ORIGINAL RESEARCH
published: 15 February 2016
doi: 10.3389/fmicb.2016.00025
Assembly and Succession of Iron
Oxide Microbial Mat Communities in
Acidic Geothermal Springs
Edited by:
Tanja Bosak,
Massachusetts Institute of
Technology, USA
Reviewed by:
Susan Childers,
Colby College, USA
William D. Leavitt,
Washington University in St. Louis,
USA
*Correspondence:
William P. Inskeep
binskeep@montana.edu
†
Present Address:
Jacob P. Beam,
Bigelow Laboratory for Ocean
Sciences, East Boothbay, USA
Mark A. Kozubal,
Sustainable Bioproducts LLC.,
Bozeman, USA
Ryan deM. Jennings,
Mercer University, Macon, USA
Specialty section:
This article was submitted to
Microbiological Chemistry and
Geomicrobiology,
a section of the journal
Frontiers in Microbiology
Received: 07 October 2015
Accepted: 11 January 2016
Published: 15 February 2016
Citation:
Beam JP, Bernstein HC, Jay ZJ,
Kozubal MA, Jennings Rd, Tringe SG
and Inskeep WP (2016) Assembly and
Succession of Iron Oxide Microbial
Mat Communities in Acidic
Geothermal Springs.
Front. Microbiol. 7:25.
doi: 10.3389/fmicb.2016.00025
Jacob P. Beam 1 † , Hans C. Bernstein 2, 3 , Zackary J. Jay 1, 2 , Mark A. Kozubal 1 † ,
Ryan deM. Jennings 1 † , Susannah G. Tringe 4 and William P. Inskeep 1*
1
Department of Land Resources and Environmental Sciences, Thermal Biology Institute, Montana State University,
Bozeman, MT, USA, 2 Department of Chemical and Biological Engineering, Center for Biofilm Engineering, Montana State
University, Bozeman, MT, USA, 3 Biodetection Science and Biological Science Division, Pacific Northwest National
Laboratory, Richland, WA, USA, 4 United States Department of Energy Joint Genome Institute, Walnut Creek, CA, USA
Biomineralized ferric oxide microbial mats are ubiquitous features on Earth, are common
in hot springs of Yellowstone National Park (YNP, WY, USA), and form due to direct
interaction between microbial and physicochemical processes. The overall goal of
this study was to determine the contribution of different community members to the
assembly and succession of acidic high-temperature Fe(III)-oxide mat ecosystems.
Spatial and temporal changes in Fe(III)-oxide accretion and the abundance of relevant
community members were monitored over 70 days using sterile glass microscope
slides incubated in the outflow channels of two acidic geothermal springs (pH = 3–3.5;
temperature = 68–75◦ C) in YNP. Hydrogenobaculum spp. were the most abundant
taxon identified during early successional stages (4–40 days), and have been shown
to oxidize arsenite, sulfide, and hydrogen coupled to oxygen reduction. Iron-oxidizing
populations of Metallosphaera yellowstonensis were detected within 4 days, and reached
steady-state levels within 14–30 days, corresponding to visible Fe(III)-oxide accretion.
Heterotrophic archaea colonized near 30 days, and emerged as the dominant functional
guild after 70 days and in mature Fe(III)-oxide mats (1–2 cm thick). First-order rate
constants of Fe(III)-oxide accretion ranged from 0.046 to 0.05 day−1 , and in situ
microelectrode measurements showed that the oxidation of Fe(II) is limited by the
diffusion of O2 into the Fe(III)-oxide mat. The formation of microterracettes also implicated
O2 as a major variable controlling microbial growth and subsequent mat morphology. The
assembly and succession of Fe(III)-oxide mat communities follows a repeatable pattern
of colonization by lithoautotrophic organisms, and the subsequent growth of diverse
organoheterotrophs. The unique geochemical signatures and micromorphology of extant
biomineralized Fe(III)-oxide mats are also useful for understanding other Fe(II)-oxidizing
systems.
Keywords: Hydrogenobaculum, Metallosphaera, lithoautotroph, organoheterotroph, archaea, biomineralization,
oxygen
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Succession of Thermoacidic Iron Oxide Mats
INTRODUCTION
Aquificales) are versatile chemolithoautotrophs that inhabit
sulfur and iron-dominated acidic hot springs, and have been
shown to utilize several different electron donors (e.g., As(III),
H2 , and H2 S) coupled with the reduction of O2 to drive
the fixation of CO2 via the reductive TCA cycle (DonahoeChristiansen et al., 2004; D’Imperio et al., 2007; Hamamura
et al., 2009; Romano et al., 2013; Takacs-Vesbach et al., 2013).
Hydrogenobaculum spp. and Metallosphaera yellowstonensis are
both implicated as early colonizing populations (Macur et al.,
2004) and carbon dioxide (CO2 ) fixing organisms (Jennings
et al., 2014) in high-temperature Fe(III)-oxide mats of YNP;
however, the role of these lithoautotrophs in early stages of
Fe(III)-oxide mat formation has not been elucidated.
Prior work using stable carbon isotopes (13 C) has shown
that CO2 -derived microbial C contributes a minimum of 40%
of the total biomass C in mature Fe(III)-oxide mats (Jennings
et al., 2014), which provides a significant organic C source
for organoheterotrophic thermoacidophiles. Furthermore,
Hydrogenobaculum spp. have also been shown to uptake
radiolabeled bicarbonate (14 CO2 ) in high sulfide zones of
the same acidic geothermal springs (Boyd et al., 2009). Thus,
the growth and assembly of early-colonizing lithoautotrophic
populations (i.e., Hydrogenobaculum spp. and Metallosphaera
yellowstonensis) will likely influence the subsequent succession
and activity of organoheterotrophic organisms. Consequently,
the primary objectives of the current study were to (i) determine
the spatiotemporal dynamics of key community members
involved in Fe(III)-oxide mat assembly in acidic geothermal
springs of Norris Geyser Basin (YNP), (ii) quantify the amount
of Fe(III)-oxides accreted and oxygen consumed in situ as a
function of time, (iii) monitor temporal changes in community
composition using 16S rRNA gene sequencing, and (iv)
integrate laboratory measurements and field observations across
different scales to develop a conceptual model of Fe(III)-oxide
assembly and succession. A combination of geochemical,
microscopic, and molecular methods were employed to reveal
that Hydrogenobaculum spp. exhibit rapid growth rates in situ
and are the first colonizers in high-temperature acidic Fe mats,
followed by the accretion of Fe(III)-oxides due to the rise of
Fe(II)-oxidizing populations of Metallosphaera yellowstonensis.
Other heterotrophic archaea, which include several novel
groups and additional Crenarchaeota (e.g., Sulfolobales,
Desulfurococcales) colonize at later stages of mat development
and likely utilize organic C produced by lithoautotrophs. Distinct
stages of mat development were associated with specific micromorphological features that provide a basis for understanding
the assembly and succession of thermoacidic Fe(III)-oxide
microbial mats.
Microbial mat communities are ubiquitous geobiological features
(e.g., stromatolites; Riding, 1999) in contemporary and past
environments on Earth, are often stratified due to gradients
in key geochemical constituents (e.g., oxygen; de Beer et al.,
1994; Bernstein et al., 2013), and often leave biological
signatures (biomarkers) preserved in the rock record (e.g., lipids;
Peters and Moldowan, 1993). Biogeochemical stratification of
microbial mats may produce distinct morphological features
that can be preserved (e.g., iron formations and marine
carbonates), and provide insights into past geochemical and
hydrodynamic conditions (e.g., Kappler and Straub, 2005; Fouke,
2011). Microbial mat communities often produce extracellular
polymeric substances (EPS), which may serve as sites for mineral
nucleation and growth (e.g., Mann, 1988; Kandianis et al.,
2008). Understanding mechanisms of assembly and succession
in modern-day mat communities provides clues regarding past
environmental conditions and biogeophysical controls that lead
to the biomineralization of specific solid phases (Reid et al.,
2000; Fouke, 2011). Moreover, modern microbial mat ecosystems
can be observed in real-time and monitored over spatial and
temporal scales to elucidate mechanisms of formation under
various geochemical and hydrologic conditions.
Iron is the fourth most abundant element in the Earth’s crust
and is an essential cofactor in numerous proteins across all
domains of life (e.g., iron-sulfur proteins). Microorganisms may
also gain energy via the oxidation of Fe(II) to Fe(III) under
aerobic or anaerobic conditions, which usually results in the
precipitation of insoluble solid-phase Fe(III)-oxides (Konhauser,
1998, 2006; Kappler and Straub, 2005; Ehrlich and Newman,
2009; Emerson, 2012). The reduction of Fe(III)-oxides to Fe(II)
can be coupled with the oxidation of inorganic (e.g., hydrogen)
or organic (e.g., acetate) compounds by other microorganisms,
completing the Fe cycle. Uncatalyzed abiotic rates of Fe(II)oxidation are extremely slow at pH values less than 4 (Singer
and Stumm, 1970; Kappler and Straub, 2005); consequently,
the deposition of Fe(III)-oxides under acidic conditions can
often be attributed to the activity of Fe(II)-oxidizing microbial
populations. Low pH (<4) acid-mine drainage (Denef et al.,
2010) and acidic geothermal springs in YNP (Langner et al., 2001;
Kozubal et al., 2012) exhibit significant amounts of Fe(III)-oxide
biomineralization.
