Fast “Feast/Famine” Cycles for Studying Microbial Physiology Under Dynamic Conditions: A Case Study with Saccharomyces cerevisiae

Abstract

Microorganisms are constantly exposed to rapidly changing conditions, under natural as well as industrial production scale environments, especially due to large-scale substrate mixing limitations. In this work, we present an experimental approach based on a dynamic feast/famine regime (400 s) that leads to repetitive cycles with moderate changes in substrate availability in an aerobic glucose cultivation of Saccharomyces cerevisiae. After a few cycles, the feast/famine produced a stable and repetitive pattern with a reproducible metabolic response in time, thus providing a robust platform for studying the microorganism’s physiology under dynamic conditions. We found that the biomass yield was slightly reduced (−5%) under the feast/famine regime, while the averaged substrate and oxygen consumption as well as the carbon dioxide production rates were comparable. The dynamic response of the intracellular metabolites showed specific differences in comparison to other dynamic experiments (especially stimulus-response experiments, SRE). Remarkably, the frequently reported ATP paradox observed in single pulse experiments was not present during the repetitive perturbations applied here. We found that intracellular dynamic accumulations led to an uncoupling of the substrate uptake rate (up to 9-fold change at 20 s.) Moreover, the dynamic profiles of the intracellular metabolites obtained with the feast/famine suggest the presence of regulatory mechanisms that resulted in a delayed response. With the feast famine setup many cellular states can be measured at high frequency given the feature of reproducible cycles. The feast/famine regime is thus a versatile platform for systems biology approaches, which can help us to identify and investigate metabolite regulations under realistic conditions (e.g., large-scale bioreactors or natural environments).

Disorders in NADPH generation via pentose phosphate pathway influence the reproductive potential of the Saccharomyces cerevisiae yeast due to changes in redox status

“…NADPH, nicotinamide adenine dinucleotide phosphate; TH-L cycle, transhydrogenase-like cycle NADP + ratio is 3.52 ± 0.03 in the BY4742 WT strain ( Figure 5) and it is similar to 4.86 and 5.26 previously reported for CEN.PK113-7D and TN1 strains, respectively. 34,35 These results confirm a larger level of the reduced form as opposed to the oxidised form of the cofactor, which is associated with the significant role that NADPH plays in the cells. The Δzwf1 mutant which is devoid of glucose-6phosphate dehydrogenase activity (Figure 3) decreased its NADPH/NADP + ratio to 1.96 ± 0.24 ( Figure 5).…”

Comparative performance of different scale‐down simulators of substrate gradients in Penicillium chrysogenum cultures: the need of a biological systems response analysis

“…In the TCR2, the PEP level and the pyruvate level are not much reduced compared to other glycolytic intermediates such as G6P, F6P, suggesting there might be a buffering capacity of the closely related amino acid, alanine (Suarez‐Mendez et al ., ), which is indeed observed to be 14% lower in the non‐feed compartment (Table S3). Besides this, no significant differences are observed regarding the intracellular levels of amino acids between the continuous feeding regime, the IFRs and the TCR system (Table S3).…”

Matrices (re)loaded: Durability, viability, and fermentative capacity of yeast encapsulated in beads of different composition during long‐term fed‐batch culture

“…42 The latter observation could be attributed to the feast-and-famine nature of repeated fed-batch culture, which introduces variability in culture conditions not experienced by continuously fed cells. 92 Among different matrix types, Pr beads contained more total cells than at least one other matrix type at 8 out of 16 timepoints (one-way ANOVA, p < .05), and fewer cells than any another matrix type at only one (Table S6). This higher cell number may have contributed to the higher ethanol yields observed for Pr beads, but does not explain this difference in and of itself.…”