Oxygen-dependent niche formation of a pyrite-dependent acidophilic consortium built by archaea ...

13 Pages·2013·3.54 MB·English

by  Sibylle Ziegler

TheISMEJournal(2013)7,1725–1737
&2013InternationalSocietyforMicrobialEcology Allrightsreserved 1751-7362/13
www.nature.com/ismej
ORIGINAL ARTICLE
Oxygen-dependent niche formation of a
pyrite-dependent acidophilic consortium built by
archaea and bacteria
Sibylle Ziegler1,2, Kerstin Dolch2, Katharina Geiger2, Susanne Krause2,
Maximilian Asskamp1, Karin Eusterhues3, Michael Kriews4, Dorothee Wilhelms-Dick5,
Joerg Goettlicher6, Juraj Majzlan3 and Johannes Gescher2
1DepartmentofMicrobiology,Albert-LudwigsUniversity,Freiburg,Germany;2DepartmentofAppliedBiology,
Karlsruhe Institute of Technology, Karlsruhe, Germany; 3Department of Mineralogy, Friedrich Schiller
University, Jena, Germany; 4Department of Geosciences/Glaciology, Alfred Wegener Institute for Polar and
Marine Research, Bremerhaven, Germany; 5Department of Geoscience, University of Bremen, Bremen,
Germany and 6Institute for Synchrotron Radiation, Karlsruhe Institute of Technology, Karlsruhe, Germany
Biofilms can provide a number of different ecological niches for microorganisms. Here, a
multispecies biofilm was studied in which pyrite-oxidizing microbes are the primary producers.
Its stability allowed not only detailed fluorescence in situ hybridization (FISH)-based characteri-
zationofthemicrobialpopulationindifferentareasofthebiofilmbutalsotointegratetheseresults
with oxygenand pH microsensor measurements conductedbefore. TheO concentration declined
2
rapidly from the outside to the inside of the biofilm. Hence, part of the population lives under
microoxic or anoxic conditions. Leptospirillum ferrooxidans strains dominate the microbial
populationbutareonlylocatedintheoxicperipheryofthesnottitestructure.Interestingly,archaea
wereidentifiedonlyintheanoxicpartsofthebiofilm.Thearchaealcommunityconsistsmainlyofso
far uncultured Thermoplasmatales as well as novel ARMAN (Archaeal Richmond Mine Acidophilic
Nanoorganism) species. Inductively coupled plasma analysis and X-ray absorption near edge
structure spectra provide further insight in the biofilm characteristics but revealed no other major
factors than oxygen affecting the distribution of bacteria and archaea. In addition to catalyzed
reporterdepositionFISHandoxygenmicrosensormeasurements,microautoradiographicFISHwas
usedtoidentifyareasinwhichactiveCO fixationtakesplace.Leptospirillaaswellasacidithiobacilli
2
were identified as primary producers. Fixation of gaseous CO seems to proceed only in the outer
2
rim of the snottite. Archaea inhabiting the snottite core do not seem to contribute to the primary
production.Thisworkgivesinsightintheecologicalnichesofacidophilicmicroorganismsandtheir
rolein aconsortium.The dataprovidedthe basisfor the enrichmentof unculturedarchaea.
TheISME Journal(2013)7, 1725–1737;doi:10.1038/ismej.2013.64; published online25 April2013
SubjectCategory: microbial population and community ecology
Keywords: acid mine drainage; Leptospririllum; Acidithionbacillus; ecological niche; ARMAN;
thermoplasmatales
Introduction activity of acidophilic aerobic chemolithotrophic
iron-oxidizing microorganisms (Singer and Stumm,
Acidification occurs often in mining areas where
1970). The end products of pyrite dissolution are
due to mining activities sulfide ores are exposed to
sulfuric acid and ferric iron. Hence, acidic waters
an oxic environment. Interestingly, ferric iron and
containing high concentrations of metals are
not oxygen is the most influentialoxidantfor pyrite
formed. This process, called acid mine drainage, is
(FeS ), the most abundant sulfide mineral.
2 a major threat for the environment in mining areas
Theabioticreoxidationoftheproducedferrousiron
(Nordstrom, 2000; Johnson and Hallberg, 2005).
is a rather slow process under low pH conditions.
