Enrichment and isolation.
Ten milliliters of freshwater medium containing 100 mM poorly crystalline Fe(III) as an electron acceptor and H2 (as H2-CO2; 80:20, vol/vol; 101 kPa) as the electron donor was inoculated with 1 ml of sample from Obsidian Pool, Yellowstone National Park. After 2 days at 80°C, the color of the poorly crystalline Fe(III) oxide changed from brick red-brown to a black magnetic mineral (probably magnetite). These enrichments were then used to inoculate (10%, vol/vol) 10 ml of the same medium in sets of five so that a member of each set could then be incubated at 75, 80, 85, 90, and 100°C. Only one of the original enrichments (designated FW-1a) was successfully transferred past the third transfer at 85°C. After the fifth successful transfer, serial dilutions (10−1 to 10−9) were made from this enrichment. The highest dilution (10−8) that reduced Fe(III) served as the inoculum for an additional series of dilutions. This procedure was then repeated a third time, and the highest positive dilution was selected for isolation of a pure culture.
Individual colonies were obtained in roll tube dilutions of hydrogen-Fe(III) oxide medium solidified with Gelrite. After 10 days at 85°C, single black colonies (0.5 to 1.0 mm) appeared in the more dilute tubes. Single colonies were picked from the highest dilutions and transferred into 2 ml of the freshwater medium containing 100 mM poorly crystalline Fe(III) oxide and H2
; 80:20, vol/vol; 101 kPa), supplemented with 0.25 mM cysteine and 1.3 mM FeCl2
O, and were incubated at 85°C. Cultures with the highest rate of Fe(III) reduction were serially diluted into fresh medium, and then the highest positive dilution was serially diluted again. This process was repeated a total of three times, at which point the culture was considered to be pure. The culture, designated strain FW-1a, was morphologically uniform and contained only one 16S rDNA sequence.
By epifluorescence microscopy, cells of strain FW-1a were rod shaped, ca. 1.0 to 1.2 μm in length and 0.5 μm in diameter, and usually observed as single cells or in pairs (not shown). The cells were highly motile, even at room temperature, when examined by phase-contrast microscopy. The electron micrograph of the cells exhibited monotrichous flagellation (a single flagellum about 12 nm thick and up to 8 μm long) (Fig. ). Several attempts to Gram stain the cells of strain FW-1a were unsuccessful, probably due to its exclusive growth on poorly crystalline Fe(III) oxide as the electron acceptor, which appears as dark deposits (electron-dense granules) on the cell surface (Fig. ) and interferes with the Gram staining reaction. However, electron micrographs from ultrathin sections of cells of strain FW-1a are typical of a gram-negative organism (Fig. ). Spore formation was not observed.
FIG. 1. (A) Electron micrograph of strain FW-1a, showing a single flagellum. (B) Electron micrograph of strain FW-1a grown in freshwater medium with poorly crystalline Fe(III) oxide as the sole electron acceptor and H2 as the sole electron donor in early log (more ...) Hydrogen oxidation coupled to Fe(III) oxide reduction.
Strain FW-1a grew in defined medium at 85°C and under strictly anaerobic conditions, with H2
as the sole electron donor and poorly crystalline Fe(III) oxide as the sole electron acceptor, without the addition of an organic carbon source. In the presence of hydrogen, Fe(III) was reduced, as evidenced by an accumulation of Fe(II) in the medium over the incubation period (Fig. ). There was no Fe(III) reduction or cell growth in the absence of added hydrogen. For each mole of H2
consumed, 1.97 ± 0.16 mol (mean ± standard deviation; n
= 5) of Fe(II) was produced, which is in agreement with the expected stoichiometry according to the equation H2
+ 2Fe(III) → 2H+
+ 2Fe(II). This is only the second example of an Fe(III)-reducing microorganism capable of growing autotrophically on hydrogen (17
Growth of strain FW-1a at 85°C with H2 as the electron donor and poorly crystalline Fe(III) oxide as the electron acceptor. The results are the means from duplicate cultures.
Other electron donors and acceptors.
