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.     

Microelectrode studies of oxygen transfer in trickling filter slimes*

Slime-covered rocks and samples of process waters from two trickling filters for treatment of municipal wastes were brought to the laboratory for probing with microelectrodes to determine dissolved oxygen (DO). Slime thickness was 0.4–1.5 mm. Flow rate of medium over the slime had a minor effect on slime respiration, but pH 5 or below was strongly inhibitory. Increasing temperature showed lower oxygen concentration throughout a slime, although 27°C had results little different from those at 22°C. Medium concentration had a profound effect on oxygen concentration profiles, and either oxygen-limited or substrate limited respiration could be demonstrated. Illumination of slimes from the top of the trickling filter developed oxygen supersaturation because oxygen from photosynthesis could not diffuse away rapidly.     

Involvement of Glutathione in the Differentiation of the Slime Mold Physarum polycephalum

The effects of experimentally-altered glutathione concentration on differentiation of the slime mold, Physarum polycephalum were examined. Spherulation was induced by transfer of Physarum from growth medium to a salts-only starvation medium. As differentiation proceeded, superoxide dismutase (SOD) activity in control cultures increased by as much as 21-fold. This increase in SOD activity paralleled the rate of differentiation. Glutathione (GSH) concentration decreased during differentiation by more than 80% in all cultures, regardless of the initial concentration. The rate of differentiation was inversely related to the initial GSH concentration and directly proportional to the SOD activity. These observations suggest that a free radical mechanism may be involved in the differentiation of Physarum microplasmodia into spherules.     

