Role of plant associated bacteria to improve phytoremediation of organic pollutants

Abstract

Phytoremediation, the use of plants for the in situ treatment of contaminated soils and sediments, is an emerging technology that promises effective and inexpensive clean-up of certain contaminated sites. Phytoremediation is most suited for sites with shallow contamination (<5m depth), moderately hydrophobic pollutants such BTEX compounds, chlorinated solvents, nitrotoluene ammunition wastes or excess nutrients (nitrate, ammonium and phosphate) (Schnoor et al., 1995). Although many organic pollutants are metabolized in plants, xenobiotics or their metabolites can be toxic to plants, and this could limit the applicability of phytoremediation. Alternatively, in case of volatile pollutants, plants can release the compounds or their metabolites, through the stomata, which could question the merits of phytoremediation (Schwitzguebel et at., 2002). A possible solution for these problems could be the use of endophytic bacteria, equipped with the necessary degradation pathway(s), which reside within living plant tissue, without doing substantive harm to the plant. Yellow lupine (Lupinus tuteus L.) and their associated endophytic bacteria (Burkhotdena cepacia), equipped with a toluene degradation pathway, were used as a laboratory and greenhouse test species. Toluene was chosen as a model of a highly water soluble and volatile organic compounds. Inoculation with the endophytic bacterial strain B. cepacia VM1330 resulted in a drastic decrease of toluene phytotoxicity and a 3-4 times reduction of toluene evapotranspiration through the leaves. Poplar cells cultures were used to demonstrate that poplar cells do not posses the capability for toluene degradation. Those results supported our hypothesis that for successful phytoremediation collaboration between plants (in our case poplar trees) and their associated microorganisms, with appropriate degradation pathways is necessary. Subsequently, two poplar cultivars (Poputus trichocarpa x dettoides cv. "Hoogvorst" and "Hazendans") have been studied as representative model plants for phytoremediation in the field, with emphasis on the role of their associated bacteria. Plant material was prepared aseptically and the natural rhizosphere and endophytic bacterial communities were characterised and identified by comparative sequence analysis of 16S rDNA. One hundred and forty six morphologically, cultivable distinct isolates were obtained, belonging to twenty one different genera. The most dominant endophytic strains were chosen for further investigations. Re-colonization capability of four different natural endophytic strains, all belonging to Pseudomonas genus, was studied showing that three of them could colonize poplar rhizosphere and roots, two were active colonizers of stem and only one could colonize the leaves. Furthermore, poplar cuttings were inoculated with a chosen endophytic strain (B. cepacia VM1468) equipped, via natural gene transfer, with the TOM plasmid, which is able to degrade toluene. The effect of a 10 weeks application of toluene on the growth of poplar cuttings, inoculated with B. cepacia VM1468, was studied. Toluene evapotraspiration from the aerial parts of the poplar cuttings was investigated. Horizontal transfer of pTOM between introduced strain and naturally occurring endophytic bacteria in poplar cuttings was observed in this experiment. These bacteria were capable to assist their host plant in overcoming toluene toxicity, and at the same time lowered toluene phyto-volatilization. The effect of poplar trees and their associated bacteria was monitored on a field site contaminated with BTEX compounds. During five years of study it was observed that poplar trees, together with their associated rhizosphere and endophytic bacteria, can play an active role in the phytoremediation process. After reaching the groundwater with their roots they stopped further dispersion of the pollutants plume.