The influence of intracellular storage material on bacterial identification by means of Raman spectroscopy.

Previous studies dealing with bacterial identification by means of Raman spectroscopy have demonstrated that micro-Raman is a suitable technique for single-cell microbial identification. Raman spectra yield fingerprint-like information about all chemical components within one cell, and combined with multivariate methods, differentiation down to species or even strain level is possible. Many microorganisms may accumulate high amounts of polyhydroxyalkanoates (PHA) as carbon and energy storage materials within the cell and the Raman bands of PHA might impede the identification and differentiation of cells. To date, the identification by means of Raman spectroscopy have never been tested on bacteria which had accumulated PHA. Therefore, the aim of this study is to investigate the effect of intracellular polymer accumulation on the bacterial identification rate. Combining fluorescence imaging and Raman spectroscopy, we identified polyhydroxybutyrate (PHB) as a storage polymer accumulating in the investigated cells. The amount of energy storage material present within the cells was dependent on the physiological status of the microorganisms and strongly influenced the identification results. Bacteria in the stationary phase formed granules of crystalline PHB, which obstructed the Raman spectroscopic identification of bacterial species. The Raman spectra of bacteria in the exponential phase were dominated by signals from the storage material. However, the bands from proteins, lipids, and nucleic acids were not completely obscured by signals from PHB. Cells growing under either oxic or anoxic conditions could also be differentiated, suggesting that changes in Raman spectra can be interpreted as an indicator of different metabolic pathways. Although the presence of PHB induced severe changes in the Raman spectra, our results suggest that Raman spectroscopy can be successfully used for identification as long as the bacteria are not in the stationary phase.