The formation of a multispecies denitrifying biofilm was monitored by confocal scanning laser microscopy (CSLM). The time series of tri-dimensional reconstitutions was used to develop a methodology for biofilm structural characterization based on the identification of axis of development. The algorithm is an adaptation of the standard medial axis transform technique (MAT) and was applied to both 2D sections and to the 3D stack. The use of development axis as the primary structural feature is a well-established practice to the study of morphogenesis of superior organisms. The extension of the concept to microbial aggregates is justified by the experimental realization of its highly structured nature. The determination of development axis (DA) is scalable and can be applied to structures with time resolution (effectively corresponding to four dimension structures) in order to define unifying measures of biofilm morphogenesis. Practical applications of DA characterization include the determination of how density of specific biofilms will be reflected in pore turtuosity, which in turn will condition the internal mass transfer limitations.

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