~ Pergamon 0043-1354(95)00054-2 Wat. Res. Vol. 29~ No. 10, pp. 2227-2245, 1995 Elsevier ScienceLtd. Printed in Great Britain REVIEW PAPER BIOFILM CHARACTERIZATION AND ACTIVITY ANALYSIS IN WATER AND WASTEWATER TREATMENT V. LAZAROVA *@ and J. MANEM Centre of International Research for Water and Environment (CIRSEE), Lyonnaise des Eaux, 38 rue du Pr6sident Wilson, 78230 Le Pecq, France (First received March 1994; accepted in rerised form Februa O, 1995) Abstract--Biofilm composition and activity are two important parameters for the successful operation and control of fixed film processes in water and wastewater treatment. Widely used parameters for biofilm characterization are biofilm thickness, total dry weight and total cell count. These parameters are, however, not sufficient to describe biofilm activity. Improved analytical methods and procedures are needed in order to understand the physiological behaviour of the fixed biomass. Common methods used to analyse biofilms are critically reviewed in terms of their principle, complexity, reproducibility, sensitivity and cost. Conventional and advanced analytical methods are classifed in three main categories: ( 1 ) biofilm formation and structure; (2) biofilm composition described in terms of total biofilm amount, specific biofilm components and cellular components; and (3) biomass activity descriptors. The discussion focused on the potential of these methods for accurate biofilm characterization and biomass activity estimation in fundamental research and for the practical needs of wastewater treatment operation and control. Key words--biofilm, biofilm analysis, biofilm formation, external structure, biofilm components, cellular components, biomass activity, water and wastewater treatment INTRODUCTION Biofilms have been successfully used in water treatment for over a century (Atkinson, 1975). It was not until the early 1980s, however, that the advantages of this type ofbioprocess became a focus of interest for a considerable number of researchers, not only in the field of water and wastewater treatment, but also in many other areas of biotechnology (Adler, 1987). A large number of research projects are currently being conducted on biofilm reactors for the production of bioactive substances, for plant and animal cell cultures, drinking water production and wastewater treatment. One key advantage of biofilms is the positive influence of solid surfaces on bacterial activity observed 50 years ago by ZoBell (1943) and confirmed more recently by other researchers (Hattori and Edeline, 1981; Audic et al., 1984; Doran and Bailey, 1986; Klein and Ziehr, 1990). There is considerable discussion about the mechanism, direct or indirect, which induces the greater activity of the fixed biomass (Loosdrecht et al., 1990). Some authors (Fletcher, 1986, Klein and Ziehr, 1990) attribute this phenom- enon to physiological modifications of attached cells: e.g. the ~switching on" of some genes at the surface as described by Dagostino et al. (1991). Some other *Author to whom all correspondence should be addressed. authors stated that changes in the cell surrounding increase either the local concentration of nutrients and enzymes (Rittmann et al., 1986; Manem, 1988) or the "selective" effect of the biofilm exo- polymer matrix (molecular sieve or ion exchanger role) towards toxic or inhibiting substances (Blenkinsopp and Costerton, 1991). It has been demonstrated that fixed cultures are less strongly affected than suspended cultures by changes in environmental conditions (temperature, pH, nutrient concentrations, metabolic products and toxic substances). This effect has been observed in biofilms in drinking water distribution systems (Pedersen, 1990) and in autotrophic cultures of nitrifying bacteria (Duddles et al., 1974; Olem and Unz, 1980) and ferro-oxidizing bacteria (Nakamura et al., 1986; Karamanev and Nikolov, 1988). Biofilm activity is not proportional to the quantity of fixed biomass, but increases with the thickness of biofilm up to a determined level, termed the "active thickness" (Kornegay and Andrews, 1968; LaMotta, 1976). Above this level, the diffusion of nutrients becomes a limiting factor, thus differentiating an ~active" biofilm from an "inactive" biofilm. A stable, thin and active biofilm thus offers numerous advantages in the water and wastewater treatment. In order to achieve this objective, it is important to develop methods for fixed biomass activity estimation which are not only simple and rapid, but also sensitive, precise and representative. 2227