O2 partitioning of sulfur oxidizing bacteria drives acidity and thiosulfate distributions in mining waters.

The acidification of water in mining areas is a global environmental issue primarily catalyzed by sulfur-oxidizing bacteria (SOB). Little is known about microbial sulfur cycling in circumneutral pH mine tailing impoundment waters. Here we investigate biological sulfur oxidation over four years in a mine tailings impoundment water cap, integrating aqueous sulfur geochemistry, genome-resolved metagenomics and metatranscriptomics. The microbial community is consistently dominated by neutrophilic, chemolithoautotrophic SOB (relative abundances of ~76% in 2015, ~55% in 2016/2017 and ~60% in 2018). Results reveal two SOB strategies alternately dominate across the four years, influencing acid generation and sulfur speciation. Under oxic conditions, novel Halothiobacillus drive lower pH conditions (as low as 4.3) and lower [S2O32-] via the complete Sox pathway coupled to O2. Under anoxic conditions, Thiobacillus spp. dominate in activity, via the incomplete Sox and rDSR pathways coupled to NO3-, resulting in higher [S2O32-] and no net significant acidity generation. This study provides genomic evidence explaining acidity generation and thiosulfate accumulation patterns in a circumneutral mine tailing impoundment and has significant environmental applications in preventing the discharge of sulfur compounds that can impact downstream environments. These insights illuminate opportunities for in situ biotreatment of reduced sulfur compounds and prediction of acidification events using gene-based monitoring and in situ RNA detection.

Alum Addition Triggers Hypoxia in an Engineered Pit Lake.

Here, we examine the geobiological response to a whole-lake alum (aluminum sulfate) treatment (2016) of Base Mine Lake (BML), the first pilot-scale pit lake established in the Alberta oil sands region. The rationale for trialing this management amendment was based on its successful use to reduce internal phosphorus loading to eutrophying lakes. Modest increases in water cap epilimnetic oxygen concentrations, associated with increased Secchi depths and chlorophyll-a concentrations, were co-incident with anoxic waters immediately above the fluid fine tailings (FFT) layer post alum. Decreased water cap nitrate and detectable sulfide concentrations, as well as increased hypolimnetic phospholipid fatty acid abundances, signaled greater anaerobic heterotrophic activity. Shifts in microbial community to groups associated with greater organic carbon degradation (i.e., SAR11-LD12 subclade) and the SRB group Desulfuromonodales emerged post alum and the loss of specialist groups associated with carbon-limited, ammonia-rich restricted niches (i.e., MBAE14) also occurred. Alum treatment resulted in additional oxygen consumption associated with increased autochthonous carbon production, watercap anoxia and sulfide generation, which further exacerbate oxygen consumption associated with on-going FFT mobilized reductants. The results illustrate the importance of understanding the broader biogeochemical implications of adaptive management interventions to avoid unanticipated outcomes that pose greater risks and improve tailings reclamation for oil sands operations and, more broadly, the global mining sector.

Phage-encoded ribosomal protein S21 expression is linked to late-stage phage replication.

The ribosomal protein S21 (bS21) gene has been detected in diverse viruses with a large range of genome sizes, yet its in situ expression and potential significance have not been investigated. Here, we report five closely related clades of bacteriophages (phages) represented by 47 genomes (8 curated to completion and up to 331 kbp in length) that encode a bS21 gene. The bS21 gene is on the reverse strand within a conserved region that encodes the large terminase, major capsid protein, prohead protease, portal vertex proteins, and some hypothetical proteins. Based on CRISPR spacer targeting, the predominance of bacterial taxonomic affiliations of phage genes with those from Bacteroidetes, and the high sequence similarity of the phage bS21 genes and those from Bacteroidetes classes of Flavobacteriia, Cytophagia and Saprospiria, these phages are predicted to infect diverse Bacteroidetes species that inhabit a range of depths in freshwater lakes. Thus, bS21 phages have the potential to impact microbial community composition and carbon turnover in lake ecosystems. The transcriptionally active bS21-encoding phages were likely in the late stage of replication when collected, as core structural genes and bS21 were highly expressed. Thus, our analyses suggest that the phage bS21, which is involved in translation initiation, substitutes into the Bacteroidetes ribosomes and selects preferentially for phage transcripts during the late-stage replication when large-scale phage protein production is required for assembly of phage particles.

