ia

Cl a

, Qlde, Ql9, A

triazine herbicides are of particular concern because of potential adverse effects, both on photosynthetic

human health problems. Triazine herbicides are toxic to non-targetphotosynthetic species, including phototropic bacteria, freshwateralgae, mangrove trees and corals (Bell and Duke, 2005; Jones andKerwell, 2003; Lockert et al., 2006; Sutton et al., 1984). In addi-tion, it has been claimed that atrazine may be carcinogenic (Huff,

Sutherland et al., 2004). Indeed, free-enzyme systems are consid-ered to be sufciently safe for use in the clinical treatment ofpesticide poisoning (Bird et al., 2008), and are currently usedcommercially for organophosphate insecticide remediation (http://www.orica-landguard.com/).

Despite the advantages of free-enzyme bioremediation, thereare also constraints, and the requisite characteristics required ofenzymes used in free-enzyme remediation restrict its applicability

* Corresponding author. Tel.: 61 2 6246 4090.

Contents lists availab

ls

Journal of Environmental Management 91 (2010) 2075e2078E-mail address: colin.scott@csiro.au (C. Scott).Herbicide runoff from agricultural lands is a major issue world-wide. It is of particular concern in the sugar cane growing regionwithin theGreat Barrier Reef catchment area andhasbeen identiedas a threat to receiving freshwater andmarine environments (Lewiset al., 2009). In particular, herbicides designed to inhibit thephotosystem II in plants (e.g. triazines, phenyl ureas) exceed fresh-water and marine guidelines during both low ow and event/highow conditions (Lewis et al., 2009). These ecosystems includefreshwater wetlands of national or international signicance andthe Great Barrier Reef World Heritage Area (Lewis et al., 2009).

Atrazine, the most commonly used triazine, has been usedextensively since its introduction in 1958 (Tomlin, 2006), and ofparticular concern as it has been linked to environmental and

in vertebrate species (Hayes et al., 2002). These issues are com-pounded by atrazines relatively long environmental half-life offour to fty seven weeks (Belluck et al., 1991) and its high level ofmobility; it has been detected in both surface and ground waters inseveral countries (Gavrilescu, 2005; Thurman andMeyer, 1996; vander Meer, 2006), at concentrations up to 1 ppm.

Several bioremediation strategies have been suggested fordecontaminating atrazine-contaminated water, including the useof transgenic plants and bacteria (Kawahigashi et al., 2006; Stronget al., 2000; Wang et al., 2005). However, technical and regulatoryimpediments prevent the use of live transgenic organisms inenvironmental settings (Watanabe, 2001). Free-enzyme bioreme-diation is an attractive alternative to the use live organisms, as it isnot constrained by these (Alcalde et al., 2006; Scott et al., 2008;Received in revised form28 April 2010Accepted 6 May 2010Available online 31 May 2010

Keywords:ChlorohydrolaseHydrolaseTriazineField trialHerbicide remediation

1. Introduction0301-4797/$ e see front matter Crown Copyright 2doi:10.1016/j.jenvman.2010.05.007organisms and upon vertebrate development. To date a number of bioremediation strategies have beenproposed for triazine herbicides, but are unlikely to be implemented due to their reliance upon therelease of genetically modied organisms. We propose an alternative strategy using a free-enzymebioremediant, which is unconstrained by the issues surrounding the use of live organisms. Here wereport an initial eld trial with an enzyme-based product, demonstrating that the technology is tech-nically capable of remediating water bodies contaminated with the most common triazine herbicide,atrazine.

Crown Copyright 2010 Published by Elsevier Ltd. All rights reserved.

2002; Huff and Sass, 2007), and may cause endocrine dysfunctionArticle history:Received 22 March 2010Herbicide contamination from agriculture is a major issue worldwide, and has been identied as a threatto freshwater and marine environments in the Great Barrier Reef World Heritage Area in Australia. TheShort communication

A free-enzyme catalyst for the bioremedatrazine contamination

Colin Scott a,*, Steve E. Lewis b, Rob Milla c, MatthewJon E. Brodie b, John G. Oakeshott a, Robyn J. RusselaCSIRO Division of Entomology, GPO Box 1700, Canberra, ACT 2601, AustraliabAustralian Centre for Tropical Freshwater Research, James Cook University, TownsvillecQueensland Department of Primary Industries and Fisheries, GPO Box 1085, TownsvilldCSIRO Division of Molecular and Health Technologies, Private Bag 10, Clayton, VIC 316

a r t i c l e i n f o a b s t r a c t

Journal of Environm

journal homepage: www.e010 Published by Elsevier Ltd. Alltion of environmental

. Taylor a, Andrew J.W. Rodgers d, Geoff Dumsday d,

4811, Australiad 4810, Australiaustralia

le at ScienceDirect

ental Management

evier .com/locate/ jenvmanrights reserved.

