Solvent toxicity in organic-aqueous systems analysed by multivariate analysis
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The effect of several solvents present in a biphasic reaction system on cells of Rhodococcus erythropolis DCL14, Xanthobacter Py2, Arthrobacter simplex and Mycobacterium sp. NRRL B-3805 was evaluated. These four strains have been widely exploited, from bioremediation to the production of fine chemicals, in two-phase reaction media. The solvents tested were ethyl butyrate, n-hexane, cyclohexane, iso-octane, n-dodecane, DMSO, bis(2-ethylhexyl) phthalate and fluorinert FC-70. The cell population was monitored by fluorescence microscopy and analysis of the images captured provided single-cell-level information on cell viability, morphological factors of both viable and non-viable cells, and on the number of viable cells in clusters. These data, and those concerning the initial carveol concentration, the carbon source used during growth, the adaptation time to the solvent prior to substrate addition, and the properties of the organic solvent, were interpreted using principal components analysis (PCA). For R. erythropolis, X. Py2, and A. simplex, between 70.1 and 80.4% of the variability of the data could be explained by six principal components, while 86.7% of the variance in the results obtained with Mycobacterium sp. could be represented by seven principal components. In all cases, solvent toxicity could explain over a third of the variability in the data. R. erythropolis cells were able to maintain their viability under harsh conditions. A period of contact between cells and solvent, prior to the addition of substrate, was prejudicial for R. erythropolis and A. simplex cells, at least for the most toxic solvents, but was beneficial for X. Py2 cells. The number of Mycobacterium sp. cells in clusters was lower after an adaptation period compared to the number of cells in aggregates when the substrate was added at time zero. The substrates transformed by R. erythropolis and A. simplex cells increased the toxicity of the system by decreasing the log P of the reaction mixture. Hydrocortisone was responsible for a reduction in the ability of A. simplex to respond to stress conditions. Mycobacterium sp. cells were apparently unaffected by β-sitosterol. The results obtained are useful for the design of more efficient two-phase reaction systems where solvent toxicity may be overcome in order to increase cell productivity.
KeywordsPCA Cell behaviour Bacteria Organic solvent Cell morphology Viability
The authors would like to thank: Prof. Marie-Nöelle Pons from the CNRS, Nancy, France, for the development of the image analysis programs to be used with Visilog 5; the French Embassy in Lisbon for financing C. Carvalho during a 1-month project in the Laboratoire des Sciences du Génie Chimique, Nancy, France, for the development of the image analysis programs; A. Cruz, P. Fernandes and F. van Keulen for providing resting cells of Mycobacterium sp., A. simplex and Xanthobacter PY2, respectively.This study was partially supported by a PhD. grant (PRAXIS XXI/BD/21574/99) awarded to Carla da C. C. R. de Carvalho by Fundação para a Ciência e a Tecnologia, Portugal.
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