Nutritional control of nikkomycin and juglomycin production by Streptomyces tendae in continuous culture

Summary Continuous cultures with Streptomyces tendae revealed some interesting facts. In a continuous culture running for more than 2500 h the production of either nikkomycins or juglomycins could be selected by varying the feed composition. Decreasing the phosphate supply in the feed broth from the initial concentration of 2.5 mm to 1.0 mm enhanced the productivity of nikkomycins and decreased the productivity of juglomycins. When switching back to the initial conditions of the experiment after 2000 h nearly the same production behaviour as at the beginning of the fermentation could be observed. This indicated a stable behaviour of the population with regard to nikkomycin productivity. The long continuous fermentation showed the ability of S. tendae Tü 901/8c to produce nikkomycin at a high level for at least 1500 h. In a second continuous culture it was shown that the productivity of the nikkomycins and juglomycins decreased and increased, respectively, with increasing dilution rate. Comparing batch cultures with continuous fermentations, higher juglomycin productivity was found in the latter. These facts indicate that the strain responds to complex interacting physiological controls, by producing either nikkomycins or juglomycins in a higher amount. Offprint requests to: D. Hege-Treskatis     

Production of nikkomycin by immobilized Streptomyces cells — Physiological properties

Summary The antibiotic nikkomycin can be produced by calcium alginate immobilized mycelium of Streptomyces tendae Tü 901 in batch and continuous culture.Scanning electron micrographs show the porous structure of the matrix and the distribution of the cells in the gel.Some physiological properties of free and immobilized mycelia were compared. Immobilization does not change the relative amounts of nikkomycin compounds in the culture broth. DNA and protein content were the same in free and immobilized cells. The specific activity of fructosediphosphate aldolase dropped during fermentation and was lower for entrapped than for free cells. The specific activity of mannitol dehydrogenase increased up to the end of the fermentation and was the same for free and immobilized mycelium.In continuous culture the relative amount of mannitol consumed decreased with increasing flow rate. When the medium was supplemented with amino acids mannitol consumption increased significantly.Dedicated to Prof. Dr. L. Acker on the occasion of his 70th birthday     

Enhanced continuous production of lovastatin using pellets and siran supported growth of Aspergillus terreus in an airlift reactor

Lovastatin, a hypocholesterolemic agent, is a secondary metabolite produced by filamentous microorganism Aspergillus terreus in submerged batch cultivation. Lovastatin production by pellets and immobilized siran cells was investigated in an airlift reactor. The process was carried out by submerged cultivation in continuous mode with the objective of increasing productivity using pellet and siran supported growth of A terreus. The continuous mode of fermentation improves the rate of lovastatin production. The effect of dilution rate and aeration rate were studied in continuous culture. The optimum dilution rate for pellet was 0.02 h⁻¹ and for siran carrier was 0.025 h⁻¹. Lovastatin productivity using immobilized siran carrier (0.0255 g/L/h) was found to be greater than pellets (0.022 g/L/h). The productivity by both modes of fermentation was found higher than that of batch process which suggests that continuous cultivation is a promising strategy for lovastatin production.     

Effects of oxygen supply on the production of nikkomycin with immobilized cells of Streptomyces tendae

Summary For continuous production of the antibiotic nikkomycin immobilized cells have been used in a fluidized bed bioreactor. Cells of Streptomyces tendae were immobilized on sintered glass particles. Different biomass concentrations on the particles correspond to different thicknesses of mycelial layers because growth occurs only on the outer surface of the particles. The antibiotic productivity decreased with increasing layer thickness. In fermentations with higher concentrations of both biomass on the particles and dissolved oxygen levels of about 70% the productivity was also limited because of limited oxygen diffusion in the layers. Offprint requests to: H. U. Trück     

Chlortetracycline production with immobilized Streptomyces aureofaciens

Summary The semicontinuous production of chlortetracycline by immobilized cells of Streptomyces aureofaciens ATCC 10762 was compared with that of free cells. Immobilized cells transferred repeatedly to a new production medium, showed a fourfold increase in the half life time of antibiotic production.In an air bubble column a high chlortetracycline productivity was obtained with a high aeration rate.A semicontinuous production of chlortetracycline by immobilized S. aureofaciens could be improved by varying the fermentation conditions.For continuous chlortetracycline production by immobilized cells, no improvement was detected.     

