Long Term Impacts of a Genetically Engineered Microorganism (GEM) and Polycyclic Aromatic Hydrocarbons (PAHs) on Soil Bacterial Communities

Microbes capable of polyaromatic hydrocarbon (PAH) biodegradation can be used to remediate soils contaminated with these persistent pollutants. To monitor in situ PAH-biodegradation, the bioluminescent bio-reporter Pseudomonas fluorescens HK44, containing a lux luminescent gene cassette inserted into its naphthalene degradation operon, was released into PAH-contaminated soil in lysimeters in 1996. Three treatments were imposed: strain HK44 mixed with PAH-contaminated soil (PAH+, HK44+; n=3); strain HK44 mixed with uncontaminated soil (PAH–, HK44+; n=2) and PAH-contaminated soil alone (PAH+, HK44–; n=1). The objective of this study was to assess the long term impacts of these treatments on the indigenous soil bacterial community structure in the lysimeters. In 2010, 14 years after experiment initiation, replicate soil cores were taken from each lysimeter. Soil bacterial community structures were determined by 454 pyrosequencing of 16S rRNA gene amplicons. Even though PAH concentrations fell below detectable levels within the first couple years of the lysimeter experiment, PAH+ lysimeters showed significantly higher soil organic matter content (1.30 ± 0.23%) than PAH– lysimeters (0.81 ± 0.08%). 16S rRNA gene libraries reveal that there was a change in the bacterial community structure in PAH+ compared to PAH– lysimeters: 9.59% of OTUs (operational taxonomic units) were shared between PAH+ lysimeters while only 4.08% were shared between PAH+ and PAH– lysimeters. Multivariate ordination and cluster analysis, and phylogenetic tree-based analysis indicate that communities fell into three clusters: lysimeter 1 and 2 (both PAH+, HK44+); lysimeter 4 (PAH+, HK44+) and 6 (PAH+, HK44–); and lysimeter 3 and 5 (both PAH–, HK44+). Therefore over the long term, the addition of PAHs was more influential on bacterial community structure than the introduction of a GEM.