Research Goals: To understand the genetic structure and activity of microbial populations found in PAH- and metal-contaminated soils and sediments, and how they interact with each other to transform these contaminants in the soil environment.

Overview: Our knowledge about microorganisms that degrade PAHs and transform high levels of heavy metals in the environment is overwhelming based on studies that have been done under laboratory conditions using single microbial species and single compounds. However, most contaminated environments have a wide array of microbial species and complex mixtures of contaminants. Microbial transformations of soil contaminants occur through ecological interactions and may be affected by the transfer of genetic material between microorganisms. Degradation processes traditionally have been studied by classical culturing techniques, which rely on bacterial growth on conventional media. However, close to 99% of the microorganisms in nature are non-culturable. In practical terms, most bacteria species that play a role in the degradation of PAHs and the transformation of heavy metals remain uninvestigated. One aspect of this project is to study microorganisms in their natural environment and to understand their role in contaminant transformation by combining molecular techniques with more classical microbiology techniques. This project integrates basic genetics, biochemistry, physiology, and ecology to investigate the in situ structure and activity of bacteria communities involved in transformation PAHs and metals.

Progress to Date: Using SR-DVC, the proportion of soil bacteria that degrade pyrene (used as a model PAH) has been determined for a soil historically contamination with PAHs. Preliminary work has begun on determining the community structure of metabolically active bacteria that respond to heavy metal contamination.

Application of Knowledge: The SR-DVC technique is applicable to a wide range of PAHs and environments (e.g. groundwater and biofilms). Combining the techniques of SR-DVC and FISH is a powerful approach for studying degradative bacteria in contaminated soils and sediments. The diversity of active bacteria in heavy metal contaminated soils will indicate how the bacterial community adapts to the environmental stress.

Future Directions: We intend to identify the bacteria that degrade PAHs in soil up to the species level using oligonucleotide probes that target specific regions within the 16s rRNA, which allows for the quantification of major bacterial degraders in situ. The next step will be determining if active bacteria possess genes that are important in detoxifying metal contaminants.

Techniques Incorporated:
Substrate-Responsive Direct Viable Counting (SR-DVC)
Fluorescent in situ Hybridization (FISH)
Bromodeoxyuridine (BrdU) Labeling of DNA
Denaturing Gradient Gel Electrophoresis (DGGE) Bacterial Community Analysis

Keywords:
Direct viable count (DVC)
FISH
Oligonucleotide probe
Pyrene
DGGE