Estimation of fermentation biomass concentration by measuring oxygen uptake off-line with an oxygen electrode

Summary An indirect method for biomass determination was developed for aerobic cultivation in media containing solid substrates. This method involved off-line estimation of oxygen uptake with an oxygen electrode using a calibration factor and was applied to penicillin fermentation with Penicillium chrysogenum. The volumetric oxygen uptake rate was independent of the amount of sample sealed in the measuring cell. Storage of the sample for a period up to 2.5 h also had no influence on the rate of O₂ uptake. The specific O₂ uptake rate at 70 to 75 h fermentation time was used as the calibration factor to calculate the respiration-active part of the biomass. Specific penicillin V formation rates related to net dry weight, respiration-active biomass (X _A )and RNA content were compared. Only the penicillin V production rate related to X _A showed a similar behaviour as the penicillin V production rate related to RNA content. For this reason, it is assumed that X _A ,like RNA content, is more closely related to the metabolic state of the cells than the net dry weight. This method, therefore, provides a more suitable reference parameter for fermentation control than the generally used net dry weight.Offprint requests to: D. Siegmund     

Phenological mapping in a topographically complex landscape by combining field survey with an irradiation model

Abstract. A phenological map has been prepared for the Podyjí/Thayatal National Park, located on the border between the Czech Republic and Austria. The area is characterized by V-shaped river valleys deeply incised into a gently undulating landscape. Five stages of phenological development were defined, based upon phenological spectra of plant communities in late April; 96 sites, more or less regularly spaced over the area, were assigned to one of these stages using field observations of these spectra. Potential direct solar irradiation was calculated from a digital elevation model for a 25 m x 25 m grid extending over the study area. A method was developed for interpolation of phenological observations by weighted regression of phenology on the irradiation model. This interpolation enables the prediction of the local stage of phenological development across the study area without levelling out phenological patterns dependent upon small-scale topographic variation. Spring phenological events appear to be more advanced in the valleys (except for steep north-facing slopes) than in the adjacent landscape. Possible climatic processes underlying this pattern are discussed with emphasis on temperature inversions in the river valleys. Cold-air-drainage inversions reported from the valleys are probably too infrequent and of too short duration to delay plant phenology. The combined effects of advanced phenology and the increased risk of spring frost injury due to these inversions in the valleys may be an explanation for local distributional patterns of flora and vegetation.     

Unhindered copper uptake by glutaraldehyde-polyethyleneimine coatings in an artificial seawater model system with adsorbed swollen polysaccharides and competing ligand EDTA

Shortly after a surface is submerged in the sea, a conditioning film is generally formed by adsorption of organic molecules, such as polysaccharides. This could affect transport of molecules and ions between the seawater and the surface. An artificial seawater model system was developed to understand how adsorbed polysaccharides impact copper binding by glutaraldehyde-crosslinked polyethyleneimine coatings. Coating performance was also determined when competed against copper-chelating EDTA. Polysaccharide adsorption and copper binding and distribution were investigated using advanced analytical techniques, including depth-resolved time-of-flight secondary ion mass spectroscopy, grazing incidence X-ray absorption near-edge spectroscopy, quartz crystal microbalance with dissipation monitoring and X-ray photoelectron spectroscopy. In artificial seawater, the polysaccharides adsorbed in a swollen state that copper readily penetrated and the glutaraldehyde-polyethyleneimine coatings outcompeted EDTA for copper binding. Furthermore, the depth distribution of copper species was determined with nanometre precision. The results are highly relevant for copper-binding and copper-releasing materials in seawater.     

