WO2018072472A1 - Method for reducing inhibiting effect of byproducts in lignocellulose alkaline pretreatment liquid and preparation of cellulosic ethanol based on the method - Google Patents
Method for reducing inhibiting effect of byproducts in lignocellulose alkaline pretreatment liquid and preparation of cellulosic ethanol based on the method Download PDFInfo
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- WO2018072472A1 WO2018072472A1 PCT/CN2017/091475 CN2017091475W WO2018072472A1 WO 2018072472 A1 WO2018072472 A1 WO 2018072472A1 CN 2017091475 W CN2017091475 W CN 2017091475W WO 2018072472 A1 WO2018072472 A1 WO 2018072472A1
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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
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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
- C12P2201/00—Pretreatment of cellulosic or lignocellulosic material for subsequent enzymatic treatment or hydrolysis
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- 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
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- the invention relates to the field of biochemical engineering and fermentation engineering, in particular to a method for reducing the inhibitory effect of by-products in a lignocellulosic alkali pretreatment liquid and preparing ethanol based on the method.
- Lignocellulose is an ideal raw material for replacing fossil energy because of its wide source, rich content and renewable.
- the production of bioethanol, biobutanol, biosynthesis and other alternative energy sources from lignocellulose has become a research hotspot in the world. Since ethanol can be directly used in existing engines in various proportions and can be produced by microbial fermentation, the production of fuel ethanol from lignocellulose has become one of the research focuses.
- the lignocellulose that can be used as a raw material for producing fuel ethanol is mainly agricultural and forestry waste, such as crop straw, bagasse, wood processing waste, and the like. As the largest renewable energy on the earth, lignocellulose accounts for about 90% of the world's annual biomass production. Lignocellulose is mainly composed of three high polymers of cellulose (40% to 50%), hemicellulose (20% to 30%) and lignin (20% to 28%) by covalent and non-covalent combination. . In the lignocellulose in the natural state, cellulose is generally embedded in a polymer formed of lignin, hemicellulose, or the like to form a complex network structure.
- This structure plays an active role in maintaining plant morphology and resisting foreign invasion, but since the substrate of cellulase is a non-crystalline single-chain cellulose molecule, the structure of natural lignocellulose becomes an obstacle in the process of cellulose degradation. . Therefore, the lignocellulosic feedstock must undergo a pretreatment step to remove the cellulose-encapsulated lignin and hemicellulose while opening the crystalline region of the cellulose, thereby exposing the cellulose to cellulase for hydrolysis and saccharification.
- furan derivatives derived from pentose and hexose including furfural. (furfural) and 5-hydroxymethylfurfural (5-HMF);
- organic acids also known as fatty acids, also known as fatty acids
- hemicellulose-derived acetic acid including hemicellulose-derived acetic acid and further degradation by furfural or hydroxymethylfurfural Forming formic acid, levulinic acid, etc.
- lignin-derived phenols and aromatic compounds are mainly classified into three categories: i) furan derivatives derived from pentose and hexose, including furfural. (furfural) and 5-hydroxymethylfurfural (5-HMF);
- organic acids also known as fatty acids, also known as fatty acids
- hemicellulose-derived acetic acid including hemicellulose-derived acetic acid and further degradation by furfural or hydroxymethylfurfural Forming formic acid, levulinic acid, etc.
- furfural and hydroxymethylfurfural mainly affect the redox balance of fermentation process and inhibit the activity of glycolytic enzyme; organic acid mainly inhibits yeast growth and ethanol production; phenolic compounds affect cell membrane permeability. Affect the growth and fermentation of yeast. Microorganisms such as yeast can metabolize organic acids, furfural, and HMF, and convert them into less toxic compounds, so they are relatively less toxic, but they still have an inhibitory effect in the presence of high concentrations.
- the phenolic compound has a much lower toxicity than the by-products derived from cellulose and hemicellulose, although the concentration is low.
- ferulic acid produces a concentration inhibitory effect on yeast that is two orders of magnitude lower than the required concentration of formic acid, acetic acid, and levulinic acid (1 mM vs 100 mM), while coniferylaldehyde requires only 0.1 mM.
- Yeast produces inhibition.
- a detoxification step to detoxify the pretreated raw materials.
- Common methods of detoxification include physical methods, chemical methods, and biological methods. By vacuum evaporation, more than 98% of furfural and part of acetic acid in the wood hydrolyzate can be removed; activated carbon adsorption can remove 95% of the phenolic compound; and 60% of acetic acid in the corn stover hydrolysate is removed by membrane adsorption.
- Over-liming is a common chemical detoxification method that removes most of the furfural in the lignocellulosic hydrolysate; however, it causes about 10% loss of fermentable sugar due to adsorption.
- a major disadvantage of most physical or chemical detoxification methods is the need for additional processing steps, which not only increases the complexity of the process, but also introduces additional consumption of reagents and energy.
- the biological method uses microorganisms such as laccase and peroxidase or white rot fungi to degrade the phenolic compounds in the hydrolyzate by oxidation, without adding new equipment and operations.
- the current biological method takes a long time and the conditions are harsh. widely used.
- the present invention proposes a novel biological detoxification of the lignocellulosic alkali pretreatment liquid.
- the method requires only one step of enzymatic hydrolysis to complete the detoxification and eliminate the inhibition of the fermentation ability of by-products on cellulase and yeast.
- an object of the present invention is to overcome the deficiencies of the prior art, and to provide a method for reducing the by-product inhibition effect in a lignocellulosic alkali pretreatment liquid and a process for preparing cellulosic ethanol based on the method.
- the present invention is implemented by the following scheme:
- the enzyme treatment of lignocellulosic alkali pretreatment liquid pretreats the lignocellulose for the lye used in the prior art to remove the cellulose-coated lignin and hemicellulose, and opens the crystalline region of the cellulose to expose the cellulose.
- the cellulase is hydrolyzed and saccharified.
- lignocellulose is subjected to an alkali treatment to produce various by-products which have an inhibitory effect on subsequent cellulase enzymatic hydrolysis and yeast fermentation, and the present invention innovatively adds a glucose oxidase (GOD) pair.
- the biological detoxification of the pretreatment liquid not only degrades the phenolic compounds in the pretreatment liquid, but also metabolizes most of the by-products such as organic acids, furfural, hydroxymethylfurfural, and greatly reduces the content of by-products.
- the invention has simple process, and only needs to add glucose oxidase to react after treating the lignocellulose by the alkali method, thereby completeing detoxification and eliminating inhibition of cellulase enzymatic hydrolysis and yeast fermentation by by-products.
