Kinetic study of ozonation of molasses fermentation wastewater

A kinetic study of molasses wastewater ozonation was carried out in a stirred tank reactor to obtain the rate constants for the decolorization reaction and the regime through which ozone is absorbed. First, fundamental mass transfer parameters such as ozone solubility, volumetric mass transfer coefficients and ozone decomposition kinetics were determined from semi-batch experiments in organic-free solutions with an ionic composition similar that of industrial wastewater. The influence of operating variables such as the stirring rate and gas flow rate on the kinetic and mass transfer parameters was also studied. The application of film theory allows to establish that the reactions between ozone and colored compounds in wastewater take place in the fast and pseudo-first-order regime, within the liquid film. The decolorization rate constants were evaluated at pH 8.7 and 25 degrees C, varying from 0.6 x 10(7) to 3.8 x 10(7)L mol(-1)s(-1), depending on the stirring rate and the inlet gas flow.     

Electrochemical study of reversible hydrogenase reaction of Desulfovibrio vulgaris cells with methyl viologen as an electron carrier.

An electrode modified with immobilized whole cells of Desulfovibrio vulgaris (Hildenborough) produces an S-shaped voltammogram with both cathodic- and anodic-catalytic-limiting currents in a methyl viologen-containing buffer saturated with H2. Methyl viologen penetrates into the bacterial cells to serve as an electron carrier in the reversible reaction of hydrogenase in the cells and functions as an electron-transfer mediator between the bacterial cells and the electrode, thus producing the catalytic currents for the evolution and consumption of H2. An equation for the catalytic current that takes into account the reversible hydrogenase reaction explains well the shape of the voltammogram. The potential at null current on the voltammogram agrees with the potential determined by potentiometry with the same electrode, which is equal to the redox potential of the H+/H2 couple in the solution--the standard potential of a hydrogen electrode at the pH of the solution. When D. vulgaris cells are suspended in an argon-saturated buffer containing methyl viologen, the suspension produces a catalytic current at a bare glassy carbon electrode for the evolution of H2. Analysis of the current by a theory for a catalytic current for a unidirectional nonlinear enzyme catalysis allows us to determine the kinetic parameters of the reaction between methyl viologen and hydrogenase in intact D. vulgaris cells. Thus we obtain the apparent Michaelis constant for methyl viologen cation radical, K'MV.+ = 0.16 mM, and the apparent catalytic constant (that is, the turnover number per D. vulgaris cell), zkcat,H+ = 1.2 x 10(7) s-1, for the H2 evolution reaction at pH 5.5 and at 25 degrees C, z being the number of hydrogenases contained in a D. vulgaris cell. The bimolecular reaction rate constant, kcat,H+/K'MV.+, of the reaction between methyl viologen cation radical and oxidized hydrogenase in intact D. vulgaris cells is estimated as 4.2 x 10(7) M-1 s-1. Similarly, the bimolecular reaction rate constant, kcat,H2/K'MV2+, of the reaction between methyl viologen and reduced hydrogenase is estimated to be 1.2 x 10(7) M-1 s-1 at pH 9.5 and 25 degrees C. Both rate constants are large enough for the reactions to be diffusion-limited processes.     

Mechanistic studies on enzymatic reactions by electrospray ionization MS using a capillary mixer with adjustable reaction chamber volume for time-resolved measurements

Mass spectrometry (MS)-based techniques have enormous potential for kinetic studies on enzyme-catalyzed processes. In particular, the use of electrospray ionization (ESI) MS for steady-state measurements is well established. However, there are very few reports of MS-based studies in the pre-steady-state regime, because it is difficult to achieve the time resolution required for this type of experiment. We have recently developed a capillary mixer with adjustable reaction chamber volume for kinetic studies by ESI-MS with millisecond time resolution (Wilson, D. J.; Konermann, L. Anal. Chem. 2003, 75, 6408-6414). Data can be acquired in kinetic mode, where the concentrations of selected reactive species are monitored as a function of time, or in spectral mode, where entire mass spectra are obtained for selected reaction times. Here, we describe the application of this technique to study the kinetics of enzyme reactions. The hydrolysis of p-nitrophenyl acetate by chymotrypsin was chosen as a simple chromophoric model system. On-line addition of a "makeup solvent" immediately prior to ionization allowed the pre-steady-state accumulation of acetylated chymotrypsin to be monitored. The rate constant for acetylation, as well as the dissociation constant of the enzyme-substrate complex obtained from these data, is in excellent agreement with results obtained by conventional stopped-flow methods. Bradykinin was chosen to illustrate the performance of the ESI-MS-based method with a nonchromophoric substrate. In this case, the unfavorable rate constant ratio for acylation and deacylation of the enzyme precluded measurements in the pre-steady-state regime. Steady-state experiments were carried out to determine the turnover number and the Michaelis constant for bradykinin. The methodologies used in this work open a wide range of possibilities for future ESI-MS-based kinetic assays in enzymology.     

