WO2020206863A1 - 木质纤维素密化过程中加入碱或酸性试剂进行预处理及生物转化的方法 - Google Patents

木质纤维素密化过程中加入碱或酸性试剂进行预处理及生物转化的方法 Download PDF

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WO2020206863A1
WO2020206863A1 PCT/CN2019/096329 CN2019096329W WO2020206863A1 WO 2020206863 A1 WO2020206863 A1 WO 2020206863A1 CN 2019096329 W CN2019096329 W CN 2019096329W WO 2020206863 A1 WO2020206863 A1 WO 2020206863A1
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acid
pretreatment
lignocellulose
densification
alkali
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PCT/CN2019/096329
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English (en)
French (fr)
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金明杰
陈相雪
袁鑫川
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南京理工大学
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Priority claimed from CN201910277078.0A external-priority patent/CN111793662A/zh
Priority claimed from CN201910536652.XA external-priority patent/CN112111540B/zh
Application filed by 南京理工大学 filed Critical 南京理工大学
Priority to US17/252,168 priority Critical patent/US11643674B2/en
Priority to BR112021020112A priority patent/BR112021020112A2/pt
Priority to EP19924538.2A priority patent/EP3954779A4/en
Publication of WO2020206863A1 publication Critical patent/WO2020206863A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/06—Ethanol, i.e. non-beverage
    • C12P7/08—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate
    • C12P7/10—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate substrate containing cellulosic material
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/14—Pretreatment of feeding-stuffs with enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/02—Monosaccharides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P2201/00—Pretreatment of cellulosic or lignocellulosic material for subsequent enzymatic treatment or hydrolysis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P2203/00—Fermentation products obtained from optionally pretreated or hydrolyzed cellulosic or lignocellulosic material as the carbon source
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

  • the invention belongs to the technical field of biorefining, and relates to a method for pretreatment and biotransformation by adding alkali or acidic reagents in the densification process of lignocellulose.
  • Lignocellulose is one of the most common renewable resources in nature, widely distributed and easy to obtain.
  • the biorefinery of lignocellulose converts it into various bio-based products, which not only guarantees energy security, promotes rural economic development, but also reduces carbon emissions, protects the environment, and promotes sustainable development.
  • Lignocellulose pretreatment is a necessary step in the process of biorefining and producing target products such as biofuels. Its role is to reduce the natural resistance of lignocellulose and facilitate the subsequent biotransformation.
  • Traditional pretreatment methods have problems such as serious corrosion of pretreatment reaction equipment, low safety, high energy consumption, and cumbersome operation (Theerarattananoon K, Xu F, Wilson J, et al.
  • Densification of lignocellulose into a dense structure can effectively increase the density of lignocellulose (density can be increased by 5-15 times).
  • the storage cost and transportation cost of dense lignocellulose can be reduced by more than 50% and 90%, respectively.
  • the dense lignocellulose has a uniform size and shape, which is convenient for industrial production operations (Zhang PF, Zhang Q, Obviouslys TW, et al.
  • densified lignocellulose can greatly increase the loading capacity of raw materials and greatly increase the utilization rate of equipment volume (Venkatesh Balan, Sugarland, TX (US); Leonardo da Costa Sousa, Brighton, MI (US), De- esterification of biomass prior to ammonia pretreatment and systems and products related thereto[P],Appl.NO.:16/029,452,2018-07-06).
  • it is convenient to operate in the subsequent biotransformation process and is beneficial to increase the concentration of the substrate (Bals B D, Gunawan C, Moore J, et al. Enzymatic Hydrolysis of Pelletized AFEX TM- Treated Corn Stover at High Solid Loadings[J].
  • Densification can also be performed after the pretreatment of the lignocellulosic feedstock.
  • a study by Bruce E. Dyer found that corn stalks were densified after AFEX (Ammonia Fiber Explosion) pretreatment, which is conducive to enzymatic hydrolysis of high substrate concentrations and convenient feeding (Brian Bars, Fazzanet Tamori, Timothy J. Campbell, Bruce E. Dell, Method for hydrolyzing pretreated densified biomass particles and related systems [P], Chinese Patent: 201380022053.7, 2013-04-26).
  • densified lignocellulose is beneficial to increase the loading of raw materials during pretreatment, it will also lead to higher requirements for pretreatment conditions, such as temperature increase and reaction time extension (Venkatesh Balan, Sugarland, TX (US); Leonardo da Costa Sousa,Brighton,MI(US),De-esterification of biomass prior to ammonia pretreatment and systems and products related thereto[P],Appl.NO.:16/029,452,2018-07-06).
  • temperature increase and reaction time extension Venkatesh Balan, Sugarland, TX (US); Leonardo da Costa Sousa,Brighton,MI(US),De-esterification of biomass prior to ammonia pretreatment and systems and products related thereto[P],Appl.NO.:16/029,452,2018-07-06).
  • the present invention provides a method for adding alkali during densification of lignocellulose. Or a method for pretreatment and biotransformation with acidic reagents, by adding alkaline reagents or acidic reagents during the densification process of lignocellulose to achieve pretreatment of lignocellulose.
  • the alkaline reagents or acidic reagents in the densified lignocellulose further carry out a relatively mild pretreatment on the lignocellulose raw materials during subsequent transportation and storage, so as to make full use of the transportation and storage time to further improve the pretreatment effect.
  • Densified lignocellulose containing alkali or acid is not suitable for moldy deterioration or decay, and the increase in density makes it more suitable for storage and transportation.
  • the invention avoids the requirement of traditional technology on pretreatment equipment and energy consumption.
  • the severity of the further pretreatment conditions can be reduced to a certain extent, and the mass transfer problem of chemical reagents in the pretreatment process of densified lignocellulose is solved.
  • the alkali-densified lignocellulose When the alkali-densified lignocellulose is further pretreated with water, it will release heat (alkali meets water to release heat), which solves the heat transfer problem during the pretreatment of densified lignocellulose to a certain extent. Therefore, the effect of further pretreatment will also be greatly improved.
  • alkaline reagents or acidic reagents during the densification of lignocellulosic raw materials; or add alkaline reagents or acidic reagents to lignocellulose raw materials, mix well and perform densification treatment; or add alkaline reagents to lignocellulose raw materials Reagents or acidic reagents are densified after reaction to obtain densified lignocellulose containing alkali or acid.
  • the present invention also provides a method for bioconversion of alkali-containing or acid-containing densified lignocellulose, and the specific steps are as follows:
  • alkaline reagents or acidic reagents during the densification of lignocellulosic raw materials; or add alkaline reagents or acidic reagents to lignocellulose raw materials, mix well and perform densification treatment; or add alkaline reagents to lignocellulose raw materials Reagents or acidic reagents are subjected to densification treatment after reaction, and the obtained densified lignocellulose containing alkali or acid is directly subjected to biotransformation or undergoes further selective pretreatment and then biotransformation.
  • the lignocellulose is wheat straw, corn straw, agricultural and forestry waste, rice straw, sorghum straw, soybean straw, forestry waste, recycled wood pulp fiber, wood chips, softwood, hardwood, aquatic plants, aquatic plants, algae, and One or more of animal feces; the moisture content of lignocellulose is 0% to 90%.
  • the alkaline reagent is sodium hydroxide, calcium hydroxide, potassium hydroxide, sodium sulfite, sodium bisulfite, sodium acetate, sodium carbonate, sodium bicarbonate, sodium sulfate, ethylenediamine, triethylamine One or more of, ammonia, liquid ammonia, calcium oxide and sodium oxide.
  • the acid reagent is sulfuric acid, nitric acid, nitrous acid, hydrochloric acid, phosphoric acid, sulfurous acid, perchloric acid, chloric acid, chlorous acid, hypochlorous acid, oxalic acid, formic acid, acetic acid, propionic acid, benzoic acid , Phenylacetic acid, benzenesulfonic acid, oxalic acid, succinic acid, one or more of carbon dioxide, sulfur dioxide, sodium bisulfate and sodium bisulfite.
  • the amount of the alkaline reagent or the acidic reagent accounts for 0.5% to 30.0% of the mass of the lignocellulose.
  • the alkaline or acidic reagent can be added directly in the form of a pure alkaline or acidic reagent, or in the form of an aqueous solution of the alkaline or acidic reagent; the adding method is direct pouring or adding, spraying, or spraying. Bring in steam, spray in alkaline or acidic gas, etc., so that the alkaline or acid reagent and lignocellulose are fully mixed, and then the lignocellulose is densified; or the alkaline or acid reagent is mixed with the lignocellulose during the densification process.
  • the cellulose is added to the densification apparatus to make a dense shape of densified lignocellulose.
  • the acid or alkali-containing densified lignocellulose without further pretreatment is subjected to biotransformation immediately or after being placed for a period of time; the biotransformation is carried out after further selective pretreatment, the further selection
  • the sexual pretreatment is spraying water, steaming, immersion, exposure, low temperature freezing, high temperature treatment, water cooking, high temperature sterilization, microwave and ultrasonic crushing on the surface of densified lignocellulose containing acid or alkali; or steam explosion pretreatment , Hot water pretreatment, steam pretreatment and other hydrothermal pretreatment; or dilute acid pretreatment, weak acid pretreatment, oxidizing acid pretreatment and mixed acid pretreatment and other acid pretreatment; or dilute alkali pretreatment, weak alkali pretreatment , Alkaline pretreatment such as ammonia pretreatment, oxidizing alkali pretreatment or mixed alkali pretreatment; or organic solvent pretreatment, etc.
