3.4 Summary
The results of the research presented in this chapter showed that:
• Zinc uptake by industrial yeast strains was generally very fast and the element entirely disappeared from the medium. Zinc supplementations in the first hours of growth also resulted in quick uptake by the yeast cells.
• Zinc uptake was influenced by temperature (e.g. reduced at lower temperatures).
• Zinc uptake was metabolism dependent. Dead cells failed to take up zinc while uptake in protein synthesis inhibited cells was unaltered.
• Calcium concentrations (range 16-76 ppm) did not significantly influence zinc uptake.
• Various zinc salts (22 ppm of zinc) added did not significantly influence zinc uptake.
• Zinc uptake was metabolism-dependent. During brewing processes, it is suggested to add zinc at time of pitching, when optimal sugar levels are available as sources of driving energy. For the same reason it is discouraged to add zinc at low temperatures (~0°C) and when sugar sources are minimal (e.g. during acid-washing or yeast storage).
4.4 Summary
The results of the research presented in this chapter showed that:
• The yeast cell wall does not play a key role in zinc uptake during fermentation.
• Zinc is rapidly stored into the vacuole and shared from mother to daughter cells during cell division.
• In brewing and bioethanol industries, where yeast biomass is recycled for several fermentations, particular attention must be paid to the practice of removing part of the yeast crop. This may lead to the depletion of an important reservoir of zinc from the yeast biomass.
5.4 Summary
The results of the research presented in this chapter showed that:
• Zinc concentration in the range 0.4- 1 ppm gave fastest fermentation rates in brewing fermentation and using lager strain B. At lower concentrations, fermentations were sluggish. High levels of zinc (eg. 23 ppm) were not toxic to yeast, but did slow fermentation rates. The optimal zinc level was yeast strain dependent.
• Various zinc concentrations in the range 0-23 ppm did not adversely affect yeast cell viability in both brewing and wine yeasts.
• Irrespective of zinc availability, yeast membrane fluidity varied with culture age. There was a complex relationship between yeast cell zinc status and membrane fluidity, but it appeared that cells with low zinc content exhibited low GP levels indicating high membrane fluidity. Such cells would be more susceptible to membrane fluidisation by ethanol.
• Brewing yeast cells preconditioned with zinc performed very well, even in very low zinc wort.
• Initial zinc levels influenced esters and higher alcohol profiles in green beer.
• At pilot plant brewing scale, the intracellular zinc content of yeast may be different at various fractions of the yeast cone. Attention should be paid to selectively remove appropriate parts of the yeast cone when the yeast cells are recycled for the following fermentation.
• Some yeast strains may release zinc back into the medium. This phenomenon was observed in the wine strains and may depend on the ethanol stress imposed on cells during the fermentation process.
6.4 Summary
The results of the research presented in this chapter showed that:
• Yeast cells of the lager strain B insulted by oxidative and acid-washing stresses were not affected in terms of viability and intracellular zinc content, at the conditions herein studied.
• Severe and prolonged exposure to ethanol, temperature and osmotic stresses decreased both viability and intracellular zinc content of the lager strain B.
• Freeze-drying treatments, using the procedure described in this Thesis, killed the cells of the lager strain B but did not alter dramatically the intracellular zinc content.
• Cells of the lager strain B stored in liquid nitrogen maintained high viability, although they lose some of their intracellular zinc, if they were pre-conditioned in high malt wort zinc levels.
• Ethanol and heat stresses have a synergistic and accelerated effect on mortality and on released zinc cell content of the lager strain B.
• Yeast cells of the lager strain B killed by temperature, ethanol and temperature/ethanol stresses lose magnesium and reduced their intracellular ATP levels. This phenomenon might have depended on alterations in plasma membrane permeability.• Generally, a relationship appeared to exist between cell zinc loss and decrease in viability.
• Control and correct management of the physico-chemical properties of the environment are essential to avoid stresses leading to diminished fermentation performance and zinc loss.
• Further investigations are necessary to establish the role of zinc in stress protection, including the determination of the optimal intracellular zinc content for plasma membrane stability during chemical and physical stresses.
7.4 Summary
The results of the research presented in this chapter showed that:
• In batch culture, in conditions of zinc depletion, the growth of the brewing strain B was not arrested but considerably impaired as was ethanol production.
• In chemostat continuous culture, in conditions of zinc limitation, the fermentative profiles of zinc limited cultures of the haploid strain CEN.PK-113D were similar to those found in nitrogen or carbon limited cultures.
• In anaerobic chemostat continuous culture, acetate and glycerol production rates were higher compared with nitrogen and carbon-limitations. This was probably due to a redox problem. Higher production of acetate may indicate a limited capacity of ADH for ethanol production.
• In zinc-limited chemostat continuous culture, in both anaerobic and aerobic conditions, zinc supplementation to cells in steady state produced an immediate increase in biomass yield, glucose consumption and CO2 production. These changes were presumably due to immediate uptake of zinc ions by yeast cells from the growth medium.
• The establishment of zinc-limitation conditions in chemostat continuous culture has allowed the identification of Zn-signature transcripts obtained by DNA microarrays. The determination of up and down regulated genes during zinc-limitation will generate important information for both fundamental research and practical applications in biotechnology. The former is aimed at understanding the role of zinc in yeast physiology. The latter is aimed at determining specific sets of genes (zinc-responsive molecular biomarkers), to be used to detect zinc cellular deficiency in yeast during industrial processes.