THE USE OF PLANT ROOT SYSTEMS IN BIOREMEDIATION

Kristina Lindström¹, Leena Suominen¹, Minna Jussila¹, Anna Kaksonen¹, Alexander Kraft¹, Katri Mäkeläinen¹, Sirpa Tiikkainen¹, Inga Sarand², Teija Koivula², Rainer Peltola^1,2, Kielo Haahtela² and Martin Romantschuk². ¹Department of Applied Chemistry and Microbiology and ²Department of Biosciences, Biocenter 1, P.O.Box 56, FIN-00014 University of Helsinki, Finland

The plant rhizosphere is in microbial ecology traditionally recognized as a niche rich in growth substrates in comparison with the surrounding bulk soil. Since the harsh conditions prevailing in soil have been one of the obstacles in microbial bioremediation, a new concept, rhizoremediation, has been introduced. In rhizoremediation plant roots sustain the degrading microflora by supplying it with nutrients other than the pollutant, and also help spreading the degrading microorganisms to new sites in the soil. We have studied how the perennial legume goat’s rue (Galega orientalis), which fixes nitrogen in symbiosis with Rhizobium galegae, and Scots pine (Pinus sylvestris) in symbiosis with mycorrhiza and mycorrhiza colonizing bacteria could be used to clean soil contaminated with BTEX and other compounds, which are common in oil and gasoline.

In an axenic model system we first demonstrated that both the rhizobia and goat’s rue can survuve high (500-2000 ppm) levels of m-toluate in the medium. When transferred into clean medium the plant commenced growth and nodulation. When Pseudomonas putida carrying the TOL plasmid pWW0 was added to the system, m-toluate was consumed. In pot experiments in the greenhouse a good bioremediation effect was achieved with these organisms added, but in addition to the inoculated pseudomonad, indigenous rhizosphere bacteria were active. Bacteria growing on m-toluate were isolated from the rhizosphere of pot-grown plants in oil or m-toluate contaminated soil and from a field experiment in oil soil. After one growing season in the field, the density of bacteria tolerating m-toluate was significantly higher in the rhizosphere of goat’s rue inoculated with R. galegae and P. putida (pWW0) compared with the bulk oil soil. Most isolates from both experiments were Gram negative and many grew in the presence of 9000 ppm m-toluate. The diversity of the isolates was studied by rep-PCR with 5GTG primers, and PCR-RFLP and sequencing of 16S rDNA. The genomic diversity was high. The presence of the xylE gene as an indicator of the meta pathway of aromatics degradation present on the TOL plasmid was studied by PCR and by addition of substrate for the xylE encoded enzyme 2,3-dioxygenase. Both tests showed that xylE was present in only a small proportion of the cultivated population.

In experiments using the pine rhizosphere for bioremediation purposes it was shown that bacteria isolated from the mycorrhizosphere and equipped, by in vitro conjugation with the TOL-plasmid pWW0 survived, colonized well, and retained the plasmid when a selective pressure was maintained. Furthermore, the plasmid was efficiently transferred to indigenous bacteria in the soil, thus apparently improving the degradation rate of the model contaminant m-toluate. When the soil in a microcosm was contaminated with radioactively labelled pyrene the contaminant was metabolized only when the microcosm was pre-inoculated with soil from an old oil landfarming site. Pine roots, stems and needles were found to contain some of the radioactive label. Whether this uptake was via mineralization followed by CO₂ assimilation or by root uptake of degradation products is under investigation.

Our studies show that the plant rhizospheres used show good potential for bioremediation and that the rhizosphere microorganisms adapt to contaminants. Inoculation with selected inoculants might enhance degradation.