  • the density of the densified lignocellulose is 200-1500kg/m 3 ;
  • the shape of the densified lignocellulose is rod-like, granular, block or pellet; the rod-like specification: diameter 1mm- 30cm; granular specifications: diameter 0.1cm-50cm; block specifications: length 0.1cm-200cm, width 0.1cm-200cm, height 0.1cm-200cm; pellet specifications: diameter 0.1cm-50cm, thickness 0.01cm-20cm.
  • the biological transformation is one or more of enzymatic reaction and microbial transformation;
  • the enzymatic reaction is the addition of enzymes or enzymes to the pretreatment substrate, and the enzymes are selected from cellulose.
  • the conversion of the microorganisms into microbial fermentation is to add fermenting microorganisms to the pretreatment substrate or enzymatic hydrolysate.
  • the fermenting microorganisms are fermenting microorganisms used in conventional biorefining, and can be one of yeast, bacteria, and mold. Many kinds.
  • the products produced by the method of biotransformation are biofuels, bulk chemicals, fine chemicals, animal feed, food additives, medicines, etc., such as ethanol, butanol, acetone, acetic acid, lactic acid, aliphatic hydrocarbons, oils , Protein, amino acids, enzymes, antibiotics, vitamins, antibodies, biogas.
  • the present invention adds alkaline reagents or acidic reagents in the densification process of lignocellulose, which not only utilizes the mechanical action in the densification process and the heat (combined with acid or alkali) in the compression process to destroy the lignocellulose structure, but also During transportation and storage at different temperatures (-40°C ⁇ 100°C), both alkali and acid reagents can exert a chemical effect on lignocellulose, and lignocellulose can be pretreated, which can be directly used in the next step. Biotransformation, or further pretreatment (at this time the alkali or acidic reagent has been fully mixed with lignocellulose, which is beneficial to further pretreatment at a high lignocellulose loading) before biotransformation.
  • the present invention has the following advantages:
  • the densified lignocellulose containing alkaline reagents or acidic reagents prepared by the present invention is more convenient for transportation and storage;
  • Figure 1 (a) is the surface morphology of the corn stalk particles densified without alkaline agent in Example 1 on the first day, and (b) is the corn stalk particles densified without alkaline agent on the 6th day at room temperature Surface morphology map, (c) is the surface morphology map of corn stover particles pretreated with sodium hydroxide (0.3g/g dry weight of corn stalk) on the first day, (d) is sodium hydroxide ( 0.3g/g dry weight of corn stalks) The surface morphology map of the corn stalk pellets that have been densified and placed at room temperature for 6 days.
  • Figure 2 (a) is the growth state diagram of the microorganisms in the LB solid medium of the loose corn stalk supernatant without chemical reagents in Example 1, (b) is the microorganisms in the LB solid medium of the corn stalk supernatant pretreated by densification without chemical reagents The growth state diagram on the solid medium, (c) is the growth state diagram of the microorganisms on the LB solid medium containing the corn stalk supernatant pretreated with alkali densification, (d) is the corn stalk supernatant containing the acid densification pretreatment The growth state diagram of liquid microorganisms on LB solid medium.
  • Figure 3 (A) is a diagram of the diameter of corn stalks pretreated by densification containing alkaline reagents or acid reagents in Example 2, and (B) is a diagram of the length of corn stalks pretreated by densification containing alkaline reagents or acid reagents.
  • Figure 4 (A) is a view of the loose corn stalks in a 100 ml plastic cup in Example 2, and (B) is a view of the corn stalks containing acid densification pretreatment in a 100 ml plastic cup.
  • Example 5 is a graph showing the effect of different storage days (0, 6, 12 days) of corn stover pretreated with calcium hydroxide densification (DLL) on the enzymatic hydrolysis effect in Example 3.
  • DLL calcium hydroxide densification
  • Example 6 is a graph showing the effect of different storage days (2, 6, 12) of corn stover pretreated with sodium hydroxide (DLS) on the enzymatic hydrolysis effect in Example 4.
  • DLS sodium hydroxide
  • Figure 7 shows the substrate concentration of 10% (wt/wt, corn stalks in the total weight of straw, water, hydrolase and chemical reagents) in Example 5, the traditional dilute acid (DA) pretreatment of corn stalks and acid Map of sugar concentration of corn stover pretreated with hot water (DLA+LHW) after enzymatic hydrolysis for 72h after densification pretreatment.
  • DA dilute acid
  • DLA+LHW hot water
  • Figure 8 shows the traditional dilute alkali (AL) pretreatment, at a concentration of 6% (wt/wt, glucan in the straw accounts for the total weight of the straw, water, hydrolase and chemical reagents) in Example 6
  • PCS+AL dilute alkali
  • DLS+Auctoclave alkali densification pretreatment and sterilization
  • Figure 9 shows the substrate concentration of 20% (wt/wt, corn stalks in the total weight of straw, water, hydrolase and chemical reagents) in Example 7, and further hot water (DLA+LHW) after acid densification pretreatment.
  • Figure 10 shows the substrate concentration of 20% (wt/wt, corn stover in the total weight of the straw, water, hydrolase and chemical reagents) in Example 8, and further hot water (DLL) after densification pretreatment with calcium hydroxide +LHW) Ethanol fermentation curve of the enzymatic hydrolysate obtained by enzymatic hydrolysis of pretreated corn stover without detoxification and batch feeding.
  • Example 11 is a graph showing the sugar concentration of the corn stover that was further sterilized (DLL+Auctoclave) after densification pretreatment with sodium hydroxide in Example 9 under different substrate concentrations for 72 hours of enzymatic hydrolysis.
  • the reagents, raw materials, instruments and equipment used in the present invention can all be purchased in the market or prepared by existing methods.
  • PCS Portized Corn stover pelletized corn stover
  • DA Dilute sulfuric Acid pretreatment
  • AL Alkali pretreatment
  • LHW Liquid Hot Water
  • DLL Densifying Lignocellulose with Lime pretreatment (due to the addition of water during the densification process, lime becomes calcium hydroxide)
  • DLS Densifying Lignocellulose with Sodium hydroxide) pretreatment
  • DLA Densifying Lignocellulose with sulfuric Acid pretreatment
  • PCS+AL Pelletized Corn Stover pretreated bydilute ALkali pretreatment
  • DLA+LHW Densifying Lignocellulose with sulfuric acid After pretreatment, further hot water (DensifyingLignocellulosewithsulfuricAcidfollowed by LiquidHot Water) pretreatment
  • DLS+Autoclave DensifyingLignocellulosewithSodiumhydroxidefollowed by autoclave treatment
  • DLL+LHW Calcium hydroxide pretreatment
  • a pellet machine is used for densification during the densification process.
  • Densification pretreatment spray the crushed corn stalks with sodium hydroxide solution, sulfuric acid solution and pure water respectively, the content of sodium hydroxide is 0.3g/g corn stalk dry matter, and the content of sulfuric acid is 0.1g/g corn stalk Dry matter, the water content of the three densification methods is 0.5g/g corn stalk dry matter, and a pelletizer is used to perform densification operations on the corn stalks that are uniformly mixed with reagents. After the reaction, it was left at room temperature for 6 days.
  • Coating process mix the corn stalks containing alkali densification pretreatment (DLS), acid densification pretreatment (DLA) corn stalks and chemical reagent-free densification (PCS) corn stalks with sterile water evenly , The ratio of dry straw to water is 1:20. Take 10 ⁇ L of the supernatant and place it on the LB solid medium. After dilution and coating, place it in a constant temperature incubator at 30°C for 3 days.
  • LDS alkali densification pretreatment
  • DLA acid densification pretreatment
  • PCS chemical reagent-free densification
  • Figure 1(a) shows the corn stalk densified without alkaline reagents on the first day.
  • the surface phenomenon Figure 1(b) is the surface phenomenon of corn stalk particles densified without alkaline reagents on the 6th day at room temperature
  • Figure 1(c) is the dried corn stalk containing sodium hydroxide (0.3g/g) Weight) the surface phenomenon of corn stover pellets pretreated by densification on the first day
  • Figure 1(d) shows the corn stover pellets pretreated by densification containing sodium hydroxide (0.3g/g dry weight of corn stover) at room temperature Surface phenomenon after 6 days.
  • Figure 2(a) shows the growth state of microorganisms on the LB solid medium of loose corn stalk supernatant without chemical reagents
  • Figure 2(b) shows the corn stalk supernatant without chemical reagent densification pretreatment
  • the growth status of microorganisms on LB solid medium shows the growth status of microorganisms in the supernatant of corn stalk pretreated with alkali densification on LB solid medium.
  • Figure 2(d) shows the pretreatment with acid densification.
  • the microorganisms in the supernatant of corn stover were grown on LB solid medium. It can be seen from Fig.
