Karel Ch. A. M. Luyben

Physiological responses to mixing in large scale bioreactors

Escherichia coli fed-batch cultivations at 22 m3 scale were compared to corresponding laboratory scale processes and cultivations using a scale-down reactor furnished with a high-glucose concentration zone to mimic the conditions in a feed zone of the large bioreactor. Formate accumulated in the large reactor, indicating the existence of oxygen limitation zones. It is suggested that the reduced biomass yield at large scale partly is due to repeated production/re-assimilation of acetate from overflow metabolism and mixed acid fermentation products due to local moving zones with oxygen limitation. The conditions that generated mixed-acid fermentation in the scale-down reactor also induced a number of stress responses, monitored by analysis of mRNA of selected stress induced genes. The stress responses were relaxed when the cells returned to the substrate limited and oxygen sufficient compartment of the reactor. Corresponding analysis in the large reactor showed that the concentration of mRNA of four stress induced genes was lowest at the sampling port most distant from the feed zone. It is assumed that repeated induction/relaxation of stress responses in a large bioreactor may contribute to altered physiological properties of the cells grown in large-scale bioreactor. Flow cytometric analysis revealed reduced damage with respect to cytoplasmic membrane potential and integrity in cells grown in the dynamic environments of the large scale reactor and the scale-down reactor.

Mixing in large-scale vessels stirred with multiple radial or radial and axial up-pumping impellers: modelling and measurements

Mixing phenomena are regarded as one of the major factors responsible for the failure to successfully scale up some bioprocesses. Such phenomena have been investigated within the framework of an EC project ‘Bioprocess Scale-up Strategy’. Mixing in bioreactors depends on energy input, impeller type, reactor configuration and impeller geometry. Here, two different reactors of volumes 12 and 30m3 were used, and they were equipped with either multiple Rushton turbines or with a combination of a Scaba 6SRGT radial impeller with multiple 3SHP axial up-pumping hydrofoils above it. Mixing time, power consumption, gas hold-up and liquid velocities were measured at different stirrer speeds and aeration rates in water. At the same total specific power input, aeration did not influence the mixing time much unless it changed the bulk flow pattern. A considerable reduction of mixing time was achieved if the upper impellers were axial instead of radial Rushtons at the same power consumption. The improvement with the axial impellers could be related to the reduction of axial flow barriers due to different circulation flow patterns. The Compartment Model Approach (CMA) was used to develop a flow model based on the general knowledge of the hydrodynamics of both unaerated and aerated stirred vessels. The model was successfully verified for different impeller and reactor configurations and different scales with measured pulse response curves, using either a fluorescent or a hot water tracer. The model can be used for process design purposes.

Effect of Agitation Intensities on Fungal Morphology of Submerged Fermentation

Both parallel fermentations with Aspergillus awamori (CBS 115.52) and a literature study on several fungi have been carried out to determine a relation between fungal morphology and agitation intensity. The studied parameters include hyphal length, pellet size, surface structure or so-called hairy length of pellets, and dry mass per-wet-pellet volume at different specific energy dissipation rates. The literature data from different strains, different fermenters, and different cultivation conditions can be summarized to say that the main mean hyphal length is proportional to the specific energy dissipation rate according to a power function with an exponent of -0.25 +/- 0.08. Fermentations with identical inocula showed that pellet size was also a function of the specific energy dissipation rate and proportional to the specific energy dissipation rate to an exponent of -0.16 +/- 0.03. Based on the experimental observations, we propose the following mechanism of pellet damage during submerged cultivation in stirred fermenters. Interaction between mechanical forces and pellets results in the hyphal chip-off from the pellet outer zone instead of the breakup of pellets. By this mechanism, the extension of the hyphae or hair from pellets is restricted so that the size of pellets is related to the specific energy dissipation rate. Hyphae chipped off from pellets contribute free filamentous mycelia and reseed their growth. So the fraction of filamentous mycelial mass in the total biomass is related to the specific energy dissipation rate as well.To describe the surface morphology of pellets, the hyphal length in the outer zone of pellets or the so-called hairy length was measured in this study. A theoretical relation of the hairy length with the specific energy dissipation rate was derived. This relation matched the measured data well. It was found that the porosity of pellets showed an inverse relationship with the specific energy dissipation rate and that the dry biomass per-wet-pellet volume increased with the specific energy dissipation rates. This means that the tensile strength of pellets increased with the increase of specific energy dissipation rate. The assumption of a constant tensile strength, which is often used in literature, is then not valid for the derivation of the relation between pellet size and specific energy dissipation rate. The fraction of free filamentous mycelia in the total biomass appeared to be a function of the specific energy dissipation in stirred bioreactors. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 715-726, 1997.

A hydrodynamic model for an airlift-loop bioreactor with external loop

Abstract A simple model is introduced for the hydrodynamic description of an airlift-loop bioreactor with external loop. The model is based on the drift flux model of Zuber and Findlay for a two-phase flow and predicts the liquid velocity and the local gas hold-up in both the upflow and downflow region in relation to the gas input rate and the reactor dimensions. The model is non-isobaric and takes into account non-uniform flow profiles. Liquid velocity and local gas hold-up in airlift-loop reactors from laboratory to pilot plant scales are predicted to within 5% – 10% accuracy.

