Quantitative Physiology of Non-Energy-Limited Retentostat Cultures of Saccharomyces cerevisiae at Near-Zero Specific Growth Rates

Abstract

So far, the physiology of Saccharomyces cerevisiae at near-zero growth rates has been studied in retentostat cultures with a growth-limiting supply of the carbon and energy source. Despite its relevance in nature and industry, the near-zero growth physiology of S. cerevisiae under conditions where growth is limited by the supply of non-energy substrates remains largely unexplored. This study analyzes the physiology of S. cerevisiae in aerobic chemostat and retentostat cultures grown under either ammonium or phosphate limitation. To compensate for loss of extracellular nitrogen- or phosphorus-containing compounds, establishing near-zero growth rates (μ < 0.002 h−1) in these retentostats required addition of low concentrations of ammonium or phosphate to reservoir media. In chemostats as well as in retentostats, strongly reduced cellular contents of the growth-limiting element (nitrogen or phosphorus) and high accumulation levels of storage carbohydrates were observed. Even at near-zero growth rates, culture viability in non-energy-limited retentostats remained above 80% and ATP synthesis was still sufficient to maintain an adequate energy status and keep cells in a metabolically active state. Compared to similar glucose-limited retentostat cultures, the nitrogen- and phosphate-limited cultures showed aerobic fermentation and a partial uncoupling of catabolism and anabolism. The possibility to achieve stable, near-zero growth cultures of S. cerevisiae under nitrogen or phosphorus limitation offers interesting prospects for high-yield production of bio-based chemicals. IMPORTANCE The yeast Saccharomyces cerevisiae is a commonly used microbial host for production of various biochemical compounds. From a physiological perspective, biosynthesis of these compounds competes with biomass formation in terms of carbon and/or energy equivalents. Fermentation processes functioning at extremely low or near-zero growth rates would prevent loss of feedstock to biomass production. Establishing S. cerevisiae cultures in which growth is restricted by the limited supply of a non-energy substrate therefore could have a wide range of industrial applications but remains largely unexplored. In this work we accomplished near-zero growth of S. cerevisiae through limited supply of a non-energy nutrient, namely, the nitrogen or phosphorus source, and carried out a quantitative physiological study of the cells under these conditions. The possibility to achieve near-zero-growth S. cerevisiae cultures through limited supply of a non-energy nutrient may offer interesting prospects to develop novel fermentation processes for high-yield production of bio-based chemicals.

Physiological responses of Saccharomyces cerevisiae to industrially relevant conditions: Slow growth, low pH, and high CO2 levels

“…White bars: pH 5, gray bars: pH 3 (and 50% CO 2 for glucose‐limited cultures). Data for glucose‐limited cultures grown at pH 5 are from Vos et al (), data for ammonium‐limited cultivation at pH 5 are from Liu et al ()…”

“…To maintain intracellular pH homeostasis, S. cerevisiae can expel protons via the plasma-membrane ATPase Pma1 (Carmelo et al, 1996;Eraso & Gancedo, 1987), a process that is an intrinsic part of maintenanceenergy metabolism (Figure 4). In glucose-limited chemostat cultures, no changes in the expression of genes encoding for proteins involved in proton homeostasis, including PMA1 and genes encoding subunits Vos et al (2016), data for ammonium-limited cultivation at pH 5 are from Liu et al (2019) HAKKAART ET AL.…”

“…Ammonium‐limited retentostats grown at pH 3 were preceded by a chemostat phase under the same nutrient limitation, essentially as described before (Liu et al, ). Details on bioreactor operation and media composition of these nitrogen‐limited cultures are given in Supporting Information Appendix 1.…”

“…Despite its industrial relevance, quantitative understanding of maintenance‐related processes in S. cerevisiae and their sensitivity to industrially relevant process conditions is far from complete. Previous studies showed that, while growth‐rate independent (Boender, de Hulster, van Maris, Daran‐Lapujade, & Pronk, ; Vos et al, ), the maintenance‐energy requirement ( m S ; mmol glucose/g biomass/h) of S. cerevisiae can be affected by the cultivation conditions (Lahtvee, Kumar, Hallstrom, & Nielsen, ; Liu, el Bouhaddani, Pronk, & van Gulik, ; Vos et al, ). For example, growth at pH 2.5 substantially reduces the maximum specific growth rate in batch cultures (Carmelo et al, ; Della‐Bianca & Gombert, ; Della‐Bianca et al, ; Orij et al, ) and increases activity of the plasma‐membrane proton pumps, suggesting that low pH also affects m S (Carmelo et al, ; Eraso & Gancedo, ).…”

“…During ammonium-limited growth, additional mechanisms might explain the increase in nongrowth associated energy requirements (Figure 4). Futile cycling of ammonia and ammonium across the plasma membrane could require addition proton pumping via Pma1 (Cueto-Rojas et al, 2017;Liu et al, 2019) and might be aggravated at low pH. Additionally, presence of ethanol in the ammonium-limited cultures (up to 15 g/L) might stimulate proton leakage across the plasma membrane and thus trigger an increase in ATP-mediated proton export (Lindahl et al, 2017;Madeira et al, 2010) Together, the results of this study indicate that high death rates of slow-growing cultures at low pH cannot be directly attributed to energy-limited growth or increased maintenance energy-requirements.…”