Monitoring Oxygen Consumption During a Microbial Toxicity Test
Background
The Arthrobacter globiformis sediment contact test is an incubation with a standardised culture of a ubiquitous bacterium to assess possible toxicity of sediments or other solid materials in a dose-response manner. Inhibition of dehydrogenase activity is measured over one hour in a standard microtiter format. Gentle shaking before measurements ensures homogenisation and some oxygen input, but it is unclear how much oxygen levels decrease during the incubation. We used the 96 well FirePlate O2 to find out if oxygen consumption during the test correlates with observed enzymatic activity, and if critical oxygen concentrations may have an effect on the outcome of the test.
Set-Up/Sample
The typical Arthrobacter globiformis sediment contact test uses fluorescence-based detection of resorufin which is formed by reduction of resazurin. As this fluorescence was assumed to interfere with the oxygen measurement, a duplicate plate was prepared that received the buffer only without resazurin. Both were incubated at 30 °C (Figure 1). We chose a river sediment rich in organic matter (7.6%) as test sample and included 3,5dichlorophenol as a positive control.
Results
96 well FirePlate O2
Figure 1 Sensor setup in temperature controlled shaking chamber
Figure 2 Exemplary oxygen evolution data (liquid and gas phase) with fits from kinetic model. Oxygen concentrations in Arthrobacter globiformis toxicity test in 60 minute incubations
Figure 3 Exemplary oxygen and hydrogen evolution data Snapshot from the PyroScience software during the A. globiformis incubation. Oxygen concentrations are given in values of µmol/L on the left. An automatically color-coded map of the well plate is displayed on the right, allowing live monitoring of relative changes in oxygen concentrations between the wells
Oxygen levels fluctuated during the first 15 min of incubation and then decreased linearly in all wells, allowing calculation of respiration rates (Figure 2). Regarding the 3,5-dichlorophenole controls, dehydrogenase inhibition and oxygen consumption were not correlated. Despite dose-dependent inhibition from 35-80% the oxygen consumption rates were all between 4 and 5 µmol min-1. The tested sediment also inhibited the dehydrogenase activity. While trends were unclear for 25-100% sediment, inhibition decreased from 40-20% with 12.5-3.125% sediment, and at the same time oxygen consumption rate doubled from 3-4 µmol min-1 to 7-8 µmol min-1. We explain this pattern by opposed effects of different sediment constituents: while inhibitory substances were diluted, the remaining carbon sources could be metabolized more quickly and intensely.
Conclusion
The setup provided by PyroScience allowed us to quantify oxygen consumption in sediment contact tests and elucidate its relation to observed toxicity which is not a simple linear correlation. We could also show that oxygen is unlikely to compromise the validity of the contact test with oxidized sediments.
Katrin Wendt-Potthoff, Corinna Völkner, Patrick Aurich
Helmholtz-Centre for Environmental Research, UFZ
Department Lake Research
4/6/26