Rapid monitoring of cell size, vitality and lipid droplet development in the oleaginous yeast Waltomyces lipofer.
The aim of this work was the development of rapid methods suitable for monitoring the growth of the oleaginous yeast Waltomyces lipofer by means of cell size, vitality and the development of internal lipid droplets throughout different growth phases. Oleaginous yeasts are of interest for the industrial production of lipids and therefore precise monitoring of growth characteristics is needed. This paper provides information about both the method development as well as about examples for their use in monitoring applications. Cell size and shape were determined using FPIA (Flow Particle Image Analysis). Vitality and internal lipid droplets were measured using two independent staining methods for Flow Cytometry. Double staining with cFDA & PI was used for the distinction between "vital", "sublethal" and "dead" subpopulations, whereas Nile Red allowed the monitoring of lipid accumulation. In this approach the method for vitality measurement was optimized focussing on the staining buffer. An addition of 25 mM citric acid and pH 4.8 revealed to be optimal. The cells in the growth experiment showed a constantly high vitality, which was always above 90%, but slowly decreasing over time. In the course of lipid droplet development it could be seen that the cell size and the Nile Red fluorescence intensity increased. It was demonstrated that the tested method combination provides a powerful tool for rapid fermentation monitoring of the oleaginous yeast W. lipofer, which allows gaining information about the desired growth characteristics in less than 45 min. Further applications for the two methods will be discussed in this article.
Raschke
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Department of Food Biotechnology and Food Process Engineering, Technische Universität Berlin, Königin-Luise-Strasse 22, Berlin, Germany. dennis.raschke@tu-berlin.de
Knorr
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D004396:Coloring Agents / Q000494:pharmacology; D005434:Flow Cytometry / Q000379:methods*; D005452:Fluoresceins / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000494:pharmacology; D050356:Lipid Metabolism; D055160:Lipomyces / Q000166:cytology / Q000254:growth & development / Q000378:metabolism / Q000502:physiology*; D050296:Microbial Viability; D009172:Mycology / Q000379:methods*; D010078:Oxazines / Q000494:pharmacology; D011419:Propidium / Q000494:pharmacology; D013194:Staining and Labeling / Q000379:methods; D013997:Time Factors
D004396:Coloring Agents; D005452:Fluoresceins; D005456:Fluorescent Dyes; D010078:Oxazines; C027780:carboxyfluoresceindiacetate; D011419:Propidium; C044808:nile red