The relationship between transport kinetics and glucose uptake by Saccharomyces cerevisiae in aerobic chemostat cultures

The steady-state residual glucose concentrations in aerobic chemostat cultures of Saccharomyces cerevisiae ATCC 4126, grown in a complex medium, increased sharply in the respiro-fermentative region, suggesting a large increase in the apparent k_s value. By contrast, strain CBS 8066 exhibited much lower steady-state residual glucose concentrations in this region. Glucose transport assays were conducted with these strains to determine the relationship between transport kinetics and sugar assimilation. With strain CBS 8066, a high-affinity glucose uptake system was evident up to a dilution rate of 0.41 h^–1, with a low-affinity uptake system and high residual glucose levels only evident at the higher dilution rates. With strain ATCC 4126, the high-affinity uptake system was present up to a dilution rate of about 0.38 h^–1, but a low-affinity uptake system was discerned already from a dilution rate of 0.27 h^–1, which coincided with the sharp increase in the residual glucose concentration. Neither of the above yeast strains had an absolute vitamin requirement for aerobic growth. Nevertheless, in the same medium supplemented with vitamins, no low-affinity uptake system was evident in cells of strain ATCC 4126 even at high dilution rates and the steady-state residual glucose concentration was much lower. The shift in the relative proportions of the high and low-affinity uptake systems of strain ATCC 4126, which might have been mediated by an inositol deficiency through its effect on the cell membrane, may offer an explanation for the unusually high steady-state residual glucose concentrations observed at dilution rates above 52% of the wash-out dilution rate.     

Induction of apoptosis in oxygen-deprived cultures of hybridoma cells

It is now well documented that apoptosis represents the prevalent mode of cell death in hybridoma cultures. Apoptotic or programmed cell death occurs spontaneously in late exponential phase of batch cultures. Until lately, no specific triggering factors had been identified. Recently, we observed that glutamine, cystine or glucose deprivation induced apoptosis in both hybridoma and myeloma cell lines whereas accumulation of toxic metabolites induced necrotic cell death in these cells. Other triggering factors such as oxygen deprivation might also be responsible for induction of apoptosis. In the present study, induction of cell death by exposure to anoxia was examined in batch culture of the SP2/0-derived hybridoma D5 clone. The mode of cell death was studied by morphological examination of acridine orange-ethidium bromide stained cells in a 1.5 L bioreactor culture grown under anoxic conditions for 75 hours. Under such conditions, viable cell density levelled off rapidly and remained constant for 25 hours. After 45 hours of anoxia, cell viability had decreased to 30% and the dead cell population was found to be 90% apoptotic. In terms of cellular metabolism, anoxia resulted in an increase in the utilization rates of glucose and arginine, and in a decrease in the utilization rate of glutamine. The lactate production rate and the yield of lactate on glucose increased significantly while the MAb production rate decreased. These results demonstrate that glycolysis becomes the main source of energy under anoxic conditions.Cells incubated for 10 hours or less under anoxic conditions were able to recuperate almost immediately and displayed normal growth rates when reincubated in oxic conditions whereas cells incubated for 22 hours or more displayed reduced growth rates. Nonetheless, even after 22 h or 29 h of anoxia, cells reincubated in oxic conditions showed no further progression into apoptosis. Therefore, upon removal of the triggering signal, induction of apoptosis ceased.Abbreviations VNA Viable non-apoptotic cells - VA Viable apoptotic cells - NVNA Nonviable non-apoptotic or necrotic cells - NVA Nonviable apoptotic cells - CF Chromatin-free cells (late nonviable apoptotic cells) - AO Acridine orange - EB Ethidium Bromide - MAb Monoclocnal antibody - D.O. Dissolved oxygen - qMAb Specific MAb production rate (mg. (10⁹ cells)^–1.day^–1) - Specific growth rate (h^–1) - X_v Viable cell number (10⁵ cells.mL^–1) - X_t Total cell number (10⁵ cells.mL^–1) - Y_lac/glc Yield coefficient of lactate on glucose (mM lactate produced/mM glucose consumed)     

Effect of the dilution rate on the biomass yield ofBacillus thuringiensis and determination of its rate coefficients under steady-state conditions

