Miniaturized growth systems for heterogeneous culture collections are not only attractive in reducing demands for incubation space and medium but also in making the parallel handling of large numbers of strains more practicable. We report here on the optimization of oxygen transfer rates in deep-well microtiter plates and the development of a replication system allowing the simultaneous and reproducible sampling of 96 frozen glycerol stock cultures while the remaining culture volume remains frozen. Oxygen transfer rates were derived from growth curves of Pseudomonas putida and from rates of oxygen disappearance due to the cobalt-catalyzed oxidation of sulfite. Maximum oxygen transfer rates (38 mmol liter 21 h 21 , corresponding to a mass transfer coefficient of 188 h 21 ) were measured during orbital shaking at 300 rpm at a shaking diameter of 5 cm and a culture volume of 0.5 ml. A shaking diameter of 2.5 cm resulted in threefold-lower values. These high oxygen transfer rates allowed P. putida to reach a cell density of approximately 9 g (dry weight) liter 21 during growth on a glucose mineral medium at culture volumes of up to 1 ml. The growth-and-replication system was evaluated for a culture collection consisting of aerobic strains, mainly from the genera Pseudomonas, Rhodococcus, and Alcaligenes, using mineral media and rich media. Cross-contamination and excessive evaporation during vigorous aeration were adequately prevented by the use of a sandwich cover of spongy silicone and cotton wool on top of the microtiter plates.

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Methods for Intense Aeration, Growth, Storage, and Replication of Bacterial Strains in Microtiter Plates

Publisher Summary At the heart of the liquid-phase processes, gas scrubbing process, manufacturing of pure products, and biological systems, there exists the absorber or the reactor of a particular configuration best suited to the chemical absorption or reaction being carried out. Its selection, design, sizing, and performance depend on the hydrodynamics and axial dispersion, mass and heat transfer, and reaction kinetics. This chapter focuses on the subject of mass transfer with chemical reaction. It presents the techniques, results, and opinions on mass-transfer coefficients and interfacial areas in most types of absorbers and reactors. To study gas–liquid mass-transfer phenomena, it is convenient to consider steady-state situations in which the composition of the gas and the liquid are statistically constant when averaged over time in a specified region, such as a short, vertical slice of a tubular column or the entire volume of a single-compartment agitated vessel. Useful predictions have been developed for describing the behavior of complicated systems, using highly simplified models that simulate the situation for practical purposes without introducing a large number of parameters. The procedure differs depending on whether physical or chemical absorption is involved.

Mass-Transfer Rates in Gas-Liquid Absorbers and Reactors

Chemical Engineering Use of Catalyzed Sulfite Oxidation Kinetics for the Determination of Mass Transfer Characteristics of Gas-Liquid Contactors

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.

Characterization of gas–liquid mass transfer phenomena in microtiter plates

A critical review and experimental verification of the correct use of the dynamic method for the determination of oxygen transfer in aerated agitated vessels to water, electrolyte solutions and viscous liquids

The kinetics of oxidation of aqueous sodium sulphite solution

The influence of diffusivity on liquid phase mass transfer in solutions of electrolytes

The chemical method for the determination of the interfacial area: The influence of absorption rate on the hold-up and on the interfacial area in a heterogeneous gas—liquid system

V. Linek

Papers

The kinetics of oxidation of aqueous sodium sulphite solution

The influence of diffusivity on liquid phase mass transfer in solutions of electrolytes

The chemical method for the determination of the interfacial area: The influence of absorption rate on the hold-up and on the interfacial area in a heterogeneous gas—liquid system

The kinetics of oxidation of aqueous sulphite solutions in the presence of cobalt(II) sulphate

The influence of surface films of electrolyte solutions upon mass transfer between gas and liquid