Thesis (MScAgric)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: The herbicide atrazine (ATZ, 2-chloro-4-ethylamino-6-isopropylamino-1,3,5-triazine) has been identified as an environmental endocrine disruptor and possible human carcinogen. The presence of atrazine, along with its degradation products, in soils and water supplies therefore raises concern. Atrazine biodegradation in soils is well-covered to date, however, atrazine degradation by abiotic mineral surfaces, and the chemical mechanism by which it occurs, is not fully understood. Furthermore, with a changing global climate, the effects of wetting and drying cycles on soil processes (e.g. atrazine degradation) is largely unknown, but increasing in importance. This study therefore investigated atrazine degradation on six common soil mineral surfaces, namely birnessite, goethite, ferrihydrite, gibbsite, Al3+-saturated smectite and quartz, as well as the effects that drying these surfaces has on atrazine degradation. In the first part, a comparison was conducted between the reactivity of fully hydrated and drying mineral surfaces toward atrazine, by reacting atrazine-mineral mixtures under both moist and ambient drying conditions, in parallel, for 14 days. Under moist conditions, none of the mineral surfaces degraded atrazine, but under drying, birnessite and goethite degraded atrazine to non-phytotoxic hydroxyatrazine (ATZ-OH, 2-hydroxy-4-ethylamino-6-isopropylamino-1,3,5-triazine) as major product and phytotoxic deethylatrazine (DEA, 2-chloro-4-amino-6-isopropylamino-1,3,5-triazine) as minor product. The mineral surface reactivity was birnessite (66% degradation) > goethite (18% degradation) >> other mineral surfaces (negligible degradation), indicating possible atrazine oxidation. In the second part, the effects of drying rate were investigated on birnessite only (the most reactive surface), by conducting the drying (1) gradually at ambient rates, (2) rapidly under an air stream, and (3) gradually in the absence of water using only organic solvent. After 30 days of ambient drying, 90% of the atrazine was degraded to ATZ-OH and DEA, but the same extent of degradation was achieved after only 4 days of rapid drying with an air stream. Thirty days of gradual drying using only organic solvent did not increase atrazine degradation compared to the water-moist drying surface. In each case, degradation initiated at a critical moisture content of 10% of the original moisture content. In the third part, the degradation mechanism was further investigated. To test for the possible oxidation of atrazine by the birnessite surface, moist atrazine-birnessite mixtures were dried under a nitrogen (N2) stream to eliminate possible oxidation by atmospheric oxygen (O2). Dissolved Mn2+ was extracted at the end of the experiment to observe any reduction of birnessite. Under N2, the same products were formed as before, with no appreciable Mn2+ production, indicating non-oxidative atrazine degradation by birnessite. The final part investigated the effects ultraviolet (UV) radiation has on the degradation of atrazine by drying mineral surfaces. The UV-radiation enhanced the degradation of atrazine, but no other degradation products were formed. It was therefore concluded that atrazine degradation on redox-active soil mineral surfaces is enhanced by drying, via a net non-oxidative mechanism. Furthermore, this drying-induced degradation is an atrazine detoxification mechanism which could be easily applied through agricultural practices such as windrowing, ploughing and any other practice that (rapidly) dries a Mn- or Fe-oxide rich agricultural soil. / AFRIKAANSE OPSOMMING: Die onkruiddoder atrasien (ATS, 2-chloro-4-etielamino-6-isopropielamino-1,3,5-triasien) is as 'n omgewings endokriene versteurder en moontlike menslike karsinogeen geidentifiseer. Die teenwoordigheid van atrasien, tesame met sy afbreekprodukte, in grond en water toevoere wek dus kommer. Die bio-afbreking van atrasien in gronde is tot dusver goed