Large Bioreactors Research Articles

Incomplete mixing in large bioreactors ? a study of its role in the fermentative production of streptokinase

The production of streptokinase in a batch fermentation has been analysed for the role of incomplete macromixing of the broth. The analysis is based on a kinetic model exhibiting inhibition by the substrate and a primary metabolite (lactic acid), and a mixing model comprising two continuous flow reactors (CFRs) with closed-loop recycle. The inoculum is introduced into one region (one CFR) and the mixing process determines its distribution, growth and reactivity. By varying the dilution rates of the CFRs, any degree of macromixing can be simulated. For dilution rates larger than 1.0 h−1 almost complete macromixing is achieved, for which an analogy has been drawn with micromixing. Increasing the volume of the inoculated region relative to the noninoculated region improves the maximum attainable activity of streptokinase and shortens the time for this. In such a situation an imperfectly mixed bioreactor is superior to a perfectly mixed one, implying that good productivity requires a large inoculated region and incomplete macromixing. These inferences are supported by earlier studies of fluid mixing and relaxation times in bioreactors.

The investigation of transient multidimensional heat transfer in solid state fermentation

A mathematical model is presented which predicts the temperature profiles for solid state fermentation in a packed bed in two dimensions, radial and axial. Heat transfer within the bed occurs by conduction through the solid material and convection by the air used for aeration. The temperature at the wall of the reactor is fixed at 298 K. Simulations with the model indicate that the air velocity and air temperature will significantly influence the temperature and biomass concentration profiles. The effect of the bioreactor geometry is also discussed. This fundamental model of solid state fermentation can be used to predict the effects of a range of parameters and can be used a a tool to aid scale-up to large bioreactors.

Influence of substrate oscillations on acetate formation and growth yield in Escherichia coli glucose limited fed‐batch cultivations

A Large bioreactor is an inhomogenous system with concentration gradients which depend on the fluid dynamics and the mass transfer of the reactor, the feeding strategy, the saturation constant, and the cell density. The responses of Escherichia coli cells to short-term oscillations of the carbon/energy substrate in glucose limited fed-batch cultivations were studied in a two-compartment reactor system consisting of a stirred tank reactor (STR) and an aerated plug flow reactor (PFR) as a recycle loop. Short-term glucose excess or starvation in the PFR was simulated by feeding of glucose to the PFR or to the STR alternatively. The cellular response to repeated short-term glucose excess was a transient increase of glucose consumption and acetate formation. But, there was no accumulation of acetate in the culture, because it was consumed in the STR part where the glucose concentration was growth limiting. However, acetate accumulated during the cultivation if the oxygen supply in the PFR was insufficient, causing higher acetate formation. The biomass yield was then negatively influenced, which was also the case if the PFR was used to simulate a glucose starvation zone. The results suggest that short-term heterogeneities influence the cellular physiology and growth, and can be of major importance for the process performance. (c) 1995 John Wiley & Sons, Inc.

A fluid dynamic study of the retrofitting of large agitated bioreactors: Turbulent flow

Studies were conducted in three 19-m(3) fermentors (14 m(3) working volume, aspect ratio = 3:1), one fitted with four Rushton turbines (D/T = 0.35), one with three Lightnin' A315 hydrofoil impellers (D/T = 0.46). The power drawn under the same aerated conditions relative to the unaerated ones was always greater with the hydrofoils, which gives them the potential for enhanced mass transfer rates under practical operating conditions. However, the power draw was also sensitive to the magnitude of the unaerated power. Indeed, at low unaerated specific power ( approximately 0.6 W-kg) and high air flow rates ( approximately 1vvm), the relative power draw with the hydrofoils could be even greater than 1. The hold-up with each of the impellers was broadly similar at the same aeration rate and power input, though the later had a much smaller impact in these large vessels than has been reported in the literature based on smaller scale work. As usual, repressed coalescence caused increased hold-up, and, with the hydrofoils, this increase was associated with a lower power draw. Because of the greater mechanical vibration of the reactors with the hydrofoils, vibration characteristics of the vessels were measured and they were very similar. The results showed that provided care is taken in the mechanical design of the system, such impellers can operate reliably in large-scale fermentations with the potential for enhanced biological performance. (c) 1994 John Wiley & Sons, Inc.

Vortex flow filtration of mammalian and insect cells.

