Heterotrophic Uptake Experiments with 14C-Labeled Histidine in a Histidine-Limited Chemostat

The histidine uptake by bacterial strain HIS 42 was determined with [U-¹⁴C]histidine and through oxygen uptake experiments on samples taken from a histidine-limited chemostat. The uptake of [U-¹⁴C]histidine was characterized by a saturation constant of 12.8 to 78.6 nM histidine. At higher growth rates, the measured maximum uptake rate of histidine was lower than the actual uptake rate in the culture. The percentage of respired substrate (76 to 93%) was about 30 to 40% higher than the comparable value for the culture. The uptake of histidine as analyzed through the measurement of oxygen uptake rates was characterized by a saturation constant of 1.7 to 10.5 μM histidine; the maximum uptake rate was always greater than the actual histidine uptake rate in the culture. By the application of the two cited methods, set up to determine the histidine uptake kinetics, two different uptake processes were analyzed. It appeared that the determination of the histidine uptake through measurement of the oxygen uptake rate showed a better reflection of the actual uptake process of histidine in the culture. With the available data it was impossible to assess a correlation between the uptake of histidine, as determined with [U-¹⁴C]histidine, and the actual metabolism of the bacterial population.     

Response of an antarctic lake heterotrophic community to high dissolved oxygen

The upper waters of Lake Hoare, Antarctica, contain dissolved oxygen at about three times the normal saturation (>/=42 mg liter). The response of the heterotrophic plankton community to this high dissolved oxygen was evaluated by the criteria of CFU and d-[U-C]glucose assimilated-respired. High dissolved oxygen was not inhibitory to d-[U-C]glucose assimilation-respiration compared with normal atmospheric dissolved oxygen in Lake Hoare water. The d-[U-C]glucose was assimilated and respired optimally at 12 degrees C in Lake Hoare. The d-[U-C]glucose assimilated-respired in the upper saturated atmospheric dissolved oxygen waters of Mountain Lake, Va., was inhibited in contrast to Lake Hoare (P < 0.05). CFU formation was inhibited in both lakes. CFU represent <1% of the fluorochrome-stained direct counts in Lake Hoare. Lake Hoare planktobacteria are smaller than the planktobacteria in Mountain Lake. ATP size fractionation revealed that 39% of the ATP biomass was <0.5 mum in Lake Hoare.     

Sensitivity of an oligotrophic lake planktonic bacterial community to oxygen stress

Dissolved oxygen at approximately four times normal saturation (42 mg liter) inhibited the growth and metabolism of summer planktonic bacteria in the surface water of alpine oligotrophic Mountain Lake (Giles County, Va.). Data were derived from growth of CFU on membrane filters, d-[U-C]glucose incorporation into the extractable lipid of these CFU, and respiration and assimilation of d-[U-C]glucose by lake water samples. Statistically significant (alpha < 0.05) differences were not detected in either CFU or C incorporation in lipid when superoxide dismutase (30 U ml) or catalase (130 U ml) was added to the medium. Thus, exogenous oxygen by-products apparently are not responsible for the observed inhibition of growth and metabolism.     

Investigation of liquid-solid hydrodynamic boundary layers and oxygen requirements in hairy root cultures.

Rates of oxygen uptake, growth and alkaloid production by hairy roots in submerged culture were investigated using a recirculation reactor allowing operation at high liquid velocities for removal of hydrodynamic boundary layers. Measurements were performed at dissolved oxygen tensions of 31-450% air saturation. Critical oxygen concentrations for Atropa belladonna hairy roots were above air saturation, viz. 100-125% air saturation for oxygen uptake and 150% air saturation for growth, demonstrating that these roots cultivated in reactors with air sparging are oxygen-limited. The critical oxygen tension for oxygen uptake by Solanum aviculare hairy roots was 75% air saturation. Both the specific oxygen uptake rate and specific growth rate of A. belladonna hairy roots were dependent on the mass (g dry weight) of roots present; even in the absence of boundary layers, growth did not remain exponential over the entire culture period. Cryo-scanning electron microscopy showed that hairy roots grown submerged in liquid medium were covered with thick layers of hydrated mucilage and root hairs, representing a significant additional barrier to oxygen transfer. Roots protruding out of the liquid medium showed no evidence of mucilage accumulation. The specific oxygen demand of A. belladonna root tips was 3.3-11.5 times higher than for the remainder of the roots, the ratio increasing as the dissolved oxygen tension was reduced. Specific growth rates, biomass yields from sugar, and atropine levels were maximum at around 150% air saturation, but decreased significantly with oxygen concentrations above ca. 200%.     

