Biomineralization of atrazine and analysis of 16S rRNA and catabolic genes of atrazinede-graders in a former pesticide mixing and machinery washing area at a farm site and in a constructed wetland

Atrazine is one of the most widely used herbicides in the world. It is primarily used in the production of corn in the United States. Although it may marginally increase crop yields, atrazine is also an endocrine disruptor in non-target organisms. Its moderate solubility in water allows for atrazine to contaminate surface and ground waters far removed from the point of application to soil. Although atrazine can be degraded abiotically, its primary mode of attenuation in natural environments is through bacterial degradation. Full mineralization of atrazine to CO2, H2O, Cl- and NH4+ has been demonstrated in Pseudomonas ADP, which contains the complete suite of atz atrazine catabolic genes.;The overall hypothesis of this study is that the microorganisms and catabolic pathways reported in the literature do not universally account for the atrazine biodegradation observed in different natural environments. Furthermore, it is hypothesized that in situ pre-enrichment methods yield atrazine degraders uncultivable by classical laboratory enrichment, including anaerobic bacteria. The discovery of atrazine catabolic genes other than those in the atz pathway and the demonstrated involvement of consortia of bacteria in atrazine biodegradation suggest that the full diversity of environmental atrazine biodegradation has yet to be elucidated.;In order to further elucidate the bacteria and genes responsible for atrazine biodegradation in different environments, locations with different exposures to atrazine were chosen for study. Among them were a farm location that was the site of multiple pesticide spill events throughout its history and a constructed wetland that receives river water containing agricultural runoff. Samples from these sites were assessed for atrazine biomineralization via biometer studies. In addition to traditional environmental sampling, these sites were also sampled with Bio-Sep beads to allow for in situ pre-enrichment for atrazine-degrading microbes. Soil and sediment samples were enriched for atrazine-degraders in liquid media, which included atrazine as the sole source of energy, C and N. Atrazine-degrading mixed cultures were screened via 16S-rDNA-PCR-DGGE, and mixed cultures displaying unique banding patterns were chosen for 16S rDNA clone library formation and sequencing. These isolates were also screened for eight atrazine catabolic genes via PCR.;Atrazine mineralization was demonstrated in the sampling sites, but never exceeded 30% total mineralization of the added [U-ring-14C]-atrazine. Atrazine degrading pure and mixed cultures were successfully retrieved, with Acidovorax, Hydrogenophaga, Ralstonia, Variovorax, and Xanthobacter most prevalent among the mixed culture clone libraries and Arthrobacter spp. dominating the isolate collection.