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.

Biogeochemical Processes and Microbial Dynamics Governing Phosphorus Retention and Release in Sediments: A Case Study in Lower Great Lakes Headwaters.

The ability of headwater bed and suspended sediments to mitigate non-point agricultural phosphorus (P) loads to the lower Great Lakes is recognized, but the specific biogeochemical processes promoting sediment P retention or internal P release remain poorly understood. To elucidate these mechanisms, three headwater segments located within priority watersheds of Southern Ontario, Canada, were sampled through the growing season of 2018-2020. The study employed equilibrium P assays along with novel assessments of legacy watershed nutrients, nitrogen (N) concentrations, sediment redox, and microbial community composition. 20-year data revealed elevated total P (TP) and total Nitrogen (TN) at an inorganic fertilizer and manure fertilizer-impacted site, respectively. Overall, sampled sites acted as P sinks; however, agricultural sediments exhibited significantly lower buffering capacity compared to a reference forested watershed. Collection of fine suspended sediment (<63 µm) through time-integrated sampling showed the suspended load at the inorganic-fertilized site was saturated with P, indicating a greater potential for P release into surface waters compared to bed sediments. Through vertical microsensor profiling and DNA sequencing of the sediment microbial community, site-specific factors associated with a distinct P-source event were identified. These included rapid depletion of dissolved oxygen (DO) across the sediment water interface (SWI), as well as the presence of nitrate-reducing bacterial and ammonia-oxidizing archaeal (AOA) genera. This research provides valuable insights into the dynamics of P in headwaters, shedding light on P retention and release. Understanding these processes is crucial for effective management strategies aimed at mitigating P pollution to the lower Great Lakes.

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.

Identifying chemolithotrophic and pathogenic-related gene expression within suspended sediment flocs in freshwater environments: A metatranscriptomic assessment.

The introduction and proliferation of pathogenic organisms in aquatic systems is a serious global issue that consequently leads to economic, financial, and health concerns. Health and safety related to recreational water use is typically monitored through water quality assessments that are outdated and can be misleading. These traditional methods focus on broad taxa groups, provide no insight into the active community or source of contamination, and the sediment compartments (bed and suspended) are often overlooked. To bridge this knowledge gap, our study aimed to 1) examine the metatranscriptome of the microbial community associated with suspended sediment (SS) in freshwater systems; 2) explore the influence of SS in tributaries to the littoral zone of the receiving lake; and 3) compare the SS fraction with previously reported nearshore bed sediment data. Samples were collected seasonally from Lake St. Clair and Lake Erie. Beaches in this region are influenced by both agriculture runoff and continued urban expansion. Results show that both adjacent tributary and beach SS have similar microbial functional diversity and are strongly correlated by site and season. We identified expression of transcripts encoding sequences with similarities to genes involved in nine bacterial infectious disease pathways, including legionellosis (sdhA) and Vibrio cholerae pathogenesis. According to MG-RAST gene categories, lake samples typically showed higher overall expression (p < 0.05) of transcripts with similarities to genes involved in infectious disease pathways compared to the tributaries, with summer upregulated (p < 0.05) compared to fall. Our data suggests SS acts as a strong vector for pathogen transport, making this facet an important area for further research as it pertains to human health regarding recreational water use. To our knowledge, this work is the first to investigate SS in aquatic microbial communities using metatranscriptomic analyses and has significant potential to help address growing issues of microbial contamination impacting freshwater security.

Averting an Outbreak of SARS-CoV-2 in a University Residence Hall through Wastewater Surveillance.

A wastewater surveillance program targeting a university residence hall was implemented during the spring semester 2021 as a proactive measure to avoid an outbreak of COVID-19 on campus. Over a period of 7 weeks from early February through late March 2021, wastewater originating from the residence hall was collected as grab samples 3 times per week. During this time, there was no detection of SARS-CoV-2 by reverse transcriptase quantitative PCR (RT-qPCR) in the residence hall wastewater stream. Aiming to obtain a sample more representative of the residence hall community, a decision was made to use passive samplers beginning in late March onwards. Adopting a Moore swab approach, SARS-CoV-2 was detected in wastewater samples just 2 days after passive samplers were deployed. These samples also tested positive for the B.1.1.7 (Alpha) variant of concern (VOC) using RT-qPCR. The positive result triggered a public health case-finding response, including a mobile testing unit deployed to the residence hall the following day, with testing of nearly 200 students and staff, which identified two laboratory-confirmed cases of Alpha variant COVID-19. These individuals were relocated to a separate quarantine facility, averting an outbreak on campus. Aggregating wastewater and clinical data, the campus wastewater surveillance program has yielded the first estimates of fecal shedding rates of the Alpha VOC of SARS-CoV-2 in individuals from a nonclinical setting. IMPORTANCE Among early adopters of wastewater monitoring for SARS-CoV-2 have been colleges and universities throughout North America, many of whom are using this approach to monitor congregate living facilities for early evidence of COVID-19 infection as an integral component of campus screening programs. Yet, while there have been numerous examples where wastewater monitoring on a university campus has detected evidence for infection among community members, there are few examples where this monitoring triggered a public health response that may have averted an actual outbreak. This report details a wastewater-testing program targeting a residence hall on a university campus during spring 2021, when there was mounting concern globally over the emergence of SARS-CoV-2 variants of concern, reported to be more transmissible than the wild-type Wuhan strain. In this communication, we present a clear example of how wastewater monitoring resulted in actionable responses by university administration and public health, which averted an outbreak of COVID-19 on a university campus.

