CN102586342A - Method for lowering fermentation inhibitor from source - Google Patents

Method for lowering fermentation inhibitor from source Download PDF

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CN102586342A
CN102586342A CN2012100147967A CN201210014796A CN102586342A CN 102586342 A CN102586342 A CN 102586342A CN 2012100147967 A CN2012100147967 A CN 2012100147967A CN 201210014796 A CN201210014796 A CN 201210014796A CN 102586342 A CN102586342 A CN 102586342A
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陈洪章
张玉针
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Institute of Process Engineering of CAS
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Abstract

本发明公开了一种从源头上降低木质纤维素来源的发酵抑制物的方法,该方法将蒸汽爆破技术和气流分级技术组合起来应用于木质纤维原料的预处理。二段汽爆分梳工艺有效的针对纤维原料各组织的特征选择性的进行预处理,既能保证纤维组织达到较好的预处理效果,又能避免薄壁组织的过度降解,从而提高纤维原料的酶解率的同时,又能有效控制副反应的发生,即降低抑制物含量,省去了脱毒单元操作的引入,简化了工艺。另外,该工艺中,纤维细胞和薄壁细胞的分离和选择性预处理,为木质纤维原料的分层多级转化和生物量权利用提供了新途径,且二段汽爆分梳工艺还有助于纤维原料的复水均匀,降低了第二段物料的汽爆条件且减少了生料含量,提高了原料的利用率。The invention discloses a method for reducing fermentation inhibitors derived from lignocellulose from the source. The method combines steam explosion technology and air classification technology and applies it to the pretreatment of lignocellulosic raw materials. The two-stage steam explosion carding process effectively pretreats the characteristics of each tissue of the fiber raw material selectively, which can not only ensure that the fiber tissue achieves a better pretreatment effect, but also avoid excessive degradation of the parenchyma, thereby improving the quality of the fiber raw material. At the same time, it can effectively control the occurrence of side reactions, that is, reduce the content of inhibitors, save the introduction of detoxification unit operation, and simplify the process. In addition, in this process, the separation and selective pretreatment of fiber cells and parenchyma cells provide a new way for the hierarchical and multi-stage conversion of lignocellulosic raw materials and the utilization of biomass rights, and the two-stage steam explosion carding process also has It helps the rehydration of fiber raw materials evenly, reduces the steam explosion conditions of the second-stage materials and reduces the content of raw materials, and improves the utilization rate of raw materials.

Description

一种从源头上降低发酵抑制物的方法A method for reducing fermentation inhibitors from the source

技术领域 technical field

本发明涉及一种降低木质纤维素来源的发酵抑制物的方法,特别涉及以蒸汽爆破技术为核心的预处理方法。The invention relates to a method for reducing fermentation inhibitors derived from lignocellulose, in particular to a pretreatment method with steam explosion technology as the core.

背景技术 Background technique

木质纤维素的结构与组成复杂,其中,主要以葡萄糖基组成的纤维素结构致密,较难水解,而以多种杂糖基组成的半纤维素,结构比较松散,预处理可以被水解成单糖。目前正在研究的有多种木质纤维素原料预处理方法,例如酸水解、蒸汽爆破、氨纤维爆破及湿氧化法等。各种不同的预处理过程都产生多种化合物,其种类及含量随纤维素原料性质及预处理条件的不同而不同,主要形成3类微生物生长抑制物:1)弱酸:乙酸、甲酸、乙酰丙酸等。乙酸由半纤维素脱乙酰生成,甲酸和乙酰丙酸是5-羟甲基糠醛(HMF,5-Hydroxymethylfurfural)的降解产物,同时甲酸也可由糠醛在酸性环境下降解产生;2)呋喃醛类:主要是糠醛(Furfural)和HMF,分别由戊糖和己糖在酸性环境下脱水生成;3)酚类化合物:主要由木素降解形成。The structure and composition of lignocellulose are complex. Among them, the cellulose composed mainly of glucose groups has a dense structure and is difficult to hydrolyze, while the hemicellulose composed of various heterosaccharide groups has a loose structure and can be hydrolyzed into single sugar. A variety of lignocellulosic raw material pretreatment methods are currently being studied, such as acid hydrolysis, steam explosion, ammonia fiber explosion, and wet oxidation. Various pretreatment processes produce a variety of compounds, the types and contents of which vary with the properties of cellulose raw materials and pretreatment conditions, and mainly form three types of microbial growth inhibitors: 1) Weak acids: acetic acid, formic acid, acetylpropane Acid etc. Acetic acid is produced by deacetylation of hemicellulose. Formic acid and levulinic acid are degradation products of 5-hydroxymethylfurfural (HMF, 5-Hydroxymethylfurfural). At the same time, formic acid can also be produced by the degradation of furfural in an acidic environment; 2) furan aldehydes: Mainly furfural (Furfural) and HMF, which are produced by dehydration of pentose and hexose in acidic environment respectively; 3) Phenolic compounds: mainly formed by lignin degradation.

