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

Abstract

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 [S₂O₃^2-] 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 [S₂O₃^2-] 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.

Fig. 1. Microbial community and aqueous geochemical distributions over time.

a Bray Curtis dissimilarity clustering of microbial communities and bubble plot representation of relative abundances of bacterial genera (showing only genera present at >1% within at least two samples). Non-metric dimensional scaling (NMDS) (k = 3) representation of the microbial community composition b, and functionality (c) (derived as the relative abundance of sulfur cycling, nitrogen cycling, hydrogen cycling, carbon fixation, photosynthetic and aerobic respiration genes (Supplementary Dataset 5), (n = 30) stress = 0.065, non-metric fit = 0.996, linear fit, R² = 0.97). d The pH depth profiles from July 2015 to 2018. e Spring dissolved oxygen (mM) depth profiles in 2015, 2016 and 2018. f Box-plot and statistical (ANOVA and subsequent Tukey pairwise statistical comparison) of annual [H⁺]/[SO₄²⁻] values (omitting May 2017 0.5 m and May 2018 21 m where both pH and SO₄²⁻ were unavailable). g Box-plot and statistical (ANOVA and subsequent Tukey pairwise statistical comparison) of annual observed [S₂O₃²⁻] (mM) in tailings impoundment waters inclusive of March 2015 to August 2018 with the exception of May 2018 (0.5 m), July 11, 2018 (2.8 m) July 23, 2018 (0.5 m) when data were not available. Two outliers included in the statistical (ANOVA and subsequent Tukey pairwise statistical comparison statistical comparisons were excluded from the graph for visualization purposes (March 2015 at 21 m (0.59 mM) and June 2018 at 0.5 m (1.7 mM); see Supplementary Dataset 3). In f and g, box plots enclose the first to third quartiles of data values, with a black line at the median value.

Fig. 2. Metabolic capacity for sulfur oxidative strategies over time in the tailings impoundment/pit lake, significant sulfur oxidizers and acidity and thiosulfate outcomes.

a Schematic of potential sulfur oxidation pathways based on identified genes encoding sulfur metabolic enzymes. A black circle with “X” indicates the gene was not detected in the genomes of the listed genera. This pathway representation was adapted from Watanabe et al.. b Community level sulfur oxidative pathway gene distributions (TsdA, soxABCDXYZ, ttrABC dsrABEFH, aprAB and sat.). c Relative abundances of Halothiobacillus (complete Sox pathway genes) and Thiobacillus (incomplete Sox and rDSR pathway genes). d The distribution of S₂O₃²⁻. e [H⁺]/[SO₄²⁻] ratios from 2015 to 2018 in the tailings impoundment.

Fig. 3. Oxic, hypoxic and anoxic zonations of SOB communities, their respective gene based sulfur oxidative and TEA reduction strategies and aqueous geochemical outcomes.

a SOB relative abundances in August 2015, May 2018 and August 2018 through depth. b August 2018 dissolved oxygen concentrations (mM) through depth. c August 2018 H⁺/SO₄²⁻ ratios throughout depths. d August 2018 thiosulfate concentration (mM) through depth. SOB, sulfur oxidizing bacteria.

Fig. 4. RNA expression activities and sulfur oxidation strategies revealed by metatranscriptomics.

a August 2018 RNA expression levels of sulfur oxidative and oxygen/nitrogen compound reductase genes of Halothiobacillus and Thiobacillus groups through depth. b Oxic, hypoxic and anoxic zonations of dominant sulfur oxidative pathways based on the RNA expression levels.

Fig. 5. Conceptual model of differential microbial sulfur oxidative strategies throughout steep oxygen zonations.

a Dissolved O₂ zones, and dominant microbial oxidative strategy, based on gene expression, in the oxic zone (Sox driven oxidation coupled to O₂), hypoxic zone (Sox-driven oxidation coupled to O₂ and rDSR-driven oxidation coupled to NO₃⁻) and anoxic zone (rDSR-driven oxidation coupled to NO₃⁻) along with the different expected environmental water geochemical outcomes (acidity generation and thiosulfate abundance). b Sulfur oxidative strategy biochemical pathways and cellular location. Scientific illustration and visualization by Mark Belan (www.artscistudios.com).