Energetics of Saccharomyces cerevisiae CBS 426: Comparison of anaerobic and aerobic glucose limitation

Saccharomyces cerevisiae CBS 426 was grown aerobically and anaerobically in a glucose-limited chemostat. The flows of biomass, glucose, ethanol, carbon dioxide, oxygen, glycerol, and the elemental composition of the biomass were measured. Models for anaerobic and aerobic growth are constructed. Values for Y_ATP and P/O are obtained from continuous culture data for aerobic growth; this Y_ATP value is compared with that obtained from the anaerobic growth results. The ratio between the heat produced and the oxygen consumed increases if more glucose in fermented to ethanol and carbon dioxide. An equation for ?_H/?_O as a function of the respiratory quotient is given.     

Continuous and biomass recycle fermentations of Clostridium acetobutylicum

Using experimental data from continuous cultures of Clostridium acetobutylicum with and without biomass recycle, relationships between product formation, growth and energetic parameters were explored, developed and tested. For glucose-limited cultures the maintenance models for, the Y _ATP and biomass yield on glucose, and were found valid, as well as the following relationships between the butanol (Y _B/G) or butyrate (Y _BE/G) yields and the ATP ratio (R _ATP, an energetic parameter), Y _B/G =0.82-1.35 R _ATP, Y _BE/G =0.54 + 1.90 R _ATP. For non-glucose-limited cultures the following correlations were developed, Y _B/G =0.57-1.07 , Y _B/G =0.82-1.35 R _ATPATP and similar equations for the ethanol yield. All these expressions are valid with and without biomass recycle, and independently of glucose feed or residual concentrations, biomass and product concentrations. The practical significance of these expressions is also discussed.List of Symbols D h^–1 dilution rate - m _e mol g^–1 h^–1 maintenance energy coefficient - m _G mol g^–1 h^–1 maintenance energy coefficient - R biomass recycle ratio, (dimensionless) - R _ATP ATP ratio (eqs.(5), (10) and (11)), (dimensionless) - X kg/m³ biomass concentration - Y _ATP g biomass per mol ATP biomass yield on ATP - Y _ATP ^max g biomass per mol ATP maximum Y _ATP - Y _A/G mol acetate produced per mol glucose consumed molar yield of acetate - y _an/g mol acetone produced per mol glucose consumed molar yield of acetone - Y _B/G mol butanol produced per mol glucose consumed molar yield of butanol - y _be/g mol butyrate produced per mol glucose consumed molar yield of butyrate - Y _E/G mol ethanol produced per mol glucose consumed molar yield of ethanol - Y _X/G g biomass per mol glucose consumed biomass yield on glucose - Y _ATP ^max g biomass per mol maximum Y _X/G glucose consumed - h^–1 specific growth rate     

A mathematical model for the aerobic growth of Saccharomyces cerevisiae with a saturated respiratory capacity

A mathematical model for the aerobic growth of Saccharomyces cerevisiae in both batch and continuous culture is described. It was based on the experimental observation that the respiratory capacity of organism may become saturated and exhibit a maximum specific oxygen uptake rate after suitable adaptation. This experimental observation led to the possibility that transport into and out of the mitochondrion was of major importance in the overall metabolism of S. cerevisiae and was subject to long-term adaptation. Consistent with this observation a distributed model was proposed which. as its basis, assumed the control of repression or inhibition of the uptake rates of other substrates. No other regulation of fermentation and respiration was assumed. The model provided a suitable structure allowing precise quantification of the changes in rate and stoichiometry of energy production. The model clearly indicated that growth under the wide range of experimental conditions reported could not be predicted using constant values for the maximum specific respiratory rate of constant values of Y_ATP (g biomass/mol ATP) and PO ratio of (mol ATP/atom oxygen). The causes of the variation in the respiratory rate were not determined and it was concluded that a more detailed analysis (reported subsequently) was required. The variation of Y_ATP and PO ratio with specific growth rate implied that the efficiency of ATP generation or ATP utilization decreased with increasing specific growth rate. It was concluded that it was not possible to quantify the individual effect of Y_ATP and PO ratio until independent means for their reliable estimation is available.     

