Introduction

Microbial communities or aggregates also known as biofilm systems may be divided into stationary ones and mobile ones. Stationary ones are the classical microbial films usually on solid surfaces. Mobile ones have been named with a variety of terms such as assemblages, aggregates, flocs, snow, or mobile biofilms.1 The techniques described in the following chapter apply to both biofilms and flocs. Aquatic aggregates (river, lake, marine, technical) may be very different in terms of size, composition, density, and stability.2 Lotic aggregates are structurally very stable as they are exposed to a constant shear force resulting in relatively small aggregates (« 5 to 300 ^m), whereas lake or marine snow may be very fragile and much larger (millimeters to meters). Both environmental aggregates are colonized to a certain degree by prokaryotic and euka-ryotic microorganisms (bacteria, algae, fungi, protozoa). The bacterial composition of environmental aggregates was studied in situ, for example, by Weiss et al.3 In comparison to natural aggregates, technical aggregates are heavily colonized mainly by bacteria, for example, in activated sludge.4 The microbial population structure of activated sludge was first analyzed in situ by Wagner et al.5 Another example for man-made aggregates are mobile biofilms growing on carrier material, for example, in fluidized bed reactors. Due to high shear force, these immobilized aggregates are extremely dense and stable.6 A major understudied component of all these microbial systems is their exopolymeric matrix.

Exopolymeric substances have correctly been referred to as the mystical substance of biofilms and aggregates7 and a challenge to properly characterize.8 The extracellular polymeric substances (EPS) are defined as organic polymers of biological origin which in biofilm systems are responsible for the interaction with interfaces.7 Although EPS are understood as extracellular polymers mainly composed of microbial polysaccharides, by definition other extracellular polymeric substances may also be present, for example, proteins, nucleic acids and polymeric lipophilic compounds.8-11 In biofilm systems we can expect two types of structural polymeric carbohydrate structures. First, those associated with cell surfaces and second, those located extracellularly throughout the extracellular biofilm matrix. The importance of EPS in flocs and biofilm systems is fundamentally twofold: (i) they represent a major structural component of flocs and (ii) they are responsible for sorption processes.12,13 Particularly in complex environmental systems, the EPS are difficult if not impossible to chemically characterize on the traditional basis of isolating single polymer species. Chemical approaches are limited to pure culture, chemically defined systems. Despite this problem, chemical quantification of EPS constituents in biofilm systems have been reported.14 These confirm the complex nature of the material and the extensive range of polymers present. Increasingly attempts have been made to examine natural biofilm and floc polysaccharides in situ.1,8,15-18

The critical need for in situ analyses and visualization of EPS is due to its complex chemical nature and the importance of its molecular structure in its behavior. Indeed, the challenge remains to characterize its chemical composition in the context of its biological form. To do this we have proposed a variety of in situ methods based on the application of chemical probes and 1P (1-photon) and 2P (2-photon) laser microscopy. In addition, synchrotron radiation using the interaction of x-rays with the molecular structure of intact hydrated biofilms has proven an effective approach. In this overview we assess in situ analyses of EPS using light of various wavelengths ultraviolet, visible, infrared, and x-ray in combination with targeted probes to assess the structure of biofilms and flocs.

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