Using nonequilibrium capillary electrophoresis of equilibrium mixtures for the determination of temperature in capillary electrophoresis

Until now, all methods for temperature sensing in capillary electrophoresis (CE) relied on molecular probes with temperature-dependent spectral/optical properties. Here we introduce a nonspectroscopic approach to determining temperature in CE. It is based on measuring a temperature-dependent rate constant of complex dissociation by means of a kinetic CE method known as nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM). Conceptually, a calibration curve of "the rate constant versus temperature" is built using NECEEM and a CE instrument with a reliable temperature control or, alternatively, a nonelectrophoretic method, such as surface plasmon resonance. The calibration curve is then used to find the temperature during CE in the same buffer but with another CE apparatus or under otherwise different conditions (cooling efficiency, length and diameter of the capillary, electrical field, etc.). In this proof-of-principle work, we used the dissociation of a protein-DNA complex to demonstrate that the NECEEM-based temperature determination method allows for temperature determination in CE with a precision of 2 degrees C. Then, we applied the NECEEM-based temperature determination method to study heat dissipation efficiency in CE instruments with active and passive cooling of the capillary. The nonspectroscopic nature of the method makes it potentially applicable to nonspectroscopic detection schemes, e.g. electrochemical detection. A "kinetic probe" can be coloaded into the capillary along with a sample for in situ temperature measurements. Higher order chemical reactions can also be used for temperature sensing, provided a kinetic CE method for measuring a corresponding rate constant is available.     

The size refinement of Cu crystallites under mechanical processing conditions: a phenomenological modeling approach

A phenomenological model is developed for describing the kinetics of the crystallite size refinement process of Cu powder under mechanical treatment conditions. Based on the evidence that collisions represent the elementary events of energy transfer, the rate of crystallite size decrease is related on a statistical basis to the amount of powder trapped at each collision, to the number of collisions and to the collision energy. The mathematical approach allows for identifying the approximate functional form of the kinetic curves obtained at largely different impact energies. The values of the apparent kinetic constants and of the model parameters involved can be thus estimated by fitting the model curves to the experimental data. The results obtained provide a deeper insight into the details of the crystallite size refinement process.     

Prediction of surface oxidation weight gain on 7.8 wt% Cr-containing stainless steel electrode during electroslag remelting

The surface oxidation weight gains on 7.8 wt% Cr-containing stainless steel at different temperatures were studied by thermogravimetric analysis and scanning electron microscope (SEM) equipped with energy-dispersive spectroscopy (EDS). The results show that there are two layers of oxide scales covering the surface of steel matrix after 100 min high-temperature oxidation. The inner oxide scale consists of (Fe, Cr) oxide and the outer oxide scale is composed of Fe oxide due to the stronger affinity of Cr with O. The growth of outer oxide scale is dominated by the combined effects of chemical reaction rate and mass transfer. Based on the study of the oxidation kinetics at different temperatures, a prediction model for surface oxidation weight gain on 7.8 wt% Cr-containing stainless steel electrode during electroslag remelting is acquired, providing a reference for the determination of the amount of deoxidiser added into the molten slag during electrode remelting.     

