A plate assay to screen manganese-tolerant Aspergillus niger strains

Two different physiological effects of Mn[II] interactions with Aspergillus niger are known. Excess Mn[II] levels are toxic and restrict fungal radial growth on plates and second, an Mn[II] limited fungal growth is the key to successful citric acid fermentation. While studying Mn[II] toxicity we noted that the Mn[II]-supplemented minimal medium (MM) agar plates were translucent. Also, clearing zones around the fungal colonies owing to organic acid release were observed. Simultaneous scoring of these two parameters was exploited to screen for Mn[II] tolerant/ insensitive A. niger strains with improved acid production. Clear halos were best viewed on MM plates supplemented with ≥ 5.0 mM of Mn[II]. Accordingly, an acid unit (AU; the ratio of diameter of the clear zone to that of colony diameter) was defined. Colonies that produced larger zones of clearing (and higher AUs) were picked up and sub-cultured. A Mn[II] tolerant/ insensitive spontaneous mutant of A. niger was isolated by this screen and characterized. It produced significantly higher amounts of citric acid and the yield was not adversely affected by the added Mn[II]. While separate screens for metal tolerance and citric acid production were reported earlier, the method presented here is simple and directly combines the two features for strain selection/screening.