Fluorescent proteins as in-vivo and in-situ reporters to study the development of a Saccharomyces cerevisiae yeast biofilm and its invasion by the bacteria Escherichia coli

Abstract

This work deals with the bacterial contamination of yeast, both as biofilm and in the planktonic phase. A model continuous system using self-fluorescent microorganisms was proposed to perform in vivo and in situ studies of a mixed biofilm. The yeast strain was inoculated first while the bacteria were added few days later to mimic a contamination. Supports sampled during the experiment were observed by scanning confocal laser microscopy. The behaviour of the microorganisms in real process conditions was then analysed without any treatment that could modify their physiology and/or damage the community structure. Using image analysis, the characteristics of biofilm development (microorganism ratio, 3D-organisation, growth rates) were studied and compared to the behaviour of the suspended cells in the bioreactor. Based on the biovolumes (volume occupied by each microorganism), the growth rates in biofilm for the bacteria and the yeasts were determined at 0.10 and 0.03 h(-1) respectively, while the imposed dilution rate was 0.10 h(-1). Even though the ability of yeast to develop biofilm was demonstrated, its capacity remained very low compared to that of the bacteria which quickly invaded and covered the whole yeast biofilm. This approach makes an original and powerful tool to study the competition phenomena occurring in model biofilms.