蒸汽爆破技术被认为是植物纤维预处理的一项最具前景和最经济高效的技术。原料用蒸汽加热至180-235℃,维压一定时间,在突然减压喷放时,体积猛增,受机械力的作用,其固体物料结构被破坏。蒸汽爆碎的几个优点可归纳如下:(1)可应用于各种植物生物质,预处理条件容易调节控制。(2)半纤维素、木质素和纤维素三种组分会在三个不同的流程中分离,分别为水溶组分、碱溶组分和碱不溶组分。(3)纤维素的酶解转化率可达到理论最大值。(4)经过蒸汽处理后的木质素仍能够用于其他化学产品的转化。(5)半纤维素产生的糖可以被全利用,转化为液体燃料。(6)汽爆过程中产生的发酵抑制物可通过控制汽爆条件而大大降低。该预处理方法适用于硬木、软木、农业废弃物,如蔗渣、麦草、稻草、玉米秸杆和其他非纤维素原料等各种植物生物质,而且正在这方面发挥越来越大的作用。但是,蒸汽爆破预处理技术最大的缺点就是仍会产生一些含量相对其他预处理技术少的发酵抑制物。Steam explosion technology is considered to be one of the most promising and cost-effective techniques for plant fiber pretreatment. The raw material is heated to 180-235°C with steam, and the pressure is maintained for a certain period of time. When it is suddenly decompressed and sprayed, the volume increases sharply, and its solid material structure is destroyed by the action of mechanical force. Several advantages of steam explosion can be summarized as follows: (1) It can be applied to various plant biomass, and the pretreatment conditions are easy to adjust and control. (2) The three components of hemicellulose, lignin and cellulose will be separated in three different processes, namely water-soluble components, alkali-soluble components and alkali-insoluble components. (3) The enzymatic conversion rate of cellulose can reach the theoretical maximum. (4) The lignin after steam treatment can still be used for conversion of other chemical products. (5) The sugar produced by hemicellulose can be fully utilized and converted into liquid fuel. (6) The fermentation inhibitors produced in the steam explosion process can be greatly reduced by controlling the steam explosion conditions. This pretreatment method is applicable to, and is playing an increasing role in, various plant biomasses such as hardwoods, softwoods, agricultural wastes such as bagasse, wheat straw, straw, corn stover and other non-cellulosic feedstocks. However, the biggest disadvantage of steam explosion pretreatment technology is that it still produces some fermentation inhibitors with less content than other pretreatment technologies.

这些抑制物对随后发酵微生物的抑制,已成为木质纤维素生物加工过程的主要瓶颈之一。须采取必要的应对抑制物的措施以减少或消除其抑制作用。人们尝试了多种方法对木质纤维素水解液进行发酵前的脱毒处理,包括生物学、物理及化学方法等及不同措施组合使用。生物学方法包括利用特定酶(例如漆酶laccase)或微生物对水解液进行脱毒处理。漆酶是一类含铜氧化还原酶,专一性地对酚类底物分子进行单电子氧化,生成相应的活性自由基,活性的中间物随后转变为二聚体、寡聚体和高聚物。在这一过程中小分子量酚类化合物发生了氧化聚合反应,生成了毒性较低的高分子量化合物,从而减少了木质纤维素预处理液中酚类物质的毒性。Mussatto等用只能利用乙酸生长的酿酒酵母突变株处理水解液,使其中的乙酸浓度由6.8g/L降低至0.4g/L。物理方法有真空干燥浓缩、蒸煮、活性炭吸附、离子交换吸附及溶剂萃取等,真空浓缩及蒸煮可以使挥发性抑制物大量减少,离子交换及溶剂萃取可有效降低乙酸、呋喃醛及酚类化合物含量;化学方法包括利用各种碱(NH4OH、NaOH、Ca(OH)2等)及过量石灰法等对水解液处理,其中NH4OH能有效去除呋喃醛类物质,过量石灰处理可以有效提高水解液中单糖利用率。另外,水洗预处理原料也是一种简单的脱毒过程。将2种或多种方法联合使用可以达到更好的脱毒效果。Inhibition of these inhibitors on subsequent fermentation microorganisms has become one of the major bottlenecks in lignocellulose bioprocessing. Necessary measures to deal with inhibitors shall be taken to reduce or eliminate their inhibitory effects. Various methods have been tried to detoxify the lignocellulose hydrolyzate before fermentation, including biological, physical and chemical methods and combinations of different measures. Biological methods include detoxification of the hydrolyzate using specific enzymes (such as laccase) or microorganisms. Laccase is a class of copper-containing oxidoreductases, which specifically perform one-electron oxidation on phenolic substrate molecules to generate corresponding active free radicals, and the active intermediates are then transformed into dimers, oligomers and polymers. thing. In this process, the oxidative polymerization reaction of small molecular weight phenolic compounds occurs, and high molecular weight compounds with low toxicity are generated, thereby reducing the toxicity of phenolic compounds in lignocellulose pretreatment liquid. Mussatto et al. treated the hydrolyzate with a mutant strain of Saccharomyces cerevisiae that could only use acetic acid to reduce the concentration of acetic acid from 6.8 g/L to 0.4 g/L. Physical methods include vacuum drying and concentration, cooking, activated carbon adsorption, ion exchange adsorption and solvent extraction, etc. Vacuum concentration and cooking can greatly reduce volatile inhibitors, and ion exchange and solvent extraction can effectively reduce the content of acetic acid, furan aldehyde and phenolic compounds The chemical method includes utilizing various alkalis (NH4OH, NaOH, Ca(OH)2, etc.) and excess lime method to treat the hydrolyzate, wherein NH4OH can effectively remove furan aldehydes, and excessive lime treatment can effectively improve the single sugar utilization. In addition, washing pretreated raw materials is also a simple detoxification process. Combining two or more methods can achieve better detoxification effect.