HETEROTROPHIC SLIMES IN FLOWING WATERS

1. While much attention has been paid to the ecology of macro-invertebrates in flowing water, the microbial ecology of such systems has been largely ignored and our knowledge of heterotrophic slimes in particular remains far from complete. Slime-forming organisms are ubiquitous in their distribution and are part of the normal riverine flora. Slime outbreaks occur in all types of organically enriched flowing fresh waters, regardless of their chemical nature. Slimes are predominantly of heterotrophs which require a constant supply of (i) a suitable carbon source, (ii) inorganic nutrients and in particular nitrogen and phosphorous, and possibly (iii) growth factors such as vitamins. Phosphorous is not a limiting factor for growth, with slimes developing in rivers with < 0·02 mg P l^-1. Other inorganic nutrients such as nitrogen can be used in various forms and are usually present in adequate amounts, even in unpolluted streams. Therefore occurrence appears to be most closely correlated with the presence of a source of available carbon. 2. The severity of outbreaks are not closely associated with soluble organic carbon content although there is a tendency for heavy growths to occur more frequently in more severely polluted waters. Low-molecular-weight sugars are clearly the causative agents of Sphaerotilus natans dominated slimes with higher molecular weight material such as starches not immediately effective as growth promoters. Mono- to penta-saccharides are mainly used by bacterial slimes while fungal components utilize fatty acids up to C₈. It is not possible to adopt a nationwide BOD₅ standards to control slime outbreaks as even small increases in river BOD₅ (< 1·0 mg l_-1) can support slime growth. There is a need to develop new methods of assessing the slime-promoting capability of effluents such as measuring the readily degradable low-molecular-weight carbon compounds, so that threshold concentrations of soluble organic carbon below which slime will not develop can be determined. 3. The effect of effluent enrichment on slime growth diminishes downstream as there is a tendency for the soluble carbohydrate to mix and dilute. The slime also metabolizes the carbohydrate, reducing the concentration by up to 60 % depending on the stage of slime development, thus limiting its own proliferation. This is the typical pattern of self-purification in flowing waters. 4. The taxonomy of all the slime-forming species are poorly understood as is the ecology of slimes. Species composition of slimes vary temporally and spatially within individual rivers. The primary factors affecting composition are nutrient type and water velocity, although pH determines whether a slime is either predominantly fungal or bacterial. The rate of transfer of oxygen and nutrients is dependent on water velocity with zoogloeal forms predominating as velocity falls to < 0·05 m s_-1. More details of the effects of water velocity and other environmental factors on all the slime-forming organisms is required. 5. The effects of specific environmental factors on slime growth have been determined primarily from laboratory-based studies, often using pure cultures on solid medium or batch culture methods. Clearly it has been difficult to relate these results to what is happening in the field. Little quantitative information on the productivity of slimes exist and the energy budgets or role of slimes in the self-purification process of rivers is largely unknown. The effects of temperature, pH, dissolved oxygen concentration, water velocity, solar energy input and grazing on the relationship between organic nutrient concentration and slime growth needs to be fully understood. Therefore there is a need for both field-based flow channel and field studies using mixed slime populations to accurately model the effects of environmental factors on slime development. Formation of such models is a prerequisite to the development of control strategies. 6. Due to slower oxidation rates at lower temperatures and reduced sloughing resulting in more luxuriant growths, slimes are generally considered to be more frequent and extensive in winter. However this is clearly not the case with outbreaks far more abundant in the summer due to the smaller flows reducing the dilution factor of effluents, and the enhanced temperature and suppressed oxygen concentration of the water, reducing competition and grazing. 7. The presence of slimes is not always detrimental, the major effect is unsightly appearance and reduced amenity value. Slime-forming organisms are predominantly aerobic and the rate of oxygen consumption of slimes is directly related to the dissolved oxygen concentration of the water. It would appear that slimes rarely cause deoxygenation, although sudden increases in water temperature which lowers the solubility of oxygen or enhanced chemical oxygen demand of the effluent due to reduced dilution, may cause total oxygen utilization. 8. The effects of slime growths on the aquatic environment are numerous and cumulative, especially in relation to salmonid fisheries. Nearly all pollutants that are released into the environment will enter surface waters at some stage, so the ability of heterotrophic slimes to rapidly accumulate heavy metals and perhaps other pollutants by various sorption processes will result in metals being concentrated and transferred along the food chain. This will eventually result in increased residual metal concentrations in fish or toxic concentrations of metals being accumulated in macro-invertebrates normally eaten by fish. Metals can be transported out of the polluted zone via sloughed floes and be released back in to solution downstream of the site originally affected. 9. The severity of problems associated with outbreaks increase with the length of the slime growth, with the majority of the longer outbreaks (> 5 km) resulting in oxygen depletion, increased siltation, alteration in flow pattern, increase in sloughed biomass, reduction in species diversity, destruction and reduction in habitat diversity and the elimination of fisheries. Case studies on the effects of slime growth, especially those causing fish kills, need to be carefully analysed and published. 10. The potential of heterotrophic slimes in biotechnology and wastewater treatment has yet to be fully realized. The ability to grow rapidly, producing considerable biomass rich in protein could be utilized. The sorption of heavy metals by all the slime-forming organisms but especially by the iron and manganese bacteria, could be used for the removal of low concentrations of metals in wastes, treatment of metal-rich effluents or for metal recovery. The property of removing phosphorous and nitrogen from solution should also be further considered. 11. No adequate control measures are available except for full treatment of effluents prior to discharge. Even traces of low-molecular-weight carbon compounds will result in slime development. Inadequate partial treatment may enhance slime growth by partially breaking down the effluent and releasing slime-promoting compounds. Intermittent discharge can reduce the standing crop of slime per unit surface area but as the total biomass supported by an effluent will remain the same, the slime will be extended over a greater length of river. Bacterial slimes are assemblages of filamentous and dispersed bacteria, and are far more common than fungal or algal dominated slimes. The two slime-forming organisms S. natans and zoogloeal bacteria are the major components of the majority of heterotrophic slimes, therefore any attempt at control should be aimed at these two species.     

Nutrient-split feeding strategy for dialysis cultivation of Escherichia coli

Reduction in nutrient loss during dialysis cultivation of Escherichia coli on a glycerol medium was investigated. A dialysis reactor with an inner fermentation and an outer dialysis chamber was used. Aerobic condition was maintained by limiting the glycerol feed rate to an optimum value which was estimated from the oxygen requirements for glycerol oxidation and oxygen transfer capacity of the reactor. High reduction in nutrient loss was achieved by using water as the dialyzing fluid. However, osmotic movement of water from the dialysis to the fermentation chamber was observed, and the final cell concentration was low. With a nutrient-split feeding strategy (feeding glycerol directly to the fermentation chamber and dialyzing with salt solution), glycerol loss was small, there was no osmotic flux of water to the fermentation chamber, and the cell concentration was high. Both glycerol and salt loss could be avoided, and a cell concentration of 170 g/L was obtained when the dialysis process was substituted by addition of XAD adsorbents to the dialysis chamber. Application of this nutrient-split feeding strategy to cell cultivation in a stirred tank reactor, coupled with dialysis in external dialyzer modules, resulted in low cell concentrations. (c) 1993 Wiley & Sons, Inc.     

