P. Ayazi Shamlou

Design of a prototype miniature bioreactor for high throughput automated bioprocessing

Abstract A new miniature bioreactor with a diameter equal to that of a single well of a 24-well plate is described and its engineering performance as a fermenter assessed. Mixing in the miniature bioreactor is provided by a set of three impellers mechanically driven via a microfabricated electric motor and aeration is achieved with a single tube sparger. Parameter sensitive fluorophors are used with fibre optic probes for continuous monitoring of dissolved oxygen tension and an optical based method is employed to monitor cell biomass concentration during fermentation. Experimental measurements are provided on volumetric mass transfer coefficient for air–water and bacterial fermentation data are presented for Escherichia coli. The local and average power input, energy dissipation rate and bubble size are derived from an analysis of the multiphase flow in the miniature bioreactor using computational fluid dynamics (CFD). Volumetric mass transfer coefficients are predicted using Higbies penetration model with the contact time obtained from the CFD simulations of the turbulent flow in the bioreactor. Comparative data are provided from parallel experiments carried out in a 20 l ( 15 l working volume) conventional fermenter. Predicted and measured volumetric mass transfer coefficients in the miniature bioreactor are in the range 100– 400 h −1 , typical of those reported for large-scale fermentation.

The influence of mechanical forces on the morphology and penicillin production of Penicillium chrysogenum

The influence of mechanical forces resulting from the rotation of (multiple) turbine impellers on the morphology and penicillin production of Penicillium chrysogenum Panlabs P-1 was investigated in batch fermentations using semi-defined media. Experiments were carried out at three different scales of fermentation, 5 dm3,100 dm3 and 1000 dm3 working volume, with the impeller tip speed ranging from 2.5 to 6.3 m/s. Throughout all fermentations, the dissolved oxygen concentration never fell below the critical value for maximum penicillin production. Morphological measurements using image analysis showed that the mean main hyphal length and mean hyphal growth unit increased during the rapid growth period and then decreased to a relatively constant value dependent on the agitation intensity. The specific rate of penicillin production (qpen)and the average main hyphal length during the linear penicillin production phase were lower at high agitation speed, which promoted more rapid mycelial fragmentation and a higher branching frequency. Comparison of the results from the three scales showed that impeller tip speed is a poor scale up parameter whereas a term based on mycelial circulation through the zone of high energy dissipation fitted the data well.

Turbulent breakage of filamentous microorganisms in submerged culture in mechanically stirred bioreactors

Abstract A discussion is presented suggesting that hyphal breakage in mechanically stirred fermenters is likely to occur by direct tensile stresses acting on the opposite ends of the hypha of filamentous microorganisms. These stresses originate from the dynamic pressure fluctuations of eddies with scales in the inertial convection subrange of the turbulent energy spectrum. The maximum strain energy theory of failure of an elastic material under this mode of stress is used to set up a relationship between the stable length of hyphae and some of the physical parameters affecting it, including the mean energy dissipation rate in the bioreactor. Experimental data are reported for the rate of breakage of hyphae obtained under different operating conditions during the fermentation of Penicillium chrysogenum in a 7 and 150 1 mechanically stirred bioreactor. Data on the stable size of hyphae agree well with the model based on the maximum energy criterion. The experimental data further suggest that hyphal breakage is approximately a first-order kinetic process with a rate constant which appears to be moderately dependent on the product of the mean energy dissipation rate and the reciprocal of the impeller circulation time.

Characterization of flow intensity in continuous centrifuges for the development of laboratory mimics

Predicting the recovery of “delicate” biological materials by centrifugation using laboratory centrifuges has been a major challenge to biochemical engineers partly because of the difficulty in accurately quantifying the shear stresses in continuous-flow industrial centrifuges and partly because the clarification and dewatering conditions in the laboratory units do not represent those occurring in industrial centrifuges. In this paper, the flow field in the feed zone of an industrial multichamber-bowl centrifuge is mapped and its profile of energy dissipation rate established using computational fluid dynamics (CFD). A small high-speed rotating-disc device is designed with the capacity to reproduce the CFD-predicted energy dissipation rates in the feed zone. Milliliter quantities of the process material are shear-treated in the device operating at a speed that mimics the local critical flow conditions in the industrial centrifuge. The results are used to assess the impact of flow conditions in the feed zone of the centrifuge on the physical properties of protein precipitates and on their predicted recovery using a laboratory centrifuge. This approach was used to explain the reduction in the performance of the centrifuge from the predicted 88% clarification to the observed 39% clarification of the precipitate particles. The combination of the small high-speed disc device and the laboratory centrifuge is particularly advantageous when dealing with biological products at the early stages of process development for which only small quantities of test material are often available.