Isotopic and Chemical Assessment of the Dynamics of Methane Sources and Microbial Cycling during Early Development of an Oil Sands Pit Lake.

Water-capped tailings technology (WCTT) is a key component of the reclamation strategies in the Athabasca oil sands region (AOSR) of northeastern Alberta, Canada. The release of microbial methane from tailings emplaced within oil sands pit lakes, and its subsequent microbial oxidation, could inhibit the development of persistent oxygen concentrations within the water column, which are critical to the success of this reclamation approach. Here, we describe the results of a four-year (2015-2018) chemical and isotopic (δ13C) investigation into the dynamics of microbial methane cycling within Base Mine Lake (BML), the first full-scale pit lake commissioned in the AOSR. Overall, the water-column methane concentrations decreased over the course of the study, though this was dynamic both seasonally and annually. Phospholipid fatty acid (PLFA) distributions and δ13C demonstrated that dissolved methane, primarily input via fluid fine tailings (FFT) porewater advection, was oxidized by the water column microbial community at all sampling times. Modeling and under-ice observations indicated that the dissolution of methane from bubbles during ebullition, or when trapped beneath ice, was also an important source of dissolved methane. The addition of alum to BML in the fall of 2016 impacted the microbial cycling in BML, leading to decreased methane oxidation rates, the short-term dominance of a phototrophic community, and longer-term shifts in the microbial community metabolism. Overall, our results highlight a need to understand the dynamic nature of these microbial communities and the impact of perturbations on the associated biogeochemical cycling within oil sands pit lakes.

Large freshwater phages with the potential to augment aerobic methane oxidation.

There is growing evidence that phages with unusually large genomes are common across various microbiomes, but little is known about their genetic inventories or potential ecosystem impacts. In the present study, we reconstructed large phage genomes from freshwater lakes known to contain bacteria that oxidize methane. Of manually curated genomes, 22 (18 are complete), ranging from 159 kilobase (kb) to 527 kb in length, were found to encode the pmoC gene, an enzymatically critical subunit of the particulate methane monooxygenase, the predominant methane oxidation catalyst in nature. The phage-associated PmoC sequences show high similarity to (>90%), and affiliate phylogenetically with, those of coexisting bacterial methanotrophs, including members of Methyloparacoccus, Methylocystis and Methylobacter spp. In addition, pmoC-phage abundance patterns correlate with those of the coexisting bacterial methanotrophs, supporting host-phage relationships. Future work is needed to determine whether phage-associated PmoC has similar functions to additional copies of PmoC encoded in bacterial genomes, thus contributing to growth on methane. Transcriptomics data from Lake Rotsee (Switzerland) showed that some phage-associated pmoC genes were highly expressed in situ and, of interest, that the most rapidly growing methanotroph was infected by three pmoC-phages. Thus, augmentation of bacterial methane oxidation by pmoC-phages during infection could modulate the efflux of this potent greenhouse gas into the environment.

Isomer-specific monitoring of naphthenic acids at an oil sands pit lake by comprehensive two-dimensional gas chromatography-mass spectrometry.