Stoichiometric flux balance models quantitatively predict growth and metabolic by-product secretion in wild-type Escherichia coli W3110.

Flux balance models of metabolism use stoichiometry of metabolic pathways, metabolic demands of growth, and optimality principles to predict metabolic flux distribution and cellular growth under specified environmental conditions. These models have provided a mechanistic interpretation of systemic metabolic physiology, and they are also useful as a quantitative tool for metabolic pathway design. Quantitative predictions of cell growth and metabolic by-product secretion that are experimentally testable can be obtained from these models. In the present report, we used independent measurements to determine the model parameters for the wild-type Escherichia coli strain W3110. We experimentally determined the maximum oxygen utilization rate (15 mmol of O2 per g [dry weight] per h), the maximum aerobic glucose utilization rate (10.5 mmol of Glc per g [dry weight] per h), the maximum anaerobic glucose utilization rate (18.5 mmol of Glc per g [dry weight] per h), the non-growth-associated maintenance requirements (7.6 mmol of ATP per g [dry weight] per h), and the growth-associated maintenance requirements (13 mmol of ATP per g of biomass). The flux balance model specified by these parameters was found to quantitatively predict glucose and oxygen uptake rates as well as acetate secretion rates observed in chemostat experiments. We have formulated a predictive algorithm in order to apply the flux balance model to describe unsteady-state growth and by-product secretion in aerobic batch, fed-batch, and anaerobic batch cultures. In aerobic experiments we observed acetate secretion, accumulation in the culture medium, and reutilization from the culture medium. In fed-batch cultures acetate is cometabolized with glucose during the later part of the culture period.(ABSTRACT TRUNCATED AT 250 WORDS)     

Temperature compensation of [u-C]glucose incorporation by microbial communities in a river with a fluctuating thermal regime

In summer, the river Saar in the southwest of Germany exhibits distinct temperature fluctuations from 8 degrees C at the source to nearly 30 degrees C in the middle region. Temperature optima for bacterial plate counts and the uptake velocity of [U-C]glucose by the natural microbial communities of different regions ranged from 20 to 30 degrees C, which is significantly above the mean annual water temperature. A correlation between temperature optima and different seasons or habitats was not observed. Despite the relatively high temperature optima, the turnover time for glucose was shortest at temperatures around the mean annual water temperature, due to changes in the substrate affinity. At limiting substrate concentrations, the higher substrate affinity at lower temperatures may lead to a higher real activity at in situ temperatures, and a compensatory stabilization of uptake rates at fluctuating temperatures is possible.     

On-line estimation of viable cells in a hybridoma culture at various DO levels using ATP balancing and redox potential measurement

Two on-line methods for the estimation of viable cell number in hybridoma cultivation were investigated. One used an empirical correlation between redox potential and animal cell density. The other was based on an ATP balance with ATP steady-state assumption. Oxygen uptake rate measurement provided the amount of ATP which was produced by oxidation of NADH. Oxygen uptake rate was measured either by stationary liquid phase balance with surface aeration or by gas balance during bubble aeration with headspace flushing with an inert gas. The amount of ATP produced through the glycolysis was estimated based on the amount of lactate produced. In cultures, in which pH was controlled via manipulation of the gas phase composition, the flow of CO(2) was linearly correlated with the lactate concentration. At constant dissolved oxygen levels, the viable cell density was proportional to the estimated ATP production rate, during exponential growth and during later phases. The estimated specific ATP production rate, however, varied from 2.2 pmol cell(-1) h(-1) at 10% air saturation to 4.5 pmol cell(-1) h(-1) at 100% air saturation. Specific rates of glutamine, glucose, and lactate followed the shape of the specific ATP production rate, whereas the specific oxygen uptake rate was minimal at around 50% air saturation. (c) 1996 John Wiley & Sons, Inc.     

