Fed-batch cultivation of Bordetella pertussis: Metabolism and Pertussis Toxin production

The production of acellular pertussis in comparison with whole cell pertussis vaccines demands 5-25 times the amount of Bordetella pertussis' virulence factors, such as Pertussis Toxin (PT), to produce the same number of vaccine doses. An increase in the volumetric productivity by employing fed-batch rather than the currently used batch cultivations of B. pertussis could reduce the cost price of acellular pertussis vaccines. This study defined the conditions that enable fed-batch cultivations at high specific PT production. A solution containing lactate and glutamate was fed to the cultures at various rates. The feed rate and whether or not the fed substrates were completely consumed, significantly influenced cellular metabolism. If lactate was detectable in the culture broth while glutamate was not, poly-hydroxy-butyrate (PHB) was formed. Any PHB present was metabolized when glutamate became detectable again in the culture liquid. At higher lactate and glutamate concentrations, free fatty acids were produced. Though toxic, free fatty acids were not the reason the cultures stopped growing. By choosing appropriate conditions, a cell density of 6.5 g/L dry weight was reached, i.e. a 7-fold increase compared to batch culture. The metabolic mechanisms behind the formation of PHB and fatty acids are discussed, as well as how to increase the cell density further. The PT production stopped at 12 mg/L, well before growth stopped, indicating that regulatory mechanisms of PT production may be involved.     

Experimental design and metabolic flux analysis tools to optimize industrially relevant Haemophilus influenzae type b growth medium

Haemophilus influenzae type b (Hib), a Gram‐negative capsulated bacterium, is a causative agent of meningitis worldwide. The capsular polysaccharide, a high molecular mass polymer consisting of the repeated units of the polyribosyl‐ribitol‐phosphate, is considered the main virulence factor and it is used as an antigen to vaccines, conjugated to a carrier protein. The industrial production of the polysaccharide requires the cultivation of Hib in rich medium, which impacts process costs and product recovery. In this study, a central composite rotational experimental design strategy was used to access the influence of key components of culture medium (soy peptone, yeast extract and glucose) on biomass formation and polysaccharide production in shake‐flasks. The optimized medium formulation, containing half of the usual yeast extract and soytone concentrations, was further validated in batch bioreactor cultivations. High polysaccharide production (～500 mg/L) was obtained in a cheaper and more competitive production process for use in Hib vaccine production. In addition, simulations of a metabolic model describing Hib central metabolism were used to assess the role of key amino acids on growth. A chemically defined medium supplemented only with amino acids from α‐ketoglutarate and oxaloacetate families as well as phenylalanine was suggested as a promising alternative for reduced acetate accumulation and enhanced polysaccharide production in Hib cultures. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1508–1519, 2017     

Process intensification for production of Streptococcus pneumoniae whole-cell vaccine

The available pneumococcal conjugate vaccines provide protection against only those serotypes that are included in the vaccine, which leads to a selective pressure and serotype replacement in the population. An alternative low-cost, safe and serotype-independent vaccine was developed based on a nonencapsulated pneumococcus strain. This study evaluates process intensification to improve biomass production and shows for the first time the use of perfusion-batch with cell recycling for bacterial vaccine production. Batch, fed-batch, and perfusion-batch were performed at 10 L scale using a complex animal component-free culture medium. Cells were harvested at the highest optical density, concentrated and washed using microfiltration or centrifugation to compare cell separation methods. Higher biomass was achieved using perfusion-batch, which removes lactate while retaining cells. The biomass produced in perfusion-batch would represent at least a fourfold greater number of doses per cultivation than in the previously described batch process. Each strategy yielded similar vaccines in terms of quality as evaluated by western blot and animal immunization assays, indicating that so far, perfusion-batch is the best strategy for the intensification of pneumococcal whole-cell vaccine production, as it can be integrated to the cell separation process keeping the same vaccine quality.     

