MATTHEW J. DAVIES, DARREN N NESBETH, NICOLAS SZITA*
*Corresponding author
University College London, Department of Biochemical Engineering, Gower St, London, United Kingdom

Abstract

Microbioreactor technology has made rapid advances in the last decade and the capability of these tiny reactors to monitor fermentation variables in situ, such as optical density, dissolved oxygen, pH and fluorescent protein expression, provide real-time and quantitative data from microlitre volumes. Several systems are nowadays commercially available and are being applied for early stage bioprocess development. Whilst a majority of the microbioreactors has been designed for batch and fed-batch fermentation, there are still only few efforts directed at developing microbioreactors for chemostat mode operation. Chemostat operation, however, could not only be employed for the rapid optimization of bioprocess conditions, but would also lend itself for rigorous characterization of de novo engineered cells for applications in synthetic biology. The microbioreactor design presented here was developed to enable different operation modes, such as batch and continuous culture. The design offers versatility while maintaining key aspects of single-use: fluidic connections, for example, can easily be adapted, such as to introduce different culture medium or for the time-controlled delivery of an inducer, and the microbioreactor can be made disposable to reduce sterilization and cleaning loads.

INTRODUCTION

Since the pioneering work of Kostov et al. (2001) (1), microbioreactor technology has made rapid advances, and fermentation of prokaryotic and eukaryotic microbes with relevant biomass densities and with oxygen transfer characteristics comparable to bench and pilot-scales have been successfully demonstrated in several microbioreactors (2). While the small volumes limit the ability to take samples and, concomitant with that, perform analyses, methods have been developed for incorporating many of the analyses relevant to understanding fermentation processes. For instance, integration of commercially available sensor spots enables the external optical interrogation of the dissolved oxygen concentration, pH and CO2 concentration (3). Measurements of optical density provide an indirect measurement of the biomass and incorporation of fluorescence detection can be used to measure the concentration of a fluorescent protein such as GFP as a marker of the expression of the desired product (4). Due to these non-invasive detection methods, quantitative data on the growth kinetics have been obtained in real time from culture volumes as small as 5 microli ...