next-generation-sequencing-of-16s-ribosomal-rna-gene-amplicons

Next-generation Sequencing of 16S Ribosomal RNA Gene Amplicons

One of the major questions in microbial ecology is &#8220;who is there?&#8221; This question can be answered using various tools, but one of the ...

National Research Council Canada

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.

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

The Athabasca oil sands deposit is the largest reservoir of crude bitumen in the world. Recently, the soaring demand for oil and the availability of modern bitumen extraction technology have heightened exploitation of this reservoir and the potential unintended consequences of pollution in the Athabasca River. The main objective of the present study was to evaluate the potential impacts of oil sands mining on neighboring aquatic microbial community structure. Microbial communities were sampled from sediments in the Athabasca River and its tributaries as well as in oil sands tailings ponds. Bacterial and archaeal 16S rRNA genes were amplified and sequenced using next-generation sequencing technology (454 and Ion Torrent). Sediments were also analyzed for a variety of chemical and physical characteristics. Microbial communities in the fine tailings of the tailings ponds were strikingly distinct from those in the Athabasca River and tributary sediments. Microbial communities in sediments taken close to tailings ponds were more similar to those in the fine tailings of the tailings ponds than to the ones from sediments further away. Additionally, bacterial diversity was significantly lower in tailings pond sediments. Several taxonomic groups of Bacteria and Archaea showed significant correlations with the concentrations of different contaminants, highlighting their potential as bioindicators. We also extensively validated Ion Torrent sequencing in the context of environmental studies by comparing Ion Torrent and 454 data sets and by analyzing control samples.

Next-generation sequencing of microbial communities in the Athabasca River and its tributaries in relation to oil sands mining activities.

Surface waters worldwide are contaminated by pharmaceutical products that are released into the environment from wastewater treatment plants. Here, we hypothesize that pharmaceutical products have effects on organisms as well as genes related to nutrient cycling in complex microbial communities. To test this hypothesis, biofilms were grown in reactors and subjected low concentrations of three antibiotics [erythromycin, ER, sulfamethoxazole, SL and sulfamethazine, SN) and a lipid regulator (gemfibrozil, GM). Total community RNA was extracted and sequenced together with PCR amplicons of the 16S rRNA gene using 454 pyrosequencing. Exposure to pharmaceutical products resulted in very little change in bacterial community composition at the phylum level based on 16S rRNA gene amplicons, even though some genera were significantly affected. In contrast, large shifts were observed in the active community composition based on taxonomic affiliations of mRNA sequences. Consequently, expression of gene categories related to N, P and C cycling were strongly affected by the presence of pharmaceutical products, with each treatment having specific effects. These results indicate that low pharmaceutical product concentrations rapidly provoke a variety of functional shifts in river bacterial communities. In the longer term these shifts in gene expression and microbial activity could lead to a disruption of important ecosystem processes like nutrient cycling.

Sub-inhibitory concentrations of different pharmaceutical products affect the meta-transcriptome of river biofilm communities cultivated in rotating annular reactors.

Sediments from the Athabasca River and its tributaries naturally contain bitumen at various concentrations, but the impacts of this variation on the ecology of the river are unknown. Here, we used controlled rotating biofilm reactors in which we recirculated diluted sediments containing various concentrations of bituminous compounds taken from the Athabasca River and three tributaries. Biofilms exposed to sediments having low and high concentrations of bituminous compounds were compared. The latter were 29% thinner, had a different extracellular polysaccharide composition, 67% less bacterial biomass per μm2, 68% less cyanobacterial biomass per μm2, 64% less algal biomass per μm2, 13% fewer protozoa per cm2, were 21% less productive, and had a 33% reduced content in chlorophyll a per mm2 and a 20% reduction in the expression of photosynthetic genes, but they had a 23% increase in the expression of aromatic hydrocarbon degradation genes. Within the Bacteria, differences in community composition were also observed, with relatively more Alphaproteobacteria and Betaproteobacteria and less Cyanobacteria, Bacteroidetes, and Firmicutes in biofilms exposed to high concentrations of bituminous compounds. Altogether, our results suggest that biofilms that develop in the presence of higher concentrations of bituminous compounds are less productive and have lower biomass, linked to a decrease in the activities and abundance of photosynthetic organisms likely due to inhibitory effects. However, within this general inhibition, some specific microbial taxa and functional genes are stimulated because they are less sensitive to the inhibitory effects of bituminous compounds or can degrade and utilize some bitumen-associated compounds.

Aerobic biofilms grown from Athabasca watershed sediments are inhibited by increasing concentrations of bituminous compounds.

The goal of phytoremediation is to use plants to immobilize, extract or degrade organic and inorganic pollutants. In the case of organic contaminants, plants essentially act indirectly through the stimulation of rhizosphere microorganisms. A detailed understanding of the effect plants have on the activities of rhizosphere microorganisms could help optimize phytoremediation systems and enhance their use. In this study, willows were planted in contaminated and non-contaminated soils in a greenhouse, and the active microbial communities and the expression of functional genes in the rhizosphere and bulk soil were compared. Ion Torrent sequencing of 16S rRNA and Illumina sequencing of mRNA were performed. Genes related to carbon and amino-acid uptake and utilization were upregulated in the willow rhizosphere, providing indirect evidence of the compositional content of the root exudates. Related to this increased nutrient input, several microbial taxa showed a significant increase in activity in the rhizosphere. The extent of the rhizosphere stimulation varied markedly with soil contamination levels. The combined selective pressure of contaminants and rhizosphere resulted in higher expression of genes related to competition (antibiotic resistance and biofilm formation) in the contaminated rhizosphere. Genes related to hydrocarbon degradation were generally more expressed in contaminated soils, but the exact complement of genes induced was different for bulk and rhizosphere soils. Together, these results provide an unprecedented view of microbial gene expression in the plant rhizosphere during phytoremediation.

Article	Year
Metagenomic analysis of the bioremediation of diesel-contaminated Canadian high arctic soils. PloS one\| PubMed ID: 22253877	2012
Next-generation sequencing of microbial communities in the Athabasca River and its tributaries in relation to oil sands mining activities. Applied and environmental microbiology\| PubMed ID: 22923391	2012
Sub-inhibitory concentrations of different pharmaceutical products affect the meta-transcriptome of river biofilm communities cultivated in rotating annular reactors. Environmental microbiology reports\| PubMed ID: 23760799	2012
Aerobic biofilms grown from Athabasca watershed sediments are inhibited by increasing concentrations of bituminous compounds. Applied and environmental microbiology\| PubMed ID: 24056457	2013
Microbial expression profiles in the rhizosphere of willows depend on soil contamination. The ISME journal\| PubMed ID: 24067257	2014

Sylvie Sanschagrin

Biography

JoVE Journal Publications

Other Publications