Biomineralization of atrazine and analysis of 16S rRNA and catabolic genes of atrazine degraders in a former pesticide mixing site and a machinery washing area

Abstract

Purpose: The purpose of this study was to determine the first-order rate constants and half-lives of aerobic and anaerobic biomineralization of atrazine in soil samples from an agricultural farm site that had been previously used for mixing pesticide formulations and washing application equipment. Atrazine catabolic genes and atrazine-degrading bacteria in the soil samples were analyzed by molecular methods. Materials and methods: Biomineralization of atrazine was measured in soil samples with a [U-ring-14C]-atrazine biometer technique in soil samples. Enrichment cultures growing with atrazine were derived from soil samples and they were analyzed for bacterial diversity by constructing 16S rDNA clone libraries and sequencing. Bacterial isolates were also obtained and they were screened for atrazine catabolic genes. Results and discussion: The soils contained active atrazine-metabolizing microbial communities and both aerobic and anaerobic biomineralization of [U-ring-14C]-atrazine to 14CO2 was demonstrated. In contrast to aerobic incubations, anaerobic biometers displayed considerable differences in the kinetics of atrazine mineralization between duplicates. Sequence analysis of 16S rDNA clone libraries constructed from the enrichment cultures revealed a preponderance of Variovorax spp. (51 %) and Schlesneria (16 %). Analysis of 16S rRNA gene sequences from pure cultures (n = 12) isolated from enrichment cultures yielded almost exclusively Arthrobacter spp. (83 %; 10/12 isolates). PCR screening of pure culture isolates for atrazine catabolic genes detected atzB, atzC, trzD, trzN, and possibly atzA. The presence of a complete metabolic pathway was not demonstrated by the amplification of catabolic genes among these isolates. Conclusions: The soils contained active atrazine-metabolizing microbial communities. The anaerobic biometer data showed variable response of atrazine biomineralization to external electron acceptor conditions. Partial pathways are inevitable in soil microbial communities, with metabolites linking into other catabolic and assimilative pathways of carbon and nitrogen. There was no evidence for the complete set of functional genes of the known pathways of atrazine biomineralization among the isolates.