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.

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.

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.

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.

Identification of individual thiophene-, indane-, tetralin-, cyclohexane-, and adamantane-type carboxylic acids in composite tailings pore water from Alberta oil sands.

RATIONALE: Naphthenic acids (NAs) accumulate in oil sands process-affected water (OSPW) as a result of the water-based extraction processes, and represent one of the toxic fractions in OSPW. They exist as a complex mixture and so the development of an analytical method to characterize and quantify individual acids has been an on-going challenge. The multidimensional separation technique of two-dimensional gas chromatography (GC × GC) has the potential to provide a fingerprint of the sources of NAs and can potentially resolve individual analytes for target analysis. However, the identity and toxicity of a large proportion of the acids present in tailing waters are still unknown. METHODS: Comprehensive two-dimensional gas chromatography/time-of-flight mass spectrometry (GC × GC/TOFMS) was used to characterize NAs in a pore water sample from a Syncrude composite tailings (CT) deposit in Fort McMurray, Alberta, Canada. The extractable organic acid fraction was derivatized with diazomethane and the structures of selected resolved esters were elucidated through interpretation of their electron ionization (EI) mass spectra and, if available, confirmed by comparison with the spectra of reference standards. RESULTS: The high resolving power of the GC × GC/TOFMS technique allowed for the structural elucidation of numerous as yet unidentified acids in the CT pore water sample such as carboxylic acids containing a thiophene, indane, tetralin or cyclohexane moiety. Seventeen members of the previously reported class of adamantane-type carboxylic acids in oil sands process water could also be identified in the sample. CONCLUSIONS: This study underlines the complexity of naphthenic acid isomer distributions in composite tailings and provides a useful inventory of individual acids.

Diurnal floc generation from neuston biofilms in two contrasting freshwater lakes.

Selective adaptation of biofilm-forming bacteria to the nutrient-rich but environmentally challenging conditions of the surface microlayer (SML) or neuston layer was evident in littoral regions of two physically and geochemically contrasting freshwater lakes. SML bacterial communities (bacterioneuston) in these systems were depleted in Actinobacteria, enriched in either Betaproteobacteria or Gammaproteobacteria, and either unicellular Cyanobacteria were absent or microbial mat forming Cyanobacteria enriched relative to communities in the underlying shallow water column (0.5 m depth). Consistent with the occurrence of biofilm-hosted, geochemically distinct microhabitats, As-, Fe-, and S-metabolizing bacteria including anaerobic taxa were detected only in the SML in both systems. Over diurnal time scales, higher wind speeds resulted in the generation of floc from SML biofilms, identifying a transport mechanism entraining SML accumulated microorganisms, nutrients, and contaminants into the underlying water column. The energy regime experienced by the SML was more important to floc generation as larger flocs were more abundant in the larger, oligotrophic lake (higher relative energy regime) compared to the sheltered, smaller lake, despite relatively higher concentrations of bacteria, organic carbon, Fe, and PO4(3-) in the latter system.

Microbial engineering of floc Fe and trace element geochemistry in a circumneutral, remote lake.

Evaluation of lacustrine floc Fe, Pb, and Cd biogeochemistry over seasonal (summer, winter) and water column depth (metalimnetic, hypolimnetic) scales reveals depth-independent seasonally significant differences in floc Fe biominerals and trace element (TE: Pb, Cd) sequestration, driven by floc microbial community shifts. Winter floc [TE] were significantly lower than summer [TE], driven by declining abundance and reactivity of floc amorphous Fe((III))-(oxy)hydroxide (FeOOH) phases under ice ([FeOOH](summer) = 37-77 mgg(-1) vs [FeOOH](winter) = 0.3-7 mgg(-1)). Further, while high summer floc [FeOOH] was observed at both water column depths, winter floc was dominated by Fe((II)) phases. However, the observed seasonal change in the nature and concentrations of floc Fe-phases was independent of water column [Fe], O2, and pH and, instead, significantly correlated to floc bacterial community membership. Bioinformatic modeling (Unifrac, PCA analyses) of in situ and experimental microcosm results identified a temperature-driven seasonal turnover of floc microbial communities, shifting from dominantly putative Fe metabolisms within summer floc to wintertime ancillary Fe reducing and S metabolizing bacteria. This seasonal shift of floc microbial community functioning, significantly the wintertime loss of microbial Fe((II))-oxidizing capability and concomitant increases of sulfur-reducing bacteria, alters dominant floc Fe minerals from Fe((III)) to Fe((II)) phases. This resulted in decreased winter floc [TE], not predicted by water column geochemistry.

