Observing Microbial Diversity and Horizontal Gene Transfer in a Shallow Contaminated Aquifer

Cornell Univ - State: Awds Made Prior May 2010, Ithaca NY

Investigators

Abstract

This project is designed to advance knowledge about the diversity and activity of microorganisms residing in subsurface water and sediments in a shallow naphthalene-contaminated aquifer in South Glens Falls, NY. This site has been studied hydrologically, chemically, and microbiologically for a decade and features an extensive infrastructure and both hydrogeochemical and microbiological data bases for developing and testing hypotheses about factors controlling the identity, activity, and genetic exchange processes of resident microorganisms. The four main project objectives are: (i) to assess the diversity of general heterotrophic microorganisms residing in water and sediments; (ii) to assess the diversity of microorganisms' metabolic pathways, and dioxygenase genes responsible for metabolizing the naphthalene (the major environmental contaminant on site); (iii) to discover the mechanisms of horizontal gene transfer causing the subsurface microorganisms to adapt to naphthalene exposure; and (iv) to assemble a heterotrophic geochemical budget for the site that quantifies the role of microorganisms in carbon and energy flow through the system. These objectives will be achieved by sampling site waters and sediments quarterly for 5 years. Sampling locations will be inside and outside the contaminated zone and key climatic and biogeochemical parameters [O2, other electron acceptors, bicarbonate, dissolved organic carbon (DOC), N, P, and organic contaminants] will be measured and used for interpretive purposes. Microbial characterization procedures will include growth and isolation on ecologically-relevant carbon sources (site DOC and naphthalene) -- as well as nucleic acid-based procedures [e.g., DNA extraction, cloning, sequencing, PCR, and Terminal-Restriction Fragment Length Polymorphism (T-RFLP)]. Measures of diversity will be based on phenotypic (colony type, Gram stain, physiology, etc.) and genotypic [e.g., 16S rRNA sequences, naphthalene dioxygenase (nahAc and analogues)] traits. Microscopic examination of site populations will play a critical role in achieving this project's goals. Fluorescent In situ Hybridization (FISH) will be used in conjunction with Substrate Specific Direct Viable Count (SS-DVC), and a general respiratory assay (electron transport reduction of a tetrazolium compound to its fluorescent form) to independently assess the identity, physiological potential, and gene expression of individual microorganisms residing in this site. Prior studies at the site have indicated that naphthalene catabolic plasmids within the native populations play a role in metabolic adaptation. Factors controlling plasmid transfer will be examined in laboratory and field experiments. This project will advance knowledge of the identities and roles that microorganisms play in this subsurface habitat. To date, no complete census of microorganisms residing in any habitat has been achieved. By systematically applying both culture-based and nucleic acid-based procedures at this already well-studied site, a complete census may be approached. But more importantly, the diversity of microbial metabolic function will be determined in this unique setting where introduced naphthalene has strongly influenced the local biogeochemistry. Because of its relatively simple biogeochemical characteristic and the preexisting database, this site is ideal for the ambitious task of cataloging the presence, heterotrophic activity, and genetic exchange mechanisms of naturally occurring microorganisms. Discoveries of the roles of individual microbial populations in biogeochemical cycling will be conveyed to the public through written documents, an internet web site, and site visits.