Depending on the biomass yield on glucose and the cell morphology ofBacillus thuringiensis, three different metabolic states were observed in continuous culture. At dilution rates between 0.18 h^–1 and 0.31 h^–1 vegetative cells, sporulating bacteria and spores coexisted, while glucose and amino acids were consumed. Only vegetative cells were observed at dilution rates between 0.42 h^–1 and 0.47 h^–1 and glucose was used as the main carbon and energy source. AtD = 0.50 h^–1 the biomass yield on glucose decreases sharply. To define better the specific growth rate range in which the microorganism uses mainly glucose, a dilution rate of 0.25–0.45 h^–1 was studied. The experimental data could be adjusted to a Monod model and the following rate coefficients and growth yields were determined: maximum specific growth rate 0.54 h^–1, saturation constant 0.56 mg glucose ml^–1, biomass growth yields 0.43 g cells (g glucose)^–1, and 0.76 g cells (g oxygen)^–1, and maintenance coefficients 0.065 g glucose (g cells)^–1 h^–1 and 0.039 g oxygen (g cells)^–1 h^–1.     

Influence of dilution rate on the morphology and technological properties ofLactobacillus delbrueckii subsp.bulgaricus

Lactobacillus delbrueckii subsp.bulgaricus ATCC 11842 was grown in a chemostat at 45°C and pH 5.5 using glucose as the carbon source, with the aim of optimizing biomass production. Cells were grown in a complex medium under nitrogen. At dilution rates lower than 0.18h^–1, it was difficult to keep steady-state conditions and pleomorphic forms were observed. The addition of 30mM Ca²⁺ and Mn²⁺ reverted the cells to normal shape: 30mM Mg²⁺ had no effect. Increasing the dilution rate resulted in normal morphology without the addition of any cations. Under these conditions, a maximum productivity of 1.24g dry biomass 1^–1 h^–1 was obtained. The maximum growth yield, corrected for maintenance, was 30g biomass mol^–1 glucose and the maintenance energy was 0.26g glucose g^–1 biomass h^–1. Lactate was the main fermentation product at all glucose concentrations used in the fed medium. Cells grown at high dilution rates had normal technological properties (acid production and proteolysis) when tested in milk.     

An analysis of the growth of Gluconobacter oxydans in chemostat cultures

Gluconobacter oxydans was grown successively in glucose and nitrogen-limited chemostat cultures. Construction of mass balances of organisms growing at increasing dilution rates in glucose-limited cultures, at pH 5.5, revealed a major shift from extensive glucose metabolism via the pentose phosphate pathway to the direct pathway of glucose oxidation yielding gluconic acid. Thus, whereas carbon dioxide production from glucose accounted for 49.4% of the carbon input at a dilution rate (D)=0.05 h^-1, it accounted for only 1.3% at D=0.26 h^-1. This decline in pentose phosphate pathway activity resulted in decreasing molar growth yields on glucose. At dilution rates of 0.05 h^-1 and 0.26 h^-1 molar growth yields of 19.5 g/mol and 3.2 g/mol, respectively, were obtained. Increase of the steady state glucose concentration in nitrogen-limited chemostat cultures maintained at a constant dilution rate also resulted in a decreased flow of carbon through the pentose phosphate pathway. Above a threshold value of 15–20 mM glucose in the culture, pentose phosphate pathway activity almost completely inhibited. In G. oxydans the coupling between energy generation and growth was very inefficient; yield values obtained at various dilution rates varied between 0.8–3.4 g/cells synthesized per 0.5 mol of oxygen consumed.     

n-3 Polyunsaturated fatty acid productivity of the marine microalgaIsochrysis galbana. Growth conditions and phenotypic selection

Two set of isolates were obtained in an isolation/selection programme to select eicosapentaenoic acid (EPA) and/or docosahexaenoic acid (DHA) rich strains ofIsochrysis galbana. EPA content was improved up to an average of 5.3% (d.wt) for the second set of isolates. On the other hand, with the aeration rate, pH and irradiance kept at low levels, the growth rate was slow and EPA synthesis was enhanced, but productivity increased when growth rates were maximum. A model relating steady-state dilution rates in chemostat cultures ofI. galbana to internal average irradiance is proposed. The greatest productivities were obtained between 0.0295 h^–1 and 0.0355 h^–1 with 300 mg m^–3 h^–1 for EPA and 130 mg m^–3 h^–1 for DHA.     