High-temperature (65–80◦ C), acidic (pH = 2–3.5) Fe(III)oxide microbial mats in Yellowstone National Park (YNP)
represent hydrodynamically-controlled model systems for
studying microbial interactions and biogeochemical processes
in situ (Kozubal et al., 2008, 2012; Inskeep et al., 2010,
2013). Metallosphaera yellowstonensis (order Sulfolobales,
Crenarchaeota) is the primary chemolithoautotroph responsible
for Fe(II)-oxidation in these systems, which results in the
precipitation of copious amounts of Fe(III)-oxides and/or
jarosite, depending on spring geochemistry (Kozubal et al.,
2008, 2012). Protein-coding genes responsible for Fe(II)oxidation in M. yellowstonensis (e.g., foxC) have been elucidated,
and are highly expressed in Fe(III)-oxide microbial mats of
YNP (Kozubal et al., 2011). Hydrogenobaculum spp. (order
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MATERIALS AND METHODS
Site Descriptions
Two acidic geothermal springs in Norris Geyser Basin (NGB),
YNP were chosen for this study based on long-term microbial
and geochemical data obtained over the last 10–15 years
(Jackson et al., 2001; Langner et al., 2001; Inskeep et al.,
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Succession of Thermoacidic Iron Oxide Mats
2004, 2005, 2010, 2013; Macur et al., 2004; Inskeep and
McDermott, 2005; Ackerman, 2006; Kozubal et al., 2008, 2012,
2013; Beam et al., 2014; Jay et al., 2014; Jennings et al., 2014),
which provides an excellent biological and physicochemical
context for integrating results from the current study. Discharge
source waters of “Beowulf ” Spring (Thermal Inventory Number
NHSP035; Lat/Lon = 44.731519, −110.711357) and an unnamed
spring in One Hundred Spring Plain (OSP) (referred to here
and in prior studies as OSP; Thermal Inventory Number
NHSP115; Lat/Lon = 44.733044, −110.709012) exhibit low pH
values (3–3.5), high temperatures (80–85◦ C) (Figure 1; Table 1),
and contain reduced chemical species and high concentrations
of dissolved inorganic carbon (DIC) formed by water-rock
interactions occurring deep in the hydrothermal reservoir
(Fournier, 1989). The concentrations of Fe(II), As(III), H2 S, H2 ,
and CH4 are significant and can potentially serve as electron
donors for lithotrophic microbial populations (Macur et al., 2004;
Inskeep et al., 2005; Kozubal et al., 2012). There is also an
abundance of potential electron acceptors, which include O2 ,
−
SO2−
4 , NO3 , Fe(III), and As(V) (Inskeep et al., 2005).
Iron Accretion Rates
Iron-oxide accretion rates were measured in situ by inserting acid
washed (2% HCl) and autoclaved borosilicate glass microscope
slides (2.5 × 7.5 cm) into the main outflow channels of OSP
and Beowulf Springs (Figure 1). Glass slides were chosen for
the growth substrate for multiple reasons: (1) they mimic the
native siliceous sinter that these iron oxide mats grow on, (2)
they are easy to clean and sterilize, (3) glass is inexpensive,
and (4) they are easy to deploy and sample in the hot spring
study sites. Slides were inserted and removed at various time
points during four field seasons (2010–2013) with the most
extensive sampling occurring in 2012–2013 (Table S1). The
number of slides inserted at any time point (∼6–8) was limited
to a small area in the outflow channel that exhibited the desired
physicochemical environment (temperature range = 70–75◦ C in
FIGURE 1 | Photographs of One Hundred Spring Plain (A) and Beowulf (B) Springs located in Norris Geyser Basin, Yellowstone National Park, WY, USA
(scale bar = 30 cm). The black arrows represent the approximate location of slide placement in the primary outflow channel. Glass slides incubated for 70 days in
OSP (C) and Beowulf (D) Springs (scale bar = 1 cm). The dashed white arrows represent direction of flow.
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TABLE 1 | Physical and geochemical parameters measured over four field seasons corresponding to in situ slide incubations.
Spring (Position)
Temperature (◦ C)
pH
Channel velocity
(cm s−1 )
Reynolds
number
O2 (aq)
Fe (TS)
As (TS)
CO2 (aq)
DOC
µM
H2 (aq)
nM
One Hundred
Spring Plain (B)
72.8 (1.4)
3.5 (0.1)
2–5
1.5 • 103
33 (11)
25 (7)
24 (6)
100 (40)
109 (55)
39 (40)
Beowulf (D)
67.6 (1.8)
2.9 (0.1)
20–30
1.4 • 104
44 (6)
30 (3)
28 (4)
200 (180)
52 (12)
13 (7)
Numbers in parentheses are equal to ± 1 standard deviation from the mean.
Reynolds Number = inertial forces/viscous forces, ρVD/µ: ρ = density of fluid (kg/m−3 ), V = fluid velocity (ms−1 ), D = channel depth (m), and µ = dynamic fluid viscosity [kg (ms)−1 ].
DOC, dissolved organic carbon.
OSP Spring and 65–70◦ C in Beowulf Spring). Inclusion of two
different geothermal springs provided a direct comparison of
general trends in iron accretion and deposition. Iron oxides were
removed from glass slides using a razor blade, dried overnight
at 70◦ C, then transferred to 50 mL of 0.175 M ammonium
oxalate buffer (pH = 3) to dissolve poorly-crystalline iron
(oxyhydr)oxides (Loeppert and Inskeep, 1996). Slides without
visible Fe(III)-oxide deposition were placed directly into the
0.175 M ammonium oxalate buffer. The extracting solutions
were shaken (Model E600, Eberbach Co. Ann Arbor, MI,
USA) for ∼2 h to promote Fe(III)-oxide dissolution. All Feoxalate extractions were filtered (0.22 µm) into 15 mL Falcon
tubes, and analyzed for Al, Ag, As, Ba, Be, B, Cd, Ca, Cr,
Co, Cu, Fe, P, Pb, Mg, Mn, Mo, Na, Ni, K, S, Sb, Se, Si,
Sr, Tl, Ti, V, W, and Zn using inductively coupled plasmaoptical emission spectroscopy (OPTIMA 5300, Perkin-Elmer,
Waltham, MA, USA). Rate constants of Fe(III)-oxide accretion
were estimated in R using nonlinear least squares fits to
the exponential growth rate equation x = xo ekt , where x
= Fe accreted (µmol cm−2 ), xo = initial Fe (µmol cm−2 ),
k = first-order rate constant (day−1 ), and t = time (day).
Different growth substrates including polyether ether ketone,
titanium, polypropylene, polycarbonate, polytetrafluoroethylene,
and ultra-high-molecular-weight polyethylene were also tested to
determine if the rate of Fe(III)-oxide accretion on glass could
also be observed on other substrates (substrates were deployed in
Beowulf Spring from October 24 to November 6, 2013 (13 days)
and total iron was measured as above (Figure S1).
2.0 fluorometer and Qubit R dsDNA High Sensitivity Assay Kit
(range 0.2–100 ng total dsDNA) (Life Technologies Co.). DNA
quantification (expressed as ng DNA cm−2 day−1 ) provided an
estimate of biomass production as a function of time. Nonlinear
model fits were generated in R as described above, where x and
xo are equal to DNA concentrations (ng cm−2 ).
Scanning Electron Microscopy
Slides incubated in situ were also used for direct examination
of microbial colonization with scanning electron microscopy.
A subset of slides removed from the springs were fixed in 1%
(final concentration) filter-sterilized (0.22 µm) glutaraldehyde. A
Zeiss SUPRA 55VP field emission scanning electron microscope
(Image and Chemical Analysis Laboratory, Montana State
University) was used to image colonized slide surfaces, which
were sputter-coated with iridium to minimize charging at low
voltage (1 keV). Direct imaging of cells also provided estimates
of in situ growth rates (3–5 random field views per estimate) on
slides incubated for 4–15 days.
™
Fluorescence In situ Hybridization
Glass microscope slides with 0.3 cm2 round Teflon printed wells
(SPI Supplies/Structure Probe, Inc. West Chester, PA, USA)
were acid washed and autoclaved as above, incubated in situ
for 6 days (October 24–30, 2013), removed and fixed with 1%
paraformaldehyde (final concentration) for 5 min at 4◦ C. The
fixative was removed from the slide by rinsing in a 1:1 solution of
1 X phosphate buffered saline (PBS): 100% ethanol (EtOH) and
stored in a 50 mL canonical tube containing 1:1 1 X PBS: 100%
EtOH at −20◦ C until hybridization.
