Flocculation onset, growth phase, and genealogical age in Saccharomyces cerevisiae Eduardo V. Soares and Manuel Mota Abstract: Flocculation onset, the time during the fermentative cycle at which the strains of Saccharomyces cerevisiae become flocculent, is an important factor in the brewing industry. The flocculation ability of Flol phenotype (strain NCYC 869) remained practically unchanged throughout the growth and seems to be insensitive to the presence of nutrients of the culture medium. On the contrary, the flocculation of NewFlo phenotype (strain NCYC 1195) exhibited a cyclic behaviour. It was found that the loss of flocculation in the early growth was the result of two combined effects: the dismantling of the flocculation mechanism of the cells coming from the inoculum and the nonflocculent state of the new cells produced after growth has started. The onset of flocculation of strain NCYC 1195 in the cultural conditions used in this work coincided with the end of the exponential growth, when the minimum glucose level in the culture medium was attained. It was demonstrated that it is possible to manipulate the flocculation onset by changing the initial glucose concentration in the culture medium. Key words: yeast, flocculation inhibition, glucose limitation, flocculation onset, genealogical age. Resume : Le seuil de flocculation, soit le moment ou, durant le cycle de fermentation, les souches de Saccharomyces cerevesiae commencent B flocculer, est un parambtre important dans l'industrie du brassage de la bibre. La capacitk de flocculation du phCnotype Flol (souche NCYC 869) demeure pratiquement intacte au cours de la croissance et ne semble pas influencCe par les ClCments nutritifs prCsents dans le milieu de culture. Par contre, chez le phCnotype NewFlo (souche NCYC1195), la flocculation apparait de f a ~ o n clyclique. La perte de la capacitk de flocculation en dCbut de croissance semble due 2 deux effets combinCs : I'altCration du mCcanisme de flocculation des cellules qui viendrait de I'inoculum et 1'Ctat de non-flocculation des nouvelles cellules produites aprbs le dCmarrage de la croissance. Dans les conditions de culture utilisCes dans le prCsent travail, le seuil de flocculation de la souche NCYC1195 coyncide avec la fin de la phase exponentielle de croissance au moment ou la concentration de glucose dans le milieu de culture atteint sont minimum. I1 a CtC dCmontrC qu'il est possible de modifier le seuil de flocculation en changeant la concentration initiale du glucose dans le milieu de culture. Mots clLs : levure, inhibition de la flocculation, limitation en glucose, seuil de flocculation, bge gCnCalogique. [Traduit par la rCdaction] Introduction culent cells) recognizes and interacts with carbohydrate resi- Flocculation can be defined as an asexual aggregation (Johnson et al. 1988) of single yeast cells in clumps with subsequent fast sedimentation from the medium in which they were suspended (Stewart 1975). The ability of yeast cells to adhere and form flocs is exploited in fermentation processes such as brewing and wine making (Stewart and Russell 1981) and in the pro- duction of ethanol with high density cultures (Comberbach and Bu'Lock 1984). According to the lectin-like theory, initially suggested by Eddy and Rudin (1958) and subsequently developed by Taylor and Orton (1978) and Miki et al. (1982a, 1982b), in flocculent bonds of Saccharomyces cerevisiae a specific lectin (on floc- Received October 13, 1995. Revision received January 12, 1996. Accepted January 16, 1996. 1 E.V. Soars. Departamento de Engenharia Quimica, Instituto Superior de Engenharia do Porto, Rua de S. TomC, 4200 Porto, Portugal. M. M0ta.l Departamento de Engenharia BioMgica, Universidade do Minho, Largo do P a ~ o 4709 Braga Codex, Portugal. Author to whom all correspondence should be addressed. dues of the a-mannan (receptor) on the neighbouring cell walls, using calcium ions to ensure correct conformation of the lect- ins. Using mnn mutants and concanavalin A (ConA), it was shown that flocculation receptors are the outer-chain mannan side branches of two or three mannose residues in length (Stratford 1992). Two flocculation phenotypes (Flol and New- Flo) were distinguished according to their different sensitivity to sugar inhibition and protease sensitivity (Stratford and Assinder 1991). These two phenotypes have also different sensitivity to cultural conditions, namely to the pH of culture medium and growth temperature (Soares et al. 1994). Flocculation of Saccharomyces cerevisiae is genetically con- trolled. Several FLO genes, their suppressors, and several mutations have been implicated in this phenomenon (Johnston and Reader 1983; Stratford 1994; Teunissen et al. 1995a, 1995b). Recently, the FLOl gene was cloned and its complete nucleotide sequence was determined (Teunissen et al. 1993; Watari et al. 1994). The FLOl gene product has been localized at yeast cell surface (Bidard et al. 1995) and might be anchored in the plasma membrane at the C-terminal region by an integral membrane domain or a glycosylphosphatidylinositol (GPI) anchor (Teunissen et al. 1993; Watari et al. 1994; Bidard et al. 1995). Can. J. Microbial. 42: 539-547 (1996). Printed in Canada 1 Imprimt au Canada