Extremely acidic, pendulous cave wall biofilms from the Frasassi cave system, Italy

Extremely acidic, pendulous cave wall biofilms fromthe Frasassi cave system, Italy

Jennifer L. Macalady,* Daniel S. Jones andEzra H. LyonDepartment of Geosciences, Pennsylvania StateUniversity, University Park, PA 16802, USA.

Summary

The sulfide-rich Frasassi cave system hosts anaphotic, subsurface microbial ecosystem includingextremely acidic (pH 0–1), viscous biofilms (snot-tites) hanging from the cave walls. We investigatedthe diversity and population structure of snottitesfrom three locations in the cave system using fullcycle rRNA methods and culturing. The snottiteswere composed primarily of bacteria related toAcidithiobacillus species. Other populations presentin the snottites included Thermoplasmata grouparchaea, bacteria related to Sulfobacillus, Acidimi-crobium, and the proposed bacterial lineage TM6,protists, and filamentous fungi. Based on fluores-cence in situ hybridization population counts,Acidithiobacillus are key members of the snottitecommunities, accompanied in some cases bysmaller numbers of archaea related to Ferroplasmaand other Thermoplasmata. Diversity estimates showthat the Frasassi snottites are among the lowest-diversity natural microbial communities known, withone to six prokaryotic phylotypes observed depend-ing on the sample. This study represents the firstin-depth molecular survey of cave snottite microbialdiversity and population structure, and contributesto understanding of rapid limestone dissolution andcave formation by microbially mediated sulfuric acidspeleogenesis.

Introduction

Sulfidic caves form in carbonate rocks where sulfide-richwaters interact with oxygen at the water table or atsubterranean springs. Limestone dissolves as a result ofsulfuric acid production (Eq. 1) from microbial or abioticsulfur oxidation. The sulfuric acid reacts with carbonatehost rock to form gypsum and carbonic acid (Eq. 2).

H S O H SO2 2 2 42+ → (1)

CaCO H SO CaSO H CO3 2 4 4 2 3+ → + (2)

Some of the longest caves known have formed by thisprocess, including Lechugilla Cave in New Mexico, USAwith 184 km of passages (Hill, 1995).

The Grotta Grande del Vento-Grotta del Fiume(Frasassi) cave complex in central Italy is a large, activelyforming sulfidic cave, hosting more than 23 km ofpassages (Fig. 1). The cave system is hosted in Jurassicplatform limestone (Calcare Massiccio Formation) in theApennine Mountains of the Marches Region, Central Italy(Fig. 1). A thick sequence of Triassic evaporites andorganic-rich limestones (Burano Formation, > 1000 mthick) underlies the platform limestone and is the likelysource of high salinity and dissolved sulfur in the aquiferintersecting the cave system. The active, sulfidic level ofthe cave is at the elevation of the Sentino River, whichflows 600–700 m below mountains on either side of theFrasassi gorge. Based on uranium series dating ofcarbonate speleothems, the highest cave level beganforming approximately 200 000 years ago (Taddeucciet al., 1992). Large gypsum deposits throughout theupper levels of the cave are analogous to gypsum crustscurrently forming and depositing in the lowest level, sug-gesting a similar mode of cave formation throughout thehistory of the cave. Sulfidic water and air are currentlyfound only in the lowest cave level. The cave streams andsprings have geochemical compositions that vary season-ally and spatially between well-defined end members dueto mixing between rising sulfidic water and oxygenatedmeteoric water percolating downward through ~600 m oflimestone (Sarbu et al., 2000; Cocchioni et al., 2003).Abundant sulfur-cycling microbial biofilms coating sur-faces below the water table have recently been described(Macalady et al., 2006). Variations in the cave air andwater temperatures are within 1–2°C throughout the yearand humidity is consistently near 100%.

Snottites are extremely acidic (pH 0–1), viscousbiofilms. Few studies on cave snottites have beenpublished previously. Vlasceanu and colleagues (2000)obtained two bacterial clones related to Sulfobacillusand Acidithiobacillus species and a Halothiobacillus-related isolate from a Frasassi snottite sample (Vlas-ceanu et al., 2000). Hose and colleagues (2000)reported 19 bacterial clones retrieved from snottites in

Received 13 July, 2006; accepted 17 December, 2007. *Forcorrespondence. E-mail [email protected]; Tel. (+1) 814 8656330; Fax (+1) 814 863 7823.

Environmental Microbiology (2007) doi:10.1111/j.1462-2920.2007.01256.x

© 2007 The AuthorsJournal compilation © 2007 Society for Applied Microbiology and Blackwell Publishing Ltd

mailto:[email protected]

Cueva de la Villa Luz, Mexico as related to ‘Thiobacillus’spp. (probably Acidithiobacillus) and Acidimicrobium fer-rooxidans, but the sequences are not available in publicdatabases and phylogenies of the clones were notdescribed in detail (Hose et al., 2000). Archaea in cavesnottites have not previously been reported. Here wedescribe the seasonal population structure of bacteria,archaea and eukaryotes in snottite communities fromthree sample locations within the Frasassi cave system.Our results show that bacteria closely related toAcidithiobacillus thiooxidans are ubiquitous and abun-dant members of the snottite communities, accompaniedin some cases by acidophilic archaea in the Thermo-plasmata clade and other acidophiles.