Batch cultivation of Methylosinus trichosporium OB3b: II. Production of particulate methane monooxygenase

The obligatory methanotroph, Methylosinus trichosporium OB3b, was studied to optimize the batch culture conditions for the formation of particulate methane monooxygenase (pMMO) in a nitrate minimal salts medium. The important medium components investigated were copper, carbon dioxide, and nitrate. The whole-cell specific pMMO activity decreased sharply with increasing copper concentrations in the range of 10-40 muM and remained constant upon further increases of the copper concentration to 120 muM. The cell growth rate (mu), on the other hand, decreased over the entire range (10-120 muM) of copper concentrations tested. When pMMO was produced in a bioreactor with an optimal initial copper concentration of 10 muM, M. trichosporium OB3b exhibited a much faster overall growth rate and a higher whole-cell propene epoxidation activity compared to our earlier study, in which soluble methane monooxygenase (sMMO) was produced with copper-deficient medium. The addition of external carbon dioxide to the bioreactor culture eliminated an initial lag period in the cell growth. When the standard culture medium nitrate concentration (10 mM) was depleted, the pMMO activity, but not the growth rate, decreased rapidly. The whole-cell specific pMMO activity could be maintained by subsequent supplementation of nitrate. A 4-fold higher initial culture medium nitrate concentration of 40 mM, however, resulted in slower cell growth and lower pMMO activity. These observations demonstrate that, in addition to affecting the exclusive production of pMMO, copper also has an important previously unrecognized role in enhancing the growth rate of M. trichosporium OB3b. They also indicate that for the optimal batch production of pMMO with the minimal medium under study, nitrate should be supplied intermittently during the course of cultivation until other culture medium components become growth-limiting.     

Scaling-up vaccine production: implementation aspects of a biomass growth observer and controller

This study considers two aspects of the implementation of a biomass growth observer and specific growth rate controller in scale-up from small- to pilot-scale bioreactors towards a feasible bulk production process for whole-cell vaccine against whooping cough. The first is the calculation of the oxygen uptake rate, the starting point for online monitoring and control of biomass growth, taking into account the dynamics in the gas-phase. Mixing effects and delays are caused by amongst others the headspace and tubing to the analyzer. These gas phase dynamics are modelled using knowledge of the system in order to reconstruct oxygen consumption. The second aspect is to evaluate performance of the monitoring and control system with the required modifications of the oxygen consumption calculation on pilot-scale. In pilot-scale fed-batch cultivation good monitoring and control performance is obtained enabling a doubled concentration of bulk vaccine compared to standard batch production.     

Overview of recent clinical trials of acellular pertussis vaccines.

The evidence from pre-licensure studies does not suggest that there are clinically important differences in reactogenicity between acellular vaccines. The merits of different acellular products will therefore have to be compared on efficacy criteria. Ideally, acellular vaccines with the minimum antigen content necessary to ensure optimum protection should be used in order to avoid administration of superfluous antigens to children and to simplify licensing and batch release procedures.On the basis of the evidence so far available it seems unlikely that monocomponent pertussis toxin (PT) vaccines provide optimal protection and that multicomponent vaccines are needed to achieve a level of disease control that approaches that of a good whole-cell vaccine. It is unclear whether all two component vaccines containing PT and filamentous haemagglutinin (FHA) have similar efficacy but on the available evidence the safest option for policy makers would seem to be to use a vaccine with at least three components, PT+FHA+pertactin. There is now good evidence that the five component vaccine which contains agglutinogens 2 and 3 in addition to PT/FHA and pertactin provides the best protection and is the only acellular vaccine whose efficacy matches that of a good whole cell vaccine. However, the public health advantage of the five component vaccine over other acellular vaccines may not become apparent until they have been in routine use for some decades and their ability to protect against transmission as well as clinical pertussis has emerged.The decision to replace an effective whole-cell vaccine by an acellular vaccine for primary immunisation needs careful consideration. Apart from the probable sacrifice of efficacy for reduced reactogenicity (at least for vaccines which do not contain agglutinogens 2 and 3) there is the question of value for money and the ease with which acellular DTP vaccines can be combined with conjugate polysaccharide vaccines such as Haemophilus influenzae type b.Whatever the decision of policy makers, the need for continued follow up of trial cohorts and active surveillance of the efficacy and safety of those acellular vaccines that are introduced into routine use must be accorded a high priority.     

