Variation Of Substrate Concentration Research Articles

Comparison of real‐time methods for maximizing power output in microbial fuel cells

AbstractMicrobial fuel cells (MFCs) constitute a novel power generation technology that converts organic waste to electrical energy using microbially catalyzed electrochemical reactions. Since the power output of MFCs changes considerably with varying operating conditions, the online optimization of electrical load (i.e., external resistance) is extremely important for maintaining a stable MFC performance. The application of several real‐time optimization methods is presented, such as the perturbation and observation method, the gradient method, and the recently proposed multiunit method, for maximizing power output of MFCs by varying the external resistance. Experiments were carried out in two similar MFCs fed with acetate. Variations in substrate concentration and temperature were introduced to study the performance of each optimization method in the face of disturbances unknown to the algorithms. Experimental results were used to discuss advantages and limitations of each optimization method. © 2010 American Institute of Chemical Engineers AIChE J, 2010

Continuum Simulations of Acetylcholine Consumption by Acetylcholinesterase: A Poisson−Nernst−Planck Approach

The Poisson-Nernst-Planck (PNP) equation provides a continuum description of electrostatic-driven diffusion and is used here to model the diffusion and reaction of acetylcholine (ACh) with acetylcholinesterase (AChE) enzymes. This study focuses on the effects of ion and substrate concentrations on the reaction rate and rate coefficient. To this end, the PNP equations are numerically solved with a hybrid finite element and boundary element method at a wide range of ion and substrate concentrations, and the results are compared with the partially coupled Smoluchowski-Poisson-Boltzmann model. The reaction rate is found to depend strongly on the concentrations of both the substrate and ions; this is explained by the competition between the intersubstrate repulsion and the ionic screening effects. The reaction rate coefficient is independent of the substrate concentration only at very high ion concentrations, whereas at low ion concentrations the behavior of the rate depends strongly on the substrate concentration. Moreover, at physiological ion concentrations, variations in substrate concentration significantly affect the transient behavior of the reaction. Our results offer a reliable estimate of reaction rates at various conditions and imply that the concentrations of charged substrates must be coupled with the electrostatic computation to provide a more realistic description of neurotransmission and other electrodiffusion and reaction processes.

Maximization of metabolite in fed-batch cultures: Sufficient conditions for singular arc and optimal feed rate profiles

Fed-batch fermentation for maximization of metabolite production at a final time is normally a singular control problem. The singular optimal feed rate strategy depends on relative position of peaks in the specific rates of cell growth μ, substrate consumption σ and product formation π. Regions in which singular feed rates exist are identified and variation of substrate concentration in the singular region is elucidated. Sufficient conditions for the existence of a singular arc are developed, which can be incorporated into an efficient and stable computational scheme to obtain the optimal feed rate profiles.

Metabolite uptake, stoichiometry and chemoautotrophic function of the hydrothermal vent tubeworm Riftia pachyptila: responses to environmental variations in substrate concentrations and temperature

The hydrothermal vent tubeworm Riftia pachyptila is a dominant member of many hydrothermal vent communities along the East Pacific rise and is one of the fastest growing metazoans known. Riftia flourish in diffuse hydrothermal fluid flows, an environment with high spatial and temporal heterogeneity in physical and chemical conditions. To date, physiological and biochemical studies of Riftia have focused on Riftia's adaptations to its chemoautotrophic bacterial symbionts. However the relation between in situ physico-chemical heterogeneity and Riftia host and symbiont metabolism, in particular symbiont chemoautotrophic function, remain poorly understood. Accordingly, we conducted experiments using shipboard high-pressure respirometers to ascertain the effect of varying substrate concentrations and temperature on Riftia metabolite uptake and symbiont carbon fixation. Our results show that substrate concentrations can strongly govern Riftia oxygen and sulfide uptake rates, as well as net carbon uptake (which is a proxy for chemoautotrophic primary production). However, after sufficient exposure to sulfide and oxygen, Riftia were capable of sustaining symbiont autotrophic function for several hours in seawater devoid of sulfide or oxygen, enabling the association to support symbiont metabolism through brief periods of substrate deficiency. Overall, temperature had the largest influence on Riftia metabolite uptake and symbiont autotrophic metabolism. In sum, while Riftia requires sufficient availability of substrates to support symbiont chemoautotrophic function, it is extremely well poised to buffer the temporal and spatial heterogeneity in environmental substrate concentrations, alleviating the influence of environmental heterogeneity on symbiont chemoautotrophic function.

