Journal of Environmental Management 91 (2010) 2075e2078

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Journal of Environmental Management

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Short communication

A free-enzyme catalyst for the bioremediation of environmental

atrazine contamination
Colin Scott a, *, Steve E. Lewis b, Rob Milla c, Matthew C. Taylor a, Andrew J.W. Rodgers d, Geoff Dumsday d,
Jon E. Brodie b, John G. Oakeshott a, Robyn J. Russell a
a

CSIRO Division of Entomology, GPO Box 1700, Canberra, ACT 2601, Australia
Australian Centre for Tropical Freshwater Research, James Cook University, Townsville, Qld 4811, Australia
Queensland Department of Primary Industries and Fisheries, GPO Box 1085, Townsville, Qld 4810, Australia
d
CSIRO Division of Molecular and Health Technologies, Private Bag 10, Clayton, VIC 3169, Australia
b
c

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 22 March 2010
Received in revised form
28 April 2010
Accepted 6 May 2010
Available online 31 May 2010

Herbicide contamination from agriculture is a major issue worldwide, and has been identied as a threat
to freshwater and marine environments in the Great Barrier Reef World Heritage Area in Australia. The
triazine herbicides are of particular concern because of potential adverse effects, both on photosynthetic
organisms and upon vertebrate development. To date a number of bioremediation strategies have been
proposed for triazine herbicides, but are unlikely to be implemented due to their reliance upon the
release of genetically modied organisms. We propose an alternative strategy using a free-enzyme
bioremediant, which is unconstrained by the issues surrounding the use of live organisms. Here we
report an initial eld trial with an enzyme-based product, demonstrating that the technology is technically capable of remediating water bodies contaminated with the most common triazine herbicide,
atrazine.
Crown Copyright 2010 Published by Elsevier Ltd. All rights reserved.

Keywords:
Chlorohydrolase
Hydrolase
Triazine
Field trial
Herbicide remediation

1. Introduction
Herbicide runoff from agricultural lands is a major issue worldwide. It is of particular concern in the sugar cane growing region
within the Great Barrier Reef catchment area and has been identied
as a threat to receiving freshwater and marine environments (Lewis
et al., 2009). In particular, herbicides designed to inhibit the
photosystem II in plants (e.g. triazines, phenyl ureas) exceed freshwater and marine guidelines during both low ow and event/high
ow conditions (Lewis et al., 2009). These ecosystems include
freshwater wetlands of national or international signicance and
the Great Barrier Reef World Heritage Area (Lewis et al., 2009).
Atrazine, the most commonly used triazine, has been used
extensively since its introduction in 1958 (Tomlin, 2006), and of
particular concern as it has been linked to environmental and
human health problems. Triazine herbicides are toxic to non-target
photosynthetic species, including phototropic bacteria, freshwater
algae, mangrove trees and corals (Bell and Duke, 2005; Jones and
Kerwell, 2003; Lockert et al., 2006; Sutton et al., 1984). In addition, it has been claimed that atrazine may be carcinogenic (Huff,
* Corresponding author. Tel.: 61 2 6246 4090.
E-mail address: colin.scott@csiro.au (C. Scott).

2002; Huff and Sass, 2007), and may cause endocrine dysfunction
in vertebrate species (Hayes et al., 2002). These issues are compounded by atrazines relatively long environmental half-life of
four to fty seven weeks (Belluck et al., 1991) and its high level of
mobility; it has been detected in both surface and ground waters in
several countries (Gavrilescu, 2005; Thurman and Meyer, 1996; van
der Meer, 2006), at concentrations up to 1 ppm.
Several bioremediation strategies have been suggested for
decontaminating atrazine-contaminated water, including the use
of transgenic plants and bacteria (Kawahigashi et al., 2006; Strong
et al., 2000; Wang et al., 2005). However, technical and regulatory
impediments prevent the use of live transgenic organisms in
environmental settings (Watanabe, 2001). Free-enzyme bioremediation is an attractive alternative to the use live organisms, as it is
not constrained by these (Alcalde et al., 2006; Scott et al., 2008;
Sutherland et al., 2004). Indeed, free-enzyme systems are considered to be sufciently safe for use in the clinical treatment of
pesticide poisoning (Bird et al., 2008), and are currently used
commercially for organophosphate insecticide remediation (http://
www.orica-landguard.com/).
Despite the advantages of free-enzyme bioremediation, there
are also constraints, and the requisite characteristics required of
enzymes used in free-enzyme remediation restrict its applicability

