Diversity and Functional Analysis of Bacterial Communities Associated with Natural Hydrocarbon Seeps in Acidic Soils at Rainbow Springs, Yellowstone National Park

Abstract

In this paper we describe the bacterial communities associated with natural hydrocarbon seeps in nonthermal soils at Rainbow Springs, Yellowstone National Park. Soil chemical analysis revealed high sulfate concentrations and low pH values (pH 2.8 to 3.8), which are characteristic of acid-sulfate geothermal activity. The hydrocarbon composition of the seep soils consisted almost entirely of saturated, acyclic alkanes (e.g., nalkanes with chain lengths of C 15 to C 30 , as well as branched alkanes, predominately pristane and phytane). Bacterial populations present in the seep soils were phylogenetically characterized by 16S rRNA gene clone library analysis. The majority of the sequences recovered (>75%) were related to sequences of heterotrophic acidophilic bacteria, including Acidisphaera spp. and Acidiphilium spp. of the ␣-Proteobacteria. Clones related to the iron-and sulfur-oxidizing chemolithotroph Acidithiobacillus spp. were also recovered from one of the seep soils. Hydrocarbon-amended soil-sand mixtures were established to examine [ 14 C]hexadecane mineralization and corresponding changes in the bacterial populations using denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene fragments. Approximately 50% of the [ 14 C]hexadecane added was recovered as 14 CO 2 during an 80-day incubation, and this was accompanied by detection of heterotrophic acidophile-related sequences as dominant DGGE bands. An alkane-degrading isolate was cultivated, whose 16S rRNA gene sequence was identical to the sequence of a dominant DGGE band in the soil-sand mixture, as well as the clone sequence recovered most frequently from the original soil. This and the presence of an alkB gene homolog in this isolate confirmed the alkane degradation capability of one population indigenous to acidic hydrocarbon seep soils.The occurrence of natural hydrocarbon seeps in thermal areas of northwestern Wyoming, including the Yellowstone National Park (YNP) region, was reported by explorers as early as 1807 (33). Three hydrocarbon seeps located near Calcite Springs, Rainbow Springs, and Tower Bridge in the northeast corner of YNP were described by Love and Good (32). All of the seeps had abundant sulfur and were surrounded and underlain by volcanic rocks. Thermally active springs and vents were closely associated with the seeps at the time of investigations by Good in 1963 and 1968 (33). The Calcite Springs oils are composed primarily of aromatic hydrocarbons, polar compounds, and asphaltenes and contain less than 1% saturated hydrocarbons, whereas the oils from Rainbow Springs consist almost entirely of n-alkanes (6). The chemical composition of Tower Bridge oils has not been reported. Of the three seeps, Rainbow Springs is the least accessible, and it is located about 32 km northeast of Lake Village, YNP.The association of aliphatic hydrocarbons with acidic, sulfate-rich geothermal environments distinguishes the Rainbow Springs sites from other natural hydrocarbon seeps. The extreme acidity (pH Ͻ3) associated with geothermal areas of ...

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