Kinetic behaviour of free lipase and mica-based immobilized lipase catalyzing the synthesis of sugar esters

The utilization of natural mica as a biocatalyst support in kinetic investigations is first described in this study. The formation of lactose caprate from lactose sugar and capric acid, using free lipase (free-CRL) and lipase immobilized on nanoporous mica (NER-CRL) as a biocatalyst, was evaluated through a kinetic study. The apparent kinetic parameters, K(m) and V(max), were determined by means of the Michaelis-Menten kinetic model. The Ping-Pong Bi-Bi mechanism with single substrate inhibition was adopted as it best explains the experimental findings. The kinetic results show lower K(m) values with NER-CRL than with free-CRL, indicating the higher affinity of NER-CRL towards both substrates at the maximum reaction velocity (V(max,app)>V(max)). The kinetic parameters deduced from this model were used to simulate reaction rate data which were in close agreement with the experimental values.     

Influence of reactor geometry on mixing characteristics in a down flow jet loop bioreactor: newtonian and non-newtonian fluids

Mixing characteristics in the downcomer and the riser of a continuous down-flow jet loop bioreactor was studied with Newtonian and non-Newtonian fluids. The mixing parameters were determined through the curve fitting of the experimental impulse response data with the solution of one dimensional axial dispersion model. It was found that circulation number and axial dispersion coefficient increased with an increase in liquid flow rate and draft tube to column diameter ratio and the axial dispersion coefficient was comparatively higher in the riser. The circulation number increased with decrease in nozzle diameter. The model predicted the experimental data well within 8% deviation for both the systems (water and CMC). Correlations were obtained to predict axial dispersion coefficients in the riser and downcomer of the reactor.     

Expanded bed adsorption of protein with DEAE Spherodex M

In this article, the bed expansion behavior and the hydrodynamic and protein adsorption properties of the DEAE Spherodex M in expanded bed with mobile phases of different viscosities have been studied. The axial liquid-phase dispersion coefficient is found to be on the order of 10(-6) m(2)/s, falling into the common range from 1.0 x 10(-6) to 1.0 x 10(-5) m(2)/s observed previously in expanded bed operation. Because of the small size of the adsorbent, the high pore diffusivity of protein and the favorable column efficiency (low dispersion coefficient), the dynamic binding capacity (DBC) of bovine serum albumin (BSA) at 5% breakthrough in the expanded bed reaches over 80% that of the equilibrium adsorption density (EAD). Moreover, a theoretical model with unadjustable model parameters is used for the prediction of the breakthrough curves. Computer simulations show that the model agrees well with the experimental results at breakthrough less than about 50%. It indicates that the model is promising in the prediction of protein breakthrough behavior because breakthrough profiles at 5-50% breakthrough points are more important in practical applications.     

Breakthrough performance of plasmid DNA on ion-exchange membrane columns

Breakthrough performance of plasmid DNA adsorption on ion-exchange membrane columns was theoretically and experimentally investigated using batch and fixed-bed systems. System dispersion curves showed the absence of flow non-idealities in the experimental arrangement. Breakthrough curves (BTC) were significantly affected by inlet flow rate and solute concentration. In the theoretical analysis, a model was integrated by the serial coupling of the membrane transport model and the system dispersion model. A transport model that considers finite kinetic rate and column dispersed flow was used in the study. A simplex optimization routine, coupled to the solution of the partial differential model equations, was employed to estimate the maximum adsorption capacity constant, the equilibrium desorption constant, and the forward interaction rate constant, which are the parameters of the membrane transport model. The analysis shows that as inlet concentration or flow rate increases, the deviation of the model from the experimental behavior decreases. The BTCs displacement as inlet concentration increases was explained in terms of a greater degree of column saturation reached and more efficient operation accomplished. The degree of column saturation was not influenced by inlet flow rate. It was necessary to consider in the column model the slight variation in the BTC produced by the axial dispersion, in order to accomplish the experimental curve dispersion. Consequently, the design criteria that for Pe > 40 the column axial dispersion can be neglected should be taken with precaution.     

Empirical modeling of batch fermentation kinetics for poly(glutamic acid) production and other microbial biopolymers

An empirical kinetic model is proposed for the batch production of poly(glutamic acid) from Bacillus subtilis IFO 3335. In addition, the proposed model was used to fit the kinetic data of poly(glutamic acid) production from other bacterial strains using different media, as well as kinetic data from different strains for the production of the exocellular biopolymers dextran, hyaluronic acid, xanthan, alginate, and the endocellular biopolymer polyhydroxybutyrate. The empirical model treats the biopolymer as a component of the biomass and fits the experimental biomass data using a sigmoidal relationship that includes the maximum specific growth rate, mu(max), and the substrate saturation parameter, K(S). An empirical parameter, the relative coefficient (r), quantifies, in relative terms, the degree of nongrowth-associated biopolymer formation.     

