【作者】 陈海燕；

【导师】 秦玉琪； 赵建；

【作者基本信息】 山东大学 ， 生物化学与分子生物学， 2022， 博士

【摘要】 木质纤维素生物转化主要包括预处理、酶解糖化和发酵三个步骤。在众多预处理方法中,由于乙醇-水(EtOH)预处理具有可以有效脱除木质素、保护纤维素,乙醇毒性低且可以蒸馏回收再利用等优点,被越来越多的学者所关注。但该预处理方法需要高温环境才能有效脱除木质素和改善纤维原料的酶解糖化效率,并且在预处理过程中半纤维素损失严重,影响了可发酵糖的总产量。另外,由于传质、木质素对酶的吸附、产物抑制等因素,导致酶解过程中的酶用量高和糖化效率低。生物转化过程的高成本,特别是预处理和酶解糖化过程的高成本是当前阻碍木质纤维素生物转化工业化的主要障碍。基于以上背景,本论文以我国储量丰富的玉米秸秆为原料,针对EtOH预处理过程中目前存在的上述不足,研究并提出了KMnO4辅助的EtOH预处理新工艺;进而通过优化酶系组成、筛选优良表面活性剂将其添加到酶解体系等方式,在减少酶用量的同时提高了酶解糖化产可发酵糖得率;并利用酶解残渣成功制备出了木质纤维素纳米纤丝(Lignocellulosicnanofibrils,LCNFs)。研究可为进一步促进EtOH预处理工艺的应用和纤维糖液的生产,实现木质纤维素的高效生物转化和综合利用提供理论参考。论文主要研究内容和取得的结果如下:1.KMnO4辅助的EtOH预处理对玉米秸秆酶解性能的影响及机制研究为了改善EtOH预处理对玉米秸秆的作用效果,引入了一步简单的KMnO4预处理过程。研究显示,与单独EtOH预处理相比,KMnO4辅助的EtOH预处理(KMnO4+EtOH)能够有效减少半纤维素的降解,增加预处理后碳水化合物,特别是半纤维素的回收率,减少抑制剂的形成。在较低的预处理温度下就可以达到有效改善玉米秸秆酶解性能的目的,酶解后可发酵糖得率显著增加。对KMnO4辅助的EtOH预处理工艺及条件进行了优化。使用1%(w/v)KMnO4在40℃下预处理玉米秸秆60 min,然后再采用50%乙醇浓度在185℃预处理90min后,玉米秸秆中纤维素和半纤维素的回收率分别达到94.28%和58.45%。利用草酸青霉生产的商业纤维素酶(Commercial Enzyme,CE)对预处理后原料进行水解,酶解72 h时纤维素和半纤维素的转化率分别达到89.45%和85.30%。进一步采用多种分析方法,探讨了KMnO4辅助的EtOH预处理破除原料“抗降解屏障”的机制。研究发现,相对仅用EtOH预处理,经KMnO4辅助的EtOH预处理后,玉米秸秆表面暴露出更多的碳水化合物,原料表面含有更少的木质素和抽出物,因此增加了酶对底物的可及性,有利于酶对底物的作用。此外,由于KMnO4辅助的EtOH预处理后玉米秸秆中含有更多的半纤维素,预处理后原料的热稳定性和结晶度相对EtOH预处理后原料要低。KMnO4辅助的EtOH预处理工艺,可以在保留EtOH预处理工艺优势的同时,减少预处理过程能耗和半纤维素损失,提高酶解后可发酵糖的总产量。2.表面活性剂对预处理后玉米秸秆酶解糖化效率的影响及机理研究已知,添加表面活性剂可以促进高固形物含量下的酶解糖化过程,提高酶解效率,但目前仍需要进一步寻找廉价且更高效的表面活性剂,以降低酶解过程成本。论文探讨了非离子表面活性剂AEO-23和LAE-24、阴离子表面活性剂SSL、阳离子表面活性剂CTAB等,对KMnO4辅助的EtOH预处理玉米秸秆(KEPCS)、稀硫酸预处理玉米秸秆(HPCS)和NaOH预处理玉米秸秆(NPCS)等三种原料纤维素酶解的促进效果,并与文献中常用的非离子表面活性剂Tween-80进行比较。结果显示,表面活性剂对底物酶解糖化的改善效果,不仅与表面活性剂的类型、纤维原料特性等因素有关,还与表面活性剂添加量、底物浓度、酶解工艺等有关。其中,非离子表面活性剂AEO-23对上述三种方法预处理后的玉米秸秆均表现出良好的促进酶解作用,且促进效果优于Tween-80,显示出良好的应用潜力。进一步对AEO-23促进纤维素酶解过程的可能机制进行了分析。发现添加AEO-23可以减少酶在木质素上的非生产性吸附,提高酶解反应体系中游离的酶蛋白浓度和不同酶活力,特别是游离的内切纤维素酶和外切纤维素酶的活性,降低结晶度,且效果优于Tween-80。从AEO-23和木质素的官能团结构及电子效应的角度分析推测,在反应体系中添加AEO-23,AEO-23上的亲水性聚氧乙烯基(-(CH2CH2O)23-)中带负电的氧原子可能与木质素结构单元上的甲氧基、苯丙烷基和羟基上的氢通过多种氢键结合方式联结,增加木质素的亲水性、同时结合在木质素上的AEO-23大分子形成的空间位阻效应会阻碍酶与木质素的结合,减少木质素对酶的无效吸附。以上结果为表面活性剂在酶解糖化过程的工业化应用提供了更多可能性。3.KMnO4辅助的EtOH预处理玉米秸秆酶解工艺的初步优化研究比较了 3种里氏木霉来源和4种草酸青霉来源的不同菌株发酵产生的酶系与商业纤维素酶对KEPCS.的酶解糖化效果。对比发现,里氏木霉SCB18酶系降解KEPCS时纤维素和半纤维素的转化率最高。进一步研究发现里氏木霉QXC和草酸青霉CXy13-4产酶系对SCB18降解KEPCS具有良好的促进作用,在等滤纸酶活添加量条件下(12 FPU/g底物),当SCB18、QXC和CXyl3-4的酶系按照FPU比例8:2:2(总12 PFU)进行混合时,对KEPCS的酶解糖化效果最优。进一步通过添加内切纤维素酶和木聚糖酶,酶解效率进一步提高。在高固形物浓度下水解时采用分批补料方式有助于提高糖化效率,增加水解液中的总糖量。最终,预处理玉米秸秆在13 FPU/g底物的总酶量(来自菌株SCB18、QXC、CXy13-4、cEES-XM的酶系按比例8:2:2:1进行混合),20%固形物浓度下经过酶水解,获得了葡萄糖浓度84.47g/L和木糖浓度23.50 g/L,总可发酵糖浓度达107.97g/L的酶解糖液。4.酶解后固体残渣制备木质纤维素纳米纤丝(LCNFs)的研究从生物炼制的理念出发,以增加整个生物转化过程的总收益为目的,成功地利用KEPCS酶解糖化后的固体残渣制备出了形貌、直径和热稳定性等特征不同的多种木质纤维素纳米纤丝(LCNFs)产品。研究发现,直接使用均质处理制备的LCNFs的直径分布范围宽,不均一现象较明显,且纳米纤丝间存在较严重的絮聚、叠加和交织等现象。对于木质素含量较高的残渣,增加均质强度可以降低LCNFs的平均直径、有利于改善LCNFs的分散性,且直径分布变得更加均一。但对纤维素含量较高的残渣,增加均质强度虽可以降低LCNFs的平均直径,但对分散性和直径分布的改善效果不明显。在均质处理前对酶解残渣进行一定的超声预处理,在实验条件下并没有对LCNFs的形貌和直径分布产生明显影响。但在均质前采用酶预处理影响了 LCNFs的形貌和直径分布。合适的酶预处理可使均质后获得的LCNFs的平均直径更细、直径分布更均一,且纳米纤丝的分散性能好。在纤维素酶中添加合适量的木聚糖酶,会促进纤维素酶的预处理效果,制备出直径更细、分布更均一和热稳定性更好的LCNFs,且可以改善LCNFs的分散性能,减少LCNFs的交织和絮聚现象。更多还原

