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Australasian Biotechnology, Vol. 11 No. 3, 2001, pp. 24-26 BIOPROCESSING ENZYMATIC BIOREMEDIATION OF CHEMICAL PESTICIDESRJ Russell, TD Sutherland, I Horne and JG Oakeshott, CSIRO Entomology, Australian Cotton CRC and CRC for Sustainable Rice Production, GPO Box 1700, Canberra, ACT 2601, Australia; M Zachariou, CSIRO Molecular Science, Bag 10, Clayton South, Vic. 3169, Australia; HV Nguyen, ML Selleck and M Costello, Orica Australia Ltd, Gate 4 Stanford Street, Ascot Vale, Vic, 3032, Australia. Code Number: au01040 Abstract Enzyme-based bioremediation technologies are potentially capable of qualitatively quicker action than traditional microbial bioremediation processes. This in turn qualitatively expands their potential applications. CSIRO and Orica Australia are developing an enzyme technology for remediating pesticide residues in applications as diverse as waste irrigation waters, surface contaminated fruit and vegetables, and spent dip liquors for a variety of agricultural productions and processing industries. Effective and affordable remediation under these circumstances requires some highly specialised enzymes. However early trials on the team's first organophosphate degrading enzyme have shown it is feasible to achieve remediation in such circumstances. Introduction Growing community conviction that industry must meet social and environmental as well as economic objectives is creating opportunities for a range of new biotechnological products. One set of opportunities lies in the field of bioremediation, which can be defined as remediation of contaminants by biological means. This article shows how modern biology is extending the scope of bioremediation technologies by outlining the progress of a joint CSIRO-Orica Australia Ltd initiative to use enzymes in the clean-up of pesticide residues. Historically, bioremediation has largely involved the clean-up of contaminated soils or sludges by in situ culture of micro-organisms selected for their ability to degrade the particular toxicants. Often it has involved little more than effective composting techniques, albeit more sophisticated systems are now also under development overseas involving the use of genetically modified bacteria that can degrade otherwise intractable contaminants (Timmis and Peiper 1999; Pieper and Reineke 2000; Sayler and Ripp 2000). Whether the organisms are genetically modified or not, microbial bioremediation relies on microbial growth to metabolise the toxicants. It is therefore generally a relatively slow process - a few weeks through to several months, depending on the application. Over the last decade there has been increasing interest in the use of formulated enzymes rather than live bacteria as bioremediation agents. These enzymes may be isolated from bacteria or other natural sources or developed in the laboratory using the various techniques of in vitro enzyme evolution now available (Olsen et al. 2000; Walsh 2001; Arnold 2001). Appropriately formulated they could be used in the remediation of soils or sludges. However their major use seems likely to be in the remediation of contaminated liquids and, in some cases, commodities. They are particularly suited to situations where rapid remediation is needed - at most a few hours, perhaps as little as a few minutes. CSIRO and Orica Australia Ltd have recognised that an enzymatic bioremediation technology capable of rapidly detoxifying residues of chemical pesticides would have several applications in modern agriculture. One major application is in the clean-up of waste waters from agricultural production and processing industries. These industries are struggling to meet water-quality regulations that are becoming increasingly stringent through much of the developed world (Naidu et al. 1996; Kennedy 1998). The issue is particularly sensitive in Australia because of the scarcity of our water resources and the concentration of our agricultural industries in sensitive catchments like the Murray-Darling Basin and periurban areas upstream of major populations centres. A second major application for the technology lies in the detoxification of surface-localised residues on fruit and vegetables. Consumer tolerance of such residues is diminishing, as are the maximum residue limits (MRLs) permitted by governments (Rowland 1998; ARMCANZ 1998). End users for this application could be producers/packers, or consumers. The research team is focussing first on enzymes to detoxify several major insecticides that are most problematic in respect of residue issues. These include endosulfan, organophosphates (OPs), carbamates and synthetic pyrethroids (Table 1: representatives with structures , m.o.a., major markets). These compounds in fact account for a majority of insecticide sales, both in Australia and worldwide. Enzyme Discovery Enzymes that might effectively remediate these pesticides in the field must first meet several exacting performance criteria in CSIRO's laboratory trials (Russell et al. 1998, 1999). First and foremost they must obviously break down the pesticides into essentially non-toxic products. Moreover they must achieve this efficiently in terms of their kinetics, because this will impact directly on field performance and price. The team has therefore calculated threshold kinetic criteria for cost-effective use in the major potential markets. Also relevant here is the possible need for cofactors. Many potentially useful enzymes have a requirement for small-molecule cofactors that would be prohibitively expensive to provide in a commercial setting. In addition the enzymes must be robust to the range of temperature, pH and ionic environments that would occur in the various applications targeted. Another complex set of performance criteria concerns substrate range. Versatility of candidate enzymes within a particular pesticide class is crucial. There are perhaps twenty OPs which are widely used in Australian and overseas agriculture. There are fewer carbamates and pyrethroids but the carbamates are structurally very diverse and almost all the commercial pyrethroids are complex racemic mixtures. Even commercial endosulfan is also a mixture of two isomers. There is also a second issue in respect of substrate range. In the field many pesticides are broken down chemically or biologically to products that are also toxic. Endosulfan for example is converted to endosulfan sulfate and it is the latter that accumulates in animal fat and has caused major residue incidents in the beef exports of several producer countries. An effective bioremediation technology for endosulfan should therefore encompass the detoxification