Kinetics of biofilm formation by drinking water isolated Penicillium expansum

Abstract

Current knowledge on drinking water (DW) biofilms has been obtained mainly from studies on bacterial biofilms. Very few reports on filamentous fungi (ff) biofilms are available, although they can contribute to the reduction in DW quality. This study aimed to assess the dynamics of biofilm formation by Penicillium expansum using microtiter plates under static conditions, mimicking water flow behaviour in stagnant regions of drinking water distribution systems. Biofilms were analysed in terms of biomass (crystal violet staining), metabolic activity (resazurin, fluorescein diacetate and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide [MTT]) and morphology (epifluorescence [calcofluor white M2R, FUN-1, FDA and acridine orange] and bright-field microscopies). Biofilm development over time showed the typical sigmoidal curve with noticeable different phases in biofilm formation (induction, exponential, stationary, and sloughing off). The methods used to assess metabolic activity provided similar results. The microscope analysis allowed identification of the involvement of conidia in initial adhesion (4 h), germlings (8 h), initial monolayers (12 h), a monolayer of intertwined hyphae (24 h), mycelial development, hyphal layering and bundling, and development of the mature biofilms (≥48 h). P. expansum grows as a complex, multicellular biofilm in 48 h. The metabolic activity and biomass of the fungal biofilms were shown to increase over time and a correlation between metabolism, biofilm mass and hyphal development was found.

Keywords: biofilm analysis; biomass determination; filamentous fungi; metabolic stains; microscopy; microtiter plate.