The kinetics of penicillin-V deacylation on an immobilized enzyme

An immobilized Penicillin-V-acylase (commercial name, Novozym 217) with high specificity for the phenoxyacetyl-(V)- side chain was investigated in a recycle reactor and in a batch reactor to find the enzymatic reaction rate as a function of conversion, x, substrate concentration, c(A) (0) and pH. The reaction rate depends strongly on pH, and both products, phenoxy-acetic acid and 6-APA, inhibit the reaction. Nonspecific side reactions amount to only a few per cent when c(A) (0) <150mM and pH& gt; 6.5. The effectiveness factor for commercial-size particles is found to be about 0.65, and a value of 1.3mM is obtained for the equilibrium constant, K(eq), of the deacylation reaction. A kinetic model for the deacylation process which includes the effect of pH and of the reverse (acylation) reaction is proposed. Rate data for particles of different size are fitted to the nonlinear model. Five kinetic parameters and an effective diffusivity for the immobilized enzyme particles are determined.     

Modeling and simulation of a pressure-swing reactor for the conversion of poorly soluble substrate by immobilized enzyme: the case of d-hydantoinase reaction

An immobilized enzyme reactor system for converting poorly soluble substrate is proposed. In this stirred batch reactor, the solid substrate and immobilized enzyme suspensions are separated by a microporous filter. The advantage of separating the solid substrate from immobilized enzyme is that the fouling and breakage of the immobilized enzyme usually encountered in the stirred tank reactor can be prevented. Pressure swing can be applied to enhance the mass transfer between the two compartments. The hydrolytic reaction converting the poorly soluble substrate p-hydroxyphenylhydantoin (pHPH) into soluble N-carbamoyl-p-d-hydroxyphenylglycine (CpHPG) by immobilized d-hydantoinase is carried out in this reactor. The performance of this pressure-swing reactor is studied by simulation using a simple kinetic model. The pressure-swing operation increases the overall production rate significantly. The pressure swing also makes the reactor perform better for converting the solid substrate at higher concentration.     

Biodegradation of 3,4-dichloroaniline in a fluidized bed bioreactor and a steady-state biofilm Kinetic model

Mixed culture of microorganisms immobilized onto Celite diatomaceous earth particles were used to degrade 3,4-dichloroaniline (34DCA) in a three-phase draft tube fluidized bed bioreactor. Biodegradation was confirmed as the dominant removal mechanism by measurements of the concomitant chloride ion evolution. Degradation efficiencies of 95% were obtained at a reactor retention time of 1.25 h. A mathematical model was used to describe the simultaneous diffusion and reaction of 34DCA and oxygen in the biofilms on the particles in the reactor. The parameters describing freely suspended cell growth on 34DCA were obtained in batch experiments. The model was found to describe the system well for three out of four steady states and to predict qualitatively the experimentally observed transition in the biofilm kinetics from 34DCA to oxygen limitation.     

Enantioselective synthesis of (S)-ibuprofen ester prodrug in cyclohexane by Candida rugosa lipase immobilized on Accurel MP1000

An enantioselective esterification process was developed for the synthesis of 2-N-morpholinoethyl (S)-ibuprofen ester prodrug from racemic ibuprofen by using Candida rugosa lipase immobilized on Accurel MP1000 in cyclohexane. Compared with the performance of Lipase MY, the immobilized lipase possesses a higher enzyme activity and thermal stability, but with a slightly suppressed enantioselectivity. A kinetic model was proposed and confirmed from experiments, for the simulation of time-course conversions of both enantiomers at various combinations of substrate concentrations in a batch reactor. Preliminary results of employing the proposed model and the immobilized lipase in a continuous packed-bed reactor were also reported and discussed.     

Denitrification of high nitrate wastewater in a cloth strip bioreactor with immobilized sludge

Denitrification of synthetic high nitrate wastewater containing 40,000?ppm NO₃ (9,032?ppm NO₃-N) was achieved using immobilized activated sludge in a column reactor. Active anoxic sludge adsorbed onto Terry cloth was used in the denitrification of high nitrate wastewater. The operational stability of the immobilized sludge system was studied both in a batch reactor and in a continuous reactor. The immobilized sludge showed complete degradation of different concentrations of NO₃-N (1,129, 1,693, 3,387, 6,774, and 9,032?ppm) in a batch process. The reactors were successfully run for 90?days without any loss in activity. The immobilized cell process has yielded promising results in attaining high denitrifying efficiency.     

