Biology is full of complexities, and the more we learn, the more we realize how much remains unknown. A major debate in microbiology is whether DNA alone dictates an organism’s function or if metabolism and energy flows play an equally fundamental role. This question is particularly relevant for microbes in dynamic environments, where survival depends on metabolic adaptability.

This thesis focuses on “Candidatus Accumulibacter”, a key microorganism in wastewater treatment that removes excess phosphorus from water. These bacteria endure feast-famine cycles by storing and utilizing energy reserves as conditions change. While extensively studied, much remains unknown about their metabolic strategies and how environmental factors shape their function. This research combines computational models, laboratory cultivation, and multi-omics analysis to explore how “Ca. Accumulibacter” optimizes its metabolism.

Chapter 1 introduces the central debate: Is DNA the sole blueprint for microbial function, or do metabolism and energy constraints shape microbial behavior? It traces the shift from biochemical models to genome-centric approaches and highlights the potential of a metabolism-first perspective. It also contextualizes “Ca. Accumulibacter” within existing research, outlining its role in biological phosphorus removal and summarizing past findings.

Chapter 2 investigates extracellular polymeric substances (EPS) produced by “Ca. Accumulibacter”, revealing novel glycans and glycoproteins that challenge genome-based predictions. These biomolecules are crucial for biofilm formation and microbial interactions, emphasizing the need for direct biochemical analysis alongside genetic data.

Chapter 3 uses elementary flux mode analysis (EFMA) to map the metabolic potential of “Ca. Accumulibacter”. While genome annotations suggest flexibility, thermodynamic constraints limit feasible metabolic strategies, highlighting the role of energy availability in shaping microbial function.

Chapter 4 introduces the development of the Conditional Flux Balance Analysis (cFBA) Toolbox, an open-source Python framework for modeling metabolism in fluctuating environments. Unlike conventional models that assume steady-state conditions, cFBA enables dynamic predictions of resource allocation over time.

Chapter 5 explores the impact of temperature on “Ca. Accumulibacter” metabolism using cFBA. The findings confirm that biomass synthesis is mainly aerobic but also uncover metabolic shifts at lower temperatures that influence phosphorus removal efficiency and microbial competition.

Chapter 6 examines how “Ca. Accumulibacter” metabolizes multiple substrates simultaneously, revealing unexpected synergies that enhance survival in microbial communities. Combining experimental enrichment cultures with cFBA, this study identifies key metabolic trade-offs and resource optimization strategies.

Finally, Chapter 7 synthesizes the thesis findings, advocating for a shift beyond genome-based interpretations toward a metabolism-centric understanding of microbial function. It discusses broader implications for microbial ecology, wastewater engineering, and metabolic modeling, emphasizing the need for multi-omics approaches and potential applications in synthetic biology.
By integrating experimental and computational approaches, this research deepens our understanding of how “Ca. Accumulibacter” thrives in fluctuating environments. More broadly, it highlights the importance of metabolism and energy flows in shaping microbial function, offering insights that extend beyond wastewater treatment to microbial ecology and engineered bioprocesses.

Saccharomyces cerevisiae, also known as baker’s yeast, is a robust microorganism frequently used in industrial biotechnology. The scale of its applications ranges from several millilitres for research and process development in the lab to hundreds of cubic meters for cultivation in industrial production processes. In large-scale reactors mixing limitations inherently lead to physiochemical gradients in substrate and oxygen concentrations, pH or temperature. Such inhomogeneous environment in production processes can cause a reduced yield or titer compared to the small-scale development processes. Such scale performance differences can lead to significant worse process economics and increase costs and development time.
The scope of this thesis is to study and understand the regulation of Saccharomyces cerevisiae metabolism under dynamic substrate conditions, using both experimental and modelling approaches.

Phosphate accumulating organisms (PAOs) perform a storage polymer metabolism within an anaerobic-aerobic cycle. Anaerobically, PAOs take up volatile fatty acids (VFA) and store them as poly-ẞ-hydroxyalkanoates (PHA). The energy (mainly ATP) necessary for the transport and storage of VFA (and general maintenance) is obtained through the cleavage of polyphosphate. While the reducing equivalents (e.g. NADH) for VFA storage are obtained through the cleavage of glycogen and/or from the anaerobic operation of the TCA cycle. Aerobically, PAOs replenish their reserves of polyphosphate and glycogen, resulting in P uptake, whilst degrading PHA to obtain a carbon and energy supply for growth.
PAOs have the metabolic flexibility to adapt the synthesis of each polymer according to the resources available in the environment, and thus affecting the growth of the organism. Hence, within a PAOs metabolism, there is a trade-off between the use of glycogen and polyphosphate. This trade-off is dependent on the cell's requirements to obtain ATP and NADH for PHA storage. In turn, ATP and NADH amounts can be obtained in different ratios depending on the active metabolic routes. This thesis aims to determine what is the mechanism controlling this trade-off and if there are limits to this relationship.
To investigate this trade-off, a metabolic model for PAOs was created and simulated through a conditional flux balance analysis (cFBA) approach. The resulting amounts of the metabolites simulated with this approach were comparable to those obtained experimentally (figure 6). Additionally, this model was simulated to different sets of starting amounts of glycogen and polyphosphate at a constant acetate feed of 3.84 mCmol/gdw, and the resulting growth was compared between each simulation. This led to an optimal range of initial polyphosphate amounts [2.1-23.5 mPmol/gdw] and initial glycogen amounts [0.3-1 mCmol/gdw]. In reality, these glycogen amounts were never observed experimentally and to the extent of our knowledge never have been reported in PAOs literature. This suggests a glycogen minimal limit amount (e.g. 1 mCmol/gdw), that might reveal a robustness mechanism employed by PAOs to guarantee survivability in uncertain environments. In parallel, a thermodynamic analysis was performed on the malate dehydrogenase reaction, which led to the conclusion that this reaction is not feasible in an anaerobic environment, potentially highlighting a control mechanism.