Production of the macrolide antibiotic tylosin in cyclic fed-batch culture

Tylosin-producing Streptomyces fradiae was cultured on a synthetic medium with a high glutamate-glucose ratio. Tylosin batch fermentations with this medium were characterized by a high initial specific production rate of tylosin (q(tylosin), mg/g h) that decreased as the fermentation progressed. Continuous feeding of glutamate, glucose, and methyloleate at a constant feed rate initiated during the period of high q(tylosin) had been shown to produce some increase in tylosin productivity. By using a cyclic feeding strategy, it was possible to increase tylosin productivity further. Tylosin fed-batch fermentations with glutamate and glucose being fed to the culture in cyclic square-wave profiles with methyloleate in excess showed several-fold increase in final q(tylosin) and tylosin titers. By varying cycle amplitudes and period of the substrates, it was found that maximum tylosin productivity occurred when the glutamate cycle amplitude was 600 mg/L and that of glucose was 42.5 mg/L per cycle period of 24 h. With these cycle amplitudes of glutamate and glucose, the tylosin cyclic fed-batch culture also showed high cellular uptake of methyloleate. Decreasing or increasing glucose cycle amplitude at fixed glutamate amplitude lowered tylosin production, and no further stimulation of tylosin synthesis was observed when alpha-ketoglutarate was supplemented to the cyclic substrate feeds. Under optimum cyclic conditions it was possible to maintain linear tylosin accretion and a constant value of q(tylosin) up to 240 h.     

Influence of ammonium on the biosynthesis of the macrolide antibiotic tylosin

The effect of ammonium ions on growth and tylosin biosynthesis in Streptomyces fradiae NRRL 2702 cultured on a chemically defined medium was studied. Mycelial growth and tylosin production were not affected when ammonium sulphate was added to idiophase cultures to a final concentration of 10 mm or 20 mm; however, when ammonium sulphate was added to tylosin cultures to a final concentration of 20 mm before the onset of antibiotic biosynthesis (trophophase), tylosin production was severely suppressed while mycelial growth was stimulated. The activities of propionyl-coenzyme A carboxylase (EC 6.4.1.3) and methylmalonyl-coenzyme A carboxyltransferase (EC 2.1.3.1), enzymes involved in the synthesis of tylonolide precursors, were depressed in high ammonium cultures. The activity of macrocin 3′-o-methyltransferase, which catalyses the methylation of macrocin to form tylosin, was also affected by high concentrations of ammonium ions added in the trophophase.     

Production of neutral and alkaline proteases in batch and chemostat cultures by bacillus mesentericus 76

The kinetics of the bacterial extracellular protease synthesis (neutral and alkaline protease of Bacillus mesentericusstrain 76, R-form) in batch and chemostat cultures under conditions of glucose limitation were investigated. When the medium was supplemented with casein the production of the proteases was significantly higher. Optimal dilution rates for obtaining of two proteases are fixed. The synthesis of both alkaline and neutral proteases is controlled by catabolite repression and induction.     

Heterotrophic growth of Thiobacillus acidophilus in batch and chemostat cultures

Heterotrophic growth of the facultatively chemolithoautotrophic acidophile Thiobacillus acidophilus was studied in batch cultures and in carbon-limited chemostat cultures. The spectrum of carbon sources supporting heterotrophic growth in batch cultures was limited to a number of sugars and some other simple organic compounds. In addition to ammonium salts and urea, a number of amino acids could be used as nitrogen sources. Pyruvate served as a sole source of carbon and energy in chemostat cultures, but not in batch cultures. Apparently the low residual concentrations in the steady-state chemostat cultures prevented substrate inhibition that already was observed at 150 M pyruvate. Molar growth yields of T. acidophilus in heterotrophic chemostat cultures were low. The Y _max and maintenance coefficient of T. acidophilus grown under glucose limitation were 69 g biomass · mol^–1 and 0.10 mmol · g^–1 · h^–1, respectively. Neither the Y _max nor the maintenance coefficient of glucose-limited chemostat cultures changed when the culture pH was increased from 3.0 to 4.3. This indicates that in T. acidophilus the maintenance of a large pH gradient is not a major energy-requiring process. Significant activities of ribulose-1,5-bisphosphate carboxylase were retained during heterotrophic growth on a variety of carbon sources, even under conditions of substrate excess. Also thiosulphate- and tetrathionate-oxidising activities were expressed under heterotrophic growth conditions.     

