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Speciation of sulfur from ¢lamentous microbial mats from sul¢dic
cave springs using X-rayabsorption near-edge spectroscopy
Annette Summers Engel
1
, Henning Lichtenberg
2,3
, Alexander Prange
2,4
& Josef Hormes
2,3
1
Department of Geology and Geophysics, Louisiana State University, Baton Rouge, LA, USA;
2
Center for Advanced Microstructures & Devices (CAMD),
Louisiana State University, Baton Rouge, LA, USA;
3
Institute of Physics, University of Bonn, Nußallee, Bonn, Germany; and
4
Microbiology and Food
Hygiene, Niederrhein University of Applied Sciences, M ¨
onchengladbach, Germany
Correspondence: Annette Summers Engel,
Department of Geology and Geophysics,
Louisiana State University, Baton Rouge, LA
70803, USA. Tel.: 1225 578 2469; fax: 1225
578 2302; e-mail: aengel@geol.lsu.edu
Received 2 November 2006; revised 30
November 2006; accepted 4 December 2006.
DOI:10.1111/j.1574-6968.2006.00600.x
Editor: Christiane Dahl
Keywords
microbial mats; sulfidic spring; XANES
spectroscopy; sulfur K-edge;
Epsilonproteobacteria
.
Abstract
Most transformations within the sulfur cycle are controlled by the biosphere, and
deciphering the abiotic and biotic nature and turnover of sulfur is critical to
understand the geochemical and ecological changes that have occurred throughout
the Earth’s history. Here, synchrotron radiation-based sulfur K-edge X-ray
absorption near-edge structure (XANES) spectroscopy is used to examine sulfur
speciation in natural microbial mats from two aphotic (cave) settings. Habitat
geochemistry, microbial community compositions, and sulfur isotope systematics
were also evaluated. Microorganisms associated with sulfur metabolism domi-
nated the mats, including members of the Epsilonproteobacteria and Gammapro-
teobacteria. These groups have not been examined previously by sulfur K-edge
XANES. All of the mats consisted of elemental sulfur, with greater contributions of
cyclo-octasulfur (S
8
) compared with polymeric sulfur (S
m
). While this could be a
biological fingerprint for some bacteria, the signature may also indicate prefer-
ential oxidation of S
m
and S
8
accumulation. Higher sulfate content correlated to
less S
8
in the presence of Epsilonproteobacteria. Sulfur isotope compositions
confirmed that sulfur content and sulfur speciation may not correlate to microbial
metabolic processes in natural samples, thereby complicating the interpretation of
modern and ancient sulfur records.
Introduction
Accumulations of organic matter and rocks containing
the reactive element sulfur have been used to interpret
past changes on Earth (e.g. Sinninghe Damste et al., 1990;
Strauss, 1999; Canfield, 2004; Huston & Logan, 2004;
Bottrell & Newton, 2005), and might even be useful for
understanding the Martian past (e.g. Banfield et al.,
2001). Most sulfur cycle transformations are fundamentally
controlled by biosphere processes, especially by the specia-
lized metabolisms of microorganisms. The oxidation of
reduced inorganic sulfur compounds, such as hydrogen
sulfide, is carried out by phylogenetically diverse micro-
organisms (herein referred to as ‘sulfur-oxidizing bacteria’).
For a vast majority of these bacteria, sulfate is the end
product. For others, a variety of intermediate products
form, including thiosulfate, tetrathionate, and elemental
sulfur as sulfur globules. The formation of sulfur globules
and the assimilation of sulfur into biomolecules (e.g.
proteins) result in the storage of sulfur within a microbial
community, which could become preserved in the rock
record.
Therefore, to interpret past changes from ancient deposits,
it is critical to understand the factors (e.g. environmental,
metabolic, phylogenetic) that govern biological sulfur specia-
tion, the chemical nature and turnover rates of inorganic and
organic sulfur compounds, and any possible alteration path-
ways and processes that occur when sulfur becomes preserved
in the rock record. Unfortunately, many of these key factors
are poorly understood and constrained, due in part to a
paucity of suitable techniques that can characterize sulfur
speciation, the flux and transformation rates of sulfur, or
microbiological effects (e.g. Pasteris et al., 2001; Urich et al.,
2006). Geologic processes (e.g. burial and uplift of sulfur-
bearing rocks) that control the distribution of sulfur on Earth
(e.g. Bottrell & Newton, 2005) can be interpreted using sulfur
isotope systematics, and these methods have been used, almost
to the exclusion of any other method, to describe biogeo-
chemical transformations (e.g. B¨
ottcher & Thamdrup, 2001;
Mandernack et al., 2003; Huston & Logan, 2004).
