A tubular segmented-flow bioreactor for the infection of insect cells with recombinant baculovirus

A continuous process of insect cell (S f9) growth and baculovirus infection is tested with the sequential combination of a CSTR and a tubular reactor. A tubular infection reactor enables continuous introduction of baculovirus and therefore avoids the ‘passage effect’ observed in two-stage CSTR systems. Moreover, a tubular reactor can be used to test cell infection kinetics and the subsequent metabolism of infected insect cells. Unlike batch and CSTR culture, cells in a horizontally positioned tubular reactor settle due to poor mixing. We have overcome this problem by alternately introducing air bubbles and media and by maintaining a linear velocity sufficient to keep cells suspended. This article addresses the development of the tubular reactor and demonstrates its use as an infection system that complements the two-stage CSTR. This revised version was published online in July 2006 with corrections to the Cover Date.     

Comparative study of reactor performance for the resolution of d,l-amino acids

The racemic resolution of l-valine and l-serine by fungal aminoacylase has been evaluated by comparing the performance of various reactor configurations including an anion exchange nylon tangential flow membrane reactor, a tubular reactor with aminoacylase adsorbed onto DEAE-Sephadex as support and a continuous stirred tank reactor with enzyme recycling using a flat ultrafiltration module (CSTR/UF). Among the substrates tested, the N-chloroacetyl-d,l-amino acids were the preferred substrates, showing the highest catalytic efficiency (V_m/K_m).Optimum reactor operational conditions obtained in discontinuous assays were selected to study the behaviour of the reactors in a continuous mode. DEAE-Sephadex loaded six-fold more enzyme than anion exchange nylon (60 and 10 gE/litre, respectively, related to reactor volume), whereas enzyme concentration within the CSTR/UF reactor was limited only by enzyme solubility.The tangential flow membrane reactor configuration with a 10 g/litre enzyme concentration produced higher productivity values (0·35 kg l-valine/litre per day, and 80% conversion degree) and operational stability (t = 161 days) than the CSTR/UF reactor (0·24 kg l-valine/litre per day, and 80% conversion degree) performing with the same enzyme concentration. The tubular reactor with the enzyme adsorbed onto DEAE-Sephadex (60 g/litre enzyme load) showed higher productivity values (1·9 kg l-valine/litre per day, and 80% conversion degree) and operational stability (t = 70 days) than the CSTR/UF reactor (1·05 kg l-valine/litre per day, and 80% conversion degree). However, the CSTR/UF reactor was the preferred configuration, as it had the highest enzyme load and productivity (1·95 kg l-valine/litre per day of reactor volume, and 80% conversion degree), a half-life of 55 days at 50°C, and the possibility of easy continuous enzyme addition.     

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.     

Modeling and simulation of enzymatic gluconic acid production using immobilized enzyme and CSTR–PFTR circulation reaction system

Objectives

Production of gluconic acid by using immobilized enzyme and continuous stirred tank reactor-plug flow tubular reactor (CSTR–PFTR) circulation reaction system.

Results

A production system is constructed for gluconic acid production, which consists of a continuous stirred tank reactor (CSTR) for pH control and liquid storage and a plug flow tubular reactor (PFTR) filled with immobilized glucose oxidase (GOD) for gluconic acid production. Mathematical model is developed for this production system and simulation is made for the enzymatic reaction process. The pH inhibition effect on GOD is modeled by using a bell-type curve.

Conclusions

Gluconic acid can be efficiently produced by using the reaction system and the mathematical model developed for this system can simulate and predict the process well.

     

Sustained oscillations in glycolysis: an experimental and theoretical study of chaotic and complex periodic behavior and of quenching of simple oscillations

We report sustained oscillations in glycolysis conducted in an open system (a continuous-flow, stirred tank reactor; CSTR) with inflow of yeast extract as well as glucose. Depending on the operating conditions, we observe simple or complex periodic oscillations or chaos. We report the response of the system to instantaneous additions of small amounts of several substrates as functions of the amount added and the phase of the addition. We simulate oscillations and perturbations by a kinetic model based on the mechanism of glycolysis in a CSTR. We find that the response to particular perturbations forms an efficient tool for elucidating the mechanism of biochemical oscillations.     

Consecutive immobilized enzymatic reactions in continuous stirred tank reactor systems

The performance characteristics of two-enzyme reaction in a continuous stirred tank reactor (CSTR) are analytical investigated in this work. A model is formulated to describe the substrate concentration variations by taking into account the external and internal diffusion resistances. It is found that the reaction system exhibits the characteristics of reaction control or diffusion control depending on the operating conditions. The single CSTR model is also extended to describe the multiple CSTR system. The latter model enables the prediction of the number of CSTRs in series required to achieve a prescribed substrate conversion.     