Results

Snottite occurrence and geochemistry

Cave walls near sulfidic streams in the lower level of thecave were coated with crusts of microcrystalline gypsum(CaSO4·6H2O), which reached up to several centimetresin thickness. Snottites hung from the surface of themicrocrystalline gypsum, sometimes associated with S°,or from clusters of macroscopic gypsum needles. Thesnottites developed between 0.5 and 4 m above thewater table. The biofilms were hydrophobic and pinkcoloured, with pH values ranging between 0 and 1.8.Three snottite sample locations (PC1, RS1, RS2) werechosen to encompass the mineralogical and morphologi-cal diversity observed in snottites throughout the sulfidiclevel of the cave system (Fig. 2). Snottites from allsample locations were more acidic in August, when RS1and RS2 snottites had pH values < 1, and PC1 snottiteshad pH values between 0 and 1.5 (Table 1). MeasuredH2S concentrations at snottite sample locations werebetween 0.2 and 24 parts per million (p.p.m.). Replicategas measurements made in a given location during thesame day were within 20%. In contrast, fivefold differ-ences in H2S concentrations were typically measuredover less than 1 m of vertical distance and over less than3 m horizontal distance along stream channels. Generalcharacteristics of Frasassi snottite environments werecompared against a control site lacking snottites(Table 1).

Clone libraries

Population counts using bacterial (EUBMIX) andarchaeal (ARCH915) probes in fluorescence in situhybridization (FISH) experiments indicated that snottitesfrom PC1 contained only bacterial cells, whereas RS1and RS2 snottites contained a mixture of bacteria andarchaea. Bacterial 16S rDNA clone libraries were con-structed for PC1 and RS2 snottites. No PCR productswere obtained from the RS2 sample with the archaea-specific primer set 21f (5′-TTCCGGTTGATCCYGCCGGA-3′) and 1492r. However, a clone library constructed

Grotta SulfureaSentino River

Grotta Grande del Vento

Grotta del Fiume

Grotta Bella

Ramo Sulfureo

PC1

RS2RS1

500 m

Fig. 1. Map of the Frasassi cave system showing major (named)caves in different shades of grey. Topographic lines and elevations(in meters) refer to the surface topography above the cave.Sampling locations in Grotta del Fiume (including Ramo Sulphureo)are shown as open circles annotated with sample names. Basemap courtesy of Sandro Mariani.

Table 1. Physical and chemical characteristics of snottite sample locations.

Sample location PC1 RS1 RS2 Control site

Cave wall mineralogy Microcrystalline gypsum Gypsum needles Microcrystalline gypsum with S° CalcitepH (May/August) nd/< 1.5 1.8/< 1.0 1.5/< 1.0 5.6H2S (p.p.m.) 0.2–2 3.4 24 < 0.2CO2 (p.p.m.) 1200 > 3000 > 3000 1300Height above water table (m) 0.5–1.5 4 1 40

Data from a nearby site at higher elevation without snottites or gypsum crusts are given for comparison. NH3 and N2O were also sampled, but werebelow detection limits (< 0.25 and < 0.5 p.p.m. respectively).

2 J. L. Macalady, D. S. Jones and E. H. Lyon

© 2007 The AuthorsJournal compilation © 2007 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology

using a universal forward primer (533f) yielded both bac-terial and archaeal clones. Clone library results are sum-marized in Table 2.

All clones in the PC1 library (71 sequences) were asso-ciated with a single phylotype related to Acidithiobacillusspecies. The majority of clones in the RS2 snottite librar-ies also belonged to this phylotype (~65% of clones). Thesecond most abundant phylotype in the RS2 bacteriallibrary (17 clones, 31%) was closely related to Acidimicro-bium ferrooxidans. Rare phylotypes were affiliated withSulfobacillus species, environmental clones in the pro-posed TM6 lineage, putative mitochondrial sequences(alphaproteobacteria), and archaea in the Thermoplas-mata clade.

Phylogenetic relationships among the snottite bacteriaand closely related isolates and environmental clones areshown in Fig. 3. Bacterial sequences retrieved usinguniversal primers were identical to sequences in the bac-terial library, and were not used for phylogenetic treeconstruction. Most Frasassi sequences were closelyrelated to isolates and environmental clones from acidmine and acid rock drainage, bioleaching environments,and thermal sulfidic springs and vents. Frasassi clonesin the Acidithiobacilli grouped closely with A. thiooxi-dans and A. albertensis, and more distantly withA. ferrooxidans, and A. caldus, regardless of phylogeneticmethods. Acidithiobacillus and Sulfobacillus clonesretrieved by Vlasceanu and colleagues (2000) in anearlier study of a Frasassi snottite (Fras 2 and Fras 1respectively) were closely related but not identical toclones retrieved in the PC1 and RS2 clone libraries. Wealso retrieved sequences that grouped with environmentalclones in the poorly known and uncultivated TM6 lineage.Close relatives of these clones were sequences fromdiverse, near neutral-pH environments such as sewagesludge, marine mud volcanoes, peat bogs andgroundwater.

Two archaeal phylotypes were identified in the RS2clone library constructed using universal primers. Thephylogeny of the archaeal clones is shown in Fig. 4. Bothphylotypes fell within a clade containing the wall-lessarchaeal genera Ferroplasma and Thermoplasma. Themore abundant phylotype was related to F. acidiphilumand other isolates and clones from acid mine drainage,hot spring and bioleaching environments. The secondphylotype was most closely related to environ-mental clones from acid mine drainage, the RioTinto (Spain), and a Yellowstone hot spring endolithcommunity.

In order to obtain statistical estimates of taxonomicdiversity, rarefaction curves and non-parametric diversityestimators were computed for the two bacterial 16SrDNA libraries and for the combined sequences in bac-terial and universal libraries constructed from sample

RS1

RS2

PC1

Fig. 2. Snottites sampled in this study photographed in situ (seeFig. 1 for sample locations). PC1 snottites were suspended frommicrocrystalline gypsum crusts. RS1 snottites were developed onlarge gypsum needles. RS2 snottites were sampled frommicrocrystalline gypsum crusts associated with elemental sulfur.Scale bars are 2 cm.

