Mixing power, external convection, and effectiveness in bioreactors

The phenomena of mixing and mass transfer of substrates to microorganisms greatly affect the biochemical reactions which take place in fermentation processes. The effect that agitation power has on the observable reaction kinetics involved in beer fermentation has been studied in different types of bioreactors, from laboratory to industrial scale. With this aim in mind, an effectiveness factor, eta, is introduced which is defined as the relation between the existing rate of reaction, whichever bioreactor is considered, and the reaction rate in the well-mixed, and therefore presumably homogeneous, bioreactor with no diffusional limits. The limitation to homogeneously supplying nutrient material to the cells produces a decrease in this effectiveness factor, which has been correlated to the energy dissipation rate with a similar slope to that which appears in an existing correlation in the literature between this energy and the mass transfer coefficient. Additionally, a dimensionless reaction-convection number, N(RC), which is a function of the power input per unit volume, is proposed, which has been appropriately employed in correlating the effectiveness factor for the types of processes in which convection may be the key resistance factor. (c) 1996 John Wiley & Sons, Inc.     

A biological system for studies on mixing in bioreactors

A method for studying bioreactor inhomogeneity is suggested. Oxygen depletion in an E. coli cultivation leads to mixed acid fermentation with hydrogen gas production. Using a palladium metal-oxide semiconductor (Pd-MOS) hydrogen sensor on line in the effluent gas, it is shown that in a batch culture of 1 m³ hydrogen gas was evolved even before the dissolved oxygen tension (DOT) as measured by the probe reached zero. This suggests an appreciable degree of inhomogeneity with respect to DOT. Using measurements performed off line, it was observed that the lag time for hydrogen to appear became greater if the cells had been subjected to oxygen depletion. Lack of oxygen also resulted in a lowered hydrogen evolution capacity of the cells.     

Theoretical and methodological studies of continuous microbial bioreactors

This article reviews most of the author's studies on process development and reactor design for continuous microbial reactions. (1) Enzyme reactions of growing and non-growing microbial cells immobilized in agar gel beads were analyzed pertaining to the effects of external and internal diffusion of substrate on reaction kinetics. (2) Experimental correlations of production rates of beta-fructosidase and acid phosphatase with dilution rate of continuous culture were simulated based on an operon model for enzyme regulation. (3) Population dynamics of an amylase-producing bacteria and their mutant were discussed in relation to enzyme productivity in a continuous culture of spore-forming bacteria. (4) Plasmid mobilization in a mixed population of donor, recipient, and helper cells was investigated in a continuous culture as a model study of accidental release of a genetically modified plasmid into a natural environment. (5) A production rate increase of up to 100-fold was achieved by cell-recycle culturing of continuous acetic acid fermentation using a filter module with a hollow fiber membrane. (6) The feasibility of a continuous surface culture for the biooxidation of organic substances was ascribed to an enhanced oxygen absorption rate in the presence of a microbial film on a liquid surface. (7) Simultaneous separation of inhibitory products using an electrodialysis module during some organic acid fermentations was effective for increasing production in a continuous culture.     

Some studies on UASB bioreactors for the stabilization of low strength industrial effluents

The suitability of two stage biomethanation process using upflow anaerobic sludge blanket (UASB) bioreactors was studied for the treatment of low strength industrial effluents like rice mill wastewater. Maximum VFA yield was 0.75 mg (as acetic acid) per mg of COD consumed at a flow rate of 25 ml/min. Hydraulic retention time (HRT) of 1 hr was found suitable for acidification process. In the methanogenic reactor, the overall BOD and COD reductions were 89% and 78% respectively at loading rate of 3 kg COD m^х d^у, and HRT of 30 hrs. Gas yield in methanogenic reactor was 0.56 lits. per kg COD consumed which contains 62% v/v methane.     

Control in bioreactors showing gradients

In large-scale bioreactors gradients often occur as a result of non-ideal mixing. This phenomenon complicates design and control of large-scale bioreactors. Gradients in the oxygen concentration can be modeled with a two-compartment model of the liquid phase. Application of this model had been suggested for the control of the dissolved oxygen concentration with a batch gluconic acid fermentation process as the model system. The control system consists of a controller, an observer and a parameter estimator. In this work, the controller design is reconsidered and, in simulation experiments, the performance of the control system has been investigated. When the parameter values are known, the controller in combination with the observer works adequately. The parameter estimator, however, yields incorrect parameters, which are caused by a coupling between two parameters. This causes a deviation of the estimated states from the process states. The simulation results suggest that a priori knowledge of the parameters is required for application of the model for control and state estimation.     

