Extreme calorie restriction and energy source starvation in Saccharomyces cerevisiae represent distinct physiological states

Cultivation methods used to investigate microbial calorie restriction often result in carbon and energy starvation. This study aims to dissect cellular responses to calorie restriction and starvation in Saccharomyces cerevisiae by using retentostat cultivation. In retentostats, cells are continuously supplied with a small, constant carbon and energy supply, sufficient for maintenance of cellular viability and integrity but insufficient for growth. When glucose-limited retentostats cultivated under extreme calorie restriction were subjected to glucose starvation, calorie-restricted and glucose-starved cells were found to share characteristics such as increased heat-shock tolerance and expression of quiescence-related genes. However, they also displayed strikingly different features. While calorie-restricted yeast cultures remained metabolically active and viable for prolonged periods of time, glucose starvation resulted in rapid consumption of reserve carbohydrates, population heterogeneity due to appearance of senescent cells and, ultimately, loss of viability. Moreover, during starvation, calculated rates of ATP synthesis from reserve carbohydrates were 2-3 orders of magnitude lower than steady-state ATP-turnover rates calculated under extreme calorie restriction in retentostats. Stringent reduction of ATP turnover during glucose starvation was accompanied by a strong down-regulation of genes involved in protein synthesis. These results demonstrate that extreme calorie restriction and carbon starvation represent different physiological states in S. cerevisiae.     

Quantitative physiology of Lactococcus lactis at extreme low‐growth rates

This paper describes the metabolic adaptation of Lactococcus lactis during the transition from a growing to a non‐growing state using retentostat cultivation. Under retentostat cultivation, the specific growth rate decreased from 0.025 h^?1 to 0.0001 h^?1 in 42 days, while doubling time increased to more than 260 days. Viability of the overall culture was maintained above 90% but included approximately 20% damaged cells, which had lost their colony forming capacity on solid media. Although culture biomass and viability had reached a steady‐state after 14 days of retentostat cultivation, the morphology of the cells changed from coccus‐to‐rod shape at later stages of retentostat cultivation, by which the cell's surface to volume ratio was estimated to increase 2.4‐fold. Furthermore, the metabolic patterns switched between homolactic and mixed‐acid fermentation during the retentostat cultivation. Retentostat cultivation enabled the calculation of accurate substrate‐ and energy‐related maintenance coefficients and biomass yields under non‐growing conditions, which were in good agreement with those calculated by extrapolation from chemostat cultivations at high dilution rates. In this study, we illustrate how retentostat cultivation allows decoupling of growth and non‐growth associated processes in L. lactis, enabling the analysis of quantitative physiological responses of this bacterium to near zero‐specific growth rates.     

Maintenance energy demand and starvation recovery dynamics of Nitrosomonas europaea and Nitrobacter winogradskyi cultivated in a retentostat with complete biomass retention.

Nitrosomonas europaea and Nitrobacter winogradskyi (strain "Engel") were grown in ammonia-limited and nitrite-limited conditions, respectively, in a retentostat with complete biomass retention at 25 degrees C and pH 8. Fitting the retentostat biomass and oxygen consumption data of N. europaea and N. winogradskyi to the linear equation for substrate utilization resulted in up to eight-times-lower maintenance requirements compared to the maintenance energy demand (m) calculated from chemostat experiments. Independent of the growth rate at different stages of such a retention culture, the maximum specific oxygen consumption rate measured by mass spectrometric analysis of inlet and outlet gas oxygen content always amounted to approximately 45 micromol of O2 mg-1 of biomass-C x h-1 for both N. europaea and N. winogradskyi. When bacteria were starved for different time periods (up to 3 months), the spontaneous respiratory activity after an ammonia or nitrite pulse decreased with increasing duration of the previous starvation time period, but the observed decrease was many times faster for N. winogradskyi than for N. europaea. Likewise, the velocity of resuscitation decreased with extended time periods of starvation. The increase in oxygen consumption rates during resuscitation referred to the reviving population only, since in parallel no significant increase in the cell concentrations was detectable. N. europaea more readily recovers from starvation than N. winogradskyi, explaining the occasionally observed nitrite accumulation in the environment after ammonia becomes available. From chloramphenicol (100 microg x ml-1) inhibition experiments with N. winogradskyi, it has been concluded that energy-starved cells must have a lower protein turnover rate than nonstarved cells. As pointed out by Stein and Arp (L. Y. Stein and D. J. Arp, Appl. Environ. Microbiol. 64:1514-1521, 1998), nitrifying bacteria in soil have to cope with extremely low nutrient concentrations. Therefore, a chemostat is probably not a suitable tool for studying their physiological properties during a long-lasting nutrient shortage. In comparison with chemostats, retentostats offer a more realistic approach with respect to substrate provision and availability.     

