ABSTRACT: The present study aims to explore chemostat-based transcriptome analysis of mixed cultures by investigating interactions between the yeast S. cerevisiae and the lactic acid bacterium Lb. bulgaricus . S. cerevisiae and Lb. bulgaricus are both frequently encountered in kefir, a fermented dairy product (25). In the context of this study, this binary culture serves as a model for the many traditional food and beverage fermentation processes in which yeasts and lactic acid bacteria occur together (19,26-30). The design of the cultivation conditions was based on the observation that Lb. bulgaricus, but not S. cerevisiae, can use lactose as a carbon source for growth and that S. cerevisiae, but not Lb. bulgaricus, can grow on galactose that is released upon hydrolysis of lactose by the bacterial β-galactosidase. Mixed populations of yeasts and lactic acid bacteria occur in many dairy, food and beverage fermentations, but knowledge about their interactions is incomplete. In the present study, interactions between Saccharomyces cerevisiae and Lactobacillus delbrueckii subsp. bulgaricus, two microorganisms that co-occur in kefir fermentations, were studied during anaerobic growth on lactose. By combining physiological and transcriptome analysis of the two strains in the co-cultures, five mechanisms of interaction were identified. 1. Lb. bulgaricus hydrolyses lactose, which cannot be metabolized by S. cerevisiae, to galactose and glucose. Subsequently, galactose, which cannot be metabolized by Lb. bulgaricus, is excreted and provides a carbon source for yeast. 2. In pure cultures, Lb. bulgaricus only grows at increased CO2 concentrations. In anaerobic mixed cultures, the yeast provides this CO2 via alcoholic fermentation. 3. Analysis of amino acid consumption from the defined medium indicated that S. cerevisiae supplied alanine to the bacteria. 4. A mild but significant low-iron response in the yeast transcriptome, identified by DNA microarray analysis, was consistent with the chelation of iron by the lactate produced by Lb. bulgaricus. 5. Transcriptome analysis of Lb. bulgaricus in mixed cultures showed an overrepresentation of transcripts involved in lipids metabolism suggesting either a competition of the two microorganisms for fatty acids, or a response to the ethanol produced by S. cerevisiae. To our knowledge, this is the first transcriptome study of a cross-kingdom binary mixed culture that analyses responses of both microorganisms. This study demonstrates that chemostat-based transcriptome analysis is a powerful tool to investigated microbial interaction in mixed populations.
