Isolation of high acid-yielding mutants of Aspergillus niger by a paper culture selection technique.

SUMMARY: A method for isolating and identifying high acid-yielding mutants of Aspergillus niger is described. This involves cultivation of the organisms on absorbent paper soaked in an indicator medium. The advantages of the technique and the criteria used for selecting biochemically interesting mutants are briefly described. The greater acid production of mutants selected by paper culture has been confirmed by comparing yields of citric acid in surface and submerged fermentations with those given by the wild type strain. The selection of acid-producing fungi described by Foster & Davis (1949) and modified by Quilico, Panizzi & Visconti (1949) has been used frequently for isolating fungi showing marked differences in acid production. Their methods of cultivation on indicator media are unsatisfactory for selection of colonies which produce large amounts of acid because of the extensive diffusion of the acid zones around the colonies. With such strains large quantities of media are required since each colony to be screened would have to be tested on a separate agar plate, and there is no simple means of determining whether such colonies arise from one or more spores. METHODS The irradiation techniques used, and a description of the mutant strains isolated by the technique to be described, are given in the following paper The method depends on the ability of Aspergillus niger to grow as compact discrete colonies surrounded by clearly defined acid zones on absorbent paper previously soaked in liquid culture medium. There are a number of ways of carrying out this paper culture technique, and these can be modified to suit the purpose of the experiment. So far the following procedure has been used. Circular sheets of Eucalypt viscose pulp, 1 mm. thick and 10 in. in diameter were marked with pencil in fifty 1 in. squares and soaked in liquid medium (see below). The moistened paper was supported at four points in a 10 in. Petri dish containing 5 ml. 20 % (vlv) glycerol in water to prevent drying of the paper during incubation. The dish and its contents were autoclaved at 116" (10 lb./sq.in. pressure) for 15 min. The medium contained (g./l.