Naphthenic acids (NAs) are persistent, toxic contaminants that are found to accumulate in oil sands process-affected water (OSPW) and tailings after bitumen extraction. A number of strategies for the reclamation of oil sands tailings are currently being tested, including the development of the first demonstration pit lake by Syncrude Canada, Base Mine Lake (BML). An important component of reclamation activities is understanding the source and cycling of NAs in such reclamation systems. However, NAs exist as a highly complex mixture of thousands of compounds which makes their analysis an ongoing challenge. Herein, comprehensive two-dimensional gas chromatography coupled to time of flight mass spectrometry (GC × GC/TOFMS) was used to analyze the methylated extracts of water samples from the water cap and fluid fine tailings (FFT) deposit of BML to characterize the variations in NA distributions between geochemical zones. A collection of (alkylated) monocyclic-, bicyclic-, adamantane-, and thiophene-type carboxylic acids were identified. Total relative abundances were calculated for each NA class (by summation of peak areas of all detected isomers) and minimal variability was detected in the water cap. Total relative abundances for each NA class were either similar or higher in the FFT, relative to the water cap. Examination of isomer distributions indicated that differences in abundance values were generally driven by variations in only one or two isomers of a given NA class. Furthermore, GC × GC revealed distinct isomer profiles were observed between two FFT samples and between the FFT and water cap. While it is not yet clear whether these differences are due to differences in sources of NAs or in their environmental processing, these results illustrate the capability of GC × GC to investigate these questions and thus contribute to the management of these compounds within reclamation or environmental systems.

New Insights Into Acidithiobacillus thiooxidans Sulfur Metabolism Through Coupled Gene Expression, Solution Chemistry, Microscopy, and Spectroscopy Analyses.

Here, we experimentally expand understanding of the reactions and enzymes involved in Acidithiobacillus thiooxidans ATCC 19377 S0 and S 2 ⁢ O 3 2 - metabolism by developing models that integrate gene expression analyzed by RNA-Seq, solution sulfur speciation, electron microscopy and spectroscopy. The A. thiooxidans S 2 ⁢ O 3 2 - metabolism model involves the conversion of S 2 ⁢ O 3 2 - to SO 4 2 - , S0 and S 4 ⁢ O 6 2 - , mediated by the sulfur oxidase complex (Sox), tetrathionate hydrolase (TetH), sulfide quinone reductase (Sqr), and heterodisulfate reductase (Hdr) proteins. These same proteins, with the addition of rhodanese (Rhd), were identified to convert S0 to SO 3 2 - , S 2 ⁢ O 3 2 - and polythionates in the A. thiooxidans S0 metabolism model. Our combined results shed light onto the important role specifically of TetH in S 2 ⁢ O 3 2 - metabolism. Also, we show that activity of Hdr proteins rather than Sdo are likely associated with S0 oxidation. Finally, our data suggest that formation of intracellular S 2 ⁢ O 3 2 - is a critical step in S0 metabolism, and that recycling of internally generated SO 3 2 - occurs, through comproportionating reactions that result in S 2 ⁢ O 3 2 - . Electron microscopy and spectroscopy confirmed intracellular production and storage of S0 during growth on both S0 and S 2 ⁢ O 3 2 - substrates.

Clades of huge phages from across Earth's ecosystems.

Bacteriophages typically have small genomes1 and depend on their bacterial hosts for replication2. Here we sequenced DNA from diverse ecosystems and found hundreds of phage genomes with lengths of more than 200 kilobases (kb), including a genome of 735 kb, which is-to our knowledge-the largest phage genome to be described to date. Thirty-five genomes were manually curated to completion (circular and no gaps). Expanded genetic repertoires include diverse and previously undescribed CRISPR-Cas systems, transfer RNAs (tRNAs), tRNA synthetases, tRNA-modification enzymes, translation-initiation and elongation factors, and ribosomal proteins. The CRISPR-Cas systems of phages have the capacity to silence host transcription factors and translational genes, potentially as part of a larger interaction network that intercepts translation to redirect biosynthesis to phage-encoded functions. In addition, some phages may repurpose bacterial CRISPR-Cas systems to eliminate competing phages. We phylogenetically define the major clades of huge phages from human and other animal microbiomes, as well as from oceans, lakes, sediments, soils and the built environment. We conclude that the large gene inventories of huge phages reflect a conserved biological strategy, and that the phages are distributed across a broad bacterial host range and across Earth's ecosystems.

