Comparison of a batch, fed-batch and continuously operated stirred-tank reactor for the enzymatic synthesis of (R)-mandelonitrile by using a process model

The suitability of a batch, fed-batch and continuously operated stirred-tank reactor for the enzymatic production of (R)-mandelonitrile in an aqueous-organic biphasic system was investigated by using a process model. The considered biphasic system is 10-50% (v/v) 100 mM sodium citrate buffer of pH 5.5 dispersed in methyl tert-butyl ether. The constraints were that 750 moles of benzaldehyde per cubic meter should react towards (R)-mandelonitrile with an enantiomeric excess of 99% and a conversion of 98%. A continuously operated stirred-tank reactor could not meet the constraints, but the production in a batch or fed-batch reactor was feasible. The choice for a batch or fed-batch reactor is dependent on the influence of the costs for reactor operation and for the enzyme on the product costs. The choice for operating at a small or large aqueous-phase volume fraction is dependent on the costs and reusability of the enzyme. The volumetric productivity is maximal when operating as batch. The enzymatic productivity and turnover are maximal when operating as fed batch. In the fed-batch mode, the enzymatic productivity increased by 24-37%, the turnover increased by 50-60% and the volumetric productivity decreased by 33-71% as compared to a batch reactor. By enhancement of mass transfer both the volumetric and enzymatic productivity can be increased considerably, while the turnover is only slightly decreased.     

Industrial choices for protein production by large-scale cell culture

Traditional barriers to large-scale mammalian culture have now been addressed, with the standard stirred-tank reactor emerging as industry's technology of choice. The issues of adapting cells to suspension culture, shear sensitivity and oxygen supply have been largely resolved. But for many low-volume and specialty applications, such as the production of viral vaccines and gene therapies, reactor technology remains diversified, with reactor types ranging from roller bottles to stacked plates and hollow fibers.     

Deacetylation of cephalosporins in batch and continuous reactors

We wish to report here the principal observations and conclusions concerning the deacetylation of three substrates, 7-ACA¹), cephalosporin C and 2-methoxyethyl acetate by the immobilized esterase from B. subtilis (Konecny and Voser, 1977) in three types of reactors: A stirred tank and recirculation reactor, both operated in the batch mode under pH-stat conditions, and a continuous packed bed, operated with excess buffer in the feed. The results, formulated in terms of simple empirical equations, are discussed in relation to the choice of carriers, reactor efficiency, scale up and other factors governing the choice of reactor. Certain general considerations apply to all reactions which generate acid and involve a catalyst whose activity and life are adversely effected by low pH.     

Lipase kinetics: hydrolysis of triacetin by lipase from Candida cylindracea in a hollow-fiber membrane reactor

The aptitude of a hollow-fiber membrane reactor to determine lipase kinetics was investigated using the hydrolysis of triacetin catalyzed by lipase from Canadida cylindracea as a model system. The binding of the lipase to the membrane appears not to be very specific (surface adsorption), and probably its conformation is hardly altered by immobilization, resulting in an activity comparable to that of the enzyme in its native form. The reaction kinetics defined on the membrane surface area were found to obey Michaelis-Menten kinetics. The specific activity of the lipase in the membrane reactor was found to be significantly higher than in an emulsion reactor. The activity and stability of the enzyme immobilized on a hydrophilic membrane surface seem not to be influenced significantly by the choice of the membrane material. The hollow-fiber membrane reactor is a suitable tool to assess lipase kinetics in a fast and convenient way.     

New materials and technology for cell immobilization.

The choice of support materials for immobilizing cells is rapidly expanding. The literature that has appeared over the past year suggests that hydrogels will remain the first choice for the forseeable future, even though they are associated with many widely recognized problems. There is increasing interest in the use of tougher polymeric materials, and especially of inorganic ceramic supports. However, the most suitable cell support can be selected only after the process or form of reactor in which it is to be used has been assessed.     

Immobilization of acetyl-CoA:arylamine N-acetyltransferase and the preparation of an enzyme reactor for the synthesis of N-[11C]acetylserotonin

The enzyme arylamine acetyltransferase (acetyl-CoA:arylamine N-acetyltransferase, EC 2.3.1.5) from pigeon liver is immobilized onto differently derivatized controlled pore glass beads. Different silanes, spacer arms and reactive end-groups were tested, and immobilized enzyme stability tests were performed. From these experiments, the method of choice was selected: immobilization on controlled pore glass beads (24 nm pore size, 75-125 microns particle size) derivatized with gamma-aminopropyl and glutaraldehyde as the reactive end group. The kinetic properties of an enzyme reactor were investigated and optimized. The goal was to obtain a rapid high-yield conversion of 0.5-1 mumol acetyl-CoA to N-acetylserotonin, so that the reactor is useful for the 11C-labelling of N-acetylserotonin. Using an enzyme reactor (9.8 x 0.5 cm i.d.) containing 4.6 U active arylamine acetyltransferase immobilized onto 930 mg carrier, a 70% conversion of acetyl-CoA was obtained within 4 min.     

