Aims:  The aim of this work was to enrich stable mixed cultures from atrazine‐contaminated soil. The cultures were examined for their atrazine biodegradation efficiencies in comparison with J14a, a known atrazine‐degrading strain of Agrobacterium radiobacter. The cultures were also characterized to identify community structure and bacterial species present. Methods and Results:  The cultures were enriched and then stabilized in bacterial media. The stable mixed cultures and J14a were tested in a medium containing 100 μg l−1 of atrazine. For all cultures, atrazine was removed 33–51% within 7 days and the cell optical density increased from 0·05 to between 0·50 and 0·70. Four isolates designated ND1, ND2, ND3 and ND4 were purified from the mixed cultures and identified based on sequence analysis of the 16 S rRNA gene as Alcaligenes faecalis, Klebsiella ornithinolytica, Bacillus megaterium and Agrobacterium tumefaciens, respectively. An atrazine‐degrading gene, atzA, was present in ND2 and ND4. Conclusions:  The stable mixed cultures obtained could degrade atrazine. Klebsiella ornithinolytica ND2 and Ag. tumefaciens ND4 are atrazine degraders. Significance and Impact of the Study:  The novel stable mixed cultures could be used for bioremediating crop fields contaminated with atrazine. This is the first report of the atzA gene in Kl. ornithinolytica.

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