- the lignocellulose is added to the lye to be processed to obtain a pretreatment liquid;
- the lye includes, but not limited to, sodium hydroxide, potassium hydroxide, sodium ethoxide, potassium ethoxide, etc., which can be selected according to the prior art;
- the conditions of the dosage ratio, temperature, time and the like involved in the treatment are also selected according to the prior art;
- the pretreatment liquid is cooled to room temperature and the pH is adjusted to 4.5 to 5.5 before the glucose oxidase is added to the pretreatment liquid.
- the alkali-treated pretreatment liquid has a higher temperature and a higher alkalinity, and inhibits the activity of glucose oxidase. Therefore, it is necessary to adjust the temperature and acidity and alkalinity to a reasonable range to provide a more suitable environment.
- step S2 the pH is adjusted using concentrated phosphoric acid.
- Glucose oxidase can degrade the phenolic compounds in the pretreatment liquid, but the phenolic compound is only one of the by-products in the pretreatment liquid, and also contains a large amount of organic acids, furfural, hydroxymethylfurfural and the like.
- the subsequent microbial fermentation process such as yeast, it can metabolize organic acids, furfural, hydroxymethylfurfural, etc., but when the concentration of organic acid, furfural, hydroxymethylfurfural, etc. is high, microorganisms such as yeast cannot completely metabolize them, and then Fermentation inhibition.
- the inventors have found that the pH adjustment of concentrated phosphoric acid has a better pH buffer capacity relative to sulfuric acid, which is beneficial to maintain pH stability, thereby facilitating the maintenance of glucose oxidase and subsequent cellulase activity, and facilitating organic Acid, furfural, hydroxymethylfurfural and other degradation and metabolism, to the greatest extent eliminate the inhibition effect of this part of by-products; compared to dilute phosphoric acid, the amount is small, can reduce the volume as much as possible, thereby improving the subsequent cellulose enzymatic hydrolysis
- the concentration of fermentable sugar can save energy in the ethanol distillation process.
- step S2 the mixed glucose oxidase and the pretreatment liquid are reacted for 24 to 48 hours.
- the ratio of glucose oxidase to pretreatment liquid, reaction temperature, stirring speed, etc. can be selected according to a limited number of tests, and it is because of the use of glucose oxidase to treat the pretreatment liquid in a short time. Degradation of by-products is accomplished to the greatest extent (24 to 48 h).
- a method for producing ethanol using lignocellulose comprising the following steps:
- lignocellulosic alkali pretreatment and detoxification using the above method for reducing the inhibitory effect of by-products in the lignin alkali pretreatment liquid;
- step ii) Microbial fermentation: The system in step ii) is inoculated with Saccharomyces cerevisiae for fermentation.
- step ii) the enzyme is hydrolyzed for 60 to 80 hours.
- the amount of cellulase and Saccharomyces cerevisiae, the temperature of the reaction process, the stirring speed, etc. can be selected according to the prior art, and after detoxification in the pretreatment of lignocellulose, the by-products of the cellulase are reduced.
- the inhibition of fermentation and fermentation of Saccharomyces cerevisiae accelerates the speed of enzymatic hydrolysis and fermentation, so that the purpose of enzymatic hydrolysis and fermentation can be achieved in a short time; in addition, due to the detoxification treatment using glucose oxidase, no special removal is required.
- the product and the washing step, compared with the existing lignocellulose production process of ethanol, the invention has the advantages of simple operation, simple equipment demand, low energy consumption and water consumption.
- the present invention Compared with the prior art, the present invention has the following beneficial effects: the present invention firstly applies glucose oxidase (GOD) to a lignocellulosic alkali pretreatment liquid for biological detoxification, and removes by-products from the pretreatment liquid to cellulose. Enzyme and inhibition of S. cerevisiae.
- the invention utilizes the detoxification ability of glucose oxidase to construct a kind of inhibition effect of by-products can be largely eliminated by adding a certain amount of glucose oxidase after alkali pretreatment without replacing the reaction container.
- the same reaction system realizes all processes of lignocellulose pretreatment, detoxification, enzymatic hydrolysis and fermentation to produce ethanol. Compared with the existing commonly used methods such as water elution toxicity and chemical detoxification, the process is simple and environmentally friendly. The low water consumption, easy automation, and easy expansion of the reaction scale help to promote the industrial application of lignocellulosic ethanol.
- Figure 1 is a flow chart for producing ethanol from lignocellulose
- FIG. 2 is a data diagram of Embodiment 1;
- FIG. 3 is a data diagram of Embodiment 2;
- FIG. 4 is a data diagram of Embodiment 3.
- Figure 5 is a data plot of Comparative Example 1.
- step S4 then adding peptone to a final concentration of 20 g/L, and adding the pre-activated Saccharomyces cerevisiae suspension to the hydrolysate in step S3 in an amount of 20 g wet weight/L, 30 Fermentation was carried out at °C, 200 rpm for 48 h, and the fermentation process was sampled to detect the ethanol fermentation.
- step S4 then adding peptone to a final concentration of 20 g/L, and adding the pre-activated Saccharomyces cerevisiae suspension to the hydrolysate in step S3 in an amount of 20 g wet weight/L, 30 Fermentation was carried out at °C, 200 rpm for 48 h, and the fermentation process was sampled to detect the ethanol fermentation.
- step S4 then adding peptone to a final concentration of 20 g/L, and adding the pre-activated Saccharomyces cerevisiae suspension to the hydrolysate in step S3 in an amount of 20 g wet weight/L, 30 Fermentation was carried out at °C, 200 rpm for 48 h, and the fermentation process was sampled to detect the ethanol fermentation.
- This comparative example is similar to Example 3 except that in step S3, 40 U and 100 U laccase were used instead of GOD, respectively. This example was repeated three times, and the sample treated without laccase was used as a control group to use a high-efficiency liquid.
- Fig. 5 The enzymatic hydrolysis of sugarcane bagasse and ethanol yield were as shown in Fig. 5 (the hollow 100 U laccase-treated sample, the cross-over 40 U laccase-treated sample, and the solid control group). Due to the high concentration of NaOH, the by-products produced completely inhibited the fermentation of cellulose hydrolase and Saccharomyces cerevisiae, and the formation of glucose was not detected at all, and the production of ethanol was not detected. Even after 40 U of laccase treatment, there was no improvement in inhibition. ,still However, glucose and ethanol were not detected; after 100 U laccase treatment, the activity of cellulase was recovered. After 48 h of enzymatic hydrolysis, the glucose concentration was 17.4 g/L, and the final ethanol concentration reached 7.9 g/L. Both glucose and ethanol production were less than the results of the 25U GOD treatment.
