Metabolic profiling of Staphylococcus aureus cultivated under aerobic and anaerobic conditions with (1)H NMR-based nontargeted analysis

Staphylococcus aureus is a major pathogen in the medical area and food-producing sector. Detailed analyses of its basic cell physiology will help comprehensively understand this pathogen, which will be useful for developing novel diagnostic and treatment tools. Oxygen is one of the most crucial growth-limiting factors for S. aureus. In this study, to characterize and distinguish metabolic profiles of S. aureus cultivated under aerobic and anaerobic conditions, nontargeted analyses of both types of cultures were carried out using (1)H nuclear magnetic resonance spectroscopy. Fifty compounds were identified by Chenomx software. Characteristics of metabolic profiles were achieved by using principal components analysis. During aerobic growth, S. aureus mainly consumed glucose, alanine, arginine, glycine, isoleucine, leucine, phenylalanine, and acetate. Meanwhile, it accumulated 17 metabolites, mainly 2-oxoglutarate, isobutyrate, isovalerate, succinate, and ethanol. Under anaerobic condition, S. aureus mainly consumed glucose, arginine, and threonine. Meanwhile, it accumulated 13 metabolites, mainly ethanol, lactate, and ornithine. The representative metabolites that could most significantly differentiate metabolic profiles of S. aureus were isobutyrate, isovalerate, and succinate in aerobic cultivation; and lactate, ethanol, and ornithine in anaerobic cultivation. Among these metabolites, isobutyrate and ornithine were present only in aerobic and anaerobic culture, respectively.     

Use of carbon and energy balances in the study of the anaerobic metabolism of Enterobacter aerogenes at variable starting glucose concentrations

The anaerobic metabolism of Enterobacter aerogenes was studied in batch culture at increasing initial glucose levels (9.0< S(o) <72 g l(-1)). The ultimate concentrations of fermentation products were utilized to check a metabolic flux analysis based on simple carbon mass and energy balances that promise to be suitable for the study of different fermentation processes, either under aerobic or anaerobic conditions. The stoichiometric coefficients of products collected at increasing starting glucose concentrations under anaerobic conditions suggest: (a) little influence of starting glucose level on the formation of the main fermentation products (2,3-butanediol and ethanol); (b) possible inhibition of 2,3-butanediol and lactate formations by increased ethanol concentration; (c) consequent increase in carbon flux through the remaining metabolic pathways with increased molar productions of succinate, acetate and hydrogen; (d) relative constancy of the molar production of ATP and CO(2).     

Kinetics of biphasic growth of yeast in continuous and fed-batch cultures

The influence of dilution rate on the production of biomass, ethanol, and invertase in an aerobic culture of Saccharomyces carlsbergensis was studied in a glucose-limited chemostat culture. A kinetic model was developed to analyze the biphasic growth of yeast on both the glucose remaining and the ethanol produced in the culture. The model assumes a double effect where glucose regulates the flux of glucose catabolism (respiration and aerobic fermentation) and the ethanol utilization in yeast cells. The model could successfully demonstrate the experimental results of a chemostat culture featuring the monotonic decrease of biomass concentration with an increase of dilution rate higher than 0.2 hr^?1 as well as the maximum ethanol concentration at a particular dilution rate around 0.5 hr^?1. Some supplementary data were collected from an ethanol-limited aerobic chemostat culture and a glucose-limited anaerobic chemostat culture to use in the model calculation. Some parametric constants of cell growth, ethanol production, and invertase formation were determined in batch cultures under aerobic and anaerobic states as summarized in a table in comparison with the chemostat data. Using the constants, a prediction of the optimal control of a glucose fed-batch yeast culture was conducted in connection with an experiment for harvesting a high yield of yeast cells with high invertase activity.     

Transport of glucose intoSaccharomyces cerevisiae RXII

Evidence is submitted that glucose is found in a chemically unaltered form in cells ofSaccharomyces cerevisiae RXII incubated with glucose, if the culture is grown under aerobic conditions at 30°C and is in the logarithmic phase of growth at the moment of harvesting. Under these conditions, the course of formation of a glucose steady state can be studied under aerobic and anaerobic incubation conditions. The steady state glucose concentration in the cells is the linear function of the glucose concentration in the medium.     

In vivo nuclear magnetic resonance studies of glycolytic kinetics in Lactococcus lactis.

