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A bacterial mixed culture, which was obtained from sewage by a special enrichment procedure, utilized EDTA as the sole source of carbon and nitrogen for growth. High concentrations of mineral salts, particularly CaCl2, or the use of a mineral base without nitrogen protected the cells from inactivation after transfer into fresh medium containing 200-mg/liter (0.67 mM) EDTA. The chemical speciation did not influence the biodegradability of EDTA. However, when resting cells of the mixed culture were incubated with EDTA in the presence of an equivalent molar amount of FeCl3, the reaction came to a halt before the complete consumption of the substrate. A gram-negative isolate from the mixed population, BNC1, also metabolized EDTA in monoculture. Growth of the pure culture was promoted by biotin or folic acid but was always accompanied by the accumulation of unidentified metabolites and was slow (μmax, 0.024 h-1) compared with that of the original community (μmax, 0.036 h-1).