Transverse diffusion of laminar flow profiles to produce capillary nanoreactors

We introduce transverse diffusion of laminar flow profiles (TDLFP), the first generic method for mixing two or more reactants inside capillaries. Conceptually, solutions of reactants are injected inside the capillary by pressure as a series of consecutive plugs. Due to the laminar nature of flow inside the capillary, the nondiffused plugs have parabolic profiles with predominantly longitudinal interfaces between them. After injection, the plugs are mixed by transverse diffusion; longitudinal diffusion does not contribute to mixing. To prove the principle, we used TDLFP to mix two reactants-an enzyme and its substrate. After mixing the reactants by TDLFP, we incubated reaction mixtures for different periods of time and measured the reaction kinetics. We found that the reaction proceeded in time- and concentration-dependent fashion, thus confirming that the reactants were mixed by TDLFP. Remarkably, the experimental reaction kinetics were not only in qualitative agreement but also in good quantitative agreement with theoretically predicted ones. TDLFP has a number of enabling features. By facilitating the preparation of reaction mixtures inside the capillary, TDLFP lowers reagent consumption to nanoliters (microliters are required for conventionally mixing reagents in a vial). The reaction products can be then analyzed "on-line" by capillary separation coupled with optical, electrochemical, or mass spectrometric detection. The combination of TDLFP with capillary separation will be an indispensable tool in screening large combinatorial libraries for affinity probes and drug candidates: a few microliters of a target protein will be sufficient to screen thousands of compounds. The new method paves the road to a wide use of capillary nanoreactors in different areas of physical and life sciences.     

Comparison of solution chemistries of orthophthalaldehyde and 2,3-naphthalenedicarboxaldehyde

Polarography was used to obtain the concentrations of the dialdehydic (10%), monohydrated acyclic (5%), and cyclic hemiacetal form (85%) of orthophthalaldehyde (OPA). For 2,3-naphthalenedicarboxaldehyde (NDA) these values were estimated to be 15, 7, and 78%. Addition of water in unbuffered solutions followed first-order kinetics with rate constants 0.0018 s-1 for OPA and 0.0012 s-1 for NDA. Dehydration to form both the dialdehyde and the monohydrate is both acid- and base-catalyzed. Both dialdehydes yield on reaction with OH- ions geminal diol anion, which is electro-oxidized to a carboxylic acid. In the most frequently used pH range for the determination of amino acids, NDA can undergo reaction with OH- ions, but OPA does not. In aqueous solutions, NDA is less strongly hydrated than OPA.     

Functional probing of arginine residues in proteins using mass spectrometry and an arginine-specific covalent tagging concept

The reactivity of arginine residues in model proteins (ubiquitin, cytochrome c, myoglobin, ribonuclease A, lysozyme) was examined using a selective tagging reaction in combination with on-line monitoring of the reaction progress by electrospray ionization mass spectrometry (ESI-MS). The kinetics of this reaction, based on the cyclization of the guanidine group of arginine with 2,3-butanedione and phenylboronic acid at pH 8-10, allow the grouping of arginines in "exposed" or "partially buried" residues, because they differ substantially in their reaction rate constants for the conversion of the guanidine groups. The method allows one to differentiate between different protein conformations as shown for myoglobin and its apo form and native and reduced ribonuclease A: Removal of the heme group in myoglobin resulted in an increased reactivity for the two partially buried arginines. For RNAse A, quantitative reduction of the disulfide bonds lead to the exposure of an additional arginine residue and two different conformations of the reduced protein were observed by ESI-MS that could be distinguished according to their charge-state distribution. Experimentally obtained accessibilities were compared with solvent-accessibility data calculated from 3D structures and substantial agreement between both techniques was observed.     

