The SFR vario offers online monitoring of oxygen, biomass and pH or CO₂ - simultaneously. Online measured biomass data can be correlated with parameters like optical density, cell dry weight, or cell concentration. This way it is possible to get real-time information on e. g. OD₆₀₀ development. The device optics can read out one oxygen and one pH or CO₂ (CO₂ Technical Data) sensor spots - integrated in the ready-to-use cultivation vessels - and also comprise a dedicated optical set-up for biomass monitoring. The oxygen uptake rate (OUR) can be calculated from the slope of the online oxygen measurements. The system has two long-lasting, rechargeable batteries, and is compatible with any standard shaking incubators. Up to 4 SFR vario can be controlled from the SFR vario software. Measurement data are transferred wirelessly via Bluetooth to a PC / notebook.

· Simultaneous real-time measurement of O₂, biomass & pH or CO₂

· Automatic OUR calculation

· Online measurement of optical density, cell dry weight, cell concentration by correlation with biomass measurements

· Parallel measurements in up to 4 shake flasks

· Wireless data transfer enables easy integration

· Bioprocess development & media optimization

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Applications

Biomass, O₂ & pH Monitoring during Growth of E. coli

E. coli K12 shows distinct diauxic growth in medium containing glucose and lactose, which could be monitored with the SFR vario. Measurements were stopped several times for offline sampling - to determine substrate concentration - showing in gaps in the graphs. In the first growth phase glucose is consumed (1). The system reveals less precision in the lower biomass range, but this improves with increasing cell density. While the acetic acid produced by the bacteria lowers the pH of the medium the strong decrease of oxygen concentration down to 10 % a. s. indicates high metabolic activity. When the glucose concentration drops below 0.1 g L^-1 after about 6 h (determined offline, data not shown here) growth stops and the oxygen level rises rapidly. This also shows in a small plateau (zoomed insert) in the online measured biomass curve (2). During this phase the bacteria adjust their metabolism to lactose. Recording this exact match in stop of oxygen consumption and growth could only be realized with the new online monitoring system. In the third phase E. coli grow on lactose until it is consumed and the culture turns into stationary phase (3).

Yeast Growth Phases in Complex Medium

K. marxianus shows two growth phases when cultured in shake flasks with glucose as substrate. In the first phase glucose is metabolized under aerob conditions (1), and the oxygen content decreases continuously down below 5 % a. s. . Due to metabolic products the pH decreases from  a value of 7 to 6.1. After 9 hours cultivation period the glucose is completely consumed (determined offline, data not shown here), and pH increases again. During the second - oxygen limited - phase K. marxianus shows significantly slower growth, which can be clearly observed in the scattered light (= biomass) measurements (2), while metabolizing the glucose products generated in the first phase under high oxygen demand. The simultaneous monitoring of oxygen, pH and biomass with the SFR vario offers whole new possibilities for culture monitoring and evaluation of metabolic processes.

Oxygen Uptake Rate Measurements

Measuring the oxygen uptake rate of microorganisms is a very good indicator for metabolic activity and can be used to predict the state of growth or evaluate the rate at which metabolic processes take place. The graph on the left shows the OUR of S. cerevisiae in YPD medium during shake flask cultivation. The oxygen uptake rate rises constantly during the first hours of cultivation due to exponential growth. A short drop in oxygen uptake rate indicates that substrate became limiting, and the microorganisms reduce metabolic activity while switching to metabolise another substrate. Then another phase of exponential growth sets in. The period of maximum metabolic activity with highest OUR, as well as the time point when death phase sets in (due to oxygen limitation) and OUR abruptly drops to minimum values, can be clearly determined from the graph.

Methanol Independent Expression by Pichia pastoris

Anton Glieder and his colleagues from bisy e. U. and the Institute of Molecular Biology of the Technical University of Graz published a video article (J. Vis. Exp. (143), e58589 (2019)) describing a simple shake flask cultivation system for methanol free expression by P. pastoris. Online monitoring with the SFR vario and constant slow glycerol feed were used to simulate bioreactor conditions. This approach contributed to come closer to real bioreactor conditions compared to mostly applied small scale batch cultivations.