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

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.