A combined method for determining inhibition type, kinetic parameters, and inhibition coefficients for aerobic cometabolism of 1,1,1-trichloroethane by a butane-grown mixed culture.

A combined method for determining inhibition type, kinetic parameters, and inhibition coefficients is developed and presented. The method was validated by applying it to data obtained from batch kinetics of the aerobic cometabolism of 1,1,1-trichloroethane (1,1,1-TCA) by a butane-grown mixed culture. The maximum degradation rates (k(max)) and half-saturation coefficients (K(s)) were independently determined in single compound tests, and compared with those obtained from inhibition tests. The inhibition type was determined using direct linear plots at various substrate and inhibitor concentrations. Kinetic parameters (k(max) and K(s)) and inhibition coefficients (K(ic) and K(iu)) were determined by nonlinear least squares regression (NLSR) fits of the inhibition model determined from the direct linear plots. Initial guesses of the kinetic parameters for NLSR were determined from linearized inhibition equations that were derived from the correlations between apparent maximum degradation rates (k(app)(max)) and/or the apparent half-saturation coefficient (K(app)(s)) and the k(max), K(s), and inhibitor concentration (I(L)) for each inhibition equation. Two different inhibition types were indicated from the direct linear plots: competitive inhibition of 1,1,1-TCA on butane degradation, and mixed inhibition of 1,1,1-TCA transformation by butane. Good agreement was achieved between independently measured k(max) and K(s) values and those obtained from both NLSR and the linearized inhibition equations. The initial guesses of all the kinetic parameters determined from linear plots were in the range of the values estimated from NLSR analysis. Overall the results show that use of the direct linear plot method to identify the inhibition type, coupled with initial guesses from linearized plots for NLSR analysis, results in an accurate method for determining inhibition types and coefficients. Detailed studies with pure cultures and purified enzymes are needed to further demonstrate the utility of this method.     

Substrate inhibition during bio-filtration of TCE using diazotrophic bacterial community

The kinetics of biodegradation of TCE in the biofilter packed with wood charcoal and inoculated with diazotrophic bacterial community had been investigated. Use of Michaelis-Menten type model showed that substrate inhibition was present in the system. The kinetic model proposed by Edwards (1970) was used to calculate kinetic parameters-maximum elimination capacity (EC(max)), substrate constant (K(s)), and inhibition constant (K(I)). The model fitted well with the experimental data and the EC(max) was found to be in the range of 10.8-6.1 g/m(3) h. The K(s) values depended upon substrate concentration and ranged from 0.024 to 0.043 g/m(3) indicating the high affinity of diazotrophs for TCE. The K(I) values were low and nearly constant (0.011-0.015 g/m(3)) indicating a moderate substrate inhibition.     

Quantitative comparison of lactose and glucose utilization in Bifidobacterium longum cultures

In batch cultures, Bifidobacterium longum SH2 has a higher final cell concentration and greater substrate consumption when grown on lactose versus glucose. Continuous cultures were used to compare lactose and glucose utilization by B. longum quantitatively. In the continuous culture, the estimated maintenance coefficients (m) were similar when on lactose and glucose; the maximum cell yield coefficient (Y(X/S)(max)) was higher on lactose; and the specific consumption rate of lactose (q(S)) was lower than that of glucose. Assuming that cell growth followed the Monod model, the maximum specific growth rates (mu(max)) and saturation constants (K(S)) in lactose and glucose media were determined using the Hanes-Woolf plots. The respective values were 0.40 h(-)(1) and 78 mg/L for lactose and 0.46 h(-)(1) and 697 mg/L for glucose. The kinetic parameters of the continuous cultures showed that B. longum preferred lactose to glucose, although the specific consumption rate of glucose was higher than that of lactose.     