Countercurrent multistage fluidized bed reactor for immobilized biocatalysts: III. Hydrodynamic aspects

In Parts I and II of this series we described the modelling, design, and operation of a multistage fluidized bed reactor (MFBR) for immobilized biocatalysts. This article deals with those aspects of the MFBR which are different from single-stage fluidized beds which are operated in batch mode with respect to the solids. The semicontinuous transport of the particles requires perfect mixing of the particles in the reactor compartments, because particles are mainly transported from the bottom of these compartments. A large spread in the physical properties of the biocatalyst particles, especially of both size and density, may cause the particles to segregate into layers with different diameter and/or density. This affects the efficient use of the biocatalyst. The properties of the particles are dependent on the immobilization method. The suitability of different methods for possible future application in the MFBR is therefore compared. Because of segregation, successful use of a biofilm catalyst with a nonuniform thickness of the biofilm is doubtful. Experiments in a small scale reactor (+/- 0.1 m diameter) demonstrated that perfect particle mixing is possible using commercially available biocatalyst particles of uniform density. Co-immobilization of the biocatalyst with glass powder in a gel is a simple and effective method of increasing gel density. High density particles allow high liquid flow rates, and thus an improved external mass transfer can be achieved.The distributor plates, which separate the reactor compartments, must allow unhindered transport of particles. Therefore, the holes in these plates must have a diameter of at least 4.5 times that of the largest particles which are present in the particle mixture used. Furthermore, the plates must be designed such that, when scaling-up the reactor, a uniform liquid distribution over the cross-sectional area of the reactor occurs. Large-scale experiments were not carried out, but published correlations, indicate that particle mixing and a uniform liquid distribution can be accomplished in a large-scale reactor under similar flow conditions.     

A new conception of enzymatic membrane reactor for the production of whey hydrolysates with low contents of phenylalanine

A novel concept of enzymatic membrane reactor (EMR) is presented, aiming at the production of cheese whey protein hydrolysates with low contents of phenylalanine (Phe) for the diet of phenylketonuria patients. Whey proteins were first hydrolyzed by chimotrypsin, followed by the action of carboxypeptidase A (CPA), immobilized on agarose gel particles, which were retained inside the reactor using a filter. The liquid medium passes through hollow fiber ultrafiltration unit (1 kDa cut-off), and the retentate is recycled to the reactor. The innovation here is that the membrane is not used to retain the enzyme neither physically nor as an immobilization support. The EMR provided a higher performance than the classical, sequential approach: batch reaction followed by ultrafiltration. It was confirmed that the removal of products promoted by the EMR enhances reaction rates, due to the reduction of inhibition effects. A mathematical model of the EMR is also presented and validated.     

A new method for cell immobilization.

A new method for producing particles and membranes containing immobilized bacteria is presented. These immobilized bacteria display good stability over time making them well suited for use in a packed-bed reactor. Such a reactor is tested as a function of the different parameters of the system. The results are qualitatively similar to those obtained with purified enzyme reactors, but some discrepancies with the plug-flow model are noted. It is necessary to use a more sophisticated model in order to fit the experimental data.     

Conversion of benzylpenicillin to 6-aminopenicillanic acid in a batch reactor and continuous feed stirred tank reactor using immobilized penicillin amidase

A rate equation has been derived to describe the hydrolysis of benzylpenicillin to 6-aminopenicillanic acid by penicillin amidase. The integrated from of the rate equation has been shown to predict satisfactorily the progress of the reaction in a batch reactor using either soluble or immobilized penicillin amidase. The rate equation was also used to predict the performance of a continuous feed stirred tank reactor containing immobilized enzyme. There was good agreement with experimental measurements.     

Process and technoeconomics of ethanol production by immobilized cells

Summary A two-stage fermentation process has been developed for continuous ethanol production by immobilized cells of Zymomonas mobilis. About 90–92 kg/m³ ethanol was produced after 4 h of residence time. Entrapped cells of Zymomonas mobilis have a capability to convert glucose to ethanol at 93% of the theoretical yield. The immobilized cell system has functioned for several weeks, and experience indicates that the carrageenan gel apparently facilitates easy diffusion of glucose and ethanol.The simplicity and the high productivity of the plug-flow reactor employing immobilized cells makes it economically attrative. An evaluation of process economics of an immobilized cell system indicates that at least 4 c/l of ethanol can be saved using the immobilized cell system rather than the conventional batch system. The high productivity achieved in the immobilized cell reactor results in the requirement for only small reactor vessels indicating low capital cost. Consequently, by switching from batch to immobilized processing, the fixed capital investment is substantially reduced, thus increasing the profitability of ethanol production by fermentation.     