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.

Diversity of integron- and culture-associated antibiotic resistance genes in freshwater floc.

Clinically important antibiotic resistance genes were detected in culturable bacteria and class 1 integron gene cassettes recovered from suspended floc, a significant aquatic repository for microorganisms and trace elements, across freshwater systems variably impacted by anthropogenic activities. Antibiotic resistance gene cassettes in floc total community DNA differed appreciably in number and type from genes detected in bacteria cultured from floc. The number of floc antibiotic resistance gene cassette types detected across sites was positively correlated with total (the sum of Ag, As, Cu, and Pb) trace element concentrations in aqueous solution and in a component of floc readily accessible to bacteria. In particular, concentrations of Cu and Pb in the floc component were positively correlated with floc resistance gene cassette diversity. Collectively, these results identify suspended floc as an important reservoir, distinct from bulk water and bed sediment, for antibiotic resistance in aquatic environments ranging from heavily impacted urban sites to remote areas of nature reserves and indicate that trace elements, particularly Cu and Pb, are geochemical markers of resistance diversity in this environmental reservoir. The increase in contamination of global water supplies suggests that aquatic environments will become an even more important reservoir of clinically important antibiotic resistance in the future.

Comparative floc-bed sediment trace element partitioning across variably contaminated aquatic ecosystems.

Significantly higher concentrations of Ag, As, Cu, Ni and Co are found in floc compared to bed sediments across six variably impacted aquatic ecosystems. In contrast to the observed element and site-specific bed sediment trace element (TE) partitioning patterns, floc TE sequestration is consistently dominated by amorphous oxyhydroxides (FeOOH), which account for 30-79% of floc total TE concentrations, irrespective of system physico-chemistry or elements involved. FeOOH consistently occur in significantly higher concentrations in floc than within bed sediments. Further, comparative concentration factors indicate significantly higher TE reactivity of floc-FeOOH relative to sediment-FeOOH in all systems investigated, indicating that both the greater abundance and higher reactivity of floc-FeOOH contribute to enhanced floc TE uptake. Results indicate that floc-organics (live cells and exopolymeric substances, EPS) directly predict floc-FeOOH concentrations, suggesting an organic structural role in the collection/templating of FeOOH. This, in turn, facilitates the sequestration of TEs associated with floc-FeOOH formation, imparting the conserved FeOOH "signature" on floc TE geochemistry across sites. Results demonstrate that the organic rich nature of floc exerts an important control over TE geochemistry in aquatic environments, ultimately creating a distinct solid with differing controls over TE behavior than bed sediments in close proximity (<0.5 m).

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.

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.

Physical and ecological controls on freshwater floc trace metal dynamics.

Significantly higher concentrations of Ag, As, Cu, Co, Ni, and Pb are found in suspended floc compared to surficial bed sediments for a freshwater beach in Lake Ontario. Contrasting observed element-specific bed sediment metal partitioning patterns, floc sequestration for all elements is dominated by one substrate: amorphous oxyhydroxides. More specifically, floc metal scavenging is controlled by floc biogeochemical architecture. Floc organics, largely living microbial cells and associated exopolymeric substances (EPS), act as scaffolds for the collection and/or templating of amorphous Fe oxyhydroxides. While interactions between floc organics and amorphous Fe oxyhydroxides affected floc sorption behavior, specific element affinities and competition for these limited substrates was important for overall floc partitioning. Further, assessment of metal dynamics during stormy conditions indicated energy-regime driven shifts in floc and bed sediment partitioning that were specifically linked to the exchange of floc and bed sedimentary materials. These novel results demonstrate that the microbial nature of floc formation exerts an important control on floc metal dynamics distinguishable from surficial bed sediments and that hydrologic energy-regime is an important factor to consider in overall floc metal behavior, especially in beach environments.

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.

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.

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.

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.