The use of vortex flow filtration for harvesting cells or conditioned medium from large scale bioreactors has proven to be an efficient, low shear method of cell concentration and conditioned medium clarification. Several 8-10 L batches of the human histiocytic lymphoma U-937 cell line (ATCC CRL 1593) were concentrated to less than 1 L by vortex flow filtration through a 3.0 microns membrane. An aggressive filtration regimen caused a 17% loss of cell viability and a 32% loss of IL-4 receptor binding capacity when compared to a batch centrifuged control. A reduction of the rotor speed from 1500 to 500 RPM and reduction of system back pressure from 10 to 0 PSIG resulted in cell viability and IL-4 binding capacity comparable to the control. Several 10 L batches of baculovirus infected Sf-9 cells were also concentrated to less than 1 L by vortex flow filtration through a 3.0 microns membrane. SDS-PAGE analysis of filtrate samples showed that aggressive filtration caused cell damage which led to contamination of the process stream by cellular lysate. When rotor speed was reduced to 500 RPM and system back pressure was reduced to 0 PSIG, the amount of contaminating lysate proteins in filtrate samples was comparable to a batch centrifuged control.

Immobilization of cyclodextrin glucanotransferase on capillary membrane

A bioreactor system with the enzyme immobilized on a capillary membrane is a promising tool for the mass production of valuable substances, because of the good productive efficiency. To investigate the kinetics of immobilized cyclodextrin glucanotransferase ([EC 2.4.1.19]; CGTase) on a capillary membrane in a bioreactor system, the amount of immobilized CGTase and the operating conditions, such as pressure and the reaction temperature, were examined under a constant substrate concentration (1.0%) and a constant flow rate (0.12 m/s). When the CGTase was immobilized at a concentration of 0.04 to 0.62 mg per membrane area (cm 2), the decrease in the immobilized amount of CGTase resulted in an increase in the cyclodextrin production rate (g of CD/h·m 2; CPR) and the CPR correlated well with the flux of the CGTase-immobilized membrane. Although a higher reaction temperature caused an increase in the CPR within a short operating time of the bioreactor, repeated operation at 60°C led to a reduction in the CPR due to the denaturation of the immobilized CGTase. The percentage of cyclodextrin (CD) to total sugar obtained in the permeate was slightly more than 60% under most operating conditions, but immobilization of the excess amount of CGTase (0.42–0.62 mg/cm 2) reduced the CD yield as well as the ratio of α-CD to β-CD, suggesting that it led to a CGTase side-reaction such as intermolecular transglycosylation. These data suggest that the conditions under which the bioreactor with 0.04–0.40 mg/cm 2 was operated; a reaction temperature of 50°C, a residence time of 1–2 min and adjustable pressure, could be employed to obtain a high CPR using a large scale CGTase-immobilized membrane bioreactor.

Mass propagation of shoots of Stevia rebaudiana using a large scale bioreactor

A procedure for the mass propagation of multiple shoots of Stevia rebaudiana is described. Isolated shoot primordia were used as the inoculum to obtain clusters of shoot primordia. Such clusters were grown in a 500 liter bioreactor to obtain shoots. A total of 64.6 Kg of shoots were propagated from 460 g of the inoculated shoot primordia. These shoots were easily acclimatized in soil.

Hepatocyte Aggregate Culture Technique for Bioreactors in Hybrid Liver Support Systems

Utilizing a modified culture technique for hepatocytes, a high performance suspension culture is possible in which hepatocytes spontaneously form cell aggregates. The aggregates of 20-100 cells have been histologically confirmed to hold a three-dimensional structure, they show a long-term external metabolism and a survival time comparable with standard adhesion cultures. This technique has several advantages in the construction of large scale bioreactors for hybrid liver support systems.

Purification and characterization of recombinant human thyrotropin (TSH) isoforms produced by Chinese hamster ovary cells: the role of sialylation and sulfation in TSH bioactivity.

