Journal of Industrial Microbiology & Biotechnology (1997) 18, 1–3  1997 Society for Industrial Microbiology 0169-4146/97/$12.00 Accumulation of magnesium ions during fermentative metabolism in Saccharomyces cerevisiae GM Walker and AI Maynard 1 School of Molecular and Life Sciences, University of Abertay Dundee, Dundee DD1 1HG, Scotland, UK When cells of Saccharomyces cerevisiae were grown aerobically under glucose-repressed conditions, ethanol pro- duction displayed a hyperbolic relationship over a limited range of magnesium concentrations up to around 0.5 mM. A similar relationship existed between available Mg 2+ and ethanol yield, but over a narrower range of Mg 2+ concen- trations. Cellular demand for Mg 2+ during fermentation was reflected in the accumulation patterns of Mg 2+ by yeast cells from the growth medium. Entry of cells into the stationary growth phase and the time of maximum ethanol and minimum sugar concentration correlated with a period of maximum Mg 2+ transport by yeast cells. The timing of Mg 2+ transport fluxes by S. cerevisiae is potentially useful when conditioning yeast seed inocula prior to alco- hol fermentations. Keywords: yeast; magnesium ions; fermentation Introduction 1883; University of Abertay Dundee culture collection number YO36) originally provided by Mauri Yeast Pro- Magnesium ions play essential roles in the growth and ducts Ltd, Hull, UK. Active colonies of the yeast were metabolism of yeast cells [14]. With regard to carbohydrate grown on Sabouraud Dextrose Agar (SDA) slopes at 30°C catabolism and fermentation, Mg 2+ ions are required as for 24 h then maintained at 4°C. Experimental fermen- cofactors for the activity of key glycolytic and alcoholo- tations were carried out by inoculating yeast cells in modi- genic enzymes and may also play a regulatory role at the fied Edinburgh Minimal Medium (EMM) [6] in which the level of pyruvate-metabolizing enzymes when cells are level of glucose was increased to 25 g L -1 . The calcium grown respirofermentatively [14,16]. Magnesium also plays concentration was constant at 0.1 mM but the magnesium roles in protecting yeast cells against environmental stresses concentration was varied as described in the Results. during fermentation such as those caused by ethanol [3,4], Deionized glassware, AnalaR-grade reagents and ultrapure high temperature, or high osmotic pressure [2]. Dombek (18 megaohm conductivity) water were employed in prep- and Ingram [4] demonstrated that Mg 2+ deficiencies in a aration of growth media to control magnesium concen- yeast extract-peptone based fermentation broth were prim- trations during yeast culture. To generate suitable inocula arily responsible for the decline in yeast fermentative for experimental cultures, 10 ml sterile water was added to activity. In addition, a general stimulation of ethanol pro- an SDA slope of the yeast which was resuspended prior to duction by yeast is observed when complex organic feed- transfer to 100 ml EMM in an Erlenmeyer flask. This pri- stocks like molasses, wine must or malt wort are sup- mary inoculum was incubated overnight at 30°C with shak- plemented with Mg 2+ , indicating that such media may be ing at 180 rpm prior to transfer to 400 ml fresh EMM. This deficient in available Mg 2+ for optimal fermentation per- secondary inoculum was incubated as before for 10 h prior formance [16–18]. Therefore, media bioavailability, cellu- to harvesting 100-ml aliquots by centrifugation (1500 × g, lar uptake and subsequent metabolic utilization of Mg 2+ 10 min). Cell pellets were washed by resuspension in warm ions by yeast cells appear to be prerequisites for achieve- sterile water before re-centrifugation and a second resus- ment of maximum fermentative activity. pension to provide the inoculum for the main experimental This paper investigates the metabolic demands by yeast fermentations which were carried out at 30°C in 2-L fer- for Mg 2+ during fermentation. Knowledge of Mg 2+ accumu- menters (Life Science Laboratories, Luton, UK) with a lation by yeast cells may be usefully exploited in biotechno- working volume of 800 ml. Inocula prepared in this way logies concerned with production of ethanol. ensured removal of interstitial and loosely-bound cell sur- face magnesium. Materials and methods Cell numbers and biomass determination Organisms, media and culture conditions Yeast cell numbers and mean cell volumes were determined The yeast employed in this study was an industrial bakers’ using a Coulter Counter Model D (Coulter Electronics, strain of Saccharomyces cerevisiae (Meyen ex-Hansen, Luton, UK). To minimize errors due to cell aggregation, culture samples were ultra-sonicated (4 min at maximum setting in a Camlab T310 sonicator) prior to dilution and Correspondence: GM Walker, School of Molecular and Life Sciences, triplicate analysis. For yeast biomass determinations, cell University of Abertay Dundee, Dundee DD1 1HG, Scotland, UK pellets from duplicate 10-ml culture samples were washed, 1 Present address: Quest International, Menstrie FK11 7ES, Scotland, UK Received 4 March 1996; accepted 21 August 1996 dried and weighed. Downloaded from https://academic.oup.com/jimb/article/18/1/1/5989144 by guest on 10 December 2023