Dominant Microbial Populations in Limestone-Corroding Stream Biofilms, Frasassi Cave System, Italy

Abstract

Waters from an extensive sulfide-rich aquifer emerge in the Frasassi cave system, where they mix with oxygen-rich percolating water and cave air over a large surface area. The actively forming cave complex hosts a microbial community, including conspicuous white biofilms coating surfaces in cave streams, that is isolated from surface sources of C and N. Two distinct biofilm morphologies were observed in the streams over a 4-year period. Bacterial 16S rDNA libraries were constructed from samples of each biofilm type collected from Grotta Sulfurea in 2002. beta-, gamma-, delta-, and epsilon-proteobacteria in sulfur-cycling clades accounted for > or = 75% of clones in both biofilms. Sulfate-reducing and sulfur-disproportionating delta-proteobacterial sequences in the clone libraries were abundant and diverse (34% of phylotypes). Biofilm samples of both types were later collected at the same location and at an additional sample site in Ramo Sulfureo and examined, using fluorescence in situ hybridization (FISH). The biomass of all six stream biofilms was dominated by filamentous gamma-proteobacteria with Beggiatoa-like and/or Thiothrix-like cells containing abundant sulfur inclusions. The biomass of epsilon-proteobacteria detected using FISH was consistently small, ranging from 0 to less than 15% of the total biomass. Our results suggest that S cycling within the stream biofilms is an important feature of the cave biogeochemistry. Such cycling represents positive biological feedback to sulfuric acid speleogenesis and related processes that create subsurface porosity in carbonate rocks.

Full text

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 2006, p. 5596–5609 Vol. 72, No. 8
0099-2240/06/$08.00⫹0 doi:10.1128/AEM.00715-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Dominant Microbial Populations in Limestone-Corroding Stream
Biofilms, Frasassi Cave System, Italy
Jennifer L. Macalady,
1
* Ezra H. Lyon,
1
Bess Koffman,
2
Lindsey K. Albertson,
1
Katja Meyer,
1
Sandro Galdenzi,
3
and Sandro Mariani
4
Department of Geosciences, Pennsylvania State University, University Park, Pennsylvania 16802
1
; Geology Department,
Carleton College, Northfield, Minnesota 55057
2
; Istituto Italiano di Speleologia, Frasassi Section, Viale Verdi 10,
60035 Jesi, Italy
3
; and Gruppo Speleologico CAI, Via Alfieri 9, 60044 Fabriano, Italy
4
Received 28 March 2006/Accepted 14 June 2006
Waters from an extensive sulfide-rich aquifer emerge in the Frasassi cave system, where they mix with
oxygen-rich percolating water and cave air over a large surface area. The actively forming cave complex hosts
a microbial community, including conspicuous white biofilms coating surfaces in cave streams, that is isolated
from surface sources of C and N. Two distinct biofilm morphologies were observed in the streams over a 4-year
period. Bacterial 16S rDNA libraries were constructed from samples of each biofilm type collected from Grotta
Sulfurea in 2002. ␤-, ␥-, ␦-, and ␧-proteobacteria in sulfur-cycling clades accounted for >75% of clones in both
biofilms. Sulfate-reducing and sulfur-disproportionating ␦-proteobacterial sequences in the clone libraries
were abundant and diverse (34% of phylotypes). Biofilm samples of both types were later collected at the same
location and at an additional sample site in Ramo Sulfureo and examined, using fluorescence in situ hybrid-
ization (FISH). The biomass of all six stream biofilms was dominated by filamentous ␥-proteobacteria with
Beggiatoa-like and/or Thiothrix-like cells containing abundant sulfur inclusions. The biomass of ␧-proteobac-
teria detected using FISH was consistently small, ranging from 0 to less than 15% of the total biomass. Our
results suggest that S cycling within the stream biofilms is an important feature of the cave biogeochemistry.
Such cycling represents positive biological feedback to sulfuric acid speleogenesis and related processes that
create subsurface porosity in carbonate rocks.
Sulfidic caves form in carbonate rocks where sulfide-rich
waters interact with oxygen at the water table or at subterra-
nean springs. The caves form as a result of sulfuric acid pro-
duction (equation 1) from microbial or abiotic sulfur oxidation.
The sulfuric acid reacts with carbonate host rock to form gyp-
sum and carbonic acid (equation 2).
H2S⫹2O23H2SO4(1)
CaCO3⫹H2SO43CaSO4⫹H2CO3(2)
Some of the longest caves known are thought to have formed
by this process, including Lechugilla Cave in New Mexico, with
184 km of passages (17). Actively forming sulfidic caves are
uncommon but intensely valuable as natural laboratories to
understand factors influencing cave formation and resulting
biological, geochemical, and isotopic signatures. Active sulfidic
caves can host biogeochemically isolated ecosystems based en-
tirely on microbial lithoautotrophic primary productivity (16,
38). These ecosystems are aphotic, terrestrial, subsurface en-
vironments comparable to sulfureta at hot springs and deep
sea vents (10) and are of considerable interest as analogs for
microbially dominated, early earth biotic communities such as
those that might have developed after the initial rise of oxygen
in the early Proterozoic era.
Available information from culturing, fluorescence in situ
hybridization (FISH), and 16S rDNA libraries suggests that ε-
and ␥-proteobacteria are important biofilm-forming groups in
the sulfidic cave waters studied to date. Microbial biofilms in
Lower Kane Cave (Wyoming) springs and streams are domi-
nated by filamentous ε-proteobacteria (6, 8). Microbial bio-
films are also present in other active sulfidic caves, such as
Movile Cave (Romania), Parker Cave (Kentucky), and Cess-
pool Cave (Virginia). Floating biofilms in Movile Cave contain
aerobic and anaerobic sulfide-oxidizing lithoautotrophs (35,
39), methanotrophs (22), sulfate reducers, and organohetero-
trophic bacteria and fungi (35). A 48-clone 16S rDNA library
from a Sulfur River stream biofilm in Parker Cave included
primarily ε-, ␥-, and ␤-proteobacteria related to known sulfur-
oxidizing bacteria, with over half of the reported sequences
related to the ε-proteobacterium Thiomicrospira denitrificans
(2). A 70-clone 16S rDNA library from a shallow pool below a
Cesspool Cave spring was split roughly in half between ε-pro-
teobacterial and Thiothrix-related ␥-proteobacterial clones (7).
The abundances of microbial populations in the Movile,
Parker, and Cesspool Cave biofilms have not been determined.
The Grotta Grande del Vento-Grotta del Fiume (Frasassi)
cave complex in central Italy hosts more than 23 km of pas-
sages and is a rare example of a large, actively forming sulfidic
cave system. An important role for microorganisms in sulfide
oxidation in the cave has been proposed (14). However, the
microbial ecology of conspicuous microbial biofilms in sulfidic
stream waters at Frasassi is unexplored. Here we report the
molecular phylogeny and population structure of perennially
abundant white microbial biofilms sampled from two cave
streams over a 4-year period. Our data show that filamentous
␥-proteobacteria dominate the biomass of the biofilms and that
␦-proteobacteria are also abundant.
* Corresponding author. Mailing address: Geosciences Department,
Pennsylvania State University, University Park, PA 16802. Phone:
(814) 865-6330. Fax: (814) 863-7823. E-mail: jmacalad@geosc.psu.edu.
5596

