Subsurface cycling of nitrogen and anaerobic aromatic hydrocarbon biodegradation revealed by nucleic Acid and metabolic biomarkers

Abstract

Microbial processes are crucial for ecosystem maintenance, yet documentation of these processes in complex open field sites is challenging. Here we used a multidisciplinary strategy (site geochemistry, laboratory biodegradation assays, and field extraction of molecular biomarkers) to deduce an ongoing linkage between aromatic hydrocarbon biodegradation and nitrogen cycling in a contaminated subsurface site. Three site wells were monitored over a 10-month period, which revealed fluctuating concentrations of nitrate, ammonia, sulfate, sulfide, methane, and other constituents. Biodegradation assays performed under multiple redox conditions indicated that naphthalene metabolism was favored under aerobic conditions. To explore in situ field processes, we measured metabolites of anaerobic naphthalene metabolism and expressed mRNA transcripts selected to document aerobic and anaerobic microbial transformations of ammonia, nitrate, and methylated aromatic contaminants. Gas chromatography-mass spectrometry detection of two carboxylated naphthalene metabolites and transcribed benzylsuccinate synthase, cytochrome c nitrite reductase, and ammonia monooxygenase genes indicated that anaerobic metabolism of aromatic compounds and both dissimilatory nitrate reduction to ammonia (DNRA) and nitrification occurred in situ. These data link formation (via DNRA) and destruction (via nitrification) of ammonia to in situ cycling of nitrogen in this subsurface habitat, where metabolism of aromatic pollutants has led to accumulation of reduced metabolic end products (e.g., ammonia and methane).

FIG. 1.

Map of field study site, showing the locations of monitoring wells (MW), the boundary of groundwater contamination, and the direction of groundwater movement. MW 60 is the site used for determining background geochemical conditions and microbiology. MW 36 is the site where there was the greatest contamination. (Adapted from reference .)

FIG. 2.

Naphthalene biodegradation in microcosms containing well 36 groundwater and subsurface sediment from a location adjacent to well 36 in treatments (10°C) designed to favor aerobic metabolism (A), methanogenesis (B), nitrate reduction (C), sulfate reduction (D), manganese oxide reduction (E), and iron oxide reduction (F). ▴, data for viable treatments; •, data for poisoned controls. The symbols indicate the averages for triplicate microcosms; the error bars indicate one standard deviation.

FIG. 3.

Phylogenetic analysis of mRNA transcripts found in water from the site for three key site metabolic processes: anaerobic degradation of methylated aromatic compounds (bssA) (A), aerobic metabolism of ammonia (bacterial amoA) (B), and anaerobic dissimilatory reduction of nitrate to ammonia (nrfA) (C). The alignments are based on data for 250, 137, and 209 deduced amino acid sites, respectively. Neighbor-joining analyses were performed with CLUSTALX alignments using the ARB analysis package. Sequences obtained in this study are indicated by bold type, while reference sequences are indicated by light type. The distribution of clones in wells 36 and 12 is shown in Table 4. Bootstrap values of ≥50% for 100 replicates are indicated at the nodes. The GenBank accession numbers for reference sequences are indicated after the clone and organism designations. Scale bars = 0.10 change per nucleotide position.