Optimisation of nutrient removal processes requires a detailed understanding of the microbial physiologies actually occurring within the biomass of different treatment zones, and also knowledge of how these communities respond to environmental factors. This paper describes a suite of four independent and complementary, microbiological techniques, utilised to obtain detailed assessments of wastewater bacterial biomass. Examples of these techniques are shown as applied to biofilms from two surface-flow wetlands used for treating sewage effluent. Intact biofilms were prepared via freezing, followed by cryosectioning and direct microscopic observation. This approach was combined with prior, in situ incubation of biofilm with radiolabelled thymidine to assess in situ bacterial growth rates. Individual bacterial cells were assessed microscopically for in situ respiratory activity and phylogenetic identity, respectively through use of an inducibly-fluorescent redox stain (CTC), and fluorescent in situ hybridisation (FISH) of ribosomal RNA (rRNA). These complementary techniques enabled assessment of individual biofilm clusters according to their in situ status (growth, respiration, spatial location and phylogenetic affiliation). This approach detected significant spatial variations in biofilm; limited the bias inherent in data obtained using any one technique, and provided greater detail of subpopulations within the microbial community.

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