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Phylogeny and biogeochemistry of chemotrophic microorganisms

Of the numerous geothermal features distributed throughout Yellowstone National Park (YNP), many are very acidic (pH<3) due to the oxidation of S in the subsurface hydrothermal system and or contact of geothermal water with buried solfataras (Fournier et al. 1992; Xu et al. 1998). Many of these acidic geothermal sites exhibit similar chemical characteristics to nonthermal acid-mine drainage generated from pyrite oxidation (Nordstrom 1982; LeBlanc et al. 1997; Morin et al. 2004); thus their thorough study and characterization has application beyond the study of microbial ecology in extreme habitats. Acidic geothermal springs in YNP represent unique environments, harboring microorganisms with physiologies specific to such habitats and that have yet to be discovered and characterized. The low pH of these environments precludes colonization by major groups of photosynthetic bacteria such as cyanobacteria, although at moderate thermophilic temperatures (40-55°C), acid-tolerant eukaryotic algae such as Cyanidium, Galdieria, and Cyanidioschyzon find near exclusive niche opportunities at pH values below 4 (Albertano et al. 2000). Consequently, primary production in many acidic thermophilic habitats is likely dominated by chemolithotrophic prokaryotes that obtain energy from the oxidation of inorganic constituents. Many acidic geothermal features also contain what would normally be considered toxic levels of various trace elements (Phelps and Buseck 1980; Stauffer et al. 1980; Langner et al. 2001; Ball et al. 2002)—including mercury (Hg), arsenic (As), boron (B), and antimony (Sb)—suggesting that many organisms adapted to such environments possess resistance mechanisms, or perhaps utilize these constituents for metabolic functions (Silver 1996; Rosen 2002; Oremland and Stolz 2003). These extreme habitats with unique chemical signatures may be analogous to early earth or non-earth environments, and serve as models for studying functional relationships among microorganisms and biogeochemical cycling (Newman and Banfield 2002).

Acid-sulfate-chloride (ASC) springs in Yellowstone are thought to arise from the mixing of chloride water with acid-sulfate water, where the ultimate source of sulfate may come from the oxidation of reduced forms of S from buried solfataras or the hydrolysis of magmatic SO₂(g) noted in Fournier et al. 1992, and Xu et al. 1998. A common ASC spring type in Norris Basin exhibits chemical signatures dominated by mM levels of Na⁺, Cl^-, SO₄^2-, and H⁺ (pH ~3), along with µM levels of Fe(II), As(III), and NH₄⁺. Ratios of Cl^-/SO₄^2- can vary dramatically in geothermal features throughout Norris Basin; however, the springs discussed in this review exhibit consistent Cl^-/SO₄^2- molar ratios of ~10-12 (Langner et al. 2001). Furthermore, the concentrations of dissolved gases such as H₂S, H₂, CO₂, and CH₄ in the source waters of these springs are significantly oversaturated with respect to earth surface atmospheric conditions. Consequently, the source waters contain a suite of reduced chemical species that may serve as energy sources for diverse and novel microbial populations colonizing the outflow channels. As we will highlight below, geochemistry and temperature co-vary in these outflow channels as near super-imposable gradients originating from the spring source, and together act to establish niche opportunities for suitably adapted microorganisms.

During the last four years, one of our primary goals has been to define functional relationships among geochemical processes and the microorganisms that inhabit ASC springs of Norris Basin, YNP. These sites are the primary subject of a National Science Foundation (NSF) Microbial Observatory project focused on the discovery and characterization of chemolithoautotrophic, thermophilic microorganisms. Our approach has combined thorough geochemical analysis of aqueous and solid phases with molecular investigations that thus far have targeted the 16S rRNA gene. Our studies have revealed microbial communities that are suitably complex, yet simple enough to offer a reasonable opportunity for understanding the role of, and possible interactions among, specific microorganisms. The fundamental hypothesis of our work is that the significant phylogenetic and functional diversity in these geothermal systems is defined by geochemical and temperature regimes throughout the outflow channels. The data presented below provide a current summary of our understanding of the geomicrobiology of ASC Springs in Yellowstone National Park.