MATERIALS AND METHODS
Site description, sample collection, and geochemistry. The Frasassi cave sys-
tem is forming in the Jurassic Calcare Massiccio Formation (platform limestone)
in the Appennine Mountains of the Marches Region, Central Italy (Fig. 1.). The
active, sulfidic level of the cave is roughly at the elevation of the Sentino River,
which flows 600 to 700 m below mountains on either side of the Frasassi gorge.
A large area of outcropping sulfidic water in ramifying passages is accessible via
technical caving routes inside the cave system. The water includes that of shallow
cave streams, pools, and lakes and exhibits geochemical compositions that vary
spatially and seasonally between well-defined end members (3, 37). Two biofilm
samples with differing morphology (cottony versus feathery) were collected for
clone library construction during a reconnaissance trip to Grotta Sulfurea in
August 2002. Additional samples for FISH were collected in Grotta Sulfurea and
Ramo Sulfureo on subsequent trips (samples RS03-46, RS03-10, GS04-15,
RS05-6, RS05-21, and RS05-22). Samples for nucleic acid and elemental analyses
were collected in sterile tubes, using sterile syringes or Pasteur pipettes, stored
on ice, and processed or frozen within 4 to 6 h after collection. Water samples
were collected in acid-washed polypropylene bottles. The conductivity, pH, and
temperature of the stream waters were measured in situ using probes (WTW,
Weilheim, Germany). Dissolved sulfide (methylene blue method), oxygen (in-
digo carmine method), and ammonium (salicylate method) concentrations were
measured in the field, using a portable spectrophotometer according to the
manufacturer’s instructions (Hach Co., Loveland, Colo.). Oxygen measurements
are reported as means of duplicate or triplicate tests and were reproducible
within 10 to 15%. Anions were measured at the Osservatorio Geologico di
Coldigioco Geomicrobiology lab, using a portable spectrophotometer within 12 h
of collection (samples stored at 4°C) according to the manufacturer’s instructions
(Hach Co., Loveland Colo.). Total organic carbon (TOC) measurements were
determined on triplicate samples, using a Shimadzu TOC-V series analyzer.
Uncoated biofilm samples were examined with an FEI Quanta 200 ESEM with
an Oxford INCA X-ray analytical system. Imaging and energy dispersive spec-
troscopy (EDS) analyses were performed in low vacuum mode at an accelerating
voltage of 10 KeV.
Clone library construction. Small subunit rDNA clone libraries were con-
structed from two white biofilm samples with differing morphologies (cottony
versus feathery) collected within several meters of each other in the Grotta
Sulfurea stream in August 2002 (Fig. 1). DNA was extracted using the MoBio
Soil DNA extraction kit (MoBio) according to the manufacturer’s instructions.
Bacterial 16S rRNA genes were amplified by PCR from purified environmental
DNA. Each 50-␮l reaction mixture contained DNA template, 1.25 U ExTaq
polymerase (TaKaRa Bio Inc., Shiga, Japan), 0.2 mM (each) deoxynucleoside
triphosphate, 1⫻ExTaq PCR buffer, 0.2 ␮M 1492r universal primer (5⬘-GGTT
ACCTTGTTACGACTT-3⬘), and 0.2 ␮M 27f bacterial domain primer (5⬘-AG
AGTTTGATCCTGGCTCAG-3⬘). Reactions were incubated in a thermal cycler
as follows: 5 min at 94°C for initial denaturation, followed by 25 cycles of PCR
consisting of 1 min at 94°C, annealing for 45 s at 45°C, and elongation for 1 min
at 72°C, with a final elongation for 20 min at 72°C. Nonspecific amplification of
DNA from the feathery biofilm DNA extract necessitated the use of touchdown
PCR. Reactions were incubated in a thermal cycler as follows: 5 min at 94°C for
initial denaturation, followed by 32 cycles of touchdown PCR consisting of 45 s
at 94°C, annealing for 1 min at temperatures ranging from 64 to 48°C, and
elongation for 90 s at 72°C, with a final elongation for 20 min at 72°C. PCR
products were checked for size and concentration by gel electrophoresis, cloned
into the pCR4-TOPO plasmid, and used to transform chemically competent
OneShotTOP10 Escherichia coli cells as specified by the manufacturer (TOPO
TA cloning kit; Invitrogen). Colonies containing plasmids with inserts were
isolated by streak plating onto LB agar containing 50 ␮g/ml of kanamycin and
subsequently inoculated into LB plus kanamycin liquid broth. Plasmids were
isolated from overnight liquid cultures, using the Wizard Plus SV Minipreps
DNA purification kit (Promega Corp., Wis.). An aliquot of each culture was
preserved in 15% (wt/vol) sterile glycerol and stored at ⫺80°C.
Sequencing and phylogenetic analysis. Plasmid inserts were sequenced at the
Penn State University Biotechnology Center or the University of Wisconsin
Biotechnology Center, using T3 and T7 plasmid-specific primers and a 536f
internal SSU rDNA primer (5⬘-CAGCMGCCGCGGTAATWC-3⬘). Partial se-
quences were assembled using CodonCode Aligner, version 1.2.4 (CodonCode
Corp.), with Phred base calling and were manually checked. Sequences were
aligned using ARB (31) and manually checked. Possible chimeras were identified
by a partial treeing approach as described in reference 20. Briefly, aligned
sequences were added to a database of 341 representative bacterial species and
minimized with the Lane mask (27). The minimized alignment was split into 5⬘
and 3⬘halves, and neighbor-joining trees were constructed from each half, using
ARB. These partial trees were checked for branching incongruities. Partial
treeing results were compared with the online analyses Bellerophon (18) and
CHIMERA_CHECK version 2.7 at the Ribosomal Database Project II website
(4). Putative chimeras were excluded from subsequent analyses.
Nearly full-length 16S rDNA sequences were grouped into phylotypes with ⬎98%
nucleotide similarity, and one representative of a phylotype was included in each
phylogenetic analysis. Analyses included closely related BLAST matches (1),
full-length sequences of clones from sulfidic caves and springs, and cultured
representatives of major clades within bacterial subdivisions (aligned in ARB).
Alignments were minimized using Lane’s bacterial mask as described above
(1,286 nucleotide positions) (27). Phylogenetic trees were constructed using the
Bayesian analysis implemented in MrBayes, version 3.0b4 (19), using the Kimura
two-parameter substitution model (25) and a gamma distribution of site-to-site
rate variation with six discrete rate categories and run for 500,000 generations.
The first 20% of sampled trees were discarded, and consensus trees were com-
puted by 50% majority rule, using version 4.0b10 of PAUP* (42). Bayesian trees
were compared against parsimony bootstrap consensus trees (heuristic search;
500 bootstrap replicates; PAUP*, version 4.0b10) and maximum likelihood anal-
yses (fastDNAml algorithm implemented in ARB). Rarefaction analyses were
computed using EstimateS version 7.5.0 (5).
Probe design and FISH. Samples for FISH were collected in sterile microcen-
trifuge tubes, stored on ice or at 4°C, and fixed within 24 h after collection.
Samples and isolates grown in the lab for use as control cells were fixed in three
volumes of freshly prepared 4% (wt/vol) paraformaldehyde in 1⫻phosphate-
buffered saline (PBS) for 3 to 4 h and stored in a 1:1 PBS/ethanol solution at 4°C
or ⫺20°C. Fixed samples and control cells were applied to multiwell, Teflon-
coated glass slides, air dried, and dehydrated by successive immersion in 50%,
80%, and 90% ethanol washes (3 min each). Hybridizations were carried out in
8␮l/well of buffer containing 0.9 M NaCl, 20 mM Tris/HCl (pH 7.4), 0.01%
FIG. 1. Map of the Frasassi cave system showing major (named)
caves in different shades of gray. Topographic lines and elevations (in
meters) refer to the surface topography above the cave. Sampling
locations in Grotta Sulfurea and at Ramo Sulfureo in Grotta del
Fiume are shown as open circles.
VOL. 72, 2006 LIMESTONE-CORRODING CAVE STREAM BIOFILMS 5597