Anumberofmicrobialspecieshaveadaptedtoacid
This process is enormously accelerated by the
mine drainage conditions. These are characterized
by a high concentration in metalloids and sulfuric
Correspondence: J Gescher, Department of Applied Biology, acid as well as the availability of only inorganic
Karlsruhe Institute of Technology, Fritz Haber Weg 2, Karlsruhe carbon and oxygen. Leptospirillum as well as
76131,Germany. Acidithiobacillus strains are well-known members
E-mail:[email protected]
of the microbial population in mining areas
Received 31 October 2012; revised 1 March 2013; accepted 11
March2013;publishedonline25April2013 and fulfill the above mentioned chemolithotrophic
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requirements. The element cycling carried out by 29m deeper than the adit level. Therefore, oxygen
thesepioneerorganismsgivesrisetootherecological concentrations and pH values were recorded using
niches, which can be colonized by further groups of microsensor measurement systems. These experi-
microorganisms. Typically, biofilms are the macro- ments were accompanied by cryo-laser ablation
scopicmanifestationsofsuchcoexistingpopulations. inductively coupled plasma mass spectrometry
In acidic habitats, these biofilms are not only found (LA-ICP-MS), ICP optical emission spectrometry
often in the form of acid streamers but also as (OES) and X-ray absorption near edge structure
snottites, which are morphologically similar to (XANES) analysis to specify the chemical composi-
stalactites but have a rather gel-like character. tion of the potential niches in a greater detail. The
Examples of such acidic biofilms can be found in a microbial composition of the biofilm was evaluated
number of field sites (Bond et al., 2000; Hallberg using catalyzed reporter deposition fluorescence
etal.,2006;Garc´ıa-Moyanoetal.,2007;Kimuraetal., in situ hybridization (CARD FISH). Furthermore,
2011). Inside those biofilms, possible niches are primary production in the biofilm was assessed
defined, for instance, by the availability of inorganic using microautoradiography (MAR). Results of this
and organic carbon as well as terminal electron studygiveinsightsintophysiologicalcapabilitiesof
acceptors. The characterization of these niches is acidophilic microorganisms under environmental
important to understand the role of microorganisms conditions. They additionally suggest an anaerobic
inthebiofilmsandpotentiallyenablesisolationofso lifestyleformembersoftheARMAN,whichwereso
far uncultivated microorganisms. farbelievedtogrowaerobically.Inordertoproofthe
Organisms that were found in acid mine drainage assumptions regarding the lifestyle of Archaea and
biofilms are primarily iron- and/or sulfur-oxidizing ARMAN in the biofilm, enrichment experiments
bacteria and archaea like Leptospirillum spp., using anoxic culture techniques were started.
Acidithiobacillus spp., Acidimicrobium spp., Ferri-
microbium spp. and Ferroplasma spp., as well as
iron- or sulfur-reducing organisms like Acidithio- Materials and methods
bacillus spp., Acidiphilium spp., Acidimicrobium
spp., Ferrimicrobium spp., Ferroplasma spp. or Field site
Thermoplasma spp. (Bond et al., 2000; Dopson All samples used in this work were exclusively
et al., 2004; Garcı´a-Moyano et al., 2007; Ziegler snottitebiofilmsandcollectedfromorstudiedatthe
etal.,2009;Kimuraetal.,2011).Interestingly,some firstleveloftheabandonedpyritemineDreiKronen
of the members of the Thermoplasmatales found by und Ehrt in the Harz Mountains in Germany. The
culture-independentmethodsinacidicminingareas minehasonlythreeaccessiblelevels.Lowerpartsof
are—judging from the 16S rDNA sequences—quite the mine were flooded. The adit level is located
distinct from the isolated representatives (Rowe 428m above sea level and was studied in the work
et al., 2007; Gonzalez-Toril et al., 2011). Hence, it from Ziegler et al. (2009). The first level is 399m
ispossibletotaxonomicallyclassifythem,butithas above sea level. Differences in those two levels are
to be questioned whether these organisms have the in pH and iron concentrations as stated in the
same capabilities as the known isolates. Further- results of this work.
more, novel archaea, called ARMAN (Archaeal
Richmond Mine Acidophilic Nanoorganism) were
recentlydiscoveredbyshotgunsequencingofspeci- Metagenomic analysis
mens that were sampled at the Iron Mountain DNAwasisolatedfrom1gsnottitematerialfromthe
Superfund Site in California.They belong to deeply first level according to the protocol from Lo et al.
branching lineages that were not found previously (2007) with the following modifications: frozen
owing to mismatches of the hitherto believed biofilm material was homogenized under liquid
universal archaea primer sets (Baker et al., 2006). nitrogen using a mortar. Cells were then washed in
Using metagenomic and proteomic data, it was 15ml phosphate-buffered saline pH 2.1. After
assumed that these novel archaea are aerobic centrifugation, the pellet was resuspended in 5ml
chemoheterotrophs. 50mMTrispH7.5/50mMEDTApH8.0.Inall,150ml
This study focuses on acidic biofilms that were lysozyme solution (100mgml(cid:2)1) was added and
found in an abandoned pyrite mine. Only three incubation took place at 371C for 1.5h. Also, 1ml
levels of this mine are accessible. Previously, STEPbuffercontaining20mgml(cid:2)1ProteinaseKwas
biofilmsfromtheaditleveloftheminewerestudied added and incubated over night at 501C. The
(Ziegler et al., 2009). Ziegler et al. (2009) described phenol/chloroform/isoamylDNAextractionmethod
the bacterial community and mineralogy of this was used as described before (Lo et al., 2007). The
snottites.LeptospirillumferrooxidansandFerrovum shotgunmetagenomicsequencingwasdonebya454
sp. species dominated the microbial population, GS Titanium (Roche, Branford, CT, USA). It pro-
whereas archaea could not be detected. The aim duced the totality of 754643 reads, with an average
ofthisstudywastoevaluatethedifferentecological length of 366bp. The contig assembly generated
niches that are occupied and formed by the 10573continuoussequenceswithanaveragelength
microorganisms in biofilms of the first level, of 1571bp and a GC (guanine-cytosine) content of
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52.2%. Reads were analyzed using MetaSAMS formaldehyde in Leptospirillum-medium (132mgl(cid:2)1
(Zakrzewski et al., 2012). Detected 16S rRNA gene (NH ) SO ,53mgl(cid:2)1MgCl (cid:3)6H O,27mgl(cid:2)1KH PO
4 2 4 2 2 2 4
sequences were further analyzed using the classifi- and 147mgl(cid:2)1 CaCl (cid:3)2H O).The pHofthe medium
2 2
cationtoolfromtheRibosomalDatabaseProjectweb was adjusted to 2.5 with 10N H2SO4. Fixation was
interface (RDP Naive Bayesian rRNA Classifier carriedoutfor6–8hat41C.Thereafter,biofilmswere
Version 2.5, May 2012, confidence value of 80%) washed twice in the medium and three times in
(Cole et al., 2009). Using this binary identification 1(cid:4)phosphate-bufferedsalineineachcasefor10min
procedure, 692 reads with classifiable 16S rDNA (Zielinski et al., 2009). Afterwards, samples were
information were gained. subdividedinfivepieces,embeddedintoSteedman’s
wax and cut with a microtome (Leica Microsystems,
Wetzlar,Germany)in5–6mmslicesandtransferredto
Restriction fragment length polymorphism analysis Polysine slides (Thermo Scientific, Braunschweig,
DNA was isolated from a frozen, homogenized Germany) (Steedman, 1957).
