木质纤维素底物对纤维素酶吸附脱附规律及预处理同步制备纳米纤维素的研究
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摘要
利用凝胶排阻法和荧光蛋白吸附法测定了纤维素酶对不同程度角质化处理和不同预处理方法得到的两组木质纤维素底物中纤维素的可及性，研究了纤维素可及性与酶解效率的关系。利用紫外光谱法测定了木质纤维素底物对纤维素内切酶的吸附。凝胶排阻法以及TGC荧光蛋白吸附法测定了纤维素酶对木质纤维素底物中纤维素的可及性发现两者有很好的线性相关性。不同程度角质化的底物的酶解效率与纤维素酶对纤维素的可及性也呈线性关系。90%的酶解效率是由木质纤维素底物中纤维素酶可及的孔洞吸附纤维素酶水解贡献的；木质纤维素底物表面积吸附的纤维素酶对纤维素可及性以及酶解效率的贡献有限。预处理底物吸附的纤维素酶的吸附量与底物中纤维素的可及性呈线性关系。
利用紫外光谱法适时定量地测定了纤维素内切酶在木质纤维素混合液中的吸附、脱附以及重吸附，定义了纤维素内切酶吸附和脱附的竞争因子、准结合和脱附常数、纤维素酶的吸附和脱附容量以及纤维素酶结合的可逆性和纤维素酶的可回收率。木质纤维素底物的化学成分和结构对纤维素的吸附、脱附有重要影响。漂白桉木浆对内切纤维素酶的吸附基本可逆；而纤维素酶在木质素含量高的底物上的吸附几乎不可逆。木素的存在对纤维素酶的吸附有极大的促进作用，底物中木素种类、结构的差异使得对纤维素酶的吸附量也不同。木素的存在不利于纤维素酶循环利用。角质化的底物中纤维细胞发生了不可逆的塌陷，纤维素酶对纤维素的可及性下降，增加了纤维素酶结合的可逆性，利于纤维素酶的循环利用。增加洗脱温度或者调高pH促进纤维素酶从底物上脱附，利于纤维素酶结合的可逆性和纤维素酶的可回收率。研究结果为纤维素酶的循环利用奠定了一定的理论基础。
利用漂白针叶木浆，同步制取了可发酵糖和纳米纤丝纤维素。生物质制备生物燃料的瓶颈在于酶解过程中木质纤维素底物的顽抗性，彻底将底物中纤维素转化为可溶性糖很困难。木质纤维素底物中的“快糖”水解完，即终止酶解反应。结晶度高的纤维素残渣留在酶解液中。用高压微射流均质机处理分离的纤维残渣制备纳米纤丝纤维素，实现了同步生产生物燃料和纳米纤维素。实验优化了纤维素酶/内切纤维素酶的用量，探讨了酶解条件与纤维残渣得率及制备的纳米纤丝纤维素性能的关系，包括纳米纤丝纤维素的结晶度、聚合度、不透明度和机械强度。
强酸水解漂白桉木浆同步制备了纳米微晶纤维素和纳米纤丝纤维素。优化的酸水解条件下，原料的损失很少，纳米纤维素的总得率很高。文献中优化的酸水解制备纳米纤维素的条件较为剧烈，大量的纤维素被水解成葡萄糖，难以回收。本文采用较为缓和的水解条件，避免了纤维素过度降解，制备了纳米微晶纤维素，而纤维残渣经过简单的分离，便可回收用于纳米纤丝纤维素的制备。纳米微晶纤维素与纤维残渣共存范围很窄，因此同步制备纳米微晶纤维素和纳米纤丝纤维素对水解条件要求苛刻。缓和条件下制备的纳米微晶纤维素的得率与和文献中优化结果相当，同时额外得到大量的纤维残渣。纤维残渣上引入了大量的磺酸基团，通过简单机械的处理，在能耗很小的情况下，便可制备成具有很好光学性能和机械强度的纳米纤丝纤维素。在优化条件下，纳米微晶纤维素和纳米纤丝纤维素的总得率为84%
以商品漂白浆为原料，利用超微粒粉碎机，分离制备了纳米纤丝纤维素。采用扫描电镜、透射电镜等手段详细检测纳米纤维素的微米、纳米尺寸及形貌变化。超微粒粉碎机碾磨浆料产生了两种主要结构：(1)高度弯曲、自然螺旋，无扭结的骨架结构；(2)相互交织、扭曲，结构复杂的层状结构。这两种结构形成了如树状、网状、花状结构以及单根纳米纤丝纤维素等其它具体结构。经过长时间的碾磨，浆料中发现棒状结构的纳米微晶纤维素。根据其形态推测纳米微晶纤维素源于未扭结自然状态的骨架纳米纤维素。超微粒粉碎机制备纳米纤丝纤维素的能耗约为5-30kWh/kg。离心分离证实，随着碾磨能耗的增加，小尺寸的纳米纤维素的比例增加。然而，即便在碾磨后期，仍然能观察到较大尺寸的纤维结构。
纳米纤维素来源广泛，强度高，具有很好的可降解性，作为潜在的结构材料，具有广泛用途。纳米纤维素材料的性能受纳米纤维素本身的性质影响。采用扫描电镜、透射电镜以及原子力显微镜等现代测试技术深入地研究了各种方法制备的纳米微晶纤维素和纳米纤丝纤维素。纳米微晶纤维素为棒状纳米晶须结构；纳米纤丝纤维素则一般为网状结构。采用了多种便携式的商品粒度仪快速表征了纳米纤维素。依据离心分离以及布朗运动制备的两种仪器快速表征纳米纤维素得到较好的结果，实验重复性也很好。这两种仪器非常适合半定量快速表征非网状结构的纳米微晶纤维素。
Cellulose accessibilities of a set of hornified lignocellulosic substrates derived by dryingthe never dried pretreated sample and a set of differently pretreated lodgepople pine substrateswere evaluated using solute exclusion and protein adsorption methods. Direct measurementsof cellulase adsorption onto cellulose surface of the set of pretreated substrates were alsocarried out using an in-situ UV-Vis spectrophotometric technique. The celluloseaccessibilties measured by the solute exclusion and a CBM-containing green fluorescentprotein (TGC) adsorption methods correlate well for both sets of samples. The substrateenzymatic digestibilities of the hornified substrates are proportional