Co-immobilized Nitrosomonas europaea and Nitrobacter agilis cells: validation of a dynamic model for simultaneous substrate conversion and growth in kappa-carrageenan gel beads

A dynamic model for two microbial species immobilized in a gel matrix is presented and validated with experiments. The model characterizes the nitrification of ammonia with Nitrosomonas europaea and Nitrobacter agilis co-immobilized in K-carrageenan gel beads. The model consists of kinetic equations for the microorganisms and mass transfer equations for the substrates and products inside and outside the gel beads. The model predicts reactor bulk concentrations together with the substrate consumption rate, product formation, and biomass growth inside the gel beads as a function of time. A 50-day experiment with immobilized cells in a 3.3-dm(3) air-lift loop reactor was carried out to validate the model. The parameter values for the model were obtained from literature and separate experiments. The experimentally determined reactor bulk concentrations and the biomass distribution of the two microorganisms in the gel beads were well predicted by the model. A sensitivity analysis of the model for the given initial values indicated the most relevant parameters to be the maximum specific growth rate of the microorganisms, the diffusion coefficient of oxygen, and the radius of the beads. The dynamic model provides a useful tool for further study and possible control of the nitrification process. (c) 1994 John Wiley & Sons, Inc.     

Kluyveromyces lactis cells entrapped in Ca-alginate beads for the continuous production of a heterologous glucoamylase

Viable cells of Kluyveromyces lactis, transformed with the glucoamylase gene from Arxula adeninivorans, were entrapped in beads of Ca-alginate and employed on a lab scale in a continuous stirred and a fluidised bed reactor (FBR), both fed with a rich medium (YEP) containing lactose as carbon source. Experiments with freely suspended cells in batch and chemostat had demonstrated that glucoamylase production was favoured in the presence of lactose and YEP medium. Employing controlled-sized beads having a 2.13 mm diameter, specific glucoamylase productivity was higher in the stirred reactor (CSTR) than in the FBR; in the latter a higher volumetric productivity was achieved, due to the lower void degree. The performance of the immobilised cell systems, in terms of specific glucoamylase productivity, was strongly affected by mass transfer limitations occurring throughout the gel due to the high molecular weight of the product. In the perspective to improve and scale-up the immobilised cell system proposed, a mathematical model, which takes into account substrate transfer limitations throughout the gel, has been developed. The effective lactose diffusivity was related to the bead reactive efficiency by means of the Thiele modulus. The regression of the model parameters on the experimental data of substrate consumption obtained both in the CSTR and in the FBR allowed to estimate lactose diffusivity and the kinetic parameters of the immobilised yeast.     

The effect of whole cell immobilisation on the biotransformation of benzonitrile and the use of direct electric current for enhanced product removal

The nitrilase of Rhodococcus rhodochrous performs a one-step biotransformation of nitriles to their corresponding carboxylic acids. Application of a direct electric current moves the charged carboxylic acid towards an anode, across an anion exchange membrane, into a separate compartment. Cells encapsulated within alginate beads (2.9 mm diameter) for protection against the current biotransformed benzonitrile to benzoic acid with a 26% reduction in the biotransformation rate, from 0.054 mmol/min/g dcw with free cells to 0.040 mmol/min/g dcw with immobilised cells. When the electric current was applied, the biotransformation rate increased to 0.047 mmol/min/g dcw and product recovery increased from 19% to 79%.     

Rhizopus oryzae fungus cells producing L(+)-lactic acid: kinetic and metabolic parameters of free and PVA-cryogel-entrapped mycelium

Spores of the filamentous fungus Rhizopus oryzae were entrapped in macroporous poly(vinyl alcohol) cryogel (PVA-cryogel). To prepare immobilised biocatalyst capable of producing L(+)-lactic acid (LA), the fungus cells were cultivated inside the carrier beads. The growth parameters and metabolic activity of the suspended (free) and immobilised cells producing LA in a batch process were comparatively investigated. The immobilised cells possessed increased resistance to high concentrations of accumulated product and gave much higher yields of LA in the iterative working cycle than the free cells did. Detailed kinetic analysis of the changes in the intracellular adenosine triphosphate concentration, specific rate of growth, substrate consumption and LA production showed that the fungus cells entrapped in PVA-cryogel are more attractive for biotechnological applications compared to the free cells.     