Preparation of specifically labeled C-(lignin)- and C-(cellulose)-lignocelluloses and their decomposition by the microflora of soil

Microbial decomposition of lignocellulose in soil was studied using radioisotope techniques. Natural lignocelluloses containing C in either their lignin or cellulose (glucan) components were prepared by feeding plants l-[U-C]phenylalanine or d-[U-C]glucose, respectively, through their cut stems. Detailed chemical and chromatographic characterization of labeled lignocelluloses from three hardwood and three softwood species showed that those labeled by the [C]glucose incorporation method contained specifically labeled cellulosic components, whereas those labeled by the [C]phenylalanine incorporation method contained specifically labeled lignin components. Microbial degradation of these differentially labeled lignocelluloses was followed by monitoring CO(2) evolution from selected soil samples incubated with known amounts of radiolabeled lignocelluloses. The lignin components of the six woods were shown to be decomposed in soil 4 to 10 times more slowly than their cellulosic components. These rates of mineralization were comparable to the generalized patterns previously reported in the literature. The present technique, however, was thought to be simpler, more sensitive, and less prone to interference than methods previously available.     

Physiological Characterization of Adaptive Clones in Evolving Populations of the Yeast, SACCHAROMYCES CEREVISIAE

Populations of a diploid strain of S. cerevisiae were grown in glucose-limited continuous culture for more than 260 generations. A series of seven sequential adaptive changes were identified by monitoring the frequency of cycloheximide resistance in these populations. Samples were taken from the continuous cultures following each adaptive shift and characterized physiologically to determine (1) the range of phenotypes that can be selected in a precisely defined constant environment and (2) the order and predictability of the occurrence of the adaptive mutations in evolving populations. The clones were characterized with respect to the growth parameters, maximum growth rate, saturation coefficient and yield, as well as for changes in cell size and geometry and rate of glucose uptake. The maximum growth rates of the seven adaptive clones were very similar, but in contrast the saturation coefficients differed substantially. Surprisingly, not all clones showed reductions in the saturation coefficients, in comparison to the immediately preceding clones, as would be predicted from classical continuous culture kinetics. In addition, yield estimates first increased and then decreased for later isolated adaptive clones. In general, the results suggest epistatic interactions between the adaptive clones, consistent with earlier published results. The rate of glucose uptake, as measured by 14C-xylose uptake, increased dramatically after the selection and fixation of seven adaptive clones. Progressive decreases in cell volume and changes in cell geometry, resulting in increased surface area to volume ratios, were also observed in the adaptive clones, but these changes were not always seen in other haploid and diploid yeast populations evolving under the same conditions. Such changes may be easily explainable in terms of the characteristics of the glucose-limited environment. The significance of the results to the evolution of microorganisms under nutrient-limiting conditions is discussed.     

STUDIES OF THE GROWTH OF PENICILLIUM CHRYSOGENUM IN CONTINUOUS FLOW CULTURE WITH REFERENCE TO PENICILLIN PRODUCTION

SUMMARY: A filamentous mould was cultured by the continuous flow method in which medium is supplied at a constant rate and the culture volume is kept constant. Flow rates up to 0·1 culture volumes/hr were used. The mycelial dry weight concentration and the yield of mycelium/g of carbon source used were equal to or slightly greater than the maximum obtained in batch culture. With glucose concentrations up to 80 g/1. at a flow rate of 0·05 culture volumes/hr, about 45% of the substrate carbon was converted into mycelial carbon and the remainder oxidized to CO₂.
With unlimited amounts of all nutrients available growth of the mould followed the exponential law, as does bacterial growth, and therefore the mould had a constant doubling time.
The oxygen demand of the mould as function of growth rate was determined.
Conditions were found under which the rate of penicillin production/g of mycelium remained at its maximum value for 1000 hr.     

Agitator speed and dissolved oxygen effects in xanthan fermentations

Agitation speed affects both the extent of motion in Xanthan fermentation broths because of their rheological complexity and the rate of oxygen transfer. The combination of these two effects causes the dissolved oxygen concentration and its spatial uniformity also to change with agitator speed. Separating these complex interactions has been achieved in this study in the following way. First, the influence of agitation speeds of 500 and 1000 rpm has been investigated at a constant nonlimiting dissolved oxygen concentration of 20% of air saturation using gas blending. Under these controlled dissolved oxygen conditions, the results demonstrate that the biological performance of the culture was independent of agitation speed as long as broth homogeneity could be ensured. With the development of increasing rheological complexity lending to stagnant regions at Xanthan concentrations >20 g/L, it is shown that the superior bulk mixing achieved at 1000 rpm, compared with 500 rpm, leading to an increased proportion of the cells in the fermentor to be metabolically active and hence higher microbial oxygen uptake rates, was responsible for the enhanced performance. Second, the effects of varying dissolved oxygen are compared with a control in each case with an agitator speed of 1000 rpm to ensure full motion, but with a fixed, nonlimiting dissolved oxygen of 20% air saturation. The specific oxygen uptake rate of the culture in the exponential phase, determined using steady-state gas analysis data, was found to be independent of dissolved oxygen above 6% air saturation, whereas the specific growth rate of the culture was not influenced by dissolved oxygen, even at levels as low as 3%, although a decrease in Xanthan production rate could be measured. In the production phase, the critical oxygen level was determined to be 6% to 10%, so that, below this value, both specific Xanthan production rate as well as specific oxygen uptake rate decreased significantly. In addition, it is shown that the dynamic method of oxygen uptake determination is unsuitable even for moderately viscous Xanthan broths. Copyright 1998 John Wiley & Sons, Inc.     