Formation of secondary metabolism enzymes in the tylosin producerStreptomyces T59-235

Production of the macrolide antibiotic tylosin byStreptomyces T59-235 was inhibited in cultures containing high phosphate concentrations (30 mM P_i). Vegetative growth (dry weight increase, DNA and RNA synthesis) was hardly affected. Tylosin production began when macromolecule synthesis had slowed down to minimum level; in cultures with 30 mM P_i the onset of antibiotic production was retarded compared to cultures with low phosphate concentration (5 mM). The activities of three enzyme systems involved in tylosin biosynthesis (dTDP-D-glucose-4,6-dehydratase; dTDP-mycarose-forming enzyme system; SAM: macrocin-O-methyl transferase) were measured and found to be significantly lower in cultures with 30 mM P_i than in low phosphate cultures.Chloramphenicol, but not rifampicin, caused a rapid decrease of both tylosin formation rate and dTDP-D-glucose-4,6-dehydratase activity when added to tylosin producing cultures.Abbreviations P_i inorganic phosphate - dTDB 2-deoxythymidine diphosphate - SAM S-adenosyl-L-methionine - LP low phosphate (5 mM) - HP high phosphate (30 mM)     

Regulation of tylosin synthesis in Streptomyces: Effects of glucose analogs and inorganic phosphate

Summary Glucose, 2-deoxy glucose and inorganic phosphate inhibited tylosin production and fatty acid oxidation in Streptomyces T 59–235. Glucose-6-phosphate was accumulated in high-phosphate cultures. The possible function of glucose phosphate as a common mediator of both glucose and phosphate effects is discussed.     

Production of pullulanase by free and immobilized cells of <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> NRC22 in batch and continuous cultures

The production of extracellular pullulanase by Bacillus licheniformis NRC22 was investigated using different fermentation modes. In batch culture maximal enzyme activity of 18 U/ml was obtained after 24 h of growth. In continuous fermentation by the free cells, maximal reactor productivity (4.15 KU/l/h) with enzyme concentration of 14.8 U/ml and specific productivity of 334.9 U/g wet cells/h was attained at a dilution rate of 0.28/h, over a period of 25 days. B. licheniformis NRC22 cells were immobilized on Ca-alginate. The immobilization conditions with respect to matrix concentration and cell load was optimized for maximal enzyme production. In repeated batch operation, the activity of the immobilized cells was stable during the 10 cycles and the activity remained between 9.8 and 7.7 U/ml. Continuous production of pullulanase by the immobilized cells was investigated in a packed–bed reactor. Maximal reactor productivity (7.0 KU/h) with enzyme concentration of 16.8 U/ml and specific productivity of 131.64 U/g wet cells/h was attained at dilution rate of 0.42/h. The enzyme activity in the effluent started to decline gradually to the level of 8.7 U/ml after 25 days of the operation.     

Continuous neomycin production by immobilized cells of Streptomyces marinensis NUV-5 in an airlift bioreactor

Neomycin production by free and calcium alginate immobilized cells was investigated in an airlift reactor. The average volumetric productivity with continuous fermentation (72.97 mg/l/h) was greater than with free cells (45.05 mg/l/h). The total neomycin produced with continuous fermentation was 62% greater than with that of free cells. Immobilized Streptomyces particles showed a half-life of 42 days during continuous fermentation under airlift conditions.     

A study of N- and P-dependence of nikkomycin production in continuous culture with immobilized cells

Summary The influence of nitrogen and phosphate on the biosynthesis of nikkomycin was studied in chemically defined medium. Cells of Streptomyces tendae were immobilized on porous glass particles in a fluidized-bed reactor for continuous production of nikkomycin. Phosphate had no significant influence on the biosynthesis of nikkomycin. However, even a very low concentration of phosphate in the production medium (00.0125 mmol/l) resulted in microbial growth on the particles. The concentration of nitrogen was highly effective in the regulation of the biosynthesis of nikkomycin. A high level of antibiotic production (maximum 3.05 mg/g dry cell weight per hour) was maintained for a period of about 200 h in a medium that contained nitrogen at a concentration of 0.2 g NH₄NO₃/l. Offprint requests to: H. U. Trück     

Continuous alpha-amylase production using Bacillus amyloliquefaciens adsorbed on an ion exchange resin

