A CSTR-hollow fiber system for continuous hydrolysis of proteins. Performance and kinetics

The effect of four operating variables (enzyme concentration, substrate concentration, flow rate, and reaction volume) on the performance of CSTR-hollow fiber membrane reactor was studied for the continuous hydrolysis of a soy protein isolate using Pronase. Based on a residence time distribution study, the reactor system was modeled as an ideal CSTR in combination with the Michaelis-Menten equation of enzyme kinetics. This kinetic model correlated conversion with a space-time parameter modified to include all four independent variables. An empirical model based on curvilinear regression analysis was also developed. Both models predicted conversion fairly well, although the kinetic model slightly underpredicts at high conversion.     

A theoretical study on the effect of surface roughness on mass transport and transformation in biofilms

This modeling study evaluates the influence of biofilm geometrical characteristics on substrate mass transfer and conversion rates. A spatially two-dimensional model was used to compute laminar fluid flow, substrate mass transport, and conversion in irregularly shaped biofilms. The flow velocity above the biofilm surface was varied over 3 orders of magnitude. Numerical results show that increased biofilm roughness does not necessarily lead to an enhancement of either conversion rates or external mass transfer. The average mass transfer coefficient and Sherwood numbers were found to decrease almost linearly with biofilm area enlargement in the flow regime tested. The influence of flow, biofilm geometry and biofilm activity on external mass transfer could be quantified by Sh-Re correlations. The effect of biofilm surface roughness was incorporated in this correlation via area enlargement. Conversion rates could be best correlated to biofilm compactness. The more compact the biofilm, the higher the global conversion rate of substrate. Although an increase of bulk fluid velocity showed a large effect on mass transfer coefficients, the global substrate conversion rate per carrier area was less affected. If only diffusion occurs in pores and channels, then rough biofilms behave as if they were compact but having less biomass activity. In spite of the fact that the real biofilm area is increased due to roughness, the effective mass transfer area is actually decreased because only biofilm peaks receive substrate. This can be explained by the fact that in the absence of normal convection in the biofilm valleys, the substrate gradients are still largely perpendicular to the carrier. Even in the cases where convective transport dominates the external mass transfer process, roughness could lead to decreased conversion rates. The results of this study clearly indicate that only evaluation of overall conversion rates or mass fluxes can describe the correct biofilm conversion, whereas interpretation of local concentration or flow measurements as such might easily lead to erroneous conclusions.     

Mass transport in a microchannel enzyme reactor with a porous wall: Hydrodynamic modeling and applications

A two-dimensional flow model, incorporating mass transport, has been developed to simulate a microchannel enzyme reactor with a porous wall. A two-domain approach based on the finite volume method was implemented. Two parameters are defined to characterize the mass transports in the fluid and porous regions: the porous Damkohler number and the fluid Damkohler number. For reactions close to first-order type (enzyme reactor), the concentration results are found to be well correlated by the use of a reaction–convection distance parameter which incorporates the effects of axial distance, substrate consumption and convection. The reactor efficiency reduces with reaction–convection distance parameter because of reduced reaction (or flux) due to the lower concentration. Increased fluid convection improves the efficiency but it is limited by the diffusion in the fluid region. The correlated results can find applications for the design of enzyme reactors with a porous wall.     

Mass transport in a microchannel bioreactor with a porous wall

A two-dimensional flow model has been developed to simulate mass transport in a microchannel bioreactor with a porous wall. A two-domain approach, based on the finite volume method, was implemented. For the fluid part, the governing equation used was the Navier-Stokes equation; for the porous medium region, the generalized Darcy-Brinkman-Forchheimer extended model was used. For the porous-fluid interface, a stress jump condition was enforced with a continuity of normal stress, and the mass interfacial conditions were continuities of mass and mass flux. Two parameters were defined to characterize the mass transports in the fluid and porous regions. The porous Damkohler number is the ratio of consumption to diffusion of the substrates in the porous medium. The fluid Damkohler number is the ratio of the substrate consumption in the porous medium to the substrate convection in the fluid region. The concentration results were found to be well correlated by the use of a reaction-convection distance parameter, which incorporated the effects of axial distance, substrate consumption, and convection. The reactor efficiency reduced with reaction-convection distance parameter because of reduced reaction (or flux), and smaller local effectiveness factor due to the lower concentration in Michaelis-Menten type reactions. The reactor was more effective, and hence, more efficient with the smaller porous Damkohler number. The generalized results could find applications for the design of bioreactors with a porous wall.     