sodium dodecyl sulfate (SDS), 25 to 50 ng of each oligonucleotide probe, and the
formamide concentrations given in Table 1. Oligonucleotide probes were syn-
thesized and labeled at the 5⬘end with fluorescent dyes (Cy3, Cy5, and FLC) at
Sigma-Genosys. The slides were incubated for2hat46°C in sealed chambers
equilibrated with the hybridization buffer. The slides were immersed for 15 min
at 48°C in wash buffer (20 mM Tris/HCl [pH 7.4], 0.01% SDS, 5 mM EDTA, and
NaCl concentrations determined by Lathe’s formula [29]). The slides were then
rinsed with distilled water, air dried, and counterstained with 4⬘,6⬘-diamidino-2-
phenylindole (DAPI). The slides were mounted with Vectashield (Vectashield
Laboratories) and viewed on a Nikon E800 epifluorescence microscope.
The probes used in this study are described in Table 1. Probes EUBMIX,
ARCH 915, GAM42a (including competitor probe cGAM42a), and SRB385
have been used extensively to characterize environmental samples and were used
as described in the references in Table 1. One new probe (BEG811) and two
modified probes (DELTA495a and EP404) were designed using the Probe_De-
sign function of the ARB software package. Probes were designed and tested
exactly as described in reference 21, including searches against publicly available
sequences, using BLAST, the greengenes workbench (http://greengenes.lbl.gov/),
and the online database probeBase (http://www.microbial-ecology.de/probeBase).
Hybridization stringencies were determined using positive and negative controls in
experiments with formamide concentrations from 0 to 50%. Optimal formamide
concentrations for BEG811 were determined using Beggiatoa alba (negative control;
1-bp mismatch). The EP404 probe was adapted from the EP402-423 probe targeting
ε-proteobacteria described by Takai et al. (44) but was shortened for use with the
lower hybridization temperature in our FISH protocol. Optimal formamide concen-
trations were determined using Thiomicrospira denitrificans (positive control), an
environmental sample containing a Thiovulum species with a 1-bp mismatch to the
probe (negative control), and a fluorescently labeled oligonucleotide (EP404mis)
with a 1-bp mismatch (negative control). Probe DELTA495a was designed to target
most ␦-proteobacteria in a microarray reverse hybridization protocol (30). The
probe was modified for use with our FISH protocol, including design of a competitor
oligonucleotide (cDELTA495a) with a 1-bp mismatch. The optimal formamide
concentration for the DELTA495a probe was determined in the presence of
equimolar cDELTA495a, using Desulfovibrio gigas and Desulfosarcina variabilis as
positive controls and E. coli as a negative control (1-bp mismatch).
Microscopy. Samples fixed with 4% paraformaldehyde as described above
were viewed using phase contrast, differential interference contrast, and epiflu-
orescence microscopy using a Nikon E800 epifluorescence microscope. The
presence of sulfur inclusions was assayed on samples which had not been exposed
to ethanol during preservation or dehydration. The samples were examined with
phase contrast optics before and after incubation in 90% ethanol (3 or 10 min,
25°C) to check for sulfur dissolution (34). For each FISH experiment (sample-
probe combination), between four and six microscope slide wells were examined.
Because the biofilms were composed primarily of long filaments, the biomass of
cells hybridizing with each probe relative to DAPI-stained cells was estimated
visually and recorded as one of five categories: not detected, ⬍5%, 5 to 15%, 15
to 60%, or 60 to 100%.
Nucleotide sequence accession numbers. The 16S rRNA gene sequences de-
termined in this study have been submitted to GenBank with accession numbers
DQ133908 to DQ133940 and DQ415745 to DQ415869.
RESULTS
Biofilm morphology and geochemistry. The cave stream bio-
films that we observed over a 4-year period in Grotta Sulfurea
and Ramo Sulfureo had two distinct morphologies. Cottony
biofilms were found on the surface of fine, dark gray sediment
lining slow-flowing reaches or margins of streambeds (Fig. 2A
and B). Feathery biofilms were found attached to coarse lime-
stone particles (coarse sand to boulder sized) in rapidly flowing
reaches of the same streams (Fig. 2C and D). Cottony biofilms
commonly had a tufted appearance, as in Fig. 2A, and were
observed to reorganize themselves within minutes after a sam-
pling disturbance, concentrating at the sediment/water inter-
face in collection tubes. This behavior was not observed for
feathery biofilms. The two biofilm morphologies formed a
patchwork in the stream channels, with patches ranging in size
from 10 to 100 cm in diameter. Geochemical data for bulk
stream water surrounding the biofilms are shown in Table 2.
Biofilms in the cave streams were white, presumably due to
the presence of small particles of elemental sulfur. Large
bright inclusions were visible under phase contrast microscopy
inside microbial filaments in both biofilm types, exemplified by
the images shown in Fig. 3. Bright inclusions less than 1.5 ␮m
in diameter disappeared after the microscope slide was soaked
in 90% ethanol for 3 min. Soaking for 10 min removed even
the largest (⬃5-␮m diameter) inclusions. EDS analyses con-
firmed the presence of sulfur accumulations within microbial
filaments. The intensity of sulfur peaks in EDS spectra col-
lected at intervals along the length of filaments ranged from
background levels (measured outside the filaments) to more
than an order of magnitude higher, indicating localized accu-
mulations of sulfur within the cells (data not shown).
Clone libraries. A total of 87 (feathery biofilm) and 70
(cottony biofilm) nonchimeric 16S rDNA sequences were re-
trieved from the Grotta Sulfurea stream biofilms collected in
TABLE 1. Oligonucleotide probes used in this study
Probe Target group Sequence (5⬘to 3⬘)
c
% Formamide Target site Reference
EUB338
a
Most bacteria GCT GCC TCC CGT AGG AGT 0–50 16S (338–355) 2
EUB338-II
a
Planctomycetales GCA GCC ACC CGT AGG TGT 0–50 16S (338–355) 7
EUB338-III
a
Verrucomicrobiales GCT GCC ACC CGT AGG TGT 0–50 16S (338–355) 7
ARCH915 Archaea GTG CTC CCC CGC CAA TTC CT 20 16S (915–934) 44
GAM42a ␥-Proteobacteria GCC TTC CCA CAT CGT TT 35 23S (1027–1043) 34
cGAM42a Competitor GCC TTC CCA CTT CGT TT 35 23S (1027–1043) 34
DELTA495a Most ␦-proteobacteria, some
Gemmatimonas
AGT TAG CCG GTG CTT CCT45
b
16S (495–512) 31
cDELTA495a Competitor AGT TAG CCG GTG CTT CTT45 16S (495–512) This study
SRB385 Some ␦-proteobacteria,
some Actinobacteria, and
Gemmatimonas
CGG CGT CGC TGC GTC AGG 35 16S (385–402) 2
EP404 ε-Proteobacteria AAA KGY GTC ATC CTC CA 30
b
16S (404–420) Adapted from ref. 49
EP404mis Negative control for EP404 AAA KGY GTC TTC CTC CA 30 16S (404–420) This study
BEG811 Frasassi Beggiatoa clade CCT AAA CGA TGG GAA CTA 35
b
16S (811–828) This study
a
Combined in equimolar amounts to make EUBMIX.
b
Stringency optimized in this study.
c
Letters in bold indicate mismatches between probes and competitors or controls.
5598 MACALADY ET AL. APPL.ENVIRON.MICROBIOL.