An efficient model development strategy for bioprocesses based on neural networks in macroscopic balances

In the serial gray box modeling strategy, generally available knowledge, represented in the macroscopic balance, is combined naturally with neural networks, which are powerful and convenient tools to model the inaccurately known terms in the macroscopic balance. This article shows, for a typical biochemical conversion, that in the serial gray box modeling strategy the identification data only have to cover the input-output space of the inaccurately known term in the macroscopic balances and that the accurately known terms can be used to achieve reliable extrapolation. The strategy is demonstrated successfully on the modeling of the enzymatic (repeated) batch conversion of penicillin G, for which real-time results are presented. Compared with a more data-driven black box strategy, the serial gray box strategy leads to models with reliable extrapolation properties, so that with the same number of identification experiments the model can be applied to a much wider range of different conditions. Compared to a more knowledge-driven white box strategy, the serial gray box model structure is only based on readily available or easily obtainable knowledge, so that the development time of serial gray box models still may be short in a situation where there is no detailed knowledge of the system available. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 549-566, 1997.

Effect of the degree of substitution of (2-hydroxy)propyl-β-cyclodextrin on the enantioseparation of organic acids by capillary electrophoresis

Optical isomers of nine organic acids were separated by high-performance capillary electrophoresis using (2-hydroxy)propyl-β-cyclodextrins, with a degree of the substitution between 3.0 and 7.3 (2-hydroxy)propyl groups/cyclodextrin molecule. The degree of substitution has a significant influence on the resolution of the enantiomers and is therefore an important tool in the optimisation of chiral separations. Accordingly, a proper description of derivatized cyclodextrins should include the degree of substitution.

Effects of glutamine supply on growth and metabolism of mammalian cells in chemostat culture

Glutamine is a major source of energy, carbon, and nitrogen for mammalian cells. The amount of glutamine present in commercial mammalian cell media is, however, not necessarily balanced with cell requirements. Therefore, the effects of glutamine limitation on the physiology of two mammalian cell lines were studied in steady-state chemostat cultures fed with IMDM medium with 5% serum. The cell lines used were MN12, a mouse-mouse hybridoma, and SP2/0-Ag14, a mouse myeloma often used in hybridoma fusions. Cultures, grown at a fixed dilution rate of 0.03 h(-1), were fed with media containing glutamine concentrations ranging from 0.5 to 4 mmol L(-1). Biomass dry weight and cell number were linearly proportional to the glutamine concentrations fed, between 0.5 and 2 mmol L(-1), and glutamine was completely consumed by both cell lines. From this it was concluded that glutamine was the growth-limiting substrate in this concentration range and that the standard formulation of IMDM medium contains a twofold excess of glutamine. In glutamine-limited cultures, the specific rates of ammonia and alanine production were low compared to glutamine-excess cultures containing 4 mmol L(-1) glutamine in the feed medium. The specific consumption rates of nearly all amino acids decreased with increasing glutamine feed, indicating that, in their metabolic function, they may partially be replaced by glutamine. Both cell lines reacted similarly to differences in glutamine feeding in all aspects investigated, except for glucose metabolism, In SP2/0-Ag14 glutamine feed concentrations did not affect the specific glucose consumption, whereas in MN12 this parameter increased with increasing amounts of glutamine fed. This systematic study using controlled culture conditions together with a detailed analysis of culture data shows that, although cells may react similarly in many aspects, cell-line-specific characteristics may be encountered even with respect to fundamental physiological responses like the interaction of the glutamine and glucose metabolism.

Poly(ethylene glycol)–salt aqueous two-phase systems with easily recyclable volatile salts

Aqueous two-phase systems (ATPSs) have great potential in the downstream processing of fermentation products. However, the consumption of large amounts of auxiliary materials limits application in industrial practice. Promising alternatives to the salts used so far are volatile salts such as ammonium bicarbonate and ammonium carbamate, which can be recycled to the extraction system as gaseous carbon dioxide and ammonia. In this work, it is demonstrated that ammonium carbamate in combination with poly(ethylene glycol) (PEG, molecular masses of 2000, 4000 and 10000) indeed produces aqueous two-phase systems (ATPSs) at a temperature of 25 degrees C and atmospheric pressure. Ammonium bicarbonate is clearly not suitable as a phase-forming salt, because of its too-low solubility in water.

Modelling the dynamic behaviour ofSaccharomyces cerevisiae and its application in control experiments

SummaryA simple structured model describing the response of a bakers yeast culture to a glucose pulse is presented. The model is based on a limited oxidation capacity of yeast leading to a switchover from oxidative to oxido-reductive metabolism. The maximum specific consumption rates of glucose, ethanol and oxygen are modelled by first order transfer functions. These maximum rates have also been estimated experimentally as a function of the dilution rate in a continuous culture. The rates predicted by the model correspond to the measured maximum specific consumption rates. So, the model can describe a continuous culture of bakers yeast very well. The applicability of the model is tested by inserting it in a control loop and comparing the response of the model with the results of a controlled fermentation.The model behaved satisfactorily for the description of the pulse experiment as well as during the simulation of the control experiments. Although the model for balanced growth was rejected in simulating pulse responses, its results were conveniently when inserted in a control loop.

Measurement of oxygen concentration gradients in gel-immobilized recombinantEscherichia coli

SummaryIn this study, an oxygen microsensor was used to measure oxygen concentration profiles in carrageenan gel particles containing growing, immobilizedEcherichia coli B (pTG201). Profiles, which were measured at intervals during continuous culture of gel slabs and beads, became increasingly steep with time. The oxygen penetration depth in the gel decreased with time, eventually reaching a steady state value of approximately 100 μm for both gel beads and slabs. A reaction-diffusion model employing zero-order cell growth kinetics was found to provide an excellent fit to the experimental concentration data. Growth rates estimated from profiles obtained during the first few hours of culture were 0.24h−1 (gel slabs) and 0.18 h−1 (beads), compared to a value of 0.30 h−1 measured in free-cell suspensions at 25° C.