Transient molecular markers of in situ naphthalene biodegradation at a coal tar-contaminated field site

The study's objectives were to: (i) identify molecular markers of in situ naphthalene biodegradation that were transient and specific, and therefore indicative of real time activity; (ii) develop methods for the extraction and measurement of the markers at a coal tar-contaminated site, incorporating procedures to minimize artifacts associated with sampling and laboratory incubations; and (iii) investigate the genetic and metabolic diversity of naphthalene catabolic processes among bacteria isolated from the contaminated site. RNA transcripts encoding naphthalene dioxygenase (ndo) were extracted from contaminated water, reverse-transcribed, and amplified, using the polymerase chain reaction and degenerate primers targeted at ndo genes. The amplified products corresponded in size to the equivalent products from characterized naphthalene-degrading organisms. A probe, produced from site mRNA using PCR, hybridized under stringent conditions with PCR products produced from naphthalene-degrading bacteria that represent the range of described ndo diversity. Dilutions of the reverse-transcribed RNA were amplified, cloned and sequenced. The eleven resulting sequences clustered tightly into two groups related to described ndo sequences. This was the first study to identify and sequence genes actively expressed by microorganisms involved in biodegradation in a field site, and these results indicate that in situ transcription of diverse naphthalene catabolic genes was occurring at the time of sampling. 1,2-Dihydroxy-1,2-dihydronaphthalene (1,2-DHDN), the first intermediary metabolite of naphthalene catabolism, was extracted from contaminated waters. 1,2-DHDN concentration was highest in water from the center of the contaminant plume, lower near the plume boundary, and absent in adjacent uncontaminated water. Measurement of 1,2-DHDN in contaminated samples was interpreted as real time documentation of in situ naphthalene biodegradation. Metabolic and genetic diversity of naphthalene catabolism was investigated using 18 naphthalene-degrading site isolates. GC/MS analysis of metabolites produced under normal- and 18{dollar}rm {lcub}sb{lcub}Osb2{rcub}{rcub}{dollar}-containing atmospheres indicated that the naphthalene catabolic pathway is likely conserved between these isolates and model organisms. PCR analyses suggested these isolates use dioxygenases that are distinct from those previously characterized. The study adds to our understanding of how natural microbiological processes may destroy organic pollutants.