Glucose as an energy donor in acetate growing Acinetobacter calcoaceticus

Since glucose can be oxidized but not assimilated by Acinetobacter calcoaceticus 69-V the question arose whether energy generated by glucose oxidation can help incorporate carbon from heterotrophic substrates and, if so, what the efficiency of ATP production is like. For this reason this species was grown in the chemostat on acetate. After having reached steady state conditions an increasing concentration of glucose was added. This led to an increase in the biomass level from about 0.4 g/g for growth on acetate alone to 0.6–0.65 g/g in the presence of glucose, independently of either the growth rate or the steepness of the glucose gradient used. This upper value approximates about the limit of the carbon conversion efficiency calculated for non-glycolytic substrates. Glucose was almost exclusively oxidized to gluconic acid, 2- and 5-ketogluconates, and pentose 5-phosphates were found only in traces. These results demonstrate that glucose functions as an additional energy source in Acinetobacter calcoaceticus 69-V. From the transient behaviour of biomass increase and the mixing proportion at which the maximum growth yield on acetate in the presence of glucose was obtained it followed that two mol of ATP must have been generated per mol of glucose oxidized. This property is discussed in terms of coupling glucose dehydrogenase with the respiratory chain.Abbreviations G _ox glucose oxidized to gluconic acid - G _t amount of glucose necessary for complete substitution of S _d - S _o inlet concentration of the limiting carbon substrate - S _a and S _d assimilated and dissimilated part respectively of the carbon substrate - PQQ pyrrolo-quinoline-quinone - V _ATP ^Ac ATP gain from complete oxidation to CO₂ of acetate (P/O=2) - V _ATP ^Glc ATP gain from oxidation of glucose to gluconic acid     

The growth of Saccharomyces cerevisiae CBS 426 on mixtures of glucose and ethanol: a model

Saccharomyces cerevisiae CBS 426 was grown in continuous culture in a defined medium with a mixture of glucose and ethanol as carbon source. Growth on ethanol as the sole carbon source was only possible after the addition of a small amount of glutamic acid. The flows of glucose, ethanol, oxygen, carbon dioxide and biomass to and from the system were measured and a model for the growth of the yeast on the carbon sources constructed. The model is shown to allow independent estimation of Y_ATP and P/O. Y_ATP is not independent of the substrate used, but the amount of ATP used in the production of biomass from the monomers is approximately the same for growth on ethanol and on glucose.Nomenclature C chemical state vector - C_i component of the chemical state vector (C-mol) - C_x biomass present in the system (C-mol biomass) - H₂ reduction equivalents (NAD(P)H + H⁺ and FADH₂) - k the amount of ATP required in the production of 1 C-mol of biomass from the monomers (mol ATP/C-mol biomass) - m_ATP maintenance requirement for ATP (mol ATP/C-mol biomass·h) - P/O (=), efficiency of the oxidative phosphorylation (mol ATP/atom O) - r vector of reaction rates - r_i component of the vector of reaction rates (C-mol/h) - r_ATP rate of ATP production (mol ATP/h) - r_x rate of biomass production (C-mol biomass/h) - Y_ATP Y_ATP growth yield on ATP (C-mol biomass/mol ATP) - (Y_ATP)_max maximum growth yield on ATP - stoichiometry matrix - P/O - vector of the flows to the system - _s flow of glucose to the system (C-mol glucose/h) - _o flow of oxygen to the system (mol O₂/h) - _c flow of carbon dioxide to the system (mol CO₂/h) - _x flow of biomass to the system (C-mol biomass/h) - _e flow of ethanol to the system (C-mol ethanol/h) - _w flow of water produced during metabolism (mol H₂O/h)     

