Elucidating microbial mechanisms of microcystin-LR degradation in Lake Erie beach sand through metabolomics and metatranscriptomics.

As one of five Laurentian Great Lakes, Lake Erie ranks among the top freshwater drinking sources and ecosystems globally. Historical and current agriculture mismanagement and climate change sustains the environmental landscape for late summer cyanobacterial harmful algal blooms, and consequently, cyanotoxins such as microcystin (MC). Microcystin microbial degradation is a promising mitigation strategy, however the mechanisms controlling the breakdown of MCs in Lake Erie are not well understood. Pelee Island, Ontario, Canada is located in the western basin of Lake Erie and the bacterial community in the sand has demonstrated the capacity of metabolizing the toxin. Through a multi-omic approach, the metabolic, functional and taxonomical signatures of the Pelee Island microbial community during MC-LR degradation was investigated over a 48-hour period to comprehensively study the degradation mechanism. Cleavage of bonds surrounding nitrogen atoms and the upregulation of nitrogen deamination (dadA, alanine dehydrogenase, leucine dehydrogenase) and assimilation genes (glnA, gltB) suggests a targeted isolation of nitrogen by the microbial community for energy production. Methylotrophic pathways RuMP and H4MPT control assimilation and dissimilation of carbon, respectively and differential abundance of Methylophilales indicates an interconnected role through electron exchange of denitrification and methylotrophic pathways. The detected metabolites did not resolve a clear breakdown pathway, but rather the diversity of products in combination with taxonomic and functional results supports that a variety of strategies are applied, such as epoxidation, hydroxylation, and aromatic degradation. Annual repeated exposure to the toxin may have allowed the community to adaptatively establish a novel pathway through functional plasticity and horizontal gene transfer. The culmination of these results reveals the complexity of the Pelee Island sand community and supports a dynamic and cooperative metabolism between microbial species to achieve MC degradation.

Host species and habitat shape fish-associated bacterial communities: phylosymbiosis between fish and their microbiome.

BACKGROUND: While many studies have reported that the structure of the gut and skin microbiota is driven by both species-specific and habitat-specific factors, the relative importance of host-specific versus environmental factors in wild vertebrates remains poorly understood. The aim of this study was to determine the diversity and composition of fish skin, gut, and surrounding water bacterial communities (hereafter referred to as microbiota) and assess the extent to which host habitat and phylogeny predict microbiota similarity. Skin swabs and gut samples from 334 fish belonging to 17 species were sampled in three Laurentian Great Lakes (LGLs) habitats (Detroit River, Lake Erie, Lake Ontario). We also collected and filtered water samples at the time of fish collection. We analyzed bacterial community composition using 16S metabarcoding and tested for community variation. RESULTS: We found that the water microbiota was distinct from the fish microbiota, although the skin microbiota more closely resembled the water microbiota. We also found that environmental (sample location), habitat, fish diet, and host species factors shape and promote divergence or convergence of the fish microbiota. Since host species significantly affected both gut and skin microbiota (separately from host species effects), we tested for phylosymbiosis using pairwise host species phylogenetic distance versus bacterial community dissimilarity. We found significant phylogenetic effects on bacterial community dissimilarity, consistent with phylosymbiosis for both the fish skin and gut microbiota, perhaps reflecting the longstanding co-evolutionary relationship between the host species and their microbiomes. CONCLUSIONS: Analyzing the gut and skin mucus microbiota across diverse fish species in complex natural ecosystems such as the LGLs provides insights into the potential for habitat and species-specific effects on the microbiome, and ultimately the health, of the host. Video Abstract.

Functional gene transcription variation in bacterial metatranscriptomes in large freshwater Lake Ecosystems: Implications for ecosystem and human health.

