Production of tylosin and nikkomycin by immobilized Streptomyces cells

Summary Mycelia of Streptomyces sp. T 59-235 and Streptomyces tendae Tü 901 (producing the antibiotics tylosin and nikkomycin, resp.) were immobilized in different carriers. With both organisms best antibiotic production was observed in calcium alginate gel.Influence of aeration, cell density and flow rate on antibiotic production was investigated in batch fermentation and in a continuous system (air-bubbled reactor).In batch fermentation, immobilization prolongued the production phase from 72 to 120 h (Streptomyces T 59-235) and from 72 to 96 h (S. tendae). The relative productivity of immobilized cells was 40 to 50% compared to that of free mycelia in both cases.In continuous tylosin fermentation highest production rate was observed in a medium nearly saturated with oxygen.Nikkomycin production by immobilized S. tendae could be maintained for longer than 350 h in a continuous system. The production rate depended on cell density and flow rate of the medium. The maximum specific productivity was 100% compared to that of free mycelium in batch culture.     

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     

Nikkomycin quantitation in fermentation process by high-performance liquid chromatography

Summary Nikkomycins are nucleoside-peptide antibiotics, produced by Streptomyces tendae. In contrast to any biological evaluation, high-performance liquid chromatography provided the quantitation of five nikkomycin components from the culture filtrate of the fermentation broth.     

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     

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     

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 cultivation of Streptomyces tendae in different media

Summary The strain Streptomyces tendae is well suited for continuous cultivation because of its ability to grow and produce secondary metabolites simultaneously. Continuous culture experiments on defined medium show that growth is limited by nitrogen during steady state for the given medium composition. It is supposed that this also holds for complex medium. Production of antibiotics (several nikkomycins) occurs simultaneously with exponential growth. After switching from batch to continuous operation the fraction of biomass, consisting of pellets, decreases permanently.     

Development of an immobilized cell reactor for the production of 1,3-propanediol by Citrobacter freundii

Citrobacter freundii DSM 30040 immobilized on modified polyurethane carrier particles PUR 90/16 was used for continuous glycerol fermentation in an anaerobic fixed bed reactor with effluent recycle and pH control (fixed bed loop reactor). The fermentor was run with buffered mineral medium under growth conditions resulting in the permanent renewal of active biomass. The effects of glycerol concentration in the feed, dilution rate (D), pH and temperature (T) were investigated to optimize the process. With 400 mm glycerol in the feed, pH 6.9, T = 36°C and D = 0.5 h^–1 the maximum productivity could be determined as 8.2 g/l per hour of 1,3-propanediol.     

In-situ recovery of butanol during fermentation

End-product inhibition in the acetone-butanol fermentation was reduced by using extractive fermentation to continuously remove acetone and butanol from the fermentation broth. In situ removal of inhibitory products from Clostridium acetobutylicum resulted in increased reactor productivity; volumetric butanol productivity increased from 0.58 kg/(m³h) in batch fermentation to 1.5 kg/(m³h) in fed-batch extractive fermentation using oleyl alcohol as the extraction solvent. The use of fed-batch operation allowed glucose solutions of up to 500 kg/m³ to be fermented, resulting in a 3.5- to 5-fold decrease in waste water volume. Butanol reached a concentration of 30–35 kg/m³ in the oleyl alcohol extractant at the end of fermentation, a concentration that is 2–3 times higher than is possible in regular batch or fed-batch fermentation. Butanol productivities and glucose conversions in fed-batch extractive fermentation compare favorable with continuous fermentation and in situ product removal fermentations.List of Symbols C _g kg/m³ concentration of glucose in the feed - C _w dm³/m³ concentration of water in the feed - F(t) cm³/h flowrate of feed to the fermentor at time t - V(t) dm³ broth volume at time t - V _i dm³ initial broth volume - V _si dm³ volume of the i-th aqueous phase sample - effective fraction of water in the feed Part 1. Bioprocess Engineering 2 (1987) 1–12     

Limitation of growth and lactic acid production in batch and continuous cultures of Lactobacillus helveticus

