Dermot M. Malone

Plant cell suspension cultures: some engineering considerations

Higher plants are the source of a vast array of biochemicals which are used as drugs, pesticides, flavourings and fragrances. For some of these compounds, plant cell culture can provide a potential production alternative to traditional cultivation methods or chemical synthesis routes. Many systems have been patented and the last 20 years have seen considerable industrial and academic interest in the development of large scale cultures to produce pharmaceutically active, high value substances. However, the industrial application of plant cell suspension cultures has, to date, been limited. Commercialisation has essentially been impeded by economic feasibility, arising from both biological and engineering considerations. This paper reviews the commercial development of the technology to date and focuses on the impact of specific engineering-related factors, in particular, the shear sensitivity of plant cell suspension cultures. Evidence of sensitivity to hydrodynamic shear in bioreactors has generally been attributed to the physical characteristics of the suspended cells. Recent studies indicate that shear sensitivity may not be as important, in some cases, as initially anticipated.

Effects of Hydrodynamic and Interfacial Forces on Plant Cell Suspension Systems

Plant cells are perceived to be sensitive to the hydrodynamic environment in conventional bioreactors. Heightened sensitivity, relative to most bacterial cultures, is frequently attributed to larger plant cell sizes, extensive vacuolization and aggregation patterns. Early studies of shear sensitivity focused on cell lysis and/or loss of viability. More recently, an extensive array of sub-lethal responses has been identified. A fuller understanding of these sub-lytic effects may assist in the optimization of large-scale cultivation conditions. This paper reviews recent work on the hydrodynamic shear sensitivity of plant cell systems, under cultivation conditions and in purpose-built shearing devices. The relevance of different approaches to the characterization of the intensity of a given hydrodynamic environment is discussed. Indicators of cell response to hydrodynamic stress are evaluated. The potential significance of cellular defense mechanisms, observed in response to mechanical stimulants, is identified.

Modelling the effects of particle polydispersity in crossflow filtration

Abstract A mathematical model of crossflow filtration which accounts for particle polydispersity is developed. The model combines classical filtration theory with the concept of the cut-off particle diameter (i.e., the diameter below which particles will deposit on the membrane), to yield a single differential equation for the filter cake thickness as a function of time. Using an analysis of the hydrodynamic forces acting on a depositing particle, the cut-off diameter is expressed as a function of cake thickness. The differential equation is then solved numerically by considering the cake to be incompressible and to be composed of discrete layers of different average particle size, the average particle size being greatest in the layer adjacent to the membrane, and lowest in the outermost layer. Steady state is shown to be attained when the fraction of suspension particles which are below the cut-off diameter, approaches zero. It is demonstrated that the steady state filtrate flux increases with transmembrane pressure and crossflow velocity, and decreases with increasing membrane resistance. The average specific cake resistance is found to increase with membrane resistance and crossflow velocity (due to deposition of smaller particles), but to decrease with increasing transmembrane pressure (due to deposition of larger particles). Thus, the particle polydispersity effects modelled here cannot explain experimental observations of fluxes which decrease with increasing crossflow velocity, because the decrease in cake-height more than compensates for the increase in specific cake resistance.

Growth patterns of Saccharomyces cerevisiae microcolonies in alginate and carrageenan gel particles: Effect of physical and chemical properties of gels

Abstract When Saccharomyces cerevisiae cells were inoculated at low density (1 · 103–1.5 · 105 cells [g gel]−1) in alginate gel beads and cylinders, cells grew in the form of distinct microcolonies throughout the gel matrix. Alginate gel beads and cylinders, formed by external gelation with Ca2+, gave rise to microcolonies which became elongated and lens-shaped, with their major axes aligned with the gel surface. The aspect ratio (major axis/minor axis length) of the microcolonies and the local concentration of alginate increased with increasing distance from the center of the gel particles. In contrast, spherical microcolonies were observed in alginate cylinders formed by internal gelation and no significant local concentration gradients of alginate were detected in these gels. Nonspherical microcolonies were also observed in carrageenan gel beads. However, the colonies were irregularly shaped, and their major axes demonstrated no preferential alignment.

Cell growth patterns in immobilization matrices

Within an immobilized cell matrix, mass transfer limitations on substrate delivery or product removal can often lead to a wide range of local chemical environments. As immobilized living cell populations actively grow and adapt to their surroundings, these mass transfer effects often lead to strong, time-dependent spatial variations in substrate concentration and biomass densities and growth rates. This review focuses on the methods that have been devised, both experimentally and theoretically, to study the non-uniform growth patterns that arise in the mass transfer limited environment of an immobilization matrix, with particular attention being paid to cell growth in polysaccharide gels.

The effects of turbulent jet flows on plant cell suspension cultures

Cell suspensions of Morinda citrifolia were subjected to turbulent flow conditions in a submerged jet apparatus, to investigate their hydrodynamic shear susceptibility. The suspensions were exposed to repeated, pressure-driven passages through a submerged jet. Two nozzles, of 1 mm and 2 mm diameter, were employed. Average energy dissipation rates were in the range 10(3)-10(5) W/kg and cumulative energy dissipation in the range 10(5)-10(7) J/m3. System response to the imposed conditions was evaluated in terms of suspension viability (determined using a dye exclusion technique) and variations in both chain length distribution and maximum chain length. Viability loss was well-described by a first-order model, and a linear relationship was identified between the specific death rate constant and the average energy dissipation rate. This relationship was consistent with results obtained using the same suspension cultures in a turbulent capillary flow device. Morphological measurements indicated that exposure to the hydrodynamic environment generated in the jet resulted in a significant reduction in both the average and maximum chain lengths, and the reduction in the maximum chain length was identified as an appropriate measure of sustained damage. Analysis of both viability and chain length in terms of cumulative energy dissipated revealed good agreement with results reported by other authors for morphologically different plant cell systems. Copyright 1998 John Wiley & Sons, Inc.

Preferential deposition of smaller cells during cross-flow microfiltration of a yeast suspension

By examining the size distribution of cells that do not deposit during cross-flow microfiltration of a yeast suspension, we demonstrate that the smaller cells in the suspension are preferentially deposited on the membrane. The extent to which the deposition process favours smaller cells was found to be unaffected by cell concentration or membrane type over the range of concentrations examined.

Membrane Fouling during Constant Flux Crossflow Microfiltration of Dilute Suspensions of Active Dry Yeast

Abstract Fouling of microporous and ultrafiltration membranes during crossflow microfiltration of rehydrated active dry yeast (ADY) was investigated using measurements of the transmembrane pressure as a function of time at constant flux. By centrifuging the suspensions and comparing the increase in transmembrane pressure produced by both the original suspensions and the supernatant alone, it was determined that this increase was mainly caused by soluble components in the supernatant. This finding is consistent with previous observations that considerable quantities of intracellular matter leak from cells of ADY when they are rehydrated. The increase in transmembrane pressure caused by the supernatant alone was found to be independent of tangential flow rate. suggesting that the underlying mechanism was one of internal membrane fouling. Fouling was found to be enhanced by increasing the transmembrane flux and reducing the membrane pore size. Membrane fouling by the supernatant was modeled as a process invo...

Determination of the radial distribution of Saccharomyces cerevisiae immobilised in calcium alginate gel beads

The dissolution of alginate gel beads in 20 g sodium citrate /l produces a linear decrease in bead diameter. The rate of dissolution is dependent on the concentration of CaCl2 within the gel beads. This method allows the controlled release of Saccharomyces cerevisiae from alginate gel beads and permits the simple and rapid determination of the radial distribution of cell concentration.