The metabolism of glucose by nongrowing cells of L. lactis strain MG5267 was studied under controlled conditions of pH, temperature, and gas atmosphere (anaerobic and aerobic) using a circulating system coupled to nuclear magnetic resonance (NMR) detection that allowed a noninvasive determination of intracellular pools of intermediate metabolites by 13C-NMR with a time resolution of 30 seconds. In addition, intracellular parameters, such as pH, NTP levels, and concentration of inorganic phosphate in the cytoplasm, could be monitored on-line by 31P-NMR with a time resolution of approx. 3 min. The time course for the concentrations of intracellular fructose 1,6-bisphosphate (FBP), 3-phosphoglycerate (3-PGA), and phosphoenolpyruvate (PEP), together with kinetic measurements of substrate consumption and endproducts formation, were used as a basis for the construction of a mechanistic model for glycolysis. In vivo measurements were complemented with determinations of phosphorylated metabolites in perchloric acid extracts. A top-down model was developed by simplifying the metabolism to the resolution allowed by the experimental data collected by in vivo NMR (grouped in seven metabolic steps). This simplified mechanistic model was adjusted to the metabolite concentrations determined by in vivo NMR. The results obtained led to the rationalization of the dynamics of glucose metabolism as being driven largely by ATP surplus. This excess causes accumulation of FBP due to NAD+ limitation, whose regeneration is dependent on downstream pyruvate reduction. The model was capable of predicting qualitative shifts in the metabolism of glucose when changing from anaerobic to aerobic conditions.     

The influence of conditions of growth on the endogenous metabolism of Saccharomyces cerevisiae: effect on protein,carbohydrate, sterol and fatty acid content and on viability

The nature of the endogenous reserves of Saccharomyces cerevisiae was examined with respect to conditions of growth, specifically extremes of oxygen tension and carbon source. Cells were grown in batch culture at 30 C under aerobic conditions on a galactose or glucose carbon source and under anaerobic conditions on glucose. The greatest effect of growth conditions on the chemical composition of the cells was on their fatty acid and sterol content.Cells grown under both aerobic and anaerobic conditions mobilised concurrently protein, glycogen, trehalose and fatty acids during a period of 72 hours' starvation under aerobic conditions. The viability of both types of the aerobically grown cells declined to 75% during this period and was not influenced by the initial fatty acid and sterol content of the cells. Cells grown anaerobically showed a more rapid decline in viability which was only 17% after 72 hours' starvation. This loss of viability was not due to a lack of available endogenous reserves but was probably due to an impaired membrane function caused by a deficiency of sterols and unsaturated fatty acids.     

Stoichiometric flux balance models quantitatively predict growth and metabolic by-product secretion in wild-type Escherichia coli W3110.

Flux balance models of metabolism use stoichiometry of metabolic pathways, metabolic demands of growth, and optimality principles to predict metabolic flux distribution and cellular growth under specified environmental conditions. These models have provided a mechanistic interpretation of systemic metabolic physiology, and they are also useful as a quantitative tool for metabolic pathway design. Quantitative predictions of cell growth and metabolic by-product secretion that are experimentally testable can be obtained from these models. In the present report, we used independent measurements to determine the model parameters for the wild-type Escherichia coli strain W3110. We experimentally determined the maximum oxygen utilization rate (15 mmol of O2 per g [dry weight] per h), the maximum aerobic glucose utilization rate (10.5 mmol of Glc per g [dry weight] per h), the maximum anaerobic glucose utilization rate (18.5 mmol of Glc per g [dry weight] per h), the non-growth-associated maintenance requirements (7.6 mmol of ATP per g [dry weight] per h), and the growth-associated maintenance requirements (13 mmol of ATP per g of biomass). The flux balance model specified by these parameters was found to quantitatively predict glucose and oxygen uptake rates as well as acetate secretion rates observed in chemostat experiments. We have formulated a predictive algorithm in order to apply the flux balance model to describe unsteady-state growth and by-product secretion in aerobic batch, fed-batch, and anaerobic batch cultures. In aerobic experiments we observed acetate secretion, accumulation in the culture medium, and reutilization from the culture medium. In fed-batch cultures acetate is cometabolized with glucose during the later part of the culture period.(ABSTRACT TRUNCATED AT 250 WORDS)     

Anaerobiosis and Release from Dormancy in Apple Embryos: Leaching of (+/-) [C]Abscisic Acid and its Metabolites under Aerobic and Anaerobic Conditions

An anaerobic treatment released Pyrus malus L. cv Golden Delicious embryos from their primary dormancy. It also suppressed the inhibitory effect induced by exogenous abscisic acid (ABA) on after-ripened embryos. For the study of ABA metabolism, a two-step culture method was developed. Embryos in primary dormancy were cultivated aerobically in the presence of [¹⁴C]ABA (first culture). Some were directly analyzed to evaluate metabolism of absorbed ABA. The remaining embryos were cultivated on moist cotton without ABA, either in aerobic or anaerobic conditions (second culture). The amounts of ABA and its metabolites were measured both in the embryos and the water-leachates. After the second culture, the embryos showed a spectacular decrease in ABA content, with no difference between anaerobic and aerobic cultures. The amount of ABA glucose ester increased slightly in aerobiosis but diminished markedly in anaerobiosis. Radioactivity of the butanol fraction, which corresponded to polar conjugates, decreased considerably in anaerobiosis, whereas it increased in aerobiosis.