Oxygen transfer in slurry bioreactors

The oxygen transfer in bioreactors with slurries having a yield stress was investigated. The volumetric mass transfer coefficients in a 40-L bubble column with simulated fermentation broths, the Theological properties of which were represented by the Casson model, were measured. Experimental data were compared with a theoretical correlation developed on the basis of a combination of Higbie's penetration theory and Kolmogoroff's theory of isotropic turbulence. Comparisons between the proposed correlation and data for the simulated broths show good agreement. The mass transfer data for actual mycelial fermentation broths reported previously by the authors were re-examined. Their Theological data was correlated by the Bingham plastic model. The oxygen transfer rate data in the mycelial fermentation broths fit the predictions of the proposed theoretical correlation.     

Glycoprotein production in moss bioreactors

Complex multimeric recombinant proteins such as therapeutic antibodies require a eukaryotic expression system. Transgenic plants may serve as promising alternatives to the currently favored mammalian cell lines or hybridomas. In contrast to prokaryotic systems, posttranslational modifications of plant and human proteins resemble each other largely, among those, protein N-glycosylation of the complex type. However, a few plant-specific sugar residues may cause immune reactions in humans, representing an obstacle for the broad use of plant-based systems as biopharmaceutical production hosts. The moss Physcomitrella patens represents a flexible tissue-culture system for the contained production and secretion of recombinant biopharmaceuticals in photobioreactors. The recent synthesis of therapeutic proteins as a scFv antibody fragment or the large and heavily modified complement regulator factor H demonstrate the versatility of this expression system. A uniquely efficient gene targeting mechanism can be employed to precisely engineer the glycosylation machinery for recombinant products. In this way, P. patens lines with non-immunogenic optimized glycan structures were created. Therapeutic antibodies produced in these strains exhibited antibody-dependent cellular cytotoxicity superior to the same molecules synthesized in mammalian cell lines.     

Sulfate reduction in methanogenic bioreactors

Abstract: In the anaerobic treatment of sulfate-containing wastewater, sulfate reduction interferes with methanogenesis. Both mutualistic and competitive interactions between sulfate-reducing bacteria and methanogenic bacteria have been observed. Sulfate reducers will compete with methanogens for the common substrates hydrogen, formate and acetate. In general, sulfate reducers have better growth kinetic properties than methanogens, but additional factors which may be of importance in the competition are adherence properties, mixed substrate utilization, affinity for sulfate of sulfate reducers, relative numbers of bacteria, and reactor conditions such as pH, temperature and sulfide concentration. Sulfate reducers also compete with syntrophic methanogenic consortia involved in the degradation of substrates like propionate and butyrate. In the absence of sulfate these methanogenic consortia are very important, but in the presence of sulfate they are thought to be easily outcompeted by sulfate reducers. However, at relatively low sulfate concentrations, syntrophic degradation of propionate and butyrate coupled to HZ removal via sulfate reduction rather than via methanogenesis may become important. A remarkable feature of some sulfate reducers is their ability to grow fermentatively or to grow in syntrophic association with methanogens in the absence of sulfate.     

Concentric-tube airlift bioreactors

Mass transfer coefficients were measured in three concentric-tube airlift reactors of different scales (RIMP, V _L =0.07 m³;RIS?1,V _L =2.50 m³;RIS?2, V _L =5.20 m³). The effects of top and bottom clearance and flow resistances at downcorner entrance were studied in water-air system. Experimental results show that h _s ,h _B and A _d /A _R ratio affect K _L a values as a result of their influence on gas holdup and liquid velocity. The gas-liquid mass-transfer coefficients for all the geometric variables were successfully correlated as Sherwood number with Froude and Galilei numbers, the bottom spatial ratio (B=h _B /D _R ), the top spatial ratio , the gas separation ratio and the downcomer flow resistance ratio (R=A _d /A _R ). The proposed empirical model satisfactorily fitted the experimental data obtained in large airlift reactors and some data presented in literature.     

