技术领域technical field
本发明公开了一种利用多氢键供体低共熔溶剂预处理玉米秸秆及其循环利用方法，属于化学工程领域。The invention discloses a method for pretreating corn stalks with a multi-hydrogen bond donor deep eutectic solvent and a recycling method thereof, belonging to the field of chemical engineering.
背景技术Background technique
我国是农业大国，木质纤维素原料相当丰富，仅每年的农作物秸秆产量就有7亿吨，约占全世界秸秆总量的30％，折合标准煤量约3.5亿吨。农作物秸秆含有丰富的纤维素及半纤维素，是一种宝贵的可再生资源，但在我国，有相当多的农作物秸秆被弃置或者焚烧，没有得到合理利用。据报道，我国秸秆利用率约为33％，其中大部分没有经过处理，经过处理后利用的仅占2.6％。因此，如果能够综合利用这些农作物秸秆资源，不仅能增加农民收入，而且对节约资源、保护环境、促进农业的可持续发展具有重要意义。但是由于秸秆的天然结构非常复杂，在高效利用秸秆中的可发酵糖之前必须对秸秆进行预处理。预处理的目的主要是破坏秸秆的整体结构，脱除秸秆中的木质素并破坏纤维素的晶体结构，增加生物质的孔隙率，提高比表面积和纤维素酶对纤维素的可及性，从而提高纤维素和半纤维素的水解率。目前，预处理方法主要有物理法、化学法、生物法以及前三种方法的组合法。其中化学法和物理法是各国研究者的研究重点。化学法主要是利用酸、碱、有机溶剂等化学试剂的催化作用或溶解作用，破坏纤维素的晶体结构，使半纤维素降解或溶解，并使木质素膨胀以消除对纤维素的保护作用，增加木质纤维素生物质的孔隙率和比表面积，提高纤维素与酶的接触几率，获得较高的酶解效率。my country is a large agricultural country with rich lignocellulose raw materials. The annual output of crop straw alone is 700 million tons, accounting for about 30% of the world's total straw, equivalent to about 350 million tons of standard coal. Crop straw is rich in cellulose and hemicellulose, and is a valuable renewable resource. However, in our country, quite a lot of crop straw is discarded or burned, and has not been rationally utilized. According to reports, the utilization rate of straw in my country is about 33%, most of which have not been treated, and only 2.6% have been used after treatment. Therefore, if these crop straw resources can be comprehensively utilized, it will not only increase farmers' income, but also be of great significance to saving resources, protecting the environment, and promoting the sustainable development of agriculture. However, because the natural structure of straw is very complex, the straw must be pretreated before the fermentable sugars in the straw can be efficiently utilized. The purpose of pretreatment is mainly to destroy the overall structure of straw, remove lignin in straw and destroy the crystal structure of cellulose, increase the porosity of biomass, improve the specific surface area and the accessibility of cellulase to cellulose, thereby Improve the hydrolysis rate of cellulose and hemicellulose. At present, the pretreatment methods mainly include physical method, chemical method, biological method and the combination of the first three methods. Among them, chemical and physical methods are the research focus of researchers from various countries. The chemical method mainly uses the catalysis or dissolution of chemical reagents such as acids, alkalis, and organic solvents to destroy the crystal structure of cellulose, degrade or dissolve hemicellulose, and expand lignin to eliminate the protective effect on cellulose. Increase the porosity and specific surface area of lignocellulosic biomass, increase the contact probability of cellulose and enzymes, and obtain higher enzymatic hydrolysis efficiency.