Slides were dehydrated in an increasing ethanol series of
50, 80, and 100% for 3 min each, and then air-dried at room
temperature. Hybridization buffer containing 40% formamide,
0.9 M NaCl, 20 mM Tris HCL, and 0.1% sodium dodecyl sulfate
was added to multiple wells (30 µL) and 1 µL of each probe
(6-FAM labeled Aqi338, Kubo et al., 2011; Cy5 labeled Arch915,
Stahl and Amann, 1991) was added directly to the hybridization
buffer on the wells (probe working solutions were 50 ng/µL
for 6-FAM and 30 ng/µL for Cy5). The slide was placed in a
50 mL conical tube with tissue paper soaked in hybridization
buffer (∼1 mL) and incubated for 1.5 h in a 46◦ C hybridization
oven. The slide was then washed for exactly 10 min in buffer
that was pre-warmed to 48◦ C containing 46 mM NaCl, 20 mM
Tris HCl, and 5 mM EDTA. The slide was then rinsed with room
DNA Extraction
DNA was extracted from slides grown over various time points to
determine temporal microbial community composition. Biomass
and mineralized iron oxides were removed from the slides by
either scrapping off a known area with a sterile razor blade
(when visible iron oxides were present), or by vortexing the slide
in a 50 mL conical tube for ∼30 s containing a sterile solution
(autoclaved and 0.22 µm filtered) of 17.5 mM ammonium oxalate
buffer (pH = 3), followed by cell collection on a 0.22 µm filter.
The direct extraction method was performed when there were
no visible Fe(III)-oxides present, which was common for slides
incubated for <10 days. DNA was extracted from scraped Fe(III)oxides or from cell-enriched filters using the FastDNA Spin
Kit for Soil DNA extraction kit and protocol (MP Biomedicals,
LLC, Solon, OH, USA). DNA was quantified using a Qubit R
™
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Succession of Thermoacidic Iron Oxide Mats
75◦ C, pH = 3.5, O2 (aq) = 55 µM) to identify variation
in net areal O2 fluxes compared to prior measurements at
OSP and Beowulf Spring Fe(III)-oxide mats (Bernstein et al.,
2013). Custom Clark-type oxygen electrodes (tip diameter =
50 µm) designed with a high-temperature resistant electrolyte
solution (Unisense A/S, Aarhus, Denmark) were used to make
replicate measurements at OSP Spring (O2 microelectrode
measurements were not repeated at Beowulf Spring Fe(III)-oxide
mats due to difficulties (i.e., breaking microsensors) making these
measurements on the harder Fe(III)-oxide mats present in this
spring. Details on flux and reaction-diffusion modeling were
discussed in Bernstein et al. (2013). Briefly, O2 microprofiles
(n = 11) were modeled (first-order) using the dimensionless
equation, u = (ϕ2 • ζ2 )/2 − ϕ2 • ζ + 1, where u = CO 2 /CO 2 ’,
ζ = z/Lf , and ϕ2 = k1 L2f /De (z = mat depth, CO 2 = oxygen
concentration, CO 2 ’ = bulk O2 concentration, k1 = first-order
rate constant, Lf = mat thickness, and De = diffusion coefficient
of O2 ). The solution of this equation provides an estimate of
the relative contribution of the rate of O2 diffusion vs. the
rate of O2 consumption (ϕ = Thiele Modulus), where ϕ > 0
indicates that diffusion is limiting and ϕ < 0 indicates that the
reaction rate is limiting the observed rate of consumption (Thiele,
1939).
temperature distilled water and dried with laboratory air. The
slide was then immediately visualized with a Leica SP5 inverted
confocal scanning laser microscope (Leica Microsystems Inc.,
Buffalo Grove, IL, USA) at the Montana State University Center
for Biofilm Engineering Confocal Microscopy Laboratory, or
stored at −20◦ C for up to 2–3 days without fluorescence signal
loss.
Archaeal and Bacterial 16S rRNA Gene
Illumina Sequencing and Analysis
Archaeal and bacterial 16S rRNA gene sequences were amplified
with the universal 515F (5′ -GTG CCA GCM GCC GCG
GTA A-3′ )/806R (5′ -GGA CTA CHV GGG TWT CTA AT-3′ )
(Caporaso et al., 2010) primer pair at the Department of Energy—
Joint Genome Institute (Walnut Creek, CA, USA) and sequenced
on an Illumina MiSeq (NCBI Bioproject ID PRJNA306640).
A custom pipeline was utilized to screen Illumina Tag (iTag)
16S rRNA gene sequences (average length = 250 bp) with
a database of relevant 16S rRNA gene sequences from YNP
hot springs. Briefly, chimeras were removed from the iTag
dataset, the sequences grouped into operational taxonomic units
at 97% identity, then identified based on comparison to a
curated group of long fragment (>1200 bp) 16S rRNA gene
sequences from these and similar sites. Heatmaps showing
the relative abundance of phylotypes were generated in R
with the heatmap.plus package. Bray-Curtis dissimilarities were
calculated with the vegan community ecology package in R
(Oksanen et al., 2016). The abundance of phylotypes from mature
Fe(III)-oxide mats were compared using Illumina 16S rRNA
gene barcodes vs. Illumina random metagenome sequencing
from matching samples. Barcoded 16S rRNA gene amplification
using universal archaeal and bacterial primers (515F/806R;
Caporaso et al., 2010; Earth Microbiome Project, http://www.
earthmicrobiome.org/) on mature Fe(III)-oxide mats resulted in
overestimation of Hydrogenobaculum spp. and underestimation
of M. yellowstonensis-like organisms (see Table 2 in Results).
The under- and overestimation of these phylotypes is caused
by a mismatch of the 515F primer to the 16S rRNA gene
in M. yellowstonensis (phylum Crenarchaeota), and is an
important consideration for universal primer-based studies of
Fe(III)-oxide microbial mats that contain abundant members
of the Crenarchaeota. Although the relative abundance of
Hydrogenobaculum spp. was corrected for two copies of the 16S
rRNA gene, this adds more sequences to the PCR pool, and could
result in additional overestimation. The 16S rRNA gene PCR
amplification step using universal primers on these relatively
simple communities illustrates how a single primer mismatch
to the target sequence can cause a large discrepancy in relative
abundance estimates of an important community member (i.e.,
M. yellowstonensis). Metagenome data for One Hundred Spring
Plain (OSP_B) is located under the Integrated Microbial Genome
Submission IDs 10386 (Illumina) and 1781 (454), and Beowulf
Spring (BE_D) 10390 (Illumina), 2254 (454), and 278 (Sanger).
RESULTS
Early Colonization
Rod-shaped bacteria colonized slides rapidly, and significant
cell densities of these organisms were observed using scanning
electron microscopy (SEM) within 4–7 days of incubation in both
OSP and Beowulf Springs (Figure 2). The taxonomic identity
of these bacteria was confirmed to be Hydrogenobaculum spp.
using 16S rRNA gene specific fluorescence in situ hybridization
(FISH) probes (Figure 3). After confident taxonomic assignment
of these bacteria, SEM images were useful for obtaining growth
estimates during early incubation times (<14 days), prior to
the extensive deposition of Fe-oxides and exogenous debris that
precluded accurate cell counting. Colonization rate estimates of
Hydrogenobaculum spp. were 3.7 ± 1.8 • 106 and 6.8 ± 3.4 •
106 cells cm−2 day−1 in OSP and Beowulf Springs, respectively.
Coccus-shaped archaea were also identified at early time points
(e.g., within 4–7 days) using both SEM and FISH (Figure 3),
but were considerably less abundant than Hydrogenobaculum
spp. (Figures 2, 3). Although M. yellowstonensis probes have
proven difficult in Fe(III)-oxide samples in situ (Kozubal et al.,
2008), archaeal probes were positive and molecular data (below)
indicated that early-colonizing archaea were M. yellowstonensislike organisms. Early-colonizing archaea were often found as
individuals within 4–6 days, and as microcolonies containing up
to 50 cells within 15 days (Figures 2C–E). These cocci colonized
at rates of approximately 9.2 ± 5.1 • 105 and 8.6 ± 4.1 • 105
cells cm−2 day−1 in OSP and Beowulf Springs, respectively,
which is nearly five times slower than the colonization rates by
Hydrogenobaculum spp. Visible Fe-oxide staining from 7 to 14
days also corresponded to the detection and proliferation of these
archaea. Hydrogenobaculum spp. and M. yellowstonensis were
Oxygen Microsensor Measurements
Oxygen microsensor measurements were made on May 21, 2013
at OSP Spring Fe(III)-oxide microbial mats (temperature =
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Succession of Thermoacidic Iron Oxide Mats
FIGURE 2 | Scanning electron micrographs of slides incubated for 7 and 15 days in Fe(III)-oxide mats from One Hundred Spring Plain (A,C,E from
7/13/2011 and 7/21/2013) and Beowulf Spring (B,D,F from 7/13/2011 and 7/21/2011). Archaeal microcolonies from Beowulf Spring (F) are highlighted with
dashed circles.