但是,脱毒步骤无疑增加了生物基产品的发酵成本,使工艺过程更为复杂,同时也导致一部分可发酵性糖的损失,所以在发酵过程中须尽量减少发酵前的脱毒环节。可行的方法有选育高抗性菌株,提高其自身内在的耐受能力,或者从源头上优化水解工艺控制抑制物的产生,降低其含量。木质纤维素原料具有复杂的、不均一的多级结构。从化学成分上看,木质纤维原料的化学成分包括纤维素、半纤维素和木质素。从细胞组成上看,包括纤维状的纤维细胞和杂细胞(包括导管、薄壁细胞、表皮细胞等)。纤维细胞是植物纤维原料中最主要、最基本的细胞,是植物原料的支持组织,纤维细胞一般具有发达的次生壁(即厚度较大)。植物原料中另一类比较重要的细胞是薄壁细胞,在植物生长中起着储存营养的作用,薄壁细胞腔大、壁薄、长度短的特点。鉴于2类细胞的结构和形态上的差异,可知这2类细胞所要求的预处理条件不一样。纤维细胞,细胞壁木质化程度高,结构致密,受热过程中其传质传热阻力大,且不易被撕裂;薄壁细胞,壁薄而腔大,即利于传质传热,又利于水蒸汽闪蒸对其物理撕裂。不同原料中,这两类细胞的含量、结构各不相同,这就从本质上决定了其所需的处理条件的差异。因此,可以针对不同组织细胞分别优化处理条件,从而达到各自最好的水解效果并且副反应程度控制在最小。However, the detoxification step will undoubtedly increase the fermentation cost of bio-based products, make the process more complicated, and also lead to the loss of a part of fermentable sugars, so the detoxification process before fermentation must be minimized during the fermentation process. Feasible methods include breeding highly resistant strains to improve their inherent tolerance, or optimizing the hydrolysis process from the source to control the production of inhibitors and reduce their content. Lignocellulosic raw materials have complex, heterogeneous and multi-level structures. In terms of chemical composition, the chemical composition of lignocellulosic raw materials includes cellulose, hemicellulose and lignin. From the perspective of cell composition, it includes fibrous fibroblasts and miscellaneous cells (including ducts, parenchyma cells, epidermal cells, etc.). Fibroblasts are the most important and basic cells in plant fiber raw materials, and are the supporting tissues of plant raw materials. Fibroblasts generally have well-developed secondary walls (that is, thicker). Another important type of cells in plant materials is parenchyma cells, which play a role in storing nutrients in plant growth. Parenchyma cells are characterized by large lumens, thin walls, and short lengths. In view of the structural and morphological differences of the two types of cells, it can be seen that the pretreatment conditions required by the two types of cells are different. Fibrous cells have a high degree of lignification of the cell wall and a dense structure, which has a large mass and heat transfer resistance during the heating process and is not easily torn; parenchyma cells have thin walls and large cavities, which are not only conducive to mass and heat transfer, but also conducive to water vapor Flashing physically tears it apart. In different raw materials, the content and structure of these two types of cells are different, which essentially determines the difference in the processing conditions they require. Therefore, the treatment conditions can be optimized for different tissue cells, so as to achieve the best hydrolysis effect and minimize the degree of side effects.

本发明开发的二段汽爆分梳工艺,不同于传统所指的二段汽爆工艺,这里是指采用较温和的汽爆条件进行第一段汽爆,通过气流分级装置将第一段物料分级,得到薄壁组织和纤维组织,再将纤维组织在合适的条件下进行第二段汽爆。二段汽爆分梳工艺有效的针对其各组织的特征要求选择性的进行预处理,既能保证纤维组织达到较好的预处理效果,又能避免薄壁组织的过度降解,从而提高纤维原料的酶解率的同时,又能有效控制副反应的发生,即降低抑制物含量,省去了脱毒单元操作的引入,简化了工艺。另外,该工艺中,纤维细胞和薄壁细胞的分离和选择性预处理,为木质纤维原料的分层多级转化和生物量权利用提供了新途径,且二段汽爆分梳工艺还有助于纤维原料的复水均匀,降低了第二段物料的汽爆条件且减少了生料含量,提高了原料的利用率。The two-stage steam explosion combing process developed by the present invention is different from the traditional two-stage steam explosion process. Here, it refers to the use of milder steam explosion conditions to carry out the first stage steam explosion, and the first stage material is separated by an air classification device. Grading to obtain parenchyma and fibrous tissue, and then the fibrous tissue is subjected to the second stage of steam explosion under suitable conditions. The two-stage steam explosion carding process can effectively perform selective pretreatment according to the characteristics of each tissue, which can not only ensure that the fiber tissue achieves a better pretreatment effect, but also avoid excessive degradation of the parenchyma tissue, thereby improving the quality of fiber raw materials. At the same time, it can effectively control the occurrence of side reactions, that is, reduce the content of inhibitors, save the introduction of detoxification unit operation, and simplify the process. In addition, in this process, the separation and selective pretreatment of fiber cells and parenchyma cells provide a new way for the hierarchical and multi-stage conversion of lignocellulosic raw materials and the utilization of biomass rights, and the two-stage steam explosion carding process also has It helps the rehydration of fiber raw materials evenly, reduces the steam explosion conditions of the second-stage materials and reduces the content of raw materials, and improves the utilization rate of raw materials.