of both the parental endosulfan isomers and endosulfan sulfate. Whilst the selection criteria might appear daunting the team now has enzymes for all the insecticide classes above in various stages of development, and an enzyme effective against most OPs is proceeding through downstream production and formulation research and field trialling. Process Development and Implementation Commercial enzyme production will be by industrial fermentation. Depending on the enzyme this might involve fermentation of the source organism or of a standard industrial vector manipulated to express the relevant gene. In the latter event production and downstream processing will need to meet the requirements of Office of the Gene Technology Regulator (OGTR) or equivalent regulatory bodies overseas. This could mean the removal of unlysed cells and transformable recombinant DNA during downstream processing. On the other hand, for most uses under consideration there should be no need for purification of the enzymes away from other soluble materials in the ferment liquor. Application technologies will clearly vary with the application. One early application is expected to be the clean-up of run-off water after pesticide sprays in irrigated industries like cotton, some horticulture and possibly rice. In some of these cases where the volumes of water are large and the time available for treatment is small the requirement for kinetically efficient enzymes is particularly important. Equally however it is critical to achieve rapid mixing of the enzyme(s) through the water. Accordingly several simple physical mixing devices and membrane technologies (Feng et al. 1997; LeJeune et al. 1998; Gordon et al. 1999; Giorno and Drioli 2000) are being evaluated. Different issues arise in implementing the technology in the clean-up of surface-localised residues on fruit and vegetables. The goal for use by packers and processors is to integrate enzyme use where possible into the prevailing washing conditions for the particular commodity. These conditions vary greatly with the commodity but can involve relatively high temperatures, brief exposure times and the presence of detergents. For use by consumers and, in some cases, by packers and processors a further issue is that the identity of potential pesticide contaminants may not be known, so a mixture of enzymes covering a range of possible contaminants may be required. Notwithstanding all the above challenges for effective implementation, early trials with our most advanced OP enzymes are giving extremely promising results for both the clean-up of irrigation run-off and fruit and vegetables. In the case of the former, our first field trial achieved a 10-fold reduction in methyl-parathion residues in 84,000 L of fast flowing run-off water in drainage channels on a cotton farm with a treatment time of only ten minutes. In the case of the latter, early laboratory trials achieved similar levels of reduction in OP residues even on commodities like leafy vegetables, whose complex surface structure might be expected to be problematic for effective remediation. The kinetic and stability characteristics of this enzyme give us confidence that it would perform well in a variety of the commodity washing processes employed by packers and processors. Several other potential uses are also being evaluated. These include the clean-up of used pesticide containers and spent dip liquors. These might be dips used for ectoparasite control in livestock industries or for fruit-fly disinfestation in horticulture. While individually small as market opportunities, technical feasibility for applications like these is relatively high. This is because treatment times can generally be much longer, and treatment volumes much smaller, than for clean-up of waste water flows from broad-acre irrigation operations. Several of our enzymes have now been tested and shown to be effective in cleaning up pesticide 'spikes' in dip liquors from various industries. Table 1. Representative structures of the four major classes of insecticides
Conclusions Chemical pesticides will be a major component of food and fibre production systems throughout the world for the foreseeable future. It has been estimated that agricultural outputs would diminish by 30-40% without them (Oakeshott and Whitten 1993; Kennedy 1998). The introduction of transgenic crops expressing effective insect resistance and other input traits could significantly reduce the dependence on chemical pesticides in the medium term. Nevertheless, quite apart from any market acceptance issues, there will be many production systems where the diversity of pest and disease problems makes transgenic control unfeasible in the foreseeable future. Balanced against the ongoing need for chemical pesticides is a growing concern about their non-target toxicity and unintended side effects. This has been addressed to some extent by the efforts of the agrochemical industry to develop more target-specific pesticides and by improvements in the pesticide and water management practices of producers and processors. As yet however the companies have had only modest success in developing new chemicals which are more target-specific but still cheap and efficacious. And even with significant improvements in their pesticide and water management practices, several production and processing industries still struggle to meet the increasingly stringent residue requirements of government regulatory bodies. There are therefore major market opportunities for effective pesticide remediation technologies in a range of industries and applications. Since many of the potential applications require rapid action many of the traditional microbial bioremediation strategies are inappropriate. Enzyme formulations can in theory act with the necessary speed, although there are still significant practical challenges both in the development and implementation of the requisite enzymes. The CSIRO-Orica Australia team is exploiting a range of modern biotechnologies to meet these challenges and trial products are now meeting exacting performance criteria in a range of field and laboratory trials. Acknowledgements The authors gratefully acknowledge the other members of the bioremediation team, including in particular, Susan Dorrian, Michelle Williams, Rama Heidari, Kahli Weir, Narelle Dryden and Geoff Dumsday, Kylie Henderson and Miriam Eldridge. Also acknowledged is the financial support for the project from Horticulture Australia Ltd, the Cotton and Rural Industries Research and Development Corporations, and the International Wool Secretariat. References
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