Of microparticles and bacteria identification – (resonance) Raman micro-spectroscopy as a tool for biofilm analysis

Confocal resonance Raman microscopy is a powerful tool for the non-invasive analysis of complex biological aggregates without preparation and prior knowledge of the samples. We present the capabilities of confocal resonance Raman microscopy with a spatial resolution of 350 nm2 × 2.0 μm and excitation times of 1 s and less per recorded spectrum. Granules sampled from two sequencing batch reactors (SBR) for anaerobic ammonium oxidization (anammox) were regularly mapped in vivo for three months after SBR startup. Uncultured microorganisms and mineral particles were tracked throughout operation and identified in situ by their (resonance) Raman spectra. Co-existing microcolonies of Nitrosomonae formed the outer layer of anammox granules. Polymorph TiO2 microparticles were found embedded in the outer layer of granules overgrown with purple bacteria, indicating bacterial response to the variant toxicity of the mineral phase.

► Analysis of native bacterial aggregates with confocal (resonance) Raman microscopy.
► In vivo mapping, tracking, and identification of uncultured microorganisms in situ.
► Examining microbial and mineral content without prior knowledge of the sample.
► Polymorph TiO2 microparticles embedded in purple bacteria biofilm.
► Coexisting microcolonies of Nitrosomonae in the outer layer of anammox granules.