Caroline M. McFarlane

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.

Small bubble formation via a coalescence dependent break-up mechanism

Abstract A mechanism has been elucidated for the coalescence-mediated break-up of bubbles in gas–liquid systems. Images taken from dynamic systems (a coalescence cell and laboratory-scale bubble columns) show that in some instances the coalescence of two bubbles is accompanied by the formation of a much smaller daughter bubble. Following the coalescence process an annular wave is formed due to the very rapid expansion of the hole following the instant of film rupture. As the wave moves along the length of the bubble, away from the point of rupture it causes a rippling effect which distorts the newly coalesced bubble and may result in the formation of an unstable extension. Instabilities due to the annular wave pinch off a portion of this extension, resulting in the generation of a small daughter bubble. In coalescence dominated systems the process results in the generation of significant numbers of bubbles much smaller (100– 200 μm diameter) than the Sauter mean diameter (3– 4 mm ).

The use of flow cytometry to study the impact of fluid mechanical stress on Escherichia coli W3110 during continuous cultivation in an agitated bioreactor

Continuous culture fermentations of Escherichia coli W3110 have been carried out at controlled dissolved oxygen levels of 40% and 10% of saturation. Satisfactory and reproducible results were obtained. Agitation speeds of 400 and 1200 rpm at an aeration rate of 1 vvm have been used as well as an aeration rate of 3 vvm at 400 rpm. The upper levels of these variables represent much higher agitation and aeration intensities than those normally used in practical fermentations. The fermentations were monitored by mass spectrometry and optical density, and cell samples were studied by flow cytometry, SEM, and TEM. Protocols were developed so the state of both cell membranes and cell size could be measured by flow cytometry. Under all the conditions of agitation and aeration, flow cytometric analysis indicated that both cell membranes were intact and that a cytoplasmic membrane potential existed; also the cell size did not change, results confirmed by SEM and TEM. There were no detectable changes in off-gas analysis or optical density during the continuous fermentation nor in the cell structure as revealed by SEM or TEM, except at the highest agitation intensity. Under the latter conditions, after 7 h, the outer polysaccharide layer on the cell was stripped away. It is concluded that any changes in biological performance of this E. coli cell line due to variations in agitation or aeration intensity or scale of operation cannot be attributed to fluid dynamic stresses associated with the turbulence generated by impellers or with bursting bubbles.

Growth and enumeration of the meat spoilage bacterium Brochothrix thermosphacta.

Brochothrix thermosphacta is a common meat spoilage bacterium. The morphology of this bacterium changes from coccobacilli and short rods to chains during growth, which may give a false estimation in numbers using some enumeration techniques. Methods for the quantification of this bacterium have been compared. Turbidimetric readings showed good agreement with cell dry weight indicating that the former provides a good measure of the change in cell mass during growth. The turbidimetric method also correlated well with bacterial numbers determined by plate counts, flow cytometry and manual counts (by microscope) over a limited range of 10(7)-10(9) cells/ml. Flow cytometry and manual counts gave a linear relationship over a wider range of 10(5)-10(9) cells/ml. The sensitivity of analysis, growth rates and lag time attained using these methods were also compared. As a consequence of changes in bacterial cell size during growth, turbidimetry over-estimated the growth rate. The plate count method proved unable to detect the difference between bacteria existing as chains or single cells. The sensitivity of analysis and the calculated growth related parameters were similar for flow cytometry and manual counts. This suggests that flow cytometry is capable of counting individual cells in a chain. Further investigation showed that passage of B. thermosphacta cells through the flow cytometer resulted in the breakage of chains into single cells. The reliability, low error and rapidity of this technique make it attractive for bacterial enumeration, something which has been demonstrated using B. thermosphacta, a bacterium which exhibits complex morphologies.

Visualisation of bubble coalescence in a coalescence cell, a stirred tank and a bubble column

Clear pictures of coalescence events are presented for the first time in a coalescence cell, in a stirred tank and in a bulle column. These pictures also show that coalescence can occur when a pair of bubbles are forced together as they emerge from opposing horizontal nozzles even in electrolyte solutions at concentrations which are normally considered to be non-coalescing in the more gentle contact found in the bulk of agitated vessels and in bubble columns.

