Department of Biotechnology, Institute of Chemical Technology Prague, Technická 5, 166 28, Prague, Czech Republic.
Folia Microbiol (Praha). 2012 Jul;57(4):307-11. doi: 10.1007/s12223-012-0131-8. Epub 2012 Apr 13.
We endeavored to develop a method for viability determination of solventogenic clostridia and to apply it for monitoring acetone-butanol-ethanol (ABE) fermentation. Six fluorescent probes (propidium iodide [PI], ethidium bromide, fluorescein diacetate, carboxyfluorescein diacetate [cFDA], rhodamine 123, bis-(1,3-dibutylbarbituric acid)trimethine oxonol [BOX]) were tested in order to distinguish two subpopulations of live and dead clostridial cells in suspension. Three of them were found to be appropriate (PI, BOX and cFDA) for this purpose. Developed fluorescent staining methods were applied to batch fermentation processes of Clostridium pasteurianum and C. beijerinckii carried out in a laboratory bioreactor under anaerobic conditions. Whereas PI was found to be applicable to both strains, BOX was convenient only for viability determination of C. pasteurianum. Although cFDA can distinguish two cell subpopulations in suspension, it was found to be unsuitable for viability determination under tested conditions, since it reflected more variable esterase activity during sporulation cell cycle than viability. Flow cytometry in combination with convenient fluorescent probe has been proved to be a valuable tool for viability determination. We assume this rapid and simple method can help to obtain more complex and precise information about ABE fermentation.
我们致力于开发一种用于鉴定溶剂梭菌活力的方法，并将其应用于丙酮丁醇乙醇（ABE）发酵的监测。为了区分悬浮液中活细胞和死细胞的两个亚群，我们测试了六种荧光探针（碘化丙啶[PI]、溴化乙锭、荧光素二乙酸酯、羧基荧光素二乙酸酯[cFDA]、罗丹明 123、双-（1,3-二丁基巴比妥酸）三嗪氧基[BOX]）。其中三种被发现适合用于此目的（PI、BOX 和 cFDA）。开发的荧光染色方法应用于在厌氧条件下在实验室生物反应器中进行的巴氏梭菌和拜氏梭菌的分批发酵过程。PI 被发现适用于两种菌株，而 BOX 仅适用于巴氏梭菌的活力测定。虽然 cFDA 可以区分悬浮液中的两个细胞亚群，但在测试条件下发现它不适合用于活力测定，因为它在孢子形成细胞周期中反映的酯酶活性比活力更具可变性。流式细胞术与方便的荧光探针相结合已被证明是一种用于活力测定的有价值的工具。我们假设这种快速简单的方法可以帮助获得关于 ABE 发酵的更复杂和更精确的信息。
