DENSELY PACKED YEAST: A TOOL TO STUDY EVOLUTION OF GROUP DYNAMICS AND TO ENHANCE BIOMANUFACTURING.
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Yeast can thrive in dense assemblages of its own, or our making, resulting in patterns of behavior that differ markedly from that of single planktonic cells. We first studied unicellular yeast that create their own assemblage upon selection for rapid settling in liquid medium. In these experiments, large clonal clusters, composed of hundreds to thousands of cells, began to form even larger macroscopic aggregates composed of hundreds of potentially unrelated clusters. The matrix of these aggregates includes extracellular proteins, and is likely produced via apoptosis. We found that such aggregates increase survival in environments that favor fast settling, but are evolutionarily unstable because they do not discriminate between aggregate-producers and non-producers. Next, we examined unicellular yeast maintained at high density via immobilization in a Ca2+-alginate matrix. Immobilization uncouples reproduction from metabolism, resulting in metabolically active but growth-arrested cells. Because immobilized yeast allocates little substrate to biomass, we investigated its potential to enhance ethanol production under biorefinery-like conditions. We found that over a 7-week course of fed-batch culture immobilized yeast is able to produce more ethanol from the same amount of substrate than planktonic yeast. We further tested the resilience of different immobilization matrices to support long-term, fed-batch cultures, and determined that Protanal alginate has the lowest rates of cell escape and the highest ethanol yields. Lastly, we tested whether immobilization, by inducing replication arrest, could stabilize the genomes of dikaryons created by protoplast fusion of different yeast species: Saccharomyces cerevisiae and Pichia stipitis. In the absence of selection, planktonic dikaryons quickly revert to their parental genotypes via nuclear segregation during replication. We discovered that genome content of growth-arrested immobilized dikaryons is stable in 19-day, fed-batch cultures, and that stable dikaryons retain the metabolic capacity of different the fused species to ferment 6-carbon and 5-carbon sugars. Taken together, our results demonstrate the utility of densely-packed yeasts to address issues related to the evolution of group dynamics as well as to enhance efficiency and longevity in biomanufacturing.