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
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. The knowledge 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 2005 continued to focus on studying the regulation of the tbu regulon of Ralstonia pickettii PKO1, a model microorganism capable of degrading aromatic fuel hydrocarbons and chlorinated ethene solvents under suboxic conditions. The tbu regulon encodes the monooxygenase enzymes necessary for hydrocarbon degradation, and previous studies demonstrated enhanced expression of the catechol 2,3-dioxygenase gene (tbuE) with nitrate under suboxic conditions (<2 mg/L O2). A DNA microarray-based approach is being employed to understand the compensatory mechanisms of gene expression in PKO1 during hypoxia. In the absence of genome sequence data for R. pickettii PKO1, Dr. Abriola’s group relied on the availability of genome sequence data for Ralstonia metallidurans CH34, Ralstonia eutropha JMP134, and Ralstonia solanacearum GMI1000 for microarray design. A set of 37 genes was targeted that included genes from six broad functional categories: denitrification, stress response, solvent resistance, oxidative phosphorylation, exopolysaccharide production, and the tbu regulon. PCR products for each candidate target gene were generated using primers designed from DNA alignments of the respective gene sequences from each of the sequenced Ralstonia genomes. Genomic DNA from R. solanacearum GMI1000 was used to assess PCR primer utility and fidelity. The resulting PKO1 amplicons were purified, cloned and sequenced. Based on PKO1 amplicon sequence analysis, this methodology produced amplicons with sequence similarities as low as 51.5% (narH of R. metallidurans CH34) to the corresponding Ralstonia genome sequences. This work indicates the efficacy of applying known genome sequence data to access genome information in phylogenetically related microorganisms for which the genome sequence is unavailable.
Solvent-exposure stress has also been investigated in batch and column experiments during this project period. Representative strains of toluene oxidizing bacteria: Ralstonia pickettii PKO1 (Wild type and tbuX knockedout), Burkholderia vietnamiensis G4 were screened for the composition and amount of extracellular polymeric substances (EPS) produced during exposure to dissolved-phase TCE. All strains produce EPS, but composition and amount of EPS were found to depend on the concentration of TCE. After exposure to sub-toxic levels of TCE, R. pickettii PKO1 produced dramatically more EPS which was richer in uronic acid. This model organism was also used in column experiments to determine the influence of TCE on EPS content and organism transport in sandy media. Attached TCE induced R. pickettii PKO1 cells were exposed to a pulse of dissolved phase TCE. Results indicate that the cells extracted from the sandy medium following the experiment had produced a greater amount of EPS than was observed in the batch experiments. Furthermore, the uronic acid to carbohydrate ratio of the extracted cells was observed to be significantly greater than that observed in the influent. While uronic acid enrichment has been reported to reduce cell adhesion, concentrations of biomass in the column effluent and soil profile (at the conclusion of the experiment) were not indicative of macroscopic changes in the amount of biomass retained in the column. These findings suggest an interaction between the presence of solid phase (increased production of EPS components shown to facilitate attachment) and presence of a solvent stressor (increased production of EPS components shown to limit attachment).