Abstract
The use of in-line analytics for monitoring and understanding biological
processes has been promoted since 2004 through the Process Analytical
Technology (PAT) initiative. Two such technologies are Raman and
dielectric spectroscopies that are well established but deployed
independently. For the very first time, simultaneous usage of in-line
Raman and dielectric spectroscopic analytics for tracking progress of a
lycopene overproducer E. coli 322 engineered strain was studied
herein, alongside scale-up experimentation from shake flask to 0.5L and
10L bioreactor cultures. An intracellular lycopene extraction protocol
was optimised for off-line quantification by Raman or UV-Vis
spectroscopies, which aids the estimation of lycopene over-production in
a high cell density E. coli fed-batch culture via in-line Raman.
Correlating dielectric capacitance recordings and flow cytometry viable
cell count measurements allows for real-time cell culture viability
monitoring, which is more informative for bioprocess dynamic control in
contrast to conventional in-/off-line biomass optical density
(OD600) or dry cell weight (gDCW). This
enabled fed-batch culture revitalisation prior to inducing intracellular
lycopene production. The final biomass OD600 was ca.
81.8 corresponding to in-line dielectric capacitance estimated culture
viability 43.38 gVCC/L. Intracellular lycopene yield was
estimated at 495.1 mg/gVCC via in-line Raman, which was
12.3 times increase compared to initial trial reported for E.
coli 322. The successful implementation of dual in-line dielectric
capacitance and Raman monitoring exemplifies the quality by design (QbD)
approach espoused by PAT framework for expediting bioprocess
understanding, development and scale-up of engineered microbial strains.