发明内容 Contents of the invention

本发明的目的是针对目前木质纤维素预处理过程中产生发酵抑制物已成为木质纤维素生物加工过程的主要瓶颈之一,而常采用的脱毒工序不仅增加了生物基产品的发酵成本,使工艺过程更为复杂,而且也导致一部分可发酵性糖的损失等问题,开发了新型的二段汽爆分梳工艺。这里的二段汽爆分梳工艺,不同于传统所指的二段汽爆工艺,是指采用较温和的汽爆条件进行第一段汽爆,通过气流分级装置将第一段物料分级,得到薄壁组织和纤维组织,再将纤维组织在合适的条件下进行第二段汽爆。The purpose of the present invention is to aim at the production of fermentation inhibitors in the current lignocellulose pretreatment process has become one of the main bottlenecks in the lignocellulose bioprocessing process, and the commonly used detoxification process not only increases the fermentation cost of bio-based products, but also makes The process is more complicated, and it also leads to the loss of part of the fermentable sugar. A new two-stage steam explosion carding process has been developed. The two-stage steam explosion carding process here is different from the traditional two-stage steam explosion process, which means that the first stage steam explosion is carried out under milder steam explosion conditions, and the first stage materials are classified by an air classification device to obtain Parenchyma and fibrous tissue, and then the fibrous tissue is subjected to the second stage of steam explosion under suitable conditions.

本发明的技术方案如下:Technical scheme of the present invention is as follows:

本发明提供的从源头上降低木质纤维素来源的发酵抑制物含量的新型二段汽爆分梳工艺,其包括以下步骤:1)在压力为0.5~1.5MPThe novel two-stage steam explosion carding process for reducing the content of fermentation inhibitors derived from lignocellulose from the source provided by the present invention comprises the following steps: 1) at a pressure of 0.5 to 1.5MP

a,时间为1~10min的汽爆条件进行对秸秆原料进行第一段蒸汽爆破处理;2)通过气流分级装置将第一段汽爆物料进行分级,得到薄壁组织和纤维组织;3)将分梳得到的纤维组织在压力为0.5~1.5MPa,时间为1~10min的条件下进行第二段蒸汽爆破处理;4)分别对第一段汽爆、分梳以及第二段汽爆后的物料进行酶解和发酵;5)分别测定秸秆原料、第一段汽爆物料、分梳后的薄壁组织和纤维组织以及第二段汽爆物料的含水率、水洗液中的抑制物含量、酶解率以及发酵产物的转化率。a, steam explosion conditions of 1 to 10 minutes are used to carry out the first-stage steam explosion treatment on straw raw materials; 2) classify the first-stage steam-exploded materials through an airflow classification device to obtain parenchyma and fibrous tissue; 3) put The fibrous tissue obtained by carding is subjected to the second stage of steam explosion treatment under the condition of a pressure of 0.5-1.5MPa and a time of 1-10min; 4) the first stage of steam explosion, carding and the second stage of steam explosion The material is subjected to enzymolysis and fermentation; 5) The moisture content of the straw raw material, the steam-exploded material in the first stage, the parenchyma and fibrous tissue after carding, the steam-exploded material in the second stage, the inhibitor content in the washing liquid, Enzymolysis rate and conversion rate of fermentation products.

本发明中,酶解条件为汽爆秸秆与pH 4.850mM柠檬酸缓冲液的固液比为1∶10-1∶30,加酶量为15IU/g-110/g底物,在50℃,150rpm的水浴摇床中酶解24-120h。然后,离心过滤得酶解液。In the present invention, the enzymolysis conditions are that the solid-to-liquid ratio of steam-exploded straw and pH 4.850mM citric acid buffer is 1:10-1:30, the amount of enzyme added is 15IU/g-110/g substrate, at 50°C, Enzymolysis in a water-bath shaker at 150rpm for 24-120h. Then, centrifuge and filter to obtain the enzymatic hydrolyzate.

