Microplates are widely used to increase throughput while decreasing sample volume. The multidishes, available in 24- or 6-well format, contain either an oxygen sensor (OxoDish®) or pH sensor (HydroDish®) at the bottom of each well. Filled with the sample the multidish is put onto the Sensor Dish® Reader (SDR) and the sensors are read out noninvasively. The SDR can be used in incubators and on shakers. (For more information on the SDR, see GEN, March 15, 2011, p. 52).
Shake flask cultures are used across a broad field, including industrial bioprocess development. PreSens has developed disposable sensors that are mounted inside Erlenmeyer flasks that are used with the Shake Flask Reader (SFR).
The system, which enables simultaneous online monitoring of both oxygen and pH values within up to nine shake flasks, also allows continuous insight into oxygen supply and metabolic activity of a particular culture. The system is mounted on a conventional shaker and is compatible with nearly all shaking systems.
To test the performance quality of the SFR system and reproducibility of retrieved data, E. coli DH5α was cultivated in baffled glass and unbaffled plastic shake flasks. The flasks were equipped with an oxygen sensor and, in the case of the plastic flasks, with an additional pH sensor.
Shake flasks with similar kLa values were used for parallel experiments to investigate the reproducibility of measurements. (The kLa is a specific coefficient that quantifies oxygen mass transfer in a given cultivation system.)
All cultivations were carried out in duplicate at 37°C and at an initial shaking frequency of 90 rpm. During cultivation in baffled glass shake flasks an accelerating decline of DO could be observed just 2 h after inoculation with an optical density (OD600 nm) of 0.1 (Figure 1A).
In order to avoid oxygen limitation, the shaking rate was increased: from 90 to 120 rpm after 2 hours of cultivation, and again from 120 to 170 rpm shaking rate after 3.5 hours. During the first two hours exponential growth was evident. In the subsequent phase different substrates, available in the used LB medium, were consumed.
Within that period rearrangement of the cellular metabolism could be observed, where DO concentration showed some distinct peaks. After a total of five hours the shaking frequency was reduced to 120 rpm. Due to the gradually decreasing respiratory activity of the culture, DO slowly increased.
In contrast, the profiles of DO and pH of cultures in unbaffled shake flasks (Figure 1B) revealed significant differences: the supply of oxygen was clearly insufficient. Unbaffled shake flasks were not so well suited for aerobic cultivation of fast-growing E. coli.
The chemical optical measurement technology can also be used for improving process quality in bioreactors. Integrated flow-through cells or optical sensor patches, attached to the transparent bioreactor wall, make it possible to take noninvasive measurements.
Flow-through cells are integrated via Luer-lock connectors at external conduits. The additional monitoring of CO2, as well as oxygen and pH measurements, in bioreactors allows adjustments to be made. For example, if the CO2 level is too high, oxygen could be added to the headspace of the sample.