Kinetics of thermal inactivation of oleandomycin in aqueous and native alkaline solutions

The process of oleandomycin inactivation in aqueous alkaline solutions with their heating was studied by using the microbiological method of the antibiotic content assay. The initial specific rate of inactivation of crystalline oleandomycin in buffer solutions and oleandomycin in the fermentation broth filtrate was evaluated. It was shown that the inactivation was retarded by the reaction products and the components of the fermentation broth filtrate. The production rate of oleandomycin anhydro derivatives amounting to 3-40 per cent of the total mass of the inactivation product was estimated by UV spectrophotometry.     

Effect of the products of the vital activity of yeast-like fungi on levorin synthesis

The effect on levorin synthesis of the cells and fermentation broth filtrates of Candida tropicalis after their cultivation in the fermentation medium was studied. It was found that the yeast-like fungi belonging to Candida excreted during their development some products capable of stimulating the synthesis of levorin by 40--60 per cent. When the actinomycete producing levorin was grown on the medium containing 80 per cent of the filtrate the level of levorin synthesis was the same as that observed with mixed cultivation of the actinomycete and C. tropicalis. The study on the conditions providing accumulation of the stimulating substances showed the following: production of the stimulating substances started during the first hours of the yeast growth and reached its maximum by the 48th hour, these substances being consumed by the actinomycete during the fermentation process. Aeration is required for production of the stimulating substances but its high levels are not necessary.     

The role of succinic acid in the biosynthesis of levorin

The influence of succinic acid as a component of media for biosynthesis of levorin, a polyenic antibiotic was studied. It was shown that with the use of the soybean-corn medium supplemented with succinic acid (0.05-0.4 per cent) the antibiotic content in the fermentation broth was higher than that in the control. The highest stimulating effect (135 per cent) was observed with addition of 0.1 per cent of succinic acid. For providing optimal antibiotic production in the synthetic medium supplemented with succinic acid (0.4 per cent) addition of acetic acid (0.05 per cent) was required. Studies with the soybean-corn medium with and without succinic acid revealed differences in the level of p-aminoacetophenone, an aromatic fragment of the levorin molecule. Under the conditions of the medium with succinic acid the content of p-aminoacetophenone in the mycelium was higher by 10 to 18 per cent as compared to that in the control and depended on the fermentation period. The role of succinic acid in biosynthesis of levorin is discussed.     

Effect of different pH values on the medium on the synthesis of antibiotics in the joint cultivation of Actinomyces levoris with yeasts]

Changes in the pH level of the fermentation medium used for preliminary cultivation of C. tropicalis were studied with respect to its initial aciditv or alkalinitv. When C. tropicalis was grown on the medium used for levorin fermentation with ph 5.1--10.3, the yeast changed it in 24 hours to the level of 6.2--7.9. As dependent on the initial pH values for cultivation of C. trophicalis, production of levorin on subsequent inoculation of Act. levoris changed. The antibiotic activity increased and ranged within 120--178% of the control. Synthesis of levoristatin, a non-polyenic antibiotic equally increased under such conditions and ranged within 153--163% of the control. The pH values of 9.4--10.3 of the initial fermentation medium were optimal for mixed cultivation of Act levoris and C. tropicalis and maxium production of levorin and levoristatin.     

Variability of the levorin producer, Actinomyces levoris Krass]

Natural variation of the levorin-producing organism Act. levoris, strain 28 was studied with respect to the colony morphology and production of levorin and levoristatin. The population of strain 28 consisted of 3 morphological colony types, the main type amounting to 99.7 percent. The strain variation with respect to production of levorin and levoristatin ranged from 20 to 180 and from 0 to 300 percent respectively as compared to the control. Mutant M-28 differing from the initial strain by the colony morphology, moderate phage titer and preferable production of levoristatin was isolated as a result of repeated passages of strain 28 onto agarized Chapek media with starch without maintaining selection. Variants differing from the population of strains 28 and M-28 by the ratio of levorin and levoristatin in the culture fluid were selected. No correlation in production of the above antibiotics by strain 28 was noted. Preparations obtained with strain M-28 differed from those obtained with strain 28 in a significant content of levoristatin.     

