The influence of poly-β-hydroxybutyrate accumulation on cell volume and buoyant density in alcaligenes eutrophus

Accumulation of poly--hydroxybutyrate (PHB) was studied in Alcaligenes eutrophus strain N9A. Under nitrogen limitation and heterotrophic conditions, the cells accumulated PHB at a rate of 50 fg cell^-1 h^-1. Volume increased from 1.208 to 3.808 m³ and buoyant density from 1.110 to 1.145 pg m^-3 with an increase in PHB from 0 up to 1.699 pg cell^-1. Volume was found to change linearly with PHB content. The changes were due to increases in cell width and not in cell length. PHB explained 93% of the changes in cellular volume. The relationship between density and PHB was hyperbolic. PHB explained 96% of the changes in density. When a mutant strain unable to accumulate PHB was analyzed together with the wild type, the PHB-less mutant and the wild type showed densities of 1.100 pg m^-3 and 1.120 pg m^-3, respectively, in gradients of 65% Percoll. In sucrose gradients, nevertheless, the results were reversed. This discrepancy was explained by the high osmolarity of sucrose which gives artificial results. Thus, we conclude that Percoll is a more suitable medium than sucrose to measure the density of live bacterial cells.Abbreviation PHB poly--hydroxybutyrate     

Long term shear effects on a hybridoma cell line by dynamic perfusion devices

The long term shear effects on a hybridoma cell line were studied by the simulation of a hollow fiber perfusion system. Various mechanical/environmental stress conditions were applied and steady state concentrations of live, dead and lysed cells were measured or calculated in a continuous culture. From mathematical modeling, it is shown that inclusion of a lysed cell index (LCI) renders a better fit to the material balance equation at steady state. The specific cell death rate increased with increasing shear force as expected only when the LCI was included. Without the inclusion of the LCI, the calculated specific cell growth rates are about 25–60% of the value when included. The results reported may lend some insight to design improvements since most perfusion devices add shear stresses to the cells in the reactor.List of Symbols b ml/hr continuous culture flow rate - D hr^–1 dilution rate (b/V) - m g glucose/10⁹ cells/hr specific maintenance coefficient - S ₀ g/l feed substrate concentration - S g/l reactor substrate concentration - t hr time - V ml reactor volume - X ⁺ cells/ml live cell concentration - X ^– cells/ml dead cell concentration - X ⁰ cells/ml lysed cell concentration - Y _x/s 10⁹ cells/g glucose cell/substrate yield coefficient - hr^–1 specific growth rate - hr^–1 specific death rate - hr^–1 specific lysis rate - hr^–1 specific lysis rate for simultaneous death and lysis     

A modified unstructured mathemathical model for the penicillin G fed-batch fermentation

Summary In this paper, an updated unstructured mathematical model for the penicillin G fed-batch fermentation is proposed, in order to correct some physical and biochemical shortcomings in the model of Heijnen et al. (1979,Biotechnol. Bioeng.,21, 2175–2201) and the model of Bajpai and Reuß (1980,J. Chem. Tech. Biotechnol.,30, 332–344). Its main features are the consistency for all values of the variables, and the ability to adequately describe different metabolic conditions of the mould. The model presented here can be considered as the translation of the latest advances in the biochemical knowledge of the penicillin biosynthesis.Nomenclature t time (h) - S amount of substrate in broth (g) - X amount of cell mass in broth (g) - P amount of product in broth (g) - V fermentor volume (L) - F input substrate feed rate (L/hr) - C _s S/V substrate concentration in broth (g/L) - C _x X/V cell mass concentration in broth (g/L) - C _P P/V product concentration in broth (g/L) - s _F substrate concentration in feed stream (g/L) - E _m parameter related to the endogenous fraction of maintenance (g/L) - E _p parameter related to the endogenous fraction of production (g/L) - K _x Contois saturation constant for substrate limitation of biomass production (g/g DM) - K _s Monod saturation constant for substrate limitation of biomss production (g/L) - K _p saturation constant for substrate limitation of product formation (g/L) - K _i substrate inhibition constant for product formation (g/L) - m _s maintenance constant (g/g DM hr) - k _h penicillin hydrolysis or degradation constant (hr^–1) - Y _x/s cell mass on substrate yield (g DM/g) - Y _p/s product on substrate yield (g/g) - specific substrate consumption rate (g/g DM hr) - specific growth rate (hr^–1) - _substr specific substrate to biomass conversion rate (hr^–1) - _x maximum specific substrate to biomass conversion rate (hr^–1) - specific production rate (g/g DM hr) - _p specific production constant (g/g DM hr)     