FEMS Microbiol Lett xx (2007) 000–000 c2007 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
The motivation for this study was driven by the need to
evaluate processes affecting the inorganic and organic speciation
of sulfur in naturally occurring microbial mats to identify
possible sulfur biosignatures. Various approaches have been
adopted to differentiate sulfur stores in pure cultures (e.g.
Hageage et al., 1970; Lawry et al., 1981; Prange et al., 1999;
Pasteris et al., 2001; Pickering et al., 2001). Mats from two
aphotic (cave), sulfidic spring habitats were sampled. Sulfur
K-edge X-ray absorption near-edge structure (XANES) spectro-
scopy because it is one of the most efficient tools to study the
speciation of sulfur in situ from biological and geological
systems (e.g. Prange & Modrow, 2002; Kemner et al., 2005).
XANES spectroscopy is nondestructive, can be performed in situ
from solid or liquid samples, and allows for the determination
of the electronic (e.g. valence, types of chemical bonds) and
geometric configuration (e.g. length of sulfur chains) of the
sulfur atoms (e.g. Chauvistr´
eet al., 1997; Rompel et al., 1998).
Moreover, XANES spectra from various sulfur-containing com-
pounds are additive, and by comparing known spectra of
reference compounds with unknown spectra from samples,
spectra can be fitted and quantitative analyses can be carried
out (e.g. Prange et al., 2002a). To place the XANES results in an
ecological context, spectra were compared to habitat conditions,
bulk sulfur content, sulfur isotope systematics, and general
microbial community compositions.
Materials and methods
Sample collection, habitat geochemistry, and
stable isotope analyses
Water was collected for dissolved constituent analyses ac-
cording to Engel et al. (2004a, b). Fresh mat samples were
aseptically acquired from springs that discharge into the
cave passages, including two springs from Lower Kane Cave
(LKC), Wyoming (Fissure and Upper Spring; FS and US,
respectively) (Engel, 2004), and spring-fed streams in
the Frasassi Caves (FC), Italy (Ramo Sulfureo, Pozzo di
Cristalli, and Grotta Bella; RS, PC, and GB, respectively)
(Galdenzi & Menichetti, 1995; Fig. 1a and b). Samples were
placed in sterile, nonreactive polystyrene or polypropylene
(with O-rings) tubes, which were stored under aerobic
or anaerobic (BBL gaspak anaerobic system, Becton-
Dickinson) conditions. Tubes remained sealed and were
maintained at 4 1C for up to c. 5–7 months before analyses.
Aliquots (2 mL wet material) of each sample to be
analyzed were homogenized by vortexing, freeze-drying,
and powdering with a sterile mortar and pestle. Sulfur
isotope ratio analyses and determination of total
sulfur content (as inorganic and organic sulfur compounds)
for the FC samples were performed by Coastal Science
Laboratories (Austin, Texas) on a VG (Micromass)
isotope ratio mass spectrometer; sulfur isotope ratio
analyses and sulfur content for the LKC samples are
described in Engel (2004).
XANES spectroscopy
Wet samples were spread uniformly on a sulfur-free, self-
adhesive kapton
s
film and/or sulfur-free filter paper. From
experience with other microbial samples (e.g. Prange et al.,
1999; Prange et al., 2002a), including anaerobic photosyn-
thetic sulfur bacteria, no oxidation artifacts are expected
from sample storage or preparation because zero-valent
sulfur is relatively stable for long periods (although poly-
sulfides may not be). In a preliminary study, a natural sample
Epsilonproteobacteria
Deltaproteobacteria
Gammaproteobacteria
Betaproteobacteria
Chlorobi-Bacteriodes
Planctomycetes
Chloroflexi
Verrucomicrobia
OP-11 & other
(a) (b)
(c) (d)
Fig. 1. (a) Microbial mats in sulfidic stream
channel flowing from Upper Spring, Lower Kane
Cave, Wyoming. (b) Microbial mats from Ramo
Sulfureo, Frassassi Caves, Italy; carabineer at
center is c. 10 cm long (photograph courtesy of
M. Menichetti). (c) Summary of 16S rRNA gene
sequences from clone libraries constructed from
white filament bundles from Lower Kane Cave
(Engel et al., 2004a). (d) Summary of 16S rRNA
gene sequences from clone libraries constructed
from white filaments from the Ramo Sulfuro,
based on results from Reger et al. (2006). Stars
refer to microbial groups involved in cycling
sulfur.
