Groundwater is the major source of drinking water in many European countries and in Denmark alone it accounts for more than 99% of the drinking water supply. Within the past decade pesticide residues have more frequently been detected in the groundwater, and in many cases at levels exceeding the 0.1 µg/l limit set by the European Community, raising public concern related to potential health problems. As a consequence, drinking water wells have had to be closed, and vast amounts of money is spent on establishing new wells and for conventional non-biological cleaning processes. Phenylurea and
-triazine herbicides are among the most commonly used groups of pesticides in Europe and probably also globally. Furthermore, representatives of the phenylurea herbicides are intensively used as anti-fouling components in paints and as broad-range herbicides in urban areas
broadening the environmental exposure. Both groups are represented among suspected endocrine-disrupting contaminants, where especially the
-triazine herbicide atrazine and phenylurea herbicide linuron have attracted attention. For all these pesticides stable metabolites have been reported, and some of these are detected in surface- and groundwater more frequently than the pesticides themselves. Another example of this phenomenon is the herbicide dichlobenil, where its metabolite 2,6-dichlorbenzamid (BAM) has been the most frequently encountered contaminant in Danish groundwater since 1993. Clean drinking water is a limited and decreasing resource and there is a demand for development of efficient and cost-effective methods for purifying soil and water resources contaminated with the most heavily used pesticides.
Bioremediation, defined as "the process of judiciously exploiting biological processes to minimize an unwanted environmental impact; usually it is the removal of a contaminant from the biosphere", offers a cost-effective alternative to more common non-biological approaches for treatment of pesticide-contaminated water or soil. It is well-known that most pesticides are microbially degraded in different environments, and the metabolic plasticity enabling this is only beginning to be known on a detailed genetic level. Pesticide-degrading bacteria have been isolated from various natural environments, and especially the high microbial diversity in soil has served as a rich source of efficient degradative strains. Bacterial strains with the ability to use the beneficiary characteristics of pesticides, i.e. as an alternative source of essential nutrients, facilitating the complete mineralisation to harmless inorganic components, offer a potential for finding efficacious bioremediation agents for purifying contaminated environments. To evaluate the bioremediation potential of pesticide-degrading strains accurate and rapid methods for monitoring population size and specific activity in environmental samples need to be available. Such methods will create a deeper understanding of the microbial ecology of
pesticide degradation and eventually facilitate development of new more successful bioremediation strategies.
The overall objective of this project is to develop a molecular toolbox using state-of-the-art DNA-techniques enabling monitoring of selected pesticide-degrading bacteria in environmental samples, with the aim of providing a thorough understanding of the processes underlying bacterial-based bioremediation. The project involves selection and molecular characterisation of pesticide-degrading bacteria in regards to phylogeny, metabolic pathways, functional genes and degradation efficiency under various environmental conditions. Genetic markers suitable for detecting abundance and specific catabolic activity will be elucidated among the degradative strains, with the aim of applying microchip array technologies for rapid and extensive analysis of environmental samples. Potential genetic markers will involve genes encoding catabolic enzymes as well as gene fragments specific for different phylogenetic groups. Additionally, quantitative PCR methods will be employed for quantifying functional gene abundance and expression.
The overall objective of this research project is to characterise pesticide-degrading bacteria with the aim of developing molecular methods for monitoring their numbers and activity when serving as bioremediation agents in contaminated environments. Focus will be given to bacterial strains metabolising the phenylurea herbicides isoproturon and linuron, the benzonitrile herbicide dichlobenil and its stable metabolite BAM. The project is divided into three sections and the specific objectives of these are:
Selection of candidate strains.
Different pesticide-degrading bacteria will be compared with the aim of selecting efficient strains serving as basis for the remaining part of the research project. Strains available for culture collection, international collaborators and other sources will be included in the selection. The selection of the strains will be based on comparison of the stability of their metabolic activity, the catabolic pathways, the range of related compounds degraded, and their degradation efficiency in experiments with artificially contaminated soil and groundwater. Preferably the candidate strains should have a stable catabolic pathway and mineralise the pesticide without significant transient accumulation of metabolites.
Genetic characterisation of pesticide-degrading strains.
Candidate strains will be subject to a detailed molecular characterisation with the aim of finding appropriate genetic markers for monitoring their presence and specific catabolic activity in-situ. New isolates will be characterised by 16S rRNA gene sequencing and biochemical tests allowing a phylogenetic analysis. In addition, other phylogenetic markers from the strains will be sequenced where possible, to enable construction of gene probes that will selectively detect closely related organisms. Presence of known catabolic genes in the candidate strains will be detected by PCR amplification using specific primers. Attempts will be made to determine whether the catabolic genes are associated with plasmids or are located in the chromosome, and whether they are constitutively expressed or inducible. Novel genes coding catabolic enzymes involved in the bacterial pesticide metabolism will be attempted elucidated with the aim of increasing the amounts of potential metabolic markers available.
Development and evaluation of microarray methods for in-situ detection.
With the aim of developing molecular tools for monitoring the candidate strains in bioremediation environments microchip arrays and quantitative PCR techniques targeting the selected genes will be applied. These will focus on detecting presence and specific catabolic activity. Microchip arrays will be applied with the aim of developing "phylogenetic and functional gene arrays" defining the community structure and the expression of genes involved in bacterial pesticide degradation in bioremediation environments. Real-time PCR will be included with the aim of quantifying the functional genes and their expression. The level of gene expression will be monitored by reverse transcription PCR (RT-PCR) specific for target mRNA, and the functional genes will be quantified by targeting specific PCR-amplified DNA-sequences. The developed molecular toolbox will finally be evaluated by batch and column experiments using the strains as bioremediation agents in contaminated groundwater while monitoring presence, activity and pesticides residues over time. Here fluorescence in situ hybridisation (FISH) using universal and specific probes will be included for quantifying overall community size and the specific degraders.