Countercurrent multistage fluidized bed reactor for immobilized biocatalysts: II. Operation of a laboratory-scale reactor

In Part I of this series,(1) we derived a model and made simulations for a multistage fluidized bed reactor (MFBR). It was concluded that the MFBR can be an attractive alternative for a fixed bed reactor when operated with a deactivating biocatalyst. In Part II of this series, the design of a laboratory-scale MFBR and its evaluation to investigate the practical feasibility of this reactor type, will be described. Experiments with a duration as long as 10 days were carried out successfully using immobilized glucose isomerase as a model reaction system. The results predicted by the model are in good agreement with the measured glucose concentration and biocatalyst activity gradients, indicating perfect mixing of the particles in the reactor compartments.The diameters of the biocatalyst particles used in the experiments showed a large spread, with the largest being 1.7 times the smallest. Therefore, an additional check was carried out, to make sure that the particles were not segregating according to size. Particles withdrawn from the reactor compartments were investigated using an image analyzer. Histograms of particle size distribution do not indicate segregation and it is concluded that the particles used have been mixed completely within the compartments. As a result, transport of biocatalyst is nearly plug flow.     

Removal and recovery of uranium (VI) from aqueous solutions by immobilized Aspergillus niger powder beads

The immobilized Aspergillus niger powder beads were obtained by entrapping nonviable A. niger powder into Ca-alginate gel. The effects of pH, contact time, initial uranium (VI) concentration and biomass dosage on the biosorption of uranium (VI) onto the beads from aqueous solutions were investigated in a batch system. Biosorption equilibrium data were agreeable with Langmuir isotherm model and the maximum biosorption capacity of the beads for uranium (VI) was estimated to be 649.4?mg/g at 30?°C. The biosorption kinetics followed the pseudo-second-order model and intraparticle diffusion equation. The variations in enthalpy (26.45?kJ/mol), entropy (0.167?kJ/mol?K) and Gibbs free energy were calculated from the experimental data. SEM and EDS analysis indicated that the beads have strong adsorption capability for uranium (VI). The adsorbed uranium (VI) on the beads could be released with HNO₃ or HCl. The results showed that the immobilized A. niger powder beads had great potential for removing and recovering uranium (VI) from aqueous solutions.     

Enzymatic removal of flatulence-inducing sugars in chickpea milk using free and polyvinyl alcohol immobilized α-galactosidase from <Emphasis Type="Italic">Aspergillus oryzae</Emphasis>

The treatment of chickpea milk was carried out in batch, repeated batch and continuous reaction by soluble and polyvinyl alcohol (PVA) immobilized Aspergillus oryzae alpha-galactosidase for the removal of raffinose family oligosaccharides (RFOs). In the batch mode of treatment 96 and 92% of RFOs hydrolysis was observed by soluble and immobilized enzyme, respectively. In repeated batch experiments, immobilized enzyme showed 70% RFOs hydrolysis up to sixth cycle. Polyvinyl alcohol immobilized alpha-galactosidase in fluidized bed reactor showed highest reduction of 94% at a flow rate of 30 ml/h. The results obtained from the present study are very interesting for industrial use of PVA-immobilized enzyme.     

Effects of solid-phase mass transfer on the performance of a stirred anaerobic sequencing batch reactor containing immobilized biomass

This work reports on experiments for an anaerobic sequencing batch reactor containing immobilized biomass which aimed at verifying the effects of solid-phase mass transfer on the reactor's overall performance. Four experiments were carried out at 30 degrees C with cubic polyurethane foam particles previously inoculated with anaerobic biomass. Different solid-phase mass transfer conditions were reached in each experiment by varying the size of the bioparticle from 0.5 to 3.0 cm. The reactor was fed with a low-strength synthetic wastewater containing protein, carbohydrates and lipid and the effects of mass transfer were evaluated through dynamic substrate concentration profiles during 8-hour batch cycles. A modified first-order kinetic model provided a good representation of the behavior of the dynamic concentration profiles. The solid-phase mass transfer was found to slightly affect the concentration of effluent organic matter expressed as chemical oxygen demand (COD). The concentration of residual effluent substrate increased as the size of the bioparticle was increased. The cycle time was not affected as the size of the bioparticle was increased from 0.5 to 2.0 cm. However, it was found that the cycle time in a reactor with 3.0-cm cubic particles should be higher than that required in systems with smaller particles. The apparent first-order kinetic parameter was estimated as 0.59+/-0.01 h(-1) for experiments with bioparticle sizes ranging from 0.5 to 2.0 cm, while a value of 0.48 h(-1) was obtained in the experiment with 3.0-cm bioparticles.     

beta-Galactosidase immobilization for milk lactose hydrolysis: a simple experimental and modelling study of batch and continuous reactors

The experiment described in this paper introduces students to the practical use of an enzyme (beta-galactosidase, or lactase) acting on a natural substrate. The enzyme is immobilized onto a cheap support, and the immobilized derivative is used in a packed-bed reactor for continuous milk lactose hydrolysis. The results are compared to those obtained for discontinuous batch reactors with soluble enzyme. A mathematical model of the two types of reactors is run, and its results are compared with the experimental data obtained.     