Optimum temperature operation mode for glucose isomerase reactor operating at constant glucose conversion

The optimum temperature operation mode required to achieve constant outlet glucose conversion is determined for immobilized glucose isomerase continuous packed bed reactor. The reactor design equation assumes reversible Michaelis-Menten kinetics with both enzyme deactivation and substrate protection. An increasing temperature profiles are determined for different operating periods, residence times and glucose conversions. The temperature increase with time is very small at low degree of glucose conversion and at relatively long residence time. The temperature rise with time increases at high degree of conversion and at relatively short residence time.     

Non-porous magnetic micro-particles: comparison to porous enzyme carriers for a diffusion rate-controlled enzymatic conversion

In this study the kinetics of conversion of a low-soluble substrate by an immobilized enzyme was investigated with respect to the diffusion limitation within porous and non-porous carriers. Non-porous micro-magnetic beads in comparison to conventional porous supports like Eupergit and Sepharose were tested. Due to their small diameters and their magnetic properties, micro-magnetic beads are especially applicable in diffusion rate-controlled processes in biological suspensions. The enzymatic reaction studied was the conversion of emulsified dirhamnolipid by immobilized Naringinase from Penicillium decumbens to monorhamnolipid and L-rhamnose. Taking into account mass transfer phenomena, the variation of the reaction effectiveness factor with increasing enzyme loading was estimated and compared with experimental efficiencies utilizing different enzyme loaded immobilized preparations. For comparison, carrier activities were also determined with the model substrate p-nitro-phenyl-rhamnoside. Intrinsic enzyme activities were thereby evaluated for porous supports. Highest specific activities were obtained with the micro-magnetic beads. These non-porous micro-beads demonstrated to be the most suitable carrier for bioconversion of a low-soluble substrate like rhamnolipids, where mass diffusional resistances in the three-phase reaction process are completely overcome. However, the smaller particle surface available limited the specific activity obtained at high protein loadings.     

Application of two component biodegradable carriers in a particle-fixed biofilm airlift suspension reactor: development and structure of biofilms

Two component biodegradable carriers for biofilm airlift suspension (BAS) reactors were investigated with respect to development of biofilm structure and oxygen transport inside the biofilm. The carriers were composed of PHB (polyhydroxybutyrate), which is easily degradable and PCL (caprolactone), which is less easily degradable by heterotrophic microorganisms. Cryosectioning combined with classical light microscopy and CLSM was used to identify the surface structure of the carrier material over a period of 250 days of biofilm cultivation in an airlift reactor. Pores of 50 to several hundred micrometers depth are formed due to the preferred degradation of PHB. Furthermore, microelectrode studies show the transport mechanism for different types of biofilm structures, which were generated under different substrate conditions. At high loading rates, the growth of a rather loosely structured biofilm with high penetration depths of oxygen was found. Strong changes of substrate concentration during fed-batch mode operation of the reactor enhance the growth of filamentous biofilms on the carriers. Mass transport in the outer regions of such biofilms was mainly driven by advection.     