An evaluation of the anti-bacterial action of ceramic powder slurries using multi-parameter flow cytometry

Multi-parameter flow cytometric techniques have been used to study the effects of three ceramic powders CaO, MgO and ZnO on the physiology of individual, exponentially growing E. coli cells. Whilst all three powders inhibited reproductive growth, depending on their concentration, the mechanism of action of CaO and MgO was different to that of ZnO as shown by fluorescent staining techniques developed in our laboratory.

Using the Microcyte Flow Cytometer To Monitor Cell Number, Viability, and Apoptosis in Mammalian Cell Culture

The Microcyte is a novel, portable flow cytometer based on diode laser technology whose use has been established for yeast and bacterial analysis. We present data that demonstrate its suitability for routine mammalian cell counting and viability determination. To extend its range of applications in the field of animal cell culture biotechnology, a test to determine the number of apoptotic cells present has been developed for use with the instrument. Apoptosis was induced in hybridoma cell cultures by treatment with camptothecin. Apoptotic cells were labeled with biotinylated Annexin V and then visualized using a streptavidin‐allophycocyanin conjugate. Their numbers were counted, and the cell size of the apoptotic cell population was determined using the Microcyte.

Studies of High Solidity Ratio Hydrofoil Impellers for Aerated Bioreactors. 4. Comparison of Impeller Types

This paper compares the performance of the Prochem Maxflo T and Lightnin A315 hydrofoil impellers, a Rushton disc turbine, and a mixed flow impeller in water, electrolyte solution, and viscous shear thinning sodium carboxymethylcellulose solution. Impeller performance was compared on the basis of (a) the reduction in power drawn on aeration, for operation at equal ungassed specific power inputs, (b) the power required to disperse gas at a given gas flow rate, (c) the gas holdup generated at a given gassed specific power input and superficial gas velocity, and (d) flow stability. The equipment, techniques, and model fluids used are described in parts 2 and 3 of this series of papers. Hydrofoil impellers can maintain a higher relative power than either the Rushton turbine or mixed flow impeller over a wide range of gas flow rates, and the overall fall in power drawn is much less (typically ∼30−50%, as compared with 50−65%). This is advantageous in terms of operating efficiency and implies a higher gas−liquid mass transfer potential since the latter is directly related to the gassed specific power input. Both classes of downward pumping impeller are prone to flow and associated torque fluctuations, while operation with the Rushton turbine is much more stable. The A315 is more energy efficient in dispersing gas than the Prochem. Although in this respect the efficiencies of the A315 and Rushton turbine are similar in water, the latter is superior at low and moderate gas flow rates in CMC solution. Gas holdup can be correlated with gassed specific power input and superficial gas velocity, and the holdups produced by the different impeller types in a given model fluid are broadly similar.

Alternative ways of applying the hydrogen peroxide steady state method of KLa measurement

Activated manganese dioxide can replace the enzyme catalase as catalyst of the hydrogen peroxide decomposition in the steady state peroxide feeding method for K L a determination in aeration equipment. The equivalence of both techniques was experimentally demonstrated in deionized water. Manganese dioxide is thus a reliable alternative for the application of the method in media like strong electrolyte solutions where catalase is quickly deactivated, but some limitations of its own have also to be considered with oxidizable fluids, e.g. polypropylene glycol.

Studies of High Solidity Ratio Hydrofoil Impellers for Aerated Bioreactors. 3. Fluids of Enhanced Viscosity and Exhibiting Coalescence Repression

This paper reports on the influence of coalescence inhibition and the enhanced viscosity associated with shear thinning behavior on the fluid dynamics and power characteristics of the Prochem Maxflo T and Lightnin A315 hydrofoil impellers. The impellers, vessel, and techniques used are described in part 2 of this four‐part series of papers. Coalescence inhibition by the addition of sodium sulphate has little effect on the power drawn and by inference on the local impeller hydrodynamics although the power required to disperse gas at a given gas flow rate is reduced relative to water. The presence of shear thinning behavior and an increased viscosity (μA up to approximately 0.1 Pa s) results in a marked reduction in the ability of hydrofoil impellers to disperse gas and a reduction in the relative power drawn by the impeller. In comparison with water, the power drawn is less sensitive to operating conditions and fluid dynamic and torque instabilities are reduced for the specific geometry of a large ring sparger (DS = 0.8D) at a large absolute sparger−impeller separation.