Almost half of the medicines on the market are natural products including polyketides, fatty acids, amino
acids, terpenoids and steroids. Though many of the natural products are discovered in plants, mass
production of these molecules relies on culturing microorganisms in bioreactors. However, it is often a
challenge to develop and maintain high biomanufacturing productivity and yield to enable low-cost and high-
quality production at large scales. One unmet need is an ability to rapidly and accurately measure the
physiological status of microbes at the cellular level within bioreactors. Measuring the cellular metabolic state,
especially energetic and redox parameters, is key to developing improved biosynthesis processes and to
guiding feeding strategies and other operational actions. Due to the complex nature of bioreactor cell culture
systems, currently the capability of measuring cellular metabolic parameters in real time is unavailable, and
metabolic assessment relies on time-consuming, periodic removal of culture samples for off-line analysis.
During this R&D program, Physical Sciences Inc. (PSI), in collaboration with the University of
Massachusetts Lowell (UML) and Northeastern University (NEU), will develop a novel two-photon excitation
(TPE) fluorescence redox sensor for on-line, real-time measurement of cell metabolism in bioreactors for
natural products fermentation. A miniaturized optical probe will be developed that can be sterilized and
inserted into bioreactor cultures for continuous measurement of dynamic changes of important intracellular
metabolites. The technology is equivalent to an online cytometer within the bioreactor, providing critical
cellular-level physiology data that would otherwise be only available from off-line measurements. The
intracellular redox ratio will be monitored using the autofluorescence of endogenous fluorophores, without
the need (but not excluding the option) for exogenous fluorescence labeling.
During the Phase I program, the PSI led team successfully demonstrated the feasibility of the proposed
technology. A Gen-I prototype redox sensor was fabricated and tested during continuous metabolism
measurements in operational bioreactors, mitigating the critical technical risks of the proposed Phase II
research. During the Phase II program, the research effort will be focused on optimizing the technology,
demonstrating its value in advancing biomanufacturing process development and operations, and preparing
for commercialization. In particular, a Gen-II prototype with significantly improved signal to noise ratio and
system robustness will be fabricated. The Gen-II instrument will then be tested in a large-number of
bioreactor operations to demonstrate how the new real-time cellular metabolism data will be utilized to guide
the development and operation of novel and more effective bioreactor processes for improved product yield.
The experimental data will also be input into theoretical bioprocess models to promote improved
understanding and control of the processes.