A. Řičicová

Laboratory fermentation of gibberellic acid

SummaryIn studying the fermentation conditions suitable for gibberellic acid production, 6 collection strains ofFusarium moniliforme andGibberella fujikuroi were used. The strain used was of decisive importance for the yield and composition of the effective metabolite.After verification of methods described in the literature a new medium for gibberellic acid production was developed to be used for fermentation on a shaker and in laboratory fermentors, the medium containing sucrose and corn steep liquor. By using a reciprocal shaker, maximum production of gibberellic acid was obtained after 14 days, by using three-stage propagations after 12 days of fermentation.Maximum values achieved during fermentation of the most suitable strains amounted to 150–240 μg./ml. Maximum production in laboratory fermentors reached 212 μg./ml.The metabolite isolated after fermentation under the described conditions was identified as gibberellic acid.AbstractПри изучении условий ферментации пригодные для gibberellic кислоты 6 коллекция штаммов Fusarium moniliforme и Gibberella fujikuroi были использованы Используется штамм имеет решающее значение за урожайностью и состав эффективный метаболит. После проверки методов, описанных в литературе нового средства массовой информации для gibberellic кислоты был разработан, которые будут использоваться для брожения на шейкер и в лабораторных fermentors, средних содержащие сахарозы и кукуруза крутых спиртными напитками. С помощью взаимных шейкер, максимум производство gibberellic кислота была получена после 14 дней, используя три этапа propagations по истечении 12 дней после брожения Максимальные значения, достигнутые в ходе ферментации из наиболее подходящие штаммы составил 150-240 μ g. / мл. Максимум производства в лаборатории fermentors достигло 212 μ g. / мл Метаболит изолированных после брожения в рамках описанных условиях определены в качестве gibberellic кислота

Taxonomic characteristic of the strain ETH 7437 producing granaticin

The streptomycete strain ETH 7437, producing granaticin, was characterized according to present taxonomic standards and compared with the strainsStreptomyces olivaceus (Waksman) Waksman and Henrici,Streptomyces coelicolor (Müller) andStreptomyces violaceruber (Waksman & Curtis). The strain ETH 7437 formed gray aerial mycelium, straight and flexuos sporophores, even single hooks and open spirals. Normally the amount of spores in chains was over 50, the surface of spores being smooth. The strain utilizedd-glucose,d-arabinose,d-xylose andd-fructose. The strain was capable of forming antibiotic pigment stably. For the strain ETH 7437 the termStreptomyces granaticolor was proposed.

Preparation of inoculum from sclerotia of the ascomyceteClaviceps purpurea

Slices of pleetenchymatous tissues of the purified sclerotia are used for preparation of saprophytic cultures of Claviceps purpurea producing ergot alkaloids (Kirchhoff, 1929; GrSger & Tyler, 1963). This procedure is often accompanied by contamination which decreases the number of the obtained cultures available for inoculation purposes. In this paper we wish to describe a procedure according to which it is possible to prepare more of the inoculation material and at the same time to reduce the hazard of contamination considerably. Sclerotia of the strain Claviceps purpurea (Fr.) Tul. were used in our work. For the disinfection of their surface following procedures were employed: 1) Soaking of sclerotia in water followed by 25 min submersion in a sublimate solution (0.1%) with an admixture of tween (0.1%); 2) plunging of sclerotia (30 min) into a solution of famosept containing tween (0.1%); 3) mechanical cleaning with scalpel, dipping into a 50% solution of propanol (2 min) and a 4% formaldehyde solution (2 min) (GrSger & Tyler, 1963). The efficiency of the disinfection procedures was examined after a 7 day incubation of the sclerotia washed in water on malt agar (8 ~ Bal.) at 28 ~ C. The best results have been shown by procedure 3 yielding a massive myeelial growth proving tha t the germinating ability of the cultures remained unaffected. For the preparation of the inoculum the disinr

Quantitative changes of the alkaloid complex in a submerged culture ofClaviceps paspali

Claviceps paspali FA produced high concentrations of alkaloid under submerged conditions. Their production was found to depend on the developmental stage and treatment of the filamentous culture inoculum. A medium containing Bacto-peptone with a constant composition of amino acids was selected for the preparation of the inoculum. A two-week fermentation in a synthetic medium with mannitol at 24 ± 1 °C resulted in an increased production of total alkaloids from the original value of 100–200 μg/mL to more than 2 000 μg/mL. Addition of tryptophan did not further increase the production of alkaloids but resulted in changes of the spectrum of some metabolites. 2,3-Dihydroxybenzoic acid accompanied the alkaloids in the fermentation medium. α-Hydroxyethyllysergamide was the predominant component of extracellular alkaloids (80 % in the first days of fermentation). During fermentation the level of this alkaloid continuously decreased while the concentration of the accompanying alkaloids,i.e. lysergamide and the corresponding minor isomers, increased.

Physiological activities of ergoline alkaloids in submerged cultures ofClaviceps paspali andClaviceps purpurea

Ergoline alkaloids do not belong to a group of physio ogically inert secondary metabolites. Ergometrine as well as ergotamine increased the acetyl-CoA carboxylase activity and inhibited the activities of citrate synthase and isocitrate lyase. The slightly inhibitory effect of ergotamine and stimulatory effect of ergometrine on malate synthase was established. Supplied ergometrine brought about a change in the culture growth rate,i.e. inhibition (10−4m) or stimulation (10−3m). Addition of 10−4m ergometrine to the culture ofClaviceps paspali MG-6 increased the total alkaloid yield by 120%. The stimulatory influence was inversely proportional to the concentration of added alkaloid. It is assumed that accumulating alkaloids suppress attainment of the yield that would be achieved by continuous control of the alkaloid level. A positive relationship is assumed between the process of alkaloid synthesis and the viability of the culture.