Putative Mixotrophic Nitrifying-Denitrifying Gammaproteobacteria Implicated in Nitrogen Cycling Within the Ammonia/Oxygen Transition Zone of an Oil Sands Pit Lake.

Anthropogenically-impacted environments offer the opportunity to discover novel microbial species and metabolisms, which may be undetectable in natural systems. Here, a combined metagenomic and geochemical study in Base Mine Lake, Alberta, Canada, which is the only oil sands end pit lake to date, revealed that nitrification was performed by members from Nitrosomonadaceae, Chloroflexi and unclassified Gammaproteobacteria "MBAE14." While Nitrosomonadaceae and Chloroflexi groups were relatively abundant in the upper oxygenated zones, MBAE14 dominated the hypoxic hypolimnetic zones (approximately 30% of total microbial communities); MBAE14 was not detected in the underlying anoxic tailings. Replication rate analyses indicate that MBAE14 grew in metalimnetic and hypolimnetic water cap regions, most actively at the metalimnetic, ammonia/oxygen transition zone consistent with it putatively conducting nitrification. Detailed genomic analyses of MBAE14 evidenced both ammonia oxidation and denitrification into dinitrogen capabilities. However, the absence of known CO2-fixation genes suggests a heterotrophic denitrifying metabolism. Functional marker genes of ammonia oxidation (amo and hao) in the MBAE14 genome are homologous with those conserved in autotrophic nitrifiers, but not with those of known heterotrophic nitrifiers. We propose that this novel MBAE14 inhabits the specific ammonia-rich, oxygen and labile organic matter-limited conditions occurring in Base Mine Lake which selectively favors mixotrophic coupled nitrifier denitrification metabolism. Our results highlight the opportunities to better constrain biogeochemical cycles from the application of metagenomics to engineered systems associated with extractive resource sectors.

Wide Distribution of Phage That Infect Freshwater SAR11 Bacteria.

Fonsibacter (LD12 subclade) is among the most abundant bacterioplankton in freshwater ecosystems. These bacteria belong to the order Pelagibacterales (SAR11) and are related to Pelagibacter (marine SAR11), which dominates many marine habitats. Although a few Pelagibacter phage (Pelagiphage) have been described, no phage that infect Fonsibacter have been reported. In this study, we describe two groups of Podoviridae phage that infect Fonsibacter A complete Fonsibacter genome containing a prophage was reconstructed from metagenomic data. A circularized and complete genome related to the prophage, referred to as uv-Fonsiphage-EPL (lysogenic strategy), shows high similarity to marine Pelagiphage HTVC025P. Additionally, we reconstructed three complete genomes and one draft genome of phage related to marine Pelagiphage HTVC010P and predicted a lytic strategy. The similarity in codon usage and cooccurrence patterns of HTVC010P-related phage and Fonsibacter suggested that these phage infect Fonsibacter Similar phage were detected in Lake Mendota, Wisconsin, where Fonsibacter is also present. A search of related phage revealed the worldwide distribution of some genotypes in freshwater ecosystems, suggesting their substantial role in shaping indigenous microbial assemblages and influence on biogeochemical cycling. However, the uv-Fonsiphage-EPL and one group of HTVC010P-related phage have a more limited distribution in freshwater ecosystems. Overall, the findings provide insights into the genomic features of phage that infect Fonsibacter and expand understanding of the ecology and evolution of these important bacteria.IMPORTANCE Fonsibacter represents a significant microbial group of freshwater ecosystems. Although the genomic and metabolic features of these bacteria have been well studied, no phage infecting them has been reported. In this study, we reconstructed complete genomes of Fonsibacter and infecting phage and revealed their close relatedness to the phage infecting marine SAR11 members. Also, we illustrated that phage that infect Fonsibacter are widely distributed in freshwater habitats. In summary, the results contribute new insights into the ecology and evolution of Fonsibacter and phage.

The Potential Role of Halothiobacillus spp. in Sulfur Oxidation and Acid Generation in Circum-Neutral Mine Tailings Reservoirs.