Modeling of diffusion and concurrent metabolism in cutaneous tissue

Clearance by cutaneous metabolism can shield the body from penetration of environmental and therapeutic xenobiotics. Here we report on a physical model to relate Fickian diffusion and concurrent Michaelis-Menten metabolism of drugs in the viable epidermis of human skin. For this purpose, we numerically generated substrate concentration profiles within the metabolizing tissue and the resulting donor-to-receiver substrate fluxes through the tissue for various mass transport and metabolism parameters. To validate the model, permeation and concurrent metabolism of a peptidomimetic compound, L -Ala-4-methoxy-2-naphthylamide (Ala-MNA), across both stripped human skin and HaCaT cell culture sheets were compared to numerical simulations. Parameter estimates for those calculations were extracted from independent experiments. Experimental data and numerical predictions were in excellent agreement. Also, numerical fits and independently validated parameters correlated closely, indicating the principal validity of the physical model. Numerical simulations and theoretical derivations illustrate the kinetic impact of the factors involved, i.e. the diffusion coefficient D, substrate donor concentration C(S,D), substrate partition coefficient P, tissue thickness L and maximum metabolic rate V(max), on drug permeation, with L having the strongest effect. In the steady state, the coefficient 2 alpha, i.e. the dimensionless ratio of the residence time term (L(2)/D) of a substrate in the tissue to the metabolic half-life term (C(S,D)P/2 V(max)), allows to estimate concentration gradients within the tissue and the extent of metabolism. High 2 alpha values represent practically complete metabolic cleavage upon penetration. Epidermis ( approximately 40 microm thick) of stripped human skin and HaCaT sheets ( approximately 10 microm) had 2 alpha values of 43 and 2.7, respectively, indicating that intact Ala-MNA could only permeate HaCaT sheets, but not skin. Independent permeation experiments confirmed this outcome. This physical model may be applicable to other metabolizing tissues as well.     

Kinetically controlled enzymatic synthesis of dipeptide precursor of L-alanyl-L-glutamine

An important nutritional dipeptide precursor, benzoyloxycarbonyl protected L-alanyl-L-glutamine (Z-Ala-Gln), was successfully prepared through a kinetically controlled enzymatic peptide synthesis method. A commercially available and low-cost protease (papain) was used as biocatalyst with Z-Ala-OMe and Gln as acyl donor and nucleophile, respectively. The dipeptide yield was 35.5% under the optimized reaction conditions: 35°C, pH 9.5, and the ratio of acyl donor/nucleophile is 1:10. Based on the reaction mechanism and experimental data, the kinetic model was established, which was in accordance with the Michaelis-Menten equation, and the apparent Michaelis constant K(m)(app) and the apparent maximum reaction rate r(max)(app) were calculated as 1.71 mol/L and 6.09 mmol/(L Min), respectively.     

Modeling nitrate-nitrogen removal process in first-flush reactor for stormwater treatment

Stormwater runoff is one of the most common non-point sources of water pollution to rivers, lakes, estuaries, and coastal beaches. While most pollutants and nutrients, including nitrate-nitrogen, in stormwater are discharged into receiving waters during the first-flush period, no existing best management practices (BMPs) are specifically designed to capture and treat the first-flush portion of urban stormwater runoff. This paper presents a novel BMP device for highway and urban stormwater treatment with emphasis on numerical modeling of the new BMP, called first-flush reactor (FFR). A new model, called VART-DN model, for simulation of denitrification process in the designed first-flush reactor was developed using the variable residence time (VART) model. The VART-DN model is capable of simulating various processes and mechanisms responsible for denitrification in the FFR. Based on sensitivity analysis results of model parameters, the denitrification process is sensitive to the temperature correction factor (b), maximum nitrate-nitrogen decay rate (K (max)), actual varying residence time (T (v)), the constant decay rate of denitrifiying bacteria (v (dec)), temperature (T), biomass inhibition constant (K (b)), maximum growth rate of denitrifiying bacteria (v (max)), denitrifying bacteria concentration (X), longitudinal dispersion coefficient (K (s)), and half-saturation constant of dissolved carbon for biomass (K (Car-X)); a 10% increase in the model parameter values causes a change in model root mean square error (RMSE) of -28.02, -16.16, -12.35, 11.44, -9.68, 10.61, -16.30, -9.27, 6.58 and 3.89%, respectively. The VART-DN model was tested using the data from laboratory experiments conducted using highway stormwater and secondary wastewater. Model results for the denitrification process of highway stormwater showed a good agreement with observed data and the simulation error was less than 9.0%. The RMSE and the coefficient of determination for simulating denitrification process of wastewater were 0.5167 and 0.6912, respectively, demonstrating the efficacy of the VART-DN model.     