ENZYPLOTW: A USEFUL TOOL TO CALCULATE ENZYME KINETIC PARAMETERS

Assays of enzyme activity are among the most frequently employed procedures in biochemistry. They are used to estimate the amount of a given enzyme in a cell or tissue, to quantify kinetic parameters or to investigate a catalytic mechanism. The purpose of enzyme kinetics obtained under steady-state conditions is to estimate KM and VM values by fitting initial rate (v) and substrate concentration (S) values to the Michaelis-Menten equation, permitting a convenient graphical representation and an accurate estimation of KM and VM. However, there is no ideal assay for any particular enzyme and, since the assay of enzyme activity is essentially a kinetic measurement, various pitfalls await the unwary investigator. Despite difficulties consequent to intrinsic enzyme properties, kinetic parameters can be estimated accurately when steady-state conditions (<10% variation in substrate concentration during activity assay) and initial rate measurements (catalytic enzyme concentration) are guaranteed.EnzyplotW is an application developed for use on any Intel-based computer running Windows 98 or later, and uses a non-linear regression method to fit steady-state kinetic data for Michaelian enzymes. EnzyplotW can be employed not only for educational purposes but also in routine laboratory work, and includes three important features: teaching students how to obtain and recognize sound data for the best estimation of kinetic parameter values; aid the investigator at the bench in gauging the limitations of the various equivalent plots of the Michaelis-Menten equation used to estimate KM and VM; and allow the student to manipulate the mathematical formula of the corresponding plot to comprehend the qualitative and quantitative issues that govern the relationship between substrate concentration and reaction rate.

OPTIMASI ANALISIS AMILASE DAN GLUKANASE YANG DIEKSTRAK DARI MISELIUM Pleurotus ostreatus DENGAN ASAM 3,5 DINITROSALISILAT

Enzymatic activities of amylase (1.4-a-D-glucan glucano hydrolase) and glucanase (1.4-b-glucanase) that extracted from Pleurotus ostreatus mycelium determined through the reducing sugar accumulation. The enzymes assayed in amorphous cellulose (carboxymethylcellulose = CMC) and starch substrate, respectively. Complexity reaction of the sugar to DNS (3.5-dinitrosalicylic acid) become sensitive caused of adding up oxidative agent of sodium sulfite and phenol. Variation in substrate concentration, combine with incubation period and its temperature was able to promote optimum activities of amylase enzymes. The best possible activity of amylase was turn out on 25° C temperature incubation along 20 minutes in 5 percent starch substrate assessment. Incubation period more then 60 minutes on amylase and cellulase caused those activities leaved to 40 and 70 percent down, correspondingly. Modified DNS fastened on reducing sugar twice effectively compared to unmodified one.

Estimation of Substrate Effective Diffusivities in Anaerobic Bioparticles

A simple and rapid method to estimate effective diffusivities (De) of substrates in bioparticles containing anaerobic sludge is proposed. The method is based on substrate mass balances under unsteady-state conditions utilizing data obtained from ‘finite-bath’ assays performed in an incubator shaker. The experiments were carried out under conditions of negligible liquid-phase mass transfer resistance and absence of biochemical reactions. The first condition was obtained by the application of high agitation speeds. The biomass was inactivated with ethanol solution 50% (v/v) to reach the second condition. Values of the effective diffusivity (De) of glucose, sucrose and acetate were obtained from the temporal variation of substrate concentration in the bulk liquid in assays utilizing alginate gel and polyurethane foam bioparticles. Additionally, the specific methanogenic activity technique was used to evaluate the effectiveness of the method proposed for biomass inactivation. The method used to estimate the substrate diffusivity coefficient was found to provide suitable values of De for substrates at very low concentrations. Values of De for glucose, sucrose and acetate in alginate gel bioparticles were estimated as 0.58 × 10−5, 1.06 × 10−5 and 1.49 × 10−5 cm2 s−1, respectively. The value of De for glucose in polyurethane foam particles was found to correspond to the value of glucose diffusion in water at infinite dilution. The method applied for cell inactivation was found to be adequate for intraparticle mass transfer experiments, making the temporary sludge inactivity long enough to avoid interference due to biochemical reactions.