The biological significance of glycosylation variants of pituitary glycoprotein hormones remains controversial because of the indirect methods usually employed to determine carbohydrate composition or structure as well as the use of unreliable biological/immunological ratio to determine bioactivity. We have previously characterized recombinant human TSH (rhTSH) secreted by Chinese hamster ovary cells attached to microcarrier beads in a large scale bioreactor after stable transfection of hCG alpha and hTSH beta minigenes. In the present study rhTSH has been used as a model to determine structure-function relationships of different isoforms of glycoprotein hormones. We have now produced greater than 200 mg rhTSH using a hollow fiber bioreactor. The highly purified rhTSH produced in the hollow fiber bioreactor (rhTSH-N) as well as rhTSH commercially produced in a large scale bioreactor (rhTSH-G) were quantitated by immunoassays, receptor binding assay, and amino acid analysis and further characterized by a variety of physico-biochemical methods, including chromatofocusing and carbohydrate analysis. rhTSH-G, rhTSH-N, as well as pituitary human TSH (phTSH) have been separated by chromatofocusing on a Mono P column into several isoforms with different pI values. Compositional analysis of the fractions showed higher sialic acid content in the more acidic rhTSH-G fractions. phTSH acidic isoforms showed higher total sulfate and sialic acid contents than the more basic fractions. The bioactivities of various TSH isoforms based on rigorous quantitation of mass by amino acid analysis determined in three different FRTL-5 cell bioassays showed that the more basic and less sialylated fractions of rhTSH-G were more active than the more acidic fractions. In contrast to the in vitro data, highly sialylated and acidic rhTSH-G isoforms showed longer plasma half-lives and higher in vivo bioactivity than the basic forms. These results indicate that secreted rhTSH, similar to intrapituitary phTSH, exists as a mixture of charge isoforms that are related at least in part to the degree of sialylation. The degree of sialylation, highly dependent on the bioreactor production conditions, appears to be the major factor affecting the charge heterogeneity, MCR, and bioactivity of rhTSH.

Animal cell culture in stirred bioreactors: Observations on scale-up

Scale-up of stirred tank bioreactors from 0.02 m3 to 0.3 m3 commercial plant is discussed for hybridoma suspension culture. Schemes for dissolved oxygen control with sparged air in serum containing media are described as well as mechanical breakage of foam in small and large bioreactors. Porous metal spargers (180−200×10−6 m) are found to produce foams which are hard to control. Aeration with larger (> 0.001 m) multihole spargers is recommended. Combined cell damage due to foam formation and control, and possible damage at mechanical seals or submerged bearings, are found to have no measurable effect on cell growth relative to roller bottle production. Hybridomas are shown to withstand significant impeller tip speed (> 1 ms−1) and fluid turbulence as evidenced by impeller Reynolds numbers in excess of 105. The size of the energy dissipating terminal eddies is calculated to be > 10-fold that of the hybridoma cells. Specific fluid turnover rate is employed as the scale-up criterion.

Animal cell culture in stirred bioreactors: observations on scale-up

The scale-up of stirred tank bioreactors from 0·02 m 3 to a 0·3 m 3 commercial plant is discussed for hybridoma suspension cultures. Schemes for dissolved oxygen control with sparged air in serum containing media are described, as well as mechanical breakage of foam in small and large bioreactors. Porous metal spargers (180–200 × 10 −6 m) were found to produce foams which were hard to control. Aeration with larger (≥ 0·001 m) multihole spargers is recommended. Combined cell damage due to foam formation and control, and possible damage at mechanical seals or submerged bearings, were found to have no measurable effect on cell growth relative to roller bottle production. Hybridomas are shown to withstand significant impeller tip speed ( > 1 m s −1) and fluid turbulence as evidenced by impeller Reynolds numbers in excess of 10 5. The size of the energy-dissipating terminal eddies was calculated to be greater than ten-fold that of the hybridoma cells. The specific fluid turnover rate was employed as the scale-up criterion.

Calorimetry of technical microbial reactions

Abstract

Animal cells in turbulent fluids: details of the physical stimulus and the biological response.

Animal cells in large scale bioreactors are subjected to a variety of fluid forces for which they are not adapted by evolution. In severe cases the result is cell death, but under more modest agitation conditions an increasing number of nonlethal responses affecting growth rate, metabolism, and product formation have been reported. The forces causing these responses have not been characterized because particle-turbulence interactions are extremely complex. The current understanding of the microscopic structure of turbulence in an infinite liquid and in boundary layers shows that an average shear stress alone is not likely to be adequate to describe the bioreactor environment. Combining knowledge of the physical stimuli and the biological responses will lead to better ways of limiting cell damage and possibly to using physical stresses as a means of specifically modifying cell behavior.