Kinetic Behaviour of Free Lipase and Mica-Based Immobilized Lipase Catalyzing the Synthesis of Sugar Esters

The utilization of natural mica as a biocatalyst support in kinetic investigations is first described in this study. The formation of lactose caprate from lactose sugar and capric acid, using free lipase (free-CRL) and lipase immobilized on nanoporous mica (NER-CRL) as a biocatalyst, was evaluated through a kinetic study. The apparent kinetic parameters, K _m and V _max, were determined by means of the Michaelis-Menten kinetic model. The Ping-Pong Bi-Bi mechanism with single substrate inhibition was adopted as it best explains the experimental findings. The kinetic results show lower K _m values with NER-CRL than with free-CRL, indicating the higher affinity of NER-CRL towards both substrates at the maximum reaction velocity (V _max,app>V _max). The kinetic parameters deduced from this model were used to simulate reaction rate data which were in close agreement with the experimental values.     

Phenomenological model of fungal biofilters for the abatement of hydrophobic VOCs

This work describes the growth of filamentous fungi in biofilters for the degradation of hydrophobic VOCs. The study system was n-hexane and Fusarium solani B1. The system is mathematically described and the main physical, kinetic data and morphological parameters were obtained by independent experiments and validated with data from laboratory experiments. The model describes the increase in the transport area by the growth of the filamentous cylindrical mycelia and its relation with n-hexane elimination in quasi-stationary state in a biofilter. The model describing fungal growth includes Monod-Haldane kinetic and hyphal elongation and ramification. A specific surface area of transport (SSAT) of 1.91 x 10(5) m(2) m(-3) and a maximum elimination capacity (EC) of 248 g m(-3) h(-1) were obtained by the mathematical model simulation, with a 10% of error with respect to the experimental EC.     

Kinetic study of the concentration dependence of the mass transfer rate coefficient in anion-exchange chromatography of bovine serum albumin.

The experimental results of a previous study of the mass transfer kinetics of bovine serum albumin (BSA) in ion-exchange chromatography under nonlinear conditions are reevaluated. The analysis of the concentration dependence of the lumped mass-transfer rate coefficient (k(m,L)) provides information on the kinetics of axial dispersion, fluid-to-particle mass transfer, intraparticle mass transfer, and adsorption/desorption. The new analysis shows that the contribution of intraparticle mass transfer is the dominant one. Similar to k(m,L), the surface diffusivity (D(s)) of BSA increases with increasing concentration. The linear concentration dependence of k(m,L) seems to originate in a similar dependence of D(s). The use of an heterogeneous-surface model for the anion-exchange resin provides an explanation of the positive concentration dependence of D(s). This work illustrates how frontal analysis data can be used for a detailed investigation of the kinetics of mass transfer between the phases of a chromatographic column, in addition to its conventional use in the determination of the thermodynamic characteristics of the phase equilibrium.     

Variation in the divalent cation requirements of influenza A virus N1 neuraminidases

Enzymatic kinetic parameters of influenza A virus N1 neuraminidases (NA) chromatographically purified from several vaccine candidate strains were tested. With ionic strength held constant, Ca2+ or Mg2+ increased the initial rate of enzymatic activity. The 1934 and 1943 strains had statistically significant highest initial velocities, V(max)/K(m) and V(max). There were no significant differences among the influenza virus strains from 1947 to 1991. Measured K(m) for the 1943 strain (6.2 x 10(-5) M) was significantly higher than other strains (3.1-4.7 x 10(-5) M). V(max)/K(m) varied from 0.78 M(-1) s(-1) to 0.91 M(-1) s(-1) and V(max) varied from 3.0 s(-1) to 5.5 s(-1) before the addition of a divalent cation and increased approximately 2-fold for each of these kinetic parameters for each strain after the addition of exogenous Ca2+ or Mg2+. Dialysis reduced the initial velocity and immunogenicity of each strain with significant differences found among strains. Enzymatic activity and immunogenicity were partially restored by the addition of exogenous Ca2+. Nucleic acid sequence analysis could not predict these differences. Selection of vaccine strains must include analysis of antigenic changes, but also enzymatic studies and determination of the requirement of divalent cations to maintain immunogenicity and activity during production.     