Investigating the microbial dynamics of microcystin-LR degradation in Lake Erie sand.

Cyanobacterial blooms and the associated hepatotoxins produced (e.g., microcystins, MCs) create a significant human health risk in freshwater lakes around the world, including Lake Erie. Though various physical and chemical treatment options are utilized, these are costly and their effectiveness decreases when other organics are present. Laboratory studies have identified a remediation option based on a mlr gene operon that can systematically degrade this toxin; however, studies on Lake Erie have been unable to amplify mlr genes from MC-degrading bacteria. These results suggest that either existing primers may be inefficient for broad identification of the mlr genes or that MC degradation genes and/or pathways may vary among bacterial taxa. To investigate the dynamics of the Lake Erie microbial community involved in the degradation of microcystin-LR (MC-LR), a flow-through column experiment using collected beach sand was conducted over a period of six weeks. Increasing concentrations of lake water spiked with MC-LR were continuously delivered to both biotic and abiotic (sterilized) sand columns, with influent and effluent MC-LR concentrations measured by LC-MS/MS. Despite the toxin concentrations far exceeding natural conditions during a bloom event (maximum dosage = 15.4 µg/L), MC-LR was completely removed within 21 h of contact time in the biotic columns. Stimulation of community taxa during the degradation process included Burkholderiaceae, Illumatobacteraceae, Pseudomonadaceae, Rhodocyclaceae and Nitrosomonadaceae. The overall results suggest several critical species may be required for the most complete and effective degradation of MC-LR.

Nitrate assimilation, dissimilatory nitrate reduction to ammonium, and denitrification coexist in Pseudomonas putida Y-9 under aerobic conditions.

The specific nitrate reduction pathway in Pseudomonas putida Y-9 under aerobic conditions was studied. Strain Y-9 removed 82% of the nitrate accompanied by an accumulation of ammonium and a decrease of total nitrogen. Ammonium inhibited nitrate transformation (removal efficiency was 22.65%), illustrating that nitrate assimilation exists in strain Y-9. The detectable ammonium in the supernatant during the nitrate reduction process came from intracellular locations in strain Y-9. The nirBD that encodes nitrite reductase had an important role in strain growth and ammonium production. A 15N isotope experiment demonstrated that strain Y-9 can conduct dissimilatory nitrate reduction to ammonium (DNRA) and nirBD controls this process. This further indicated that the loss of total nitrogen is due to denitrification. All results highlighted that strain Y-9 performs simultaneous nitrate assimilation, DNRA, and denitrification under aerobic conditions, and nirBD controls the assimilation and DNRA process. Thereinto, nitrate assimilation dominates the removal of nitrate.

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.

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.

Bio-physicochemical effects of gamma irradiation treatment for naphthenic acids in oil sands fluid fine tailings.

Naphthenic acids (NAs) are persistent compounds that are components of most petroleum, including those found in the Athabasca oil sands. Their presence in freshly processed tailings is of significant environmental concern due to their toxicity to aquatic organisms. Gamma irradiation (GI) was used to reduce the toxicity and concentration of NAs in oil sands process water (OSPW) and fluid fine tailings (FFT). This investigation systematically studied the impact of GI on the biogeochemical development and progressive reduction of toxicity using laboratory incubations of fresh and aged tailings under anoxic and oxic conditions. GI reduced NA concentrations in OSPW by up to 97% in OSPW and in FFT by 85%. The GI-treated FFT exhibited increased rates of biogeochemical change, dependent on the age of the tailings source. Dissolved oxygen (DO) flux was enhanced in GI-treated FFT from fresh and aged source materials, whereas hydrogen sulfide (HS(-)) flux was stimulated only in the fresh FFT. Acute toxicity to Vibrio fischeri was immediately reduced following GI treatment of fresh OSPW. GI treatment followed by 4-week incubation reduced toxicity of aged OSPW to V. fischeri.

Simultaneous release of Fe and As during the reductive dissolution of Pb-As jarosite by Shewanella putrefaciens CN32.