gedek, maar die afbreking van atrasien deur abiotiese mineraaloppervlaktes, en die chemiese meganisme waarmee dit plaasvind, word nie heeltemal verstaan nie. Verder, met 'n veranderende globale klimaat, is die effekte van benatting- en drooging-siklusse op grondprosesse (bv. atrasien afbreking) grootliks onbekend, maar toenemend belangrik. Daarom het hierdie studie atrasien afbreek op ses algemene mineraaloppervlaktes, naamlik birnessiet, goethiet, ferrihidriet, gibbsiet, Al3+-versadigde smektiet en kwarts, ondersoek, asook die effekte wat drooging van hierdie oppervlaktes op atrasien afbreking het. In die eerste deel, was 'n vergelyking gedoen tussen die reaktiwiteit van volgehidreerde en droëende mineraaloppervlaktes teenoor atrasien, deur atrasien-mineraal mengsels, in parallel, onder albei nat en omliggende droogings toestande te reageer vir 14 dae. Onder nat toestande, het geeneen van die mineraaloppervlaktes atrasien afgebreek nie, maar onder drooging het birnessiet en goethiet atrasien afgebreek na nie-fitotoksiese hidroksieatrasien (ATS-OH, 2-hidroksie-4-etielamino-6-isopropielamino-1,3,5-triasien) as hoofproduk en fitotoksiese deetielatrasien (DEA, 2-chloro-4-amino-6-isopropielamino-1,3,5-triasien) as minder-produk. Die mineraaloppervlakte-reaktiwiteit was birnessiet (66% afbreking) > goethiet (18% afbreking) >> ander mineraaloppervlaktes (geringe afbreking), wat moontlike atrasien oksidasie aandui. In die tweede deel, is die effekte van droogingstempo ondersoek, op birnessiet alleenlik (die mees reaktiewe oppervlak) deur drooging by (1) 'n omliggende geleidelike tempo, (2) 'n versnelde tempo onder 'n lugstroom, en (3) 'n geleidelike tempo in die afwesigheid van water, deur slegs gebruik te maak van 'n organiese oplosmiddel. Na 30 dae se geleidelike drooging, is 90% van die atrasien afgebreek na ATS-OH en DEA, maar dieselfe hoeveelheid afbreking is bereik na slegs 4 dae onder versnelde drooging met die lugstroom. Dertig dae van geleidelike drooging met slegs organiese oplosmiddel het nie atrasien afbreking vermeerder in vergelyking met die water-nat droëende oppervlak nie. In elke geval, is afbreking geïnisieer by 'n kritiese water inhoud van 10% van die oorspronklike water inhoud. In die derde deel is die afbrekingsmeganisme verder ondersoek. Om te toets vir die moontlike oksidasie van atrasien deur die birnessiet oppervlak, is nat atrasien-birnessiet mengsels onder stikstof (N2) gedroog, om die moontlike oksidasie deur atmosferiese suurstof (O2) te verhoed. Opgeloste Mn2+ was teen die einde van die eksperiment geekstraëer om enige reduksie van birnessiet waar te neem. Onder N2 is dieselfde produkte as voorheen gevorm, met geen aansienlike Mn2+ produksie nie, aanduidend van 'n nie-oksideerende afbreek van atrasien deur birnessiet. Die laaste deel het die effekte van ultraviolet (UV) straling op die afbreek van atrasien op droëende mineraaloppervlaktes ondersoek. Die UV-straling het atrasien afbreek vermeerder, maar geen ander afbreek-produkte is gevorm nie. Die gevolgtrekking is dus dat atrasien afbreking op redoks-aktiewe mineraal-oppervlaktes verhoog word met drooging, deur 'n netto nie-oksidasie meganisme. Verder is hierdie drooging-geinduseerde afbreking 'n atrasien ontgiftingsmeganisme wat eenvoudig toegepas kan word deur landboupraktyke soos windrying, ploeg en ander praktyke wat (vinnig) 'n Mn- of Fe-oksied ryke landbou grond verdroog. / National Research Foundation (NRF)

