Acetophenone tolerance, chemical adaptation, and residual bioreductive capacity of non-fermenting baker's yeast (Saccharomyces cerevisiae) during sequential reactor cycles

Bioreduction of acetophenone (ACP) to phenethyl alcohol (PEA) by baker's ye ast (Saccharomyces cerevisiae), which is highly enantioselective, can be ca rried out entirely in a resting state using stored carbohydrate, suggesting that a high degree of chemical tolerance might be possible. However, viabi lity and catalytic activity of precultured cells decline steeply within 24 h at initial ACP concentrations >0.2% (17 mM). Viability of cells at 0.4% A CP was 1/4 the viability At 0.2% ACP as determined by vital staining, and < 1% based on colony-forming ability. This sensitivity was observed in suspen sions with a cell content of nearly 30% (w/v), Longterm PEA production is s trongly dependent on viability, indicating that the cumulative yield per ba tch of cells is maximized by maintaining a very low concentration of substr ate (similar to 0.2%). However, nonviable cells (CFU ml(-1) <1% cells ml(-1 )) can achieve PEA yields up to 1/3 the maximum, an amount representing ini tial absorption of ACP without further uptake, Regarding population adaptab ility, when cells surviving the most selective (toxic) concentration of ACP (0.6%) were subcultured in an ACP-free medium and re-reacted, the 24-h per cent viabilities (vital staining) and colony-forming frequencies exceeded t hose of non-selected cells. However, the surviving cells represented only a small fraction (similar to 1%) of the recultured progeny. Even at ACP conc entrations as low as 0.25% (w/v), surviving cells were unreliable in transm itting and maintaining ACP-tolerance. In addition, there was no evidence th at the chemical yield of recultured ACP-tolerant cells (amount of PEA relat ive to initial amount of ACP) can consistently exceed the maximum yield of an equivalent density of previously unreacted (non-selected) cells. These r esults indicate that over a broad range of substrate concentrations, rapid replacement of cells may be more cost-effective than maintenance or reuse o f viable cells.