3D reconstruction of Artificially Structured Microbial Consortia (ASMC) by image analysis

A biofilm is formed when microorganisms are attached to a surface and, within the whole biofilm; microorganisms are only a small volume fraction, which is complemented by the extracellular matrix of the structure. Inside the structure, fluids flow carrying, in and out, dissolved nutrients and intermediate metabolic products, which can be consumed by other bacterial species in the biofilm. In order to study these phenomena, Artificially Structured Microbial Consortia (ASMC) were constructed using Acinetobacter sp. C6 and Pseudomonas putida R1, this strain is a Green Fluorescent Protein (GFP) producer that was monitored. ASMC were constructed for degrading benzyl alcohol, which is initially transformed into benzoic acid, by Acinetobacter sp. C6, and then it is consumed until full aerobic degradation by Pseudomonas putida R1. The progress in time of ASMC was followed using confocal laser scanning microscopy (CLSM) obtaining cross-section micrographies for image analysis. 3D reconstruction of these images was achieved using information from the plane and confocal cross-sections of the ASMC. This image analysis technique takes into account the edge of each cross-section, which is defined by the GFP producer species. Pixels intensity was estimated using data from bitmap files that were obtained after processing the confocal images; this information was useful for studying biofilm morphology and carrying out 3D image reconstruction.