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
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.
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.
KeywordsArthrobacter spp. Atz and trz genes Biodegradation of atrazine Herbicide biodegradation Pesticide biodegradation
- Abigail MEA, Lakshmi V, Das N (2012) Biodegradation of atrazine by Cryptococcus laurentii isolated from contaminated agricultural soil. J Microbiol Biotechnol Res 2:450–457Google Scholar
- Abigail MEA, Salam JA, Das N (2013) Atrazine degradation in liquid culture and soil by a novel yeast Pichia kudriavzevii strain Atz-EN-01 and its potential application for bioremediation. J Appl Pharmaceut Sci 3(6):35–40Google Scholar
- Boundy-Mills KL, de Souza ML, Mandelbaum RT, Wackett LP, Sadowsky MJ (1997) The atzB gene of Pseudomonas sp. strain ADP encodes the second enzyme of a novel atrazine degradation pathway. Appl Environ Microbiol 63:916–923Google Scholar
- de Souza ML, Wackett LP, Boundy-Mills KL, Mandelbaum RT, Sadowsky MJ (1995) Cloning, characterization, and expression of a gene region from Pseudomonas sp. strain ADP involved in the dechlorination of atrazine. Appl Environ Microbiol 61:3373–3378Google Scholar
- de Souza ML, Seffernick J, Martinez B, Sadowsky MJ, Wackett LP (1998) The atrazine catabolism genes atzABC are widespread and highly conserved. J Bacteriol 180:1951–1954Google Scholar
- Donnelly PK, Entry JA, Crawford DL (1993) Degradation of atrazine and 2,4-dichlorophenoxyacetic acid by mycorrhizal fungi at three nitrogen concentrations in vitro. Appl Environ Microbiol 59:2642–2647Google Scholar
- Eaton RW, Karns JS (1991) Cloning and analysis of s-triazine catabolic genes from Pseudomonas sp. strain NRRLB-12227. J Bacteriol 173:1215–1222Google Scholar
- Guerin WF, Boyd SA (1992) Differential bioavailability of soil-sorbed naphthalene to two bacterial species. Appl Environ Microbiol 58:1142–1152Google Scholar
- Kulichevskaya IS, Ivanova AO, Belova SE, Baulina OI, Bodelier PLE, Rijpstra WIC, Damsté JSS, Zavarzin GA, Dedysh SN (2007) Schlesneria paludicola gen. nov., sp. nov., the first acidophilic member of the order Planctomycetales, from Sphagnum-dominated boreal wetlands. Int J System Evol Microbiol 57:2680–2687CrossRefGoogle Scholar
- Lane DJ (1991) 16S/23S rRNA sequencing. In: Stackebrandt E, Goodfellow M (eds) Nucleic Acid Techniques in Bacterial Systematics. Wiley, Chichester, pp 115–175Google Scholar
- Mougin C, Laugero C, Asther M, Dubroca J, Frasse P, Asther M (1994) Biotransformation of the herbicide atrazine by the white rot fungus Phanerochaete chrysosporium. Appl Environ Microbiol 60:705–708Google Scholar
- Muyzer G, de Waal EC, Uitterlinden AG (1993) Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA. Appl Environ Microbiol 59:695–700Google Scholar
- Radosevich M, Traina SJ, Hao Y-L, Tuovinen OH (1995) Degradation and mineralization of atrazine by a soil bacterial isolate. Appl Environ Microbiol 61:297–302Google Scholar
- Romero MC, Urrutia MI, Reinoso EH, Vedoval RD, Reynaldi FJ (2014) Atrazine degradation by wild filamentous fungi. Global Res J Microbiol 4:10–16Google Scholar
- Sadowsky MJ, Tong Z, de Souza M, Wackett LP (1998) AtzC is a new member of the amidohydrolase protein superfamily and is homologous to other atrazine-metabolizing enzymes. J Bacteriol 180:152–158Google Scholar
- Sajjaphan K, Heepngoen P, Sadowsky MJ, Boonkerd N (2010) Arthrobacter sp. strain KU001 isolated from a Thai soil degrades atrazine in the presence of inorganic nitrogen sources. J Microbiol Biotechnol 20:602–608Google Scholar
- Singh B, Singh K (2016) Microbial degradation of herbicides. Crit Rev Microbiol 42:245–261Google Scholar
- Spanoghe P, Maes A, Steurbaut W (2009) Limitation of point source pesticide pollution: results of bioremediation system. Comm Appl Biol Sci Ghent Univ 74(2):1–14Google Scholar
- Udiković N, Hršak D, Mendaš G, Filipčić D (2003) Enrichment and characterization of atrazine degrading microbial communities. Food Technol Biotechnol 41:211–217Google Scholar
- Udiković-Kolić N, Hršak D, Devers M, Klepac-Ceraj V, Petrič I, Martin-Laurent F (2010) Taxonomic and functional diversity of atrazine-degrading bacterial communities enriched from agrochemical factory soil. J Appl Microbiol 109:355–367Google Scholar