Since their first introduction in the mid 1950s, man-made s-triazine herbicides such as atrazine have extensively been used in agriculture to control broadleaf weed growth in different crops, and thus contributed to improving crop yield and quality. Atrazine is the most widely used s-triazine herbicide for the control of weeds in crops such as corn and sorghum. Although atrazine was initially found to be slowly and partially biodegradable, predominantly by nonspecific P450 monoxygenases which do not sustain microbial growth, microorganisms gradually evolved as a result of repeated exposure, started using it as a growth substrate and eventually succeeded in mineralizing it. Within three decades, an entirely new hydrolase-dependent pathway for atrazine mineralization emerged and rapidly spread worldwide among genetically different bacteria. This review focuses on the enzymes involved in atrazine mineralization and their evolutionary histories, the genetic composition of microbial populations involved in atrazine degradation and the biotechnologies that have been developed, based on these systems, for the bioremediation of atrazine contamination in the environment.

https://www.bib.irb.hr/657979/download/657979.Udikovic-Kolic_2012_review.pdf

Evolution of atrazine-degrading capabilities in the environment

Novel catabolic pathways enabling rapid detoxification of s-triazine herbicides have been elucidated and detected at a growing number of locations. The genes responsible for s-triazine mineralization, i.e. atzABCDEF and trzNDF, occur in at least four bacterial phyla and are implicated in the development of enhanced degradation in agricultural soils from all continents except Antarctica. Enhanced degradation occurs in at least nine crops and six crop rotation systems that rely on s-triazine herbicides for weed control, and, with the exception of acidic soil conditions and s-triazine application frequency, adaptation of the microbial population is independent of soil physiochemical properties and cultural management practices. From an agronomic perspective, residual weed control could be reduced tenfold in s-triazine-adapted relative to non-adapted soils. From an environmental standpoint, the off-site loss of total s-triazine residues could be overestimated 13-fold in adapted soils if altered persistence estimates and metabolic pathways are not reflected in fate and transport models. Empirical models requiring soil pH and s-triazine use history as input parameters predict atrazine persistence more accurately than historical estimates, thereby allowing practitioners to adjust weed control strategies and model input values when warranted.

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Agronomic and environmental implications of enhanced s-triazine degradation

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.

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

Methoxychlor is an organochlorine pesticide used worldwide against several insect pests, resulting in human exposure. This pesticide mimics endocrine hormone functions, interfering with normal endocrine activity in humans and wildlife. For this reason, it is imperative to develop methods to remove this pesticide from the envi- ronment, and though, bioremediation using microorgan- isms results as an excellent strategy. Five Streptomyces spp. strains previously isolated from organochlorine-pol- luted sites and capable to grow and remove methoxychlor were combined as different mixed cultures to increase methoxychlor removal. From the 39 consortia tested, one consortium (Streptomyces spp. A6, A12, A14, M7) was selected because of its high pesticide removal and specific dechlorinase activity to be assayed on slurry and soil sys- tems. This consortium showed higher biomass values (8.3 9 10 6 ± 5.7 9 10 5 CFU mL -1 ) and methoxychlor removal (56.2 ± 2.3 %) on enriched slurry than in non- enriched slurry (7.3 9 10 5 ± 1.2 9 10 5 CFU mL -1 and 45.6 ± 7.4 % of pesticide removal). In soil systems, Streptomyces consortium showed higher growth (1.0 9 10 11 ± 5.0 9 10 10 CFU g -1 ) than in enriched slurry, although differences in methoxychlor removal between both culture conditions were not statistically sig- nificant. Therefore, the selected Streptomyces consortium may be suitable for the development of in situ (soil) and ex situ (slurry bioreactor) bioremediation methods because of their potential to remove methoxychlor from different systems.

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Methoxychlor bioremediation by defined consortium of environmental Streptomyces strains

Mineralization of atrazine in the river water intake and sediments of a constructed flow-through wetland

Agricultural waste water containing pesticides can reach the sea via rivers and estuaries, including brackish lakes. We studied the metabolic fate of methoxychlor [MXC; 1,1,1-trichloro-2,2-bis(4-methoxyphenyl)ethane] in a model system consisting of sediment and associated water collected from two sampling sites: a brackish lake and a freshwater river. MXC degraded rapidly and was finally mineralized in both sediment systems. The first step of degradation was dechlorination to yield 1,1-dichloro-2,2-bis(4-methoxyphenyl)ethane [de-Cl-MXC] or CN-replacement to yield 2,2-bis(4-methoxyphenyl)acetonitrile [MXC-CN], followed by O-demethylation. Although the metabolites were common to the two sediments, the dynamics of the metabolites over time were clearly distinct. In the brackish lake sediment, de-Cl-MXC accumulated transiently, whereas in the river sediment, it was rapidly converted to its demethylated metabolite. We also found that dechlorination and CN-replacement proceeded in autoclave-sterilized river sed...