近年来离子液体对木质纤维素生物质的预处理情况得到了人们的广泛关注。但是，离子液体的“绿色方面”最近受到了极大的质疑，其可能的毒性以及非常低的生物降解性目前文献里都有所报道。为了克服这些缺点并保持离子液体的优良特性，Abbott等人(Journal of the American Chemical Society,2004,126:9142-9147)报道了一种新型的离子液体：低共熔溶剂。相对于传统酸碱预处理方法而言，低共熔溶剂作为一种绿色溶剂，在预处理玉米秸秆的过程中不仅减少了环境污染，而且低共熔溶剂还具有易于回收的特点，这就极大地降低了预处理成本。2014年，Xia等人(RSC Advances,2014,4:10586-10596)首先用胆碱类低共熔溶剂来处理纤维素和柳枝稷，这是首次将低共熔溶剂应用于木质纤维素生物质的预处理。我们课题组建立了多氢键供体低共熔溶剂(氯化胆碱:天冬氨酸:谷氨酸)预处理玉米秸秆的方法，处理效果要比一般低共熔溶剂明显。并且经过多氢键供体低共熔溶剂预处理的玉米秸秆的水解液可以用来发酵生产丁醇，对丁醇生产菌株没有抑制作用。In recent years, the pretreatment of lignocellulosic biomass by ionic liquids has received widespread attention. However, the "green aspect" of ionic liquids has recently been greatly questioned, with possible toxicity and very low biodegradability currently reported in the literature. In order to overcome these disadvantages and maintain the excellent properties of ionic liquids, Abbott et al. (Journal of the American Chemical Society, 2004, 126:9142-9147) reported a new type of ionic liquid: deep eutectic solvent. Compared with the traditional acid-base pretreatment method, the deep eutectic solvent, as a green solvent, not only reduces environmental pollution in the process of pretreatment of corn stalks, but also has the characteristics of easy recycling, which is extremely Greatly reduces the cost of pretreatment. In 2014, Xia et al. (RSC Advances, 2014, 4:10586-10596) first treated cellulose and switchgrass with a choline-based deep eutectic solvent, which was the first time that a deep eutectic solvent was applied to lignocellulosic biomass. preprocessing. Our research group established a multi-hydrogen bond donor deep eutectic solvent (choline chloride: aspartic acid: glutamic acid) pretreatment method for corn stalks, and the treatment effect is more obvious than that of general deep eutectic solvents. Moreover, the hydrolyzate of corn stalks pretreated by the multi-hydrogen bond donor deep eutectic solvent can be used to ferment and produce butanol, and has no inhibitory effect on butanol-producing strains.
发明内容Contents of the invention
本发明需要解决的技术问题是公开一种利用多氢键供体低共熔溶剂预处理玉米秸秆及其循环利用方法，实现玉米秸秆的高效糖化。The technical problem to be solved in the present invention is to disclose a method for pretreating corn stalks with a multi-hydrogen bond donor deep eutectic solvent and a recycling method thereof, so as to realize high-efficiency saccharification of corn stalks.
本发明的一种利用多氢键供体低共熔溶剂预处理玉米秸秆及其循环利用方法，按照如下步骤进行：A method for pretreating corn stalks using a multi-hydrogen bond donor deep eutectic solvent and its recycling method according to the present invention is carried out according to the following steps:
在多氢键供体低共熔溶剂体系(氯化胆碱:天冬氨酸:谷氨酸)中，加入玉米秸秆，90～130℃预处理玉米秸秆0.5～3h后，将混合物离心，收集上清液，并将沉淀用去离子水洗涤三次。取上述上清液加入一定量的玉米秸秆，在一定温度下预处理玉米秸秆一定时间，重复上述步骤。将预处理后的玉米秸秆与柠檬酸-柠檬酸钠缓冲液按一定固液比混合，然后用纤维素酶进行酶解，在150rpm的水浴摇床上反应，即可达到酶解玉米秸秆的目的。In the multi-hydrogen bond donor deep eutectic solvent system (choline chloride:aspartic acid:glutamic acid), add corn stalks, pretreat the corn stalks at 90-130°C for 0.5-3h, centrifuge the mixture, and collect supernatant, and the pellet was washed three times with deionized water. Take the above supernatant and add a certain amount of corn stalks, pretreat the corn stalks at a certain temperature for a certain period of time, and repeat the above steps. The pretreated corn stalks are mixed with citric acid-sodium citrate buffer according to a certain solid-to-liquid ratio, then enzymatically hydrolyzed with cellulase, and reacted on a water bath shaker at 150 rpm to achieve the purpose of enzymolyzing corn stalks.
其中，氯化胆碱、天冬氨酸与谷氨酸的摩尔比为2:0.5:0.5～2:1.5:1.5。Wherein, the molar ratio of choline chloride, aspartic acid and glutamic acid is 2:0.5:0.5˜2:1.5:1.5.
其中，多氢键供体低共熔溶剂与玉米秸秆的质量比为7:1～30:1。Wherein, the mass ratio of the multi-hydrogen bond donor deep eutectic solvent to the corn stalk is 7:1-30:1.
其中，酶解玉米秸秆时的固液比为1:10。Wherein, the solid-liquid ratio when enzymatically hydrolyzing corn stalks is 1:10.