Summary A method for the continuous production of extracellular alpha amylase by surface immobilized cells of Bacillus amyloliquefaciens NRC 2147 has been developed. A large-pore, macroreticular anionic exchange resin was capable of initially immobilizing an effective cell concentration of 17.5 g DW/1 (based on a total reactor volume of 160 ml). The reactor was operated continuously with a nutrient medium containing 15 g/l soluble starch, as well as yeast extract and salts. Aeration was achieved by sparging oxygen enriched air into the column inlet. Fermentor plugging by cells was avoided by periodically substituting the nutrient medium with medium lacking in both soluble starch and yeast extract. This fermentor was operated for over 200 h and obtained a steady state enzyme concentration of 18700 amylase activity units per litre (18.7 kU/l), and an enzyme volumetric productivity of 9700 amylase activity units per litre per hour (9.7 kU/l-h). Parallel fermentations were performed using a 2 l stirred vessel fermentor capable of operation in batch and continuous mode. All fermentation conditions employed were identical to those of the immobilized cell experiments in order to assess the performance of the immobilized cell reactor. Batch stirred tank operation yielded a maximum amylase activity of 150 kU/l and a volumetric productivity of 2.45 kU/l-h. The maximum cell concentration obtained was 5.85 g DW/l. Continuous stirred tank fermentation obtained a maximum effluent amylase activity of 6.9 kU/l and a maximum enzyme volumetric productivity of 2.73 kU/l-h. Both of these maximum values were observed at a dilution rate of 0.345 l/h. The immobilized cell reactor was observed to achieve larger volumetric productivities than either mode of stirred tank fermentation, but achieved an enzyme activity concentration lower than that of the batch stirred tank fermentor.     

Process and technoeconomics of ethanol production by immobilized cells

Summary A two-stage fermentation process has been developed for continuous ethanol production by immobilized cells of Zymomonas mobilis. About 90–92 kg/m³ ethanol was produced after 4 h of residence time. Entrapped cells of Zymomonas mobilis have a capability to convert glucose to ethanol at 93% of the theoretical yield. The immobilized cell system has functioned for several weeks, and experience indicates that the carrageenan gel apparently facilitates easy diffusion of glucose and ethanol.The simplicity and the high productivity of the plug-flow reactor employing immobilized cells makes it economically attrative. An evaluation of process economics of an immobilized cell system indicates that at least 4 c/l of ethanol can be saved using the immobilized cell system rather than the conventional batch system. The high productivity achieved in the immobilized cell reactor results in the requirement for only small reactor vessels indicating low capital cost. Consequently, by switching from batch to immobilized processing, the fixed capital investment is substantially reduced, thus increasing the profitability of ethanol production by fermentation.     

Semicontinuous production of red pigment by immobilized cells of<Emphasis Type="Italic">Bacillus</Emphasis> sp. BH-99 using column bioreactor

The semicontinuous production of red pigment by immobilized cells ofBacillus sp. BH-99 was investigated in comparison with free cells. The red pigment produced highest productivity under the conditions of aeration of 0.2 mL/min and 2 mm diameter of gel beads by using 3.0% sodium alginate. Semicontinuous production by immobilized cells showed the highest productivity with replacement of fresh production medium in every 72 h for fourth fermentation cycle following the conditions of red pigment productivity.     

Investigations on neomycin production with immobilized cells of<Emphasis Type="Italic">Streptomyces marinensis</Emphasis> Nuv-5 in calcium alginate matrix

The purpose of this investigation was to study the effect ofStreptomyces marinensis NUV-5 cells immobilized in calcium alginate for the production of neomycin. The effect of various parameters, such as the effect of alginate concentration (1%, 2%, 3%, 4%, and 5% wt/vol), the effect of cation (caCl₂, BaCl₂, and SrCl₂), the concentration of cation (0.01M, 0.125M, 0.25M, 0.375M, and 0.5M), the curing times (1, 6, 11, 16, and 21 hours), and the diameter of the bead (1.48, 2.16, 3.24, 4.46, and 5.44 mm), on neomycin production and bead stability were studied. The effect of maltose (4%, 3%, 2%, and 1% wt/vol) and sodium glutamate (0.6%, 0.3%, 0.15%, and 0.075%) wt/vol) concentration on neomycin production was also studied. Better neomycin production was achieved with optimized parameters, such as alginate at 2% wt/vol, 0.25M CaCl₂, 1-hour curing time, and 3.24 mm bead diameter. Effective neomycin production was achieved with 3% wt/vol maltose and 0.6% wt/vol sodium glutamate concentration. The repeated batch fermentations were conducted (every 96 hours) using the optimized alginate beads, employing the production medium with 3% wt/vol maltose and 0.6% wt/vol sodium glutamate along with minerals salts solution. The increase in antibiotic production was observed up to the 5th cycle, and later gradual decrease in antibiotic production was observed. Comparison of the total antibiotic production with free cells and immobilized cells was also done. An enhanced antibiotic productivity of 32% was achieved with immobilized cells over the conventional free-cell fermentation, while 108% more productivity was achieved over the washed free-cell fermentation. From these results it is concluded that the immobilized cells ofS marinensis NUV-5 in calcium alginate are more efficient for the production of neomycin with repeated batch fermentation.     