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An environmental fate study of methoxychlor using water-sediment model system.

A multiple-member microbial colony (designated A14N) capable of mineralizing the herbicide atrazine has been isolated from Japanese riverbed sediment. We have determined the enzymes involved in the degradation route and the species composition of the colony. A14N converted atrazine to cyanuric acid via hydroxyatrazine and N-isopropylammelide as intermediates. Furthermore, A14N characteristically had the ability to cleave the triazine ring of cyanuric acid. A14N harbours genes coding atrazine-degrading enzymes, which are highly similar to those previously described: trzN, atzB and atzC. At least three phylogenetically different species, Nocardioides sp., Mycobacterium sp. and Leptospira sp. were present in A14N. Among these, Nocardioides sp. was closely related to the atrazine-degrading Nocardioides sp. strains previously isolated from agricultural soils. Thus, Nocardioides sp. in A14N could also be responsible for the degradation of atrazine to cyanuric acid.

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

In a simple laboratory microcosm constituted of riverbed sediment and its associated water, rapid degradation of herbicide atrazine mainly to cyanuric acid occurred through microbial degradation. To elucidate the role and behavior of microbes involved in atrazine degradation, comparative microcosms constituted of different sediment/water conditions and constitutions were employed. Fluorescence microscopic observation and direct bacterial count by DAPI (4',6-diamidino-2-phenylindole)-staining and CFU (colony forming unit) count by the plate method were also conducted to supplement the comparative study. As a result, planktonic microbes in the aqueous phase appeared not to be responsible for the initial degradation of atrazine, at least during the present experimental period of 70 days. The sediment was found to be important as a source of degrading microbes. However, once the degradation was initiated, the degrading activity in the aqueous phase was maintained without the sediment. Although CFU in aqueous phase increased with time, this increase in cell number seemed not to reflect the degrading activity. Large, glittering and whole particles, supposed to be microbial cells, were observed by DAPI-staining in the aqueous phase under a microscopic field only when active atrazine-degradation occurred. Therefore, they might be involved in the degrading activity.

Role and Behavior of Benthic Microbes Able to Degrade Herbicide Atrazine in Naturally Derived Water/sediment Microcosm

A water-miscible solvent, such as acetone, acetonitrile or methanol, is often employed as a co-solvent to dissolve an organic test chemical of low water solubility in an environmental fate study using a laboratory model microcosm. These co-solvents, however, may disrupt the microflora in the water/sediment tested, and affect the biodegradation of the target compound. In the present study, a 0.1% concentration of acetonitrile as a co-solvent greatly suppressed the microbial degradation of herbicide atrazine [2-chloro-4-(ethylamino)-6-(isopropylamino)-s-triazine] throughout the experimental period (84 days). The rapid growth of specific microbes was considered to deprive atrazine-degrading microbes of their habitat (mainly the surface area of sediment particles) in the microcosm.

A Negative Effect of Co-solvent on Atrazine Biodegradation in Experimental River Microcosms

To obtain basic information on the metabolic fate of xenobiotics in the brackish water, bivalve Corbicula japonica, bioconcentration and biotransformation experiments were performed using methoxychlor (MXC) as a model compound. Bivalves were exposed to [ring-U-14C]MXC (10 µg L−1) for 28 days under semi-static conditions followed by a 14-day depuration phase.The 14C concentration in the bivalves rapidly increased and reached a steady state after exposure for 7 days (BCFss = 2010); however, it rapidly decreased with a half-life of 2.2 days in the depuration phase.Mono- and bis-demethylated MXC, and their corresponding sulphate conjugates, were identified as minor metabolites. No glycoside conjugates (including glucuronide and glucoside) were detected.Despite this biotransformation system, bivalves were found to excrete retained MXC mostly unchanged although its relatively hydrophobic nature.

Koji Satsuma

Papers

An environmental fate study of methoxychlor using water-sediment model system.

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

Role and Behavior of Benthic Microbes Able to Degrade Herbicide Atrazine in Naturally Derived Water/sediment Microcosm

A Negative Effect of Co-solvent on Atrazine Biodegradation in Experimental River Microcosms

Bioconcentration and biotransformation of [¹⁴C]methoxychlor in the brackish water bivalve Corbicula japonica.