Superfund Research Program

Gene Enhanced Remediation of Co-Contaminated Soils

Project Leaders: Christopher G. Rensing, Ian L. Pepper
Grant Number: P42ES004940
Funding Period: 1995-2005

Project-Specific Links

Final Progress Reports

Year:   2004  1999 

Specific Aim 1:To Evaluate the Potential for Gene Transfer of a Metal Resistance Gene from an Introduced Donor Organism to Indigenous Soil Bacterial Recipients.

Researchers have engineered an E. coli strain that contains plasmid pJP4, which codes for mercury resistance as well as partial 2,4 B dichlorophenoxyacetic acid (2,4-D) degradation. Since this strain does not contain the chromosomal genes for complete degradation of 2,4-D, plating on media with 2,4-D as a sole carbon source allows for donor counter selection during gene transfer studies in which this strain is used as a donor of the plasmid to indigenous soil bacteria. Such bacteria that do contain the appropriate chromosomal degradative genes and that receive the plasmid, can degrade 2,4-D and are readily identified as transconjugates on the 2,4-D plates. Using this system, 10⁷ transconjugates/gram soil ere found when Madera Canyon soil was amended with 500 ppm 2,4-D. This number increased to 10⁸/gram soil when the amendment was 1000 ppm 2,4-D. Transconjugates were differentiated via ERIC PCR and identified by BIOLOG as Burkholderia, and Pseudomonas spp. These studies illustrate the potential for successfully transferring metal resistance genes or degradative genes into indigenous soil recipients.

Specific Aim 2:To Evaluate Metal Resistant Populations in Co-contaminated Soils and to Evaluate the Influence of Metals on the Ability of Bacteria to Degrade Organics in Metal Contaminated Soil.

An intermediate field scale study was conducted in bioreactors to assess the impact of bioaugmentation with two plasmid pJP4 bearing microorganisms: the natural host, Ralstonia eutropha JMP134, and a laboratory generated strain amenable to donor-counter selection, E. coli D11. The Ralstonia contained chromosomal genes necessary for mineralization of 2,4-D, while the E. coli did not. Accordingly, inoculation with Ralstonia was considered cell bioaugmentation and inoculation with E. coli D11, gene augmentation. The soil system was contaminated with 2,4-D alone or co-contaminated with 2,4-D and Cd. Plasmid transfer to indigenous populations, plasmid persistence in soil, and degradation of 2,4-D were monitored over a 63 day period in the bioreactors. In order to assess the impact of contaminant re-exposure, aliquots of bioreactor soil were re-amended with additional 2,4-D. Both introduced donors remained culturable and transferred plasmid pJP4 to indigenous recipients, although to different extents. Isolated transconjugants were members of the Burkholderia and Ralstonia genera, suggesting multiple if not successive plasmid transfers. Upon a second exposure to 2,4-D, enhanced degradation was observed in all treatments, suggesting microbial adaptation to 2,4-D. Degradation was most rapid in the gene augmented treatments. Cd did not significantly impact 2,4-D degradation or transconjugant formation. In addition, the establishment of an array of stable indigenous plasmid hosts may be particularly useful at sites with potential for re-exposure or long term contamination.

Specific Aim 3:To Evaluate the Potential for Transport of Degradative Genes or bacteria containing Degradative Genes.

Microbial inocula for bioaugmentation often contain plasmids that encode for metal resistance or contaminant degradative genes. However, little is known regarding plasmid fate within contaminated soils. Column studies were used to evaluate dissemination of plasmid pJP4 under unsaturated or saturated flow conditions in a 2,4-dichlorophenoxyacetic acid (2,4-D) contaminated soil. Dissemination occurred not only through transport of the donor organism, but also via gene transfer to indigenous soil recipients. Conjugation is believed to be the mode of gene transfer of this conjugative plasmid; however, transformation may also play a role. Subsequent leaching of transconjugants, growth of transconjugants, and/or gene transfer from transconjugants to additional indigenous recipients may also have facilitated plasmid dissemination. Plasmid pJP4 was introduced into 2,4-D amended soil via inoculation of a surface layer with an E. coli donor organism containing plasmid pJP4 at a concentration of 10⁶ CFU g dry soil^-1. Columns were destructively sampled following one week of percolation by removal of the soil in layers to assess the vertical distribution of donors and transconjugants within the soil. Donor microorganisms and indigenous recipients of plasmid pJP4 within each layer were enumerated via plating on selective media. Concentration of 2,4-D within each soil layer and in the column effluent was monitored via HPLC analysis. In unsaturated soil, pJP4 was detected in both culturable donor and transconjugant cells within soil 10.6 cm from the inoculated end of the column (approximately 20% the transport distance of an ideal solute). In soil subjected to saturated flow conditions, no transconjugants were detected; however, donors were found throughout the entire length of the column (30.5 cm). These results suggest that donor transport in conjunction with plasmid transfer to indigenous recipients allows for significant dissemination of introduced genes through contaminated soil, which in turn may promote enhanced degradation of organic contaminants.