本发明的有益效果为:(1)二段汽爆分梳工艺有效的针对其各组织的特征要求选择性的进行预处理,既能保证纤维组织达到较好的预处理效果,又能避免薄壁组织的过度降解,从而提高纤维原料的酶解率的同时,又能有效控制副反应的发生,即降低抑制物含量,省去了脱毒单元操作的引入,简化了工艺。(2)该工艺中,纤维细胞和薄壁细胞的分离和选择性预处理,为木质纤维原料的分层多级转化和生物量权利用提供了新途径。(3)二段汽爆分梳工艺有助于纤维原料的复水均匀,降低了第二段物料的汽爆条件且减少了生料含量,提高了原料的利用率。The beneficial effects of the present invention are as follows: (1) The two-stage steam explosion carding process effectively requires selective pretreatment according to the characteristic requirements of each tissue, which can ensure that the fibrous tissue achieves a better pretreatment effect and avoid thinning Excessive degradation of the wall tissue, thereby increasing the enzymatic hydrolysis rate of fiber raw materials, can effectively control the occurrence of side reactions, that is, reduce the content of inhibitors, save the introduction of detoxification unit operations, and simplify the process. (2) In this process, the separation and selective pretreatment of fibroblasts and parenchyma cells provide a new way for the hierarchical and multi-stage conversion of lignocellulosic raw materials and the utilization of biomass rights. (3) The second-stage steam explosion carding process helps to rehydrate the fiber raw materials evenly, reduces the steam explosion conditions of the second-stage materials and reduces the content of raw materials, and improves the utilization rate of raw materials.

具体实施方式 Detailed ways

下面通过实施例对本发明做进一步说明。The present invention will be further described below by embodiment.

本发明实施例中使用的纤维素酶制剂购自夏盛公司,110FPAIU/ml,1200IU xylanase/ml。The cellulase preparation used in the embodiment of the present invention was purchased from Xiasheng Company, 110FPAIU/ml, 1200IU xylanase/ml.

实施例1:称取200g干玉米秸秆放入汽爆罐中,密闭后通入水蒸气,使罐压迅速升至1.1MPa,在此压力下维持4min后,迅速放压,从而制得第一段汽爆秸秆。取适量第一段汽爆秸秆,在离心式气流分级装置上进行分梳,分别得到薄壁组织和纤维组织。称取适量分梳后的纤维组织,放入汽爆罐中,密闭后通入水蒸气,使罐压迅速升至1.2MPa,在此压力下维持4min后,迅速放压,从而制得第二段汽爆秸秆。预处理条件为1.2MPa、8min、不分梳为预处理对照组。Example 1: Weigh 200g of dry corn stalks and put them into a steam explosion tank, and then pass water vapor into it after airtight to make the tank pressure rise to 1.1MPa rapidly, and after maintaining this pressure for 4 minutes, release the pressure quickly to obtain the first stage Steam-exploded straw. Take an appropriate amount of the first-stage steam-exploded straw, and comb it on a centrifugal airflow classification device to obtain parenchyma and fibrous tissue respectively. Weigh an appropriate amount of combed fibrous tissue, put it into a steam explosion tank, and then inject water vapor after sealing it to make the tank pressure rise to 1.2MPa rapidly. After maintaining this pressure for 4 minutes, release the pressure quickly to obtain the second stage Steam-exploded straw. The pretreatment conditions were 1.2MPa, 8min, and no carding as the pretreatment control group.

分别称取第一段汽爆秸秆、分梳后的汽爆秸秆、第二段汽爆秸秆以及对照组汽爆秸秆1g(dry base),溶入20ml水于70℃浸提2h,取上清液,采用高效液相(Agilent 1200HPLC,美国)测定水解液中的糠醛和羟甲基糠醛(HMF)含量,采用紫外分光光度计在280nm测定水解液中的可溶性木质素含量。糠醛、羟甲基糠醛(HMF)以及可溶性木质素含量之和即为水解液中抑制物含量。Weigh 1g (dry base) of steam-exploded straw in the first stage, steam-exploded straw after carding, steam-exploded straw in the second stage and the steam-exploded straw in the control group respectively, dissolve them in 20ml of water and extract at 70°C for 2 hours, and take the supernatant The content of furfural and hydroxymethylfurfural (HMF) in the hydrolyzate was measured by high performance liquid phase (Agilent 1200HPLC, USA), and the content of soluble lignin in the hydrolyzate was measured by a UV spectrophotometer at 280nm. The sum of the contents of furfural, hydroxymethylfurfural (HMF) and soluble lignin is the inhibitor content in the hydrolyzate.

分别称取第一段汽爆秸秆、分梳后的汽爆秸秆以及第二段汽爆秸秆以及预处理对照组汽爆秸秆10g,加入200ml pH 4.850mM柠檬酸缓冲液中,加入0.5ml纤维素酶,在50℃,150rpm的水浴摇床中酶解48h。采用DNS法测酶解液中还原糖含量。然后,离心过滤得酶解液。取20ml酶解液,补水至40ml,加入0.24g磷酸二氢钾、0.4g蛋白胨,补加葡萄糖至酶解液中初始糖浓为25g/L,加入2mL serratiamarcescens种子液,调节pH为5,在37℃厌氧发酵48h。只以葡萄糖为碳源的发酵对照组。以采用HPLC测定2,3-丁二醇含量。最终折合抑制物含量为分梳后的薄壁组织中的含量与第二段汽爆后的物料的含量之和,2,3-丁二醇含量同理可算。Weigh 10g of the first stage of steam-exploded straw, combed steam-exploded straw, second-stage steam-exploded straw and pretreatment control group steam-exploded straw, add 200ml pH 4.850mM citric acid buffer solution, add 0.5ml cellulose Enzyme, hydrolyze at 50°C, 150rpm in a water-bath shaker for 48h. The content of reducing sugar in the enzymatic hydrolyzate was measured by DNS method. Then, centrifuge and filter to obtain the enzymatic hydrolyzate. Take 20ml of enzymolysis solution, add water to 40ml, add 0.24g of potassium dihydrogen phosphate, 0.4g of peptone, add glucose until the initial sugar concentration in the enzymolysis solution is 25g/L, add 2mL of serratiamarcescens seed solution, adjust the pH to 5, and Anaerobic fermentation at 37°C for 48h. Fermentation control group using only glucose as carbon source. The content of 2,3-butanediol was determined by HPLC. The final equivalent inhibitor content is the sum of the content in the carded parenchyma and the content of the material after the second steam explosion, and the content of 2,3-butanediol can be calculated in the same way.