Physiological characterisation of a pyruvate-carboxylase-negative Saccharomyces cerevisiae mutant in batch and chemostat cultures

A prototrophic pyruvate-carboxylase-negative (Pyc-) mutant was constructed by deleting the PYC1 and PYC2 genes in a CEN.PK strain of Saccharomyces cerevisiae. Its maximum specific growth rate on ethanol was identical to that of the isogenic wild type but it was unable to grow in batch cultures in glucose-ammonia media. Consistent with earlier reports, growth on glucose could be restored by supplying aspartate as a sole nitrogen source. Ethanol could not replace aspartate as a source of oxaloacetate in batch cultures. To investigate whether alleviation of glucose repression allowed expression of alternative pathways for oxaloacetate synthesis, the Pyc- strain and an isogenic wild-type strain were grown in aerobic carbon-limited chemostat cultures at a dilution rate of 0.10 h-1 on mixtures of glucose and ethanol. In such mixed-substrate chemostat cultures of the Pyc- strain, steady-state growth could only be obtained when ethanol contributed 30% or more of the substrate carbon in the feed. Attempts to further decrease the ethanol content of the feed invariably resulted in washout. In Pyc- as well as in wild-type cultures, levels of isocitrate lyase, malate synthase and phospho-enol-pyruvate carboxykinase in cell extracts decreased with a decreasing ethanol content in the feed. Nevertheless, at the lowest ethanol fraction that supported growth of the Pyc- mutant, activities of the glyoxylate cycle enzymes in cell extracts were still sufficient to meet the requirement for C4-compounds in biomass synthesis. This suggests that factors other than glucose repression of alternative routes for oxaloacetate synthesis prevent growth of Pyc-mutants on glucose.     

Stimulation of tylosin productivity resulting from cyclic feeding profiles in fed batch cultures

Summary Batch fermentations for the production of the macrolide antiobiotic tylosin were carried out with a medium in which the maximum specific rate of tylosin synthesis (q_tylosin) occurred while the producer organism Streptomyces fradiae, was actively growing. With this medium, the value of q_tylosin decreased rapidly throughout the fermentation. By using a microprocessor to control the feeding of monosodium glutamate, and glucose in cyclic square wave profiles, it was possible to minimize the decrease in q_tylosin observed in the control and to maintain a linear volumetric accretion rate of tylosin resulting in a 114% increase over control in the 250 hour tylosin titre.     

Nutritional requirements of Brettanomyces bruxellensis: growth and physiology in batch and chemostat cultures

The nutritional requirements of Brettanomyces bruxellensis have been investigated. Batch culture and chemostat pulse techniques were used to identify growth-limiting nutrients. The study included determination of the essential components of the culture medium and quantification of the effects of the components. Among the components tested, ammonium sulfate and yeast extract had a significant effect on glucose consumption, growth, and ethanol production. However, if the ammonium sulfate concentration is above 2 g/L, an inhibitory effect on B. bruxellensis growth is observed. The yeast extract appears to be the most important and significant component for growth. The maximum amount of synthesized biomass is proportional to the concentration of yeast extract added to the culture broth (in the tested range). Magnesium and phosphate ions are probably not essential for B. bruxellensis. These ions appear to be supplied in sufficient amounts by the yeast extract in the culture medium. Brettanomyces bruxellensis appears to have very low nutritional requirements for growth.     

