Inhibition of sulfate-reducing bacteria with formate.

Despite hostile environmental conditions, microbial communities have been found in µL-sized water droplets enclosed in heavy oil of the Pitch Lake, Trinidad. Some droplets showed high sulfate concentrations and surprisingly low relative abundances of sulfate-reducing bacteria in a previous study. Hence, we investigated here whether sulfate reduction might be inhibited naturally. Ion chromatography revealed very high formate concentrations around 2.37 mM in 21 out of 43 examined droplets. Since these concentrations were unexpectedly high, we performed growth experiments with the three sulfate-reducing type strains Desulfovibrio vulgaris, Desulfobacter curvatus, and Desulfococcus multivorans, and tested the effects of 2.5, 8, or 10 mM formate on sulfate reduction. Experiments demonstrated that 8 or 10 mM formate slowed down the growth rate of D. vulgaris and D. curvatus and the sulfate reduction rate of D. curvatus and D. multivorans. Increasing formate concentrations delayed the onsets of growth and sulfate reduction of D. multivorans, which were even inhibited completely while formate was added constantly. Contrary to previous studies, D. multivorans was the only organism capable of formate consumption. Our study suggests that formate accumulates in the natural environment of the water droplets dispersed in oil and that such levels are very likely inhibiting sulfate-reducing microorganisms.

Densely Populated Water Droplets in Heavy-Oil Seeps.

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.