结果表明实验组的抑制物含量比预处理对照组的抑制物含量低33%,且实验组的2,3-丁二醇含量比只以糖为碳源的2,3-丁二醇发酵对照组高5.1%,而预处理对照组的2,3-丁二醇含量比只以糖为碳源的2,3-丁二醇发酵对照组低66.7%。The results showed that the inhibitor content of the experimental group was 33% lower than that of the pretreatment control group, and the 2,3-butanediol content of the experimental group was higher than that of the 2,3-butanediol fermentation control with sugar as the carbon source. The 2,3-butanediol content of the pretreatment control group was 66.7% lower than that of the 2,3-butanediol fermentation control group with only sugar as carbon source.

实施例2:称取200g干玉米秸秆放入汽爆罐中,密闭后通入水蒸气,使罐压迅速升至1.2MPa,在此压力下维持4min后,迅速放压,从而制得第一段汽爆秸秆。取适量第一段汽爆秸秆,在离心式气流分级装置上进行分梳,分别得到薄壁组织和纤维组织。称取适量分梳后的纤维组织,放入汽爆罐中,密闭后通入水蒸气,使罐压迅速升至1.2MPa,在此压力下维持4min后,迅速放压,从而制得第二段汽爆秸秆。预处理条件为1.2MPa、8min、不分梳为预处理对照组。Example 2: Weigh 200g of dry corn stalks and put them into a steam explosion tank, and then pass water vapor into it after airtight to make the tank pressure rise to 1.2MPa rapidly, and after maintaining this pressure for 4 minutes, release the pressure quickly to obtain the first stage Steam-exploded straw. Take an appropriate amount of the first-stage steam-exploded straw, and comb it on a centrifugal airflow classification device to obtain parenchyma and fibrous tissue respectively. Weigh an appropriate amount of combed fibrous tissue, put it into a steam explosion tank, and then inject water vapor after sealing it to make the tank pressure rise to 1.2MPa rapidly. After maintaining this pressure for 4 minutes, release the pressure quickly to obtain the second stage Steam-exploded straw. The pretreatment conditions were 1.2MPa, 8min, and no carding as the pretreatment control group.

分别称取第一段汽爆秸秆、分梳后的汽爆秸秆、第二段汽爆秸秆以及对照组汽爆秸秆1g(dry base),溶入20ml水于70℃浸提2h,取上清液,采用高效液相(Agilent 1200HPLC,美国)测定水解液中的糠醛和羟甲基糠醛(HMF)含量,采用紫外分光光度计在280nm测定水解液中的可溶性木质素含量。糠醛、羟甲基糠醛(HMF)以及可溶性木质素含量之和即为水解液中抑制物含量。Weigh 1g (dry base) of steam-exploded straw in the first stage, steam-exploded straw after carding, steam-exploded straw in the second stage and the steam-exploded straw in the control group respectively, dissolve them in 20ml of water and extract at 70°C for 2 hours, and take the supernatant The content of furfural and hydroxymethylfurfural (HMF) in the hydrolyzate was measured by high performance liquid phase (Agilent 1200HPLC, USA), and the content of soluble lignin in the hydrolyzate was measured by a UV spectrophotometer at 280nm. The sum of the contents of furfural, hydroxymethylfurfural (HMF) and soluble lignin is the inhibitor content in the hydrolyzate.

分别称取第一段汽爆秸秆、分梳后的汽爆秸秆以及第二段汽爆秸秆以及预处理对照组汽爆秸秆10g,加入200ml pH 4.850mM柠檬酸缓冲液中,加入0.5ml纤维素酶,在50℃,150rpm的水浴摇床中酶解48h。采用DNS法测酶解液中还原糖含量。然后,离心过滤得酶解液。取20ml酶解液,补水至40ml,加入0.24g磷酸二氢钾、0.4g蛋白胨,补加葡萄糖至酶解液中初始糖浓为25g/L,加入2mL serratiamarcescens种子液,调节pH为5,在37℃厌氧发酵48h。只以葡萄糖为碳源的发酵对照组。以采用HPLC测定2,3-丁二醇含量。最终折合抑制物含量为分梳后的薄壁组织中的含量与第二段汽爆后的物料的含量之和,2,3-丁二醇含量同理可算。Weigh 10g of the first stage of steam-exploded straw, combed steam-exploded straw, second-stage steam-exploded straw and pretreatment control group steam-exploded straw, add 200ml pH 4.850mM citric acid buffer solution, add 0.5ml cellulose Enzyme, hydrolyze at 50°C, 150rpm in a water-bath shaker for 48h. The content of reducing sugar in the enzymatic hydrolyzate was measured by DNS method. Then, centrifuge and filter to obtain the enzymatic hydrolyzate. Take 20ml of enzymolysis solution, add water to 40ml, add 0.24g of potassium dihydrogen phosphate, 0.4g of peptone, add glucose until the initial sugar concentration in the enzymolysis solution is 25g/L, add 2mL of serratiamarcescens seed solution, adjust the pH to 5, and Anaerobic fermentation at 37°C for 48h. Fermentation control group using only glucose as carbon source. The content of 2,3-butanediol was determined by HPLC. The final equivalent inhibitor content is the sum of the content in the carded parenchyma and the content of the material after the second steam explosion, and the content of 2,3-butanediol can be calculated in the same way.