Production of hantavirus Puumala nucleocapsid protein in Saccharomyces cerevisiae for vaccine and diagnostics

The production of hantavirus Puumala nucleocapsid (N) protein for potential applications as a vaccine and for diagnostic purposes was investigated with Saccharomyces cerevisiae as a recombinant host. The N protein gene and the hexahistidine tagged N (h-N) protein gene were expressed intracellular from a 2-microm plasmid vectors under the control of a fused galactose inducible GAL10-PYK promoter. For monitoring the recombinant gene expression, a h-N and a GFP fusion protein was used. Different cultivation strategies and growth media compositions were tested in shake flasks and a 5 l bioreactor. When using defined YNB growth medium, we found the biomass yield to be unsatisfactorily low. Higher concentrated YNB medium, promoted cell growth but showed a pronounced inhibitory effect on heterologous gene expression. This phenomenon could not be attributed to plasmid losses, as we could demonstrate high stability of the vector under the applied cultivation conditions. Supplementation of YNB medium with extracts of plant origin resulted in increased biomass yields with concomitant high expression levels of the recombinant gene. The modified medium was used for fed-batch cultivations where basic metabolic features as well as growth parameters were determined in addition to recombinant gene expression. The maximal volumetric yield of N protein was 316 mg l(-1), the respective yield of h-N protein was 284 mg l(-1). Our study provides a basis for large-scale production of hantavirus vaccines, which satisfies economic efficiency as well as biosafety regulations for human applications.     

Sodium chloride, potassium chloride, and virulence in Listeria monocytogenes.

Virulence, as determined in a mouse model, and the virulence factor activities of catalase, superoxide dismutase, and listeriolysin O were examined in a parental strain (10403S) and in a nonhemolytic mutant strain (DP-L224) of Listeria monocytogenes. The cells were propagated in media containing various concentrations of sodium chloride or potassium chloride. Strains 10403S and DP-L224 exhibited significant increases in catalase activity and listeriolysin O activity when grown in medium containing either salt at 428 mM. The superoxide dismutase activities for both strains increased when they were grown in medium containing either salt. The superoxide dismutase activity was significantly increased only when cells were propagated in medium containing no salt compared with that when they were propagated in medium containing either salt at 1,112 mM. In addition, the listeriolysin O activity was highest for cells propagated in medium containing KCl at 428 mM, while the activity was significantly less for cells propagated in medium containing NaCl at an equal concentration. Virulence was examined in mouse livers and spleens after intravenous infection, and approximate 50% lethal doses were determined after intragastric and intraperitoneal infection. Each method of infection indicated that listeriolysin O is required for virulence, while growth in salt-containing medium or the production of higher levels of catalase, superoxide dismutase, and listeriolysin O do not appear to enhance the virulence of L. monocytogenes.     

Substitution of glutamine by pyruvate to reduce ammonia formation and growth inhibition of mammalian cells

In mammalian cell culture technology glutamine is required for biomass synthesis and as a major energy source together with glucose. Different pathways for glutamine metabolism are possible, resulting in different energy output and ammonia release. The accumulation of ammonia in the medium can limit cell growth and product formation. Therefore, numerous ideas to reduce ammonia concentration in cultivation broths have been developed. Here we present new aspects on the energy metabolism of mammalian cells. The replacement of glutamine (2 mM) by pyruvate (10 mM) supported cell growth without adaptation for at least 19 passages without reduction in growth rate of different adherent commercial cell lines (MDCK, BHK21, CHO-K1) in serum-containing and serum-free media. The changes in metabolism of MDCK cells due to pyruvate uptake instead of glutamine were investigated in detail (on the amino acid level) for an influenza vaccine production process in large-scale microcarrier culture. In addition, metabolite profiles from variations of this new medium formulation (1-10 mM pyruvate) were compared for MDCK cell growth in roller bottles. Even at very low levels of pyruvate (1 mM) MDCK cells grew to confluency without glutamine and accumulation of ammonia. Also glucose uptake was reduced, which resulted in lower lactate production. However, pyruvate and glutamine were both metabolized when present together. Amino acid profiles from the cell growth phase for pyruvate medium showed a reduced uptake of serine, cysteine, and methionine, an increased uptake of leucine and isoleucine and a higher release of glycine compared to glutamine medium. After virus infection completely different profiles were found for essential and nonessential amino acids.     