The biogeochemistry of acid mine drainage (AMD) derived from waste rock associated sulfide mineral oxidation is relatively well-characterized and linked to Acidithiobacillus spp.. However, little is understood about the microbial communities and sulfur cycling before AMD develops, a key component of its prevention. This study aimed to examine circum-neutral mining impacted water (MIW) communities and its laboratory enrichments for sulfur oxidizing bacteria (SoxBac). MIW in situ microbial communities differed in diversity, structure and relative abundance consistent with site specific variations in total aqueous sulfur concentrations (TotS; ~2-17 mM), pH (3.67-7.34), and oxygen (22-93% saturation). However, the sulfur oxidizer, Halothiobacillus spp. dominated seven of the nine total SoxBac enrichment communities (~76-100% relative abundance), spanning three of the four mines. The presence and relative abundance of the identified sixteen known and five unclassified Halothiobacillus spp. here, were the important clustering determinants across parent MIW and enrichment communities. Further, the presence of Halothiobacillus spp. was associated with driving the pH <4 in enrichment experiments, and the combination of specific Halothiobacillus spp. in the enrichments affected the observed acid to sulfate ratios indicating differential sulfur cycling. Halothiobacillus spp. also dominated the parent communities of the two acidic MIWs providing corroborating evidence for its active role in net acid generation within these waters. These results identify a putative indicator organism specific to mine tailings reservoirs and highlight the need for further study of tailings associated sulfur cycling for better mine management and environmental stewardship.

Profiling of individual naphthenic acids at a composite tailings reclamation fen by comprehensive two-dimensional gas chromatography-mass spectrometry.

Naphthenic acids (NAs) are naturally occurring in the Athabasca oil sands region (AOSR) and accumulate in tailings as a result of water-based extraction processes. NAs exist as a complex mixture, so the development of an analytical technique to characterize them has been an on-going challenge. The aim of this study was to use comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry to monitor individual NAs within a wetland reclamation site in the AOSR. Samples were collected from four monitoring wells at the site and the extracts were found to contain numerous resolved isomers of classical (monocyclic-, bicyclic-, adamantane-, indane-, and tetralin-type carboxylic acids) and sulfur-containing NAs (thiamonocyclic- and thiophene-type carboxylic acids). The absolute abundances of the monitored NAs were compared between four monitoring wells and unique profiles were observed at each well. Few significant changes in absolute abundances were observed over the sampling period, with the exception of one well (Well 6A). In addition, isomeric percent compositions were calculated for each set of structural isomers, and one-way analysis of variance (ANOVA) and two-dimensional hierarchical cluster analysis revealed high spatial variation at the site. However, consistent distributions were observed at each of the monitoring wells for some sets of NA isomers (such as: adamantane NAs), which may be useful for forensic applications, such as identifying sources of contamination or demonstrating biodegradation. The methods and results presented in this study demonstrate the utility of monitoring individual NAs, since both changes in absolute abundances of individual NAs and the distribution of NA isomers have the ability to provide insight into their sources and the processes controlling their concentrations that are not only of relevance to the Alberta Oil Sands, but also to other petroleum deposits and environmental systems.

Expanded diversity of microbial groups that shape the dissimilatory sulfur cycle.