August 2002. Rarefaction analyses and abundances of clones in
major bacterial lineages are compared in Fig. 4A and B, re-
spectively. The large majority of clones from both biofilms
were related to sulfur-cycling Proteobacteria, with fewer clones
from Verrucomicrobia,Cytophaga-Flexibacter-Bacteroides, and
other lineages (Fig. 4B). ␤-Proteobacterial sequences were
related to Thiomonas species (99% identity) and Thiobacillus
denitrificans (97% identity). ␥-Proteobacterial clones included
close relatives of Thiothrix species, Beggiatoa species, and a
sulfidic cave and spring clone group, as well as a distant relative
of Coxiella burnetii (Fig. 5). Beggiatoa-related clones were
present in both biofilms and formed a monophyletic clade
within the Thiotrichaceae. This clade accounted for almost half
of the total sequences retrieved from the cottony biofilm.
FIG. 2. Examples of cave stream biofilm morphologies in situ at sampling sites. Cottony biofilms are shown in panels A (Ramo Sulfureo;
RS05-21) and B (Grotta Sulfurea; GS04-15). Dark gray, fine sediment is visible underneath the biofilms. Note the tufted appearance of the biofilm
in panel A. Numerous red worms visible in panel A extend approximately 0.5 cm above the dark gray sediment surface. Feathery biofilms are shown
in panels C (Ramo Sulfureo; RS05-22) and D (Grotta Sulfurea; cottony biofilm used for clone library construction). The mottled appearance of
the images in panels C and D is due to the turbulent water surface interacting with the camera flash. The large objects in panel C are biofilm-coated
limestone boulders. In panel D, an 8-cm-long biofilm is attached to a limestone pebble in the streambed at the arrow.
VOL. 72, 2006 LIMESTONE-CORRODING CAVE STREAM BIOFILMS 5599

