-to-liquid ratio of steam puffed stalk and pH 4.850mM citrate buffer solution is 1: 10-1: 30, and enzyme concentration is the 15IU/g-110/g substrate, at 50 ℃, enzymolysis 24-120h in the shaking bath of 150rpm.Then, centrifuging gets enzymolysis solution.
Beneficial effect of the present invention is: (1) two section quick-fried carding technology of vapour effectively optionally carries out pre-treatment to the feature request of its each tissue; Can guarantee that fibrous tissue reaches pretreating effect preferably, can avoid parenchymatous excessive degradation again, thereby when improving the enzymatic hydrolyzation of fibrous material; Can effectively control the generation of side reaction again; Promptly reduce inhibitor contents, saved the introducing of detoxification unit operation, simplified technology.(2) in this technology, the isolation and selection property pre-treatment of fibrocyte and parenchyma cell, for the layered multi-stage of lignocellulose raw material transform and the living weight right with new way is provided.(3) two sections quick-fried carding technologies of vapour help the rehydration of fibrous material even, have reduced the quick-fried condition of vapour of second section material and have reduced raw material content, have improved utilization ratio of raw materials.
Embodiment
Through embodiment the present invention is further specified below.
The cellulase preparation that uses in the embodiment of the invention is available from Xia Sheng company, 110FPA IU/ml, 1200IU xylanase/ml.
Embodiment 1: take by weighing the dried corn straw of 200g and put into steam-explosion jar, airtight back feeds water vapour, makes tank pressure rise to 1.1MPa rapidly, after keeping 4min under this pressure, puts pressure rapidly, thereby makes first section steam puffed stalk.Get an amount of first section steam puffed stalk, on centrifugal airflow classification device, carry out the branch comb, obtain parenchyma and fibrous tissue respectively.Take by weighing the fibrous tissue after an amount of branch is combed, put into steam-explosion jar, airtight back feeds water vapour, makes tank pressure rise to 1.2MPa rapidly, after keeping 4min under this pressure, puts pressure rapidly, thereby makes second section steam puffed stalk.Pretreatment condition is 1.2MPa, 8min, be regardless of comb is the pre-treatment control group.
Take by weighing first section steam puffed stalk respectively, divide steam puffed stalk behind the comb, second section steam puffed stalk and control group steam puffed stalk 1g (dry base); Dissolve in 20ml water in 70 ℃ of lixiviate 2h; Get supernatant; Adopt performance liquid (Agilent 1200HPLC, the U.S.) to measure furfural and hydroxymethylfurfural (HMF) content in the hydrolyzed solution, adopt ultraviolet spectrophotometer to measure the solubility content of lignin in the hydrolyzed solution at 280nm.Furfural, hydroxymethylfurfural (HMF) and solubility content of lignin sum are inhibitor contents in the hydrolyzed solution.
Take by weighing first section steam puffed stalk respectively, divide steam puffed stalk and second section steam puffed stalk and pre-treatment control group steam puffed stalk 10g behind the comb; Add in the 200ml pH 4.850mM citrate buffer solution; Add the 0.5ml cellulase, at 50 ℃, enzymolysis 48h in the shaking bath of 150rpm.Adopt the DNS method to survey reducing sugar content in the enzymolysis solution.Then, centrifuging gets enzymolysis solution.Get the 20ml enzymolysis solution, moisturizing adds 0.24g potassium primary phosphate, 0.4g peptone to 40ml, adds the dense 25g/L of being of initial sugar in glucose to the enzymolysis solution, adds 2mL serratia marcescens seed liquor, and regulating pH is 5, at 37 ℃ of anaerobically fermenting 48h.Be the fermentation control group of carbon source only with glucose.To adopt HPLC to measure 2,3-butyleneglycol content.Finally amount to the content sum of inhibitor contents for the material after dividing content and second section vapour in the parenchyma behind the comb quick-fried, 2,3-butyleneglycol content in like manner can be regarded as.
The result shows the inhibitor contents low 33% of the inhibitor contents of experimental group than pre-treatment control group; And 2 of experimental group; 3-butyleneglycol content is than being 2 of carbon source with sugar only, and 3-butyleneglycol fermentation control group is high by 5.1%, and the pre-treatment control group 2; 3-butyleneglycol content is than being 2 of carbon source with sugar only, and 3-butyleneglycol fermentation control group hangs down 66.7%.