Several attempts to grow strain FW-1a on a wide variety of electron donors and electron acceptors other than H2 and poorly crystalline Fe(III) oxide were unsuccessful (Table ). Strain FW-1a was unable to reduce Fe(III) with a number of other organic electron donors, such as acetate, pyruvate, lactate, formate, fumarate, and aromatic compounds (Table ). No growth was observed with complex organic compounds such as yeast extract, peptone, tryptone, or Casamino Acids as substrates (Table ). None of the amino acids tested (Table ) could serve as a sole electron donor for Fe(III) reduction. Growth on sugars (e.g., glucose, fructose, and ribose) could not be investigated because the sugars reacted abiotically with Fe(III) oxide at high temperatures.
A wide variety of commonly considered electron acceptors, including sulfate, thiosulfate, sulfite, S0, nitrate, nitrite, oxygen (0.6 to 1.0% evaluated), anthraquinone-2,6-disulfonate, Mn(IV), fumarate, Fe(III)-citrate, and Fe(III)-pyrophosphate did not support growth with hydrogen as the electron donor (Table ).
Temperature, salt, and pH optima.
With hydrogen as the electron donor and Fe(III) oxide as the electron acceptor, FW-1a grew at between 65 and 100°C, with an optimum temperature at around 85 to 90°C (with a doubling time of about 14 to 15 h) (Fig. ). The doubling time at 65°C was about 35 h, while the doubling time at 100°C was around 109 h. No growth was detected at 63°C or above 100°C. At 85°C strain FW-1a grew in medium containing 0 to 0.75% (wt/vol) NaCl, but growth was slow (doubling time, 97 h) at the highest concentration of NaCl, and no growth was detected with 0.8% (wt/vol) NaCl in the medium (Fig. ).
Optimal growth temperature of isolate FW-1a during growth on H2 as the electron donor and poorly crystalline Fe(III) oxide as the electron acceptor. Doubling times were calculated from the slopes of the growth curves (not shown) at pH 7.0.
FIG. 4. Effect of NaCl concentration on growth of isolate FW-1a at 85°C with H2 as the electron donor and poorly crystalline Fe(III) oxide as the electron acceptor. Doubling times were calculated from the slopes of the growth curves (not shown) at pH (more ...)
The effect of pH on the growth of strain FW-1a was not investigated due to the inability of the isolate to utilize electron acceptors other than Fe(III), which is abiotically reduced with MES, PIPES, HEPES, and Tris buffers, particularly at the optimum growth temperature.
Sensitivity to antibiotics.
Growth of strain FW-1a was inhibited by chloramphenicol (100 μg ml−1), puromycin (100 μg ml−1), rifampin (100 μg ml−1), erythromycin (150 μg ml−1), kanamycin (200 μg ml−1), phosphomycin (200 μg ml−1), vancomycin-HCl (200 μg ml−1), and trimethoprim (300 μg ml−1). The growth of strain FW-1a was not inhibited, however, by cycloheximide (100 μg ml−1), kanamycin (100 μg ml−1), neomycin sulfate (100 μg ml−1), novobiocin (100 μg ml−1), tetracycline (150 μg ml−1), ampicillin (200 μg ml−1), penicillin G (200 μg ml−1), and streptomycin (200 μg ml−1).
16S rDNA sequence analysis.
Phylogenetic analysis of the 16S rDNA sequence of strain FW-1a (GenBank accession number AF411013
) indicated that its closest relatives are the environmental clones SRI-93 (33
) (99.0% similar), SRI-27 (33
) (96.2% similar), OPT4 (11
) (98.1% similar), OPT53 (96.5% similar), OPB45 (96.8% similar), and OPS7 (97.6% similar) (11
) (1,000 bases were considered in each case). The most closely related microorganisms available in culture are the sulfate-reducing microorganisms, T. commune
strain YSRA-1 (42
) (92% similar [1,200 bases considered]) and T. hveragerdense
) (94.5% similar [1,200 bases considered]), which are members of Thermodesulfobacteriaceae
FIG. 5. Phylogenetic tree generated by maximum-likelihood analysis of 16S rDNA sequences, showing the relationship of G. ferrireducens to previously described eubacterial sequences. Pyrococcus abyssi was used as the outgroup, and the branching order was identical (more ...) Evaluation of ability of T. commune and T. hveragerdense to reduce Fe(III).
