Biokinetics modelling of lycopene-producing E. coli fermentation using PAT methodology

Abstract

Process analytical technology (PAT), which espouses the use of in-line analytics to monitor critical process parameters (CPPs), critical quality attributes (CQAs) and mathematical analyses, was employed to elucidate underlying carbon-source metabolism of a lycopene-producing E. coli strain during non-induced and induced batch fermentations. In-line dielectric and Raman spectroscopies were deployed to monitor real-time culture biomass viability (X), primary substrate glucose (S), and intracellular lycopene production (P), together with off-line HPLC quantification of broth acetate (A). In-line PAT monitoring enabled identification of two distinct metabolic regimes during induced batch run with lycopene accumulation. Biokinetics mathematical models using differential-algebraic equations and Monod-type conversions were formulated to explain experimentally measured dynamic trends of the state variables X, S, A and P during the batch runs. Their optimized model parameters and seven dimensionless ratios reflected changes in concentrations or rates relative to glucose, acetate, or lycopene. The calculated rates of culture growth, glucose utilization, acetate accumulation, and lycopene production revealed salient underlying carbon metabolic shifts and transient trends that explained phenomenological observations: such as growth patterns, carbon source usage, overflow mechanism, cellular affinity or inhibitory effects, and lycopene cytotoxicity. These parameters and dimensionless ratios could be used to compare novel strains, develop in silico simulations for scale-up or troubleshoot batch-to-batch variations.