The effect of the oxygen transfer rate (OSR) on the formation of cellulases by Trichoderma viride in submersion culture

The formation of cellulases by Trichoderma viride in a medium containing cellulose as a sole source of carbon depends on the oxygen transfer rate (OSR); the OSR, on the other hand, depends on the concentration of cellulose in the medium because the concentration of cellulose strongly affects the viscosity of the medium. In the work presented here, the dependence has been determined for the oxygen transfer rate on geometric relations and viscosity in cellulose-containing media during cultivation in shaken flasks, and the oxygen transfer rate on N(Re') N(G') and N(a) during cultivation in a laboratory fermentor of 3000-mL volume. Two cellulosic materials have been compared with a different effect on viscosity: Microcrystalline beach cellulose and fibrous cellulose. It has been found that, in an applicable range of concentration, microcrystalline cellulose does not affect the oxygen transfer rate (at concentrations up to 3%). Fibrous cellulose increases the OSR during cultivation in shake flasks but decreases it during civilization in fermentors. On the basis of these results, the optimization has been carried out on the cultivation conditions in fermentors.     

Performance of mammalian cell culture bioreactor with a new impeller design

To improve the oxygen transfer in a mammalian cell bioreactor, a new type of impeller consisting of a double-screen concentric cylindrical cage impeller (annular cage impeller in short) was designed and its mass transfer rate evaluated. This new impeller design increases the specific screen area, and the convective mass transfer rate through the annular cage was significantly increased. The oxygen transfer rates with the new impeller and the commercially available cell-lift impeller (CelliGen by New Brunswick Scientific Co.) were evaluated and their performance compared at various rates of aeration and agitation. The results showed that with the new impeller, the oxygen transfer rate was increased by 19% in water and 21% in cell-free culture medium supplemented with 10% horse serum, the total hybridoma cell concentration was increased to 3.4 x 10(7) cells/mL, and the IgG(1) subtype monoclonal antibody (MAb) product concentration was also increased to 512 mg/L in perfusion culture of murine hybridoma cell line 62'D3. These improvements in oxygen transfer rate, cell concentration, and MAb product concentration are all very significant. The mass transfer resistance in the cell-lift impeller system was found to be mainly due to the surface area of the single-screen cage impeller. The new annular cage impeller not only provided the increased surface area for convective oxygen transfer but also protected cells from hydrodynamic shear damage, thereby achieving a significant bioprocess improvement in terms of higher viable cell concentration, higher product concentration, and higher oxygen transfer rate in the mammalian cell bioreactor system.     

Bimodal distribution of motility and cell fate in Dictyostelium discoideum

Pre-starvation amoebae of Dictyostelium discoideum exhibit random movements. Starved cells aggregate by directed movements (chemotaxis) towards cyclic AMP and differentiate into live spores or dead stalk cells. Many differences between presumptive spore and stalk cells precede differentiation. We have examined whether cell motility-related factors are also among them. Cell speeds and localisation of motility-related signalling molecules were monitored by live cell imaging and immunostaining (a) in nutrient medium during growth, (b) immediately following transfer to starvation medium and (c) in nutrient medium that was re-introduced after a brief period of starvation. Cells moved randomly under all three conditions but mean speeds increased following transfer from nutrient medium to starvation medium; the transition occurred within 15 min. The distribution of speeds in starvation medium was bimodal: about 20% of the cells moved significantly faster than the remaining 80%. The motility-related molecules F-actin, PTEN and PI3 kinase were distributed differently in slow and fast cells. Among starved cells, the calcium content of slower cells was lower than that of the faster cells. All differences reverted within 15 min after restoration of the nutrient medium. The slow/fast distinction was missing in Polysphondylium pallidum, a cellular slime mould that lacks the presumptive stalk and spore cell classes, and in the trishanku (triA(-)) mutant of D. discoideum, in which the classes exist but are unstable. The transition from growth to starvation triggers a spontaneous and reversible switch in the distribution of D. discoideum cell speeds. Cells whose calcium content is relatively low (known to be presumptive spore cells) move slower than those whose calcium levels are higher (known to be presumptive stalk cells). Slow and fast cells show different distributions of motility-related proteins. The switch is indicative of a bistable mechanism underlying cell motility.     