Measurements of kinetic parameters in a microfluidic reactor

Continuous flow microfluidic reactors that use immobilized components of enzymatic reactions present special challenges in interpretation of kinetic data. This study evaluates the difference between mass-transfer effects and reduced efficiencies of an enzyme reaction. The kinetic properties of immobilized alkaline phosphatase (AP) were measured by the dephosphorylation of 6,8-difluoro-4-methylumbelliferyl/phosphate to a fluorescent 6,8-difluoro-4-methylumbelliferone. A glass microfluidic chip with an in-channel weir was created for the capture of solid silica microbeads functionalized with enzyme. The input substrate concentrations and flow rates across the bed were varied to probe the flow-dependent transport and kinetic properties of the reaction in the microreactor bed. Unlike previous reactors, substrate was titrated directly over the fixed enzyme bed by controlling the air pressure over the chip reservoirs. The reactor explored substrate conversions from near zero to 100%. The average bed porosity, residence time, and bed resistance were measured with dye pulses. A simple criterion was derived to evaluate the importance of flow-dependent mass-transfer resistances when using microreactors for calculating kinetic rate constants. In the absence of mass-transfer resistances, the Michaelis-Menten kinetic parameters are shown to be flow independent and are appropriately predicted using low substrate conversion data. A comparison of the kinetic parameters with those obtained using solution-phase enzymatic reactions shows a significant decrease in enzyme activity in the immobilized conformation. The immobilized Km of AP is approximately 6 times greater while the kcat is reduced by approximately 28 times. Contradictions found in literature on the evaluation of Michaelis-Menten kinetic parameters for immobilized enzymes in microfluidic reactors are addressed. When product molecules occupy a significant number of enzymatic sites or modify the enzyme activity, the assumed Michaelis-Menten mechanism can no longer be valid. Under these conditions, the calculations of "apparent" kinetic rate constants, based on Michaelis-Menten kinetics, can superficially show a dependence on flow rate conditions even in the absence of mass-transfer resistances. High substrate conversions are shown to depend on flow rate. A kinetic model based on known mechanisms of the alkaline phosphatase enzyme reaction is tested to predict the measurements for high substrate conversion. The study provides a basis for appropriate use of mass-transfer and reaction arguments in successful application of enzymatic microreactors.     

Dynamic kinetic capillary isoelectric focusing: a powerful tool for studying protein-DNA interactions

A new method called dynamic kinetic capillary isoelectric focusing (DK-CIEF) is presented for the study of protein-DNA interactions. The method is based on CIEF with laser-induced fluorescence-whole column imaging detection in which protein-DNA complexes are separated with spatial resolution while dissociations of the complexes are dynamically monitored using a CCD camera with temporal resolution. This method allows for the discrimination of different complexes and the measurement of the individual dissociation rate constants.     

Viscoelastic modeling of template-directed DNA synthesis

In the present study, we have used the QCM-D technology to study the replication of surface attached oligonucleotide template strands using Escherichia coli DNA polymerase I (Klenow fragment, KF). Changes in resonance frequency (F) and energy dissipation (D) for DNA hybridization and polymerization were recorded at multiple harmonics. Formation of the polymerase/DNA complex led to a significant decrease in energy dissipation, which is consistent with a conformational change induced upon enzyme binding. This interpretation was further strengthened by a data analysis using a Voigt-based viscoelastic model. The analysis revealed a significant increase in shear viscosity and shear modulus during KF binding, whereas the viscoelastic properties of single- and double-stranded templates were almost identical. During the actual DNA synthesis, an initial increase in rigidity (shear viscosity) was followed by a gradual decrease that has two components corresponding to the release of enzyme and to the presence of the catalytically active enzyme/substrate complex. The corresponding decrease in surface concentration was found to underestimate the rate of enzyme release due to viscously coupled water that compensates for the loss in enzyme mass. Furthermore, the modeling elucidates that significant changes in both F and D originate from variations in the viscoelastic properties, which means that changes in F alone should be used with care for estimations of coupled mass and kinetics. Therefore, the modeled temporal variation in effective thickness, being proportional to coupled mass and, thus, independent of structural changes, was used to estimate the catalytic constants of the polymerization reaction. The reported work is the first example providing this type of structural information for the catalytic action of an enzyme, thereby demonstrating the potential of the technique for advanced analysis of complex biological reactions, including proper analysis of enzyme kinetics.     

Reactivity and interface characteristics of titanium-alumina composites

The reaction kinetics between -Ti alloys and single crystal sapphire, the phase composition and morphology of the reaction-zone, and the phase compatibility in the system Ti-Al-O were investigated as part of a study to determine the feasibility of fabricating useful Al₂O₃-reinforced titanium matrix composites. In the temperature range 650 to 1000° C titanium reduces Al₂O₃ to form a complex reaction layer consisting of two distinct zones; an inner zone adjacent to the Al₂O₃ of a TiO phase containing isolated particles of (Ti, Al)₂O₃, presumably, and an outer zone of a Ti₃Al phase adjacent to the Ti matrix. The isothermal growth of the reaction layer follows a parabolic rate law. The temperature dependence of the rate constants fits an Arrhenius equation yielding activation energies of 50 to 52 kcal/mol. The high Al alloys, except Ti-6Al-2Sn-4Mo-2Zr, reacted more rapidly than pure Ti indicating that Al diffusion through the reaction zone may be the rate-limiting step.     