␦-Proteobacterial clones related to sulfur-reducing and dispro-
portionating isolates were abundant and diverse in both bio-
films, comprising 19 phylotypes in the feathery biofilm and 14
phylotypes in the cottony biofilm (Fig. 6). Roughly half of
␦-proteobacterial clones from each library were close relatives
of Desulfocapsa species. ε-Proteobacterial sequences (Fig. 7)
were retrieved only from the feathery biofilm. They comprised
eight phylotypes and were phylogenetically related to Arco-
bacter species or to a symbiont/sulfidic cave clone group that
includes the isolate Sulfurovum lithotrophicum.
FISH probe design and optimization. Probe BEG811 was
designed to target a narrow clade containing Beggiatoa-related
sequences retrieved in the two stream biofilm 16S rDNA clone
libraries (35 sequences total) (Fig. 5). Other Thiotrichaceae,
including Beggiatoa and Thioploca species, have one or more
mismatches to the probe. Based on comparisons with all avail-
able sequences in public databases, the probe is specific for the
Frasassi Beggiatoa clade, with the exception that it matches one
Leptospirillum-related environmental phylotype (Nitrospira lin-
eage) retrieved from an extremely acidic environment. At 35%
formamide stringency, the probe hybridized with Beggiatoa-
like cells in both feathery and cottony biofilms but not with
Beggiatoa alba control cells (1-bp mismatch).
Probe EP404 targets diverse environmental and sulfur-cy-
cling ε-proteobacteria and is complementary to all ε-pro-
teobacteria retrieved in Frasassi stream biofilm clone libraries
(Fig. 7), as well as to ⬎90% of ε-proteobacteria in sulfur-
oxidizing clades that are available from public databases, in-
cluding the Arcobacter,Sulfurospirillum,Thiomicrospira, and
symbiont clades and unaffiliated sequences from sulfur-rich
environments. At 30% formamide stringency, EP404 produced
bright fluorescence from positive-control Thiomicrospira deni-
trificans cells. Negative-control experiments using fluorescently
labeled probe EP404mis (1-bp mismatch to the control cells)
(Table 1) produced no signal at 30% formamide. EP404 was
also tested with an environmental sample containing a Thiovulum
population with a 1-bp mismatch to the probe. No fluorescence
was detected from the negative-control Thiovulum cells at 30%
formamide.
Probe DELTA495a is identical to the microarray oligonu-
cleotide with the same name described in reference 30 but has
not previously been adapted for FISH. The probe targets all
major groups of sulfate-reducing ␦-proteobacteria as well as
environmental clones within the newly proposed bacterial lin-
eage Gemmatimonadetes. BLAST searches indicated that nu-
merous nontarget Proteobacteria and Actinobacteria have a
1-bp mismatch to the probe near the 3⬘end. A competitor
oligonucleotide (cDELTA495a) was designed in order to elim-
inate hybridization of the probe with these groups (Table 1). In
the presence of an equimolar competitor probe at 45% form-
amide, DELTA495a produced bright fluorescence from positive-
control cells (Desulfovibrio gigas and Desulfosarcina variabilis) and
no fluorescence from negative-control E. coli cells (1-bp mis-
match).
Probe SRB385 targets a broad range of ␦-proteobacteria
(50% of matches in public databases) but also matches some
Actinobacteria (20% of matches), some Gemmatimonadetes
(7% of matches), and isolated sequences scattered throughout
the bacterial tree (23% of matches).
TABLE 2. Morphology, FISH results, and geochemical parameters for stream biofilms used in FISH experiments
a
Sample Date
taken
Biofilm
morphology
Biomass of cells for indicated probe: Temp
(°C)
Cond
(␮S/cm) pH S
2⫺
(␮M)
O
2
(␮M)
NH
4⫹
(␮M)
SO
42⫺
(␮M)
NO
2⫺
(␮M)
NO
3⫺
(␮M)
TOC
(mg/liter)
ARCH915 EUBMIX GAM42a BEG811 EP404 SRB385 DELTA495a
RS03-46 10/28/03 Cottony ⫹ ⫹⫹⫹⫹ ⫹ ⫹⫹⫹⫹ ⫹⫹ ⫹ ⫹⫹⫹ 13.6 1,964 7.3 NM NM NM NM NM NM NM
RS03-10 10/28/03 Feathery ND ⫹⫹⫹⫹ ⫹⫹⫹⫹ ⫹ ⫹ ⫹ ⫹ 13.6 1,964 7.3 NM NM NM NM NM NM NM
GS04-15 7/07/04 Cottony ND ⫹⫹⫹⫹ ⫹⫹ ⫹⫹⫹⫹ ⫹⫹ ⫹ ⫹⫹⫹ 13.6 1,693 7.4 NM 6.0 74 1333 ⬍2.0 ⬍0.7 NM
RS05-6 5/27/05 Feathery ⫹ ⫹⫹⫹⫹ ⫹⫹⫹⫹ ⫹⫹ ND ⫹ND 13.4 1,370 7.4 0.2 12.6 35 1427 ⬍2.0 NM 2.8
RS05-21 8/07/05 Feathery ⫹ ⫹⫹⫹⫹ ⫹⫹⫹⫹ ⫹⫹ ⫹ ⫹ ND 13.5 1,823 7.3 212 2.6 67 1302 ⬍2.0 ⬍0.7 2.5
RS05-22 8/07/05 Cottony ND ⫹⫹⫹⫹ ⫹⫹ ⫹⫹⫹⫹ ⫹⫹ ⫹ ⫹ 13.5 1,823 7.3 212 2.6 67 1302 ⬍2.0 ⬍0.7 2.5
a
The biomass of cells hybridizing to each probe relative to DAPI-stained cells in FISH experiments is indicated by ND, not detected; ⫹,⬍5%; ⫹⫹, 5 to 15%; ⫹⫹⫹, 15 to 60%; or ⫹⫹⫹⫹, 60 to 100%. Cond, specific
conductivity; TOC, total organic carbon; NM, not measured.
5600 MACALADY ET AL. APPL.ENVIRON.MICROBIOL.

