ATP Yield Research Articles

Continuous lactose fermentation by Clostridium acetobutylicum—Assessment of energetics and product yields of the acidogenesis

An assessment of both the growth and the metabolism of acidogenic cells Clostridium acetobutylicum DSM 792 is reported in the paper. Tests were carried out in a CSTR under controlled pH conditions. Cultures were carried out using a semi-synthetic medium supplemented with lactose as carbon source. Acids and solvents, that represent products of the ABE process, have been purposely added in controlled amounts to the culture medium to investigate their effects on the product yields. The mass fractional yield of biomass and products were expressed as a function of the specific growth rate taking into account the Pirt model. The maximum ATP yield and the maintenance resulted 29.1gDM/molATP and 0.012molATP/gDMh, respectively. Quantitative features of the C. acetobutylicum growth model were in good agreement with experimental results. The model proposes as a tool to estimate the mass fractional yield even for fermentations carried out under conditions typical of the solventogenesis.

Digestible nutrients and available (ATP) energy contents of two varieties of kiwifruit (Actinidia deliciosa and Actinidia chinensis)

A model, which combines a dual in vivo–in vitro digestibility assay and stoichiometric relationships describing nutrient catabolism, has been recently developed to allow prediction of the available energy (AE) content of a food in terms of its ATP yield. The model uses the growing pig as an in vivo model for upper gastrointestinal tract digestion in humans. Terminal ileal digesta from the pig are incubated with human faecal inocula (in vitro fermentation model) to simulate human hindgut fermentation. The respective in vivo and in vitro digestibility assays provide predictions of the ileal absorbed and hindgut-fermented nutrient contents of a food which are then used to predict ATP production post-absorption, based on known stoichiometric relationships. In this study, the model was used to determine the AE contents of fresh, ripe Hayward (Actinidia deliciosavar Hayward) and Hort16A (Actinidia chinensis var Hort16A) kiwifruit. Kiwifruit pulp, containing 3gkg−1 of titanium dioxide, included as an indigestible marker, was fed to growing pigs and terminal ileal digesta were collected. Ileal nutrient digestibilities were determined. A sample of digesta was incubated in vitro with a fresh human faecal inoculum and the fermentable organic matter determined. The predicted available (ATP) energy contents of the Hayward and Hort16A kiwifruits were 5.9 and 6.2kJg−1 dry matter, respectively, approximately 44–47% of the determined apparent digestible energy (ADE) content. The AE contents of the kiwifruit, expressed relative to the AE content of dextrin (a highly digestible source of glucose) were 0.57 and 0.61 for Hayward and Hort16A, respectively. Comparable ratios for metabolisable energy (ME) were 0.74 and 0.73. The predicted AE from kiwifruit was much lower than the predicted ME from kiwifruit when compared to dextrin. The ME values overestimate the energy content of kiwifruit that is available to the cell. AE was not only lower than ME but the two energy systems ranked the kiwifruit types differently in terms of energy supply to the body. The relatively low energy content per unit of dry matter and high water content of kiwifruit make kiwifruit an ideal weight loss food.

Increasing free-energy (ATP) conservation in maltose-grown Saccharomyces cerevisiae by expression of a heterologous maltose phosphorylase

Increasing free-energy conservation from the conversion of substrate into product is crucial for further development of many biotechnological processes. In theory, replacing the hydrolysis of disaccharides by a phosphorolytic cleavage reaction provides an opportunity to increase the ATP yield on the disaccharide. To test this concept, we first deleted the native maltose metabolism genes in Saccharomyces cerevisiae. The knockout strain showed no maltose-transport activity and a very low residual maltase activity (0.03 μmol mg protein −1 min −1). Expression of a maltose phosphorylase gene from Lactobacillus sanfranciscensis and the MAL11 maltose-transporter gene resulted in relatively slow growth ( μ aerobic 0.09±0.03 h −1). Co-expression of Lactococcus lactis β-phosphoglucomutase accelerated maltose utilization via this route ( μ aerobic 0.21±0.01 h −1, μ anaerobic 0.10±0.00 h −1). Replacing maltose hydrolysis with phosphorolysis increased the anaerobic biomass yield on maltose in anaerobic maltose-limited chemostat cultures by 26%, thus demonstrating the potential of phosphorolysis to improve the free-energy conservation of disaccharide metabolism in industrial microorganisms.