Chemostat Production of Plantaricin C By Lactobacillus plantarum LL441

Plantaricin C, a bacteriocin synthesized by Lactobacillus plantarum LL441, was optimally produced in chemostats kept at pH 5.0, 30°C, 150 rpm, and a dilution rate of 0.05 h⁻¹ when glucose was used as carbon source and a dilution rate of 0.10 to 0.12 h⁻¹ when sucrose or fructose was used instead. Production was abolished at high dilution rates, i.e., when the cells grew rapidly in all carbon sources.     

Nonlinear Dependency of Intracellular Fluxes on Growth Rate in Miniaturized Continuous Cultures of Escherichia coli

A novel mini-scale chemostat system was developed for the physiological characterization of 10-ml cultures. The parallel operation of eight such mini-scale chemostats was exploited for systematic ¹³C analysis of intracellular fluxes over a broad range of growth rates in glucose-limited Escherichia coli. As expected, physiological variables changed monotonously with the dilution rate, allowing for the assessment of maintenance metabolism. Despite the linear dependence of total cellular carbon influx on dilution rate, the distribution of almost all major fluxes varied nonlinearly with dilution rate. Most prominent were the distinct maximum of glyoxylate shunt activity and the concomitant minimum of tricarboxylic acid cycle activity at low to intermediate dilution rates of 0.05 to 0.2 h⁻¹. During growth on glucose, this glyoxylate shunt activity is best understood from a network perspective as the recently described phosphoenolpyruvate (PEP)-glyoxylate cycle that oxidizes PEP (or pyruvate) to CO₂. At higher or extremely low dilution rates, in vivo PEP-glyoxylate cycle activity was low or absent. The step increase in pentose phosphate pathway activity at around 0.2 h⁻¹ was not related to the cellular demand for the reduction equivalent NADPH, since NADPH formation was 20 to 50% in excess of the anabolic demand at all dilution rates. The results demonstrate that mini-scale continuous cultivation enables quantitative and parallel characterization of intra- and extracellular phenotypes in steady state, thereby greatly reducing workload and costs for stable-isotope experiments.     

Improvement of the culture stability of non-anchorage-dependent animal cells grown in serum-free media through immobilization

A murine hybridoma cell line producing a monoclonal antibody against penicillin-G-amidase and a murine transfectoma cell line secreting a monovalent chimeric human/mouse Fab-antibody fragment were cultivated in three different media (serum-containing, low protein serum-free, and iron-rich protein-free) in flask cultures, stirred reactors and a fixed bed reactor. In static batch cultures in flasks both cell lines showed similar good growth in all three media.In suspension in a stirred reactor, the hybridoma cell line could be cultivated satisfactory only in serum-containing medium. In low protein serum-free medium, Pluronic F68 had to be added to protect the hybridoma cells against shear stress. But even with this supplement only batch, not chemostat mode was possible. In iron-rich protein-free medium the hybridoma cells grew also in continuous chemostat mode, but the stability of the culture was low. The transfectoma cell line did not grow in stirred reactors in any of the three media.Good results with both cell lines were obtained in fixed bed experiments, where the cells were immobilized in macroporous Siran^®-carriers. The media, which were optimized in flask cultures, could be used without any further adaptation in the fixed bed reactor. Immobilization improved the stability and reliability of cultures of non-adherent animal cells in serum-free media tremendously compared to suspension cultures in stirred reactors. The volume-specific glucose uptake rate, an, indicator of the activity of the immobilized cells, was similar in all three media. Deviations in the metabolism of immobilized and suspended cells seem to be mainly due to low oxygen concentrations within the macroporous carriers, where the cells are supplied with oxygen only by diffusion.List of symbols c substrate or product concentration mmol l^–1 - c₀ substrate or product concentration in the feed mmol l^–1 - c_Glc glucose concentration mmol l^–1 - c_Gln glutamine concentration mmol l^–1 - c_Amm ammonia concentration mmol l^–1 - c_Lac lactate concentration mmol l^–1 - c_FAB concentration of Fab# 10 antibody fragment g l^–1 - c_MAb monoclonal antibody concentration mg l^–1 - D dilution rate d^–1 - q cell-specific substrate uptake or metabolite production rate mmol cell^–1 h^–1 - q_Glc cell-specific glucose uptake rate mmol cell^–1 h^–1 - q_Gln cell-specific glutamine uptake rate mmol cell^–1 h^–1 - q_MAb cell-specific MAb production rate mg cell^–1 h^–1 - q^* volume-specific substrate uptake or metabolite production rate mmol l^–1 h^–1 - q^*FB volume-specific substrate uptake or metabolite production rate related to the fixed bed volume mmol l_FB ^–1 h^–1 - q^*FB,Glc volume-specific glucose uptake rate related to the fixed bed volume mmol l_FB ^–1 h^–1 - q^*FB,Gln volume-specific glutamine uptake rate related to the fixed volume mmol l_FB ^–1 h^–1 - q^*FB,MAb volume-specific MAb production rate related to the fixed volume mg l_FB ^–1 h^–1 - q^*FB,02 volume-specific oxygen uptake rate related to the fixed bed volume mmol l_FB ^–1 h^–1 - t time h - U superficial flow velocity mm s^–1 - V medium volume in the conditioning vessel of the fixed bed reactor l - V_FB volume of the fixed bed l - x_v viable cell concentration cells ml^–1 - y_Amm,Gln yield of Ammonia from glutamine - y_Lac,Glc yield of lactate from glucose - specific growth rate h^–1 - _d specific death rate h^–1     