biofilmusingtheinnuSPEEDSoilDNAkit(Analytic
Jena, Jena, Germany) according to the manu-
facturer’s instructions. Three different primer pairs CARD FISH
were used for archaeal 16S rDNA amplification. Embedded samples were first dewaxed and re-
General archaea primer (Supplementary Table S2, hydrated.Hybridizationandamplificationwasdone
primers1þ2)andARMANprimers(Supplementary as described by Pernthaler et al. (2004) with minor
Table S2, primers 3þ4) were used. For further modifications as described in the Supplementary
identification of archaea, the forward primer section SI Materials and Methods.
was deduced from the ARCH915 probe and
applied together with an universal reverse primer
MAR FISH
(Supplementary Table S2, primers 6þ5).
The above described CARD FISH procedure was
alsousedforthedetectionofCO fixation.TheMAR
2
Sequence analysis wasperformedinthedarkroomaccordingtoRogers
16S rDNA sequence analysis and similarity compar- (1979) and Okabe et al. (2004). Hypercoat LM1
ison were performed using the Ribosomal Database (GE Healthcare, Freiburg, Germany) coated slides
Project (http://rdp.cme.msu.edu) and the BLAST were incubated for 4 months.
program of the National Center for Biotechno-
logy Information (http://www.ncbi.nlm.nih.gov).
Determination of elemental compositions
The phylogenetic tree was calculated using the
neighbor-joining method. For further information Cryo-laser ablation ICP-MS. LA-ICP-MS was per-
see Supplementary ‘Materials and Methods’. formed by coupling the Elan6000 (Perkin Elmer/
Sciex, Rodgau, Germany) to the Indi-40 Spectra
Physics LA system (basis wavelength 1064nm).
Microsensor measurements Within this work, the quadrupled wavelength of
Oxygen concentrations and pH values were 266nm was used. As LA chamber, the cryo chamber
determined directly in the hanging snottites in the describedbyKriewsetal.(2001;2004)andReinhardt
mine. The biofilm was moved slightly by the etal.(2001)wasusedwithminormodifications that
microsensor but stayed stable. The profiles were are listed together with scanning procedure in the
measured using a microprofiling setup from Pyro Supplementary section SI Materials and Methods.
Science (Aachen, Germany).
ICP-OES and ICP-MS. Sample preparation was
conducted with whole flashfrozen snottite material
14C labeling from which the oxic parts (E1mm) were dissected
Snottites were labeled with 14CO directly in the
2 off using a razor blade. The samples from oxic and
mine; 3MBq (81mCi) of NaH14CO were used per
3 anoxicpartswerecentrifuged,thereafterfilteredand
snottite. A 1-L incubation chamber was mounted
subjected to ICP-MS measurement. The analyses
and sealed around the biofilms. The radioactive
were conducted in triplicate with ICP-OES Spectro-
bicarbonate was dissolved in 100mM Tris pH 10.5.
flame(Spectro,Kleve,Germany)orICP-MSX-Series
BicarbonatewasconvertedtoCO gasbyadditionof
2 II (ThermoFisher Scientific, Schwerte, Germany)
10N H2SO4 through an injection spot from the instruments.
outside. The incubation time was 22h. A pasteur-
ized snottite was used as a negative control. It was
treated as described above and incubated for 23h. Iron and sulfate quantification. Iron und sulfate
quantification was conducted from whole snottite
biofilms from the first level of the pyrite mine.