A atrazina é um herbicida amplamente utilizado no Brasil e no mundo em diferentes culturas agrícolas, principalmente em culturas de milho, sorgo, soja e cana-de-açúcar, entretanto, pode se tornar um contaminante de águas superficiais e subterrâneas e do solo, gerando uma preocupação ambiental, pois desequilibra e interfere no ecossistema. A atrazina pode sofrer biodegradação, tornando o processo de biorremediação uma alternativa viável e ecologicamente aceitável para o tratamento de ambientes contaminados por esse herbicida. O microrganismo de referência nesse processo de biodegradação é a Pseudomonas sp. ADP que possui o plasmídio pADP-1, no qual se localizam os genes atzA, atzB, atzC, atzD, atzE e atzF, que codificam enzimas atuantes na via de degradação da atrazina. O presente estudo teve como objetivo o isolamento e a caracterização de bactérias do gênero Pseudomonas quanto à presença de genes de degradação da atrazina e quanto à capacidade de degradação desse herbicida. No presente trabalho foram isoladas 123 cepas de amostras de solo de diferentes regiões do Brasil, as quais foram caracterizadas através de provas bioquímicas e moleculares e identificadas como Pseudomonas aeruginosa (74,8%) e outras espécies (25,2%) pertencentes aos gêneros Pseudomonas, Achromobacter, Burkholderia, Cupriavidus e Rhizobium. A variabilidade genética dos isolados pertencentes ao gênero Pseudomonas foi analisada através da técnica de ERIC-PCR e demonstrou que todos os isolados apresentam alta diversidade genética (<80%). Os genes atzA, atzB, atzC, atzD, atzE e atzF foram detectados em seis isolados provenientes de amostras de solo das regiões Norte, Nordeste, Sudeste e Centro-Oeste do Brasil, sendo três deles da espécie Pseudomonas aeruginosa, um Cupriavidus pauculus, uma Burkholderia cepacea e um Rhizobium radiobacter. Apenas três isolados contendo os genes atz apresentaram plasmídios. Os isolados bacterianos que apresentaram os genes atz foram testados quanto à capacidade de crescimento utilizando a atrazina como única fonte de nitrogênio e quanto à capacidade de degradação desse herbicida. Todos os isolados testados apresentaram crescimento em meio ATZ-R, mas não foi possível observar a mineralização do herbicida, tanto em meio sólido quanto em meio líquido, todavia observou-se a degradação da atrazina por uma espécie de Pseudomonas aeruginosa (isolado P86), com redução de 44%, em meio líquido ATZ-R contendo 33 ppm de atrazina. / Atrazine - the herbicide widely used in Brazil and all over the world in different agricultural crops mainly in corn, sorghum, soy, and sugar cane can contaminate the superficial and subterraneous water, and soil creating an environmental concern due to imbalance and interference caused in ecosystem. Atrazine can suffer biodegradation, which can make the bioremediation process a viable and ecologically acceptable alternative for the treatment of the environment contaminated with this herbicide. The microorganism referred on that biodegradation process is Pseudomonas sp. ADP which has pADP-1 plasmid, where are the genes atzA, atzB, atzC, atzD, atzE, and atzF that codify active enzymes on atrazine degradation way. The aim of this present study was the isolation and the characterization of Pseudomonas genus bacteria due to the presence of the atrazine degradation genes, and the capacity of degradation of this herbicide. In the present work 123 samples of soil bacteria from different Brazilian regions were isolated, and characterized by biochemical and molecular tests, and identified as Pseudomonas aeruginosa (74.8%) and other species (25.2%) of Pseudomonas, Achromobacter, Burkholderia, Cupriavidus, and Rhizobium genus. Genetic variability of the isolated of Pseudomonas genus was analyzed by the ERIC-PCR technique, and demonstrate that all the isolated ones presented high genetic diversity (<80%). atzA, atzB, atzC, atzD, atzE, and atzF genes were detected in six isolated of soil samples from North, Northeast, Southeast, and Midwest of Brazil being 3 Pseudomonas aeruginosa, 1 Cupriavidus pauculus, 1 Burkholderia cepacea, and 1 Rhizobium radiobacter. Only 3 isolated with atz genes presented plasmids. Bacterial isolates that presented atz genes were tested for growth and degradation capacity using only atrazine as nitrogen source. All tested isolates presented growth on ATZ-R, but mineralization of this herbicide in solid or liquid media was not possible to observe. However, 1 specie Pseudomonas aeruginosa (isolate P86) presented atrazine degradation (reduction of 44%) in ATZ-R liquid media with 33 ppm of atrazine.