Effect of light emitting diodes on the cultivation of Spirulina platensis using NPK‐10:26:26 complex fertilizer

NPK‐10:26:26 complex fertilizer based culture medium was studied for the mass production of Spirulina platensis using different light emitting diodes (LEDs). First, cultivation was carried out under white LED to formulate the optimum fertilizer loading for which Spirulina growth was maximized. Optimum composition for newly formulated fertilizer medium was NPK fertilizer ?0.76 g L^?1 and sodium bicarbonate ?10.0 g L^?1 and corresponding biomass productivity was found to be 76.67 mg L^?1 day^?1. The effect of different LEDs (for example, blue, white, red, green and yellow) on Spirulina growth kinetics and the accumulation of chlorophyll, protein and lipid content was determined using the optimum NPK fertilizer medium. Kinetic parameters (i.e., biomass productivity, maximum specific growth rate, maximum biomass concentration, nitrogen‐to‐biomass conversion factor and phosphorus‐to‐biomass conversion factor) and chlorophyll accumulation were affected by the use of different LEDs and follow the following trend: blue > white > red > green > yellow, whereas protein and lipid accumulation was almost independent of LEDs used. Elemental C, N, P and K concentrations were measured to find the effects of nutrients for the growth of Spirulina platensis. Physicochemical parameters (pH and conductivity) were also monitored during biomass growth under different LEDs. Finally, biomass growth using NPK‐10:26:26 fertilizer under different LEDs was compared with standard Zarrouk medium and better growth results were obtained using optimally formulated NPK‐10:26:26 fertilizer medium.     

Effect of the nitrogen source on caffeine degradation by Aspergillus tamarii

AIMS: To evaluate caffeine degradation and nitrogen requirements during Aspergillus tamarii growth in submerged culture. METHODS AND RESULTS: Aspergillus tamarii spores produced on a coffee infusion agar medium added with sucrose were used. Several caffeine and ammonium sulphate concentrations (0-1 and 0-1.36 g l-1, respectively) were tested simultaneously on fungal biomass production and caffeine degradation. An additional caffeine pulse (4 g l-1) was added for all experiments after 48 h of fermentation. Results revealed that when using 0.90 g l-1 of caffeine and 0.14 g l-1 of ammonium sulphate, biomass production and caffeine degradation were enhanced. Highest biomass production (Xmax = 9.87 g l-1) with a specific growth rate (micro) of 0.073 h-1 and caffeine degradation rate of 0.033 g l-1 h-1, was observed under these conditions. CONCLUSIONS: Caffeine degradation as well as biomass production were characterized. SIGNIFICANCE AND IMPACT OF THE STUDY: These studies set the stage for future characterization studies of intracellular enzymes involved in caffeine degradation. Moreover, results observed may help in the biotreatment of residues from the coffee agroindustry.     

Optimization of culture medium and growth conditions for production of L-arabinose isomerase and D-xylose isomerase by Lactobacillus bifermentans

Lactobacillus bifermentans was used to produce the intracellular enzymes L-arabinose isomerase and D-xylose isomerase. Various factors of cultivation (temperature, pH, or incubation period) and culture medium composition (mineral salts, carbon and nitrogen source) were studied to select the conditions that maximize production of these enzymes. Arabinose isomerase and xylose isomerase activities were 9.4 and 7.24 U/ml, respectively. They were highest at 9 h of cultivation in the optimized medium, 1.6 times higher than that in the basic MRS broth. The optimal medium composition and cultivation conditions were determined. On the other hand, the strain required for growth Tween 80 (1 g/l) and a source of inorganic nitrogen (e.g. ammonium citrate). The bacterium had no requirement for sodium acetate for both growth and production of isomerases. The production rate of enzymes was increased when metal ions were added and mainly manganese (2.5 mM).     

Effect of environmental factors and precursors on oxalic acid production,mycelial biomass and virulence of a potential bioherbicide isolate of Sclerotium rolfsii SC64 produced in liquid culture