was modeled using a first-order rate equation (Fex = Feo ekt )
where Fex is Fe(III)-oxide accreted (µmol Fe cm−2 ), Feo is the
initial Fe(III)-oxide concentration (µmol Fe cm−2 ), and k is the
empirical first-order rate constant (day−1 ). A lag phase of Fe(III)oxide accretion occurred from 0 to ∼30 days (Figures 4A,B),
which corresponded to the slower growth rate of Fe(II)-oxidizing
microorganisms. The fitted first-order rate constants for Fe(III)oxide accretion were 0.05 day−1 (std. error = 0.003, p = 2.5 •
10−15 ) and 0.047 day−1 (std. error = 0.005, p = 2.98 • 10−10 )
at OSP and Beowulf, respectively. Similar rate constants (within
10%) describing the accumulation of Fe-oxides were observed
for OSP and Beowulf springs across multiple field seasons and
suggest that similar processes control the deposition of Fe(III)oxides in these habitats. The lower amount of Fe(III)-oxide
accreted by 70 days of incubation in Beowulf Spring relative to
OSP was significant (p = 0.0018, student’s two-tailed T-test)
and may be attributed to differences in spring geochemistry (e.g.,
often observed in close spatial proximity (Figure 2D), which
suggests that these populations are interacting in situ.
Iron Oxide Accretion
The biomineralization of poorly-crystalline Fe(III)-oxide phases
occurred as visible crusts on Hydrogenobaculum rods and
filaments at times >7 days (Figure 2). These arsenate-rich,
poorly-crystalline Fe(III)-oxide phases (Inskeep et al., 2004)
begin to dominate the available surface area and form larger
(>1 µm) crusts on outer cell surfaces of Hydrogenobaculum
spp. and other inorganic templates (e.g., SiO2 and alunite)
at incubation times greater than 14 days (Figure 2E). Direct
observations of temporal changes in Fe(III)-oxide deposition
using SEM were corroborated with data obtained on Fe(III)oxide accretion as a function of time.
Iron oxide deposition increased exponentially with time
(days) in both OSP and Beowulf Springs (Figures 4A,B), and
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Succession of Thermoacidic Iron Oxide Mats
103 , respectively) resulted in lower amounts of Fe deposition
(Table 1). The vertical growth of Fe(III)-oxide mats in acidic
geothermal springs occurred at a rate of ∼15–30 µm day−1
(0.5–1 mm month−1 ), although the maximum vertical growth is
ultimately limited by channel water depths. Maximum rates of
Fe(III)-oxide accretion in acidic geothermal springs (∼0.9 µmol
Fe cm−2 day−1 ) fall within the range observed for other systems
(0.09–9 µmol Fe cm−2 day−1 ), which includes estimates of Fe
deposition rates in banded iron formations (Konhauser, 1998) as
well as observations in circumneutral pH environments (Hanert,
1974; Emerson and Revsbech, 1994).
lower pH and temperature) and local hydrodynamic conditions.
Specifically, the higher flow velocities and higher Reynolds
numbers in Beowulf vs. OSP Spring (R = 1.4 • 104 and 1.5 •
Temporal Changes in Microbial Community
Composition
The amount of microbial biomass (expressed as ng DNA cm−2 )
increased as a function of time in OSP and Beowulf Springs, and
also followed a first-order rate equation (Figures 4C,D), similar
to Fe(III)-oxide accretion. A lag phase in the accumulation
of total community DNA was also observed up to ∼30
days, after which DNA increased exponentially. The fitted
rate constants for DNA accumulation were 0.028 day−1
(std. error = 0.0063, p = 0.0062) and 0.043 day−1 (std.
error = 0.0087; p = 0.0026) for OSP and Beowulf Springs,
respectively. These values are essentially similar to the fitted
FIGURE 3 | Fluorescence in situ hybridization image of slides
incubated in Beowulf Spring for 6 days (A,B). Green rods are
Hydrogenobaculum spp. (6FAM-Aqi338) and red cocci are archaea
(Cy5-Arch915). Scale bar = 20 µm (A) and 1 µm (B).
FIGURE 4 | Iron oxide accretion measured on glass slides incubated as a function of time in One Hundred Spring Plain (OSP) (A) and Beowulf (B)
Springs outflow channels in Norris Geyser Basin, Yellowstone National Park. The dashed black line represents a model fit to the exponential rate expression
Fex = Feo ekt , where Fex is iron oxide accreted (µmol Fe cm−2 ), Feo is the initial iron oxide concentration (µmol Fe cm−2 ), k is the first-order rate constant (day−1 ),
and t is time (day). Total DNA concentrations determined from slides incubated in One Hundred Spring Plain (OSP) (C) and Beowulf Spring (D) over a time period of
70 days. The solid black line represents a model fit to the exponential rate expression DNAx = DNAo ert , where DNAx is DNA accumulated (ng DNA cm−2 ), DNAo is
the initial DNA concentration (ng DNA cm−2 ), r is the first-order rate constant (day−1 ), and t is time (day).
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February 2016 | Volume 7 | Article 25
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Succession of Thermoacidic Iron Oxide Mats
rate constants for Fe accretion, and it is clear from the time
series behavior that Fe accumulation and DNA accumulation
are also correlated (Figure 4). Molecular data confirmed that
the lithoautotrophic populations of Hydrogenobaculum spp. and
Metallosphaera yellowstonensis were the dominant community
members (>90%) at early stages of mat development (<30 days)
(Figure 5). The abundance of M. yellowstonensis was relatively
constant (10–20%) throughout mat development. In contrast,
Hydrogenobaculum spp. abundance was highest during early
stages of colonization, and declined after 15–30 days (Figure 5) as
mat depth increased and other heterotrophic archaea contributed
to the total mat community.
Dendrograms of microbial community structure as a function
of time were compared to population abundances of mature
Fe(III)-oxide mats (Figure 6). The population abundances of
Fe(III)-oxide mat communities from early incubation times
(<40 days) were more similar to one another compared to
later time points (>70 days), which reflects progression toward
“mature” 0.5–2 cm thick Fe(III)-oxide mats (Table 2, Figure 6).
The dominant microbial population observed from 4 to 70 days
was Hydrogenobaculum spp., which was consistent with direct
observations using SEM and FISH. Although Hydrogenobaculum
spp. were especially dominant at times <14 days (Figure 6),
their abundance declined on average of ∼1% per day with
increasing mat depths. Using this estimate, relative abundances
of Hydrogenobaculum spp. would reach ∼1–3% after 100 days,
which is within the range observed for “mature” Fe(III)oxide mats of 0.5–2 cm thickness (Table 2). Metallosphaera
yellowstonensis was the only other population detected in
significant numbers at early time points and remained relatively
constant over the time series, representing ∼10–20% of the total
microbial community (Figure 6). Iron-oxide accretion increased
FIGURE 6 | Relative abundance (16S rRNA gene Illumina barcodes) of
different phylotypes determined on slides incubated in One Hundred
Spring Plain (A) and Beowulf (B) Springs from 4 to 70 days. The
dendrograms on the y and x axes represent the Bray-Curtis dissimilarity matrix
between taxon abundance at different time points and taxon abundances,
respectively.
FIGURE 5 | Relative abundance (16S rRNA gene iTags) of key
lithoautotrophic microbial populations over the time course of slide
incubations in One Hundred Spring Plain (A) and Beowulf (B) Springs
(Hydrogenobaculum spp. = open squares; Metallosphaera
yellowstonensis = closed black circles).
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Succession of Thermoacidic Iron Oxide Mats
significantly after 40 days and this corresponded to the detection
of several heterotrophic archaea, which represented the majority
of the total community (in aggregate) by 70 days (Table 2,
Figure 6), as well as in “mature” Fe-oxide mats (reaching depths
of 0.5–2 cm).
Although the predominant lithoautotrophs responsible for
Fe(III)-oxide mat formation are the same in the two geothermal
springs studied, different heterotrophic assemblages between
sites were also evident. For example, populations of a novel
archaeal Group 3 (NAG3), which are phylogenetically related
to the archaeal order Thermoplasmatales (Inskeep et al., 2010;
Kozubal et al., 2012), were especially important in Beowulf
Spring at later time points (Figure 6). Furthermore, members
of Sulfolobales Group 3, which includes a novel heterotrophic
Fe(II)-oxidizing species (Kozubal et al., 2012), appeared later in
community succession (∼20 days) at Beowulf Spring, and may
contribute to Fe(III)-oxide accretion in this site. Several other
heterotrophic archaea were observed in mid to later stages of
Fe(III)-oxide mat succession, and although these phylotypes were
present in lower abundance (<1–2%), all have been observed in
acidic Fe(III)-oxide mats from YNP (Kozubal et al., 2012), and
these chemoorganotrophs do not have genes required for the
fixation of CO2 (Jennings et al., in review), which suggests that
they rely (in part) on organic C produced by Hydrogenobaculum
spp. and M. yellowstonensis.