Analysis of the water-leachates indicated that, compared to aerobic conditions, anaerobiosis increased total leaching of radioactive materials (× 4.2) as well as leaching of ABA (× 1.4). In addition, anaerobiosis induced leaching of conjugates, such as ABA glucose ester and butanol-soluble metabolites. We concluded that the anaerobic treatment affects mainly membrane permeability.

     

Effects of Oxygen and Glucose on Energy Metabolism and Dimorphism of Mucor genevensis Grown in Continuous Culture: Reversibility of Yeast-Mycelium Conversion

Mucor genevensis was grown in both glucose-limited and glucose-excess continuous cultures over a range of dissolved oxygen concentrations (<0.1 to 25 muM) to determine the effects of glucose and the influence of metabolic mode (fermentative versus oxidative) on dimorphic transformations in this organism. The extent of differentiation between yeast and mycelial phases has been correlated with physiological and biochemical parameters of the cultures. Under glucose limitation, oxidative metabolism increased as the dissolved oxygen concentration increased, and this paralleled the increase in the proportion of the mycelial phase in the cultures. Filamentous growth and oxidative metabolism were both inhibited by glucose even though mitochondrial development was only slightly repressed. However, the presence of chloramphenicol in glucose-limited aerobic cultures inhibited mitochondrial respiratory development but did not induce yeast-like growth, indicating that oxidative metabolism is not essential for mycelial development. Once mycelial cultures had been established under aerobic, glucose-limited conditions, subsequent reversal to anaerobic conditions or treatment with chloramphenicol caused only a limited reversal (<35%) to the yeast-like form. Glucose, however, induced a complete reversion to yeast-like form. It is concluded that glucose is the most important single culture factor determining the morphological status of M. genevensis; mitochondrial development and the functional oxidative capacities of the cell appear to be less important factors in the differentiation process.     

Microbial decolorization of reactive black-5 in a two-stage anaerobic–aerobic reactor using acclimatized activated textile sludge

A two-stage anaerobic–aerobic treatment process based on mixed culture of bacteria isolated from textile dye effluent was used to degrade reactive black 5 dye (RB-5). The anaerobic step was studied in more detail by varying the dye concentration from 100 to 3000 mg l⁻¹. The results showed that major decolorization was achieved during the anaerobic process. The time required for decolorization by > 90% increased as the concentration of the dye increased. It was also found that maintaining dissolved oxygen (DO) concentration below 0.5 mg l⁻¹ and addition of a co-substrate viz., glucose, facilitates anaerobic decolorization reaction remarkably. An attempt was made to identify the metabolites formed in anaerobic process by using high performance liquid chromatography (HPLC) and UV–VIS spectrophotometry. A plate assay was performed for the detection of dominant decolorizing bacteria. Only a few bacterial colonies with high clearing zones (decolorization zones) were found. The results showed that under anaerobic condition RB-5 molecules were reduced and aromatic amines were generated. The aromatic amine metabolite was partly removed in subsequent aerobic bio-treatment. It was possible to achieve more than 90% decolorization and approximately 46% reduction in amine metabolite concentration through two-stage anaerobic–aerobic treatment after a reaction period of 2 days.     

Stoichiometric model of Escherichia coli metabolism: incorporation of growth-rate dependent biomass composition and mechanistic energy requirements

