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 CHSO ₃ - and CHSO ₄ -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.