RESULTS:

The structural organization of four microbial communities was analysed by a novel computer program COMSTAT which comprises ten features for quantifying three-dimensional biofilm image stacks. Monospecies biofilms of each of the four bacteria Pseudomonas putida P. aureofaciens P. fluorescens and P. aeruginosa tagged with the green fluorescent protein (GFP) were grown in flow chambers with a defined minimal medium as substrate. Analysis by the COMSTAT program of four variables describing biofilm structure – mean thickness roughness substratum coverage and surface to volume ratio – showed that the four Pseudomonas strains represent different modes of biofilm growth. P. putida had a unique developmental pattern starting with single cells on the substratum growing into micro-colonies which were eventually succeeded by long filaments and elongated cell clusters. P. aeruginosa colonized the entire substratum and formed flat uniform biofilms. P. aureofaciens resembled P. aeruginosa but had a stronger tendency to form micro-colonies. Finally the biofilm structures of P. fluorescens had a phenotype intermediate between those of P. putida and P. aureofaciens. Analysis of biofilms of P. aureofaciens growing on 0·03 mM 0·1 mM or 0·5 mM citrate minimal media showed that mean biofilm thickness increased with increasing citrate concentration. Moreover biofilm roughness increased with lower citrate concentrations whereas surface to volume ratio increased with higher citrate concentrations.