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Abstract
A method for reducing an inhibiting effect of byproducts in lignocellulose alkaline pretreatment liquid. The method is achieved by treating lignocellulose alkaline pretreatment liquid by means of glucose oxidase without changing a reaction container and comprises the following steps: S1, adding lignocellulose to alkali liquor for treatment to obtain pretreatment liquid; and S2, adding glucose oxidase to the pretreatment liquid obtained in step S2 for reaction.
Description
本发明涉及生化工程及发酵工程领域,尤其涉及一种降低木质纤维素碱法预处理液中副产物抑制效应的方法及基于此方法制备乙醇。The invention relates to the field of biochemical engineering and fermentation engineering, in particular to a method for reducing the inhibitory effect of by-products in a lignocellulosic alkali pretreatment liquid and preparing ethanol based on the method.
木质纤维素由于其来源广泛、含量丰富、可再生,成为一种理想的替代化石能源的原料物质。以木质纤维素为原料生产生物乙醇、生物丁醇、生物合成气等替代能源成为世界各国的研究热点。由于乙醇可以以各种比例与汽油混合直接用于现有发动机,而且可以经由微生物发酵生产,因此以木质纤维素生产燃料乙醇成为研究的焦点之一。Lignocellulose is an ideal raw material for replacing fossil energy because of its wide source, rich content and renewable. The production of bioethanol, biobutanol, biosynthesis and other alternative energy sources from lignocellulose has become a research hotspot in the world. Since ethanol can be directly used in existing engines in various proportions and can be produced by microbial fermentation, the production of fuel ethanol from lignocellulose has become one of the research focuses.
可以用做生产燃料乙醇原料的木质纤维素主要为农林业废弃物,如农作物秸秆、甘蔗渣、木材加工废弃物等。木质纤维素作为地球上储量最大的可再生能源,占全球每年生物质产量约90%。木质纤维素主要由纤维素(40%~50%)、半纤维素(20%~30%)和木质素(20%~28%)三种高聚物通过共价和非共价结合而成。天然状态下的木质纤维素中,纤维素一般被包埋在木质素、半纤维素等形成的聚合物中,共同组成复杂的网状结构。这种结构在维持植物形态及抵御外来侵害中发挥积极作用,但是由于纤维素酶的底物是非结晶态的单链纤维素分子,所以天然木质纤维素的结构反而在纤维素降解过程中成为障碍。因此木质纤维素原料必须经过预处理步骤,以去除包裹纤维素的木质素和半纤维素,同时打开纤维素的结晶区,从而使纤维素暴露给纤维素酶进行水解糖化。The lignocellulose that can be used as a raw material for producing fuel ethanol is mainly agricultural and forestry waste, such as crop straw, bagasse, wood processing waste, and the like. As the largest renewable energy on the earth, lignocellulose accounts for about 90% of the world's annual biomass production. Lignocellulose is mainly composed of three high polymers of cellulose (40% to 50%), hemicellulose (20% to 30%) and lignin (20% to 28%) by covalent and non-covalent combination. . In the lignocellulose in the natural state, cellulose is generally embedded in a polymer formed of lignin, hemicellulose, or the like to form a complex network structure. This structure plays an active role in maintaining plant morphology and resisting foreign invasion, but since the substrate of cellulase is a non-crystalline single-chain cellulose molecule, the structure of natural lignocellulose becomes an obstacle in the process of cellulose degradation. . Therefore, the lignocellulosic feedstock must undergo a pretreatment step to remove the cellulose-encapsulated lignin and hemicellulose while opening the crystalline region of the cellulose, thereby exposing the cellulose to cellulase for hydrolysis and saccharification.
综合考虑各种方法的操作难易程度以及经济成本因素,稀酸和碱处理是目前获得关注最多的处理方法,但这两种预处理方法,都会导致木质纤维素的三种主要成分不同程度的降解和产生一系列化学反应,从而生成多种副产物,而这些副产物往往对酶解和发酵过程具有抑制作用,限制了其推广应用。因此,探索消除副产物对酶解和发酵的抑制的方法,是当前国内外木质纤维素预处理领域的研究热点。Considering the ease of operation of various methods and economic cost factors, dilute acid and alkali treatment are currently the most concerned treatment methods, but both pretreatment methods lead to different levels of three main components of lignocellulose. Degradation and production of a series of chemical reactions, resulting in a variety of by-products, and these by-products often inhibit the enzymatic hydrolysis and fermentation process, limiting its promotion and application. Therefore, exploring the method of eliminating the inhibition of enzymatic hydrolysis and fermentation by by-products is a research hotspot in the field of lignocellulose pretreatment at home and abroad.
木质纤维素的预处理过程中产生的副产物抑制物大多由半纤维素和木质素的分解产生,主要分为三类:i)来源于戊糖和己糖的呋喃衍生物,包括糠醛
(furfural)和羟甲基糠醛(5-hydroxymethylfurfural,5-HMF);ii)有机酸(又称脂肪酸,aliphatic carboxylic acids),包括来源于半纤维素的乙酸和由糠醛或羟甲基糠醛进一步降解形成甲酸、乙酰丙酸等;iii)来源于木质素的酚类和芳香类化合物。研究表明,糠醛和羟甲基糠醛主要影响发酵过程的氧化还原平衡以及抑制糖酵解酶的活性;有机酸主要抑制酵母的生长及乙醇的生成;酚类化合物则通过影响细胞膜的通透性从而影响酵母的生长及发酵。酵母等微生物能够代谢有机酸及糠醛、HMF,并将其转换为毒性更低的化合物,因此相对毒性较低,但高浓度存在时仍会产生抑制作用。Most of the by-product inhibitors produced during the pretreatment of lignocellulose are produced by the decomposition of hemicellulose and lignin. They are mainly classified into three categories: i) furan derivatives derived from pentose and hexose, including furfural.
(furfural) and 5-hydroxymethylfurfural (5-HMF); ii) organic acids (also known as fatty acids, also known as fatty acids), including hemicellulose-derived acetic acid and further degradation by furfural or hydroxymethylfurfural Forming formic acid, levulinic acid, etc.; iii) lignin-derived phenols and aromatic compounds. Studies have shown that furfural and hydroxymethylfurfural mainly affect the redox balance of fermentation process and inhibit the activity of glycolytic enzyme; organic acid mainly inhibits yeast growth and ethanol production; phenolic compounds affect cell membrane permeability. Affect the growth and fermentation of yeast. Microorganisms such as yeast can metabolize organic acids, furfural, and HMF, and convert them into less toxic compounds, so they are relatively less toxic, but they still have an inhibitory effect in the presence of high concentrations.