The relative abundance of different phylotypes in “mature”
(0.5–2 cm thick) Fe(III)-oxide microbial mats was estimated from
random shotgun metagenome and 16S rRNA gene sequencing
(Table 2, Figure 6). Metallosphaera yellowstonensis populations
ranged from ∼4 to 16% of the microbial community across
temperatures of 65–75◦ C (Table 2), and is consistent with
the reported optimum growth temperature (Kozubal et al.,
2008). Other Sulfolobales populations were also detected, and
a representative of Sulfolobales Group 3 (strain MK5) has
been shown to oxidize Fe(II) heterotrophically (Kozubal et al.,
2012). Hydrogenobaculum spp. were less abundant in mature
Fe(III)-oxide mats (Table 2), which is consistent with the
measured decline in these populations over time (Figures 5, 6).
Members of the Geoarchaeota (Kozubal et al., 2013) represent
the dominant heterotrophic phylotype in OSP Spring, whereas
members of a novel archaeal Group 2 (NAG2; Kozubal et al.,
2012) were the dominant heterotrophs in Beowulf Spring.
Deeply-branching Thaumarchaeota (Beam et al., 2014) were also
observed in mature Fe mats, and were abundant in Beowulf
Spring (Table 2). Differences in temperature and pH between
the two sites likely influence the relative abundance(s) of these
heterotrophic archaeal populations (Kozubal et al., 2012).
In situ Oxygen Consumption and
Formation of Microterracettes
Oxygen microelectrode measurements in OSP Spring Fe(III)oxide mats revealed an areal O2 (aq) consumption rate of
1.14 • 10−4 µmol cm−2 s−1 (Figure 7). The concentration of
O2 (aq) ranged from ∼55 µM at the mat-aqueous interface and
dropped to below detection (<0.3 µM) within the top 1 mm.
These observations are consistent with prior O2 microelectrode
measurements in the same Fe(III)-oxide mat systems (Table 3).
Dimensionless reaction-diffusion model fits (see Materials and
Methods) of oxygen microprofiles resulted in an estimate of 28
for the Thiele modulus (ϕ), which indicates that the rate of biotic
O2 consumption was at least an order of magnitude faster than
O2 diffusion into the Fe(III)-oxide mats (Bernstein et al., 2013).
Thus, the microbial consumption of O2 is limited by the rate of
O2 diffusion; an average flux of O2 into these poorly-crystalline
Fe(III)-oxide mats was 1.2 ± 0.5 • 10−4 µmol cm−2 s−1 at
temperatures ranging from 60 to 75◦ C (Table 3).
TABLE 2 | The relative abundance of different phylotypes from “mature” Fe(III)-oxide mats of One Hundred Spring Plain and Beowulf Spring (YNP)
determined using random DNA sequencing over multiple years (and sequencing technologies), or short-fragment 16S rRNA gene sequencing (Illumina
iTag).
Spring
Sequencing technology
pH/Temperature (◦ C)
One Hundred Spring Plain Spring
Beowulf Spring
Random
Random
16S Tags
Random
454
Illumina
Illumina
3.6/72
3.5/75
3.5/75
PHYLOTYPE
Random
Random
16S Tags
Sanger
454
Illumina
Illumina
3/65
2.9/66
2.9/68
2.9/68
RELATIVE ABUNDANCE (%)
M. yellowstonensis
10.3
16
0.51
3.9
6.6
12
–
Hydrogenobaculum spp.
3.4
3
24.2
2
1.2
4.3
14
Geoarchaeota
43.4
33
41
1
3
6
4
4
6
24
26
38
27
42
3.2
Novel archaeal group 2
Thaumarchaeota
0.4
0.4
3
19
4.4
2
Novel archaeal group 3
0.1
0.12
–
4
3
5
26
Other Sulfolobales
2.5
4
3.3
3.9
10
11
8.5
Nanoarchaeota
0.2
0.6
–
0
0.1
0.6
–
–, not identified, Sample dates for One Hundred Spring Plain 454 (July 15, 2010) and Illumina (October 11, 2011), and Beowulf Sanger (August 7, 2006), 454 (July 15, 2010), and
Illumina (November 16, 2011).
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Succession of Thermoacidic Iron Oxide Mats
Microterracettes containing both Hydrogenobaculum spp.
and M. yellowstonensis were observed within 6 days, already
reaching heights up to 10 µm and spaced at ∼10–20 µm intervals
(Figure 8). These smaller microterracettes may coalesce to form
larger (∼1 mm high) structures (Figure 8), which evolve into
visible cm-scale microterracettes common in mature Fe(III)oxide mats (Figure 1). These structures increase the overall
surface area and flux of O2 into the mat system, similar to the
formation of “wrinkles” in microbial communities, which have
been shown to form as a result of O2 mass transfer limitations
to microorganisms (Okegbe et al., 2014). The relative size and
periodicity of microterracettes is likely correlated with the extent
and size of the mass transfer boundary layer, which is a function
of water velocity and character (i.e., turbulent vs. laminar flow),
as well as physical properties of the biomineralized mat.
Specifically, M. yellowstonensis and Hydrogenobaculum spp. were
more abundant in the top 1 mm of the Fe(III)-oxide surface
relative to middle or bottom positions, which is consistent with
the high O2 requirements of these lithoautotrophs. Microaerobic
chemoorganotrophs (i.e., NAG2 and Geoarchaeota) were more
abundant in middle and bottom mat sections (Kozubal et al.,
2012). The relative abundance of hypoxic community members
such as Acidilobus spp. (order Desulfurococcales) increased in
bottom mat positions, which is consistent with the metabolic
attributes of these organisms (Jay et al., 2014) and the low O2
concentrations (<0.3 µM) observed at mat depths >1 mm (e.g.,
Table 3, Figure 8). These data are also consistent with prior
results showing that active Fe(II)-oxidizing M. yellowstonensis
populations are higher in the upper (∼1 mm) mat layer
(Bernstein et al., 2013).
Vertical Stratification of Microbial
Populations
Adsorption of Oxyanions
The biomineralization of Fe(III)-oxides in acidic mats of NGB
results in the concomitant adsorption, coprecipitation and/or
biomass uptake of oxyanions such as arsenate, phosphate and
tungstate over time (Figure 10) (e.g., Leblanc et al., 1996; Karl
et al., 1988). Molar ratios of As:Fe over all time points ranged
from 0.5 (OSP) to 0.67 (Beowulf), which is consistent with
prior measurements of As:Fe ratios in “mature” Fe(III)-oxide
mats (Langner et al., 2001; Inskeep et al., 2004; Macur et al.,
2004). The co-accumulation of As, P, W and Fe could provide
evidence of microbiological activity under acidic conditions [e.g.,
Fe(II) and As(III) oxidation and subsequent biomineralization].
Molar ratios of P (0.01) and W (0.002) to Fe were similar
between OSP and Beowulf Springs (Figure 10). Tungsten may
substitute for molybdenum in enzymes (e.g., dimethyl sulfoxide
molybdopterins) utilized by thermoacidophilic organisms to
perform specific functions such as arsenite oxidation or
degradation of organic matter (Kletzin and Adams, 1996).
Oxygen gradients result in the stratification of Fe(III)-oxide
mat community members. We dissected “mature” Fe(III)-oxide
mats into discrete zones (top 1 mm, middle, bottom) for 16S
rRNA gene (iTag) analysis, which revealed stratification of the
microbial community as a function of mat depth (Figure 9).
DISCUSSION
A conceptual model describing the assembly and succession
of thermoacidic Fe(III)-oxide microbial mats was developed by
integrating geochemical, imaging, and molecular measurements
across multiple scales of observation. The life cycle of a
high-temperature Fe(III)-oxide mat in the acid-sulfate-chloride
springs of Norris Geyser Basin (YNP) can be represented by
four stages (Figure 11). Early colonization (Stage I) by aerobic,
FIGURE 7 | Oxygen microprofiles (n = 11, electrode tip diameter = 50
µm) measured in One Hundred Spring Plain Fe(III)-oxide mats (May
2013; temperature = 75◦ C, pH = 3.5, O2 (aq) ∼55 µM). The diffusive flux
of oxygen, JO 2 , was estimated from the measured concentration profiles as a
function of Fe(III)-oxide mat depth. Position zero refers to the
aqueous-Fe(III)-oxide mat interface.
TABLE 3 | Oxygen flux estimates, penetration depths, and Thielea moduli describing oxygen diffusion in thermoacidic Fe(III)-oxide mats from Norris
Geyser Basin, Yellowstone National Park.