  • the corn stover pretreated with acid or alkali densification reduces the cost of transportation and storage, including the following steps:
  • Densification pretreatment spray the crushed corn stalks with sodium hydroxide solution or sulfuric acid solution respectively, the sodium hydroxide content is 0.3g/g corn stalk dry weight, or the sulfuric acid content is 0.1g/g corn stalk dry weight, The water content of the two densification methods is 0.5 g/g corn stalk dry weight, and a granulator is used to uniformly mix the acidic or alkaline reagent corn stalks for densification.
  • the corn stover pretreated with acid or alkali densification reduces the cost of transportation and storage.
  • Figure 3A shows the diameter of corn stalks densified pretreated with alkaline or acidic reagents
  • Figure 3B shows the length of corn stalks densified pretreated with alkaline or acidic reagents
  • Figure 4A shows the loose corn stalks in a 100ml plastic cup.
  • Figure 4B shows the state of containing the corn stover pretreated by acid densification in a 100ml plastic cup. It can be obtained from Figure 3 that the diameter of the corn stalk pretreated with acid densification is 0.8cm, the length is 2.5cm, and the mass is 1.85g.
  • the volume of the corn stalk pretreated with acid densification is about:
  • the density of corn stover pretreated with acid densification is about: As can be seen in Figure 4, the mass of loose corn stalks in a 100 mL plastic cup is 12.54 g, while the mass of corn stalks pretreated with acid densification is 81.81 g in a 100 mL plastic cup. Therefore, the density of corn stover pretreated with acid densification is about 6.5 times the density of loose corn stover. It shows that the corn stalk pretreated with acid or alkali densification has great advantages over loose straw in transportation and storage.
  • the effect of different storage days of corn stover pretreated with calcium hydroxide densification (DLL) on enzymatic hydrolysis includes the following steps:
  • DLL pretreatment place the crushed corn stalks in a sealed bag, and evenly spray calcium hydroxide solution.
  • the amount of calcium hydroxide and water is determined by the dry weight of the corn stalks, and the content of calcium hydroxide is 0.15g/ g corn stalk dry matter, water content is 0.5 g/g corn stalk dry matter, the densification operation is performed at room temperature, and the densified straw is placed at room temperature for 0, 6, 12 days.
  • the effect of different storage days of corn stover pretreated with sodium hydroxide (DLS) on the enzymatic hydrolysis effect includes the following steps:
  • DLS pretreatment place the crushed corn stalks in a sealed bag, and spray sodium hydroxide solution evenly.
  • the amount of sodium hydroxide and water is determined by the dry weight of the corn stalks, and the content of sodium hydroxide is 0.3 g/g corn stalk dry matter, water content is 0.5 g/g corn stalk dry matter, the densification operation is performed at room temperature, after treatment, the densified straw is placed at room temperature for 2, 6, 12 days.
  • DLA pretreatment spray the crushed corn stalks with sulfuric acid solution.
  • the added amount of sulfuric acid is 0.1g/g straw dry weight, and the added water content is 0.5g/g straw dry weight.
  • Use a granulator to uniformly mix acid The corn stover of the reagent is densified. After the densification, the acid-containing densified corn stalks are bagged, sealed, and placed at room temperature for more than 1 day.
  • DLA+LHW pretreatment acid-containing densification pretreatment of corn stalks with an acid concentration of 0.1g/g dry matter, the dry weight of the straw: the total water volume is 1:9; put it in a high temperature and high pressure reactor for heating and heating The temperature is 160°C, and the reaction time is 10 minutes.
  • (4)DA traditional acid pretreatment method pretreatment: raw material is loose straw, add sulfuric acid solution to it, the amount of sulfuric acid is 0.1g/g corn stover dry matter, the dry weight of straw: the total water amount is 1:9, It is placed in a high temperature and high pressure reactor for heating, the heating temperature is 160°C, and the reaction time is 10 minutes.
  • Enzymatic hydrolysis process the concentration of hydrolyzed substrate is 10% (wt/wt, the percentage of corn stalks accounting for the total weight of straw, water, hydrolase and chemical reagents), and hydrolase and citric acid-sodium citrate buffer are added, Place the culture in a shaking incubator (set rotation speed at 250 rpm, temperature at 50°C) for a hydrolysis time of 72 hours.
  • DLA+LHW damages the structure of corn stalks more thoroughly than traditional dilute acid pretreatment (DA), and releases more hydrolyzable polysaccharides, which is beneficial to subsequent operations. It shows that the acid-containing densification pretreatment (DLA) has great potential in industrial applications.
  • DLS pretreatment spray the crushed corn stalks with sodium hydroxide solution, add sodium hydroxide at 0.1g/g corn stalk dry matter, add water at 0.5g/g corn stalk dry matter, use a granulator Perform densification operation by uniformly mixing sodium hydroxide reagent and corn stover. After the densification is completed, bag it, seal it, and place it at room temperature for more than 1 day.
  • DLS+Autoclave pretreatment the concentration of sodium hydroxide is 0.1g/g dry matter corn stalks densified pretreated with sodium hydroxide, 30% (wt/wt, the percentage of the dry weight of corn stalks to the total weight) corn stalks Dry weight, place it in a sterilization pot for heating, the heating temperature is 120°C, and the reaction time is 60 minutes.
  • PCS+AL pretreatment the corn stalks without sodium hydroxide densification and the sodium hydroxide solution are uniformly mixed, and they are placed in a sterilization pot.
  • concentration of the dry matter is 20% (wt/wt, corn stalk dry
  • the weight is the percentage of the total weight)
  • the content of sodium hydroxide is 0.1 g/g corn stover dry matter.
  • the heating temperature is 120°C, and the reaction time is 120 minutes.
  • Enzymatic hydrolysis process the concentration of hydrolyzed substrate is 6% (wt/wt, glucan accounts for the percentage of the total weight of straw, water, hydrolase and chemical reagents) glucan, and hydrolase and citric acid-citric acid are added Sodium buffer solution, cultured in a shaking incubator (set rotation speed is 250 rpm, temperature is 50° C.), the hydrolysis time is 24 hours.
  • DLS+Autoclave has a higher pretreatment substrate concentration than PCS+AL, and the pretreatment reaction time is 50% of the PCS+AL reaction time.
  • AL and PCS+AL are the traditional dilute alkali pretreatment methods, and DLS+Autoclave as the pretreatment method of the present invention not only increases the concentration of the pretreatment substrate, but also reduces the usage of chemical reagents and the pretreatment intensity. Promotion is of great significance.
  • DLA pretreatment spray the crushed corn stalks with sulfuric acid solution, add 0.1g/g dry corn stalks with sulfuric acid, add 0.5g/g dry corn stalks with water, use a granulator to uniformly mix acid
  • the corn stover of the reagent is densified. After the densification, the acid-containing densified corn stalks are bagged, sealed, and placed at room temperature for more than 1 day.
  • DLA+LHW pretreatment acid densification pretreated corn stalk with an acid concentration of 0.1g/g dry matter, dry straw weight: total water volume of 1:5; put it in a high temperature and high pressure reactor for heating, The heating temperature is 160°C, and the reaction time is 10 minutes.
  • the raw material is corn stalks without acid densification, to which sulfuric acid solution is added, the amount of sulphuric acid is 0.1g/g corn stalk dry matter, the dry weight of straw: the total water volume is 1:5; Placed in a high temperature and high pressure reactor for heating, the heating temperature is 160°C, and the reaction time is 10 minutes.
  • DA pretreatment raw material is loose straw, add sulfuric acid solution to it, the amount of sulfuric acid is 0.1g/g corn stalk dry matter, the dry weight of straw: the total amount of water is 1:9; put it in a high temperature and high pressure reactor Heating, the heating temperature is 160°C, and the reaction time is 10 minutes.
  • Enzymatic hydrolysis process the concentration of hydrolyzed substrate is 20% (wt/wt, the percentage of corn stalks in the total weight of straw, water, hydrolase and chemical reagents), add hydrolytic enzyme and pure water, and place in a shaking incubator Cultivation (set rotation speed at 250 rpm, temperature at 50°C) hydrolysis time is 72 hours.
  • DLA+LHW After 72 hours of hydrolysis, the final glucose concentration of DLA+LHW was 73g/L, slightly higher than PCS+DA (70g/L) and DA (68g/L). The final xylose concentration of DLA+LHW is up to 32g/L.
  • the enzymatic hydrolysis effect of the three pretreatment methods was close to the maximum concentration, and within 12h, the hydrolysis rate of DLA+LHW was the fastest. The above shows that, compared with PCS+DA and DA, DLA+LHW has the advantages of faster conversion rate and higher yield of hydrolyzed sugars in the process of substrate enzymatic hydrolysis, and has broad application prospects.
  • (2) DLL pretreatment spray the crushed corn stalks with calcium hydroxide solution, add calcium hydroxide at 0.15g/g corn stalk dry matter, add water at 0.5g/g corn stalk dry matter, use a pelletizer Densify the corn stalks uniformly mixed with calcium hydroxide reagent. After the densification is completed, the calcium hydroxide-containing densified corn stalks are bagged, sealed, and placed at room temperature for more than 1 day.