【Abstract】 There are three main steps for lignocellulose bioconversion,that is,pretreatment,enzymatic saccharification and fermentation.Among them,the high costs of pretreatment and saccharification process are the key factors hindering the industrialization of lignocellulose bioconversion.Due to the high delignification efficiency,high cellulose recovery,lack of toxicity,and ease of recovery,ethanol was frequently used as solvent for lignocellulose pretreatment.However,the pretreatment with ethanol was generally conducted under high reaction temperature for effective dissolving out lignin and achieving high saccharification efficiency,resulted in serious degradation of hemicellulose during the pretreatment,which affected the total yields of fermentable sugars and economic benefit.In addition,high enzyme dosage and low saccharification efficiency led to the high cost in subsequent enzymatic saccharification process.Based on the above background,paper proposed a new pretreatment process,KMnO4 assisted EtOH pretreatment(KMnO4+EtOH),to overcome the disadvantages of EtOH pretreatment process.At the same time,the enzyme system components used in enzymatic hydrolysis were optimized and effect of surfactants on enzymatic hydrolysis was studied to enhance enzymatic hydrolysis.Using solid residue from enzymatic saccharification as raw material,lignocellulosic nanofibrils(LCNFs)were also prepared.This study provides theoretical reference for further promoting the application of EtOH pretreatment process and production of high concentration sugar,thus help to comprehensive utilization of lignocellulose biomass.The main research contents and results of this paper were as follows:1.Effect of KMnO4 assisted EtOH pretreatment on enzymatic hydrolysis of corn stover and mechanism of improving enzymatic digestibilityA KMnO4 assisted EtOH pretreatment process was proposed to improving enzymatic digestibility of corn stover.In the process,a simple KMnO4 pretreatment was used before EtOH pretreatment.The results showed that the KMnO4 assisted EtOH pretreatment effectively reduced the degradation of hemicellulose and increased the recovery of carbohydrates,at the same time,decreased the formation of inhibitors.The enzymatic digestibility of corn stover was also effectively improved by the pretreatment under lower temperature compared to the single EtOH pretreatment.Process conditions of the KMnO4 assisted EtOH pretreatment were optimized and found that,when corn stover was firstly treated by 1%(w/v)KMnO4 at 40℃ for 60 min,then treated by 50%EtOH at 185℃ for 90 min,the recoverys of cellulose and hemicellulose reach to 94.28%and 58.45%respectively.Using commercial cellulase produced by Penicillium oxalicum to hydrolyze the pretreated corn stover,the conversion of cellulose and hemicellulose at 72 h of