Microscopy and FISH. Both cottony and feathery biofilms
were composed primarily of long filamentous bacteria. The
biomass of cells that hybridized with specific oligonucleotide
probes in FISH experiments is shown in Table 2. Archaeal cells
detected using the ARCH915 probe were rare (typically ⬍3%
of the biomass) or not detected. In contrast, almost all cells
in the biofilms fluoresced brightly in experiments with the
EUBMIX probe set. DAPI staining confirmed that all cells in
the biofilms hybridized with either EUBMIX or ARCH915
probes.
Further FISH experiments revealed two distinct microbial
communities correlated with the macroscopic morphologies of
the biofilms. Cottony biofilms (GS04-15, RS03-46, and RS05-
22) contained abundant large filaments (5 to 7 ␮m in diameter;
20 to 500 ␮m long) that hybridized with the newly designed
probe BEG811, targeting Beggiatoa-related Frasassi clones
(Fig. 8). The large filaments lacked vacuoles and had rounded
termini and abundant sulfur inclusions (Fig. 3A and 8). Inter-
estingly, the Beggiatoa-like filaments did not hybridize with the
GAM42a probe targeting 23S rRNA of ␥-proteobacteria. Two
of the cottony biofilm samples (GS04-15 and RS03-46) had an
additional filamentous bacterial population closely intertwined
with the Beggiatoa-like filaments. The smaller filaments (1 to 4
␮m in diameter; 50 to 500 ␮m long) hybridized with probes
EUBMIX and DELTA495a (Fig. 9A and B) and lacked sulfur
inclusions. In addition to the long filaments, DELTA495a also
hybridized with dense populations of large vibrios (3 um long)
and smaller populations of rods and cocci (0.5 to 1 um in
diameter). EP404-positive cells were relatively few, comprising
5 to 15% of the biomass (Table 2), and included short rods (⬍1
␮m in diameter) in dense colonies and rare, thin, short fila-
ments (1 ␮m in diameter; 20 to 50 ␮m long). Probe SRB385
hybridized with scattered rods and cocci cells in all cottony
biofilm samples.
Feathery biofilm samples (RS03-10, RS05-6, and RS05-21)
were dominated by long, thin filaments with sulfur inclusions
that hybridized with the GAM42a probe (1 to 2.5 ␮mindi-
ameter; 50 to 500 ␮m long) (Fig. 3B and 9). These filaments
had no observable vacuoles under phase, transmitted-light, or
fluorescence microscopy. The filaments were often observed in
rosettes (Fig. 10) or attached to mineral holdfasts at one end.
Thick filaments identical to Beggiatoa-like cells in the cottony
biofilms were also present and made up ⬃4 to 15% of the
biomass. Probe EP404, targeting ε-proteobacteria, detected
isolated rods and cocci (⬃1 um in diameter) contributing 0 to
5% of the biomass. Probe SRB385 hybridized with scattered
rods and cocci cells in all samples. Probe DELTA495a de-
tected isolated rods in sample RS03-10 but no cells in the other
two feathery biofilm samples.
DISCUSSION
Sulfur oxidizers. Neutral-pH cave streams at Frasassi are
sites of intense limestone corrosion, equivalent to roughly 15
mg CaCO
3
/cm
2
/year, or 5 cm/1,000 years (15). In the present
study, we found perennially abundant white biofilms attached
to corroding limestone surfaces and to thin organic-rich sedi-
ments in the streams. These biofilms are dominated by fila-
mentous ␥-proteobacteria with Beggiatoa-like (cottony bio-
films) or Thiothrix-like (feathery biofilms) cell morphologies
and abundant sulfur inclusions. ␤- and ε-proteobacteria related
to Thiobacillus,Arcobacter, and other sulfur-oxidizing groups
were retrieved in clone libraries but made up a small fraction
of the biomass in both biofilm types, based on FISH experi-
ments (Table 2).
Freshwater and small marine Beggiatoa species are typically
described as gradient microorganisms which colonize steep
oxygen and sulfide gradients near the sediment/water interface
(46). The newly designed probe BEG811 hybridized with 5- to
7-␮m-diameter filaments present in cottony biofilms from both
the Ramo Sulfureo and Grotta Sulfurea sample sites at
Frasassi. The morphology and behavior of the filaments were
FIG. 3. Phase contrast micrographs showing abundant bright sulfur inclusions in filamentous cells in cottony (A) and feathery (B) biofilm
samples. The scale bars are 10 microns.
VOL. 72, 2006 LIMESTONE-CORRODING CAVE STREAM BIOFILMS 5601

consistent with descriptions of freshwater Beggiatoa species
(40, 46), including evidence of gliding motility. The Beggiatoa-
like filaments were present in all of the stream biofilm samples
we analyzed but dominated in cottony biofilms collected from
the surfaces of fine, dark gray sediment near the margins or in
slowly flowing reaches of streams.
Feathery biofilms were dominated by thin, GAM42a-posi-
tive filaments, often in rosettes or attached to mineral particles
by holdfasts at one end. Compared to recently described ro-
settes of vacuolate sulfur oxidizers growing at shallow marine
hydrothermal vents (4 to 10 ␮m) (24), the feathery biofilm
filaments were smaller in diameter (1.0 to 2.5 ␮m) and non-
vacuolate. Feathery biofilms were found attached to limestone
rocks in rapidly flowing water, typical of Thiothrix habitats
where sulfide and oxygen are turbulently mixed in a strong
current (28).
Cottony and feathery biofilm types were commonly observed
adjacent to each other in the same streams, separated by dis-
tances of 10 to 100 cm. This pattern is exemplified by the two
Grotta Sulfurea biofilms used for clone library construction
and FISH sample pairs RS03-46 (cottony)/RS03-10 (feathery)
and RS05-21 (cottony)/RS05-22 (feathery). This phenomenon
FIG. 4. Rarefaction curves (A) and percentage of clones in major bacterial lineages (B) for stream biofilm 16S rDNA clone libraries. WM and
zEL are clones from this study. OTU, operational taxonomic unit.
5602 MACALADY ET AL. APPL.ENVIRON.MICROBIOL.

suggests that bulk stream water geochemical measurements
are not sensitive enough to detect ecologically meaningful vari-
ations in sulfide and oxygen concentrations which may control
biofilm spatial and temporal distributions. Measured sulfide
concentrations at Ramo Sulfureo ranged from 0.2 ␮M (May)
to 212 ␮M (August), consistent with the seasonal range of
sulfide concentrations observed in previous long-term hydro-
logic studies at the Ramo Sulfureo stream sample site (3).
Oxygen concentrations in the bulk stream waters varied over
a much smaller range (2.6 to 12.6 ␮M). Data from Lower Kane
Cave (8) show that Thiothrix-related 16S rDNA clones are not
common in biofilm communities until dissolved oxygen con-
FIG. 5. Bayesian phylogenetic analysis of Grotta Sulfurea white biofilm 16S rDNA clones grouping within the ␥-proteobacteria. Clones from
this study are indicated in large bold type (WM and zEL clones). Filled black circles indicate nodes supported by Bayesian posterior probabilities
of ⱖ90% and maximum parsimony bootstrap values of ⱖ90%. Filled gray circles indicate nodes supported by Bayesian posterior probabilities
of ⱖ75% and maximum parsimony bootstrap values of ⱖ75%. Open circles indicate nodes supported by Bayesian posterior probabilities of ⱖ75%
or maximum parsimony bootstrap values of ⱖ75%. CFB, Cytophaga-Flexibacter-Bacteroides.
VOL. 72, 2006 LIMESTONE-CORRODING CAVE STREAM BIOFILMS 5603

FIG. 6. Bayesian phylogenetic analysis of Grotta Sulfurea white biofilm 16S rDNA clones grouping within sulfur-reducing clades of the
␦-proteobacteria. Clones from this study are indicated in large bold type (WM and zEL clones). Filled black circles indicate nodes supported by
Bayesian posterior probabilities of ⱖ90% and maximum parsimony bootstrap values of ⱖ90%. Filled gray circles indicate nodes supported by
Bayesian posterior probabilities of ⱖ75% and maximum parsimony bootstrap values of ⱖ75%. Open circles indicate nodes supported by Bayesian
posterior probabilities of ⱖ75% or maximum parsimony bootstrap values of ⱖ75%.
5604 MACALADY ET AL. APPL.ENVIRON.MICROBIOL.