to the measured celluloseaccessibilities. Approximately over90%of substrate enzymatic digestibility was contributedby the accessible pore surfaces of the hornified substrates, suggesting that the substrateexternal surface plays a minor role contributing to cellulose accessibility and substrateenzymatic digestibilities. The cellulose accessibilities of the pretreated substrates arecorrelated well with the amounts of cellulase adsorbed. The substrate enzymaticdigestibilities directly are correlated with the amounts of adsorbed cellulase.
Quantitative kinetic modeling and in-situ and temporally resolved measurements ofadsorption, desorption, and re-adsorption of a commercial endoglucanase in lignocellulosicsuspensions was conducted. The study defined a cellulase adsorption and desorptioncompetition parameter, a pseudo rate of binding and desorption, binding and desorptioncapacity, as well as cellulase binding reversibility and recyclability. The results indicate thatboth substrate chemical and physical structures play important roles in cellulase binding anddesorption. Binding of a commercial cellulase onto a cellulosic substrate was reversible.Bindings to two different lignocellulosic substrates were almost irreversible. While lignin andits structure positively affect binding capacity to substrate, they negatively affect cellulaserecyclability. Collapsing of substrate pores reduce cellulose accessibility and cellulase bindingcapacity and increase reversibility and recyclability. Increasing temperature and pH increasecellulase desorption and increase binding reversibility and capacity. This study lays thefoundation for developing effective cellulase recycling strategies.
Integrating preparation of biofuel and cellulose nanofibrils as a co-product was carriedout using softwood pulp. One key barrier to converting woody biomass to biofuel through thesugar platform is the enzymatic cellulose saccharification because of the strong recalcitranceof the crystalline cellulose. Cellulase cocktails were optimized for integrated preparation ofsugar and cellulose nanofibrils. Enzymatic hydrolysis of substrates was quenched as long as all fast sugar was degraded. Cellulosic substrate left with high crystallinity was separatedfrom hydrolysate and utilized as staring material for cellulose nanofibrils production. Highpress microfluidizer was used to liberate cellulose nanofibrils from cellulosic residual. Therelationships between enzymatic hydrolysis conditions, cellulosic residual yield, andproperties of cellulose nanofibrils, including degree of polymerization, crystallinity andmechanical performance were investigated.