Embodiment 2: take by weighing the dried corn straw of 200g and put into steam-explosion jar, airtight back feeds water vapour, makes tank pressure rise to 1.2MPa rapidly, after keeping 4min under this pressure, puts pressure rapidly, thereby makes first section steam puffed stalk.Get an amount of first section steam puffed stalk, on centrifugal airflow classification device, carry out the branch comb, obtain parenchyma and fibrous tissue respectively.Take by weighing the fibrous tissue after an amount of branch is combed, put into steam-explosion jar, airtight back feeds water vapour, makes tank pressure rise to 1.2MPa rapidly, after keeping 4min under this pressure, puts pressure rapidly, thereby makes second section steam puffed stalk.Pretreatment condition is 1.2MPa, 8min, be regardless of comb is the pre-treatment control group.
Take by weighing first section steam puffed stalk respectively, divide steam puffed stalk behind the comb, second section steam puffed stalk and control group steam puffed stalk 1g (dry base); Dissolve in 20ml water in 70 ℃ of lixiviate 2h; Get supernatant; Adopt performance liquid (Agilent 1200HPLC, the U.S.) to measure furfural and hydroxymethylfurfural (HMF) content in the hydrolyzed solution, adopt ultraviolet spectrophotometer to measure the solubility content of lignin in the hydrolyzed solution at 280nm.Furfural, hydroxymethylfurfural (HMF) and solubility content of lignin sum are inhibitor contents in the hydrolyzed solution.
Take by weighing first section steam puffed stalk respectively, divide steam puffed stalk and second section steam puffed stalk and pre-treatment control group steam puffed stalk 10g behind the comb; Add in the 200ml pH 4.850mM citrate buffer solution; Add the 0.5ml cellulase, at 50 ℃, enzymolysis 48h in the shaking bath of 150rpm.Adopt the DNS method to survey reducing sugar content in the enzymolysis solution.Then, centrifuging gets enzymolysis solution.Get the 20ml enzymolysis solution, moisturizing adds 0.24g potassium primary phosphate, 0.4g peptone to 40ml, adds the dense 25g/L of being of initial sugar in glucose to the enzymolysis solution, adds 2mL serratiamarcescens seed liquor, and regulating pH is 5, at 37 ℃ of anaerobically fermenting 48h.Be the fermentation control group of carbon source only with glucose.To adopt HPLC to measure 2,3-butyleneglycol content.Finally amount to the content sum of inhibitor contents for the material after dividing content and second section vapour in the parenchyma behind the comb quick-fried, 2,3-butyleneglycol content in like manner can be regarded as.
The result shows the inhibitor contents low 27% of the inhibitor contents of experimental group than pre-treatment control group; And 2 of experimental group; 3-butyleneglycol content is than being 2 of carbon source with sugar only, and 3-butyleneglycol fermentation control group is high by 4.3%, and the pre-treatment control group 2; 3-butyleneglycol content is than being 2 of carbon source with sugar only, and 3-butyleneglycol fermentation control group hangs down 66.5%.
Embodiment 3: take by weighing the dried corn straw of 200g and put into steam-explosion jar, airtight back feeds water vapour, makes tank pressure rise to 1.1MPa rapidly, after keeping 4min under this pressure, puts pressure rapidly, thereby makes first section steam puffed stalk.Get an amount of first section steam puffed stalk, on centrifugal airflow classification device, carry out the branch comb, obtain parenchyma and fibrous tissue respectively.Take by weighing the fibrous tissue after an amount of branch is combed, put into steam-explosion jar, airtight back feeds water vapour, makes tank pressure rise to 1.2MPa rapidly, after keeping 4min under this pressure, puts pressure rapidly, thereby makes second section steam puffed stalk.Pretreatment condition is 1.2MPa, 8min, be regardless of comb is the pre-treatment control group.
Take by weighing first section steam puffed stalk respectively, divide steam puffed stalk behind the comb, second section steam puffed stalk and control group steam puffed stalk 1g (dry base); Dissolve in 20ml water in 70 ℃ of lixiviate 2h; Get supernatant; Adopt performance liquid (Agilent 1200HPLC, the U.S.) to measure furfural and hydroxymethylfurfural (HMF) content in the hydrolyzed solution, adopt ultraviolet spectrophotometer to measure the solubility content of lignin in the hydrolyzed solution at 280nm.Furfural, hydroxymethylfurfural (HMF) and solubility content of lignin sum are inhibitor contents in the hydrolyzed solution.