Little is known regarding the temporal and spatial functional variation of freshwater bacterial community (BC) under non-bloom conditions, especially in winter. To address this, we used metatranscriptomics to assess bacterial gene transcription variation among three sites across three seasons. Our metatranscriptome data for freshwater BCs at three public beaches (Ontario, Canada) sampled in the winter (no ice), summer and fall (2019) showed relatively little spatial, but a strong temporal variation. Our data showed high transcriptional activity in summer and fall but surprisingly, 89% of the KEGG pathway genes and 60% of the selected candidate genes (52 genes) associated with physiological and ecological activity were still active in freezing temperatures (winter). Our data also supported the possibility of an adaptively flexible gene expression response of the freshwater BC to low temperature conditions (winter). Only 32% of the bacterial genera detected in the samples were active, indicating that the majority of detected taxa were non-active (dormant). We also identified high seasonal variation in the abundance and activity of taxa associated with health risks (i.e., Cyanobacteria and waterborne bacterial pathogens). This study provides a baseline for further characterization of freshwater BCs, health-related microbial activity/dormancy and the main drivers of their functional variation (such as rapid human-induced environmental change and climate change).

Regulation of host gene expression by gastrointestinal tract microbiota in Chinook Salmon (Oncorhynchus tshawytscha).

Differences in gut microbiome composition are linked with health, disease and ultimately host fitness; however, the molecular mechanisms underlying that relationship are not well characterized. Here, we modified the fish gut microbiota using antibiotic and probiotic feed treatments to address the effect of host microbiome on gene expression patterns. Chinook salmon (Oncorhynchus tshawytscha) gut gene expression was evaluated using whole transcriptome sequencing (RNA-Seq) on hindgut mucosa samples from individuals treated with antibiotic, probiotic and control diets to determine differentially expressed (DE) host genes. Fifty DE host genes were selected for further characterization using nanofluidic qPCR chips. We used 16S rRNA gene metabarcoding to characterize the rearing water and host gut microbiome (bacterial) communities. Daily administration of antibiotics and probiotics resulted in significant changes in fish gut and aquatic microbiota as well as more than 100 DE genes in the antibiotic and probiotic treatment fish, relative to healthy controls. Normal microbiota depletion by antibiotics mostly led to downregulation of different aspects of immunity and upregulation of apoptotic process. In the probiotic treatment, genes related to post-translation modification and inflammatory responses were up-regulated relative to controls. Our qPCR results revealed significant effects of treatment (antibiotic and probiotic) on rabep2, aifm3, manf, prmt3 gene transcription. Moreover, we found significant associations between members of Lactobacillaceae and Bifidobacteriaceae with host gene expression patterns. Overall, our analysis showed that the microbiota had significant impacts on many host signalling pathways, specifically targeting immune, developmental and metabolic processes. Our characterization of some of the molecular mechanisms involved in microbiome-host interactions will help develop new strategies for preventing/ treating microbiome disruption-related diseases.

The Western Lake Erie culture collection: A promising resource for evaluating the physiological and genetic diversity of Microcystis and its associated microbiome.

Cyanobacterial harmful algal blooms (cyanoHABs) dominated by Microcystis spp. have significant public health and economic implications in freshwater bodies around the world. These blooms are capable of producing a variety of cyanotoxins, including microcystins, that affect fishing and tourism industries, human and environmental health, and access to drinking water. In this study, we isolated and sequenced the genomes of 21 primarily unialgal Microcystis cultures collected from western Lake Erie between 2017 and 2019. While some cultures isolated in different years have a high degree of genetic similarity (genomic Average Nucleotide Identity >99%), genomic data show that these cultures also represent much of the breadth of known Microcystis diversity in natural populations. Only five isolates contained all the genes required for microcystin biosynthesis while two isolates contained a previously described partial mcy operon. Microcystin production within cultures was also assessed using Enzyme-Linked Immunosorbent Assay (ELISA) and supported genomic results with high concentrations (up to 900 µg L⁻¹) in cultures with complete mcy operons and no or low toxin detected otherwise. These xenic cultures also contained a substantial diversity of bacteria associated with Microcystis, which has become increasingly recognized as an essential component of cyanoHAB community dynamics. These results highlight the genomic diversity among Microcystis strains and associated bacteria in Lake Erie, and their potential impacts on bloom development, toxin production, and toxin degradation. This culture collection significantly increases the availability of environmentally relevant Microcystis strains from temperate North America.

The effects of host quantitative genetic architecture on the gut microbiota composition of Chinook salmon (Oncorhynchus tshawytscha).