Synthesis of staphylococcal enterotoxin A and nuclease under controlled fermentor conditions.

The production of enterotoxin A and nuclease by Staphylococcus aureus strain 100 was studied in a 1.0-liter fermentor. The effects of the gas flow rate, pH, and dissolved oxygen were evaluated. Toxin and nuclease secretion occurred under all conditions which permitted growth of the organism. Final yields of toxin and nuclease in cultures grown at constant air flow rates, ranging from 50 to 500 cm3 per min, were higher at successively higher flow rates. An optimum flow rate for either toxin or nuclease production was not observed. When the aeration rate alone or aeration rate and pH were held constant, the dissolved oxygen levels in the culture decreased from the initial 100% level to 0 to 5% 3 to 4 h after inoculation. The O2 demand of the culture then maintained this level for an additional 4 to 5 h. This low dissolved oxygen interval was characterized by rapid growth and extracellular protein production. Controlling the dissolved oxygen at a constant level throughout growth did not increase the final levels of toxin and nuclease above those achieved at the respective constant pH values. Growth under the influence of a constant aeration rate of 500 cm3 per min and a constant pH of 6.5 and 7.0 yielded the highest titers of nuclease (1,550 units/ml) and toxin (10.5 mug/ml) obtained in any of the fermentations conducted in this study. Sparging fermentor cultures with pure oxygen at a rate of 100 cm3 per min yielded growth and extracellular protein levels similar to those achieved at the sparge rate of 500 cm3 of air per min. Controlling the dissolved oxygen at 100% of pure oxygen saturation appeared to inhibit the culture, as the final cultural turbidity as well as the levels of toxin and nuclease were reduced. These data indicate that enterotoxin and nuclease secretions are closely associated with the growth of strain 100. Analyses of the production rates of these components indicated that early log phase was the most efficient production interval in the growth cycle and that this efficiency was increased by pH control at 6.7 to 6.8 and dissolved oxygen control at 10% of air saturation.     

Synthesis of staphylococcal enterotoxin A and nuclease under controlled fermentor conditions.

The production of enterotoxin A and nuclease by Staphylococcus aureus strain 100 was studied in a 1.0-liter fermentor. The effects of the gas flow rate, pH, and dissolved oxygen were evaluated. Toxin and nuclease secretion occurred under all conditions which permitted growth of the organism. Final yields of toxin and nuclease in cultures grown at constant air flow rates, ranging from 50 to 500 cm3 per min, were higher at successively higher flow rates. An optimum flow rate for either toxin or nuclease production was not observed. When the aeration rate alone or aeration rate and pH were held constant, the dissolved oxygen levels in the culture decreased from the initial 100% level to 0 to 5% 3 to 4 h after inoculation. The O2 demand of the culture then maintained this level for an additional 4 to 5 h. This low dissolved oxygen interval was characterized by rapid growth and extracellular protein production. Controlling the dissolved oxygen at a constant level throughout growth did not increase the final levels of toxin and nuclease above those achieved at the respective constant pH values. Growth under the influence of a constant aeration rate of 500 cm3 per min and a constant pH of 6.5 and 7.0 yielded the highest titers of nuclease (1,550 units/ml) and toxin (10.5 mug/ml) obtained in any of the fermentations conducted in this study. Sparging fermentor cultures with pure oxygen at a rate of 100 cm3 per min yielded growth and extracellular protein levels similar to those achieved at the sparge rate of 500 cm3 of air per min. Controlling the dissolved oxygen at 100% of pure oxygen saturation appeared to inhibit the culture, as the final cultural turbidity as well as the levels of toxin and nuclease were reduced. These data indicate that enterotoxin and nuclease secretions are closely associated with the growth of strain 100. Analyses of the production rates of these components indicated that early log phase was the most efficient production interval in the growth cycle and that this efficiency was increased by pH control at 6.7 to 6.8 and dissolved oxygen control at 10% of air saturation.     