Potential application of monolith packed columns as bioreactors, control of biofilm formation

Monolith reactors combine good mass transfer characteristics with low-pressure drop, the principle factors affecting the cost effectiveness of industrial processes. Recently, these specific features of the monolith reactors have drawn the attention toward the application of the monolith reactor in multiphase reaction systems. In this study, we explore the potential application of monolith reactors as bioreactor requiring gas-liquid mass transfer for substrate supply. It is demonstrated on theoretical grounds that the monolith reactor is a competitive alternative to conventional gas-liquid bioreactors such as stirred tanks, packed beds, and airlift bioreactors because it allows for a significant reduction of the energy dissipation that is normally required for gas-liquid contacting. A potential problem of monolith reactors for biological processes is clogging due to biofilm formation. This paper presents experimental results of a study into the formation and possible removal of biofilms during operation of a monolith reactor as suspended cells bioreactor. The results indicate that biofilm formation may be minimized and postponed by a proper choice of operating conditions. Periodic biofilm removal could straightforwardly be achieved by rinsing with water at moderate pressures and allows for stable operation for prolonged periods of time.     

Dynamic fuzzy model based predictive controller for a biochemical reactor

The kinetics of bioreactions often involve some uncertainties and the dynamics of the process vary during the course of fermentation. For such processes, conventional control schemes may not provide satisfactory control performance and demands extra effort to design advanced control schemes. In this study, a dynamic fuzzy model based predictive controller (DFMBPC) is presented for the control of a biochemical reactor. The DFMBPC incorporates an adaptive fuzzy modeling framework into a model based predictive control scheme to derive analytical controller output. The DFMBPC has the flexibility to opt with various types of fuzzy models whose choice also lead to improve the control performance. The performance of DFMBPC is evaluated by comparing with a fuzzy model based predictive controller (FMBPC) with no model adaptation and a conventional PI controller. The results show that DFMBPC provides better performance for tracking setpoint changes and rejecting unmeasured disturbances in the biochemical reactor.     

Optimal Bioreactor Operational Policies for the Enzymatic Hydrolysis of Sugarcane Bagasse

The consolidation of the industrial production of second-generation (2G) bioethanol relies on the improvement of the economics of the process. Within this general scope, this paper addresses one aspect that impacts the costs of the biochemical route for producing 2G bioethanol: defining optimal operational policies for the reactor running the enzymatic hydrolysis of the C6 biomass fraction. The use of fed-batch reactors is one common choice for this process, aiming at maximum yields and productivities. The optimization problem for fed-batch reactors usually consists in determining substrate feeding profiles, in order to maximize some performance index. In the present control problem, the performance index and the system dynamics are both linear with respect to the control variable (the trajectory of substrate feed flow). Simple Michaelis–Menten pseudo-homogeneous kinetic models with product inhibition were used in the dynamic modeling of a fed-bath reactor, and two feeding policies were implemented and validated in bench-scale reactors processing pre-treated sugarcane bagasse. The first approach applied classical optimal control theory. The second policy was defined with the purpose of sustaining high rates of glucose production, adding enzyme (Accellerase® 1500) and substrate simultaneously during the reaction course. A methodology is described, which used economical criteria for comparing the performance of the reactor operating in successive batches and in fed-batch modes. Fed-batch mode was less sensitive to enzyme prices than successive batches. Process intensification in the fed-batch reactor led to glucose final concentrations around 200 g/L.     

Continuous proteolysis with a stabilized protease. II. Continuous experiments

Batch proteolysis experiments were performed in order to choose a protein–protease system to prepare a correct hydrolysate suitable for the enrichment of soft-drinks. The system eventually studied was casein–Alcalase. Comparative batch and continuous proteolysis of casein by Alcalase showed that the reaction, which does not exactly follow first order kinetics with respect to the substrate concentration, is inhibited by the reaction products. Furthermore, experiments were done in order to determine the reaction conditions (pH 8.8 in the reactor, casein concentration 5%, 40°C). Determining the molecular weight of Alcalase (43,000) suggested the choice of ultrafiltration membrane PM 30. Studies of continuous proteolysis with the chemically stabilized enzyme retained by the ultrafiltration reactor showed that protease reuse for seven days at 40°C is possible and that the growth of microorganisms is practically inhibited under these conditions. Gel chromatography showed the molecular weight range of the peptides to be less than 2,000. Triangular taste tests showed that the threshold identification concentration of the dry hydrolysate in orange juice is about 0.65%.     