Competition between an Escherichia coli tyrosine auxotroph and a prototrophic revertant in glucose- and tyrosine-limited chemostats

A tyrosine-requiring strain of Escherichia coli was grown in tyrosine-limited chemostats at a range of dilution rates between 0.08 h^-1 and 0.42 h^-1, conditions which always resulted in the selection of a prototrophic revertant population able to synthesise tyrosine. Analysis of the two-membered mixed cultures which arose showed that the prototrophic population outgrew the auxotroph since its growth rate was not restricted by the growth-limiting concentrations of exogenous tyrosine. During the take-over of the culture, the prototroph population grew exponentially but the specific growth rate increased with decreasing dilution rate of the competition experiments. In glucose-limited chemostats (in the presence of non-growth-limiting concentrations of tyrosine) of the tyrosine-requiring strain, prototrophs were never detected. Constructed two-membered mixed cultures with both populations competing for limiting amounts of glucose, showed that the prototroph was less competitive than the auxotroph.This work was supported by a grant from the Science Research Council.     

Exopolysaccharide and extracellular metabolite production by Lactobacillus delbrueckii subsp. bulgaricus, grown on lactose in continuous culture

Lactobacillus delbrueckii subsp. bulgaricus NCFB 2483, when grown on lactose in continuous culture, showed increasing specific yields and volumetric productivities of exopolysaccharide (EPS) with increasing dilution rate. Specific and volumetric productivities of lactate and galactose, as extracellular metabolites, increased in response to the incremental changes in the dilution rate up to 0.4 h^–1. Elevated Y_p/s values determined for EPS (0.025 g EPSg lactose^–1) at the dilution rates of 0.3 h^–1–0.4 h^–1, relative to those determined at lower dilution rates, suggest a diversion of carbon flux towards EPS being associated with the higher rates of growth.     

Determination of cell lysis and death kinetics in continuous hybridoma cultures from the measurement of lactate dehydrogenase release

The death of the hybridoma VO 208 in a continuous culture at pH 7 and 6.8 was investigated by measuring both the appearance of visible dead cells which do not exclude the trypan blue dye and the release of lactate dehydrogenase (LDH) in the culture medium. The intracellular LDH was found to be completely released either when live cells lysed or when they were transformed into visible dead cells. No significant lysis of blue dead cells could be observed at the two different pH. Using a LDH balance over the culture system, cell lysis was found negligible at pH 7, but accounted for 20% of the total cell death at pH 6.8. A methodology is proposed to evaluate the rate constants of hybridoma lysis and total death. For the investigated cell line in continuous culture, the calculated total cell death rate constant was found to increase from 0.002 h^–1 to 0.01 h^–1 when decreasing the pH from 7 to 6.8.Abbreviations D dilution rate (h^–1) - k_b specific trypan-blue dead cells appearance rate (h^–1) - k_L specific lysis rate of viable cells (h^–1) - k_d specific death rate (h-1) - LDH₀ lactate dehydrogenase activity in the feed culture medium (IU.l^–1) - LDH lactate dehydrogenase activity in the outlet culture medium (IU.l^–1) - LDH_i intracellular lactate dehydrogenase activity of viable cells (IU.10^–9 cells) - r_LDH total rate of LDH release (IU.h^–1.L^–1) - r_b transformation rate of viable cells into blue dead cells (10⁹ cells.h^–1.L^–1) - x_v viable cell concentration (10⁹ cells.l^–1) - x_b trypan-blue dead cell concentration (10⁹ cells.l^–1)     

