Exposure to excessive nutrient concentrations can trigger remarkable responses in cells. In yeasts, such as Saccharomyces cerevisiae, high glucose availability results in less energy-efficient anaerobic combustion to ethanol, regardless of an oxygenated environment allowing for respiration. Likewise, bacteria, such as Escherichia coli, utilize the strategy of incomplete oxidation to acetate when a surplus of glucose is found in the environment, while lactate accumulation is associated with the cultivation of mammalian cells (Vazquez, 2018; Luginsland et al., 2024). This seemingly wasteful strategy results in less energy than complete oxidation via respiration; still, it occurs ubiquitously among fast-growing cells and is generally referred to as overflow metabolism. Although the exact reason for this phenomenon is still debated, it is widely accepted to result from a combination of several factors (Bernal et al., 2016). Szenk et al. argue that anaerobic combustion of C-sources is preferred in fast-growing cells due to an increased crowding of respiratory proteins caused by a reduced surface-to-volume ratio (Szenk et al., 2017). Co-cultivation experiments propose a competitive advantage of organisms exploiting an overflow metabolism by rapidly depleting nutrients and producing toxic metabolites, thereby inhibiting competing organisms (Yasir et al., 2024). Other theories suggest a protective measure against the harmful accumulation of metabolites (Li et al., 2022) or energy savings when employing fermentation (Basan et al., 2015).

Conversely, only little is known about the effect of nutrient surplus on archaea. Aerobic halophilic archaea, such as those in the genera Haloferax and Haloarcula, produce acetate as a byproduct during batch cultivation. Acetate is then consumed when the cells reach the stationary phase, similar to what occurs in E. coli (Oren and Gurevich, 1994; Kuprat et al., 2021). However, the mechanism of acetate formation in halophilic archaea differs significantly from their bacterial counterparts. Haloferax utilizes an ADP-forming acetyl-CoA synthetase to convert acetyl-CoA to acetate instead of the classical two-step reaction in bacteria (Bräsen and Schönheit, 2001). More detailed considerations regarding overflow metabolism are missing in the literature, particularly for the phylum Crenarchaeota. However, indications of incomplete glucose oxidation at high growth rates suggest the presence of overflow metabolism in crenarchaea (Rastädter et al., 2021).

Sulfolobus acidocaldarius, one of the best-characterized members of Crenarchaeota, thrives under extreme conditions, with an optimal pH = 3 and a growth temperature of 75 °C. S. acidocaldarius has been intensively investigated for several biotechnological applications, such as enzyme and lipid production (Quehenberger et al., 2017; Sedlmayr et al., 2024b). Only recently, the upscaling of a continuous cultivation process to 200 L was reported, solidifying the development toward industrial applications of extremophilic bioprocesses (Rastädter et al., 2023). Further progress in the biotechnological utilization of S. acidocaldarius requires progress in the basic understanding of metabolic behavior against stress factors to enhance process efficiency and product yields (Gecse et al., 2024). This knowledge can guide process intensification strategies, leading to improved yields, cost reductions, and enhanced sustainability in industrial processes. Apart from temperature and pH extremes, S. acidocaldarius’ natural habitat of acid thermal soils and acid hot springs is characterized by various environmental hurdles. Numerous studies have explored the extremophile’s responses to stress, revealing a versatile repertoire of metabolic, morphological, and physiological answers. Stress factors that have been studied include temperature (Baes et al., 2023), nutrient limitation (Bischof et al., 2019), solvent stress (Benninghoff et al., 2021), salt stress (Stracke et al., 2020), and pH stress (Buetti-Dinh et al., 2016).

In this study, chemostat cultivations were performed to assess the physiological and transcriptome response of S. acidocaldarius to excess nutrient concentrations. The defined Vienna Defined (VD) Medium, containing two C-sources, glucose and monosodium glutamate (MSG), was used as a substrate (Quehenberger et al., 2019). Chemostat cultivation is the direction the biopharmaceutical industry is heading, offering valuable advantages for experimental design (Kopp et al., 2019). It allows the cultivation at a precise specific growth rate and enables the manipulation of particular parameters with minimal impact on other process conditions (Mendes et al., 2013). By carefully adjusting the process parameters, we analyzed three conditions: (I) “Low,” with a moderate cell density and specific substrate uptake rate (q_s), (II) “High,” with a higher cell density and q_s, and (III) “Overfeed”: characterized by the highest cell density and q_s, resulting in the accumulation of C-source in the broth. This experimental setup allowed us to differentiate between effects caused by high cell concentrations and impacts associated with excessive nutrient availability. With the help of RNA-sequencing (RNA-seq) and computational methods using parsimonious flux balance analysis, we obtained fundamental insights into the response of S. acidocaldarius to excessive nutrient availability, including shifts in energy and carbon management, respiration, and preferential use of different metabolic pathways.