Physiology of Geobacter metallireducens under excess and limitation of electron donors. Part II. Mimicking environmental conditions during cultivation in retentostats

The strict anaerobe Geobacter metallireducens was cultivated in retentostats under acetate and acetate plus benzoate limitation in the presence of Fe(III) citrate in order to investigate its physiology under close to natural conditions. Growth rates below 0.003 h⁻¹ were achieved in the course of cultivation. A nano-liquid chromatography–tandem mass spectrometry-based proteomic approach (nano-LC–MS/MS) with subsequent label-free quantification was performed on proteins extracted from cells sampled at different time points during retentostat cultivation. Proteins detected at low (0.002 h⁻¹) and high (0.06 h⁻¹) growth rates were compared between corresponding growth conditions (acetate or acetate plus benzoate). Carbon limitation significantly increased the abundances of several catabolic proteins involved in the degradation of substrates not present in the medium (ethanol, butyrate, fatty acids, and aromatic compounds). Growth rate-specific physiology was reflected in the changed abundances of energy-, chemotaxis-, oxidative stress-, and transport-related proteins. Mimicking natural conditions by extremely slow bacterial growth allowed to show how G. metallireducens optimized its physiology in order to survive in its natural habitats, since it was prepared to consume several carbon sources simultaneously and to withstand various environmental stresses.     

Quantitative Physiology of Saccharomyces cerevisiae at Near-Zero Specific Growth Rates

Growth at near-zero specific growth rates is a largely unexplored area of yeast physiology. To investigate the physiology of Saccharomyces cerevisiae under these conditions, the effluent removal pipe of anaerobic, glucose-limited chemostat culture (dilution rate, 0.025 h⁻¹) was fitted with a 0.22-μm-pore-size polypropylene filter unit. This setup enabled prolonged cultivation with complete cell retention. After 22 days of cultivation, specific growth rates had decreased below 0.001 h⁻¹ (doubling time of >700 h). Over this period, viability of the retentostat cultures decreased to ca. 80%. The viable biomass concentration in the retentostats could be accurately predicted by a maintenance coefficient of 0.50 mmol of glucose g⁻¹ of biomass h⁻¹ calculated from anaerobic, glucose-limited chemostat cultures grown at dilution rates of 0.025 to 0.20 h⁻¹. This indicated that, in contrast to the situation in several prokaryotes, maintenance energy requirements in S. cerevisiae do not substantially change at near-zero specific growth rates. After 22 days of retentostat cultivation, glucose metabolism was predominantly geared toward alcoholic fermentation to meet maintenance energy requirements. The strict correlation between glycerol production and biomass formation observed at higher specific growth rates was not maintained at the near-zero growth rates reached in the retentostat cultures. In addition to glycerol, the organic acids acetate, d-lactate, and succinate were produced at low rates during prolonged retentostat cultivation. This study identifies robustness and by-product formation as key issues in attempts to uncouple growth and product formation in S. cerevisiae.Laboratory studies on microorganisms are often performed in batch cultures. During the initial phase of batch cultivation, all nutrients are usually present in excess. As a consequence, the initial specific growth rate, μ, of the microorganism in such cultures equals the maximum specific growth rate, μ_max. In natural environments, the specific growth rate of microorganisms is likely to be constrained by the limited availability of one or more growth-limiting nutrients, resulting in specific growth rates far below μ_max (8, 24). In chemostat cultures fed with a medium containing a single growth-limiting nutrient, the dilution rate determines the specific growth rate. Chemostat cultivation therefore offers the possibility to study microbial physiology at carefully controlled, submaximal specific growth rates and to investigate the effect of specific growth rate on cellular physiology (20). Chemostat cultivation of the yeast Saccharomyces cerevisiae has demonstrated strong effects of specific growth rate on biomass composition (26, 51), product formation (5, 37), and cell size (23). Moreover, during energy-limited growth at low specific growth rates, a relatively large fraction of the energy substrate has to be dissimilated for maintenance-related processes such as maintenance of chemi-osmotic gradients and turnover of cellular components (34). Not surprisingly, recent genome-wide studies have shown strong effects of specific growth rate on levels of mRNAs and proteins (9, 14, 38).In chemostat studies on S. cerevisiae, the steady-state specific growth rate is usually between 0.03 h⁻¹ and 0.40 h⁻¹. While this range is relevant for many industrial applications, there are several incentives to study growth of this yeast at even lower specific growth rates. In many natural environments, growth at a μ of 0.03 h⁻¹, corresponding to a doubling time of 23.1 h, probably still represents extremely fast growth. Furthermore, in industrial applications, S. cerevisiae and other microorganisms can be considered as self-replicating catalysts, and, unless biomass is the desired product, growth can be considered as undesirable by-product formation leading to nonproductive substrate consumption. This problem is further augmented when the excess yeast biomass cannot be valorized because it is genetically modified or has been used for the production of compounds that are not compatible with use as, for example, cattle feed. A third incentive for exploring the physiology of S. cerevisiae at near-zero growth rates is related to the increasing interest in this yeast as a systems biology model for human cells (16, 27, 33). At near-zero growth rates, the age of individual yeast cells becomes much higher than can be achieved in conventional batch or chemostat cultures. Studies on extremely slow growth of S. cerevisiae under defined conditions may therefore provide an interesting model for ageing of human cells.Retentostat cultivation, first proposed by Herbert (18), is a modification of chemostat cultivation that has been specifically designed to study microbial physiology at near-zero specific growth rates. In a retentostat, sometimes referred to as recycling fermentor or recyclostat, the growth-limiting energy substrate is fed at a constant rate, and biomass is retained in the fermentor by an internal filter probe connected to the effluent line or by an external filter module. Prolonged retentostat cultivation should, in theory, result in a situation where the specific growth rate becomes zero and where the specific rate of substrate consumption equals the maintenance energy requirement. This situation is fundamentally different from starvation, which involves deterioration of physiological processes, and from resting states typified by spores, which have little or no metabolic activity. Retentostat cultivation has been applied to several bacterial systems including Escherichia coli (11), Paracoccus denitrificans, and Bacillus licheniformis (49) and the autotrophs Nitrosomonas europaea and Nitrobacter winogradskyi (46, 47). These studies demonstrated that the physiology of these prokaryotes at extremely low specific growth rates could not be accurately predicted by a simple extrapolation of results obtained at higher specific growth rates. In particular, near-zero specific growth rates coincided with increased levels of ppGpp (2), which induces the stringent response, a regulatory program that diverts cellular resources from growth to amino acid biosynthesis (10, 21). Furthermore, it was concluded that extremely slow growth led to a reduction of the maintenance energy requirement of prokaryotes. A recent quantitative analysis on cell retention cultures of S. cerevisiae was performed under severely nitrogen-limited growth conditions and used incomplete cell retention (7), which precluded a quantitative comparison with maintenance energy requirements calculated from energy-limited chemostat cultures.The goal of the present study was to quantitatively analyze the physiology of S. cerevisiae at extremely low specific growth rates in glucose-limited retentostat cultures. To this end, an internal filter probe was introduced in the effluent line of standard laboratory chemostat fermentors and used in long-term cultivation runs with complete cell retention. Anaerobic conditions were chosen to facilitate quantification of catabolic fluxes and growth energetics.     