本发明的有益效果：Beneficial effects of the present invention:
本发明的一种多氢键供体低共熔溶剂预处理玉米秸秆及其循环利用方法，用两种酸性氨基酸作为氢键供体合成多氢键供体低共熔溶剂，在预处理玉米秸秆过程中能够达到较好的预处理效果。预处理玉米秸秆具有过程经济、三废少等优点。并且预处理后的玉米秸秆水解液可以用来发酵生产丁醇。A multi-hydrogen bond donor deep eutectic solvent of the present invention pretreats corn stalks and its recycling method, uses two acidic amino acids as hydrogen bond donors to synthesize a multi hydrogen bond donor deep eutectic solvent, and pretreats corn stalks A better preprocessing effect can be achieved in the process. Pretreatment of corn stalks has the advantages of process economy and less waste. And the pretreated corn stalk hydrolyzate can be used for fermentation to produce butanol.
附图说明Description of drawings
图1利用预处理玉米秸秆水解液发酵生产丁醇结果。Figure 1 The results of butanol production by fermentation of pretreated corn stalk hydrolyzate.
具体实施方式detailed description
葡萄糖含量利用高效液相色谱(HPLC)进行测定。葡萄糖产率＝酶解过程中的葡萄糖含量×100％/玉米秸秆中的理论葡萄糖含量。Glucose content was determined by high performance liquid chromatography (HPLC). Glucose yield=glucose content in the enzymatic hydrolysis process×100%/theoretical glucose content in corn stover.
实施例1不同摩尔比的氯化胆碱、天冬氨酸与谷氨酸预处理玉米秸秆Example 1 Choline chloride, aspartic acid and glutamic acid pretreated corn stalks in different molar ratios
取100g不同摩尔配比的氯化胆碱:天冬氨酸:谷氨酸(2:0.5:0.5、2:1:0.5、2:0.5:1、2:1:1和2:1.5:1.5)，加入5g玉米秸秆，在130℃下搅拌处理2h，然后将混合物离心，用去离子水洗涤沉淀3次。在烘干的沉淀中加入pH 4.8、50mM的柠檬酸-柠檬酸钠缓冲液形成混合体系，玉米秸秆最终质量浓度为10％，加入的纤维素酶进行酶解，在50℃和150rpm的水浴摇床上酶解72h，测葡萄糖产率，结果如表1。Get the choline chloride of 100g different molar proportions: aspartic acid: glutamic acid (2:0.5:0.5,2:1:0.5,2:0.5:1,2:1:1 and 2:1.5:1.5 ), adding 5 g of corn stalks, stirring at 130° C. for 2 h, then centrifuging the mixture, and washing the precipitate with deionized water for 3 times. Add pH 4.8, 50mM citric acid-sodium citrate buffer to the dried precipitate to form a mixed system, the final mass concentration of corn stalks is 10%, add The cellulase was used for enzymolysis, and the enzymolysis was carried out on a water bath shaker at 50°C and 150rpm for 72 hours, and the glucose yield was measured. The results are shown in Table 1.
表1 氯化胆碱、天冬氨酸与谷氨酸摩尔比的优化结果Table 1 The optimization results of the molar ratio of choline chloride, aspartic acid and glutamic acid
实施例2多氢键供体低共熔溶剂与玉米秸秆的质量比优化Example 2 Optimization of the Mass Ratio of Multi-Hydrogen Bond Donor Deep Eutectic Solvent and Corn Stalk
取100g摩尔比为2:1:1的氯化胆碱:天冬氨酸:谷氨酸，分别按质量比7:1、10:1、15:1、20:1和30:1加入玉米秸秆，在130℃下搅拌处理2h，然后将混合物离心，用去离子水洗涤沉淀3次。在烘干的沉淀中加入pH 4.8、50mM的柠檬酸-柠檬酸钠缓冲液形成混合体系，玉米秸秆最终质量浓度为10％，加入的纤维素酶进行酶解，在50℃和150rpm的水浴摇床上酶解72h，测葡萄糖产率，结果如表2。Get 100g of choline chloride with a molar ratio of 2:1:1: aspartic acid: glutamic acid, add corn by mass ratio 7:1, 10:1, 15:1, 20:1 and 30:1 respectively The straw was stirred at 130° C. for 2 h, then the mixture was centrifuged, and the precipitate was washed 3 times with deionized water. Add pH 4.8, 50mM citric acid-sodium citrate buffer to the dried precipitate to form a mixed system, the final mass concentration of corn stalks is 10%, add The cellulase was used for enzymolysis, and the enzymolysis was carried out on a water bath shaker at 50°C and 150rpm for 72h, and the glucose yield was measured. The results are shown in Table 2.