Oxygen transfer rate,respiration and yields in batch and chemostat cultures ofKlebsiella aerogenes

The effect was studied of oxygen supply on the changes in total and specific rate of oxygen consumption by the cells, oxygen transfer rate, saturation concentrations of dissolved oxygen and the yields of batch and continuous cultivations. Experiments were done on the microorganismKlebsiella aerogenes CCM 2318 growing on synthetic glucose medium. Continuous cultivations were carried out at dilution rates of 0.96 and 0.178 h⁻¹. The rate of oxygen transfer was determined by the sulphite method and the coefficient K_La was assessed using the dynamic method with a correction for changes in the saturations of dissolved oxygen. A lowered oxygen supply in batch cultivations caused deformations in the course of cell respiration. Comparison of results of batch and continuous cultivations showed that the highest yields Y_x/s and Y_x/o are attained at low dilution rates without oxygen limitation. Batch cultivations, on the other hand, exhibit the lowest yields and the highest cell respiration levels. In both types of cultivations, a respiration peak was ascertained under the conditions of growth limitation by oxygen.     

Stimulation of enzymes involved in tylosin biosynthesis by cyclic feeding profiles in fed batch cultures

Summary Previous reports have shown that feeding glucose and monosodium glutamate in a cyclic fashion to Streptomyces fradiae fermentations greatly stimulated the specific production rate for tylosin (q_tylosin) over that which could be obtained in fermentations where the nutrients were fed at a constant rate, all other conditions being the same. In this report it was shown that the activities of three of the enzymes involved in tylosin biosynthesis, propionyl-coenzyme A carboxylase (EC 6.4.1.3), methylmalonyl-coenzyme A carboxyltransferase (EC 2.1.3.1) and macrocin 3-0-methyl-transferase, were stimulated in the case of the cyclic fed-batch fermentation when compared with control levels i.e. the cyclic feed stimulated the activities of enzymes on the tylosin pathway as well as the q_tylosin values.     

Microcalorimetric investigations of the metabolism of yeasts. growth in batch and chemostat cultures on ethanol medium

Summary In the presence of protein, Hansenula polymorpha cultivation medium exhibits a maximum volumetric mass transfer coefficient, k_La, as function of the employed antifoam agents (soy oil and Desmophen 3600). With diminishing superficial gas velocity this maximum disappeas.Symbols E_G Relative gas holdup - k_La Volumetric mass transfer coefficient (s^–1) - w_SL Superficial liquid velocity (cm s^–1) - w_SG Superficial gas velocity (cm s^–1)     

Kinetics of biosynthesis of polyene macrolide antibiotics in batch cultures: Cell maturation time

The changes in several parameters of the candicidin fermentation (total, mycelium-associated, and extracellular product formation; growth rate; DNA content; glucose utilization; dissolved oxygen in the broth; and oxygen uptake rate) during the trophophase-idiophase transition are compared with previously reported data for the polyene macrolides, candidin and candihexin. The maturation time, t_m, and the productformation rate constant, k_p, have been calculated for each of the three polyene macrolides using a simple mathematical model. Slow-feeding of glucose, which resulted in candidin and candihexin overproduction has been shown to increase the polyene formation rate constant and toretard trophophase to idiophase transition (longer maturation time). The opposite effect is achieved by repeated feeding of soybean meal. The values of the maturation times and polyene formation rates obtained were used to predict the production of polyene macrolide antibiotics in batch cultures.     

Production and localization of beta-fructosidase in asynchronous and synchronous chemostat cultures of yeasts