Rheological properties of chromosomal and plasmid DNA during alkaline lysis reaction

The experimental apparatus for the simultaneous L-lactic acid fermentation by Rhizopus oryzae immobilized in calcium alginate beads and product separation process was set up in which a three-phase fluidized-bed bioreactor was used as a fermentor and an external electrodialyzer as a separator, and a pump was applied to recycle the fermentation broth between the bioreactor and the separator. The L-lactic acid produced in the fermentor was separated in the separator, product inhibition was alleviated without any addition of alkali or alkali salts and the product purification process could be simplified. The specific productivity and the yield in electrodialysis fermentation (ED-F) process operated in continuous feeding mode were almost the same as that in CaCO₃-buffered fermentation process. A mathematical model of L-lactic acid production in ED-F process was also suggested, in which the model equations for the bioreactor and the electrodialyzer were combined to describe the simultaneous fermentation and product separation. The model predictions were in good agreement with the experimental data.     

Wine vinasses treatments by ozone and an activated sludge system in continuous reactors

In this work wine vinasses have been treated separately by means of a chemical ozonation and a biological aerobic degradation in an activated sludge system, and later by means of a combined process which consisted of an aerobic pretreatment followed by an ozonation treatment, in continuous reactors in all cases. In the ozonation experiments, the hydraulic retention time and the ozone partial pressure were varied leading to substrate removals in the range 4.4-16%, with increases in this removal when both operating variables were increased. A kinetic study, which combines mixed flow reactor model for the liquid phase and plug flow reactor model for the gas phase, allows to determine the rate constant for the ozone reaction and the consumption ratio, which are k_O3 = 3.6 l/(g COD · h) and b = 22.5 g COD degraded/mol O₃ consumed. The aerobic degradation experiments were conducted in the activated sludge system with variations in the retention time and influent organic substrate concentration in the wastewater. A modified Contois model applied to the experimental results leads to the determination of the kinetic parameters of that model: K₁ = 5.43 l/g VSS and q_max = 6.29 g COD/(g VSS · h). Finally, the combined process reveals an improvement in the efficiency of the ozonation stage due to the previous aerobic treatment with increases in the substrate removal reached and in the rate constant obtained, the last one being k_O3 = 5.6 l/(g COD · h).     

Malolactic fermentation in chardonnay wine by immobilised Lactobacillus casei cells

The kinetics of malolactic fermentation in Chardonnay wine by immobilised Lactobacillus casei cells has been studied. Calcium pectate gel and chemically modified chitosan beads were used as supports for immobilisation. Repeated batch fermentations were carried out with different wine samples, some of which were treated with sulfur dioxide (free 19–25 mg/litre and total 80–88 mg/litre), in shake flask at 36, 25 and 20°C without any loss of activity. The degradation of malic acid obtained using immobilised cells was twice as high as that obtained with free cells. At an initial pH 3·2, decrease of malic acid of about 30% was observed at 25°C in one hour using L. casei cells immobilised either in pectate gel or on chitosan. Among the physico-chemical parameters studied, temperature was the main factor affecting metabolism of the organic acids as well as the rate of the malolactic fermentation. Operational stability of calcium pectate gel beads and chemically modified chitosan beads was 6 months after eight fermentations and 2 months after five fermentations, respectively, which proved the possibility of industrial application of the chosen supports in wine making.     