Fixation and embedding of the samples The biofilm was centrifuged at 10000g for 10min,
Snottites from the microsensor measurements and the supernatant was analyzed. Ferrous and
and the 14CO labeling were fixed directly in 4% total iron were quantified as described before
2
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Figure1 Oxygenprofilemeasurementsinthesnottite.(A)Locationofoxygenprofilesacrossasnottitebiofilm,takenatdistancesof1,
3,4.5and6cmfromtheceiling.(B)Oxygenprofiles.(a)Theprofiletakenclosesttotherocksurfaceshowsthefastestoxygendecrease:at
adepthof700mm,oxygenwasnolongerdetectable.(bandc)showanoxicsignalsat1300mm.Profile(d) fromspot4showsnoanoxic
zoneanymore.(C).Modeloftheoxygendistributioninthesnottitebiofilm.Indicatedaretheouteroxicpartandtheinneranoxicpart.
(Stookey, 1970; Ziegler et al., 2009). Sulfate quanti- addition of 2mgml(cid:2)1 vancomycin, 150mgml(cid:2)1
fication was conducted as described by Kolmert streptomycin, 50mgml(cid:2)1 kanamycin and 30mgml(cid:2)1
et al. (2000). chloramphenicol.
S and Fe K edge XANES spectroscopy. Microtome
Results
slices of the embedded biofilm that were also used
for the microsensor analysis between spots 1 and 2
Characterization of the possible niches in the biofilm
of the oxygen measurements (Figure 1A) were fixed
on a Kapton tape. S and Fe K XANES spectra have
been recorded at different sample positions across Oxygen concentrations. Oxygen is a major para-
the biofilm. Measurements were done at the X-ray meter for niche formation. If oxygen is available, it
beamline of the synchrotron radiation laboratory will be used as primary electron acceptor even if
for environmental studies (SUL-X) of the synchro- some examples of simultaneous reduction of other
tron radiation facility ANKA (Karlsruhe Institute electron acceptors are known (Johnson and
of Technology, Karlsruhe, Germany) with a wiggler McGinness, 1991). Due to this accentuated role,
as radiation source. the oxygen concentration throughout the biofilm
matrix was determined first. Oxygen measurements
Cryo-SEM. Scanning electron microscope (SEM) were conducted in situ using intact snottites that
images were taken with a Zeiss Ultra Plus SEM were still attached to the mine ceiling. Thus,
(Zeiss, Oberkochen, Germany) equipped with a dehydration or deformations were prevented since
thermal Schottky field emitter (Zeiss). Snottite the continuous water flow through the matrix was
samples were clamped on a sample holder, frozen not interrupted and the shape of the biofilm
by plunging them into liquid nitrogen and trans- maintained.Horizontalprofileswererecordedalong
ferred to the cryochamber (Alto 2500, Gatan, the longitudinal axes of the snottites to detect the
Munich, Germany). Here, surface water was spatial variations of O concentration as a function
2
removed by sublimation at (cid:2)951C before the of depth or the proximity to the pyrite-rich ceiling
sample was sputtered with Pt and introduced to (Figure 1A). Oxygen measurements were conducted
the microscope. on four different snottites and all showed large
anoxic parts inside of sections 1, 2 and 3 of the
Archaea enrichment. Parts from snottite biofilms biofilm. Oxygen concentrations declined rapidly
were inoculated in Hungate tubes containing a from the outer to the inner part of the biofilm. The
previously described anoxic medium (Huber and oxic zone at section 1 of the biofilm ended already
Stetter, 2001) to which 0.1% casein and 20mM after 700mm, indicating a high respiration rate
Fe (SO ) were added. The head space of the (Figure 1Ba). The oxic part slightly broadened at
2 4 3
tubes was flushed with an 80% CO /20% H gas theprofiles2and3.Here,thesnottiteisthinnerand
2 2
mixture. Bacterial growth was inhibited by the the concentration of reduced electron donors might
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be lower due to consumption in areas that are more biologically induced secondary mineral formation
closelylocalizedtotheceiling(Figures1BbandBc). or the simple capture of cationic species by the
In section 4 of the snottite, oxygen was detectable negatively charged microbial surface. The inten-
throughout the biofilm (Figure 1Bd). Here, the sities of some measured isotopes were too low to
biofilm thickness is about 1800mm, and the respira- differ substantially from the chamber background
tory consumption due to electron donor limitation level. Concentrations of S, Fe, K, Na and Ni were
might not be sufficient to result in an anoxic above the detection limit but showed a rather
zone, which would explain oxygen detection even constant distribution across the snottite (data not
in the biofilm center. In summary, partial areas shown). To verify whether a difference in the
of the biofilm are anoxic or contain oxygen con- concentrationofdissolvedelementsbetweenanoxic
centrations below the detection limit of 1 p.p.b. and oxic parts of the biofilm might exist, element
(Figure 1C). concentrations were determined by ICP-MS or-OES
using liquid samples from the anoxic and oxic
regionsofthebiofilm(Table1).Mostoftheanalyzed
pH values. Similar to oxygen, pH profiles
elements did not differ in their concentration
were also recorded at different positions of the
between the oxic and anoxic part of the biofilm.
biofilm using a micro-pH-electrode. In contrast to
Only Na and K concentrations differed by 410%.
