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Thermophilic Fe(III)-Reducing Bacteria from the Deep Subsurface: The Evolutionary Implications

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Science  22 Aug 1997:
Vol. 277, Issue 5329, pp. 1106-1109
DOI: 10.1126/science.277.5329.1106

Abstract

Thermophilic (45° to 75°C) bacteria that reduce amorphous Fe(III)-oxyhydroxide to magnetic iron oxides have been discovered in two geologically and hydrologically isolated Cretaceous- and Triassic-age sedimentary basins in the deep (>860 meters below land surface) terrestrial subsurface. Molecular analyses based on 16S ribosomal RNA (rRNA) gene sequences revealed that some of these bacteria represent an unrecognized phylogenetic group of dissimilatory Fe(III)-reducing bacteria. This discovery adds another dimension to the study of microbial Fe(III) reduction and biogenic magnetism. It also provides examples for understanding the history of Fe(III)-reducing microorganisms and for assessing possible roles of such microorganisms on hot primitive planets.

Dissimilatory Fe(III) reduction is proposed to be an early form of microbial respiration (1), and it may have influenced the geochemistry and the paleomagnetism of the Archaean Earth (2). Microbial Fe(III) reduction has been observed primarily in low-temperature environments that have been extensively influenced by modern surface processes (3). Previous studies on dissimilatory Fe(III)-reducing bacteria have been focused on mesophilic microorganisms within Proteobacteria (4), which are located distant from the deep branches on the phylogenetic tree (5). The paucity of information on thermophilic dissimilatory Fe(III)-reducing microorganisms (6) is striking in that thermophilic species are frequently found in many other groups of microorganisms such as methanogens, sulfate-reducing bacteria, and acetogens (7). This lack of information presents a difficulty in explaining microbial Fe(III) reduction on primitive Earth, which was reputedly warmer than Earth is now (8). Although the geological evidence for microbial Fe(III) reduction in Archaean Earth is recognized (2), the early evolution of microbial Fe(III)-reducing microorganisms on Earth has not yet been delineated in biological studies.

We studied two geologically and hydrologically separated sedimentary basins, the Triassic-age Taylorsville Basin in Virginia and the Cretaceous-age Piceance Basin in Colorado. Geological and hydrological studies have shown that the deep portions of these basins have been isolated from surface processes for millions of years (9). The current temperature at the sampling depths (2652 to 2798 m below land surface) in the Taylorsville Basin ranges from 65° to 85°C, and the pore fluid pressure ranges from 30 to 35 MPa (9). The current temperatures at the three sampling depths (856 to 862 m, 1996 m, and 2090 to 2096 m below land surface) in the Piceance Basin were estimated to be 42°, 81°, and 85°C, respectively (9). Core samples were collected from tightly cemented, low-porosity arkosic silt stone and from organic-rich, laminated shale in the Triassic Taylorsville Basin, as well as from three geologic units in the Piceance Basin: one in the Tertiary Wasatch Formation and two in the Upper Cretaceous Williams Fork Formation (9). Drilling fluids were sampled from the fluid-receiving tank at different times during the drilling operation in the Piceance Basin. The sedimentary rock and drilling fluid samples were processed anaerobically and shipped on ice overnight to our laboratory for initiation of microbial incubations (10).

Enrichment cultures for thermophilic Fe(III)-reducing microorganisms were prepared with mineral media containing amorphous Fe(III) oxyhydroxide as an electron acceptor, and hydrogen (H2) or short-chain fatty acids as the electron donors (10). Previous studies had shown that these substances are potentially available in deep terrestrial subsurface environments (11) and thus may be suitable substrates for deep-subsurface Fe(III)-reducing microorganisms. Within 1 to 2 weeks of inoculation with subsurface samples or enrichment cultures, black magnetic precipitates formed in incubation mixtures held at 60°C (Table 1). The Taylorsville Basin enrichment cultures used the fatty acids formate, acetate, and lactate as electron donors for Fe(III) reduction and magnetic mineral formation. The Piceance Basin enrichment cultures utilized H2 and pyruvate as electron donors in addition to the above three fatty acids.

