Isolation of High Acid-yielding Mutants of Aspergillus niger by a Paper Culture Selection Technique

Abstract

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. METHODSThe irradiation techniques used, and a description of the mutant strains isolated by the technique to be described, are given in the following paper (Gardner, James & Rubbo, 1956). Paper culture selection techniqueThe 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.) : cane molasses (56%, w/v, sucrose), 40; KH,PO,, 1.0; MgS04.7H,0, 0.25; NH,NO,, 2.5. To every 100 ml. of this medium (adjusted to pH 5.0) 10 ml. of bromcresol green G . Microb. XIV 1 . 5

Production of Citric Acid by Mutants of Aspergillus niger

“…With crude sugars the superiority of the new strains was most evident. On treated and untreated molasses the yields of acid with mutant V7-1 were about twice those obtained with mutant X 20, a similar ratio having been obtained in surface fermentation (James et al 1956). The results recorded in Table 3, based on those shown in Fig.…”

“…These mutants were induced by X-ray and ultraviolet irradiation of spores and selected by paper culture technique (James et al 1956). They were culturally distinct from the parent strain and stable in all characteristics studied.…”

“…A method developed for selection of such mutants is described separately (James, Rubbo & Gardner, 1956). …”

History of Industrial Biotechnology

“…Mutants producing higher concentrations were obtained by Miles Laboratories [118] , James et al . [119] and Hannan et al . [120] .…”

Wirkung von Hexacyanoferrat auf die Citronensäruregärung von Aspergillus niger im Melassemedium

“…1st dies der Fall, so muB der Effekt von Hcf auf Mycelentwicklung und Siiureproduktion durch Zugabe dieses Ions nach der Sterilisation aufgehoben werden. Es wurden Metallionen beriicksichtigt, die in der Melasse nach der Analyse verschiedener Autoren (VOGEL 1949, JAMES et al 1956 Hohe Eisenkonzentrationen zeigen eine weitgehende Hemmung der Siiureproduktion, die aber nicht mit einem morphologischen Effekt verbunden ist. Die Verminderung der Eisenkonzentration im Melassemedium durch Ausfiillung mit Hcf ist daher eine wichtige Voraussetzung fur eine gute Siiureproduktion.…”