Microbial community in black rust exposed to hot ridge flank crustal fluids

Abstract

During Integrated Ocean Drilling Program Expedition 301, we obtained a sample of black rust from a circulation obviation retrofit kit (CORK) observatory at a borehole on the eastern flank of Juan de Fuca Ridge. Due to overpressure, the CORK had failed to seal the borehole. Hot fluids from oceanic crust had discharged to the overlying bottom seawater and resulted in the formation of black rust analogous to a hydrothermal chimney deposit. Both culture-dependent and culture-independent analyses indicated that the black-rust-associated community differed from communities reported from other microbial habitats, including hydrothermal vents at seafloor spreading centers, while it shared phylotypes with communities previously detected in crustal fluids from the same borehole. The most frequently retrieved sequences of bacterial and archaeal 16S rRNA genes were related to the genera Ammonifex and Methanothermococcus, respectively. Most phylotypes, including phylotypes previously detected in crustal fluids, were isolated in pure culture, and their metabolic traits were determined. Quantification of the dissimilatory sulfite reductase (dsrAB) genes, together with stable sulfur isotopic and electron microscopic analyses, strongly suggested the prevalence of sulfate reduction, potentially by the Ammonifex group of bacteria. Stable carbon isotopic analyses suggested that the bulk of the microbial community was trophically reliant upon photosynthesis-derived organic matter. This report provides important insights into the phylogenetic, physiological, and trophic characteristics of subseafloor microbial ecosystems in warm ridge flank crusts.

FIG. 1.

(a) Location of CORK 1026B on the eastern flank of the Juan de Fuca Ridge. (b) Schematic diagram of CORK 1026B and borehole (modified from reference 16). The inset shows the black rust used in this study. msb, meters subbasement.

FIG. 2.

(a and b) Transmission electron microscopy image (a) and EDX spectrum (b) of a prokaryotic cell in the black rust that had accumulated iron and sulfur. (c and d) SAED pattern (c), showing that a crystal accumulated on the cell surface (d) is greigite.

FIG.3.

Phylogenetic relationships of isolates and representative environmental clones as determined by neighbor-joining analysis of 16S rRNA gene sequences. Trees were constructed by using 419 (a) and 411 (b) sites that could be unambiguously aligned. Some sequences are indicated as follows: red, isolates obtained in this study; blue, clones sequenced in this study; boldface black, clones and isolates obtained in previous fluid surveys (6, 23). The remaining sequences were obtained from DDBJ. The clonal frequency of each representative clone obtained in this study and DDBJ accession numbers are shown in parentheses. Branch points conserved with bootstrap values of >75% (solid circles) and with bootstrap values of 50 to 74% (open circles) are indicated. Some groups are represented by shaded trapezoids that indicate the numbers of sequences. Scale bar = 0.02 expected change per nucleotide position. (a) Tree indicating the phylogenetic relationship among members of the Bacteria. (b) Tree indicating the phylogenetic relationship among members of the Archaea. PCB, polychlorinated biphenyl.

FIG.3.

Phylogenetic relationships of isolates and representative environmental clones as determined by neighbor-joining analysis of 16S rRNA gene sequences. Trees were constructed by using 419 (a) and 411 (b) sites that could be unambiguously aligned. Some sequences are indicated as follows: red, isolates obtained in this study; blue, clones sequenced in this study; boldface black, clones and isolates obtained in previous fluid surveys (6, 23). The remaining sequences were obtained from DDBJ. The clonal frequency of each representative clone obtained in this study and DDBJ accession numbers are shown in parentheses. Branch points conserved with bootstrap values of >75% (solid circles) and with bootstrap values of 50 to 74% (open circles) are indicated. Some groups are represented by shaded trapezoids that indicate the numbers of sequences. Scale bar = 0.02 expected change per nucleotide position. (a) Tree indicating the phylogenetic relationship among members of the Bacteria. (b) Tree indicating the phylogenetic relationship among members of the Archaea. PCB, polychlorinated biphenyl.

FIG. 4.

Phylogenetic tree based on the deduced amino acid sequences of the dsrAB genes of representative clones and sulfate-reducing strains. The tree was constructed by using 356 amino acid sequences. See the legend to Fig. 3 for additional information.