A continuous membrane bioreactor for ester synthesis in organic media: I. Operational characterization and stability

The feasibility of continuous ester synthesis in a membrane bioreactor (MBR) by a recombinant cutinase from Fusarium solani pisi was investigated, using the optimal conditions previously attained by medium engineering. The objective was to analyze the MBR behavior as a differential or an integral reactor. The main component of the reactor was an anisotropic ceramic membrane with 15,000 NMWCO. The operating variables included the influence of substrates ratio and flow rate on the conversion degree and on the productivity. The highest conversion degree was obtained using 1M of hexanol and 0.1M of butyl acetate as acyl donor. The use of these substrate concentrations led to a conversion degree of 79.3% and a specific productivity of 41 g hexyl acetate/(d x g cutinase), when the permeate flow rate was 0.025 mL/min. The increase of flow rate to 0.4 mL/min decreased the conversion to 35.6%, although the productivity was enhanced to 294 g product/day x g enzyme. The MBR characterization involved the calculations of mass balance, recirculation rate, conversion per pass, number of cycles, and hydraulic residence time. The operational stability was also evaluated in a longterm experiment over 900 hours and the enzyme half-life was estimated to be approximately 2 years.     

Enhancing operational stability and exhibition of enzyme activity by removing water in the immobilized lipase‐catalyzed production of erythorbyl laurate

Erythorbyl laurate was continuously synthesized by esterification in a packed‐bed enzyme reactor with immobilized lipase from Candida antarctica. Response surface methodology based on a five‐level three‐factor central composite design was adopted to optimize conditions for the enzymatic esterification. The reaction variables, such as reaction temperature (10–70°C), substrate molar ratio ([lauric acid]/[erythorbic acid], 5–15), and residence time (8–40 min) were evaluated and their optimum conditions were found to be 56.2°C, 14.3, and 24.2 min, respectively. Under the optimum conditions, the molar conversion yield was 83.4%, which was not significantly different (P < 0.05) from the value predicted (84.4%). Especially, continuous water removal by adsorption on an ion‐exchange resin in a packed‐bed enzyme reactor improved operational stability, resulting in prolongation of half‐life (2.02 times longer compared to the control without water‐removal system). Furthermore, in the case of batch‐type reactor, it exhibited significant increase in initial velocity of molar conversion from 1.58% to 2.04%/min. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:882–889, 2013     

Kinetic modeling of cellulosic biomass to ethanol via simultaneous saccharification and fermentation: Part I. Accommodation of intermittent feeding and analysis of staged reactors

The model of South et al. [South et al. (1995) Enzyme Microb Technol 17(9): 797-803] for simultaneous saccharification of fermentation of cellulosic biomass is extended and modified to accommodate intermittent feeding of substrate and enzyme, cascade reactor configurations, and to be more computationally efficient. A dynamic enzyme adsorption model is found to be much more computationally efficient than the equilibrium model used previously, thus increasing the feasibility of incorporating the kinetic model in a computational fluid dynamic framework in the future. For continuous or discretely fed reactors, it is necessary to use particle conversion in conversion-dependent hydrolysis rate laws rather than reactor conversion. Whereas reactor conversion decreases due to both reaction and exit of particles from the reactor, particle conversion decreases due to reaction only. Using the modified models, it is predicted that cellulose conversion increases with decreasing feeding frequency (feedings per residence time, f). A computationally efficient strategy for modeling cascade reactors involving a modified rate constant is shown to give equivalent results relative to an exhaustive approach considering the distribution of particles in each successive fermenter.     

Degradation of ribonucleic acid by immobilized ribonuclease.

An immobilized enzyme (pancreatic ribonuclease bound to porous titania) was investigated for the degradation of purified yeast ribonucleic acid as a substrate. The immobilized enzyme is active and stable in the pH range 4--8. Dependence of enzymatic activity on ionic strength, pH, temperature, fluid flow rate, and substrate concentration were investigated. A cumulative fluid residence time of 6 sec is sufficient for 50% substrate conversion at 25 degrees C and pH 7.0. The critical flow rate (i.e., the fluid flow rate necessary to remove film diffusion resistance) approximately doubles with each 10 degree C rise in reaction temperature. The critical flow rates obtained in this study are about 40 times greater than those obtained for a similar study on immobilized glucose oxidase. Arrhenius plots gave activation energies of -9.6 and -7.1 kcal/g mol at pH 4.6 and 7.0, respectively. The work reported herein is a bench-scale investigation of an immobilized enzyme with primary emphasis on the mass transfer and kinetic characteristics of the system. The rapid reaction rates obtainable at relatively low temperatures offer a potential alternative method of purifying yeast single cell protein (SCP) with miminum loss of desired protein. The key questions are how such a system would react in a yeast homogenate, what conditions in such a system must be controlled, and what type of immobilized reactor should be utilized, if such further work continued to show promise.     

