Abstract：
Determination of the viability, ratio of dead and live cell populations, of Sulfolobus acidocaldarius is still being done by tedious and material-intensive plating assays that can only provide time-lagged results. Although S. acidocaldarius, an extremophilic Archaeon thriving at 75 °C and pH 3.0, and related species harbor great potential for the exploitation as production hosts and biocatalysts in biotechnological applications, no industrial processes have been established yet. One hindrance is that during development and scaling of industrial bioprocesses timely monitoring of the impact of process parameters on the cultivated organism is crucial-a task that cannot be fulfilled by traditional plating assays. As alternative, flow cytometry (FCM) promises a fast and reliable method for viability assessment via the use of fluorescent dyes. In this study, commercially available fluorescent dyes applicable in S. acidocaldarius were identified. The dyes, fluorescein diacetate and concanavalin A conjugated with rhodamine, were discovered to be suitable for viability determination via FCM. For showing the applicability of the developed at-line tool for bioprocess monitoring, a chemostat cultivation on a defined growth medium at 75 °C, pH 3.0 was conducted. Over the timeframe of 800 h, this developed FCM method was compared to the plating assay by monitoring the change in viability upon controlled pH shifts. Both methods detected an impact on the viability at pH values of 2.0 and 1.5 when compared to pH 3.0. A logarithmic relationship between the viability observed via plating assay and via FCM was observed.
摘要：
生存能力的确定，死细胞和活细胞种群的比例，仍然通过繁琐且材料密集的平板试验进行，这些试验只能提供滞后的结果。虽然S.acidocaldarius，在75°C和pH3.0下蓬勃发展的极端古细菌，相关物种具有在生物技术应用中作为生产宿主和生物催化剂的开发潜力，尚未建立工业流程。一个障碍是，在工业生物过程的开发和缩放过程中，及时监测过程参数对培养生物的影响至关重要，这是传统平板试验无法完成的任务。作为替代，流式细胞术（FCM）有望通过使用荧光染料进行活力评估的快速可靠的方法。在这项研究中,已鉴定出适用于酸乳杆菌的市售荧光染料。染料，荧光素二乙酸酯和伴刀豆球蛋白A与罗丹明结合，被发现适用于通过FCM测定活力。为了显示开发的在线生物过程监测工具的适用性，在75°C的特定生长培养基上进行恒化器培养，进行pH3.0。在800小时的时间范围内，通过监测受控pH变化后的活力变化，将这种开发的FCM方法与平板试验进行了比较。当与pH3.0相比时,两种方法都检测到在2.0和1.5的pH值对生存力的影响。观察到通过平板试验和通过FCM观察到的活力之间的对数关系。