  • DLL+LHW pretreatment Calcium hydroxide concentration of 0.15g/g dry matter contains sodium hydroxide for densification pretreatment of corn stalks, the dry weight of straw: the total water volume is 1:5; put it in the high temperature and high pressure reactor Heating is performed, the heating temperature is 140°C, and the reaction time is 60 minutes.
  • Enzymatic hydrolysis process the concentration of hydrolyzed substrate is 20% (wt/wt, the percentage of corn stalks in the total weight of straw, water, hydrolytic enzymes and chemical reagents), add hydrolytic enzymes and pure water, and place in a shaking incubator Cultivation (set rotation speed at 250 rpm, temperature at 50°C) hydrolysis time is 72 hours.
  • Fermentation process select the fermentation strain to be a strain that uses xylose, and the strain is initially inoculated with an OD of 2; it is cultured in a shaking incubator (set rotation speed is 150rpm, temperature is 30°C) and the fermentation time is 72 hours.
  • the enzymatic hydrolysis of DLL+LHW pretreated corn stover resulted in a final glucose concentration of 91.1g/L and a xylose concentration of 24.4g/L.
  • the final ethanol output reached 51.5g/L, and the final total sugar conversion rate of the fermentation reached over 97%. It is inferred that the concentration of inhibitors produced by DLL+LHW pretreatment of corn stover is low, and the conversion efficiency of sugar and the concentration of ethanol are high in the process of no detoxification and no batch feeding, so it has great advantages in the bioconversion process.
  • DLS pretreatment place the crushed corn stalks in a sealed bag, and spray sodium hydroxide solution evenly.
  • the amount of sodium hydroxide and water is determined by the dry weight of the corn stalks.
  • the content of sodium hydroxide is 0.1g/g corn stalk dry matter
  • water content is 0.1g/g corn stalk dry matter
  • the densification operation is performed at room temperature
  • the DLS pretreated corn stalk is placed at room temperature for more than 1 day.
  • Enzymatic hydrolysis process Place DLS+Auctoclave pretreated corn stalks in an enzymatic hydrolysis reactor, the substrate concentration is 3%, 6% and 9% (wt/wt, glucan accounts for straw, water, hydrolytic enzyme And the percentage of the total weight of the chemical reagents) dextran, hydrolytic enzyme and citric acid-sodium citrate buffer were added, and the enzymatic hydrolysis reaction was carried out in a shaking box (250 rpm) at 50° C. for 72 hours.

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Abstract

一种木质纤维素密化过程中加入碱或酸性试剂进行预处理及生物转化的方法。所述方法在木质纤维素原料中，加入碱性试剂或酸性试剂，进行密化处理，形成压缩致密形状的含碱或含酸密化木质纤维素，从而实现预处理。预处理后的木质纤维素中的酸或碱在后续运输、储存过程中能够对木质纤维素原料进行较温和的进一步预处理。如需后续预处理，后续预处理严苛程度可大幅度降低；并且酸或碱与木质纤维素混合均匀、原料密度大，促进了密化木质纤维素后续预处理的高效性及高装载量。本方法简单高效，得到的木质纤维素原料密度大，且含酸或碱不易变质或腐烂，利于运输和储存，后续处理过程中设备利用率高。

Description

木质纤维素密化过程中加入碱或酸性试剂进行预处理及生物转化的方法 技术领域
本发明属于生物炼制技术领域，涉及一种木质纤维素密化过程中加入碱或酸性试剂进行预处理及生物转化的方法。
背景技术
木质纤维素是自然界中最普遍的可再生资源之一，分布广泛，易于获取。木质纤维素的生物炼制将其转化为各种生物基产品，不仅能够保障能源安全、促进农村经济发展，还能减少碳排放、保护环境以及促进可持续发展。
木质纤维素生物质的低密度特性及易发霉、变质、腐烂使其运输和储存的成本偏高，一定程度上制约了木质纤维素生物炼制的工业化。木质纤维素预处理是生物炼制生产生物燃料等目标产品工艺中的必要步骤，其作用是降低木质纤维素的天然抵抗性，利于接下来生物转化的进行。传统的预处理方法存在着对预处理反应设备腐蚀严重、安全性低、耗能高和操作繁琐等问题(Theerarattananoon K,Xu F,Wilson J,et al.Effects of the pelleting conditions on chemical composition and sugar yield of corn stover,big bluestem,wheat straw,and sorghum stalk pellets[J].Bioprocess BiosystEng,2012,35(4):615-623)。另外，木质纤维素的低密度和松散特性也限制了其在预处理和生物转化过程中在设备中的装载量，致使木质纤维素生物炼制工艺生产强度偏低、设备利用率低、成本偏高。
