Department of Electrical Engineering, Federal University of São Carlos, Rodovia Washington Luís, km 235, São Carlos, SP, CEP 13565-905, Brazil.
Department of Information Technology, Mannheim University of Applied Sciences, Paul-Wittsack-Straße 10, 68163 Mannheim, Germany.
J Microbiol Methods. 2020 Aug;175:105973. doi: 10.1016/j.mimet.2020.105973. Epub 2020 Jun 6.
In industrial yeast fermentation processes, single-cell yeast suspensions are usually preferable to cells in aggregates, as single cells exhibit a larger contact area with the nutrient medium, which in many cases helps optimize the process. In addition to affecting fermentation time and efficiency, cell aggregates (e.g., pseudohyphal yeast morphology) may also impair centrifugation systems, one of the most expensive and complex steps of the production process that involves the recovery of yeast cells for subsequent fermentation cycles. To date, no standard technique allows for a systematic diagnosis of yeast morphology in real time during sugarcane biofuel fermentation. Accordingly, we investigate an in situ microscope (ISM) for online monitoring of the density and morphology of an industrial Saccharomyces cerevisiae strain widely used in Brazilian distilleries (PE-2). During batch and repeated batch sugarcane molasses fermentation, the instrument revealed single cells, budding yeast cells, and pseudohyphae, all in a variety of sizes and shapes. The ISM image analysis indicated that the volume of single yeast cells increased by roughly 40% over the lag phase before budding and remained approximately constant thereafter. Pseudohyphae with three and more cells appeared mostly during the stationary phase. Cooling problems were simulated by raising the temperature from 33 to 45 °C. During this thermal stress, single cells as well as budding cells and pseudohyphae forming cells became smaller and exhibited intracellular inhomogeneities. From these results, we conclude that an ISM is a useful tool for monitoring yeast morphology during sugarcane fermentation. Atypical morphologies can be detected early and be used as an automatic warning system.
在工业酵母发酵过程中，单细胞酵母悬浮液通常比聚集的细胞更可取，因为单细胞与营养培养基具有更大的接触面积，这在许多情况下有助于优化工艺。除了影响发酵时间和效率外，细胞聚集体（例如假菌丝酵母形态）还可能损坏离心系统，这是生产过程中最昂贵和最复杂的步骤之一，涉及回收酵母细胞以用于随后的发酵周期。迄今为止，没有标准技术可以在甘蔗生物燃料发酵过程中实时系统地诊断酵母形态。因此，我们研究了一种原位显微镜（ISM），用于在线监测广泛用于巴西酿酒厂的工业酿酒酵母（PE-2）菌株的密度和形态。在分批和重复分批甘蔗糖蜜发酵过程中，该仪器揭示了单细胞、芽殖酵母细胞和假菌丝，所有这些细胞都具有各种大小和形状。ISM 图像分析表明，在出芽前的滞后阶段，单个酵母细胞的体积增加了约 40%，此后基本保持不变。具有三个或更多细胞的假菌丝主要出现在静止期。通过将温度从 33°C 升高到 45°C 来模拟冷却问题。在这种热应力下，单细胞以及出芽细胞和形成假菌丝的细胞变得更小，并表现出细胞内不均匀性。根据这些结果，我们得出结论，ISM 是监测甘蔗发酵过程中酵母形态的有用工具。可以早期检测到异常形态，并用作自动报警系统。
