Process and Systems Engineering Center (PROSYS), Department of Chemical and Biochemical Engineering, Technical University of Denmark (DTU), Building 228A, 2800, Lyngby, Denmark.
Department of Food Science, University of Copenhagen (KU), Rolighedsvej 26, 1958, Frederiksberg C, Denmark.
Sci Rep. 2021 Mar 25;11(1):6853. doi: 10.1038/s41598-021-86135-z.
Gaining an in-depth understanding of the response of Saccharomyces cerevisiae to the different inhibitors generated during the pretreatment of lignocellulosic material is driving the development of new strains with higher inhibitor tolerances. The objective of this study is to assess, using flow cytometry, how three common inhibitors (vanillin, furfural, and acetic acid) affect the membrane potential, the membrane permeability and the concentration of reactive oxygen species (ROS) during the different fermentations. The membrane potential decreased during the detoxification phase and reflected on the different mechanisms of the toxicity of the inhibitors. While vanillin and furfural caused a metabolic inhibition and a gradual depolarization, acetic acid toxicity was related to fast acidification of the cytosol, causing an immediate depolarization. In the absence of acetic acid, ethanol increased membrane permeability, indicating a possible acquired tolerance to ethanol due to an adaptive response to acetic acid. The intracellular ROS concentration also increased in the presence of the inhibitors, indicating oxidative stress. Measuring these features with flow cytometry allows a real-time assessment of the stress of a cell culture, which can be used in the development of new yeast strains and to design new propagation strategies to pre-adapt the cell cultures to the inhibitors.
深入了解酿酒酵母对木质纤维素预处理过程中产生的不同抑制剂的反应，是开发具有更高抑制剂耐受性的新菌株的关键。本研究旨在通过流式细胞术评估三种常见抑制剂（香草醛、糠醛和乙酸）在不同发酵过程中如何影响细胞膜电位、膜通透性和活性氧（ROS）浓度。在解毒阶段，细胞膜电位下降，反映了抑制剂毒性的不同机制。香草醛和糠醛引起代谢抑制和逐渐去极化，而乙酸毒性与细胞质的快速酸化有关，导致立即去极化。在没有乙酸的情况下，乙醇增加了膜通透性，表明由于对乙酸的适应性反应，细胞可能获得了对乙醇的耐受性。在抑制剂存在的情况下，细胞内 ROS 浓度也增加，表明存在氧化应激。通过流式细胞术测量这些特征，可以实时评估细胞培养的应激状态，这可用于开发新的酵母菌株，并设计新的繁殖策略，使细胞培养物预先适应抑制剂。