与来源于纤维素和半纤维素的副产物相比,酚类化合物虽然浓度低,但是毒性却大很多。例如,阿魏酸(ferulic acid)产生对酵母抑制效应的浓度比甲酸、乙酸和乙酰丙酸所需浓度低两个数量级(1mM vs 100mM),而松柏醛(coniferylaldehyde)只需要0.1mM即可对酵母产生抑制作用。The phenolic compound has a much lower toxicity than the by-products derived from cellulose and hemicellulose, although the concentration is low. For example, ferulic acid produces a concentration inhibitory effect on yeast that is two orders of magnitude lower than the required concentration of formic acid, acetic acid, and levulinic acid (1 mM vs 100 mM), while coniferylaldehyde requires only 0.1 mM. Yeast produces inhibition.
为了解决副产物抑制的问题,通常需要采用脱毒步骤来对预处理的原料进行脱毒。常用的脱毒方法包括物理法、化学法和生物法。通过真空蒸发,可以去除木材水解液中的98%以上的糠醛及部分乙酸;活性炭吸附可以除去95%的酚类化合物;用膜吸附去除玉米秸秆水解液中60%的乙酸。饱和生石灰(over-liming)是比较常用的化学脱毒法,能去除木质纤维素水解液中大部分的糠醛;但由于吸附作用,会造成约10%的可发酵糖的损失。大多数的物理或化学脱毒方法都有一个缺点,就是需要额外的处理步骤,这不仅加大工艺的复杂性,而且带来试剂、能源上的额外消耗。生物法是使用漆酶和过氧化物酶或白腐菌等微生物来通过氧化降解水解液中的酚类化合物,不需要新增设备和操作,然而目前的生物法耗时长,条件苛刻影响了其广泛应用。In order to solve the problem of by-product inhibition, it is usually necessary to use a detoxification step to detoxify the pretreated raw materials. Common methods of detoxification include physical methods, chemical methods, and biological methods. By vacuum evaporation, more than 98% of furfural and part of acetic acid in the wood hydrolyzate can be removed; activated carbon adsorption can remove 95% of the phenolic compound; and 60% of acetic acid in the corn stover hydrolysate is removed by membrane adsorption. Over-liming is a common chemical detoxification method that removes most of the furfural in the lignocellulosic hydrolysate; however, it causes about 10% loss of fermentable sugar due to adsorption. A major disadvantage of most physical or chemical detoxification methods is the need for additional processing steps, which not only increases the complexity of the process, but also introduces additional consumption of reagents and energy. The biological method uses microorganisms such as laccase and peroxidase or white rot fungi to degrade the phenolic compounds in the hydrolyzate by oxidation, without adding new equipment and operations. However, the current biological method takes a long time and the conditions are harsh. widely used.
在此背景下,为改进木质纤维素转化乙醇的工艺,尤其针对目前脱毒工艺繁琐、耗时、废水、耗能的缺点,本发明提出对木质纤维素碱法预处理液进行新型生物脱毒的方法,只需要增加一步酶解反应,即可完成脱毒,消除副产物对纤维素酶和酵母发酵能力的抑制。In this context, in order to improve the process of converting lignocellulose into ethanol, especially for the disadvantages of cumbersome, time-consuming, waste water and energy consumption of the current detoxification process, the present invention proposes a novel biological detoxification of the lignocellulosic alkali pretreatment liquid. The method requires only one step of enzymatic hydrolysis to complete the detoxification and eliminate the inhibition of the fermentation ability of by-products on cellulase and yeast.
发明内容Summary of the invention
有鉴于此,本发明的目的在于克服现有技术的不足,提供一种能降低木质纤维素碱法预处理液中副产物抑制效应的方法及基于此方法制备纤维素乙醇的工艺。In view of the above, an object of the present invention is to overcome the deficiencies of the prior art, and to provide a method for reducing the by-product inhibition effect in a lignocellulosic alkali pretreatment liquid and a process for preparing cellulosic ethanol based on the method.
为了解决上述技术问题,本发明采用如下方案实现:In order to solve the above technical problem, the present invention is implemented by the following scheme:
一种降低木质纤维素减法预处理液中副产物抑制效应的方法,用葡萄糖氧
化酶处理木质纤维素碱法预处理液。所述的碱法预处理液为现有技术中采用的碱液对木质纤维素进行预处理,以去除包裹纤维素的木质素和半纤维素,并打开纤维素的结晶区,使得纤维素暴露给纤维素酶进行水解糖化。如背景技术中提及到的,木质纤维素经过碱法处理后会产生多种对后续纤维素酶酶解和酵母发酵产生抑制效应的副产物,本发明创新地添加葡萄糖氧化酶(GOD)对预处理液进行生物脱毒,不仅降解了预处理液中的酚类化合物,而且还能代谢有机酸、糠醛、羟甲基糠醛等大多数副产物,大大降低了副产物的含量。本发明工艺简单,只需在利用碱法处理木质纤维素后添加葡萄糖氧化酶进行反应,即可完成脱毒,消除副产物对纤维素酶酶解和酵母发酵的抑制。Method for reducing by-product inhibition effect in lignocellulosic subtraction pretreatment liquid, using glucose oxygen
The enzyme treatment of lignocellulosic alkali pretreatment liquid. The alkaline pretreatment liquid pretreats the lignocellulose for the lye used in the prior art to remove the cellulose-coated lignin and hemicellulose, and opens the crystalline region of the cellulose to expose the cellulose. The cellulase is hydrolyzed and saccharified. As mentioned in the background, lignocellulose is subjected to an alkali treatment to produce various by-products which have an inhibitory effect on subsequent cellulase enzymatic hydrolysis and yeast fermentation, and the present invention innovatively adds a glucose oxidase (GOD) pair. The biological detoxification of the pretreatment liquid not only degrades the phenolic compounds in the pretreatment liquid, but also metabolizes most of the by-products such as organic acids, furfural, hydroxymethylfurfural, and greatly reduces the content of by-products. The invention has simple process, and only needs to add glucose oxidase to react after treating the lignocellulose by the alkali method, thereby completeing detoxification and eliminating inhibition of cellulase enzymatic hydrolysis and yeast fermentation by by-products.
