The omnipresence of population heterogeneity in industrial bioprocesses originates from prevailing dynamic bioprocess conditions, which promote differences in the expression of cellular characteristics. Despite the awareness, the concrete consequences of this phenomenon remain poorly understood. Therefore, for the first time, a L-phenylalanine overproducing Escherichia coli quadruple reporter strain was established for monitoring of general stress response, growth behavior, oxygen limitation and product formation of single cells based on mTagBFP2, mEmerald, CyOFP1, and mCardinal2 expression measured by flow cytometry. This strain was applied for the fed-batch production of L-phenylalanine from glycerol and ammonia in a stirred-tank bioreactor at homogeneous conditions compared to the same process in a novel two-compartment bioreactor. This two-compartment bioreactor consists of a stirred-tank bioreactor with an initial volume of 0.9 L (homogeneous zone) with a coiled flow inverter with a fixed working volume of 0.45 L as a bypass (limitation zone) operated at a mean hydraulic residence time of 102 s. The product formation was similar in both bioreactor setups with maximum L-phenylalanine concentrations of 21.1 ± 0.6 g L−1 demonstrating the consistency of this study’s microbial L-phenylalanine production. However, cell growth was vulnerable to repetitive exposure to the dynamically changing conditions in the two-compartment bioreactor with maximum biomass yields reduced by 21%. The functionality of reporter molecules was approved in the stirred-tank bioreactor cultivation, in which expressed fluorescence levels of all four markers were in accordance with respective process state variables. Additional evaluation of the distributions on single-cell level revealed the presence of population heterogeneity in both bioprocesses. Especially for the marker of the general stress response and the product formation, the corresponding histograms were characterized by bimodal shapes and broad distributions. These phenomena were pronounced particularly at the beginning and the end of the fed-batch process. The here shown findings confirm multiple reporter strains to be a noninvasive tool for monitoring cellular characteristics and identifying potential subpopulations in bioprocesses. In combination with experiments in scale-down setups, these can be utilized for a better physiological understanding of bioprocesses and support future scale-up procedures.

中文翻译：

---

混合不充分对新型两室生物反应器中大肠杆菌报告菌株生产 L-苯丙氨酸的影响

工业生物过程中普遍存在的群体异质性源于普遍的动态生物过程条件，这促进了细胞特征表达的差异。尽管人们已经意识到这一点，但人们对这种现象的具体后果仍然知之甚少。因此，首次建立了L-苯丙氨酸过量生产的大肠杆菌四重报告菌株，用于监测单细胞的一般应激反应、生长行为、氧限制和产物形成，基于mTagBFP2、mEmerald、CyOFP1和mCardinal2表达测量流式细胞术。与新型两室生物反应器中的相同工艺相比，该菌株应用于在搅拌罐生物反应器中在均质条件下从甘油和氨分批补料生产 L-苯丙氨酸。该两室生物反应器由初始体积为 0.9 L（均质区）的搅拌槽生物反应器和固定工作体积为 0.45 L 的盘管流逆变器（作为旁路（限制区））组成，在平均水力停留时间下运行共 102 秒。两种生物反应器设置中的产物形成相似，最大 L-苯丙氨酸浓度为 21.1 ± 0.6 g L−1，证明了本研究微生物 L-苯丙氨酸生产的一致性。然而，细胞生长容易受到两室生物反应器中反复暴露于动态变化条件的影响，最大生物量产量减少了 21%。报告分子的功能在搅拌罐生物反应器培养中得到批准，其中所有四种标记物的表达荧光水平均与各自的过程状态变量一致。对单细胞水平分布的额外评估揭示了两种生物过程中存在群体异质性。特别是对于一般应力响应和产物形成的标记，相应的直方图具有双峰形状和广泛的分布特征。这些现象在补料分批过程的开始和结束时尤其明显。这里显示的发现证实多个报告菌株是监测细胞特征和识别生物过程中潜在亚群的非侵入性工具。与按比例缩小设置的实验相结合，这些可以用于更好地了解生物过程的生理学知识并支持未来的按比例放大程序。