A stoichiometric model of metabolism was developed to describe the balance of metabolic reactions during steady-state growth of Escherichia coli on glucose (or metabolic intermediates) and mineral salts. The model incorporates 153 reversible and 147 irreversible reactions and 289 metabolites from several metabolic data bases for the biosynthesis of the macromolecular precursors, coenzymes, and prosthetic groups necessary for synthesis of all cellular macromolecules. Correlations describing how the cellular composition changes with growth rate were developed from experimental data and were used to calculate the drain of precursors to macromolecules, coenzymes, and prosthetic groups from the metabolic network for the synthesis of those macromolecules at a specific growth rate. Energy requirements for macromolecular polymerization and proofreading, transport of metabolites, and maintenance of transmembrane gradients were included in the model rather than a lumped maintenance energy term. The underdetermined set of equations was solved using the Simplex algorithm, employing realistic objective functions and constraints; the drain of precursors, coenzymes, and prosthetic groups and the energy requirements for the synthesis of macromolecules served as the primary set of constraints. The model accurately predicted experimentally determined metabolic fluxes for aerobic growth on acetate or acetate plus glucose. In addition, the model predicted the genetic and metabolic regulation that must occur for growth under different conditions, such as the opening of the glyoxylate shunt during growth on acetate and the branching of the tricarboxylic acid cycle under anaerobic growth. Sensitivity analyses were performed to determine the flexibility of pathways and the effects of different rates and growth conditions on the distribution of fluxes. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 398-421, 1997.     

Metabolite profiles of the biocontrol yeast<Emphasis Type="Italic"> Pichia anomala</Emphasis> J121 grown under oxygen limitation

The biocontrol yeast Pichia anomala J121 prevents mould growth during the storage of moist grain under low oxygen/high carbon dioxide conditions. Growth and metabolite formation of P. anomala was analyzed under two conditions of oxygen limitation: (a) initial aerobic conditions with restricted oxygen access during the growth period and (b) initial microaerobic conditions followed by anaerobiosis. Major intra- and extracellular metabolites were analyzed by high-resolution magic-angle spinning (HR-MAS) NMR and HPLC, respectively. HR-MAS NMR allows the analysis of major soluble compounds inside intact cells, without the need for an extraction step. Biomass production was higher in treatment (a), whereas the specific ethanol production rate during growth on glucose was similar in both treatments. This implies that oxygen availability affected the respiration and not the fermentation of the yeast. Following glucose depletion, ethanol was oxidized to acetate in treatment (a), but continued to be produced in (b). Arabitol accumulated in the culture substrate of both treatments, whereas glycerol only accumulated in treatment (b). Trehalose, arabitol, and glycerol accumulated inside the cells in both treatments. The levels of these metabolites were generally significantly higher in treatment (b) than in (a), indicating their importance for P. anomala during severe oxygen limitation/anaerobic conditions.     

Involvement of oxygen and mitochondrial function in the metabolism of D-xylulose by Saccharomyces cerevisiae

Mitochondrial function associated with oxygen was required for growth of Saccharomyces cerevisiae on D-xylulose. The requirement was shown by (i) the inhibition of growth of a wild-type strain under anaerobic conditions, (ii) the inhibition of aerobic growth after treatment with inhibitors of mitochondrial function, and (iii) the lack of aerobic and anaerobic growth of nuclear and cytoplasmic petites. The mitochondrial function was associated with the channeling of catabolites of D-xylulose to growth processes, since ethanol was formed even when growth was inhibited. Mitochondrial function was implicated as well in determining the extent of growth and the concentration of ethanol in aerobic cultures of the wild-type. In such cultures, the concentration of ethanol decreased and growth increased concomitantly as aeration rate increased. A factor in this relation was considered to be the relatively poor ability of D-xylulose to inhibit the oxidative utilization of ethanol.     

Comparison of the performance of one stage and two stage sequential anaerobic-aerobic biological processes for the treatment of reactive-azo-dye-containing synthetic wastewaters

In the present study the performance of anaerobic-aerobic one and two stage processes for the biological treatment of synthetic wastewaters containing Reactive Black 5 (RB5) were studied and compared with each other. In both processes the majority of colour removal by biodegradation occurred under anaerobic environment. The colour change under aerobic conditions was correlated with extent of anaerobic decolourisation in the preceding phase/stage of the process. Partial mineralisation of the anaerobic dye metabolites, roughly to the same extent, was achieved aerobically in both one stage and two stage processes. The majority of COD was removed in the anaerobic stage for two stage processes and aerobic stage in one stage processes. In one stage processes, the exposure of anaerobic sludge to alternating anaerobic-aerobic environment decreased anaerobic decolourisation efficiency and COD removal; when employing activated sludge, the same exposure enhanced anaerobic substrate utilisation whereas the effect on the anaerobic decolourisation efficiency depended on RB5 concentration. The comparative performance of one and two stage processes in terms of overall dye decolourisation depended on RB5 concentration. Both types of processes brought about similar overall COD removal. Increase in RB5 concentration, in the range studied, resulted in decrease in overall COD removal for both processes.     

