Cultivation of phylogenetically diverse and metabolically novel atrazine degrading soil bacteria using Bio-Sep® beads

by Martin, Emily Catherine.

The s-triazine herbicide atrazine is among the most widely used herbicides worldwide. The human health effects of atrazine exposure remain unclear, but atrazine and its metabolites appear to cause developmental abnormalities in amphibians. A mounting body of knowledge concerning the ecology of atrazine degradation suggests the current collection of microorganisms and genetic biomarkers of atrazine degradation cannot accurately predict the natural attenuation of atrazine. To this end, a novel in situ enrichment approach using highly porous, atrazine-impregnated Bio-Sep iii ® beads was employed to isolate a taxonomically diverse group of atrazine-degrading bacteria from soil and wetland environments in Tennessee and Ohio. The study greatly increased the scope and diversity of organisms previously shown to degrade atrazine. Most notable, a novel lineage within the Bacteriodetes phylum, Dyadobacter sp. was obtained, constituting the first report of the atrazine-degrading phenotype within this division. Although not taxonomically novel, previously unreported atrazine-degrading taxa from Actinobacteria (Catellatospora, Microbacterium, and Glycomyces), Alpha-Proteobacteria (Methylobacterium, Methylopila, and Sphingomonas), Beta-Proteobacteria (Variovorax and Acidovorax), and Gamma-Proteobacteria (Acinetobacter, Rahnella, and Pantoea) were also isolated. Evidence for metabolic diversity in atrazine catabolism was observed in the collection. Most significantly, the atrazine-chlorohydrolase gene, encoded by trzN, was the only known catabolic gene detected in our collection with the exception of the Arthrobacter strains which typically also possessed atzB and atzC, that code for enzymes needed for sequential dealkylation of 2-hydroxy atrazine. No other known genes for the intermediate metabolism were detected in many of the isolates suggesting the presence of alternative degradative pathways for atrazine among soil bacteria. Previously, trzN has only been reported in high G+C Gram-positive bacteria but our results revealed that this catabolic gene is much more broadly distributed among classes including the Alpha and Beta Proteobacteria. The results demonstrate that Bio-Sep® beads are a suitable matrix for recruiting a highly diverse subset of the bacterial community involved in atrazine degradation. iv