the variations seen with oxygen concentrations, the
TheconcentrationofdissolvedNais1.6-foldhigher
observed pH values were quite constant and varied
attheanoxiccentercomparedtotheoxicarea,while
only slightly around 2.05 (Figure 2). Hence, the
the K concentration in the anoxic part is only
influence of pH on the distribution of microbial
0.7-fold of what was detected at the oxic part
species is most probably not as pronounced as the
(Table 1). Considering the amount of dissolved
influence from oxygen. pH measurements were
sulfur per liter (92–97mM) as quantified by ICP-
conducted several times in four different snottites
OES, it becomes evident that sulfate is the major
and never showed variations above the level shown
contributor of the detected dissolved sulfur as the
in Figure 2.
conducted sulfate quantifications from a whole
biofilm via the method from Kolmert et al. (2000)
Elemental composition. Cryo-LA-ICP-MS from reveal concentrations of 95mM (±3.8mM). The
frozen cross sections was conducted to analyze quantification of dissolved iron (47–50mM) by ICP
spatial variations of selected elements throughout isinagreementwiththequantificationsofdissolved
the biofilm. These variations could be due to a ferric (47.4±4.0mM) and ferrous iron (8.3±1.5mM)
by the ferrozine assay (Stookey, 1970).
XANESspectrawererecordedfromcrosssections
to analyze species of the two major elements iron
and sulfur throughout the biofilm (Supplementary
Figure S1A). The Fe K XANES spectra across
two slices differ in shape and edge positions
(Supplementary Figure S1B andC). Via principal
componentanalysis,onlytwoironspeciesoccurred
over the whole biofilm. Although one species
matches best with jarosite where Fe is trivalent,
spectra with edges shifted to lower energies could
not be identified directly (Supplementary Figures
S1BandC).TheyrepresenteitheraFe(II)speciesor
a mixture of it with jarosite. The latter is assumed
Figure2 pHprofileofasnottite.Thedatadisplayacrosssection
because the spectrum with the flank at the
asshowninFigure1.at1.5cmdistancefromthepyriteceiling.
lowest energy of the series did not match with the
ThepHrangesbetween2and2.5butthereisnotrendbetween
biofilmrimandcenter. ones of any known Fe(II) reference compound.
Table1 ICPMS/OESresults
Sample Ca(OES)mgl(cid:2)1 Cu(MS)mgl(cid:2)1 Fe(OES)mgl(cid:2)1 K(OES)mgl(cid:2)1 Mn(OES)mgl(cid:2)1
Biofilmanoxicpart 393±1 210.5±0.4 2628±6 27.8±0.1 32.54±0.06
Biofilmoxicpart 433.63±0.03 214±2 2807±5 39.73±0.01 34.79±0.07
Sample Na(OES)mgl(cid:2)1 Ni(MS)mgl(cid:2)1 P(OES)mgl(cid:2)1 S(OES)mgl(cid:2)1 V(MS)mgl(cid:2)1
Biofilmanoxicpart 251.8±0.3 527±7 8.8±0.2 2949±9 66.5±0.8
Biofilmoxicpart 157.597±0.009 578±5 9.3±0.2 3125±9 61±0.5
Abbreviations:ICP,inductivelycoupledplasma;OES,opticalemissionspectrometry;MS,massspectrometry.
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A hypothetical end member spectrum—obtained by in four to five components. Peak positions in the
subtracting a jarosite spectral fraction until the spectra are 2472.4, 2475.2, 2480.2 and 2481.5eV
white line or flank position of a typical Fe(II) (Supplementary Figure S3). The latter is in agree-
spectrum is reached—shows spectral features simi- ment with the positions known for sulfatic sulfur
lar to Fe(II) sulfate (Supplementary Figure S2). (S6þ).Especiallyattheoxicpartoftheslices,sulfate
Hence, it is likely that the second Fe species is the dominant species, which correlates with the
originates from Fe(II), most likely dissolved in the identification Fe K XANES spectra from the oxic
aqueous phase or adsorbed on the organic consti- areasthathavebeenidentifiedascontainingamajor
tuents of the biofilm. The spectra that are closely fraction of jarosite. In comparison with appropriate
relatedtojarositecouldbemeasuredintheoxicpart references, peak 2472.4 could be identified as
ofthebiofilmbutsimilarspectrawererecordedalso thiol group R-S-H and peak 2475.2 as a sulfoxide
in the anoxic center (Supplementary Figure S1 B: R-(S¼O)-R. Peak 2480.2 is related to RSO units,
3
locations1,22andC:locations2,10and25).TheS here represented by anthraquinone-2-sulfonic-acid,
K XANES spectra are more complex than the Fe K which is only found in the anoxic part
edge spectra. Principal component analysis results (Supplementary Figure S4).