Crystal break-up in dilute turbulently agitated suspensions

An analysis of particle attrition in dilute stirred suspensions is developed to predict both the impact attrition rate as a consequence of crystal collisions and the turbulent attrition rate caused by the liquid motion. Detailed attrition experiments designed for model discrimination were carried out in a 1.5 l vessel fitted with four wall baffles and agitated by means of stainless steel and silicone rubber (RTV) coated turbines, respectively, for dilute suspensions (< 3.2% v/v) in the unit power input range 0.6–1.5W/kg and parent particle size range 100–1000 μm, using potassium sulphate and potash alum crystals in saturated ethanol solutions respectively. The experimental results are consistent with predictions of attrition fragments arising from crystal-impeller collisions, with a strong effect of impeller hardness on the average attrition rate, together with turbulent fluid drag-induced particle attrition. It is also predicted, however, that the total attrition rate decreases on vessel scale-up at constant power input, with an increasing proportion of fine particles being due to turbulence, arising from the independence of turbulent forces of the vessel scale and a decrease in the corresponding collisional impact forces.

Solids suspension and distribution in liquids under turbulent agitation

Abstract Experimental data are provided for axial concentration gradients formed during particle suspension in liquids under turbulent agitation. A simple model based on a turbulent diffusion support mechanism is developed in order to interpret the observed longitudinal concentration profiles. The model is based on Kolmogoroffs theory of isotropic turbulence and the settling characteristics of the particles. The reasonable agreement between the theory and the measurements indicates a general applicability for the proposed model.

Power consumption of helical ribbon mixers in viscous newtonian and non-newtonian fluids

Abstract Power input measurements are reported for helical ribbon impellers for two scales; a 0.15 m diameter and a 0.4 m diameter tank. Data for viscous Newtonian and non-Newtonian fluids are brought together by use of the average apparent viscosity concept and the following equation: where ks is the shear rate constant, c is the clearance between vessel wall and impeller with diameter D. Power measurements from this work combined with relevant information extracted from the published literature indicate that impeller geometry has a profound effect upon power requirement, particularly in the laminar region, where the reported data can be described by: where Kp is a geometric constant and all the other symbols have their usual significance. Theoretical models which fail to allow for system geometry and fluid properties give values which may be seriously in error.

The effects of material properties and fluid flow intensity on plasmid DNA recovery during cell lysis

The disruption of recombinant E. coli cells containing a 76.8 kb plasmid DNA was achieved by the chemical lysis method in a coaxial cylinder rheometer which allowed in situ measurements of rheological changes to be carried out as the lysis reaction proceeded. For the cases studied the cell lysis time was found to be approximately 30 s. Moreover, the release of intracellular material produced a mixture with shear thinning flow properties, the extent of non-Newtonian flow was found to depend on the shear rate used during the cell lysis operation. On neutralisation, the lysate produced a highly flocculated and shear sensitive gel which floated on the top of the liquor containing the plasmid DNA. Small amplitude oscillatory data were obtained showing the viscoelastic properties of the gel matrix. Experimental data were also obtained on the shear sensitivity of the plasmid DNA recovered using a purpose-built rotating disk shear device. Shear rates of the order of 106 s-1 were generated in the device and were confirmed by CFD analysis of the prevailing flow field. Tests carried out with 20 and 29 kb plasmid DNA showed that both plasmids were susceptible to shear damage. The extent of shear damage increased with plasmid size and as the ionic strength of solution decreased.

A physical model of high-pressure disruption of bakers' yeast cells

A model is presented for the disruption of microbial cells in a high-pressure homogeniser. It assumes that cell deformation and breakage due to elongational stresses occur in the high-stress zones close to the surfaces of the valve rod and the impact ring of the homogeniser. The equation based on the proposed model predicts an inverse cube-root dependency of the maximum diameter of cells that remain unbroken upon the pressure drop across the valve, and suggests that the maximum whole cell diameter will increase with an increase in the mechanical integrity of the cell walls. Experimental data are provided for the disruption of batch and continuously grown cells of Saccharomyces cerevisiae. The results from these experiments support, and are interpreted in terms of the predictions from, the proposed model.

Hydrodynamics of secondary nucleation in suspension crystallization

Abstract Hydrodynamic models of secondary nucleus generation in mechanically agitated suspension crystallizers are analysed in detail and their predictions compared with experimental measurements using potassium sulphate crystals. The data indicate that, under operating conditions similar to those found in industrial crystallizers, secondary nuclei are produced by a particle attrition process consistent with a turbulent fluid-induced breeding mechanism having critical eddies in the viscous dissipation subrange of the turbulent energy spectrum.