Genome-resolved insights into hydrocarbon-transforming and nitrate-reducing microbial communities from deep petroleum reservoir cores of the Nashpa Oil Field, Pakistan

Abstract

Introduction Microbial communities from deep subsurface petroleum reservoirs are adapted to hydrocarbon-rich, oxygen-limited, and physicochemically extreme environments. However, genome-resolved knowledge of petroleum reservoir microbiomes from Pakistan remains largely unexplored. Methods Shotgun metagenomic sequencing was used to investigate the functional and metabolic potential of microbial communities inhabiting deep subsurface petroleum reservoir cores from the Nashpa Oil Field, Pakistan, at depths of 3,770–4,315 m. Metagenome-assembled genomes (MAGs) were reconstructed and functionally annotated to assess taxonomic composition and predicted metabolic capabilities. Results A total of 402 metagenome-assembled genomes (MAGs) were recovered, of which 216 were high-quality MAGs (≥90% completeness and ≤5% contamination). Taxonomic analysis showed dominance of Pseudomonadota and Actinobacteriota,, including genera such as Alcanivorax, Marinobacter, Pseudomonas, Rhodococcus, and Thermohalobaculum. Functional annotation revealed genes involved in hydrocarbon transformation, nitrate-linked respiration, oxygen-limited metabolism, oxidative phosphorylation, aromatic compound degradation, and cellular stress-response systems. However, markers of hydrocarbon degradation were detected only in a small subset of MAGs, suggesting taxon-specific metabolic specialization rather than broad community-wide enrichment. Genes associated with nitrate reduction and microaerophilic or anaerobic respiration suggested metabolic flexibility under the variable oxygen conditions typical of deep petroleum reservoirs. Stress-associated genes, including molecular chaperones and heat-shock proteins, further indicated putative adaptation to reservoir-associated environmental stress, although thermotolerance was not experimentally confirmed. Discussion This study provides one of the first genome-resolved insights into deep petroleum reservoir microbiomes from Pakistan and identifies candidate microbial lineages carrying MEOR-relevant genomic traits for future functional validation.