Anaerobic microorganisms alter heavy oil chemistry by oxygenation and sulfurization

Microbial transformation of heavy oil under anoxic conditions involves complex changes in hydrocarbon and heteroatom-containing molecular compositions. However, the molecular-level evolution of sulfur-containing compounds and their relationship to hydrocarbon transformation remain insufficiently resolved. In this study, long-term anaerobic microcosms containing heavy oil from Pitch Lake, Trinidad and Tobago, were analyzed using Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) and microbial community profiling to evaluate temporal changes in molecular compositions and microbiota structure over 945 days of incubation. FT-ICR MS detected 4,499 molecular formulas dominated by CHO-, CHNO-, CHOS-, and CHNOS-class formulas. Pronounced compositional shifts occurred during incubation, with sulfur-containing molecular formulas becoming increasingly abundant and resulting in a marked increase in CHOS-class formulas over time. Intensity-weighted elemental formulas showed increasing oxygen and sulfur abundances, while the average m/z increased from 344 at day 93 to 366 at day 945, corresponding to an approximately 6.4% increase in the intensity-weighted average molecular mass. Multivariate analyses revealed distinct temporal clustering of samples and parallel shifts in microbial community composition, depicting a greater contribution of anaerobic and sulfur-associated taxa. Correlation analyses further showed associations between microbial community shifts and increasing abundances of oxidized sulfur-containing compounds, particularly CHSO₃- and CHSO₄-class formulas. These findings indicate substantial reorganization of the heteroatom-containing molecular pool during anoxic incubation and highlight the potential roles of microbial and sulfur-related processes in the transformation of heavy oil under anoxic conditions.IMPORTANCEHeavy oil reservoirs contain complex mixtures of hydrocarbons and heteroatom-containing compounds that undergo transformation under anoxic conditions. Characterizing the molecular-level changes occurring during these transformations is important for understanding hydrocarbon biodegradation, the persistence of recalcitrant compounds, and the formation and fate of organic sulfur species in oil reservoirs. Using Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) and microbial community analyses, this study reveals that long-term anaerobic incubation drives enrichment of CHOS-class formulas, reduced microbial diversity, and shifts toward anaerobic, sulfur-associated taxa. These findings demonstrate that microbial and sulfur-driven processes play a key role in reorganizing oil chemistry and suggest that co-metabolic activity contributes to the accumulation of partially transformed sulfur-containing compounds. By directly linking changes in microbial community composition with ultrahigh-resolution molecular characterization, this work provides important new insights into the microbial and chemical mechanisms controlling heavy oil transformation and sulfur metabolism in subsurface oil reservoirs.