Frederic K. Pfaender

Effect of Incubation Conditions on the Enrichment of Pyrene-degrading Bacteria Identified by Stable-isotope Probing in an Aged, PAH-contaminated Soil

To determine whether the diversity of pyrene-degrading bacteria in an aged polycyclic aromatic hydrocarbon-contaminated soil is affected by the addition of inorganic nutrients or by slurrying the soil, various incubation conditions (all including phosphate buffer) were examined by mineralization studies and stable-isotope probing (SIP). The addition of nitrogen to either continuously mixed slurry or static field-wet soil incubations increased the rate and extent of mineralization of [14C]pyrene, with the most rapid mineralization observed in slurried, nitrogen-amended soil. Microcosms of slurry and static field-wet soil amended with nitrogen were also examined by SIP with [U-13C]pyrene. Recovered 13C-enriched deoxyribonucleic acid (DNA) was analyzed by denaturing-gradient gel electrophoresis (DGGE) and 16S ribosomal ribonucleic acid (rRNA) gene clone libraries. DGGE profiles of 13C-enriched DNA fractions from both incubation conditions were similar, suggesting that pyrene-degrading bacterial community diversity may be independent of treatment method. The vast majority (67 of 71) of the partial sequences recovered from clone libraries were greater than or equal to 97% similar to one another, 98% similar to sequences of pyrene-degrading bacteria previously detected by SIP with pyrene in different soil, and only 89% similar to the closest cultivated genus. All of the sequences recovered from the field-wet incubation and most of the sequences recovered from the slurry incubation were in this clade. Of the four sequences from slurry incubations not within this clade, three possessed greater than 99% similarity to the 16S rRNA gene sequences of phylogenetically dissimilar Caulobacter spp.

Impact of imposed anaerobic conditions and microbial activity on aqueous-phase solubility of polycyclic aromatic hydrocarbons from soil

The influence of anaerobic conditions on aqueous-phase polycyclic aromatic hydrocarbon (PAH) bioavailability was investigated in laboratory microcosms. Highly aged (>70 years), PAH-contaminated soil was incubated under anaerobic conditions by using various anaerobic headspaces, anaerobic headspaces with an oxygen-scavenging complex (titanium(III) citrate) in the aqueous phase, or anaerobic headspaces with electron-acceptor amendments in the aqueous phase. Incubation of soil solely under anaerobic conditions resulted in increased aqueous concentrations of all PAHs tested (fluoranthene, pyrene, benz[a]anthracene, and benzo[a]pyrene). Benz[a]anthracene and benzo[a]pyrene extractable concentrations were above aqueous solubility, by as much as an order of magnitude for the latter. The degree of solubility increase observed was a function of molecular weight of the PAH regardless of initial soil concentration, suggesting formation of stable PAH-soluble organic matter associations. The soil samples incubated aerobically for 90 d before imposition of anaerobic conditions did not release PAHs to the aqueous phase. Methanogenic organisms and sulfate-reducing bacteria were seen to have the most significant effect on increases in aqueous-phase PAHs. Polycyclic aromatic hydrocarbons made more soluble under anaerobic conditions was available to be degraded or transformed under aerobic conditions.

Effects of anaerobic incubation on the desorption of polycyclic aromatic hydrocarbons from contaminated soils.

Incubation of field-contaminated soil under anaerobic conditions can lead to increased mobilization of polycyclic aromatic hydrocarbons (PAHs) into water. In the present study, we evaluated the effects of anaerobic incubation on the rate and extent of desorption of PAH from two field-contaminated soil samples. One was a highly contaminated soil from a former wood-preserving site that had not been subject to previous treatment; the other was a soil from a former manufactured-gas plant site that had been treated in an aerobic bioreactor. A two-site desorption model was applied to quantify the fast and slowly desorbing fractions of each PAH and the corresponding first-order rate constants for each fraction. For most of the PAHs, the total amount desorbed after 18 d from anaerobically incubated samples was significantly greater than that from their counterparts not subjected to anaerobic incubation, but the overall effect was modest. The rate constant corresponding to the slowly desorbing fraction (k(2)) was much higher for the samples incubated under active anaerobic conditions than that for the controls, implying anaerobic incubation had the greatest influence on the soil compartments controlling the slow release of PAHs. Anaerobic incubation had little to no effect on the rapidly desorbing fraction.

A note on the isolation of the bacteriumVibrio parahaemolyticus from estuarine North Carolina

The potentially pathogenic bacterium,Vibrio parahaemolyticus, was isolated from water, sediment, and oyster samples collected from estuarine North Carolina. In October, when water temperatures were 15–18°C,V. parahaemolyticus was still detected in all substrates. However, the organism was detected only in one sediment sample in January when water temperatures were 4–5°C. No correlation was observed between the occurrence of the bacterium and domestic wastewater contamination.

Epifluorescence microscopy and image analysis of high‐level polycyclic aromatic hydrocarbon contamination in soils

Interactions between polycyclic aromatic hydrocarbons (PAHs) and soil are an important determinant of their chemical availability and transport. Laboratory examination of microscale PAH-soil interaction is limited by the availability of methods for particle-scale observation. Inverted epifluorescence microscopy, combined with digital photography and computer image analysis, was evaluated for specificity and linearity using dissolved PAHs. A pyrene filter (excitation wavelength, 360-400 nm; emission wavelength, 450-520 nm) gave nonspecific PAH fluorescence, and bias for fluoranthene, benzo[b]fluoranthene, benzo[g,h,i]perylene, and benz[a]anthracene was quantified in comparison to that for pyrene. Concentrations ranging from 1 to 10 mM for anthracene, fluoranthene, and pyrene and from 1 to 50 mM for naphthalene produced a linear response with low interpixel variability. Liquid-phase analyses validated use of the technique for the descriptive analysis of PAH distribution in solid samples, but liquid-phase calibration was not quantitative for spiked or field-contaminated soils. The mean luminance for three field soils was proportional to the values predicted from their chemically measured concentrations and to values from spiked, aged, uncontaminated materials. Image analysis of laboratory- and field-contaminated samples determined the area distribution of fluorescent intensity and the size of fluorescent areas exceeding a threshold luminance. These qualitative descriptions of the microscale spatial distribution of PAH contamination are presented as potential endpoints for future research on biogeochemical interactions in heavily contaminated solids.

Products of laccase catalyzed reaction of 1-hydroxypyrene

The formation of residues in soils and sediments which are not readily extractable is an important fate for hydrophobic organic contaminants. Reaction of 1-hydroxypyrene, a known fungal and terrestrial invertebrate metabolite and photo-oxidation product of pyrene, with a commercially prepared laccase (an oxidoreductive enzyme) in solution provides a test system to study the fate of residues in nature. The reaction of dissolved 1-hydroxypyrene with laccase produced a distinct color change and caused a precipitate to form. At least six products were shown by HPLC analysis with photodiode array detection. The most prominent peak was composed of 1,6- and 1,8-pyrene-dione as confirmed by proton NMR analysis. LC/MS data on remaining peaks indicate that radical coupling produces acetonitrile soluble dimers and at least one trimer. Proposed structures consistent with MS and photodiode array data indicate that pyrene subunits are linked predominantly by ether bonds. Additional products, insoluble in acetonitrile, were also formed. The formation of complex products in vitro, indicates that the same mechanisms could produce bound residues in soil.