Superfund Research Program

Bioremediation of Soil

Project Leader: Jodi R. Shann
Grant Number: P42ES004908
Funding Period: 1995 - 2001

Project-Specific Links

Final Progress Reports

Year:   1999 

Investigators have developed a set of primers specific to the "fast-growing" group of Mycobacterium spp., which includes many xenobiotic-degrading strains. These primers targeting 16S rDNA were used for temperature gradient gel electrophoresis (TGGE) analysis of soil populations in four matched pairs of pristine and PAH-contaminated soils obtained from Superfund sites. These soils were selected for the study based on their differences in textural properties, polycyclic aromatic hydrocarbon (PAH) content, and results of Microtox toxicity assays. Purified DNA extracts from soil were PCR-amplified and used directly for TGGE analysis. Results indicated that soils contaminated with moderate levels of PAHs have reduced diversity within the mycobacteria community. This may be an effect of hydrocarbon toxicity as suggested by Microtox toxicity assays. Contaminated clay-silt soils were found to be phylotypically more diverse than contaminated silt-sand soils. Inoculation with a known PAH degrader, Mycobacterium sp. RJGIII-135, increased mineralization of added pyrene.

A similar set of PCR primers were developed that target Sphingomonas spp., a group which include many degradative bacteria, and are commonly found in contaminated soil, sediment, and groundwater. The primers were used for PCR amplification of rDNA and rt-PCR amplification of rRNA, followed by TGGE analysis, to determine the diversity and activity of Sphingomonas spp. populations in a long-term, PAH-contaminated sediments present in the Little Scioto River. Low levels of Sphingomonas diversity were found in all sediments, with no apparent effect from the high levels of PAH resulting from the creosote plant.

Researchers have also continued the investigation of phytoremediation. Although numerous studies have demonstrated increased degradation of hazardous organic compounds in the soil surrounding plant roots (the rhizosphere), little is known about the basic mechanisms contributing to this phenomena. Over the last year, Project 7 has focused on the effect of root exudates on PAH degradation by soil microbial communities and on the uptake of metals and PAHs by plants.

Soluble root exudates were isolated from aseptic, hydroponically-grown corn (Zea mays L.) and used to amend soil columns. Microbial communities in the exudate-amended soil were characterized by plate counts, analysis of biomass and activity, and their ability to mineralize pyrene. Community shifts in carbon utilization were determined by Biolog^tm GN analysis. Results indicated that long term application of exudates to bulk soil enhanced microbial mineralization of ¹⁴C-pyrene. However, stimulation of biomass and activity was minimal, suggesting that mere microbial numbers or activities were not responsible for the increased mineralization. Multivariate analysis of Biolog^tm GN plates revealed functional shifts in microbial communities with exudate amendment. This data suggests exudate pressures may shift community structure in favor of the degrading populations of bacteria.

Current studies are investigating the root exudate phenomena by addressing the influence of extracted root exudates on two different genera of bacteria, a gram negative and a gram positive. Typically associated with different portions of the soil (rhizosphere vs. bulk soil), their responses to plant root exudates will help elucidate the role these exudates play for different types of bacteria.

In addition to adding material (e.g. root exudates) to the soil, plants can also remove contaminants from their environment. Uptake of metals by plants is commonly reported, but PAH uptake has been less well studied. Information on plant uptake (of metals or PAHs) from mixtures is virtually unavailable. To address this, the uptake of the heavy metals, Cd, Ni, and Zn, and the PAHs, pyrene and benzo[a]pyrene by mustard (Brassica juncea) and alfalfa was investigated. Plants were exposed to hydroponic solutions containing the individual contaminants or mixtures of them. Uptake was determined by metal and/or PAHs loss from the hydroponic solution over time and from analysis of plant tissues. Metal uptake by the test plants was found to increase significantly in the presence of either PAH. Excised root experiments indicated the uptake of both ¹⁴C pyrene and benzo[a]pyrene.