The potential of simultaneously recovering cellulosic solid residues and producingcellulose nanocrystals by strong sulfuric acid hydrolysis to achieve near zero cellulose losswas demonstrated in the3rd chapter. A set of slightly milder acid hydrolysis conditions thanthat considered as “optimal” were used to significantly minimize the degradation of celluloseinto soluble sugars that cannot be economically recovered, but resulted in cellulosic solidresidues that is easily recoverable through conventional centrifuge. It was found that thewindow for simultaneous recoveries of cellulosic solid residues and cellulose nanocrystals instrong acid hydrolysis was extremely narrow. The cellulose nanocrystals yield may not bereduced compared with that obtained under the “optimal condition”. The maximal totalcellulosic solid yield can be improved to approximately84%using a bleach eucalyptus pulpwith cellulose content of90%. The resultant cellulosic solid residues contain sulfonate groupsthat facilitate subsequent mechanical nano-fibrillation to nanowhiskers, a potential high valuenanocellulosic material for a variety of applications. The resultant cellulose nanofibrils filmsexhibit excellent optical and mechanical properties.
Cellulose nanofibrils from a bleached eucalyptus pulp were isolated using a commercialstone grinder. SEM and TEM images were used to reveal morphological development ofcellulose nanofibrils at micro and nano scales, respectively. Two major structures wereidentified:(1) highly kinked, naturally helical, and untwisted fibrils that serve as backbones ofcellulose nanofibrils networks,(2) entangled, less distinctively kinked (or curled) and twistednanofibril “meniscus” structure. These two major structures forme different types of cellulosenanofibrils structures such as “trees”,“net”,“flower”, single fibril, etc. Prolonged fibrillationcan break the nanofibrils into nanowhiskers from the untwisted fibrils with high crystallinity.Energy input for mechanical fibrillation is on the order of20-30kWh/kg. The graduatereduction in network size of cellulose nanofibrils with time is only in a statistical sense whichmay be used to fractionate cellulose nanofibrils.
The potential utility of wood nanocellulose as a building block of a variety of highlyfunctional and high value materials and products warrants a complete understanding of itsmorphological properties. The last chapter is intended to provide a relatively complete morphological picture of several kinds of wood nanocellulose including cellulose nanocrystalsand cellulose nanofibrils produced using most commonly used processes. TEM imaging aswell as AFM analysis were applied to provide visual examinations of several nanocellulosesamples. It was found that cellulose nanocrystals are mainly rodlike nano-whiskers asexpected and cellulose nanofibrils are fibril network particles. Commercial particle sizinginstruments were evaluated for quick particle sizing of these samples. The results suggest thata centrifuge and a Brownian motion based instruments show good repeatability in particlesizing. Both instruments are well suited for semi-quantitative characterizing non networkparticles even with large aspect ratios such as cellulose nanocrystals, but require further studybefore using for characterizing network particles such as cellulose nanofibrils.