The microbiota consists of microbes living in or on an organism and has been implicated in host health and function. Environmental and host-related factors were shown to shape host microbiota composition and diversity in many fish species, but the role of host quantitative architecture across populations and among families within a population is not fully characterized. Here, Chinook salmon were used to determine if inter-population differences and additive genetic variation within populations influenced the gut microbiota diversity and composition. Specifically, hybrid stocks of Chinook salmon were created by crossing males from eight populations with eggs from an inbred line created from self-fertilized hermaphrodite salmon. Based on high-throughput sequencing of the 16S rRNA gene, significant gut microbial community diversity and composition differences were found among the hybrid stocks. Furthermore, additive genetic variance components varied among hybrid stocks, indicative of population-specific heritability patterns, suggesting the potential to select for specific gut microbiota composition for aquaculture purposes. Determining the role of host genetics in shaping their gut microbiota has important implications for predicting population responses to environmental changes and will thus impact conservation efforts for declining populations of Chinook salmon.

Microbe-sediment interactions in Great Lakes recreational waters: Implications for human health risk.

Microbial assessments of recreational water have traditionally focused on culturing or DNA-based approaches of the planktonic water column, omitting influence from microbe-sediment relationships. Sediment (bed and suspended) has been shown to often harbour levels of bacteria higher than the planktonic phase. The fate of suspended sediment (SS) bacteria is extensively related to transport dynamics (e.g., deposition) of the associated sediment/floc. When hydraulic energy allows, SS will settle, introducing new (potentially pathogenic) organisms to the bed. With turbulence, including waves, currents and swimmers, the risk of human ingestion is elevated due to resuspension of bed sediment and associated microbes. This research used multiplex nanofluidic reverse transcriptase quantitative PCR on RNA of bacteria associated with bed and SS to explore the active bacteria in freshwater shorelines. Bacterial genes of human health concern regarding recreational water use were targeted, such as faecal indicator bacteria (FIB), microbial source tracking genes and virulence factors from waterborne pathogens. Results indicate avian sources (i.e., gulls, geese) to be the largest nonpoint source of FIB associated with sediment in Great Lakes shorelines. This research introduces a novel approach to microbial water quality assessments and enhances our understanding of microbe-sediment dynamics and the quality of freshwater beaches.

Variation in the diversity of bacterial communities and potential sources of fecal contamination of beaches in the Huron to Erie corridor.

Understanding the diversity of bacteria and E.coli levels at beaches is important for managing health risks. This study compared temporal changes of the bacterial communities of Belle Isle Beach (Detroit, MI) and Sand Point Beach (Windsor, ONT), both located near the Lake St. Clair origin of the Detroit River. Water samples collected 4 days/week for 12 weeks in summer, were subjected to 16S rRNA analysis of amplicon sequencing and E. coli enumeration. Bacterial communities changed over time, as determined by cluster dendrogram analysis, exhibiting different communities in July and August than in June and different communities at the two beaches. After June, alpha diversity decreased and relative abundance of Enterobacter (Gammaproteobacteria) increased at Sand Point; whereas, Belle Isle maintained its alpha diversity and dominance by Betaproteobacteria and Actinobacteria. Contamination at both beaches is dominated by birds (23% to 50% of samples), while only ∼10% had evidence of human-associated bacteria. High E. coli at both beaches was often associated with precipitation. Nearshore sampling counts were higher than waist-deep sampling counts. Despite the dynamic changes in bacterial communities between the two beaches, this analysis based on 16S rRNA amplicon sequencing is able to provide information about bacterial types associated with high E. coli levels and to use bacterial sequences to more precisely determine sources and health relevance of contaminants.

Spatio-temporal dynamics of bacterial communities in the shoreline of Laurentian great Lake Erie and Lake St. Clair's large freshwater ecosystems.