Energetics of glucose uptake in Salmonella typhimurium

We have studied the energetics of glucose uptake in Salmonella typhimurium. Strain PP418 transprots glucose via the phosphoenolpyruvate: glucose phosphotransferase system, while strain PP1705 lacks this system and can only use the galactose permease for glucose uptake. These two strains were cultured anaerobically in glucose-limited chemostats. Both strains produced ethanol and acetate in equimolar amounts but a significant difference was observed in the molar growth yield on glucose (Y _Glc). It is suggested that this difference is due to a difference in the energetics of the glucose uptake systems in the two strains.Assuming an equal Y _ATP for both strains, we could calculate that uptake of 1 mole of glucose via the galactose permease consumes the equivalent of 0.5 mole of ATP. With the additional assumption that one proton is transported in symport with one glucose molecule, these results imply a stoichiometry of two protons per ATP hydrolysed.Abbreviations PTS Phosphoenolpyruvate: carbohydrate phosphotransferase system - D dilution rate (h^-1 - DW dry weight - GalP galactose permease - EtOH ethanol - HAc acetate - Lact lactate - Suc succinate - HFo formate - Glc Glucose - Y _Glc, Y _ATP yield of cells per glucose or ATP - q specific production rate     

The growth rate control in Escherichia coli at near to maximum growth rates: the A-stat approach

The growth characteristics of Escherichia coli K-12 in the continuous culture with a smooth increase in the dilution rate (A-stat) of various carbon sources (glucose, acetate, succinate, glycerol, lactate, acetate + succinate, casamino, acids + glucose) were studied. For all substrates studied the maximum value of specific respiration rate, Q _O2, remained between 14–18 mmol O₂ h^-1 g dwt^-1 and the maximum growth rate varied from 0.22 h-¹ on acetate to 0.77 h^-1 on glucose + casamino acids. After the respiratory capacity of the cells was exhausted at growth rates µ < µ_crit, the growth yield Y_XO2, increased slightly when the dilution rate increased. The maximum growth rate of Escherichia coli K12 was dependent on growth yield, respiratory capacity and glycolytic capacity of the strain. Analysis of the cultivation data using a stoichiometric flux model indicated that ATP synthesis in E. coli exceeds by two-fold that (theoretically) required to build up biomass. The experimental value of m_ATP < 4 mmol ATP h^-1 g dwt^-1 determined from A-stat cultivation data was low compared with the calculated unproductive hydrolysis of ATP (64–103 mmole ATP g dwt^-1).     

Dependency of growth yield,maintenance and K s-values on the dissolved oxygen concentration in continuous cultures of Azotobacter vinelandii

Azotobacter vinelandii was grown diazotrophically in sucrose-limited chemostat cultures at either 12, 48, 108, 144 or 192 M dissolved oxygen. Steady state protein levels and growth yield coefficients (Y) on sucrose increased with increasing dilution rate (D). Specific rate of sucrose consumption (q) increased in direct proportion to D. Maintenance coefficients (m) extrapolated from plots of q versus D, as well as from plots of 1/Y versus 1/D exhibited a nonlinear relationship to the dissolved oxygen concentration. Constant maximal theoretical growth yield coefficients (Y ^G) of 77.7 g cells per mol of sucrose consumed were extrapolated irrespective of differences in ambient oxygen concentration. For comparison, glucose-, as well as acetate-limited cultures were grown at 108 M oxygen. Fairly identical m- and Y ^G-values, when based on mol of substrate-carbon with glucose and sucrose grown cells, indicated that both substrates were used with the same efficiency. However, acetate-limited cultures showed significantly lower m- and, at comparable, D, higher Y-values than cultures limited by either sucrose or glucose. Substrate concentrations (K _s) required for half-maximal growth rates on sucrose were not constant, they increased when the ambient oxygen concentration was raised and, at a given oxygen concentration, when D was decreased. Since biomass levels varied in linear proportion to K _s these results are interpreted in terms of variable substrate uptake activity of the culture.Abbreviations D dilution rate - K _s substrate concentration required for half maximal growth rate - m maintenance coefficient - q specific rate of substrate consumption - Y growth yield coefficient - Y ^G maximum theoretical growth yield coefficient     

Growth and fermentation responses of Selenomonas ruminantium to limiting and non-limiting concentrations of ammonium chloride