Characterization of oxygen uptake and mass transfer in flocculent yeast cell cultures with or without a flocculation additive

Summary The effect of solids concentration and the presence of the charged polymeric additive Magna Floc LT25 on gas-liquid oxygen transfer and oxygen consumption rates have been evaluated for flocculent yeast cells grown in batch fermentations. No significant differences were found for oxygen consumption due to the presence of the additive. Low biomass concentrations have a positive effect on oxygen transfer rate, when the additive was present in the medium. For biomass concentrations above 1 g/L, an increase in biomass concentration leads to a reduction on oxygen transfer rates regardless of the presence of the additive.     

The effects of several factors on the growth of pure and mixed cultures of Azotobacter chroococcum and Bacillus subtilis

We studied the effect of a clay mineral, palygorskite, on the physiological activity of Azotobacter chroococcum and the phosphate-mobilizing bacterium Bacillus subtilis, as well as their mixed cultures, under various oxygen supply conditions during the utilization of phosphorus from readily and poorly soluble compounds (K2HPO4 x 3H2O) and (Ca3(PO4)2), respectively. During cultivation of the bacteria in a nutrient medium with Ca3(PO4)2, the number of microorganisms was higher than that observed in a medium with K2HPO4. An increase in oxygen mass transfer in the nutrient medium was followed by a rise in the number of Bacillus subtilis cells and an inhibition of Azotobacter chroococcum growth. An addition of palygorskite (5 g/l) into the nutrient medium stimulated the growth of both bacteria and stopped the decreasing growth of Azotobacter chroococcum at high values of oxygen mass transfer. The number of Bacillus and, particularly, Azotobacter cells was two to five times lower in a mixed culture than in a monoculture. These differences were less significant during the cultivation of mixed cultures in medium with palygorskite.     

Identification of the limiting component of the medium during microbial continuous cultivation

The paper presents a method allowing a rapid identification of the growth limiting component of the medium. This is done with respect to the fast change in the low inertial parameter of fermentation (rate of titration or oxygen consumption) in response to an insignificant variation of the concentration of the growth limiting nutrient component in the fermentation medium.     

High viable cell concentration fed-batch cultures of hybridoma cells through on-line nutrient feeding

A hybridoma cell line was cultivated in fed-batch cultures using a low-protein, serum-free medium. On-line oxygen uptake rate (OUR) measurement was used to adjust the nutrient feeding rate based on glucose consumption, which was estimated on-line using the stoichiometric relations between glucose and oxygen consumption. Through on-line control of the nutrient feeding rate, not only sufficients were supplied for cell growth and antibody production, but also the concentrations of glucose and other important nutrients such as amino acids were maintained at low levels during the cell growth phase. During the cultivation, cell metabolism changed from high lactate production and low oxygen consumption to low lactate production and high oxygen consumption. As a result the accumulation of lactate was reduced and the growth phase was extended. In comparison with the batch cultures, in which cells reached a concentration of approximately 2 x 10(6) cells/mL, a very high concentration of 1.36 x 10(7) cells/mL with a high cell viability (>90%) was achieved in the fed-batch culture. By considering the consumption of glucose and amino acids, as well as the production of cell mass, metabolites, and antibodies, a well-closed material balance was established. Our results demonstrate the value of coupling on-line OUR measurement and the stoichiometric realations for dynamic nutrient feeding in high cell concentration fed batch cultures. (c) 1995 John Wiley & Sons, Inc.     

