Combined use of confocal laser scanning microscopy (CLSM) and Raman microscopy (RM): investigations on EPS-Matrix

Confocal laser scanning microscopy (CLSM) was applied in combination with Raman microscopy (RM) for the characterization of heterotrophic biofilms. Compared to CLSM, RM allows for a deeper insight into the chemical structure of extracellular polymeric substances (EPS) of the biofilm matrix. A low load of glucose (2 g m(-2)d(-1)) was applied as substrate to ensure small growth rates of the heterotrophic biofilm. To investigate the influence of hydrodynamic conditions on the chemical composition of EPS, a three funnel flow system was used, wherein biofilms were grown at Reynolds numbers of 1000, 2500 and 4000, respectively. 31 and 92 days after inoculation with activated sludge supernatant RM was applied as an additional technique to standard CLSM measurements for a more detailed analysis of the biofilm matrix. Polysaccharide-related Raman bands are in good agreement with the lectin binding analysis from CLSM. For the older biofilm, lectin binding analysis showed no change in the composition of EPS, whereas Raman spectra pointed out a change of EPS composition from predominantly polysaccharides to predominantly (glyco) proteins. For the applied substrate condition no significant influence of the Reynolds number on the chemical properties was observed.