Comparative kinetic behavior of nitrifiers with different growth environments

A batch feed study using nitrifiers that had been continuously acclimated under a low-ammonia environment showed that a sudden change of ammonia concentration resulted in sluggish physiological adaptation and biochemical reaction of nitrifiers (i.e., indicated by the parameter specific oxygen utilization rate). When the one-stage continuous-stirred tank reactor (CSTR) system was maintained at a short microbial cell residence time and a high volumetric loading rate, an accumulation of nitrite occurred. Under such circumstances, ammonia and nitrite oxidation both limit overall nitrification at different stages of the process. Batch studies with biomass respectively removed from the front and rear reactors (i.e., high-ammonia and low-ammonia growth environments) of a two-stage CSTR system showed that the estimated kinetic parameters for nitrifiers with the low-ammonia growth environment were 0.3-0.8-fold lower than those for nitrifiers with the high-ammonia growth environment, possibly leading to inaccurate model simulation results. Accordingly, biomass removed from a CSTR system that had been operated continuously to grow bacteria under a high-substrate environment should be loaded into the batch reactor if the batch reactor method is to be used to estimate kinetic parameters.     

A comparison between the fermentative activities of free and Ca-alginate-entrapped cells of saccharomyces cerevisiae

The continuous ethanol production by free and Ca-alginate-entrapped cells of the yeast Saccharomyces cer. IBT H 191 was investigated in a membrane reactor and in a CSTR, respectively. An empiric productivity model for free-cell fermentation was found to be valid for immobilized cells if no external diffusion resistance existed within the reactor. In such a case the fermentative power of the immobilized cells was identical with this one of the free cells. A change of the ethanol-tolerance behaviour of the cells as a result of the cell-immobilization did not occur.     

Studies on reaction techniques concerning reactor design for the anaerobic degradation of complex substrates with the example of the methanation of effluents in the fermentation industry

Two fixed-bed loop reactors were used to evaluate singleand separated-phase anaerobic treatments of a high strength waste-water from ethanol fermentation. The one-phase system consisted of an anaerobic fixed-bed loop reactor containing both acidogenic as well as methanogenic populations allowing a complete conversion of the carbon source into gaseous end products and biomass.The two-phase system consisted of a second fixed-bed loop reactor operated as a methanogenic unit, which was proceeded by a CSTR for acidification, both connected in series allowing sequential acidogenesis and methanogenesis of the organic components. The reactors were operated under steady state and variable process conditions. By gradually increasing the feed supply in both systems, maximum turnover of COD was determined.The separated-phase system consistently gave a better quality effluent with lower suspended solids and total COD. Maximum loading rates and COD elimination of the methanogenic phase of the two-phase system was over two times higher than that of the one-phase system. Process stability was also higher.On overloading the methane reactor of the two phase system accumulation of different fatty acids within the reactor was observed. Hydrogen concentration in the biogas can be used as a reliable indicator for system overloadings. At least, continuous online monitoring of hydrogen in the methanogenic reactor gas should provide a convenient alternative to other analyses for process control.     

Effect of wall growth on the kinetic modeling of nitrite oxidation in a CSTR

A simple kinetic model was developed for describing nitrite oxidation by autotrophic aerobic nitrifiers in a continuous stirred tank reactor (CSTR), in which mixed (suspended and attached) growth conditions prevail. The CSTR system was operated under conditions of constant nitrite feed concentration and varying volumetric flow rates. Experimental data from steady-state conditions in the CSTR system and from batch experiments were used for the determination of the model's kinetic parameters. Model predictions were verified against experimental data obtained under transient operating conditions, when volumetric flow rate and nitrite feed concentration disturbances were imposed on the CSTR. The presented kinetic modeling procedure is quite simple and general and therefore can also be applied to other mixed growth biological systems.     

Effect of tubular inhomogeneities on filter properties of thick ascending limb of Henle's loop

We used a simple mathematical model of rat thick ascending limb (TAL) of the loop of Henle to predict the impact of spatially inhomogeneous NaCl permeability, spatially inhomogeneous NaCl active transport, and spatially inhomogeneous tubular radius on luminal NaCl concentration when sustained, sinusoidal perturbations were superimposed on steady-state TAL flow. A mathematical model previously devised by us that used homogeneous TAL transport and fixed TAL radius predicted that such perturbations result in TAL luminal fluid NaCl concentration profiles that are standing waves. That study also predicted that nodes in NaCl concentration occur at the end of the TAL when the tubular fluid transit time equals the period of a periodic perturbation, and that, for non-nodal periods, sinusoidal perturbations generate non-sinusoidal oscillations (and thus a series of harmonics) in NaCl concentration at the TAL end. In the present study we find that the inhomogeneities transform the standing waves and their associated nodes into approximate standing waves and approximate nodes. The impact of inhomogeneous NaCl permeability is small. However, for inhomogeneous active transport or inhomogeneous radius, the oscillations for non-nodal periods tend to be less sinusoidal and more distorted than in the homogeneous case and to thus have stronger harmonics. Both the homogeneous and non-homogeneous cases predict that the TAL, in its transduction of flow oscillations into concentration oscillations, acts as a low-pass filter, but the inhomogeneities result in a less effective filter that has accentuated non-linearities.     