BACKGROUND: Long-term trends in freshwater bacterial community composition (BCC) and dynamics are not yet well characterized, particularly in large lake ecosystems. We addressed this gap by temporally (15 months) and spatially (6 sampling locations) characterizing BCC variation in lakes Erie and St. Clair; two connected ecosystems in the Laurentian Great Lakes. RESULTS: We found a spatial variation of the BCC between the two lakes and among the sampling locations (significant changes in the relative abundance of 16% of the identified OTUs at the sampling location level). We observed five distinct temporal clusters (UPGMA broad-scale temporal variation) corresponding to seasonal variation over the 15 months of sampling. Temporal variation among months was high, with significant variation in the relative abundance of 69% of the OTUs. We identified significant differences in taxonomic composition between summer months of 2016 and 2017, with a corresponding significant reduction in the diversity of BCC in summer 2017. CONCLUSIONS: As bacteria play a key role in biogeochemical cycling, and hence in healthy ecosystem function our study defines the scope for temporal and spatial variation in large lake ecosystems. Our data also show that freshwater BCC could serve as an effective proxy and monitoring tool to access large lake health.

Microbial community and abiotic effects on aquatic bacterial communities in north temperate lakes.

The aquatic bacterial community (BC) plays a vital role in determining the nature and rate of ecosystem function. However, the biotic and abiotic factors influencing BC structure and function are largely unknown. Hence, the current study characterizes the impact of biotic and abiotic factors on aquatic bacterial biodiversity to determine whether the dominant effects are biotic or abiotic by partitioning their relative effects across temperate Canadian lakes. We collected water samples from sixty southern Ontario lakes and characterized their BC and microbial eukaryotic community (MEC) compositions using high throughput metabarcode sequencing of 16S and 18S rRNA gene fragments. The diversity and richness of aquatic BCs differed considerably among our study lakes, and those differences were explained by environmental, spatial, and biotic (MEC) factors (31%, 23%, and 23% of variance explained, respectively). The relatively large contribution from biotic and abiotic factors (54%), relative to spatial effects, shows deterministic processes prevail in shaping BC assembly in freshwater lakes. However, spatial effects also contributed significantly, highlighting the role of stochastic processes (ecological drift and coupled with limited dispersal) in shaping BC structure. Furthermore, our co-occurrence network analysis showed strong positive and negative interactions within and between the BCs and MECs, indicating mutualistic or antagonistic co-occurrence patterns relationships play important roles in driving the variation in BC composition among our sampled lakes. Considered together, our community analyses show that deterministic and stochastic processes combined contribute to determining the aquatic BC composition, and hence likely function as well, across a broad array of temperate freshwater lakes.

Environmental (e)RNA advances the reliability of eDNA by predicting its age.

Environmental DNA (eDNA) analysis has advanced conservation biology and biodiversity management. However, accurate estimation of age and origin of eDNA is complicated by particle transport and the presence of legacy genetic material, which can obscure accurate interpretation of eDNA detection and quantification. To understand the state of genomic material within the environment, we investigated the degradation relationships between (a) size of fragments (long vs short), (b) genomic origins (mitochondrial vs nuclear), (c) nucleic acids (eDNA vs eRNA), and (d) RNA types (messenger (m)RNA vs ribosomal (r)RNA) from non-indigenous Dreissena mussels. Initial concentrations of eRNA followed expected transcriptional trends, with rRNAs found at > 1000 × that of eDNA, and a mitosis-associated mRNA falling below detection limits within 24 h. Furthermore, the ratio of eRNA:eDNA significantly decreased throughout degradation, potentially providing an estimate for the age of genomic material. Thus, eRNA quantification can increase detection due to the high concentrations of rRNAs. Furthermore, it may improve interpretation of positive detections through the eRNA:eDNA ratio and/or by detecting low abundant mitosis-associated mRNAs that degrade within ~ 24 h.

Diel Dynamics of Freshwater Bacterial Communities at Beaches in Lake Erie and Lake St. Clair, Windsor, Ontario.

Bacteria play a key role in freshwater biogeochemical cycling as well as water safety, but short-term trends in freshwater bacterial community composition and dynamics are not yet well characterized. We sampled four public beaches in southern Ontario, Canada; in June, July, and August (2016) over a 24-h (diel) cycle at 2-h intervals. Using high-throughput sequencing of 16S rRNA gene, we found substantial bi-hourly and day/night variation in the bacterial communities with considerable fluctuation in the relative abundance of Actinobacteria and Proteobacteria phyla. Moreover, relative abundance of Enterobacteriaceae (associated with potential health risk) was significantly high at night in some dial cycles. Diversity was significantly high at night across most of the diel sampling events. qPCR assays showed a substantial bi-hourly variation of Escherichia coli levels with a significant high level of E. coli at night hours in comparison with day hours and the lowest levels at noon and during the afternoon hours. Taken together, these findings highlighted a considerable short-term temporal variation of bacterial communities which helps better understanding of freshwater bacterial dynamics and their ecology. E. coli monitoring showed that multiple samples in different hours will provide more accurate picture of freshwater safety and human health risk. Graphical abstract.