Reaction Engineering Aspects of Microbial Mercury Removal

Mercury‐resistant microorganisms are widespread in natural environments and can effectively be used to demercurize Hg(II)‐contaminated wastewaters as was already demonstrated on an industrial scale. The aim of this paper is to find the performance limits with regard to Hg(II) loadings D c_Hg,in (dilution rate × Hg(II) inlet concentration) and residual Hg(II) at the reactor outlet and to provide a reasonable basis for an optimal and safe process design. To this end, comprehensive studies were carried out with different single microbes (natural isolates and a genetically engineered strain) as well as microbial consortia in batch and continuous stirred reactors and fixed beds with microorganisms immobilized as films. The rate of the biotransformation (reduction of inorganically and organically bound Hg(II) to elemental Hg(0)) was found to follow a uniform mechanism with inhibition kinetics (Haldane type). Both reactor types are able to cope with high Hg(II) loadings and yield conversions up to 98 %. The stirred vessel is particularly suited for high c_Hg,in but restricted to low D (D < μ_max), while the fixed bed can be operated at high D, say 10 h^–1, but can only deal with c_Hg,in < 10 mg/L due to the limited Hg(II) tolerance of microorganisms. The loading limitations can be removed by appropriate recycle flows for both reactor types. However, irrespective of reactor type used, the residual Hg at the outlet cannot be reduced below the legal discharge limit (50 μg/L) mainly owing to the adsorption of Hg(II) on biomass. Therefore, a separation step following the reactor is required (sand bed, activated carbon filter). Comparing the reactor types exhibits the superiority of the fixed bed system due to its simpler construction, easier operation and higher cost effectiveness. Furthermore, the fixed bed shows better flexibility and robustness to extreme loadings. This justifies a posteriori the choice of a fixed bed reactor applied in the technical process.     

In vitro DNA-based predator-prey system with oscillatory kinetics

A coupled system of two isothermal in vitro DNA/RNA amplification reactions using different primers is modeled kinetically with realistic rate parameters and shown to exhibit oscillatory behavior in a flow reactor. One of the two isothermal amplification reactions acts as a predator of the other, the prey. The mechanism of the oscillatory behavior is analyzed in terms of a hierarchy of kinetic models. The work provides an insight into the choice of parameters for experiments. The latter are important in providing detailed insight into the complex processes of ecological interactions and their evolution.     

Microalgal reactors: a review of enclosed system designs and performances

One major challenge to industrial microalgal culturing is to devise and develop technical apparata, cultivation procedures and algal strains susceptible of undergoing substantial increases in efficiency of use of solar energy and carbon dioxide. Despite several research efforts developed to date, there is no such thing as "the best reactor system"- defined, in an absolute fashion, as the one able to achieve maximum productivity with minimum operation costs, irrespective of the biological and chemical system at stake. In fact, choice of the most suitable system is situation-dependent, as both the species of alga available and the final purpose intended will play a role. The need of accurate control impairs use of open-system configurations, so current investigation has focused mostly on closed systems. In this review, several types of closed bioreactors described in the technical literature as able to support production of microalgae are comprehensively presented and duly discussed, using transport phenomenon and process engineering methodological approaches. The text is subdivided into subsections on: reactor design, which includes tubular reactors, flat plate reactors and fermenter-type reactors; and processing parameters, which include gaseous transfer, medium mixing and light requirements.     

Rechnergestützte Führung von Fermentationsprozessen,Teil 2

In the first part of the publication [1] an algorithm for the adaptation of the static optimum was represented. This part demonstrates the application of this algorithim to a specific problem. The problems, which are connected with the application of the choice of the object function, of making the process model available and of the realizable economic effects are elaborated. The example of the tested on-line control of a strirred tank reactor shows the advantages and disadvantages of the used algorithm.     

A novel entrapping matrix for yeast-catalyzed ethanol fermentation

Saccharomyces cerevisiae cells were immobilized by cell suspension homogenization with a soluble prepolymeric matrix, forming insoluble polymer entrapped homogeneously distributed cells. The fermentation capacity of this system was found to be maximal at 30°C and relatively pH insensitive. A packed column reactor was used to test this biocatalyst's operational sensitivity to key fermentation variables. The results obtained as well as the characteristics of the polymer, prepared by an epoxy resin and diaminopolyethylene oxide polymerization, present this method as a good choice for ethanol production.     