Kinetics of infective juvenile production of the entomopathogenic nematode Steinernema carpocapsae in submerged monoxenic culture

The effects of culture medium formulations on the kinetics of infective juvenile (IJ) production of the entomopathogenic nematode Steinernema carpocapsae in submerged monoxenic culture, were studied at the cylindrical-bottle scale using six culture media containing agave juice from Agave spp. among other ingredients. The IJ production kinetics was well modelled through a re-parameterised 3-parameter Gompertz model with kinetic parameters: IJ-lag phase lambda (IJ) (day), maximum IJ-stage production rate m (max )(day(-1)), and IJ-multiplication factor (C (IJ)/C (IJ,0))(max)(-). The variation of lambda (IJ) was not very important within fermentations (10.3-16.2 days); nonetheless, important effects were observed on m (max) (32.8-241.2 days(-1)) and (C (IJ)/C (IJ,0))(max) (66(-) to 611.4(-)). Particularly, maximum values of m (max) and (C (IJ)/C (IJ,0))(max) were obtained in medium A4 (0.276 l l(-1) agave juice, 17 g l(-1) yeast extract, 12 g l(-1) dried egg yolk, 0.025 l l(-1) corn oil). Also, the maximum IJ concentration (249,444 per ml) was achieved in A4-fermentations.     

Transient kinetics of hybridoma growth and monoclonal antibody production in serum-limited cultures

A quantitative study of the influence of initial serum concentration on hybridoma growth rate, maximum viable and total cell yield, and specific antibody production rate is presented. The specific growth rate varied in a Monod fashion with initial serum levels (2-10% FCS), giving K(m) = 1.6 v/v% and mu(max) = 0.92 d(-1). The maximum cell yields (total and viable) were linear with initial serum level, indicating stoichiometric as well as kinetic limitation by serum component(s). The specific antibody production rate for each individual run fitted well to a non-growth-associated model. However, the non-growth-associated parameter varied monotonically with initial serum concentration, suggesting the catalytic role of serum component(s) in antibody production. Also, glutamine was utilized inefficiently, with at least a third of it secreted back into the culture supernate in the form of glutamate. While very simple model equations describe the specific growth and product formation rate for an individual batch run, the larger picture indicates need for a more detailed unstructured or structured model.     

Aerobic biological treatment of black table olive washing wastewaters: effect of an ozonation stage

The present work is a study of oxidative degradation of the organic matter present in the washing waters from the black table olive industry. Pollutant organic matter reduction was studied by an aerobic biological process and by the combination of two successive steps: ozonation pretreatment followed by aerobic biological degradation. In the single aerobic biological process, the evolution of biomass and organic matter contents was followed during each experiment. Contaminant removal was followed by means of global parameters directly related to the concentration of organic compounds in those effluents: chemical oxygen demand (COD) and total phenolic content (TP). A kinetic study was performed using the Contois model, which applied to the experimental data, provides the specific kinetic parameters of this model: 4.81×10⁻² h⁻¹ for the kinetic substrate removal rate constant, 0.279 g VSS g COD⁻¹ for the cellular yield coefficient and 1.92×10⁻² h⁻¹ for the kinetic constant for endogenous metabolism. In the combined process, an ozonation pretreatment is conducted with experiments where an important reduction in the phenolic compounds is achieved. The kinetic parameters of the following aerobic degradation stage are also evaluated, being 5.42×10⁻² h⁻¹ for the kinetic substrate removal rate constant, 0.280 g VSS g COD⁻¹ for the cellular yield coefficient and 9.1×10⁻³ h⁻¹ for the kinetic constant for the endogenous metabolism.     

Temperature-dependent growth kinetics of Escherichia coli ML 30 in glucose-limited continuous culture.

Detailed comparison of growth kinetics at temperatures below and above the optimal temperature was carried out with Escherichia coli ML 30 (DSM 1329) in continuous culture. The culture was grown with glucose as the sole limiting source of carbon and energy (100 mg liter(-1) in feed medium), and the resulting steady-state concentrations of glucose were measured as a function of the dilution rate at 17.4, 28.4, 37, and 40 degrees C. The experimental data could not be described by the conventional Monod equation over the entire temperature range, but an extended form of the Monod model [mu = mu(max) x (s - s(min))/(Ks + s - s(min))], which predicts a finite substrate concentration at 0 growth rate (s(min)), provided a good fit. The two parameters mu(max) and s(min) were temperature dependent, whereas, surprisingly, fitting the model to the experimental data yielded virtually identical Ks values (approximately 33 microg liter(-1)) at all temperatures. A model that describes steady-state glucose concentrations as a function of temperature at constant growth rates is presented. In similar experiments with mixtures of glucose and galactose (1:1 mixture), the two sugars were utilized simultaneously at all temperatures examined, and their steady-state concentrations were reduced compared with to growth with either glucose or galactose alone. The results of laboratory-scale kinetic experiments are discussed with respect to the concentrations observed in natural environments.