Densely Populated Water Droplets in Heavy-Oil Seeps

Abstract

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.

Indigenous microbial communities in heavy oil show a threshold response to salinity

“…DNA extraction from 1 µL of sample was carried out according to a 2-step-lysis protocol (Pannekens et al . 2020 ). In short, DNA was treated 1 h at 37°C with 1 µL of a freshly prepared enzyme-mix, containing 2.5 U/µL lysozyme, 0.048 U/µL lysostaphin and 0.6 U/µL mutanolysin in order to lyse a broad range of Gram-positive cells.…”

“…The alkaline lysis was stopped after 5 min reaction time by the addition of Tris-HCl (pH 4) (Pannekens et al . 2020 ). In order to rule out contamination of the samples by foreign DNA, negative controls where introduced at different steps during DNA extraction and 16S rRNA gene library preparation.…”

“… 2014 ; Pannekens et al . 2020 ). The activity of microorganisms regulates the natural biodegradation of oil in reservoirs and reservoir souring during oil production, and therefore, has generated interest in understanding the environmental factors that determine the assembly of microbial communities in oil (Youssef et al .…”

“… 2014 ; Pannekens et al . 2020 , 2021 ). To investigate the relationship between environmental conditions and microbial community composition we analysed 43 water droplets that we extracted from oil that we sampled at six different sites on the Pitch Lake.…”

Soehngenia longivitae sp. nov., a Fermenting Bacterium Isolated from a Petroleum Reservoir in Azerbaijan, and Emended Description of the Genus Soehngenia

“…The strain performs dismutation of benzaldehyde to benzoate and benzyl alcohol [ 4 ]. Using 16S rRNA gene sequencing, members of the genus Soehngenia were detected in petroleum reservoirs [ 5 , 6 , 7 , 8 ] and in water droplets dispersed in heavy oil [ 9 ], in methanogenic crude oil- and propionate-degrading enrichments [ 10 , 11 ], in marine sediments contaminated with heavy metals and polycyclic aromatic hydrocarbons [ 12 ], and in cyanide-degrading enrichments [ 13 ].…”