Productivity in a CHO perfusion culture reactor was maximized when pCO(2) was maintained in the range of 30-76 mm Hg. Higher levels of pCO(2) (> 150 mm Hg) resulted in CHO cell growth inhibition and dramatic reduction in productivity. We measured the oxygen utilization and CO(2) production rates for CHO cells in perfusion culture at 5.55×10(-17) mol cell(-1) sec(-1) and 5.36×10(-17) mol cell(-1) sec(-1) respectively. A simple method to directly measure the mass transfer coefficients for oxygen and carbon dioxide was also developed. For a 500 L bioreactor using pure oxygen sparge at 0.002 VVM from a microporous frit sparger, the overall apparent transfer rates (k(L)a+k(A)A) for oxygen and carbon dioxide were 0.07264 min(-1) and 0.002962 min(-1) respectively. Thus, while a very low flow rate of pure oxygen microbubbles would be adequate to meet oxygen supply requirements for up to 2.1×10(7) cells/mL, the low CO(2) removal efficiency would limit culture density to only 2.4×10(6) cells/mL. An additional model was developed to predict the effect of bubble size on oxygen and CO(2) transfer rates. If pure oxygen is used in both the headspace and sparge, then the sparging rate can be minimized by the use of bubbles in the size range of 2-3 mm. For bubbles in this size range, the ratio of oxygen supply to carbon dioxide removal rates is matched to the ratio of metabolic oxygen utilization and carbon dioxide generation rates. Using this strategy in the 500 L reactor, we predict that dissolved oxygen and CO(2) levels can be maintained in the range to support maximum productivity (40% DO, 76 mm Hg pCO(2)) for a culture at 10(7) cells/mL, and with a minimum sparge rate of 0.006 vessel volumes per minute.A = volumetric agitated gas-liquid interfacial area at the top of the liquid, 1/mB = cell broth bleeding rate from the vessel, L/minCER = carbon dioxide evolution rate in the bioreactor, mol/min[CO(2)] = dissolved CO(2) concentration in liquid, M[CO(2)](*) = CO(2) concentration in equilibrium with sparger gas, M[CO(2)](**) = CO(2) concentration in equilibrium with headspace gas, MCO(2)(1) = dissolved carbon dioxide molecule in water[C(T)] = total carbonic species concentration in bioreactor medium, M[C(T)](F) = total carbonic species concentration in feed medium, MD = bioreactor diameter, mD(I) = impeller diameter, mD(b) = the initial delivered bubble diameter, mF = fresh medium feeding rate, L/minH(L) = liquid height in the vessel, mk(A) = carbon dioxide transfer coefficient at liquid surface, m/mink (infA) (supO) = oxygen transfer coefficient at liquid surface, m/min.

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Expression of the glucoamylase gene from Aspergillus awamori by laboratory and distiller's strains of Saccharomyces cerevisiae allowed them to ferment soluble starch. Approximately 95% of the carbohydrates in the starch were utilized. Glycerol production was significantly decreased when soluble starch was used instead of glucose. Ethanol yield on soluble starch was higher than that on glucose. The rate of starch fermentation was directly related to the level of glucoamylase activity. Strains with higher levels of glucoamylase expression fermented starch faster. The decline in starch fermentation rates toward the end of the fermentation was associated with accumulation of disaccharides and limit dextrins, poor substrates for glucoamylase. The buildup of these products in continuous fermentations inhibited glucoamylase activity and complete utilization of the starch. Under these conditions maltose-fermenting strains had a significant advantage over nonfermenting strains. The synthesis and secretion of glucoamylase showed no deleterious effects on cell growth rates, fermetation rates, and fermentation products.

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