木质纤维素预处理和酶水解回收的过程调控与强化研究
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摘要
本论文以强化纤维素乙醇核心生产过程、降低过程工艺经济成本为目的，选择农林废弃资源玉米芯为原料，考察了预处理过程中原料的综合利用方式、研究了预处理及酶解过程的强化手段、开发了多种调控纤维素酶吸/脱附的方法，最终考察了影响纤维素酶定量的操作因素。
（1）碱性过氧化氢预处理：选取玉米芯作为木质纤维素原料，提出了常压中温下H_2O_2/NaOH同步预处理-组分回收工艺，该工艺实现了玉米芯三大组份的有效分离与回收，同时得到了较高的木质素脱除率（85.2%）和纤维素保留率（81.3%）。
（2）超声辅助氨浸预处理：选择玉米芯为原料，设计并优选了超声辅助氨浸预处理方案，并对工艺参数进行了优化。最佳工艺条件下处理的玉米芯样品，酶解率可提高34.2%，而还原糖释放速率与还原糖平衡浓度分别为未超声的氨浸玉米芯样品的1.3和1.4倍。
（3）超声调控酶吸附和水解：恒温超声反应器中，Spezyme CP酶在氨浸玉米芯上的吸/脱附表现出周期性的振荡，并且这种振荡是可逆可控的。通过改变底物表面性质和脱除木质素，超声能够极大地提高氨浸玉米芯底物的酶解效率。另外，应使用较低的能量输出和超声温度，以避免超声对纤维素酶的失活效应。
（4）pH调控酶吸附行为：纤维素酶在木质纤维素底物上的吸／脱附行为可以通过改变环境pH来调控。具体表现为酸性pH有利于吸附而中性和碱性pH则更有利于脱附。虽然调节pH也会带来纤维素酶的失活，但是在低温下，可以通过将pH调回4.8的方式使纤维素酶酶活可逆地恢复。
（5）酶回收策略：采用pH7溶液对氨浸玉米芯24h酶解残渣进行脱附，90min后脱附率可达91.9%。将上述指标带入重吸附过程游离酶回收率方程，预测得到引入pH调控的重吸附首轮即可回收70.9%的初始酶活。
（6）离心影响纤维素酶定量分析：离心对纤维素酶定量分析的影响由离心方式决定，当采用“取清液离心”的方式时，基本不会为后续蛋白定量带来影响；而当采用“混合态离心”的方式时，则会因为离心场的作用造成底物-酶体系中的游离酶下降，进而导致测得的吸附酶数据偏大、而脱附酶与底物的酶可及度数据偏小。
For the purpose to intensify the core processes and to reduce the economic costsin cellulosic ethanol bioconversion, this dissertation selects corncobs, a biomassresource from agricultural and forestry waste, as the raw materials to investigate thecomprehensive utilization of lignocellulose in the pretreatment process, to study theintensification approaches of the pretreatment and enzymatic hydrolysis process, todevelop a variety of methods controlling the adsorption/desorption of the cellulase,and finally discovered the operational factors that influence the cellulase quantitation.
Alkaline peroxide pretreatment: It established a synchronous pretreating andfraction-recycling process, using corncob feedsotcks and H_2O_2/NaOH solvents, andunder mild temperature. This novel process realized the effective separation andrecovery of three key components whilst attained high lignin removal rate (85.2%)and cellulose retention rate (81.3%).
Ultrasound-assisted soaking in aqueous ammonia: Selecting corncob as rawmaterials, it designed and screened the optimum strategy of ultrasound-assistedsoaking in aqueous ammonia, as well as optimized the process parameters. Theenzymatic hydrolysis rate of corncob samples under the optimum conditions couldincrease34.2%whilst the release rate and equilibrium concentration of reducing sugarwere1.3and1.4times of the non-ultrasound treated samples, respectively.
Cellulase adsorption and lignocellulosic hydrolysis controlled by ultrasound:In the isothermal ultrasonic reactor, Spezyme CP enzymes oscillatingly adsorbed onto/desorbed from the soaking in aqueous ammonia treated corncob (SAA-CC), whichwere reversible and controllable. Through improving the accessibility and removingthe lignin, ultrasound could greatly increase the enzymatic hydrolysis efficiency of thelignocellulosic substrates (SAA-CC). In addition, the lower energy outputs and themilder ultrasonic temperatures should be employed in lignocellulosic hydrolysis toavoid the inactivation of cellulase.
Controllable cellulase adsorption by pH adjustment: Cellulase adsorption anddesorption on the lignocellulosic substrate could be controlled by pH adjustment. Indetail, the acidic pHs were benefit to cellulase adsorption while the neutral and alkaline pHs were more conducive to cellulase desorption. Moreover, adjusting thepH could inactivate cellulase. But, after adjusting the pH back to4.8as long as at lowtemperatures, the cellulase activity could recover reversibly.
Design of the strategy of cellulase recycling: After90min incubation with pH7solution and SAA-CC residues, substrate and enzyme mixtures after24h hydrolysis,the91.9%cellulase could be desorbed from solid residues. To substitute this valueinto the recovery rate predictive equation, it is estimated that70.9%of the cellulaseactivity could be recovered in first round readsorption by the employment of pHadjustment.
Factors influence the cellulase quantitation: The influence of centrifugationon cellulase quantitation was depended on the centrifugal way. In detail, whenemploying the “aliquot centrifugation” way, the centrifugation did not influencecellulase concentration; but when following the “mixture centrifugation” way, the freeenzyme concentration would decrease. This negative effect could greatly influence thesubsequent quantitation process, such as increased the measured adsorption values,decreased the measured desorption values and accessibility values.