The growth rate control in Escherichia coli at near to maximum growth rates: the A-stat approach

The growth characteristics of Escherichia coli K-12 in the continuous culture with a smooth increase in the dilution rate (A-stat) of various carbon sources (glucose, acetate, succinate, glycerol, lactate, acetate + succinate, casamino, acids + glucose) were studied. For all substrates studied the maximum value of specific respiration rate, Q _O2, remained between 14–18 mmol O₂ h^-1 g dwt^-1 and the maximum growth rate varied from 0.22 h-¹ on acetate to 0.77 h^-1 on glucose + casamino acids. After the respiratory capacity of the cells was exhausted at growth rates µ < µ_crit, the growth yield Y_XO2, increased slightly when the dilution rate increased. The maximum growth rate of Escherichia coli K12 was dependent on growth yield, respiratory capacity and glycolytic capacity of the strain. Analysis of the cultivation data using a stoichiometric flux model indicated that ATP synthesis in E. coli exceeds by two-fold that (theoretically) required to build up biomass. The experimental value of m_ATP < 4 mmol ATP h^-1 g dwt^-1 determined from A-stat cultivation data was low compared with the calculated unproductive hydrolysis of ATP (64–103 mmole ATP g dwt^-1).     

Relationship between phosphates and alkaline phosphatase of Anabaena flos-aquae in continuous culture

Summary Anabaena flos-aquae was grown in chemostats with phosphate-limiting growth and dilution rate of 0.015–0.03 h^-1. The yields of cells were dependent on dilution rate and a two-fold increase obtained by growth in the presence of 15 mM KNO₃. Alkaline phosphatase activity varied 20-fold, lowest activity with excess phosphate light-limited cells and the highest activity with cells grown in the presence of 15 mM KNO₃. There was no correlation between hot water soluble phosphate of cells and alkaline phosphatase activity.     

Continuous cultivation of the yeast Candida utilis at different dilution rates on pineapple cannery effluent

Candida utilis was grown on a pineapple cannery effluent in a chemostat at dilution rates ranging between 0.05 and 0.65 h^–1 to establish optimal conditions for biomass production and chemical oxygen demand (COD) reduction. Sucrose, fructose and glucose were the main sugars in the effluent. Maximum value for cell yield coefficient and productivity were (0.686, g_x/g_s) and (2.96, g_x/l/h) at a dilution rate of 0.425 and 0.475 h^–1, respectively, while maximum COD reduction (98%) was attained at a dilution rate of 0.1 h^–1. The maintenance coefficient attained a value of (0.093, g_s/g_x/h). An increase in dilution rate produced a higher protein content of the biomass.     

Production of xanthan by in-flow cultures of Xanthomonas campestris

The kinetics of xanthan formation in Xanthomonas campestris continuous and fed-batch fermentations was studied along with metabolic changes due to growth rate variation. A maximum growth rate within the range 0.11–0.12 h^–1 was obtained from the continuous culture data in defined medium, producing xanthan at rates up to 0.36 g l^–1 h^–1 corresponding to a maximum 67% glucose conversion at a dilution rate (D) of 0.05 h^–1. Comparatively, fed-batch cultivation was more efficient, producing maximum xanthan at 0.75 g l^–1 h^–1 and 63% glucose conversion at 0.1 h^–1. When reaching D=0.062 h^–1 in continuous cultures, a change was observed and the values of the specific rate of substrate consumption shifted, initiating an uncoupled growth region expressing a lack of balance of the catabolic and anabolic reactions. The deviation was not accompanied by a change in specific xanthan production indicating that xanthan metabolism was not affected by D. For fed-batch-grown X. campestris cells within the range D=0.03–0.1 h^–1, both metabolic parameters changed linearly with the growth rate showing a wide region coupled to growth. Outside that range, glucose accumulated and the specific xanthan production dropped, suggesting substrate inhibition. Correspondence to: J. C. Roseiro     