A Stable Urea Biosensor with Fluoride Ion Selective Electrode. 4. Steady-State Measuring Range

ABSTRACTSome problems are discussed in this article concerning measurement range of urea biosensor with fluoride ion-selective electrode. An analytic model is proposed based on a quadratic approximation of variation of substrate concentration in the active membrane. Families of calibration curves are generated by digital simulation and different variants are analysed.

Purification and Characterization of a GDP-fucose:Polypeptide Fucosyltransferase from Chinese Hamster Ovary Cells

A GDP-fucose:polypeptide fucosyltransferase was purified 5000-fold to homogeneity from Chinese hamster ovary cell extracts in the absence of detergent. The purification procedure included two affinity chromatographic steps using the acceptor substrate, a recombinant factor VII EGF-1 domain, and the donor substrate analog, GDP-hexanolamine, as ligands. The purified enzyme migrates as a single band of 44,000 daltons on SDS-polyacrylamide gel electrophoresis and is itself a glycoprotein with more than one high mannose type oligosaccharide chain with a total molecular weight of 4000. The Km values for factor VII EGF-1 domain and GDP-fucose are 15 and 6 microM, respectively. The Vmax is 2.5 micromol.min-1.mg-1. The presence of 50 mM Mn2+ increased the enzyme activity 17-fold, but Mn2+ was not absolutely required, since the enzyme exhibited some activity even in the presence of EDTA. The acceptor substrate specificity was studied using site-directed mutagenesis of human factor IX EGF domain. Only one of several differently folded species could serve as acceptor substrate, although they all had the same molecular weight as determined by liquid chromatography on-line with mass spectrometry. This indicates that the enzyme requires proper folding of the epidermal growth factor domain for its activity.

Acetate removal in sewer biofilms under aerobic conditions

Removal of acetate has been investigated in sewer biofilms by continuous-flow biofilm reactor studies simulating the conditions in a gravity sewer. Non-steady-state conditions are prevailing in sewers, due to periodic variations in substrate concentrations. In order to simulate two extreme situations in a gravity sewer, biofilms defined as high-loaded and low-loaded, respectively, were grown by continuously feeding wastewater to the reactors with and without supplementary addition of acetate. During short-term experiments with high acetate concentrations (1–2 h), surface removal rates of acetate and dissolved oxygen (DO) and observed yield coefficients were determined, as well as the influence of DO concentration on acetate removal rates. The low-loaded biofilms showed very high acetate removal rates in short-term experiments at high acetate concentrations. The DO uptake rates were low, resulting in an average observed yield coefficient of 0.79 g biomass produced per gram acetate (as chemical oxygen demand, COD) consumed. This indicated a luxury uptake by the cells probably for storage inside the cells or for production of extracellular polymeric substances. The high-loaded biofilms showed lower acetate removal rates during the short-term experiments, with an average yield coefficient of 0.49 g biomass produced per gram acetate (as COD) consumed. The level of the acetate removal rates seemed to be related to the structure of the biofilm. The highest acetate removal rates were found for the low-loaded biofilm, where the biofilm was very hairy with “streamers” with a length of 8–9 mm. At low acetate removal rates (high-loaded biofilm), the “streamer” lengths were only 3–5 mm. The surface removal rates for acetate and DO seemed to follow 1 2 order approximations to biofilm kinetics. For a DO of 0.8 and 6.0 g/m 3, the limiting acetate concentrations were about 3–4 and 20 g-COD/m 3, respectively. Under real gravity sewer conditions, the typical concentration ranges for acetate and DO are at levels where any of them may be rate-limiting for microbial acetate removal.