to a relatively narrow range of enzyme classes. The enzymes used infree-enzyme bioremediation must withstand environmentalconditions for long enough to remediate their target and must beindependent of diffusible cofactors, or cofactors that require activeregeneration (Alcalde et al., 2006; Scott et al., 2008; Sutherlandet al., 2004). Hydrolases are particularly well suited to this appli-cation, whilst other enzyme systems, such as reductive dehaloge-nases, monooxygenases and dioxygenases, are not (Gibson and

C. Scott et al. / Journal of Environmental20762. Materials and methods

2.1. Preparation of TrzN-containing homogenate

The gene encoding TrzN was cloned into pET14b (Novagen)using the NdeI and BamHI restriction sites using appropriaterestriction endonucleases (New England Biolabs). The resultantplasmid was used to transform BL21 l(DE3) (Novagen), which wasthen used as the expression strain. Claried bacterial homogenatecontaining active TrzN was prepared from a 2 L ferment of BL21lDE3 expressing TrzN, grown on a minimal medium (10.6 g/LKH2PO4, 4 g/L (NH4)2HPO4, 1.7 g/L citric acid monohydrate, 31.3 mL/L glycerol). After autoclaving 10mL/L of PTM4 salts (0.2 g/L D-biotin,2.0 g/L CuSO4$5H2O, 0.08 g/L NaI, 3.0 g/L MnSO4$H2O, 0.2 g/LNa2MoO4, 0.02 g/L Boric acid, 0.5 g/L CoCl2$6H2O, 7.0 g/L ZnCl2,22.0 g/L FeSO4$7H2O, 0.5 g/L CaSO4, 1 mL/L H2SO4) and 0.6 g/LMgSO4 was added. The fermentation was fed with glycerol, sup-plemented with 150 mg/L ampicillin and 331 mg/L thiamine, andinduced with 11.9 mg/L IPTG. The ferment yielded ca. 240 g wetweight of cell pellet (OD600 122). The cells were suspended in5.2 g/L MOPS pH 6.9, then passed through a homogeniser 3 timesand claried by centrifugation. The claried lysate was passedParales, 2000; Lofer and Edwards, 2006; Whiteley and Lee,2006; Furukawa, 2006; Field and Sierra-Alvarez, 2008).

Suitable hydrolytic enzymes for atrazine bioremediation havebeen isolated: the atrazine dechlorinase (de Souza et al., 1996),AtzA, and the triazine hydrolase, TrzN (Mulbry et al., 2002). Bothenzymes catalyse the irreversible hydrolytic dechlorination ofatrazine to produce non-herbicidal products (Fig. 1). GenerallyAtzA has been explored as the most promising bioremediant foratrazine, and the generation of transgenic plants containing theatzA gene (Wang et al., 2005), trialling of transgenic Escherichia coliin eld-scale bioremediation (Strong et al., 2000), and variousattempts at enzyme engineering and improvement (Raillard et al.,2001; Scott et al., 2009) have all been reported. However, AtzA islimited to the remediation of chlorinated triazine herbicides (e.g.atrazine, propazine and simazine) (Seffernick et al., 2000), whilstthe alternative enzyme, TrzN, acts upon a broader range oftriazines including methoxytriazines (e.g. atraton) and methyl-thiotriazine (e.g. ametryn) (Shapir et al., 2005). TrzN is also moreefcient catalytically than AtzA (de Souza et al., 1996; Scott et al.,2009; Shapir et al., 2005), making it in many ways a moreattractive bioremediant than AtzA.

Herewe report the rst eld trial of a free-enzyme bioremediantof atrazine, based on the catalytically superior enzyme TrzN.Fig. 1. Dechlorination of atrazine catalysed by the triazine hydrolase TrzN andproducing non-herbicidal hydroxyatrazine.through a 0.22 mM lter to remove intact cells and DNaseI was usedto remove intact DNA. Enzymatic activity was determined at258 19 mg of atrazine/mg of lysate/using both the UV absorbancemethod described by de Souza et al. (1996) and the colorimetricmethod described in Scott et al. (2009). The homogenatewas storedat 80 C and thawed at 4 C when required.

2.2. Preparation of test dam

Holding dams are used to collect spent irrigationwater so that itis withheld from entering the catchment, allowing the water to bereused in farm activities and providing time for pesticide residuesto degrade by biological or photolytic routes. A ca. 1.5 ML holdingdam at a sugar cane farm near Clare (Lat. 19:48, Long. 147:14) in thedry tropics of Queensland, Australia, was lled with headwaterfrom irrigation of a eld pre-treatedwith the recommended dose ofatrazine (3.3 kg per hectare). 240 g of bacterial homogenate wassuspended in 20 L of water, and applied by hand by spreadingevenly across the surface of the holding dam. Duplicate 1 L sampleswere taken before the dam was lled with atrazine-contaminatedrunoff water, before the enzyme was added and at time intervalsafter the addition of the enzyme. Samples were stored immediatelyon ice to stop the enzymatic reaction. Samples were frozen after nomore than 4 h on ice.

