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Microbial biogeography in terrestrial and freshwater ecosystems is mainly dominated by community biofilm lifestyles. Here, we describe applications of computer-assisted microscopy using CMEIAS (Center for Microbial Ecology Image Analysis System) bioimage informatics software for a comprehensive analysis of river biofilm architectures and ecology. Natural biofilms were developed for four summer days on microscope slides of plain borosilicate glass and transparent polystyrene submerged in the Red Cedar River that flows through the Michigan State University campus. Images of the biofilm communities were acquired using brightfield and phase-contrast microscopy at spatial resolutions revealing details of microcolonies and individual cells, then digitally segmented to the foreground objects of interest. Phenotypic features of their size, abundance, surface texture, contour morphology, fractal geometry, ecophysiology, and landscape/spatial ecology were digitally extracted and evaluated by many discriminating statistical tests. The results indicate that river biofilm architecture exhibits significant geospatial structure in situ, providing many insights on the strong influence that substratum hydrophobicity–wettability exert on biofilm development and ecology, including their productivity and colonization intensity, morphological diversity/dominance/conditional rarity, nutrient apportionment/uptake efficiency/utilization, allometry/metabolic activity, responses to starvation and bacteriovory stresses, spatial patterns of distribution/dispersion/connectivity, and interpolated autocorrelations of cooperative/conflicting cell–cell interactions at real-world spatial scales directly relevant to their ecological niches. The significant impact of substratum physicochemistry was revealed for biofilms during their early immature stage of development in the river ecosystem. Bioimage informatics can fill major gaps in understanding the geomicrobiology and microbial ecology of biofilms in situ when examined at spatial scales suitable for phenotypic analysis at microcolony and single-cell resolutions.

Influence of Substratum Hydrophobicity on the Geomicrobiology of River Biofilm Architecture and Ecology Analyzed by CMEIAS Bioimage Informatics