木质纤维素高固酶解过程扩散-吸附-反应的分析与调控

For enzymatic conversion of lignocellulosic biomass, conducting at high solids (substrate) concentrations is advantageous, since it increases product concentrations and plant productivity while lowering energy and water input. However, it has been shown that at increasing substrate concentration, the corresponding conversion efficiency of cellulose linearly decreases for a number of lignocellulosic substrates. The purpose of this proposal is to discover the key factors and the effective regulating ways for the decreasing efficiency of enzymatic conversion at high solids loading. Therefore, the main works are focused on multi-scale characterization, theoretical analysis and process optimization by setting the study of the whole process of cellulase diffusion, adsorption and reaction as the central research line. (1) Multi-scale charaterization: quantitatively analyzing the distribution (diffusion), transfer (adsorption/ desorption) and variation (reaction) of macroscopic substrate particles, mesoscopic cellulase and microscopic sugars; (2) Theoretical analysis: investigating the changes in the reactivity of various components of cellulases, the distribution and adsorption of cellulases together with product inhibition; discussing the influence of diffusion and adsorption/desorption on enzymatic conversion of cellulose; constructing the diffusion-adsorption-reaction kinetics for high solids cellulose hydrolysis; and discovering the process steps, the key factors and the control approaches for the decrease in cellulose conversion; (3) Process optimization: Determining the effective way to improve the hydrolysis efficiency based on the kinetic model and theoretical analysis, and optimizing the bioreactor design, the premixing of substrate and cellulase, fed-batch strategy and enzyme mixture design to enhance the hydrolysis efficiency. This research will provide the theoretical basis for the operation of high solids enzymatic hydrolysis of lignocellulose, and further promote the fundamental and application study of cellulosic ethanol and biomass-based chemicals.

木质纤维素高固酶解工艺具有耗水少、能耗低、产能大、糖浓度高等优点，但存在水解效率随固含量增加而线性下降的共性技术问题。本项目旨在揭示高固酶解效率下降的关键因素和调控途径，以酶解体系的扩散、吸附与反应为主线，开展酶解过程的多尺度表征、机理分析与过程调控。①多尺度表征：组合色谱、波谱和电镜分析与CFD模拟，定量描述宏观底物颗粒、介观纤维素酶、微观产物糖的分布（扩散）、转移（吸/脱附）与变化（反应）；②机理分析：通过研究纤维素酶分布、活力变化、产物抑制与吸附行为，探讨酶解反应受扩散、吸附/脱附的影响规律，构建扩散-吸附-反应动力学模型，明确水解效率下降的存在环节和关键因素；③过程调控：基于动力学模型和机理分析，确定提高水解效率的调控途径，优化反应器设计、酶与底物预混、补料设计、酶制剂复配等工艺。研究成果将为高固酶解体系工艺优化提供理论依据，促进纤维素乙醇和生物基化学品的基础研究并推进其开发应用。

纤维素乙醇炼制过程中较高的乙醇浓度是蒸馏工段能否经济性运行的关键参数。要实现纤维素乙醇的商业化，体系含水量必须严格控制，即必须开展高固含量的纤维素酶解糖化，才能获得较高的糖醇浓度。此外，高固酶解工艺还具有耗水少、能耗低、产能大等优点。本项目针对高固含量酶解体系酶解效率低、酶耗量大两大瓶颈问题，开展了一系列工作：1）多尺度表征：基于SEC-MALLS、QCM、SPR、太赫兹、色谱、波谱等技术，建立了一套对宏观底物颗粒、介观纤维素酶、微观产物糖的分布和变化定量表征等方法；2）新型预处理技术：建立水热处理、碱性过氧化氢、亚铁与乙醇协同处理、芬顿（或与碱法协同处理）等预处理方式，获得各种组分含量和结构的底物；3）扩散分析与调控：揭示了纤维素酶解反应过程受扩散的调控规律，建立了步降式搅拌策略，既保证了酶解效率，又降低了能量消耗；4）吸附规律与调控：考察了纤维素酶在不同pH条件下的吸附行为和酶稳定性，发现酸性有利于吸附，而碱性则有利于脱附，设计了一种基于pH调控的纤维素酶回收策略，大幅提高了纤维素酶的回收效率；构建了可分泌β-葡萄糖苷酶的重组酵母菌株，采用该菌株发酵产酶与底物重吸附法回收外切／内切纤维素酶，显著降低了β-葡萄糖苷酶的补加量；进一步耦合表面活性剂，通过重吸附法和再用发酵残渣回收固液两相中的纤维素酶，避免了纤维素酶循环使用过程中β-葡萄糖苷酶的补加。5）酶解反应机理：采用SEC-MALLS跟踪分析了木质纤维素在酶解过程中三大组份的分子量分布变化规律，发现纤维素和半纤维素在底物的各个层面上相互缠结。外切纤维素酶遵循层层剪切方式，是酶解的关键限速步骤。项目研究成果为高固含量纤维素酶的高效水解和有效回收提供实验方法和理论依据。通过本项目研究，在Biotechnol Bioeng、J Agri Food Chem、Bioresource Technol、Appl Microbiol Biot等重要期刊发表学术论文14篇，其中SCI文章10篇，申请中国发明专利2项，获得天津市自然科学二等奖。