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10 - sutherland

USING ENZYMES TO CLEAN UP PESTICIDE RESIDUES Tara Sutherland and Robyn Russell from CSIRO Entomology in Canberra, Australia, and Michael Selleck ofOrica Australia Pty Ltd. describe exciting work being done on the use of enzymes to clean up pesticideresidues Current research focuses on several major insecticide classes Increasing pesticide use in recent years has led to public including organophosphates, carbamates, synthetic pyre- concern about the social and environmental impacts of throids and the organochlorine, endosulfan. pesticide residues. Of particular concern is the contamina-tion of irrigation run-off and drainage water, agriculturalsoils and horticultural products. Detoxification of pesticide residues in contaminated soil The team has isolated a soil microorganism (an Agrobac- has been achieved by introducing, and/or encouraging, the terium) that utilises organophosphate compounds as growth of microorganisms that are capable of detoxifying nutrient sources and detoxifies them. The organophosphate- the residues on site – a technology known as bioremediation degrading enzyme isolated from this bacterium attacks the (detoxification using biological material). A well-known phosphoester bond of aromatic oxon and thion organophos- example of this is the clean up of petrochemical contami- phates, but does not have activity against the aliphatic nated Olympic sites in Sydney. This method of bioremedia- versions of these compounds. However, protein engineering tion is based on traditional composting techniques and relies technologies have been employed to create a synthetic on microbial growth to metabolise the toxicants. The detox- variant of this enzyme that also degrades the aliphatic ification process is generally slow, taking weeks to months compounds. Collectively, the original enzyme and variants to accomplish. Furthermore, the methodology is not suited are predicted to degrade 90% of registered organophos- to the generally low aeration, low nutrient state of contami- phate compounds. The performance of the original enzyme for decontami- However, the microorganisms that break down toxicants nating organophosphates has been evaluated in several field in contaminated soil can be sources of enzymes that will trials. In the first field trial, methyl parathion levels in detoxify pesticide residues in such situations. The applic- 80,000 L of fast flowing run-off water in cotton farm ation of these enzymes is particularly suited to pesticide- drainage channels were reduced by 90% in less than ten contaminated water as they can achieve rapid remediation minutes. This is a low concentration/high volume source of without the addition of nutrients or aeration. The problem of pesticide contamination of irrigation water must be resolved before it can be released into water-ways. CSIRO Entomology, in conjunction with members ofthe Advanced Water Technologies business of OricaAustralia Pty Ltd. and CSIRO Molecular Sciences, hassuccessfully developed enzyme-based bioremediation tech-nologies for detoxifying pesticides in contaminated water.
This technology is being extended to the clean up of pesticidespills, the clean up of rinsings from spray equipmentwashdown, and for treatment of horticultural produce.
The search for such enzymes can be divided into four source of enzyme is identified in bacteria or from other the gene encoding the enzyme is identified and isolated the enzyme is cloned into a common bacterium, such as Figure 1. Assembly of the enzyme dosing and mixing system.
Escherischia coli Foreground: Orica employees Michael Selleck (left) and
the enzyme is produced by industrial-scale fermentation of Andrew Dowd (right) lock the static mixer elements into
the E. coli. Once there is a sufficient volume, the E. coli is place. Following assembly, the unit was lowered into the drop
box and inserted into the culvert. Background: Pipe work for
killed off, the enzymes which have been produced by the the enzyme dosing system which was used to transfer and
bacterium are collected and applied to the contaminated inject the dilute enzyme solution to the entry point to the
This journal is The Royal Society of Chemistry 2002 soil. In contrast to the organophosphate degradingbacterium, which has the ability to degrade many differentorganophosphates, this bacterium has a narrow substraterange and does not detoxify other carbamate pesticides.
However, the enzyme that is responsible for this activity isstable under a variety of conditions, does not require any co-factors for activity and results in substantial detoxificationof carbaryl, a pesticide used worldwide on horticulturalproduce. CSIRO Entomology will use protein engineeringtechnologies in an attempt to increase the substrate range ofthis enzyme so that it can be used to detoxify othercarbamate compounds.
Pyrethroids
Another hydrolase enzyme with potential for use in biore-
mediation has been isolated from insects that are resistant to
certain pesticides. Protein technologies have again been
employed to design variants of this enzyme that can degrade
pyrethroid insecticides. These enzymes are stable against a
range of biotic and abiotic challenges (e.g. extremes of pH
and temperature, and bacterial protease digestion) with half-
lives in some agricultural waste streams of a number of
days. These enzymes can be produced on a large scale in
bacteria using fermentation techniques, and they are being
trialed for commercial use in the detoxification of pyrethroid
residues.
Figure 2. Preparation of the dilute enzyme solution. Orica
employee, Michael Selleck, mixing in 8 l of enzyme
The CSIRO Entomology team has also isolated bacteria that concentrate with 172 l of a buffer solution. The 180 l of dilute
degrade the two isomers of endosulfan and the toxic enzyme solution was subsequently used to treat 80,000 l of
metabolite of endosulfan, endosulfan sulfate. These bacteria were isolated by providing endosulfan, as the only source ofsulfur, to a soil microbial population. Sulfur is an essential pesticide-contaminated water, that also contains high levelsof silt and other particulate matter. In a second field trialconducted at the Victorian Department of Natural Resourcesand Environment, Tatura, enzyme treatment of rinsate fromthe washdown of pesticide spray equipment achieved areduction in methyl parathion concentration of 90% in 10minutes, and 99% after 1 hour. In contrast to the run-offwater in the first trial, rinsate is a high concentration/lowvolume source, which also contains organic solvents.