Comparison of diffusion and reaction rates in anaerobic microbial aggregates

The ability of hydrogen diffusion to account for the rates of methane production in microbial aggregates was studied in a defined coculture consisting of a sulfate reducer grown as a syntrophic hydrogen producer in the absence of sulfate and a methanogen. The hydrogen uptake kinetics of the methanogen were determined using the infinite dilution technique. The maximum hydrogen uptake velocity was 7.1 nmol/min/μg protein and the half saturation constant for hydrogen uptake was 386 nmol/liter. A threshold of 28 nmol/liter below which no further hydrogen consumption occurred was observed. The reconstituted co-culture was shown to produce methane at rates similar to mixed culture enrichments grown on lactate. The diffusion model demonstrated that for the particular system studied, the rates of hydrogen diffusion could account for the overall rate of methane production.     

The response by microorganisms to steady-state growth in controlled concentrations of oxygen and glucose. II. Saccharomyces carlsbergensis

The growth of Saccharomyces carlsbergensis in continuous culture has been studied when dissolved oxygen and glucose concentrations were held constant at a series of steady-state levels. Both oxygen and glucose controlled the degree of aerobic metabolism and of ethanolic fermentation. When the glucose uptake rate was low (between 1.2 and 2.8 mmoles per hour per gram of yeast) the relative distribution of glucose between ethanolic and aerobic fermentation was sensitive to oxygen: when dissolved oxygen was near to saturation, glucose metabolism was 0.98 aerobic; when dissolved oxygen was 0.01 saturated, 0.8 of intake glucose metabolism was by ethanolic fermentation. On the other hand when glucose intake was high (between 7.6 and 18.2 mmoles) metabolism was predominately by ethanolic fermentation even when dissolved oxygen concentration was at saturation. The extent, to which catabolism proceeded by an anaerobic or aerobic pathway, as judged by ethanol production, was controlled more by the uptake of glucose than of oxygen.     

The basis of the alkalophilic property of a species of bacillus.

An alkalophilic bacterium belonging to the genus Bacillus was isolated from an indigo ball. The bacterium exhibited a maximum growth rate at pH 10-0 TO 10-5. The incorporation of 14C-labelled amino acids or [14C]uracil, uptake of 14C-labelled alpha-amino isobutyric acid into the bacterium and oxygen consumption of the bacterium with amino acids as substrates were all maximum at pH 9-0 to 10-5. The uptake of [U-14C]glucose into the organism and oxygen consumption with carbohydrates, on the other hand, showed little variation of rate in the pH 8 to 10 region. The oxygen consumption of intact bacteria or protoplasts in culture medium was maximum at pH 10. The membrane of the bacterium oxidized NADH maximally at pH 7-5, and ATPase bound to the membrane exhibited maximum activity at pH 7.L-Lactate, L-alanine and malate dehydrogenases in the soluble fraction exhibited maximum activities at pH 7-4 to 8-4. The alkalophilic property of the bacterium may be due to the behaviour of the membrane towards charged substances admitted into the organisms.     

Measurements of Diel Rates of Bacterial Secondary Production in Aquatic Environments

Measurements of bacterial secondary production were carried out during 13 diel studies at one coastal marine station and in five lakes differing with respect to nutrient concentration and primary production. Bacterial secondary production was measured in situ every 3 to 5 h by [³H]thymidine incorporation into DNA. In some of the diel studies, these results were compared with results obtained from dark ¹⁴CO₂ uptake and frequency of dividing cells. Only minor diel changes were observed. The rate of [³H]thymidine incorporation into DNA and the frequency of dividing cells varied from 23 to 194% of the diel mean. The dark CO₂ uptake rate varied from 12 to 259% of the diel mean. An analysis of variance demonstrated that no specific time periods during 24 h showed significantly different production rates, supporting the idea that bacterial activities in natural assemblages are controlled by a variety of events. The best correction (r² = 0.74) was obtained between the [³H]thymidine incorporation and frequency of dividing cells procedures from the lake water samples. The actual production rates calculated by [³H]thymidine incorporation into DNA were appreciably lower than those obtained by the frequency of dividing cells and the dark CO₂ uptake techniques. Diel rates of bacterial production are discussed in relation to sampling frequency, statistical errors, and choice of method.     

