Excessive use of pesticides and herbicides is a major environmental and health concern worldwide. Atrazine, a synthetic triazine herbicide commonly used to control grassy and broadleaf weeds in crops, is a major pollutant of soil and water ecosystems. Atrazine modifies the growth, enzymatic processes and photosynthesis in plants. Atrazine exerts mutagenicity, genotoxicity, defective cell division, erroneous lipid synthesis and hormonal imbalance in aquatic fauna and nontarget animals. It has threatened the sustainability of agricultural soils due to detrimental effects on resident soil microbial communities. The detection of atrazine in soil and reservoir sites is usually made by IR spectroscopy, ELISA, HPLC, UPLC, LC–MS and GC–MS techniques. HPLC/LC–MS and GC–MS techniques are considered the most effective tools, having detection limits up to ppb levels in different matrices. Biodegradation of atrazine by microbial species is increasingly being recognized as an eco-friendly, economically feasible and sustainable bioremediation strategy. This review presents the toxicity, analytical techniques, abiotic degradation and microbial metabolism of atrazine.

Toxicity, degradation and analysis of the herbicide atrazine

Biodegradation of xenobiotic compounds is examined with s-triazines as an example and with biological treatment of wastewater containing s-triazines as an aim. s-Triazines have been termed recalcitrant, but examination of the literature indicates that a potential for biodegradation exists. Nitrogen-limited enrichment cultures yield organisms able to degrade by-products of the industrial synthesis of s-triazine herbicides as sources of nitrogen. The choice of inoculum for these enrichments is important and often allows for successful enrichment after simple batch culture, but organisms containing several degradative reactions could be obtained only after selection in extended culture. Routine, specific determinations of all s-triazines (by HPLC) were essential throughout the work. Molar growth yields show complete mass balances for the utilization of s-triazines. Kinetic experiments indicate that specific degradation rates of s-triazines in growing cells are about 0.4 mkat/kg of protein. Characterised biochemical pathways consist of a series of hydrolytic cleavages of chloro-, amino- and alkylamino-groups from the s-triazine ring. Pathways converge to cyanuric acid, which is subject to hydrolytic ring cleavage to CO2 and NH4+ via hydrolysis of biuret and urea. Our cultures degraded all significant s-triazines in real wastewater. But the system was not practicable because the specific activities of some enzymes were too low, because of inhibition by salt in the wastewater and because expensive carbon sources were necessary. Improved planning for enrichment cultures is seen to be necessary and this depends on adequate knowledge of the chemistry of the wastes.

http://kops.uni-konstanz.de/bitstream/handle/123456789/6885/Biodegradation_of_s_triazine.pdf?sequence=1

Biodegration of s-triazine xenobiotics

Atrazine, simazine, and propazine are closely related herbicides that can be used for weed control. The degradation of s‐triazines in soil, water, and sediments has received considerable attention. In this review, the literature related to degradation of atrazine and related s‐triazines is reviewed. Particular emphasis is directed toward those substances formed along the degradative pathways and to the biodegradation aspects of mineralization. The s‐triazines are unusual in that the carbon and nitrogen ring contains no available electrons for aerobic biodegradation. The only sources of energy for aerobic biodegradation of atrazine are found on the ethyl and isopropy I groups. There is some evidence to support the concept that dealkylation of s‐triazines occurs to support the bioenergetic needs of microorganisms. Biodegradation studies with simazine or atrazine as the carbon and energy source have generally shown biodegradation of the alkyl side chains with evolution of radioactive carbon dioxide from chai...