 The objective of this study was to assess fermentation product, growth rate and growth yield responses of Selenomonas ruminantium HD₄ to limiting and non-limiting ammonia concentrations. The ammonia half-inhibition constant for S. ruminantium in batch culture was 296 mM. Cells were grown in continuous culture with a defined ascorbate-reduced basal medium containing either 0.5, 5, 25, 50, 100 or 200 mM NH₄Cl and dilution rates were 0.07, 0.14, 0.24 or 0.40 h^-1. Ammonia was the growth-limiting nutrient when 0.5 mM NH₄Cl was provided and the half-saturation constant was 72 μM. Specific rates of glucose utilization and fermentation acid carbon formation were highest for 0.5 mM NH₄Cl. Lactate production (moles per mole of glucose disappearing) increased at the fastest dilution rate (0.40 h^-1) for 5.0 mM NH₄Cl while acetate and propionate decreased when compared to slower dilutions (0.07 and 0.14 h^-1). Lactate production remained low while acetate and propionate remained high for all dilution rates when NH₄Cl concentrations were 25 mM or greater. Yield (Y _Glc and Y _ATP) were nearly doubled when NH₄Cl was increased from 0.5 mM (25.1 g cells/mol glucose used and 13.9 g cells/mol ATP produced respectively) to the higher concentrations. Y _Glc was highest at 25 mM and 50 mM NH₄Cl (48.2 cells/mol and 43.1 cells/mol respectively) as was Y _ATP (23.2 cells/mol and 20.8 cells/mol respectively). Y _NH3 was highest at the lowest NH₄Cl concentration. The maximal fermentation product formation rate occurred at a growth-limiting ammonia concentration, while maximal glucose and ATP bacterial yields occurred at non-growth-limiting ammonia concentrations. Given the growth response of this ruminal bacterium, it is possible that maximization of ruminal bacterial yield may necessitate sacrificing the substrate degradation rate and vice versa. Received: 5 December 1995/Received revision: 2 April 1996/Accepted: 22 April 1996     

NMR Spectroscopy of Cultured Astrocytes: Effects of Glutamine and the Gliotoxin Fluorocitrate

Abstract: Glial synthesis of glutamine, citrate, and other carbon skeletons, as well as metabolic effects of the gliotoxin fluorocitrate, were studied in cultured astrocytes with ¹³C and ³¹P NMR spectroscopy. f2–¹³C]Acetate and [1–¹³C]glucose were used as labeled precursors. In some experiments glutamine (2.5 mM) was added to the culture medium. Fluorocitrate (20 μM) inhibited the tricarboxylic acid (TCA) cycle without affecting the level of ATP. The net export of glutamine was reduced significantly, and that of citrate increased similarly, consistent with an inhibition of aconitase. Fluorocitrate (100 μM) inhibited TCA cycle activity even more and (without addition of glutamine) caused a 40% reduction in the level of ATP. In the presence of 2.5 mM glutamine, 100 μM fluorocitrate did not affect ATP levels, although glutamine synthesis was nearly fully blocked. The consumption of the added glutamine increased with increasing concentrations of fluorocitrate, whereas the consumption of glucose decreased. This shows that glutamine fed into the TCA cycle, substituting for glucose as an energy substrate. These findings may explain how fluorocitrate selectively lowers the level of glutamine and inhibits glutamine formation in the brain in vivo, viz., not by depleting glial cells of ATP, but by causing a rerouting of 2-oxoglutarate from glutamine synthesis into the TCA cycle during inhibition of aconitase. Analysis ^; of the ¹³C labeling of the C-2 versus the C-4 positions in glutamine obtained with [2–¹³C]acetate revealed that 57% of the TCA cycle intermediates were lost per turn of the cycle. Glutamine and citrate were the main TCA cycle intermediates to be released, but a large amount of lactate formed from TCA cycle intermediates was also released, showing that recycling of pyruvate takes place in astrocytes.     

