Effect of oxygen on the growth, respiratory rate and morphology of Candida utilis cells in periodic and continuous cultures

The aim of this work was to study how the concentration of oxygen dissolved in the cultural broth influenced the respiration and morphology of the yeast Candida utilis in batch and continuous cultures. Highly effective respiration was registered in cells growing for a certain period of time at low oxygen concentrations limiting the growth; the respiration was characterized by low values of the Michaelis constant kc and the critical concentration of dissolved oxygen Ccr. When passing from the low oxygen concentration to a high one, the character of cellular respiration changed abruptly in the cells whose growth was limited with oxygen for a long time. The morphology of the culture limited with oxygen was characterized by an increase in the percentage of elongated forms in the population. The respiration of the cells cultivated at high oxygen concentrations, when their growth was either non-limited or limited by glucose, was distinguished by high Ccr values and slow respiration rates at small oxygen concentrations while the dependence of the respiration rate on the concentration of oxygen had an about S-shaped character.     

Nitrogenase activity and regeneration of the cellular ATP pool in Azotobacter vinelandii adapted to different oxygen concentrations.

The in vivo activity of nitrogenase under aerobiosis was studied with diazotrophic chemostat cultures of Azotobacter vinelandii grown under glucose- or phosphate-limited conditions at different dilution rates (Ds, representing the growth rate mu) and different dissolved oxygen concentrations. Under steady-state conditions, the concentration as well as the cellular level of ATP increased in glucose-limited cultures when D was increased. Irrespective of the type of growth limitation or the dissolved oxygen concentration, the steady-state concentrations of ATP and of dinitrogen fixed by nitrogenase increased in direct proportion to each other. Specific rates of dinitrogen fixation as well as of the regeneration of the cellular ATP pool were compared with specific rates of cellular respiration. With glucose-limited cultures, the rate of regeneration of the ATP pool and the rate of respiration varied in direct proportion to each other. This relationship, however, was dependent on the dissolved oxygen concentration. As compared to the phosphate-sufficient control, phosphate-limited cultures exhibited the same nitrogenase activity but significantly increased respiratory activities. Rates of ATP regeneration and of cellular respiration of phosphate-limited cultures did not fit into the relationship characteristic of glucose-limited cultures. However, a linear relationship between the rates of dinitrogen fixation and ATP regeneration was identified irrespective of the type of growth limitation and the dissolved oxygen concentration. The results suggest that the ATP supply rather than cellular oxygen consumption is of primary importance in keeping nitrogenase activity in aerobic cultures of A. vinelandii.     

Growth kinetics of Dioscorea deltoidea and Catharanthus roseus in batch culture

Sugar-limited batch growth of Dioscorea deltoidea and Catharanthus roseus plant cell cultures was studied in a 14-L stirred tank fermentor. With dissolved oxygen concentration monitored and maintained at nonlimiting levels, growth rates and ratios of dry weight to fresh weight were found to be strongly influenced by sugar concentration. Linear relationships between respiration rate and growth rate were observed, and respiration rate was found to drop to a maintenance level after sugar had been fully depleted from the medium. Diosgenin biosynthesis by D. deltoidea was shown to be independent of growth rate. Ajmalicine biosynthesis in C. roseus was negligible during sugar-limited growth but was induced by inoculation into an 80-g/L glucose solution.     

Effects of cell density and glucose and glutamine levels on the respiration rates of hybridoma cells

The effects of cell density as well as the concentration levels of glucose and glutamine on the specific respiration rate of a hybridoma cell line were investigated. The experimental oxygen consumption rate was found to be constant over a wide range of dissolved oxygen levels if the suspension medium contained glutamine. In glutamine-free medium, however, the rate of oxygen consumption decreased slowly with time.In a stationary flask batch culture, the specific respiration rate decreased from about 7 to 2.9 mumol/min per 10(9) cells as the cell density increased exponentially from 1 x 10(5) to 1.2 x 10(6)/mL. To isolate the effect of cell density, cells were re suspended in fresh culture medium so that nutrient concentrations were the same for all experiments. The specific respiration rate decreased with increasing cell density in the same manner as in the stationary flask culture, falling from 8 to 4 mumol/min per 10(9) cells as the cell density increased from 10(5) to 10(6) cells/mL, then declining to 2 mumol/min per 10(9) cells when the cell density reached 10(7) cells/mL.Cells suspended in Hanks balanced sale solution (HBSS) were used to elucidate the effect of glucose and glutamine levels on respiration. The addition of glucose in concentrations of 0.25, 0.50, and 0.75 g/L had no observable effect on the specific oxygen uptake rate; however, a glucose concentration of 1 g/L reduced the uptake rate by 22%. Glutamine in a concentration of 0.30 g/L increased the specific respiration rate in HBSS containing 0 and 1 g/L glucose by approximately 13%.     