Action of the products of the vital activity of yeastlike fungi on the biosynthesis of levorin, levoristatin and fatty acids by a Streptomyces levoris culture

The data on the effect of the products of vital activity of Candida tropicalis, a yeast-like fungus, on the biosynthesis of levorin, levoristatin and fatty acids by Streptomyces levoris are presented. It was shown that the effect of the biostimulators was not specific with respect to production of levorin, since in the presence of the products of vital activity of C. tropicalis an increase in the synthesis of levoristatin and fatty acids was also observed. The qualitative and quantitative composition of the fatty acids of the mycelium of S. levoris was studied. Interrelation between the biosynthesis of levorin and synthesis of unsaturated fatty acids and branched chain fatty acids was noted.     

Selective action of novobiocin on Act. levoris, the producer of levorin and levoristatin]

The effect of sodium novobiocin on Act. levoris, strain LIA 0868 producing levorin and levoristatin was studied. High lethal effect of the antibiotic on Act. levoris was found. The effect increased with a rise in the antibiotic concentration in the agar from 2 to 6 lambda/ml. The inhibitory effect of novobiocin on Act, levoris was evident from a marked increase in the number of morphologically changed colonies with a low level of levorin production. The selective effect of novobiocin on the organism producing levorin and levoristatin was evident from selection of variants with high levels of levoristatin production on media containing novobiocin.     

Oleandomycin content of mycelium and fermentation solution during cultivation of Streptomyces antibioticus

The time-course of the oleandomycin content in the mycelium and fermentation broth-filtrate was studied by the microbiological assay at different periods of cultivation of strains 471 and 961 in fermenters and flasks containing a rich soybean-corn medium. It was shown that centrifugation of the mycelium over the sucrose density gradient induced a 25-80 per cent decrease in its moist weight at the expense of removal of the admixture components of the rich medium. Addition of glucose (2 per cent) to the culture-grown in a lactose medium by the 72nd hour of fermentation had no effect on further increase of the cell biomass. However, it lowered the content of the mycelium-fixed and excreted antibiotic at all the subsequent fermentation periods. The content of oleandomycin in the untreated mycelium was only 0.36 per cent of its content in the fermentation broth filtrate. After centrifugation of the mycelium over the sucrose density gradient and its intensive washing with distilled water the content of the mycelium-fixed antibiotic decreased still more. The time-course of the content of the mycelium-fixed and excreted oleandomycin was characterized by the presence of two activity peaks; by the 80-110th and by the 140-170th hour of cultivation.     

Selection and physiological study of culture of Bacillus circulans-- producer of butirosin]

For isolating a highly active variant of the butirosin-producing culture, a strain forming trace amounts of the antibiotic substance was used. Exposure to nitrosomethylbiuret and nitrosoguanidine and the use of selective media containing streptomycin and butirosin resulted in a 30-fold increase in the strain productivity. Thin layer chromatography of the produced antibiotic substance in the solvent system developed by the authors, mass spectrometry and assay of the antimicrobial spectrum in regard to ++gram-positive and ++gram-negative bacteria by using the known aminoglycosidine-inactivating enzymes revealed that the substance was identical to butirosin. Along with the major product, the fermentation broth contained up to 5 per cent of ribostamycin.     

Nature of the stimulating substances found in the vital activity products of yeastlike fungi

The cultural broth of Candida tropicalis was shown to contain an active compound which stimulated the synthesis of levorin, a polyene antibiotic Succinic acid was found to stimulate the antibiotic synthesis. The stimulating effect of the active compound increased in proportion to the content of succinic acid in it and became maximal at the same concentration of succinic acid as in a pure preparation. However, succinic acid was not an only compound responsible for the elevated synthesis of the antibiotic since the biostimulating effect was higher than that of pure succinic acid.     