Table 1

Fe(III)-reducing and magnetic iron oxide-forming activities at 60°C in materials obtained from deep terrestrial subsurface environments (21).

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The formation of magnetic products was observed at temperatures between 45° and 75°C (Fig. 1) and at a salinity of 0 to 4% NaCl. In various uninoculated controls, significant abiotic Fe(III) reduction and magnetic product formation was not observed. Exposure of the thermophilic Fe(III)-reducing enrichment cultures to oxygen prevented the Fe(III) reduction, an indication of the anaerobic nature of the Fe(III)-reducing microorganisms. Furthermore, the thermophilic formation of magnetic iron oxides was prevented by the ferric reductase inhibitor p -chloromercuriphenyl sulfonate (pCMPS), the hydrogenase inhibitor quinacrine dihydrochloride, the protonophore carbonyl cyanide m -chlorophenylhydrazone (CCCP), and the electron transport inhibitors 2- n -heptyl-4-hydroxyquinoline- N -oxide (HOQNO) and dicumarol. The susceptibility of the thermophilic Fe(III)-reducing cultures to these metabolic inhibitors is consistent with a dissimilatory mechanism for Fe(III) reduction, which has been shown for mesophilic Fe(III)-reducing bacteria (12). The sulfate reduction inhibitor molybdate did not stop the Fe(III) reduction but shifted formation of reduced minerals from magnetite to siderite.

Figure 1

Temperature profile of the microbial Fe(III)-reducing activity of the H2-utilizing enrichment cultures from the Piceance Basin. After incubation for 5 days at the indicated temperatures, brown non-magnetic amorphous Fe(III) oxyhydroxides in the incubation tubes at 45° to 75°C became black and magnetic, and were attracted to the magnetic stirring bars taped to the outsides of incubation tubes during photographing. This caused the sloping of the precipitates toward the magnet side of the tube. Precipitates in incubation mixtures at 25°, 35°, and 83°C remained brown and non-magnetic at the end of incubation.

Magnetic precipitates (10 to 300 nm in size) were deposited extracellularly in association with microorganisms of different morphologies (Fig. 2A). The magnetic minerals were identified as a mixture of magnetite and maghemite crystallites on the basis of the lattice spacings revealed by selected area electron diffraction (Fig. 2B) and by high-resolution electron microscopy. Magnetite was the predominant phase. The existence of these two forms of magnetic iron oxides in the magnetic precipitates was also confirmed by x-ray diffraction and Mössbauer spectroscopic analysis.

Figure 2

Morphologies of bacteria and magnetic minerals and a selected area electron diffraction pattern of magnetic crystals in drilling fluid cultures from the Piceance Basin that were grown on H2-CO2-Fe(III) oxyhydroxide at 60°C. (A) SEM image of samples prepared anaerobically with minimum disturbance (22). (B) Selected area electron diffraction pattern of magnetic crystals. The presence of magnetite (Fe3O4) and maghemite (γ-Fe2O3) was indicated by black dots in the rings with characteristic d-spacings (23).

To further characterize the thermophilic Fe(III)-reducing enrichment cultures, we undertook a molecular analysis (13) of 16S rRNA genes. Community DNAs were extracted from the Fe(III)-reducing enrichment cultures incubated with nonfermentable substrates (H2 and acetate) and from those with a fermentable substrate (pyruvate). The 16S rRNA genes were amplified and cloned into plasmid vectors, and the restriction fragment length polymorphism (RFLP) patterns of the clones were analyzed (14). Three dominant RFLP patterns were observed in various enrichment cultures (Table 2). RFLP patterns 1 and 2 were shared by enrichment cultures grown with nonfermentable and fermentable substrates, and the two patterns accounted for 43.6 to 54.6% of the clones. RFLP pattern 3 was observed in clones of H2- and pyruvate-utilizing cultures but not in the acetate-utilizing culture.

