| Trichloroethylene (TCE), a widely used solvent and ubiquitous contaminant, is effectively removed from soil and groundwater by the use of plants (phytoremediation). Rapid removal has been reported at the root-zone (rhizosphere), where methanotrophs (methane-oxidizing bacteria) capable of co-oxidizing TCE are present. The objective of the study was to determine, by the development of an adequate protocol, how plant type, system design, and environmental conditions present at two phytoremediation sites impacts methanotroph's biodegradation potential. To develop characterization methods, phenotypic and genotypic analyses of an uncharacterized methanotroph, Strain CSC1, isolated from an uncontaminated groundwater aquifer, were performed. Field sites represented an engineered system, with poplar and willow trees, and a natural loblolly pine re-growth area. Laboratory studies were conducted to assess the ability of methanotrophs to oxidize pine exudates (monoterpenes) and its effects on TCE oxidation. Field samples were analyzed by culture-dependent microbial counts and enrichments, and culture-independent stable isotope probing (SIP) microcosms and molecular methods. Strain CSC1 possessed a unique spiny S-layer and was shown to be a novel strain of the genus Methylocystis and was named Methylocystis aldrichii sp. nov. Characterization methods developed with Strain CSC1 were successfully applied to phytoremediation field samples and isolates. Different types of methanotrophs were capable of oxidizing monoterpenes (alpha-pinene) and, in the presence of TCE; antagonistic and synergistic responses were observed depending on methanotroph type. Rhizosphere samples analyzed by culture-dependent methods confirmed the presence of methanotrophs at both sites; however, enrichments were biased towards type II methanotrophs and did not correspond with the active populations. Active populations were more diverse and abundant in the planted samples and strongly influenced by the design, especially the use of planting material that resulted in a dominance of thermotolerant methanotrophs. Variable results between the engineered and natural settings highlight the importance of measuring oxidation potentials and diversity of rhizosphere methanotrophs at any phytoremediation site, especially if monoterpene-releasing plants are contemplated for use. Also, study of active populations was shown to be the most accurate characterization method. Phylogenetic analysis combined with SIP microcosms offers powerful analytical tools that can ultimately aid practitioners in optimizing phytoremediation for more effective treatment. |