Influence of oxygen availability on physiology, verocytotoxin expression and adherence of Escherichia coli O157

A strain of Escherichia coli serotype O157 was grown in steady state chemostat culture under aerobic, oxygen-limited and anaerobic conditions. The growth and metabolic efficiency of oxygen-limited and anaerobic cultures was impaired, with biomass yield and the molar growth yield for glucose, Yglucose, reduced markedly in comparison with aerobic cultures. Steady state cells were typically short rods 2-3 microns long, and were encapsulated by a layer of extracellular material. The majority of cells were non-flagellated and fimbriae were not observed. Chemostat-grown cells were significantly more adhesive for HEp-2 monolayers than cells grown in aerobic batch culture. Furthermore, oxygen-limited and anaerobic cultures were significantly more adhesive for Hep-2 cells when compared with cells grown in aerobic chemostat culture, possibly reflecting increased pathogenicity associated with the induction of novel adhesins. Type 1 pili were not responsible for increased adherence. Verocytotoxins, VT1 and VT2, were expressed constitutively and were not influenced by oxygen availability. This study demonstrates that E. coli O157 is a versatile micro-organism, which responds to environmental conditions likely to be encountered during infection by inducing a phenotype which is more adhesive for human epithelial cells.     

A microcalorimetric study of the growth of Klebsiella aerogenes in simple salts/glucose media.

Heat output-time records or 'thermograms' produced during the aerobic growth of Klebsiella aerogenes in simple salts/glucose media with growth limiting glucose concentrations of 2.0, 1.0 and 0.5 g dm-3 were obtained using a flow-microcalorimeter fitted with an aerobic cell. These traces are interpreted in terms of the recorded oxygen tension, pH, glucose concentration and bacterial population of the culture. Heat output is greatest during the phase of exponential growth, indicating that here the organisms are most energetically inefficient. During the stationary phase aerobic processes, which give rise to a low oxygen tension, produce a smaller heat output until secondary metabolic processes are complete.     

Degradation of the herbicide bromoxynil inPseudomonas putida

Biological conversion of the herbicide bromoxynil (3,5-dibromo-4-hydroxybenzonitrile) was studied in a batch culture ofPseudomonas putida by using HPLC. The process had a cometabolic character and proceeded only in the presence of another, simultaneously metabolizable, carbon and energy source. The intensity of degradation correlated with the growth rate, the degradation stopping when the cosubstrate becomes exhausted or the pH value of the medium falls below 6.5. In a medium with glucose, no lag phase longer than one day was observed concerning growth, sugar and herbicide consumption and formation of metabolic herbicide derivatives (3,5-dibromo-4-hydroxybenzamide and 3,5-dibromo-4-hydroxybenzoic acid). In a medium with ribose, the initial lag of the above processes took 2 d. No formation of other degradation products was detected. Growth inhibition was proportional to the concentration of bromoxynil. Translated by Č. Novotny     

The effect of oxygen and pH on the glucose metabolism ofLactobacillus casei var.rhamnosus ATCC 7469

Growth of cultures ofLactobacillus casei ATCC 7469 without pH control under aerobic conditions resulted in very low maximum specific growth rates (0.19 hr⁻¹), exponential glucose utilization rates (0.10 log units/hr/ml of culture) and exponential lactate production rates (0.17 log units/hr/ml of culture), compared to anaerobic cultures. In anaerobic cultures glucose was converted stoichiometrically to lactate but in aerobic cultures this was never observed. It was found that aeration affects both the rate at which glucose is converted to lactate and the stoichiometry of this conversion. The investigation of a number of glucose-metabolizing enzymes suggests that an oxidative pathway for glucose breakdown becomes operative under aerated conditions. This work has been carried out with the financial help from the Commonwealth Postgraduate Award Scheme, University Research Grant and the Australian Research Grant Commission.     

Aerobic and anaerobic metabolism of Listeria monocytogenes in defined glucose medium.

A defined medium with glucose as the carbon source was used to quantitatively determine the metabolic end products produced by Listeria monocytogenes under aerobic and anaerobic conditions. Of 10 strains tested, all produced acetoin under aerobic conditions but not anaerobic conditions. Percent carbon recoveries of end products, typified by strain F5069, were as follows: lactate, 28%; acetate, 23%; and acetoin, 26% for aerobic growth and lactate, 79%; acetate, 2%; formate, 5.4%; ethanol, 7.8%; and carbon dioxide, 2.3% for anaerobic growth. No attempt to determine carbon dioxide under aerobic growth conditions was made. The possibility of using acetoin production to assay for growth of L. monocytogenes under defined conditions should be considered.