Transgenic animal bioreactors

The production of recombinant proteins is one of the major successes of biotechnology. Animal cells are required to synthesize proteins with the appropriate post-translational modifications. Transgenic animals are being used for this purpose. Milk, egg white, blood, urine, seminal plasma and silk worm cocoon from transgenic animals are candidates to be the source of recombinant proteins at an industrial scale. Although the first recombinant protein produced by transgenic animals is expected to be in the market in 2000, a certain number of technical problems remain to be solved before the various systems are optimized. Although the generation of transgenic farm animals has become recently easier mainly with the technique of animal cloning using transfected somatic cells as nuclear donor, this point remains a limitation as far as cost is concerned. Numerous experiments carried out for the last 15 years have shown that the expression of the transgene is predictable only to a limited extent. This is clearly due to the fact that the expression vectors are not constructed in an appropriate manner. This undoubtedly comes from the fact that all the signals contained in genes have not yet been identified. Gene constructions thus result sometime in poorly functional expression vectors. One possibility consists in using long genomic DNA fragments contained in YAC or BAC vectors. The other relies on the identification of the major important elements required to obtain a satisfactory transgene expression. These elements include essentially gene insulators, chromatin openers, matrix attached regions, enhancers and introns. A certain number of proteins having complex structures (formed by several subunits, being glycosylated, cleaved, carboxylated...) have been obtained at levels sufficient for an industrial exploitation. In other cases, the mammary cellular machinery seems insufficient to promote all the post-translational modifications. The addition of genes coding for enzymes involved in protein maturation has been envisaged and successfully performed in one case. Furin gene expressed specifically in the mammary gland proved to able to cleave native human protein C with good efficiency. In a certain number of cases, the recombinant proteins produced in milk have deleterious effects on the mammary gland function or in the animals themselves. This comes independently from ectopic expression of the transgenes and from the transfer of the recombinant proteins from milk to blood. One possibility to eliminate or reduce these side-effects may be to use systems inducible by an exogenous molecule such as tetracycline allowing the transgene to be expressed only during lactation and strictly in the mammary gland. The purification of recombinant proteins from milk is generally not particularly difficult. This may not be the case, however, when the endogenous proteins such as serum albumin or antibodies are abundantly present in milk. This problem may be still more crucial if proteins are produced in blood. Among the biological contaminants potentially present in the recombinant proteins prepared from transgenic animals, prions are certainly those raising the major concern. The selection of animals chosen to generate transgenics on one hand and the elimination of the potentially contaminated animals, thanks to recently defined quite sensitive tests may reduce the risk to an extremely low level. The available techniques to produce pharmaceutical proteins in milk can be used as well to optimize milk composition of farm animals, to add nutriceuticals in milk and potentially to reduce or even eliminate some mammary infectious diseases. This revised version was published online in August 2006 with corrections to the Cover Date.     

Concentric-tube airlift bioreactors

Gas holdup investigations were performed in three concentric-tube airlift reactors of different scales of operation (RIMP: 0.070 m³; RIS-1: 2.5 m³; RIS-2: 5.2 m³; nominal volumes). The influences of the top and bottom clearances and the flow resistances at the downcomer entrance were studied using tap water as liquid phase and air as gaseous phase, at atmospheric pressure. It was found that the gas holdup in the individual zone of the reactor: riser, downcomer and gas-separator, as well as that in the overall reactor is affected by the analyzed geometrical parameters in different ways, depending on their effects on liquid circulation velocity. Gas holdup was satisfactorily correlated with Fr, Ga, bottom spatial ratio (B), top spatial ratio (T), gas separation ratio (Y) and downcomer flow resistance ratio (A _d /A _R ). Correlations are presented for gas holdup in riser, downcomer, gas separator and for the total gas holdup in the reactor. All the above stressed the importance of the geometry in dynamic behaviour of airlift reactors.     

Concentric-tube airlift bioreactors

Liquid circulation velocity was investigated in three concentric-tube airlift reactors of different scales (RIMP, V _L =0.07 m³; RIS-1, V _L =2.5 m³; RIS-2, V _L =5.20 m³). The effects of top and bottom clearance and resistance in flow pathway at downcomer entrance on the riser liquid superficial velocity, the circulation time, the friction coefficient and flow radial profiles of the gas holdup and the liquid superficial velocity in riser, using water-air as a biphasic system, were studied. It was found that the riser liquid superficial velocity is affected by the analyzed geometrical parameters in different ways, depending on their effects on the pressure loss. The riser liquid superficial velocity, the friction coefficient and the parameters of the drift-flux model were satisfactorily correlated with the bottom spatial ratio (B), gas separation ratio (Y) and downcomer flow resistance ratio (A _d /A _D ), resulting empirical models, with correlation coefficients greater than 0.85.     

Living with heterogeneities in bioreactors

The presence of spatial gradients in fundamental culture parameters, such as dissolved gases, pH, concentration of substrates, and shear rate, among others, is an important problem that frequently occurs in large-scale bioreactors. This problem is caused by a deficient mixing that results from limitations inherent to traditional scale-up methods and practical constraints during large-scale bioreactor design and operation. When cultured in a heterogeneous environment, cells are continuously exposed to fluctuating conditions as they travel through the various zones of a bioreactor. Such fluctuations can affect cell metabolism, yields, and quality of the products of interest. In this review, the theoretical analyses that predict the existence of environmental gradients in bioreactors and their experimental confirmation are reviewed. The origins of gradients in common culture parameters and their effects on various organisms of biotechnological importance are discussed. In particular, studies based on the scale-down methodology, a convenient tool for assessing the effect of environmental heterogeneities, are surveyed.     