Maintenance Energy Demand and Starvation Recovery Dynamics of Nitrosomonas europaea and Nitrobacter winogradskyi Cultivated in a Retentostat with Complete Biomass Retention

Nitrosomonas europaea and Nitrobacter winogradskyi (strain “Engel”) were grown in ammonia-limited and nitrite-limited conditions, respectively, in a retentostat with complete biomass retention at 25°C and pH 8. Fitting the retentostat biomass and oxygen consumption data of N. europaea and N. winogradskyi to the linear equation for substrate utilization resulted in up to eight-times-lower maintenance requirements compared to the maintenance energy demand (m) calculated from chemostat experiments. Independent of the growth rate at different stages of such a retention culture, the maximum specific oxygen consumption rate measured by mass spectrometric analysis of inlet and outlet gas oxygen content always amounted to approximately 45 μmol of O₂ mg⁻¹ of biomass-C · h⁻¹ for both N. europaea and N. winogradskyi. When bacteria were starved for different time periods (up to 3 months), the spontaneous respiratory activity after an ammonia or nitrite pulse decreased with increasing duration of the previous starvation time period, but the observed decrease was many times faster for N. winogradskyi than for N. europaea. Likewise, the velocity of resuscitation decreased with extended time periods of starvation. The increase in oxygen consumption rates during resuscitation referred to the reviving population only, since in parallel no significant increase in the cell concentrations was detectable. N. europaea more readily recovers from starvation than N. winogradskyi, explaining the occasionally observed nitrite accumulation in the environment after ammonia becomes available. From chloramphenicol (100 μg · ml⁻¹) inhibition experiments with N. winogradskyi, it has been concluded that energy-starved cells must have a lower protein turnover rate than nonstarved cells. As pointed out by Stein and Arp (L. Y. Stein and D. J. Arp, Appl. Environ. Microbiol. 64:1514–1521, 1998), nitrifying bacteria in soil have to cope with extremely low nutrient concentrations. Therefore, a chemostat is probably not a suitable tool for studying their physiological properties during a long-lasting nutrient shortage. In comparison with chemostats, retentostats offer a more realistic approach with respect to substrate provision and availability.     

