This study explores the relation between biomass-specific succinic acid (SA) production rate and specific growth rate of an engineered industrial strain of Saccharomyces cerevisiae, with the aim to investigate the extent to which growth and product formation can be uncoupled. Ammonium-limited aerobic chemostat and retentostat cultures were grown at different specific growth rates under industrially relevant conditions, that is, at a culture pH of 3 and with sparging of a 1:1 CO₂–air mixture. Biomass-specific SA production rates decreased asymptotically with decreasing growth rate. At near-zero growth rates, the engineered strain maintained a stable biomass-specific SA production rate for over 500 h, with a SA yield on glucose of 0.61 mol mol⁻¹. These results demonstrate that uncoupling of growth and SA production could indeed be achieved. A linear relation between the biomass-specific SA production rate and glucose consumption rate indicated the coupling of SA production rate and the flux through primary metabolism. The low culture pH resulted in an increased death rate, which was lowest at near-zero growth rates. Nevertheless, a significant amount of non-viable biomass accumulated in the retentostat cultures, thus underlining the importance of improving low-pH tolerance in further strain development for industrial SA production with S. cerevisiae.

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The progress of modern biotechnology has enabled the development of fermentation processes for the production of fuels and chemicals from renewable feedstocks. The current fermentation processes for bio-based production commonly start with a growth phase of the microorganisms followed by a production phase. This implies that biomass formation competes with the production of the desired product in terms of consumption of the feedstock. In industrial fermentations, maximizing the product yield, in other words, minimizing the substrate flux to biomass, CO2 and byproducts is the primary goal. To reach this objective, the uncoupling of microbial growth from product formation seems like a feasible approach, providing that the microbial host maintains high productivity in the absence of growth. The research presented in this thesis aims to improve understanding of the physiology of microbial at near-zero growth rates and thereby provide insights for the design of industrial fermentation processes based on the zero-growth concept. Specifically, S. cerevisiae was applied as the microbial cell factory, and succinic acid, a non-catabolic product and its synthesis from sugar requires a net input of ATP, was chosen as a model product. The microbe was cultivated in the chemostat and retentostat mode under industrially relevant conditions, as reflected by aerobic cultivation at low pH and at a high dissolved CO2 level. To the end, the quantitative physiology of yeast at slow and near-zero growth states was investigated, and uncoupling S. cerevisiae growth and succinic acid production was achieved with considerable succinic acid productivity. This study illustrates the potential for high-yield production of non-dissimilatory products at near-zero growth rates, with growth being limited by nutrients other than the carbon and energy source. In addition, it highlights a requirement for further research into enhancing strain robustness under industrial conditions, with specific attention for low-pH tolerance.@en

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.

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Engineered strains of Saccharomyces cerevisiae are used for industrial production of succinic acid. Optimal process conditions for dicarboxylic-acid yield and recovery include slow growth, low pH, and high CO₂. To quantify and understand how these process parameters affect yeast physiology, this study investigates individual and combined impacts of low pH (3.0) and high CO₂ (50%) on slow-growing chemostat and retentostat cultures of the reference strain S. cerevisiae CEN.PK113-7D. Combined exposure to low pH and high CO₂ led to increased maintenance-energy requirements and death rates in aerobic, glucose-limited cultures. Further experiments showed that these effects were predominantly caused by low pH. Growth under ammonium-limited, energy-excess conditions did not aggravate or ameliorate these adverse impacts. Despite the absence of a synergistic effect of low pH and high CO₂ on physiology, high CO₂ strongly affected genome-wide transcriptional responses to low pH. Interference of high CO₂ with low-pH signaling is consistent with low-pH and high-CO₂ signals being relayed via common (MAPK) signaling pathways, notably the cell wall integrity, high-osmolarity glycerol, and calcineurin pathways. This study highlights the need to further increase robustness of cell factories to low pH for carboxylic-acid production, even in organisms that are already applied at industrial scale.

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