Long-term biofilm processes are influenced by the interplay of biofilm accumulation and detachment, which in turn depend partially on the biofilm structure and composition. In this study a combination of confocal laser scanning microscopy (CLSM) and nuclear magnetic resonance (NMR) spectroscopy was applied to analyze biofilm structure, composition and molecular mobility. Whereas CLSM delivers information about the structure of biofilms the NMR measurement provides detailed but not locally resolved information about the chemical composition of biofilm constituents. Heterotrophic mixed-species biofilms were cultivated in rotating annular reactors exposed to different flow conditions and glucose concentrations in order to obtain biofilms with diverse architectural structures. The growth state of the biofilms appeared to influence the composition of biofilm and detached biomass. The difference in the C-13 NMR spectra between the differently structured biofilms or between biofilm and detached biomass was small, except for the still exponential growing biofilm supplied with the highest glucose concentration. More information was gained from the mobility of specific molecular groups within the biofilm biomass. Molecules within the biofilm biomass of the non-filamentous biofilms were more strongly bound than the molecules within the respective detached biomass. Glucose starvation resulted in a reduction in the biofilm molecular mobility. The opposite was observed in the filamentous biofilm. In this case, the molecular mobility in the biofilm increased after starvation and the molecules in the detached biomass were bound more strongly than in the respective biofilm biomass. It could be shown that the combination of CLSM and C-13 NMR spectroscopy is a promising approach to analyze the interactions between biofilm architecture, composition or growth state and biofilm detachment. (C) 2010 Elsevier Ltd. All rights reserved.