Summary Continuous and batch cultures of Lactobacillus helveticus operated under different conditions were studied with respect to the limitation of growth and lactic acid production by increasing undissociated lactic acid and hydrogen ion concentrations, respectively. In a single-stage continuous culture without pH control a final pH of 3.8 and 65 mm undissociated lactic acid was obtained. In two-stage continuous cultures provided with different growth media and run at different pH values, 65–70 mm free acid was obtained in the second stage. Further batch-culture experiments showed growth limitation at 60–70 mm lactic acid. After growth ceased, production of lactate continued until a lactic acid concentration of about 100 mm was reached; obviously an uncoupling of growth and acid production had occurred. Examining the effect of different concentrations of either lactic acid or hydrochloric acid, added to growing batch cultures of L. helveticus, it was shown that the undissociated lactic acid concentration was responsible for growth limitation and lactic acid production in this organism, whereas the pH value had only an indirect effect.     

Molecular characterization of co-transcribed genes from Streptomyces tendae Tü901 involved in the biosynthesis of the peptidyl moiety of the peptidyl nucleoside antibiotic nikkomycin

Six genes (nikA, nikB, nikD, nikE, nikF, and nikG) from Streptomyces tendae Tü901 were identified by sequencing the region surrounding the nikC gene, which encodes L-lysine 2-aminotransferase, previously shown to catalyze the initial reaction in the biosynthesis of hydroxypyridylhomothreonine, the peptidyl moiety of the peptidyl nucleoside antibiotic nikkomycin. These genes, together with the nikC gene, span a DNA region of 7.87 kb and are transcribed as a polycistronic mRNA in a growth-phase–dependent manner. The sequences of the deduced proteins NikA and NikB exhibit significant similarity to those of acetaldehyde dehydrogenases and 4-hydroxy-2-oxovalerate aldolases, respectively, which are involved in meta-cleavage degradation of aromatic hydrocarbons. The predicted NikD gene product shows sequence similarity to monomeric sarcosine oxidases, and the deduced NikE protein belongs to the superfamily of adenylate-forming enzymes. The nikF gene and the nikG gene encode a cytochrome P450 monooxygenase and a ferredoxin, respectively. Disruption of any of the genes nikA, nikB, nikD, nikE and nikF by insertion of a kanamycin resistance cassette abolished formation of the biologically active nikkomycins I, J, X, and Z. The nikA, nikB, nikD, and nikE mutants accumulated the nucleoside moieties nikkomycins C_x and C_z. In the nikD and nikE mutants nikkomycin production (nikkomycins I, J, X, Z) could be restored by feeding with picolinic acid and hydroxypyridylhomothreonine, respectively. The nikF mutant exclusively produced novel derivatives, nikkomycins L_x and L_z, which contain pyridylhomothreonine as the peptidyl moiety. Our results indicate that the nikA, nikB, nikD, nikE, nikF, and nikG genes, in addition to nikC, function in the biosynthetic pathway leading to hydroxypyridylhomothreonine; the putative activities of each of their products are discussed. Received: 1 February 1999 / Accepted: 29 April 1999     

Evaluation ofl-lactic acid production in batch, fed-batch, and continuous cultures ofRhizopus sp. MK-96-1196 using an airlift bioreactor

Various processes which producel-lactic acid using ammonia-tolerant mutant strain,Rhizopus sp. MK-96-1196, in a 3 L airlift bioreactor were evaluated. When the fed-batch culture was carried out by keeping the glucose concentration at 30 g/l, more than 140 g/l ofl-lactic acid was produced with a product yield of 83%. In the case of the batch culture with 200 g/l of initial glucose concentration, 121 g/L ofl-lactic acid was obtained but the low product yield based on the amount of glucose consumed. In the case of a continuous culture, 1.5 g/l/h of the volumetric productivity with a product yield of 71% was achieved at dilution rate of 0.024 h⁻¹. Basis on these results three processes were evaluated by simple variable cost estimation including carbon source, steam, and waste treatment costs. The total variable costs of the fed-batch and continuous cultures were 88% and 140%, respectively, compared to that of batch culture. The fed-batch culture with highl-lactic acid concentration and high product yield decreased variable costs, and was the best-suited for the industrial production ofl-lactic acid.     