将木质纤维素密化成致密结构可以有效增加木质纤维素的密度(密度可提高5-15倍)。致密的木质纤维素存储成本和运输成本可分别降低50％和90％以上。另外，致密的木质纤维素都有统一的尺寸和形状，方便工业生产的操作(Zhang P F,Zhang Q,Deines T W,et al.Ultrasonic Vibration-Assisted Pelleting of Wheat Straw:A Designed Experimental Investigation on Pellet Quality and Sugar Yield，Asme International Manufacturing Science&Engineering Conference.2012；Theerarattananoon K,Xu F,Wilson J,et al.Effects of the pelleting conditions on chemical composition and sugar yield of corn stover,big bluestem,wheat straw,and sorghum stalk pellets[J].Bioprocess BiosystEng,2012,35(4):615-623.)。密化木质纤维素在预处理过程中可大幅度提高原料的装载量、大幅提高设备容积的利用率 (VenkateshBalan,Sugarland,TX(US)；Leonardo da Costa Sousa,Brighton,MI(US),De-esterification of biomass prior to ammonia pretreatment and systems and products related thereto[P],Appl.NO.:16/029,452,2018-07-06)。同时在后续的生物转化过程中操作方便、有利于提高底物的浓度(Bals B D,Gunawan C,Moore J,et al.Enzymatic Hydrolysis of Pelletized AFEX  TM-Treated Corn Stover at High Solid Loadings[J].Biotechnology&Bioengineering,2013,111(2):264-271.)。密化也可在木质纤维素原料预处理之后进行。布鲁斯·E·戴尔研究发现玉米秸秆经过AFEX(Ammonia Fiber EXplosion)预处理后进行密化，有利于高底物浓度的酶水解以及方便进料(布莱恩·巴尔斯,法札内·塔莫里,蒂莫西·J·坎贝尔,布鲁斯·E·戴尔，水解经预处理的密化生物质颗粒的方法及其相关系统[P],中国专利：201380022053.7，2013-04-26)。
密化木质纤维素虽然有利于提高预处理时的原料装载量，但也会导致预处理条件要求的提高，如温度的提升和反应时间的延长(VenkateshBalan,Sugarland,TX(US)；Leonardo da Costa Sousa,Brighton,MI(US),De-esterification of biomass prior to ammonia pretreatment and systems and products related thereto[P],Appl.NO.:16/029,452,2018-07-06)。同时，预处理过程中化学试剂与密化的木质纤维素存在传质问题：因致密的结构，化学试剂渗透进密化秸秆中较为困难，往往导致预处理效果不理想或需要更为苛刻的预处理条件。另外，传热问题也可能导致密化木质纤维素原料内部和外部的温度差异，从而影响预处理效果。
发明内容
针对现有的木质纤维素运输储存成本高、预处理设备要求高、耗能高、密化木质纤维素预处理传质传热等问题，本发明提供一种木质纤维素密化过程中加入碱或酸性试剂进行预处理及生物转化的方法，通过在木质纤维素密化过程中添加碱性试剂或酸性试剂对木质纤维素实现预处理。密化木质纤维素中的碱性试剂或酸性试剂在后续运输、储存过程中进一步对木质纤维素原料进行较温和的预处理，从而充分利用运输存储的时间进一步提升预处理效果。含碱或酸的密化木质纤维素不宜发霉变质或腐烂，加上密度的提高更利用存储和运输。本发明避免了传统技术对预处理设备和能耗的要求。另外，如需进一步预处理，进一步预处理的条件严苛性可一定程度的降低，同时解决了密化木质纤维素预处理过程中化学试剂的传质问题。加碱密化的木质纤维素在进一步预处理时加水会放热(碱遇水放热)， 一定程度上解决了密化木质纤维素预处理过程中传热的问题。因此，进一步预处理的效果也将大大提升。
本发明的技术方案如下：
木质纤维素密化过程中加入碱或酸性试剂进行预处理的方法，具体步骤如下：
在木质纤维素原料密化过程中加入碱性试剂或酸性试剂；或在木质纤维素原料中加入碱性试剂或酸性试剂，混合均匀后进行密化处理；或在木质纤维素原料中加入碱性试剂或酸性试剂，反应后进行密化处理，得到含碱或含酸的密化木质纤维素。
进一步地，本发明还提供含碱或含酸的密化木质纤维素的生物转化方法，具体步骤如下：
在木质纤维素原料密化过程中加入碱性试剂或酸性试剂；或在木质纤维素原料中加入碱性试剂或酸性试剂，混合均匀后进行密化处理；或在木质纤维素原料中加入碱性试剂或酸性试剂，反应后进行密化处理，得到的含碱或含酸的密化木质纤维素直接进行生物转化或经进一步选择性预处理后再进行生物转化。
本发明中，所述的木质纤维素为小麦秸秆、玉米秸秆、农林废料、水稻秸秆、高粱秸秆、大豆秸秆、林业废料、回收木浆纤维、木屑、软木、硬木、水草、水生植物、藻类以及动物粪便中的一种或者多种；木质纤维素的水分含量为0％～90％。
本发明中，所述的碱性试剂为氢氧化钠、氢氧化钙、氢氧化钾、亚硫酸钠、亚硫酸氢钠、醋酸钠、碳酸钠、碳酸氢钠、硫酸钠、乙二胺、三乙胺、氨水、液氨、氧化钙和氧化钠中的一种或者多种。
本发明中，所述的酸性试剂为硫酸、硝酸、亚硝酸、盐酸、磷酸、亚硫酸、高氯酸、氯酸、亚氯酸、次氯酸、草酸、甲酸、乙酸、丙酸、苯甲酸、苯乙酸、苯磺酸、乙二酸、丁二酸，二氧化碳、二氧化硫、硫酸氢钠和亚硫酸氢钠中的一种或者多种。
本发明中，所述的碱性试剂或酸性试剂用量占木质纤维素质量的0.5％～30.0％。
本发明中，所述的碱性或酸性试剂的加入方式可以直接以纯碱性或酸性试剂加入，或以碱性或酸性试剂的水溶液的形式加入；加入方式为直接倒入或加入、喷洒、通入蒸汽带入、喷入碱性或酸性气体等方式，使碱性或酸性试剂和木质纤 维素充分混合后进行木质纤维素密化处理；或在密化过程中将碱性或酸性试剂与木质纤维素一起加入密化仪器中制成致密形状的密化木质纤维素。
本发明中，不进行进一步预处理的含酸或碱的密化木质纤维素立即进行生物转化或放置一段时间后进行生物转化；经进一步选择性预处理后再进行生物转化，所述的进一步选择性预处理为在含酸或碱的密化木质纤维素表面喷洒水、通入蒸汽、浸泡、暴晒、低温冷冻、高温处理、加水蒸煮、高温灭菌、微波和超声破碎；或蒸汽爆破预处理、热水预处理、蒸汽预处理等水热预处理；或稀酸预处理、弱酸预处理和氧化性酸预处理及混合酸预处理等酸性预处理；或稀碱预处理、弱碱预处理、氨预处理、氧化性碱预处理或混合碱预处理等碱性预处理；或有机溶剂预处理等。
本发明中，所述的密化木质纤维素的密度为200-1500kg/m 3；所述的密化木质纤维素的形状为棒状、粒状、块状或丸粒状；棒状的规格：直径1mm-30cm；粒状的规格：直径0.1cm-50cm；块状的规格：长度为0.1cm-200cm，宽度为0.1cm-200cm，高度为0.1cm-200cm；丸粒状的规格：直径0.1cm-50cm，厚度0.01cm-20cm。
本发明中，所述的生物转化为酶促反应和微生物转化中的一种或多种；所述的酶促反应，为在预处理基质中加入酶或者酵素，所述的酶选自纤维素酶、半纤维素酶、果胶酶、木聚糖酶、淀粉酶、糖化酶中的一种或者多种。所述的微生物转化为微生物发酵，为在预处理基质或酶水解液中加入发酵微生物，所述的发酵微生物为常规生物炼制使用的发酵微生物，可以为酵母、细菌、霉菌中的一种或多种。
本发明中，所述的方法生物转化生产的产品为生物燃料、大宗化学品、精细化学品、动物饲料、食品添加剂、药品等，如乙醇、丁醇、丙酮、乙酸、乳酸、脂肪烃、油脂、蛋白、氨基酸、酶、抗生素、维生素、抗体、沼气。
本发明在木质纤维素密化过程中加入碱性试剂或酸性试剂，不仅利用了密化过程中的机械作用和压缩过程中的热量(结合酸或碱)对木质纤维素结构进行破坏，还能够在不同温度下(-40℃～100℃)的运输和存储过程中，使碱或酸性试剂均能对木质纤维素发挥化学作用，对木质纤维素进行预处理，后续可直接用于下一步的生物转化，或进行进一步的预处理(此时的碱或酸性试剂已与木质纤维 素充分混合，有利于进一步预处理在高木质纤维素装载量下进行)后再进行生物转化。
与现有技术相比，本发明具有以下优点：
(1)本发明制得的含碱性试剂或酸性试剂密化木质纤维素更方便运输和储存；
(2)将密化过程与预处理过程合二为一、降低成本；
(3)预处理条件温和，充分利用运输和存储时间；
(4)如需进一步预处理，预处理强度(严苛度)降低，传质传热效果好；
(5)由于预处理强度降低，产生的预处理抑制物可能比较少，更利于生物转化。
附图说明
图1(a)为实施例1中无碱性试剂密化的玉米秸秆颗粒在第1天表面形貌图，(b)为在常温下放置第6天无碱性试剂密化的玉米秸秆颗粒表面形貌图，(c)为含氢氧化钠(0.3g/g玉米秸秆的干重)密化预处理的玉米秸秆颗粒在第1天表面形貌图，(d)为含氢氧化钠(0.3g/g玉米秸秆的干重)密化预处理的玉米秸秆颗粒在常温下放置6天表面形貌图。