Molecular Crowding Defines a Common Origin for the Warburg Effect in Proliferating Cells and the Lactate Threshold in Muscle Physiology

Aerobic glycolysis is a seemingly wasteful mode of ATP production that is seen both in rapidly proliferating mammalian cells and highly active contracting muscles, but whether there is a common origin for its presence in these widely different systems is unknown. To study this issue, here we develop a model of human central metabolism that incorporates a solvent capacity constraint of metabolic enzymes and mitochondria, accounting for their occupied volume densities, while assuming glucose and/or fatty acid utilization. The model demonstrates that activation of aerobic glycolysis is favored above a threshold metabolic rate in both rapidly proliferating cells and heavily contracting muscles, because it provides higher ATP yield per volume density than mitochondrial oxidative phosphorylation. In the case of muscle physiology, the model also predicts that before the lactate switch, fatty acid oxidation increases, reaches a maximum, and then decreases to zero with concomitant increase in glucose utilization, in agreement with the empirical evidence. These results are further corroborated by a larger scale model, including biosynthesis of major cell biomass components. The larger scale model also predicts that in proliferating cells the lactate switch is accompanied by activation of glutaminolysis, another distinctive feature of the Warburg effect. In conclusion, intracellular molecular crowding is a fundamental constraint for cell metabolism in both rapidly proliferating- and non-proliferating cells with high metabolic demand. Addition of this constraint to metabolic flux balance models can explain several observations of mammalian cell metabolism under steady state conditions.

A specific role of 43Ca in the enzymatic ATP synthesis

This is the first report ever on the nuclear spin selective 43 Ca 40 Ca isotope effect expressed in the Calcium affected enzymatic ATP synthesis. Once the creatine kinase (CK) catalytic sites were loaded with 43 Ca 2+ , the resulting enzyme activity goes up (2.0±0.3 – fold) compared to the CKdirected ATP synthesis rate occurred in the presence of nonmagnetic, spinless, 40 Ca nuclei. The effect manifests itself predominantly within a high concentration range (Calcium chloride,40 mM) indicating that the ATP synthesis is a spin-selective ion-radical process. Primary reaction of this path is an electron transfer between the reaction partners, С a(H2O)n 2+ ( n ≤ 4) and Ca(ADP) 3- , generating an ion-radical pair where the singlet -triplet spin conversion takes place as a result of the nuclear spin selectivity and a consequent magnetic isotope effect. This, in turn, makes an essential impact on the final reaction’s ATP yield. To the contrast, there is no any isotope effect found within a low Ca 2+ concentration range showing that the ATP synthesis proceeds in a most common nucleophylic way. The submolecular mechanisms of phenomena described and their possible applied significance are under discussion.

Characteristics of hydrogen production of an Enterobacter aerogenes mutant generated by a new atmospheric and room temperature plasma (ARTP)

A novel atmospheric and room temperature plasma (ARTP) which used helium as the working gas was employed to generate mutants of Enterobacter aerogenes for improving the hydrogen production. For the mutation, 50 μl of the E. aerogenes culture (OD 600 = 2.0) was dipped onto a sterilized stainless steel plate (5 mm in diameter). The plate was then treated for 3 min by ARTP at the gas flow rate of 15.0 slpm (standard liters per minute) and 100 W of radio-frequency power input. The positive mutation rate defined by the ratio of the mutants with the increase of hydrogen productivity to the total mutants reached about 10%, and a mutant with improved cell growth and hydrogen productivity was then selected. The total hydrogen yield per mole glucose was increased by 26.4%, mainly dependent on the increase of hydrogen production by the NADH pathway. Two important parameters of the ATP yield and oxidation state balance indicated that the cellular metabolism was changed in the mutant. The genetic stability of the mutant for hydrogen production was maintained after more than 25 subcultures.