Spring/Temperature (◦ C)
Sample date
Net areal O2 flux (µmol cm−2 s−1 )
Penetration depth (µm)
Thiele modulus
One Hundred Spring Plain/75
May 21, 2013
1.14 • 10−4
950
28
This study
One Hundred Spring Plain/75
August 18, 2010
1.41 • 10−4
750
30
Bernstein et al., 2013
Beowulf/68
July 6, 2011
1.64 • 10−4
250–1000
60
Bernstein et al., 2013
Beowulf/60
July 13, 2004
500
nd
Kühl and Kozubal,
unpublished
a see
5 • 10−5
References
Methods for definition of Thiele modulus.
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Succession of Thermoacidic Iron Oxide Mats
FIGURE 8 | Growth and formation of microterracettes from slides incubated in Beowulf Spring. Scanning electron micrographs (A–C) of ridge structures:
scale bar = 1 µm (A), and 10 µm (B,C). Photograph of larger, 1 mm ridges (D) present in 70 days Fe(III)-oxide mats incubated for 70 days (scale bar = 1 mm). The
flow direction (arrows) is perpendicular to microterracettes.
chemolithoautotrophic populations of Hydrogenobaculum spp.
and Metallosphaera yellowstonensis provides critical founder
populations for initial surface roughness and continued mat
growth. Hydrogenobaculum spp. exhibit significantly greater
colonization rates than M. yellowstonensis (factor of 5) in hightemperature acidic Fe(III)-oxide mats. The aerobic oxidation of
arsenite (and/or reduced sulfur species) by Hydrogenobaculum
spp. (D’Imperio et al., 2007, 2008; Hamamura et al., 2009) and
Fe(II) by M. yellowstonensis (Kozubal et al., 2008) is coupled
with the fixation of DIC as a primary C source via the reductive
tricarboxylic acid and 3-hydroxypropionate/4-hydroxybutyrate
cycles, respectively (Berg et al., 2007; Takacs-Vesbach et al.,
2013; Jennings et al., 2014), and provides the Fe(III) and As(V)
necessary for the formation of high-As, poorly-crystalline Fe(III)oxides characteristic of these springs (Langner et al., 2001;
Inskeep et al., 2004; Macur et al., 2004).
Appreciable amounts of Fe(III)-oxides are produced
between ∼7 and 30 days (Stage II), which correlates with the
establishment of M. yellowstonensis (e.g., microcolonies of 10–50
cells), and which results in the encrustation of rapidly-growing
Hydrogenobaculum rods and filaments (Figure 2). The cell
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surface of Hydrogenobaculum may contain macromolecules that
aid in the nucleation of poorly-crystalline Fe(III)-oxide phases,
because these cells are encrusted preferentially (also see Macur
et al., 2004). Moreover, Fe(III)-oxides do not accumulate on the
cell surface(s) of M. yellowstonensis under culture conditions
(Kozubal et al., 2008), or in situ (Figure 2). Hydrogenobaculum
spp. inhabit a large reach (several meters) within the outflow
channels of OSP and Beowulf Springs (Figures 1A,B), and data
obtained during the first 4 days of slide placement suggest that
these organisms are continually colonizing the upper surface
of iron mats where O2 concentrations are highest. Higher
rates of cell growth occur along microscale ridges as early as
6–7 days (Figure 8). These structures reflect the microbial
control of microterracette formation in acidic Fe(III)-oxide
mats, and are likely formed in response to O2 mass transfer
limitations (Figure 7) to growing chemolithotrophic populations
(i.e., Hydrogenobaculum and M. yellowstonensis). Cell growth
proceeds upward into the shallow, high-velocity (∼20–30 cm
s−1 ) aqueous phase where O2 (aq) concentrations range from 20
to 60 µM (Table 1), and results in Fe(III)-oxide mat heights of
0.5–1 mm within 30 days.
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Beam et al.
Succession of Thermoacidic Iron Oxide Mats
FIGURE 9 | Relative abundance of taxa (16S rRNA gene Illumina
barcodes) as a function of depth in mature thermoacidic iron oxide
mats from One Hundred Spring Plain (A) and Beowulf (B) Springs. The
top position refers to the upper 1 mm oxygenated zone, the middle position
represents depths of ∼2–7 mm, and the bottom position represents hypoxic
regions > 10 mm.
Continued Fe(III)-oxide accretion and cell growth >30 days
results in significant accumulation of biomass C and niche
diversity (e.g., O2 gradients) that allow for the colonization
of heterotrophic archaea (Stage III). Moreover, the probability
of trapping exogenous detritus and/or debris from landscape
sources increases as the surface roughness increases due to
cell growth and Fe(III)-oxide formation. For example, plant
material, diatoms, and wind-blown solid-phases can be trapped
by the complex series of ridges and valleys established during
mat development. The later succession of heterotrophs also
suggests that they require specific metabolites and/or cofactors
produced by autotrophic populations. The primary archaeal
organoheterotrophs observed from 30 to 70 days using iTag
analysis (16S rRNA) included members of the candidate phylum
Geoarchaeota (Kozubal et al., 2013), novel archaeal Groups 2 and
3 (Inskeep et al., 2010, 2013; Kozubal et al., 2012) and members
from two additional Sulfolobales lineages, which have also been
observed at these lower abundances (1–2%) in prior Fe-mat
studies (Inskeep et al., 2010; Kozubal et al., 2012). By 70 days,
Fe(III)-oxide mat depths can reach 2–4 mm, and have developed
initial O2 concentration gradients (Figure 7) that support further
niche diversification to include hypoxic populations. Although
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FIGURE 10 | Molar ratios of arsenic (A), phosphorous (B), and tungsten
(C) to total solid phase iron extracted from slides incubated in One
Hundred Spring Plain (open triangles, dotted black line) and Beowulf
(filled circles, black line) Springs (Figure 1). Numbers in parentheses are
the standard error.
the fixation of CO2 by autotrophic Hydrogenobaculum spp. and
M. yellowstonensis populations represents a significant fraction of
the biomass C in “mature” Fe(III)-oxide mats (no less than ∼40%
DIC signature; Jennings et al., 2014), the relative abundance
of Hydrogenobaculum spp. is actually negatively correlated with
Fe(III)-oxide accretion over time (Figures 5, 6) due to the rise
of other heterotrophic populations; although Hydrogenobaculum
spp. are constantly colonizing and growing in the active O2
consuming layer (i.e., the upper 1 mm of mat) (Figure 3).
Ultimately, the shallow water depth in the outflow channels
(<2 cm) becomes a limiting factor for the maximum thickness
of these Fe(III)-oxide mats (Stage IV). Several metagenomes
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Beam et al.
Succession of Thermoacidic Iron Oxide Mats
FIGURE 11 | Conceptual model of iron oxide mat development (four stages) in high-temperature, acidic geothermal springs of Yellowstone National
Park. Stage I is defined by the primary colonization of lithoautotrophic populations of Hydrogenobaculum spp. and Metallosphaera yellowstonensis. Stage II is
represented by visible iron oxide accretion, encrustation of Hydrogenobaculum spp. by Fe(III)-oxides, and initial formation of microterracettes. Heterotrophic archaea
begin colonizing in Stage III and gradients in O2 are apparent. As iron oxide depth increases to form “mature” 0.5–2 cm thick mats (Stage IV) the microbial community
undergoes succession toward organoheterotrophic archaea, where steep gradients in oxygen, and macroscale ridges and pools are observed.
The succession of early colonizing autotrophs to later colonizing
heterotrophs is directly applicable to other Fe(III)-oxide mat hot
spring ecosystems; autotroph-heterotroph successional patterns
are a common theme in other microbial mat communities, as well
as larger-scale ecosystems (Odum, 1969).
High-temperature Fe(III)-oxide mats of Norris Geyser Basin
are modern-day stromatolites (Riding, 1999), which form as
a direct consequence of microbial metabolism and associated
hydrogeochemical controls. The preferential biomineralization
of Fe(III)-oxides on cell surfaces of Hydrogenobaculum spp. may
be related to enhanced nucleation rates of poorly-crystalline
Fe(III)-oxide phases. Other members of the Aquificales are also
were obtained from “mature” Fe(III)-oxide mats of this thickness
in both spring positions used in the current study, and
these samples were also analyzed using iTags (Table 2). The
integration and comparison of these datasets with the temporal
incubation studies provides an opportunity to understand the
cycle of Fe(III)-oxide mat development. Larger microterracettes
(∼1 mm), which are visible even after ∼70 days (Figure 8),
continue to grow to form a series of centimeter-scale terracettes
over a temperature range of ∼55–75◦ C (Figure 1). These thicker
Fe(III)-oxide terracettes reveal changes in population abundance
as a function of mat depth (Figure 9) that are consistent with
expected gradients in O2 observed in “mature” mats (Figure 7).
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Beam et al.
Succession of Thermoacidic Iron Oxide Mats
0654336) (fellowship support to JB, HB, ZJ, and RJ). The work
conducted by the U.S. Department of Energy Joint Genome
Institute, a DOE Office of Science User Facility, is supported
by the Office of Science of the U.S. Department of Energy
under Contract No. DE-AC02-05CH11231. HB is also grateful
for support from the Linus Pauling Distinguished Postdoctoral
Fellowship, a Laboratory Directed Research and Development
Program at PNNL. JB appreciates assistance from M. Wagner
and M. Schmid (University of Vienna) for FISH training,
C. Hendrix and S. Gunther (YNP Center for Resources) for
research permitting in YNP (YELL-SCI-5068), C. Carey and A.