结果表明实验组的抑制物含量比预处理对照组的抑制物含量低27%,且实验组的2,3-丁二醇含量比只以糖为碳源的2,3-丁二醇发酵对照组高4.3%,而预处理对照组的2,3-丁二醇含量比只以糖为碳源的2,3-丁二醇发酵对照组低66.5%。The results show that the inhibitor content of the experimental group is 27% lower than that of the pretreatment control group, and the 2,3-butanediol content of the experimental group is higher than that of the 2,3-butanediol fermentation control with sugar as the carbon source. The 2,3-butanediol content of the pretreatment control group was 66.5% lower than that of the 2,3-butanediol fermentation control group with only sugar as carbon source.

实施例3:称取200g干玉米秸秆放入汽爆罐中,密闭后通入水蒸气,使罐压迅速升至1.1MPa,在此压力下维持4min后,迅速放压,从而制得第一段汽爆秸秆。取适量第一段汽爆秸秆,在离心式气流分级装置上进行分梳,分别得到薄壁组织和纤维组织。称取适量分梳后的纤维组织,放入汽爆罐中,密闭后通入水蒸气,使罐压迅速升至1.2MPa,在此压力下维持4min后,迅速放压,从而制得第二段汽爆秸秆。预处理条件为1.2MPa、8min、不分梳为预处理对照组。Example 3: Weigh 200g of dry corn stalks and put them into a steam explosion tank, and then inject water vapor after airtight to make the tank pressure rise rapidly to 1.1MPa, and after maintaining this pressure for 4 minutes, release the pressure quickly, thereby obtaining the first stage Steam-exploded straw. Take an appropriate amount of the first-stage steam-exploded straw, and comb it on a centrifugal airflow classification device to obtain parenchyma and fibrous tissue respectively. Weigh an appropriate amount of combed fibrous tissue, put it into a steam explosion tank, and then inject water vapor after sealing it to make the tank pressure rise to 1.2MPa rapidly. After maintaining this pressure for 4 minutes, release the pressure quickly to obtain the second stage Steam-exploded straw. The pretreatment conditions were 1.2MPa, 8min, and no carding as the pretreatment control group.

分别称取第一段汽爆秸秆、分梳后的汽爆秸秆、第二段汽爆秸秆以及对照组汽爆秸秆1g(dry base),溶入20ml水于70℃浸提2h,取上清液,采用高效液相(Agilent 1200HPLC,美国)测定水解液中的糠醛和羟甲基糠醛(HMF)含量,采用紫外分光光度计在280nm测定水解液中的可溶性木质素含量。糠醛、羟甲基糠醛(HMF)以及可溶性木质素含量之和即为水解液中抑制物含量。Weigh 1g (dry base) of steam-exploded straw in the first stage, steam-exploded straw after carding, steam-exploded straw in the second stage and the steam-exploded straw in the control group respectively, dissolve them in 20ml of water and extract at 70°C for 2 hours, and take the supernatant The content of furfural and hydroxymethylfurfural (HMF) in the hydrolyzate was measured by high performance liquid phase (Agilent 1200HPLC, USA), and the content of soluble lignin in the hydrolyzate was measured by a UV spectrophotometer at 280nm. The sum of the contents of furfural, hydroxymethylfurfural (HMF) and soluble lignin is the inhibitor content in the hydrolyzate.