Extremely acidic cave snottites 3


RS2. Results based on operational taxonomic units(OTUs) grouped by > 98% sequence similarity areshown in Table 3. Results for alternative OTU cut-offswere similar (data not shown). The observed diversity ofall three libraries was extremely low, with nine OTUs insix lineages detected in total. Diversity estimates werealso very low, with good agreement between observedand estimated OTUs. For the RS2 combined bacterialand universal libraries, the Chao1 estimate for 107clones sampled was 10 OTUs, only slightly higher thanthe estimate for 54 clones sampled. The low diversity ofsnottite clone libraries contrasted with a clone libraryconstructed using identical methods from a neutralbiofilm present at a nearby cave wall control site lackingsnottites (see Table 1 for geochemistry). Chao1 andACE diversity estimates for the neutral pH control biofilmwere more than an order of magnitude higher than forthe snottites (Table 3).

Fluorescence in situ hybridization

4′,6′-diamidino-2-phenylindole (DAPI) staining and FISHexperiments were employed to evaluate bacterial andarchaeal population dynamics in the snottite samples.PC1 and RS2 clone library sequences were comparedagainst previously published probes specific for Acidi-thiobacillus species (THIO1), Acidimicrobium species(ACM732) and Ferroplasma species (FER656). Theseprobes were found to target the Frasassi clones with nomismatches. The probes were also compared againstpublic databases at NCBI to evaluate their specificity.Probe THIO1 targets all known environmental and isolatesequences within the Acidithiobacillus clade with no mis-matches, and has two mismatches to the next nearestneighbour, Thermithiobacillus tepidus. Other sequenceshave at least three mismatches to the probe. The ACM732probe targets close relatives of Acidimicrobium

Table 2. Summary of three Frasassi 16S rDNA clone libraries.

Snottite PCR forward primer Taxonomy of nearest relatives No. of clones Representative clone(s) Inferred physiology

PC1 Bacterial 27f BacteriaGammaproteobacteria

Genus Acidithiobacillus 71 FS45 Sulfur oxidizer, autotrophRS2 Bacterial 27f Bacteria

GammaproteobacteriaGenus Acidithiobacillus 34 DSJB72 Sulfur oxidizer, autotroph

ActinobacteriaGenus Acidimicrobium 17 DSJB22 Sulfur oxidizer?Sulfobacilli

Genus Sulfobacillus 1 DSJB13 Sulfur oxidizerTM6 lineage 3 DSJB94, DSJB30 Unknown

Universal 533f BacteriaGammaproteobacteria

Genus Acidithiobacillus 34 DSJA15 Sulfur oxidizer, autotrophActinobacteria

Genus Acidimicrobium 5 DSJA12 Sulfur oxidizer?TM6 lineage 2 DSJA63 UnknownAlphaproteobacteria

Mitochondria 3 DSJA62 OrganelleArchaeaThermoplasmata

Genus Ferroplasma 6 DSJA4 Sulfur/Corg oxidizer?‘Cplasma’ 1 DSJA51 Sulfur/Corg oxidizer?

Taxonomy and inferred physiology were determined based on phylogenetic analyses.

Table 3. Characteristics of 16S rDNA clone libraries, including observed diversity and diversity calculated using rarefaction and non-parametricestimators.

Sample location Clone names Total clones OTUsObserved OTUs(rarefaction)a � SD

Estimated OTUs(Chao1)a � SD

Estimated OTUs(ACE)a � SD

PC1 FS 71 1 1 � 0 1 � 0 1 � 0RS2 DSJB 54 5 5 � 0 6 � 1 8 � 0RS2 DSJB, DSJA 107 9 7 � 1 10 � 5 11 � 4Control – 67 48 41 � 2 135 � 74 159 � 42

a. Values shown are for 54 clones, the highest shared level of sampling among libraries.



ferrooxidans. Sequences outside the Acidimicrobidaehave at least three mismatches. The FER656 probetargets all environmental sequences and isolates in theFerroplasma clade, but has at least three mismatcheswith other sequences within the Thermoplasmata andother prokaryotic and eukaryotic ribosomal sequences.

The genus-specific probes described above were usedin combination with domain-specific probes for bacteria(EUBMIX) and archaea (ARCH915), and with probe

GAM42a targeting gammaproteobacteria. A complete listof probes and hybridization stringencies used in this studyis shown in Table 4. Snottite samples collected in May(high water table) and August (low water table) were com-pared in order to identify the effects of changing streamgeochemistry and hydrology on snottite ecology (Figs 5and 6). Population counts using FISH probes are shownin Fig. 5. Results for probe GAM42a were identical toprobe THIO1 for all samples, indicating that all gam-

Fig. 3. Bayesian phylogenetic analysis ofbacterial 16S rRNA genes. Clones from thisstudy are shown in bold type, followed by thenumber of clones with similarity values > 98%shown in parentheses. Bayesian posteriorprobabilities (left) and maximum parsimonybootstrap values (right) are shown at eachnode. Dashes indicate bootstrap values below50%.