Chemo-enzymatic epoxidation-process options for improving biocatalytic productivity

The reactor choice is crucial when designing a process where inactivation of the biocatalyst is a problem. The main bottleneck for the chemo-enzymatic epoxidation has been found to be enzyme inactivation by the hydrogen peroxide, H(2) O(2) , substrate. In the work reported here, the effect of reaction parameters on the reaction performance have been investigated and used to establish suitable operating strategies to minimize the inactivation of the enzyme, using rapeseed methyl ester (RME) as a substrate in a solvent-free system. The use of a controlled fed-batch reactor for maintaining H(2) O(2) concentration at 1.5 M resulted in increased productivity, up to 76 grams of product per gram of biocatalyst with higher retention of enzyme activity. Further investigation included a multistage design that separated the enzymatic reaction and the saturation of the RME substrate with H(2) O(2) into different vessels. This setup showed that the reaction rate as well as enzyme inactivation is strongly dependent on the H(2) O(2) concentration. A 20-fold improvement in enzymatic efficiency is required for reaching an economically feasible process. This will require a combination of enzyme modification and careful process design.     

Thermophilic vs. mesophilic bioleaching process performance

Abstract: Mesophilic and thermophilic mineral oxidising microbial cultures were compared for their capacity to leach both a complex pyritic and an arsenopyritic ore, aiming at copper and gold recovery, respectively. The mesophilic cultures are primarily based on the activity of Thiobacillus -type microorganisms, while the thermophile characteristics place them in the sulphur metabolizers branch df Archaebacteria. Study of key process variables and determination of their optimum values were carried out to provide a basis for significant physiological and technical comparison. Each microbial culture finds a preferential process application, depending on the choice of the reactor and ore.     

State-of-the-art of the production of retroviral vectors

Retroviral vectors are still the vectors that are used in the majority of gene therapy trials for treatment of acquired or inherited diseases. In this review, the present state-of-the-art of the production of retroviral vectors and the most important parameters, such as the choice of the producer cell line, stability issues, medium additives, serum, type of bioreactor, that influence production issues is presented and discussed in light of an optimal vector production. The available literature data clearly indicate that, on one hand, the choice of the producer cell line is of utmost importance for obtaining a high level producer cell line, and that, on the other hand, the optimization of the medium, e.g. the replacement of glucose by fructose, has a potential for improving vector production rates and titers. Finally, the use of high-density perfusion culture systems for adherent as well as for suspension cells presents the best choice for a production system, because high cell densities imply high reactor specific production rates, which must be associated with a rapid harvest of the produced vector, thus avoiding vector inactivation due to an extended residence time. The overall optimization of the cultivation and production parameters will have a significant impact on the use of retroviral vectors for gene therapy purposes.     

Mammalian cell retention devices for stirred perfusion bioreactors

Within the spectrum of current applications for cell culture technologies, efficient large-scale mammalian cell production processes are typically carried out in stirred fed-batch or perfusion bioreactors. The specific aspects of each individual process that can be considered when determining the method of choice are presented. A major challenge for perfusion reactor design and operation is the reliability of the cell retention device. Current retention systems include cross-flow membrane filters, spin-filters, inclined settlers, continuous centrifuges and ultrasonic separators. The relative merits and limitations of these technologies for cell retention and their suitability for large-scale perfusion are discussed. This revised version was published online in August 2006 with corrections to the Cover Date.     

Immunoaffinity reactors for prion protein qualitative analysis

The cellular prion protein (PrPc) represents the substrate for generation of conformational aberrant PrP isoforms which occur in human and animal prion diseases. The published two-dimensional maps of human PrPc show a vast microheterogeneity of this glycoprotein. The main goal of this project was to develop a highly specific immunoaffinity reactor for qualitative analysis of PrP cellular isoforms isolated from brain homogenate, cerebrospinal fluid and other tissues. New techniques for affinity proteomics, carriers and immobilization chemistry were applied. The choice of matrix (chemical and magnetic properties, particle size and distribution, porosity) was the key factor that influenced the quality of the reactor and the nature of final applications. Mouse anti-prion IgGs directed to N-terminal and C-terminal epitopes (residues 23-40 and 147-165) were grafted in different manners to magnetic micro- and nanoparticles particularly developed for micro-CHIP application. High operational and storage stability of affinity reactors with minimized nonspecific absorption were achieved. The quality of the immunoreactors was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblotting and by two-dimensional electrophoresis.