Determination of specific monoclonal antibody secretion rate during very slow hybridoma growth

A total cell recycling suspension perfusion reactor has been constructed for investigation of specific monoclonal antibody secretion rate of the 9.2.27 murine hybridoma under conditions of a very low growth rate. By rapidly recycling hybridoma cells using a thermostated tangential flow filter, 3.6 mg cell dry weight/cm³ could be maintained at growth rate of <0.05 _max for over 250 h. Under these conditions, secretion of lactate, ammonia and l-alanine were directly related to the rate of l-glutamine supply. Monoclonal antibody accumulated in the reactor to levels in excess of 1400 g/ cm³. Surprisingly, as specific growth rate decreased, the specific immunoglobulin secretion rate remained constant, implying that monoclonal antibody synthesis could be uncoupled from growth.List of Symbols CMF cm³/(min · cm²) cross membrane flow rate - D h^–1 dilution rate - DOT % air saturation dissolved oxygen tension - F _R cm³/min perfusion rate - GlcPR mg/min glucose provision rate - GlnPR mg/min l-glutamine provision rate - N _A mmoles O₂/(dm³ · h) oxygen transfer rate - q _ala mmoles/h l-alanine secretion rate - q _MAB mg MAB · 10^–8 viable cells ^–1 · day^–1 specific MAB secretion rate by viable cells - ¯q _MAB (dimensionless) ¯q _MAB/¯q _{MAB MAX} - ¯q _{NH 3} mmoles/h ammonia secretion rate - S _R mg/cm³ limiting substrate concentration - h^–1 specific growth rate - _app h^–1 apparent growth rate - ¯ (dimensionless) / _MAX - VC cells/cm³ viable cell number     

Effect of endogenous proteins on growth and antibody productivity in hybridoma batch cultures

It has been shown that some B-cell hybridomas secrete autocrine factorsin vitro which can influence cell metabolic processes. Rather than screen specifically for suspected cytokines, that may or may not affect our cell line, we have examined the lumped effects of intracellular and secreted factors on cell proliferation and monoclonal productivity in hybridoma batch cultures. Firstly, supplements of total soluble intracellular proteins combined with other intracellular metabolites were found to both decrease the specific growth rate and increase the antibody production rate at higher concentrations in batch culture. This is an important consideration in high cell density cultures, such as perfusion systems, where a reduction of growth by the presence of intracellular factors may be compensated by an increase in MAb production. In addition, flow cytometry data revealed that the average cell cycle G₁ phase fraction was unaffected by the variation in the maximum specific growth rates during the exponential growth phase, caused by the addition of intracellular factors; this suggests that higher MAb productivity at lower growth rates are not a result of cell arrest in the G₁ phase. Secondly, secreted extracellular proteins larger than 10,000 Daltons, which were concentrated from spent culture supernatant, were shown to have no significant effect on growth and specific MAb productivity when supplemented to batch culture at levels twice that encountered late in normal batch culture. This indicates that endogenous secreted cytokines, if at all present, do not play a major autocrine role for this cell line.Abbreviations FBS fetal bovine serum - MAb monoclonal antibody - MWCO molecular weight cut off - SDS-PAGE sodium dodecyl-sulphate-polyacrylamide gel electrophoresis - k _d exponential phase death rate, h^–1 - q _MAb exponential phase specific monoclonal antibody productivity, pg/(cell·h) - t time, h - X _d dead cell density, cells/mL - X _v viable cell density, cells/mL - specific growth rate, h^–1 - _{max app} apparent maximum specific growth rate, h^–1 - _max maximum specific growth rate, h^–1 _max = _{max app + Kd}      

Enhancement of the attachment on microcarriers and tPA production by fibroblast cells in a serum-free medium by the addition of the extracts ofCentella asiatica

The addition of ethanol extracts ofCentella asiatica showed a remarkable enhancement of fibroblast cells attachment to Cytodex beads in serum-free (SF) medium. It also improves tPA production in both batch and perfusion cultivations. The optimal concentration for SF medium was determined as 2 ppm of the extracts when using Cytodex III. In batch cultivation a high specific tPA production rate was obtained, compared to that from 5% FBS containing medium. However, a fast specific growth rate was observed in 5% FBS medium. In perfusion cultivation a reasonably good cell density and tPA production was achieved at a perfusion rate of 2.4×10⁶ (viable cell/ml) and 0.65 (g/ml), respectively at 22 ml/min.     