Acidithiobacillus caldus (Z29975)Acidithiobacillus thiooxidans ATCC 19377 (AJ459803)

FS45 (71)Frasassi snottite clone Fras2 (AF213056)

DSJB72 (68)Acidithiobacillus thiooxidans (Y11596)Acidithiobacillus albertensis (AJ459804)Acidithiobacillus ferrooxidans (AJ278719)

Thermithiobacillus tepidarius (459801)

Agrobacterium tumefaciens (D01261)Azospirillum brasilense (Z29617)

Wolinella succinogenes (M26636)

Arcobacter nitrofigilis (L14627)Thiomicrospira denitrificans (L40808)

Desulfococcus multivorans (AF418173)

Peat bog clone TM6 (X97099)

Sludge clone Ebpr6 (AF255637)

DSJB30 (1)DSJB94 (4)Freshwater clone PRD01a004B (AF289152)Humic lake clone CrystalBog2E1 (AY792307)

Manure water clone 144ds20 (AY212595)

Contamin. water clone NABIR-FRC (AY661981)Mud volvano clone BC20-2B-15 (AY592375)

Pelobacter propionicus (AAJH01000022)

Rhodococcus ruber (X80625)

Nocardia asteroidea (X57949)Acidimicrobium ferrooxidans (U75647)

Hot spring clone SK300 (AY882849)

DSJB22 (22)Forested wetland clone RCP1-77 (AF523906)

Ferromicrobium acidophilum (AF251436)

Acid streamer CS11 (AY765999)

Hot spring clone OPB55 (AF026993)

Acid mine drainage clone BA84 (AF225451)

Sulfobacillus acidophilus (M79375)

Bioleaching clone JTC05 (AY805540)

Sulfobacillus sp. str. 4G (AY213055)

Sulfobacillus thermosulfooxidans (AB089844)Sulfobacillus thermotolerans (DQ124681)

Frasassi snottite clone Fras1 (AF213055)DSJB13 (1)

Thermal soil clone YNPFFP2 (AF391985)

Methanosarcina barkeri (M59144)Halorubrum saccharovorum (X82167)

Acidianus brierleyi (X90477)0.05 substitutions/site

100/100

54/-

95/98

100/100

100/100

100/100

100/96

100/72

100/100

100/88

100/62

100/-

100/-

100/100

83/-

65/-

100/-

100/100

100/-

100/85

87/-

100/-

100/100

100/97

92/82

100/96

100/100

100/63

93/-

100/-

100/96

95/72

100/99

92/-

99/-

100/98

100/100

100/100

99/85

100/100

Actinobacteria

δ

TM6

ε

α

γ

Sulfobacilli

Archaea

Pro

teo

ba

cte

ria

Table 4. Oligonucleotide probes used in this study.

Probe Target group Sequence (5′ → 3′) % formamide Reference

EUB338a Most Bacteria GCT GCC TCC CGT AGG AGT 0–50% Amann et al. (1990)EUB338-IIa Planctomycetales GCA GCC ACC CGT AGG TGT 0–50% Daims et al. (1999)EUB338-IIIa Verrucomicrobiales GCT GCC ACC CGT AGG TGT 0–50% Daims et al. (1999)ARCH915 Archaea GTG CTC CCC CGC CAA TTC CT 20% Stahl and Amann (1991)GAM42ab g-Proteobacteria GCC TTC CCA CAT CGT TT 35% Manz et al. (1992)THIO1 Acidithiobacillus spp. GCG CTT TCT GGG GTC TGC 35% Gonzalez-Toril et al. (2003)ACM732 Acidimicrobium ferrooxidans

and close relativesGTA CCG GCC CAG ATC GCT G 35% Bond and Banfield (2001)

FER656 Ferroplasma spp. CGT TTA ACC TCA CCC GAT C 25% Edwards et al. (2000)

a. Combined in equimolar amounts to make EUBMIX.b. Requires competitor oligonucleotide cGAM42a (GCC TTC CCA CTT CGT TT).



maproteobacteria detected by GAM42a also hybridizewith probe THIO1 (data not shown).

Three bacterial (EUBMIX) morphologies wereobserved, including THIO1-hybridizing short rods(1–2 mm long, 1 mm thick), ACM732-hybridizing thinvibrios (3–6 mm long), and thicker ovoid cells not targetedby genus-level probes (1–2 mm long, 2 mm thick). THIO1-labelled cells varied little in size and shape, but occurredeither as isolated cells, loose chains, or nearly continuouschains approximating filaments. No sheath structureassociated with chains or filaments was observed underepifluorescence, phase, or differential interference con-trast microscopy. In addition, two archaeal (ARCH915)morphologies were observed. Large, irregularly shapedcells (1–3 mm) hybridized with the FER656 probe.Coccoid cells of varying sizes (< 1–2 mm) only sometimeshybridized with FER656, consistent with the presence ofat least one additional archaeal population. We alsoobserved large ovoid cells (> 5 mm) with nuclei, presum-ably protists, and weakly autofluorescing fungal filamentswith evenly spaced, DAPI-staining nuclei. Fungal fila-ments were branching and were associated with autofluo-rescent spores (> 5 mm) and fruiting structures.

THIO1-hybridizing cells were the most abundant popu-lations at all three snottite locations. PC1 had the simplestcommunity structure. Nearly all prokaryotic cells in thesample hybridized with probe THIO1. Very few archaeawere detected using probes ARCH915 or FER656 (< 2%of DAPI-stained cells). The May sample lacked eukary-

otes, but in the August sample fungal filaments, fungalspores and abundant protists were observed. DAPIcounts suggested a ‘missing’ population not detected bythe probes, which could account for up to 20% of cells,although no unique DAPI-staining morphologies wereobserved. The ‘missing’ population could be revealed bysequencing more clones, but may also contain 16S rDNAgenes which do not hybridize with the probes and primersused in this study.