Metabarcoding of native and invasive species in stomach contents of Great Lakes fishes.

As aquatic invasive species (AIS) proliferate worldwide, a better understanding of their roles in invaded habitats is needed to inform management and introduction prevention strategies and priorities. Metabarcoding of stomach content DNA (scDNA) shows considerable promise in such regard. We thus metabarcoded scDNA from two non-native fish species (alewife (Alosa pseudoharengus) and rainbow smelt (Osmerus mordax)), and three native ones (bloater (Coregonus hoyi), ninespine stickleback (Pungitius pungitius), and slimy sculpin (Cottus cognatus)). Fishes (N = 376) were sampled in spring 2009 and 2010 from 73-128 m depths at three Lake Michigan sites. Four mitochondrial cytochrome oxidase 1 (CO1) primer sets designed to target five potential AIS prey, and a universal aquatic invertebrate CO1 primer set targeting both native and AIS prey were used. Quality controlled prey amplicons were matched to three AIS prey: Bythotrephes longimanus (mean percent frequency occurrence, all samples = 7%), Cercopagis pengoi (5%), and Dreissena rostriformis bugensis (11%). Neither invasive prey Dreissena polymorpha nor Hemimysis anomala were detected. Native prey Leptodiaptomus sicilis, Limnocalanus macrurus, and Mysis diluviana were relatively common in scDNA (respective mean percent occurrences, all samples: 48%, 25%, 42%). Analysis of variation in prey occurrences for sample site, predator species, sample year, sample depth, and predator total length (TL) indicated site and predator species were most important. However, B. longimanus occurrence in scDNA depended upon predator TL, perhaps indicative of its unique defensive spine limiting susceptibility to predation until fishes exceed species-specific gape-based limitations. Our analysis of native and invasive prey species indicated possible indirect AIS impacts such as native predators switching their diet due to AIS-driven losses of preferred native prey. Metabarcoding demonstrated that AIS are integrated components of the offshore Lake Michigan food web, with both native and non-native predators, and both invasive and native prey are affecting species interactions across multiple trophic levels.

'Nitrification kinetics and microbial community dynamics of attached biofilm in wastewater treatment'.

A comparative bench-scale and field site analysis of BioCord was conducted to investigate seasonal microbial community dynamics and its impact on nitrogen removal in wastewater. This was assessed using metabolite (NO3 -) stable isotope analysis, high-throughput sequencing of the 16S rRNA gene, and RT-qPCR of key genes in biological treatment representing nitrification, anammox, and denitrification. Bench-scale experiments showed an increase in nitrifiers with increasing ammonia loading resulting in an ammonia removal efficiency up to 98 ± 0.14%. Stable isotope analysis showed that 15ɛ and δ18ONO3 could be used in monitoring the efficiency of the enhanced biological nitrification. In the lagoon field trials, an increase in total nitrogen promoted three principle nitrifying genera (Nitrosomonas, Nitrospira, Candidatus Nitrotoga) and enhanced the expression of denitrification genes (nirK, norB, and nosZ). Further, anaerobic ammonia oxidizers were active within BioCord biofilm. Even at lower temperatures (2-6°C) the nitrifying bacteria remained active on the BioCord.

Exploring bacterial pathogen community dynamics in freshwater beach sediments: A tale of two lakes.