Production of human-mouse chimeric antibody by high cell density perfusion culture

Two mouse myeloma cell lines which were transfected with chimeric mouse variable-human constant immunoglobulin heavy and light chain genes have been cultured at high cell density in a settling perfusion culture vessel to produce chimeric antibody specific for human common acute lymphocytic leukemia antigen (cALLA).J558L transfectant proliferated well in a serum-free medium (ITES-eRDF) to a viable cell density of 3.7×10⁷ cells/ml and produced chimeric antibody to a maximum value of 60 g/ml in 120 ml scale vessel. X63Ag8.653 transfectant reached a density of 1.9×10⁷ cells/ml in 1.2 I scale vessel in serum supplemented medium (10% FCS-eRDF) and produced chimeric antibody which consisted of chimeric gamma and chimeric kappa chains to a maximum value of 5.8 g/ml.     

The economic production of anti-microbial factor from human promyelocytes in low serum containing medium under chemostat cultivation

It proves that a purifed Anti-Microbial Factor (AMF) from human promyelocytes has strong activity on Gram(–) and Gram(+) bacteria, showing 0.5 (g/ml) of Minimal Bacterical Concentration (MBC) on bothE. coli andS. aureus. For mass production of AMF, chemostat cultivation is recommended to accumulate cells out of the reactor since it is an intracellular protein and its system requires only 1% serum in the medium. Its production process proves to be closely growth-related. 1.7×10^–8 (g/viable cell/day) of maximum specific AMF production rate is estimated at 0.026 h^–1 of dilution rate, maintaining 6×10⁶ (viable cell/ml). Ca. 300 (mg/ml) of crude AMF can be obtained for 50 days of continuous cultivation under optimal conditions. The cell growth reaches relatively fast steady state.     

Plasmid instability in an industrial strain ofBacillus subtilis grown in chemostat culture

Summary A pUB110-derived plasmid/Bacillus subtilis host combination was segregationally unstable when grown in chemostat culture with complex or minimal medium and under starch, glucose or magnesium limitation. The kinetics of plasmid loss were described in terms of the difference in growth rates between plasmid-containing and plasmid-free cells (d) and the rate at which plasmid-free cells were generated from plasmid-containing cells (R). Loss of plasmid-containing cells from the population was d dominated. Changes in medium composition and the nature of growth limitation caused variations in both d and R. The plasmid was most stable in glucose-limited chemostat cultures with minimal medium and least stable under starch limitation with complex complex medium. R and d were smaller for cultures in complex media than those in minimal media. Limitation by starch induced expression of the plasmid-encoded HT amylase gene and was associated with increased values of R and d. Magnesium limitation in minimal medium caused a significant increase in d and a decrease in R.Abbreviations Cm chloramphenicol - Kan kanamycin - Cm^r cells resistant to chloramphenicol (5 mg L^–1) - Kan^r cells resistant to kanamycin (5 mg L^–1) - Cm^sKan^s cells sensitive to chloramphenicol and kanamycin     

Diagnosing lactic acid fermentation based on specific rates of growth,substrate consumption and product formation

The on-line calculated specific rates of growth, substrate consumption and product formation were used to diagnose microbial activities during a lactic acid fermentation. The specific rates were calculated from on-line measured cell mass, and substrate and product concentrations. The specific rates were more sensitive indicators of slight changes in fermentation conditions than such monitored data as cell mass or product concentrations.List of Symbols 1/h specific rate of cell growth - 1/h specific rate of substrate consumption - 1/h specific rate of product formation - ^* dimensionless specific rate of cell growth - ^* dimensionless specific rate of substrate consumption - ^* dimensionless specific rate of product formation - _max 1/h maximum specific rate of cell growth - _max 1/h maximum specific rate of substrate consumption - _max 1/h maximum specific rate of product formation - X g/l cell mass concentration - S g/l substrate concentration - S ^* dimensionless substrate concentration - S ₀ g/l initial substrate concentration - P g/l product concentration     