Continuous <Emphasis Type="SmallCaps">d</Emphasis>-lactic acid production by a novelthermotolerant <Emphasis Type="Italic">Lactobacillus delbrueckii</Emphasis> subsp. <Emphasis Type="Italic">lactis</Emphasis> QU 41

We isolated and characterized a d-lactic acid-producing lactic acid bacterium (d-LAB), identified as Lactobacillus delbrueckii subsp. lactis QU 41. When compared to Lactobacillus coryniformis subsp. torquens JCM 1166 ^T and L. delbrueckii subsp. lactis JCM 1248 ^T, which are also known as d-LAB, the QU 41 strain exhibited a high thermotolerance and produced d-lactic acid at temperatures of 50 °C and higher. In order to optimize the culture conditions of the QU 41 strain, we examined the effects of pH control, temperature, neutralizing reagent, and initial glucose concentration on d-lactic acid production in batch cultures. It was found that the optimal production of 20.1 g/l d-lactic acid was acquired with high optical purity (>99.9% of d-lactic acid) in a pH 6.0-controlled batch culture, by adding ammonium hydroxide as a neutralizing reagent, at 43 °C in MRS medium containing 20 g/l glucose. As a result of product inhibition and low cell density, continuous cultures were investigated using a microfiltration membrane module to recycle flow-through cells in order to improve d-lactic acid productivity. At a dilution rate of 0.87 h⁻¹, the high cell density continuous culture exhibited the highest d-lactic acid productivity of 18.0 g/l/h with a high yield (ca. 1.0 g/g consumed glucose) and a low residual glucose (<0.1 g/l) in comparison with systems published to date.     

Identification of cellular proteins involved in nikkomycin production in Steptomyces tendae Tü901

Expression of genes involved in nikkomycin production in Streptomyces tendae was investigated by two-dimensional gel electrophoresis of cellular proteins. Ten gene products (P1–P10) were identified that were synthesized when nikkomycin was produced; these proteins were not detected in non-producing mutants. N-terminal sequences of six of the 10 proteins were obtained by microsequencing of protein spots excised from preparative two-dimensional gels. Protein P8 was identified as l -histidine amino-transferase (HisAT), which has been previously correlated with nikkomycin production. By using oligo-nucleotide probes deduced from the N-terminal sequences of protein P2 and P6, we isolated an 8 kb Bam HI fragment and a 6.5 kb Pvu II fragment, respectively, from the genome of Streptomyces tendae Tü901. Restriction analyses revealed that both fragments overlapped within a region of 1.5 kb. Mapping of the oligonucleotide probe hybridizing sites indicated that the genes encoding protein P2 and P6 are closely spaced on the 8 kb Bam HI fragment, and the latter is located on the overlapping region. DNA sequence analysis revealed that proteins P1 and P2 are encoded by a single gene, orfP1, that is translated at two initiation codons. The orfP1 gene was interrupted by homologous recombination using the integrating vector pWHM3. The gene-disrupted transformants did not produce nikkomycin, indicating that proteins P1 and P2 are essential for nikkomycin production. The data presented show that reverse genetics was successfully used to isolate genes Involved in nikkomycin production.     