A critical step in the biogeochemical cycle of sulfur on Earth is microbial sulfate reduction, yet organisms from relatively few lineages have been implicated in this process. Previous studies using functional marker genes have detected abundant, novel dissimilatory sulfite reductases (DsrAB) that could confer the capacity for microbial sulfite/sulfate reduction but were not affiliated with known organisms. Thus, the identity of a significant fraction of sulfate/sulfite-reducing microbes has remained elusive. Here we report the discovery of the capacity for sulfate/sulfite reduction in the genomes of organisms from 13 bacterial and archaeal phyla, thereby more than doubling the number of microbial phyla associated with this process. Eight of the 13 newly identified groups are candidate phyla that lack isolated representatives, a finding only possible given genomes from metagenomes. Organisms from Verrucomicrobia and two candidate phyla, Candidatus Rokubacteria and Candidatus Hydrothermarchaeota, contain some of the earliest evolved dsrAB genes. The capacity for sulfite reduction has been laterally transferred in multiple events within some phyla, and a key gene potentially capable of modulating sulfur metabolism in associated cells has been acquired by putatively symbiotic bacteria. We conclude that current functional predictions based on phylogeny significantly underestimate the extent of sulfate/sulfite reduction across Earth's ecosystems. Understanding the prevalence of this capacity is integral to interpreting the carbon cycle because sulfate reduction is often coupled to turnover of buried organic carbon. Our findings expand the diversity of microbial groups associated with sulfur transformations in the environment and motivate revision of biogeochemical process models based on microbial community composition.

Improved coverage of naphthenic acid fraction compounds by comprehensive two-dimensional gas chromatography coupled with high resolution mass spectrometry.

This study reports the first application of comprehensive two-dimensional gas chromatography coupled to a high-resolution quadrupole time-of-flight mass spectrometer (GC×GC/HRQTOF-MS) for the characterization of naphthenic acid fraction compounds (NAFCs) from the Alberta Oil Sands. High resolution mass spectrometry (HRMS) significantly increased the coverage of NAFCs in the mixture and allowed the differentiation of NAFCs from several chemical classes. It was demonstrated that GC×GC, in combination with the high mass accuracy and precision of the HRQTOF-MS, could distinguish chemical species with the C3 vs SH4 mass split at a much lower resolving power than required with direct infusion experiments. Mass defect plots were useful for visualizing the complex datasets generated by GC×GC/HRQTOF-MS and led to the identification of 1105 chemical species with unique elemental compositions (<5ppm mass accuracy). Mass defect plots were shown to be a powerful screening tool and enabled the detection of extensive isomer series from the SO2 chemical class, some of which have not been previously reported in oil sands related samples. The GC×GC/HRQTOF-MS approach is expected to improve NAFC monitoring programs since the technique allows the qualitative analysis of individual NAFCs and provides unique fingerprints via isomer distributions which may assist in future fingerprinting studies.

Elucidating carbon sources driving microbial metabolism during oil sands reclamation.

Microbial communities play key roles in remediation and reclamation of contaminated environments via biogeochemical cycling of organic and inorganic components. Understanding the trends in in situ microbial community abundance, metabolism and carbon sources is therefore a crucial component of effective site management. The focus of this study was to use radiocarbon analysis to elucidate the carbon sources driving microbial metabolism within the first pilot wetland reclamation project in the Alberta oil sands region where the observation of H2S had indicated the occurrence of microbial sulphate reduction. The reclamation project involved construction of a three compartment system consisting of a freshwater wetland on top of a sand cap overlying a composite tailings (CT) deposit. Radiocarbon analysis demonstrated that both dissolved and sediment associated organic carbon associated with the deepest compartments (the CT and sand cap) was primarily fossil (Δ14C = -769 to -955‰) while organic carbon in the overlying peat was hundreds to thousands of years old (Δ14C = -250 to -350‰). Radiocarbon contents of sediment associated microbial phospholipid fatty acids (PLFA) were consistent with the sediment bulk organic carbon pools (Peat: Δ14CPLFA = -257‰; Sand cap Δ14CPLFA = -805‰) indicating that these microbes were using sediment associated carbon. In contrast, microbial PLFA grown on biofilm units installed in wells within the deepest compartments contained much more modern carbon that the associated bulk carbon pools. This implied that the transfer of relatively more modern carbon was stimulating the microbial community at depth within the system. Correlation between cellular abundance estimates based on PLFA concentrations and the Δ14CPLFA indicated that the utilization of this more modern carbon was stimulating the microbial community at depth. These results highlight the importance of understanding the occurrence and potential outcomes of the introduction of relatively bioavailable carbon to mine wastes in order to predict and manage the performance of reclamation strategies.

pH and Organic Carbon Dose Rates Control Microbially Driven Bioremediation Efficacy in Alkaline Bauxite Residue.