Influence of growth rate and starvation on fluorescent in situ hybridization of Rhodopseudomonas palustris

In situ hybridization with a fluorescently labeled 16S rRNA-targeted probe was examined using Rhodopseudomonas palustris as a model organism, which had been grown at different rates and under different conditions of growth and starvation. The specific growth rate did not affect the percentage of hybridized cells in aerobically grown R. palustris cultures. However, significant changes in the percentage of hybridized cells occurred during extended periods of starvation. These changes were observed both in batch cultures grown and starved aerobically in the dark, and in cultures grown phototrophically and starved anaerobically in the dark. Aerobic growth in batch culture and subsequent starvation resulted in a complete lack of detectable hybridization after 20 days of starvation. In contrast, even after 30 days of starvation, 50% of all cells were still detectable in cultures grown aerobically at growth rates <0.06 h(-1) and then starved aerobically in the dark. The same was true for phototrophically grown cells that were starved anaerobically in the light. During starvation there was a clear, though non-linear, positive correlation between the percentage of hybridized cells and the RNA content. In contrast, no direct correlation was observed between the number of hybridized cells in a culture and the viability of this culture. Thus, in habitats with growing, non-growing, and starving bacteria, data on quantitative detection of populations based on 16S rRNA-targeted probing should be used with extreme caution as the detectability of the individual cells is strongly influenced by their physiological history and current physiological state.     

Physiological and Transcriptional Responses of Different Industrial Microbes at Near-Zero Specific Growth Rates

The current knowledge of the physiology and gene expression of industrially relevant microorganisms is largely based on laboratory studies under conditions of rapid growth and high metabolic activity. However, in natural ecosystems and industrial processes, microbes frequently encounter severe calorie restriction. As a consequence, microbial growth rates in such settings can be extremely slow and even approach zero. Furthermore, uncoupling microbial growth from product formation, while cellular integrity and activity are maintained, offers perspectives that are economically highly interesting. Retentostat cultures have been employed to investigate microbial physiology at (near-)zero growth rates. This minireview compares information from recent physiological and gene expression studies on retentostat cultures of the industrially relevant microorganisms Lactobacillus plantarum, Lactococcus lactis, Bacillus subtilis, Saccharomyces cerevisiae, and Aspergillus niger. Shared responses of these organisms to (near-)zero growth rates include increased stress tolerance and a downregulation of genes involved in protein synthesis. Other adaptations, such as changes in morphology and (secondary) metabolite production, were species specific. This comparison underlines the industrial and scientific significance of further research on microbial (near-)zero growth physiology.     

Cultivation of nitrifying bacteria in the retentostat, a simple fermenter with internal biomass retention

Abstract: The retentostat was developed for long-term continuous, axenic cultivation of microorganisms at those low growth rates which prevail in most natural habitats and which cannot be established properly in chemostats. How a microbial population approaches 'zero-growth' was studied in axenic cultures of Nitrosomonas europaea with complete biomass retention at 25°C and constant input of a nutrient solution containing ammonium (0.57 mM) as energy source. Since only cell-free filtrate left the reactor, biomass accumulated until a stable maximum of 2.7 × 10⁹ cells ml⁻¹ (398 mg l⁻¹ dry matter) was reached after about 5 weeks. In this state, growth rate approached zero, and the ammonium input just met the substrate demand required for maintenance energy (1.43 μmol NH₃–N mg dm⁻¹ h⁻¹). The potential of the retentostat for studying interactions between different microorganisms was demonstrated with a cascade of cultures of Nitrosomonas, Nitrobacter , and a denitrifying Pseudomonas . Thereby the ammonia was completely eliminated from artificial wastewater.     

