Process Engineering Considerations For Scaleup

A. Fluid mechanical stress or so-called "shear damage''

Anecdotal reference to the damaging effects on cells of fluid mechanical stress or so-called ''shear damage'' is frequently made to explain poor process performance when mechanical agitation and aeration are introduced into a bioreactor as compared to the nonagitated and nonsparged conditions in a shake flask or microtitre plate (Thomas, 1990). Thomas (1990) suggested that cells might be considered to be unaffected by fluid dynamic stresses if they were of a size smaller than the Kolmogoroff microscale of turbulence, 1K. The microscale of turbulence is related to the local specific energy dissipation rate eT by Eq. (5.7). Therefore, if eT is 1 W/kg in a water-like medium, 1K ' 30 mm. However, even though bacterial cells, of size ~1-2 mm, are well below the Kolmogoroff micro-scale of turbulence, it has been reported that the mean cell volume of two strains of E. coli and of two other species of bacteria increased linearly with impeller speed during continuous cultivation with a concomitant increase in intracellular potassium and sodium ion concentration (Wase and Patel, 1985; Wase and Rattwatte, 1985). Toma et al. (1991) also studied the effect of mechanical agitation on two species, Brevibacterium flavium and Trichoderma reesei. In each case, they found that under conditions of high agitation intensity during batch culture, both growth and metabolism were inhibited. They even coined the term ''turbohypobiosis'' to describe this phenomenon and suggested that excessive turbulence may cause this inhibition by damaging the membranes of the cell. However, in these cases, the results are difficult to interpret because any changes in agitation and aeration rate will also affect levels of dissolved oxygen (dO2) via Eqs. (5.1) and (5.2) and depending on the critical dO2 value, this parameter may also affect biological performance. Thus, any experimental protocol for investigating the impact of fluid dynamic stress on cell response should be undertaken under steady state (continuous culture) conditions, including the control of dO2, if the cause of the change is to be determined conclusively. Therefore, in the cases discussed above, the results were probably based on poor experimental design and their controversial findings may have been due to the lack of controlled dO2 (Wase and Patel, 1985; Wase and Rattwatte, 1985) or the use of the constantly changing conditions experienced during batch culture (Toma et al., 1991).

Studies concerning the impact of agitation and aeration [because animal cells are potentially more easily damaged by bursting bubbles rather than rotating impellers (Nienow, 2006)] on microbial fermentations have been carried out in a stirred tank bioreactor. The bioreactor was operated as a chemostat, with blending of sparged air and nitrogen to control the driving force. Thus, again via Eqs. (5.1) and (5.2), the dO2 was controlled to a constant value. First, the impact of high levels of agitation and aeration intensity (fluid mechanical stress) on E. coli fermentation performance were addressed as measured by standard microbiological techniques and the physiologically sensitive technique of multiparameter flow cytometry (Hewitt and Nebe-von-Caron, 2001, 2004; Hewitt et al., 1998). The initial work in glucose-limited continuous culture at the 5-liter scale showed that agitation intensities, expressed as mean specific energy dissipation rates eT up to 30 W/kg and aeration rates up to 3 vvm, served only to strip away the outer polysaccharide layer (endotoxin) of the cells but did not lead to any significant change in the physiological response of individual cells, which could lead to a detrimental change in biopro-cessing. Estimates of the Kolmogoroff microscale of turbulence based on et at 30 W/kg gives 1K = 13.5 mm, well above the size of the cell (~1-2 mm). Even if the maximum local specific energy dissipation rate is used (^30 eT), to estimate 1K, a value of ^6 mm is obtained, still greater than the cell size. This agitation intensity is an order of magnitude or more greater than those typically found on the industrial scale, and the range of aeration rates tested was much higher than those normally used, thus eliminating the possibility that damage due to fluid mechanical stresses may occur under the normal range of operating conditions.

Further studies were also undertaken during continuous cultivation with the Gram-positive bacterium Corynebacterium glutamicum (Chamsartra et al., 2005) with essentially similar results. In this case, it was shown that variations in agitation, aeration rate, or dO2 concentrations down to ~1% of saturation do not cause a significant change in physiological response of C. glutamicum even though the mean cell size was slightly reduced (Figs. 5.4 and 5.5).

Similar work with the larger (~7 mm) S. cerevisiae showed that under steady state conditions, specific power inputs in the range 0.04-5 kW/m3 (1K = 16 mm) were found to have little effect on either cellular morphology or physiology even though at the upper end of the agitation range there was a small, but transient measurable effect on cell division

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