Xylose metabolism by Candida shehatae in continuous culture

Summary Xylose metabolism by Candida shehatae in continuous culture was examined under both fully-aerobic and semi-aerobic conditions. Growth did not occur in the absence of respiration. Under fully-aerobic conditions, the cell yield was constant at 0.51 g/g and the specific respiration rate Q _{o 2} was linearly related to the specific growth rate with a slope of 15 mmol g^-1 and an intercept of 1.2 mmol g^-1 h^-1. Under semi-aerobic conditions, Q _{O 2} was proportional to with a slope consistent with a cell yield from oxygen Y _{O 2} of 35±7 g mol^-1. The specific xylose uptake rate was a constant independent of and equal to the maximum rate of xylose transport. Ethanol production was observed under semi-aerobic conditions only, and the specific fermentation rate was inversely related to .The use of trade, firm, or corporation names in this publication is for the information and convenience of the reader. Such use does not constitute an official endorsement or approval by the U.S. Department of Agriculture of any product or service to the exclusion of others which may be suitableMaintained in cooperation with the University of Wisconsin-Madison     

Effect of dilution rate on the release of pertussis toxin and lipopolysaccharide ofBordetella pertussis

Summary The kinetics ofBordetella pertussis growth was studied in a glutamate-limited continuous culture. Growth kinetics corresponded to Monod's model. The saturation constant and maximum specific growth rate were estimated as well as the energetic parameters, theoretical yield of cells and maintenance coefficient. Release of pertussis toxin (PT) and lipopolysaccharide (LPS) were growth-associated. In addition, they showed a linear relationship between them. Growth rate affected neither outer membrane proteins nor the cell-bound LPS pattern.Nomenclature X cell concentration (g L^–1) - specific growth rate (h^–1) - _m maximum specific growth rate (h^–1) - D dilution rate (h^–1) - S concentration of growth rate-limiting nutrient (glutamate) (mmol L^–1 or g L^–1) - Ks substrate saturation constant (mol L^–1) - m_s maintenance coefficient (g g^–1 h^–1) - Y_x/s theoretical yield of cells from glutamate (g g^–1) - Y_x/s yield of cells from glutamate (g g^–1) - Y_PT/s yield of soluble PT from glutamate (mg g^–1) - Y_KDO/s yield of cell-free KDO from glutamate (g g^–1) - Y_PT/x specific yield of soluble PT (mg g^–1) - Y_KDO/x specific yield of cell-free KDO (g g^–1) - q_PT specific soluble PT production rate (mg g^–1 h^–1) - q_KDO specific cell-free KDO production rate (g g^–1 h^–1)     

Product inhibition during the feeding phasein fed-batch ethanol fermentation of sugar-cane blackstrap molasses

Summary The following equations represent the influence of the ethanol concentration (E) on the specific growth rate of the yeast cells () and on the specific production rate of ethanol () during the reactor filling phase in fed-batch fermentation of sugar-cane blackstrap molasses: = ₀ - k · E and v = v ₀ · K/(K +E) Nomenclature E ethanol concentration in the aqueous phase of the fermenting medium (g.L^–1) - E_m value of E when = 0 or = 0 (g.L^–1) - F medium feeding rate (L.h^–1) - k empirical constant (L.g^–1.h^–1) - K empirical constant (g.L^–1) - M_as mass of TRS added to the, reactor (g) - M_cs mass of consumed TRS (g) - M_e mass of ethanol in the aqueous phase of the fermenting medium (g) - M_s mass of TRS in the aqueous phase of the fermenting medium (g) - M_x mass of yeast cells (dry matter) in the fermenting medium (g) - r correlation coefficient - S TRS concentration in the aqueous phase of the fermenting medium (g.L^–1) - S_m TRS concentration of the feeding medium (g.L^–1) - t time (h) - T temperature (° C) - TRS total reducing sugars calculated as glucose - V volume of the fermenting medium (L) - V₀ volume of the inoculum (L) - X yeast cells concentration (dry matter) in the fermenting medium (g.L^–1) - filling-up time (h) - specific growth rate of the yeast cells (h^–1) - ₀ value of when E=0 - specific production rate of ethanol (h^–1) - ₀ value of when E=0 - density of the yeast cells (g.L^–1) - dry matter content of the yeast cells     

The growth of Pseudomonas putida on m-toluic acid and on toluene in batch and in chemostat cultures