Physiological similarity and bioreactor scale-up

An economically effective transfer of biological processes from laboratory to production scale is the main task of microbial process engineering. In contrast to the principle of geometrical, chemical, thermal, hydrodynamic or chemical similarity, recommended for scale-up of chemical reactors we propose the principle of physiological similarity. According to this principle same the microenvironment of the living cell must be established to reproduce the same physiological function (e.g. growth, product formation or substrate consumption rates) in the large scale bioreactor as in the laboratory one.

Large-scale calorimetry and biotechnology

Heat dissipation in biotechnical processes is often a very conspicuous phenomenon in large scale bioreactors. These reactors could hence be considered as a form of calorimeter in that the heat dissipation rate could be measured without sophisticated probes or instrumentation. Such measurements are scarce in large scale fermenters, but literature exists on calorimetric measurements carried out in the laboratory under conditions closely resembling those maintained in industrial reactors. The calorimetric techniques used for such experiments are reviewed briefly, followed by a description of a few examples of the results obtained. Published reports are described which illustrate how the knowledge gained by such experiments could be applied to industrial bioprocesses in order to control the reactor, based on monitoring the heat dissipation rate of the culture.

Some effects of heat shocks on bacterial growth

In large industrial scale bioreactors process cultures are frequently exposed to supperoptimal temperatures for short intervals of time. Data concerning the physiologica effects of such exposures are scant. Here, experiments involving short term heat shocks on two bacteria, Klebsiella pneumoniae, a non-fastidious mesophile growing on glucose and Bacillus sp. NCIB 12522, a fastidious thermotolerant methylotroph growing on methanol, are described. Markedly different response patterns for the two bacteria are evident, clearly indicating not only the dangers of making generalizations with respect to the effects of superoptimal temperatures on growing bacterial cultures but also, the significance of scale related segregation phenomena such as wall growth.

Improved Phycocatalysis of Carotene Production by Flow Cytometry and Cell Sorting

Using flow cytometry, strains of Dunaliella salina that produce large amounts of beta-carotene were sorted from mixed populations. When grown in large outdoor bioreactors, one carotene-producing strain thrived and was phenotypically stable throughout the three year trial. The use of this high-yield strain in industrial-scale processes doubled beta-carotene productivity. Flow cytometry provided a rapid and precise method to identify and isolate algal strains of potential commercial value.

Economic Mass Cultivation of Paramecium tetraurelia on a 200‐Liter Scale1

ABSTRACT. A new and inexpensive medium is described for axenic mass cultivation of Paramecium tetraurelia stock 51s and the double mutant pawn A/pawn B. Skim milk powder is the major carbon and nitrogen source in this medium. Growth characteristics (proliferation rate, final cell density, and cell size) are similar to those observed with other axenic culture media. Cultures were run in one‐liter erlenmeyer flasks, a 20‐liter, and a 250‐liter airlift bioreactor. The yield of a large bioreactor is 750 g (wet wt.) Paramecium or 5 × 109 cells. This easy, economical culture technique will greatly facilitate the use of Paramecium as a model organism for extensive biochemical studies.

Modelling and Control of a Bubble Free Aerated Bioreactor for Mammalian Cell Cultivation

A relatively new field in biotechnology is the cultivation of animal cells in large scale bioreactors. For this, particular requirements for reactor design, process control and careful treatment of the cells must be met.In this paper, modelling and control strategy are presented for a 30 1 and a 70 l-bioreactor with a special bubble-free membrane aeration system. The plants are used for repeated batch and continuous cultivations of modified mouse L 929 Tk-cells.The cells are growing on micro-carriers and producing glycosilated human interferon-B constitutively. For a mathematical description of the plant, a lumped parameter model of the reactor liquid phase and a distributed parameter model of the bubble-free aeration system was developed. The latter includes mass balances of the gas phase and of gas exchange with the fermentation broth. The control strategy is based on system analysis with this model and the design steps of the control are explained. The control algorithm was implemented on a freely programmable DDC-process-station MICON P-200. (Manufacturer: VDO, Hannover, Germany.

The Use of Automated Analyzers to Control Microbial Processes

Analytical methods used to describe fermentation processes in laboratory experiments have been automated and have been transferred to large scale bioreactors. Therefore information from laboratory experiments can be made available in process control. More substrates and products can be measu red and open new opportunities to describe large-scale processes and improve process control and performance.