Production of <Emphasis Type="SmallCaps">l</Emphasis>-lactic acid by <Emphasis Type="Italic">Rhizopus oryzae</Emphasis> using semicontinuous fermentation in bioreactor

Semicontinuous fermentation using pellets of Rhizopus oryzae has been recognized as a promising technology for l-lactic acid production. In this work, semicontinuous fermentation of R. oryzae AS 3.819 for l-lactic acid production has been developed with high l-lactic acid yield and volumetric productivity. The effects of factors such as inoculations, CaCO₃ addition time, and temperature on l-lactic acid yield and R. oryzae morphology were researched in detail. The results showed that optimal fermentation conditions for the first cycle were: inoculation with 4% spore suspension, CaCO₃ added to the culture medium at the beginning of culture, and culture temperature of 32–34°C. In orthogonal experiments, high l-lactic acid yield was achieved when the feeding medium was (g/l): glucose, 100; (NH₄)₂SO₄, 2; KH₂PO₄, 0.1; ZnSO₄·7H₂O, 0.33; MgSO₄·7H₂O, 0.15; CaCO₃, 50. Twenty cycles of semicontinuous fermentation were carried out in flask culture. l-lactic acid yield was 78.75% for the first cycle and 80–90% for the repeated cycles; the activities of lactate dehydrogenases (LDH) were 7.2–9.2 U/mg; fermentation was completed in 24 h for each repeated cycle. In a 7-l magnetically stirred fermentor, semicontinuous fermentation lasted for 25 cycles using pellets of R. oryzae AS 3.819 under the optimal conditions determined from flask cultures. The final l-lactic acid concentration (LLAC) reached 103.7 g/l, and the volumetric productivity was 2.16 g/(l·h) for the first cycle; in the following 19 repeated cycles, the final LLAC reached 81–95 g/l, and the volumetric productivities were 3.40–3.85 g/(l·h).     

Continuous 2-keto-<Emphasis Type="SmallCaps">l</Emphasis>-gulonic acid fermentation from <Emphasis Type="SmallCaps">l</Emphasis>-sorbose by <Emphasis Type="Italic">Ketogulonigenium vulgare</Emphasis> DSM 4025

A single-stage continuous fermentation process for the production of 2-keto-l-gulonic acid (2KGA) from l-sorbose using Ketogulonigenium vulgare DSM 4025 was developed. The chemostat culture with the dilution rate that was calculated based on the relationship between the 2KGA production rate and the 2KGA concentration was feasible for production with high concentration of 2KGA. In this system, 112.2 g/L of 2KGA on the average was continuously produced from 114 g/L of l-sorbose. A steady state of the fermentation was maintained for the duration of more than 110 h. The dilution rate was kept in the range of 0.035 and 0.043 h⁻¹, and the 2KGA productivity was 3.90 to 4.80 g/L/h. The average molar conversion yield of 2KGA from l-sorbose was 91.3%. Under the optimal conditions, l-sorbose concentration was kept at 0 g/L. Meanwhile, the dissolved oxygen level was changing in response to the dilution rate and 2KGA concentration. In the dissolved oxygen (DO) range of 16% to 58%, it was revealed that the relationship between DO and D possessed high degree of positive correlation under the l-sorbose limiting condition (complete consumption of l-sorbose). Increasing D closer to the critical value for washing out point of the continuous fermentation, DO value tended to be gradually increased up to 58%. In conclusion, an efficient and reproducible continuous fermentation process for 2KGA production by K. vulgare DSM 4025 could be developed using a medium containing baker’s yeast without using a second helper microorganism.     

Potential of solid state fermentation for production of L(+)-lactic acid by Rhizopus oryzae

Production of l(+)-lactic acid by Rhizopus oryzae NRRL 395 was studied in solid medium on sugar-cane bagasse impregnated with a nutrient solution containing glucose and CaCO₃. A comparative study was undertaken in submerged and solid-state cultures. The optimal concentrations in glucose were 120 g/l in liquid culture and 180 g/l in solid-state fermentation corresponding to production of l(+)-lactic acid of 93.8 and 137.0 g/l, respectively. The productivity was 1.38 g/l per hour in liquid medium and 1.43 g/l per hour in solid medium. However, the fermentation yield was about 77% whatever the medium. These figures are significant for l(+)-lactic acid production.     

