Microfluidic reactor for continuous cultivation of Saccharomyces cerevisiae

Abstract

A diffusion-based microreactor system operated with a reaction volume of 8 μL is presented and characterized to intensify the process understanding in microscale cultivations. Its potential as screening tool for biological processes is evaluated. The advantage of the designed microbioreactor is the use for the continuous cultivation mode by integrating online measurement technique for dissolved oxygen (DO) and optical density (OD). A further advantage is the broaden application for biological systems. The bioreactor geometry was chosen to achieve homogeneous flow during continuous process operation. The device consisted of a microstructured top layer made of poly(dimethylsiloxane) (PDMS), which was designed and fabricated using UV-depth and soft lithography assembled with a glass bottom. CFD simulation data used for geometry design were verified via microparticle-image-velocimetry (μPIV). In the used microreactor geometry no concentration gradients occurred along the entire reaction volume because of rapid diffusive mixing, the homogeneous medium flow inside the growth chamber of the microreactor could be realized. Undesirable bubble formation before and during operation was reduced by using degassed medium as well as moistened and moderate incident air flow above the gas permeable PDMS membrane. Because of this a passive oxygen supply of the culture medium in the device is ensured by diffusion through the PDMS membrane. The oxygen supply itself was monitored online via integrated DO sensors based on a fluorescent dye complex. An adequate overall volumetric oxygen transfer coefficient K(L)a as well as mechanical stability of the device were accomplished for a membrane thickness of 300 μm. Experimental investigations considering measurements of OD (online) and several metabolite concentrations (offline) in a modified Verduyn medium. The used model organism Saccharomyces cerevisiae DSM 2155 tended to strong reactor wall growth resembling a biofilm.

Quantification of the oxygen uptake rate in a dissolved oxygen controlled oscillating jet‐driven microbioreactor

“…Continuous cultivation of S . cerevisiae in a µBR with monitoring of the dissolved oxygen was previously reported . Very recently, monitoring of OUR was demonstrated in a µBR configured as a vertical microbubble column .…”

Characterization of oxygen transfer in vertical microbubble columns for aerobic biotechnological processes

“…For all experiments, a chemically defined cultivation medium was used. Except for the trace element solution, this medium was composed as previously reported (Edlich et al, ). The trace element solution (pH 4) contains the following (g/L): 16.6 Na 2 EDTA·2 H 2 O, 4.5 ZnSO 4 ·7 H 2 O, 1.57 MnCl 2 ·4 H 2 O, 0.3 CuSO 4 ·5 H 2 O, 0.4 Na 2 MoO 4 ·2 H 2 O, 3.39 CaCl 2 , 3.0 FeSO 4 ·7 H 2 O, and 0.1 KI.…”

Evolutionary engineering of Saccharomyces cerevisiae for improved industrially important properties

“…It is an analytical system that allows processing and manipulation of fluids in small amounts (Vinuselvi et al, 2011). A diffusion-based microreactor system for continuous cultivation was also described (Edlich et al, 2010). Microfluidic bioreactors are microfluidic devices that can be used for evolutionary adaptation during long-term cultivation, microbial growth, and cell quantification (Vinuselvi et al, 2011).…”

“…The advantages of this system are low cost and labor, high resolution and precision, and high-throughput continuous and batch processing of multiple samples. The oxygen supply was monitored online by integrated DO sensors, on the basis of a fluorescent dye complex (Edlich et al, 2010). Examples include a microfluidic chemostat for bacterial and yeast cell cultivations (Groisman et al, 2005); the 'Envirostat', a microfluidic bioreactor that allows constant environmental conditions (Kortmann et al, 2009); and the bacteria-algae -yeast (BAY) microbioreactor without valves, mixers, and pumps, powered by digital microfluidics, where a fluorescent viability assay and the use of a fluorescent reporter gene allowed measurements of cell growth and density (Au et al, 2011).…”