Greg Potter (1), Suzanne M. Budge (1), Alex Speers (2), Chantel Swart (3), Hendrik Swart (3), Pieter Van Wyk (3); (1) Dalhousie University, Halifax, NS, Canada; (2) The International Center for Brewing and Distilling, Edinburgh, U.K.; (3) University of the Free State, Bloemfontein, South Africa
Yeast, Fermentation, and Microbiology
Carbon dioxide (CO2) is a well-established and industrially important by-product of fermentative growth in Saccharomyces cerevisiae and Saccharomyces pastorianus, yet the specific mechanism and site of CO2 generation within the cell has not been elucidated. In this study we have analyzed the SMA strain of Saccharomyces pastorianus using another application of nano-scanning Auger microscopy (NanoSAM), where specific cells can be targeted by argon-etching to expose the interior and then subsequently viewed with a high-resolution scanning electron microscope (SEM). These high-resolution images of the interior of argon-etched fermenting SMA yeast cells uncovered a maze of coalescing CO2 bubbles that increased in complexity and number with fermentation duration. Similar cell preparations were also visualized using transmission electron microscopy (TEM), and smaller networks of CO2 bubbles were identified that provide information on the formation and origin of intracellular bubbles. An unusual group of oxygenated fatty acids, 3-hydroxy (OH) oxylipins, are presumed to play a potential role in flocculation during fermentation, so the effect of gas bubble formation and, thus, fermentation on 3-OH oxylipin production was also of interest. To investigate this, fermentatively and non-fermentatively grown SMA cells were examined using time-of-flight secondary ion mass spectrometry (TOF-SIMS) and the negative atomic ions C–, NH–, O–, OH–, P–, and S– and negative molecular ions 160– (3-hydroxy 8:0) and 188– (3-hydroxy 10:0) were monitored. Distinctly different cellular compositions were found in the fermenting and non-fermenting cells. In particular, both 3-OH 8:0 and 3-OH 10:0 were constitutively produced in respiring cells, while there was a delayed onset of 3-OH 8:0 production in fermenting cells. Thus, the novel application of these nanotechnological techniques has revealed a correlation between metabolic state (fermentation vs. respiration), bubble production and 3-OH oxylipin profile. Further investigations using the biological applications of these material science techniques in conjunction with more conventional lipid analyses will uncover the role of 3-OH oxylipins in flocculation and the origin of these hydroxylated fatty acids in fermentation yeasts.