Project Details
Projekt
ProPHet2Con: Towards control of population heterogeneity induced variations in process performance during L-phenylalanine production with Escherichia coli
Subject Area
Biological Process Engineering
Term
since 2019
Project identifier
Deutsche Forschungsgemeinschaft (DFG) - Project number 428015958
Upscaling of bioprocesses is often accompanied by loss in productivity compared to well-mixed lab-scale processes. One reason is the formation of population heterogeneity whose mechanistic understanding is still comparably low. During the first funding period (project ProPHet) quantitative understanding of population heterogeneity originating from cell-bioreactor interaction in fed-batch processes with E. coli FUS4 (pF81kan) converting glycerol to L-phenylalanine (L-Phe) was raised. Experimental investigation was done with a quadruple reporter strain monitoring growth, general stress response, oxygen availability and product formation of single cells. Cultivations in a well-mixed stirred-tank bioreactor (STR) were compared with large-scale bioprocess conditions simulated in a scale-down reactor system consisting of a STR and a novel by-pass reactor that is designed as a coiled flow inverter (CFI). Theoretical investigation was done with population balance equations and agent based modeling after generating a non-segregated coarse-grained model. Building upon results from ProPHet, ProPHet2Con is aiming at controlling population heterogeneity induced variations in process performance in the fed-batch processes for L-Phe production. A population heterogeneity level that is beneficial for process performance will be maintained by promoting the abundance of subpopulations with advantageous characteristics. A special focus will be put on the process in the STR-CFI. Prio to implementation of process control, the L-Phe production process will be optimized applying the coarse-grained model and stoichiometric modeling to improve final product titer and productivity, especially in the STR-CFI setup. As a prerequisite for influencing the level of population heterogeneity by automated process control, manual at-line flow cytometry measurement will be replaced by automated real-time flow cytometry (ART-FCM). The ART-FCM setup will be advanced by the previously published process concept segregostat, that allows to control the degree of diversification of a bioreactor population at a predefined setpoint by single-cell physiology-based feeding of the carbon source. Furthermore, model-based soft sensors will implemented to estimate quantities cannot be measured directly. Then, based on the outcome of experimental investigations, strategies will be formulated to influence the level of population heterogeneity by model-based process feedback control following ART-FCM evaluation of single cell physiology. To our knowledge such a coupling between ART-FCM and an advanced model-based process control has so far never been realized. The formulated strategies will be experimentally validated in the fed-batch process for L-Phe production in the STR and the STR-CFI.Once process control for L-Phe production is realized, scale-up potential of the process to pilot-scale will be investigated.
DFG Programme
Priority Programmes
Major Instrumentation
Automatisierter Probenehmer
Instrumentation Group
1060 Dilutoren, Pipettiergeräte, Probennehmer
Co-Investigator
Professor Dr.-Ing. Dirk Weuster-Botz