Jarosites are produced during metallurgical processing, on oxidized sulfide deposits, and in acid mine drainage environments. Despite the environmental relevance of jarosites, few studies have examined their biogeochemical stability. This study demonstrates the simultaneous reduction of structural Fe(III) and aqueous As(V) during the dissolution of synthetic Pb-As jarosite (PbFe(3)(SO(4),AsO(4))(2)(OH)(6)) by Shewanella putrefaciens using batch experiments under anaerobic circumneutral conditions. Fe(III) reduction occurred immediately in inoculated samples while As(V) reduction was observed after 72 h. XANES spectra showed As(III) (14.7%) in the solid phase at 168 h coincident with decreased aqueous As(V). At 336 h, XANES spectra and aqueous speciation analysis demonstrated 20.2% and 3.0% of total As was present as As(III) in the solid and aqueous phase, respectively. In contrast, 12.4% of total Fe was present as aqueous Fe(II) and was below the detection limits of XANES in the solid phase. TEM-EDS analysis at 336 h showed secondary precipitates enriched in Fe and O with minor amounts of As and Pb. Based on experimental data and thermodynamic modeling, we suggest that structural Fe(III) reduction was thermodynamically driven while aqueous As(V) reduction was triggered by detoxification induced to offset the high As(V) (328 µM) concentrations released during dissolution.

Reductive dissolution of Tl(I)-jarosite by Shewanella putrefaciens: providing new insights into Tl biogeochemistry.

Thallium (Tl) is emerging as a metal of concern in countries such as China due to its release during the natural weathering of Tl-bearing ore deposits and mining activities. Despite the high toxicity of Tl, few studies have examined the reductive dissolution of Tl mineral phases by microbial populations. In this study we examined the dissolution of synthetic Tl(I)-jarosite, (H(3)O)(0.29)Tl(0.71)Fe(2.74)(SO(4))(2)(OH)(5.22)(H(2)O)(0.78), by Shewanella putrefaciens CN32 using batch experiments under anaerobic circumneutral conditions. Fe(II) concentrations were measured over time and showed Fe(II) production (4.6 mM) in inoculated samples by 893 h not seen in mineral and dead cell controls. Release of aqueous Tl was enhanced in inoculated samples whereby maximum concentrations in inoculated and cell-free samples reached 3.2 and 2.1 mM, respectively, by termination of the experiment. Complementary batch Tl/S. putrefaciens sorption experiments were conducted under experimentally relevant pH (5 and 6.3) at a Tl concentration of 35 µM and did not show significant Tl accumulation by either live or dead cells. Therefore, in contrast to many metals such as Pb and Cd, S. putrefaciens does not represent a sink for Tl in the environment and Tl is readily released from Tl-jarosite during both abiotic and biotic dissolution.

Intracellular precipitation of Pb by Shewanella putrefaciens CN32 during the reductive dissolution of Pb-jarosite.

Jarosites (MFe(3)(SO(4))(2)(OH)(6)) are precipitated in the Zn industry to remove impurities during the extraction process and contain metals such as Pb and Ag. Jarosite wastes are often confined to capped tailings ponds, thereby creating potential for anaerobic reductive dissolution by microbial populations. This study demonstrates the reductive dissolution of synthetic Pb-jarosite (PbFe(6)(SO(4))(4)(OH)(12)) by a subsurface dissimilatory Fe reducing bacterium (Shewanella putrefaciens CN32) using batch experiments under anaerobic circumneutral conditions. Solution chemistry, pH, Eh, and cell viability were monitored over time and illustrated the reduction of released structural Fe(III) from the Pb-jarosite to Fe(II). Inoculated samples containing Pb-jarosite also demonstrated decreased cellular viability coinciding with increased Pb concentrations. SEM images showed progressive nucleation of electron dense nanoparticles on the surface of bacteria, identified by TEM/EDS as intracellular crystalline precipitates enriched in Pb and P. The intracellular precipitation of Pb by S. putrefaciens CN32 observed in this study provides potential new insight into the biogeochemical cycling of Pb in reducing environments.

Field column study using zerovalent iron for mercury removal from contaminated groundwater.

Passive in situ remediation technologies, for example, permeable reactive barriers, PRBs, are an attractive and less expensive alternative compared to conventional pump and treat systems for groundwater remediation. Field column experiments were conducted to evaluate the removal of dissolved mercury from groundwater using zerovalent iron as the reactive media. Two column tests were conducted over a 6-week period, which simulated 2 and 10 years of groundwater flow through a potential full-scale treatment system. The influent groundwater pH was 7.8-9.5. The groundwater was reduced with an Eh, corrected to the standard hydrogen electrode, ranging from 0 to 120 mV over the trial period. Prior to treatment the total mercury concentration of the groundwater was approximately 40 microg L(-1). Effluent from the 10-year simulation contained approximately 0.5 microg/L of mercury during the first 3 weeks and increased to as much as 4 microg L(-1) by the end of the testing period. Effluent from the 2-year simulation was generally < 0.1 microg L(-1). Profile sampling of the 2-year simulation suggests that most of the mercury removal occurred in the initial 50% of the 20 cm column. Mineralogical studies, conducted using SEM/EDS and X-ray absorption spectroscopy (XAS), confirm the accumulation of mercury onto a zerovalent iron surface in this 20-cm zone. These analyses indicate that mercury accumulated as a mercury sulfide with a stoichiometery similar to those of cinnabar and metacinnabar (HgS).