enzymatic hydrolysis reached 89.45%and 85.30%respectively.Further,mechanism of improvement of enzymatic hydrolysis of corn stover after the KMnO4 assisted EtOH pretreatment was investigated.It was found that,after the KMnO4 assisted EtOH pretreatment,the cell wall surface contained more carbohydrates,less lignin and extract,which increased the accessibility of enzyme to substrate,thus improving the efficiency of enzymatic saccharification.In general,compared to single EtOH pretreatment,the KMnO4 assisted EtOH pretreatment process can reduce energy consumption and hemicellulose loss while improve the total output of fermentable sugar.2.Effect and mechanism of surfactants on enzymatic saccharification of pretreated corn stoverIt is known that the addition of surfactant can improve the efficiency of saccharification during enzymatic hydrolysis of lignocellulose.However,it is necessary to search for cheap and efficient surfactants for reducing the cost of saccharification process.In this study,effects of several surfactants including nonionic surfactants AEO-23 and LAE-24,anionic surfactants SSL and cationic surfactants CTAB on the saccharification process of KMnO4 assisted EtOH pretreated corn stover(KEPCS),diluted H2SO4 pretreated corn stover(HPCS)and NaOH pretreated corn stover(NPCS)were studied.The results showed that the effect of adding surfactants on the improvement of saccharification efficiency was not only related to the type of surfactant and the properties of substrate,but also related to the dosage of surfactant,the concentration of substrate and saccharification process.Among them,nonionic surfactant AEO-23 showed better promoting effect on the saccharification of the three pretreated corn stover than tween-80,showing a good application potential.The possible mechanism of surfactant AEO-23 promoting enzymatic saccharification was further analyzed.It was found that addition of AEO-23 during enzymatic hydrolysis could reduce the unproductive adsorption of enzymes onto lignin,increase free enzyme concentration and activities of free endoglucanase and free cellobiohydrolase in hydrolysis system.From the perspective of the functional group structure and electronic effect of AEO-23 on lignin,it is speculated that negatively charged oxygen atoms in the hydrophilic polyoxyethylene((CH2CH2O)23-)of AEO-23 may linked to the methoxyl,phenylpropanyl and hydroxyl groups of lignin through various hydrogen bond bonding modes,thus increasing the hydrophilicity of lignin.The steric hindrance formed by the AEO macromolecule bound to lignin could also hinder the binding of enzymes to lignin and reduce the nonproductive adsorption of enzymes onto lignin.3.Optimization of saccharification process of pretreated corn stover by KMnO4 assisted EtOH processThe saccharification efficiency of pretreated corn stover using commercial cellulase,cellulases from three Trichoderma reesei strains and four