Most of the microbial degradation in oil reservoirs is believed to take place at the oil-water transition zone (OWTZ). However, a recent study indicates that there is microbial life enclosed in microliter-sized water droplets dispersed in heavy oil of Pitch Lake in Trinidad and Tobago. This life in oil suggests that microbial degradation of oil also takes place in water pockets in the oil-bearing rock of an oil leg independent of the OWTZ. However, it is unknown whether microbial life in water droplets dispersed in oil is a generic property of oil reservoirs rather than an exotic exception. Hence, we took samples from three heavy-oil seeps, Pitch Lake (Trinidad and Tobago), the La Brea Tar Pits (California, USA), and an oil seep on the McKittrick oil field (California, USA). All three tested oil seeps contained dispersed water droplets. Larger droplets between 1 and 10 μl revealed high cell densities of up to 109 cells ml−1. Testing for ATP content and LIVE/DEAD staining showed that these populations consist of active and viable microbial cells with an average of 60% membrane-intact cells and ATP concentrations comparable to those of other subsurface ecosystems. Microbial community analyses based on 16S rRNA gene amplicon sequencing revealed the presence of known anaerobic oil-degrading microorganisms. Surprisingly, the community analyses showed similarities between all three oil seeps, revealing common OTUs, although the sampling sites were thousands of kilometers apart. Our results indicate that small water inclusions are densely populated microhabitats in heavy oil and possibly a generic trait of degraded-oil reservoirs. IMPORTANCE Our results confirmed that small water droplets in oil are densely populated microhabitats containing active microbial communities. Since these microhabitats occurred in three tested oil seeps which are located thousands of kilometers away from each other, such populated water droplets might be a generic trait of biodegraded oil reservoirs and might be involved in the overall oil degradation process. Microbial degradation might thus also take place in water pockets in the oil-bearing oil legs of the reservoir rock rather than only at the oil-water transition zone.

Microorganisms are present in nearly every oil or bitumen sample originating from temperate reservoirs. Nevertheless, it is very difficult to obtain reliable estimates about microbial processes taking place in deep reservoirs, since metabolic rates are rather low and differ strongly during artificially cultivation. Here, we demonstrate the importance and impact of microorganisms entrapped in microscale water droplets for the overall biodegradation process in bitumen. To this end, we measured degradation rates of heavily biodegraded bitumen from the Pitch Lake (Trinidad and Tobago) using the novel technique of reverse stable isotope labeling, allowing precise measurements of comparatively low mineralization rates in the ng range in microcosms under close to natural conditions. Freshly taken bitumen samples were overlain with artificial brackish water and incubated for 945 days. Additionally, three-dimensional distribution of water droplets in bitumen was studied with computed tomography, revealing a water bitumen interface of 1134 cm 2 per liter bitumen, resulting in an average mineralization rate of 9.4−38.6 mmol CO 2 per liter bitumen and year. Furthermore, a stable and biofilm-forming microbial community established on the bitumen itself, mainly composed of fermenting and sulfate-reducing bacteria. Our results suggest that small water inclusions inside the bitumen substantially increase the bitumen−water interface and might have a major impact on the overall oil degradation process.

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