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.     

Substrate gradients in bioreactors: origin and consequences

Gradients of glucose in time and space are shown in a 30 m³ cultivation of Saccharomyces cerevisiae grown in minimal medium to a cell density of 20 gl^–1. The fed-batch concept was used with glucose as the limiting component which was fed continuously to the process. As the mean glucose concentration declined throughout the process, the level of glucose was at all times different in three sampling ports (bottom/middle/top) of the reactor. These gradients were furthermore shown to depend on the feed position. This means that if the feed was supplied in the relatively stagnant mixing zone above the top impeller, the gradients were more pronounced than by feed in the well mixed bottom impeller zone. A rapid sampling system was constructed, and continuous glucose samples of every 0.15 s were analysed from a point of the reactor. Fifty samples were collected with this system, but the amount and frequency is possible to change. The results of these series show a variance of the glucose concentration where at one stage, a peak appeared of a relative difference in concentration of 40 mgl^–1. The pattern of these rapid glucose fluctuations was shown to depend on the turbulence level at the location of the feed. It was shown, that the fluctuations were more pronounced when the feed was localised in a relatively stagnant area than in the well-mixed impeller area, where the deviation from the mean was negligible. The fluid flow, in the impeller (gassed and ungassed) and bulk area (ungassed) of the reactor, was characterised by turbulence measurements using thermal anemometry. These types of areas resembles well the different areas of sampling as mentioned above. The turbulent frequencies in these areas were in the range of 10^–1 to 10⁴ Hz with the highest amplitudes at low frequencies. The spectra depicts a uniform time scale for all zones, especially at the low frequencies. The dominance of low frequency, high amplitude flow variations and the observed short-time oscillations in substrate concentration support the hypothesis of substrate transport over fairly long distances without substantial mixing both in the impeller, but especially, in the bulk zone of the reactor.Simulations with an integrated CFD and biokinetic model were performed. The predictions of the glucose gradients of this model were compared to measurements.This project was supported by a grant from the Nordic Programme on Bioprocess Engineering under the auspices of NI, the Nordic Fund for Technology and Industrial Development and NUTEK, The Swedish Board for Technical and Industrial Development. This investigation was possible to perform due to the support from Statoil Biosentrum in Stavanger, Norway, that provided their pilot plant. Especially the cooperation with Lars Aasberg and Andreas Rag is greatly acknowledged.     

Oxygen gradients in small and big sparged insect-cell bioreactors

Conclusions It should be clear from the above that the calculations described here are at best rough estimations yielding order-of-magnitude values. Even though, the following general conclusions can be drawn. The gradients in stagnant layers surrounding the particles which are characteristic for animal-cell bioreactors are relatively small as compared to the gradients which can be expected in the bulk-liquid phases of the three bioreactors considered, in particular to the gradients in the stagnant layer surrounding the air bubbles. It can be concluded that under almost all circumstances gradients are likely to exist and can be very steep in larger vessels and in particular at high cell densities. The effects of gradients, however, are largely unknown; therefore research on the effects of gradients on specific and volumetric productivities and product quality seems to be an interesting area.     

Hydraulic conductance and water potential gradients in squash leaves showing mycorrhiza-induced increases in stomatal conductance

Stomatal conductance (g _s) and transpiration rates vary widely across plant species. Leaf hydraulic conductance (k _leaf) tends to change with g _s, to maintain hydraulic homeostasis and prevent wide and potentially harmful fluctuations in transpiration-induced water potential gradients across the leaf (ΔΨ _leaf). Because arbuscular mycorrhizal (AM) symbiosis often increases g _s in the plant host, we tested whether the symbiosis affects leaf hydraulic homeostasis. Specifically, we tested whether k _leaf changes with g _s to maintain ΔΨ _leaf or whether ΔΨ _leaf differs when g _s differs in AM and non-AM plants. Colonization of squash plants with Glomus intraradices resulted in increased g _s relative to non-AM controls, by an average of 27% under amply watered, unstressed conditions. Stomatal conductance was similar in AM and non-AM plants with exposure to NaCl stress. Across all AM and NaCl treatments, k _leaf did change in synchrony with g _s (positive correlation of g _s and k _leaf), corroborating leaf tendency toward hydraulic homeostasis under varying rates of transpirational water loss. However, k _leaf did not increase in AM plants to compensate for the higher g _s of unstressed AM plants relative to non-AM plants. Consequently, ΔΨ _leaf did tend to be higher in AM leaves. A trend toward slightly higher ΔΨ _leaf has been observed recently in more highly evolved plant taxa having higher productivity. Higher ΔΨ _leaf in leaves of mycorrhizal plants would therefore be consistent with the higher rates of gas exchange that often accompany mycorrhizal symbiosis and that are presumed to be necessary to supply the carbon needs of the fungal symbiont.     

