Superfund Research Program
Processes Influencing the Natural Attenuation of Organic Contaminant Plumes: Transport, Enzymatic Regulation and Microbial Transformation Rates in Flowing Groundwater Systems
Project Leader: Linda M. Abriola (Tufts University)
Grant Number: P42ES004911
Funding Period: 1995 - 2006
Final Progress Reports
Year: 2004 1999
The goal of this research is to quantify the relationship between contaminant exposure history, growth conditions, and regulation of microbial biodegradative capabilities in natural porous media, under conditions representative of typical field-scale plume contamination scenarios. Knowledge gained here will provide a foundation for improved Superfund site remediation and characterization methods, as well as development of a scientifically sound approach for assessment of alternative endpoint clean-up standards under natural (intrinsic) and engineered remediation conditions.
Research efforts during this funding period have continued to focus on Ralstonia pickettii PKO1 as a model microorganism capable of degrading aromatic fuel hydrocarbons and chlorinated ethene solvents under suboxic conditions. A small-scale DNA microarray-based approach is being employed to investigate the hypothesis that, under oxygen-limiting conditions, the upregulation of the oxygen-requiring catabolic enzymes of the tbu regulon of strain PKO1 will occur with a compensatory decrease in other oxygen-intensive cellular processes. In the absence of genome sequence data for R. pickettii PKO1, a comparative genomics strategy was used to design DNA microarray targets that relied on the genome sequence data for Ralstonia metallidurans CH34, Ralstonia eutropha JMP134, and Ralstonia solanacearum GMI1000. Microarray targets included genes from six broad functional categories - denitrification, stress response, solvent resistance, oxidative phosphorylation, exopolysaccharide production, and the tbu regulon. The process of DNA microarray development has also enabled several other avenues of investigation. The sequence information garnered during microarray development allowed the development of two separate gene knockout strains of PKO1. These knockouts were made in the fnr gene, a global coordinator of the cellular response to changes in redox status, and the epsC gene, a component necessary for the synthesis of exopolysaccharide. Additionally, chemostat growth studies under a range of dissolved oxygen conditions have been completed to provide baseline parameters that can be extrapolated to future sand column experiments.
Efforts this funding period have also focused on organism transport under carbon starvation-induced and solvent-exposure stresses at the column-scale. Five representative strains of trichloroethene (TCE) degrading toluene oxidizing bacteria: Ralstonia pickettii PKO1 (Wild type and tbuX knockedout), Burkholderia vietnamiensis G4, Pseudomonas putida F1, and Pseudomonas sp. strain W31, were screened for their ability to synthesize extracellular polymeric substances (EPS) that microbes use to adhere to solid surfaces. All five strains produce EPS, but composition and amount of EPS were found to depend upon the type of carbon source (lactate, glucose, or toluene). Batch experimental results further revealed that cells under carbon-starvation produce more EPS than non-starved cells. Solvent stress-mitigation was also observed to be one of the functions of EPS production in these bacteria. For example, R. pickettii PKO1 produced dramatically more EPS at higher toluene concentrations. In addition, batch experimental studies that exposed R. pickettii PKO1 to a range of TCE concentrations indicate that solvent-exposure stress can result in uronic acid enrichment of EPS, potentially resulting in reduced adhesion. These findings provide information for the current investigation of the relationship between EPS composition and microbial transport in porous media during periods of stress mitigation.