Molecular crowding defines a common origin for the Warburg effect in proliferating cells and the lactate threshold in muscle physiology

Aerobic glycolysis is a seemingly wasteful mode of ATP production that is seen both in rapidly proliferating mammalian cells and highly active contracting muscles, but whether there is a common origin for its presence in these widely different systems is unknown. To study this issue, here we develop a model of human central metabolism that incorporates a solvent capacity constraint of metabolic enzymes and mitochondria, accounting for their occupied volume densities, while assuming glucose and/or fatty acid utilization. The model demonstrates that activation of aerobic glycolysis is favored above a threshold metabolic rate in both rapidly proliferating cells and heavily contracting muscles, because it provides higher ATP yield per volume density than mitochondrial oxidative phosphorylation. In the case of muscle physiology, the model also predicts that before the lactate switch, fatty acid oxidation increases, reaches a maximum, and then decreases to zero with concomitant increase in glucose utilization, in agreement with the empirical evidence. These results are further corroborated by a larger scale model, including biosynthesis of major cell biomass components. The larger scale model also predicts that in proliferating cells the lactate switch is accompanied by activation of glutaminolysis, another distinctive feature of the Warburg effect. In conclusion, intracellular molecular crowding is a fundamental constraint for cell metabolism in both rapidly proliferating- and non-proliferating cells with high metabolic demand. Addition of this constraint to metabolic flux balance models can explain several observations of mammalian cell metabolism under steady state conditions.

Molecular crowding defines a common origin for the Warburg effect in proliferating cells and the lactate threshold in muscle physiology

AbstractAerobic glycolysis is a seemingly wasteful mode of ATP production that is seen both in rapidly proliferating mammalian cells and highly active contracting muscles, but whether there is a common origin for its presence in these widely different systems is unknown. To study this issue, here we develop a model of human central metabolism that incorporates a solvent capacity constraint of metabolic enzymes and mitochondria, accounting for their occupied volume densities, while assuming glucose and/or fatty acid utilization. The model demonstrates that activation of aerobic glycolysis is favored above a threshold metabolic rate in both rapidly proliferating cells and heavily contracting muscles, because it provides higher ATP yield per volume density than mitochondrial oxidative phosphorylation. In the case of muscle physiology, the model also predicts that before the lactate switch, fatty acid oxidation increases, reaches a maximum, and then decreases to zero with concomitant increase in glucose utilization, in agreement with the empirical evidence. These results are further corroborated by a larger scale model, including biosynthesis of major cell biomass components. The larger scale model also predicts that in proliferating cells the lactate switch is accompanied by activation of glutaminolysis, another distinctive feature of the Warburg effect. In conclusion, intracellular molecular crowding is a fundamental constraint for cell metabolism in both rapidly proliferating- and non-proliferating cells with high metabolic demand. Addition of this constraint to metabolic flux balance models can explain several observations of mammalian cell metabolism under steady state conditions.

In silico characterization of microbial electrosynthesis for metabolic engineering of biochemicals

BackgroundA critical concern in metabolic engineering is the need to balance the demand and supply of redox intermediates such as NADH. Bioelectrochemical techniques offer a novel and promising method to alleviate redox imbalances during the synthesis of biochemicals and biofuels. Broadly, these techniques reduce intracellular NAD+ to NADH and therefore manipulate the cell's redox balance. The cellular response to such redox changes and the additional reducing power available to the cell can be harnessed to produce desired metabolites. In the context of microbial fermentation, these bioelectrochemical techniques can be used to improve product yields and/or productivity.ResultsWe have developed a method to characterize the role of bioelectrosynthesis in chemical production using the genome-scale metabolic model of E. coli. The results in this paper elucidate the role of bioelectrosynthesis and its impact on biomass growth, cellular ATP yields and biochemical production. The results also suggest that strain design strategies can change for fermentation processes that employ microbial electrosynthesis and suggest that dynamic operating strategies lead to maximizing productivity.ConclusionsThe results in this paper provide a systematic understanding of the benefits and limitations of bioelectrochemical techniques for biochemical production and highlight how electrical enhancement can impact cellular metabolism and biochemical production.

