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

Abstract

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.

Illuminating the Tiny World: A Navigation Guide for Proper Raman Studies on Microorganisms

“…Environmental changes could be the short- or long-term transportation from one point to another, or even plain storage in a fridge or freezer [ 68 , 69 ]. Factors such as the cultivation medium [ 70 , 71 , 72 ], the cultivation temperature [ 71 ], cultivation time [ 6 ], or even the CO 2 and O 2 levels [ 73 , 74 , 75 , 76 , 77 ] may have an impact on the phenotype of the bacterium. A genetically homogeneous colony on a Petri dish may result in a totally different outcome than a genetically heterogeneous liquid culture ( Figure 4 ).…”

“…This resulted in the same Raman bands as cytochrome c, yet cytochrome c was found to be elevated in the oxygen-deprived cells. Furthermore, cells with a high ferrous cytochrome c concentration were observed in deprived Nitrosomonas eutropha and Nitrosospira samples, which might indicate continuous electron storage at the time of assessment [ 73 ].…”

Analysis and differentiation of toxic and non-toxic cyanobacteria using Raman spectroscopy

“…Proteins and other components from the inner structure of cells can be analyzed non-invasively and label-free with Raman spectroscopy. [7][8][9][10][11][12] Signals from algae and bacteria can be measured and used for analysis or differentiation of algae, bacteria or cyanobacteria. [13][14][15][16] Also cyanotoxins can be investigated in detail and distinguished using Raman spectroscopy.…”

Raman-based analysis and structural differentiation of potentially harmful algae and cyanobacteria

“…Raman spectroscopy is a promising tool to enable such an analysis as well. It is capable to analyze components, proteins and inner structures of bacteria or cells with high specificity [6][7][8][9][10]. The method is non-invasive, label-free and enables a cost and time effective measurement.…”