Prediction of microbial growth rate versus biomass yield by a metabolic network with kinetic parameters

Identifying the factors that determine microbial growth rate under various environmental and genetic conditions is a major challenge of systems biology. While current genome-scale metabolic modeling approaches enable us to successfully predict a variety of metabolic phenotypes, including maximal biomass yield, the prediction of actual growth rate is a long standing goal. This gap stems from strictly relying on data regarding reaction stoichiometry and directionality, without accounting for enzyme kinetic considerations. Here we present a novel metabolic network-based approach, MetabOlic Modeling with ENzyme kineTics (MOMENT), which predicts metabolic flux rate and growth rate by utilizing prior data on enzyme turnover rates and enzyme molecular weights, without requiring measurements of nutrient uptake rates. The method is based on an identified design principle of metabolism in which enzymes catalyzing high flux reactions across different media tend to be more efficient in terms of having higher turnover numbers. Extending upon previous attempts to utilize kinetic data in genome-scale metabolic modeling, our approach takes into account the requirement for specific enzyme concentrations for catalyzing predicted metabolic flux rates, considering isozymes, protein complexes, and multi-functional enzymes. MOMENT is shown to significantly improve the prediction accuracy of various metabolic phenotypes in E. coli, including intracellular flux rates and changes in gene expression levels under different growth rates. Most importantly, MOMENT is shown to predict growth rates of E. coli under a diverse set of media that are correlated with experimental measurements, markedly improving upon existing state-of-the art stoichiometric modeling approaches. These results support the view that a physiological bound on cellular enzyme concentrations is a key factor that determines microbial growth rate.     

Dual substrate model for novel approach towards a kinetic study of acetylcholinesterase inhibition by diazinon

Limited reports as compared to other insecticides appear in the literature for acetylcholinesterase (AChE) inhibition by diazinon. In the current study, new kinetic parameters of AChE inhibition by diazinon have been investigated. The assay was done with bovine retinal AChE using two different substrate (ASCh) concentrations in the absence and presence of diazinon (0.08-1.28 mM). The optical density was monitored up to 25 min (reaction time) for the assay. New kinetic parameters k'(oms), K'(sms), k(oms), K(sms), K'(asms) and K(asms) ) were calculated from these experimental data.     

Approximation of a physiologically structured population model with seasonal reproduction by a stage-structured biomass model

Seasonal reproduction causes, due to the periodic inflow of young small individuals in the population, seasonal fluctuations in population size distributions. Seasonal reproduction furthermore implies that the energetic body condition of reproducing individuals varies over time. Through these mechanisms, seasonal reproduction likely affects population and community dynamics. While seasonal reproduction is often incorporated in population models using discrete time equations, these are not suitable for size-structured populations in which individuals grow continuously between reproductive events. Size-structured population models that consider seasonal reproduction, an explicit growing season and individual-level energetic processes exist in the form of physiologically structured population models. However, modeling large species ensembles with these models is virtually impossible. In this study, we therefore develop a simpler model framework by approximating a cohort-based size-structured population model with seasonal reproduction to a stage-structured biomass model of four ODEs. The model translates individual-level assumptions about food ingestion, bioenergetics, growth, investment in reproduction, storage of reproductive energy, and seasonal reproduction in stage-based processes at the population level. Numerical analysis of the two models shows similar values for the average biomass of juveniles, adults, and resource unless large-amplitude cycles with a single cohort dominating the population occur. The model framework can be extended by adding species or multiple juvenile and/or adult stages. This opens up possibilities to investigate population dynamics of interacting species while incorporating ontogenetic development and complex life histories in combination with seasonal reproduction.     

Determining RuBisCO activation kinetics and other rate and equilibrium constants by simultaneous multiple non-linear regression of a kinetic model

The forward and reverse rate constants involved in carbamylation, activation, carboxylation, and inhibition of D-ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) have been estimated by a new technique of simultaneous non-linear regression of a differential equation kinetic model to multiple experimental data. Parameters predicted by the model fitted to data from purified spinach enzyme in vitro included binding affinity constants for non-substrate CO2 and Mg2+ of 200+/-80 microM and 700+/-200 microM, respectively, as well as a turnover number (k(cat)) of 3.3+/-0.5 s(-1), a Michaelis half-saturation constant for carboxylation (K(M,C)) of 10+/-4 microM and a Michaelis constant for RuBP binding (K(M,RuBP)) of 1.5+/-0.5 microM. These and other constants agree well with previously measured values where they exist. The model is then used to show that slow inactivation of RuBisCO (fallover) in oxygen-free conditions at low concentrations of CO2 and Mg2+ is due to decarbamylation and binding of RuBP to uncarbamylated enzyme. In spite of RuBP binding more tightly to uncarbamylated enzyme than to the activated form, RuBisCO is activated at high concentrations of CO2 and Mg2+. This apparent paradox is resolved by considering activation kinetics and the fact that while RuBP binds tightly but slowly to uncarbamylated enzyme, it binds fast and loosely to activated enzyme. This modelling technique is presented as a new method for determining multiple kinetic data simultaneously from a limited experimental data set. The method can be used to compare the properties of RuBisCO from different species quickly and easily.     