Bioremediation of alkaline tailings, based on fermentative microbial metabolisms, is a novel strategy for achieving rapid pH neutralization and thus improving environmental outcomes associated with mining and refining activities. Laboratory-scale bioreactors containing bauxite residue (an alkaline, saline tailings material generated as a byproduct of alumina refining), to which a diverse microbial inoculum was added, were used in this study to identify key factors (pH, salinity, organic carbon supply) controlling the rates and extent of microbially driven pH neutralization (bioremediation) in alkaline tailings. Initial tailings pH and organic carbon dose rates both significantly affected bioremediation extent and efficiency with lower minimum pHs and higher extents of pH neutralization occurring under low initial pH or high organic carbon conditions. Rates of pH neutralization (up to 0.13 mM H+ produced per day with pH decreasing from 9.5 to ≤6.5 in three days) were significantly higher in low initial pH treatments. Representatives of the Bacillaceae and Enterobacteriaceae, which contain many known facultative anaerobes and fermenters, were identified as key contributors to 2,3-butanediol and/or mixed acid fermentation as the major mechanism(s) of pH neutralization. Initial pH and salinity significantly influenced microbial community successional trajectories, and microbial community structure was significantly related to markers of fermentation activity. This study provides the first experimental demonstration of bioremediation in bauxite residue, identifying pH and organic carbon dose rates as key controls on bioremediation efficacy, and will enable future development of bioreactor technologies at full field scale.

S reactivity of an oil sands composite tailings deposit undergoing reclamation wetland construction.

This study is the first to characterize the S stability of a composite tailings (CT) deposit undergoing pilot wetland reclamation in the Athabasca Oil Sands Region (AOSR, Alberta, Canada). As CT is sulfur, organic carbon and bacterially rich, the goal of this study was to characterize the in situ aqueous distribution of sulfur compounds across the wetland, sand cap and underlying CT zones of the deposit, in an effort to establish the potential for microbial sulfur cycling and generation of H2S, an explosive, corrosive and toxicity risk. Porewater samples from three depths spanning the different layers of the deposit, as well as wetland surface ponded water samples were collected for geochemical analyses (July and Sept 2013), and for microbial enrichments (both S reducing and S oxidizing bacteria) in June 2014. While porewater ΣH2S(aq) was detected at all depths across the three zones of the deposit, results identify that the sand cap layer required for construction, acts as a mixing zone generating the highest solution H2S concentrations (>500 uM or 18 mg/L) and H2S gas levels (over 100 and up to 180 ppm) observed. Porewater dissolved sulfate concentrations (0.14-6.97 mM) were orders of magnitude higher and did not correlate to the observed distribution of ΣH2S concentrations throughout the deposit. Unique to the sandcap, dissolved organic carbon positively correlated with the observed maxima of ΣH2S(aq) seen in this layer. The water management of the deposit is a critical factor in the observed S trends. Active dewatering of the CT resulted in migration of S rich water up into the sandcap, while downwelling labile organic carbon from the developing wetland acted in concert to stimulate microbial generation of the H2S in this structural layer to the highest levels observed. Functional enrichments identified that diverse S reducing and oxidizing microbial metabolisms are widespread throughout the deposit, indicating that these waste materials are biogeochemically reactive with implications for longterm stability. These results are of relevance to both the oil sands region, as well as other mine contexts where S rich wastes occur, identifying the need to consider the potential bacterially driven cycling of S and C in the generation of constituents of concern, as well as the water management of such waste deposits to minimize risk.

Influence of wave action on the partitioning and transport of unattached and floc-associated bacteria in fresh water.