The advantages of periodically operated biofilm reactors for the treatment of highly variable wastewater

Many wastes generated by industry, at hazardous wastes sites and at landfill experience shock loads of inhibitory organic compounds which can upset biological treatment systems. Biofilm reactors can be used to treat these wastes. However, the continuous flow mode operation normally used results in biofilm stratification and uneven biomass distribution that limits the ability of biofilm processes to treat shock loads. Periodic operation of biofilm reactors is advantageous because even biomass distribution can be obtained. In addition, data from membrane sequencing batch biofilm reactors indicates that periodic operation imposes regular variations in substrate concentration on the biofilm. As a result, organisms throughout the film achieve maximum growth rates which cause physiological adaptations that can improve biofilm response to shock loads.

The advantages of periodically operated biofilm reactors for the treatment of highly variable wastewater

Many wastes generated by industry, at hazardous wastes sites and at landfills experience shock loads of inhibitory organic compounds which can upset biological treatment systems. Biofilm reactors can be used to treat these wastes. However, the continuous flow mode operation normally used results in biofilm stratification and uneven biomass distribution that limits the ability of biofilm processes to treat shock loads. Periodic operation of biofilm reactors is advantageous because even biomass distribution can be obtained. In addition, data from membrane sequencing batch biofilm reactors indicates that periodic operation imposes regular variations in substrate concentration on the biofilm. As a result, organisms throughout the film achieve maximum growth rates which cause physiological adaptations that can improve biofilm response to shock loads.

Effect of sinusoidal variation of feed concentration and temperature on the performance of a packed‐bed biological reactor – a theoretical study

AbstractThe performance of a packed‐bed biological reactor has been analysed under sinusoidal variations of substrate concentration and temperature for zero‐order, first‐order and Michaelis‐Menten kinetics. The model equations were solved by the method of orthogonal collocation. The results show that the cyclic steady‐state conversion is not affected by cyclic variations in the feed concentration. However, cyclic temperature variations with an amplitude of 20°C significantly decrease the mean exit concentration for zero‐order and Michaelis‐Menten kinetics compared to the constant‐temperature case. The approach to cyclic steady‐state conditions is estimated to be somewhat flower for zero‐order kinetics than for the other kinetics models investigated. We conclude that temperature variations during the day or changes in the performance of upstream plant will not adversely affect the performance of a packed‐bed biological reactor.

Purification and characterization of clavaminate synthase from Streptomyces clavuligerus: an unusual oxidative enzyme in natural product biosynthesis

A pivotal step in the biosynthetic pathway to the beta-lactamase inhibitor clavulanic acid is the conversion of proclavaminic acid to clavaminic acid in a reaction requiring Fe2+, alpha-ketoglutarate, and oxygen [Elson, S. W., Baggaley, K. H., Gillett, J., Holland, S., Nicholson, N. H., Sime, J. T., & Woroniecki, S. R. (1987) J. Chem. Soc., Chem. Commun., 1736-1738]. Clavaminate synthase, the enzyme that catalyzes this oxidative cyclization/desaturation, has been purified to homogeneity from clavulanic acid producing cells of Streptomyces clavuligerus (ATCC 27064). The enzyme behaved as a monomer during gel filtration and migrated with Mr 47,000 during denaturing gel electrophoresis. After ion-exchange FPLC two active forms of the protein were resolved that differed slightly in kinetic constants and apparent molecular weight. Kinetic comparisons with the four possible diastereomers of proclavaminate confirmed the absolute configuration of the substrate to be 2S,3R. The stoichiometry of the overall transformation was determined to be proclavaminate + 2(alpha-ketoglutarate) + 2O2----clavaminate + 2(succinate) + 2CO2 + 2H2O. In the absence of proclavaminate a slow decarboxylation of alpha-ketoglutarate to succinate and CO2 was observed in an uncoupled reaction which resulted in enzyme inactivation. Steady-state kinetic studies were undertaken for an initial description of the enzyme's catalytic cycle. The double-reciprocal plot with alpha-ketoglutarate as the variable substrate was linear; this supports the proposal that two stepwise oxidations of proclavaminate occur, each with the consumption of alpha-ketoglutarate and oxygen and the release of succinate, CO2, and H2O. The intersecting initial velocity plots obtained from pairwise variation of substrate concentrations were consistent with a sequential kinetic mechanism for the first oxidation. Similarities observed between clavaminate synthase and alpha-ketoglutarate-dependent dioxygenases argue for a common mechanism of oxygen activation. However, the nature of the interactions of the substrates in the active site of clavaminate synthase apparently redirects the conventional hydroxylase activity of dioxygenases to the construction of a strained bicyclic skeleton driven by the overall reduction of dioxygen.