Microgradients of Microbial Oxygen Consumption in a Barley Rhizosphere Model System

A microelectrode technique was used to map the radial distribution of oxygen concentrations and oxygen consumption rates around single roots of 7-day-old barley seedlings. The seedlings were grown in gel-stabilized medium containing a nutrient solution, a soil extract, and an inert polymer. Oxygen consumption by microbial respiration in the rhizosphere (<5 mm from the root) and in bulk medium (>30 mm from the root) was determined by using Fick's laws of diffusion and an analytical approach with curve fitting to measured microprofiles of oxygen concentration. A marked increase of microbial respiration was observed in the inner 0- to 3-mm-thick, concentric zone around the root (rhizosphere). The volume-specific oxygen consumption rate (specific activity) was thus 30 to 60 times higher in the innermost 0 to 0.01 mm (rhizoplane) than in the bulk medium. The oxygen consumption rate in the root tissue was in turn 10 to 30 times higher than that in the rhizoplane. Both microbial respiration and oxygen uptake by the root varied between different roots. This was probably due to a between-root variation of the exudation rate for easily degradable carbon compounds supporting the microbial oxygen consumption.     

Oxygen transfer effects in serine alkaline protease fermentation by Bacillus licheniformis: use of citric acid as the carbon source

The effects of oxygen transfer on serine alkaline protease (SAP) production by Bacillus licheniformis on a defined medium with C_c = 9.0 kg m⁻³ citric acid as sole carbon source were investigated in 3.5 dm³ batch bioreactor systems. The concentrations of the product (SAP) and by-products, i.e., neutral protease, amylase, amino acids, and organic acids were determined in addition to SAP activities. At Q_o/V = 1 vvm air flow rate, the effect of agitation rate on DO concentration, pH, product, and by-product concentrations and SAP activity were investigated at N = 150, 500, and 750 min⁻¹; these are named as low-(LOT), medium-(MOT), and high oxygen transfer (HOT) conditions. LOT conditions favor biomass concentration; however, substrate consumption was highest at HOT conditions. MOT was optimum for maximum SAP activity which was 441 U cm⁻³ at t = 37 h. The total amino acid concentration was maximum in LOT and minimum in MOT conditions; lysine had the highest concentration under all oxygen transfer conditions. Among organic acids, acetic acid had the highest concentration and its concentration increased with oxygen transfer rate. The oxygen transfer coefficient increases with the agitation rate and the oxygen consumption rate increased almost linearly with the biomass concentration.     

The Relationship between Transpiration and the Absorption of Inorganic Ions by Intact Plants

The relationship between the rate at which water and the rubidiumand phosphate ions are absorbed by intact plants, and transferredto their shoots has been investigated in water culture undervarying conditions of transpiration and nutrient supply. When the external concentration and the nutrient status of theplants are sufficient low, wide variations in the rate of transpirationhave little effect on the transfer of nutrients to shoots; whenlittle water is being lost by transpiration the concentrationin the transpiration stream may exceed that in the externalmedium by factors exceeding 100. In contrast when the externalconcenration and the nutrient status of the plants are highthe rate of transfer of ions to shoots may vary closely withthe rate of transpiration and the concentration in the transpirationstream may be similar to, or less then, that in the externalmedium. The occurrence of concentrations of ions in the roots is transpirationstream which greatly exceed those in the medium external tothe roots is regarded as evidence that ions not transferredpassively across the roots of intact plants to a significantextent.     

Alteration of mammalian cell metabolism by dynamic nutrient feeding

The metabolism of hybridoma cells was controlled to reduce metabolic formation in fed-batch cultures by dynamically feeding a salt-free nutrient concentrate. For this purpose, on-line oxygen uptake rate (OUR) measurement was used to estimate the metabolic demand of hybridoma cells and to determine the feeding rate of a concentrated solution of salt-free DMEM/F12 medium supplemented with other medium components. The ratios among glucose, glutamine and other medium components in the feeding nutrient concentrate were adjusted stoichiometrically to provide balanced nutrient conditions for cell growth. Through on-line control of the feeding rate of the nutrient concentrate, both glucose and glutamine concentrations were maintained at low levels of 0.5 and 0.2 mM respectively during the growth stage. The concentrations of the other essential amino acids were also maintained without large fluctuations. The cell metabolism was altered from that observed in batch cultures resulting in a significant reduction of lactate, ammonia and alanine production. Compared to a previously reported fed-batch culture in which only glucose was maintained at a low level and only a reduced lactate production was observed, this culture has also reduced the production of other metabolites, such as ammonium and alanine. As a result, a high viable cell concentration of more than 1.0 × 10⁷ cells/mL was achieved and sustained over an extended period. The results demonstrate an efficient nutrient feeding strategy for controlling cell metabolism to achieve and sustain a high viable cell concentration in fed-batch mammalian cell cultures in order to enhance the productivity. This revised version was published online in July 2006 with corrections to the Cover Date.     