Take by weighing first section steam puffed stalk respectively, divide steam puffed stalk and second section steam puffed stalk and pre-treatment control group steam puffed stalk 10g behind the comb; Add in the 200ml pH 4.850mM citrate buffer solution; Add the 0.5ml cellulase, at 50 ℃, enzymolysis 48h in the shaking bath of 150rpm.Adopt the DNS method to survey reducing sugar content in the enzymolysis solution.Then, centrifuging gets enzymolysis solution.Get the 40ml enzymolysis solution, add 0.2g potassium primary phosphate, 0.08g ammonium sulfate, 0.016g sal epsom, 0.008g calcium chloride, 0.08g yeast powder.Add the dense 25g/L of being of initial sugar in glucose to the enzymolysis solution, add 2mL Angel Yeast seed liquor, regulating pH is 5.5, at 32 ℃ of anaerobically fermenting 48h.Be the fermentation control group of carbon source only with glucose.Adopt HPLC to measure ethanol content.Finally amount to the content sum of inhibitor contents for the material after dividing content and second section vapour in the parenchyma behind the comb quick-fried, ethanol content in like manner can be regarded as.
The result shows the inhibitor contents low 33% of the inhibitor contents of experimental group than pre-treatment control group.The ethanol content of experimental group is higher by 13.8% than the ethanol content of pre-treatment control group, and respectively than the ethanol fermentation control group low 22.4% and 33% that only with sugar is carbon source.
Claims (4)
1. method that reduces the fermentation inhibitor in lignocellulose source from the source may further comprise the steps:
1) stalk being carried out first section steam explosion handles;
2) first section gas-explosive material carried out the branch comb, obtain parenchyma and fibrous tissue;
3) fibrous tissue that branch is combed carries out second section steam explosion to be handled;
4) material quick-fried to first section vapour respectively, after dividing comb and second section vapour quick-fried carries out enzymolysis and fermentation;
5) measure respectively stalk, first section gas-explosive material, divide parenchyma and water ratio, the inhibitor contents in the water lotion and the tunning content of fibrous tissue and second section gas-explosive material behind the comb.
2. method according to claim 1, wherein first of step 1) section steam explosion processing is in steam-explosion jar, under the vapor pressure of 0.5～1.5MP a, to carry out 1～10min.
3. method according to claim 1, wherein second of step 3) section steam explosion processing is in steam-explosion jar, under the vapor pressure of 0.5～1.5MP a, to carry out 1～10min.
4. method according to claim 1; Wherein the enzymatic hydrolysis condition in the step 4) is: steam puffed stalk and pH 4.8 and concentration are that the solid-to-liquid ratio of 50mM citrate buffer solution is 1: 10～1: 30; Enzyme concentration is 15IU/g～110/g substrate; At 50 ℃, enzymolysis 24～120h in the shaking table of 100～200rpm.Then, the centrifugal enzymolysis solution that gets.
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| CN104878055A (en) * | 2015-05-26 | 2015-09-02 | 华南理工大学 | Method for pretreating ethyl alcohol produced from corn straws |
| CN112553261A (en) * | 2020-11-06 | 2021-03-26 | 华南理工大学 | Method for high-solid enzymolysis of lignocellulose |
| CN115627277A (en) * | 2022-11-18 | 2023-01-20 | 中国科学院过程工程研究所 | Method for performing solid state fermentation on 2, 3-butanediol by high-solid-state enzymolysis of straws |
| CN117051055A (en) * | 2023-10-12 | 2023-11-14 | 中国科学院过程工程研究所 | Method for preparing low inhibitor straw sugar suitable for fermentation and catalysis by using uniform graded straw |
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| CN104878055A (en) * | 2015-05-26 | 2015-09-02 | 华南理工大学 | Method for pretreating ethyl alcohol produced from corn straws |
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| CN115627277A (en) * | 2022-11-18 | 2023-01-20 | 中国科学院过程工程研究所 | Method for performing solid state fermentation on 2, 3-butanediol by high-solid-state enzymolysis of straws |
| CN117051055A (en) * | 2023-10-12 | 2023-11-14 | 中国科学院过程工程研究所 | Method for preparing low inhibitor straw sugar suitable for fermentation and catalysis by using uniform graded straw |
| CN117051055B (en) * | 2023-10-12 | 2024-02-06 | 中国科学院过程工程研究所 | Method for preparing low inhibitor straw sugar suitable for fermentation and catalysis by using uniform graded straw |
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Application publication date: 20120718 |