Pathogenic bacteria associated with freshwater ecosystems can pose significant health risks particularly where recreational water use is popular. Common water quality assessments involve quantifying indicator Escherichia coli within the water column but neglect to consider physical and geochemical factors and contributions from the sediment. In this study, we used high-throughput sequencing to investigate sediment microbial communities at four freshwater public beaches in southern Ontario, Canada and analysed community structure, function, and gene expression with relation to geographical characteristics. Our results indicate that beach sediments at the sediment-water interface could serve as potential sources of bacterial contamination under low-energy environments with tightly packed small sediment particles compared with high-energy environments. Further, the absence of pathogens but expression of pathogenic transcripts suggests occurrence of alternate gene acquisition. Pathogenicity at these locations included expression of Salmonella virulence factors, genes involved in pertussis, and antimicrobial resistance. Finally, we introduce a proposed universal bacterial pathogen model to consider the combined and synergistic processes used by these microbes. To our knowledge, this is the first study of its kind to investigate chemolithotrophic activity related to pathogens within bed sediment at freshwater beaches. This work helps advance current understanding of health risks in these environments.

Phytoplankton growth characterization in short term MPN culture assays using 18S metabarcoding and qRT-PCR.

The most probable number dilution-culture assay (MPN) is used to enumerate viable phytoplankton in regulatory tests of ballast water treatment systems. However the United States Coast Guard has not yet accepted MPN, in part due to concerns of biased results due to cells being viable but not growing. MPN does not assess the fate of every cell, and thus the bias can only be evaluated by a companion method that assesses the ability of the various taxa to grow. This growth ability ("growability") is the complement of the bias, and has been evaluated by microscopic taxonomy of before-culture and after-culture samples. However, microscopic taxonomy is extremely laborious and few data have been produced for phytoplankton growability in MPN assays. To address the need for more and more reliable growability data, a method was developed using next-generation sequencing (NGS) and quantitative real time PCR (qRT-PCR) techniques that target the V9 region of the 18S rRNA gene for the taxonomic identification and growth assessment of eukaryotic phytoplankton, respectively. This growability method was applied to MPN samples from a ballast water management system test that were incubated with two different enrichment media at two different temperatures. DNA was extracted from filters of before-culture and after-culture samples, and assessed for taxonomy by NGS and for PCR template DNA concentration by qRT-PCR. Growth ratios based on changes in 18S template concentration over the incubation period were calculated for each taxon, and dead-cell DNA persistence through a 14 day incubation was verified to be <1% and did not influence the growth calculations. In total, 95 of 97 eukaryotic phytoplankton in the before-culture sample demonstrated growth, with definitive growth ratios ranging from 4.0 × 101-2.6 × 105. An additional 13 taxa demonstrated growth from non-detect in before-culture samples. Taxa-based growability values were 87-88% in individual incubation conditions with no statistical differences among conditions, and 98% for all conditions combined. When growability was weighted by the before-culture abundance of each taxa, relevant to regulations based on all organisms regardless of taxa, community-based growability was >99% in each condition and in all conditions combined because the most abundant taxa all exhibited growth. This study verifies that conventional phytoplankton MPN assays produce accurate results with low bias from undetected viable cells, regardless of enrichments and incubation temperatures. This work can provide regulatory confidence for broader acceptance of MPN assays without limitations.

Inhibition of anaerobic biological sulfate reduction process by copper precipitates.

The single-stage biological sulfate reduction process for treatment of heavy metal laden wastewater is a promising treatment method, but the formation of metal precipitates has been suggested to be inhibitory to the activity of sulfate reducers. The present study examined the impact of copper (Cu) precipitates on anaerobic biological sulfate reduction in semi continuous stirred tank reactors (SCSTRs) at 35 ±â€¯2 °C. The results show that Cu precipitates significantly affected the sulfate reduction process. At an HRT of 50 days, steady-state sulfate reduction was approximately 55% at influent Cu concentrations of 0 (control) and 200 mg/L, which reduced to approximately 39% at influent Cu concentration of 400 mg/L. Microbial population (measured as volatile suspended solids) and rate of sulfate reduction were also affected, with reductions observed even at influent Cu of 200 mg/L. Copper precipitates also affected the microbial community diversity distribution.

Assessing high-throughput environmental DNA extraction methods for meta-barcode characterization of aquatic microbial communities.

