Investigating Overflow Metabolism in Heterotrophic Cultures of the Green Alga Chromochloris zofingiensis.

Phytoplankton growth and death depend on interactions with heterotrophic bacteria, yet the underlying mechanisms remain mostly unclear. Here we ask whether mathematical models explicitly representing four putative mechanisms of interaction (overflow metabolism, mixotrophy, exoenzymes and reactive oxygen species detoxification) can recapitulate diverse dynamics observed in laboratory co-cultures between the cyanobacterium Prochlorococcus and eight heterotrophic bacteria. Two distinct modes of interaction emerge from our models: (1) organic carbon and nitrogen recycling through exoenzymes or an overflow metabolism, in which the high biomass of both organisms leads to more productivity and recalcitrant organic matter, and (2) reactive oxygen species detoxification, in which a small number of 'exploited' heterotrophs are sufficient to support Prochlorococcus survival. Recycling is probably the main process in laboratory co-cultures. Models do not reproduce total inhibition of Prochlorococcus, suggesting that additional mechanisms such as allelopathy may be involved. The models highlight cell death and biomass recycling as unconstrained, key processes that could enhance our understanding of how interactions impact ecologically and biogeochemically important processes.

Cyanobacteria such as Synechocystis sp. PCC 6803 are promising chassis for sustainable bioproduction. During nitrogen starvation, Synechocystis redirects fixed carbon from biomass growth toward glycogen accumulation as a carbon and energy reserve. Inhibiting glycogen synthesis results in the excretion of excess carbon as organic acids, predominantly pyruvate and 2-oxoglutarate. Efficiently rerouting this carbon toward the formation of value-added products such as the plastic alternative polyhydroxybutyrate requires a deeper understanding of carbon partitioning and overflow metabolism. To investigate this, we quantified intra- and extracellular metabolites in Synechocystis wild-type and mutant strains with altered glycogen metabolism (Δpgm, ΔglgC, ΔglgA1, ΔglgA2), nitrogen signaling (ΔglnB), and carbon allocation (ΔpirC), including the double mutant ΔglgCΔpirC. Metabolites were analyzed after two days of nitrogen-replete or -depleted growth using enzymatic glycogen quantification and liquid chromatography-mass spectrometry. Excretion was primarily triggered by inhibition of glycogen synthesis but modulated by other changes in carbon flow, such as pirC deletion. Besides pyruvate and 2-oxoglutarate, small amounts of glutamate, succinate, and malate were excreted. Our findings suggest that, rather than a passive consequence of metabolite accumulation, excretion is a selective, threshold-dependent process that limits intracellular metabolite buildup, revealing an additional layer of metabolic control relevant to cyanobacterial bioengineering.

Bacillus subtilis is one of the most important production organisms in industrial biotechnology. However, there is still limited knowledge about the kinetics of fed-batch processes in bioreactors, as well as a lack of biological performance indicators, such as production yields, particularly regarding their variation over time. Understanding these kinetics and changes is crucial for optimizing the productivity in fed-batch processes. Fed-batch bioreactor cultures of Bacillus subtilis BMV9 in high cell density processes for surfactin production have been characterized with a kinetic model composed of first-order ordinary differential equations, describing the time course of biomass, substrate, surfactin and acetate. This model contributes to understanding critical restrictions and the knowledge gained was used to design and implement a model-based process. The model integrates biomass growth based on Monod kinetics, substrate consumption, surfactin synthesis and formation of the by-product acetate. After the model was parameterized for B. subtilis BMV9 using 12 different fed-batch bioreactor experiments, the kinetic model was able to accurately describe biomass accumulation, substrate consumption, product formation rates and, to some extent, the overflow metabolism involving acetate. Based on this, the kinetic model was used for a process design, in which the batch was omitted, which led to a product titre of 46.33g/L and a space-time-yield of 2.11g/(L*h) was achieved. The kinetic model developed in this study enables the description of the time course of biomass growth, substrate consumption and product formation and thus significantly improves process understanding. The computation of process parameters, which are not analytically accessible at any time, could be realized. A sensitivity analysis identified the maximum specific growth rate, substrate-related maintenance and the maximum acetate formation rate as key parameters influencing model outputs.