Animal-cell damage in sparged bioreactors

The gas sparging of culture broth causes damage to suspended animal cells. However, despite this, sparged bioreactors remain the preferred means of cell culture because sparging is a robust method of supplying oxygen, especially on a large scale. This article examines the underlying mechanisms involved in bubble-associated cell damage and the methods available for controlling such damage.     

Stem cell cultivation in bioreactors

Cell-based therapies have generated great interest in the scientific and medical communities, and stem cells in particular are very appealing for regenerative medicine, drug screening and other biomedical applications. These unspecialized cells have unlimited self-renewal capacity and the remarkable ability to produce mature cells with specialized functions, such as blood cells, nerve cells or cardiac muscle. However, the actual number of cells that can be obtained from available donors is very low. One possible solution for the generation of relevant numbers of cells for several applications is to scale-up the culture of these cells in vitro. This review describes recent developments in the cultivation of stem cells in bioreactors, particularly considerations regarding critical culture parameters, possible bioreactor configurations, and integration of novel technologies in the bioprocess development stage. We expect that this review will provide updated and detailed information focusing on the systematic production of stem cell products in compliance with regulatory guidelines, while using robust and cost-effective approaches.     

Bubble coalescence in air-sparged bioreactors

Coalescence frequency has been measured in an air-sparged bubble column by observing the rate with which insoluble tracer gases were mixed within the dispersed gas phase. The effects of gas-sparge rate and salt concentration were examined. Coalescence frequencies increased with increasing gas-sparge rates approximately in proportion to the increase in gas-phase hold up. The coalescence frequency decreased with NaCI concentration to a minimum frequency about half the frequency observed in pure water. Bubble size distributions were also measured in the air-sparged column. The size distribution changed significantly over the same range of NaCI concentration as variations in the coalescence frequency were observed. The gas-sparge rate showed no effect on the bubble size.     

Oxygen gradients in animal-cell bioreactors

An estimation is made of oxygen gradients in animal-cell bioreactors, using straightforward engineering calculations. Three types of bioreactor are considered: stirred vessel, bubble column and air lift, of sizes between 0.01 and 10 m³. First, the gradient is estimated in the stagnant layer surrounding a cell (15 m), a microcarrier (185 m) with 300 cells attached to it, a macroporous support (1.25 mm) containing 185,00 cells and one (6 mm) containing 4.25 million cells. It is assumed that oxygen consumption is 10^–16 mole O₂·cell^–1·s^–1, while mass transfer coefficients are obtained from Sherwood relations. Circulation and liquid-retention times of the bioreactors are compared with the oxygen-exhaust times of suspensions with 10¹², 10¹³ and 10¹⁴ cells/m³ to estimate if oxygen gradients are likely to exist in the bulk-liquid phase. Finally, the gradient in the liquid film surrounding air bubbles is estimated using k _l A-values obtained from empirical correlations. It is clear from all these estimations that in many situations severe gradients can be expected. The question remains, however, whether gradients should be avoided as much as possible, or may be tolerated to a certain extent or even created on purpose because of possible beneficial effects.     

Gas-inducible product gene expression in bioreactors

Inducible transgene expression technologies are of unmatched potential for biopharmaceutical manufacturing of unstable, growth-impairing and cytotoxic proteins as well as conditional metabolic engineering to improve desired cell phenotypes. Currently available transgene dosing modalities which rely on physical parameters or small-molecule drugs for transgene fine-tuning compromise downstream processing and/or are difficult to implement technologically. The recently designed gas-inducible acetaldehyde-inducible regulation (AIR) technology takes advantage of gaseous acetaldehyde to modulate product gene expression levels. At regulation effective concentrations gaseous acetaldehyde is physiologically inert and approved as food additive by the Federal Drug Administration (FDA). During standard bioreactor operation, gaseous acetaldehyde could simply be administered using standard/existing gas supply tubing and eventually eliminated by stripping with inducer-free air. We have determined key parameters controlling acetaldehyde transfer in three types of bioreactors and designed a mass balance-based model for optimal product gene expression fine-tuning using gaseous acetaldehyde. Operating a standard stirred-tank bioreactor set-up at 10 L scale we have validated AIR technology using CHO-K1-derived serum-free suspension cultures transgenic for gas-inducible production of human interferon-beta (IFN-beta). Gaseous acetaldehyde-inducible IFN-beta production management was fully reversible while maintaining cell viability at over 95% during the entire process. Compatible with standard bioreactor design and downstream processing procedures AIR-based technology will foster novel opportunities for pilot and large-scale manufacturing of difficult-to-produce protein pharmaceuticals.