An evaluation of soil bioaugmentation with microorganisms bearing plasmidpJP4: Plasmid dissemination and impact on remediation

Abstract

The objective of this research was to evaluate the impact of bioaugmentation of soil with microorganisms harboring plasmid pJP4 on remediation, plasmid transfer, and plasmid dispersal. Divided into three sections, this research showed that use of microbial inocula harboring self-transmissible plasmids holds promise as an applicable bioremediation approach. In the first study, a pJP4 donor that could readily be counter-selected due to a lack of chromosomal genes necessary for 2,4-dichlorophenoxyacetic acid (2,4-D) mineralization was generated to allow detection of transconjugants in soil. Plasmid pJP4 was introduced into Escherichia coli (ATCC 15224), via plate mating with Ralstonia eutropha JMP134 to create such a donor (E. coli D11). Transfer of Plasmid pJP4 to diverse indigenous populations was detected in soils, and under conditions, where it had not been observed previously. Plexiglass columns were used in the second study to evaluate dissemination of plasmid pJP4 under unsaturated or saturated flow conditions in a 2,4-D contaminated soil. In unsaturated soil, pJP4 was detected in both culturable donor and transconjugant cells extending to 10.5 cm from the inoculated layer. 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). Thus, donor transport in conjunction with plasmid transfer to indigenous recipients allowed for significant dissemination of introduced genes through contaminated soil. The last study was conducted using soil contaminated with 2,4-D alone or co-contaminated with 2,4-D and cadmium (Cd). This study assessed the impact of introduction of the pJP4 genes via cell bioaugmentation (R. eutropha JMP134 donor), or via gene augmentation (E. coli D11 donor). Both introduced donors remained culturable and transferred plasmid pJP4 to diverse indigenous recipients. Cell bioaugmentation resulted in the most rapid 2,4-D degradation; however, upon a second exposure to 2,4-D, gene augmentation of indigenous populations was more successful. The presence of Cd (100 μg g dry soil⁻¹) had a minimal impact on 2,4-D degradation and transconjugant formation. The establishment of an array of stable indigenous plasmid hosts may be particularly useful in sites with potential for re-exposure or extensive, and thus, long term contamination.