Metagenomic analysis of the bioremediation of diesel-contaminated Canadian high arctic soils.

As human activity in the Arctic increases, so does the risk of hydrocarbon pollution events. On site bioremediation of contaminated soil is the only feasible clean up solution in these remote areas, but degradation rates vary widely between bioremediation treatments. Most previous studies have focused on the feasibility of on site clean-up and very little attention has been given to the microbial and functional communities involved and their ecology. Here, we ask the question: which microorganisms and functional genes are abundant and active during hydrocarbon degradation at cold temperature? To answer this question, we sequenced the soil metagenome of an ongoing bioremediation project in Alert, Canada through a time course. We also used reverse-transcriptase real-time PCR (RT-qPCR) to quantify the expression of several hydrocarbon-degrading genes. Pseudomonas species appeared as the most abundant organisms in Alert soils right after contamination with diesel and excavation (t = 0) and one month after the start of the bioremediation treatment (t = 1m), when degradation rates were at their highest, but decreased after one year (t = 1y), when residual soil hydrocarbons were almost depleted. This trend was also reflected in hydrocarbon degrading genes, which were mainly affiliated with Gammaproteobacteria at t = 0 and t = 1m and with Alphaproteobacteria and Actinobacteria at t = 1y. RT-qPCR assays confirmed that Pseudomonas and Rhodococcus species actively expressed hydrocarbon degradation genes in Arctic biopile soils. Taken together, these results indicated that biopile treatment leads to major shifts in soil microbial communities, favoring aerobic bacteria that can degrade hydrocarbons.

Influence of nutrients, hexadecane, and temporal variations on nitrification and exopolysaccharide composition of river biofilms.

Biofilms were cultivated on polycarbonate strips in rotating annular reactors using South Saskatchewan River water during the fall of 1999 and the fall of 2001. The reactors were supplemented with carbon (glucose), nitrogen (NH(4)Cl), phosphorus (KH(2)PO(4)), or combined nutrients (CNP), with or without hexadecane. The impact of these treatments on nitrification and on the exopolysaccharide composition of river biofilms was determined. The results showed that the biofilms had higher NH4(+) oxidation, NO3(-) production, and N2O production activities in fall 1999 than fall 2001 when grown with CNP but had higher activities in fall 2001 than fall 1999 when grown with individual nutrients. The exopolysaccharide amounts and proportions were generally higher in fall 1999 than fall 2001, as a consequence of the higher nutrient levels in the river water in the first year of this study. The addition of P and especially CNP stimulated NH4(+) oxidation by the biofilms, showing a P limitation in this river ecosystem. The presence of hexadecane negatively affected these activities and lowered the amounts of exopolysaccharides in CNP and P biofilms in fall 1999 but increased the biofilm activities and exopolysaccharide amounts in CNP biofilm in fall 2001. Antagonistic, synergistic, and independent effects between nutrients and hexadecane were also observed. This study demonstrated that the biofilm autotrophic nitrification activity in the South Saskatchewan River was limited by P, that this activity and the exopolysaccharide amounts and proportions were dependent on the nutrient concentrations in the river water, and suggested that exopolysaccharides may play a protective role for biofilm microorganisms against toxic pollutants.

Soil washing improves the recovery of total community DNA from polluted and high organic content sediments.

Treatment of soil with surfactants and chelating agents is used in bioremediation studies to desorb and solubilize contaminants to increase their bioavalability to microorganisms. In the same way that pollutants are made more bioavailable to microorganisms, the procedure can be used to remove potential interfering materials from soil prior to cell lysis and extraction of DNA from indigenous microorganisms. The effect of soil washing was evaluated by extracting DNA from sediments of an intertidal freshwater wetland contaminated with hydrocarbons and from highly contaminated marine sediments from Sydney Harbour, Nova Scotia, Canada. Sediment samples had total organic carbon (TOC) contents that varied between 0.2% and 13%. The chemical lysis technique was also examined by comparison of an ammonium acetate precipitation of proteins and humic acids with a hexadecyltrimethylammonium bromide (CTAB) incubation and phenol:chloroform extraction. In this study, the incorporation of soil washing steps facilitated the desorption of contaminants from sediment surfaces and improved the recovery of DNA of amplifiable quality from both freshwater and marine sediments. CTAB contributed only slightly to the recovery of DNA of higher quality in the most contaminated sample from Sydney Harbour and was concomitant with a decrease in DNA yield in both sediment types. The incorporation of a soil washing step prior to the extraction of DNA from polluted environments may be important to solubilize and remove contaminants when high-quality DNA is required for subsequent analyses.

Impact of seasonal variations and nutrient inputs on nitrogen cycling and degradation of hexadecane by replicated river biofilms.

Biofilm communities cultivated in rotating annular bioreactors using water from the South Saskatchewan River were assessed for the effects of seasonal variations and nutrient (C, N, and P) additions. Confocal laser microscopy revealed that while control biofilms were consistently dominated by bacterial biomass, the addition of nutrients shifted biofilms of summer and fall water samples to phototrophic-dominated communities. In nutrient-amended biofilms, similar patterns of nitrification, denitrification, and hexadecane mineralization rates were observed for winter and spring biofilms; fall biofilms had the highest rates of nitrification and hexadecane mineralization, and summer biofilms had the highest rates of denitrification. Very low rates of all measured activities were detected in control biofilms (without nutrient addition) regardless of season. Nutrient addition caused large increases in hexadecane mineralization and denitrification rates but only modest increases, if any, in nitrification rates, depending upon the season. Generally, both alkB and nirK were more readily PCR amplified from nutrient-amended biofilms. Both genes were amplified from all samples except for nirK from the fall control biofilm. It appears that bacterial production in the South Saskatchewan River water is limited by the availability of nutrients and that biofilm activities and composition vary with nutrient availability and time of year.