高浓度乙醇连续发酵系统振荡条件下动态动力学

Very high gravity (VHG) fermentation can significantly increase ethanol titer in the fermentation broth, and correspondingly save energy consumption, not only for ethanol distillation, but also for the treatment of waste distillage discharged from the distillation system, which has thus being pursuing endlessly in industry for ethanol production from sugar- and starch-based feedstocks. However, preliminary research indicated that continuous ethanol fermentation by Saccharomyces cerevisiae under VHG conditions triggeried oscillation that was characterized by long oscillation period and large oscillation amplitude, which not only risks more sugars remained unfermented,and thus compromises ethanol yield, the most important aspect for fuel ethanol production, but also affects the operation of the downstream ethanol distillation process. ..It is hypothesized that the mechanisms underlying this process oscillation is the lag response of the intracellular metabolism of S.cerevisiae to ethanol inhibition. Therefore, the severer the ethanol inhibition, the longer the oscillation period and the larger the oscillation amplitude will be, which presents a challenge to the metabolic flux analys that was developed based on the quasi-steady state assumption of intracellular enzymatic reactions and mass balance principle at key nodes of the metabolic network. In this project, metabolites of the Embden-Meyerhof-Parnas (EMP) pathway will be minotored during the oscillation of continuous ethanol fermentation by S. cerevisiae under VHG conditions, using advanced analytical technologies such as LC-MS and GC-MS, with an expectation to identify key enzymes that regulate the oscillation to support the theoretical hypothesis for the process oscillation, and in the meantime modify the metabolic flux analysis strategy by incorporating the lag response or time delay for intracellular enzymatic reactions to explore the intrinsic dynamic nature of intracellular metabolism. ..At the end, dynamic kinetic models to predict oscillation is expected to be established, and process engineering strategies that can attenuate or augment the oscillation, depending on its impact on the fermentation system, will be developed.

超高浓度（Very High Gravity,VHG）发酵可以显著降低能耗，是糖质和淀粉质原料生产燃料乙醇技术开发的主要方向。然而酿酒酵母（Saccharomyce cerevisiae）在VHG条件下进行乙醇连续发酵时，呈现长周期、大振幅的振荡行为，不仅影响发酵系统的自身的性能，而且不利于乙醇精馏装置的稳定运行。初步研究工作表明，诱发这一现象的机理是酵母胞内代谢对高浓度乙醇抑制的延迟反应，对在胞内代谢拟稳态假说前提下建立的酵母细胞糖酵解及相关代谢途径通量分析模型的适用性提出了挑战。本项目拟借助LC-MS和GC-MS等现代分析技术手段，定量分析酵母细胞VHG乙醇连续发酵糖酵解及相关代解途径的动态特征，寻找关键酶并研究其对细胞乙醇抑制应激反应滞后调节的机制，构建动态代谢通量分析模型。在此基础上，研究VHG乙醇连续发酵系统的动态动力学，为开发振荡行为前置干预调控策略奠定基础。

乙醇是最重要的生物燃料，与汽油配混使用可以降低车辆尾气有害组分，减轻对石油基燃料的依赖，促进经济和社会的可持续发展。提高发酵过程终点乙醇浓度，不仅可以节省乙醇精馏能耗，而且减少废糟液的排放，是燃料乙醇生产节能减排，降低成本的主要策略。然而，酿酒酵母高浓度乙醇连续过程诱发振荡行为，影响生产装置的稳定运行。针对酿酒酵母高浓度乙醇连续发酵条件下诱发的振荡行为，提出了发酵过程逐渐积累的高浓度乙醇的抑制以及胞内代谢对这种抑制的延迟反映是诱发这一现象分子机理的科学假说。.基于高浓度乙醇发酵条件下底物糖的渗透压胁迫与产物乙醇抑制偶联的特点，首先通过分别向稳态运行的低浓度乙醇连续发酵系统分别添加酿酒酵母不能代谢利用的木糖和乙醇，模拟高浓度乙醇连续发酵工况条件，使酵母细胞经受的底物糖渗透压胁迫和高浓度乙醇抑制解耦，在过程工程水平验证了乙醇抑制是诱发酿酒酵母高浓度乙醇发酵系统振荡行为的根本原因，并推断高浓度抑制性产物积累诱发振荡行为对微生物细胞连续培养与发酵过程具有普适性。建立了高浓度乙醇连续发酵系统振荡条件下酿酒酵母细胞内代谢活动快速淬灭后代谢物液质定量分析方法，深入研究了过程振荡完整周期内酿酒酵母细胞内代谢物组学和全局基因转录学特征，借助代谢通量分析构建了独特的动态代谢网络，诊断了关键代谢途径、酶和代谢物，并通过相位分析，比较了从上游糖的转运和吸收到下游乙醇发酵生成的时间滞后，从分子水平验证了关键代谢途径对乙醇抑制响应的滞后。在此基础上，建立了动态动力学模型。.研究工作取得的学术成果在Elsevier出版 SCI IF9.848的Biotechnology Advances及Springer出版SCI IF 6.444的Biotechnology for Biofuels等本领域高水平国际期刊正式发表论文六篇，为酿酒酵母高浓度乙醇连续发酵过程非稳态行为预测和前置干预策略创新技术开发奠定了科学基础。