A atrazina é um herbicida amplamente utilizado no Brasil e no mundo em diferentes culturas agrícolas, principalmente em culturas de milho, sorgo, soja e cana-de-açúcar, entretanto, pode se tornar um contaminante de águas superficiais e subterrâneas e do solo, gerando uma preocupação ambiental, pois desequilibra e interfere no ecossistema. A atrazina pode sofrer biodegradação, tornando o processo de biorremediação uma alternativa viável e ecologicamente aceitável para o tratamento de ambientes contaminados por esse herbicida. O microrganismo de referência nesse processo de biodegradação é a Pseudomonas sp. ADP que possui o plasmídio pADP-1, no qual se localizam os genes atzA, atzB, atzC, atzD, atzE e atzF, que codificam enzimas atuantes na via de degradação da atrazina. O presente estudo teve como objetivo o isolamento e a caracterização de bactérias do gênero Pseudomonas quanto à presença de genes de degradação da atrazina e quanto à capacidade de degradação desse herbicida. No presente trabalho foram isoladas 123 cepas de amostras de solo de diferentes regiões do Brasil, as quais foram caracterizadas através de provas bioquímicas e moleculares e identificadas como Pseudomonas aeruginosa (74,8%) e outras espécies (25,2%) pertencentes aos gêneros Pseudomonas, Achromobacter, Burkholderia, Cupriavidus e Rhizobium. A variabilidade genética dos isolados pertencentes ao gênero Pseudomonas foi analisada através da técnica de ERIC-PCR e demonstrou que todos os isolados apresentam alta diversidade genética (<80%). Os genes atzA, atzB, atzC, atzD, atzE e atzF foram detectados em seis isolados provenientes de amostras de solo das regiões Norte, Nordeste, Sudeste e Centro-Oeste do Brasil, sendo três deles da espécie Pseudomonas aeruginosa, um Cupriavidus pauculus, uma Burkholderia cepacea e um Rhizobium radiobacter. Apenas três isolados contendo os genes atz apresentaram plasmídios. Os isolados bacterianos que apresentaram os genes atz foram testados quanto à capacidade de crescimento utilizando a atrazina como única fonte de nitrogênio e quanto à capacidade de degradação desse herbicida. Todos os isolados testados apresentaram crescimento em meio ATZ-R, mas não foi possível observar a mineralização do herbicida, tanto em meio sólido quanto em meio líquido, todavia observou-se a degradação da atrazina por uma espécie de Pseudomonas aeruginosa (isolado P86), com redução de 44%, em meio líquido ATZ-R contendo 33 ppm de atrazina. / Atrazine - the herbicide widely used in Brazil and all over the world in different agricultural crops mainly in corn, sorghum, soy, and sugar cane can contaminate the superficial and subterraneous water, and soil creating an environmental concern due to imbalance and interference caused in ecosystem. Atrazine can suffer biodegradation, which can make the bioremediation process a viable and ecologically acceptable alternative for the treatment of the environment contaminated with this herbicide. The microorganism referred on that biodegradation process is Pseudomonas sp. ADP which has pADP-1 plasmid, where are the genes atzA, atzB, atzC, atzD, atzE, and atzF that codify active enzymes on atrazine degradation way. The aim of this present study was the isolation and the characterization of Pseudomonas genus bacteria due to the presence of the atrazine degradation genes, and the capacity of degradation of this herbicide. In the present work 123 samples of soil bacteria from different Brazilian regions were isolated, and characterized by biochemical and molecular tests, and identified as Pseudomonas aeruginosa (74.8%) and other species (25.2%) of Pseudomonas, Achromobacter, Burkholderia, Cupriavidus, and Rhizobium genus. Genetic variability of the isolated of Pseudomonas genus was analyzed by the ERIC-PCR technique, and demonstrate that all the isolated ones presented high genetic diversity (<80%). atzA, atzB, atzC, atzD, atzE, and atzF genes were detected in six isolated of soil samples from North, Northeast, Southeast, and Midwest of Brazil being 3 Pseudomonas aeruginosa, 1 Cupriavidus pauculus, 1 Burkholderia cepacea, and 1 Rhizobium radiobacter. Only 3 isolated with atz genes presented plasmids. Bacterial isolates that presented atz genes were tested for growth and degradation capacity using only atrazine as nitrogen source. All tested isolates presented growth on ATZ-R, but mineralization of this herbicide in solid or liquid media was not possible to observe. However, 1 specie Pseudomonas aeruginosa (isolate P86) presented atrazine degradation (reduction of 44%) in ATZ-R liquid media with 33 ppm of atrazine.