图2(a)为实施例1中无化学试剂松散玉米秸秆上清液微生物在LB固体培养基上生长状态图，(b)为无化学试剂密化预处理的玉米秸秆上清液微生物在LB固体培养基上生长状态图，(c)为含碱密化预处理的玉米秸秆上清液微生物在LB固体培养基上生长状态图，(d)为含酸密化预处理的玉米秸秆上清液微生物在LB固体培养基上生长状态图。
图3(A)为实施例2中为含碱性试剂或酸性试剂密化预处理的玉米秸秆直径图，(B)为含碱性试剂或酸性试剂密化预处理的玉米秸秆长度图。
图4(A)为实施例2中为松散玉米秸秆在100ml塑料杯盛装状态图，(B)为含酸密化预处理的玉米秸秆在100ml塑料杯中的盛装状态图。
图5为实施例3中含氢氧化钙密化(DLL)预处理的玉米秸秆不同储存天数(0，6，12天)对酶水解效果的影响结果图。
图6为实施例4中含氢氧化钠密化(DLS)预处理的玉米秸秆不同储存天数(2，6，12)对酶水解效果的影响结果图。
图7为实施例5中在10％(wt/wt,玉米秸秆占秸秆、水、水解酶和化学试剂总重的百分数)底物浓度下，传统稀酸(DA)预处理玉米秸秆和含酸密化预处理后进一步热水(DLA+LHW)预处理的玉米秸秆酶水解72h后的糖浓度图。
图8为实施例6中在6％(wt/wt，秸秆中葡聚糖占秸秆、水、水解酶和化学试剂总重的百分数)葡聚糖浓度下，传统稀碱(AL)预处理、秸秆密化后进行稀碱(PCS+AL)预处理和含碱密化预处理并灭菌(DLS+Auctoclave)的玉米秸秆酶水解24小时的糖浓度图。
图9为实施例7中20％(wt/wt，玉米秸秆占秸秆、水、水解酶和化学试剂总重的百分数)底物浓度下，含酸密化预处理后进一步热水(DLA+LHW)预处理、秸秆密化后稀酸(PCS+DA)预处理和传统稀酸(DA)预处理的玉米秸秆酶水解糖浓度随水解时间的变化曲线。
图10为实施例8中20％(wt/wt，玉米秸秆占秸秆、水、水解酶和化学试剂总重的百分数)底物浓度下，含氢氧化钙密化预处理后进一步热水(DLL+LHW)预处理的玉米秸秆在无脱毒和无分批加料情况下进行酶水解后获得的酶水解液的乙醇发酵曲线。
图11为实施例9中含氢氧化钠密化预处理后进一步灭菌(DLL+Auctoclave)的玉米秸秆在不同底物浓度下酶水解72小时的糖浓度图。
具体实施方式
为了便于理解本发明，以下将结合说明书附图和较佳的实施例来对本发明做更全面，细致的描述，但本发明的保护范围并不限于以下具体的实施例。
下文所用到的所有专业术语与本领域技术人员通常理解的含义相同，除非另有定义。本文中所使用的专业术语只是为了描述具体事例的目的，并不是旨在限制本发明的保护范围。
除非另有特别说明，本发明中用到的试剂，原料，仪器和设备等均可市场购买得到或者已经有的方法制备得到。
实施例中使用以下的缩写：PCS：(Pelletized Corn stover)颗粒化的玉米秸秆；DA：稀硫酸(Dilute sulfuric Acid)预处理；AL：稀碱(ALkali)预处理；LHW：热水(Liquid Hot Water)预处理；DLL：含氢氧化钙密化(DensifyingLignocellulosewith Lime)预处理(由于密化过程中添加水分， 石灰变成了氢氧化钙)；DLS：含氢氧化钠密化(Densifying Lignocellulose with Sodium hydroxide)预处理；DLA：含硫酸密化(Densifying Lignocellulose with sulfuric Acid)预处理；PCS+AL：秸秆颗粒化后进行稀碱(Pelletized Corn Stover pretreated bydiluteALkali)预处理；DLA+LHW：含酸密化预处理后进一步热水(DensifyingLignocellulosewithsulfuricAcidfollowed by Liquid Hot Water)预处理；DLS+Autoclave：含氢氧化钠密化预处理后进行灭菌(DensifyingLignocellulosewithSodiumhydroxidefollowed by autoclave)处理；DLL+LHW：含氢氧化钙预处理后进一步热水(DensifyingLignocellulosewithLimefollowed by Liquid Hot Water)预处理。
下述实施例密化过程中采用颗粒机进行密化。
实施例1
在玉米秸秆密化过程中加入碱或酸对原料储存或运输的影响，包括以下步骤：
1、原料准备：将农田里的玉米秸秆收集后，自然晾干后粉碎成1-4mm的颗粒。
2、密化预处理：将粉碎后的玉米秸秆分别喷洒氢氧化钠溶液，硫酸溶液和纯水，氢氧化钠的含量为0.3g/g玉米秸秆干物，硫酸的含量为0.1g/g玉米秸秆干物，三种密化方式的水含量为0.5g/g玉米秸秆干物，使用造粒机对加试剂均匀混合的玉米秸秆进行密化操作。反应后，在常温下放置6天。
3、涂平板过程：将含碱密化预处理(DLS)玉米秸秆、含酸密化预处理(DLA)的玉米秸秆和无化学试剂密化(PCS)的玉米秸秆和无菌水均匀的混合，秸秆干物与水的比例为1：20，取10μL的上清液置于LB固体培养基上，稀释涂布后，置于30℃的恒温培养箱3天。
本实施例中，有无化学试剂密化的玉米秸秆在不同的储存时间表面微生物的生长状态如图1所示，图1(a)为无碱性试剂密化的玉米秸秆颗粒在第1天表面的现象；图1(b)为在常温下放置第6天无碱性试剂密化的玉米秸秆颗粒表面的现象；图1(c)为含氢氧化钠(0.3g/g玉米秸秆的干重)密化预处理的玉米秸秆颗粒在第1天表面的现象；图1(d)为含氢氧化钠(0.3g/g玉米秸秆的干重)密化预处理的玉米秸秆颗粒在常温下放置6天表面的现象。从图(a)和(b)可以看出未加入碱性试剂密化预处理的玉米秸秆在第六天有微生物在表面上生 长，而图(c)和(d)的加入碱性试剂密化的玉米秸秆表面上无微生物的生长；有无化学试剂密化预处理的玉米秸秆加入一定比例无菌水后，上清液中的微生物在LB固体培养基生长状态。如图2所示，图2(a)为无化学试剂松散玉米秸秆上清液微生物在LB固体培养基上生长状态，图2(b)为无化学试剂密化预处理的玉米秸秆上清液微生物在LB固体培养基上生长状态，图2(c)为含碱密化预处理的玉米秸秆上清液微生物在LB固体培养基上生长状态，图2(d)为含酸密化预处理的玉米秸秆上清液微生物在LB固体培养基上生长状态。从图2中可以看出含碱或酸密化预处理的玉米秸秆平板上无菌落生长，而松散秸秆和无化学试剂密化的玉米秸秆都有不同程度的菌落生长。表明加入酸性试剂或碱性试剂密化预处理的玉米秸秆在储存和运输过程中，抑制微生物的生长，从而防止原料的腐烂或者发霉，减少对工业生产的不利影响。
实施例2
含酸或碱密化预处理的玉米秸秆降低运输和储存的成本，包括以下步骤：
1、原料准备：将农田里的玉米秸秆收集后，自然晾干后粉碎成1-4mm的颗粒。
2、密化预处理：将粉碎后的玉米秸秆分别喷洒氢氧化钠溶液或硫酸溶液，氢氧化钠含量为0.3g/g玉米秸秆干重，或硫酸含量为0.1g/g玉米秸秆干重，两种密化方式的水含量为0.5g/g玉米秸秆干重，使用造粒机对均匀混合酸性试剂或碱性试剂玉米秸秆进行密化操作。
本实施例中，含酸或碱密化预处理的玉米秸秆降低运输和储存的成本。图3A为含碱性试剂或酸性试剂密化预处理的玉米秸秆直径，图3B为含碱性试剂或酸性试剂密化预处理的玉米秸秆长度；图4A为松散玉米秸秆在100ml塑料杯盛装状态；图4B为含酸密化预处理的玉米秸秆在100ml塑料杯中的盛装状态。由图3可以得到，含酸密化预处理的玉米秸秆直径为0.8cm，长度为2.5cm，质量为1.85g，则，含酸密化预处理的玉米秸秆体积约为：
Figure PCTCN2019096329-appb-000001
含酸密化预处理的玉米秸秆密度约为：
Figure PCTCN2019096329-appb-000002
图4可以看出，松散玉米秸秆在100mL塑料杯质量为12.54g，而含酸密化预处理的玉米秸秆在100mL塑料杯质量为81.81g。所以含酸密化预处理的玉米秸秆是松散玉米秸秆密度的大约6.5倍。表明含酸或碱密化预处理的玉米秸秆在运输和储存中比松散秸 秆具有很大的优势。
实施例3
含氢氧化钙密化(DLL)预处理的玉米秸秆不同储存天数对酶水解效果的影响，包括以下步骤：
1、原料准备：将农田里的玉米秸秆收集后，自然晾干后粉碎成1-4mm的颗粒。
2、DLL预处理：将粉碎后的玉米秸秆置于封口袋中，均匀的喷洒氢氧化钙溶液，氢氧化钙和水的量根据玉米秸秆的干重决定，氢氧化钙的含量为0.15g/g玉米秸秆干物，水的含量为0.5g/g玉米秸秆干物，在常温下进行密化操作，密化秸秆放置在室温条件下0，6，12天。
3、将不同天数的密化玉米秸秆置于酶水解反应器中，底物质量浓度为3％(wt/wt，玉米秸秆占秸秆、水、水解酶和化学试剂总重的百分数)，加入水解酶和柠檬酸-柠檬酸钠缓冲液，在50℃的振荡箱(250rpm)内进行酶解反应，酶水解24小时。
本实施例中，含氢氧化钙密化预处理的玉米秸秆在不同储存天数对酶水解效果的影响。如图5所示，
Figure PCTCN2019096329-appb-000003