Other applications of enzyme technology are also being investigated. One application with considerable potential isthe treatment of used sheep dip liquor, which contains theorganophosphate diazinon. Laboratory studies have providedproof of concept and planning for a field trial is in progress. Asecond application concerns the treatment of permethrincontaining effluent from the wool dyeing operations. Laboratory studies aimed at establishing proof of concept are underway. Enzymes also have the potential to help in Figure 3. Treatment of irrigation run-off water. A small volume
decontamination of polluted soils where they could be used of the run-off water leaving the field was pumped out of the
in solutions applied directly to damp soil.
drop box by a submersible pump (lower left). To this volume,
the dilute enzyme solution was dosed proportionally and pre-
mixed prior to being injected into the bulk of the run-off
water via the injection line (vertical pipe, centre). The static
mixer that was previously inserted into the culvert provided
A bacterium that degrades the carbamate insecticide, brief, but intense mixing of the injected enzyme/run-off mix
carbaryl, has been isolated from carbamate-contaminated with the bulk of the run-off.
component of living matter. Therefore only the bacteria that Lyndall Briggs, Erica Crone, Sue Dorrian, Robyn Russell could release the sulfur from endosulfan could survive.
and John Oakeshott (CSIRO Entomology); Michael Selleck, Removal of sulfur from either endosulfan or endosulfan sulfate Mel Costello and Hung Nguyen (Orica Australia Pty Ltd); results in substantial detoxification of these compounds. The Geoff Dumsday and Michael Zachariou (CSIRO Molecular enzymes responsible for this activity are monooxygenases.
Science) and is funded by Horticulture Australia Limited, They differ from the hydrolase enzymes described above in Cotton Research and Development Corporation, Australian requiring co-factors for activity. CSIRO and Orica Australia Cotton CRC, CRC for Sustainable Rice Production and are currently investigating the use of these systems for biore- Orica Australia Ltd. Orica also wishes to thank the Victorian Department of Natural Resources andEnvironment (NRE), Tatura, for hosting the latest field trial. Contacts: Robyn Russell, CSIRO Entomology Ph 02 6246 4 Commercialisation of the enzymatic bioremediation Fax 02 6246 4 Email [email protected] technology could involve three types of products.
Michael Selleck, Orica Australia Pty Ltd Ph 03 9283 6342 A freeze-dried enzyme powder for treatment of contami- Fax 03 9283 6266 Email [email protected] nated water. This will have a shelf life of several monthsand will be reconstituted prior to application to the conta- minated water.
Russell, R. J.; Harcourt, R. L.; Oakeshott, J. G. (1998). Bioremedi- A polyurethane pad containing immobilised enzymes.
ation of pesticides using enzymes. In ACIAR Proceedings No. Prototypes of this product have proven effective for 85: Seeking Agricultural Produce Free of Pesticide Residues. I.
soaking up and decontaminating pesticide spills and R. Kennedy, J. H. Skerritt, G. I. Johnson and E. Highley (eds), wiping down spraying equipment.
pp 341–348.
Russell, R. J.; Harcourt, R.; Sutherland, T.; Nguyen, H.; A self-contained user-friendly device containing Oakeshott, J. G. (1999) Can we clean water with microbes? immobilised enzyme that will allow the detection of Microbiology Australia, 20, 14–17.
pesticide residues at concentrations as low as 20 parts per Russell, R. J., Sutherland, T. D., Horne, I., Oakeshott, J. G., billion is being developed. Zachariou, M., Nguyen, H. V., Selleck, M. L.; Costello, M.
(2001) Enzymatic bioremediation of chemical pesticides.
Chemical pesticide usage is unlikely to decline substantially Australasian Biotechnology, 11, 24–26.
in the near future, despite the development of transgeniccrops and other alternative biological controls. Environmentaland safety concerns are leading to increasingly stringentresidue requirements by regulatory authorities. Whilst theseconcerns are being addressed through better pesticide andwater management practices, there is an increasing need for Tara Sutherland is currently doing a second post-doc in the Biopro- rapid and effective remediation technologies in many cessing and Enzymology section at CSIRO Entomology, Canberra,Australia, following post-doc work in Arizona, USA.
industrial and agricultural processes. The CSIRO-OricaAustralia joint initiative is developing biotechnologies to Robyn Russell, who has 23 years post-doctoral experience in address this need.
molecular biology and biochemistry, leads the Bioprocessing andEnzymology project at CSIRO Entomology.
Michael Selleck is a chemical engineer and molecular biologist, and has spent the past two and a half years working on enzymatic The research is being carried out by Tara Sutherland, Irene bioremediation technology for Orica Australia Pty Ltd.
Horne, Kahli Weir, Christopher Coppin, Michelle Williams, EVER THOUGHT OF WRITING AN ARTICLE FOR PESTICIDE OUTLOOK?
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