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.

Microbial degradation influences the quality of oil resources. The environmental factors that shape the composition of oil microbial communities are largely unknown because most samples from oil fields are impacted by anthropogenic oil production, perturbing the native ecosystem with exogenous fluids and microorganisms. We investigated the relationship between formation water geochemistry and microbial community composition in undisturbed oil samples. We isolated 43 microliter-sized water droplets naturally enclosed in the heavy oil of the Pitch Lake, Trinidad and Tobago. The water chemistry and microbial community composition within the same water droplet were determined by ion chromatography and 16S rRNA gene amplicon sequencing, respectively. The results revealed a high variability in ion concentrations and community composition between water droplets. Microbial community composition was mostly affected by chloride concentration, which ranged from freshwater to brackish-sea water. Remarkably, microbial communities did not respond gradually to increasing chloride concentration but showed a sudden change to less diverse and uneven communities when exceeding a chloride concentration of 57.3 mM. The results reveal a threshold-regulated response of microbial communities to salinity, offering new insights into the microbial ecology of oil reservoirs.

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. Concerning D. multivorans, increasing formate concentrations delayed the onsets of growth and sulfate reduction, 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.

Natural and oil surface slicks are widespread phenomena at the air-water interface and represent visible surface films with distinct physicochemical and biological properties compared to non-slick surface films and underlying marine water. Both types of slicks have major functions in nourishing and dispersing surface-dwelling micro- and macroorganisms, contribute to particle generation and carbon cycling, and are known to accumulate pollutants. Despite these functions and the high frequency of slicks in the marine environment, slicks are little understood considering their microbial inhabitants and contributions of these organisms for natural and oil slick establishment, function, and ecology. Our mini review summarizes the current knowledge of microbial life in natural slicks, natural and anthropogenic oil slicks, including the major knowledge gaps and perspectives for future research. Overall, we aim for an increased awareness about the existence of natural slicks, the differentiation between natural and anthropogenic oil slicks and an improved reporting of slick events during sampling of surface film and seawater from the epipelagic zone.

Anthropogenic carbon dioxide (CO2) emissions drive climate change and pose one of the major challenges of our century. The effects of increased CO2 in the form of ocean acidification (OA) on the communities of marine planktonic eukaryotes in tropical regions such as the Timor Sea are barely understood. Here, we show the effects of high CO2 (mean ± SD pCO2 = 1823 ± 161 μatm and pHT = 7.46 ± 0.05) versus in situ CO2 (504 ± 42 µatm, 7.95 ± 0.04) seawater on the community composition of marine planktonic eukaryotes after 3 and 48 h of treatment exposure in a shipboard microcosm experiment. Illumina sequencing of the V9 hypervariable region of 18S rRNA (gene) was used to study the eukaryotic community composition. Increased CO2 significantly suppressed the relative abundances of different eukaryotic operational taxonomic units (OTUs), including important primary producers, although the chlorophyll a concentration remained constant. OA effects on eukaryotes were consistent between total (DNA-based) and active (cDNA-based) taxa after 48 h, e.g. for the diatoms Trieres chinensis and Stephanopyxis turris. Effects of OA on the relative abundances of OTUs were often species- or even ecotype-specific, and the incubation selectively allowed for detection of the OA-sensitive OTUs that benefitted the most from incubation in a closed bottle, as containment effects on the community structure were evident after 48 h. Many OTUs were adversely affected by sudden decreases of seawater pH, suggesting high sensitivity to OA at the base of the tropical marine biodiversity and difficult-to-predict outcomes for food-web functioning in the future ocean.

Ocean acidification effects on planktonic eukaryotes ABSTRACT Anthropogenic carbon dioxide (CO2) emissions drive climate change and pose one of the major challenges of our century. The effects of increased CO2 in the form of ocean acidification (OA) on the communities of marine planktonic eukaryotes in tropical regions such as the Timor Sea are barely understood. Here, we show the effects of high CO2 (pCO2=1823±161 µatm, pHT=7.46±0.05) versus in situ CO2 (pCO2=504±42 µatm, pHT=7.95±0.04) seawater on the community composition of marine planktonic eukaryotes immediately and after 48 hours of treatment exposure in a shipboard microcosm experiment. Illumina sequencing of the V9 hypervariable region of 18S rRNA (gene) was used to study the eukaryotic community composition. Down-regulation of extracellular carbonic anhydrase occurred faster in the high CO2 treatment. Increased CO2 significantly suppressed the relative abundances of eukaryotic operational taxonomic units (OTUs), including important primary producers. These effectswere consistent between abundant (DNA-based) and active (cDNA-based) taxa after 48 hours, e.g., for the diatoms Trieres chinensis and Stephanopyxis turris. Effects were also very speciesspecific among the different diatoms. The microbial eukaryotes showed adaptation to the CO2 treatment over time, but many OTUs were adversely affected by decreasing pH. OA effects might fundamentally impact the base of marine biodiversity, suggesting unpredictable outcomes for food web functioning in the future ocean.

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