A Large bioreactor is an inhomogenous system with concentration gradients which depend on the fluid dynamics and the mass transfer of the reactor, the feeding strategy, the saturation constant, and the cell density. The responses of Escherichia coli cells to short-term oscillations of the carbon/energy substrate in glucose limited fed-batch cultivations were studied in a two-compartment reactor system consisting of a stirred tank reactor (STR) and an aerated plug flow reactor (PFR) as a recycle loop. Short-term glucose excess or starvation in the PFR was simulated by feeding of glucose to the PFR or to the STR alternatively. The cellular response to repeated short-term glucose excess was a transient increase of glucose consumption and acetate formation. But, there was no accumulation of acetate in the culture, because it was consumed in the STR part where the glucose concentration was growth limiting. However, acetate accumulated during the cultivation if the oxygen supply in the PFR was insufficient, causing higher acetate formation. The biomass yield was then negatively influenced, which was also the case if the PFR was used to simulate a glucose starvation zone. The results suggest that short-term heterogeneities influence the cellular physiology and growth, and can be of major importance for the process performance. (c) 1995 John Wiley & Sons, Inc.

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Insect cell metabolism was studied in substrate-limited fed batch cultures of Spodoptera frugiperda (Sf-9) cells. Results from a glucose-limited culture, a glutamine-limited culture, a culture limited in both glucose and glutamine, and a batch culture were compared. A stringent relation between glucose excess and alanine formation was found. In contrast, glucose limitation induced ammonium formation, while, at the same time, alanine formation was completely suppressed. Simultaneous glucose and glucosamine limitation suppressed both alanine and ammonium formation. Although the metabolism was influenced by substrate limitation, the specific growth rate was similar in all cultures. Alanine formation must involve incorporation of free ammonium, if ammonium formation is mediated by glutaminase and glutamate dehydrogenase, as our data suggest. On the basis of the results, two possible pathways for the formation of alanine in the intermediary metabolism are suggested. The cellular yield on glucose was increased 6.6 times during glucose limitation, independently of the cellular yield on glutamine, which was increased 50-100 times during glutamine limitation. The results indicate that alanine overflow metabolism is energetically wasteful and that glutamine is a dispensable amino acid for cultured Sf-9 cells. Preliminary data confirm that glutamine can be synthesized by the cells themselves in amounts sufficient to support growth.

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Cell cycle progression was studied in serum-free batch cultures of Spodoptera frugiperda (Sf9) insect cells, and the implications for proliferation and productivity were investigated. Cell cycle dynamics in KBM10 serum-free medium was characterized by an accumulation of 50-70% of the cells in the G(2)/M phase of the cell cycle during the first 24 h after inoculation. Following the cell cycle arrest, the cell population was redistributed into G(1) and in particular into the S phase. Maximum rate of proliferation (micro(N, max)) was reached 24-48 h after the release from cell cycle arrest, coinciding with a minimum distribution of cells in the G(2)/M phase. The following declining micro(N) could be explained by a slow increase in the G(2)/M cell population. However, at approximately 100 h, an abrupt increase in the amount of G(2)/M cells occurred. This switch occurred at about the same time point and cell density, irrespective of medium composition and maximum cell density. An octaploid population evolved from G(2)/M arrested cells, showing the occurrence of endoreplication in this cell line. In addition, conditioned medium factor(s) were found to increase micro(N,max), decrease the time to reach micro(N,max), and decrease the synchronization of cells in G(2)/M during the lag and growth phase. A conditioned medium factor appears to be a small peptide. On basis of these results we suggest that the observed cell cycle dynamics is the result of autoregulatory events occurring at key points during the course of a culture, and that entry into mitosis is the target for regulation. Infecting the Sf9 cells with recombinant baculovirus resulted in a linear increase in volumetric productivity of beta-galactosidase up to 68-75 h of culture. Beyond this point almost no product was formed. Medium renewal at the time of infection could only partly restore the lost hypertrophy and product yield of cultures infected after the transition point. The critical time of infection correlated to the time when the mean population cell volume had attained a minimum, and this occurred 24 h before the switch into the G(2)/M phase. We suggest that the cell density dependent decrease in productivity ultimately depends on the autoregulatory events leading to G(2)/M cell cycle arrest.

Carbon mass balances were calculated in fed-batch cultures of E. coli W3110, using mineral medium with glucose as the limiting substrate. The carbon recovery, based on biomass, CO(2), and acetate was approximately 90% at the end of the culture (25 h, 27 g L(-1) dw). The missing carbon remained as soluble organic compounds in the medium. Outer membrane (OM) constituents, such as lipopolysaccharides (LPS), phospholipids (PL), and carbohydrates (each at approximately 1 g L(-1)) contributed to 63% of the extracellular carbon. The amount of released LPS and PL equaled the total amount of OM bound to the cells in the culture. Small amounts of DNA and protein detected in the medium indicated that no cell lysis had occurred. Acetate, lactate, ethanol, formate, succinate and amino acids (Glu, Gln, Asp, Asn, Ala, Gly, Ser) were detected in the culture medium, but made up only a few percent of the extracellular carbon mass. The remaining 30% was not identified, but was assumed to constitute complex carbohydrates.