Bacterial communities associated with subsurface geochemical processes in continental serpentinite springs

Abstract

Reactions associated with the geochemical process of serpentinization can generate copious quantities of hydrogen and low-molecular-weight organic carbon compounds, which may provide energy and nutrients to sustain subsurface microbial communities independently of the photosynthetically supported surface biosphere. Previous microbial ecology studies have tested this hypothesis in deep sea hydrothermal vents, such as the Lost City hydrothermal field. This study applied similar methods, including molecular fingerprinting and tag sequencing of the 16S rRNA gene, to ultrabasic continental springs emanating from serpentinizing ultramafic rocks. These molecular surveys were linked with geochemical measurements of the fluids in an interdisciplinary approach designed to distinguish potential subsurface organisms from those derived from surface habitats. The betaproteobacterial genus Hydrogenophaga was identified as a likely inhabitant of transition zones where hydrogen-enriched subsurface fluids mix with oxygenated surface water. The Firmicutes genus Erysipelothrix was most strongly correlated with geochemical factors indicative of subsurface fluids and was identified as the most likely inhabitant of a serpentinization-powered subsurface biosphere. Both of these taxa have been identified in multiple hydrogen-enriched subsurface habitats worldwide, and the results of this study contribute to an emerging biogeographic pattern in which Betaproteobacteria occur in near-surface mixing zones and Firmicutes are present in deeper, anoxic subsurface habitats.

Fig 1

The bacterial diversity of Tablelands serpentinite springs decreases as the f_UB increases. The freshwater end member (f_UB, 0) has the highest number of TRFLP fragment clusters and OTUs produced by the CoDL and JGI tag sequencing projects. The most ultrabasic spring for which tag sequences were available yielded the fewest OTUs.

Fig 2

Dendrogram illustrating bacterial community similarity (as measured by the presence/absence of TRFLP fragment clusters) among samples collected from Tablelands serpentinite springs and surface freshwater. Dotted lines represent samples that could not be distinguished by a SIMPROF test. Subscript numbers indicate replicate samples from the same spring. The abundance levels of Hydrogenophaga and Erysipelothrix in the underlying table reflect the percent relative abundance of the corresponding TRFLP fragment cluster in each sample. Color coding of the table is an arbitrary visual aid to identify the highest and lowest numbers for each variable. Sample names and associated geochemical characteristics correspond to data provided in Table 1.

Fig 3

Proportions of each genus in representative samples of an ultrabasic spring (WHC2B), a mixing site (WHC2C), and surface freshwater (WHB).White dots indicate sampling locations for WHC2B and WHC2C. The sampling location for WHB is located approximately 10 m upstream from the pool depicted in this photograph. Arrows indicate subsurface fluid exiting from WHC2B and surface freshwater entering the pool at WHC2C from upstream. The white lines indicate the width (∼1 m) and length (∼3 m) of the pool. Results in this figure are derived from JGI 16S rRNA tag sequencing. A full taxonomic summary is provided in Table S1 of the supplemental material.

Fig 4

Maximum-likelihood phylogenetic trees of Hydrogenophaga-related (A) and Erysipelothrix-related (B) 16S rRNA sequences. For each tree, the representative tag sequence was placed next to its most likely neighbor in the reference phylogeny of nearly full-length sequences by using the evolutionary placement algorithm (32). Each tree represents the best topology after 20 maximum-likelihood references, and all nodes in the reference phylogeny received 100% bootstrap support.

Fig 5

Association network of the relative abundance of TRFLP fragment clusters and geochemical variables. Each circle is a TRFLP fragment cluster, and its color corresponds to the type of sample in which its relative abundance was greatest (see figure legend). Positive correlations are black lines; negative correlations are red lines; the width of each line is proportional to the r value. All correlations with P values of <0.05 and q levels of <0.05 are shown. The inset shows geochemical correlations only for Hydrogenophaga and Erysipelothrix, which were identified by linking observed TRFLP fragment sizes to predicted restriction sizes of tag sequences. For clarity, taxonomic assignments for other TRFLP fragment clusters are not shown. The six blue circles other than Erysipelothrix represent unidentified TRFLP fragment clusters, which did not constitute more than 3% of the total TRFLP signal in any sample.