具体的,包括如下步骤:Specifically, the following steps are included:
S1:木质纤维素加入碱液进行处理得预处理液;所述的碱液包括但不限于氢氧化钠、氢氧化钾、乙醇钠、乙醇钾等,均可根据现有技术进行选择;碱法处理涉及到的用量配比、温度、时间等条件,同样根据现有技术进行选择即可;S1: the lignocellulose is added to the lye to be processed to obtain a pretreatment liquid; the lye includes, but not limited to, sodium hydroxide, potassium hydroxide, sodium ethoxide, potassium ethoxide, etc., which can be selected according to the prior art; The conditions of the dosage ratio, temperature, time and the like involved in the treatment are also selected according to the prior art;
S2:将葡萄糖氧化酶加入步骤S2中的预处理液中进行反应。S2: Glucose oxidase is added to the pretreatment liquid in the step S2 to carry out the reaction.
上述步骤S2中,在葡萄糖氧化酶加入预处理液前先将预处理液冷却至室温并调节pH至4.5-5.5。碱法处理后的预处理液具有较高的温度以及较高碱性,会抑制葡萄糖氧化酶的活性,因此需要将温度以及酸碱性调节至合理范围以提供更适宜的环境。In the above step S2, the pretreatment liquid is cooled to room temperature and the pH is adjusted to 4.5 to 5.5 before the glucose oxidase is added to the pretreatment liquid. The alkali-treated pretreatment liquid has a higher temperature and a higher alkalinity, and inhibits the activity of glucose oxidase. Therefore, it is necessary to adjust the temperature and acidity and alkalinity to a reasonable range to provide a more suitable environment.
进一步的,步骤S2中,使用浓磷酸对pH进行调节。葡萄糖氧化酶能降解预处理液中的酚类化合物,但是酚类化合物只是预处理液中副产物中的一种,其中还含有大量的有机酸、糠醛、羟甲基糠醛等。虽然在后续酵母等微生物发酵过程中,能代谢有机酸、糠醛、羟甲基糠醛等,但是有机酸、糠醛、羟甲基糠醛等浓度较高时,酵母等微生物不能完全将它们代谢,继而对发酵产抑制。发明人发现,使用浓磷酸对pH进行调节后,相对于硫酸,具有更好的pH缓冲容量,有利于维持pH的稳定,从而有利于葡萄糖氧化酶及后续纤维素酶的活性保持,便于将有机酸、糠醛、羟甲基糠醛等进行降解代谢,最大程度上消除这部分副产物的抑制效应;相比于稀磷酸,用量少,可以尽可能减少体积,从而提高后续纤维素酶解所得的可发酵糖的浓度,可以节省乙醇蒸馏过程的能耗。Further, in step S2, the pH is adjusted using concentrated phosphoric acid. Glucose oxidase can degrade the phenolic compounds in the pretreatment liquid, but the phenolic compound is only one of the by-products in the pretreatment liquid, and also contains a large amount of organic acids, furfural, hydroxymethylfurfural and the like. Although in the subsequent microbial fermentation process such as yeast, it can metabolize organic acids, furfural, hydroxymethylfurfural, etc., but when the concentration of organic acid, furfural, hydroxymethylfurfural, etc. is high, microorganisms such as yeast cannot completely metabolize them, and then Fermentation inhibition. The inventors have found that the pH adjustment of concentrated phosphoric acid has a better pH buffer capacity relative to sulfuric acid, which is beneficial to maintain pH stability, thereby facilitating the maintenance of glucose oxidase and subsequent cellulase activity, and facilitating organic Acid, furfural, hydroxymethylfurfural and other degradation and metabolism, to the greatest extent eliminate the inhibition effect of this part of by-products; compared to dilute phosphoric acid, the amount is small, can reduce the volume as much as possible, thereby improving the subsequent cellulose enzymatic hydrolysis The concentration of fermentable sugar can save energy in the ethanol distillation process.
再进一步的,步骤S2中,相混合的葡萄糖氧化酶和预处理液反应24~48h。葡萄糖氧化酶和预处理液的用量比例、反应温度、搅拌速度等都可根据有限次试验作出选择,而正是由于使用了葡萄糖氧化酶来处理预处理液,能在较短时间内
最大程度上(24~48h)完成对副产物的降解。Further, in step S2, the mixed glucose oxidase and the pretreatment liquid are reacted for 24 to 48 hours. The ratio of glucose oxidase to pretreatment liquid, reaction temperature, stirring speed, etc. can be selected according to a limited number of tests, and it is because of the use of glucose oxidase to treat the pretreatment liquid in a short time.
Degradation of by-products is accomplished to the greatest extent (24 to 48 h).
一种利用木质纤维素生产乙醇的方法,包括如下步骤:A method for producing ethanol using lignocellulose, comprising the following steps:
ⅰ)木质纤维素碱法预处理及脱毒:采用上述降低木质素碱法预处理液中副产物抑制效应的方法进行处理;i) lignocellulosic alkali pretreatment and detoxification: using the above method for reducing the inhibitory effect of by-products in the lignin alkali pretreatment liquid;
ⅱ)纤维素酶酶解:往步骤ⅰ)中的体系加入纤维素酶进行酶解;Ii) Cellulase enzymatic hydrolysis: adding cellulase to the system in step i) for enzymatic hydrolysis;
ⅲ)微生物发酵:往步骤ⅱ)中的体系接种酿酒酵母进行发酵。Iii) Microbial fermentation: The system in step ii) is inoculated with Saccharomyces cerevisiae for fermentation.
步骤ⅱ)中酶解60~80h。In step ii), the enzyme is hydrolyzed for 60 to 80 hours.
步骤ⅲ)中发酵20~30h。Ferment in step iii) for 20 to 30 hours.
纤维素酶和酿酒酵母的用量、反应过程的温度、搅拌速度等都可根据现有技术作出选择,经过对木质纤维素预处理中进行脱毒后,降低了其中的副产物对纤维素酶酶解和酿酒酵母发酵的抑制,加速了酶解与发酵的速度,从而可以在短时间内达到酶解和发酵的目的;再者,由于使用葡萄糖氧化酶进行脱毒处理,不需要专门的去除副产物以及水洗等步骤,与现有的木质纤维素生产乙醇的工艺相比,本发明具有操作更为简便、设备需求更简单、能耗及水耗低等优点。The amount of cellulase and Saccharomyces cerevisiae, the temperature of the reaction process, the stirring speed, etc. can be selected according to the prior art, and after detoxification in the pretreatment of lignocellulose, the by-products of the cellulase are reduced. The inhibition of fermentation and fermentation of Saccharomyces cerevisiae accelerates the speed of enzymatic hydrolysis and fermentation, so that the purpose of enzymatic hydrolysis and fermentation can be achieved in a short time; in addition, due to the detoxification treatment using glucose oxidase, no special removal is required. The product and the washing step, compared with the existing lignocellulose production process of ethanol, the invention has the advantages of simple operation, simple equipment demand, low energy consumption and water consumption.
