Nearly all bacteria live in microbial assemblages such as biofilms and granules with a continuum of chemical gradients. Each of these layers provide a niche selective for specific bacteria and functions. A gentle extraction approach based on a cation exchange resin and defined shear was employed to collect single layers in microbial granules for microbial and chemical analysis. The use of an existing cation-exchange resin for gradual biomass disintegration, combined with NGS and proteome analysis have revealed a promising approach for investigating the identity and functions in multilayered biofilm structures.