Antibiotics production in a fluidized bed reactor utilizing a transverse magnetic field

A mathematical model is developed to describe the performance of a three-phase fluidized bed reactor utilizing a transverse magnetic field. The model is based on the axially dispersed plug flow model for the bulk of liquid phase and on the Michaelis-Menten kinetics. The model equations are solved by the explicit finite difference method from transient to steady state conditions. The results of the numerical simulation indicate that the magnetic field increases the degree of bioconversion. The mathematical model is experimentally verified in a three-phase fluidized bed reactor with Penicillium chrysogenum immobilized on magnetic beads. The experimental results are well described by the developed model when the reactor operates in the stabilized regime. At low and relatively high magnetic field intensities certain discrepancy in the model solution is observed when the model over estimates the product concentration.     

Propionic acid production by whole cells of Propionibacterium freudenreichii

The microbial production of propionic acid by Propionibacterium freudenreichii NCIM 2111, has been studied in this communication. Shake-flask studies were carried out to determine the optimum combination of various process parameters like stab age, inoculum age, inoculum level, medium constituents, temperature, and the initial pH for maximizing the production of propionic acid by using central composite design method. The system was found to exhibit product inhibition and hence the product inhibition kinetics was studied. A two parameter kinetic model, taking into account of the product inhibition, was proposed. Leudeking and Piret model was used to describe the production kinetics. The result from the shake-flask studies were compared with that obtained from mechanically stirred batch bioreactor and total recycle batch bioreactor.     

Metapopulation structure and movements in five species of checkerspot butterflies

We studied the patterns and rates of migration among habitat patches for five species of checkerspot butterflies (Lepidoptera: Melitaeini) in Finland: Euphydryas aurinia, E. maturna, Melitaea cinxia, M. diamina and M. athalia. We applied the virtual migration (VM) model to mark-release-recapture data collected from multiple populations. The model includes parameters describing migration and survival rates and how they depend on the areas and connectivities of habitat patches. The number of individuals captured varied from 73 to 1,123, depending on species and sex, and the daily recapture probabilities varied between 0.09-0.52. The VM model fitted the data quite well. The results show that the five species are broadly similar in their movement rates and patterns, though, e.g. E. maturna tends to move shorter distances than the other species. There is no indication of any phylogenetic component in the parameter values. The parameter values estimated for each species suggest that a large percentage (80-90%) of migration events were successful in the landscapes that were studied. The area of the habitat patch had a substantial effect on emigration and immigration rates, such that butterflies were more likely to leave small than large patches and large patches were more likely than small patches to receive immigrants.     

Joint aerobic biodegradation of wastewater from table olive manufacturing industries and urban wastewater

Wastewater generated in the elaboration of table olives has been treated using activated sludge from a municipal wastewater plant after adequate acclimation. To avoid bactericide properties of some chemical structures present in this type of effluents, synthetic urban wastewater has been used to dilute the original wastewater. The main parameters affecting efficiency of biological processes have been studied. Thus, initial biomass concentration, temperature up to 303 K (upper working temperature limit = 313 K) and initial substrate concentration exerted a positive influence on COD degradation rate. The optimum pH was found to be around 7, experiencing a slight inhibition on cell activity at pH 4. Under the experimental conditions investigated other parameters like polyphenol content, absorbance at 254 nm and total organic carbon were also reduced to some extent. Only nitrates amount was increased after the biological process took place. A kinetic model based on Monod equation was proposed and applied to experimental results. The maximum specific growth rate was calculated by means of the aforementioned kinetic model. The value of this parameter as a function of temperature was fitted to an Arrhenius expression, w_max = 9.43 2 10¹⁰ exp(72021/RT) h^у (R in J mol^у K^у283 K < T < 303 K, pH , 7-10).     

