Microbioreactors for bioprocess development

Abstract

This thesis presents the design, fabrication, and characterization of a microbioreactor integrated with automated sensors and actuators as a step towards high-throughput bioprocess development. In particular, this thesis demonstrates the feasibility of culturing microbial cells in microliter-volume reactors in batch, continuous, fed-batch operations. The microbioreactor is fabricated out of poly(methylmethacrylate) and poly(dimethylsiloxane). Active mixing is made possible by a miniature magnetic stir bar. Online optical measurements for optical density, pH, and dissolved oxygen are integrated. Oxygenation in the microbioreactor is characterized and reproducible batch fermentation of Escherichia coli and Saccharomyces cerevisiae are demonstrated and benchmarked with benchscale bioreactors. Global gene expression analysis of S. cerevisiae exhibits physiological and molecular characteristics which parallel those of large-scales. A microchemostat, continuous culture of microbial cells, is realized in the microbioreactor. E. coli cells are fed by pressure-driven single phase flow of fresh medium through a microchannel. Chemotaxis, the back growth of bacterial cells into the medium feed channel, is prevented by local heating.

(cont.) Using poly(ethylene glycol) -grafted poly(acrylic acid) copolymer films, PMMA and PDMS surfaces are modified to generate bio-inert surfaces resistant to nonspecific protein adsorption and cell adhesion. These advances enable cell growth kinetics and stoichoimetry to be obtained in the microchemostat consistent with reported phenomena from conventional stirred-tank bioreactors, as indicated by the time profiles of OD600nm, pH, and DO measurements at steady states. Water evaporation from the microbioreactor allows feeding of base and glucose solutions into the small reactor to realize fed-batch operations. Commercial microvalves are integrated to obtain closed-loop pH control. pH value in the microbioreactor is successfully maintained within a physiological scale during the time course of E. coli cell cultivation in rich media. One key issue for high-throughput bioprocessing is the parallel operation of multiple microbial fermentations while keeping each single microbioreactor disposable. Plug-in-and-flow microfluidic connectors and fabricated polymer micro-optical lenses/connectors are integrated in the microbioreactor "cassettes" for fast set-up and easy operation.

(cont.) A protocol multiplexed system for the parallel operation of four microbioreactors is demonstrated. The demonstrated functionality of the microbioreactor with integrated measurements and flexible operations could potentially have a large impact in bioprocess developments.