Technical Session 22: Yeast IV Session
Johnathon B Layfield, NC State University, Raleigh, NC, USA
Co-author(s): Lucas Vann and John Sheppard, NC State University, Raleigh, NC, USA
ABSTRACT: In conventional batch and continuous fermentation, the cell cycles of individual yeast are randomized within the population, and the observed metabolic performance is the result of an averaging effect. Synchronous cellular growth is characterized by cells in a population aligned with respect to their metabolic processes traversing the cell cycle and dividing mostly in unison. Thus, synchronized populations of cells can be used as a tool to reveal more precisely how an individual cell reacts under different environmental conditions (Sheppard et al, 1999). S. cerevisiae is a unique organism in that it serves as a model eukaryote for academic and industrial research. Thus, a method for inducing and storing a synchronous yeast culture for rapid use in metabolic studies is advantageous to both academia and industry. In this study, a novel method for inducing and retaining cell cycle synchronization in yeast cells (diploid- and polyploid-type cells) was developed. This technique is derived from the continuous phased-culture induction method (Dawson, 1969). The original induction method was based on a cyclical process in which one-half of the cell culture was harvested and a fresh nutrient solution added to replace the harvested volume at a period corresponding to cell doubling. This replenishment of sufficient nutrients only for cell doubling resulted in the growth and division of a single division of cells prior to the beginning of a new cycle. After about six such cycles, the cells became aligned with respect to their cell cycles and began dividing synchronously. Our new method begins with a small volume and doubles it each cycle by periodically adding fresh nutrient solution, without having to remove any cells. This adaptation is better suited for industrial applications, such as seed expansion, due to its relative simplicity and equivalent effectiveness in producing cell synchrony. This was demonstrated by measuring the synchrony index of both S. cerevisiae 288C (diploid) and the brewing strain London ESB 1986 (polyploid), which matched that produced using the conventional continuous phased method (71 and 83%, respectively). We have also shown that synchronized cells can be stored for later use in glycerol at –80°C for at least 2 weeks without significant loss in synchrony. Small volumes (1.5 and 10 mL) of both S. cerevisiae 288C and London ESB 1986 showed no loss of synchrony from the original synchrony procedure. However, as the volume of a synchronous stock increased to 50 mL, certain aspects of synchrony (depending on the strain) seemed to degrade. The extra time required for both freezing and thawing the larger synchronous stocks is thought to be the cause. However, for most metabolic studies, freezing at –80°C is a viable approach for retaining cell synchronization in S. cerevisiae.
Johnathon Blake Layfield received a bachelor’s degree in food science (2003) and master’s degree in food science, with a minor in biotechnology (2009), from North Carolina State University in Raleigh, NC. He is currently pursuing a Ph.D. degree in food science at NC State University under John D. Sheppard. Johnathon has interned for Smithfield Foods Ltd. (quality assurance) and Novozymes (biofuel R&D) and was a co-op student with Campbell Soup Co. (beverage product development). He is a member of both the Institute of Food Technologists (IFT) and the American Society of Brewing Chemists (ASBC). Johnathon has published in JASBC, where his work on desiccation tolerance in lager yeast was selected as an “Editor’s Pick” (August 2011). He also gave an oral presentation at the 2009 ASBC Annual Meeting in Tuscon, AZ.