Kinetics and modeling of autotrophic thiocyanate biodegradation

The biokinetic parameters for autotrophic systems are difficult to obtain and are often mistakenly determined because the size of the autotrophic population in mixed (i.e., heterotrophic and autotrophic) cultures cannot be accurately estimated. This article presents a systematic approach, combining bioenergetic calculations and experimental data, to obtain values of the biokinetic parameters pertinent to the aerobic, autotrophic biodegradation of thiocyanate. Nonlinear regression techniques were employed using both initial thiocyanate utilization rate data and single thiocyanate depletion curves. Both types of data were necessary to overcome the problems arising from the linear nature of the substrate depletion curves and the high correlation of the biokinetic model parameters inherent in nonlinear regression analysis. The aerobic biodegradation of thiocyanate followed a substrate inhibition pattern that was successfully described by the Haldane-Andrews model. Although regression analysis did not yield unique biokinetic parameter estimates, the following parameter value ranges were obtained: maximum specific substrate utilization rate (k), 0.26 to 0.44 mg SCN-/mg biomass h; half-saturation coefficient (Ks), 2.3 to 7.1 mg SCN-/L; and inhibition coefficient (Ki), 28 to 109 mg SCN-/L. Based on the estimated biokinetic parameter values, a design and operation diagram was constructed that depicts the steady-state thiocyanate concentration as a function of solids retention time for a completely mixed, continuous-flow reactor.     

Kinetic study and modelling of the xylitol production using Candida parapsilosis in oxygen-limited culture conditions

Kinetic studies are presented for xylitol production and growth of the yeast Candida parapsilosis ATCC 28474. The oxygen supply influence on xylitol production from xylose was investigated. No metabolic activity was detected in anaerobic conditions. In contrast, it was found that under low aeration rates (0.1-0.2 vvm), xylitol is produced. Xylitol production decreases when air flow to reactor is augmented. An unstructured model is proposed for the kinetic behaviour analysis of yeast growing in batch culture. A simplex method was used for the estimation of model parameters. The parameter confidence intervals were also calculated.     

Combined immobilized cultures producing fibrinolytic proteinases

The authors obtained combined immobilised systems composed of a culture producing fibrinolytic proteinases and a stimulating strain. The optimal ratio between the two cultures in gel was selected at a high starting cell density. Highly stable immobilised cultures were produced by growing the cells in gel particles. The interrelationship of the partners was studied in the binary immobilised culture. The biosynthetic activity of the system fell down to the level of a monoculture when the cells of the stimulating strain were eliminated from gel. The producing and stimulating strains are at equilibrium in associative immobilised cultures obtained by growing the cells in gel, and Arthrobacter is not eliminated. The mechanism of biosynthesis stimulation in a combined immobilised culture has been studied. Apparently, the procedure of immobilisation and the action of a stimulating compound exert the synergistic effect.     

A bioprocess for the remediation of anaerobically digested molasses spentwash from biogas plant and simultaneous production of lactic acid

A bacterial isolate LAB-1 identified as Lactobacillus casei was cultivated in a fermentation medium containing biogas plant effluent. This effluent was generated after anaerobic digestion of molasses spentwash in biogas fermenters. In the bioremediation process of this very dark coloured effluent through the cultivation of L. casei, decrease in effluent's colour of 49 and 52% and reduction in COD level up to 54 and 57% were achieved using bacterial cells in free and immobilised system, respectively, in 5 days batch cultivation. During this process of bioremediation, a metabolite lactic acid was produced by this bacterial isolate with the yield of 10.9 and 11.3 mg/ml, in free and immobilised cells fermentation, respectively.     

Modelling of continuous microbial cultivation taking into account the memory effects

The problem of chemostat dynamics modelling for the purpose of control is considered. The "memory" of the culture is explicitly taken into account. Two possibilities for improving the quality of the proposed modelling approaches are discussed. A general model that accounts for the culture `memory' by means of different `memory' functions in the expressions of the specific growth rate and of the specific consumption rate and a polynomial function of the substrate concentration for the yield factor is proposed. The case where the maintenance energy is taken into account is also discussed. Two modifications of the general model (w-type and S-type) are presented. A zero-order `memory' function and a i-function with delay are applied in order to describe the `memory' effects. Continuous growth of the strain Saccharomyces cerevisiae on a glucose limited medium is considered as a case study. Detailed investigations of the variety of models, derived from the general model by applying different `memory' functions and different assumptions are carried out. The results are compared with those previously reported for the same process. It is shown that a significant improvement in predicting the substrate dynamics (not accompanied by any decrease in the quality of the model with respect to the biomass concentration) could be achieved, involving a first- or second-order polynomial function for the yield factor. It is also shown that the quality of the model mainly depends on the way that `memory' function is incorporated. The detailed investigations give priority to the w-type models. In this case past values of both biomass and substrate variables are considered. The time delay models with pure (constant) delay and those which account for the culture `memory' by zero-order `memory' function (adaptability parameter) are compared with respect to their utilization for the purpose of model-based control.     

