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.

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.

Here, we experimentally expand understanding of the reactions and enzymes involved in Acidithiobacillus thiooxidans ATCC 19377 S⁰ and ${\text{S}}_2{\text{O}}_3^{2-}$ metabolism by developing models that integrate gene expression analyzed by RNA-Seq, solution sulfur speciation, electron microscopy and spectroscopy. The A. thiooxidans ${\text{S}}_2{\text{O}}_3^{2-}$ metabolism model involves the conversion of ${\text{S}}_2{\text{O}}_3^{2-}$ to ${\text{SO}}_4^{2-}$, S⁰ and ${\text{S}}_4{\text{O}}_6^{2-},$ 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 ${\text{SO}}_3^{2-}$, ${\text{S}}_2{\text{O}}_3^{2-}$ and polythionates in the A. thiooxidans S⁰ metabolism model. Our combined results shed light onto the important role specifically of TetH in ${\text{S}}_2{\text{O}}_3^{2-}$ 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 ${\text{S}}_2{\text{O}}_3^{2-}$ is a critical step in S⁰ metabolism, and that recycling of internally generated ${\text{SO}}_3^{2-}$ occurs, through comproportionating reactions that result in ${\text{S}}_2{\text{O}}_3^{2-}$. Electron microscopy and spectroscopy confirmed intracellular production and storage of S⁰ during growth on both S⁰ and ${\text{S}}_2{\text{O}}_3^{2-}$ substrates.

The biogeochemistry of acid mine drainage (AMD) derived from waste rock associated sulphide mineral oxidation is relatively well characterized and linked to Acidithiobacillus spp.. However, little is understood about the microbial communities and sulphur cycling before AMD develops, key component on its prevention. This study aimed to examine circum-neutral mining impacted water (MIW) communities and its laboratory enrichments for sulphur oxidizing bacteria (SoxBac). MIW in situ microbial communities differed in diversity, structure and relative abundance consistent with site specific variations in total aqueous sulphur concentrations (TotS; ~2 to 17 mM), pH (3.67 – 7.34), and oxygen (22% - 93% saturation). However, the sulphur oxidizer, Halothiobacillus spp. dominated seven of the nine total SoxBac enrichment communities (~76 to ~100% relative abundance), spanning three of the four mines. Indeed, 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 sulphate ratios indicating differential sulphur 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 sulphur cycling for better mine management and environmental stewardship.

Sulfur oxidizing bacteria (SOB) play a key role in sulfur cycling in mine tailings impoundment (TI) waters, where sulfur concentrations are typically high. However, our understanding of SOB sulfur cycling via potential S oxidation pathways (sox, rdsr, and S₄I) in these globally ubiquitous contexts, remains limited. Here, we identified TI water column SOB community composition, metagenomics derived metabolic repertoires, physicochemistry, and aqueous sulfur concentration and speciation in four Canadian base metal mine, circumneutral-alkaline TIs over four years (2016 – 2019). Identification and examination of genomes from nine SOB genera occurring in these TI waters revealed two pH partitioned, metabolically distinct groups, which differentially influenced acid generation and sulfur speciation. Complete sox (csox) dominant SOB (e.g., Halothiobacillus spp., Thiomonas spp.) drove acidity generation and S₂O₃^2- consumption via the csox pathway at lower pH (pH ~5 to ~6.5). At circumneutral pH conditions (pH ~6.5 to ~8.5), the presence of non-csox dominant SOB (hosting the incomplete sox, rdsr, and/or other S oxidation reactions; e.g. Thiobacillus spp., Sulfuriferula spp.) were associated with higher [S₂O₃^2-] and limited acidity generation. The S₄I pathway part 1 (tsdA; S₂O₃^2- to S₄O₆^2-), was not constrained by pH, while S4I pathway part 2 (S₄O₆^2- disproportionation via tetH) was limited to Thiobacillus spp. and thus circumneutral pH values. Comparative analysis of low, natural (e.g., hydrothermal vents and sulfur hot springs) and high (e.g., Zn, Cu, Pb/Zn, and Ni tailings) sulfur systems literature data with these TI results, reveals a distinct TI SOB mining microbiome, characterized by elevated abundances of csox dominant SOB, likely sustained by continuous replenishment of sulfur species through tailings or mining impacted water additions. Our results indicate that under the primarily oxic conditions in these systems, S₂O₃^2- availability plays a key role in determining the dominant sulfur oxidation pathways and associated geochemical and physicochemical outcomes, highlighting the potential for biological management of mining impacted waters via pH and [S₂O₃^2-] manipulation.

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.