Alterations in superoxide dismutase, glutathione, and peroxides in the plasmodial slime mold Physarum polycephalum during differentiation

Changes in the level of antioxidant defenses and the concentration of free radical by-products were examined in differentiating (M3cVII and LU897 X LU863), non-differentiating (LU887 X LU897), and heterokaryon microplasmodia of the slime mold Physarum polycephalum during spherulation in salts-only medium. As differentiation proceeded, superoxide dismutase activity increased by as much as 46 fold; glutathione concentration and the rate of oxygen consumption decreased; cyanide-resistant respiration, hydrogen peroxide, and organic peroxide concentrations increased. The non-differentiating culture failed to exhibit any of these changes. A heterokaryon obtained by the fusion of differentiating and non-differentiating strains was observed to differentiate at a very retarded rate and to exhibit the changes observed in the spherulating strains at a correspondingly slower rate. These observations suggest that a free radical mechanism may be involved in the differentiation of Physarum microplasmodia into spherules.     

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%.     

Role of nutrient supply on cell growth in bioreactor design for tissue engineering of hematopoietic cells

In the present study, a dynamic mathematical model for the growth of granulocyte progenitor cells in the hematopoietic process is developed based on the principles of diffusion and chemical reaction. This model simulates granulocyte progenitor cell growth and oxygen consumption in a three-dimensional (3-D) perfusion bioreactor. Material balances on cells are coupled to the nutrient balances in 3-D matrices to determine the effects of transport limitations on cell growth. The method of volume averaging is used to formulate the material balances for the cells and the nutrients in the porous matrix containing the cells. All model parameters are obtained from the literature. The maximum cell volume fraction reached when oxygen is depleted in the cell layer at 15 days and is nearly 0.63, corresponding to a cell density of 2.25 x 10(8) cells/mL. The substrate inhibition kinetics for cell growth lead to complex effects with respect to the roles of oxygen concentration and supply by convection and diffusion on cell growth. Variation in the height of the liquid layer above the cell matrix where nutrient supply is introduced affected the relative and absolute amounts of oxygen supply by hydrodynamic flow and by diffusion across a gas permeable FEP membrane. Mass transfer restrictions of the FEP membrane are considerable, and the supply of oxygen by convection is essential to achieve higher levels of cell growth. A maximum growth rate occurs at a specific flow rate. For flow rates higher than this optimal, the high oxygen concentration led to growth inhibition and for lower flow rates growth limitations occur due to insufficient oxygen supply. Because of the nonlinear effects of the autocatalytic substrate inhibition growth kinetics coupled to the convective transport, the rate of growth at this optimal flow rate is higher than that in a corresponding well-mixed reactor where oxygen concentration is set at the maximum indicated by the inhibitory kinetics.     

Gratuitous Synthesis of β-Lactamase in Staphylococcus aureus

The synthesis of beta-lactamase in response to 2-(2'-carboxyphenyl)-benzoyl-6-aminopenicillanic acid as inducer was studied in Staphylococcus aureus. The inducer was not detectably hydrolyzed by beta-lactamase and had minimal antibacterial activity. The kinetics of induction showed a lag of 4 to 6 min in a nutrient broth medium and 8 to 12 min in a defined medium, followed by constant differential rates of synthesis of beta-lactamase. The differential rate of beta-lactamase synthesis in nutrient broth was unaltered by supplementing the medium with glucose, galactose, lactose, arabinose, glycerol, or sucrose. Variations in the partial pressure of oxygen did not alter the differential rate of synthesis of beta-lactamase over the range 18 to 50% oxygen in nitrogen. Even when the rate of growth was considerably reduced by high-oxygen tension, the differential rate of synthesis of the enzyme remained the same. The differential rate of beta-lactamase synthesis at low inducer concentration increased after a shift down in growth rate. The effect was observed with several inducers and under different nutritional conditions, but was always preceded by a change in growth rate. It is suggested that the change in growth rate itself causes the increase in differential rate of beta-lactamase synthesis.