Description of the work
The work is divided into three successive work packages, where each has its own objective and research methodology:
Selection of candidate strains
. GEUS has a collection of pesticide-degrading bacteria including three strains mineralising isoproturon, three strains able to mineralise linuron and several promising enrichment cultures metabolising the persistent metabolite BAM.
Molecular characterisation of pesticide-degrading strains.
Several of the pesticide-degrading bacteria included in this project are newly isolated strains. These will initially be characterised by sequencing of the 16S rRNA gene and additional phylogenetic markers, such as DNA gyrase, to facilitate the development of gene probes that will selectively detect very closely related bacteria. New catabolic genes will be elucidated by using gene knock-out techniques (e.g. using mini-Tn5 transposons and standard vectors) and by employing transcriptome and proteome techniques. This approach will be used to detect differential expression of genes that are up-regulated in response to pesticide degradation i.e. identify transcripts by hybridisation with non-induced RNA and proteins by 2D electrophoresis. The sequence of novel genes involved in pesticide degradation will be determined by a combination of cloning and PCR followed by sequence analysis. This analysis will finally enable selection of proper markers for the development of the molecular methods for in-situ detection.
Development of metabolic and taxonomic microarrays for environmental applications.
The specific phylogenetic and metabolic probes characterised among the degradative strains will serve as the basis for preparation of microchip arrays for detecting presence and activity of the strains in-situ. This will be enabled using the facilities available at QUESTOR. Quantitative PCR techniques will be used on DNA and mRNA extracted from the cultures allowing determination of gene number and expression. The level of gene expression will be monitored by RT-PCR specific for target mRNA, and the functional genes will be quantified by targeting specific PCR-amplified DNA-sequences.
Research facilities and partners
This project is financed by the Danish Technical Research Council covering a total of two man-years research together with related consumables and travel expenses. The project will be managed and performed by Senior Researcher Sebastian R. Sørensen, Department of Geochemistry, GEUS (
firstname.lastname@example.org). The research will be carried out as a two-year project (Nov. 2004 - Nov. 2006). The research will be performed at the Microbiology Laboratory at the Department of Geochemistry, GEUS and at the Environmental Genomics and Microbiology Laboratory within the Queen`s University Environmental Science and Technology Research (QUESTOR) Centre at the Queen`s University Belfast, United Kingdom. Moreover, the project will involve other international collaborations and aims at educating 1 - 2 new MSc students within the fields of environmental biotechnology and applied microbiology.
Within the last decade GEUS has emphasised research dealing with the fate of pesticides in soil and groundwater. At GEUS the research integrates classical geology research areas with disciplines such as hydrology, microbiology, and new research areas such as immunochemistry and environmental genomics. GEUS has well-equipped laboratory facilities, databases and research experience from mapping and monitoring activities related to contaminated sites. GEUS has several fully equipped experimental field sites and extensive expertise in advanced subsurface field work. GEUS has the facilities for measuring low concentrations of pesticides in groundwater with a range of different techniques including HPLC-MS/MS, GC-MS/MS, 14 C-tracer techniques and immunoassays. The Department has a unique expertise in studies of microbial pesticide degradation and a extensive record of high-quality international publications within this area. Additionally, the Department is well-equipped for DNA and RNA work including facilities for Real-Time PCR, DNA-sequencing, DGGE-fingerprinting, RT-PCR, GFP- and luxAB-labelling of bacterial isolates and FISH using oligonucleotide probes.
Parts of the project will be performed at the Environmental Genomics and Microbiology Laboratory within the industry-university collaborative centre QUESTOR at The Queens University Belfast (UK) under the guidance of Dr. Michael J. Larkin . QUESTOR is an interdisciplinary centre that is acknowledged as one of the leading in the UK. The disciplins include molecular microbiology and ecology, post-genomic technology, geochemistry and tracer technology and chemistry, on-site remediation, hydrogeology and waste treatment engineering linked to developments in computer modelling and environmental communications. An in-house probe database based on published rRNA sequence data for microbial community profiling applications, including primers for functional gene detection, is available in this laboratory.
QUESTOR has integrated this database with a laboratory information management system, which will support the production of custom microarrays for a variety of environmental applications. QUESTOR also has a "clean- air" laboratory custom built to run a BioRobotics MicroGrid II TAS array spotting robot, for the production of DNA "microarrays" and "macroarrays". The instrument can immobilise different DNA probes as arrays of discrete spots on either glass microscope slides (at a density of>
100,000 spots per slide), or on nylon membranes. Following hybridisation to samples of appropriately labelled DNA or RNA, glass slide arrays can be scanned using a Tecan LS-200 confocal laser scanner, and image analysis performed using TecanArray software. A liquid-handling robot and a multifunctional microplate reader are also available to support development work using microarrays - either on glass slides / membranes, or using alternative formats such as microspheres / microtitre plates. The facility can produce DNA microarrays that have applications for microbial community profiling, and for detection of specific microbes or functional genes in environmental samples. In addition, a quantitative PCR instrument is available for applications that require PCR-based detection, or estimation of target gene copy number.