Impact of sample collection on prokaryotic and eukaryotic diversity of niche environments of the oil-sand mining impacted Athabasca River.

Microbial communities are an important aspect of overall riverine ecology; however, appreciation of the effects of anthropogenic activities on unique riverine microbial niches, and how the collection of these samples affects the observed diversity and community profile is lacking. We analyzed prokaryotic and eukaryotic communities from surface water, biofilms, and suspended load niches along a gradient of oil sands-related contamination in the Athabasca River (Alberta, Canada), with suspended load or particle-associated communities collected either via Kenney Sampler or centrifugation manifold. At the phylum level, different niche communities were highly similar to each other and across locations. However, there were significant differences in the abundance of specific genera among the different niches and across sampling locations. A generalized linear model revealed that use of the Kenney Sampler resulted in more diverse bacterial and eukaryotic suspended load community than centrifugal collection, though suspended load communities collected by any means remained stably diverse across locations. Although there was an influence of water quality parameters on community composition, all sampled sites support diverse bacterial and eukaryotic communities regardless of the degree of contamination, highlighting the need to look beyond ecological diversity as a means of assessing ecological perturbations, and consider collecting samples from multiple niche environments.

In situ microcosms deployed at the coast of British Columbia (Canada) to study dilbit weathering and associated microbial communities under marine conditions.

Douglas Channel and the adjacent Hecate Strait (British Columbia, Canada) are part of a proposed route to ship diluted bitumen (dilbit). This study presents how two types of dilbit naturally degrade in this environment by using an in situ microcosm design based on dilbit-coated beads. We show that dilbit-associated n-alkanes were microbially biodegraded with estimated half-lives of 57-69 days. n-Alkanes appeared to be primarily degraded using the aerobic alkB, ladA and CYP153 pathways. The loss of dilbit polycyclic aromatic hydrocarbons (PAHs) was slower than of n-alkanes, with half-lives of 89-439 days. A biodegradation of PAHs could not be conclusively determined, although a significant enrichment of the phnAc gene (a marker for aerobic PAH biodegradation) was observed. PAH degradation appeared to be slower in Hecate Strait than in Douglas Channel. Microcosm-associated microbial communities were shaped by the presence of dilbit, deployment location and incubation time but not by dilbit type. Metagenome-assembled genomes of putative dilbit-degraders were obtained and could be divided into populations of early, late and continuous degraders. The majority of the identified MAGs could be assigned to the orders Flavobacteriales, Methylococcales, Pseudomonadales and Rhodobacterales. A high proportion of the MAGs represent currently unknown lineages or lineages with currently no cultured representative.

Metatranscriptomic Insights Into the Response of River Biofilm Communities to Ionic and Nano-Zinc Oxide Exposures.

Manufactured Zn oxide nanoparticle (ZnO-NP) are extensively used world-wide in personal care and industrial products and are important contaminants of aquatic environments. To understand the overall impact of ZnO-NP contamination on aquatic ecosystems, investigation of their toxicity on aquatic biofilms is of particular consequence, given biofilms are known sinks for NP contaminants. In order to assess alterations in the functional activity of river microbial biofilm communities as a result of environmentally-relevant ZnO-NP exposure, biofilms were exposed to ionic zinc salt or ZnOPs that were uncoated (hydrophilic), coated with silane (hydrophobic) or stearic acid (lipophilic), at a total concentration of 188 µg l-1 Zn. ICP-MS analyses of biofilms indicated ZnO-NP concentrated in the biofilms, with hydrophilic, hydrophobic, and lipophilic treatments reaching 0.310, 0.250, and 0.220 µg Zn cm-2 of biofilm, respectively, while scanning transmission X-ray microspectroscopy (STXM) analyses of biofilms confirmed that Zn was extensively- and differentially-sorbed to biofilm material. Microbial community composition, based on taxonomic affiliation of mRNA sequences and enumeration of protozoa and micrometazoa, was not affected by these treatments, and the total transcriptional response of biofilms to all experimental exposures was not indicative of a global toxic-response, as cellular processes involved in general cell maintenance and housekeeping were abundantly transcribed. Transcripts related to major biological processes, including photosynthesis, energy metabolism, nitrogen metabolism, lipid metabolism, membrane transport, antibiotic resistance and xenobiotic degradation, were differentially expressed in Zn-exposures relative to controls. Notably, transcripts involved in nitrogen fixation and photosynthesis were decreased in abundance in response to Zn-exposure, while transcripts related to lipid degradation and motility-chemotaxis were increased, suggesting a potential role of Zn in biofilm dissolution. ZnO-NP and ionic Zn exposures elicited generally overlapping transcriptional responses, however hydrophilic and hydrophobic ZnO-NPs induced a more distinct effect than that of lipophilic ZnO-NPs, which had an effect similar to that of low ionic Zn exposure. While the physical coating of ZnO-NP may not induce specific toxicity observable at a community level, alteration of ecologically important processes of photosynthesis and nitrogen cycling are an important potential consequence of exposure to ionic Zn and Zn oxides.

