Integrated methodology to characterize microbial populations and functions across small spatial scales in an oil production facility

Petroleum reservoir microorganisms directly influence the economics of energy recovery operations through deleterious activities such as souring and biocorrosion. We investigated potential differences in chemistry and microbial populations between stagnant areas and bulk process fluids from three processing modules (low-pressure separator, desalter, and water separator) within an oil-processing facility. Samples were collected before and after the flushing of a sample valve at each site. Microbial community surveys were conducted through 16S rRNA gene sequencing and paired with analyses of fluid chemical composition through chromatographic methods and mass spectrum-based metabolite profiling. The production fluids were high saline brines (1.5-4.0 M [Cl⁻]) with a pH range of 4.5-6.2, varying both within and between sampling sites. Sulfate (0.1-8.0 mM) and acetate (0.5-9.3 mM) were present in all samples, but nitrate was below detection levels. Marked chemical differences were observed between bulk fluids and stagnant deadleg fluids at each sampling point. Stagnant dead-leg samples exhibited chemical stratification with a chloride gradient of ~3 M in the desalter module as well as sulfate and acetate gradients in the water separator module. Molecular surveys revealed that the microbial communities were predominantly composed of three taxonomic groups: Halanaerobiales, Campylobacterales, and Desulfovibrionales. An increase in species diversity was found in the stagnant fluids from each dead leg relative to the respective bulk fluid. MPN determinations of heterotrophic fermenting, sulfate-reducing, and thiosulfatereducing organisms were all greater than 1 × 10⁴ cells mL^-1. Targeted mass spectral analysis of the production fluids revealed the presence of catechols, phenols, and dihydrodiols, indicative of aerobic hydrocarbon biodegradation. Metabolites associated with anaerobic hydrocarbon biodegradation were not detected. Untargeted metabolomic screening revealed over 1000 identified compounds that mapped primarily to known lipid, carbohydrate, and amino acid metabolic pathways. Through the pairing of molecular microbial surveys and advanced metabolite profile analyses, these results suggest a systemic colonization of the facility by anaerobic halophiles, commonly associated with petroleum reservoirs. These organisms were likely cross-fed by organic electron donors produced during the transformation of hydrocarbons by aerobic microorganisms. Furthermore, these results highlight the importance of interrogating the small volumes of stagnated fluids within dead legs for an accurate assessment of the chemical and biological processes occurring within these problematic sites.