FEMS Microbiol Lett xx (2007) 000–000
c2007 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
2A.S. Engel et al.
was prepared under both ambient air conditions and an
argon atmosphere in a glove box. XANES spectra were
measured in a helium atmosphere. No differences in the
sample measurements were observed. XANES spectra at the
sulfur K-edge were recorded under reduced air pressure or in
an helium atmosphere at the DCM beamline of the Center
for Advanced Microstructures & Devices (CAMD) (Hormes
et al., 2006) operated at an energy of 1.3 GeV with electron
currents between 200 and 80 mA. The synchrotron radiation
was monochromatized by a modified Lemonnier-type
double-crystal X-ray monochromator (Lemonnier et al.,
1978) equipped with InSb (111) crystals. The monochromatic
flux rate per second on the sample was about 5 10
8
photons
(at 100mA). Measurements of most samples were performed
in transmission mode using ionization chambers (60mbar air
pressure inside). Samples with low sulfur concentrations were
measured in the fluorescence mode. The sample chamber and
ionization chamber in front (on the upstream side) of the
sample were flushed with helium to minimize the attenuation
of the fluorescence radiation.
For energy calibration of the spectra, the spectrum of zinc
sulfate was used as a ‘secondary standard’ setting the max-
imum of the first resonance (white line) to an energy of
2481.44 eV. According to the step width, this value is repro-
ducible to 0.1 eV. Spectra were scanned (generally, repeated
twice) with step widths of 0.5 eV between 2440 and 2468eV,
0.1 eV between 2468 and 2485 eV, and 0.3 eV between 2485
and 2520 eV (integration time: 1 s per point). Using the Origin
program (Origin Lab Corporation, Northampton, MA), a
linear background determined in the pre-edge region was
subtracted from the raw data (logarithmic ratio of I
0
and I in
case of transmission data and I
F
– averaged over nine detector
elements – divided by I
0
in case of fluorescence data) to correct
the spectra from contributions of higher shells and from
supporting materials. Spectra were normalized at 2510 eV.
Reference compounds, XANES spectral fitting,
and quantitative analysis
A set of reference spectra (cyclo-octasulfur, polymeric sulfur,
methionine sulfone, cysteic acid, and zinc sulfate) was used
(Fig. 2) as representatives of the atomic environment of the
sulfur atom (cf., Prange et al., 2002a), but also to represent
the most common elemental sulfur species (sulfur rings and
polymeric sulfur) and three oxidized forms (methionine
sulfone, sulfonate, sulfate) of sulfur that are found in
biological systems (Prange et al., 2002a, b). The formal
valence of elemental sulfur is zero (‘S
0
’); however, elemental
sulfur tends to catenate and to form chains with various
lengths (S
8
or S
m
) and ring sizes (S
n
) (Steudel & Eckert,
2003). Polymeric sulfur, which is frequently used in the
rubber industry for vulcanization of natural and synthetic
rubbers, consists of chain-like macromolecules, as well as
the possible additional presence of large S
n
(n450) rings
(e.g. Steudel & Eckert, 2003). Reagent-grade methionine
sulfone (Met-O
2
), cysteic acid (Cya), and zinc sulfate were
purchased from Sigma (Sigma, Deisenhofen, Germany) and
the cyclo-octasulfur (S
8
rings) and polymeric sulfur (S
m
)
were both kindly provided by Prof. Dr R. Steudel, Technical
University of Berlin (Germany). Zinc sulfate is representa-
tive of the basic sulfate species. Takahashi et al. (2006)
analyzed various sulfate compounds using XANES and
found that cations bound to the sulfate ion shifted the
postedge region of the peaks, but there was no effect on the
main sulfate K-edge peak within 0.5 eV. All reference
compounds were ground into fine powder and placed
homogeneously on a sulfur-free, self-adhesive kapton
s
film
and were measured in the transmission mode.