Photographic tour of the ASC springs discussed in this review, scaling from a panorama of Norris Geyser Basin to the micromorphology of microorganisms found in these springs. A. One Hundred Springs Plain, looking north from Ragged Hills. B&C. Source and outflow channels of Dragon Spring showing prominent solid phase zonation, including the yellow S and hydrous ferric oxide (HFO) depositional zones and green bands of the eukaryotic algae Galdieria (temperature limit ca. 50 °C). D. Scanning electron micrograph (SEM) of yellow streamers in C reveal filamentous organisms associated with rhombohedral S. E. SEM of filamentous organisms present in Fe mats containing As-rich HFO. F. Terraced Fe mats of Beowulf Spring adjacent to eukaryotic acid-tolerant algae (presumed Galdieria) with an observed upper temperature limit of ~ 51 °C . G. Optical microscopic image of an HFO mat thin section from Beowulf Spring shows banding patterns of Fe deposition parallel to flow direction. H-K. SEM and transmission electron micrographs of HFO mats from Beowulf Spring show biomineralization of As(V)-HFO phases intimately associated with microbial cells.

Photo Gallery

S deposition zone in Dragon Spring. Temperatures here range from 72-60°C
Elemental S crystals from the zone of elemental S deposition in Dragon Spring.	Closeup of Elemental S rhombs. From Dragon	Close up of Microbial filaments coated with As-rich Fe oxide phases from Dragon Spring.
Fe deposition zone in Dragon Spring Temperatures range from 60-50 C
Microbial filaments coated with Fe oxide phase From Dragon Springs.	Examples of the acid chloride spring environment at Succession Spring in Norris Geyser Basin.
Beowulf Spring in Norris Geyser Basin.
Microbial filaments showing Fe oxide sheaths. From Beowulf Spring Fe mats in Norris Geyser Basin.

Publications

Biomineralization of As(V)-hydrous ferric oxyhydroxide in microbial mats of an acid-sulfate-chloride
geothermal spring, Yellowstone National Park
W.P Inskeep, B.C. Bostick, G. Harrison, R. E. Macur and S. Fendorf

Abstract—Acid-sulfate-chloride (pH_3) geothermal springs in Yellowstone National Park (YNP) often contain Fe(II), As(III), and S(-II) at discharge, providing several electron donors for chemolithotrophic metabolism. The microbial populations inhabiting these environments are inextricably linked with geochemical processes controlling the behavior of As and Fe. Consequently, the objectives of the current study were to (i) characterize Fe-rich microbial mats of an ASC thermal spring, (ii) evaluate the composition and structure of As-rich hydrous ferric oxides (HFO) associated with these mats, and (iii) identify microorganisms that are potentially responsible for mat formation via the oxidation of Fe(II) and or As(III). Aqueous and solid phase mat samples obtained from a spring in Norris Basin, YNP (YNP Thermal Inventory NHSP35) were analyzed using a complement of chemical, microscopic and spectroscopic techniques. In addition, molecular analysis (16S rDNA) was used to identify potentially dominant microbial populations within different mat locations.
The biomineralization of As-rich HFO occurs in the presence of nearly equimolar aqueous As(III) and As(V) (_12 _M), and _ 48 _M Fe(II), forming sheaths external to microbial cell walls. These solid phases were found to be poorly ordered nanocrystalline HFO containing mole ratios of As(V):Fe(III) of 0.62 _ 0.02. The bonding environment of As(V) and Fe(III) is consistent with adsorption of arsenate on edge and corner positions of Fe(III)-OH octahedra. Numerous archaeal and bacterial sequences were identified (with no closely related cultured relatives), along with several 16S sequences that are closely related to Acidimicrobium, Thiomonas, Metallosphaera and Marinithermus isolates. Several of these cultured relatives have been implicated in Fe(II) and or As(III) oxidation in other low pH, high Fe, and high As environments (e.g. acid-mine drainage). The unique composition and morphologies of the biomineralized phases may be useful as modern-day analogs for identifying microbial life in past Fe-As rich environments.
Copyright © 2004Elsevier Ltd

Fig. 1. Beowulf Spring located in Norris Geyser Basin, Yellowstone National Park, showing the east geothermal source (0 m) and aqueous sampling points Ba (3 m) and Bb (6 m) (A), and a view down gradient from (A) showing Fe microbial mats at sampling sites B1L (9 m) and B1D (9 m) (B). A 50–51°C isotherm is defined by the interface of Fe mats and an acid-tolerant algae, likely Cyanidium caldarium. Photograph taken in March 2002.