Penicillium oxalicum strains were compared.It was found that the conversions of cellulose and hemicellulose were highest when the cellulase from Trichoderma reesei SCB18 was used to hydrolyze the pretreated corn stover compared to above other cellulases.It was also found that the cellulases from T.reesei QXC and P.oxalicum CXy13-4 could enhance hydrolysis efficiency of the cellulase from Trichoderma reesei SCB18,and the crude enzyme solution from strain SCB18,QXC and CXy13-4 respectively was mixed in the ratio of 8:2:2(based on PFU,and total dosage was 12 FPU/g-substrate)and used to hydrolyze pretreated corn stover,high sugar yield was obtained.By further adding endoglucanase from strain cEES-XM and xylanase,the saccharification efficiency of pretreated corn stover was further improved.Using fed batch saccharification process,the saccharification efficiency of substrate at high solid loading was improved,and the total fermentable sugars yield increased.The total fermentable sugar of 107.97 g/L,in which,the glucose concentration and xylose concentration was 84.47 g/L and 23.50 g/L respectively,was obtained in enzymatic saccharification stage under the conditions of 13 FPU/g-substrate of total enzyme dosage and 20%solid loading,4.Preparation of Lignocellulosic nanofibers(LCNFs)from solid residue from saccharification stepUsing the solid residues from enzymatic saccharification of KEPCS as raw materials,lignocellulosic nanofibers(LCNFs)with different morphology,diameter and thermal stability were successfully prepared.It was found that the LCNFs prepared by homogenization treatment had a wide diameter distribution and obvious heterogeneity,and there were phenomena such as serious flocculation,superposition and interweaving among nanofibers.For the solid residues with high lignin content,increasing homogenization strength can reduce the average diameter of LCNFs,improve the dispersion of LCNFs,and the diameter distribution becomes more uniform.For the solid residues with high cellulose content,however,increasing homogenization strength reduced the average diameter of LCNFs,but the improvement effect on dispersion and diameter distribution was not obvious.Ultrasonic pretreatment prior to homogenization treatment had little influence on the morphology and diameter distribution of LCNFs under experiment conditions used in the paper.However,enzymatic pretreatment before homogenization affected the morphology and diameter distribution of LCNFs,and the LCNFs with more small average diameter,more uniform diameter distribution and good dispersion performance was obtained by suitable enzymatic treatment.Addition of appropriate amount of xylanase during enzymatic pretreatment with cellulase enhanced enzymatic pretreatment effect,and improved diameter distribution uniformity,thermal stability and dispersion performance of LCNFs,and reduce the interweaving and flocculation phenomenon among LCNFs.更多还原