Superfund Research Program
Mechanisms of Bioavailability Regulation in Soil
Project Leader: Frederic K. Pfaender
Grant Number: P42ES005948
Funding Period: 1995 - 2006
Final Progress Reports
Year: 2005 1999
Drs. Pfaender and Christman along with their research teams have continued to make progress on several specific aims related to understanding the interaction between microorganisms, soil organic materials, and PAH.
The microbial communities in a PAH-contaminated soil and its uncontaminated counterpart have been compared. Using profiles generated by denaturing gradient gel electrophoresis, the dominant species from each community appear to be very different. However, DNA sequencing on excised bands from the uncontaminated soil has proven difficult, most likely due to a high level of diversity resulting in comigration of bands. Therefore, clone libraries of the two communities have been created. DNA sequence results from these are still pending.
Incubations of three PAH-contaminated soils (ranging from 400 mg/kg to 10,000 mg/kg total PAH) are underway to compare two methods for in situ hybridization--a slurry-based method and an aggregate method developed in this lab. Both methods appear to be adequate even at the highest level of PAH contamination (10,000 mg/kg). Further incubations of the uncontaminated soil spiked with pyrene are being monitored for community changes corresponding to pyrene compartmentalization. Ribosomal RNA probes may be designed from the first part of this project to help examine the spatial relationship of pyrene degraders to the contaminant.
Another goal of this research is to develop a new method for visualizing, using fluorescence microscopy, where individual soil constituents, biological, carbonaceous, humic and anthropogenic substances, reside on mineral particles in relation to each other and how this association influences soil structure and pyrene sorption. Studies from the past year determined that as concentrations of humic acid and peptidoglycan bound to mineral phases were increased, the amount of pyrene sorbed also increased as did soil particle aggregation. The biological material showed a significantly greater capacity for pyrene sorption and particle aggregation over humic acid alone.
During the past year experiments using a small scale flow cell (total volume 0.9 µm3) have examined the interaction of a solid naphthalene crystal and the bacterium Pseudomonas putida. The flow cell, containing a solid crystal of naphthalene, is viewed at 100x magnification while either M9 media (abiotic) or a killed or live culture of P. putida is introduced via a syringe pump. Dissolution and degradation rates are estimated by image analysis, and concentration of dissolved naphthalene in the flow cell effluent is determined by a fluorescent detector. It has been observed that over time P. putida (stained blue by the conversion of indole to indigo) accumulates on the crystal (as seen in Figure 1) and affects the rate of disappearance of the naphthalene crystal. A small volume flow cell, combined with microscopic digital photography and image analysis represents a novel way to investigate bacterial colonization of surfaces, and subsequent biodegradation.
Figure 1: (a) Crystal at the beginning of the experiment before P. putida introduction.(b) Crystal after 5 hours of P. putida being flown through at a rate of 0.01 mL/min.