Small Sample Stress: Probing Oxygen-Deprived Ammonia-Oxidizing Bacteria with Raman Spectroscopy In Vivo

The stress response of ammonia-oxidizing bacteria (AOB) to oxygen deprivation limits AOB growth and leads to different nitrification pathways that cause the release of greenhouse gases. Measuring the stress response of AOB has proven to be a challenge due to the low growth rates of stressed AOB, making the sample volumes required to monitor the internal stress response of AOB prohibitive to repeated analysis. In a proof-of-concept study, confocal Raman microscopy with excitation resonant to the heme c moiety of cytochrome c was used to compare the cytochrome c content and activity of stressed and unstressed Nitrosomonas europaea (Nm 50), Nitrosomonas eutropha (Nm 57), Nitrosospira briensis (Nsp 10), and Nitrosospira sp. (Nsp 02) in vivo. Each analysis required no more than 1000 individual cells per sampling; thus, the monitoring of cultures with low cell concentrations was possible. The identified spectral marker delivered reproducible results within the signal-to-noise ratio of the underlying Raman spectra. Cytochrome c content was found to be elevated in oxygen-deprived and previously oxygen-deprived samples. In addition, cells with predominantly ferrous cytochrome c content were found in deprived Nitrosomonas eutropha and Nitrosospira samples, which may be indicative of ongoing electron storage at the time of measurement.