Characterisation of new strains of atrazine‐degrading Nocardioides sp. isolated from Japanese riverbed sediment using naturally derived river ecosystem

Abstract

A Gram-positive bacterial strain able to degrade the herbicide atrazine was isolated using a simple model ecosystem constituted with Japanese riverbed sediment and its associated water (microcosm). Treatment of the water phase of the microcosm with 1 mg litre-1 [ring-14C]atrazine resulted in the rapid degradation of atrazine after a 10 day lag phase period. The [ring-14C]cyanuric acid formed was transiently accumulated as an intermediary metabolite in the water phase and was subsequently mineralised through triazine ring cleavage. Possible atrazine-degrading microbes suspended in the water phase of the microcosm were isolated by the plating method while rapid degradation of atrazine was in progress. Among the 48 strains that were isolated, 47 exhibited atrazine-degrading activity. From these 47 isolates, 12 strains that were randomly selected were found to identically convert atrazine to cyanuric acid via hydroxyatrazine. Polymerase chain reaction (PCR) amplification of the genes corresponding to atrazine degradation revealed that these strains at least carried the genes trzN (atrazine chlorohydrolase from Nocardioides C190) and atzC (N-isopropylammelide isopropyl amidohydrolase from Pseudomonas ADP). Physiological characteristics and 16S rDNA partial sequences of six strains that were further selected strongly suggested that all these isolates originated from the same Nocardioides sp. strain. Additionally, only one isolate could mineralise the triazine ring of cyanuric acid. Based on microscopic observations, this strain appears to be a two-membered microbial consortium consisting of Nocardioides sp. and a Gram-negative bacterium. In conclusion, atrazine biodegradation in the microcosm appeared to occur predominantly by Nocardioides sp. to yield cyanuric acid, which could be mineralised by the other relatively ubiquitous microbes.

Removal of atrazine from river waters by indigenous microorganisms

“…Strong et al (2002) detected the atrazine metabolites hydroxyatrazine, N-isopropylammelide and cyanuric acid, via ESI-MS, following incubation of atrazine with the bacterium Arthrobacter aurescens TC1.). Elsewhere, in a microcosm containing river water and sediment, atrazine was degraded by sediment-bound bacteria to cyanuric acid, presumably via de-chlorination and subsequent hydrolysis to give 2-hydroxyatrazine, followed by removal of the alkyl substituents on the triazine ring (Satsuma et al 2002;Satsuma 2006). The cyanuric acid was then mineralised to CO 2 .…”

Evolution of Catabolic Pathways: Genomic Insights into Microbial s -Triazine Metabolism

“…Many of the recently isolated atrazine-degrading bacteria contain TrzN rather than AtzA for initiating the metabolism of s-triazines (1,56,70,72). In one recent example with simazine as the herbicide and a number of different bacteria, TrzN, but not AtzA, was detected in the strains (55).…”

“…In the period between 1960 and 1990, many studies reported bacterial metabolism of atrazine to occur via dealkylation of the N-alkyl substituents on the s-triazine ring (20,21,34), a pathway that does not typically lead to s-triazine ring cleavage (44,62). Since 1995, most reports of metabolic pathways for atrazine degradation have described metabolism via hydroxyatrazine and not involving dealkylation (18,49,53,56,70,72,73,75,76). Earlier reports expressed the view that isolating bacteria able to grow on atrazine as a sole nitrogen or carbon source was difficult (13,20).…”

Characterization of a Nocardioides-based, atrazine-mineralizing microbial colony isolated from Japanese riverbed sediment

“…28,29) Among them, atrazinemineralizing colonies were then further screened by the following procedures. A 100-fold-diluted nutrient broth (Difco) containing 1 mg/l [ 14 C]atrazine was prepared.…”

“…At appropriate intervals, atrazine degradation was monitored by HPLC, 29) and the amount of evolved 14 CO 2 was determined. Intermediary metabolites were identified by TLC-and HPLC-co-chromatography and compared with the reference standards.…”