Kinetic Analysis of the Inhibitory Effect of Glibenclamide on KATP Channels of Mammalian Skeletal Muscle

We investigated the block of K_ATP channels by glibenclamide in inside-out membrane patches of rat flexor digitorum brevis muscle. (1) We found that glibenclamide inhibited K_ATP channels with an apparent K _i of 63 nm and a Hill coefficient of 0.85. The inhibition of K_ATP channels by glibenclamide was unaffected by internal Mg²⁺. (2) Glibenclamide altered all kinetic parameters measured; mean open time and burst length were reduced, whereas mean closed time was increased. (3) By making the assumption that binding of glibenclamide to the sulphonylurea receptor (SUR) leads to channel closure, we have used the relation between mean open time, glibenclamide concentration and K _D to estimate binding and unbinding rate constants. We found an apparent rate constant for glibenclamide binding of 9.9 × 10⁷ m ⁻¹ sec⁻¹ and an unbinding rate of 6.26 sec⁻¹. (4) Glibenclamide is a lipophilic molecule and is likely to act on sulfonylurea receptors from within the hydrophobic phase of the cell membrane. The glibenclamide concentration within this phase will be greater than that in the aqueous solution and we have taken this into account to estimate a true binding rate constant of 1.66 × 10⁶ m ⁻¹ sec⁻¹. Received: 7 July 1996/Revised: 4 October 1996     

Opening of Cardiac Sarcolemmal KATP Channels by Dinitrophenol Separate from Metabolic Inhibition

Opening of ATP-sensitive K⁺ (K_ATP) channels by the uncoupler of oxidative phosphorylation, 2,4 dinitrophenol (DNP), has been assumed to be secondary to metabolic inhibition and reduced intracellular ATP levels. Herein, we present data which show that DNP (200 μm) can induce opening of cardiac K_ATP channels, under whole-cell and inside-out conditions, despite millimolar concentrations of ATP (1–2.5 mm). DNP-induced currents had a single channel conductance (71 pS), inward rectification, reversal potential, and intraburst kinetic properties (open time constant, τ_open: 4.8 msec; fast closed time constant, τ_closed(f): 0.33 msec) characteristic of K_ATP channels suggesting that DNP did not affect the pore region of the channel, but may have altered the functional coupling of the ATP-dependent channel gating. A DNP analogue, with the pH-titrable hydroxyl replaced by a methyl group, could not open K_ATP channels. The pH-dependence of the effect of DNP on channel opening under whole-cell, cell-attached, and inside-out conditions suggested that transfer of protonated DNP across the sarcolemma is essential for activation of K_ATP channels in the presence of ATP. We conclude that the use of DNP for metabolic stress-induced K_ATP channel opening should be reevaluated. Received: 10 September 1996/Revised: 27 December 1996     

Oxygen uptake kinetics of Pseudomonas chlororaphis grown in glucose- or glutamate-limited continuous cultures

Oxygen uptake and glucose and glutamate oxidation kinetics of the heterotrophic bacterium Pseudomonas chlororaphis grown in glucose- or glutamate-limited cultures under oxygen-saturating or oxygen-limiting conditions were determined. K _m values for oxygen were 1.4– 5.6 μM. Only in the case of glucose were significantly lower K _m values and enhanced specific oxygen affinity (V _max/K _m) per cell found under oxygen-limiting conditions. Both K _m and specific affinity values for glucose and glutamate oxidation were apparently affected by oxygen concentration, although a statistically significant enhancement of the oxidation kinetics was found only for glutamate. The kinetic data found for P. chlororaphis support the conclusion that the outcome of competition for oxygen with Nitrosomonas europaea in the rhizosphere of oxygen-releasing macrophytes will primarily be determined by oxidation kinetics of the electron donor instead of the oxygen uptake kinetics of the respective organisms. Received: 20 September 1996 / Accepted: 5 February 1997     

Physiological characteristics of chemostatically grownCitrobacter freundii as a function of the specific growth rate and type of nutrient limitation