The population structures of RS1 and RS2 snottiteswere similar to each other and slightly more complex thanPC1. RS1 and RS2 snottites both contained significantarchaeal populations (40% and 17% of DAPI-stained cellsrespectively). Roughly half of archaeal cells in RS1hybridized with FER656. In RS2, most archaea hybridizedwith FER656, with other archaea comprising the remain-ing 6%. RS1 and RS2 snottites also contained isolatedclusters of cells that hybridized with probe ACM732 (~4%and 7% of DAPI-staining cells respectively). Protists andfungal filaments and spores were abundant in the RS1and RS2 snottites with the exception of the May RS1sample which contained few protists and lacked fungi.DAPI and probe counts of both RS1 and RS2 snottitesshow that all major prokaryotic populations are accountedfor. Counts made using the EUBMIX probe are equivalentwithin error to counts made using THIO1 and ACM732combined. Likewise, counts made using DAPI are equiva-lent to counts made using EUBMIX and ARCH915combined.

Fig. 4. Bayesian phylogenetic analysis ofThermoplasmata group archaeal 16S rRNAgenes. Clones from this study are shown inbold type, followed by the number of cloneswith similarity values > 98% shown inparentheses. Bayesian posterior probabilities(left) and maximum parsimony bootstrapvalues (right) are shown at each node.Dashes indicate bootstrap values below 50%.

Picrophilus oshimae (X84901)

copper bioleach clone Aglo120 (AJ003138)Picrophilus torridus (NC_00587)

Ferroplasma acidophilum (AJ224936)

Ferroplasma sp. DR1 (AY222042)Ferroplasma sp. MT17 (AF513710)

DSJA4 (6)

Ferroplasma acidarmanus (AABC0400)

Ferroplasma cyprexacervatum (AY907888)hot spring clone MS14 (AF232925)

Ferroplasma sp. str. JTC3 (AY907888)AMD clone IMRP42 (AY789589)coal wetland clone ARCP160 (AF523941)

AMD clone AS4 (AF544223)

coal wetland clone ARCP127 (AF523941)

Thermoplasma sp. 739-a6 (AY350607)Thermoplasma acidophilum (NC_00257)Thermoplasma volcanium (AF339746)

DSJA51 (1)AMD clone AS1 (AF544221)

Tinto River biofilm clone (DQ303249)Yellowstone endolith clone (AY911452)

acidic hot spring clone M7 (AY627859)hot spring clone S207 (AY882761)

hot spring clone SK154 (AY882711)

hydrothermal vent clone plSA12 (AB019741)

Edmond vent field clone (AY251065)

Guaymas Basin clone G26_C56 (AF356635)S. solfataricus (X90483)

P. aerophilum (L07510)P. abyssi (X99559)


AMD clone ASL32 (AF544222)


Glenwood spring clone (AY702823)

Glenwood spring clone (AY702824)

AMD clone AS10 (AF544221)

0.01 substitutions/site

100/100

59/-

100/100

75/-

100/-

100/99

100/100

100/99

100/100

100/100

51/-

100/-100/64

100/60

100/68

100/82

78/66

59/-

100/-

75/83

100/100

100/100

100/100

100/100

100/100

93/98

97/88

100/95

outgroup

Ferro

pla

sm

a

“alphabet

plasmas”



Fluorescence in situ hybridization micrographs docu-menting cell morphologies and biofilm architecture areshown in Fig. 6. No strong differences were notedbetween the May and August samples from each loca-tion, except for the presence or absence of eukaryotes(as described above), and variations in the arrangementof Acidithiobacillus cells in the snottite matrix. In the MayPC1 and RS1 samples, THIO1-hybridizing cells occurredexclusively as isolated rods. August samples from thesetwo locations contained some cells arranged in filament-like chains. THIO1-labelled cells in the RS2 snottiteoccurred as a mixture of isolated cells and filaments

in both May and August (see Fig. 6 panels A, B, Eand F).

Discussion

Snottite diversity, phylogeny and population structure

Based on rarefaction and non-parametric diversityestimates Chao1 and ACE, Frasassi cave snottites areamong the simplest natural microbial communities known.Chao1 gives a lower bound for species richness becauseit does not account for rare OTUs (Hughes et al., 2001). Inaddition, PCR and cloning biases have the potential tocreate artefacts in diversity estimates based on clonelibraries. With these caveats, several interesting compari-sons can be made. The snottites had much lower diversitythan neutral pH biofilms developed nearby on the lime-stone of the cave host rock (Table 3), suggesting that pHplays a role in limiting species richness. An alternativehypothesis is that the higher energy flux due to sulfideavailability near sulfidic streams results in lower diversity,as has been observed in some macroscopic communities.The relatively low diversity of even the neutral pH cavewall biofilm compared with above-ground environmentssuch as soils (Hughes et al., 2001) suggests that thespatial isolation and relatively low geochemical complex-ity of the cave wall environment also play a role in limitingthe number of microbial species found there. The speciesrichness of the snottites is comparable with that reportedfor bioleaching and extreme acid mine drainage environ-ments, which typically host a low-diversity assemblage ofacidophilic microbial taxa including protists, fungi, bacte-ria and wall-less acidophilic archaea (Baker and Banfield,2003).

The physiologies of Frasassi clones, as far as they canbe inferred from close relatives, are consistent with theextremely acidic, oxic, aphotic, iron-poor and sulfur-richgeochemistry of the environment. Related Acidithiobacil-lus species such as A. thiooxidans and A. albertensis areautotrophs specializing in the oxidation of reduced sulfurspecies, including thiosulfate and elemental sulfur.Archaea closely related to Frasassi snottite clones includeboth autotrophs (Ferroplasma acidiphilum) and heterotro-phs, some of which also oxidize reduced sulfur com-pounds (Okibe et al., 2003). Related Sulfobacilli such asS. acidophilus are able to grow autotrophically or mix-otrophically on elemental sulfur, or as heterotrophs (Norriset al., 1996). Acidimicrobium ferrooxidans is not known tooxidize sulfur compounds, but grows as a heterotroph inextremely acidic environments. Previous studies haveshown that heterotrophs and mixotrophs may play animportant role in acidophilic communities by removing theorganic waste products of the primary producers (Bacelar-Nicolau and Johnson, 1999).