表示含氢氧化钙密化预处理的玉米秸秆酶水解葡萄糖的浓度；
Figure PCTCN2019096329-appb-000004
表示含氢氧化钙密化预处理的玉米秸秆酶水解木糖的浓度。从图5可以看出玉米秸秆经过密化后，随着含氢氧化钙密化玉米秸秆储存天数的增加，酶水解的葡萄糖和木糖浓度都是的增加的，且在储存的第12天，3％的底物酶水解葡萄糖的浓度达到6.9g/L，木糖的浓度为2.8g/L。表明含氢氧化钙密化预处理的玉米秸秆在储存过程中，氢氧化钙破坏玉米秸秆的原有致密结构。
实施例4
含氢氧化钠密化(DLS)预处理的玉米秸秆不同储存天数对酶水解效果的影响，包括以下步骤：
1、原料准备：将农田里的玉米秸秆收集后，自然晾干后粉碎成1-4mm的颗粒。
2、DLS预处理：将粉碎后的玉米秸秆置于封口袋中，均匀的喷洒氢氧化钠溶液，氢氧化钠和水的量是根据玉米秸秆的干重决定的，氢氧化钠的含量为0.3g/g玉米秸秆干物，水的含量为0.5g/g玉米秸秆干物，在常温下进行密化操 作，处理后，密化秸秆放置在室温条件下2，6，12天。
3、将不同储存时间的含氢氧化钠密化预处理的玉米秸秆置于酶水解反应器中，底物质量浓度为3％(wt/wt，玉米秸秆占秸秆、水、水解酶和化学试剂总重的百分数)，加入水解酶和柠檬酸-柠檬酸钠缓冲液，在50℃的振荡箱(250rpm)内进行酶解反应，酶水解24小时。
本实施例中，含氢氧化钠密化预处理的玉米秸秆不同储存天数对酶水解效果的影响。如图6所示，
Figure PCTCN2019096329-appb-000005
表示含氢氧化钠密化预处理的玉米秸秆酶水解葡萄糖的浓度；
Figure PCTCN2019096329-appb-000006
表示含氢氧化钠密化预处理里的玉米秸秆酶水解木糖的浓度。从图6可以看出玉米秸秆经过密化预处理后，随着储存天数的增加，酶水解的葡萄糖和木糖浓度都是增加的，且在储存的第12天，3％的低物酶水解葡萄糖的浓度达到8.0g/L，木糖的浓度为3.0g/L。表明含氢氧化钠密化预处理的玉米秸秆在储存过程中，氢氧化钠对玉米秸秆的原有结构进行破坏，达到提高酶水解效果的目的。
实施例5
在相同的预处理条件下，DLA+LHW和传统的DA预处理玉米秸秆的酶水解效果的比较。包含下列步骤：
(1)原料准备：将农田里的玉米秸秆收集后，自然晾干后粉碎成1-4mm的颗粒。
(2)DLA预处理：将粉碎后的玉米秸秆喷洒硫酸溶液，硫酸加入量为0.1g/g秸秆干重，加入水的含量为0.5g/g秸秆干重，使用造粒机对均匀混合酸性试剂的玉米秸秆进行密化操作。密化完成后，将含酸密化玉米秸秆装袋、密封，在常温下放置1天以上。
(3)DLA+LHW预处理：酸浓度为0.1g/g干物的含酸密化预处理玉米秸秆，秸秆干重:总水量为1:9；将其置于高温高压反应釜进行加热，加热温度为160℃，反应时间为10分钟。
(4)DA(传统酸预处理的方法)预处理：原料为疏松秸秆，向其中加入硫酸溶液，硫酸的量为0.1g/g玉米秸秆干物，秸秆干重:总水量为1:9，将其置于高温高压反应釜进行加热，加热温度为160℃，反应时间为10分钟。
(5)酶水解过程：水解底物浓度为10％(wt/wt，玉米秸秆占秸秆、水、 水解酶和化学试剂总重的百分数)，加入水解酶和柠檬酸-柠檬酸钠缓冲液，置于震荡培养箱中培养(设置转速为250rpm，温度为50℃)水解时间为72小时。
本实施例中，在相同的预处理条件下，DLA+LHW和DA预处理玉米秸秆酶水解效果的比较。如图7所示，
Figure PCTCN2019096329-appb-000007
表示酶水解过程葡萄糖的浓度；
Figure PCTCN2019096329-appb-000008
表示玉米秸秆酶水解过程木糖的浓度。由图7所示，DLA+LHW预处理玉米秸秆酶水解液葡萄糖浓度最高，达到40g/L，远高于DA(28g/L)。木糖浓度也达到12g/L，与DA的玉米秸秆酶水解液木糖浓度无明显性差异。总之，DLA+LHW比传统稀酸预处理(DA)对玉米秸秆结构破坏更加彻底，释放更多可以水解的多聚糖，利于后续操作。表明含酸密化预处理(DLA)在工业上的应用具有较大的潜力。
实施例6
在6％(wt/wt，葡聚糖占秸秆、水、水解酶和化学试剂总重的百分数)葡聚糖底物浓度下，AL、PCS+AL和DLS+Autoclave预处理的玉米秸秆酶水解效果的比较。包含以下步骤：
(1)原料准备：将农田里的玉米秸秆收集后，自然晾干后粉碎成1-4mm的颗粒。
(2)DLS预处理：将粉碎后的玉米秸秆喷洒氢氧化钠溶液，氢氧化钠加入量为0.1g/g玉米秸秆干物，加入水的含量为0.5g/g玉米秸秆干物，使用造粒机对均匀混合氢氧化钠试剂和玉米秸秆进行密化操作。密化完成后，装袋，密封，在常温下放置1天以上。
(3)DLS+Autoclave预处理：氢氧化钠浓度为0.1g/g干物含氢氧化钠密化预处理的玉米秸秆，30％(wt/wt，玉米秸秆干重占总重的百分数)玉米秸秆干重，将其置于灭菌锅进行加热，加热温度为120℃，反应时间为60分钟。
(4)PCS+AL预处理：无氢氧化钠密化的玉米秸秆和氢氧化钠溶液均匀的混合，将其置于灭菌锅中，干物的浓度为20％(wt/wt，玉米秸秆干重占总重的百分数)，氢氧化钠的含量为0.1g/g玉米秸秆干物。加热温度为120℃，反应时间为120分钟。
(5)AL预处理：原料为疏松秸秆，向其中加入氢氧化钠溶液，氢氧化钠的量为0.2g/g秸秆干重，干物的浓度为10％(wt/wt，玉米秸秆干重占总重的百分数)。将其置于灭菌锅进行加热，加热温度为120℃，反应时间为20分钟。
(6)酶水解过程：水解底物浓度为6％(wt/wt，葡聚糖占秸秆、水、水解酶和化学试剂总重的百分数)葡聚糖，加入水解酶和柠檬酸-柠檬酸钠缓冲液，置于震荡培养箱中培养(设置转速为250rpm，温度为50℃)水解时间为24小时。
本实施例中，在6％(wt/wt，葡聚糖占秸秆、水、水解酶和化学试剂总重的百分数)葡聚糖底物条件下，AL、PCS+AL和DLS+Autoclave预处理的玉米秸秆酶水解效果的比较。如图8所示，
Figure PCTCN2019096329-appb-000009
表示酶水解液中葡萄糖的浓度；
Figure PCTCN2019096329-appb-000010
表示酶水解液中木糖的浓度。AL、PCS+AL和DLS+Autoclave三种预处理方式酶水解效果无明显性差异。但是PCS+AL和DLS+Autoclave比AL在预处理过程中，氢氧化钠添加量减少50％和预处理的底物浓度分别提高100％和200％。DLS+Autoclave比PCS+AL的预处理底物浓度高，同时预处理反应时间是PCS+AL反应时间的50％。AL和PCS+AL为传统的稀碱预处理方法，而DLS+Autoclave作为本发明的预处理方法不仅提高预处理的底物浓度，而且降低化学试剂的使用量和预处理强度，对燃料乙醇的推广具有重要的意义。
实施例7
在20％(wt/wt，玉米秸秆占秸秆、水、水解酶和化学试剂总重的百分数)底物浓度下，DLA+LHW、PCS+DA和DA预处理的玉米秸秆酶水解效果的比较。包含以下步骤：
(1)原料准备：将农田里的玉米秸秆收集后，自然晾干后粉碎成1-4mm的颗粒。
(2)DLA预处理：将粉碎后的玉米秸秆喷洒硫酸溶液，硫酸加入量为0.1g/g玉米秸秆干物，加入水的含量为0.5g/g玉米秸秆干物，使用造粒机对均匀混合酸性试剂的玉米秸秆进行密化操作。密化完成后，将含酸密化玉米秸秆装袋、密封，在常温下放置1天以上。
(3)DLA+LHW预处理：酸浓度为0.1g/g干物的含酸密化预处理的玉米秸秆，秸秆干重:总水量为1:5；将其置于高温高压反应釜进行加热，加热温度为160℃，反应时间为10分钟。
(4)PCS+DA预处理：原料为无酸密化的玉米秸秆，向其中加入硫酸溶液，硫酸的量为0.1g/g玉米秸秆干物，秸秆干重:总水量为1:5；将其置于高温高压反应釜进行加热，加热温度为160℃，反应时间为10分钟。
(5)DA预处理：原料为疏松秸秆，向其中加入硫酸溶液，硫酸的量为0.1g/g玉米秸秆干物，秸秆干重:总水量为1:9；将其置于高温高压反应釜进行加热，加热温度为160℃，反应时间为10分钟。
(6)酶水解过程：水解底物浓度为20％(wt/wt，玉米秸秆占秸秆、水、水解酶和化学试剂总重的百分数)，加入水解酶和纯水，置于震荡培养箱中培养(设置转速为250rpm，温度为50℃)水解时间为72小时。
本实施例中，在20％(wt/wt，玉米秸秆占秸秆、水、水解酶和化学试剂总重的百分数)酶水解底物条件下，DLA+LHW、PCS+DA和DA预处理玉米秸秆酶水解效果的比较。据图9所示，
Figure PCTCN2019096329-appb-000011
表示DLA+LHW预处理玉米秸秆酶水解液糖的浓度；
Figure PCTCN2019096329-appb-000012
表示PCS+DA预处理玉米秸秆酶水解液糖的浓度；
Figure PCTCN2019096329-appb-000013