In-situ recovery of butanol during fermentation

End product inhibition can be reduced by the in situ removal of inhibitory fermentation products as they form. Extractive fermentation, in which an immiscible organic solvent is added to the fermentor in order to extract inhibitory products, was applied to the acetone-butanol fermentation. Six solvents or solvent mixtures were tested in batch extractive fermentations: kerosene, 30 wt% tetradecanol in kerosene, 50 wt% dodecanol in kerosene, oleyl alcohol, 50 wt% oleyl alcohol in a decane fraction and 50 wt% oleyl alcohol in benzyl benzoate. The best results were obtained with oleyl alcohol or a mixture of oleyl alcohol and benzyl benzoate. In normal batch fermentation of Clostridium acetobutylicum, glucose consumption is limited to about 80 kg/m³ due to the accumulation of butanol in the broth. In extractive fermentation using oleyl alcohol or a mixture of oleyl alcohol and benzyl benzoate, over 100 kg/m³ of glucose can be fermented. Removal of butanol from the broth as it formed also increased the rate of butanol production. Maximum volumetric butanol productivity was increased by as much as 60% in extractive fermentation compared to batch fermentation. Butanol productivities obtained in extractive fermentation compare favorably with other in situ product removal fermentations.     

Distribution of oleandomycin between the native broth and butylacetate depending on the biosynthesis conditions]

Distribution of the active substance contained in the fermentation broth of Act. antibioticus between the acqueous phase and butylacetate depended on the fermentation conditions and the procedure for the fermentation broth treatment before filtration. Increase in pH values during the fermentation process resulted in lower antibiotic distribution coefficients which may be explained by the presence of oleandomycin-X, a biologically active substance in the fermentation broth filtrates. This substance differed from oleandomycin and did not pass into butylacetate from the acqueous alkaline solution. For transference of oleandomycin-X into oleandomycin exposition of the fermentation broth filtrate at pH 5.0--5.5 is required.     

Determining the conditions for stimulation of levorin biosynthesis in the presence of succinic acid

Conditions promoting the stimulating effect of succinic acid on biosynthesis of levorin were determined. The optimal concentration of succinate was shown to be equal to 0.05-0.3 per cent and the best time for the addition was before inoculation. The most pronounced stimulating effect was observed when the initial pH value of the fermentation medium was equal to 7.0. The important role of ammonium nitrogen in manifestation of the succinic acid stimulating effect on biosynthesis of levorin was suggested.     

Use of a polarographic method for determining trichothecin]

The polarographic behaviour of trichothecin was studied. It was shown that the antibiotic could be detected in solutions at concentrations of 7.10(-7) moles with the help of the polarographic method. Conditions for the polarographic determination of trichothecin in fermentation broth were developed. The error was not more than 3 per cent. The reliability of the results was shown by statistical treatment of data performed in accordance with the requirement of the USSR State Pharmacopeia, X ed., prescribing that the precision of the assay is such that the fiducial limits at p = 95 per cent deviate from the average value by not more than 5 per cent. Comparison of the results of trichothecin determination in the fermentation broth with the polarographic and biological methods showed no significant difference. Therefore, the polarographic method may be recommended for trichothecin determination in the fermentation broth.     

Selection of Penicillium chrysogenum--producer of penicillin with several valuable technologic features]

The efficiency of the routine methods for improvement of P. chrysogenum providing specific selection by several features with testing one of them was studied. A new highly potent strain of P. chrysogenum producing phenoxymethylpenicillin was isolated. The strain is characterized by a shorter fermentation cycle, lower viscosity of the fermentation broth and capacity for synthesizing 32,600 units/ml of phenoxymethylpenicillin under industrial conditions (in a lactose medium) by the 97th hour of the cultivation in fermenters with energy consumption of 1.3 kW. The increased amylolytic activity of the strain and the decreased viscosity of the fermentation broth provided using of the fermentation media containing 3.5 per cent of corn meal.     

Extractive fermentation for butyric acid production from glucose by Clostridium tyrobutyricum

A novel extractive fermentation for butyric acid production from glucose, using immobilized cells of Clostridium tyrobutyricum in a fibrous bed bioreactor, was developed by using 10% (v/v) Alamine 336 in oleyl alcohol as the extractant contained in a hollow-fiber membrane extractor for selective removal of butyric acid from the fermentation broth. The extractant was simultaneously regenerated by stripping with NaOH in a second membrane extractor. The fermentation pH was self-regulated by a balance between acid production and removal by extraction, and was kept at approximately pH 5.5 throughout the study. Compared with conventional fermentation, extractive fermentation resulted in a much higher product concentration (>300 g/L) and product purity (91%). It also resulted in higher reactor productivity (7.37 g/L. h) and butyric acid yield (0.45 g/g). Without on-line extraction to remove the acid products, at the optimal pH of 6.0, the final butyric acid concentration was only approximately 43.4 g/L, butyric acid yield was 0.423 g/g, and reactor productivity was 6.77 g/L. h. These values were much lower at pH 5.5: 20.4 g/L, 0.38 g/g, and 5.11 g/L. h, respectively. The improved performance for extractive fermentation can be attributed to the reduced product inhibition by selective removal of butyric acid from the fermentation broth. The solvent was found to be toxic to free cells in suspension, but not harmful to cells immobilized in the fibrous bed. The process was stable and provided consistent long-term performance for the entire 2-week period of study.     