0

10

20

30

40

50

60

70

80

90

May August

0

10

20

30

40

50

60

70

80

90

% o

f D

AP

I-sta

ined c

ells

RS2

PC1

0

10

20

30

40

50

60

70

80

90

100RS1

not dete

rmin

ed

not dete

cte

d

not dete

cte

d

EUBMIX

THIO1

ACM732

ARCH915

FER656

Fig. 5. Snottite microbial community composition based on FISHcell counts for three locations sampled in May and August 2005.Cell counts are expressed as percentages of total DAPI-stainedcells. Error bars represent one standard deviation. Stars indicatepresence of Acidithiobacillus cells arranged into filament-like chains(see Fig. 6E), with larger stars where chains were more abundant(described in the text).



RS1

FER656

EUBMIX

DAPI

RS1

THIO1

DAPI

RS2

ARCH915

EUBMIX

DAPI

RS2

ARCH915

EUBMIX

DAPI

A

D

B

C

RS2

THIO1

EUBMIX

DAPI

G

E

RS2

ACM732

EUBMIX

DAPI

H

F

RS2

GAM42a

EUBMIX

DAPI

RS1

ACM732

EUBMIX

DAPI



Relatives of five Frasassi snottite clones are membersof the proposed bacterial lineage TM6. No cultivatedrepresentatives have been isolated from this lineage todate. Environmental clones closely related to Frasassiclones are neutrophilic and were retrieved from diverseanoxic and oxic environments including Antarctic marinesediments, anaerobic digesters, and peat and forestsoils (Rheims et al., 1996; Bowman et al., 2000; Liuet al., 2001). Previous surveys of acidic environmentshave not retrieved TM6 sequences, and therefore theFrasassi clones are the first extremely acidophilic popu-lations associated with this lineage. An alternativehypothesis is that the TM6-related clones are intracellu-lar symbionts not exposed to the extremely acidic pH ofthe snottite matrix. Ongoing work is aimed at identifyingthe metabolic role of TM6 lineage populations in thesnottites.

In a previous investigation of snottite microbiology atFrasassi, PCR amplification of bulk DNA with archaea-specific 16S rDNA primers (23f, 1492r) yielded no prod-ucts (Vlasceanu et al., 2000). The same primers alsofailed to amplify products from snottite DNA in our study,but both FISH experiments and PCR with universalprimers (533f, 1492r) demonstrated that archaea areabundant and important members of snottite communi-ties, comprising up to 40% of cells (Fig. 5). Clone DSJA51is related to a diverse group of wall-less euryarchaeotawith no cultivated relatives that have only recently beenisolated from acid mine drainage environments (Bakerand Banfield, 2003). The metabolic characteristics ofthese archaeal populations remain uncertain, but basedon environments where they have been retrieved, mayinclude organotrophy and/or iron and sulfur oxidation.Clone DSJA4 and five closely related snottite clonesgroup with Ferroplasma species including F. acidiphilum,which is able to grow as an autotroph (Golyshina et al.,2000). However, metabolic studies of other Ferroplasmastrains as well as environmental genomic data for Ferro-plasma type II species populations inhabiting the Rich-mond mine AMD site suggest that most members of the

wall-less euryarchaeota are adapted for heterotrophic ormixotrophic metabolism (Tyson et al., 2004).

Research on AMD has revealed an apparent correlationbetween lower pH environments and the dominance ofFerroplasma over Acidithiobacillus populations (Goly-shina and Timmis, 2005). In extreme AMD environmentswhere Ferroplasma are abundant, Leptospirillum popula-tions may fill the role of primary producers (Bond et al.,2000), suggesting that Ferroplasma and other wall-lessarchaea may have a similar ecological role in AMD andsnottites. Although we cannot say with certainty whatdetermines the relative abundances of archaea andAcidithiobacillus cells in the snottites, it is clear that lowerpH does not favour Ferroplasma or other archaea overthe pH range 0–2 in snottites we have examined to date(Table 1, Fig. 5). Factors in addition to pH which may beimportant include redox potential, which is dependent onmetabolic oxygen consumption rates and on the viscosityand size of snottites, predation and the amount of tempo-ral environmental variability (Bond et al., 2000).

Implications for speleogenesis

Abiotic sulfide oxidation rates are generally slow, withmarkedly slower reaction rates at pH values below 4(Millero et al., 1987). Half-lives for sulfide at 13°C inoxygen-saturated solutions of ionic strengths betweenfresh and seawater range between 50 and 250 h, sug-gesting an important role for microbial sulfide oxidation,especially at low pH. In addition to direct enzymatic oxi-dation by microorganisms, sulfide oxidation is enhancedin the presence of hydrogen peroxide (Hoffmann, 1977), acommon by-product of aerobic metabolisms.