a b
c
d e
Figure3 CARDFISHpicturesofacrosssectionofthesnottite.(a)Intheupperpart(Spot1,Figure1A)ofthesnottitebacteria(EUB338,
Alexa546inred)canbeseenthroughoutthewholecrosssectionwhilethearchaealsignal(ARCH915,Alexa488ingreen)isonlyinthe
middlepart,whichismostlikelyfullyanoxic.(b)Innerareaoftheupperpartofthebiofilm(Spot1,Figure1A).Archaea(ARCH915,
Alexa488ingreen)arefoundinhighabundance.(c)Bacteria(EUB338,Alexa546inred)andarchaea(ARCH915,Alexa488ingreen)in
thelowerpartofthebiofilm(Spot4,Figure1A).Theimageshowsthatthewholebiofilmispopulatedbybacteriabutalmostnoarchaea-
derived signal is detectable. (d) Image of the upper part (Spot 1, Figure 1A) of the snottite. The Acidithiobacillus species (THIO1,
Alex488ingreen)areintheveryouterandintheinnerpartwhileweseeLeptospirillumferrooxidans(LEP636,Alexa546inred)ina
broadouterarea.DAPI(4,6-diamidino-2-phenylindole)inblueshowsallcells.(e)Inthelowerpartofthesnottite(Spot4,Figure1A)
wherenoanoxiczonewasdetectable(Spot4,Figure1A),anunspecificdistributionoftheLeptospirillum(LEP636,Alexa546inred)and
Acidithiobacillus(THIO1,Alex488ingreen)speciesisfound.DAPIstainshowsallorganismsinblue.
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Microorganisms in the biofilm The probes for Leptospirillum group II (probe
First, hybridizations with general probes for bacteria LEP154) didnotresultinany signalwhile wefound
(probe EUB 338, Supplementary Table S1) occasional signals from Leptospirillum group III
andarchaea(probeARCH915)wereconducted.Cross (probe LEP634) (data not shown). L. ferrooxidans
sections from Figure 1A were analyzed by CARD species inhabit a major part of the oxic part of the
FISH.Atsection1(Figure1A),alldetectablearchaea biofilmrim.Acidithiobacillicanbedetectednotonly
inhabittheanoxiczoneofthesnottiteandcannotbe in the oxic but also in the anoxic part of
spotted in the oxic areas (Figures 3a and b). the cross sections (Figures 3d and e). In summary,
Correlating with this observation, the cross section as shown by pyro-sequencing and CARD FISH
from area 4 (Figure 1A) of the biofilm contains very experiments, the microbial population is mainly
few to almost no archaeal signals (Figure 3c). There- composed by bacteria, more precisely by Leptospir-
after, further identification of the microbial popula- illum spp. and Acidithiobacillus spp. Interestingly,
tion was conducted using 454 pyro-sequencing of neither in the CARD FISH experiments nor in the
isolated snottite DNA from biofilms collected in the sequencing data we could detect Ferrovum sp.,
first level. Using this approach, 647 reads with 16S which were found in former studies from the adit
rRNAsequenceinformationwereabletobeclassified level of the mine (Ziegler et al., 2009). In the
into a phylum. A total of 96.3% of these sequences metagenomic data also, other typical acidophilic
(reads)couldbeattributedtothebacteria,whileonly bacteria were detected in minor abundance—mainly
3.7% archaeal sequences were obtained. Within the Proteobacteria and Actinobacteria—like the iron-
bacterialsequences,about74.1%couldbeascribedto reducing bacterium Ferrimicrobium sp. (Figure 4).
thegenusLeptospirillum.AscanbeseeninFigure4, In former studies, no archaea were detected in
the next most abundant organisms are species biofilmsofthismine(Ziegleretal.,2009).Duetothe
belonging to the genus Acidithiobacillus (10.5% of low coverage of the archaeal sequences from the
the bacterial community). These results are sup- metagenome, we used restriction fragment length
ported by the CARD FISH experiments. Using more analysisandsubsequentsequencingtogainadeeper
specific bacterial probes, we found that the bacterial understanding of the archaeal community. General
community consists mainly of Acidithiobacillus sp. archaea primers failed to amplify an archaeal 16S
(probe THIO1) and L. ferrooxidans (probe LEP636). rRNA gene signal. By contrary, primers deduced
Figure4 454pyro-sequencing16SrDNAresultsfromthesecondlevelofthepyritemine‘DreiKronenundEhrt’.Speciesclassifiedby
RibosomalDatabaseProjectclassificationofthemetagenomereads.Theabsolutenumberofreadsisshownatthetipofthebarandthe
amountinpercentageofallreadswith16Sinformationisdisplayedalongthexaxis.Leptospirillumspp.buildsthemainpopulation
ofthemicroorganismcommunity.Acidithiobacilliarethenextmostabundantspecies.
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from the archaeal probe ARCH915 combined with a represent the major group of archaea in the biofilm
universal primer and specific primers for the that is also corroborated by the 454 sequencing
recently discovered ARMAN-enabled PCR product results (Figure 4 and Supplementary Figure S5A).
formation (Supplementary Table S2) (Baker et al., The ARMAN signal does not clearly correspond to
2006). Sequence analysis revealed the presence of the general archaeal probe but is always located in
clones most closely related to the ARMAN species the same area (Supplementary Figure S5B). In
described by Baker et al. (2006) as well as clones summary, archaea present in the biofilm are related
belonging to the Thermoplasmatales. The detected to uncultured Thermoplasmatales and novel
ARMAN clones (ARMAN-Clone_HM_4–46% abun- ARMAN species and prefer the anoxic part of the
dant;ARMAN-Clone_HM_7–50%andARMAN-Clo- biofilm.