Identification of possible two-reactant sources of oscillations in the Calvin photosynthesis cycle and ancillary pathways

A systematic search for possible sources of experimentally observed oscillations in the photosynthetic reaction system has been performed by application of recent theoretical results characterizing the transient-state rate behaviour of metabolic reactions involving two independent concentration variables. All subsystems involving two independent reactants in metabolically fundamental parts of the Calvin cycle and the ancillary pathways of starch and sucrose synthesis have been examined in order to decide on basis of their kinetic and stoichiometric structure whether or not they may trigger oscillations. The results show that no less than 20 possible oscillators can be identified in the examined reaction system, only three of which have been previously considered as potential sources of experimentally observed oscillations. This illustrates the superiority of the method now applied over those previously used to identify possible two-reactant sources of metabolic oscillations and indicates that there should be no difficulty in complex metabolic pathways to point to a multitude of interactions that may trigger an oscillatory rate behaviour of the system.     

Simultaneous nitrification-denitrification achieved by an innovative internal-loop airlift MBR: comparative study

This study assessed the performance of different single-stage continuous aerated submerged membrane bioreactors (MBR) for nitrogen removal. Almost complete nitrification was achieved in each MBR irrespective of operating mode and biomass system. Denitrification was found to be the rate-limiting step for total nitrogen (T-N) removal. The MBR with internal-loop airlift reactor (ALR) configuration performed better as regards T-N removal compared with continuous stirred-tank reactor (CSTR). It was demonstrated that simultaneous nitrification and denitrification (SND) is the mechanism leading to nitrogen removal and the contribution of microenvironment on SND is more remarkable for the MBRs with hybrid biomass. Macroenvironment analyses showed that gradient distribution of dissolved oxygen (DO) level in airlift MBRs imposed a significant effect on SND. Higher mixed liquor suspended solid (MLSS) concentration led to the improvement in T-N removal by enhancing anoxic microenvironment. Apparent nitrite accumulation coupled with higher nitrogen reduction was accomplished at MLSS concentration exceeded 12.6 g/L.     

Two-stage anaerobic fermentation of organic waste in CSTR and UFAF-reactors.

The mechanically separated liquid fraction of organic waste from households was used as a substrate for anaerobic fermentation. A two-step system consisting of a 2001 continuously stirred tank reactor (CSTR) and a 501 upflow anaerobic filter filled with glass foam pearls was constructed. The CSTR was operated for 5 months with a loading rate of 9.8 kg CSB m(-3) day(-1). At a resulting hydraulic retention time (HRT) of 24 days, 68% COD was degraded and a gas productivity of 4.0 m3 m(-3) day(-1) was achieved. Further digestion of the CSTR output was separately optimised in a 20 l-UFAF and based on these results a 50 l-UFAF was connected to the CSTR. At a resulting hydraulic retention time (HRT) of 6 days 38% COD was degraded and a gas productivity of 1.8 m3 m(-3) day(-1) was achieved with the 50 l-UFAF. Thus, the overall degradation efficiency of the two-phase system was 80%. The methane content (61%) of the biogas produced in the 50 l-UF     

Enzymatic resolution of (S)-(+)-naproxen in a trapped aqueous-organic solvent biphase continuous reactor

A trapped aqueous-organic biphase system for the continuous production of (S)-(+)-2-(6-methoxy-2-naphthyl) propionic acid (Naproxen) has been developed. The process consists of a stereoselective hydrolysis of the racemic Naproxen methyl ester by Candida rugosa lipase in a trapped aqueous-organic biphase system. The reaction has been carried out in a laboratory-scale continuous-flow stirred tank reactor (CSTR). The staring material has been supplied in and remaining substrate recovered by organic phase. YWG-C(6)H(5), a poorly polar synthetic support, has been employed to immobilize the lipase and to restrict the aqueous phase. Lipase immobilized on YWG-C(6)H(5) containing aqueous phase has been added into the CSTR to catalyze the hydrolysis. A dialysis membrane tube containing a continuous flow closed-loop buffer has been applied in the CSTR for the extraction of product and recruiting of the aqueous part consumed. Various reaction conditions have been studied. The activity of immobilized enzyme was effected by the polarity of support, the substrate concentration, logP value of organic phase and the product inhibition. At steady-state operating conditions, an initial conversion of 35% has been obtained. The CSTR was allowed to operate continuously for 60 days at 30 degrees C with a 30% loss of activity. The hydrolysis reaction yielded (S)-(+)-Naproxen with >90% enantiomeric excess and overall conversion of 30%.     