In caves where sulfide is present at concentrationsabove a few parts per million, snottites are abundant oncave walls above and near sulfidic water. Snottites havebeen observed in caves including Frasassi (Galdenzi andMaruoka, 2003), Grotta di Rio Garrafo (Jones et al.,2006), Cueva de la Villa Luz (Hose et al., 2000) andCueva Luna Azufre (Hose and Macalady, 2006). Where

Fig. 6. Fluorescence in situ hybridization micrographs of Frasassi snottites. Scale bars are 5 mm. False colour images were assigned greenfor EUBMIX, blue for DAPI, and red for other probes.A. Coccoid archaeal cells in sample RS2. Archaea appear pink (red + blue), bacteria appear blue-green. Chains of bacterial rods, probablyAcidithiobacillus, are visible at red arrows. Large round white objects at white arrow, oversaturated in the DAPI image, are protist nuclei.B. Coccoid archaea binding to probe FER656 (Ferroplasma spp.) in sample RS1.C. Coccoid and irregularly shaped archaeal cells in sample RS2. Weakly autofluorescing fungal filaments with evenly spaced nuclei (whitearrows) and protists (red arrow) are visible as well as chains of bacteria.D. Acidithiobacillus cells hybridizing with probe THIO1 in sample RS1. Cells stained with DAPI only are mainly coccoid and likely archaea.E. Acidithiobacillus cells in sample RS2 occur as isolated rods and chains of rods. A few non-Acidithiobacillus bacterial rods are also visible(white arrows).F. Bacterial cells, probably Acidithiobacillus, hybridizing with probe GAM42a in sample RS2. Isolated rods and chains of rods are visible, aswell as non-GAM42a bacterial rods, non-bacterial cells stained with DAPI, and weakly autofluorescing fungal filaments (red arrow showsnucleus).G. Cluster of Acidimicrobium cells in sample RS1.H. Cluster of Acidimicrobium cells in sample RS2. Weakly fluorescing fungal filaments with evenly spaced nuclei (white arrows) are alsovisible. Red arrow indicates autofluorescent protist with bright nucleus.



snottites drip onto limestone surfaces, dissolution pits andrills are observed on cave floors (Hose et al., 2000). Directevidence for the importance of cave wall biofilms in caveformation comes from a 5 year limestone dissolutionexperiment conducted at Frasassi sample site RS1(Fig. 1) by Galdenzi and colleagues (1997). The authorsfound that limestone dissolution proceeds at approxi-mately the same rate for tablets submerged in sulfidicwater as for those suspended in the air above streamsurfaces. Scanning electron microscopy images of thetablets suspended in the air indicated the presence ofmicrobial cells, although no further microbiological analy-ses were reported. Cave atmosphere H2S concentrationsat the elevation of the suspended limestone tablets variedbetween 1 and 8 p.p.m., including seasonal changes (S.Galdenzi, pers. comm.).

Future work

Snottites in sulfidic caves separated by vast geographicaldistances have strikingly similar community structures. Aswe have shown for Frasassi, Cueva de la Villa Luz(Mexico) snottites appear to be dominated by sulfur-oxidizing Acidithiobacillus species, with minor populationsrepresenting Acidimicrobium species, other acidophilicbacteria, acidophilic archaea, fungi and protists (Hoseet al., 2000). Our FISH investigation of snottites from theRio Garrafo caves (Acquasanta Terme, Italy) indicatesthat Acidithiobacillus and Ferroplasma are the mostnumerous populations (Jones et al., 2006). The extremeand isolated nature of snottite habitats around the worldprovide an excellent opportunity to study ecological andgeochemical controls on microbial diversity. The simplicityof snottite community structures also makes them ideallysuited for genetic and metagenomic studies, currently inprogress, aimed at mapping the spatial and temporal his-tories of microbial evolutionary events such as adapta-tions and gene transfers.

Experimental procedures

Field work, sample collection and geochemistry

Outcropping sulfidic water in the Frasassi cave system isaccessible in ramifying passages via technical cavingroutes. Biofilms were collected from cave walls at locationsindicated on Fig. 1 in May and August 2005. Biofilms werecollected either directly into sterile tubes or using steriletweezers. Each sample was collected from roughly0.1–0.5 m2 of wall surface area and consisted of snottiteswith the same morphology (size, colour, mineralogy). Sub-samples for clone library construction and FISH were storedon ice and processed within 4–6 h of collection. Watersamples were collected in acid-washed polypropylenebottles. Conductivity, pH and temperature of the stream

waters were measured in the field using probes and a WTWmultimeter. Dissolved sulfide (methylene blue method),oxygen (indigo carmine method) and ammonium (salicylatemethod) concentrations were measured in the field using aportable spectrophotometer according to the manufacturer’sinstructions (Hach, Loveland, CO). Anions in 0.2 mm filteredwater samples were measured at the Osservatorio Geo-logico di Coldigioco Geomicrobiology Laboratory using aportable spectrophotometer within 12 h of collection (storedat 4°C) according to the manufacturer’s instructions (Hach,Loveland, CO).

Clone library construction

Clone libraries were constructed from two snottite samplescollected in May 2005. DNA was extracted at the Osservato-rio Geologico di Coldigioco Geomicrobiology Laboratory or inUS laboratories using the MoBio Soil DNA extraction kit(MoBio Laboratories, USA) according to the manufacturer’sinstructions. Bacterial small subunit ribosomal RNA geneswere amplified by PCR. Each 50 ml reaction mixture con-tained: environmental DNA template, 1.25 U ExTaq DNApolymerase and 1¥ PCR buffer (TaKaRa Bio, Shiga, Japan),0.2 mM each dNTPs, 0.2 mM 1492r universal reverse primer(5′-GGTTACCTTGTTACGACTT-3′) and 0.2 mM 27f primer(5′-AGAGTTTGATCCTGGCTCAG-3′). Thermal cycling wasas follows: initial denaturation 5 min at 94°C, 25 cycles of94°C for 1 min, hybridization at 50°C for 25 s and elongationat 72°C for 2 min followed by a final elongation at 72°C for20 min. Additional 16S libraries were constructed using uni-versal forward and reverse primers 533f (5′-GTG CCA GCCGCC GCG GTA A-3′) and 1492r. Thermal cycling was asabove except for hybridization at 45°C for 45 s. PCR productswere cloned into the pCR4-TOPO plasmid and used to trans-form chemically competent OneShot TOP10 E. coli cells asspecified by the manufacturer (TOPO TA cloning kit, Invitro-gen, Carlsbad, CA). Colonies containing inserts were isolatedby streak-plating onto Luria–Bertani agar containing50 mg ml-1 kanamycin. Individual colonies were inoculatedinto Terrific Broth + kanamycin. Plasmid DNA was isolatedfrom overnight liquid cultures using the UltraClean StandardMini Plasmid Prep Kit (MoBio Laboratories, USA) accordingto the manufacturer’s instructions.