The dynamic interaction of bacteria within bed sediment and suspended sediment (i.e., floc) in a wave-dominated beach environment was assessed using a laboratory wave flume. The influence of shear stress (wave energy) on bacterial concentrations and on the partitioning and transport of unattached and floc-associated bacteria was investigated. The study showed that increasing wave energy (0.60 and 5.35 N/s) resulted in a 0.5 to 1.5 log increase in unattached cells of the test bacterium Pseudomonas sp. strain CTO7::gfp-2 in the water column. There was a positive correlation between the bacterial concentrations in water and the total suspended solids, with the latter increasing from values of near 0 to up to 200 mg/L over the same wave energy increase. The median equivalent spherical diameter of flocs in suspension also increased by an order of magnitude in all experimental trials. Under both low (0.60 N/s) and high (5.35 N/s) energy regime, bacteria were shown to preferentially associate with flocs upon cessation of wave activity. The results suggest that collecting water samples during periods of low wave action for the purpose of monitoring the microbiological quality of water may underestimate bacterial concentrations partly because of an inability to account for the effect of shear stress on the erosion and mobilization of bacteria from bed sediment to the water column. This highlights the need to develop a more comprehensive beach analysis strategy that not only addresses presently uncharacterized shores and sediments but also recognizes the importance of eroded flocs as a vector for the transport of bacteria in aquatic environments.

Microbial Diversity in Engineered Haloalkaline Environments Shaped by Shared Geochemical Drivers Observed in Natural Analogues.

Microbial communities in engineered terrestrial haloalkaline environments have been poorly characterized relative to their natural counterparts and are geologically recent in formation, offering opportunities to explore microbial diversity and assembly in dynamic, geochemically comparable contexts. In this study, the microbial community structure and geochemical characteristics of three geographically dispersed bauxite residue environments along a remediation gradient were assessed and subsequently compared with other engineered and natural haloalkaline systems. In bauxite residues, bacterial communities were similar at the phylum level (dominated by Proteobacteria and Firmicutes) to those found in soda lakes, oil sands tailings, and nuclear wastes; however, they differed at lower taxonomic levels, with only 23% of operational taxonomic units (OTUs) shared with other haloalkaline environments. Although being less diverse than natural analogues, bauxite residue harbored substantial novel bacterial taxa, with 90% of OTUs nonmatchable to cultured representative sequences. Fungal communities were dominated by Ascomycota and Basidiomycota, consistent with previous studies of hypersaline environments, and also harbored substantial novel (73% of OTUs) taxa. In bauxite residues, community structure was clearly linked to geochemical and physical environmental parameters, with 84% of variation in bacterial and 73% of variation in fungal community structures explained by environmental parameters. The major driver of bacterial community structure (salinity) was consistent across natural and engineered environments; however, drivers differed for fungal community structure between natural (pH) and engineered (total alkalinity) environments. This study demonstrates that both engineered and natural terrestrial haloalkaline environments host substantial repositories of microbial diversity, which are strongly shaped by geochemical drivers.

Microbially-driven strategies for bioremediation of bauxite residue.

Globally, 3 Gt of bauxite residue is currently in storage, with an additional 120 Mt generated every year. Bauxite residue is an alkaline, saline, sodic, massive, and fine grained material with little organic carbon or plant nutrients. To date, remediation of bauxite residue has focused on the use of chemical and physical amendments to address high pH, high salinity, and poor drainage and aeration. No studies to date have evaluated the potential for microbial communities to contribute to remediation as part of a combined approach integrating chemical, physical, and biological amendments. This review considers natural alkaline, saline environments that present similar challenges for microbial survival and evaluates candidate microorganisms that are both adapted for survival in these environments and have the capacity to carry out beneficial metabolisms in bauxite residue. Fermentation, sulfur oxidation, and extracellular polymeric substance production emerge as promising pathways for bioremediation whether employed individually or in combination. A combination of bioaugmentation (addition of inocula from other alkaline, saline environments) and biostimulation (addition of nutrients to promote microbial growth and activity) of the native community in bauxite residue is recommended as the approach most likely to be successful in promoting bioremediation of bauxite residue.