For quantitative analysis, the fitting and plotting package
WinXAS was used (Ressler, 1997, 1998); additional details
concerning XANES spectra quantitative analysis have been
published elsewhere (Prange & Modrow, 2002; Prange et al.,
2002a). Errors for contributions are estimated at o10%
(absolute value) (Prange & Modrow, 2002; Prange et al.,
2002a).
Results and discussion
As an extension of work to evaluate microbial sulfur
fingerprints using XANES (e.g. Prange et al., 1999), sulfur
2465 2470 2475 2480 2485 2490
Absorption (a.u.)
Energy (eV)
(a)
(b)
(c)
(d)
(e)
Fig. 2. Sulfur K-edge XANES spectra of (a) cyclo-octasulfur, (b) poly-
meric sulfur, (c) methionine sulfone, (d) cysteic acid and (e) zinc sulfate.
FEMS Microbiol Lett xx (2007) 000–000 c2007 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
3Sulfur K-edge XANES spectroscopy of cave microbial mats
speciation for natural microbial mats was examined.
Although sulfur speciation using XANES has been per-
formed previously from humic substances extracted from
natural marine sediments (e.g. Vairavamurthy et al., 1997),
from pure microbial cultures (e.g. Prange et al., 1999;
Pickering et al., 2001; Prange et al., 2002a), and from
individual filaments colonizing artificial microcolonizers
from a deep-sea vent (Lopez-Garcia et al., 2003), there are
limited sulfur speciation studies of naturally occurring mat
communities (i.e. where there is more than a ‘monocul-
ture’). Moreover, because caves are aphotic, it was possible
to evaluate sulfur speciation without overlapping photo-
trophic signatures.
For the current study, the samples originated from
3–10 cm thick microbial mats consisting of white filament
bundles interconnected with thin web-like biofilms (Fig. 1a
and b), both of which covered a gelatinous gray-brown mat
interior (Engel et al., 2004a). In both caves, the spring and
stream waters had low dissolved oxygen, and were all of a
calcium-bicarbonate-sulfate water type (i.e., dominated by
those three ions). The pH of the cave waters was buffered to
circum-neutral by ongoing carbonate dissolution due to
active sulfuric acid speleogenesis (Galdenzi & Menichetti,
1995; Engel et al., 2004a).
The microbial mat phylogenetic diversity has previously
been described from the full-cycle 16S rRNA gene approach
(Engel et al., 2003; Engel et al., 2004a; Meisinger et al., 2005;
Macalady et al., 2006; Reger et al., 2006) (Fig. 1c and d).
Similar taxonomic groups were present in both systems,
including organisms capable of metabolizing sulfur com-
pounds, either as sulfur-oxidizers or as sulfate-reducers.
Briefly, the communities from the surface of LKC mats
consisted of white filament bundles dominated by the
uncultured Epsilon- (e-) proteobacteria,Gamma (g-) proteo-
bacteria (specifically Thiothrix spp.), Beta (b-) proteobacteria
(including various thiobacilli), and Delta (d-) proteobacteria
classes, with Acidobacteria and Bacteriodes/Chlorobi rarely
being encountered (Fig. 1c). In comparison, the LKC ‘gray’
mats (in most cases, being covered by the white mat
component) had higher diversity, with sulfate-reducing
bacteria affiliated to the d-proteobacteria and members of
the Chloroflexi phylum. From FISH, 70% of the biovolume
(predominantly from areas with low oxygen content) was
dominated by e-proteobacteria. Bundles from downstream
reaches, where higher concentrations of dissolved oxygen
existed, had lower abundances of e-proteobacteria but great-
er abundances of Thiothrix spp. (up to 90% of the mat
biovolume). The ‘white’ mats from the FC also consisted of
e-proteobacteria, with (in order of relative abundance) d-
proteobacteria,Bacteroides/Chlorobi,Verrucomicrobia,g-pro-
teobacteria (Thiothrix and Beggiatoa spp.), b-proteobacteria,
Chloroflexi,Plancotmycetes, and different OP candidate
groups (Reger et al., 2006) (Fig. 1d). These results were
replicated by Macalady et al. (2006) from mats from the
Grotta Sulfurea, a system proximal to the RS, PC, and GB
areas. Using FISH, Macalady et al. (2006) also found that
filamentous g-proteobacteria affiliated with Beggiatoa and/or
Thiothrix spp. dominated the samples, with low relative
abundances (0 to o15%) of e-proteobacteria.