ne_HM_29–4%) cluster in the ARMAN branch The described experiments identified the differ-
together with other uncultured archaea clones from ent niches within the biofilms and their microbial
the Rio Tinto (Amaral-Zettler et al., 2011). The population. However, what was missing was a clear
Thermoplasmatales sequences are mainly related to picture how this community might look under
a branch with uncultured representatives of this insituconditions.Cryo-SEMwasconductedtogain
order, whereas one type (Archaea-Clone_HM_ thisinformationandtheimagesdisplaymicroorgan-
19–40% from all archaea clones) is related isms embedded in a complex mixture of exopoly-
to Ferroplasma (Figure 5). The closest relatives to saccharide structures and secondary minerals
Archaea-Clone_HM_2 (42%), Archaea-Clone_ (Figure 6). Jarosite crystals with their typical
HM_20 (6%) and Archaea-Clone_HM_21 (13%) are pseudocubic habitus dominate the secondary
so far uncultured representatives from other acidic minerals.
environments found in the Iberian Pyrite Belt
(Figure 5) (Rowe et al., 2007; Amaral-Zettler et al.,
2011; Gonzalez-Toril et al., 2011). Based on the Enrichment of ARMAN
restriction fragment length polymorphism results, It was surprising to see that microbes belonging to
more specific probes for the detection of ARMAN the recently described ARMAN group were found
and Thermoplasmatales were used. We see clearly exclusively in the anoxic parts of the biofilm, as it
the Thermoplasmatales signal correlating with the wasassumedbeforethattheyratherhaveanaerobic
archaeal signal. Thermoplasmatales seem to lifestyle (Baker et al., 2010). To corroborate this
Figure5 16SrDNA-basedphylogenetictreeofthedetectedarchaeaandrelatedsequences.Greenindicatessequencesfromthearchaeal
primerpair(SupplementaryTableS2,primers1þ2),redsequencesgainedfromtheARMANprimers(SupplementaryTableS2,primers
3þ4).Theneighbor-joiningmethodwasusedandbootstrapwascalculatedfrom1000replicates.Bootstrapvalues450areindicatedin
thegraph.
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Ecologicalnichesofacidophilicmicroorganisms
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to be present in the medium, otherwise microbial
growth cannot be detected. Furthermore, ferric iron
is a necessary constituent of the medium.
Primary production
14CO labeling experiments were conducted to
2
localize the uptake of atmospheric CO in the
2
biofilm. Furthermore, CARD FISH analyses were
used to trace this activity back to microbial genera.
These experiments were conducted in situ. The
MAR FISHimagesshow silvercrystals intherimof
the snottite that are due to 14CO incorporation by
2
autotrophic organisms (Figure 8). Correlations with
fluorescent images reveal the exclusive influence of
the bacterial community in CO -based biomass
2
production.Archaea(probeARCH915)appearagain
onlyinthecentralpartofthecrosssectionandshow
no detectable sign of fixation of atmospheric 14CO
2
(Figures8aandb).Ofnote,silvercrystalsshouldnot
be mistaken for secondary minerals in the center of
the cross section. Mineral crystals shine very bright
in different foci while the silver crystals stay dark
(Figures 8b and c). Further analysis revealed that
Nitrospira (probe NTR712) and Acidithiobacillus
spp.(probeTHIO1)arethemainprimaryproducers.
The CARD FISH signals correlate with the MAR
spots in the outer part (Figure 8c). The Nitrospira
signal is derived from Leptospirillum spp. These
organisms and the acidithiobacilli are known for
their autotrophic lifestyle (Balashova et al., 1973;
Gale and Beck, 1967). Notably, fixation of atmo-
spheric CO is detectable only at the rim of
2
Figure6 Cryo-SEMsecondaryelectronimagesfromthebiofilm. the biofilm and rangesB30mm in the inner part.
(a) The image shows microorganisms and some jarosite crystals However, Nitrospira and Acidithiobacillus spp.
embedded in the matrix. Noticeable are rod like structures and
inhabit an area that is by far larger than these
cocciaswellassomecurvedrods(indicatedbyarrows).(b)High
30mm, which raises the question whether dissolved
amountofjarositecrystalsinthematrix.Inbetweentherearerod
like structures and also coccoid microorganisms (indicated by H CO is the carbon source for Leptospirillum and
2 3
arrows). Acidithiobacillus species that are localized to the
inner part of the biofilm or whether the MAR
FISH method was not sensitive enough to detect a
finding, it was tried to enrich ARMAN from probably lower rate of CO fixation by these
2
the biofilms using anoxic culturing techniques. A organisms.
samplefromthebiofilmwasinoculatedinananoxic
medium containing casein and yeast extract as
possible sources for heterotrophic growth. Ferric Discussion
iron sulfate was added as electron acceptor. The
medium was further supplemented with a mixture Microbial niches in the biofilm
of antibiotics to hamper bacterial growth. Mean- The most surprising result of this study is the
while, we were able to transfer the culture up to exclusive localization of archaea in anoxic areas of
12 times. Oxygen contaminations were excluded the consortium. Quantification of pH and the
using periodic microsensor measurements. Figure 7 concentration of several elements did not point
shows the results of the enrichment experiments. towardotherfactorscontributingtonicheformation.
