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Our areas of interest include the upgrade of waste streams and surplus products through bacterial cultivation for the production of biopolyesters, the use of multiphase bioreactors (modeling of hydrodynamics and mass transfer; scaling-down for fast screening), and study of biocatalysis in aqueous-organic media with whole cells. The latter naturally requires an understanding of the mechanisms of bacterial cell adaptation to stressful conditions, such as the presence of solvents. The cell population, either in the form of attached or freely suspended cells, should maintain a high viability through cofactor regeneration dependent processes, such as the production of terpenes in multistep reactions. Most of the bacterial cells used in our laboratory are highly hydrophobic in character and adhere easily both to surfaces and other cells. Our studies on biofilms aim at developing strategies to promote and/or prevent the formation of biofilms. Work to help the understanding of cell partition (between the aqueous and the organic phases and the interface), morphology (cell size and shape, cell clustering), and physiology (viability, membrane potential, intracellular pH) are in progress.
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The Technique
At a certain stage we were in need of fast, simple, and reliable methods for monitoring the microbial population, during the time-course of biotransformations and biodegradations. During the studies carried out with biofilms, it was necessary to develop a quantification method to assess the extent of cell aggregation and analysis of the structure of the biofilm formed. Some of the solvents tested as growth substrate for the cells in biofilms are known to interfere with techniques such as fluorescence in situ hybridization. A confocal laserscanning microscope, usually used to acquire three-dimensional (3-D) data, was not available at our laboratory. Because we could not afford sophisticated equipment, we decided “to keep it simple.” Therefore, we've developed a technique that allows the assessment of 3-D data on the structure of biofilms from two-dimensional (2-D) images obtained with an optical microscope equipped with fluorescence light. A relation between the amount of light that passes through the biofilm and the number of cells in the vertical plane allows the construction of 3-D images showing the surface of the biofilm. The effect of organic solvents on the type of biofilm formed can thus be studied. We had previously achieved significant advances (e.g., a method to observe 3-D organic solvent drops with a standard optical microscope) using fluorescence microscopy and image analysis. The simplicity of the techniques and the equipment required is likely to be helpful to other laboratories.
Assessment of three-dimensional biofilm structure using an optical microscope, p. 616.
