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Appl Environ Microbiol. 2009 January; 75(1): 242–245.
Published online 2008 October 31. doi:  10.1128/AEM.01462-08
PMCID: PMC2612199

Abundances of Hyperthermophilic Autotrophic Fe(III) Oxide Reducers and Heterotrophs in Hydrothermal Sulfide Chimneys of the Northeastern Pacific Ocean [down-pointing small open triangle]


The abundances of hyperthermophilic heterotrophs, methanogens, and autotrophic reducers of amorphous Fe(III) oxide in 18 samples of deep-sea hydrothermal vent sulfide chimneys of the Endeavour Segment were measured. The results indicate that conditions favor the growth of iron reducers toward the interiors of these deposits and that of heterotrophs toward the outer surfaces near high-temperature polychaete worms (Paralvinella sulfincola).

Hyperthermophiles that inhabit deep-sea geothermal environments serve as tracers of the in situ chemical and physical conditions in these systems since they are generally not found in surrounding seawater and their metabolisms are reflections of the chemistry and temperature of their environment (12). Hydrothermal sulfide chimneys form at deep-sea vents when upwelling high-temperature hydrothermal fluids mix with cold seawater, resulting in the precipitation of metal sulfides (26). The resultant deposits host diverse microbial communities that thrive within their warm, porous interiors (23).

One of the best-studied deep-sea hydrothermal systems is the Endeavour Segment of the Juan de Fuca Ridge in the northeastern Pacific Ocean (see Fig. S1 in the supplemental material), where massive sulfide chimneys currently form (3, 7, 21). In one of these chimneys, approximately 65% of the total microbial population in the interior wall was archaeal on the basis of fluorescent in situ hybridization (22). The proportions of Methanocaldococcaceae and Thermococcaceae (both hyperthermophilic families within the Euryarchaeota) were very low (≤4%) in numerous samples taken across the structure, and 16S rRNA phylogenetic analyses indicate that the majority of the Archaea are unknown crenarchaeota (22). A novel hyperthermophilic member of the Crenarchaeota was isolated from the same deposit that is an obligately autotrophic reducer of amorphous Fe(III) oxide with an optimum growth temperature of 106°C (14). In 2004, our efforts to culture hyperthermophilic archaea at 95°C on eight types of media [NO3, S°, Fe(III) reduction, and methanogen media for autotrophs and NO3, S°, SO42−, and Fe(III) reduction media for heterotrophs] from six sulfide deposits collected from Endeavour resulted only in the growth of autotrophic dissimilatory iron reducers (four samples) and heterotrophic sulfur reducers (five samples). There are few other reports of hyperthermophilic reducers of amorphous Fe(III) oxide from deep-sea hydrothermal environments (15, 16); therefore, determining their potential relevance at Endeavour was a goal of this study.

While molecular assessments of microbial diversity in submarine hydrothermal sulfide deposits are common (see reference 23 for a review), our understanding of the distribution of metabolic activities and microbial abundances of indicator organisms in submarine vents is in a nascent state. The purpose of this study was to determine the relative abundances of hyperthermophilic autotrophic Fe(III) oxide reducers, methanogens, and heterotrophs in the interiors of seven actively venting sulfide chimneys (Fig. 1A and B), three low-temperature (5 to 140°C) hydrothermal fluid samples that were a mixture of end member hydrothermal fluid and seawater (Fig. (Fig.1C),1C), and two collections of 25 Paralvinella sulfincola polychaete worms from the exteriors of two active deposits (Fig. (Fig.1D).1D). The samples were collected in 2006 and 2008 from the Main, Mothra, and High Rise vent fields along the Endeavour Segment by using the deep-sea research submarine Alvin.

FIG. 1.
Samples collected from the Endeavour Segment. (A) Black smoker chimney on the Boardwalk sulfide deposit (scale bar = 10 cm). (B) Wurtzite-sphalerite-rich material from the Hulk sulfide deposit used for culturing (scale bar = 4 cm). (C) ...

Once onboard the ship, 12 to 24 g of the soft, porous wurtzite-sphalerite-rich material from the interiors of the chimneys (2 to 10 cm below the hard silicate-enriched outer crust) were added to 50 ml of sterile, anoxic artificial seawater composed of the salts in DSMZ medium 141 ( The serum bottle containing the sample was sealed, flushed with N2-CO2 (70%:30%), and reduced with 0.025% (wt/vol) each of cysteine-HCl·H2O and Na2S·9H2O. Two batches of 25 P. sulfincola worms were collected, halved, added to DSMZ medium 141 salts, and processed as described above. Low-temperature, diffuse hydrothermal fluids (50 ml) collected using 750-ml titanium syringes were transferred immediately into a sealed serum bottle that had been flushed with N2-CO2 and reduced with the cysteine-sulfide solution as before.

Three-tube most-probable-number (MPN) analyses (8, 10) were performed at 90°C (Table (Table1).1). A complete description of the growth media is available in the supplemental material. Up to 99 iron reducer cells per gram (dry weight) of sulfide material in each of the seven black smoker chimneys examined were measured. In six of these, the estimated numbers of autotrophic iron reducers were higher than those for the heterotrophs. Up to 33,000 iron reducer cells per liter in diffuse fluids emitted from the interiors of the sulfide deposits were measured. Similarly, in these fluids the estimated numbers of iron reducers were higher than those for the heterotrophs. For the worm samples, up to 2,580 heterotroph cells per worm were measured, which greatly exceeded the numbers of iron reducers in these samples. Similar to results from 2004, methanogens were not detected in any of the samples.