Table 2

The dominant RFLP patterns and 16S rRNA gene sequence similarity of the 16S rDNA clones in thermophilic Fe(III)-reducing enrichment cultures grown with different electron donors. The numbers in parentheses are percentages of the total clones.

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The 16S rRNA gene sequences of representative clones bearing the three dominant RFLP patterns were analyzed (15). All of the sequences were affiliated with Thermoanaerobacter in the Gram-positive bacteria (Fig. 3) and (Table 2). The clones of RFLP patterns 1 and 3 were closely related to the Thermoanaerobacter species, bearing ∼98% similarity (Table 2). The clones of the RFLP pattern 2, however, were most closely related to T. ethanolicus but had a 13% dissimilarity (Table 2). Such a degree of divergence in the 16S rRNA gene sequences suggests that the clones with RFLP pattern 2 may represent a previously unrecognized genus or family of bacteria (16).

Figure 3

The phylogenetic relation between the newly discovered thermophilic Fe(III)-reducing bacteria (bold Roman letters connected with a thick line) and other examples of Fe(III)-reducing bacteria (italic letters connected with thick lines). This relation is deduced from 16S rRNA gene sequencesof the bacteria. The letters H, A, and P indicate the hydrogen-, acetate-, and pyruvate-utilizing cultures, respectively. The numbers after the hyphens specify the clones (Table 2). The scale bar represents 0.05 nucleotide change per position.

From the analysis of the RFLP patterns in various cultures, we hypothesize that the microorganisms directly responsible for the dissimilatory Fe(III) reduction are those bearing RFLP pattern 2. This pattern was shared by Fe(III)-reducing microbial populations grown on three different electron donors. RFLP pattern 1 was also common to enrichment cultures grown on all three substrates. However, isolated bacteria such as TOR-39 (6) whose 16S rRNA sequences are highly similar to pattern 1 clones were unable to catalyze nonfermentative dissimilatory Fe(III) reduction. Bacteria bearing the RFLP pattern 3 might play some role in the Fe(III)-reduction in the hydrogen- and pyruvate-grown cultures. However, bacteria with this RFLP pattern are unlikely candidates for producing the observed dissimilatory Fe(III) reduction; this RFLP pattern was absent from acetate-grown cultures and the clones with this pattern were highly similar in 16S rRNA gene sequences to those of fermentative Thermoanaerobacter species. However, further studies are necessary to delineate the respective roles of the various bacteria in thermophilic Fe(III) reduction.

The discovery of these forms of thermophilic Fe(III)-reducing bacteria described above has broad implications regarding the evolution of Fe(III)-reducing microorganisms and biogenic contributions to paleomagnetism. A wide phylogenetic distribution of Fe(III)-reducing bacteria, including mesophilic and thermophilic species, could reflect the early evolution of Fe(III) respiration because early evolved characteristics tend to be conserved among widely distributed descendants (17). Furthermore, the existence of thermophilic Fe(III)-reducing bacteria in geologically isolated, millions-of-years-old thermal regimes suggests that thermophily may be an ancestral feature associated with Fe(III)-reduction. The ability of microorganisms to produce magnetic iron oxides from amorphous Fe(III) oxyhydroxide at high temperatures expands the biotope boundary for microbial Fe(III)-reducing activities. This capability also supports the theory that biogenic magnetism may have occurred in Archaean banded iron formations that formed at high temperatures (55° to 76°C) (18).

The discovery of thermophilic Fe(III)-reducing bacteria on Earth may also have implications for studying exobiology on, for example, Mars: the recent finding of putative biogenic magnetite in an ancient martian meteorite (19) raises the possibility that Fe(III)-reducing microorganisms evolved not only on Earth but also elsewhere. Early Mars and early Earth might have exhibited similar hydrothermal activities compatible with life (20). Thus, thermophily may have been a common feature of the early evolved forms of Fe(III)-reducing bacteria.

  • * Present address: Department of Microbiology and Immunology, Allegheny University of the Health Sciences, Philadelphia, PA 19129, USA.

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