Mazurie (MSU) for iTag data processing, and B. Pitts (MSU)
for instruction and helpful comments on confocal microscopy.
FISH images were taken at microscope facilities in the Center
for Biofilm Engineering (Montana State University), which is
supported by the NSF-MRI Program and the M.J. Murdock
Charitable Trust. JB and WI appreciate helpful comments and
discussion with E. N. J. Brookshire, B. W. Fouke, and M.
W. Fields. The authors also appreciate the comments of an
anonymous reviewer that improved the quality of the final
manuscript.
thought to promote the nucleation and growth of aragonite
(CaCO3 ; Kandianis et al., 2008; Fouke, 2011). Stable carbon
isotopes (i.e., 13 C) also provide signatures indicative of biological
activity, such as evidence for significant CO2 fixation in extant
Fe(III)-oxide mats (Jennings et al., 2014). Micro-morphological
changes across different stages of Fe(III)-oxide mat development
including cell encrustation by Fe(III)-oxides and the formation of
microterracettes also reveal microbiological signatures that may
provide a reference for comparison to other extant and ancient
Fe(III)-oxide hot spring ecosystems on Earth, or Fe(III)-oxide
mineral deposits identified on other planetary systems such as
Mars (Madden et al., 2004).
AUTHOR CONTRIBUTIONS
JB and WI conceived and designed experiments. JB, HB, ZJ,
MK, RJ, and WI performed experiments. ST contributed Illumina
sequencing and support. JB, HB, ZJ, and WI analyzed data. JB and
WI wrote the manuscript.
ACKNOWLEDGMENTS
This work was supported by the Department of Energy (DOE)Pacific Northwest National Laboratory Foundational Science
Focus Area in Biological Interactions (subcontract 112443), the
DOE-Joint Genome Institute Community Sequencing Projects
(CSP 787081 and 787701), and the National Science FoundationIntegrative Graduate Education and Training Program (DGE
SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found
online at: http://journal.frontiersin.org/article/10.3389/fmicb.
2016.00025
REFERENCES
in a geothermal spring. Appl. Environ. Microbiol. 73, 7067–7074. doi:
10.1128/AEM.01161-07
D’Imperio, S., Lehr, C. R., Oduro, H., Druschel, G., Kühl, M., and McDermott, T.
R. (2008). Relative Importance of H2 and H2 S as Energy Sources for Primary
Production in Geothermal Springs. Appl. Environ. Microbiol. 74, 5802–5808.
doi: 10.1128/AEM.00852-08
Donahoe-Christiansen, J., D’Imperio, S., Jackson, C., Inskeep, W., and McDermott,
T. (2004). Arsenite-oxidizing Hydrogenobaculum strain isolated from an
acid-sulfate-chloride geothermal spring in Yellowstone National Park. Appl.
Environ. Microbiol. 70, 1865–1868. doi: 10.1128/AEM.70.3.1865-1868.2004
Ehrlich, H. L., and Newman, D. K. (2009). “Geomicrobiology of iron,” in
Geomicrobiology, 5th Edn. eds H. L. Ehrlich and D. K. Newman (New York,
NY: CRC Press; Taylor and Francis Group, LLC), 279–329.
Emerson, D. (2012). Biochemistry and microbiology of microaerobic Fe(II)
oxidation. Biochem. Soc. Trans. 40, 1211–1216. doi: 10.1042/BST201
20154
Emerson, D., and Revsbech, N. P. (1994). Investigation of an iron-oxidizing
microbial mat community located near Aarhus, Denmark: field studies. Appl.
Environ. Microbiol. 60, 4022–4031.
Fouke, B. (2011). Hot-spring Systems Geobiology: abiotic and biotic influences
on travertine formation at Mammoth Hot Springs, Yellowstone National Park,
USA. Sedimentology 58, 170–219. doi: 10.1111/j.1365-3091.2010.01209.x
Fournier, R. O. (1989). Geochemistry and dynamics of the Yellowstone National
Park hydrothermal system. Ann. Rev. Earth Planet. Sci. 17, 13–53. doi:
10.1146/annurev.ea.17.050189.000305
Hamamura, N., Macur, R. E., Korf, S., Ackerman, G., Taylor, W. P.,
Kozubal, M., et al. (2009). Linking microbial oxidation of arsenic
with detection and phylogenetic analysis of arsenite oxidase genes in
diverse geothermal environments. Environ. Microbiol. 11, 421–431. doi:
10.1111/j.1462-2920.2008.01781.x
Hanert, V. H. (1974). In situ-untersuchungen zur analyse und intensitat der
Eisen(III)-fallung in dranungen. Z f Kulturtechnik und Flurbereinigung 15,
80–90.
Ackerman, G. G. (2006). Biogeochemical Gradients and Energetics in Geothermal
Systems of Yellowstone National Park. Master of Science thesis, Montana State
University, Montana State University.
Beam, J., Jay, Z., Kozubal, M., and Inskeep, W. (2014). Niche specialization
of novel Thaumarchaeota to oxic and hypoxic acidic geothermal springs
of Yellowstone National Park. ISME J. 8, 938–951. doi: 10.1038/ismej.
2013.193
Berg, I. A., Kockelkorn, D., Buckel, W., and Fuchs, G. (2007). A 3hydroxypropionate/4-hydroxybutyrate
autotrophic
carbon
dioxide
assimilation pathway in archaea. Science 318, 1782–1786. doi:
10.1126/science.1149976
Bernstein, H. C., Beam, J. P., Kozubal, M. A., Carlson, R. P., and Inskeep, W.
P. (2013). In situ analysis of oxygen consumption and diffusive transport in
high-temperature acidic iron-oxide microbial mats. Environ. Microbiol. 15,
2360–2370. doi: 10.1111/1462-2920.12109
Boyd, E. B., Leavitt, W. D., and Geesey, G. G. (2009). CO2 uptake and fixation by
a thermoacidophilic microbial community attached to precipitated elemental
sulfur in a geothermal spring. Appl. Environ. Microbiol. 75, 4289–4296. doi:
10.1128/AEM.02751-08
Caporaso, J. G., Lauber, C. L., Walters, W. A., Berg-Lyons, D., Lozupone, C. A.,
Turnbaugh, P. J., et al. (2010). Global patterns of 16S rRNA diversity at a
depth of millions of sequences per sample. Proc. Natl. Acad. Sci. U.S.A. 108,
4516–4522. doi: 10.1073/pnas.1000080107
de Beer, D., Stoodley, P., Roe, F., and Lewandowski, Z. (1994). Effects of biofilm
structures on oxygen distribution and mass-transport. Biotechnol. Bioenerg. 43,
1131–1138. doi: 10.1002/bit.260431118
Denef, V., Mueller, R., and Banfield, J. (2010). AMD biofilms: using model
communities to study microbial evolution and ecological complexity in nature.
ISME J. 4, 599–610. doi: 10.1038/ismej.2009.158
D’Imperio, S., Lehr, C., Breary, M., and McDermott, T. (2007). Autecology of
an arsenite chemolithotroph: sulfide constraints on function and distribution
Frontiers in Microbiology | www.frontiersin.org
14
February 2016 | Volume 7 | Article 25
Beam et al.
Succession of Thermoacidic Iron Oxide Mats
spring, Japan. Syst. Appl. Microbiol. 34, 293–302. doi: 10.1016/j.syapm.2010.
12.002
Langner, H., Jackson, C., McDermott, T., and Inskeep, W. (2001). Rapid oxidation
of arsenite in a hot spring ecosystem, Yellowstone National Park. Environ. Sci.
Technol. 35, 3302–3309. doi: 10.1021/es0105562
Leblanc, M., Achard, B., Othman, D. B., Luck, J. M., Bertrand-Sarfati, J., and
Personné, J. C. (1996). Accumulation of arsenic from acidic mine waters by
ferruginous bacterial accretions (stromatolites). Appl. Geochem. 11, 541–554.
doi: 10.1016/0883-2927(96)00010-8
Loeppert, R. H., and Inskeep, W. P. (1996). “Iron,” in Methods of Soil Analysis:
Part 3 Chemical Methods, ed J. M. Bartels (Madison, WI: Soil Science Society of
America and American Society of Agronomy), 639–664.
Macur, R. E., Langner, H. W., Kocar, B. D., and Inskeep, W. P. (2004).
Linking geochemical processes with microbial community analysis:
successional dynamics in an arsenic-rich, acid-sulphate-chloride
geothermal spring. Geobiology 2, 163–177. doi: 10.1111/j.1472-4677.2004.