This study assessed the physiological, transcriptome, and metabolic response of S. acidocaldarius to different nutrient availability. While the behavior of mammalian, fungal, and bacterial cells in nutrient-rich environments has been extensively studied for several decades, the effects on archaeal organisms, especially Crenarchaeota, have remained largely unexplored. This highlights the significant challenges and limitations in archaeal biotechnology. Despite recent advances in genetic manipulation techniques for archaea, there are still considerable obstacles compared to those for their eukaryotic and bacterial counterparts. These difficulties are partly due to the extreme conditions required and the relatively low growth rates associated with many archaeal species. This underscores the importance of improving cultivation methods for archaea and emphasizes the relevance of our efforts in this field (Quehenberger et al., 2019; Rastädter et al., 2021; Sedlmayr et al., 2024a).

Therefore, we decided to perform chemostat cultivations, allowing the study of cells under strictly defined physiological steady states with theoretical unchanging concentrations of intra-and extracellular molecules (Adamberg et al., 2015). Consequently, the samples derived from equal process parameters are associated with a high degree of homogeneity, allowing the almost unaltered study of the temporal dynamics of cells when compared to samples derived from simple batch experiments (Nieto-Taype et al., 2020). The bioprocessing of extremophilic archaea has experienced significant progress in recent years, mainly due to their enormous potential for the biopharmaceutical industry (Quehenberger et al., 2017). This development is exemplified by the development of a defined medium for the cultivation of several Sulfolobus strains (Quehenberger et al., 2019) and the determination of critical process parameters for the continuous cultivation of S. acidocaldarius (Rastädter et al., 2021).

Building on these achievements, we analyzed three states during chemostat cultivations—Low, High, and Overfeed—each characterized by adjustments in total flow rate and nutrient concentration in the feed, resulting in different C-source availability in the bioreactor. “Low” represents the baseline condition with minimal stress on the organism, allowing for the examination of the metabolic state without overflow metabolism (40% of μ_max). In “High,” stress was introduced to the system by increasing both TFR and nutrient concentration, enhancing the specific growth rate to almost 50% of μ_max. This allowed for the examination of the cellular response toward higher nutrient availability without causing C-source accumulation and provided a middle ground to understand the transition from normal to stressed metabolic states. “Overflow” represents a high stress condition, where maximization of the nutrient availability leads to C-source accumulation and significant overflow metabolism. Here, cultivations were conducted at approximately 70% of μ_max.

By adjusting the respective process parameters, cell density was enhanced from 5.0 g/L (Low) up to 18.5 g/L (Overfeed). The significantly increased μ also reflects the increased biomass concentration. Consequently, with enhanced nutrient availability, the specific substrate uptake rates for glucose and MSG increased. Noteworthy, the ratio between q_MSG and q_Glc was decreasing from Low to Overfeed. With increasing nutrient availability, the preference for the two C-sources changed, eventually leading to an almost 40-fold higher accumulation rate for MSG than for glucose. This observation is striking, as the Monod substrate affinity constant of S. acidocaldarius toward MSG has been described to be 7-fold higher than for glucose (Rastädter et al., 2021).

Two transporters, putatively involved in glutamate transport, were upregulated, whereas saci_1835 was differentially transcribed only for the Overfeed condition. Reasons for the enhanced MSG accumulation might be a saturation of transporters for MSG or a changing regulatory mechanism. While we cannot conclude the degree of saturation, the reason behind the accumulation of MSG is likely a saturated TCA cycle and/or the accumulation of harmful metabolites. While glucose utilization exploits flexibility due to the branching of the ED pathway, the incorporation of MSG into the metabolism is directly linked to its transformation to AKG and utilization via the TCA cycle, resulting in the production of ATP. ATP has already been identified as harmful at elevated concentrations (Li et al., 2022). As predicted by pFBA, the cells performed substantial efforts in the depletion of intermediates of the TCA cycle with increasing nutrient availability, with higher fluxes and more active reactions to deplete intermediates of the TCA cycle. The putative glucose ABC transporter was upregulated in Overfeed, potentially explaining the efficient glucose utilization and its low accumulation rate. S. acidocaldarius utilizes a modified ED pathway for glucose utilization, including two branches. In Sa. solfataricus, the semi-phosphorylative pathway is used for glycogenetic functions, while the glycolytic branch is completed in the non-phosphorylative pathway (Kouril et al., 2013). Thereby, the formation of thermolabile intermediates of the semi-phosphorylative ED pathway, such as GAP, DHAB and 1,3 BPG, is avoided (Schmerling et al., 2022). According to pFBA, the flux toward the non-phosphorylative ED pathway was decreased in Overfeed, increasing glycogenetic utilization of glucose. Thereby, the incorporation of additional carbon into the TCA cycle and additional ATP production was prevented. It can only be speculated what the consequence higher fluxes of semi-phosphorylative ED pathway have on intracellular methylglyoxal concentration, a non-enzymatical decomposition product of GAP and DHAB. Methylglyoxal is known to cause dicarbonyl stress (Schmerling et al., 2022).

