Microbial flocs, which occur in most freshwater and marine environments, are built by numerous bacterial species that grow as single cells, filaments, or cell aggregates. Flocs also contain extracellular polymeric substances (EPS), which are synthesized by the microorganisms, as well as particulate organic and inorganic matter and cavities between these components. Flocculation is advantageous for microorganisms: the matrix of dense flocs provides some protection from grazing protozoa. Floc bacteria involved in mutualistic symbiotic interactions benefit from the close proximity to their partners. Changes of the environmental conditions outside of flocs may have attenuated effects inside of flocs, where microorganisms to a certain degree create and maintain their own microenvironments. Concentration gradients of soluble nutrients, waste compounds, and gases form within flocs due to diffusion barriers

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and microbial metabolic activity. The combination of a complex physical structure with chemical gradients creates in the same floc microhabitats for different functional groups of microorganisms, which may be involved in essential nutrient recycling processes. Flocs also provide niches for species that would otherwise not survive in a particular environment. For example, in aerobic habitats, oxygen is consumed by aerobic organisms, which are located in the outer layers of flocs. Therefore, the central floc regions are depleted in oxygen and can be colonized by microaerophilic or even oblig-ately anaerobic bacteria, which are sensitive to oxygen and do not occur in the same habitat outside of flocs or biofilms.1 Consequently, flocculation promotes a higher microbial biodiversity in ecosystems.

Any attempt to determine how flocculation influences the performance and stability of ecosystems requires a detailed knowledge of the structure and function of the microbial communities in flocs. Traditional microbiological methods, which are based on the isolation and cultivation of bacteria, fail to provide such insight because most microorganisms in nature are recalcitrant to cultivation.2 The following text explains how cultivation-independent molecular techniques can be applied to study complex microbial communities in flocs. Since an encompassing description of all available molecular tools would be beyond the scope of this chapter, special emphasis is given to the rRNA approach2 and to commonly used in situ methods, which allow monitoring microorganisms directly in their habitats. The section on methods is followed by a case study, which deals with the discovery and functional analysis of uncultured Nitrospira-like bacteria. These organisms are important nitrite oxidizers in natural habitats and in wastewater treatment plants — engineered systems where flocculation plays a critical role.

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