Degradation of atrazine and related s‐triazines

We previously reported the isolation of a 21.5-kb genomic DNA fragment from Pseudomonas sp. strain ADP, which contains the atzA gene, encoding the first metabolic step for the degradation of the herbicide atrazine (M. de Souza, L. P. Wackett, K. L. Boundy-Mills, R. T. Mandelbaum, and M. J. Sadowsky, Appl. Environ. Microbiol. 61:3373-3378, 1995). In this study, we show that this fragment also contained the second gene of the atrazine metabolic pathway, atzB. AtzB catalyzed the transformation of hydroxyatrazine to N-isopropylammelide. The product was identified by use of high-performance liquid chromatography, mass spectrometery, and nuclear magnetic resonance spectroscopy. Tn5 mutagenesis of pMD1 was used to determine that atzB was located 8 kb downstream of atzA. Hydroxyatrazine degradation activity was localized to a 4.0-kb ClaI fragment, which was subcloned into the vector pACYC184 to produce plasmid pATZB-2. The DNA sequence of this region was determined and found to contain two large overlapping divergent open reading frames, ORF1 and ORF2. ORF1 was identified as the coding region of atzB by demonstrating that (i) only ORF1 was transcribed in Pseudomonas sp. strain ADP, (ii) a Tn5 insertion in ORF2 did not disrupt function, and (iii) codon usage was consistent with ORF1 being translated. AtzB had 25% amino acid identity with TrzA, a protein that catalyzes a hydrolytic deamination of the s-triazine substrate melamine. The atzA and atzB genes catalyze the first two steps of the metabolic pathway in a bacterium that rapidly metabolizes atrazine to carbon dioxide, ammonia, and chloride.

https://aem.asm.org/content/aem/63/3/916.full.pdf

The atzB gene of Pseudomonas sp. strain ADP encodes the second enzyme of a novel atrazine degradation pathway.

We performed the first field-scale atrazine remediation study in the United States using chemically killed, recombinant organisms This field study compared biostimulation methods for enhancing atrazine degradation with a novel bioaugmentation protocol using a killed and stabilized whole-cell suspension of recombinant Escherichia coli engineered to overproduce atrazine chlorohyrolase, AtzA AtzA dechlorinates atrazine, producing non-toxic and non-phytotoxic hydroxyatrazine Soil contaminated by an accidental spill of atrazine (up to 29,000 ppm) supported significant populations of indigenous microorganisms capable of atrazine catabolism Laboratory experiments indicated that supplementing soil with carbon inhibited atrazine biodegradation, but inorganic phosphate stimulated atrazine biodegradation A subsequent field-scale study consisting of nine (075m3) treatment plots was designed to test four treatment protocols in triplicate Control plots contained moistened soil; biostimulation plots received 300ppm phosphate; bioaugmentation plots received 05% (w/w) killed, recombinant E coli cells encapsulating AtzA; and combination plots received phosphate plus the enzyme-containing cells After 8 weeks, atrazine levels declined 52% in plots containing killed recombinant E coli cells, and 77% in combination plots In contrast, atrazine levels in control and biostimulation plots did not decline significantly These data indicate that genetically engineered bacteria overexpressing catabolic genes significantly increased degradation in this soil heavily contaminated with atrazine

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Field-scale remediation of atrazine-contaminated soil using recombinant Escherichia coli expressing atrazine chlorohydrolase

The purpose of this paper is to elucidate the final degradative pathways of atrazine (I) by a Nocardia strain through experiments on the degradation of its metabolite, 4-amino-2-chloro-6-isopropylamino-1,3,5-triazine (IIa). This compound, in the bacterial medium, was transformed into several products identified as 2-chloro-4,6-diamino-1,3,5-triazine (IIc), 4-amino-2-hydroxy-6-isopropylamino-1,3,5-triazine (VIII), 4-amino-1,2-dihydro-1,3,5-triazin-2-one (IV) and dicyanodi-amidine (VII). The formation of (IIc) was attributed to a microbial N-dealkylation, while the detection of (IV) and (VII) was attributed to a chemical degradation of the intermediate 4-amino-2-chloro-1,3,5-triazine (III). Compound (III) undergoes rapid hydrolysis and does not accumulate in the culture medium. On the basis of the microbial and chemical results, we propose a degradative pathway for atrazine.

G. Filacchioni

Papers

Chemical and Biological Degradation of Primary Metabolites of Atrazine by a Nocardia Strain