表示DA预处理玉米秸秆酶水解液糖的浓度。经过72h小时水解之后，DLA+LHW的最终葡萄糖浓度为73g/L，略高于PCS+DA(70g/L)和DA(68g/L)。DLA+LHW的最终木糖浓度最高为32g/L。此外，在水解17h时，三种预处理方法酶水解效果都已经接近最大浓度，且在12h内，DLA+LHW的水解速率最快。综合以上表明，与PCS+DA和DA相比，DLA+LHW在底物酶水解过程中具有转化率快，水解糖产率高等优势，具有广阔的运用前景。
实施例8
在20％(wt/wt，玉米秸秆占秸秆，水，水解酶和化学试剂总重的百分数)酶水解底物条件下，无脱毒和无分批加料过程DLL+LHW预处理玉米秸秆酶水解和发酵的效果。包含以下步骤：
(1)原料准备：将农田里的玉米秸秆收集后，自然晾干后粉碎成1-4mm的颗粒。
(2)DLL预处理：将粉碎后的玉米秸秆喷洒氢氧化钙溶液，氢氧化钙加入量为0.15g/g玉米秸秆干物，加入水的含量为0.5g/g玉米秸秆干物，使用造粒机对均匀混合氢氧化钙试剂的玉米秸秆进行密化操作。密化完成后，将含氢氧化钙密化玉米秸秆装袋、密封，在常温下放置1天以上。
(3)DLL+LHW预处理：氢氧化钙浓度为0.15g/g干物的含氢氧化钠密化预处理玉米秸秆，秸秆干重:总水量为1:5；将其置于高温高压反应釜进行加热，加热温度为140℃，反应时间为60分钟。
(4)酶水解过程：水解底物浓度为20％(wt/wt，玉米秸秆占秸秆、水、水解酶和化学试剂总重的百分数)，加入水解酶和纯水，置于震荡培养箱中培养(设置转速为250rpm，温度为50℃)水解时间为72小时。
(5)发酵过程：选择发酵菌种为利用木糖的菌株，菌种初始接种时OD为2；置于震荡培养箱中培养(设置转速为150rpm，温度为30℃)发酵时间为72小时。
本实施例中，在20％(wt/wt，玉米秸秆占秸秆，水，水解酶和化学试剂总重的百分数)酶水解底物条件下，无脱毒和无补料过程DLL+LHW预处理玉米秸秆酶水解和发酵的效果。如图10所示，
Figure PCTCN2019096329-appb-000014
表示DLL+LHW发酵液葡萄糖的浓度；
Figure PCTCN2019096329-appb-000015
表示DLL+LHW发酵液木糖的浓度；
Figure PCTCN2019096329-appb-000016
表示DLL+LHW发酵液乙醇的浓度。经过72h小时水解之后，DLL+LHW预处理玉米秸秆酶水解最终葡萄糖浓度为91.1g/L，木糖的浓度为24.4g/L。此外，36h发酵后，乙醇终产量达到51.5g/L，发酵最终总糖转化率达到了97％以上。由此推测，DLL+LHW预处理玉米秸秆产生的抑制物浓度低，在无脱毒和无分批加料过程，糖的转化效率和乙醇的浓度很高，故在生物转化过程具有很大优势。
实施例9
不同酶水解底物DLS预处理的玉米秸秆对酶水解效果的影响，包括以下步骤：
(1)原料准备：将农田里的玉米秸秆收集后，自然晾干后粉碎成1-4mm的颗粒。
(2)DLS预处理：将粉碎后的玉米秸秆置于封口袋中，均匀的喷洒氢氧化钠溶液，氢氧化钠和水的量是根据玉米秸秆的干重决定的，氢氧化钠的含量为0.1g/g玉米秸秆干物，水的含量为0.1g/g玉米秸秆干物，在常温下进行密化操作，处理后DLS预处理玉米秸秆放置在室温条件下1天以上。
(3)DLS+Auctoclave预处理：将含碱密化的玉米秸秆，氢氧化钠的含量为0.1g/g玉米秸秆干物、置于灭菌锅中，加入纯水后，干物浓度为30％(基于总质量)，高温高压反应釜进行加热，加热温度为120℃，反应时间为60分钟。
(4)酶水解过程：将DLS+Auctoclave预处理玉米秸秆置于酶水解反应器中，底物浓度为3％，6％和9％(wt/wt，葡聚糖占秸秆、水、水解酶和化学试剂总重的百分数)的葡聚糖，加入水解酶和柠檬酸-柠檬酸钠缓冲液，在50℃的振荡箱(250rpm)内进行酶解反应，酶水解72小时。
本实施例中，不同酶水解底物DLS预处理的玉米秸秆对酶水解效果的影响。如图11所示，
Figure PCTCN2019096329-appb-000017
表示DLS+Auctoclave预处理密化玉米秸秆酶水解葡萄糖的浓度；
Figure PCTCN2019096329-appb-000018
表示DLS+Auctoclave预处理玉米秸秆酶水解木糖的浓度。从图9可以看出玉米秸秆经过含氢氧化钠密化预处理后，随着酶水解底物浓度的增加，酶水解的葡萄糖和木糖浓度增加，且9％的葡聚糖底物条件下的酶水解葡萄糖的浓度达到105.1g/L，木糖的浓度为40.1g/L。总糖浓度达到145.2g/L，具有生物转化成高乙醇浓度的潜力。

Claims (14)

  1. 木质纤维素密化过程中加入碱或酸性试剂进行预处理的方法，其特征在于，具体步骤如下：
    在木质纤维素原料密化过程中加入碱性试剂或酸性试剂；或在木质纤维素原料中加入碱性试剂或酸性试剂，混合均匀后进行密化处理；或在木质纤维素原料中加入碱性试剂或酸性试剂，反应后进行密化处理，得到含碱或含酸的密化木质纤维素。
  2. 含碱或含酸的密化木质纤维素的生物转化方法，其特征在于，具体步骤如下：
    在木质纤维素原料密化过程中加入碱性试剂或酸性试剂；或在木质纤维素原料中加入碱性试剂或酸性试剂，混合均匀后进行密化处理；或在木质纤维素原料中加入碱性试剂或酸性试剂，反应后进行密化处理，得到的含碱或含酸的密化木质纤维素直接进行生物转化或经进一步选择性预处理后再进行生物转化。
  3. 根据权利要求1或2所述的方法，其特征在于，所述的木质纤维素为小麦秸秆、玉米秸秆、农林废料、水稻秸秆、高粱秸秆、大豆秸秆、林业废料、回收木浆纤维、木屑、软木、硬木、水草、水生植物、藻类以及动物粪便中的一种或者多种；木质纤维素的水分含量为0％～90％。
  4. 根据权利要求1或2所述的方法，其特征在于，所述的碱性试剂为氢氧化钠、氢氧化钙、氢氧化钾、亚硫酸钠、亚硫酸氢钠、醋酸钠、碳酸钠、碳酸氢钠、硫酸钠、乙二胺、三乙胺、氨水、液氨、氧化钙和氧化钠中的一种或者多种。
  5. 根据权利要求1或2所述的方法，其特征在于，所述的酸性试剂为硫酸、硝酸、亚硝酸、盐酸、磷酸、亚硫酸、高氯酸、氯酸、亚氯酸、次氯酸、草酸、甲酸、乙酸、丙酸、苯甲酸、苯乙酸、苯磺酸、乙二酸、丁二酸，二氧化碳、二氧化硫、硫酸氢钠和亚硫酸氢钠中的一种或者多种。
  6. 根据权利要求1或2所述的方法，其特征在于，所述的碱性试剂或酸性试剂用量占木质纤维素质量的0.5％～30.0％。
  7. 根据权利要求1或2所述的方法，其特征在于，所述的碱性或酸性试剂的加入方式可以直接以纯碱性或酸性试剂加入，或以碱性或酸性试剂的水溶液的形式加入。
  8. 根据权利要求1或2所述的方法，其特征在于，所述的碱性或酸性试剂的加入方式为直接倒入或加入、喷洒、通入蒸汽带入、喷入碱性或酸性气体的方式，使碱性或酸性试剂和木质纤维素充分混合后进行木质纤维素密化处理；或在密化过程中将碱性或酸性试剂与木质纤维素一起加入密化仪器中制成致密形状的密化木质纤维素。
  9. 根据权利要求2所述的方法，其特征在于，不进行进一步预处理的含酸或碱的密化木质纤维素立即进行生物转化或放置一段时间后进行生物转化。
  10. 根据权利要求2所述的方法，其特征在于，经进一步选择性预处理后再进行生物转化，所述的进一步选择性预处理为在含酸或碱的密化木质纤维素表面喷洒水、通入蒸汽、浸泡、暴晒、低温冷冻、高温处理、加水蒸煮、高温灭菌、微波、或超声处理；或蒸汽爆破预处理、热水预处理、蒸汽预处理的水热预处理；或稀酸预处理、弱酸预处理和氧化性酸预处理及混合酸预处理的酸性预处理；或稀碱预处理、弱碱预处理、氨预处理、氧化性碱预处理或混合碱预处理的碱性预处理；或有机溶剂预处理。
  11. 根据权利要求1或2所述的方法，其特征在于，所述的密化木质纤维素的密度为200-1500kg/m 3；所述的密化木质纤维素的形状为棒状、粒状、块状或丸粒状；棒状的规格：直径1mm-30cm；粒状的规格：直径0.1cm-50cm；块状的规格：长度为0.1cm-200cm，宽度为0.1cm-200cm，高度为0.1cm-200cm；丸粒状的规格：直径0.1cm-50cm，厚度0.01cm-20cm。
  12. 根据权利要求2所述的方法，其特征在于，所述的生物转化为酶促反应和微生物转化中的一种或多种；所述的酶促反应，为在预处理基质中加入酶或者酵素，所述的酶选自纤维素酶、半纤维素酶、果胶酶、木聚糖酶、淀粉酶、糖化酶中的一种或者多种；所述的微生物转化为微生物发酵，为在预处理基质或酶水解液中加入发酵微生物，所述的发酵微生物为酵母、细菌、霉菌中的一种或多种。
  13. 根据权利要求2所述的方法，其特征在于，所述的生物转化生产的产品为生物燃料、大宗化学品、精细化学品、动物饲料、食品添加剂或药品。
  14. 根据权利要求13所述的方法，其特征在于，所述的生物转化生产的产品为乙醇、丁醇、丙酮、乙酸、乳酸、脂肪烃、油脂、蛋白、氨基酸、酶、抗生素、维生素、抗体或沼气。
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