Simulation of oxygen transfer in microbial slimes

A model based on diffusion mechanisms accounts for oxygen transfer from flowing nutrient medium into a film of organisms in slime. Profiles of dissolved oxygen concentration are generated at distances downstream from the start of the slime. In these studies, the nutrient medium was assumed rich, thus the rate of oxygen transport limited microbial respiration. Parameter sensitivity tests were performed for flow rate, oxygen concentration in the medium, and mass transfer coefficients.     

Microprobe techniques for determining diffusivities and respiration rates in microbial slime systems

A microprobe electrode was used to determine dissolved oxygen concentrations near the surface and within a bacterial slime mass supplied with a continuous flow of nutrient solution. With dilute medium, the oxygen profile became level at high concentrations within the film, indicating substrate-limited respiration. More concentrated medium caused the profile to fall to low oxygen concentrations characteristic of oxygen-limited respiration. Oxygen responses to sudden changes in concentration of nutrient medium were measured. Estimates of microbial respiration rate and of diffusivity of oxygen were based on well-known diffusion equations.     

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%.     

Induction of a nitrogenase activity rhythm inSynechococcus and the protection of its nitrogenase against photosynthetic oxygen

All oxygen levels are detrimental to the nitrogenase activity ofSynechococcus RF-1 cells. In continuous light, cultures maintain a high dissolved oxygen concentration and a continuous but usually low rate of nitrogenase activity.Cultures adapted to a light-dark regimen will reduce acetylene almost exclusively during the dark periods. When switched to continuous light, they continue to exhibit a diurnal rhythm in nitrogenase activity. While in continuous light, each upsurge of nitrogenase activity coincides with a marked drop in the net oxygen production rate; this drop is due largely to a concomitant increase in the dark respiration rate of the culture.The endogenous nitrogenase activity rhythm can be induced in continuous light by periodically lowering the oxygen concentration of the culture by either bubbling nitrogen through it or by treating the culture with 3(3,4-dichlorophenol)-1,1-dimethylurea (DCMU or diuron).     

Nitrogenase activity and respiration of cultures of Rhizobium spp. with special reference to concentration of dissolved oxygen

Studies of nitrogenase in culture of the cowpea rhizobia (Rhizobium spp.) strains 32H1 and CB756 are reported. Preliminary experiments extablished that, even agar cultures were grown in air, suspension of bacteria prepared anaerobically from them were most active at low concentrations of free dissolved O₂. Consequently, assay for activity usd low concentrations of O₂, stabilized by adding the nodule pigment leghaemoglobin.In continous, glutamine-limited cultures of 32H1, nitrogenase activity appeared only when the concentration of dissolved O₂ in the cultures approached 1 μM. Lowering the glutamine concentration in the medium supplied to the cultured from 2 to 1 mM halved the cell yield and nitrogenase activity was also deminished. Omitting succinate from the medium caused the concentration of dissolved O₂ to rise and nitrogenase activity was lost. Upon restoration of the succinate supply, The O₂ concentration immediately fell and nitrogenase was restored. The activity doubled in about 8 h, whereas the doubling time of this culture was 14 h. Sonic extracts of 32H1 cells from continous cultures with active nitrogenase contained components reacting with antiserum against nitrogenase Mo-Fe protein from soybean bacteroids. Continous cultures grown at higher O₂ concentration, with only a trace of active nitrogenase, contained less of these antigens and they were not detected in highly aerobic cultures. Nitrogenase activity of a continous cultures was repressed by NH₄⁺; the apparent half-life was about 90 min.Cells of 32H1 from a continous culture growing at between 30 and 100 μM dissolved O₂ possessed a protective mechanism which permitted respiration to increase following exposure to a rapid increase in O₂ concentration from low levels (O₂ shock). This effect disappeared as disappeared as the O₂ concentration for growth was reduced towards 1 μM.     

