Diversity, Abundance, and Consistency of Microbial Oxygenase Expression and Biodegradation in a Shallow Contaminated Aquifer

Abstract

The diversity of Rieske dioxygenase genes and short-term temporal variability in the abundance of two selected dioxygenase gene sequences were examined in a naphthalene-rich, coal tar waste-contaminated subsurface study site. Using a previously published PCR-based approach (S. M. Ní Chadhain, R. S. Norman, K. V. Pesce, J. J. Kukor, and G. J. Zylstra, Appl. Environ. Microbiol. 72:4078-4087, 2006) a broad suite of genes was detected, ranging from dioxygenase sequences associated with Rhodococcus and Sphingomonas to 32 previously uncharacterized Rieske gene sequence clone groups. The nag genes appeared frequently (20% of the total) in two groundwater monitoring wells characterized by low (ϳ10 2 ppb; ϳ1 M) ambient concentrations of naphthalene. A quantitative competitive PCR assay was used to show that abundances of nag genes (and archetypal nah genes) fluctuated substantially over a 9-month period. To contrast short-term variation with long-term community stability, in situ community gene expression (dioxygenase mRNA) and biodegradation potential (community metabolism of naphthalene in microcosms) were compared to measurements from 6 years earlier. cDNA sequences amplified from total RNA extracts revealed that nah-and nag-type genes were expressed in situ, corresponding well with structural gene abundances. Despite evidence for short-term (9-month) shifts in dioxygenase gene copy number, agreement in field gene expression (dioxygenase mRNA) and biodegradation potential was observed in comparisons to equivalent assays performed 6 years earlier. Thus, stability in community biodegradation characteristics at the hemidecadal time frame has been documented for these subsurface microbial communities.Polycyclic aromatic hydrocarbons (PAHs), derived from both natural and anthropogenic sources, are toxic pollutants formed during the incomplete combustion of organic matter (58). Phylogenetically and physiologically diverse microbial populations contribute to biodegradation of PAHs in soil, sediment, and groundwater (22, 52). The most widely studied bacterial degradation pathways for naphthalene, the simplest and most soluble PAH, and a model compound for PAH metabolism, are initiated by the catalytic activity of an evolutionarily conserved naphthalene dioxygenase (NDO) system (22, 52). The two primary NDO-mediated degradation pathways are distinguished by conversion of naphthalene, via salicylate, to either catechol (e.g., nah genes) or gentisate (e.g., nag genes [22]). Many bacteria harbor well-characterized NDO systems, including gram-negative species (e.g., Pseudomonas spp., nah genes; Burkholderia spp., phn genes; and Ralstonia and Comamonas spp., nag genes) and gram-positive species (e.g., Mycobacteria spp., nid genes; and Rhodococcus spp., nar genes) (22,44).A variety of culture-independent, biomarker-based approaches for characterizing microbial assemblages and their activity in naturally occurring communities have been applied in PAH-contaminated environments. Analyses have ranged from metagenomic libraries (62), flu...

Soil Type-Dependent Responses to Phenanthrene as Revealed by Determining the Diversity and Abundance of Polycyclic Aromatic Hydrocarbon Ring-Hydroxylating Dioxygenase Genes by Using a Novel PCR Detection System

“…Without exposure to phenanthrene, no PAH-RHD ␣ gene amplicons were detected by PCR and qPCR, and thus, the copy number was below the estimated detection limit of 10 4 copies g of soil Ϫ1 . Other studies using different primer systems also reported the absence of PCR amplicons when TC DNA from noncontaminated soils or groundwater was used (3,9,14,35). In contrast to the only previously published primer system (24) that also targeted RHD ␣ genes of Gram-positive and Gramnegative bacteria, the amplified sequence that can be used for sequence analysis is much longer (approximately 280 bp compared to approximately 47 bp) and the primers specifically target PAH-RHD ␣ genes.…”

Benzene Degradation by a Variovorax Species within a Coal Tar-Contaminated Groundwater Microbial Community

“…The study site is a coal tar-contaminated aquifer located in South Glens Falls, New York. The site's microbiology and geochemistry are well documented (13,54,55). Well water samples were taken from two monitoring wells (well 12 and well 36) as described by Bakermans and Madsen (55).…”

“…To identify the members of the aquifer microbial community that were active in benzene metabolism, a stable isotope probing (SIP) experiment was performed in parallel with the degradation assays. The treatments were prepared in a manner identical to the above degradation assay, except 13 C-labeled liquid benzene was added to a set of microcosms. After 70% of the 13 C-labeled benzene was degraded, the microcosms were sacrificed.…”

“…Numerous studies have applied assays with molecular biomarker approaches to contaminated sites, with the objective of exploring the diversities of biodegradation genes and of the naturally occurring populations involved in metabolizing hydrocarbons (10)(11)(12)(13)(14)(15)(16). In addition, cultivation efforts have successfully isolated hydrocarbon-degrading organisms from habitats, including from soil, groundwater, marine waters, and deep sea sediments (12,(17)(18)(19)(20)(21)(22).…”

“…Stable isotope probing (SIP) is a promising tool for targeting and identifying active populations within microbial communities without requiring cultivation (25)(26)(27)(28). Studies using SIP dose substrates labeled with heavy isotopes (e.g., 13 C and 15 N) into mixed microbial populations. Then, assimilative metabolic processes incorporate the heavy atoms into the metabolically active biomass (typically a small subset of the total).…”