The fungus Sclerotium rolfsii is presently under development as a bioherbicide for broadleaf weed species using fungus-infested substrates as application material in this laboratory. The effect of environmental factors and three precursors (citric acid, ascorbic acid, and sodium succinate) on mycelial growth, oxalic acid production, and virulence by SC64 in liquid culture were investigated. The results showed that for mycelia growth the optimum liquid medium was Modified Richard's solution (MRS) among the five tested media, but potato dextrose broth (PDB) produced the maximum oxalic acid production and virulence on detached Solidago canadensis leaves. When PDB was used as the basic medium, the oxalic acid/mycelial dry weight (mg g^–1) ratio reached the peak 4 days after inoculation. The optimum temperature for oxalic acid production was at 27°C, but increased mycelial dry weight and virulence were observed at 30°C. The optimum range of initial pH value for oxalic acid accumulation was 4.0–6.0, with the optimal pH 5.0; highest mycelial growth was with an initial pH 3.5–6.0 (optimum pH 5.0) and subsequently pH 3.5–5.5 (maximum at pH 3.5). Both mycelial dry weight and oxalic acid production showed a decreasing trend as a result of the precursor of oxalic acid being added to PDB. Among the three precursors, the greatest decrease in mycelial dry weight, and oxalic acid production was caused by sodium succinate. This clarification of optimal conditions for production of mycelial biomass while insuring high concentrations of oxalic acid and high virulence should be useful for further development of this fungus as biocontrol agent.     

Effect of some metabolic inhibitors on citric acid production by Aspergillus niger

Stationary cultures of Aspergillus niger grown on a synthetic medium have been used to study the effect of some metabolic inhibitors on citric acid production. Addition of 0.05 to 1 mM sodium malonate or 0.01 to 0.1 mM potassium ferricyanide, iodoacetate, sodium azide, sodium arsenate or sodium fluoride stimulated citric acid production (3.6 to 45%), but not total titratable acids. Addition of higher concentrations (0.2 to 10 mM) of later inhibitors caused a marked inhibition of fungal growth and citric acid production. The implications of these preliminary findings are discussed.     

Enzyme-based glucose delivery as a high content screening tool in yeast-based whole-cell biocatalysis

The influence of glucose release on growth and biotransformation of yeasts was examined by using the medium EnBase^® Flo in shake flasks. The medium contains a polysaccharide acting as substrate, which is degraded to glucose by the addition of an enzyme. In the present paper, this medium was adapted for the cultivation of yeasts by increasing the complex components (booster) and the enzyme concentrations to guarantee a higher glucose release rate. Important changes were an increase of the complex component booster to 10–15% and an increased glucose release by increasing the enzyme content to 15 U L^?1. The 20 yeasts investigated in the present work showed an improvement of growth and biomass production when cultivated with the EnBase medium in comparison to yeast extract dextrose (YED) medium. Values of optical densities (OD₆₀₀) of approximately 40 AU (corresponding to over 60 g L^?1 wet cell weight) were achieved for all 20 yeast strains tested. During the following screening of the yeasts in whole-cell biotransformation, an improvement of the conversion for 19 out of the 20 yeasts cultivated with the EnBase Flo medium could be observed. The biomass from the EnBase Flo cultivation showed a higher conversion activity in the reduction of 2-butanone to (R/S)-2-butanol. The enantioselectivity (ee) of 15 yeast strains showed an improvement by using the EnBase medium. The number of yeasts with an ee >97% increased from zero with YED to six with EnBase medium. Thus, the use of a glucose release cultivation strategy in the screening process for transformation approaches provides significant benefits compared to standard batch approaches.     

Optimization of culture medium and growth conditions for production of L-arabinose isomerase and D-xylose isomerase by <Emphasis Type="Italic">Lactobacillus bifermentans</Emphasis>

Lactobacillus bifermentans was used to produce the intracellular enzymes L-arabinose isomerase and D-xylose isomerase. Various factors of cultivation (temperature, pH, and incubation period) and culture medium composition (mineral salts, carbon source, and nitrogen source) were studied to select the conditions that maximize production of these enzymes. Arabinose isomerase and xylose isomerase activities were 9.4 and 7.24 U/ml, respectively. They were highest at 9 h of cultivation in the optimized medium, 1.6 times higher than that in the basic MRS broth. The optimal medium composition and cultivation conditions were determined. For optimal growth, the strain required Tween 80 (1 g/l) and a source of inorganic nitrogen (e.g., ammonium citrate). The bacterium had no requirement for sodium acetate for either growth or production of isomerases. The production rate of enzymes was increased when metal ions were added, primarily manganese (2.5 mM). The text was submitted by the authors in English.     