Description of the anomalous diffusion of fast electrons by a kinetic equation with a fractional spatial derivative

It is proposed to use fractional spatial derivatives to describe the effect of anomalous diffusion of fast electrons in a stochastic magnetic field on the shape of the distribution function. A self-similar kinetic equation is considered. The use of self-similar variables makes it possible to determine the velocity range in which the distribution function is distorted to the greatest extent. Calculations show that the quantities associated with the stochasticity of the magnetic field lines can be estimated from the experimentally measured characteristic energies of suprathermal electrons in the energy range in which the behavior of the distribution function changes substantially.     

Biosorption of a textile dye (Acid Blue 40) by cone biomass of Thuja orientalis: estimation of equilibrium, thermodynamic and kinetic parameters

Biosorption of Acid Blue 40 (AB40) onto cone biomass of Thuja orientalis was studied with variation in the parameters of pH, contact time, biosorbent and dye concentration and temperature to estimate the equilibrium, thermodynamic and kinetic parameters. The AB40 biosorption was fast and the equilibrium was attained within 50 min. Equilibrium data fitted well to the Langmuir isotherm model in the studied concentration range of AB40 and at various temperatures. Maximum biosorption capacity (q(max)) for AB40 was 2.05 x 10(-4)mol g(-1) or 97.06 mg g(-1) at 20 degrees C. The changes of Gibbs free energy, enthalpy and entropy of biosorption were also evaluated for the biosorption of AB40 onto T. orientalis. The results indicate that the biosorption was spontaneous and exothermic. Kinetics of biosorption of AB40 was analyzed and rate constants were also derived and the results show that the pseudo-second-order kinetic model agrees very well with the experimental data.     

Characterization of articular cartilage by combining microscopic analysis with a fibril-reinforced finite-element model

Load-bearing characteristics of articular cartilage are impaired during tissue degeneration. Quantitative microscopy enables in vitro investigation of cartilage structure but determination of tissue functional properties necessitates experimental mechanical testing. The fibril-reinforced poroviscoelastic (FRPVE) model has been used successfully for estimation of cartilage mechanical properties. The model includes realistic collagen network architecture, as shown by microscopic imaging techniques. The aim of the present study was to investigate the relationships between the cartilage proteoglycan (PG) and collagen content as assessed by quantitative microscopic findings, and model-based mechanical parameters of the tissue. Site-specific variation of the collagen network moduli, PG matrix modulus and permeability was analyzed. Cylindrical cartilage samples (n=22) were harvested from various sites of the bovine knee and shoulder joints. Collagen orientation, as quantitated by polarized light microscopy, was incorporated into the finite-element model. Stepwise stress-relaxation experiments in unconfined compression were conducted for the samples, and sample-specific models were fitted to the experimental data in order to determine values of the model parameters. For comparison, Fourier transform infrared imaging and digital densitometry were used for the determination of collagen and PG content in the same samples, respectively. The initial and strain-dependent fibril network moduli as well as the initial permeability correlated significantly with the tissue collagen content. The equilibrium Young's modulus of the nonfibrillar matrix and the strain dependency of permeability were significantly associated with the tissue PG content. The present study demonstrates that modern quantitative microscopic methods in combination with the FRPVE model are feasible methods to characterize the structure-function relationships of articular cartilage.     

A model to calculate the uptake by a developing root system or root hair system of solutes with concentration variable diffusion coefficients

Summary Two modifications to a method for calculating the uptake of solutes by a developing root system are presented. The method is investigated in the cases which would give rise to the greatest error, and this was found to be less than 10 per cent. The modifications may be of particular interest in studies of the absorption of phosphate and potassium from soils. Paper No. IV: Uptake of solute by multiple root systems in soil. Paper No. IV: Uptake of solute by multiple root systems in soil.     