Molecular characterization of co-transcribed genes from Streptomyces tendae Tü901 involved in the biosynthesis of the peptidyl moiety of the peptidyl nucleoside antibiotic nikkomycin

Six genes (nikA, nikB, nikD, nikE, nikF, and nikG) from Streptomyces tendae Tü901 were identified by sequencing the region surrounding the nikC gene, which encodes L-lysine 2-aminotransferase, previously shown to catalyze the initial reaction in the biosynthesis of hydroxypyridylhomothreonine, the peptidyl moiety of the peptidyl nucleoside antibiotic nikkomycin. These genes, together with the nikC gene, span a DNA region of 7.87?kb and are transcribed as a polycistronic mRNA in a growth-phase–dependent manner. The sequences of the deduced proteins NikA and NikB exhibit significant similarity to those of acetaldehyde dehydrogenases and 4-hydroxy-2-oxovalerate aldolases, respectively, which are involved in meta-cleavage degradation of aromatic hydrocarbons. The predicted NikD gene product shows sequence similarity to monomeric sarcosine oxidases, and the deduced NikE protein belongs to the superfamily of adenylate-forming enzymes. The nikF gene and the nikG gene encode a cytochrome P450 monooxygenase and a ferredoxin, respectively. Disruption of any of the genes nikA, nikB, nikD, nikE and nikF by insertion of a kanamycin resistance cassette abolished formation of the biologically active nikkomycins I, J, X, and Z. The nikA, nikB, nikD, and nikE mutants accumulated the nucleoside moieties nikkomycins C_x and C_z. In the nikD and nikE mutants nikkomycin production (nikkomycins I, J, X, Z) could be restored by feeding with picolinic acid and hydroxypyridylhomothreonine, respectively. The nikF mutant exclusively produced novel derivatives, nikkomycins L_x and L_z, which contain pyridylhomothreonine as the peptidyl moiety. Our results indicate that the nikA, nikB, nikD, nikE, nikF, and nikG genes, in addition to nikC, function in the biosynthetic pathway leading to hydroxypyridylhomothreonine; the putative activities of each of their products are discussed.     

Metabolic products of microorganisms

The effect of the nucleoside-peptide antibiotics nikkomycin Z, nikkomycin X, and polyoxin A was tested on chitosomal chitin synthetase from yeast cells of the dimorphic fungus Mucor rouxii. The K _i was 0.6 M for polyoxin A and 0.5 M for nikkomycin X; nikkomycin Z was slightly less inhibitory (K _i=3.5M). Whereas the minimum inhibitory concentrations of the nikkomycins for growth and germination were quite low (about 1M, or lower), polyoxin A displayed no antifungal activity against yeast cells and sporangiospores of the test organism, even when present in high concentrations. These results are discussed with respect to structure/activity relationships.Abbreviations MIC minimum inhibitory concentration (i.e. concentration required to completely suppress growth: cf. Drews, 1979) - GlcNAc N-acetyl-d-glucosamine - UDP-GlcNAc uridine 5-diphospho-N-acetyl-d-glucosamine Metabolic products of microorganisms. 202. H. P. Kaiser and W. Keller-Schierlein: Strukturaufklärung von Elaiophylin: Spektroskopische Untersuchungen und Abbau. Helv. Chim. Acta 64: 407–424 (1981)     

Biological acidification during grape must fermentation using mixed cultures of <Emphasis Type="Italic">Kluyveromyces thermotolerans</Emphasis> and <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Four mixed culture fermentations of grape must were carried out with Kluyveromyces thermotolerans strain TH941 and Saccharomyces cerevisiae strain SCM952. In the first culture, both yeasts were added together, whereas in the remaining three cultures S. cerevisiae was added 1, 2, and 3 days after the inoculation of K. thermotolerans. The growth and survival of the K. thermotolerans strain and the amount of the produced l-lactic acid depend on the time of inoculation of the S. cerevisiae strain and provided an effective acidification during alcoholic fermentation. The four cultures contained, respectively, at the end of fermentation 0.18, 1.80, 4.28, and 5.13 g l-lactic acid l⁻¹. The grape must with an initial pH of 3.50 was effectively acidified (70% increase in titratable acidity, 0.30 pH unit decrease) by the production of 5.13 g l-lactic acid l⁻¹.