Factors influencing the production of a novel compound, 7,10-dihydroxy-8(E)-octadecenoic acid, by Pseudomonas aeruginosa PR3 (NRRL B-18602) in batch cultures

Pseudomonas aeruginosa PR3 (NRRL B-18602) converts oleic acid to a novel compound, 7,10-dihydroxy-8(E)-octadecenoic acid (DOD). Parameters that included medium volume, cell growth time, gyration speed, pH, substrate concentration, and dissolved oxygen concentration were evaluated for a scale-up production of DOD in batch cultures using Fernbach flasks and a bench-top bioreactor. Maximum production of about 2 g DOD (38% yield) was attained in Fernbach flasks containing 500 ml medium when cells were grown at 28 degrees C and 300 rpm for 16-20 h and the culture was adjusted to pH 7 prior to substrate addition. Increases of medium volume and substrate concentration failed to enhance yield. When batch cultures were initially conducted in a reactor, excessive foaming occurred that made the bioconversion process inoperable. This was overcome by a new aeration mechanism that provided adequate dissolved oxygen to the fermentation culture. Under the optimal conditions of 650 rpm, 28 degrees C, and 40-60% dissolved oxygen concentration, DOD production reached about 40 g (40% yield) in 4.5 L culture medium using a 7-L reactor vessel. This is the first report on a successful scale-up production of DOD.     

Alternative respiration and antibiotic production of Acremonium chrysogenum

The influence of potassium cyanide (KCN), dissolved O₂ concentration and medium composition on alternative respiration (AR) of Acremonium chrysogenum were investigated. The respiration of the fungus was only partially inhibited by KCN, but completely inhibited by the combination of KCN with salicylhydroxamic acid. It has been proved by in-situ measurements of the NADH-dependent fluorescence that the AR is active at low dissolved O₂ concentrations. The influence of the medium composition and the age of the fungus on the specific oxygen uptake rate is considered. Correpondence to: K. Schügerl     

Submerged organ culture: An improved method

Summary Explants of whole ovaries and oviducts from postnatal rats were completely submerged during cultivation as organ cultures in chemically defined medium. The oxygen concentration in the culture chamber was raised to over 90%, and the stainless steel platform, used for cultures at the surface of the medium, was abandoned and excluded from further use. Thus, all of the periphery of an explant had equal access to nutrients and oxygen. Throughout the ovarian explants the tissues appeared uniformly viable, and mitotic figures were distributed evenly. These observations are in contrast to those on organs cultivated at the surface of the medium where a lack of structural uniformity had been detected. The method has general application to a variety of organs.     

Submerged organ culture: An improved method

Summary Explants of whole ovaries and oviducts from postnatal rats were completely submerged during cultivation as organ cultures in chemically defined medium. The oxygen concentration in the culture chamber was raised to over 90%, and the stainless steel platform, used for cultures at the surface of the medium, was abandoned and excluded from further use. Thus, all of the periphery of an explant had equal access to nutrients and oxygen. Throughout the ovarian explants the tissues appeared uniformly viable, and mitotic figures were distributed evenly. These observations are in contrast to those on organs cultivated at the surface of the medium where a lack of structural uniformity had been detected. The method has general application to a variety of organs.     

High density cultivation of Anchusa officinalis in a stirred-tank bioreactor with in situ filtration

Previously, Su et al. [Biotechnol Bioeng 42: 884–890 (1993)] reported improved production of rosmarinic acid by Anchusa officinalis in shake-flask cultures using a cultivation strategy that involved intermittent medium exchange. Implementation of this cultivation strategy in 2.5-1 stirred-tank bioreactor cultures is investigated in the present study. Intermittent cell/medium separation in the bioreactor was accomplished by means of automated in situ culture filtration. In the bioreactor culture, rosmarinic acid production was found very sensitive to agitation and aeration conditions as well as dissolved oxygen concentration. A maximum cell density of 35 g dry weight/l and a rosmarinic acid concentration of 3.7 g/l were obtained by maintaining the dissolved oxygen concentration above 30% air saturation, gradually raising the impeller tip speed from 34 cm/s to 72 cm/s, and keeping the aeration rate at 0.44 vvm while increasing the O₂: air ratio in the gas feed stream to 4:1. This result is comparable with the data obtained from shake-flask cultures using the same culture strategy.     