The uptake of radiocolloids by macrophages in vitro; a kinetic analysis with radioactive colloidal gold

Macrophages isolated from the rabbit peritoneal cavity extract radioactive colloidal gold from solutions in vitro. This reaction (ultraphagocytosis) involves two phases: the reversible adsorption of gold on the cell surface and the subsequent irreversible removal of surface-bound colloid into the cell. The latter process (called ingestion) appears to proceed at a rate which is proportional at any moment to the amount of gold attached to the cell surface; the latter in turn can be related to the concentration in extracellular fluid by a simple adsorption isotherm. In terms of rate, therefore, ingestion is related to the extracellular gold concentration in the same way that many enzyme reactions are related to the substrate concentration. Although enzyme kinetics are useful in describing rates of ultraphagocytosis, there is no evidence that enzymes participate in either adsorption or ingestion or that metabolic energy is required of the macrophage. Exudative leucocytes of the heterophilic series show little or no interaction with these finely dispersed gold sols (mean particle diameter 6 to 9 millimicrons). 37°C. three parameters are sufficient to characterize the reaction between gold and a suspension of macrophages, namely an affinity constant (1/K_s), an adsorption maximum (L), and a rate constant of ingestion (k₃). Although numerical values differed markedly among cells of different exudates, all three parameters were estimated in three instances. In these suspensions between 2 and 20 per cent of the surface-bound gold was ingested each minute (37°C., pH 7.4). Under conditions of surface saturation, it was estimated that tens of thousands of gold particles were attached to the surface of an average macrophage; this amount of colloid, however, occupied less than 1 per cent of the geometric area of the cell surface. Although surface saturation imposed an upper limit on the rate of ingestion, no practical limit was noted in the capacity of macrophages to continue the reaction. Optical measurements imply that within the cell agglutination of colloidal gold began promptly after its ingestion. These data are compared with published kinetic studies on the phagocytosis of microscopic particulates and on the parasitism of bacteria by virus.     

Hydrolysis of urea by gelatin-immobilized urease: separation of kinetic and diffusion phenomena in a model immobilized-enzyme reactor system.

Experiments and appropriate mathematical models are presented in an attempt to elucidate and separate the effects of mass transfer and immobilization on the apparent kinetics of hydrolysis of urea by urease immobilized within a crosslinked gelatin film. Diffusion of urea through the gelatin matrix appears to exert the major influence on the observed kinetics. Diffusion coefficients are measured, and a model for the "effectiveness factor" is presented, accounting for this aspect of mass transfer control. A secondary, but significant, influence on apparent kinetics arises because the reaction products lead to an increased pH level which, because of diffusion resistance, remains high within the gelatin matrix. For pH levels in the 6.7 to 9.0 range the activity of urease is a strongly decreasing function of pH. An approximate model accounting for ionic equilibrium allows this pH-diffusion effect to be introduced in such a way as to lead to predictions of the apparent kinetics that are compared with experimental observations. Examination of these results indicates that the immobilization procedure leads to some loss of activity due to an interaction of the gelatin crosslinking reaction with the enzyme itself.     

Phosphate uptake by blue green algae in vitro and in a lake during an algal bloom: Useful application of a force-flow relationship

A bioenergetic model, developed by M. Thellier, was applied to the ³²P-phosphate uptake data obtained with blue green algae and allowed the determination of a threshold concentration for the absorption of phosphate. Data obtained during an algal bloom of Oscillatoria rubescens showed that algal growth ceased when the phosphate concentration in the lake dropped below this threshold value. The resulting starvation led to an increase in the permeability of the membrane for phosphate.     