T. commune could be routinely transferred in medium with hydrogen as the sole electron donor and Fe(III) as the sole electron acceptor (Fig. ). However, growth was much slower than that of strain FW-1a under the same conditions (doubling times of ≈ 20 versus ≈ 10 h). T. commune did not grow with lactate as the electron donor and Fe(III) as the electron acceptor. However, it grows with lactate and sulfate in the same medium. Multiple attempts to adapt T. hveragerdense to grow with poorly crystalline Fe(III) oxide as the electron acceptor with hydrogen and/or lactate as the electron donor were unsuccessful.
Growth of T. commune at 70°C with lactate as an electron donor and poorly crystalline Fe(III) oxide as an electron acceptor. The results are the means from duplicate cultures.
In two recent studies of terrestrial hot springs, 16S rDNA sequences closely related to strain FW-1a were found to be important components of the microbial community (8
). In both cases, it was inferred that the microorganisms with these sequences were sulfate-reducing microorganisms. Furthermore, in one instance (8
) it was also inferred that the microorganisms had a heterotrophic metabolism. These inferences were valid based on the information available at that time, because the closest known relatives for these sequences were T. commune
and T. hveragerdense
, two thermophilic sulfate-reducing microorganisms (34
). However, all of the 16S rDNA sequences recovered from these hydrothermal environments are more closely related to the 16S rDNA sequence of strain FW-1a than to that of either T. commune
or T. hveragerdense
. This, coupled with the finding that strain FW-1a cannot reduce sulfate and is able to use only hydrogen as the electron donor, suggests that the microorganisms which were predicted to be sulfate reducers may in fact not have the capacity for sulfate reduction or for heterotrophic metabolism. Rather they may have been autotrophic hydrogen-oxidizing Fe(III)-reducing microorganisms, with a physiology similar to that of strain FW-1a.
It was suggested that in Obsidian Pool, the environment from which strain FW-1a was isolated, a source of hydrogen for the microbial community might be the reduction of water with Fe(II) (8
). If so, this would produce not only hydrogen but also Fe(III), providing both the electron acceptor and the electron donor needed to support the growth of FW-1a and possibly the other organisms (8
) which were previously thought to be sulfate-reducing microorganisms. It is also expected that Fe(III) will be available in this and other hydrothermal environments, as Fe(II)-rich waters contact oxygen which will abiotically oxidize Fe(II) to Fe(III) with the precipitation of Fe(III) oxide (6
). Thus, the possibility that not only strain FW-1a but also the microorganisms associated with previously recovered 16S rDNA sequences from Obsidian Pool (8
) may be growing via Fe(III) reduction is consistent with the geochemistry of this environment.
These results demonstrate the limitations to inferring physiology and likely biogeochemical reactions in hydrothermal environments based on analysis of 16S rDNA sequences. Ideally, the biogeochemical reactions in such environments should be measured directly, and the activity of different microbial populations should be assessed by monitoring the expression of genes related to the physiology of interest. However, the ability to infer physiology from 16S rDNA sequences will continue to improve as more organisms are recovered in pure culture and their physiology is characterized. Although it has been asserted that it is difficult or impossible to isolate most of the organisms from hydrothermal environments (8
), the study reported here represents only the second (17
) attempt to isolate a microorganism from a hydrothermal environment with Fe(III) oxide as the electron acceptor. Given the widespread ability of hyperthermophilic microorganisms to reduce Fe(III) and the fact that some of these organisms, such as FW-1a, can use only Fe(III) oxide as an electron acceptor (17
), further attempts to recover more of the as-yet-uncultured organisms from hydrothermal environments with Fe(III) oxide as the electron acceptor seem warranted.
Comparison with Thermodesulfobacterium species.
