Monoclonal antibody productivity and the metabolic pattern of perfusion cultures under varying oxygen tensions

The metabolic pattern and cell culture kinetics of high-cell-density perfusion cultures were compared under two different oxygen transfer conditions: oxygen limiting and not limiting. When oxygen was a limiting factor during perfusion culture, both specific glucose uptake and lactate production rates increased, compared to non-oxygen-limited condition, by about 60% and 30%, respectively. The specific glutamine uptake rate under oxygen-limited conditions was almost 4.0 times higher than that under non-oxygen-limited conditions. The activity of lactate dehydrogenase (LDH) released into the medium by the dead cells can be used as an indicator for the metabolic and physiological conditions related to oxygen limitation. There was a 3.2 times higher specific rate of LDH activity released by dead cells in oxygen-limited cultures than those in non-oxygen-limited cultures. The specific production rate of monoclonal antibody was not significantly affected by the oxygen transfer conditions during the rapid cell growth period, but it rapidly increased toward the end of perfusion cultures. The higher perfusion rate may have limited further cell growth during high-cell-density perfusion culture, because cell damage was caused by the hydrodynamic shear within a hollow fiber microfiltration cartridge installed to withdraw the spent medium and the waste metabolites. (c) 1993 John Wiley & Sons, Inc.     

Alterations in the characteristics of 2-deoxy D-glucose transport into cultured human glioma cells during cell growth.

The uptake of 2-deoxy-d-glucose (2-DG) into human glioma cells (138 MG) was related to cell growth. The uptake of 2-DG was high at confluency but low in both rapidly growing sparse cultures and in growth-inhibited dense cultures. Lineweaver-Burke plots of uptake at different cell densities showed changes in V_max; K_m, however, remained constant. Dibutyryl cyclic-AMP (db-cAMP) doubled the uptake of 2-DG into rapidly growing sparse cultures but lacked effect at higher cell density. Independent of their density, cells treated with db-cAMP attained the characteristic morphology of differentiated glial cells.     

Kinetics of microbial oxidation of n-heptane. I. Characterization of the process

Utilization of n-heptane by a Pseudomonad was studied in pilot-size butch cultures. Optimal pH and temperature were determined by a factorial design and a medium based upon mineral uptake rates was formulated. High cell yields were obtained by volatilizing heptane in the incoming air and thereby achieving good hydrocarbon dispersion. Hydrocarbon carried by effluent gases was recovered and recycled. In cultures where pH is not controlled, decrease in the electrolytic conductivity of the medium was found to be indicative of viable cells and was used in monitoring bacterial propagation. If not checked, increase in salinity in pH controlled cultures was found to affect cell production negatively. Viscosity changes were not very significant. Heptane to aqueous medium ratio was found to affect oxygen supply to the system due to higher dissolved oxygen concentrations associated with hydrocarbons.     

Properties ofAzotobacter vinelandii in phosphate-limited batch cultures

Batch cultures ofA. vinelandii in ammonium phosphate-limited and N-free phosphate-limited media were compared with control cultures (N-free phosphate-sufficient media). The effects of phosphate limitation on growth were determined by viable cells counts. Under phosphate-limitation conditions, growth inhibition and decreased viability were observed. Intracellular levels of RNA, poly-3-hydroxybutyrate, phosphate and oxygen uptake were significantly affected by phosphate limitation. When phosphate-limited cultures were examined microscopically, pleomorphism was more marked than in control cultures. Also phosphate-limited cells showed an increase in resistance to UV irradiation, mechanical disruption, desiceation and the combined action of ethylenediaminetetraacetie acid and lysozyme.     

