L U P H U UD N pH FLYL H DL 101 H FH E I U S LPL L I UHF PEL D SU HL V E H Abstract Phenotypic heterogeneity provides the microbial population with fast adaptive response (i.e., in the range of several minutes) against various kinds of stress factors, mainly for improving the natural fitness of the microbial population in the presence of environmental fluctuations The stability and reproducibility of such productive phenotype became more critical when applying continuous cultivation in industrial production Here we report using a single-cell toolbox to study the impact of nutrient fluctuations on microbial population dynamics, and this platform can be used to ensure the long-term stability of phenotypic diversification The setup, design, and operation of the so-called segregostat combined with an automated sampling platform and online flow cytometry were developed in order to implement real-time analysis and feedback control with a very high time resolution This closed-loop controller is able to drive phenotypic diversification employing defined pulsefrequency modulation within continuously running bioreactor setups Two exemplary case studies have been discussed, i.e., phenotypic diversification dynamics based on outer membrane permeabilization and the response of a coherent feedforward loop motif in E coli, highlighting the applicability and flexibility of the proposed approach In the first case, glucose pulses were added to maintain a predefined diversification ratio between non-permeablized and permeabilized cells based on an exogenous biomarker (propidium iodide) Our segregostat platform offered the possibility to adjust the environmental fluctuation profile based on the state of the population and automated this dynamic control in real-time The second case study was focused on the induction of the arabinose operon as a model system; the GFP level prompted by an arabinose-inducible promoter has been tracked by flow cytometry The bimodal distribution in the GFP expression pattern and the co-existence of GFP positive and negative subpopulations were observed in the glucose/arabinose co-feeding chemostat Furthermore, adjusting the arabinose/glucose transitions based on the phenotypic switching under a bang-bang controller provides a fully predictable, but oscillatory, gene expression dynamics Forcing the expression level of the arabinose system by increasing the inducer stimulation frequency led to harmonic oscillation, a more homogenous response of the cell population, and alleviated the bimodal behavior in gene expression Such dynamic control could enable the repeated and reversible switching between two gene expression states, allowing individual cells to switch around a predefined threshold Periodic inducer addition could be applied in bioprocesses for driving microbial population into a predefined phenotypic trajectory Taken altogether, these results pointed out that our control platform is a major step in quantitatively controlling phenotypic heterogeneity among microbial population Keywords: Segregostat, Single cell analysis, Flow cytometry, Stress response, Arabinose operon, Biological noise, Plasmid stability, GFP reporter, P-II ... segregostat platform offered the possibility to adjust the environmental fluctuation profile based on the state of the population and automated this dynamic control in real-time The second case... provides the microbial population with fast adaptive response (i.e., in the range of several minutes) against various kinds of stress factors, mainly for improving the natural fitness of the microbial... focused on the induction of the arabinose operon as a model system; the GFP level prompted by an arabinose-inducible promoter has been tracked by flow cytometry The bimodal distribution in the GFP