Despite genetic variability, ‘white’ mats (where 450% of
the diversity was dominated by e- and/or g-proteobacteria;
Fig. 1c and d) had similar XANES spectra and higher bulk
sulfur content compared with spectra for ‘gray’ samples (Fig.
3; Table 1). Although the concentration of dissolved Fe in all
of the cave waters was o0.01 mmoL L
1
(data not shown),
it was hypothesized that the gray- to black-colored mats had
high mineral content (e.g. pyrite, Engel et al., 2004a).
Instead, the chosen nonsulfide reference compounds pro-
vided better fits of the XANES spectra from the gray mats
(Table 1; Fig. 2) than sulfide reference compounds that were
tested (data not shown). Sulfur-containing minerals, such as
pyrite, have an S K-edge white line at significantly lower
energies than those of the sample spectra (e.g. Kataby et al.,
1998), indicating the absence of these mineral phases in the
mats analyzed.
All samples had more S
8
(ring sulfur) than any other
sulfur compound, and more than half of the spectra could
be ascribed to Z95% elemental sulfur (as both S
8
1S
m
).
High S
8
content correlated to high sulfur content. Only one
spectrum (ITY-05-005D) could be fit with o50% S
8
content (Fig. 3, spectra i). In most cases, especially for the
white mats, the quality of eight of 10 fits could be improved
by including spectra representing C–S–C bonds, e.g. repre-
sented by monosulfane (C–S–C-compound) (data not
shown). In those cases, the ratio of S
8
–S
m
shifted slightly to
a higher S
m
content. From previous studies of pure cultures
(e.g. Prange et al., 1999), this monosulfane contribution
probably does arise come from amino acids, as the low
relative sulfur content of amino acids gives rise to a very
weak sulfur K-edge signal. However, for some samples, there
might be a significant (10%) monosulfane contribution,
although the origin and final speciation is not yet known.
Sulfur K-edge XANES spectra have not been generated
for Thiothrix spp. or the uncultured, environmental
e-proteobacteria. While Thiothrix spp. store intracellular
globules of elemental sulfur (e.g. Dahl & Prange, 2006), it is
not clear whether the e-proteobacteria found from the caves
form intracellular sulfur (Engel et al., 2004b).The spectra
generated for the cave samples showing a prevalence of S
8
,
are comparable to the ‘sulfur signature’ associated with
intracellular sulfur globule formation for Beggiatoa alba,
Thiomargarita namibiensis, and from sulfur-depositing en-
dosymbionts (Pasteris et al., 2001; Dahl & Prange, 2006), as
well as for environmental Thioploca spp. samples (Pasteris
et al., 2001). The cave sample XANES spectra are distinct
from spectra acquired for purple and green sulfur bacteria or
FEMS Microbiol Lett xx (2007) 000–000
c2007 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
4A.S. Engel et al.
Tabl e 1. Results from fitting the sulfur K-edge XANES spectra of the microbial community samples to the sum of different reference spectra
Sample Number
Description and
prevalent microbial
groups
Microbial community habitat physicochemistry Percentage contribution of sulfur species from XANES
pH
Temperature
(1C)
Oxygen
(mmol L
1
)
Sulfide
(mmol L
1
)
Sulfate
(mmol L
1
)
d
34
S
(%)%S
S
8
cyclo-
octasulfur
S
m
polymeric
sulfur
Met-O
2
methionine
sulfone
Cya
cysteic
acid
SO
4
2
zinc
sulfate
LKC-04A-03
w
US; white filaments; thick mat 7.3 21.6 26.6 18.5 1.33 23.3 50 71 27 – – –
LKC-05-203 US; white filaments 7.3 21.6 24.7 18.5 1.33 24.1 50 91 8 – – –
LKC-05-123 FS; white, thin webs 7.5 21.1 46.9 22.5 1.28 22.1 18 63 34 – – –
ITY-05-013 GB; white–gray; thin film 7.3 13.8 10.2 153 1.03 ND ND 66 31 – – –
ITY-05-005E RS; white filaments 7.4 13.7 6.9 201 1.27 12.4 490 78 17 – – 3
ITY-05-010D PC; white–gray thick mat 7.3 13.5 12.5 345 2.03 15.7 490 60 36 2 – 2
LKC-05-201 US; white–gray; thick mat
and biofilms
7.4 21.5 19.4 18.5 1.31 22.3 26 58 20 3 8 11
LKC-04A-04 US; gray; orifice sediment 7.4 21.3 o0.1 35 1.27 23.5 o262 21 2 2 13
ITY-05-005D RS; gray filaments 7.4 13.7 6.9 201 1.27 11.6 o5 35 28 2 15 20
ITY-05-011A PC; white–gray thick mat, 5 m
downstream
7.4 13.8 15 230 2.07 ND o553 14 2 4 28
Total dissolved ion concentration.
w
LKC samples from Lower Kane Cave (USA) and ITY samples from the Frasassi Caves (Italy); see methods for location name abbreviations.