0301-4797/$ e see front matter Crown Copyright 2010 Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.jenvman.2010.05.007

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C. Scott et al. / Journal of Environmental Management 91 (2010) 2075e2078

to a relatively narrow range of enzyme classes. The enzymes used in

free-enzyme bioremediation must withstand environmental
conditions for long enough to remediate their target and must be
independent of diffusible cofactors, or cofactors that require active
regeneration (Alcalde et al., 2006; Scott et al., 2008; Sutherland
et al., 2004). Hydrolases are particularly well suited to this application, whilst other enzyme systems, such as reductive dehalogenases, monooxygenases and dioxygenases, are not (Gibson and
Parales, 2000; Lofer and Edwards, 2006; Whiteley and Lee,
2006; Furukawa, 2006; Field and Sierra-Alvarez, 2008).
Suitable hydrolytic enzymes for atrazine bioremediation have
been isolated: the atrazine dechlorinase (de Souza et al., 1996),
AtzA, and the triazine hydrolase, TrzN (Mulbry et al., 2002). Both
enzymes catalyse the irreversible hydrolytic dechlorination of
atrazine to produce non-herbicidal products (Fig. 1). Generally
AtzA has been explored as the most promising bioremediant for
atrazine, and the generation of transgenic plants containing the
atzA gene (Wang et al., 2005), trialling of transgenic Escherichia coli
in eld-scale bioremediation (Strong et al., 2000), and various
attempts at enzyme engineering and improvement (Raillard et al.,
2001; Scott et al., 2009) have all been reported. However, AtzA is
limited to the remediation of chlorinated triazine herbicides (e.g.
atrazine, propazine and simazine) (Seffernick et al., 2000), whilst
the alternative enzyme, TrzN, acts upon a broader range of
triazines including methoxytriazines (e.g. atraton) and methylthiotriazine (e.g. ametryn) (Shapir et al., 2005). TrzN is also more
efcient catalytically than AtzA (de Souza et al., 1996; Scott et al.,
2009; Shapir et al., 2005), making it in many ways a more
attractive bioremediant than AtzA.
Here we report the rst eld trial of a free-enzyme bioremediant
of atrazine, based on the catalytically superior enzyme TrzN.

2. 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 appropriate
restriction endonucleases (New England Biolabs). The resultant
plasmid was used to transform BL21 l(DE3) (Novagen), which was
then used as the expression strain. Claried bacterial homogenate
containing active TrzN was prepared from a 2 L ferment of BL21
lDE3 expressing TrzN, grown on a minimal medium (10.6 g/L
KH2PO4, 4 g/L (NH4)2HPO4, 1.7 g/L citric acid monohydrate, 31.3 mL/
L glycerol). After autoclaving 10 mL/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/L
Na2MoO4, 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/L
MgSO4 was added. The fermentation was fed with glycerol, supplemented with 150 mg/L ampicillin and 331 mg/L thiamine, and
induced with 11.9 mg/L IPTG. The ferment yielded ca. 240 g wet
weight of cell pellet (OD600 122). The cells were suspended in
5.2 g/L MOPS pH 6.9, then passed through a homogeniser 3 times
and claried by centrifugation. The claried lysate was passed

Fig. 1. Dechlorination of atrazine catalysed by the triazine hydrolase TrzN and

producing non-herbicidal hydroxyatrazine.