与现有技术相比,本发明具有如下有益效果:本发明首次将葡萄糖氧化酶(GOD)应用于木质纤维素碱法预处理液中进行生物脱毒,去除预处理液中副产物对纤维素酶以及酿酒酵母的抑制。本发明利用葡萄糖氧化酶的脱毒能力构建一种在不更换反应容器的情况下,仅仅通过在碱法预处理后加入一定量的葡萄糖氧化酶便能很大程度消除副产物的抑制效应,在同一反应体系中实现木质纤维素预处理、脱毒、酶解及发酵生产乙醇等全部工艺,相比于现有常用的水洗脱毒、化学脱毒等方法,此工艺具有操作简单、环境友好、水耗低、易于实现自动化控制以及易于扩大反应规模等优点,有助于推动木质纤维素乙醇的工业化应用。Compared with the prior art, the present invention has the following beneficial effects: the present invention firstly applies glucose oxidase (GOD) to a lignocellulosic alkali pretreatment liquid for biological detoxification, and removes by-products from the pretreatment liquid to cellulose. Enzyme and inhibition of S. cerevisiae. The invention utilizes the detoxification ability of glucose oxidase to construct a kind of inhibition effect of by-products can be largely eliminated by adding a certain amount of glucose oxidase after alkali pretreatment without replacing the reaction container. The same reaction system realizes all processes of lignocellulose pretreatment, detoxification, enzymatic hydrolysis and fermentation to produce ethanol. Compared with the existing commonly used methods such as water elution toxicity and chemical detoxification, the process is simple and environmentally friendly. The low water consumption, easy automation, and easy expansion of the reaction scale help to promote the industrial application of lignocellulosic ethanol.
图1为由木质纤维素生产乙醇流程图;Figure 1 is a flow chart for producing ethanol from lignocellulose;
图2为实施例1数据图;Figure 2 is a data diagram of Embodiment 1;
图3为实施例2数据图;Figure 3 is a data diagram of Embodiment 2;
图4为实施例3数据图;Figure 4 is a data diagram of Embodiment 3;
图5为对比例1数据图。Figure 5 is a data plot of Comparative Example 1.
为了让本领域的技术人员更好地理解本发明的技术方案,下面结合附图对本发明作进一步阐述。
In order to enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further described below in conjunction with the accompanying drawings.
实施例1Example 1
GOD处理1%NaOH预处理的甘蔗渣并发酵生产乙醇GOD treatment of 1% NaOH pretreated bagasse and fermentation to produce ethanol
S1:精确称取甘蔗渣2.5g,以10:1的液固比(底物浓度10%(w/v))加入1%NaOH溶液,在121℃下处理甘蔗渣1h,于室温冷却得预处理液;S1: accurately weigh 2.5g of bagasse, add 1% NaOH solution with a liquid-solid ratio of 10:1 (substrate concentration 10% (w/v)), treat bagasse at 121 °C for 1 h, and cool at room temperature. Treatment liquid
S2:无菌条件下,往预处理液中添加市售浓磷酸,调节pH约为5,然后添加10U GOD,30℃,200rpm脱毒反应24h;S2: under sterile conditions, adding commercially available concentrated phosphoric acid to the pretreatment liquid, adjusting the pH to about 5, then adding 10 U GOD, 30 ° C, detoxification reaction at 200 rpm for 24 h;
S3:无菌条件下,往上述预处理液中加入纤维素酶,酶添加量和底物的比值固定为6mg酶/g底物,然后置于恒温摇床内,45℃,160rpm水解72h;水解过程中取样测定甘蔗渣酶解情况;S3: under aseptic conditions, the cellulase is added to the above pretreatment liquid, the ratio of the enzyme addition amount to the substrate is fixed to 6 mg enzyme / g substrate, and then placed in a constant temperature shaker, hydrolyzed at 45 ° C, 160 rpm for 72 h; Sampling and measuring the enzymatic hydrolysis of bagasse during the hydrolysis process;
S4:然后补加蛋白胨至终浓度为20g/L,并按菌体量为20g湿重/L的量将预先活化的酿酒酵母菌体悬液接入到步骤S3中的酶解液中,30℃,200rpm下进行发酵48h,发酵过程取样检测乙醇发酵的情况。S4: then adding peptone to a final concentration of 20 g/L, and adding the pre-activated Saccharomyces cerevisiae suspension to the hydrolysate in step S3 in an amount of 20 g wet weight/L, 30 Fermentation was carried out at °C, 200 rpm for 48 h, and the fermentation process was sampled to detect the ethanol fermentation.
本实施例重复三次,以不加GOD处理的样品为对照组,以高效液相色谱检测甘蔗渣酶解情况及乙醇产量,结果如图2(空心的为GOD处理的样品,实心为对照组)。由于预处理所用NaOH浓度较低,产生的副产物对纤维素水解酶及酿酒酵母发酵的抑制不明显;不经过GOD处理,也能有葡萄糖(16.6g/L)和乙醇(7.1g/L)生成,但加入GOD可以在一定程度提高了葡萄糖(20.6g/L)和乙醇(8.3g/L)的产量。This example was repeated three times, and the sample treated without GOD was used as a control group, and the enzymatic hydrolysis of bagasse and ethanol yield were detected by high performance liquid chromatography. The results are shown in Fig. 2 (hollow GOD-treated sample, solid control group) . Due to the low concentration of NaOH used in the pretreatment, the by-products produced have no obvious inhibition on the fermentation of cellulose hydrolase and Saccharomyces cerevisiae; glucose (16.6g/L) and ethanol (7.1g/L) can be obtained without GOD treatment. Generated, but the addition of GOD can increase the yield of glucose (20.6g / L) and ethanol (8.3g / L) to some extent.