Citrobacter freundii was grown aerobically in a chemostat on a mineral medium witn galactose or glucose as carbon and energy sources under limitation by carbon or nitrogen source respectively. At various specific growth rates ranging from 7 to 95% μ_max the culture in steady state was analysed and growth yield, specific metabolic rate of substrate utilization, intracellular concentration of pyruvate, ATP, ADP, AMP and energy charge were determined and plotted as functions of dilution rate. In all four types of experiments the physiological state of cells remained practically independent of dilution rate up toD = 0.6 μ_max, and at a given specific growth rate nearly independent on μ_max and type of limitation. At approximatelyD = 0.6 μ_max, which is close to the maximum output dilution rateD _m, the physiological state of the cells changed: growth yields decreased and intr cellular pyruvate and adenylates concentrations increased. Consequently, in a given medium two dilution rates exist at which growth rate dx/dt is the same but the physiology of the population is quite different.     

Yield regulation of lysine biosynthesis in Brevibacterium flavum

A scheme for lysine biosynthesis using variants of the Brevibacterium flavum intermediary metabolite synthesis is discussed. The main precursor of lysine that we are concerned with here is oxalacetate, which can be synthesized through the TCA or glyoxylate cycles or by carboxylation of PEP. Material energy balances for the main pathways of lysine biosynthesis from glucose and acetate have been formulated. Energy consumption, in the from of ATP – P_ATP (number of mol ATP consumed/1 mol lysine synthesized), was calculated for the main pathways of lysine biosynthesis. Theoretical conversion yields Y_p^max (g product/g substrate) were estimated. Experimental data were presented concerning the increase of Y_p by means of metabolism regulation: (a) by TCA-and glyoxylate-cycle enzyme induction; (b) by maintaining PEP carboxylase activity; (c) by eliminating by-product synthesis.     

A theoretical study on the amount of ATP required for synthesis of microbial cell material

The amount of ATP required for the formation of microbial cells growing under various conditions was calculated. It was assumed that the chemical composition of the cell was the same under all these conditions. The analysis of the chemical composition of microbial cells of Morowitz (1968) was taken as a base. It was assumed that 4 moles of ATP are required for the incorporation of one mole of amino acid into protein. The amount of ATP required on account of the instability and frequent regeneration of messenger RNA was calculated from data in the literature pertaining to the relative rates of synthesis of the various classes of RNA molecules in the cell. An estimate is given of the amount of ATP required for transport processes. For this purpose it was assumed that 0.5 mole of ATP is necessary for the uptake of 1 g-ion of potassium or ammonium, and 1 mole of ATP for the uptake of 1 mole of phosphate, amino acid, acetate, malate etc. The results of the calculations show that from preformed monomers (glucose, amino acids and nucleic acid bases) 31.9 g cells can be formed per g-mole of ATP when acetyl-CoA is formed from glucose. When acetyl-CoA cannot be formed from glucose and must be formed from acetate, Y _ATP ^MAX is only 26.4. For growth with glucose and inorganic salts a Y _ATP ^MAX value of 28.8 was found. Addition of amino acids was without effect on Y _ATP ^MAX but addition of nucleic acid bases increased the Y _ATP ^MAX value to that for cells growing with preformed monomers. Under these conditions 15–20% of the total ATP required for cell formation is used for transport processes. Much lower Y _ATP ^MAX values are found for growth with malate, lactate or acetate and inorganic salts. During growth on these substrates a greater part of the ATP required for cell formation is used for transport processes. The calculated figures are very close to the experimental values found. The interrelations between Y _ATP ^MAX and Y_ATP, the specific growth rate (μ), the maintenance coefficient (m_e) and the P/O rate are given. From a review of the literature evidence is presented that these parameters may vary under different growth conditions. It is concluded that in previous studies on the relation between ATP production and formation of cell material these effects have unjustly been neglected.     

Coordination of energy and carbon metabolism in lysine producing Brevibacterium strains

The present paper deals with the coordination of energy metabolism, glucose consumption rate, glycolytic and TCA cycle enzyme activities in the lysine-producing bacterium Brevibacterium flavum. It is shown, that inhibition of the elctron transport chain causes changes of the following sequence:

at first, TCA cycle enzymes are activated;

secondly, TCA cycle enzyme activity decreases, and glycolytic enzyme activities as well as glucose transport rate increase; there is a slight increase in Q_o2 and a considerable one of O₂ consumption in cyanide-resistant respiration pathway;

thirdly, TCA cycle enzyme activities and glucose transport rate decrease.