The constant biomass yield observed across the three conditions suggested that nutrient availability and potential overflow metabolism did not impact biomass production, indicating a robust metabolic regulation. Previous studies showed that process parameters, such as pH and temperature, are critical determinants of biomass yield in S. acidocaldarius (Cobban et al., 2020; Rastädter et al., 2021). The constant Y_x/s implies that the biomass concentration of S. acidocaldarius could potentially be boosted if a different feeding strategy was applied, such as exponentially increasing the substrate concentration. Additionally, since the MSG concentration in the VD Medium was close to the maximal metabolic capacity, increasing the glucose concentration in the feed might help sustain high carbon influx. In contrast to the biomass yield, CO₂ yield dropped severely with increasing nutrient availability from 0.64 Cmol/Cmol (Low) to 0.36 Cmol/Cmol (Overfeed). Such observations strongly indicate an active overflow metabolism, where an increased anabolic activity favors the production of various excreted metabolites, reducing the amount of carbon funneled through oxidative pathways (Valgepea et al., 2010). Accordingly, trehalose production, a common secretion product associated with the cultivation of Sulfolobales (Martins et al., 1997), was enhanced. Hence, flux for the glycogenetic ED pathway was increased. However, no difference between High and Overfeed could be obtained for q_Trehalose. A lagging formation and secretion of trehalose might be the reason for similar trehalose production rates (Stracke et al., 2020). In addition to trehalose, amino acids—particularly valine, glycine, and alanine—were found as secretion products in the cell broth. The secretion of amino acids may serve multiple purposes: Firstly, as products of pyruvate and other metabolic intermediates, they help reduce intracellular concentrations of these metabolites, alleviating flux toward the already saturated TCA cycle. Secondly, their synthesis and secretion help to manage increased NADPH levels derived from the ED pathway due to the high glucose flux, thereby maintaining redox balance within the cell. Thirdly, the secretion of these amino acids helps regulate intracellular nitrogen balance by removing excess nitrogen in the form of amino groups, thus maintaining nitrogen homeostasis. The non-closing C-balance indicates the presence of other compounds that are produced. As the HPLC method can detect several metabolites, we checked our chromatograms and compared the retention times of common secretion products. Therefore, we could exclude the secretion of metabolites, such as lactate, acetate, ethanol, glycerol, oxaloacetate, malate, pyruvate and citrate.

Apart from the secretion of metabolites, intracellular carbon storage may help explain the non-closing C-balance. Glycogen has been reported to serve as a central carbon storage in several Sulfolobales species (König et al., 1982). No differential expression of the glg operon could be detected by RNA-seq (Lee et al., 2021). However, pFBA predicts substantial flux for the glycogen pathway for High and Overfeed. Apart from polysaccharides, lipids are commonly used energy reservoirs in nature. Although the incorporation of ether lipids into the membrane is one distinct feature of the archaeal domain, the abundance of fatty acid components has long been known (Langworthy et al., 1974). Their exact role in archaea is still a matter of debate, while a vital role in the metabolism is likely, as the association of fatty acids to specific proteins is crucial for functionality (Corcelli et al., 1996). Recently, the pathways for fatty acid synthesis and β-oxidation in S. acidocaldarius have been described (Schmerling et al., 2024). As cultivation of S. acidocaldarius on short-chain fatty acids (C₄ and C₆) has been demonstrated experimentally (Wang et al., 2019), the utilization of fatty acids as an energy reservoir is likely. Therefore, fatty acid anabolism could be a plausible contributor to the missing carbon. Genes associated with the reconstructed fatty acid synthesis have not been differentially transcribed (Schmerling et al., 2024), however other genes with putative involvement in the fatty acid metabolism (saci_1103-saci_1126) (Dibrova et al., 2014; Wang et al., 2019). Besides, genes from the cluster saci_2231–2235 were upregulated, encoding acyl-CoA dehydrogenases, acetyl-CoA acetyltransferase and acyl-CoA synthetase. This cluster was also associated with downregulation during cultivations on xylose compared to a mixture of xylose and NZ-amines (Wagner et al., 2018). One can speculate on the role of excess amino acids in the formation of fatty acids in archaea. Additionally, the biosynthesis of fatty acids is accompanied by the depletion of acetyl-CoA, which alleviates the oversaturation of the TCA cycle by depleting the intermediate and hinders its accumulation. Furthermore, due to the consumption of NADPH during fatty acid synthesis, fatty acid synthesis can serve as a measurement to counteract redox imbalance.