A numerical method for determining the kinetic constants of gas–liquid metal interactions in N–Ni–20Cr and N–ASTM F-75 alloy systems

In gas–liquid interaction systems, under non-equilibrium degassing conditions, the rate of a particular process often follows a mixed control regime where serial-acting mechanisms affect the global rate of the process and, therefore, the determination of real kinetic constants is difficult. In this paper a numerical method for determining these kinetic constants is presented and applied to the cases of denitrogenation of liquid Ni–20Cr alloys at 1873 K and nitrogen absorption in liquid ASTM F-75 alloys at 1773 and 1823 K. The results showed that interaction kinetics of nitrogen with both alloys was limited by both first and second order mechanisms. This results permit manufacturing engineers to act on the process parameters in order to obtain the nitrogen content that allows the best performance of mechanical parts.     

A millisecond infrared stopped-flow apparatus

In this paper, we have described a stopped-flow apparatus that is capable of measuring infrared kinetics in the amide I' region of a protein's vibrational spectrum. The dead time of this setup, determined by the reducing reaction of 2,6-Dichlorophenolindophenol by L-ascorbic acid, is between 6 to 15 ms, depending on the flow rate. Therefore, this stopped-flow IR method provides a means of measuring infrared kinetics in a time window that is not easily accessible to other mixing-based IR techniques. Using this apparatus, we have studied the alkaline transition of cytrochrome c and have found that this conformational event proceeds in a biphasic manner. The characteristic time constants of these two phases were determined to be 68 +/- 20 ms and 624 +/- 37 ms, respectively.     

Benefits and limitations of porous substrates as biosensors for protein adsorption

Porous substrates have gained widespread interest for biosensor applications based on molecular recognition. Thus, there is a great demand to systematically investigate the parameters that limit the transport of molecules toward and within the porous matrix as a function of pore geometry. Finite element simulations (FES) and time-resolved optical waveguide spectroscopy (OWS) experiments were used to systematically study the transport of molecules and their binding on the inner surface of a porous material. OWS allowed us to measure the kinetics of protein adsorption within porous anodic aluminum oxide membranes composed of parallel-aligned, cylindrical pores with pore radii of 10-40 nm and pore depths of 0.8-9.6 μm. FES showed that protein adsorption on the inner surface of a porous matrix is almost exclusively governed by the flux into the pores. The pore-interior surface nearly acts as a perfect sink for the macromolecules. Neither diffusion within the pores nor adsorption on the surface are rate limiting steps, except for very low rate constants of adsorption. While adsorption on the pore walls is mainly governed by the stationary flux into the pores, desorption from the inner pore walls involves the rate constants of desorption and adsorption, essentially representing the protein-surface interaction potential. FES captured the essential features of the OWS experiments such as the initial linear slopes of the adsorption kinetics, which are inversely proportional to the pore depth and linearly proportional to protein concentration. We show that protein adsorption kinetics allows for an accurate determination of protein concentration, while desorption kinetics could be used to capture the interaction potential of the macromolecules with the pore walls.     

Porous SiO2 interferometric biosensor for quantitative determination of protein interactions: binding of protein A to immunoglobulins derived from different species