It is supposed, that coordination of carbon and energy metabolism in B. flavum depends on intracellular ATP concentration or energy charge value.     

Energetic aspects of anaerobic growth of Aerobacter aerogenes in complex medium

Molar growth yields for anaerobic growth of Aerobacter aerogenes in complex medium were much higher than for growth in minimal medium. In batch cultures the molar growth yield for glucose varied from 44 to 50 and Y _ATP from 17.1 to 18.8. For glucose-limited chemostat cultures a value of 17.5 g/mole was found for Y _ATP ^max and a value of 2.3 mmoles ATP/g dry weight h for the maintenance coeficient. Growth dependent pH changes were used to control the addition of fresh medium, containing excess of glucose to a continuous culture. The specific growth rate and the population density were dependent on the pH difference between the inflowing medium and the culture. At a value of 1.44 h^-1 the molar growth yield for glucose was about 70 and Y _ATP about 28.5. An-equation is presented, which gives the relation between theoretical and experimental Y _ATP ^max values.     

Genome‐derived minimal metabolic models for Escherichia coli MG1655 with estimated in vivo respiratory ATP stoichiometry

Metabolic network models describing growth of Escherichia coli on glucose, glycerol and acetate were derived from a genome scale model of E. coli. One of the uncertainties in the metabolic networks is the exact stoichiometry of energy generating and consuming processes. Accurate estimation of biomass and product yields requires correct information on the ATP stoichiometry. The unknown ATP stoichiometry parameters of the constructed E. coli network were estimated from experimental data of eight different aerobic chemostat experiments carried out with E. coli MG1655, grown at different dilution rates (0.025, 0.05, 0.1, and 0.3 h^?1) and on different carbon substrates (glucose, glycerol, and acetate). Proper estimation of the ATP stoichiometry requires proper information on the biomass composition of the organism as well as accurate assessment of net conversion rates under well‐defined conditions. For this purpose a growth rate dependent biomass composition was derived, based on measurements and literature data. After incorporation of the growth rate dependent biomass composition in a metabolic network model, an effective P/O ratio of 1.49 ± 0.26 mol of ATP/mol of O, K_X (growth dependent maintenance) of 0.46 ± 0.27 mol of ATP/C‐mol of biomass and m_ATP (growth independent maintenance) of 0.075 ± 0.015 mol of ATP/C‐mol of biomass/h were estimated using a newly developed Comprehensive Data Reconciliation (CDR) method, assuming that the three energetic parameters were independent of the growth rate and the used substrate. The resulting metabolic network model only requires the specific rate of growth, µ, as an input in order to accurately predict all other fluxes and yields. Biotechnol. Bioeng. 2010;107: 369–381. © 2010 Wiley Periodicals, Inc.     

Estimation of Trichoderma QM 9414 biomass and growth rate by indirect means

Trichoderma QM 9414 was aerobically grown on glucose as the sole carbon and energy sources in a chemostat culture. The specific rates of glucose consumption (Q_G), oxygen consumption (Q), and carbon dioxide production (Q) at the steady state were measured to estimate the growth and maintenance requirements. From the results it was estimated that 2 mol adenosine triphosphate (ATP) were produced when1 mol NADH was oxidized through the respiratory chain of this microorganism. The true growth yield for ATP (Y_ATP) and specific ATP consumption rate for maintenance (Q) calculated with this value were 0.0106 g dry cell/mmol ATP and 5.2 mmol ATP/g dry cell/hr, respectively. Using the relationships between specific growth rate (μ) and (Q) and between μ and Q_G obtained from chemostat-culture data, cell and glucose concentration histories were estimated from the carbon dioxide production rate during the batch culture. The estimated cell concentrations agreed with the experimentally measured values. Glucose concentration were slightly overestimated.