Identification of Nitrogen-Incorporating Bacteria in Petroleum-Contaminated Arctic Soils by Using [ ¹⁵ N]DNA-Based Stable Isotope Probing and Pyrosequencing

Abstract

Arctic soils are increasingly susceptible to petroleum hydrocarbon contamination, as exploration and exploitation of the Arctic increase. Bioremediation in these soils is challenging due to logistical constraints and because soil temperatures only rise above 0°C for ϳ2 months each year. Nitrogen is often added to contaminated soil in situ to stimulate the existing microbial community, but little is known about how the added nutrients are used by these microorganisms. Microbes vary widely in their ability to metabolize petroleum hydrocarbons, so the question becomes: which hydrocarbon-degrading microorganisms most effectively use this added nitrogen for growth? Using [15 N]DNA-based stable isotope probing, we determined which taxonomic groups most readily incorporated nitrogen from the monoammonium phosphate added to contaminated and uncontaminated soil in Canadian Forces Station-Alert, Nunavut, Canada. Fractions from each sample were amplified with bacterial 16S rRNA and alkane monooxygenase B (alkB) gene-specific primers and then sequenced using lage-scale parallel-pyrosequencing. Sequence data was combined with 16S rRNA and alkB gene C quantitative PCR data to measure the presence of various phylogenetic groups in fractions at different buoyant densities. Several families of Proteobacteria and Actinobacteria that are directly involved in petroleum degradation incorporated the added nitrogen in contaminated soils, but it was the DNA of Sphingomonadaceae that was most enriched in 15 N. Bacterial growth in uncontaminated soils was not stimulated by nutrient amendment. Our results suggest that nitrogen uptake efficiency differs between bacterial groups in contaminated soils. A better understanding of how groups of hydrocarbon-degraders contribute to the catabolism of petroleum will facilitate the design of more targeted bioremediation treatments.

Predictable bacterial composition and hydrocarbon degradation in Arctic soils following diesel and nutrient disturbance

“…This lack of success has been blamed on a failure to consider other environmental controls on hydrocarbon degraders (Thompson et al, 2005), and is not surprising in light of our results. Although hydrocarbon contamination can reduce the number of bacterial taxa in Arctic soils, there are still many that remain active (Bell et al, 2011), and competitive interactions between these remaining groups are likely to be governed by other environmental parameters. Our experimental setup controlled for contaminant composition, age and concentration, and found that differences in phylum-level community composition was best explained by soil organic matter content, especially in DSL-MAP soils.…”

“…The amendment that we selected is comparable in both type and concentration to those that have been used previously at hydrocarbon-contaminated Arctic sites (ThomassinLacroix et al, 2002;McCarthy et al, 2004;Greer, 2009;Bell et al, 2011). We expected that the success of hydrocarbon-degrading bacteria in disturbed soils would be governed by adaptations to the soil environment, and that we would observe clear differences in the relative abundance of hydrocarbon-degrading groups across soil gradients.…”

“…We selected diesel fuel as it is a common contaminant of polar soils, as is jet fuel (Aislabie et al, 2006;Bell et al, 2011;Yergeau et al, 2012c), which is similar in composition to diesel. Soils were incubated for 1 week at 1 1C to allow abiotic loss of the volatile components of diesel, as most short-term evaporative weathering can occur within 1 week of contamination (Neff et al, 2000).…”

“…In addition, despite the identification of many hydrocarbon-degrading taxa across ecosystems, it is generally not known which indigenous microorganisms should be promoted to optimize the rate and extent of bioremediation in contaminated environments. Added nutrients are not incorporated equally by bacterial taxa in hydrocarbon-contaminated soils (Bell et al, 2011), and because of the fact that many taxa are able to remain active following hydrocarbon contamination, it is not clear that the most effective hydrocarbondegrading taxa are favoured by bioremediation treatments or the natural environment.…”

Next-Generation Sequencing of Microbial Communities in the Athabasca River and Its Tributaries in Relation to Oil Sands Mining Activities

“…Amplification for 454 and for Ion Torrent sequencing was carried out using the Fusion protocol. For 454 sequencing, bacterial 16S rRNA gene amplification was carried out as described by Bell et al (1) using the forward primer Univ-9F (5=-GAG TTT GAT YMT GGC TC-3=) and the reverse primer BR534/18 (5=-ATT ACC GCG GCT GCT GGC-3=) (42) that were linked to Roche 454 adapters and the multiplex identifiers listed in Table S1 in the supplemental material. For Archaea 16S rRNA gene amplification, we used the following primers: forward primer 5=-CCA TCT CAT CCC TGC GTG TCT CCG ACT CAG XXX XXX XXX XAA TTG GAN TCA ACG CCG G-3=, first reverse primer 5=-CCT CTC TAT GGG CAG TCG GTG ATC GRC GGC CAT GCA CCW C-3=, and second reverse primer 5=-CCT CTC TAT GGG CAG TCG GTG ATC GRC RGC CAT GYA CCW C-3=, where the Xs represent the sample-specific multiplex identifier (listed in Table S1 in the supplemental material), the underlined sequences represent the template-specific sequences (958arcF, 1048arcRmajor, and 1048arcRminor, respectively, from reference 8), and the remaining sequence is the Ion Torrent adapter A (forward) and adapter trP1 (reverse).…”