The influence of cyclic glucose feeding on a continuous bakers' yeast culture

Conclusions Except for the pronounced adaptation-hysteresis effect, the pulse experiments exhibited the expected trend: deviation from the steady feed reference curve was greatest at lowest dilution rates. Under conditions of lowest glucose level the effect of pulsing would be expected to cause the largest fluctuations in glucose, causing a certain fraction of the cells to ferment. Generally over the entire dilution rate range the biomass production was decreased and the ethanol was increased by pulsing the feed stream. There is, however, some evidence that pulse feeding can trigger quite unexpected results. Point (6) at D=0.3 h^–1 in Fig. 1 exhibit a biomass productivity which was about 20% greater than the continuous feeding reference value (DX=3.6 kg m^–3 h^–1 as compared with 3.0 kg m^–3 h^–1). Such performance would be of significant commercial value, but the poor reproducibility due to adaptation, as seen here, certainly would preclude its application.The results obtained should also be applicable to fed batch operation at the corresponding glucose level. Further experiments including the variation of the glucose feeding period would be necessary to obtain a conclusive picture. The observed phenomena are likely to occur in other fermentations and could eventually explain some of the problems existing with scale up of fermentation processes.Symbols D dilution rate h^–1 - P product (ethanol) concentration kg m^–3 - Q_O2 specific oxygen uptake rate mol kg^–1 s^–1 - Q_CO2 specific CO₂ production rate mol kg^–1 s^–1 - S substrate (glucose) concentration kg m^–3 - X biomass concentration kg m^–3 - Y_P/S yield of ethanol from glucose kg kg^–1 - Y_X/S yield of biomass from glucose kg kg^–1     

Improvement of a mammalian cell culture process by adaptive,model-based dialysis fed-batch cultivation and suppression of apoptosis

Both conventional and genetic engineering techniques can significantly improve the performance of animal cell cultures for the large-scale production of pharmaceutical products. In this paper, the effect of such techniques on cell yield and antibody production of two NS0 cell lines is presented. On the one hand, the effect of fed-batch cultivation using dialysis is compared to cultivation without dialysis. Maximum cell density could be increased by a factor of ~5–7 by dialysis fed-batch cultivation. On the other hand, suppression of apoptosis in the NS0 cell line 6A1 bcl-2 resulted in a prolonged growth phase and a higher viability and maximum cell density in fed-batch cultivation in contrast to the control cell line 6A1 (100)3. These factors resulted in more product formation (by a factor ~2). Finally, the adaptive model-based OLFO controller, developed as a general tool for cell culture fed-batch processes, was able to control the fed-batch and dialysis fed-batch cultivations of both cell lines.Abbreviations A membrane area (dm²) - c _Glc,F glucose concentration in nutrient feed (mmol L^–1) - c _Glc,FD glucose concentration in dialysis feed (mmol L^–1) - c _Glc,i glucose concentration in inner reactor chamber (mmol L^–1) - c _Glc,o glucose concentration in outer reactor chamber (dialysis chamber) (mmol L^–1) - c _Lac,FD lactate concentration in dialysis feed (mmol L^–1) - c _Lac,i lactate concentration in inner reactor chamber (mmol L^–1) - c _Lac,o lactate concentration in outer reactor chamber (dialysis chamber) (mmol L^–1) - c _LS,FD limiting substrate concentration in dialysis feed (mmol L^–1) - c _LS,i limiting substrate concentration in inner reactor chamber (mmol L^–1) - c _LS,o limiting substrate concentration in outer reactor chamber (dialysis chamber) (mmol L^–1) - c _Mab monoclonal antibody concentration (mg L^–1) - F _D feed rate of dialysis feed (L h^–1) - F _Glc feed rate of nutrient concentrate feed (L h^–1) - K _d maximum death constant (h^–1) - k _d,LS death rate constant for limiting substrate (mmol L^–1) - k _Glc monod kinetic constant for glucose uptake (mmol L^–1) - k _Lac monod kinetic constant for lactate uptake (mmol L^–1) - k _LS monod kinetic constant for limiting substrate uptake (mmol L^–1) - K _Lys cell lysis constant (h^–1) - K _S,Glc monod kinetic constant for glucose (mmol L^–1) - K _S,LS monod kinetic constant for limiting substrate (mmol L^–1) - µ cell-specific growth rate (h^–1) - µ _d cell-specific death rate (h^–1) - µ _d,min minimum cell-specific death rate (h^–1) - µ _max maximum cell-specific growth rate (h^–1) - P _Glc membrane permeation coefficient for glucose (dm h^–1) - P _Lac membrane permeation coefficient for lactate (dm h^–1) - P _LS membrane permeation coefficient for limiting substrate (dm h^–1) - q _Glc cell-specific glucose uptake rate (mmol cell^–1 h^–1) - q _Glc,max maximum cell-specific glucose uptake rate (mmol cell^–1 h^–1) - q _Lac cell-specific lactate uptake/production rate (mmol cell^–1 h^–1) - q _Lac,max maximum cell-specific lactate uptake rate (mmol cell^–1 h^–1) - q _LS cell-specific limiting substrate uptake rate (mmol cell^–1 h^–1) - q _LS,max maximum cell-specific limiting substrate uptake rate (mmol cell ^–1 h^–1) - q _Mab cell-specific antibody production rate (mg cell^–1 h^–1) - q _MAb,max maximum cell-specific antibody production rate (mg cell^–1 h^–1) - t time (h) - V _i volume of inner reactor chamber (culture chamber) (L) - V _o volume of outer reactor chamber (dialysis chamber) (L) - X _t total cell concentration (cells L^–1) - X viable cell concentration (cells L^–1) - Y _Lac/Glc kinetic production constant (stoichiometric ratio of lactate production and glucose uptake) (–)     