Carrier-mediated Uptake of Arginine and Urea by Volvox carteri f. nagariensis

Volvox carteri f. nagariensis takes up arginine via a high affinity, highly specific carrier, whereas carriers for neutral and acidic amino acids cannot be detected (even in nitrogen-starved cultures). Exogenous arginine is accumulated against a steep concentration gradient and is incorporated into protein with high efficiency, but it is not catabolized to any significant extent and will not serve as a nitrogen source adequate to support growth. Urea is also taken up by a saturable carrier, but several lines of evidence indicate that the arginine and urea carriers are distinct and different. Preexposure to arginine suppresses arginine uptake while stimulating urea uptake. The K_i values observed for reciprocal, competitive inhibition of uptake by arginine and urea are orders of magnitude different from the respective K_m values for uptake. The two uptake systems show entirely different patterns of sensitivity to inhibition by structural analogs. Finally, the V_max values for arginine and urea uptake fluctuate independently (but in a regular pattern) during the asexual life cycle. The fluctuations of urea uptake activity are of considerable magnitude and appear to be linked to key phases of the developmental program.     

Microoxic-Anoxic Niche of Beggiatoa spp.: Microelectrode Survey of Marine and Freshwater Strains

Beggiatoa spp. grow optimally in media containing opposed gradients of oxygen and soluble sulfide, although some strains also require an organic substrate. By using microelectrodes, we characterized oxygen and sulfide gradients during their initial development in uninoculated media and in cultures of marine and freshwater strains. In gradient media, Beggiatoa strains always grew some distance below the air/agar interface as a dense “plate” of constantly gliding filaments with sharply demarcated upper and lower boundaries. Within established plates, the maximum oxygen partial pressure was 0.6 to 6.0% of air saturation and not significantly lower if filaments were fixing nitrogen. Oxygen penetrated only 100 to 300 μm into the plate, and the anoxic fraction increased from less than 10% to approximately 90% during later stages of growth. For lithoautotrophically grown marine strains, the linearity of the oxygen profile above the plate plus its drop to zero therein indicated that oxygen uptake for the entire tube occurred only within the Beggiatoa plate. Consequently, oxygen consumption could be predicted solely from the distance between the air/agar interface and the top of a plate, given the diffusion coefficient for oxygen. By contrast, for freshwater strains grown heterotrophically (with sulfide also in the medium), oxygen profiles were frequently nonlinear because of nonbiological reaction with sulfide which had diffused past the aggregated filaments. For all strains tested, microoxic aggregation also occurred in the absence of sulfide, apparently reflecting a step-up phobic response to oxygen.     

Determination of the respiration kinetics for mycelial pellets of Phanerochaete chrysosporium.

In mycelial pellet cultures of the white rot basidiomycete Phanerochaete chrysosporium, low oxygen concentration negatively affects the production of the extracellular lignin peroxidases and manganese peroxidases which are key components of the lignin-degrading system of this organism. To test the hypothesis that oxygen limitation in the pellets is responsible for this effect, oxygen microelectrodes were used to determine oxygen concentration gradients within the mycelial pellets of P. chrysosporium. Pellets were removed from oxygenated cultures, allowed to equilibrate with air, and probed with oxygen microelectrodes. The oxygen profiles were modelled assuming that O2 uptake follows a Michaelis-Menten relationship. The Vmax and Km values for oxygen uptake were 0.76 +/- 0.10 g/m3 of pellet per s and 0.5 +/- 0.3 g/m3, respectively. These kinetic values were used to predict respiration rates in air-flushed cultures, oxygen-flushed cultures, and cultures with large pellets (diameter greater than 6 mm). The predicted respiration rates were independently validated by experimentally measuring the evolution of carbon dioxide from whole cultures.     

Changes in eggshell permeability to oxygen during the early developmental stages in diapause eggs of Bombyx mori

An apparatus for direct measurement of the eggshell permeability to oxygen was devised to test the hypothesis that diapause initiation in the Bombyx mori egg is caused by the eggshell becoming insufficiently perrneable to air to allow further embryogenesis. Using this apparatus, it was found that permeability of the eggshell to oxygen decreased dramatically during the first 2 days of incubation in prospective diapause eggs, but no appreciable changes were found in nondiapause eggs for the first 6 days of incubation. The rates of oxygen uptake by diapause and nondiapause eggs increased in a similar pattern for 26 h after oviposition. Thereafter, the rate of oxygen uptake in the former decreased while that of latter continuously increased. The cause and physiological meaning of the rapid drop of eggshell permeability just before diapause initiation are obscure. It is suggested that the decrease in oxygen uptake may be due to decreased eggshell permeability to oxygen.     