Influence of the soya meal fractions on gibberellic acid and gibberellin A production in submerse cultivation ofGibberella fujikuroi

The relationship of different soya meal components to gibberellin (GA) production was studied. Fluorometric assay confirmed that under the given fermentation conditions, only gibberellic acid (GA3) was synthesized on medium containing corn steep. On substituting soya flour for corn steep, the same amount of GA3 was produced and in addition gibberellin A (GA1) was formed. The GA3: GA1 ratio was 1∶1. The course of fermentation in media containing the soya meal protein fraction (fraction I), the soya meal amino acid complex, the corn steep amino acid complex and individual amino acids (γ-aminobutyric acid or tryptophane) was the same as in the control medium containing soya meal. The soya meal fraction II, which is characterized by a high cellulose and carbohydrate content, raised GA production by 25% as compared with production in medium containling soya meal; it simultaneously stimulated GA3 production, so that the final GA3: GA1 ratio was 4∶1.AbstractКонтролировалось отношение различных компонентов соевой муки к образованию гиббереллинов (GA). Флуорометрическим методом было подтверждено, что при данных условиях ферментации в среде с кукурузным экстрактом возникает только гибберелловая кислота (GA3). Если же кукурузный экстракт заменяли соевой мукой, образовались такие же количества GA3, как в среде с кукурузным экстрактом, и сверх того еще гиббереллин A (GA1). Соотношение GA3: GA1 равнялось 1∶1. Ферментация в среде с белковой фракцией соевой муки (фракция ї), с комплексом аминокислот соевой муки или кукурузного экстркта и с отдельными аминокислотами, т. е. с аминомасляной кислотой или триптофаном, проходила так же, как и в контрольной среде с соевой мукой. Фракция II из соевой муки, отличающаяся высоким содержанием, целлюлозы и углеводов, повышала образование GA по сравнению с его образованием в среде с соевой мукой на 25% при одновременном повышении образования GA3, так что окончательное соотношение GA3: GA1 равнялось 4∶1.

Quantitative assay of granaticin by the microbiological diffusion method.

AbstractПроизводились количественное определение гранатицина с помощью диффузионного микробиологического метода с применением тест-микроба Bacillus subtilis ATCC 6633. Среда для тестирования содержала 1% глюкозы, 0,5% bacto-пептона, 0,3% говяжьего экстракта (Difco), 0,5% NaCl и дистиллированную воду (при pH 6,2). Образцы, содержащие гранатицин, разводили 1–15 м фосфатным буфером с pH 6. Реакция учитывалась после 16-часовой инкубации пластинок агара при 28°C. Концентрациу гранатицина определяли, вычитая размеры зон угнетения из размеров прямой стандарта.

Characteristics of the saprophytic reference strain FA ofClaviceps paspali

Growth properties and morphological features of the saprophytic collection cultureClaviceps paspali strain FA were investigated. The strain was characterized by a range of utilization of 13 carbon sources in a basic synthetic and a peptone medium. A temperature of 23 °C was more favourable for growth than 28 °C. Stages of changes of the culture cultivated for a long time on Sabouraud’s medium were detected by electron microscopy. The white culture was characterized by true septated mycelium with different types of terminal parts of hyphae. Arthrospores occurred after a 14-d cultivation, small spherical conidia released individually from hyphae were quite rare. As compared with other strains of the same species, strain FA did not form spherical clusters of conidia. On the other hand, a spontaneous rupture of the surface cell wall in different parts of hyphae and release of the cytoplasm were observed. In corn-steep containing media the formation of individual, pair and chain-like forms of arthritic conidia was stimulated. Destructive autolytic changes of hyphae were detected in the medium with potato extract.

Srinivasan’s coagulation test in taxonomically classified streptomycetes

Srinivasan’s coagulation test was performed in 18 strains of the genusStreptomyces and one strain of the genusActinoplanes. The highest coagulation activity was detected in strains systematically classified in a series of streptomycetes with pink or red aerial mycelium:S. erythreus, Streptomyces sp. AJ/22,S. roseo-luteus andS. griseofuscus. With the exception ofS. griseofuscus these three cultures also exhibited the highest inhibitory activity againstB. subtilis. When using hemoglobin as substrate it was possible to detect acid, neutral and alkaline proteinases with the highest proteolytic activity at pH 3.0 to 4.0 in the most active strain ofS. erythreus.

A psychrophilic strainActinoplanes sp. 220

The strain designatedActinoplanes sp. 220 differed in its characteristics from other strains of the genusActinoplanes listed in Sergey’s Manual (1974). The strain belongs to psychrophilic culture growing within the range of 0–30 °C. The optimal temperature for growth on yeast —mali agar is 10–23 °C. Cultures transferred at 23 and 28 °C differed in morphological and physiological properties, enzyme activity and pigmentation in standard media. Submerged culture transferred at 28 °C inhibited growth ofBacillus subtilis ATCC 6633 and ATCC 9945.ll-2,6-Diaminopimelic acid was chromatographically detected in the submerged mycelium of this culture. This compound was not found in the mycelium of the original culture transferred at 23 °C. The cultures did not substantially differ in the composition of other amino acids contained in larger quantities in the mycelium.