In synchronized continuous cultures of Saccharomyces cerevisiae CBS 8066, the production of the extracellular invertase (EC 3.2.1.26) showed a cyclic behavior that coincided with the budding cycle. The invertase activity increased during bud development and ceased at bud maturation and cell scission. The cyclic changes in invertase production resulted in cyclic changes in amounts of invertase localized in the cell wall. However, the amount of enzyme invertase present in the culture liquid remained constant throughout the budding cycle. Also, in asynchronous continuous cultures of S. cerevisiae, the production and localization of invertase showed significant fluctuation. The overall invertase production in an asynchronous culture was two to three times higher than in synchronous cultures. This could be due to more-severe invertase-repressive conditions in a synchronous chemostat culture. Both the intracellular glucose-6-phosphate concentration and residual glucose concentration were significantly higher in synchronous chemostat cultures than in asynchronous chemostat cultures. In the asynchronous and synchronous continuous cultures of S. cerevisiae, about 40% of the invertase was released into the culture liquid; it has generally been believed that S. cerevisiae releases only about 5% of its invertase. In contrast to invertase production and localization in the chemostat cultures of S. cerevisiae, no significant changes in inulinase (EC 3.2.1.7) production and localization were observed in chemostat cultures of Kluyveromyces maxianus CBS 6556. In cultures of K. marxianus about 50% of the inulinase was present in the culture liquid.     

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.     

The role of hydrogen mass transfer for the growth kinetics of Methanobacterium thermoautotrophicum in batch and chemostat cultures

Hydrogen concentration was determined in batch and chemostat cultures of Methanobacterium thermoautotrophicum, both in the headspace and in the medium using mass spectrometry. The calculated dissolved hydrogen concentration in the medium as derived from the headspace hydrogen concentration when equilibrium conditions between gas and liquid phase were assumed, was ten times higher than the experimentally determined hydrogen concentration. Variation of the partial pressure of hydrogen resulted in different values for substrate affinity for hydrogen (K_s) and yield (Y) of the cells. Upon hydrogen limitation, K_s decreased while the yield coefficient for hydrogen increased, indicating a change in the affinity of the cells towards hydrogen. Received 15 November 1996/ Accepted in revised form 21 July 1997     

Stability and expression of a plasmid-containing killer toxin cDNA in batch and chemostat cultures of saccharomyces cerevisiae

A chimeric plasmid (pYT760-ADH1) containing the yeast killer toxin-immunity cDNA was transformed into a leucine-histidine mutant (AH22) and into four industrial toxin-sensitive yeasts. The chimeric plasmid was very stable and expressed toxin production (89.5 +/- 4.8% killer cells) in two of the transformed yeasts that contained the 2mu plasmid, but was lost within 10 generations from two other transformed pickle yeasts that did not contain the 2mu plasmid. It suggested that plasmid stability was dependent on the presence of the 2mu plasmid which is naturally present in some yeasts. The plasmid was extremely stable (100% killer cells) and expressed more toxin in the mutant strain AH22. The effects of dilution rate, D(h(-1)) on plasmid stability and toxin expression were studied in transformed AH22 (AH22/T3) and Montrachet 522 (522/T1) wine yeast grown in glucose-limited chemostat cultures. The results show that killer toxin production by AH22/T3 cells increased as a function of D(h(-1)) and that plasmid stability reached 100% at D >/= 0.09 +/- 0.01 h(-1). However, with Montrachet 522/T1 transformed cells, 100% plasmid stability was seen at D >/= 0.18 +/- 0.02. h(-1). We also challenged the AH22/T3 in chemostat culture (D = 0.25 h(-1)) with an equal number of untransformed cells (AH22). Transformed cells dominated the population (100%) within 8-10 h of growth, a time equivalent to two mean residence time.     

Kinetics of Streptomyces baarnensis growth and antibiotic synthesis in batch and dialysis cultures

The kinetics of biomass and antibiotic formation in batch and dialysis culture of Streptomyces baarnensis at various initial concentrations limiting the substrate growth (glucose) has been studied. The antibiotic substances were synthesized by actively growing culture, its concentration in the cultural media was maximum in the log-phase. In continuous dialysis culture on the background of biomass lianer growth in the course of time the constant antibiotic concentration in the media proportional to the glucose input concentration has been established. The inactivation (decomposition) of antibiotic was immediately initiated after discontinuation of substrate supply and followed first kinetics order. Observed features were used for construction of kinetical model of antibiotic biosynthesis. A conclusion has been made that the dialysis culture gives opportunity for more effective antibiotic synthesis as compared with the batch one.