In situ mapping of nitrifiers and anammox bacteria in microbial aggregates by means of confocal resonance Raman microscopy.

In the present work the exploration of microbial communities by confocal resonance Raman microscopy (CRRM) is reported. Using the resonance Raman effect of cytochrome c (Cyt c) we were able to record the microbial distribution of nitrifiers and anammox bacteria directly in their natural environment without the need of sample preparation. For this new non-invasive investigation a reference database of bacteria assumed to be found in microbial aggregates obtained from biological wastewater treatment was created. Reference spectra of enriched cultures of the interesting bacteria were taken by means of optical tweezers. Significant spectra were achieved in less than 1 s (excitation wavelength 532 nm, laser power 9 mW) due to the resonance Raman effect. In addition, the impact of different parameters on the reference spectra, such as integration time and culture conditions, was analysed. We successfully demonstrate the grouping of bacteria down to strain level based on the heterogeneity of the resonance Raman spectra of the heme protein Cyt c.