Potential of willow and its genetically engineered associated bacteria to remediate mixed Cd and toluene contamination

The purpose of this study was to investigate if bacteria with beneficial properties that were isolated from willow growing on a metal-contaminated site can be further equipped with genes coding for a specific degradation pathway to finally obtain transconjugants that can be inoculated in willow to improve phytoremediation efficiency of mixed contaminations. Cultivable rhizosphere bacteria and root endophytes were isolated from willow (cv. Tora) growing on a metal-contaminated soil. All isolated strains were tested for their metal resistance and potential to promote plant growth. The two most promising strains were selected and were equipped with the pTOM plasmid coding for toluene degradation. Both transconjugants were inoculated separately and combined in willow cuttings exposed to mixed Cd-toluene contamination, and their effect on phytotoxicity, Cd uptake, and toluene evapotranspiration was evaluated. Many of the isolated strains tested positive for the production of siderophores, organic acids, and indole acetic acid (IAA) and showed increased Cd resistance. The Cd-resistant, siderophore-producing rhizosphere strain Burkholderia sp. HU001 and the Cd-resistant root endophyte Pseudomonas sp. HU002, able to produce siderophores, organic acids, and IAA, were selected as receptors for conjugation with the toluene-degrading Burkholderia vietnamiensis BU61 as a donor of the pTOM-TCE plasmid. Although inoculation with the individual transconjugant strains had no effect on plant growth and negatively affected Cd uptake, their combined inoculation resulted in an increased shoot biomass upon Cd-toluene exposure did not affect Cd uptake and strongly reduced evapotranspiration of toluene to the atmosphere. In this study, inoculation of willow with a consortium of plant-associated bacteria equipped with the appropriate characteristics resulted in an improved phytoremediation of a mixed Cd-toluene contamination: the degradation of toluene was improved leading to a decreased toxicity and evapotranspiration, while Cd uptake and translocation were not affected.