Extracellular proteolytic enzyme activity in sediments of an intertidal mudflat

Extracellular proteolytic activity (EPA) on an intertidal mudflat was examined over a 2‐yr period, with focus on characteristics of enzyme systems and the controlling influences on enzyme activity levels. EPA was primarily associated with the particulate rather than the pore‐water phase. Inhibitor studies indicated the presence of primarily metallo‐ and thiol proteases, with pH optima in the range 8–9.5. Temperature optima increased with depth in the sediment, being in the 20°–35°C range for surficial sediments and the 40°–50°C range in the subsurface. Activation energies at environmental temperatures were in the 63–67 kJ mol−1 range. The hypothesis of progressive humification of enzyme activity with depth was discarded on the basis of results from thermal denaturation experiments. EPA levels decreased with depth and hence correlated with substrate concentration and bacterial populations, but this correlation was not a tight one. Seasonal variations in EPA varied with the temperature cycle consistent with, though somewhat damped relative to, the temperature dependence of the enzymes themselves and showed no response to variations in substrate concentrations or bacterial numbers.

Kinetic mechanism of native Escherichia coli aspartate transcarbamylase

Equilibrium isotope exchange kinetics have been used to reinvestigate the kinetic mechanism of Escherichia coli aspartate transcarbamylase (aspartate carbamoyltransferase) at pH 7.0, 30 °C. K eq = 5.9 (±0.6) × 10 3, allowing variation of substrate concentrations above and below their K m values in all experiments, a condition not possible at pH 7.8 [ F. C. Wedler and F. J. Gasser (1974) Arch. Biochem. Biophys. 163, 57–68 ]. The rate of the [ 14C]Asp ⇌ N-carbamoyl l-aspartate (CAsp) exchange reaction was five times faster than that of [ 32P]carbamyl phosphate (CP) ⇌ P i , which argues strongly against the rapid equilibrium random mechanism previously proposed by E. Heyde, A. Nagabhushanam, and J. F. Morrison [ Biochemistry 12, 4718–4726 (1973) ]. Substrate concentrations were varied either as reactant-product pairs (holding the other pair constant) or together simultaneously in constant ratio at equilibrium. The resulting kinetic saturation patterns were most consistent with a preferred order random kinetic mechanism, with CP binding prior to Asp and with CAsp being released before P i. Weak inhibition effects at high substrate levels could be accounted for by multiple weak dead-end complexes or ionic strength effects. Computer-based simulations have led to a set of rate constants that fit the experimental data, are in agreement with rate constants measured previously by pre-steady-state methods, and predict the correct initial velocities in the forward and reverse directions. Simulations also show that rate constants consistent with any of the various alternative mechanisms do not provide good fit to the experimental data. A model for the kinetic mechanism is considered, in which the binding of Asp prior to CP may restrict access of CP to the active site, but CP binding prior to Asp potentiates the enzyme for the allosteric (T-R) transition, centered entirely upon the Asp binding process.