The use of multi-parameter flow cytometry to study the impact of limiting substrate, agitation intensity, and dilution rate on cell aggregation during Bacillus licheniformis CCMI 1034 aerobic continuous culture fermentations

The main objective of this work was to establish those factors either physical (power input) or chemical (limiting substrate or dilution rate) that enhance cell aggregation (biofilm or floc formation) and cell physiological state during aerobic continuous cultures of Bacillus licheniformis. Glucose-limited steady-state continuous cultures growing at a dilution rate between 0.64 and 0.87/h and 1,000 rpm (mean specific energy dissipation rate (epsilonT) = 6.5 W/kg), led to the formation of a thin biofilm on the vessel wall characterized by the presence of a high proportion of healthy cells in the broth (after aggregate disruption by sonication) defined as having intact polarized cytoplasmic membranes. An increased epsilonT (from 6.5 W/kg to 38 W/kg) was found to hinder cell aggregation under carbon limitation. The carbon recovery calculated from glucose indicated that additional extracellular polymer was being produced at dilution rates >0.87/h. B. licheniformis growth under nitrogen limitation led to floc formation which increased in size with dilution rate. Counter-intuitively the flocs became more substantial with an increase in epsilonT from 6.5 W/kg to 38 W/kg under nitrogen limitation. Indeed the best culture conditions for enhanced metabolically active cell aggregate formation was under nitrogen limitation at epsilonT = 6.5 W/kg (leading to floc formation), and under carbon limitation at a dilution rate of between 0.64 and 0.87/h, at epsilonT = 6.5 W/kg (leading to vessel wall biofilm formation). This information could be used to optimize culture conditions for improved cell aggregation and hence biomass separation, during thermophilic aerobic bioremediation processes.     

Glycogen metabolism of the anammox bacterium “Candidatus Brocadia sinica”

Presence of glycogen granules in anaerobic ammonium-oxidizing (anammox) bacteria has been reported so far. However, very little is known about their glycogen metabolism and the exact roles. Here, we studied the glycogen metabolism in “Ca. Brocadia sinica” growing in continuous retentostat cultures with bicarbonate as a carbon source. The effect of the culture growth phase was investigated. During the growing phase, intracellular glycogen content increased up to 32.6 mg-glucose (g-biomass dry wt)⁻¹ while the specific growth rate and ATP/ADP ratio decreased. The accumulated glycogen begun to decrease at the onset of entering the near-zero growth phase and was consumed rapidly when substrates were depleted. This clearly indicates that glycogen was synthesized and utilized as an energy storage. The proteomic analysis revealed that “Ca. B. sinica” synthesized glycogen via three known glycogen biosynthesis pathways and simultaneously degraded during the progress of active anammox, implying that glycogen is being continuously recycled. When cells were starved, a part of stored glycogen was converted to trehalose, a potential stress protectant. This suggests that glycogen serves at least as a primary carbon source of trehalose synthesis for survival. This study provides the first physiological evidence of glycogen metabolism in anammox bacteria and its significance in survival under natural substrate-limited habitat.Subject terms: Applied microbiology, Water microbiology     

A comparison of the growth and starvation responses of Acanthamoeba castellanii and Hartmannella vermiformis in the presence of suspended and attached Escherichia coli K12

The growth and starvation responses of Acanthamoeba castellanii and Hartmannella vermiformis were investigated in the presence and absence of Escherichia coli on an agar surface or within shaken suspensions. The amoebae perceived all the suspended systems to be unfavourable for growth, despite being challenged with high levels of prey, and as a consequence they exhibited a starvation response. However, the response differed between species, with A. castellanii producing characteristic cysts and H. vermiformis producing round bodies. These amoebic forms were reactivated into feeding trophozoites in the presence of bacterial aggregates, which formed in the suspended systems after 68 h of incubation. In contrast, both species of amoebae grew well in the presence of attached E. coli at a concentration of 1 x 10(6) cells cm(-2) of agar and yielded specific growth rates of c. 0.04 h(-1). Starvation responses were induced at the end of the growth phase, and these were equivalent to those recorded in the suspended systems. We conclude that, when suspended, amoebae in the 'floating form' cannot feed effectively on suspended prey, and hence the starvation response is initiated. Thus the majority of amoebic feeding is via trophozoite grazing of attached bacterial prey.     

Carbon starvation of Salmonella typhimurium does not cause a general increase of mutation rates.

Mutation rates in bacteria can vary depending on the genetic target studied and the specific growth conditions of the cells. Here, two different methods were used to determine how rates of mutation to antibiotic resistance, auxotrophy, and prototrophy were influenced by carbon starvation on agar plates. The rate of mutation to rifampin resistance was increased by starvation as measured by fluctuation tests, similar to what has been reported previously for Escherichia coli. In contrast, the rates of mutation to various types of auxotrophy were unaffected or decreased as measured by both fluctuation tests and a repeated-streaking procedure. Similarly, the rates of reversion to prototrophy of his and lac nonsense and missense mutations were unaffected by starvation. Thus, mutation rates of different genetic targets can be affected differently by starvation and we conclude that carbon starvation is not generally mutagenic in Salmonella typhimurium.