00032.x
Madden, M. E. E., Bodnar, R. J., and Rimstidt, J. D. (2004). Jarosite as an
indicator of water-limited weathering on Mars. Nature 431, 821–823. doi:
10.1038/nature02971
Mann, S. (1988). Molecular recognition in biomineralization. Nature 332, 119–124.
doi: 10.1038/332119a0
Odum, E. P. (1969). The strategy of ecosystem development. Science 164, 262–270.
doi: 10.1126/science.164.3877.262
Okegbe, C., Price-Whelan, A., and Dietrich, L. E. P. (2014). Redox-driven
regulation of microbial community morphogenesis. Curr. Opin. Microbiol. 18,
39–45. doi: 10.1016/j.mib.2014.01.006
Oksanen, J., Blanchet, F. G., Kindt, R., Legendre, P., Minchin, P. R., O’Hara, R. B.,
et al. (2016). Community Ecology Package. Available online at: https://github.
com/vegandevs/vegan
Peters, K. E., and Moldowan, J. M. (1993). The Biomarker Guide: Interpreting
Molecular Fossils in Petroleum and Ancient Sediments. Upper Saddle River, NJ:
Prentice Hall.
Reid, R. P., Visscher, P. T., Decho, A. W., Stolz, J. F., Bebout, B. M., Dupraz, C.,
et al. (2000). The role of microbes in accretion, lamination and early lithification
of modern marine stromatolites. Nature 406, 989–992. doi: 10.1038/350
23158
Riding, R. (1999). The term stromatolite: towards an essential definition. Lethaia
32, 321–330. doi: 10.1111/j.1502-3931.1999.tb00550.x
Romano, C., D’Imperio, S., Woyke, T., Mavromatis, K., Lasken, R., Shock, E.,
et al. (2013). Comparative genomic analysis of phylogenetically closely related
Hydrogenobaculum sp. isolates from Yellowstone National Park. Appl. Environ.
Microbiol. 79, 2932–2943. doi: 10.1128/AEM.03591-12
Singer, P. C., and Stumm, W. (1970). Acidic mine drainage: rate-determining step.
Science 167, 1121–1123. doi: 10.1126/science.167.3921.1121
Stahl, D. A., and Amann, R. (1991). “Development and application of nucleic
acid probes in bacterial systematics,” in Nucleic Acid Techniques in Bacterial
Systematics, eds E. Stackebrandt and M. Goodfellow (Chichester: Wiley & Sons
Ltd.), 205–248.
Takacs-Vesbach, C., Inskeep, W. P., Jay, Z. J., Herrgard, M., Rusch, D. B.,
Tringe, S. G., et al. (2013). Metagenome sequence analysis of filamentous
microbial communities obtained from geochemically distinct geothermal
channels reveals specialization of three Aquificales lineages. Front. Microbiol.
4:84. doi: 10.3389/fmicb.2013.00084
Thiele, E. W. (1939). Relation between catalytic activity and size of particle. Industr.
Eng. Chem. 31, 916–920. doi: 10.1021/ie50355a027
Inskeep, W., Macur, R., Harrison, G., Bostick, B., and Fendorf, S. (2004).
Biomineralization of As(V)-hydrous ferric oxyhydroxide in microbial mats
of an acid-sulfate-chloride geothermal spring, Yellowstone National Park.
Geochim. Cosmochim. Acta 68, 3141–3155. doi: 10.1016/j.gca.2003.09.020
Inskeep, W., Rusch, D., Jay, Z., Herrgard, M., Kozubal, M., Richardson, T., et al.
(2010). Metagenomes from high-temperature chemotrophic systems reveal
geochemical controls on microbial community structure and function. PLoS
ONE 5:e9773. doi: 10.1371/journal.pone.0009773
Inskeep, W. P., Ackerman, G. A., Taylor, W. P., Kozubal, M. A., Korf, S., and Macur,
R. E. (2005). On the energetics of chemolithotrophy in nonequilibrium systems:
case studies of geothermal springs in Yellowstone National Park. Geobiology 3,
297–317. doi: 10.1111/j.1472-4669.2006.00059.x
Inskeep, W. P., Jay, Z. J., Herrgard, M. J., Kozubal, M. A., Rusch, D. B., Tringe,
S. G., et al. (2013). Phylogenetic and functional analysis of metagenome
sequence from high-temperature archaeal habitats demonstrate linkages
between metabolic potential and geochemistry. Front. Microbiol. 4:95. doi:
10.3389/fmicb.2013.00095
Inskeep, W. P., and McDermott, T. R. (2005). “Geomicrobiology of acid-sulfatechloride springs in Yellowstone National Park,” in Geothermal Biology and
Geochemistry in Yellowstone National Park, eds W. P. Inskeep and T. R.
McDermott (Bozeman, Mt: Montana State University Publications), 143–162.
Jackson, C. R., Langner, H. W., Donahoe-Christiansen, J., Inskeep, W. P., and
McDermott, T. R. (2001). Molecular analysis of microbial community structure
in an arsenite-oxidizing acidic thermal spring. Environ. Microbiol. 3, 532–542.
doi: 10.1046/j.1462-2920.2001.00221.x
Jay, Z., Rusch, D., Tringe, S., Bailey, C., Jennings, R., and Inskeep, W. (2014).
Predominant Acidilobus-like populations from geothermal environments in
Yellowstone National Park exhibit similar metabolic potential in different
hypoxic microbial communities. Appl. Environ. Microbiol. 80, 294–305. doi:
10.1128/AEM.02860-13
Jennings, R., Whitmore, L., Moran, J., Kreuzer, H., and Inskeep, W. (2014). Carbon
dioxide fixation by Metallosphaera yellowstonensis and acidothermophilic ironoxidizing microbial communities from Yellowstone National Park. Appl.
Environ. Microbiol. 80, 2665–2671. doi: 10.1128/AEM.03416-13
Kandianis, M. T., Fouke, B. W., Johnson, R. W., Veysey, J., and Inskeep, W.
P. (2008). Microbial biomass: a catalyst for the CaCO3 precipitation in
advection-dominated transport regimes. Geol. Soc. Am. Bull. 120, 442–450. doi:
10.1130/B26188.1
Kappler, A., and Straub, K. L. (2005). Geomicrobiological cycling of iron. Rev.
Mineral. Geochem. 59, 85–108. doi: 10.2138/rmg.2005.59.5
Karl, D. M., McMurtry, G. M., Malahoff, A., and Garcia, M. O. (1988). Loihi
Seamount, Hawaii: a mid-plate volcano with a distinctive hydrothermal system.
Nature 335, 532–535. doi: 10.1038/335532a0
Kletzin, A., and Adams, M. W. W. (1996). Tungsten in biological systems. FEMS
Microbiol. Rev. 18, 5–63. doi: 10.1111/j.1574-6976.1996.tb00226.x
Konhauser, K. (1998). Diversity of bacterial iron mineralization. Earth Sci. Rev. 43,
91–121. doi: 10.1016/S0012-8252(97)00036-6
Konhauser, K. (ed.). (2006). “Biomineralization,” in Introduction to
Geomicrobiology (Oxford: Blackwell Science Ltd.), 139–149.
Kozubal, M. A., Dlakić, M., and Inskeep, W. P. (2011). Terminal oxidase
diversity and function in “Metallosphaera yellowstonensis”: gene expression and
protein modeling suggest mechanisms of Fe(II) oxidation in Sulfolobales. Appl.
Environ. Microbiol. 77, 1844–1853. doi: 10.1128/AEM.01646-10
Kozubal, M. A., Macur, R. E., Jay, Z. J., Beam, J. P., Malfatti, S. A., Tringe, S. G.,
et al. (2012). Microbial iron cycling in acidic geothermal springs of Yellowstone
National Park: integrating molecular surveys, geochemical processes, and
isolation of novel Fe-active microorganisms. Front. Microbiol. 3:109. doi:
10.3389/fmicb.2012.00109
Kozubal, M. A., Macur, R. E., Korf, S., Taylor, W. P., Ackermann, G. G., Nagy, A.,
et al. (2008). Isolation and distribution of a novel iron-oxidizing crenarchaeon
from acidic geothermal springs in Yellowstone National Park. Appl. Environ.
Microbiol. 74, 942–949. doi: 10.1128/AEM.01200-07
Kozubal, M. A., Romine, M., Jennings Rde, M., Jay, Z. J., Tringe, S. G., Rusch, D. B.,
et al. (2013). Geoarchaeota: a new candidate phylum in the Archaea from hightemperature acidic iron mats in Yellowstone National Park. ISME J. 7, 622–634.
doi: 10.1038/ismej.2012.132
Kubo, K., Knittel, K., Amann, R., Fukui, M., and Matsuura, K. (2011). Sulfurmetabolizing bacterial populations in microbial mats of the Nakausa hot
Frontiers in Microbiology | www.frontiersin.org
Conflict of Interest Statement: The authors declare that the research was
conducted in the absence of any commercial or financial relationships that could
be construed as a potential conflict of interest.
Copyright © 2016 Beam, Bernstein, Jay, Kozubal, Jennings, Tringe and Inskeep.
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