Continuous acetone-butanol-ethanol fermentation using SO2-ethanol-water spent liquor from spruce

SO₂–ethanol–water (SEW) spent liquor from spruce chips was successfully used for batch and continuous production of acetone, butanol and ethanol (ABE). Initially, batch experiments were performed using spent liquor to check the suitability for production of ABE. Maximum concentration of total ABE was found to be 8.79 g/l using 4-fold diluted SEW liquor supplemented with 35 g/l of glucose. The effect of dilution rate on solvent production, productivity and yield was studied in column reactor consisting of immobilized Clostridium acetobutylicum DSM 792 on wood pulp. Total solvent concentration of 12 g/l was obtained at a dilution rate of 0.21 h⁻¹. The maximum solvent productivity (4.86 g/l h) with yield of 0.27 g/g was obtained at dilution rate of 0.64 h⁻¹. Further, to increase the solvent yield, the unutilized sugars were subjected to batch fermentation.     

Optimized nikkomycin production by fed-batch and continuous fermentation

We performed fed-batch and continuous fermentations to extend the time of maximal nikkomycin production by Streptomyces tendae Tü 901/S 2566. This was achieved by the fed-batch culture technique. Furthermore, high productivity was obtained at slow growth rates in a continuous fermentation process. Different dilution rates with and without carbon limitation were done and the results were compared. Correspondence to : T. Schüz     

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.     

Itaconic acid production by immobilizedAspergillus terreus from xylose and glucose

Summary Aspergillus terreus NRRC 1960 spores were entrapped in calcium alginate gel beads or alternotely the fungal mycelium was immobilized either on Celite R-626 or in agar gel cubes, and the biocatalyst was employed both in repeated batch and in continuous column reactors to produce itaconic acid from D-xylose or D-glucose. The highest itaconic acid yield obtained in a submerged culture batch fermentation was 54.5% based on total initial glucose (55 g/l) with a volumetric productivity of 0.32 g/l h, and 44.8% from xylose (67 g/l) with a productivity of 0.20 g/l h. In a repeated batch fermentation mycelium immobilized in agar gel had a productivity of 0.112 g/l h, and mycelium grown from spores immobilized in calcium alginate gel 0.06 g/l h, both from xylose (60 g/l). With the best immobilized biocatalyst system used employing Celite R-626 as a carrier, volumetric productivities of 1.2 g/l h from glucose and 0.56 g/l h from xylose (both at 60 g/l) were obtained in continuous column operation for more than 2 weeks.     

Continuous production of bacitracin by immobilized living whole cells of Bacillus sp.

Whole cells of Bacillus sp., a bacitracin-producing bacteria, were immobilized in polyacrylamide gel. The continuous production of bacitracin by an immobilized whole-cell-containing air-bubbled reactor was examined with 0.5% peptone solution. The bacitracin productivity (28 units/ml/hr) obtained with this system was higher than that with a batch system. The effluent bacitracin concentration increased with increasing aeration rate and reached a steady-state maximum above the aeration rate of 3.0 liter/min. A high bacitracin productivity was retained for at least eight days when the gel was washed with sterilized saline at a flow rate of 250 ml/hr for 2 hr once a day. The half-life of the immobilized whole-cell system was about 10 days. Bacitracin productivity by the immobilized whole-cell reactor was higher than that by a conventional continuous fermentation process at high dilution rates.     

Batch and continuous ribonuclease production by immobilized Aspergillus clavatus cells in a bubble-collumn bioreactor

Summary Ribonuclease production using immobilized cells (IC) of Aspergillus clavatus has been studied under batch, repeated-batch and continuous fermentation conditions in a bubble-column bioreactor and compared with production by free cells, Immobilization was achieved by the method of cryostructurization in polyvinyl alcohol beads. The effect of various aeration rates in a column bioreactor has been investigated. Enzyme production by IC [42 000 units (U)·l^–1] during batch fermentations was comparable to that of a free-cell system. The specific productivity of IC was 8.5 times higher than that of free cells. In repeated batch fermentation at various aeration rates, successful reuse of IC was obtained, with comparable levels of enzyme production. Continuous ribonuclease production was achieved for 44 days at 1 vvm aeration and a dilution rate of 0.0 h^–1 with volumetric productivity (450 U·1^–1) and yield.