BioMatNet Item: QLK3-2001-00101 - Testing integrated GM-Rhizoremediation systems for soil bioremediation (GM-Rhizoremediation)

[BioMatNet Database - FP5 Quality of Life Programme]

QLK3-2001-00101
Testing integrated GM-Rhizoremediation systems for soil bioremediation (GM-Rhizoremediation)

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Biotechnology : Fine Chemicals : Quality of Life - 3. The Cell Factory

Type of Project	Research contract
Contract No	QLK3-2001-00101
Total Cost
EC Contribution	1,186,457 EUR
Start Date	01-01-2002
Duration	36 Months

Abstract

This project intends to advance rhizoremediation - the combined application of plants and rootzone bacteria for soil cleaning - in two essential aspects.

GM bacteria engineered for in situ PCB degradation will be further developed as biomonitoring strains, i.e. to signal the presence of the pollutant in plant and soil and the efficacy of the bioremediation strains.
Molecular detection methods for tracking GM degrader bacteria in the environment will be developed to detect and quantify the GM strains under environmental conditions. Molecular tools may also be used to monitor GM strains following a possible deliberate release. A detailed risk assessment of the GM strains and bioremediation technology will be prepared resulting in a set of guidelines for regulatory authorities and end users.

Objectives

This project aims to develop an integrated biological process (based on GM technology) that can degrade the pollutant and monitor the process at two levels; presence of the target pollutant (PCBs) and expression of introduced GM traits in contaminated soils. Specifically the project will target PCB contaminants as a model system.

It will use newly engineered (rhizoremediation) bacteria in conjunction with plants as a GM process to remediate contaminated soil. These bacteria contain re-regulated degradation pathways that show higher constitutive levels of expression than wild type (WT) organisms and are induced by chemicals found in plant root exudates. The project sets out to further modify these bacteria to act as biosensors to monitor in situ the levels of pollutant and the pollution degrading activity of the rhizoremediation strains. The project will also address the biosafety aspect of the use and acceptance of such GM bioprocesses by developing molecular tools to quantify GM micro-organisms in the environment. A detailed risk assessment will be carried out on the bioprocess and guidelines produced for the safe use of such technology.

Activities

This proposal will make use of genetically modified (GM) rhizoremediation strains (available within the consortium) that have been developed with the following features:

GM strain 1: Pseudomonas fluorescens strain F113rifpcb with colonisation and ecological competence in the rhizosphere of different plants e.g. willow, alfalfa and sugar beet. The strain has been genetically modified to degrade PCBs.
GM strain 2: as GM strain 1, but with boosted expression of the bph genes (BP/PCB degrading pathway).
GM strain 3: as GM strain 1, but with inducible expression of the bph genes.
GM strain 4: as GM strains 1-3 that have limited long term persistence in soil i.e. contained strains.

It is proposed to construct and introduce biomonitoring traits into GM strains 2 and 3 to allow the real-time and non-destructive monitoring of the GM strains and detection of the end-products of the bioremediation process. In addition a novel containment strategy that will tightly couple the GM strains to the plant rhizosphere will be developed and tested. As new GM strains become available molecular tools will be developed to identify and quantify the GM bacteria unambiguously using PCR based strategies. These tools will be evaluated for their efficacy in remediating contaminated soil and their environmental survival and spreading under realistic lysimeter experiments. In addition GM strain 1 is the subject of a deliberate release application by several of the proposed consortium members. If this release proceeds, the tools developed in this proposal will be used to monitor the release site.

The role of the plant in the rhizoremediation process will also be addressed in this proposal. Bioassays will be developed using plant cell cultures and sterile hydroponic whole plants to study the fate of the target pollutant (PCBs) and their end-product chlorobenzoic acids (CBAs). These studies will be extended to plants used in realistic lysimeter experiments to yield useful data on the fate of these compounds during the remediation process.

The biomonitoring tools developed and tested in this proposal will provide data on the fate of the GM bacteria, effect of end metabolites and the role of plants. This data will be used to develop a detailed risk assessment of the rhizoremediation process and will be used to design guidelines, which will be of use for EU regulatory authorities, owners of contaminated sites and users of the technology.

Deliverables

an integrated plant-microbe system for in situ bioremediation evaluated under realistic conditions.
development and evaluation of molecular tools for monitoring and quantification of GM bacteria used in bioremediation process.
the fate of the pollutant (and key intermediates) within the system, i.e. plants and soil, elucidated.
the above system extensively tested in vitro, in microcosm and in the field (lysimeter) in different plant/ soil types.
a detailed risk analysis of the GM based bioprocess prepared.
a set of best-practice procedures for regulatory authorities, sites owners/ public authorities and end users prepared.

Contacts

Coordinator

David N. DOWLING / Institute of Technology Carlow (IT Carlow) Department of Applied Biology and Chemistry / IRELAND

Participant