Solvent screening methodology for in situ ABE extractive fermentation

Solvent screening for in situ liquid extraction of products from acetone-butanol-ethanol (ABE) fermentation was carried out, taking into account biological parameters (biocompatibility, bioavailability, and product yield) and extraction performance (partition coefficient and selectivity) determined in real fermentation broth. On the basis of different solvent characteristics obtained from literature, 16 compounds from different chemical families were selected and experimentally evaluated for their extraction capabilities in a real ABE fermentation broth system. From these compounds, nine potential solvents were also tested for their biocompatibility towards Clostridium acetobutylicum. Moreover, bioavailability and differences in substrate consumption and total n-butanol production with respect to solvent-free fermentations were quantified for each biocompatible solvent. Product yield was enhanced in the presence of organic solvents having higher affinity for butanol and butyric acid. Applying this methodology, it was found that the Guerbet alcohol 2-butyl-1-octanol presented the best extracting characteristics (the highest partition coefficient (6.76) and the third highest selectivity (644)), the highest butanol yield (27.4 %), and maintained biocompatibility with C. acetobutylicum.     

从发酵液中高效提取L-缬氨酸的工艺研究

目的:利用有机膜过滤和离子交换法分离提取发酵液中的L-缬氨酸。方法:通过有机膜过滤,除去发酵液中的菌体及蛋白,滤液浓缩结晶获得L-缬氨酸产品,通过离子交换法从结晶母液中回收部分L-缬氨酸。结果:确定了有机微滤膜和超滤膜去除发酵液中菌体蛋白和色素的操作条件;确定了采用离子交换法提取L-缬氨酸的操作条件:选择732强酸性阳离子树脂,料液pH值为3.0左右,用0.4 mol/L的氨水以1.0 mL/min的速度洗脱,L-缬氨酸的收率为89.2%。结论:通过有机膜过滤和离子交换法分离提取发酵液中的L-缬氨酸,可以提高提取收率和产品质量。     

In situ recovery of 2,3-butanediol from fermentation by liquid–liquid extraction

End-product conversion, low product concentration and large volumes of fermentation broth, the requirements for large bioreactors, in addition to the high cost involved in generating the steam required to distil fermentation products from the broth largely contributed to the decline in fermentative products. These considerations have motivated the study of organic extractants as a means to remove the product during fermentation and minimize downstream recovery. The aim of this study is to assess the practical applicability of liquid–liquid extraction in 2,3-butanediol fermentations. Eighteen organic solvents were screened to determine their biocompatibility, and bioavailability for their effects on Klebsiella pneumoniae growth. Candidate solvents at first were screened in shake flasks for toxicity to K. pneumoniae. Cell density and substrate consumption were used as measures of cell toxicity. The possibility of employing oleyl alcohol as an extraction solvent to enhance end product in 2,3-butanediol fermentation was evaluated. Fermentation was carried out at an initial glucose concentration of 80 g/l. Oleyl alcohol did not inhibit the growth of the fermentative organism. 2,3-Butanediol production increased from 17.9 g/l (in conventional fermentation) to 23.01 g/l (in extractive fermentation). Applying oleyl alcohol as the extraction solvent, about 68% of the total 2,3-butanediol produced was extracted. An erratum to this article can be found at     

Micromethod for the quantitative determination of succinic acid in the fermentation media

Summary A rapid and accurate method is described for the determination of succinic acid allowing direct application of the fermentation broth filtrate to TLC plate. Subsequent chromatographic separation on silica gel thin-layer and detection of succinic acid by a copper salt reagent, permits quantitative densitometric evaluation of succinic acid in the concentration range from 10 to 40 g. The quantitative analyses are reproducible and the assay has a coefficient of variation of 3.2%.