分别称取第一段汽爆秸秆、分梳后的汽爆秸秆以及第二段汽爆秸秆以及预处理对照组汽爆秸秆10g,加入200ml pH 4.850mM柠檬酸缓冲液中,加入0.5ml纤维素酶,在50℃,150rpm的水浴摇床中酶解48h。采用DNS法测酶解液中还原糖含量。然后,离心过滤得酶解液。取40ml酶解液,加入0.2g磷酸二氢钾、0.08g硫酸铵、0.016g硫酸镁、0.008g氯化钙、0.08g酵母粉。补加葡萄糖至酶解液中初始糖浓为25g/L,加入2mL安琪酵母种子液,调节pH为5.5,在32℃厌氧发酵48h。只以葡萄糖为碳源的发酵对照组。采用HPLC测定乙醇含量。最终折合抑制物含量为分梳后的薄壁组织中的含量与第二段汽爆后的物料的含量之和,乙醇含量同理可算。Weigh 10g of the first stage of steam-exploded straw, combed steam-exploded straw, second-stage steam-exploded straw and pretreatment control group steam-exploded straw, add 200ml pH 4.850mM citric acid buffer solution, add 0.5ml cellulose Enzyme, hydrolyze at 50°C, 150rpm in a water-bath shaker for 48h. The content of reducing sugar in the enzymatic hydrolyzate was measured by DNS method. Then, centrifuge and filter to obtain the enzymatic hydrolyzate. Take 40ml of enzymolysis solution, add 0.2g of potassium dihydrogen phosphate, 0.08g of ammonium sulfate, 0.016g of magnesium sulfate, 0.008g of calcium chloride, and 0.08g of yeast powder. Glucose was added until the initial sugar concentration in the enzymolysis solution was 25g/L, 2mL Angel yeast seed solution was added, the pH was adjusted to 5.5, and anaerobic fermentation was carried out at 32°C for 48h. Fermentation control group using only glucose as carbon source. The ethanol content was determined by HPLC. The final equivalent inhibitor content is the sum of the content in the carded parenchyma and the content of the material after the second steam explosion, and the ethanol content can be calculated in the same way.

结果表明实验组的抑制物含量比预处理对照组的抑制物含量低33%。实验组的乙醇含量比预处理对照组的乙醇含量高13.8%,且分别比只以糖为碳源的乙醇发酵对照组低22.4%和33%。The results showed that the inhibitor content of the experimental group was 33% lower than that of the pretreated control group. The ethanol content of the experimental group was 13.8% higher than that of the pretreatment control group, and 22.4% and 33% lower than that of the ethanol fermentation control group with sugar as carbon source, respectively.

Claims (4)

1.一种从源头上降低木质纤维素来源的发酵抑制物的方法,包括以下步骤:1. A method for reducing the fermentation inhibitor of lignocellulose source from the source, comprising the following steps: 1)对秸秆原料进行第一段蒸汽爆破处理;1) Carry out the first-stage steam explosion treatment to the straw raw material; 2)将第一段汽爆物料进行分梳,得到薄壁组织和纤维组织;2) Combing the first steam explosion material to obtain parenchyma and fibrous tissue; 3)对分梳得到的纤维组织进行第二段蒸汽爆破处理;3) Carry out the second stage of steam explosion treatment to the fibrous tissue obtained by carding; 4)分别对第一段汽爆、分梳以及第二段汽爆后的物料进行酶解和发酵;4) Carry out enzymatic hydrolysis and fermentation to the materials after the steam explosion in the first stage, carding and the steam explosion in the second stage respectively; 5)分别测定秸秆原料、第一段汽爆物料、分梳后的薄壁组织和纤维组织以及第二段汽爆物料的含水率、水洗液中的抑制物含量、以及发酵产物含量。5) Determination of the moisture content of the straw raw material, the steam-exploded material in the first stage, the parenchyma and fibrous tissue after carding, and the steam-exploded material in the second stage, the content of inhibitors in the washing liquid, and the content of fermentation products. 2.根据权利要求1所述的方法,其中步骤1)的第一段蒸汽爆破处理是在汽爆罐中在0.5~1.5MP a的蒸汽压力下进行1~10min。2. The method according to claim 1, wherein the first stage steam explosion treatment of step 1) is to carry out 1~10min under the steam pressure of 0.5~1.5MPa in the steam explosion tank. 3.根据权利要求1所述的方法,其中步骤3)的第二段蒸汽爆破处理是在汽爆罐中在0.5~1.5MP a的蒸汽压力下进行1~10min。3. The method according to claim 1, wherein the second stage steam explosion treatment of step 3) is to carry out 1~10min under the steam pressure of 0.5~1.5MPa in the steam explosion tank. 4.根据权利要求1所述的方法,其中步骤4)中的酶解条件为:汽爆秸秆与pH 4.8且浓度为50mM柠檬酸缓冲液的固液比为1∶10~1∶30,加酶量为15IU/g~110/g底物,在50℃,100~200rpm的摇床中酶解24~120h。然后,离心得酶解液。4. The method according to claim 1, wherein the enzymolysis condition in step 4) is: steam-exploded stalks and pH 4.8 and a concentration of 50mM citric acid buffer have a solid-to-liquid ratio of 1: 10 to 1: 30, adding The amount of enzyme is 15IU/g-110/g substrate, and the enzyme is hydrolyzed in a shaker at 50°C and 100-200rpm for 24-120h. Then, centrifuge to obtain the enzymatic hydrolyzate.
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CN112553261A (en) * 2020-11-06 2021-03-26 华南理工大学 Method for high-solid enzymolysis of lignocellulose
CN115627277A (en) * 2022-11-18 2023-01-20 中国科学院过程工程研究所 Method for performing solid state fermentation on 2, 3-butanediol by high-solid-state enzymolysis of straws
CN117051055A (en) * 2023-10-12 2023-11-14 中国科学院过程工程研究所 Method for preparing low inhibitor straw sugar suitable for fermentation and catalysis by using uniform graded straw
CN117051055B (en) * 2023-10-12 2024-02-06 中国科学院过程工程研究所 A method for preparing low-inhibitor straw sugar suitable for fermentation and catalysis from homogenized graded straw

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