A remedy to oxygen limitation problem in antibiotic production: addition of perfluorocarbon

In this study, the effect of an oxygen carrier, perfluorocarbon, on actinorhodin fermentation by Streptomyces coelicolor A3(2) was investigated using a chemically defined medium in 2 and 20 l bioreactors. The inclusion of 50% (v/v) perfluorocarbon in the fermentation medium resulted in a five-fold increase in the maximum antibiotic concentration. The use of perfluorocarbon also caused remarkable increases in both glucose and oxygen consumption rates. Moreover, the increasing concentrations of perfluorocarbon improved the dissolved oxygen profile by raising the minimum dissolved oxygen concentration. It was found that observed increases in the antibiotic production were linearly related to the volumetric oxygen uptake rates. This result could perhaps be attributed to the enhancement of oxygen transfer in S. coelicolor cultures due to the higher oxygen solubilities of the fermentation medium through inclusion of perfluorodecalin.     

Determination of the respiration kinetics for mycelial pellets of Phanerochaete chrysosporium.

In mycelial pellet cultures of the white rot basidiomycete Phanerochaete chrysosporium, low oxygen concentration negatively affects the production of the extracellular lignin peroxidases and manganese peroxidases which are key components of the lignin-degrading system of this organism. To test the hypothesis that oxygen limitation in the pellets is responsible for this effect, oxygen microelectrodes were used to determine oxygen concentration gradients within the mycelial pellets of P. chrysosporium. Pellets were removed from oxygenated cultures, allowed to equilibrate with air, and probed with oxygen microelectrodes. The oxygen profiles were modelled assuming that O2 uptake follows a Michaelis-Menten relationship. The Vmax and Km values for oxygen uptake were 0.76 +/- 0.10 g/m3 of pellet per s and 0.5 +/- 0.3 g/m3, respectively. These kinetic values were used to predict respiration rates in air-flushed cultures, oxygen-flushed cultures, and cultures with large pellets (diameter greater than 6 mm). The predicted respiration rates were independently validated by experimentally measuring the evolution of carbon dioxide from whole cultures.     

FACTORS LIMITING BACTERIAL GROWTH : III. CELL SIZE AND "PHYSIOLOGIC YOUTH" IN BACTERIUM COLI CULTURES

1. Measurements of the rate of oxygen uptake per cell in transplants of Bacterium coli from cultures of this organism in different phases of growth have given results in essential agreement with the observations of others. 2. Correlations of viable count, centrifugable nitrogen, and turbidity, with oxygen consumption, indicate that the increased metabolism during the early portion of the growth period is quantitatively referable to increased average size of cells. 3. Indirect evidence has suggested that the initial rate of growth of transplants is not related to the phase of growth of the parent culture.     

Dietary influence on the biology and metabolism of Isotoma viridis (Collembola)

(1) Two groups of individually cultured Isotoma viridis were studied. They were given a diet of Tetramin fishfood and Pleurococcus algae respectively, in order to monitor individual changes in oxygen uptake during culture. (2) Growth rate, moulting frequency and the maximal live weight reached were higher when fed on Tetramin. (3) Egg production in the culture fed Pleurococcus was twice that fed on Tetramin, due to the more fertile intermoult periods without oviposition in animals fed Tetramin. The egg quality seemed to be lowered in the algae-fed animals rather less than in those fed with fishfood. (4) In both cultures, an increase of 35–40% of the metabolic rate at the start of the experiment was observed. This increase was maintained with a Tetramin diet, whilst it disappeared in cultures fed Pleurococcus. The F₁ generation of both groups showed an increased metabolic rate, that from Tetramin-fed being significantly higher than that from Pleurococcus-fed animals. (5) It is suggested that the initial increase in metabolism is a result of excess food, and the increased level of oxygen uptake in animals on a Tetramin diet the result of inadequacy of the composition of the fishfood for egg production, thus channelling the energy uptake mainly into growth and metabolism. (6) Neither Tetramin nor Pleurococcus are considered to provide an adequate diet for I. viridis, as reproductive success differed considerably from field conditions.     

