Potential of the Galega Rhizobium galegae system for bioremediation of oil-contaminated soil

Bioremediation potential of the nitrogen-fixing leguminous plant Galega orientalis Lam. and its microsymbiont Rhizobium galegae was evaluated in microcosm and mesocosm scale in oil and BTEX (benzene, toluene, ethylbenzene, xylene) contaminated soils, with m-toluate serving as a model for the latter group. G. orientalis and Rhizobium galegae remained viable in m-toluate fractions up to 3000 ppm. Plant growth and nodulation were inhibited in 500 ppm m-toluate, but were restored when plants were transferred to clean medium. In soil, G. orientalis nodulated and showed good growth in 2000 ppm m-toluate as well as in diesel-contaminated soil in the field, where the plant was stimulating bacterial growth in the rhizosphere. A collection of 52 indigenous m-toluate-tolerating bacteria isolated from oil-contaminated rhizosphere of G. orientalis was characterised and identified by classical and molecular biological methods. 16SrDNA PCR-RFLP and (GTG)(5)-PCR genomic fingerprinting combined with partial sequencing indicated the presence of five major lineages of the Bacteria domain. A TOL plasmid-specific xylE-PCR was developed in order to detect both active and potential degraders of in-toluate. The ability to degrade in-toluate in the presence of the gene xylE was detected only within the genus Pseudomonas. The isolates were tested for capacity to grow on m-toluate as their sole carbon and energy source. In laboratory experiments, the best rhizosphere isolates performed equally well to the positive control strain and are good candidates for inoculant production in the future. They have been tagged with marker genes for further studies on colonisation and persistence.