Morphological studies on Kluyveromyces Marxianus var; Marxianus NRRLy2415 in suspension culture: a study incorporating computer aided image analysis

Abstract

The morphology of the lactose fermenting organism, Kluyveromyces marxianus var. marxianus NRRLy2415 was studied in batch and continuous culture. The morphology of the organism was observed to vary significantly from a budding yeastlike form to a branched pseudohyphal form depending on the operating conditions. The following morphological classes were deemed important for the complete description of culture morphology: yeast, elongated yeast, filament, double yeast, double elongated yeast, double filament and pseudohyphae. Image analysis was used to implement the classification system, due to its objectivity in measurement of visual phenomena. The protocol developed was also capable of measuring geometric properties of the cell population including volume, length and width for all cells and hyphal growth unit length for pseudohyphae. The predominant morphology observed for the organism in fully aerobic batch culture was yeast-like. When the agitation rate was decreased, an increased heterogeneity in the morphology was observed, w ith the generation o f more elongated yeast and filamentous forms. This was attributed to partial oxygen limitation in the fermentations. In chemostat culture, the morphology observed was predominantly pseudohyphal. This morphological form was found to dominate over a large range of dilution rates (0 .1-0 .45 h ']) and substrate feed concentrations (5 to 20 g /L lactose). At both extrema of dilution rates a reversion to a yeastlike morphology was observed. The mycelial morphology was attributed to substrate limitation and was demonstrated to be of ecological advantage under substrate limited conditions. Kinetic modelling of substrate metabolism was undertaken for continuous and batch culture results. It was demonstrated that, the stoichiometry of the metabolic pathways studied was identical under all operating conditions. This is significant, as the morphology of the organism varies significantly from batch to continuous culture. A population model was developed which was capable of describing the distributions of cell geometric parameters based on summed lognormal distributions of selected morphological classes. The ultrastructure of pseudohyphae was examined. This study demonstrated significant differences in methods of formation between pseudohyphae and true hyphae and highlighted key issues that need to be addressed if successful modeling of such grow this to be undertaken