Hydrocarbon biodegradation in hypersaline environments

When mineral oil, hexadecane, and glutamate were added to natural samples of varying salinity (3.3 to 28.4%) from salt evaporation ponds and Great Salt Lake, Utah, rates of metabolism of these compounds decreased as salinity increased. Rate limitations did not appear to relate to low oxygen levels or to the availability of organic nutrients. Some oxidation of l-[U-C]glutamic acid occurred even at extreme salinities, whereas oxidation of [1-C]hexadecane was too low to be detected. Gas chromatographic examination of hexane-soluble components of tar samples from natural seeps at Rozel Point in Great Salt Lake demonstrated no evidence of biological oxidation of isoprenoid alkanes subject to degradation in normal environments. Some hexane-soluble components of the same tar were altered by incubation in a low-salinity enrichment culture inoculated with garden soil. Attempts to enrich for microorganisms in saline waters able to use mineral oil as a sole source of carbon and energy were successful below, but not above, about 20% salinity. This study strongly suggests a general reduction of metabolic rate at extreme salinities and raises doubt about the biodegradation of hydrocarbons in hypersaline environments.     

Metabolism of sulfated glycosaminoglycans in cultivated bovine arterial cells. II. Quantitative studies on the uptake of 35SO4-labeled proteoglycans.

Cultured arterial fibroblasts were used for a quantitative study on adsorption, uptake and degradation of [35S]proteoglycans derived from secretions of cultured arterial or skin fibroblasts. The following results were obtained: 1) Proteoglycans added to the culture medium are integrated into the pool of cell membrane-associated (trypsin-removable) glycosaminoglycans by a saturable process, which depends on time and temperature. 2) Up to 17% of the added proteoglycans are taken up by the cells within 24 h. The uptake exhibits saturation kinetics, characteristic for adsorptive pinocytosis. Proteoglycan concentrations required for half-maximum uptake are higher than for half-maximum saturation of the glycosaminoglycan pool associated with the cell membrane. 3) After a lag phase, inorganic 35SO4 appears in the culture medium as a degradation product of the internalized proteoglycans. Pinocytosed proteoglycans are catabolized more rapidly than proteoglycans which remain inside the cell after their biosynthesis. 4) Pinocytosis exhibits specificity, the individual proteoglycans being internalized at different rates. The highest rate of uptake was measured for a dermatan-sulfate-rich proteoglycan. No competition of uptake between a dermatan-sulfate-rich and a heparan-sulfate-rich proteoglycan was observed. 5) Optimum pinocytosis requires an intact protein moiety and, presumably, undegraded carbohydrate chains of the proteoglycans.     

Oxygen requirements and mass transfer in hairy-root culture

Oxygen mass transfer in clumps of Atropa belladonna hairy roots was investigated as a function of root density and external flow conditions. Convection was the dominant mechanism for mass transfer into root clumps 3.5 to 5.0 cm in diameter; Peclet numbers inside the clumps ranged from 1.4 x 10(3) to 7.1 x 10(4) for external superficial flow velocities between 0.4 and 1.4 cm s(-1). Local dissolved-oxygen levels and rates of oxygen uptake were measured in aflow chamber and in bubble column and stirred bioreactors. When air was used as oxygen source, intraclump dissolved-oxygen tensions ranged from90% to 100% air saturation at high external flow velocity andlow root density, to less than 20% air saturation in dense root clumps. Specific oxygen-uptake rate declined with increasing root density. When external boundary layers around individual roots were eliminated byforcing liquid through the clumps at superficial velocities between 0.2 and1.0 cm s(-1), internal dissolved-oxygen tension was maintained at 95% to 100% air saturation and rate of oxygen uptake at 1.6 x 10(-6) g g(-1) s(-1) dry weight. Liquid culture of single A. belladonna hairy roots was used to investigate the effect of dissolved-oxygen tensionon root growth and morphology. Total root length and number of root tips increased exponentially at oxygen tensions between 70% and 100%air saturation. Specific growth rate increased with oxygen tension up to 100% air saturation; this result demonstrates that hairy roots aeratedwithout oxygen supplementation are likely to be oxygenlimited. No growth occurred at 50% air saturation. Growth of hairy roots proceeded with an average length per tip of about 1 cm; this value was essentially independent of dissolved-oxygen tension between 70% and 100% air saturation. (c) 1994 John Wiley & Sons, Inc.