Enhanced adenosine triphosphate production by Saccharomyces cerevisiae using an efficient energy regeneration system

The process of ATP biosynthesis from adenosine catalyzed by Saccharomyces cerevisiae was studied using an efficient energy regeneration system. A fractional factorial design (29−5) was used to evaluate the effects of different components in the medium. Magnesium chloride, toluene, and acetaldehyde were found to significantly influence ATP production. The concentrations of the three factors were then optimized using central composition design and response surface analysis. Based on the second-order polynomial model obtained from the experiments, the optimal parameters were obtained as follows: adenosine 20 g/L; glucose 67 g/L; S. cerevisiae cells 250 g/L; magnesium chloride 4.37 g/L; potassium dihydrogen phosphate 67 g/L; toluene 1.40 mL/L; acetaldehyde 2.67 mL/L; pH 7.0; and temperature 37.0 °C. Under the condition, the yield and concentration of ATP reached 97.5% and 37 g/L, respectively. The yield was nearly 10% higher than the level before optimization and the concentration increased two-fold. In addition, the utilization efficiency of energy after optimization increased nearly 6%.

Thermodynamics of Fatty Acid Degradation

In this communication, the correlation of some thermodynamic properties of structurally similar biological compounds with items such as the number of carbon atoms or the number of its characteristic groups, such as phosphates, was applied to enzyme thermodynamics, to advance the thermodynamics of fatty acid degradation. It was shown that these concepts apply equally well for all three major thermodynamic functions: Gibbs energy of formation, enthalpy of formation, and entropy of formation. First, the standard transformed Gibbs energies of formation, Δ(f)G'(o), were calculated for the major portion of saturated fatty acids at 25 °C and pH 5, pH 7, and pH 9. Second, a total ATP yield was calculated for complete combustion of major saturated fatty acids to CO(2) and water. Third, the standard transformed Gibbs energies of reaction, Δ(r)G'(o), were calculated for enzyme reactions catalyzing complete β-oxidation of butyryl-CoA to acetyl-CoA at 25 °C and pH 7.

Revisiting the Thermodynamic Theory of Optimal ATP Stoichiometries by Analysis of Various ATP-Producing Metabolic Pathways

The stoichiometry of ATP-producing metabolic pathways had been analysed theoretically by several authors by using evolutionary arguments and optimality principles. Waddell et al. (Biochem Educ 27:12-13, 1999) analysed (lactate-producing) glycolysis and used linear irreversible thermodynamics. The result was that half of the free-energy difference should be converted into free-energy of ATP and the remaining half should be used to drive the pathway. The calculated stoichiometry is in agreement with the observed yield of two moles of ATP per mole of glucose. Using the same approach, we here analyse eight other metabolic pathways. Although the deviation is not very large, the calculated values do not fit as nicely as for glycolysis as leading to lactate. For example, for O₂ respiration, the theoretical ATP yield equals 27.9. The real value varies among organisms between 26 and 38. For mixed-acid fermentation in Escherichia coli, the theoretical and experimental values are 2.24 and 2, respectively. For arginine degradation in M. pneumoniae, the calculated value is 2.43 mol of ATP, while in vivo only one mole is produced. During evolution, some pathways may not have reached their optimal ATP net production because energy yield is not their only function. Moreover, it should be acknowledged that the approach by linear irreversible thermodynamics is a rough approximation.