FIG. 7. Bayesian phylogenetic analysis of Grotta Sulfurea white biofilm 16S rDNA clones grouping within the ε-proteobacteria. Clones
from this study are indicated in large bold type (WM clones). No ε-proteobacterial clones were retrieved from the cottony biofilm sample
(zEL clones). Filled black circles indicate nodes supported by Bayesian posterior probabilities of ⱖ90% and maximum parsimony bootstrap
values of ⱖ90%. Filled gray circles indicate nodes supported by Bayesian posterior probabilities of ⱖ75% and maximum parsimony bootstrap
values of ⱖ75%. Open circles indicate nodes supported by Bayesian posterior probabilities of ⱖ75% or maximum parsimony bootstrap values of ⱖ75%.
VOL. 72, 2006 LIMESTONE-CORRODING CAVE STREAM BIOFILMS 5605

centrations reach at least 10 to 20 ␮M. In contrast, the dis-
solved oxygen concentration in the growth zone of Beggiatoa
species is reported to be lower, in the range of 1 to 2.5 ␮M
(33). Notably, feathery Thiothrix-like biofilms were observed
only in turbulent water, where oxygen diffusion from the cave
atmosphere would be much faster than in slowly flowing water
hosting fine sediment and cottony biofilms. We also observed
thick accumulations (⬃2 cm) of Thiothrix-like filaments at a
sulfidic spring outflow draining the cave system (J. L. Macalady,
unpublished results). Oxygen concentrations in the spring water
were high (84 ␮M) compared to those for streams inside the cave.
We conclude that oxygen availability is an important factor
controlling the spatial distributions of Frasassi Beggiatoa
and Thiothrix populations and that Beggiatoa populations
thrive in microenvironments with slightly lower oxygen
availability than Thiothrix.
The ε-proteobacterial clones retrieved from the feathery
stream biofilm sample are closely related to sulfur-oxidizing
strains or clones from sulfide- and sulfur-rich environments,
suggesting that ε-proteobacteria play a role in S oxidation at
Frasassi. Although ε-proteobacteria were not retrieved in the
cottony biofilm clone library, ε-proteobacteria were consis-
tently detected in cottony biofilms in FISH experiments using
probe EP404. These results are not contradictory, since our
clone libraries did not catalog the bacterial diversity in the
samples exhaustively (Fig. 4A), and since PCR bias may have
influenced the composition of the clone libraries. Regardless,
ε-proteobacteria contributed only a minor fraction (0 to 15%)
of the biomass in the six biofilms we examined using FISH. The
EP404 probe used in this study matches more than 90% of
ε-proteobacteria sequences in sulfur-oxidizing clades that are
available from public databases, including sequences from sul-
fur-rich environments such as sulfidic caves. Although it is
FIG. 8. FISH micrographs of cottony biofilm samples GS04-15 (A),
RS03-46 (B), and RS05-22 (C) hybridized with EUBMIX (green) and
BEG811 (red) probes. Cells which hybridized with both probes appear
yellow/orange. The scale bars are 10 ␮m.
FIG. 9. FISH micrographs of cottony biofilm samples GS04-15
(A) and RS03-46 (B) hybridized with EUBMIX (green) and DELTA495a
(red) probes. Cells which hybridized with both probes appear yellow/
orange. The scale bars are 10 ␮m.
5606 MACALADY ET AL. APPL.ENVIRON.MICROBIOL.

possible that the EP404 probe failed to detect one or more
ε-proteobacteria populations in the stream biofilms, other
FISH results dictate that these populations would represent a
small fraction of the biomass compared to Beggiatoa-like or
Thiothrix-like cells (Table 2). In addition, the EP404 probe
hybridized with abundant ε-proteobacteria filaments in an
isolated veil-like biofilm sampled from a stagnant Frasassi
cave pool with very low dissolved oxygen content (0.6 ␮M)
(Macalady, unpublished), confirming that it detects popula-
tions of ε-proteobacteria in the cave system.
Previous carbon isotope studies conducted at the Ramo Sul-
fureo stream site further support the conclusion that ε-pro-
teobacteria are minor contributors to the biomass of the
stream biofilms colonizing the site. Sarbu et al. (37) found that
isotopic fractionation (⌬␦
13
C) between dissolved bicarbonate
and stream biofilm biomass carbon ranged from ⫺23 to
⫺30.5‰ (37), consistent with autotrophs detected using the
pentose phosphate cycle. The observed fractionation is too
large for autotrophs detected using the reductive tricarboxylic
acid cycle, for which ⌬␦
13
Cis⬃⫺3to⫺13 ‰ (49). Recent
studies using enzyme assays and genome sequencing methods
strongly suggest that ε-proteobacterial autotrophs use the re-
verse tricarboxylic acid cycle and lack enzymes for carbon
fixation via the pentose phosphate cycle (23, 41, 43).
A likely explanation for the dominance of ␥-proteobacteria
in the stream biofilms is that oxygen concentrations in the
streams are maintained above the range preferred by ε-pro-
teobacteria. This hypothesis is supported by hydrologic studies
showing that the cave streams are mixtures containing up-
welling sulfidic water and oxygenated percolating water, with
the oxygen-saturated water contribution ranging from 35 to
65% (3). Our results are consistent with data from Lower Kane
Cave, which hosts biofilms dominated by filamentous ε-pro-
teobacteria in spring water with ⬍0.2 ␮M dissolved oxygen (8).
Engel et al. (9) showed that microorganisms directly influ-
ence the rate of limestone dissolution in Lower Kane Cave
springs because of their close physical proximity to limestone
surfaces and because they are microaerophiles which can con-
sume sulfide before it comes into contact with high concentra-
tions of dissolved oxygen. Sulfur-oxidizers using nitrate as an
electron acceptor (equation 3) could extend the zone of sulfide
oxidation into anoxic niches but would produce one-fifth as
much acid per mole of sulfide than sulfide oxidation with ox-
ygen (equation 4):
5H2S⫹8NO3⫺34N2⫹5SO42⫺⫹4H2O⫹2H⫹(3)
5H2S⫹10O235SO42⫺⫹10H⫹(4)
Nitrate and nitrite concentrations in Frasassi cave streams are
below detection limits (⬍2 and ⬍0.7 ␮M, respectively) despite
the presence of abundant dissolved ammonium (35 to 74 ␮M)
and detectable oxygen (2.6 to 12.6 ␮M), suggesting that nitrate
is effectively scavenged by anaerobic microorganisms. Many
sulfur oxidizers are facultative anaerobes, including Beggiatoa
and Thiothrix species (34) and some ␤- and ε-proteobacteria
(45). Denitrifying sulfide-oxidizers are numerically abundant in
floating white biofilms in Movile Cave, based on culturing
experiments (35). Future work will investigate whether nitrate
plays a major role in S-oxidizer metabolism in Frasassi stream
biofilm communities.
Sulfur reducers. Sulfidic cave biofilms examined to date
using molecular methods either did not contain detectable
␦-proteobacteria (Cesspool Cave [7] and Parker Cave [2]) or
contained a small percentage of clones related to Desulfocapsa
FIG. 10. FISH micrographs of feathery biofilm samples hybridized
with EUBMIX (green) and GAM42a (red) probes. Cells which hybrid-
ized with both probes appear yellow/orange. The scale bars are 10 ␮m.
VOL. 72, 2006 LIMESTONE-CORRODING CAVE STREAM BIOFILMS 5607