A comparative study of immobilized amyloglucosidase in a packed bed reactor and a continuous feed stirred tank reactor

The catalytic activity of amyloglucosidase covalently attached to DEAE-cellulose was studied in a packed bed reactor and a continuous feed stirred tank reactor (CSTR) for the reaction maltose → glucose. At low flow rates mass-transfer limitations in the bed reactor lead to lower conversions for this reactor compared to the CSTR. Simple theoretical expressions for these reactors were compared with the experimental results. There are significant differences between the kinetic parameters and pH profile of the immobilized and free enzyme. The immobilized enzyme also showed greater stability at 50°C than did free amyloglucosidase. The temperature dependence of the reaction rate was the same for immobilized and free enzyme.     

Enzyme hydrolysis of plasma proteins in a CSTR ultrafiltration reactor: Performances and modeling

By investigating the effects of four operating variables-volume (V), Ultrafiltration flux (J), enzyme concentration (E), and substrate concentration (S)-on capacity (K) and conversion rate (epsilon) of a hollow fiber CSTR, the performances of the CSTR and the kinetic constants of the reaction were determined. A model which takes into account the course of fractional conversion (X) according to the modified space-time parameter, tau (integrated form of V, J, S, and E), was devised by employing the relationship to integrate the equation for the reaction rate of the CSTR and the expression of the modified space time. Correlation of this model and the experimentally obtained results demonstrates that the characteristics for an ultrafiltration membrane reactor for enzymatic hydrolysis by alcalase of plasma proteins are close to those of an ideal CSTR. Optimal scaling up, however, remains dependent on the compromise which may be obtained between capacity and the conversion rate.     

Turnover characteristics in continuous l-lysine fermentation

The turnover characteristics of a microbial bioreactor were comparatively investigated as a closed (batch) and a continuous stirred tank reactor (CSTR) open system, using a 2-l fermentor. Corynebacterium glutamicum (ATCC 21544) was chosen as the microorganism since it has the ability to produce l-lysine. Parameters measured were l-lysine production rates, glucose consumption rates and biomass production rates as a function of dilution rate, bioreactor volume and biomass concentration. The modes of microbial cell behaviour under steady-state and transition-state conditions were examined. Investigations on scaling properties of the CSTR system were also aimed at comparing scaling or allometry of metabolic rates in organisms that are also open energy dissipative systems.This investigation was first presented at the 10th Dechema-Jahrestagung der Biotechnologen, 1–3 June 1992, Karlsruhe, Germany     

Hydrolysis of liquefied corn starch in a membrane reactor

The objective of this study was to develop a continuous hydrolysis process for the enzymatic saccharification of liquefied corn starch using a membrane reactor. A residence time distribution study confirmed that the membrane reactor could be modeled as a simple continuous stirred tank reactor (CSTR). Kinetic studies indicated that the continuous reactor operated in the first-order region with respect to substrate concentration at substrate concentrations greater than 200 g/L. At a residence time of 1 h and an enzyme concentration of 1 g/L, the maximum reaction velocity (V(m)) was 3.86 g glucose/L min and the apparent Michaelis constant (K(m) (')) was 562 g/L. The K(m) (') value for the continuous reactor was 2-7 times greater than that obtained in a batch reactor.Kinetic data were fit to a model based on the Michaelis-Menten rate expression and the design equation for a CSTR. Application of the model at low reactor space times was successful. At space times of 6 min or less, the model predicted the reactor's performance reasonably well. Additional work on the detection and quantitation of reversion products formed by glucoamylase is required. Isolation, detection, and quantitation of reversion products by HPLC was difficult. Detailed analysis on the formation of these reversion products could lead to better reactor designs in the future.     

Quantitative comparison of stability of ANAMMOX process in different reactor configurations

A new stability evaluating system for ANAMMOX comprising three instability indices i.e. coefficient of variation ratio, coefficient of range ratio and coefficient of regression function derivative was established. Three lab-scale ANAMMOX reactors viz upflow anaerobic sludge blanket (UASB) reactor, upflow stationary fixed film (USFF) reactor and anaerobic sequencing batch reactor (ASBR) were compared for their stability based on the established criterion against the hydraulic and substrate concentration shocks. The results showed that all ANAMMOX reactors under investigation were more tolerant to the hydraulic shock than substrate concentration shock. The UASB reactor was the most stable reactor configuration towards substrate concentration shock, followed by the USFF reactor and ASBR. However, the ASBR proved the most tolerant to hydraulic shock, followed by the UASB reactor and USFF reactor. In terms of stability, UASB reactor was more suitable configuration compared with USFF reactor. The instability indices proved to be effective and explicit for the evaluation of ANAMMOX systems.