Summary The present study describes the growth of Pseudomonas putida cells (ATCC 33015) in batch and continuous cultures on two toxic substrates; toluene and m-toluic acid as sole carbon and energy sources. In fed-batch cultures on m-toluic acid up to 3.55 g cell dry weight/1 were achieved with a maximal specific growth rate (_max) of 0.1 h^-1. The average cellular yield was 1.42 g cell dry weight/g m-toluic acid utilized. When liquid toluene was added to shake-flask cultures in the presence of 0.7 g/1 m-toluic acid, the average cellular yield obtained was 1.3 g cell dry weight/g toluene utilized and the _max was 0.13 h^-1. Growth on toluene vapour in the presence of 0.7 g/l m-toluic acid in batch cultures resulted in a cellular yield of 1.28 g cell dry weight/g toluene utilized, with growth kinetics almost identical to those with liquid toluene (_max liquid=0.13 h^-1, _max vapour=0.12 h^-1). The maximal biomass concentration was 3.8 g cell dry weight/l, obtained in both cases after 100 h of incubation. Pseudomonas putida was grown in a chemostat initially on 0.7 g/l m-toluic acid and vapour toluene and then in the steady state on toluene as the sole source of carbon and energy. Toluene was added continuously to the culture as vapour with the inflowing airstream. Chemostat cultures could be maintained at steady state for several months on toluene. The maximal biomass concentration obtained in the chemostat culture was 3.2 g cell dry weight/l. The maximum specific growth rate was 0.13 h^-1, with a cellular yield of 1.05 g cell dry weight/g toluene utilized. Approximately 70% of the toluene consumed was converted into biomass, and the remainder was converted to CO₂ and unidentified byproducts.     

Feeding in the rotifer Brachionus calyciflorus

Summary The laboratory feeding behavior of Brachionus calyciflorus varies depending upon the type of food cell available in suspension. When feeding on the yeast Rhodotorula glutinis, rotifers show a continuous increase in ingestion with increased cell density between 0.01 and 1000 g dry weight ml^-1. Effective clearance rates drop from ca. 50 l animal^-1 h^-1 to less than 0.5 l animal^-1 h^-1 over this food density range. When feeding on Englena gracilis, B. calyciflorus ingestion rates are constant between 1.0 and 100 g ml^-1 of available food, averaging close to 25 ng animal^-1 h^-1. The decrease in clearance rate is more striking than with R. glutinis, dropping from 45 l animal^-1 h^-1 at 0.1 g ml^-1 to 0.13 l animal^-1 h^-1 at 100 g ml^-1. Differences between the patterns obtained with the two food types indicate fundamental dissimilarities in the feeding behavior of this rotifer species when presented with these different foods.     

High cell density fermentation of recombinant Escherichia coli expressing human interferon alpha 1

Summary A defined medium was developed which, by means of a specific fed-batch mode, allows growth of the recombinant Escherichia coli strain TG1 (pBB210) up to a cell density of 60 g dry weight/l. Apart from glucose and aqueous ammonia fed as carbon and nitrogen sources, it was unnecessary to supply other nutrients or O₂-enriched air. Aqueous ammonia also served for pH control. The pO₂ level was kept at 20% saturation via closed-loop controls operating the two output variables of stirrer speed and glucose feeding rate. This fed-batch method prevented significant accumulation of acetate and other metabolic by-products. The recombinant E. coli expressed interferon alpha 1 more efficiently at a lower specific growth rate (_Pr 0.15 h^–1) than at the maximum specific growth rate (_max = 0.45 h^–1). Therefore, fermentation in the batch phase at _max was only allowed to continue up to a medium cell density. In the succeeding fed-batch phase, the specific growth rate was reduced to _Pr by increasing the stirrer speed according to an empirically developed time scale. Offprint requests to: D. Riesenberg     

Characteristics of the ATP-ase activity of isolated neurons of rabbit

Summary Isolated and homogenised Deiters' neurons from the lateral vestibular nucleus of rabbit in a Krebs-Ringer solution containing no added Mg⁺⁺, 1.3 moles/ ml and 5 moles/ml Mg⁺⁺, broke down ATP at the maximal rate of 0.29+-0.20, 2.40+–0.20, and 5.95+–0.63 moles/cell/hr. In 1.3 mM Mg⁺⁺ solution the single cell homogenates took up phosphate at the mean rate of 2.6+–0.2 moles/cell/hr. If the rabbits were injected 1 hour before with 20 mg/kg body weight of 2-amino-1-propene-1,1,3, tricarbonitrile (triap), the breakdown of ATP in these latter media was 0.82+–0.44, 2,5+–0.60, and 6.7+– 1.1 moles/cell/hr, respectively, and the quantity of inorganic liberated did not decrease.     