Ethanol metabolism in anaerobic digestion systems for better renewable energy recovery from waste feedstocks.

Energy-carbon flow reallocation in microalgae under high CO2: Platform for carbon utilization and wastewater valorization in steel industry.

Chromochloris zofingiensis is of interest for its ability to perform a reversible trophic switch in the presence of glucose that is characterized by a shutdown of photosynthesis and an accumulation of energy storage metabolites. Previous work has shown that this trophic switch is accompanied by overflow metabolism and the production of lactate in aerobic conditions. This trophic switch is not observed in nutrient replete media. We utilized isotopically assisted metabolic flux analysis to characterize intracellular flux distributions that are associated with different metabolic phenotypes observed in this organism in different media formulations in light and dark conditions. The results of this analysis showed differences in flux through carbon fixation reactions, the TCA cycle and through the reaction catalyzed by pyruvate kinase. This analysis was complemented with transcriptomics data collected for C. zofingiensis grown in iron limited conditions to provide further evidence towards the negative impact of iron limitation on both photosynthetic and respiratory activity. Overflow metabolism allows this alga to compensate for the lower energy production that results from iron limitation. This work highlights how nutrient availability can lead to drastic changes in the metabolism of C. zofingiensis.

Bacterial overflow metabolism, where cells perform oxidative fermentation despite the availability of ample oxygen and carbon sources, remains a long-standing paradox in microbial metabolism. Traditional explanations attribute this phenomenon to bacterial physiology, including rapid growth, redox imbalances, competitive advantages in microbiomes, and catabolite repression. However, recent advances in systems biology have revealed additional contributing factors, such as thermodynamic constraints, proteome allocation efficiency, bioenergetics, and the membrane real estate hypothesis. Despite these insights, a comprehensive commentary that critically examines these perspectives is still lacking. In this mGem, we summarize key drivers of overflow metabolism, examine state-of-the-art theories, and identify unresolved questions in current understanding. By evaluating multiple viewpoints, we aim to provide a cohesive analysis of bacterial overflow metabolism and contribute to a broader understanding of microbial physiology, regulatory networks, and evolutionary adaptations shaping metabolic strategies.

Balancing the cellular budget: Lessons in metabolism from microbes to cancer.

ABSTRACTA series of Escherichia coli streamlined strains was developed by removing the expression of genes encoding extracellular structures and unessential enzymes. The streamlined strains exhibited improved metabolic performance, including lower overflow metabolism and ATP maintenance coefficient, as well as a higher growth rate, compared to their parental strain. The intracellular levels of ATP were monitored using a genetic sensor, showing the improved resource stewardship of the streamlined cells. The streamlined strains were tested as cell factories to produce plasmid DNA (pDNA) in batch cultures, exhibiting a 23% increase in the specific pDNA production rate, compared to the parental strain. Recombinant protein expression was evaluated in microbioreactors in batch and fed‐batch modes. In batch mode, recombinant protein yield from biomass was up to 82% higher in the streamlined strains than in the parental strain. Furthermore, in fed‐batch mode, the recombinant protein yield was 79% greater in the streamlined cells compared to the parental strain. Our results show the benefits of reducing cellular complexity on the biomanufacturing of pDNA and recombinant proteins in culture schemes typical of industrial settings.