ATP synthase: From sequence to ring size to the P/O ratio

There are tales of P/O battles at conferences in the 1950s. P/O is the number of ATP molecules synthesized by oxidative phosphorylation for each pair of electrons (hence, not P/O2) passing from a particular substrate, typically NADH or succinate, via a respiratory chain, to O2. Knowledge of the P/O ratio is fundamental for understanding the ATP yield from cell fuels and is a core metabolic parameter. The battles of old persisted because the P/O ratio is difficult to measure. Determination of O2 consumed became easier as manometers were replaced by electrodes, but calibration is difficult because the dissolved O2 concentration depends on temperature, ionic strength, history of the solution, and atmospheric pressure. Determining ATP made by suspensions of (usually animal) mitochondria is also error prone. Furthermore, mitochondria might be damaged, either homogeneously or heterogeneously, and thus provide underestimates of the P/O ratio because O2 consumption can proceed without full coupling to ATP synthesis, e.g., owing to leakage of protons across membranes. The combatants in the P/O battles of the 1950s could never have imagined that P/O ratios might be assessed via structural biology, as illustrated by a study (1) in PNAS. Many biochemists also have not contemplated that a highly conserved enzyme has a variable relative subunit stoichiometry. The paper (1) unexpectedly shows variation in the stoichiometry of one critical subunit, c, among ATP synthases of mitochondria from different sources.

Phosphoenolpyruvate Carboxykinase as the Sole Anaplerotic Enzyme in Saccharomyces cerevisiae

Pyruvate carboxylase is the sole anaplerotic enzyme in glucose-grown cultures of wild-type Saccharomyces cerevisiae. Pyruvate carboxylase-negative (Pyc(-)) S. cerevisiae strains cannot grow on glucose unless media are supplemented with C(4) compounds, such as aspartic acid. In several succinate-producing prokaryotes, phosphoenolpyruvate carboxykinase (PEPCK) fulfills this anaplerotic role. However, the S. cerevisiae PEPCK encoded by PCK1 is repressed by glucose and is considered to have a purely decarboxylating and gluconeogenic function. This study investigates whether and under which conditions PEPCK can replace the anaplerotic function of pyruvate carboxylase in S. cerevisiae. Pyc(-) S. cerevisiae strains constitutively overexpressing the PEPCK either from S. cerevisiae or from Actinobacillus succinogenes did not grow on glucose as the sole carbon source. However, evolutionary engineering yielded mutants able to grow on glucose as the sole carbon source at a maximum specific growth rate of ca. 0.14 h(-1), one-half that of the (pyruvate carboxylase-positive) reference strain grown under the same conditions. Growth was dependent on high carbon dioxide concentrations, indicating that the reaction catalyzed by PEPCK operates near thermodynamic equilibrium. Analysis and reverse engineering of two independently evolved strains showed that single point mutations in pyruvate kinase, which competes with PEPCK for phosphoenolpyruvate, were sufficient to enable the use of PEPCK as the sole anaplerotic enzyme. The PEPCK reaction produces one ATP per carboxylation event, whereas the original route through pyruvate kinase and pyruvate carboxylase is ATP neutral. This increased ATP yield may prove crucial for engineering of efficient and low-cost anaerobic production of C(4) dicarboxylic acids in S. cerevisiae.

Catabolic efficiency of aerobic glycolysis: The Warburg effect revisited

BackgroundCancer cells simultaneously exhibit glycolysis with lactate secretion and mitochondrial respiration even in the presence of oxygen, a phenomenon known as the Warburg effect. The maintenance of this mixed metabolic phenotype is seemingly counterintuitive given that aerobic glycolysis is far less efficient in terms of ATP yield per moles of glucose than mitochondrial respiration.ResultsHere, we resolve this apparent contradiction by expanding the notion of metabolic efficiency. We study a reduced flux balance model of ATP production that is constrained by the glucose uptake capacity and by the solvent capacity of the cell's cytoplasm, the latter quantifying the maximum amount of macromolecules that can occupy the intracellular space. At low glucose uptake rates we find that mitochondrial respiration is indeed the most efficient pathway for ATP generation. Above a threshold glucose uptake rate, however, a gradual activation of aerobic glycolysis and slight decrease of mitochondrial respiration results in the highest rate of ATP production.ConclusionsOur analyses indicate that the Warburg effect is a favorable catabolic state for all rapidly proliferating mammalian cells with high glucose uptake capacity. It arises because while aerobic glycolysis is less efficient than mitochondrial respiration in terms of ATP yield per glucose uptake, it is more efficient in terms of the required solvent capacity. These results may have direct relevance to chemotherapeutic strategies attempting to target cancer metabolism.

