Mycological studies in methods of improving the antibiotic yield of micro-organisms
Aytoun, Robert Sinclair Chadwick
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This work was carried out in an attempt to increase the efficiency of mutation programmes designed to produce strains of micro -organisms giving greater antibiotic yield. The problem was approached from two angles. First, radioactive sulphur (S35),recently shown in America to be a very effective general muta- genic agent for Neurospora crassa, was used to treat two antibiotic -producing fungi, Penicillium chrysogenum (penicillin) and P. p atulum (griseofulvin), and with both these species it proved to be an efficient agent for inducing mutations capable of increased antibiotic production. Secondly, methods of screening putative mutants were developed that enable larger numbers of isolates to be screened per unit time than has hitherto been possible by the conventional methods.Treatment of P. chrysogenum var. brevisterigma with sulphur -35 consistently yielded isolates 60% of which had mutated in their potential penicillin pro- duction and slightly more than 1% of these gave higher yields than the parent strain. A primary screening technique, designed to pick out these higher yielding strains, was evolved. This was based on the principle of using the antibiotic production of the isolate grow- ing on the surface of an agar culture as a measure of its potential yield in submerged fermentation. It is similar to one developed recently in Japan, but avoids the inaccuracies that the Japanese author ascribes to his method by maintaining continuity of the fermentatioi medium throughout. By using sulphur -35 as the mutagenic agent, and this surface-culture screening technique, an improved penicillin -producing strain (Strain B) was found among the first 50v isolates tested; on accurate testing it showed a 25 -35% improvement on the parent, although it required a slightly richer medium to achieve this increase. A second similar mutation programme with Strain B as the parent produced, among 600 isolates tested, a second mutant (Strain C) capable of the same penicillin yield as Strain B, but not requiring an enriched medium for the purpose. Both these mutants were considered to be potentially useful for industrial production.With a culture of Penicillium patùlum, which produces the antibiotic griseofulvin, there were carrie out mutation programmes involving the use of sulphur -35 and ultra - violet irradiation as separate mutagenic agents. As the surface culture screening technique did not lend itself readily to the assay of griseo- fulvin, one based on a small -volume fermentation was developed. With this technique it was found that s sulphur -35 produced a high mutation rate (about 40 %) for antibiotic yield of the treated isolates, several of which showed considerably improved yields over the parent. Two of the first 400 sulphur - treated isolates gave, on accurate testing, almost 100% improvement in the yield of griseofulvin. 'l'he ultra- violet treatment, the although producing some improved mutants, did not give as many as the sulphur method, nor were those produced as satisfactory as the ones induced by the radioactive isotope.These results strongly indicate that sulphur -35 is an efficient mutagen for producing strains of fungi yielding increased quantities of antibiotics. This is considered at length in the general discussion. The two primary screening techniques developed have been shown to be at once sufficiently accurate and capable of dealing with greater numbers of isolates than the more conventional methods.In addition, some preliminary research is des- cribed, undertaken to find out the effects of griseo- fulvin on plant pathogenic fungi of the genus Bot Otis., Experiments in vitro and in vivo showed that the presence of the antibiotic upsets the mechanism of hyphal wall in the pathogen, leading in vivo ï to inability of the hyphae to penetrate the cell membranes of the host.