through a 0.22 mM lter to remove intact cells and DNaseI was used
to remove intact DNA. Enzymatic activity was determined at
258  19 mg of atrazine/mg of lysate/using both the UV absorbance
method described by de Souza et al. (1996) and the colorimetric
method described in Scott et al. (2009). The homogenate was stored
at 80  C and thawed at 4  C when required.
2.2. Preparation of test dam
Holding dams are used to collect spent irrigation water so that it
is withheld from entering the catchment, allowing the water to be
reused in farm activities and providing time for pesticide residues
to degrade by biological or photolytic routes. A ca. 1.5 ML holding
dam at a sugar cane farm near Clare (Lat. 19:48, Long. 147:14) in the
dry tropics of Queensland, Australia, was lled with headwater
from irrigation of a eld pre-treated with the recommended dose of
atrazine (3.3 kg per hectare). 240 g of bacterial homogenate was
suspended in 20 L of water, and applied by hand by spreading
evenly across the surface of the holding dam. Duplicate 1 L samples
were taken before the dam was lled with atrazine-contaminated
runoff water, before the enzyme was added and at time intervals
after the addition of the enzyme. Samples were stored immediately
on ice to stop the enzymatic reaction. Samples were frozen after no
more than 4 h on ice.
2.3. Determination of atrazine concentration
Atrazine concentrations were determined at two independent
laboratories; 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 the
following LCMS method. Briey, 100 mL samples were acidied
with HCl to pH 2.8, then the atrazine in the samples was concentrated 1000-fold by solid phase extraction using preconditioned
Oasis SPE Max Cartridges (Waters, USA), and eluted in 3 mL of
MeOH (with ammonia). Samples were subsequently dried and
dissolved in 100 ml of MeOH. Samples were separated by HPLC and
assayed for atrazine concentrations by measuring the absorbance at
265 nm, and the analyte peak area calculated using Analyst software. Replicate samples were within 10% agreement. The identity
of the HPLC peak was conrmed by mass spectrum analysis,
whereby atrazine ions 216 m/z were extracted on an Agilent
ToFeMSD.
3. Results and discussion
The water in the holding dam contained 8e12 mg/L atrazine
before the irrigation tailwater was collected (data not shown). After
lling with irrigation tailwater the atrazine concentration rose to
157e170 mg/L (Fig. 2). There was a lag in the rate of atrazine
depletion after addition of the enzyme, which was most likely
attributable to the rate at which the enzyme mixed with the water
in the holding dam. The duration of the mixing phase for enzyme
applied in this manner is almost certainly dependent on the
volume and surface area:volume ratio of the water body to be
remediated; 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 addition
of the enzyme led to >90% depletion in the concentration of atrazine in the rst four hours after addition. This result suggests that
a TrzN-based bioremediant for triazines is technically feasible,
although the rate of remediation is slow compared with early trials
of the only other enzymatic bioremediant, OpdA. OpdA reduced
methyl-parathion concentrations by 10-fold reduction from
84,000 L in less than 10 min (Russell et al., 2001), 24-fold faster

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2077

References

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).

than TrzN removed atrazine in this trial. However, OpdA was dosed
into a smaller volume of owing water, which almost certainly
improved the rate of mixing of the enzyme and its performance
overall.
OpdA is an exceptional enzyme, and catalyses methyl-parathion
hydrolysis at near diffusion limited rates (ca. 7000 mg methyl
parathion/second/mg of OpdA) (Jackson et al., 2008). TrzN has
a considerably slower turnover rate (ca. 13 mg atrazine/second/mg
TrzN) (Shapir et al., 2005), and so greater quantities of TrzN are
required to obtain rates of atrazine hydrolysis comparable to those
obtained with OpdA-mediated methyl-parathion hydrolysis. If
required, the rate of atrazine hydrolysis by TrzN could be improved
by directed evolution, in the manner that has been reported for
AtzA (Scott et al., 2009).
4. Conclusion
The reduction of herbicide runoff from agricultural lands is a key
component of the Reef Water Quality Protection Plan (RWQPP) for
the Great Barrier Reef World Heritage Area (Anon, 2003) and
herbicide management is thus a major consideration of regionally
developed Best Management Practices through Water Quality
Improvement Plans/Reef Rescue programs in response to the
RWQPP. The commercial development and application of enzyme
products to remove herbicide residues before they enter waterways
of the Great Barrier Reef catchment area provide an alternative
strategy to reduce herbicide runoff and thus reduce their potential
impact to receiving water environs. This application may also allow
sugar cane growers and horticulturalists to continue using products
such as atrazine to maintain productivity requirements. Moreover,
this enzyme has broader applications for the removal of atrazine
from contaminated surface and ground waters around the world.
Further eld trials will be required to more accurately gauge what is
required to make this technically successful remediation technology commercially viable.
Acknowledgements
We would like to thank Wayne Dal Santo for allowing us to use
his farm in this eld trial, and Simon Christen at QHFSS for helpful
discussions.

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