Sulfide-based ATP production in Urechis unicinctus

We measured sulfide-based ATP production by isolated mitochondria from four tissues of Urechis unicinctus and the effects of inhibitors of respiratory complexes on ATP production were evaluated. The results show that these mitochondria could oxidize sulfide to produce ATP. The yield of sulfide-stimulated ATP varied from 5 nmol ATP/min/mg to 90 nmol ATP/min/mg according to the sulfide concentration and the source of the mitochondria. The maximum ATP synthesis occurred in hindgut mitochondria using 5 μmol/L sulfide as a substrate. The effects of inhibitors (Rotenone, Antimycin A, Cyanide, and Salicylhydroxamic acid) on mitochondrial ATP production varied with the source of the mitochondria. Our results indicate that sulfide-based ATP production and the associated electron transport pathway are tissue-specific in U. unicinctus.

Ion-Radical Mechanism of Enzymatic ATP Synthesis: DFT Calculations and Experimental Control

A new, ion-radical mechanism of enzymatic ATP synthesis was recently discovered by using magnesium isotopes. It functions at a high concentration of MgCl(2) and includes electron transfer from the Mg(H(2)O)(m)(2+)(ADP(3-)) complex (m = 0-4) to the Mg(H(2)O)(n)(2+) complex as a primary reaction of ATP synthesis in catalytic sites of ATP synthase and kinases. Here, the structures and electron transfer reaction energies of magnesium complexes related to ATP synthesis are calculated in terms of DFT. ADP is modeled by pyrophosphate anions, protonated (HP(2)O(7)H(2-), HP(2)O(7)CH(3)(2-)) and deprotonated (HP(2)O(7)(3-), CH(3)P(2)O(7)(3-)). The reaction generates an ion-radical pair, composed of Mg(H(2)O)(n)(+) ion and pyrophosphate anion-radical coordinated to Mg(2+) ion. The addition of the latter to the substrate P=O bond results in ATP formation. Populations of the singlet and triplet states and singlet-triplet spin conversion in the pair are controlled by hyperfine coupling of unpaired electrons with magnetic (25)Mg and (31)P nuclei and by Zeeman interaction. Due to these two interactions, the yield of ATP is a function of nuclear magnetic moment and magnetic field; both of these effects were experimentally detected. Electron transfer reaction does not depend on m but strongly depends on n. It is exoergic and energy allowed at 0 < or = n << infinity for the deprotonated pyrophosphate anions and at 0 < or = n < 4 for the protonated ones; for other values of n, the reaction is energy deficient and forbidden. The boundary between exoergic and endoergic regimes corresponds to the trigger magnitude n* (n* = 4 for protonated anions and 6 < n* << infinity for deprotonated ones). These results explain why ATP synthesis occurs only in special devices, molecular enzymatic machines, but not in water (n = infinity). Biomedical consequences of the ion-radical enzymatic ATP synthesis are also discussed.

Metabolic evolution of energy-conserving pathways for succinate production in Escherichia coli

During metabolic evolution to improve succinate production in Escherichia coli strains, significant changes in cellular metabolism were acquired that increased energy efficiency in two respects. The energy-conserving phosphoenolpyruvate (PEP) carboxykinase (pck), which normally functions in the reverse direction (gluconeogenesis; glucose repressed) during the oxidative metabolism of organic acids, evolved to become the major carboxylation pathway for succinate production. Both PCK enzyme activity and gene expression levels increased significantly in two stages because of several mutations during the metabolic evolution process. High-level expression of this enzyme-dominated CO(2) fixation and increased ATP yield (1 ATP per oxaloacetate). In addition, the native PEP-dependent phosphotransferase system for glucose uptake was inactivated by a mutation in ptsI. This glucose transport function was replaced by increased expression of the GalP permease (galP) and glucokinase (glk). Results of deleting individual transport genes confirmed that GalP served as the dominant glucose transporter in evolved strains. Using this alternative transport system would increase the pool of PEP available for redox balance. This change would also increase energy efficiency by eliminating the need to produce additional PEP from pyruvate, a reaction that requires two ATP equivalents. Together, these changes converted the wild-type E. coli fermentation pathway for succinate into a functional equivalent of the native pathway that nature evolved in succinate-producing rumen bacteria.

