Microarray and Real-Time PCR Analyses of the Responses of High-Arctic Soil Bacteria to Hydrocarbon Pollution and Bioremediation Treatments

Abstract

High-Arctic soils have low nutrient availability, low moisture content, and very low temperatures and, as such, they pose a particular problem in terms of hydrocarbon bioremediation. An in-depth knowledge of the microbiology involved in this process is likely to be crucial to understand and optimize the factors most influencing bioremediation. Here, we compared two distinct large-scale field bioremediation experiments, located at the Canadian high-Arctic stations of Alert (ex situ approach) and Eureka (in situ approach). Bacterial community structure and function were assessed using microarrays targeting the 16S rRNA genes of bacteria found in cold environments and hydrocarbon degradation genes as well as quantitative reverse transcriptase PCR targeting key functional genes. The results indicated a large difference between sampling sites in terms of both soil microbiology and decontamination rates. A rapid reorganization of the bacterial community structure and functional potential as well as rapid increases in the expression of alkane monooxygenases and polyaromatic hydrocarbon-ring-hydroxylating dioxygenases were observed 1 month after the bioremediation treatment commenced in the Alert soils. In contrast, no clear changes in community structure were observed in Eureka soils, while key gene expression increased after a relatively long lag period (1 year). Such discrepancies are likely caused by differences in bioremediation treatments (i.e., ex situ versus in situ), weathering of the hydrocarbons, indigenous microbial communities, and environmental factors such as soil humidity and temperature. In addition, this study demonstrates the value of molecular tools for the monitoring of polar bacteria and their associated functions during bioremediation.With ongoing climate warming and the possible opening of the Northwest Passage for commercial shipping in the near future, human activity will increase in the Canadian high Arctic, raising the potential for environmental contamination. The settlements in the high Arctic are using fuel for transportation and to produce electricity and heating, while spills from leaking tanks or pipelines are frequent (19,34). Bioremediation is often the only feasible cleanup option because the remoteness and unique character of these sites preclude conventional physicochemical technologies for soil treatment. Arctic soils are characterized by extremely low temperatures and the limited availability of water and nutrients, especially nitrogen (18, 26), which limit the degradation rates by indigenous soil microorganisms. One of the possible bioremediation approaches is to stimulate indigenous cold-adapted microorganisms by the application of nutrients and water. This strategy was successfully applied to diesel-contaminated soils in the Canadian high Arctic (9,33,34). However, these studies mostly focused on soil chemistry and general soil processes, with very few insights into the ecology and the functions of the microorganisms actually involved in the bioremediation processes. Although ...

The functional potential of high Arctic permafrost revealed by metagenomic sequencing, qPCR and microarray analyses

“…Real-time qPCR qPCR was performed on unamplified DNA samples (unless stated otherwise) in 25 ml volumes using the iQ SYBR green Supermix (Bio-Rad Laboratories, Hercules, CA, USA) on a Rotor-Gene 3000 apparatus (Corbett Life Science, Sydney, NSW, Australia) as previously described (Yergeau et al, 2009a). The qPCR conditions and primers are given in Supplementary Table S1.…”

“…Standards were made from 10-fold dilutions of linearized plasmids containing the gene fragment of interest that was cloned from amplified soil DNA. Phage l DNA was used to evaluate the presence of PCR inhibitors as previously described (Yergeau et al, 2009a). The inhibition was comparably low for both DNA extracts (active: 1.50%, 2-m permafrost: 2.50%).…”

“…16S rRNA genes microarray A microarray platform targeting the 16S rRNA genes of Bacteria and Archaea found in cold environments was used as previously described (Yergeau et al, 2009a). To summarize, the 16S rRNA genes were amplified from the raw DNA extracts, chemically labeled and hybridized overnight onto the microarray.…”

“…Details about the microarray and probe design are available at the National Center for Biotechnology Information (NCBI) GEO database under the platform accession number GPL8953. The presenceabsence of the different taxa targeted by the microarray was then used as a community profile to evaluate the similarity between the active layer soil and 2-m permafrost, and to contrast it with previously assessed pristine Arctic surface soil samples from the same region (Yergeau et al, 2009a).…”

“…Even though some genera were retrieved using both methods, the microarray platform is completely independent of the metagenomic data set and was used to confirm the similarity in community structure observed between the two samples. The hybridization patterns of other pristine surface soil samples from the same location (Eureka) and from another location on Ellesmere Island (Alert) from a previous study (Yergeau et al, 2009a) were used here as a comparison for the active layer soil and 2-m permafrost. The active layer DNA hybridized to 33 probes and the 2-m permafrost DNA hybridized to 23 probes.…”