Bacteria cells within biofilms are physiologically distinct from their planktonic counterparts. In particular they are more resistant to detrimental environmental conditions. In this study, we monitored the evolution of the phospholipid composition of the inner and outer membranes of P. aeruginosa during the biofilm formation (i.e., from 1-, 2-, to 6-day-old biofilm). Lipidome analyses were performed by electrospray ionization mass spectrometry. In addition to the lipidomic analysis, the fatty acid composition was analysed by gas chromatography/mass spectrometry. We found that the lipidome alterations of the inner and the outer membranes varied with the biofilm age. These alterations in phospholipid compositions reflect a higher diversity in sessile organisms than in planktonic counterparts. The diversity is characterized by the presence of PE 30∶1, PE 31∶0 and PG 31∶0 for the lower masses as well as PE 38∶1, 38∶2, 39∶1, 39∶2 and PG 38∶0, 38∶1, 38∶2, 39∶1, 39∶2 for the higher masses. However, this lipidomic feature tends to disappear with the biofilm age, in particular the high mass phospholipids tend to disappear. The amount of branched chains phospholipids mainly located in the outer membrane decreased with the biofilm age, whereas the proportion of cyclopropylated phospholipids increased in both membranes. In bacteria present in oldest biofilms, i.e., 6-day-old, the phospholipid distribution moved closer to that of planktonic bacteria.

Many studies using genetic and proteomic approaches have revealed phenotypic differences between planktonic and sessile bacteria but the mechanisms of biofilm formation and the switch between the two growth modes are not well understood yet. In this study, we focused on inner membrane lipidome modifications when Pseudomonas aeruginosa cells were grown as biofilm. Lipid analyses were performed by Electrospray Ionization Mass Spectrometry. Results showed a drastic decrease of the uneven-numbered chain phospholipids and a slight increase of long chain PEs in sessile organisms as compared with planktonic counterparts, suggesting a better lipid stability in the bilayer and a decrease in membrane fluidity. The impact of sessile growth on lipid domains was then investigated by Brewster Angle Microscopy (BAM) and Atomic Force Microscopy (AFM). Observations showed that inner membrane lipids of P. aeruginosa formed domains when the pressure was close to physiological conditions and that these domains were larger for lipids extracted from biofilm bacteria. This is coherent with the mass spectrometry analyses.

Biodeterioration can be defined as any undesired change of the properties of a material caused by biological activity of living organisms. The biodeterioration of stone materials is related to the production of pigments (aesthetic action), to cell metabolism (biochemical action) and to the mechanical action of the biomass colonizing the material during its growth (physical action). Quantification of the sessile biomass and characterization of microbial communities colonizing stone are essential first steps to ensure the diagnosis of biodeterioration processes and to implement control strategies and appropriate treatment. Different destructive and non-destructive approaches can be used to sample stone specimens from monuments: scraping, swab using, and cutting. Different analytical methods can be used depending on the type of microorganism sought: determination of chlorophyll content and color analysis for pigmented microorganisms; measurement of in situ physiological activity of surface microcolonies by applying fluorogenic substrate analogues or confocal laser scanning microscopy observations after CTC staining for active biomass; scanning or transmission electron microscopy observation for biofilm visualization; enzymelinked immunosorbent assay for the investigation of both microorganisms that can and cannot be cultured; classical microbiological methods, which consist in cultivation of microorganisms on synthetic media; and molecular methods for the study of microbial biodiversity based on the polymorphism of molecular markers using PCR, hybridization, classical or high throughput sequencing. The aim of this review is to present basics of the different biodeterioration mechanisms and to focus on the main techniques that can be used to characterize and quantify the biodeterioration biomass.