Abundances of hyperthermophilic autotrophic iron reducers, methanogens, and heterotrophic sulfur reducers grown at 90°C

Four iron reducer strains and 12 heterotroph strains were purified using three successive dilution-to-extinction transfers in liquid medium. Sequencing of their 16S rRNA genes indicates that the iron reducers are Pyrodictium and Hyperthermus species and that the heterotrophs are Pyrococcus and Thermococcus species (see Fig. S2 in the supplemental material). Among the iron reducers, only Hyperthermus strain BW06-2 grew on acetate and peptides without the addition of H2 and CO2 (see Table S1 in the supplemental material). In general, the addition of acetate or peptides to the growth medium increased neither their growth rates nor their maximum cell concentrations relative to autotrophic growth. The two Pyrodictium strains are obligate Fe(III) oxide reducers, while the two Hyperthermus strains also use nitrate as a terminal electron acceptor (see Table S2 in the supplemental material). All 12 obligate heterotroph strains grew on 0.5% (wt/vol) casein hydrolysate only, 0.5% maltose only, and 0.5% cellobiose only when elemental sulfur was present and produced H2S and H2 as end products. They appear to be well adapted for growth on polypeptides and α-1,4- and β-1,4-linked sugar polymers that likely come from vent animal secretions (13) and the biofilms of other microorganisms.

The MPN results presented in this study indicate that hyperthermophilic heterotrophs are most abundant near the outer surfaces of the chimneys in close proximity to P. sulfincola. This polychaete worm was chosen as a source of hyperthermophiles in this study because it lives in the porous outer walls of black smoker chimneys at temperatures (up to 55°C) that exceed those in areas containing other metazoans at deep-sea vents (6) and because they have previously been shown to be a source of hyperthermophilic heterotrophs (11, 20). The results also indicate that there is a transition to a more autotrophic composition among hyperthermophiles toward the interiors of the deposits. Based on MPNs, iron reducers appear to be more abundant than methanogens within the hyperthermophilic autotroph community. The absence of hyperthermophilic methanogens is curious since they have been found in 90°C MPN analyses of low-temperature, basalt-hosted hydrothermal fluids from the Endeavour Segment (10) and in molecular and fluorescent in situ hybridization analyses of Endeavour sulfide chimneys (22). We cannot exclude the possibility that there is a culturing bias or other potentially limiting factors, such as O2 exposure during sample recovery, or that the organisms are viable but not culturable. However, other possible explanations for the absence of hyperthermophilic methanogens include environmental conditions that limit their growth, such as H2 limitation, and reduction potentials that are outside the range needed for growth.

Analysis of H2 concentrations in hydrothermal fluids from the Endeavour Segment (4, 18, 19, 24) by thermodynamic modeling of hydrogenotrophic methanogenesis (9) suggests that the H2 activity of the Endeavour fluids may be below the minimum needed for growth. In contrast, the minimum H2 activity needed for growth of iron reducers should be significantly less than that for methanogenesis due to the higher free energy available from the reduction of amorphous Fe(III) oxide (1). In 2005, the dissolved H2 concentrations in hydrothermal end member fluids from the Main Endeavour Field were 0.05 to 0.1 mmol per kg, suggesting that these fluids were also only mildly reducing (4). These conditions would also likely favor the growth of hyperthermophilic Fe(III) oxide reducers, since they generally prefer mildly reducing conditions (5), over that of hydrogenotrophic methanogens that require reduction potentials below −330 mV (25).

Mildly reducing conditions would also occur at 90°C within sulfide deposits if there were a significant influx of oxygen-saturated seawater into the deposit (27). This would also provide the source of Fe(III) oxide needed to support the iron reducers. Evidence that seawater entrainment and Fe(III) oxide do occur in some Endeavour sulfide chimneys is provided by detailed petrographic and chemical analyses (17). For example, pockets of anhydrite (CaSO4), which forms during heating of seawater above 150°C (2), occur in both the exterior and the interior walls of some Endeavour sulfide chimneys, and poorly crystallized magnesium-rich clay has been documented to occur in the outer chimney walls (17). Petrographic relationships and isotopic analyses indicate that anhydrite precipitates directly from mixed hydrothermal fluid and seawater throughout much of the chimney's evolution as long as fluid temperatures remain high and high-angle fractures allow seawater ingress. Trace amorphous Fe(III) oxide has also been documented to occur in the inner and outer walls of an actively venting sulfide chimney at Endeavour (17). Additional evidence for seawater entrainment into the interiors of chimneys is provided by the presence of the chlorosulfate mineral gordaite (17).

For submarine hydrothermal systems, there is a critical need to determine the relative abundances, distributions, and metabolic characteristics of indicator organisms since they may provide insight into the chemical conditions present in the environments that are not readily accessible for study (e.g., the subsurfaces and the interiors of deep-sea chimneys). Investigation of these indicator organisms will also provide insights into the character of microbes present even when they themselves are not necessarily the most abundant or biogeochemically important organisms (12). Some of the more fundamental questions regarding vents concern the distributions of autotrophs versus heterotrophs and various autotrophs with differing growth requirements. A long-term goal is to determine what biogeochemical impacts these and other microorganisms have on hydrothermal vent systems and how this may translate into a better understanding of life deeper within the Earth's crust.

Supplementary Material

[Supplemental material]


This work was supported by grants from the NOAA West Coast and Polar Regions Undersea Research Center (UAF 06-0096 to J.F.H.), the U.S. DOT Northeast Sun Grant Institute of Excellence (NE07-030 to J.F.H.), the University of Massachusetts Faculty Research Grant program (J.F.H.), and the NSF RIDGE 2000 program (NSF OCE-0426109 to D.S.K.).


[down-pointing small open triangle]Published ahead of print on 31 October 2008.

Supplemental material for this article may be found at


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