A CSTR-hollow-fiber system for continuous hydrolysis of proteins. Factors affecting long-term stability of the reactor

Factors affecting the long-term operational stability of a CSTR-hollow-fiber reactor for continuous hydrolysis of proteins were studied. The activity declined in a stepwise manner during a run. Declining from 92% conversion to 60% conversion in about ten hours at a space time of four minutes. Initial decay appears to be due to leakage of small active fragments of the enzyme mixture (Pronase) through the membrane, and later decay due to thermal degradation and loss of activators such as calcium through the membrane. The rate of buildup of unconverted substrate in the reaction vessel was controlled by operational variables, but did not appear to affect the reactor output or the operation of the reactor. The decay of the reactor could be partially compensated for by appropriate manipulation of the space-time variables.     

Modeling biofilm accumulation and mass transport in a porous medium under high substrate loading

A packed bed biofilm reactor inoculated with pure culture Pseudomonas aeruginosa was run under high substrate loading and constant flow rate conditions. The 3.1-cm-diameter cylindrical reactor was 5 cm in length and packed with 1-mm glass beads. Daily observations of biofilm thickness, influent and effluent glucose substrate concentration, and effluent dissolved and total organic carbon were made during the 13-day experiment. Biofilm thickness appeared to rech quasi-steady-state condition after 10 days. A published biofilm process simulation program (AQUASIM) was used to analyze experimental data. Comparison of observed and simulated variables revealed three distinct phases of biofilm accumulation during the experiment: an initial phase, a growth phase, and a mature biofilm phase. Different combinations of biofilm and mass transport process variables were found to be important during each phase. Biofilm detachment was highly correlated with shear at the biofilm surface during all three phases of biofilm development. (c) 1995 John Wiley & Sons, Inc.     

Development of an ultrahigh-temperature process for the enzymatic hydrolysis of lactose. IV. Immobilization of two thermostable beta-glycosidases and optimization of a packed-bed reactor for lactose conversion.

Recombinant hyperthermostable beta-glycosidases from the archaea Sulfolobus solfataricus (Ss beta Gly) and Pyrococcus furiosus (CelB) were covalently attached onto the insoluble carriers chitosan, controlled pore glass (CPG), and Eupergit C. For each enzyme/carrier pair, the protein-binding capacity, the immobilization yield, the pH profiles for activity and stability, the activity/temperature profile, and the kinetic constants for lactose hydrolysis at 70 degrees C were determined. Eupergit C was best among the carriers in regard to retention of native-like activity and stability of Ss beta Gly and CelB over the pH range 3.0-7.5. Its protein binding capacity of approximately 0.003 (on a mass basis) was one-third times that of CPG, while immobilization yields were typically 80% in each case. Activation energies for lactose conversion by the immobilized enzymes at pH 5.5 were in the range 50-60 kJ/mol. This is compared to values of approximately 75 kJ/mol for the free enzymes. Immobilization expands the useful pH range for CelB and Ss beta Gly by approximately 1.5 pH units toward pH 3.5 and pH 4.5, respectively. A packed-bed enzyme reactor was developed for the continuous conversion of lactose in different media, including whey and milk, and operated over extended reaction times of up to 14 days. The productivities of the Eupergit C-immobilized enzyme reactor were determined at dilution rates between 1 and 12 h(-1), and using 45 and 170 g/L initial lactose. Results of kinetic modeling for the same reactor, assuming plug flow and steady state, suggest the presence of mass-transfer limitation of the reaction rate under the conditions used. Formation of galacto-oligosaccharides in the continuous packed-bed reactor and in the batch reactor using free enzyme was closely similar in regard to yield and individual saccharide components produced.     

