Microbial biodegradation of hydrocarbons in petroleum reservoirs has major consequences in the petroleum value and quality. The identification of microorganisms capable of in-situ degradation of hydrocarbons under the reservoir conditions is crucial to understand microbial roles in hydrocarbon transformation and the impact of oil exploration and production on energy resources. The aim of this study was to profile the metagenome of microbial communities in crude oils and associated formation water from two high temperature and relatively saline oil-production wells, where one has been subjected to water flooding (BA-2) and the other one is considered pristine (BA-1). The microbiome was studied in the fluids using shotgun metagenome sequencing. Distinct microbial compositions were revealed when comparing pristine and water flooded oil wells in contrast to the similar community structures observed between the aqueous and oil fluids from the same well (BA-2). The equal proportion of archaea and bacteria together with the greater anaerobic hydrocarbon degradation potential in the BA-1 pristine but degraded reservoir contrasted with the predominance of bacteria over archaea, aerobic pathways and lower frequency of anaerobic degradation genes in the BA-2 water flooded undegraded well. Our results suggest that Syntrophus, Syntrophomonas, candidatus Atribacteria and Synergistia, in association with mainly acetoclastic methanogenic archaea of the genus Methanothrix, were collectively responsible for the oil biodegradation observed in the pristine petroleum well BA-1. Conversely, the microbial composition of the water flooded oil well BA-2 was mainly dominated by the fast-growing and putatively aerobic opportunists Marinobacter and Marinobacterium. This presumable allochthonous community introduced a greater metabolic versatility, although oil biodegradation has not been detected hitherto perhaps due to in-reservoir unfavorable physicochemical conditions. These findings provide a better understanding of the petroleum reservoir microbiomes and their potential roles in biogeochemical processes occurring in environments with different geological and oil recovery histories Ver menos