The increased transcription values for acetyl-CoA acetyltransferases might indicate a regulation of ether lipids in the membrane. Acetoacetyl-CoA is a precursor for mevalonate synthesis, with mevalonate ultimately being the starting point for the biosynthesis of isoprenoids (Matsumi et al., 2011). The membrane composition in S. acidocaldarius is heavily dependent on the applied growth rate (Rastädter et al., 2020). Studies show decreased cyclopentane rings in the tetraether lipid and the share of diether lipids with increasing nutrient availability (Bischof et al., 2019; Quehenberger et al., 2020). Consequently, in a nutrient-rich environment, membrane permeability increases due to decreased rings and decreased share of diether lipids.

Proton transporters from the respiratory chain are involved in the maintenance of pH homeostasis (Baker-Austin and Dopson, 2007). As found for Overfeed, the overexpression of SoxEFGHIM terminal oxidase was accompanied by the translocation of two protons compared to only one proton translocated using the other two terminal oxidases, SoxABCDL and DoxBCE. Interestingly, this was also observed during nutrient depletion (Bischof et al., 2019), indicating increased intracellular acidification. The overexpression of SoxEFGHIM was accompanied by the overexpression of several permeases and symporters, such as saci_0383, saci_1745 (both Na⁺/proline symporter) and saci_2039 (purine-cytosine permease) in the Overfeed condition. These transporters might be involved in cytoplasmic buffering as a response to the increased acidification (Bischof et al., 2019; Helmecke, 2019). The simultaneous downregulation of cytochrome SoxNL-CbsAB-OdsN in Overfeed indicated a reduced overall respiration rate, likely due to lower energy demand due to the oversaturated TCA cycle.

Among the most upregulated genes was the cluster encoding proteins annotated to the catechol pathway (saci_2293-saci_2295). Similarly, Benninghoff and coworkers noticed a high upregulation of this cluster in S. acidocaldarius upon exposure to organic solvents (Benninghoff et al., 2021). While these genes are involved in the degradation of aromatic amino acids, in the context of this study, it is possible that they were also involved in the degradation of aromatic compounds in general, helping in the management of reactive oxygen species.

Additionally, according to RNA-seq, saci_2188 and saci_2212 were the top upregulated genes, with a fold change of 3.5/1.9 and 4.0/2.8 (Low/High) compared to Overfeed. As ribonucleotide reductases are crucial in DNA synthesis, this might indicate an increased demand for cellular repair machinery.

In this study, we gathered valuable insights into the basic metabolic and physiological responses of S. acidocaldarius upon excess nutrient concentrations. Despite a lower Monod affinity constant for MSG, its accumulation surpassed that of glucose. While glucose primarily undergoes glycogenesis, MSG integrates directly into the TCA cycle, leading to saturation and enhanced pyruvate levels. At the same time, metabolic regulation appeared to be robust, indicated by consistent biomass yields across the different conditions. Therefore, an adaption of glucose and MSG concentration in the VD Medium during high cell density cultivations might help to boost biomass concentrations of S. acidocaldarius. The non-closing C-balance speaks for the presence of other metabolites, with HPLC analysis excluding common secretion products. For the first time, we described the secretion of amino acids by S. acidocaldarius, with valine being secreted at rates similar to the common glycogenesis product trehalose. Furthermore, a strong impact on lipid metabolism was observed, suggesting intracellular carbon and energy storage and change in membrane composition. Also, changes in respiratory protein transcription suggested intracellular acidification at high nutrient concentrations, with identified transporters aiding cytoplasmic buffering. Overall, the study highlighted the metabolic flexibility and robust regulatory mechanisms in S. acidocaldarius. While in yeasts, bacteria and mammalian cells, excessive nutrient concentrations trigger different metabolic pathways, it appears that S. acidocaldarius tries to maintain its normal metabolism by reducing TCA intermediates, storing energy via fatty acid synthesis, secreting amino acids, and maintaining high glycogenesis. The absence of differentially expressed genes in key metabolic pathways prompts questions about the regulation at other levels, such as post-transcriptional, and suggests avenues for future research in understanding these regulatory mechanisms.