Stimulation of fermentation and yeast-like morphogenesis in Mucor rouxii by phenethyl alcohol

The germination of fungal spores into hyphae was inhibited by concentrations of phenethyl alcohol (PEA) from 0.05 to 0.3%. Spores of Mucor formed budding spherical cells instead of filaments. These cells were abundant in cultures of Mucor rouxii at 0.22% PEA, provided that the carbon source was a hexose at 2 to 5%. Morphology was filamentous with xylose, maltose, sucrose, or a mixture of amino acids. Removal of PEA resulted in the conversion of yeast-like cells into hyphae. PEA did not inhibit biosynthesis of cytochromes or oxygen uptake, but it stimulated CO₂ and ethyl alcohol production. PEA had no effect on the rate of oxygen uptake, but it inhibited the oxidative-phosphorylation activity of mitochondria. These results suggested that growth inhibition by PEA could result from uncoupling of oxidative phosphorylation and that, in Mucor, yeast-like morphology and fermentation were linked.     

Oxygen regulation of nitrate uptake in denitrifying Pseudomonas aeruginosa.

Oxygen had an immediate and reversible inhibitory effect on nitrate respiration by denitrifying cultures of Pseudomonas aeruginosa. Inhibition of nitrate utilization by oxygen appeared to be at the level of nitrate uptake, since nitrate reduction to nitrite in cell extracts was not affected by oxygen. The degree of oxygen inhibition was dependent on the concentration of oxygen, and increasing nitrate concentrations could not overcome the inhibition. The inhibitory effect of oxygen was maximal at approximately 0.2% oxygen saturation. The inhibition appeared to be specific for nitrate uptake. Nitrite uptake was not affected by these low levels of aeration, and nitrite reduction was only partially inhibited in the presence of oxygen. The regulation of nitrate respiration at the level of transport by oxygen may represent a major mechanism by which the entire denitrification pathway is regulated in P. aeruginosa.     

Studies on automatically aerated biosynthetic processes. II. Occurrence and elimination of CO2 during penicillin biosynthesis

Oxygen uptake of Penicillium chrysogenum hyphae growing in automatically aerated deep cultures was the subject of local and periodical change. The change depended on the concentration of carbon dioxide which accumulated in the gas phase of system during the evolution of foam bubbles, and which was suddenly liberated when the foam was destroyed. The actual concentration of sunflower oil added as an antifoaming agent also influenced the oxygen uptake of culture.     

Uptake of oxygen,release and degradation of hydrogen peroxide by Streptococcus mutans NCTC 10449

Streptococcus mutans NCTC 10499 was cultured under glucose limitation in a chemostat at varying oxygen supply. The rates of oxygen uptake and hydrogen peroxide degradation by cells from the cultures were measured polarographically using a Clark electrode. Oxygenation of the chemostat culture led to adaptation of the organism to oxygen, in that the maximum oxygen uptake rate of the cells was higher when the cells were grown at higher rate of oxygen supply. It is noted that anaerobically grown cells still exhibited significant oxygen uptake. The rate of oxygen uptake followed saturation-type kinetics and Ks values of cells for oxygen were in the micromole range. Hydrogen peroxide accumulation was not observed in aerated chemostat cultures. However, anaerobically grown cells accumulated H2O2 when exposed to oxygen. Cells from aerated cultures did not accumulate hydrogen peroxide. This may be explained by the fact that the rate of hydrogen peroxide degradation was consistently higher than the rate of oxygen uptake.     