Effectiveness factor calculation for immobilised growing cell systems

Summary Gel immobilised living cell systems represent a special form of heterogeneous catalysis. Due to mass transfer limitations on substrate delivery and product removal, time-dependent spatial variations in growth rate and biomass densities are created. A dynamic mathematical model has been developed which was used to simulate the start-up dynamics of growing yeast cells in calcium alginate beads. By coupling the transient model with the effectiveness factor calculation, the dynamic effectiveness factor could be calculated. The influence of the bead radius and the initial biomass concentration on the dynamic effectiveness factor have been investigated.     

Mathematical modeling of an aqueous film coating process in a Bohle Lab-Coater: Part 2: Application of the model

For the prediction of the air and product temperatures, the product moisture, and the air humidity during a coating process in a Bohle Lab-Coater, a model was developed. The purpose of this work was to determine the limit moisture, the critical moisture, and the constant for the exchange rate between both zones and to use these values for other sets of experiments to test the model. The adaptation of the 3 parameters (limit moisture, critical moisture, and exchange rate constant), was done by calculation of the product temperature in both zones for several sets of parameters in order to minimize the sum of square deviation between the calculated and the measured product temperatures. This set of parameters was used to test the validity of the model. By applying the model, the product temperature could be predicted based on the product, process, and equipment-related parameters. Hence, the model can be used to theoretically investigate the influence of different process paramaters. The mean difference between the predicted, and measured product temperatures in the steady state is ≈2 up to 3 K using the determined parameter set for the limit moisture, the critical moisture, and the exchange rate constant. The model is useful for the prediction of the air and product temperatures, the product moisture, and air humidity during a coating process in the Bohle Lab-Coater using round, biconvex tablets.     

Modeling of the biotransformation of crotonobetaine into L-(-)-carnitine by Escherichia coli strains

A simple unstructured model, which includes carbon source as the limiting and essential substrate and oxygen as an enhancing substrate for cell growth, has been implemented to depict cell population evolution of two Escherichia coli strains and the expression of their trimethylammonium metabolism in batch and continuous reactors. Although the model is applied to represent the trans-crotonobetaine to L-(-)-carnitine biotransformation, it is also useful for understanding the complete metabolic flow of trimethylammonium compounds in E. coli. Cell growth and biotransformation were studied in both anaerobic and aerobic conditions. For this reason we derived equations to modify the specific growth rate, mu, and the cell yield on the carbon source (glycerol), Y(xg), as oxygen increased the rate of growth. Inhibition functions representing an excess of the glycerol and oxygen were included to depict cell evolution during extreme conditions. As a result, the model fitted experimental data for various growth conditions, including different carbon source concentrations, initial oxygen levels, and the existence of a certain degree of cell death. Moreover, the production of enzymes involved within the E. coli trimethylammonium metabolism and related to trans-crotonobetaine biotransformation was also modeled as a function of both the cell and oxygen concentrations within the system. The model describes all the activities of the different enzymes within the transformed and wild strains, able to produce L-(-)-carnitine from trans-crotonobetaine under both anaerobic and aerobic conditions. Crotonobetaine reductase inhibition by either oxygen or the addition of fumarate as well as its non-reversible catalytic action was taken into consideration. The proposed model was useful for describing the whole set of variables under both growing and resting conditions. Both E. coli strains within membrane high-density reactors were well represented by the model as results matched the experimental data.