2.3. Determination of atrazine concentration

Atrazine concentrations were determined at two independentlaboratories; Queensland Health Forensic and Scientic Services(QHFSS), by the LCMSMS method described in Lewis et al. (2009),modied to use direct injection; and by CSIRO Entomology by thefollowing LCMS method. Briey, 100 mL samples were acidiedwith HCl to pH 2.8, then the atrazine in the samples was concen-trated 1000-fold by solid phase extraction using preconditionedOasis SPE Max Cartridges (Waters, USA), and eluted in 3 mL ofMeOH (with ammonia). Samples were subsequently dried anddissolved in 100 ml of MeOH. Samples were separated by HPLC andassayed for atrazine concentrations bymeasuring the absorbance at265 nm, and the analyte peak area calculated using Analyst soft-ware. Replicate samples were within 10% agreement. The identityof the HPLC peak was conrmed by mass spectrum analysis,whereby atrazine ions 216 m/z were extracted on an AgilentToFeMSD.

3. Results and discussion

The water in the holding dam contained 8e12 mg/L atrazinebefore the irrigation tailwater was collected (data not shown). Afterlling with irrigation tailwater the atrazine concentration rose to157e170 mg/L (Fig. 2). There was a lag in the rate of atrazinedepletion after addition of the enzyme, which was most likelyattributable to the rate at which the enzyme mixed with the waterin the holding dam. The duration of the mixing phase for enzymeapplied in this manner is almost certainly dependent on thevolume and surface area:volume ratio of the water body to beremediated; i.e. larger bodies and those with low surface area:volume ratios would require a longer mixing phase.

Notwithstanding the lag during the mixing phase, the additionof the enzyme led to >90% depletion in the concentration of atra-zine in the rst four hours after addition. This result suggests thata TrzN-based bioremediant for triazines is technically feasible,although the rate of remediation is slow compared with early trialsof the only other enzymatic bioremediant, OpdA. OpdA reducedmethyl-parathion concentrations by 10-fold reduction from

Management 91 (2010) 2075e207884,000 L in less than 10 min (Russell et al., 2001), 24-fold faster

ntal Management 91 (2010) 2075e2078 2077than TrzN removed atrazine in this trial. However, OpdAwas dosedinto a smaller volume of owing water, which almost certainlyimproved the rate of mixing of the enzyme and its performanceoverall.

OpdA is an exceptional enzyme, and catalyses methyl-parathionhydrolysis at near diffusion limited rates (ca. 7000 mg methylparathion/second/mg of OpdA) (Jackson et al., 2008). TrzN hasa considerably slower turnover rate (ca. 13 mg atrazine/second/mgTrzN) (Shapir et al., 2005), and so greater quantities of TrzN arerequired to obtain rates of atrazine hydrolysis comparable to thoseobtained with OpdA-mediated methyl-parathion hydrolysis. Ifrequired, the rate of atrazine hydrolysis by TrzN could be improvedby directed evolution, in the manner that has been reported forAtzA (Scott et al., 2009).

4. Conclusion

Fig. 2. Depletion of atrazine in a 1.5 ML holding dam over 10.5 h after addition of TrzN.Samples analysed by QHFSS (Squares), and CSIRO Entomology (Circles).

C. Scott et al. / Journal of EnvironmeThe reduction of herbicide runoff from agricultural lands is a keycomponent of the Reef Water Quality Protection Plan (RWQPP) forthe Great Barrier Reef World Heritage Area (Anon, 2003) andherbicide management is thus a major consideration of regionallydeveloped Best Management Practices through Water QualityImprovement Plans/Reef Rescue programs in response to theRWQPP. The commercial development and application of enzymeproducts to remove herbicide residues before they enter waterwaysof the Great Barrier Reef catchment area provide an alternativestrategy to reduce herbicide runoff and thus reduce their potentialimpact to receiving water environs. This application may also allowsugar cane growers and horticulturalists to continue using productssuch as atrazine to maintain productivity requirements. Moreover,this enzyme has broader applications for the removal of atrazinefrom contaminated surface and ground waters around the world.Further eld trials will be required tomore accurately gaugewhat isrequired to make this technically successful remediation tech-nology commercially viable.

Acknowledgements

We would like to thank Wayne Dal Santo for allowing us to usehis farm in this eld trial, and Simon Christen at QHFSS for helpfuldiscussions.References

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C. Scott et al. / Journal of Environmental Management 91 (2010) 2075e20782078

A free-enzyme catalyst for the bioremediation of environmental atrazine contaminationIntroductionMaterials and methodsPreparation of TrzN-containing homogenatePreparation of test damDetermination of atrazine concentration

Results and discussionConclusionAcknowledgementsReferences