Isolation of Methylophaga spp. from Marine Dimethylsulfide-Degrading Enrichment Cultures and Identification of Polypeptides Induced during Growth on Dimethylsulfide

Dimethylsulfide (DMS)-degrading enrichment cultures were established from samples of coastal seawater, nonaxenic Emiliania huxleyi cultures, and mixed marine methyl halide-degrading enrichment cultures. Bacterial populations from a broad phylogenetic range were identified in the mixed DMS-degrading enrichment cultures by denaturing gradient gel electrophoresis (DGGE). Sequences of dominant DGGE bands were similar to those of members of the genera Methylophaga and Alcanivorax. Several closely related Methylophaga strains were obtained that were able to grow on DMS as the carbon and energy source. Roseobacter-related populations were detected in some of the enrichment cultures; however, none of the Roseobacter group isolates that were tested were able to grow on DMS. Oxidation of DMS by Methylophaga sp. strain DMS010 was not affected by addition of the inhibitor chloroform or methyl tert-butyl ether, suggesting that DMS metabolism may occur by a route different from those described for Thiobacillus species and other unidentified marine isolates. Addition of DMS and methanethiol to whole-cell suspensions of strain DMS010 induced oxygen uptake when strain DMS010 was grown on DMS but not in cells grown on methanol. The apparent K_ms of strain DMS010 for DMS and for methanethiol were 2.1 and 4.6 μM, respectively, when grown on DMS. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the biomass of strain DMS010 and analysis of peptide bands by mass spectrometry techniques and N-terminal sequencing provided the first insight into the identity of polypeptides induced during growth on DMS. These included XoxF, a homolog of the large subunit of methanol dehydrogenase for which a biological role has not been identified previously.     

Computer-aided on-line monitoring of physiological variables in suspended cell cultures of Perilla frutescens in a bioreactor

A computer-aided on-line real-time monitoring system for plant cell bioprocesses was established and applied to the cultivation of Perilla frutescens plant cells in a bioreactor. This system calculated several informative process variables which were useful for the identification of the physiological states of the plant cells during cultivation. Some variables, such as the respiratory quotient (RQ), pH, and specific carbon dioxide evolution rate (SCER), could be used for the identification of the growing phase of cell cultures. The results also suggest that the oxygen uptake rate (OUR) and the specific OUR (SOUR) may depend on the accumulation of anthocyanin (a secondary metabolite) in P. frutescens cell cultures.     

Effects of inhibitors on respiratory oscillations in synchronised Bacillus subtilis

Respiratory activity increased in a stepwise fashion during the cell cycle of Bacillus subtilis. This was true either for cells in growth medium or for those removed by filtration and supplied with an exogenous electron donor. KCN was used to probe terminal oxidase activity and cell cycle-dependent oscillations in the degree of inhibition were measured. The extent of stimulation by an uncoupling agent (CCCP) also varied during the cycle rising to two maxima at 0.14 and 0.69, intermediate to the step rises in oxygen uptake rates (0.4 and 0.8 of the cycle). However the net effect of the uncoupler was to convert the discontinuous pattern of oxygen uptake to a continuous one. These data are consistent with control of respiratory chain activity during the cell cycle of B. subtilis, and not of the amount of its components.     

Host-parasite relationship of Angiostrongylus cantonensis in Lymnaea palustris. III. Oxygen uptake of Lymnaea palustris.

Oxygen uptake rates by Lymnaea palustris infected with Angiostrongylus cantonensis and by noninfected controls were determined at 25, 30, and 35 C over 34-day periods. Significant decreases in oxygen uptake were noted from one day to the next for both infected and noninfected groups of snails. At 25 and 30 C groups of infected snails found to have high average larval recoveries frequently had significantly higher oxygen uptake rates than for corresponding groups of noninfected snails. At 35 C readings were less reliable due to an increase in mortality for both infected and noninfected snails. Respiratory rates were not significantly altered by increasing the temperature from 25 to 30 and 35 C.