Genetic regulation of pathogenicity and virulence factors in bacteria Erwinia carotovora subsp. atroseptica: phenotypic characteristic of bacteria with the mutant kduD gene

In contrast to the closely related bacteria Erwinia chrysanthemi, bacteria Erwinia carotovora subsp. atroseptica produce lower levels of main pathogenicity and virulence factors (pectate lyases, cellulases, and proteases) in the presence of pectins. This effect was shown to be connected with the accumulation of the intermediate product of intracellular degradation of these substances, 2,5-diketo-3-deoxygluconate (DK2). The presence of DK2 in the culture broth of mutant bacteria, connected to its export in the environment, was established. The production of pectate lyases, cellulases, and proteases is repressed by DK2 only at its high concentrations in the cultivation medium, whereas low concentrations of DK2 induce the production of virulence factors. Genes involved in the intracellular catabolism of pectin substances and induced by both low and high DK2 concentrations in the cultivation medium are not repressed by this metabolite.     

The effect of copper on the growth of wood-rotting fungi and a blue-stain fungus

The effect of copper (II) ions on the growth of three brown-rot fungi, six white-rot fungi and one blue-stain fungus in solid medium was evaluated. The fungi were grown in malt extract agar with different concentrations of copper added, and the radial growth rate was determined. At the end of the incubation period, the mycelial biomass and the media pH were determined. The white-rot and blue-stain fungus grew up to 3 mM and 6 mM copper, respectively and the brown-rot fungi were the only ones that grew up to 10 mM, with higher growth rates than those shown by the other fungi. In general, the brown-rot fungi produced greater acidification in the culture media than the white-rot fungi and blue-stain fungus, and the acidification increased when the amount of copper was increased. The biomass production for the different species, in the absence or presence of copper, was not related to the radial growth rate, and the fungal species that produced the greatest biomass amounts did not correspond to those that presented the highest growth rates. The brown-rot fungi Wolfiporia cocos and Laetiporus sulfureus and blue-stain fungus Ophiostoma sp. demonstrated greater tolerance to high copper concentrations in solid medium than the white-rot fungi, determined as radial growth rate. On the other hand, the highest biomass producers in solid medium with copper added were the white-rot fungi Ganoderma australe and Trametes versicolor and the brown-rot fungus Gloeophyllum trabeum.     

A kinetic model describing Shewanella oneidensis MR-1 growth, substrate consumption, and product secretion

Aerobic growth of Shewanella oneidensis MR-1 in minimal lactate medium was studied in batch cultivation. Acetate production was observed in the middle of the exponential growth phase and was enhanced when the dissolved oxygen (DO) concentration was low. Once the lactate was nearly exhausted, S. oneidensis MR-1 used the acetate produced during growth on lactate with a similar biomass yield as lactate. A two-substrate Monod model, with competitive and uncompetitive substrate inhibition, was devised to describe the dependence of biomass growth on lactate, acetate, and oxygen and the acetate growth inhibition across a broad range of concentrations. The parameters estimated for this model indicate interesting growth kinetics: lactate is converted to acetate stoichiometrically regardless of the DO concentration; cells grow well even at low DO levels, presumably due to a very low K(m) for oxygen; cells metabolize acetate (maximum specific growth rate, micro(max,A) of 0.28 h(-1)) as a single carbon source slower than they metabolize lactate (micro(max,L) of 0.47 h(-1)); and growth on acetate is self-inhibiting at a concentration greater than 10 mM. After estimating model parameters to describe growth and metabolism under six different nutrient conditions, the model was able to successfully estimate growth, oxygen and lactate consumption, and acetate production and consumption under entirely different growth conditions.     

Production of biomass and nutraceutical compounds by Spirulina platensis under different temperature and nitrogen regimes

The cyanobacterium Spirulina platensis has been used by humans because of its nutritional and possibly medicinal effects. Our study evaluated the influence of temperature and nitrogen concentration in the medium on the production of biomass by this cyanobacterium and the biomass composition in protein, lipid and phenolic compounds. We found that at 35 degrees C there was a negative effect on biomass production but a positive effect on the production of protein, lipids and phenolics, the highest levels of these compounds being obtained in Zarrouk's medium containing 1.875 or 2.500 g l(-1) sodium nitrate. Higher biomass densities and productivity were obtained at 30 degrees C than at 35 degrees C, but nitrogen concentration appeared to have no effect on the amount of protein, lipid or phenolics, indicating that at 30 degrees C the concentration of sodium nitrate in Zarrouk's medium (2.50 g l(-1)) can be reduced without loss of productivity, an important cost-saving factor in large-scale cultivation.