实施例2Example 2
GOD处理2%NaOH预处理的甘蔗渣并发酵生产乙醇GOD treatment of 2% NaOH pretreated bagasse and fermentation to produce ethanol
S1:精确称取甘蔗渣2.5g,以10:1的液固比(底物浓度10%(w/v))加入2%NaOH溶液,在121℃下处理甘蔗渣1h,于室温冷却得预处理液;S1: accurately weigh 2.5g of bagasse, add 2% NaOH solution with a liquid-solid ratio of 10:1 (substrate concentration 10% (w/v)), treat bagasse at 121 °C for 1h, and cool at room temperature. Treatment liquid
S2:无菌条件下,往预处理液中添加市售浓磷酸,调节pH约为5,然后添加15U GOD,30℃,200rpm脱毒反应24h;S2: under sterile conditions, adding commercially available concentrated phosphoric acid to the pretreatment liquid, adjusting the pH to about 5, then adding 15 U GOD, 30 ° C, detoxification reaction at 200 rpm for 24 h;
S3:无菌条件下,往上述预处理液中加入纤维素酶,酶添加量和底物的比值固定为6mg酶/g底物,然后置于恒温摇床内,45℃,160rpm水解72h;水解过程中取样测定甘蔗渣酶解情况;S3: under aseptic conditions, the cellulase is added to the above pretreatment liquid, the ratio of the enzyme addition amount to the substrate is fixed to 6 mg enzyme / g substrate, and then placed in a constant temperature shaker, hydrolyzed at 45 ° C, 160 rpm for 72 h; Sampling and measuring the enzymatic hydrolysis of bagasse during the hydrolysis process;
S4:然后补加蛋白胨至终浓度为20g/L,并按菌体量为20g湿重/L的量将预先活化的酿酒酵母菌体悬液接入到步骤S3中的酶解液中,30℃,200rpm下进行发酵48h,发酵过程取样检测乙醇发酵的情况。S4: then adding peptone to a final concentration of 20 g/L, and adding the pre-activated Saccharomyces cerevisiae suspension to the hydrolysate in step S3 in an amount of 20 g wet weight/L, 30 Fermentation was carried out at °C, 200 rpm for 48 h, and the fermentation process was sampled to detect the ethanol fermentation.
本实施例重复三次,以不加GOD处理的样品为对照组,以高效液相色谱检
测甘蔗渣酶解情况及乙醇产量,结果如图3(空心的为GOD处理的样品,实心为对照组)。不经过GOD的处理,由于NaOH浓度增加,产生的副产物对纤维素水解酶及酿酒酵母发酵的抑制更加明显,酶解48h后葡萄糖浓度只达到2.2g/L,而且检测不到乙醇的产生;经过GOD处理后,纤维素酶的活性得到恢复,酶解48h后葡萄糖浓度达到25.2g/L,最终乙醇浓度达到9.9g/L。This example was repeated three times, and the sample which was not treated with GOD was used as a control group, and was examined by high performance liquid chromatography.
The enzymatic hydrolysis of sugarcane bagasse and ethanol production were measured. The results are shown in Fig. 3 (hollow GOD-treated samples, solid as a control group). Without the treatment of GOD, the inhibition of cellulose hydrolase and Saccharomyces cerevisiae fermentation was more obvious due to the increase of NaOH concentration. The concentration of glucose was only 2.2g/L after 48h of enzymatic hydrolysis, and the production of ethanol was not detected. After GOD treatment, the activity of cellulase was restored. After 48 h of enzymatic hydrolysis, the glucose concentration reached 25.2 g/L, and the final ethanol concentration reached 9.9 g/L.
实施例3Example 3
GOD处理3%NaOH预处理的甘蔗渣并发酵生产乙醇GOD treatment of 3% NaOH pretreated bagasse and fermentation to produce ethanol
S1:精确称取甘蔗渣2.5g,以10:1的液固比(底物浓度10%(w/v))加入3%NaOH溶液,在121℃下处理甘蔗渣1h,于室温冷却得预处理液;S1: accurately weigh 2.5g of bagasse, add 3% NaOH solution with a liquid-solid ratio of 10:1 (substrate concentration 10% (w/v)), treat bagasse at 121 °C for 1h, and cool at room temperature. Treatment liquid
S2:无菌条件下,往预处理液中添加市售浓磷酸,调节pH约为5,然后添加25U GOD,30℃,200rpm脱毒反应24h;S2: under sterile conditions, adding commercially available concentrated phosphoric acid to the pretreatment liquid, adjusting the pH to about 5, then adding 25 U GOD, 30 ° C, detoxification reaction at 200 rpm for 24 h;
S3:无菌条件下,往上述预处理液中加入纤维素酶,酶添加量和底物的比值固定为6mg酶/g底物,然后置于恒温摇床内,45℃,160rpm水解72h;水解过程中取样测定甘蔗渣酶解情况;S3: under aseptic conditions, the cellulase is added to the above pretreatment liquid, the ratio of the enzyme addition amount to the substrate is fixed to 6 mg enzyme / g substrate, and then placed in a constant temperature shaker, hydrolyzed at 45 ° C, 160 rpm for 72 h; Sampling and measuring the enzymatic hydrolysis of bagasse during the hydrolysis process;
S4:然后补加蛋白胨至终浓度为20g/L,并按菌体量为20g湿重/L的量将预先活化的酿酒酵母菌体悬液接入到步骤S3中的酶解液中,30℃,200rpm下进行发酵48h,发酵过程取样检测乙醇发酵的情况。S4: then adding peptone to a final concentration of 20 g/L, and adding the pre-activated Saccharomyces cerevisiae suspension to the hydrolysate in step S3 in an amount of 20 g wet weight/L, 30 Fermentation was carried out at °C, 200 rpm for 48 h, and the fermentation process was sampled to detect the ethanol fermentation.
本实施例重复三次,以不加GOD处理的样品为对照组,以高效液相色谱检测甘蔗渣酶解情况及乙醇产量,结果如图4(空心的为GOD处理的样品,实心为对照组)。不经过GOD的处理,由于NaOH浓度进一步增加,产生的副产物完全抑制纤维素水解酶及酿酒酵母发酵,完全检测不到葡萄糖的生成,也没有检测到乙醇的生成;经过GOD处理后,纤维素酶的活性得到恢复,酶解48h后葡萄糖浓度达到25.9g/L,最终乙醇浓度达到10.3g/L。This example was repeated three times, and the sample treated without GOD was used as a control group, and the enzymatic hydrolysis of bagasse and ethanol production were detected by high performance liquid chromatography. The results are shown in Fig. 4 (hollow GOD-treated sample, solid as a control group) . Without the treatment of GOD, the by-product produced completely inhibited the fermentation of cellulose hydrolase and Saccharomyces cerevisiae due to the further increase of NaOH concentration. The formation of glucose was not detected at all, and the production of ethanol was not detected. After GOD treatment, cellulose was produced. The activity of the enzyme was restored. After 48 hours of enzymatic hydrolysis, the glucose concentration reached 25.9 g/L, and the final ethanol concentration reached 10.3 g/L.