BackgroundAnaerobic gut fungi, known for their diverse carbohydrate-active enzymes and hydrogen production, have promising potential for the valorization of lignocellulosic materials. Despite being classified nearly 50 years ago and re-categorized into the phylum Neocallimastigomycota in 2007, their growth conditions and metabolism remain largely underexplored. This study investigates the metabolic responses of Aestipascuomyces dupliciliberans, Caecomyces churrovis, Khyollomyces ramosus, Orpinomyces joyonii, Pecoramyces ruminantium, and Neocallimastix cameroonii under various conditions, including different growth temperatures, wheat straw particle sizes, alternative carbon sources, and cultivation methods.ResultsStrain-specific differences were observed in temperature tolerance and metabolite production. Optimal growth occurred at 39 °C, while hydrogen production peaked at 41 °C in N. cameroonii, P. ruminantium, and C. churrovis. Larger wheat straw particles (2–3 mm) partially enhanced hydrogen yields, and soluble carbon sources such as glucose and cellobiose were efficiently metabolized, whereas xylose led to stress responses and low hydrogen output, particularly in K. ramosus and O. joyonii. High sugar concentrations triggered overflow metabolism, with increased lactate and formate production in A. dupliciliberans and N. cameroonii, while K. ramosus, lacking lactate dehydrogenase, accumulated formate and succinate. Fed-batch cultivation did not improve yields, likely due to substrate overfeeding and end-product inhibition. Biowaste substrates such as cucumber, carrot, and potato peels were effectively degraded and supported fungal growth. Notably, a novel morphological growth form was observed in O. joyonii under starvation conditions, suggesting a stress-induced developmental transition.ConclusionThis study provides valuable insights into the growth and physiology of anaerobic gut fungi and complements existing genomic data. The robustness of the process with respect to temperature, carbon source and substrate properties was evaluated, improving the understanding of anaerobic gut fungi cultivation and handling.

Escherichia coli is widely used in biopharmaceutical production due to its ability to grow aerobically and produce proteins intracellularly. However, the limitation of the E. coli fermentation process is acetate accumulation, a by-product of overflow metabolism during high-glucose aerobic growth, which negatively impacts cell growth and protein expression. Traditional strategies to mitigate this include genetic modifications or low-density fermentation, which have significant limitations. In the present study, a novel fed-batch fermentation strategy was developed to reduce acetate accumulation and enhance the production of recombinant pneumococcal surface adhesin A (PsaA). A design of experiments (DOE) was conducted to optimize the culture media and develop a real-time, feedback-controlled feeding strategy that prevents acetate accumulation without requiring genetic alterations. Initial runs with 20g/L glucose resulted in acetate accumulation of 7-8g/L and limited biomass growth. By lowering glucose concentration to 10g/L and inducing a carbon-limited phase via controlled feeding, E. coli cells switched from acetate production to consumption through the reverse Pta-AckA pathway. This shift led to an over 80% reduction in acetate levels. Optimized conditions consistently yielded higher cell densities. OD₆₀₀ values of 100-120 were achieved.The desired yield of the protein pneumococcal surface adhesin A (PsaA) was 3.0 g/L, representing a 2.0-fold increase over unoptimized runs. SDS-PAGE and quantitative analyses confirmed consistent robust protein expression. The strategy was validated across multiple batches, proving reproducible, scalable, and regulatory friendly. This approach offers a cost-effective and efficient alternative to genetic modification for controlling overflow metabolism and enhancing recombinant protein yields in E. coli.

High glutamate demand enables simultaneous consumption of glycerol and citrate despite carbon catabolite repression in engineered Bacillus subtilis strains.

Bacterial metabolic strategies are fundamentally linked to their physical form, yet a quantitative understanding of how cell size and shape constrain the efficiency of biomass production remains poorly understood. Here, we develop a coarse-grained whole-cell model of bacterial physiology that integrates proteome allocation, metabolic fluxes, and cell geometry with physical limits on cell surface area and intracellular diffusion. Our model shows that the efficiency of cellular growth is not monotonic with nutrient availability; instead, it peaks precisely at the onset of overflow metabolism, framing this metabolic switch as an optimal tradeoff between efficient use of imported nutrients and rapid growth. By simulating perturbations to cell morphology, we demonstrate the strong metabolic advantage of a high surface-to-volume ratio, which consistently improves growth efficiency. Finally, we show how geometric limits on growth efficiency result in a hard physical constraint: the maximum sustainable cell size is inversely related to the growth rate. This is due to a fundamental conflict between the proteomic cost of growth speed and the cost of size, which creates a budget crisis in large, fast-growing cells. Our work shows how a few physical rules define the allowable strategies for bacterial metabolism and provides a mechanistic explanation for the observed limits on microbial cell size and growth.