Fig. 2. Scanning electron micrographs (SEM) of Fe mats from Beowulf Spring at site B3 (bottom 2 mm of mat) (A, B) and site B2 (top 2 mm of mat) (C, D). Close-ups of the filamentous As-rich Fe sheaths (molar As: Fe =0.62 +-0.01) are shown in B and D, with overall diameters approaching 3 µ. Given the high Fe and As contents of these samples, it was not possible to positively confirm the composition of “web-like” material with fibers as thin as 0.04 µ shown in B.

Fig. 3. Transmission electron micrographs (TEM) of unstained cells present in Fe mats of Beowulf Spring from site B2. (A) TEM image of cell surrounded by extracellular, spheroidal solid phase. The larger spheres shown here have diameters ranging from 0.1 to 0.2 µm. The corresponding ESI images (A) were generated using a three window power law (15 eV windows) across the Fe and As edges, and confirm the importance of As coprecipitation during the mineralization of HFO. (B) Cross section of an Fe-As encrusted filament showing a
0.45 µm diameter cell surrounded by an Fe-As solid phase sheath with an average thickness of~0.5 µm. The corresponding PEELS spectra of extracellular solid phase (B) reveal prominent Fe-L_2,3 and As-L_2,3 edges.

Fig. 4. Representative X-ray diffraction patterns of Beowulf Spring samples from site B2, obtained using both conventional (B2) and synchrotron-based XRD (B2 S-XRD). The patterns are compared with diffraction patterns of arsenate-containing ferrihydrite (FeAsOH), 2-line ferrihydrite, and well crystalline scorodite. The reference patterns are based on published diffraction patterns of scorodite (Hawthorne et al., 1976), and amorphous ferric arsenate and ferrihydrite (Carlson et al., 2002).

Fig. 5. Arsenic XANES of representative As-rich Fe oxyhydroxide solid phases sampled from Beowulf Spring. The absorption edge of all Beowulf samples was near 11874 eV, indicative of arsenate [As(V)]. 3148 W. P. Inskeep et al.

Fig. 6. Arsenic K-edge EXAFS spectra of As-rich Fe oxyhydroxides from Beowulf Spring. Both the k-weighted x(k) functions (A) and their Fourier-transforms (B) are plotted. The solid lines are the experimental data, and the dotted lines are fits. 3149 Biomineralization of As(V)-hydrous ferric oxydroxides

Fig. 7. Iron K-edge EXAFS of As-rich Fe oxyhydroxides from Beowulf Spring. Both the k-weighted x(k) functions (A) and their Fourier-transforms (uncorrected for phase shift) (B) are plotted. The solid lines are the experimental data, and the dotted lines are fits.

Fig. 8. Number of Fe atoms in a cluster versus As:Fe ratio assuming complete site saturation with arsenate. The square symbols indicate site
saturation based on crystallographic limitations (the number of bridging hydroxyls present on the surface), where the error associated with
these values is based on possible variation in crystal morphology. The circle symbols assume spherical particles and an adsorption density of
3.5 sites/nm², similar to that reported by Dzombak and Morel (1990). 3150 W. P. Inskeep et al.

Fig. 9. Microbial community 16S rDNA fingerprints generated using denaturing gradient gel electrophoresis (40–70% gradient) of partial 16S rRNA genes amplified from Beowulf Spring using universal archaeal (A) and bacterial (B) primer sets. Bands that were successfully purified and sequenced are labeled with band designation, corresponding GenBank accession number, nearest GenBank neighbor, and % similarity of band sequence to nearest neighbor sequence. Dotted lines indicate comigrating bands.

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