Phylogenetic diversity of microorganisms in subseafloor crustal fluids from Holes 1025C and 1026B along the Juan de Fuca Ridge flank

Abstract

To expand investigations into the phylogenetic diversity of microorganisms inhabiting the subseafloor biosphere, basalt-hosted crustal fluids were sampled from Circulation Obviation Retrofit Kits (CORKs) affixed to Holes 1025C and 1026B along the Juan de Fuca Ridge (JdFR) flank using a clean fluid pumping system. These boreholes penetrate the crustal aquifer of young ocean crust (1.24 and 3.51 million years old, respectively), but differ with respect to borehole depth and temperature at the sediment-basement interface (147 m and 39°C vs. 295 m and 64°C, respectively). Cloning and sequencing of PCR-amplified small subunit ribosomal RNA genes revealed that fluids retrieved from Hole 1025C were dominated by relatives of the genus Desulfobulbus of the Deltaproteobacteria (56% of clones) and Candidatus Desulforudis of the Firmicutes (17%). Fluids sampled from Hole 1026B also contained plausible deep subseafloor inhabitants amongst the most abundant clone lineages; however, both geochemical analysis and microbial community structure reveal the borehole to be compromised by bottom seawater intrusion. Regardless, this study provides independent support for previous observations seeking to identify phylogenetic groups of microorganisms common to the deep ocean crustal biosphere, and extends previous observations by identifying additional lineages that may be prevalent in this unique environment.

Keywords: Juan de Fuca Ridge; Ocean Drilling Program; SSU ribosomal RNA gene; deep subsurface; diversity; microorganisms.

Figure 1

(A) Location of CORK observatory sampling sites on the Juan de Fuca Ridge flank, Pacific Ocean. (B) Cross-sectional diagram of ODP Leg 168 showing depth of basement crust and sediment thickness, basement age and associated distance from ridge axis, and locations of Holes 1025 (yellow) and 1026 (red) in boldface font (modified from Wheat et al., 2004). (C) Schematic diagram of CORKs located at Holes 1025C and 1026B (not drawn to scale). Fluids were sampled from the exit valve of the fluid delivery line (modified from Lin et al., 2012). (D) Photo of squeeze sample taken for biogeochemical analysis from top of CORK 1026B in 2008, fluid sampling device used in 2010 at Hole 1026B, and Borehole Flushing Unit and fluid sampling device used at Hole 1025C in 2010.

Figure 2

Venn diagrams showing the overlap in microbial communities between Holes 1025C, 1026B, and U1301A fluids (A,B), and between Hole 1026B fluids collected in different years (C,D). Data for “U1301A 2008–2010” appears in Jungbluth et al. (2013), “1026B 1998” in Cowen et al. (2003), and “1026B 2002” in Huber et al. (2006).

Figure 3

Phylogenetic relationships of SSU rRNA gene clones related to the phylum Deltaproteobacteria, colored according to borehole of origin. Clones derived from nearby bottom seawater are shown in blue (Jungbluth et al., 2013). Cultivated Epsilonproteobacteria were used as an outgroup (not shown). Detailed phylogenies are shown for selected lineages. Black (100%), gray (>80%), and white (>50%) circles indicate nodes with bootstrap support, from 1000 replicates. Gene clones recovered in this study are highlighted in bold font; the relative abundance of identical clones is listed in parentheses. The scale bars correspond to 0.1 substitutions per nucleotide position.

Figure 4

Phylogenetic relationships of SSU rRNA gene clones from Holes 1025C and 1026B fluids within the phylum Firmicutes, related to (A) Candidatus Desulforudis audaxviator, (B) RF3, (C) Sulfurimonas of the bacterial phylum Epsilonproteobacteria, and (D) DHVEG-6 of the domain Archaea. Short length gene clone pyrite_60 was added to the Candidatus Desulforudis audaxviator tree after tree construction and bootstrapping and is indicated by a dashed line. Other information as in Figure 3.

Figure 5

Phylogenetic relationships of SSU rRNA gene clones related to the phylum Gammaproteobacteria, colored as in Figure 3. A variety of Betaproteobacteria were used as outgroups (not shown). Detailed phylogenies are shown for selected lineages. Other information as in Figure 3.

Figure 6

Phylogenetic relationships of SSU rRNA gene clones related to the phylum Alphaproteobacteria, colored as in Figure 3. Detailed phylogenies are shown for selected lineages. Short length gene clone OCS116 was added after tree construction and bootstrapping and is indicated by a dashed line. Other information as in Figure 3.