Non-stationary processes at polymer coated rotating disk electrodes: Part I. Model and simulation

A time dependent model for the mediated oxidation of a substrate in bulk solution at a polymer modified rotating disk electrode is proposed and a numerical solution is found by orthogonal collocation. The mediation system consists of a surface bound redox species which can accept electrons from a substrate in solution. In the model considered here the substrate can enter the film freely but does not react at the electrode surface. The effect of the variation of substrate concentration, mediator concentration, effective charge transfer diffusion coefficient of the bound species and homogeneous rate constant on the electrochemical behaviour of the modified electrode is considered. Good qualitative agreement was found between simulated current-potential curves and experimental curves using [Fe(CN) 6] 4− a substrate and [Ru(bpy) 2Cl(PVP) 5]Cl as the polymer coating.

A multispecies biofilm model

Using a continuum approach and observing conservation principles, an analytical mathematical model of microbial interaction in biofilms was developed. The model predicts changes in biofilm thickness and describes the dynamics and spatial distribution of microbial species and substrates in the film. It allows for biomass detachment due to shear stress and sloughing, external mass transfer limitations, as well as variations in substrate concentrations in the bulk liquid. A computer implementation of the model is provided using an example of heterotrophicautotrophic competition to illustrate how the observed phenomena can be numerically reproduced and indicating how they might affect overall biofilm performance.

Modulation of substrate transport to the brain.

Variations of substrate transport across the cerebral capillary endothelium were examined in response to variations of the substrate demand of the brain tissue, and to variations of substrate concentration in the blood. The substrates examined included glucose and ketone bodies. The transport changes were measured in rats, using an indicator fractionation method modified by the reviewer. Four mechanisms appeared to contribute to the adjustment of substrate transport to variations in substrate demand. The first and least important mechanism was the change of concentration gradient across the endothelium that occurred when the substrate consumption rate changed. The second mechanism was the flow-dependency of the average capillary substrate concentration: the higher the perfusion rate, the higher the average capillary concentration. This mechanism failed to account for the changes of substrate transport observed during marked increases of the metabolic rate. The third and most important mechanism was a change of the capillary diffusion capacity, probably associated with a change of the number of perfused capillaries. The fourth mechanism, not previously described, was an adaptation of transport to permanent changes of substrate concentration in the blood. This mechanism appeared to reflect changes of the concentration (and affinity?) of transport proteins in the plasma membranes of endothelial cells, possibly in association with changes of cellular protein synthesis and gene expression.

Complementarity of regulation for the two glutamine synthetases from Bacillus caldolyticus, an extreme thermophile

Regulation of the two glutamine synthetases, E I and E II, from Bacillus caldolyticus was found to occur at 70 °C in a complementary fashion, so that total enzyme activity responded to the full spectrum of feedback modifiers derived from glutamine. E I was more strongly inhibited than E II by Gly, l-Ala, and l-Ser, whereas E II was more strongly inhibited than E I by l-Gln, AMP, and 5-phosphorylribose-1-pyrophosphate. Both enzymes were strongly inhibited by ADP and CTP, but less strongly by NAD, Trp, and glucosamine-6- P. Upon variation of the concentration of individual feedback modifiers, initial velocity kinetics showed that at saturation many of the inhibitors gave only partial inhibition of enzyme activity. In addition, with E II the saturation curves for AMP, ADP, and NAD were nonhyperbolic. Upon variation of substrate concentration at several fixed concentrations of modifier, it was found for E I that MnADP was noncompetitive vs MnATP and that Gly, l-Ala, l-Ser, and CTP were all noncompetitive vs l-Glu. For E II, l-Gln both raised the S 0.5 for l-Glu and changed the Hill n value from less than to greater than 1.0; AMP was noncompetitive vs ATP, increased the S 0.5 and the Hill n value for ATP binding, and also decreased V; CTP was competitive vs l-Glu, as were ADP and PRPP vs ATP. These kinetic data, along with independent heat stabilization experiments and fluorescence measurements with pairs of substrates and modifiers, were consistent with binding sites for many of the key feedback modifiers on E I and E II that are separate from the active site.