Arulrajah, PrasikaPrasikaArulrajahRiessner, Sophi KatharinaSophi KatharinaRiessnerHeins, Anna-LenaAnna-LenaHeinsWeuster-Botz, DirkDirkWeuster-Botz2025-09-302025-09-302025-07-03BioTech 14 (3): 54 (2025)https://hdl.handle.net/11420/57616Large-scale bioprocesses often suffer from spatial heterogeneities, which impact microbial performance and often lead to phenotypic population heterogeneity. To better understand these effects at the single-cell level, this study applied, for the first time, automated real-time flow cytometry (ART-FCM) to monitor L-phenylalanine production with an <i>Escherichia coli</i> triple reporter strain in a fed-batch process with glycerol as the carbon source. The strain was cultivated in both a well-mixed stirred-tank bioreactor (STR) and a scale-down two-compartment bioreactor (TCB), consisting of an STR and a coiled flow inverter (CFI) in bypass, to simulate spatial heterogeneities. ART-FCM enabled autonomous, high-frequency sampling every 20 min, allowing for real-time tracking of fluorescence signals linked to growth (<i>rrnB</i>-mEmerald), oxygen availability (<i>narGHIJ</i>-CyOFP1), and product formation (<i>aroFBL</i>-mCardinal2). The STR exhibited uniform reporter expression and higher biomass accumulation, while the TCB showed delayed product formation and pronounced phenotypic diversification depending on the set mean residence time in the CFI. Single-cell fluorescence distributions revealed that the shorter mean residence time in the CFI resulted in pronounced subpopulation formation, whereas longer exposure attenuated heterogeneity, indicating transcriptional adaptation. This finding highlights a critical aspect of scale-down studies: increased exposure duration to perturbations can enhance population robustness. Overall, this study demonstrates the relevance of ART-FCM, in combination with a multi-reporter strain, as a pioneering tool for capturing dynamic cellular behavior and correlating it to process performance, providing deeper insights into microbial heterogeneity under fluctuating bioprocess conditions.en2673-628420253MDPIhttps://creativecommons.org/licenses/by/4.0/fed-batchstirred-tank bioreactortwo-compartment bioreactorautomated real-time flow cytometryEscherichia coli reporter strainphenotypic population heterogeneityresidence timeNatural Sciences and Mathematics::570: Life Sciences, BiologyTechnology::660: Chemistry; Chemical Engineering::660.6: BiotechnologyJournal Article2025-09-26https://doi.org/10.15480/882.1593110.3390/biotech1403005410.15480/882.15931Journal Article