（1）碱性过氧化氢预处理：选取玉米芯作为木质纤维素原料，提出了常压中温下H_2O_2/NaOH同步预处理-组分回收工艺，该工艺实现了玉米芯三大组份的有效分离与回收，同时得到了较高的木质素脱除率（85.2%）和纤维素保留率（81.3%）。
（2）超声辅助氨浸预处理：选择玉米芯为原料，设计并优选了超声辅助氨浸预处理方案，并对工艺参数进行了优化。最佳工艺条件下处理的玉米芯样品，酶解率可提高34.2%，而还原糖释放速率与还原糖平衡浓度分别为未超声的氨浸玉米芯样品的1.3和1.4倍。
（3）超声调控酶吸附和水解：恒温超声反应器中，Spezyme CP酶在氨浸玉米芯上的吸/脱附表现出周期性的振荡，并且这种振荡是可逆可控的。通过改变底物表面性质和脱除木质素，超声能够极大地提高氨浸玉米芯底物的酶解效率。另外，应使用较低的能量输出和超声温度，以避免超声对纤维素酶的失活效应。
（4）pH调控酶吸附行为：纤维素酶在木质纤维素底物上的吸／脱附行为可以通过改变环境pH来调控。具体表现为酸性pH有利于吸附而中性和碱性pH则更有利于脱附。虽然调节pH也会带来纤维素酶的失活，但是在低温下，可以通过将pH调回4.8的方式使纤维素酶酶活可逆地恢复。
（5）酶回收策略：采用pH7溶液对氨浸玉米芯24h酶解残渣进行脱附，90min后脱附率可达91.9%。将上述指标带入重吸附过程游离酶回收率方程，预测得到引入pH调控的重吸附首轮即可回收70.9%的初始酶活。
（6）离心影响纤维素酶定量分析：离心对纤维素酶定量分析的影响由离心方式决定，当采用“取清液离心”的方式时，基本不会为后续蛋白定量带来影响；而当采用“混合态离心”的方式时，则会因为离心场的作用造成底物-酶体系中的游离酶下降，进而导致测得的吸附酶数据偏大、而脱附酶与底物的酶可及度数据偏小。
For the purpose to intensify the core processes and to reduce the economic costsin cellulosic ethanol bioconversion, this dissertation selects corncobs, a biomassresource from agricultural and forestry waste, as the raw materials to investigate thecomprehensive utilization of lignocellulose in the pretreatment process, to study theintensification approaches of the pretreatment and enzymatic hydrolysis process, todevelop a variety of methods controlling the adsorption/desorption of the cellulase,and finally discovered the operational factors that influence the cellulase quantitation.
Alkaline peroxide pretreatment: It established a synchronous pretreating andfraction-recycling process, using corncob feedsotcks and H_2O_2/NaOH solvents, andunder mild temperature. This novel process realized the effective separation andrecovery of three key components whilst attained high lignin removal rate (85.2%)and cellulose retention rate (81.3%).
Ultrasound-assisted soaking in aqueous ammonia: Selecting corncob as rawmaterials, it designed and screened the optimum strategy of ultrasound-assistedsoaking in aqueous ammonia, as well as optimized the process parameters. Theenzymatic hydrolysis rate of corncob samples under the optimum conditions couldincrease34.2%whilst the release rate and equilibrium concentration of reducing sugarwere1.3and1.4times of the non-ultrasound treated samples, respectively.
Cellulase adsorption and lignocellulosic hydrolysis controlled by ultrasound:In the isothermal ultrasonic reactor, Spezyme CP enzymes oscillatingly adsorbed onto/desorbed from the soaking in aqueous ammonia treated corncob (SAA-CC), whichwere reversible and controllable. Through improving the accessibility and removingthe lignin, ultrasound could greatly increase the enzymatic hydrolysis efficiency of thelignocellulosic substrates (SAA-CC). In addition, the lower energy outputs and themilder ultrasonic temperatures should be employed in lignocellulosic hydrolysis toavoid the inactivation of cellulase.
Controllable cellulase adsorption by pH adjustment: Cellulase adsorption anddesorption on the lignocellulosic substrate could be controlled by pH adjustment. Indetail, the acidic pHs were benefit to cellulase adsorption while the neutral and alkaline pHs were more conducive to cellulase desorption. Moreover, adjusting thepH could inactivate cellulase. But, after adjusting the pH back to4.8as long as at lowtemperatures, the cellulase activity could recover reversibly.
Design of the strategy of cellulase recycling: After90min incubation with pH7solution and SAA-CC residues, substrate and enzyme mixtures after24h hydrolysis,the91.9%cellulase could be desorbed from solid residues. To substitute this valueinto the recovery rate predictive equation, it is estimated that70.9%of the cellulaseactivity could be recovered in first round readsorption by the employment of pHadjustment.
Factors influence the cellulase quantitation: The influence of centrifugationon cellulase quantitation was depended on the centrifugal way. In detail, whenemploying the “aliquot centrifugation” way, the centrifugation did not influencecellulase concentration; but when following the “mixture centrifugation” way, the freeenzyme concentration would decrease. This negative effect could greatly influence thesubsequent quantitation process, such as increased the measured adsorption values,decreased the measured desorption values and accessibility values.
引文
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