利用紫外光谱法适时定量地测定了纤维素内切酶在木质纤维素混合液中的吸附、脱附以及重吸附，定义了纤维素内切酶吸附和脱附的竞争因子、准结合和脱附常数、纤维素酶的吸附和脱附容量以及纤维素酶结合的可逆性和纤维素酶的可回收率。木质纤维素底物的化学成分和结构对纤维素的吸附、脱附有重要影响。漂白桉木浆对内切纤维素酶的吸附基本可逆；而纤维素酶在木质素含量高的底物上的吸附几乎不可逆。木素的存在对纤维素酶的吸附有极大的促进作用，底物中木素种类、结构的差异使得对纤维素酶的吸附量也不同。木素的存在不利于纤维素酶循环利用。角质化的底物中纤维细胞发生了不可逆的塌陷，纤维素酶对纤维素的可及性下降，增加了纤维素酶结合的可逆性，利于纤维素酶的循环利用。增加洗脱温度或者调高pH促进纤维素酶从底物上脱附，利于纤维素酶结合的可逆性和纤维素酶的可回收率。研究结果为纤维素酶的循环利用奠定了一定的理论基础。
利用漂白针叶木浆，同步制取了可发酵糖和纳米纤丝纤维素。生物质制备生物燃料的瓶颈在于酶解过程中木质纤维素底物的顽抗性，彻底将底物中纤维素转化为可溶性糖很困难。木质纤维素底物中的“快糖”水解完，即终止酶解反应。结晶度高的纤维素残渣留在酶解液中。用高压微射流均质机处理分离的纤维残渣制备纳米纤丝纤维素，实现了同步生产生物燃料和纳米纤维素。实验优化了纤维素酶/内切纤维素酶的用量，探讨了酶解条件与纤维残渣得率及制备的纳米纤丝纤维素性能的关系，包括纳米纤丝纤维素的结晶度、聚合度、不透明度和机械强度。
强酸水解漂白桉木浆同步制备了纳米微晶纤维素和纳米纤丝纤维素。优化的酸水解条件下，原料的损失很少，纳米纤维素的总得率很高。文献中优化的酸水解制备纳米纤维素的条件较为剧烈，大量的纤维素被水解成葡萄糖，难以回收。本文采用较为缓和的水解条件，避免了纤维素过度降解，制备了纳米微晶纤维素，而纤维残渣经过简单的分离，便可回收用于纳米纤丝纤维素的制备。纳米微晶纤维素与纤维残渣共存范围很窄，因此同步制备纳米微晶纤维素和纳米纤丝纤维素对水解条件要求苛刻。缓和条件下制备的纳米微晶纤维素的得率与和文献中优化结果相当，同时额外得到大量的纤维残渣。纤维残渣上引入了大量的磺酸基团，通过简单机械的处理，在能耗很小的情况下，便可制备成具有很好光学性能和机械强度的纳米纤丝纤维素。在优化条件下，纳米微晶纤维素和纳米纤丝纤维素的总得率为84%
以商品漂白浆为原料，利用超微粒粉碎机，分离制备了纳米纤丝纤维素。采用扫描电镜、透射电镜等手段详细检测纳米纤维素的微米、纳米尺寸及形貌变化。超微粒粉碎机碾磨浆料产生了两种主要结构：(1)高度弯曲、自然螺旋，无扭结的骨架结构；(2)相互交织、扭曲，结构复杂的层状结构。这两种结构形成了如树状、网状、花状结构以及单根纳米纤丝纤维素等其它具体结构。经过长时间的碾磨，浆料中发现棒状结构的纳米微晶纤维素。根据其形态推测纳米微晶纤维素源于未扭结自然状态的骨架纳米纤维素。超微粒粉碎机制备纳米纤丝纤维素的能耗约为5-30kWh/kg。离心分离证实，随着碾磨能耗的增加，小尺寸的纳米纤维素的比例增加。然而，即便在碾磨后期，仍然能观察到较大尺寸的纤维结构。
纳米纤维素来源广泛，强度高，具有很好的可降解性，作为潜在的结构材料，具有广泛用途。纳米纤维素材料的性能受纳米纤维素本身的性质影响。采用扫描电镜、透射电镜以及原子力显微镜等现代测试技术深入地研究了各种方法制备的纳米微晶纤维素和纳米纤丝纤维素。纳米微晶纤维素为棒状纳米晶须结构；纳米纤丝纤维素则一般为网状结构。采用了多种便携式的商品粒度仪快速表征了纳米纤维素。依据离心分离以及布朗运动制备的两种仪器快速表征纳米纤维素得到较好的结果，实验重复性也很好。这两种仪器非常适合半定量快速表征非网状结构的纳米微晶纤维素。
Cellulose accessibilities of a set of hornified lignocellulosic substrates derived by dryingthe never dried pretreated sample and a set of differently pretreated lodgepople pine substrateswere evaluated using solute exclusion and protein adsorption methods. Direct measurementsof cellulase adsorption onto cellulose surface of the set of pretreated substrates were alsocarried out using an in-situ UV-Vis spectrophotometric technique. The celluloseaccessibilties measured by the solute exclusion and a CBM-containing green fluorescentprotein (TGC) adsorption methods correlate well for both sets of samples. The substrateenzymatic digestibilities of the hornified substrates are proportional to the measured celluloseaccessibilities. Approximately over90%of substrate enzymatic digestibility was contributedby the accessible pore surfaces of the hornified substrates, suggesting that the substrateexternal surface plays a minor role contributing to cellulose accessibility and substrateenzymatic digestibilities. The cellulose accessibilities of the pretreated substrates arecorrelated well with the amounts of cellulase adsorbed. The substrate enzymaticdigestibilities directly are correlated with the amounts of adsorbed cellulase.