A method for estimating oxygen availability of stationary plant cell cultures in liquid media

A method for estimating the oxygen availability in plant cell cultures grown in stationary liquid media (e.g. many protoplast cultures) was developed. The method is based on short-term measurements of respiration rate versus oxygen concentration on a sample of cells, suspended in liquid media. From such data it is possible to estimate the oxygen concentration at the bottom of a stagnant liquid culture, by calculating the amount of oxygen reaching the cells by diffusion. As an example, rape (Brassica napus L. cv. Omega) hypocotyl protoplasts were grown with different oxygen concentrations at the site of the cells, obtained by varying the cell density, the height of the liquid layer and the oxygen content of the gas phase. The number of surviving calli was positively correlated with the estimated oxygen availability in the range between 60 and 350 M O₂, below 60 M all cells died. This indicates that oxygen availability can be a limiting factor in the range usually encountered in protoplast cultures, and that the method can be useful when designing optimal growth conditions for stationary cultures of plant cells.Abbreviations C₁ bulk oxygen concentration in agitated medium - C_o oxygen concentration in medium at the gas-liquid interface, in equilibrium with the gas - C_x oxygen concentration at cell level - D diffusion constant of oxygen in water - K_La oxygen transfer rate - l height of liquid above cells - n number of cells per ml - R_x respiration rate per cell     

Correlation between steady-state cell concentration and cell death of hybridoma cultures in chemostat

In the present study, the steady-state cell density (X) of chemostat cultures of murine hybridoma was varied by the concentration of glucose and glutamine in culture medium and the dissolved oxygen partial pressure. Except at low glutamine and low oxygen levels, the specific death rate (k(d)) of the cultures was found to decrease with increasing dilution rate (D). However, the plot of k(d) vs. X/D yielded linear relation, which suggests that cell death was due to a non-growth-linked inhibitory product of the cells. The k(d) value measured at low glutamine and low oxygen levels remained practically unchanged over a wide range of D between 0.020 and 0.029 h(-1). The k(d) for low oxygen cultures was always lower than the values obtained in low glucose and low glutamine cultures. A low-molecular-weight component of possibly less than 3000 MW was detected to be cell-death-inducing in the supernatant of exponentially growing cultures. It was neither lactate nor ammonium. The autoinhibitor was not cell-line specific. (c) 1995 John Wiley & Sons, Inc.     

Design, characterization and application of a minibioreactor for the culture of human hematopoietic cells under controlled conditions

The in vitro culture of human hematopoietic cells has recently received considerable attention due to its clinical importance. Most studies of the culture and expansion of hematopoietic cells have been performed in static cultures but only very few reports exist on the use of bioreactors where strict control of environmental variables is maintained. In this work, the design, characterization and application of a fully instrumented minibioreactor for the culture of human hematopoietic cells from umbilical cord blood is presented. The system consists of a stirred- tank reactor where cells are maintained in suspension in an homogeneous environment and without the need of a stromal feeding layer. The minibioreactor was coupled to a data acquisition and control system which continuously monitored pH, dissolved oxygen and redox potential. When operated at 75 rpm with a hanging magnetic bar (impeller-to-tank diameter ratio of 0.57), the dead and mixing times were 120 and 80 s, respectively, and the maximum response rate and volumetric oxygen transfer coefficient were 0.8 mM O2 hr-1, and 1.8 hr-1, respectively. Such characteristics allowed a tight control of pH(until day 11) and dissolved oxygen at predetermined set-points, and up to a 7-fold expansion of hematopoietic progenitors was possible in cultures maintained at 20% dissolved oxygen with respect to air saturation. Growth phase and cell concentration could be inferred on- line through determinations of oxygen uptake rate and culture redox potential. Oxygen uptake rate increased during exponential growth phase to a maximum of 40 μM hr-1. Such an increase closely followed the increase in concentration of hematopoietic progenitors. In contrast, culture redox potential decreased during exponential growth phase and then increased during death phase. The designed system permits not only the maintenance of controlled environmental conditions and on-line identification of fundamental culture parameters, but also the application of control strategies for improving expansion of hematopoietic cells. This revised version was published online in August 2006 with corrections to the Cover Date.     