ND, no detection, usually due to low sulfur content.
The reference compounds for the different sulfur species are listed. Error, o10 %; dashes (–) contribution o1.0%.
FEMS Microbiol Lett xx (2007) 000–000 c2007 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
5Sulfur K-edge XANES spectroscopy of cave microbial mats
Acidothiobacillus spp. where polythionates are accumulated
(at pH 2) (Prange et al., 2002a).
Because the cave samples are far from being monocul-
tures (Fig. 1c and d), the speciation of sulfur may not reflect
the metabolism or a biosignature of the dominant microbial
groups, but instead may indicate that competing, contem-
poraneous processes are occurring. Although a high S
8
content suggests a predominance of microorganisms that
form sulfur globules (e.g. Thiothrix and/or Beggiatoa spp.), a
low S
8
1S
m
content does not necessarily indicate the lack of
these organisms (which could be supported by the 16S
rRNA gene clone library results). Enhanced microbial oxi-
dation (by non-sulfur-accumulating sulfur-oxidizers) or
reduction of S
8
1S
m
could be occurring. In the case for
oxidation, Urich et al. (2006) suggest that S
8
is biologically
unavailable compared with sulfur chains because S
8
would
have to be structurally rearranged before entry to the active
oxidation sites. These results are corroborated by XANES
spectroscopy for the phototrophic sulfur bacterium Allo-
chromatium vinosum that has been found to use only, or at
least predominantly, the polymeric chain (S
m
) fraction when
fed with elemental sulfur (a mixture of S
8
1S
m
) as a single
sulfur source (Franz et al., 2006; A. Prange et al., unpub-
lished).
Sulfate in some samples correlates to low elemental sulfur
content (S
8
1S
m
) and to the presence of e-proteobacteria
(Table 1). Engel et al. (2004b) used scanning electron
microscopy to show that gypsum (CaSO
4
2H
2
O) accumu-
lated in biofilms dominated by e-proteobacteria, despite the
waters being undersaturated with respect to gypsum. These
results indicated that the microorganisms enhanced the
precipitation of the gypsum by changing the saturation state
of the mineral in the micro-environment (Engel et al.,
2004b). It is inconclusive at this stage, however, to determine
whether sulfate in the spectra is present as gypsum or
another material, and future work will address this issue
specifically.
Although XANES spectroscopy provides information
about sulfur speciation, it does not discriminate between
biotic and abiotic processes. Sulfur isotope analyses was
conducted to determine whether there were any relation-
ships between community metabolic diversity and sulfur
speciation. The d
34
S values for the waters from both caves,
and consequently for the microbial mats, differed signifi-
cantly (Table 1), likely because the hydrogeologic source of
sulfide to the caves is slightly different (Galdenzi & Meni-
chetti, 1995; Galdenzi & Maruoka, 2003; Engel, 2004; Engel
et al., 2004a). The d
34
S value for dissolved sulfide of
incoming US water in LKC was 22.5%(n= 4), and the
d
34
S value of the water decreased downstream by approx.
1.6%(Engel, 2004). Comparatively, FC spring water was
14.2%(n= 4), with RS water being lower, at 15.03%
(Galdenzi & Maruoka, 2003). Because sulfur-oxidizing
bacteria exhibit negligible (1/2%) sulfur isotope frac-
tionation during both the transformation of sulfide to
elemental sulfur and elemental sulfur to sulfate (e.g. Fry
et al., 1986a, b; Toran & Harris, 1989), the d
34
S values for the
mats consisting of sulfur-oxidizers should reflect the spring
water sulfide isotope composition (Kaplan & Rittenberg,
1964). For the most part, the d
34
S compositions for LKC
biomass had isotopic values approximately the same as or
up to 2%lower than the allochthonous sulfide (Table 1).