Quantitative kinetic modeling and in-situ and temporally resolved measurements ofadsorption, desorption, and re-adsorption of a commercial endoglucanase in lignocellulosicsuspensions was conducted. The study defined a cellulase adsorption and desorptioncompetition parameter, a pseudo rate of binding and desorption, binding and desorptioncapacity, as well as cellulase binding reversibility and recyclability. The results indicate thatboth substrate chemical and physical structures play important roles in cellulase binding anddesorption. Binding of a commercial cellulase onto a cellulosic substrate was reversible.Bindings to two different lignocellulosic substrates were almost irreversible. While lignin andits structure positively affect binding capacity to substrate, they negatively affect cellulaserecyclability. Collapsing of substrate pores reduce cellulose accessibility and cellulase bindingcapacity and increase reversibility and recyclability. Increasing temperature and pH increasecellulase desorption and increase binding reversibility and capacity. This study lays thefoundation for developing effective cellulase recycling strategies.
Integrating preparation of biofuel and cellulose nanofibrils as a co-product was carriedout using softwood pulp. One key barrier to converting woody biomass to biofuel through thesugar platform is the enzymatic cellulose saccharification because of the strong recalcitranceof the crystalline cellulose. Cellulase cocktails were optimized for integrated preparation ofsugar and cellulose nanofibrils. Enzymatic hydrolysis of substrates was quenched as long as all fast sugar was degraded. Cellulosic substrate left with high crystallinity was separatedfrom hydrolysate and utilized as staring material for cellulose nanofibrils production. Highpress microfluidizer was used to liberate cellulose nanofibrils from cellulosic residual. Therelationships between enzymatic hydrolysis conditions, cellulosic residual yield, andproperties of cellulose nanofibrils, including degree of polymerization, crystallinity andmechanical performance were investigated.
The potential of simultaneously recovering cellulosic solid residues and producingcellulose nanocrystals by strong sulfuric acid hydrolysis to achieve near zero cellulose losswas demonstrated in the3rd chapter. A set of slightly milder acid hydrolysis conditions thanthat considered as “optimal” were used to significantly minimize the degradation of celluloseinto soluble sugars that cannot be economically recovered, but resulted in cellulosic solidresidues that is easily recoverable through conventional centrifuge. It was found that thewindow for simultaneous recoveries of cellulosic solid residues and cellulose nanocrystals instrong acid hydrolysis was extremely narrow. The cellulose nanocrystals yield may not bereduced compared with that obtained under the “optimal condition”. The maximal totalcellulosic solid yield can be improved to approximately84%using a bleach eucalyptus pulpwith cellulose content of90%. The resultant cellulosic solid residues contain sulfonate groupsthat facilitate subsequent mechanical nano-fibrillation to nanowhiskers, a potential high valuenanocellulosic material for a variety of applications. The resultant cellulose nanofibrils filmsexhibit excellent optical and mechanical properties.
Cellulose nanofibrils from a bleached eucalyptus pulp were isolated using a commercialstone grinder. SEM and TEM images were used to reveal morphological development ofcellulose nanofibrils at micro and nano scales, respectively. Two major structures wereidentified:(1) highly kinked, naturally helical, and untwisted fibrils that serve as backbones ofcellulose nanofibrils networks,(2) entangled, less distinctively kinked (or curled) and twistednanofibril “meniscus” structure. These two major structures forme different types of cellulosenanofibrils structures such as “trees”,“net”,“flower”, single fibril, etc. Prolonged fibrillationcan break the nanofibrils into nanowhiskers from the untwisted fibrils with high crystallinity.Energy input for mechanical fibrillation is on the order of20-30kWh/kg. The graduatereduction in network size of cellulose nanofibrils with time is only in a statistical sense whichmay be used to fractionate cellulose nanofibrils.
The potential utility of wood nanocellulose as a building block of a variety of highlyfunctional and high value materials and products warrants a complete understanding of itsmorphological properties. The last chapter is intended to provide a relatively complete morphological picture of several kinds of wood nanocellulose including cellulose nanocrystalsand cellulose nanofibrils produced using most commonly used processes. TEM imaging aswell as AFM analysis were applied to provide visual examinations of several nanocellulosesamples. It was found that cellulose nanocrystals are mainly rodlike nano-whiskers asexpected and cellulose nanofibrils are fibril network particles. Commercial particle sizinginstruments were evaluated for quick particle sizing of these samples. The results suggest thata centrifuge and a Brownian motion based instruments show good repeatability in particlesizing. Both instruments are well suited for semi-quantitative characterizing non networkparticles even with large aspect ratios such as cellulose nanocrystals, but require further studybefore using for characterizing network particles such as cellulose nanofibrils.
引文
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