Control of bioreactors using a neural network model

Control of a continuous bioreactor based on a artificial neural network (ANN) model is carried out theoretically. The ANN model is identified, from input-output data of a bioreactor, using a three-layer feedforward network trained by a back propagation algorithm. The performance of the controller designed on the ANN model is compared with that of a conventional PI controller.     

Mixing studies in bioreactors

Blend times and power consumptions were determined for different arrangements of two equal diameter impellers, a high efficiency A310 and a “Dumbo Ear” impeller with three large, “elephant ear” blades designed for low shear agitation. A 9 l round-bottomed, unbaffled bioreactor was used in these studies. Blend times were taken as the time for the disappearance of the pink color of a basic solution of phenolphthalein on neutralization by excess acid, and the power consumption was obtained from torque measurements. The mixing results show that the Dumbo Ear impeller gives shorter blend times than the A310?at equal rotational speeds for most of the conditions studied. As expected, the Dumbo Ear impeller consumes more power than the A310?at the same rotational speed, due to its large area blades. However, the Dumbo Ear impeller also gives shorter blend times than the A310?at equal power consumptions.     

Transgenic bioreactors

• 1.1. Although many human therapeutic proteins are currently produced in microbial fermentors using recombinant DNA techniques, it is obvious that microbial processing is not suitable for a large number of bioactive proteins owing to the inability of bacteria to carry out postsynthetic modification reactions required for full biological activity.
• 2.2. This disadvantage does not apply to animal cell bioreactors that can generate biologically fully active entities, yet the use of large-scale animal cell cultures for production purposes is prohibitively expensive.
• 3.3. With the advent of transgenic technology, the production of valuable human pharmaceuticals in large farm animals (pig, sheep, goat and dairy cattle) has become more and more attractive as a high-quantity, low-cost alternative. By employing targeted gene transfer, e.g. using mammary gland-specific regulatory sequences fused with the desired production genes, it is possible to govern the expression to occur exclusively in the mammary gland and hence the gene product is being ultimately secreted into the milk.
• 4.4. While reviewing the remarkable progress in this field that has even led to commercial exploitations, we will outline in somewhat greater detail our strategy for the use of dairy cattle as a bioreactor for valuable proteins of pharmaceutical interest.

     

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.     

Gas hold-up measurements in bioreactors

The gas hold-up of a gas-liquid dispersion is an important parameter in the fermentation industry. If it is too low, or too high, productivity can be adversely affected. Gas hold-up in fermentors cannot be calculated from physico-chemical correlations and, therefore, must be measured accurately for each fermentation.This article surveys a number of methods for measuring the gas hold-up in gas-liquid dispersions, making particular note whether these methods can be applied aseptically.     

Bubble column bioreactors

Summary The photographic and electrical conductivity methods to measure the structure of two phase flow, especially bubble size, bubble frequency, local gas hold-up and, for the latter, the bubble velocity are described.Symbols specific interfacial area - a gas/liquid interfacial area - B constant in Eq. (4) - d diameter of the bubbles - d mean diameter of the bubbles - d_S Sauter diameter - E_G relative gas hold up - I current - k_L mass transfer coefficient across the gas/liquid interface - k_L local k_L - LT^–1 - LT^–1 - 1 longitudinal distance between the start and stop sensors - 1_B pierced length of the bubble - t time - t₁ length of the square-wave signal at the start sensor - t₂ length of the square-wave signal at the stop sensor - t₁₂ time delay between start and stop signals - V volume of the bubbling layer - V_L volume of the bubble free layer - V_B bubble volume - v_B bubble velocity     

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.     

Microalgae as bioreactors

Microalgae already serve as a major natural source of valuable macromolecules including carotenoids, long-chain polyunsaturated fatty acids and phycocolloids. As photoautotrophs, their simple growth requirements make these primitive plants potentially attractive bioreactor systems for the production of high-value heterologous proteins. The difficulty of producing stable transformants has meant that the field of transgenic microalgae is still in its infancy. Nonetheless, several species can now be routinely transformed and algal biotechnology companies have begun to explore the possibilities of synthesizing recombinant therapeutic proteins in microalgae and the engineering of metabolic pathways to produce increased levels of desirable compounds. In this review, we compare the current commercially viable bioreactor systems, outline recent progress in microalgal biotechnology and transformation, and discuss the potential of microalgae as bioreactors for the production of heterologous proteins.     

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.     

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.     

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.     

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.     

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.     

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.