Hybridomas in a bioreactor cascade: modeling and determination of growth and death kinetics

Hybridomas were cultured under steady-state conditions in a series of two continuous stirred-tank reactors (CSTRs), using a serum-free medium. The substrate not completely converted in the first CSTR, was transported with the cells to the second one and very low growth rates, high death rates, and lysis of viable cells were observed in this second CSTR. These conditions are hardly accessible in a single vessel, because such experiments would be extremely time-consuming and unstable due to a low viability. In contrast to what is often observed in literature, kinetic parameters could thus be derived without the neccessity for extrapolation to lower growth rates. Good agreement with literature averages for other hybridomas was found. Furthermore, showing that the reactor series is a valuable research tool for kinetic studies under extreme conditions, the possibility to observe cell death under stable and defined steady-state conditions offers interesting opportunities to investigate apoptosis and necrosis. Additionally, a model was developed that describes hybridoma growth and monoclonal antibody production in the bioreactor cascade on the basis of glutamine metabolism. Good agreement between the model and the experiments was found.Abbreviation MAb Monoclonal antibody Nomenclature C _AConcentration of any (mol m^-3) component A D Dilution rate (s^-1) K _dDeath-rate constant (mol m^-3) K _lLysis-rate constant (mol m^-3) K _sMonod constant (mol m^-3) m Maintenance coefficient (mol cell^-1 s^-1) q Specific consumption (mol cell^-1 s^-1) or production rate t Time (s) X Cell concentration (cell m^-3) Y Yield coefficient (cell mol^-1) Greek symbols _d Specific death rate (s^-1) _l Specific lysis rate (s^-1) of viable cells _net Net specific growth (s^-1) rate _true True specific growth (s^-1) rate     

Expression of survivin mRNA and protein in gastric cancer cell line (MKN-45) during cisplatin treatment

Survivin is a member of the inhibitor of apoptosis protein (IAP) family. Survivin has been reported to be expressed in many cancers, but not in differentiated normal tissue. Recent studies revealed that survivin correlated with the chemo-resitance of cancer cells. In the present study, the changes in expression levels of survivin messenger RNA (mRNA) and survivin protein in a gastric cancer cell line (MKN-45) during cisplatin (CDDP) treatment were analyzed and compared with the occurrence of apoptotic cell death. Cell growth was inhibited even with a low dose CDDP (0.1 or 1 g/ml) 1 hr treatment. However, the percentage of apoptotic cells did not change after 48 hr incubation with low dose CDDP. Only with high dose CDDP (10 g/ml), did the percentage of apoptotic cells explosively increase between 12 and 24 hr treatment. Relative expression levels of survivin mRNA and survivin protein increased after CDDP treatment. The cell expression rates of survivin mRNA after 48 hr treatment with 0.1 and 1 g/ml of CDDP were 2 to 6 fold higher than that of the survivin mRNA of untreated cells. Also, the relative cell expression level of survivin protein after 24 hr treatment with 0.1 or 1 g/ml of CDDP was 3 to 6.5 fold higher than that of the survivin protein of untreated cells. These results indicate that survivin expression may correlate with the chemo-resistance of malignant cells.     

Selection and evaluation of astaxanthin-overproducing mutants of Phaffia rhodozyma

Mutagenesis of Phaffia rhodozyma with NTG yielded a mutant with an astaxanthin content of 1688 g (g dry biomass)^-1, a cell yield coefficient of 0.47 on glucose and a maximum specific growth rate of 0.12 h^-1. Re-mutation of the mutant decreased the cell yield and maximum specific growth rate but increased the astaxanthin content. The use of mannitol or succinate as carbon sources enhanced pigmentation, yielding astaxanthin contents of 1973 g g^-1 and 1926 g g^-1, respectively. The use of valine as sole nitrogen source also increased astaxanthin production, but severely decreased the maximum specific growth rate and cell yield coefficient. The optimum pH for growth of P. rhodozyma was between pH 4.5 and 5.5, whereas the astaxanthin content remained constant above pH 3.