Eric H. Dunlop

Effect of fluid shear forces on plant cell suspensions

Abstract The sensitivity of plant cell culture to fluid forces, or fluid-mechanical sensitivity, in a stirred bioreactor is evaluated by shearing the cells in viscometers for short duration under a range of laminar and turbulent conditions. This short-term evaluation is compared with the experiments in a stirred bioreactor under chemostat mode at various agitation speeds. Fluid-mechanical sensitivity was quantified by measuring various biological parameters including regrowth ability, membrane integrity, mitochondrial activity, aggregate size and lysis. A critical hydrodynamic variable was derived based on a model whereby the biological activity of cells after a given period in a defined shearing environment was a cumulative effect of the total work done by fluid forces on the cells. This hydrodynamic variable, calculated as total energy dissipation on cells per unit volume, was used as a common basis to quantify the agitation-based shear forces under laminar and turbulent flow conditions. Fluid-mechanical sensitivity data for plant cell suspensions under different flow conditions was successfully correlated with this hydrodynamic variable. This correlation established a hierarchy of response, ranging from a subtle biological inhibitory effect on regrowth ability or sublytic effect, to the gross physical effect of aggregate breakup and lysis. Inhibition of growth rather than lysis was found to dictate the performance of plant cell culture in the bioreactor.

Shear sensitivity of plant cells in suspensions present and future

Plant cells are a source of pharmaceuticals, fragrances, flavors, and dyes that are traditionally produced by extraction of tissues from whole plants. Recent trends in plant product research, transformed cell lines, and conservation policies place increased demand on plant cell culture technology. Unlike processing of microbial and animal cells in bioreactors, no economically viable process based on the suspension culture of plant cells in bioreactors has yet been possible in North America. It is proposed that the suspended-cell bioreactor is the method of choice and that plant cells respond to fluid forces (defined as laminar shear and turbulent eddies-based and bubble-based forces) differently from their animal cell counterparts in bioreactors. Although plant cells produce a tough cell wall, fluid forces, although not lethal within normal range, impact the membrane transport processes and metabolic function of plant cells; these effects are termed sublytic. Previous approaches to shear sensitivity of plant cells are reviewed in the context of these sublytic effects. A model for systematic evaluation of fluid-mechanical causes and physiological mechanisms behind sublytic effects is proposed. It is further proposed that, once understood, the plant cell’s sublytic responses to fluid force can be used advantageously in stirred suspension cultures.

Role of Turbulence in Fermentations

This paper raises the need for a systematic evaluation of non-linear interaction between various turbulent-driven physical processes in fermentors. Various turbulent/mixing studies published in the literature are formalized based on a multidimensional framework. This formalization leads us to the limitations of many existing approaches and scale-up rules to mixing problems. The results on the effects of micromixing and fluid shear on yeast cells will be presented as an example to show the possibility of a non-linear interaction. A micro-environmental approach is proposed whereby the effects of multiple turbulent-driven physical processes in the micro-environments of cells can be evaluated.

Evolution of a phase separated gravity independent bioreactor

The evolution of a phase-separated gravity-independent bioreactor is described. The initial prototype, a zero head-space manifold silicone membrane based reactor, maintained large diffusional resistances. Obtaining oxygen transfer rates needed to support carbon-recycling aerobic microbes is impossible if large resistances are maintained. Next generation designs (Mark I and II) mimic heat exchanger design to promote turbulence at the tubing-liquid interface, thereby reducing liquid and gas side diffusional resistances. While oxygen transfer rates increased by a factor of ten, liquid channeling prevented further increases. To overcome these problems, a Mark III reactor was developed which maintains inverted phases, i.e., media flows inside the silicone tubing, oxygen gas is applied external to the tubing. This enhances design through changes in gas side driving force concentration and liquid side turbulence levels. Combining an applied external pressure of four atmospheres with increased Reynolds numbers resulted in oxygen transfer intensities of 232 mmol O2/l/h (1000 times greater than first prototype and comparable to a conventional fermenter). A 1.0 liter Mark III reactor can potentially deliver oxygen supplies necessary to support cell cultures needed to recycle a 10 astronaut carbon load continuously.

Phase separated membrane bioreactor - Results from model system studies

The operation and evaluation of a bioreactor designed for high intensity oxygen transfer in a microgravity environment is described. The reactor itself consists of a zero headspace liquid phase separated from the air supply by a long length of silicone rubber tubing through which the oxygen diffuses in and the carbon dioxide diffuses out. Mass transfer studies show that the oxygen is film diffusion controlled both externally and internally to the tubing and not by diffusion across the tube walls. Methods of upgrading the design to eliminate these resistances are proposed. Cell growth was obtained in the fermenter using Saccharomyces cerevisiae showing that this concept is capable of sustaining cell growth in the terrestrial [correction of terrestial] simulation.

MEASUREMENT OF WATER DIFFUSIVITY IN AQUEOUS LITHIUM BROMIDE AND LITHIUM CHLORIDE SOLUTIONS

A relatively simple method was employed for measurement of water diffusivity in aqueous lithium bromide and lithium chloride solutions. The twin bulb apparatus used for these measurements was developed using an analogy between this apparatus and the conventional diaphragm cell apparatus. Tritiated water (TOH) was used as a tracer for these experiments because of its chemical similarity and proximity to the molecular weight of water. High tracer activity used at the beginning of the experiments allowed the use of relatively shorter time duration for each experiment (s;≈ 20 h) and a quasi-steady state equation to calculate the diffusivity from the observed tracer activity data Initially, the water diffusivity in lithium bromide solutions for concentrations varying from 0.5 M to 3 M (22.1 weight percent) was measured to obtain a comparison with published values. The lithium bromide concentration was further varied from 3 M to 11 M (57.4 weight percent) to obtain data in the concentration range usually employ...