A metabolic theory is presented for predicting maximum growth rate, overflow metabolism, respiration efficiency, and maintenance energy flux based on the intersection of cell geometry, membrane protein crowding, and metabolism. The presented membrane-centric theory employs biophysical properties and metabolic systems analysis to successfully predict phenotypic properties of Escherichia coli K-12 strains MG1655 and NCM3722. The strains are genetically similar but differ in surface area to volume (SA:V) ratios by up to 30%, maximum growth rates on glucose media by 40%, and overflow-inducing growth rates by 80%. The predictions were tested against experimental evidence including phenomics data, membrane proteomics data, and MG1655 SA:V mutant growth rates. The predictions were remarkably consistent with experimental data and provided a membrane-centric explanation for maximum growth rate, maintenance energy generation, respiration chain efficiency (P/O number), and optimal biomass yield of a strain. These analyses did not consider cytosolic macromolecular crowding, highlighting the distinct properties of the presented theory and gaps in current cell biology literature. Cell geometry and membrane protein crowding are significant biophysical constraints and consideration of both provide a more complete theoretical framework for improved understanding and control of cell biology.

Dynamically metabolic engineering overflow metabolism for efficient production of l-alanine in Escherichia coli.

Sugar-induced modulation of biogenic amines formation and metabolic profiles during Bacillus subtilis fermentation.

A classic problem in metabolism is that fast-proliferating cells use seemingly wasteful fermentation for energy biogenesis in the presence of sufficient oxygen. This counterintuitive phenomenon, known as overflow metabolism or the Warburg effect, is universal across various organisms. Despite extensive research, its origin and function remain unclear. Here, we show that overflow metabolism can be understood through growth optimization combined with cell heterogeneity. A model of optimal protein allocation, coupled with heterogeneity in enzyme catalytic rates among cells, quantitatively explains why and how cells choose between respiration and fermentation under different nutrient conditions. Our model quantitatively illustrates the growth rate dependence of fermentation flux and enzyme allocation under various perturbations and is fully validated by experimental results in Escherichia coli. Our work provides a quantitative explanation for the Crabtree effect in yeast and the Warburg effect in cancer cells and can be broadly used to address heterogeneity-related challenges in metabolism.

A classic problem in metabolism is that fast-proliferating cells use seemingly wasteful fermentation for energy biogenesis in the presence of sufficient oxygen. This counterintuitive phenomenon, known as overflow metabolism or the Warburg effect, is universal across various organisms. Despite extensive research, its origin and function remain unclear. Here, we show that overflow metabolism can be understood through growth optimization combined with cell heterogeneity. A model of optimal protein allocation, coupled with heterogeneity in enzyme catalytic rates among cells, quantitatively explains why and how cells choose between respiration and fermentation under different nutrient conditions. Our model quantitatively illustrates the growth rate dependence of fermentation flux and enzyme allocation under various perturbations and is fully validated by experimental results in Escherichia coli. Our work provides a quantitative explanation for the Crabtree effect in yeast and the Warburg effect in cancer cells and can be broadly used to address heterogeneity-related challenges in metabolism.

A classic problem in metabolism is that fast-proliferating cells use seemingly wasteful fermentation for energy biogenesis in the presence of sufficient oxygen. This counterintuitive phenomenon, known as overflow metabolism or the Warburg effect, is universal across various organisms. Despite extensive research, its origin and function remain unclear. Here, we show that overflow metabolism can be understood through growth optimization combined with cell heterogeneity. A model of optimal protein allocation, coupled with heterogeneity in enzyme catalytic rates among cells, quantitatively explains why and how cells choose between respiration and fermentation under different nutrient conditions. Our model quantitatively illustrates the growth rate dependence of fermentation flux and enzyme allocation under various perturbations and is fully validated by experimental results in Escherichia coli. Our work provides a quantitative explanation for the Crabtree effect in yeast and the Warburg effect in cancer cells and can be broadly used to address heterogeneity-related challenges in metabolism.