Bacteria could not be detected in the culture The detected archaeal sequences are related to the
anymore. ARMAN were abundant in all the steps recentlydiscoveredARMANorganismsaswellasto
of the enrichment procedure. It was so far not uncultured Thermoplasmatales clones and organ-
possible to separate them from other organisms that isms belonging to the genus Ferroplasma. Baker
werestillpresentintheenrichmentcultures.During etal.(2010)proposedanaerobiclifestyleofARMAN
the enrichment procedure, an attempt was made to organisms based on metagenomic and proteomic
exclude the addition of yeast extract or casein as data. This proposition is partly based on the
wellasferriciron.Eitheryeastextractorcaseinhave abundance of a protein similar to succinate
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Ecologicalnichesofacidophilicmicroorganisms
SZiegleretal
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anaerobicorganisms.Theusedcarbonsourcewould
havetobeknowninordertomakeanassumptionas
towhetherthetricarboxylicacidcyclefulfillsonlya
functioninbuildingblockproductionorfurthermore
in the generation of reducing equivalents for anaero-
bicrespiration.Additionally,othergenes,potentially
involved in a respiratory chain to oxygen, were not
detected(Bakeretal.,2010).Therevealedoccurrence
of enzymes responding to oxidative stress like
the superoxide dismutase do not necessarily have
to indicate an aerobic lifestyle as the detoxifica-
tion mechanisms operate also in strict anaerobes
(Brioukhanov and Netrusov, 2004).
The Ferroplasma-related sequences detected in
the biofilm show strong similarities to F. acidiphi-
lumand‘F.acidarmanus’.WhileF.acidiphilumisa
strict aerobic autotroph (Golyshina et al., 2000),
‘F. acidarmanus’ is a facultative anaerobe that can
respireanaerobicallyusingferricironasanelectron
acceptor (Dopson et al., 2004). Hence, organisms
related to Ferroplasma could also inhabit an anoxic
niche within the biofilm.
A number of the identified ribosomal rRNA gene
sequencesaresimilartounculturedThermoplasma-
tales and, from a phylogenetic point of view, only
distantly related to the species isolated so far. The
more related sequences from uncultured organisms
stemfromotheracidicenvironmentsliketheIberian
Pyrite Belt, including Rio Tinto (Rowe et al., 2007;
Amaral-Zettler et al., 2011; Gonzalez-Toril et al.,
2011). Due to their remote position in the phyloge-
netic tree and as there is no isolated species
belonging to these deeply branching organisms,
it is not possible to assign certain physiological
characteristics yet.
The main iron oxidizers in the studied bacterial
Figure7 CARDFISHimagesoftheARMANenrichment.(a)A
consortium are L. ferrooxidans and Acidithio-
culture that was transferred the eleventh time. The enrichment
wasgrowingfor9weeksatthetimeoffixation.ARMANcanbe bacillus. They are also the most abundant micro-
seeningreen(ARM980,Alexa488)andDAPI((4,6-diamidino-2- organisms in the biofilm (Figures 3d and e and 4).
phenylindole); blue) stained all cells in the enrichment. (b) As expected, organisms belonging to the genus
Culture transferred from (a) after 5 weeks of growth. Again
Leptospirillum can be detected solely in oxic parts
ARMANareingreen(ARM980,Alexa488)andDAPIinblue.
ofthebiofilm,whichiscertainlyduetothefactthat
theycanonlyrespireonoxygenwithferrousironas
anelectrondonor(Hippe,2000).Acidithiobacilliare
ubiquinone oxidoreductases in the protein pool. foundintheoxicaswellasintheanoxicpart.They
This enzyme is well known for its position within can thrive aerobically and anaerobically using
the citric acid cycle. The canonical enzyme oxygen or ferric iron as electron acceptors (Pronk
catalyzes the oxidation of succinate to fumarate and Johnson, 1992). Ferrovum sp. is meanwhile a
(E`1¼0±10mV) coupled to ubiquinone reduction third candidate for playing a role in ferrous iron
(Clark,1960).Itismostprobablytheredoxpotential oxidation at acidic conditions (Hallberg, 2010).
ofthisreactionandtheoftenseendownregulationof Brown et al. (2011) and Jones et al. (2011) proposed
the citric acid cycle under anoxic conditions that recently that pH and ferrous iron concentration
might have led to the assumption of an aerobic could dictate whether Ferrovum or Acidithiobacil-
metabolism of the ARMAN detected at Iron Moun- lus might dominate a microbial consortium. Their
tain. Nevertheless, succinate ubiquinone oxido- assumption is corroborated by the results from this
reductases and the anaerobic fumarate reductase and a previous study from Ziegler et al. (2009).
cannot be distinguished easily. Furthermore, it was In the adit level of the mine, the snottite biofilms
shown that succinate ubiquinone oxidoreductases contained Ferrovum species rather than acidithio-
can also function with menaquinone (Maklashina bacilli,whileFerrovumspecieswerenotdetectedin
et al., 1998). Moreover, the citric acid cycle can be the first level. This difference may be caused by
an integral part of the central metabolism also for pH (2.6 in the upper horizon, 2.05 in the lower
TheISMEJournal