Isolation and characterization of novel psychrophilic, neutrophilic, Fe-oxidizing, chemolithoautotrophic alpha- and gamma-proteobacteria from the deep sea

Abstract

We report the isolation and physiological characterization of novel, psychrophilic, iron-oxidizing bacteria (FeOB) from low-temperature weathering habitats in the vicinity of the Juan de Fuca deep-sea hydrothermal area. The FeOB were cultured from the surfaces of weathered rock and metalliferous sediments. They are capable of growth on a variety of natural and synthetic solid rock and mineral substrates, such as pyrite (FeS(2)), basalt glass ( approximately 10 wt% FeO), and siderite (FeCO(3)), as their sole energy source, as well as numerous aqueous Fe substrates. Growth temperature characteristics correspond to the in situ environmental conditions of sample origin; the FeOB grow optimally at 3 to 10 degrees C and at generation times ranging from 57 to 74 h. They are obligate chemolithoautotrophs and grow optimally under microaerobic conditions in the presence of an oxygen gradient or anaerobically in the presence of nitrate. None of the strains are capable of using any organic or alternate inorganic substrates tested. The bacteria are phylogenetically diverse and have no close Fe-oxidizing or autotrophic relatives represented in pure culture. One group of isolates are gamma-Proteobacteria most closely related to the heterotrophic bacterium Marinobacter aquaeolei (87 to 94% sequence similarity). A second group of isolates are alpha-Proteobacteria most closely related to the deep-sea heterotrophic bacterium Hyphomonas jannaschiana (81 to 89% sequence similarity). This study provides further evidence for the evolutionarily widespread capacity for Fe oxidation among bacteria and suggests that FeOB may play an unrecognized geomicrobiological role in rock weathering in the deep sea.

FIG. 1.

Location of the main Endeavour segment of the Juan de Fuca Ridge, northeast Pacific Ocean (modified from reference 13).

FIG. 2.

Examples of oxidized sulfide materials used to enrich for FeOB. (A) Weathered, brecciated sulfide rubble, collected from the vicinity of Hulk Flange (8) along the main Endeavour segment of the Juan de Fuca ridge axis. (B) Push-core sample of fine-grained sulfide sediment collected from the Middle Valley (Fig. 1).

FIG. 3.

Gradient tube (FeS-based) growth of FeOB. The four tubes on the left contain cultures of FeOB, and the single tube on the far right represents an abiotic control. In culture tubes, a distinct band of cells develops ∼1 cm from air-medium interface at the tops of the tubes. The milky region that can be seen clearly at the tops of the tubes is comprised principally of Fe oxide particles, which directly overlie the band of cell growth. In the control tube, Fe oxides develop from ∼1 cm from the top of the tube to ∼3 cm from the FeS-medium interface at the bottom.

FIG. 4.

Light (A) and electron (B) microscopic images of FeOB (strain FO10, a member of the γ-Proteobacteria). Cells were grown anaerobically with ferrous carbonate (FeCO₃) and nitrate (NO₃⁻). Bars, 20 μm (A) and 0.5 μm (B). Irregularly twisted Fe oxide particles are apparent by light microscopy in this culture. By electron microscopy, cells can be seen closely associated with Fe particles, which appear to nucleate at the cell surface or within a capsule-like structure that surrounds cells. The close association between cells and mineral particles can be seen by light microscopy; cells appear as roughly circular open areas within mineral aggregates (arrows in panel A).

FIG. 5.

Microelectrode measurements of O₂ as a function of depth within gradient tubes containing a culture (data are for strain FO10) and a corresponding abiotic control. The region of cell growth band development is approximated with shading.

FIG. 6.

(A) C fixation over time for strain FO10 grown in the presence of basaltic glass, pyrite (FeS₂), and FeS (open symbols), with corresponding control data for comparison. C fixation was greatest for the culture grown in the presence of basalt (∼0.22 μmol of C fixed/ml [total]). C fixations in the presence of FeS and FeS₂ were approximately equivalent, with a maximum of ∼0.08 μmol of C fixed/ml (total). (B) Cell growth data for strain FO10 grown in parallel cultures in the presence of basaltic glass, FeS₂, and FeS. Cell growth was highest in the presence of basalt; this was nearly double the cell yield achieved in the presence of FeS and FeS₂.

FIG. 7.

C fixation over time for strains FO1, FO2, FO8, and FO16 and a control. All data are for cells grown in gradient tubes in the presence of FeS. The final C fixation was highest for strains FO2 and FO16, at ∼0.06 μmol of C fixed/ml, and was somewhat less for strains FO1 and FO8, at ∼0.04 μmol of C fixed/ml.

FIG. 8.

Phylogenetic relationships between cultured strains of FeOB in this study (FO numbers) and other FeOB, other autotrophs, and closely related organisms within the α- and γ-Proteobacteria. Accession numbers of the sequences from databases used for tree construction are given in parentheses. Numbers given at branch notes are bootstrap values where these were greater than 50 (see text).