Distribution and behavior of sludge in upflow reactors for anaerobic treatment of wastewater

A model has been developed and experimentally checked for the physical behavior of sludge in the blanket in upflow reactors. The model is based on the mass balance for the sludge in the blanket, and can be used to predict the distribution of sludge in an upflow reactor in relation with the gas production, sludge settling characteristics, and the linear fluid velocity in the reactor. The quantitative values of the transport factors that are a measure of the efficiency of the transport of sludge by the fluid streams occurring in the reactor were determined experimentally in reactors of 30- and 200-m(3) volumes. As this was done for wastewater containing lower fatty acids as the main organic pollutants and for sludge with good settling characteristics, the predictive value of the model is limited. It may be used for the second (methane forming) step of anaerobic treatment of wastewater.     

Enzyme Deactivation in Reactors

The influence of enzyme deactivation on substrate conversion in different reactor types is examined. The influence of inter- and intra-particle diffusion on deactivation and on effectiveness factor is analyzed. Optimum temperature operations criterion and policies are presented for reactors with deactivating catalysts. Appropriate examples are provided to highlight the different concepts presented.     

Storage and growth of denitrifiers in aerobic granules: part I. model development

A mathematical model, based on the Activated Sludge Model No.3 (ASM3), is developed to describe the storage and growth activities of denitrifiers in aerobic granules under anoxic conditions. In this model, mass transfer, hydrolysis, simultaneous anoxic storage and growth, anoxic maintenance, and endogenous decay are all taken into account. The model established is implemented in the well-established AQUASIM simulation software. A combination of completely mixed reactor and biofilm reactor compartments provided by AQUASIM is used to simulate the mass transport and conversion processes occurring in both bulk liquid and granules. The modeling results explicitly show that the external substrate is immediately utilized for storage and growth at feast phase. More external substrates are diverted to storage process than the primary biomass production process. The model simulation indicates that the nitrate utilization rate (NUR) of granules-based denitrification process includes four linear phases of nitrate reduction. Furthermore, the methodology for determining the most important parameter in this model, that is, anoxic reduction factor, is established.     

Effect of immobilized enzyme deactivation in fixed- and fluid-bed reactors

A two-parameter theoretical model is developed to evaluate the effect of immobilized enzyme deactivation on substrate conversion in fixed- and fluid-bed reactors under diffusion-free conditions. The method describes a simple reaction in which three different immobilized enzyme deactivation forms are considered, and an expression is developed to evaluate the effect of immobilized enzyme deactivation on yield in a consecutive reaction. Comparison of reactor performances for the two reactor types reduces to a comparison of the appropriate dimensionless parameters. The practical implications of the development are illustrated through an example.     

Ceramic membrane microfilter as an immobilized enzyme reactor.

This study investigated the use of a ceramic microfilter as an immobilized enzyme reactor. In this type of reactor, the substrate solution permeates the ceramic membrane and reacts with an enzyme that has been immobilized within its porous interior. The objective of this study was to examine the effect of permeation rate on the observed kinetic parameters for the immobilized enzyme in order to assess possible mass transfer influences or shear effects. Kinetic parameters were found to be independent of flow rate for immobilized penicillinase and lactate dehydrogenase. Therefore, neither mass transfer nor shear effects were observed for enzymes immobilized within the ceramic membrane. Both the residence time and the conversion in the microfilter reactor could be controlled simply by regulating the transmembrane pressure drop. This study suggests that a ceramic microfilter reactor can be a desirable alternative to a packed bed of porous particles, especially when an immobilized enzyme has high activity and a low Michaelis constant.