Characterization of gas–liquid mass transfer phenomena in microtiter plates

Abstract

Gas-liquid mass transfer properties of shaken 96-well microtiter plates were characterized using a recently described method. The maximum oxygen transfer capacity (OTR(max)), the specific mass transfer area (a), and the mass transfer coefficient (k(L)) in a single well were determined at different shaking intensities (different shaking frequencies and shaking diameters at constant filling volume) and different filling volumes by means of sulfite oxidation as a chemical model system. The shape (round and square cross-sections) and the size (up to 2 mL maximum filling volume) of a microtiter plate well were also considered as influencing parameters. To get an indication of the hydrodynamic behavior of the liquid phase in a well, images were taken during shaking and the liquid height derived as a characteristic parameter. The investigations revealed that the OTR(max) is predominantly dependent on the specific mass transfer area (a) for the considered conditions in round-shaped wells. The mass transfer coefficient (k(L)) in round-shaped wells remains at a nearly constant value of about 0.2 m/h for all shaking intensities, thus within the range reported in the literature for surface-aerated bioreactors. The OTR(max) in round-shaped wells is strongly influenced by the interfacial tension, determined by the surface tension of the medium used and the surface properties of the well material. Up to a specific shaking intensity the liquid surface in the wells remains horizontal and no liquid movement can be observed. This critical shaking intensity must be exceeded to overcome the surface tension and, thus, to increase the liquid height and enlarge the specific mass transfer area. This behavior is solely specific to microtiter plates and has not yet been observed for larger shaking bioreactors such as shaking flasks. In square-shaped microtiter plate wells the corners act as baffles and cause a significant increase of OTR(max), a, and k(L). An OTR(max) of up to 0.15 mol/L/h can be reached in square-shaped wells.

Design and performance of a 24‐station high throughput microbioreactor

“…For the 24-well plate system, k L a's were determined by the sulfite oxidation method as adapted for microtiter plates by Hermann et al (2001Hermann et al ( , 2002. The reactors were filled with 1 mL of a solution of 0.5 M sodium sulfite, 0.012 M sodium phosphate, <1 mM cobalt sulfate, and the pH indicator dye HPTS, adjusted to pH 8.…”

“…This vortex would increase the surface area, which would increase the k L a. The change in surface area has been measured photographically in shaken 96-well plates and similar k L a curves were generated (Hermann et al, 2002). Because the agitation speeds can be very high, up to 2,800 rpm, the baffling effect of the heater and needles for sparging, temperature monitoring, and cell mass measurement is not sufficient to prevent vortex generation.…”

“…The plate reader has a built-in shaker, but still can only provide k L a values up to 40/h. With an improved shaker, the k L a could easily exceed 200/h (Hermann et al, 2002). Another promising study used optical measurements of dissolved oxygen and cell mass, and achieved k L a's of 100-400/h (Lamping et al, 2003).…”

“…Elmahdi et al (2003) measured pH during microscale fermentation by inserting a micro-pH probe into a 7 mL well in a microtrite plate. As mentioned earlier, Hermann et al (2002) have characterized the mass transfer characteristics of well plates. Puskeiler et al describe a 48-well milliliter scale bioreactor array.…”

Microbioreactors for Bioprocess Development

“…where (Bird et aI., 2002), the concept also applies for the flow conditions in the microbioreactor and it has been applied to similar issues in microtiter plates (Hermann et al, 2002 …”