Factors Influencing the Production of a Novel Compound, 7,10-Dihydroxy-8(<Emphasis Type="Italic">E</Emphasis>)-octadecenoic Acid,by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> PR3 (NRRL B-18602) in Batch Cultures

Pseudomonas aeruginosa PR3 (NRRL B-18602) converts oleic acid to a novel compound, 7,10-dihydroxy-8(E)-octadecenoic acid (DOD). Parameters that included medium volume, cell growth time, gyration speed, pH, substrate concentration, and dissolved oxygen concentration were evaluated for a scale-up production of DOD in batch cultures using Fernbach flasks and a bench-top bioreactor. Maximum production of about 2 g DOD (38% yield) was attained in Fernbach flasks containing 500 ml medium when cells were grown at 28°C and 300 rpm for 16–20 h and the culture was adjusted to pH 7 prior to substrate addition. Increases of medium volume and substrate concentration failed to enhance yield. When batch cultures were initially conducted in a reactor, excessive foaming occurred that made the bioconversion process inoperable. This was overcome by a new aeration mechanism that provided adequate dissolved oxygen to the fermentation culture. Under the optimal conditions of 650 rpm, 28°C, and 40–60% dissolved oxygen concentration, DOD production reached about 40 g (40% yield) in 4.5 L culture medium using a 7-L reactor vessel. This is the first report on a successful scale-up production of DOD. Received: 26 September 2002 / Accepted: 24 October 2002     

Links between morphology and physiology of Ganoderma lucidum in submerged culture for the production of exopolysaccharide

Ganoderma lucidum was grown in submerged culture in shake flasks on a medium containing peptone, yeast extract and glucose. In pre-cultures, inoculated from an agar-grown culture, morphological and metabolic events were linked: the pellets originally produced protuberances when glucose was present in the medium, although glucose was not consumed. The protuberances were then liberated into the medium as second-generation pellets, at which time glucose consumption began and the rate of exopolysaccharide (EPS) production increased. The synchrony between events was repeated in cultures fed with either glucose or peptone and yeast extract. In main cultures, inoculated from a 16-day-old pre-culture, the biomass concentration increased linearly, while glucose consumption and EPS production were initially slow but then accelerated. Protuberances were produced and liberated similarly to the pre-culture, but there was less synchrony amongst the pellets. When glucose was added to such a culture on day 10, an EPS concentration of 5.7 g L(-1) was achieved on day 13, this being the highest reliable EPS concentration yet reported for submerged culture of G. lucidum. We conclude that a greater understanding of the morphological and physiological events during the culture of G. lucidum will allow the proposal of culture strategies to improve EPS production.     

Influence of Dissolved Oxygen Levels on Production of l-Asparaginase and Prodigiosin by Serratia marcescens

The effect of dissolved oxygen concentrations on the behavior of Serratia marcescens and on yields of asparaginase and prodigiosin produced in shaken cultures and in a 55-liter stainless-steel fermentor was studied. A range of oxygen transfer rates was obtained in 500-ml Erlenmeyer flasks by using internal, stainless-steel baffles and by varying the volume of medium per flask, and in the fermentor by high speed agitation (375 rev/min) or low rates of aeration (1.5 volumes of air per volume of broth per min), or both. Dissolved oxygen levels in the fermentation medium were measured with a membrane-type electrode. Peak yields of asparaginase were obtained in unbaffled flasks (3.0 to 3.8 IU/ml) and in the fermentor (2.7 IU/ml) when the level of dissolved oxygen in the culture medium reached zero. A low rate of oxygen transfer was accomplished by limited aeration. Production of prodigiosin required a supply of dissolved oxygen that was obtainable in baffled flasks with a high rate of oxygen transfer and in the fermentor with a combination of high-speed agitation and low-rate aeration. The fermentation proceeded at a more rapid rate and changes in pH and cell populations were accelerated by maintaining high levels of dissolved oxygen in the growth medium.