A flow cytometry analysis of batch and continuous culture transient diauxic growth in Hansenula polymorpha

A flow cytometry analysis and in vitro enzyme activity study is carried out on the methylotrophic yeast, Hansenula polymorpha, during both (a) batch growth and (b) continuous cultures subjected to single perturbations in either system dilution rate or influent carbon substrate composition. Flow cytometry of yeasts growing diauxically on a glucose: methanol mixture during exponential growth, exhibit DNA and RNA distributions indicative of the S-synthesis-phase of the cell cycle. Cells at the stationary growth stage exhibit DNA and RNA distributions that indicate one portion of the population in the G ₀/G₁ resting phase and another in the M-mitosis-phase.Yeast cells grown at a steady-state of D=0.2 h¹, then shifted to D=0.35 h^–1, at a constant influent substrate mixture, are also examined with both flow cytometry and in vitro enzyme assays. Distributions of DNA, RNA, and total protein at either steady state and during the shift between dilution rates did not resemble any observed in batch culture. Flow cytometry indicates significant changes in cell composition within 20 min of the imposed dilution rate shift. In vitro enzyme assays show a response time in decreasing methanol oxidase activity of 2.5–3 h upon a dilution rate shift-up, while hexokinase activity increases to its steady-state level in less than 3 h. Similar cell compositional changes are reported for shifts in influent substrate methanol: glucose ratio at a constant dilution rate of D=0.35 h ^–1. Results suggest that an unsteady-state regime, oscillating between conditions that promote maximum enzyme activity of either glucose- or methanol-metabolizing enzymes, may allow simultaneous enhanced time-averaged production of both sets of enzymes.     

Effect of culture conditions on astaxanthin production by a mutant of Phaffia rhodozyma in batch and chemostat culture

Temperature and pH had only a slight effect on the astaxanthin content of a Phaffia rhodozyma mutant, but influenced the maximum specific growth rate and cell yield profoundly. The optimum conditions for astaxanthin production were 22°C at pH 5.0 with a low concentration of carbon source. Astaxanthin production was growth-associated, and the volumetric astaxanthin concentration gradually decreased after depletion of the carbon source. The biomass concentration decreased rapidly during the stationary growth phase with a concomitant increase in the cellular content of astaxanthin. Sucrose hydrolysis exceeded the assimilation rates of D-glucose and D-fructose and these sugars accumulated during batch cultivation. D-Glucose initially delayed D-fructose uptake, but D-fructose utilization commenced before glucose depletion. In continuous culture, the highest astaxanthin content was obtained at the lowest dilution rate of 0.043 h^–1. The cell yield reached a maximum of 0.48 g cells·g^–1 glucose utilized between dilution rates of 0.05 h^–1 and 0.07 h^–1 and decreased markedly at higher dilution rates. Correspondence to: J. C. Du Preez