The characterization of microbial community dynamics using genomic methods is rapidly expanding, impacting many fields including medical, ecological, and environmental research and applications. One of the biggest challenges for such studies is the isolation of environmental DNA (eDNA) from a variety of samples, diverse microbes, and widely variable community compositions. The current study developed environmentally friendly, user safe, economical, and high throughput eDNA extraction methods for mixed aquatic microbial communities and tested them using 16 s rRNA gene meta-barcoding. Five different lysis buffers including (1) cetyltrimethylammonium bromide (CTAB), (2) digestion buffer (DB), (3) guanidinium isothiocyanate (GITC), (4) sucrose lysis (SL), and (5) SL-CTAB, coupled with four different purification methods: (1) phenol-chloroform-isoamyl alcohol (PCI), (2) magnetic Bead-Robotic, (3) magnetic Bead-Manual, and (4) membrane-filtration were tested for their efficacy in extracting eDNA from recreational freshwater samples. Results indicated that the CTAB-PCI and SL-Bead-Robotic methods yielded the highest genomic eDNA concentrations and succeeded in detecting the core microbial community including the rare microbes. However, our study recommends the SL-Bead-Robotic eDNA extraction protocol because this method is safe, environmentally friendly, rapid, high-throughput and inexpensive.

Microbial metabolic strategies for overcoming low-oxygen in naturalized freshwater reservoirs surrounding the Athabasca Oil Sands: A proxy for End-Pit Lakes?

The success and sustainability of aquatic ecosystems are driven by the complex, cooperative metabolism of microbes. Ecological engineering strategies often strive to harness this syntrophic synergy of microbial metabolism for the reclamation of contaminated environments worldwide. Currently, there is a significant knowledge gap in our understanding of how the natural microbial ecology overcomes thermodynamic limitations in recovering contaminated environments. Here, we used in-situ metatranscriptomics and associated metataxonomic analyses on sediments collected from naturalized freshwater man-made reservoirs within the Athabasca Oil Sands region of Alberta, Canada. These reservoirs are unique since they are untouched by industrial mining processes and serve as representative endpoints for model landscape reconstruction. Results indicate that a microbial syntrophic cooperation has been established represented by the oxygenic and anoxygenic phototrophs, sustained through the efficient use of novel cellular mechanistic adaptations tailored to these unique thermodynamic conditions. Specifically, chemotaxis transcripts (cheY & MCPs-methyl-accepting chemotaxis proteins) were highly expressed, suggesting a highly active microbial response to gradients in environmental stimuli, resulting indirectly from hydrocarbon compound alteration. A high expression of photosynthetic activity, likely sustaining nutrient delivery to the similarly highly expressed methanogenic community acting in syntrophy during the breakdown of organics. Overall the more diversified functionality within sub-oxic sample locations indicates an ability to maintain efficient metabolism under thermodynamic constraints. This is the first study to holistically identify and characterize these types of in-situ, metabolic processes and address their thermodynamic feasibility within a global context for large landscape reconstruction. These characterizations of regional, natural landscapes surrounding the oil sands mining operation are severely lacking, but truly provide invaluable insight into end-point goals and targets for reclamation procedures.

Novel insights into freshwater hydrocarbon-rich sediments using metatranscriptomics: Opening the black box.

Baseline biogeochemical surveys of natural environments is an often overlooked field of environmental studies. Too often research begins once contamination has occurred, with a knowledge gap as to how the affected area behaved prior to outside (often anthropogenic) influences. These baseline characterizations can provide insight into proposed bioremediation strategies crucial in cleaning up chemical spill sites or heavily mined regions. Hence, this study was conducted to survey the in-situ microbial activity within freshwater hydrocarbon-rich environments cutting through the McMurray formation - the geologic strata constituting the oil sands. We are the first to report in-situ functional variations among these freshwater microbial ecosystems using metatranscriptomics, providing insight into the in-situ gene expression within these naturally hydrocarbon-rich sites. Key genes involved in energy metabolism (nitrogen, sulfur and methane) and hydrocarbon degradation, including transcripts relating to the observed expression of methane oxidation are reported. This information provides better linkages between hydrocarbon impacted environments, closing knowledge gaps for optimizing not only oil sands mine reclamation but also enhancing microbial reclamation strategies in various freshwater environments. These finding can also be applied to existing contaminated environments, in need of efficient reclamation efforts.