Lactic acid fermentation in a recycle batch reactor using immobilized Lactobacillus casei

Lactic acid production by recycle batch fermentation using immobilized cells of Lactobacillus casei subsp. rhamnosus was studied. The culture medium was composed of whey treated with an endoprotease, and supplemented with 2.5 g/L of yeast extract and 0.18 mM Mn(2+) ions. The fermentation set-up comprised of a column packed with polyethyleneimine-coated foam glass particles, Pora-bact A, and connected with recirculation to a stirred tank reactor vessel for pH control. The immobilization of L. casei was performed simply by circulating the culture medium inoculated with the organism over the beads. At this stage, a long lag period preceded the cell growth and lactic acid production. Subsequently, for recycle batch fermentations using the immobilized cells, the reducing sugar concentration of the medium was increased to 100 g/L by addition of glucose. The lactic acid production started immediately after onset of fermentation and the average reactor productivity during repeated cycles was about 4.3 to 4.6 g/L . h, with complete substrate utilization and more than 90% product yield. Sugar consumption and lactate yield were maintained at the same level with increase in medium volume up to at least 10 times that of the immobilized biocatalyst. The liberation of significant amounts of cells into the medium limited the number of fermentation cycles possible in a recycle batch mode. Use of lower yeast extract concentration reduced the amount of suspended biomass without significant change in productivity, thereby also increasing the number of fermentation cycles, and even maintained the D-lactate amount at low levels. The product was recovered from the clarified and decolorized broth by ion-exchange adsorption. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55:841-853, 1997.     

Immobilized Coffea arabica cell culture using a bubble-column reactor with controlled light intensity

Coffea arabica cells immobilized by calcium alginate gel were photocultured using a bubble-column reactor under controlled light intensity. This process was carried out after their alkaloid productivity was improved by increasing the cell density in the initial gel matrix and preculturing the immobilized cells in the dark prior to light irradiation. The cells were grown in the form of a biofilm on gel beads, producing 100 mg/L of purine alkaloids in a 24-day batch culture. Alkaloid production was relatively constant with respect to light intensity changes, and also cell growth was not suppressed much at high light intensity, with these behaviors being different from those obtained using suspended cells. These phenomena are explained by estimating the light intensity gradient within the cell-immobilizing particles and by measuring the viable cell distribution within them. It subsequently suggests that the subsurface cells affect both the production and growth behaviors. (c) 1993 John Wiley & Sons, Inc.     

Lipase catalysed hydrolysis of ricebran oil by free and immobilised enzyme system in batch stirred reactor

A change of the reaction rate was observed for the lipasecatalysed hydrolysis of ricebran oil in a batch stirred tank reactor using immobilized lipase enzyme as compared to free enzyme. The reactor rate was observed to be controlled mainly by factors like temperature, pH, initial enzyme concentration, initial substrate concentration and initial products concentration.     

Multiple continuous stirred tank reactors VS. Batch reactors — some design considerations

Summary A comparison of the performance of single CSTR and 2-CSTR systems against a stirred batch immobilized enzyme (IME) reactor has been made. When the downtimes in a batch reactor are not too small, the 2-CSTR system may become attractive and therefore a proper consideration should be given to the 2-CSTR system particularly when the application of a plug flow reactor is not practicable.NCL Communication No-2415     

The use of the inverted linear model of enzyme decay to plan a strategy for enzymatic batch reactions and to assess the industrial applicability of immobilized enzyme preparations

This article is concerned with the development of a model to plan a strategy for an enzymatic batch process where enzyme is subjected to deactivation described by the inverted linear decay model. The particular system studied is the enzymatic hydrolysis of penicillin to 6-amino penicillanic acid (6 APA), but the model can be utilized with other batch systems as long as the decay of the immobilized enzyme (IME) preparation is described by the inverted linear decay model. The model developed is eminently practical and simple and several example of its application are shown. Experimental data obtained in a small pilot plant batch recirculated reactor on the average are well fitted by this model. For IME systems whose decay is best described by the first-order decay model, it is not possible to use the same approach.