对比例1Comparative example 1
本对比例与实施例3类似,区别在于,步骤S3中,分别使用40U和100U漆酶替代GOD。本实施例重复三次,以不加漆酶处理的样品为对照组,以高效液This comparative example is similar to Example 3 except that in step S3, 40 U and 100 U laccase were used instead of GOD, respectively. This example was repeated three times, and the sample treated without laccase was used as a control group to use a high-efficiency liquid.
相色谱检测Chromatographic detection
甘蔗渣酶解情况及乙醇产量,结果如图5(空心的为100U漆酶处理的样品,交叉为40U漆酶处理的样品,实心为对照组)。由于NaOH浓度较高,产生的副产物完全抑制纤维素水解酶及酿酒酵母发酵,完全检测不到葡萄糖的生成,也没有检测到乙醇的生成;即使经过40U的漆酶处理,抑制情况没有任何改善,仍
然检测不到葡萄糖和乙醇;加入100U漆酶处理后,纤维素酶的活性得到一定恢复,酶解48h后葡萄糖浓度为17.4g/L,最终乙醇浓度达到7.9g/L。无论是葡萄糖还是乙醇产量,均不及25U GOD处理的结果。The enzymatic hydrolysis of sugarcane bagasse and ethanol yield were as shown in Fig. 5 (the hollow 100 U laccase-treated sample, the cross-over 40 U laccase-treated sample, and the solid control group). Due to the high concentration of NaOH, the by-products produced completely inhibited the fermentation of cellulose hydrolase and Saccharomyces cerevisiae, and the formation of glucose was not detected at all, and the production of ethanol was not detected. Even after 40 U of laccase treatment, there was no improvement in inhibition. ,still
However, glucose and ethanol were not detected; after 100 U laccase treatment, the activity of cellulase was recovered. After 48 h of enzymatic hydrolysis, the glucose concentration was 17.4 g/L, and the final ethanol concentration reached 7.9 g/L. Both glucose and ethanol production were less than the results of the 25U GOD treatment.
上述实施例仅为本发明的其中具体实现方式,其描述较为具体和详细,但并不能因此而理解为对本发明专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些显而易见的替换形式均属于本发明的保护范围。
The above embodiments are only specific implementations of the present invention, and the description thereof is more specific and detailed, but is not to be construed as limiting the scope of the invention. It should be noted that a number of variations and modifications may be made by those skilled in the art without departing from the spirit and scope of the invention.
Claims (9)
- 一种降低木质纤维素碱法预处理液中副产物抑制效应的方法,其特征在于,用葡萄糖氧化酶处理木质纤维素碱法预处理液。A method for reducing the inhibitory effect of by-products in a lignocellulosic alkali pretreatment liquid, characterized in that the lignocellulosic alkali pretreatment liquid is treated with glucose oxidase.
- 根据权利要求1所述的降低木质纤维素碱法预处理液中副产物抑制效应的方法,其特征在于,具体包括如下步骤:The method for reducing the by-product inhibition effect of the lignocellulosic alkali pretreatment liquid according to claim 1, which comprises the following steps:S1:木质纤维素加入碱液进行处理得预处理液;S1: the lignocellulose is added to the alkali solution for treatment;S2:将葡萄糖氧化酶加入步骤S2中的预处理液中进行反应。S2: Glucose oxidase is added to the pretreatment liquid in the step S2 to carry out the reaction.
- 根据权利要求2所述的降低木质纤维素碱法预处理液中副产物抑制效应的方法,其特征在于,步骤S2中,加入葡萄糖氧化酶前先将预处理液冷却至室温并调节pH至4.5-5.5。The method for reducing the by-product inhibitory effect in the lignocellulosic alkali pretreatment liquid according to claim 2, wherein in step S2, the pretreatment liquid is cooled to room temperature and the pH is adjusted to 4.5 before the glucose oxidase is added. -5.5.
- 根据权利要求3所述的降低木质纤维素碱法预处理液中副产物抑制效应的方法,其特征在于,步骤S2中,使用浓磷酸进行pH调节。The method for reducing the by-product inhibitory effect in the lignocellulosic alkali pretreatment liquid according to claim 3, wherein in step S2, pH adjustment is carried out using concentrated phosphoric acid.
- 根据权利要求4所述的降低木质纤维素碱法预处理液中副产物抑制效应的方法,其特征在于,步骤S2中,相混合的葡萄糖氧化酶和预处理液反应24~48h。The method for reducing the by-product inhibitory effect in the lignocellulosic alkali pretreatment liquid according to claim 4, wherein in step S2, the mixed glucose oxidase and the pretreatment liquid are reacted for 24 to 48 hours.
- 权利要求1至5任一项所述方法在乙醇生产中的应用。Use of the method of any one of claims 1 to 5 in ethanol production.
- 一种利用木质纤维素生产乙醇的方法,其特征在于,包括如下步骤:A method for producing ethanol using lignocellulose, comprising the steps of:ⅰ)木质纤维素碱法预处理及脱毒:采用权利要求1至5任一项所述的方法进行处理;i) lignocellulosic alkali pretreatment and detoxification: treatment by the method according to any one of claims 1 to 5;ⅱ)纤维素酶酶解:往步骤ⅰ)中的体系加入纤维素酶进行酶解;Ii) Cellulase enzymatic hydrolysis: adding cellulase to the system in step i) for enzymatic hydrolysis;ⅲ)微生物发酵:往步骤ⅱ)中的体系接种酿酒酵母进行发酵。Iii) Microbial fermentation: The system in step ii) is inoculated with Saccharomyces cerevisiae for fermentation.
- 根据权利要求7所述的利用木质纤维素生产乙醇的方法,其特征在于,步骤ⅱ)中酶解60~80h。The method for producing ethanol using lignocellulose according to claim 7, wherein the enzyme is hydrolyzed in step ii) for 60 to 80 hours.
- 根据权利要求8所述的利用木质纤维素生产乙醇的方法,其特征在于,步骤ⅲ)中发酵20~30h。 The method for producing ethanol using lignocellulose according to claim 8, wherein the fermentation in step iii) is carried out for 20 to 30 hours.
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