Objectives: The overall goal of the proposed work is to understand the microbial processes involved in the metabolism of monocyclic, bicyclic, polycyclic aromatic hydrocarbons (PAH) and alkanes in the absence of oxygen. We hypothesize that certain obligate anaerobes have the ability to metabolize these compounds, that they share a similar mechanism for the biodegradation and that the metabolites produced can serve as markers to monitor intrinsic degradation of PAHs in anoxic groundwaters. <P>We plan to evaluate specific biochemical and genetic biomarkers as a tool to assess in situ activity. Specific Objectives <OL> <LI> To determine the diversity of anaerobic communities in NJ groundwaters able to metabolize hydrocarbon contaminants using alternate electron acceptors (e.g. denitrifying and methanogenic conditions). <LI> To examine the key steps in the anaerobic metabolism of the PAH under the different reducing conditions. We hypothesize that fumarate addition and carboxylation are 2 different mechanisms and initiating steps for PAH activation by different anaerobes. The specific mechanism involved, generate novel metabolic intermediates that can serve as broadly applicable biochemical markers of activity in the environment. <LI> To use functional genes (e.g. bssA) as genetic biomarkers to monitor presence and expression (mRNA) of anaerobic hydrocarbon utilizing microorganism(s) in NJ groundwater samples. We will determine if the biomarkers developed from our strains can be used for enumerating these specific members in impacted environmental samples using qPCR. <LI> To correlate the biochemical and molecular genetic biomarkers with PAH degradation activity and to evaluate the usefulness of the two classes of biomarkers as a means of assessing intrinsic PAH metabolism. We can compare impacted and unimpacted groundwater from different sites and aquifers for both classes of biomarkers. Additionally, we will assay the levels of each biomarker in both inoculated and uninoculated sediment microcosm studies over time to test the validity of these biomarkers as indicators of the presence/absence and in situ activity of petroleum utilizing anaerobes.</OL> Expected Outputs: With this knowledge we can better manage, treat and clean up our valuable groundwater resources. On a national level 40% of the water supply comes from groundwater with agriculture using most of it, and in NJ more than 300,000 wells provide water to more than 4.3 million residents (USGS 2007). As the population of the state grows, maintaining a safe and reliable water supply for state residents, for agriculture and the environment is vital to the welfare and security of the State. With knowledge gained from this work, we provide improved remediation tools for State and local water quality officials.
Non-Technical Summary: Water quality for human, agricultural and environmental uses can be impacted by contaminants from many different sources. Groundwater is a critical resource needed for drinking water, irrigation and environmental sustainability and is being overexploited and undervalued. In New Jersey it provides almost 50% of the water supply of the state. One of the major concerns for groundwater quality is contamination by organic solvents and petroleum components. These come from leaking underground storage tanks found under gas stations and industrial areas, legal and illegal waste disposal as well as non-point and point source contamination. Removal or clean-up of the contaminants from groundwater resources include engineered solutions (pump and treat and reinjection) as well as bioremediation solutions whereby we take advantage of the naturally occurring microorganisms to biodegrade the organic contaminants. By advancing the research in biodegradation, we can expand our understanding of the microorganisms, their physiology, genetics and the biochemical mechanisms of degradation for many organic compounds of interest (including benzene, toluene, xylenes, naphthalene, phenanthrene, other polycyclic aromatic hydrocarbons and alkanes). We propose to study in detail the biodegradation by naturally occurring microorganisms of these compounds under the conditions likely to occur in groundwater. More than just serving as a treatment option, knowledge about the biodegradation of these compounds also provides insight into the natural attenuation processes that these microbes can carry out in situ. <P> Approach: Procedure: 1. Establish and maintain anaerobic cultures on BTX, PAHs and alkanes from NJ groundwater sources. Contaminated and uncontaminated groundwaters from NJ aquifers will be used to generate anaerobic cultures on specific hydrocarbons (HC). The cultures will be established under strict anaerobic techniques. To promote denitrifying, sulfate reducing and methanogenic conditions, nitrate, sulfate or carbonate, respectively, will be provided as the only available electron acceptor as previously described (Haggblom et al, 1993, Kazumi et al, 1995). 2. Examine and compare the extent of metabolism and the key initial steps under the differend reducing conditions In active cultures, mass balance measurements on carbon and/or electron acceptor can be used to assess whether degradation of the PAH is coupled to denitrification, sulfidogenesis or methanogenesis (Kazumi et al, 1995). We will look for the fumarate addition or carboxylated derivative of each NC substrate. We have identified a series of partially reduced novel bicyclic metabolites, namely, THNA and DHNA formed during the anaerobic degradation of NAP. DNA Extractions. Sediment slurry samples will be subjected to a direct, modified phenol/chloroform extraction (Kerkhof and Ward, 1993). PCR amplification of target genes. The target genes that will be used include 16S rRNA, nitrous oxide reductase (nosZ), dissimilatory sulfite reductase (dsrD), and co-enzyme M reductase (mcrA). All amplifications will be done in a Perkin-Elmer Gene Amp PCR system 2400 thermal cycler. Cloning of 16S rDNA will be done using a Cloneamp System (Gibco BRL). Once the target genes bearing the appropriate restriction site have been obtained, the sequence will be aligned using the Ribosomal Database Project (Maidak et al., 1997). 3. Quantitative analysis of specific bacteria and their activity. Quantitative Analysis of DNA targets. We will be using PCR primers that identify specific groups of anaerobes (e.g. sulfate reducers, denitrifiers) and also functional genes (e.g. bssA). The quantitative part of this analysis refers to the ability to relate the percent contribution resulting from each target within a known volume. In summary, qPCR is culture independent and not geographic specific; it is a rapid, sensitive method with the ability to isolate the targeted sequence in a complex environment. 4. Evaluate a.) the feasibility of using metabolites formed during anaerobic degradation as biomarkers of activity in anoxic groundwater. and b.) molecular genetic biomarkers for monitoring the members of the HC community as a means of assessing their presence in situ. Using the information from the studies outlined above we plan to determine if, for example, NA, THNA and/or DHNA can be observed in groundwaters where PAH metabolism is taking place. At the same time, we also will ascertain if the molecular biomarkers correlate with PAH degrading activity. Second, we will compare petroleum impacted and nonimpacted groundwater from several sites for both classes of biomarkers as well. This approach can be done for each of the target substrates, such as BTX, alkanes.