Subcellular Flux Analysis of Central Metabolism in a Heterotrophic Arabidopsis Cell Suspension Using Steady-State Stable Isotope Labeling

The presence of cytosolic and plastidic pathways of carbohydrate oxidation is a characteristic feature of plant cell metabolism. Ideally, steady-state metabolic flux analysis, an emerging tool for creating flux maps of heterotrophic plant metabolism, would capture this feature of the metabolic phenotype, but the extent to which this can be achieved is uncertain. To address this question, fluxes through the pathways of central metabolism in a heterotrophic Arabidopsis (Arabidopsis thaliana) cell suspension culture were deduced from the redistribution of label in steady-state (13)C-labeling experiments using [1-(13)C]-, [2-(13)C]-, and [U-(13)C(6)]glucose. Focusing on the pentose phosphate pathway (PPP), multiple data sets were fitted simultaneously to models in which the subcellular compartmentation of the PPP was altered. The observed redistribution of the label could be explained by any one of three models of the subcellular compartmentation of the oxidative PPP, but other biochemical evidence favored the model in which the oxidative steps of the PPP were duplicated in the cytosol and plastids, with flux through these reactions occurring largely in the cytosol. The analysis emphasizes the inherent difficulty of analyzing the PPP without predefining the extent of its compartmentation and the importance of obtaining high-quality data that report directly on specific subcellular processes. The Arabidopsis flux map also shows that the potential ATP yield of respiration in heterotrophic plant cells can greatly exceed the direct metabolic requirements for biosynthesis, highlighting the need for caution when predicting flux through metabolic networks using assumptions based on the energetics of resource utilization.

Dilution rates influence ammonia-assimilating enzyme activities and cell parameters of Selenomonas ruminantium strain D in continuous (glucose-limited) culture

The objective of this study was to examine the effect of dilution rates (Ds, varying from 0.05 to 0.42 h(-1)) in glucose-limited continuous culture on cell yield, cell composition, fermentation pattern and ammonia assimilation enzymes of Selenomonas ruminantium strain D. All glucose-limited continuous culture experiments were conducted under anaerobic conditions. Except for protein, all cell constituents including carbohydrates, RNA and DNA yielded significant cubic responses to Ds with the highest values at Ds of either 0.10 or 0.20 h(-1). At Ds higher than 0.2 h(-1), fermentation acid pattern shifted primarily from propionate and acetate to lactate production. Succinate also accumulated at the higher Ds (0.30 and 0.42 h(-1)). Glucose was most efficiently utilized by S. ruminantium D at 0.20 h(-1) after which decreases in glucose and ATP yields were observed. Under energy limiting conditions, glutamine synthetase (GS) and glutamate dehydrogenase (GDH) appeared to be the major enzymes involved in nitrogen assimilation suggesting that other potential ammonia incorporating enzymes were of little importance in ammonia assimilation in S. ruminantium D. GS exhibited lower activities than GDH at all Ds, which indicates that the bacterial growth rate is not a primary regulator of their activities. Studied dilution rates influenced cell composition, fermentation pattern and nitrogen assimilation of S. ruminantium strain D grown in glucose-limited continuous culture. Selenomonas ruminantium D is an ecologically and evolutionary important bacterium in ruminants and is present under most rumen dietary conditions. Characterizing the growth physiology and ammonia assimilation enzymes of S. ruminantium D during glucose limitation at Ds, which simulate the liquid turnover rates in rumen, will provide a better understanding of how this micro-organism responds to differing growth conditions.