Potential for Microbially Mediated Natural Attenuation of Diluted Bitumen on the Coast of British Columbia (Canada).

Western Canada produces large amounts of bitumen, a heavy, highly weathered crude oil. Douglas Channel and Hecate Strait on the coast of British Columbia are two water bodies that may be impacted by a proposed pipeline and marine shipping route for diluted bitumen (dilbit). This study investigated the potential of microbial communities from these waters to mitigate the impacts of a potential dilbit spill. Microcosm experiments were set up with water samples representing different seasons, years, sampling stations, and dilbit blends. While the alkane fraction of the tested dilbit blends was almost completely degraded after 28 days, the majority of the polycyclic aromatic hydrocarbons (PAHs) remained. The addition of the dispersant Corexit 9500A most often had either no effect or an enhancing effect on dilbit degradation. Dilbit-degrading microbial communities were highly variable between seasons, years, and stations, with dilbit type having little impact on community trajectories. Potential oil-degrading genera showed a clear succession pattern and were for the most part recruited from the "rare biosphere." At the community level, dispersant appeared to stimulate an accelerated enrichment of genera typically associated with hydrocarbon degradation, even in dilbit-free controls. This suggests that dispersant-induced growth of hydrocarbon degraders (and not only increased bioavailability of oil-associated hydrocarbons) contributes to the degradation-enhancing effect previously reported for Corexit 9500A.IMPORTANCE Western Canada hosts large petroleum deposits, which ultimately enter the market in the form of dilbit. Tanker-based shipping represents the primary means to transport dilbit to international markets. With anticipated increases in production to meet global energy needs, the risk of a dilbit spill is expected to increase. This study investigated the potential of microbial communities naturally present in the waters of a potential dilbit shipping lane to mitigate the effects of a spill. Here we show that microbial degradation of dilbit was mostly limited to n-alkanes, while the overall concentration of polycyclic aromatic hydrocarbons, which represent the most toxic fraction of dilbit, decreased only slightly within the time frame of our experiments. We further investigated the effect of the oil dispersant Corexit 9500A on microbial dilbit degradation. Our results highlight the fact that dispersant-associated growth stimulation, and not only increased bioavailability of hydrocarbons and inhibition of specific genera, contributes to the overall effect of dispersant addition.

Microbial Community Composition, Functions, and Activities in the Gulf of Mexico 1 Year after the Deepwater Horizon Accident.

Several studies have assessed the effects of the released oil on microbes, either during or immediately after the Deepwater Horizon accident. However, little is known about the potential longer-term persistent effects on microbial communities and their functions. In this study, one water column station near the wellhead (3.78 km southwest of the wellhead), one water column reference station outside the affected area (37.77 km southeast of the wellhead), and deep-sea sediments near the wellhead (3.66 km southeast of the wellhead) were sampled 1 year after the capping of the well. In order to analyze microbial community composition, function, and activity, we used metagenomics, metatranscriptomics, and mineralization assays. Mineralization of hexadecane was significantly higher at the wellhead station at a depth of ∼1,200 m than at the reference station. Community composition based on taxonomical or functional data showed that the samples taken at a depth of ∼1,200 m were significantly more dissimilar between the stations than at other depths (surface, 100 m, 750 m, and >1,500 m). Both Bacteria and Archaea showed reduced activity at depths of ∼1,200 m when the wellhead station was compared to the reference station, and their activity was significantly higher in surficial sediments than in 10-cm sediments. Surficial sediments also harbored significantly different active genera than did 5- and 10-cm sediments. For the remaining microbial parameters assessed, no significant differences could be observed between the wellhead and reference stations and between surface and 5- to 10-cm-deep sediments.

Next-generation sequencing of 16S ribosomal RNA gene amplicons.

One of the major questions in microbial ecology is "who is there?" This question can be answered using various tools, but one of the long-lasting gold standards is to sequence 16S ribosomal RNA (rRNA) gene amplicons generated by domain-level PCR reactions amplifying from genomic DNA. Traditionally, this was performed by cloning and Sanger (capillary electrophoresis) sequencing of PCR amplicons. The advent of next-generation sequencing has tremendously simplified and increased the sequencing depth for 16S rRNA gene sequencing. The introduction of benchtop sequencers now allows small labs to perform their 16S rRNA sequencing in-house in a matter of days. Here, an approach for 16S rRNA gene amplicon sequencing using a benchtop next-generation sequencer is detailed. The environmental DNA is first amplified by PCR using primers that contain sequencing adapters and barcodes. They are then coupled to spherical particles via emulsion PCR. The particles are loaded on a disposable chip and the chip is inserted in the sequencing machine after which the sequencing is performed. The sequences are retrieved in fastq format, filtered and the barcodes are used to establish the sample membership of the reads. The filtered and binned reads are then further analyzed using publically available tools. An example analysis where the reads were classified with a taxonomy-finding algorithm within the software package Mothur is given. The method outlined here is simple, inexpensive and straightforward and should help smaller labs to take advantage from the ongoing genomic revolution.

Microbial expression profiles in the rhizosphere of willows depend on soil contamination.

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.

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

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.

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

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.

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.

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

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.