thiozymogenes (Lower Kane Cave [8]). In contrast to previous
sulfidic cave studies, ␦-proteobacteria in Frasassi cave stream
biofilms were abundant and diverse and included numerous
phylotypes related to Desulfocapsa (14 phylotypes) and Desul-
fonema (7 phylotypes) species, in addition to Syntrophobacter
species, Syntrophus species, Desulfoarculum baarsii,Desulfo-
bacter postgatei,Desulfomonile species, and the Geobacter-
aceae. Probes SRB385 and DELTA495a detected bacterial
populations with diverse morphologies in both biofilm types.
Although it is possible that some of the populations identified
by these probes are not sulfur reducers, the FISH results are
consistent with the abundance and diversity of clones retrieved
in 16S rDNA libraries.
Desulfonema-related clones were the second-most-abundant
␦-proteobacterialgroup retrieved in clone libraries, and their
close relationships to sequences from cultivated Desulfonema
spp. strains is supported by phylogenetic analyses (Fig. 6). In
two of the cottony biofilm samples, Beggiatoa filaments were
associated with densely intertwined, filamentous bacteria that
hybridized with probe DELTA495a (Fig. 10). Although probe
DELTA495a hybridizes with members of the poorly known
lineage Gemmatimonadetes in addition to sulfate-reducing
␦-proteobacteria, it is highly likely, based on morphology, that
the filaments are related to members of the genus Desul-
fonema.Desulfonema species strains oxidize short-chain ali-
phatic acids and/or alcohols to CO
2
. Close physical associations
between filamentous sulfur-oxidizing bacteria and filamentous
␦-proteobacteria have been observed in organic-rich marine and
lacustrine surface sediments, where Desulfonema filaments are
epibionts on large sulfur-oxidizing filaments such as Thioploca
spp. (12). Desulfonema spp. are the dominant sulfate-reducing
bacteria in permanently oxic regions of hypersaline cyanobacte-
rial biofilms (32) and are widespread in freshwater and marine
environments, where their gliding motility allows them to exploit
oxic-anoxic interfaces (36, 47). Both cottony and feathery biofilm
clone libraries contained sequences related to Desulfonema spe-
cies. However, the greater abundance of putative Desulfonema
filaments we observed in cottony biofilms using FISH probe
DELTA495a (Table 2) is consistent with their preference for
steep oxic-anoxic interfaces, gliding motility, and previously noted
associations with Beggiatoa.
Pure cultures of Desulfocapsa spp. grow autotrophically by
disproportionation of sulfite, thiosulfate, or elemental sulfur
and require low sulfide concentrations for efficient growth (11).
Sulfide scavenging in nature can be provided by sulfide-com-
plexing metals or by sulfide-oxidizing bacteria living in close
proximity. Desulfocapsa-like bacteria have been identified in
meromictic Lake Cadagno, where they likely derive an energy
benefit by living in aggregates with sulfide-oxidizing pho-
totrophs (48). Desulfocapsa isolates can also grow as either
sulfur or sulfate reducers (11). Based on strong bootstrap and
Bayesian posterior probability support for the placement of
Frasassi clones within the Desulfocapsa clade (Fig. 6) and the
fact that many are distant from cultivated representatives of
the genus (95% identity), they likely represent novel ecological
types warranting further study.
The abundance and phylogenetic diversity of sulfate- and
sulfur-reducing bacteria in Frasassi stream biofilms indicates
that they are the locus of intensive S cycling within the cave
system. Such cycling has been suggested by previous S isotopic
studies (13). The association of sulfur oxidizers and sulfur
reducers has special implications for the microbial ecology of
Frasassi biofilms and for the geochemistry of sulfidic caves.
Based on geochemical data, sulfide is at least transiently scarce
(as low as 0.2 uM) in the bulk stream water at Ramo Sulfureo
due to seasonal changes in hydrology, whereas dissolved sul-
fate is always abundant (1 to 2 mM). The presence of sulfur-
disproportionating and sulfate-reducing bacteria in the stream
biofilms thus provides a buffer for sulfur oxidizers against tem-
poral swings in the availability of sulfide. Sulfate reduction also
represents a pathway by which primary productivity from elec-
tron donors other than sulfide (e.g., ammonium, hydrocarbons,
etc.) can be recycled to fuel sulfuric acid production and dis-
solution at limestone surfaces. An analogous process driven by
photosynthetic primary productivity causes globally significant
sulfuric acid carbonate dissolution in productive marine shelf
sediments (26). The formation of biofilms containing both S
oxidizers and S reducers therefore represents biological feed-
back to sulfuric acid cave formation and similar processes
which create subsurface porosity in limestone rocks.
ACKNOWLEDGMENTS
We thank Alessandro Montanari for logistical support and the use
of facilities and laboratory space at the Osservatorio Geologico di
Coldigioco. We also thank Laura Cleaveland, Annaliese Eipert, Paola
D’Eugenio, Dan Jones, and Ani Kameenui for field assistance, Joel
Moore for TOC analyses, and Yohey Suzuki for insightful discussions.
E.H.L. (Carleton College), L.K.A. (Brown University), B.K. (Carleton
College), and K.M. (Carleton College) contributed to this work while
undergraduate students.
B.K. and K.M. thank Carleton College for financial support. L.K.A. was
supported in part by undergraduate research funds from the Penn State
Astrobiology Research Center (PSARC; NASA award NNA04CC06A).
This study was funded by the Biogeosciences Program of the National
Science Foundation (EAR 0311854).
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