Abstract

Many microbioreactors have been developed to test growth conditions rapidly [1], [2], however aside from a few exceptions [3]-[6] most are tested using a batch fermentation. Additionally only a couple have been demonstrated with gram-positive bacteria [7], [8], with the vast majority of microbioreactors being tested with E. coli and S. cerevisiae despite the industrial relevance of gram- positive bacteria [9]. We present a microbioreactor (Figure 1 a).), tested using a gram-positive bacteria, S. carnosus, capable of operating in chemostat mode with comparable batch kinetics to shake flask fermentation. a) b) Figure 1. The Instrumented Microfluidic Chemostat (IMC) presented here is being developed as part of the SYNMOD project for the purpose of determining the growth condition test space for scaled-up fermentations in order to achieve the best yield of both biomass and product. S. carnosus, a genomically well characterized & industrially relevant bacteria, is being utilized as part of the ESF sponsored SYNMOD project as a chassis for lantibiotic producing genes. To this end a microbioreactor was developed with active stirring and capacity for independent control of multiple fluid sources at low flow rate, for introduction of medium, inoculant &, potentially, pH control and an inducer (Figure 1 b).). An electronic and optical system has been developed alongside the microbioreactor enabling constant monitoring of both biomass and GFP fluorescence, as a marker of lantibiotic production. The combination of microbioreactor and electronically controlled fluid system, optical monitoring system and environmental control results in the IMC.  a) b) Figure 2. As demonstrated in Figure 2 the IMC is capable of operating in both batch and chemostat mode. Comparative fermentations of S. carnosus in shake flask and IMC demonstrate that comparable growth rates and biomass concentration are achieved in the IMC (Figure 2 a).). The chemostat fermentation (Figure 2 b).), despite operating at low OD, demonstrates computer controlled switching between fluid sources and the initial capability to provide the precise fluid flow control necessary to maintain constant bacterial growth rate. References [1] D. Schäpper, M. N. H. Z. Alam, N. Szita, A. E. Lantz, and K. V. Gernaey, Application of microbioreactors in fermentation process development: a review, Anal Bioanal Chem, 395 (2009) 679–695. [2] Z. Zhang, G. Perozziello, P. Boccazzi, A. J. Sinskey, O. Geschke, and K. F. Jensen, Microbioreactors for Bioprocess Development, Journal of the Association for Laboratory Automation, 12 (2007) 143–151. [3] A. Groisman, C. Lobo, H. Cho, J. K. Campbell, Y. S. Dufour, A. M. Stevens, and A. Levchenko, A microfluidic chemostat for experiments with bacterial and yeast cells, Nat Methods, 2 (2005) 685–689. [4] X. Luo, K. Shen, C. Luo, H. Ji, Q. Ouyang, and Y. Chen, An automatic microturbidostat for bacterial culture at constant density, Biomed Microdevices, 12 (2010) 499–503. [5] Z. Zhang, P. Boccazzi, H.-G. Choi, G. Perozziello, A. J. Sinskey, and K. F. Jensen, Microchemostat-microbial continuous culture in a polymer-based, instrumented microbioreactor., Lab Chip, 6 (2006) 906–913. [6] K. S. Lee, P. Boccazzi, A. J. Sinskey, and R. J. Ram, Microfluidic chemostat and turbidostat with flow rate, oxygen, and temperature control for dynamic continuous culture, Lab Chip, 11 (2011) 1730–1739. [7] P. Rohe, D. Venkanna, B. Kleine, R. Freudl, and M. Oldiges, An automated workflow for enhancing microbial bioprocess optimization on a novel microbioreactor platform, Microb Cell Fact, 11 (2012) 144-156. [8] J. R. Moffitt, J. B. Lee, and P. Cluzel, The single-cell chemostat: an agarose-based, microfluidic device for high-throughput, single-cell studies of bacteria and bacterial communities, Lab Chip, 12 (2012) 1487–1494. [9] E. W. Nester, C. E. Roberts, M. E. Lidstrom, N. C. Pearsall, and M. T. Nester, Microbiology, third ed., Saunders College Publishing, USA 1983.