Molecular diagnostics of polycyclic aromatic hydrocarbon biodegradation in manufactured gas plant soils

Abstract

Traditional methods for quantifying specific catabolic bacterial populations underestimate the true population count due to the limitations of the necessary laboratory cultivation methods. Likewise, in situ activity is also difficult to assess in the laboratory without altering the sample environment. To circumvent these problems and achieve a true in situ bacterial population count and activity measurement, new methods based on molecular biological analysis of bacterial nucleic acids were applied to soils heavily contaminated with polycyclic aromatic hydrocarbons (PAH). In addition, a naphthalene-lux reporter system was used to determine bioavailability of naphthalene within these soils. DNA extracted from seven PAH-contaminated soils and hybridized with the nahA gene probe indicated that the naphthalene degradative genes were present in all samples in the range of 0.06 to 0.95 ng/100 microliters DNA extract which was calculated to represent 3.2 x 10(6) to 1.1 x 10(10) cells/g soil (assuming one copy of these genes per cell). 14C-naphthalene mineralization was observed in all contaminated soils with 14CO2 mineralization rates ranging from 3.2 x 10(-5) to 304,920.0 x 10(-5) micrograms g soil-1 h-1. Phenanthrene, anthracene, and benzo(a)pyrene were mineralized also in several soils. Messenger RNA transcripts of nahA were isolated and quantified from 4 soils. Only one soil tested, soil B, was inducible with salicylate above the in situ nahA gene transcript level. Two of the soils, C and G, were already fully induced in situ. The naphthalene mineralization rate correlated positively with the amount of nahA gene transcripts present (r = 0.99). Naphthalene was bioavailable in soils A, D, E, G, and N as determined by a bioluminescent response from the naphthalene-lux reporter system. Taken together, these data provided information on what the naphthalene-degrading bacterial population was experiencing in situ and what approaches would be necessary to increase activity.

Quantification of phnAc and nahAc in Contaminated New Zealand Soils by Competitive PCR

“…The greater numbers of copies in the PAH-contaminated soil than in the petroleum-contaminated soil may reflect higher naphthalene and phenanthrene levels. Similar observations have been made in previous studies of the molecular ecology of nah-like genotypes in PAH-contaminated soil (16,17). For the two contaminated New Zealand soils that we analyzed, the phn and nah-like genotypes were present (assuming that there was one copy of each gene per cell) at levels that were greater than or equal to the total numbers of heterotrophs determined for the same soil samples by culture techniques (Table 1).…”

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

“…4) indicated that ndo genes were actively expressed under apparent suboptimal physiological conditions (low ambient levels of oxygen and naphthalene). In situ expression of nag genes in groundwater was detected with ambient naphthalene concentrations as low as 0.8 M. Indeed, several prior reports have suggested that nag-type genes may be transcribed in response to low concentrations of substrate (12,26,59). In the present study, nag genes were more abundant than nah genes and expressed in well 12, associated with lower naphthalene concentrations than well 36.…”

Abundance of Dioxygenase Genes Similar to Ralstonia sp. Strain U2 nagAc Is Correlated with Naphthalene Concentrations in Coal Tar-Contaminated Freshwater Sediments

“…Furthermore, Sanseverino et al (31) established a threshold level of naphthalene of approximately 100 mg/kg of soil, below which the nahAc-like naphthalene-degrading population is relatively inactive.…”