Sequencing and phylogenetic analysis

Inserts were sequenced at the Penn State University Biotech-nology Center using T3 and T7 plasmid-specific primers.Partial sequences were assembled with Phred base callingusing CodonCode Aligner v.1.2.4 (CodonCode, USA) andmanually checked. Assembled sequences were submitted tothe online analyses CHIMERA_CHECK v.2.7 (Cole et al.,2003) and Bellerophon (Huber et al., 2004). For Bellerophonanalyses, a variety of window sizes (200–400 bp) andcorrections were used. Possible chimeras were excludedfrom subsequent analyses. Phylogenetic analyses includedFrasassi clones and sequences > 1250 nucleotides identifiedas close relatives using BLAST (Altschul et al., 1990) and theonline workbench green genes (DeSantis et al., 2003). Align-ments were manually checked within the program ARB



(Ludwig et al., 2004). Alignments were minimized using thebacterial mask of Lane (1286 nucleotide positions). Phyloge-netic analyses were performed using Bayesian analysis andmaximum parsimony methods. Bayesian analyses were per-formed using MrBayes v3.0b4 (Huelsenbeck, 2000) using thegeneral time-reversible substitution model and a gamma dis-tribution of site-to-site rate variation with six discrete ratecategories run for 500 000 generations. The first 20% ofsampled trees were discarded and consensus trees werecomputed by 50% majority rule using version 4.0b10 of PAUP*(Swofford, 2000). Bayesian trees were compared againstparsimony bootstrap consensus trees (heuristic search, 500bootstrap replicates, PAUP* v.4.0b10).

Fluorescence in situ hybridization

Samples for FISH were collected into sterile microcentrifugetubes, stored on ice or at 4°C, and fixed within 30 h of col-lection. Samples and isolates grown in the laboratory foruse as control cells were fixed in 3 vols of freshly prepared4% (w/v) paraformaldehyde in 1¥ PBS for 3–4 h and storedin 1:1 PBS/ethanol solution at -20°C. Fluorescence in situhybridization experiments were carried out as described inHugenholtz and colleagues (2001) using the probes listed inTable 4. Briefly, fixed samples and control cells were appliedto multiwell, Teflon-coated glass slides, air-dried, and dehy-drated by successive immersion in 50%, 80% and 90%ethanol washes (3 min each). Hybridizations were carriedout in 8 ml well-1 of buffer containing 0.9 M NaCl, 20 mMTris/HCl pH 7.4, 0.01% sodium dodecyl sulfate (SDS),25–50 ng of each oligonucleotide probe, and formamideconcentrations given in Table 1. Oligonucleotide probeswere synthesized and labelled at the 5′ ends with fluores-cent dyes (Cy3, Cy5, FLC) at Sigma-Genosys (USA). Slideswere incubated for 2 h at 46°C in chambers equilibratedwith the hybridization buffer. Slides were immersed in washbuffer [20 mM Tris/HCl pH 7.4, 0.01% SDS, 5 mM EDTAand NaCl concentrations determined by the formula ofLathe (1985)] for 15 min at 48°C. Slides were then rinsedwith distilled water, air-dried, and counterstained with DAPI.Slides were mounted with Vectashield (Vectashield Labora-tories, USA) and viewed on a Nikon E800 epifluorescencemicroscope. For population counts, roughly 1000 DAPI-stained cells (5–10 microscope fields) were counted foreach sample and probe combination.

Diversity analyses

Nearly full-length 16S rDNA sequences were grouped intoOTUs based on nucleotide similarity values. Analyses shownare based on OTUs with > 98% similarity. Rarefaction analy-ses and diversity estimates were computed using EstimateS7.5.0 (Colwell, 2005).

Nucleotide sequence accession numbers

The 16S rRNA gene sequences determined in this studyhave been submitted to GenBank with accession numbersDQ499162 to DQ499330. Only seven of the 71 nearly iden-tical bacterial sequences from the PC1 clone library have

been submitted. The complete sequences are availablefrom the authors upon request.

Acknowledgements

We thank Alessandro Montanari for logistical support and theuse of facilities and laboratory space at the OsservatorioGeologico di Coldigioco (Italy), Sandro Galdenzi for invalu-able advice, Sandro Mariani and Paola D’Eugenio for expertfield assistance, and Tess Stoffer, Lindsey Albertson, HeidiAlbrecht and Irene Schaperdoth for laboratory assistance.We thank Greg Druschel for helpful discussions on sulfurbiogeochemistry. We also thank the Banfield Laboratory forthe use of laboratory facilities at UC Berkeley during prelimi-nary FISH experiments on cave snottites. D.S.J. thanks Car-leton College for financial support of his undergraduate thesisresearch at Penn State. This work was supported by theBiogeosciences Program of the National Science Foundation(EAR 0311854) and by the NASAAstrobiology Institute (PennState Astrobiology Research Center).

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Extremely acidic, pendulous cave wall biofilms from the Frasassi cave system, Italy

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