表2 多氢键供体低共熔溶剂与玉米秸秆的质量比优化结果Table 2 Optimization results of the mass ratio of multi-hydrogen bond donor deep eutectic solvent to corn stover
实施例3回收的多氢键供体低共熔溶剂预处理玉米秸秆Example 3 Recovered multi-hydrogen bond donor deep eutectic solvent pretreatment of corn stalks
取100g摩尔比为2:1:1的氯化胆碱:天冬氨酸:谷氨酸，加入5g玉米秸秆，在130℃下搅拌处理2h，然后将混合物离心，取上清液，加入玉米秸秆(上清液与玉米秸秆质量比为20:1)，在130℃下搅拌处理2h，重复上述步骤。所有沉淀用去离子水洗涤3次。在烘干的沉淀中加入pH 4.8、50mM的柠檬酸-柠檬酸钠缓冲液形成混合体系，玉米秸秆最终质量浓度为10％，加入的纤维素酶进行酶解，在50℃和150rpm的水浴摇床上酶解72h。回收4次的氯化胆碱:天冬氨酸:谷氨酸预处理的玉米秸秆葡萄糖产率为65.4％，结果如表3。Take 100g of choline chloride:aspartic acid:glutamic acid with a molar ratio of 2:1:1, add 5g of corn stalks, stir at 130°C for 2h, then centrifuge the mixture, take the supernatant, and add corn stalks Straw (the mass ratio of supernatant to corn stalk is 20:1) was stirred at 130° C. for 2 hours, and the above steps were repeated. All precipitates were washed 3 times with deionized water. Add pH 4.8, 50mM citric acid-sodium citrate buffer to the dried precipitate to form a mixed system, the final mass concentration of corn stalks is 10%, add The cellulase was used for enzymolysis, and the enzymolysis was carried out at 50°C and 150rpm on a water bath shaker for 72h. The choline chloride recovered four times: aspartic acid: glutamic acid pretreated corn stalks had a glucose yield of 65.4%, and the results are shown in Table 3.
表3 回收的多氢键供体低共熔溶剂预处理玉米秸秆结果Table 3 Results of pretreatment of corn stover with recovered multi-hydrogen bond donors in deep eutectic solvent
0：新鲜氯化胆碱:天冬氨酸:谷氨酸预处理的玉米秸秆；1～4：循环利用1～4次的氯化胆碱:天冬氨酸:谷氨酸预处理的玉米秸秆。0: fresh choline chloride: aspartic acid: glutamic acid pretreated corn stalks; 1 to 4: choline chloride recycled 1 to 4 times: aspartic acid: glutamic acid pretreated corn straw.
实施例4利用预处理玉米秸秆水解液发酵生产丁醇。Example 4 Production of butanol by fermentation of pretreated corn stalk hydrolyzate.
取一定量氯化胆碱:天冬氨酸:谷氨酸预处理的玉米秸秆，加入pH 4.8、50mM的柠檬酸-柠檬酸钠缓冲液，并加入的纤维素酶，使最终固液比为1:10，在50℃和150rpm的水浴摇床上酶解72h，离心获得玉米秸秆水解液，并加入如下成分配成培养基：玉米浆干粉10g·L-1，CaCO3 4g·L-1，(NH4)2SO4 2g·L-1，K2HPO4 0.5g·L-1，MnSO4·H2O0.01g·L-1。调pH至6.5，115℃灭菌20min。接种活化的Clostridium saccharobutylicumDSM 13864(10％，v/v)，在37℃下厌氧培养。56h时，丁醇产量为8.6g·L-1，产率为时空产率为0.15g·L-1·h-1，结果如图1。Get a certain amount of choline chloride: aspartic acid: glutamic acid pretreated corn stalks, add pH 4.8, 50mM citric acid-sodium citrate buffer, and add The final solid-to-liquid ratio was 1:10, hydrolyzed on a water-bath shaker at 50°C and 150rpm for 72 hours, centrifuged to obtain corn stalk hydrolyzate, and added the following ingredients to form a culture medium: corn steep liquor dry powder 10g L-1 , CaCO3 4g·L-1 , (NH4 )2 SO4 2g·L-1 , K2 HPO4 0.5g·L-1 , MnSO4 ·H2 O 0.01g·L-1 . Adjust the pH to 6.5, and sterilize at 115°C for 20 minutes. Activated Clostridium saccharobutylicum DSM 13864 (10%, v/v) was inoculated and cultured anaerobically at 37°C. At 56h, the yield of butanol was 8.6g·L-1 , and the yield was The space-time yield is 0.15g·L-1 ·h-1 , the results are shown in Figure 1 .
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| WD01 | Invention patent application deemed withdrawn after publication | Application publication date:20161109 |