Mineralization of atrazine by bacteria isolated from shallow subsurface sediments

Abstract

Atrazine [2-chloro-4-ethylamino-6-isopropylamino-1,3,5-triazinel, a corn herbicide, is a common contaminant of ground water. Biodegradation in subsurface sediments represents a potential remediation for atrazine contaminated aquifers. In a previous study, atrazine was degraded within 40 days by sand from sample 3B-3.2, cored from a former agricultural chemical dealership near the water table at 2.8-3.5 meters. The purpose of this study was to isolate atrazine-degrading bacteria from a sample from 3B-3.2. Inoculation of nitrogen-limited medium with sediment from interval 3B-3.2 resulted in the isolation of a mixed bacterial culture labeled MC-067. MC-067 can degrade 20 mg L−1 atrazine as a sole carbon and nitrogen source within 50 days. MC-067 degrades deethylatrazine [2-chloro-4-amino-6-isopropylamino-1,3,5-triazine] (DEA) and deisopropylatrazine [2-chloro-4-ethylamino-6-amino-1,3,5-triazine] (DIA) as sole nitrogen sources at 20 mg L-1 within 15 and 7 days, respectively. MC-067 does not degrade 20mg L−1 deethyldeisopropylatrazine [2-chloro-4,6-diamino-1,3,5-triazine] (DEDIA) in nitrogen-limited or NH4CI-supplemented media within 20 days. MC-067 mineralized up to 92% of ring-labeled [14C]atrazine with different treatments of carbon, nitrogen, and atrazine concentration. Three members of MC-067 were identified by 16S rRNA sequence analysis as Ralstonia pickettii, Microbacterium sp., and Methylobacterium sp. Three strains of R. pickettii were isolated based on differences in atrazine degradation. Strain D required NH4CI to degrade 97.5% of 20 mg L−1 atrazine within 9 days. Strain D2 degraded atrazine as a sole nitrogen source within 5 days, but mineralized less than 5% at concentrations of 20 and 0.2 mg L−1 over a 45 day period. Strain N and Microbacterium sp. did not degrade atrazine, regardless of the nitrogen status of the medium. The degradation of DEA and DIA by Microbacterium sp. and the three R. pickettii strains resembled each individual culture's ability to degrade atrazine. None of the strains degraded DEDIA. The degradation of atrazine and metabolites by Methylobacterium sp. was not tested. Probes for the atzABC genes, which catalyze the transformation of atrazine to cyanuric acid [2,4,6-trihydroxy-1,3,-5triazine], hybridized to an atzA homolog for strain D and an atzC homolog for strain N. No atz gene homologs were detected for Microbacterium sp. Strain D2 was not probed, but anaerobic atrazine degradation suggests the presence of an atzA homolog.