However, because sulfate-reducing bacteria generate
34
S-
depleted sulfide (and more ‘negative’ sulfide compared with
2465 2470 2475 2480 248
5
2465 2470 2475 2480 2485
Ener
gy
(
eV
)
Absorption (a.u.)
Ener
gy
(
eV
)
(a)
(b)
(c)
(d)
(e)
(f) (j)
(i)
(h)
(g)
XANES spectrum
WinXAS fit
Fig. 3. Sulfur K-edge XANES spectra for samples
(solid lines), corresponding to order of informa-
tion in Table 1: (a) LKC-04A-03, (b) LKC-05-203,
(c) LKC-05-123, (d) ITY-05-013, (e) ITY-05-005E,
(f) ITY-05-010D, (g) LKC-05-201, (h) LKC-04A-
04, (i) ITY-05-005D, and (j) ITY-05-011A, with
corresponding WinXAS fits (cf. Table 1) as dotted
lines. Spectra g–j were acquired in fluorescence
mode.
FEMS Microbiol Lett xx (2007) 000–000
c2007 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
6A.S. Engel et al.
the incoming spring water) (e.g. Mandernack et al., 2003),
notable
34
S depletion for some biomass samples may
indicate that the sulfur-oxidizers utilized a mixture of
allochthonous and autochthonous sulfide (Toran & Harris,
1989; Mandernack et al., 2003). The biomass samples from
the FC had d
34
S values approximately the same as or higher
than the dissolved sulfide for the cave waters. The processes
of H
2
S volatilization and abiotic sulfide oxidation, which is
possible in the slightly oxygenated FC waters, could cause an
enrichment in the residual
34
S of the dissolved sulfide and,
consequently, an enrichment in the biomass (Fry et al.,
1986a, b). Based on the presence of Desulfocapsa spp., it is
also possible that the values reflect sulfur disproportionation
because this process would produce a lower d
34
S composi-
tion in the residual sulfide; rapid reoxidation of this sulfide
could result in large isotope fractionation (B ¨
ottcher &
Thamdrup, 2001).
Knowing the isotopic values for the source dissolved
sulfide, places these results in a unique position. The same
cannot be said for ancient deposits studied by sulfur isotope
systematics where the isotopic composition of the source
must be inferred from mineralogical and geochemical
information. The sulfur isotope values for the microbial
mats did not correlate to sulfur speciation XANES patterns.
Some samples had similar XANES spectra and species
content, but the bulk isotopic values were distinct (Table 1;
e.g. ITY-05-010D and LKC-05-201). Other samples had
similar sulfur isotope compositions but had variable sulfur
content and speciation (e.g. samples LKC-04A-04 vs. LKC-
04A-03). These results indicate that the isotopic composi-
tion of biological materials may not represent a specific type
of sulfur species recording the isotopic values, and certainly
may not signify equivalent biotic or abiotic processes.
In summary, more studies are needed to differentiate
among contemporaneous biogeochemical reactions and
microbial community structures that control sulfur specia-
tion and stable isotope signals (e.g. B¨
ottcher & Thamdrup,
2001; Bottrell & Newton, 2005). This study yields a first
approximation of the variable contributions of sulfur species
to the sulfur content of natural materials. While a biological
‘fingerprint’ may have been uncovered for sulfur-oxidizing
bacteria that accumulate intracellular sulfur, consisting pre-
dominantly of ring sulfur, this signature could also be due to
preferential oxidation of polymeric sulfur and the accumu-
lation of more ‘unfavorable’ ring sulfur. The presence of
sulfate may correlate to the presence of sulfur-oxidizing
bacterial groups that completely oxidize reduced sulfur
compounds to sulfate. Future research using cultures should
attempt to differentiate among these sulfur species and the
isotopic compositions of those components. The present
experimental findings demonstrate that XANES potentially
provides a novel insight into the complexity of modern
biogeochemical processes.
Acknowledgements
This work was partially supported for A.S.E. by the Board of
Regents Support Fund (Contract NSF/LEQSF(2005)-Pfund-
04) and the College of Basic Sciences at Louisiana State
University, and for A.P. by the Fonds der Chemischen
Industrie (grant 661209). A.P. thanks the Heinrich-Hertz-
Stiftung (Ministerium f¨
ur Innovation, Wissenschaft, For-
schung und Technologie des Landes Nordrhein-Westfalen)
for a travel grant to stay at CAMD.
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