Modelling the uptake of nitrate by a growing plant with an adjustable root nitrate uptake capacity

A new model is presented to predict the plant uptake of nitrate supplied by diffusion and mass flow to its roots. Plant growth, root-shoot ratio and the plant's nitrate uptake capacity are all set dependent on the plant's N nutrition state. By thoroughly integrating processes occurring in both plant and soil, the model enables to control the relative importance of both under a wide range of different nutritional scenarios.Soil parameters D₀ diffusion coefficient in water (m² day^-1) - D_e diffusion coefficient in soil (m² day^-1) - C nitrate concentration in soil (mol m^-3) - f tortuosity (-) - volumetric moisture content (-) - R radial distance from root axis (m) Plant parameters b₁, b₂ parameters of biomass partitioning Equation (10) - IR interroot distance (m) - K_mU Michaelis-Menten constant of the uptake system (mol m^-3) - K_mNRA Michaelis-Menten constant of nitrogen reduction system (mol g^-1) - k₁, k₂, k₃ parameters of growth model Equation (9) - L_v Root length density (m m^-3) - NO_{3 set} ^- Set point of the cytoplasmatic nitrate pool (mol g^-1 dw) - NO_{3 c} ^- cytoplasmatic nitrate concentration (mol g^-1 dw) - NO_{3 v} ^- vacuolar nitrate concentration (mol g^-1 dw) - NRA_max maximum nitrate reductase activity (mol g^-1 dw day^-1) - N_re reduced nitrogen content (mol) - N_remax maximum reduced N concentration in the plant (mol g^-1 dw) - P partitioning coefficient of nitrate between cyplasm and vacuole - R(1) root radius (m) - RGR relative growth rate (day^-1) - U uptake rate (mol day^-1 m^-2) - U_max maximum uptake rate (Eq. 6) (day^-1 m^-2) - Vo water flux at root surface (m day^-1) - W_r root dry weight (g) - W_sh shoot dry weight (g) - X model parameter: number of root compartments - Y model parameter: number of nodes     

Chaotic dynamics of a three species prey-predator competition model with bionomic harvesting due to delayed environmental noise as external driving force

We consider a biological economic model based on prey-predator interactions to study the dynamical behaviour of a fishery resource system consisting of one prey and two predators surviving on the same prey. The mathematical model is a set of first order non-linear differential equations in three variables with the population densities of one prey and the two predators. All the possible equilibrium points of the model are identified, where the local and global stabilities are investigated. Biological and bionomical equilibriums of the system are also derived. We have analysed the population intensities of fluctuations i.e., variances around the positive equilibrium due to noise with incorporation of a constant delay leading to chaos, and lastly have investigated the stability and chaotic phenomena with a computer simulation.     

Mechanism of an insect glutathione S-transferase: kinetic analysis supporting a rapid equilibrium random sequential mechanism with housefly I1 isoform

The steady-state kinetics of glutathione S-transferase I1 (GST I1) from housefly Musca domestica expressed in Escherichia coli were investigated with glutathione (GSH) and 1-chloro-2,4-dinitrobenzene (CDNB). Concentrations of the varied substrates were from 0.03 to 1 mM for GSH and 0.05 to 1 mM for CDNB. Within this range, Michaelis-Menten behaviour was observed and convergent straight lines in double reciprocal plots excluded a ping-pong kinetic mechanism. Instead, data were consistent either with rapid-equilibrium random or with steady-state ordered sequential mechanisms because of abscissa convergence. Discrimination was achieved by studying the reaction with another electrophilic partner, p-nitrophenyl-acetate (PNPA). Concentrations of PNPA and GSH varied within the ranges 0.5 to 10 mM and 0.03 to 0.6 mM, respectively. The complete set of data supports the proposal of a rapid-equilibrium random-sequential model with strictly independent sites for GSH and CDNB or PNPA. Kinetic parameters are thus true dissociation equilibrium constants with values of 0.15 mM for GSH, 0.15 mM for CDNB, and 7 mM for PNPA. Analysis of the inhibition by the product (S-(2,4-dinitrophenyl)-glutathione, 10 to 100 microM), on the coupling reaction between GSH and CDNB with either GSH (0.05 to 0.5 mM, CDNB 0.2 mM) or CDNB (0.05 to 0.5 mM, GSH 0.2 mM) varied, consistent with the proposed mechanism. Binding of product to the free enzyme excludes GSH (competitive inhibition pattern with Kp = 12 microM) but only slightly hinders binding of CDNB. Binding free energies, together with the inhibition pattern, suggest that the non-peptidic moiety of product interacts with an alternative sub-site within the large open pocket accommodating the various electrophilic substrates. These results lead us to propose a model for intra-pocket shifting of the non-peptidic moiety upon product formation which contributes to the product release.