Determination of kinetic and thermodynamic protein binding constants using interferometry from a porous Si Fabry-Perot layer is presented. A protein A capture probe is adsorbed within the pores of an oxidized porous Si matrix, and binding of immunoglobulin G (IgG) antibodies derived from different species is investigated. The relative protein A/IgG binding affinity is human > rabbit > goat, in agreement with literature values. The equilibrium binding constant (Ka) for human IgG binding to surface-immobilized protein A is determined to be (3.0 +/- 0.5) x 107 M-1 using an equilibrium Langmuir model. Kinetic rate constants are calculated to be kd = (2.1 +/- 0.2) x 10-4 s-1 and ka = (1.2 +/- 0.4) x 104 M-1 s-1 using nonlinear least-squares analysis, yielding an equilibrium binding constant of Ka = (5.5 +/- 1.5) x 107 M-1. Both steady-state and time-dependent measurements yield equilibrium binding constants that are consistent with literature values. Kinetic rate constants determined through nonlinear least-squares analysis are also in agreement with protein A/IgG binding on a surface. Dosing with a high concentration of analyte leads to deviations from ideal binding behavior, interpreted in terms of restricted analyte diffusion within the porous SiO2 matrix. It is shown that the diffusion limitations can be minimized if the kinetic measurements are performed at low analyte concentrations or under conditions in which the protein A capture probe is not saturated with analyte. Potential limitations of the use of porous SiO2 interferometers for quantitative determination of protein binding constants are discussed.     

Protein redox potential measurements based on kinetic analysis with mediated continuous-flow column electrolytic spectroelectrochemical technique. Application to TTQ-containing methylamine dehydrogenase

Kinetic determination of protein redox potentials with a mediated continuous-flow column electrolytic spectroelectrochemical technique (CFCESET) is described. In this method, the redox state of the mediator is completely regulated by the continuous-flow column electrolysis, and the homogeneous redox reaction between the mediator and a protein sample in the column is monitored spectroscopically at the downstream of the column. The protein/mediator reaction is in the pseudo-first-order kinetics, and then the rate equation is analytically solved. The kinetic analysis provides the protein redox potential as well as the homogeneous rate constant. In the kinetic measurements, equilibration of the system within the column is not required, which allows the use of increased kinds of mediators. This method was successfully applied to quinoprotein methylamine dehydrogenase containing tryptophan tryptophylquinone (TTQ) as a prosthetic group. The kinetic aspect is also valuable for the thermodynamic analysis with the mediated CFCESET. The half-life time of the kinetics can be utilized to optimize the system for the attainment of the equilibrated state within the column and can provide the assurance that the system is in equilibrium.     

Linear and non-linear regression analysis for the sorption kinetics of methylene blue onto activated carbon

Batch kinetic experiments were carried out for the sorption of methylene blue onto activated carbon. The experimental kinetics were fitted to the pseudo first-order and pseudo second-order kinetics by linear and a non-linear method. The five different types of Ho pseudo second-order expression have been discussed. A comparison of linear least-squares method and a trial and error non-linear method of estimating the pseudo second-order rate kinetic parameters were examined. The sorption process was found to follow a both pseudo first-order kinetic and pseudo second-order kinetic model. Present investigation showed that it is inappropriate to use a type 1 and type pseudo second-order expressions as proposed by Ho and Blanachard et al. respectively for predicting the kinetic rate constants and the initial sorption rate for the studied system. Three correct possible alternate linear expressions (type 2 to type 4) to better predict the initial sorption rate and kinetic rate constants for the studied system (methylene blue/activated carbon) was proposed. Linear method was found to check only the hypothesis instead of verifying the kinetic model. Non-linear regression method was found to be the more appropriate method to determine the rate kinetic parameters.     

Automated bead alignment apparatus using a single bead capturing technique for fabrication of a miniaturized bead-based DNA probe array

We have developed an automated bead alignment apparatus for fabricating a bead-based DNA probe array inside a capillary. The apparatus uses 16 micro vacuum tweezers to extract single beads from among a large amount of beads in bead stock wells. It then manipulates single beads into the probe array capillaries. Single 100-microm-diameter beads were successfully extracted from the water-contained bead-stock well by the vacuum tweezers, which have inner and outer diameters of 50 and 150 microm. An interesting aspect is that unexpected extra beads adsorbed on the outer wall of the vacuum tweezers can be removed using the surface tension force between the water and the atmosphere. In testing the total performance of this apparatus, the DNA probe arrays with 10 sets of probe-conjugated beads and 2 plain beads were produced in the intended order in the capillaries. The time needed to align the 12 beads was 10 min, and the 16 bead arrays were fabricated simultaneously. After hybridization experiments using these fabricated DNA probe arrays, fluorescence from each bead was clearly observed.