The 16S rDNA sequence of strain FW-1a indicates that its closest known relatives are T. commune (92% similar) and T. hveragerdense (94.5% similar). Although analysis of its 16S rDNA sequence suggests that strain FW-1a is most closely related to Thermodesulfobacterium species, its metabolism is completely different. Unlike the Thermodesulfobacterium species, strain FW-1a is unable to use sulfate and thiosulfate as electron acceptors. Several attempts to grow strain FW-1a on a wide variety of commonly considered electron acceptors, including sulfate (10 to 20 mM), thiosulfate (10 mM), sulfite (2 to 4 mM), and S0 (20%, wt/vol), with H2 (as H2-CO2; 80:20%, vol/vol; 101 kPa), lactate (10 mM), pyruvate (10 mM), or a combination of H2-lactate, and H2-pyruvate as electron donors were unsuccessful. Strain FW-1a grows exclusively with poorly crystalline Fe(III) oxide as the electron acceptor. In contrast to T. commune and T. hveragerdense, which are heterotrophs, strain FW-1a is unable to grow with lactate and pyruvate as electron donors; it grows exclusively with hydrogen as the electron donor. The growth temperature ranges for T. commune and T. hveragerdense are between 45 and 85°C (with an optimum of 70°C) and between 55 and 74°C (with an optimum of between 70 and 74°C), respectively. Strain FW-1a, however, grows at between 65 and 100°C (optimum temperature, between 85 and 90°C). To our knowledge this is the highest optimum growth temperature for a bacterium.
On the basis of its unique metabolic properties, morphology, and 16S rDNA sequence, we conclude that strain FW-1a represents a new genus, and the name Geothermobacterium ferrireducens is proposed.
Description of Geothermobacterium gen. nov.
Geothermobacterium (Ge.o.thermo. bacterium. Gr. n. geo, the earth; Gr. n. thermos, heat; Gr. n. bakterion, a small rod; N.L. masc.n. Geothermobacterium, a small rod from hot earth). Cells are rod shaped, 0.5 by 1.0 to 1.2 μm, occurring singly and in pairs, highly motile (even at room temperature), by means of a monotrichous flagellum. Cell wall structure typical of a gram-negative bacterium. Strictly anaerobic chemoautotroph, which conserves energy to support growth, by coupling the oxidation of hydrogen to the reduction of poorly crystalline Fe(III) oxide. Grows exclusively with hydrogen as the sole electron donor and poorly crystalline Fe(III) oxide as the sole electron acceptor. The genus Geothermobacterium is in the family Thermodesulfobacteriaceae within the division δ-Proteobacteria. Habitat, hot springs (Obsidian Pool area) in Yellowstone National Park.
Description of Geothermobacterium ferrireducens sp. nov.
Geothermobacterium ferrireducens (fer.ri.re.du′cens. L.n. ferrum, iron; L. part. adj. reducens, converting to a different state; N.L. adj. ferrireducens, reducing iron). Gram-negative rods, 0.5 by 1.0 to 1.2 μm, occurring singly and in pairs, motile (even at room temperature) by means of a monotrichous flagellum. Strictly anaerobic autotroph, grows exclusively with hydrogen as the sole electron donor and poorly crystalline Fe(III) oxide as the sole electron acceptor. No growth with sulfate, thiosulfate, sulfite, sulfur, nitrate, fumarate, ferric citrate, or ferric pyrophosphate as electron acceptor with hydrogen, dl-lactate, pyruvate, formate, fumarate, yeast extract, peptone, amino acids, aromatic compounds, short- and long-chain fatty acids, or carbohydrates. Growth optimum between 85 and 90°C, at near-neutral pH (pH 6.8 to 7.0). No growth at 58 or 102°C. Can tolerate NaCl concentration of up to 0.75%, with an optimum at 0.0 to 0.05%. Growth inhibited by 0.8% NaCl. Growth also inhibited by chloramphenicol (100 μg ml−1), puromycin (100 μg ml−1), rifampin (100 μg ml−1), erythromycin (150 μg ml−1), kanamycin (200 μg ml−1), phosphomycin (200 μg ml−1), vancomycin-HCl (200 μg ml−1), and trimethoprim (300 μg ml−1). Isolated from hot sediment samples from Obsidian Pool, Yellowstone National Park (Wyoming, United States).
The strain has been deposited in the American Type Culture Collection (ATCC BAA-426).