Table Of ContentTheISMEJournal(2012)6,1459–1468
&2012InternationalSocietyforMicrobialEcology Allrightsreserved1751-7362/12
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ORIGINAL ARTICLE
Unraveling assembly of stream biofilm communities
Katharina Besemer1,2, Hannes Peter3, Ju¨rg B Logue3, Silke Langenheder3, Eva S Lindstro¨m3,
Lars J Tranvik3 and Tom J Battin1,2
1Department of Limnology, Vienna University, Vienna, Austria; 2WasserCluster Lunz, Lunz, Austria and
3Department of Ecology and Genetics, Uppsala University, Uppsala, Sweden
Microbial biofilms assemble from cells that attach to a surface, where they develop into matrix-
enclosed communities. Mechanistic insights into community assembly are crucial to better
understandthefunctioningofnaturalbiofilms,whichdrivekeyecosystemprocessesinnumerous
aquatic habitats. We studied the role of the suspended microbial community as the source of the
biofilm community in three streams using terminal-restriction fragment length polymorphism and
454pyrosequencingofthe16SribosomalRNA(rRNA)andthe16SrRNAgene(asameasureforthe
active and the bulk community, respectively). Diversity was consistently lower in the biofilm
communitiesthaninthesuspendedstreamwatercommunities.Weproposethatthehigherdiversity
inthe suspendedcommunitiesissupported bycontinuousinflowfromvarioussourceswithinthe
catchment.Communitycompositionclearlydifferedbetweenbiofilmsandsuspendedcommunities,
whereasbiofilmcommunitiesweresimilarinallthreestreams.Thissuggeststhatbiofilmassembly
didnotsimplyreflectdifferencesinthesourcecommunities,butthatcertainmicrobialgroupsfrom
thesourcecommunityproliferateinthebiofilm.Wecomparedthebiofilmcommunitieswithrandom
samplesoftherespectivecommunitysuspendedinthestreamwater.Thisanalysisconfirmedthat
stochastic dispersal from the source community was unlikely to shape the observed community
compositionofthebiofilms,insupportofspeciessortingasamajorbiofilmassemblymechanism.
Bulk and active populations generated comparable patterns of community composition in the
biofilms and the suspended communities, which suggests similar assembly controls on these
populations.
TheISME Journal(2012)6, 1459–1468;doi:10.1038/ismej.2011.205; published online12January 2012
SubjectCategory: microbial population and community ecology
Keywords: biofilm; metacommunity ecology; pyrosequencing; streams
Introduction of local communities. By viewing biofilms as
microbial landscapes, their community assembly
Microbial biofilms develop from primary cells
canbestudiedaccordingtometacommunityecology
that attach to a surface, where they form micro-
theory (Battin et al., 2007). Mechanistic insight
colonies that eventually coalesce into matrix-
into community assembly is crucial to better under-
enclosed communities (Battin et al., 2007). Biofilm
stand the functioning of biofilms, which drive key
formationhasbeenextensivelystudiedinlaboratory
ecosystem processes in streams (Singer et al., 2010;
and medical systems that are typically composed
Peter et al., 2011).
of mono- or polycultures (Costerton et al., 1995;
Available knowledge on biofilm community as-
Hall-Stoodley et al., 2004). Such systems, while
sembly in nature is scarce and largely based on
useful to test basic concepts in microbiology,
molecular fingerprinting techniques. For instance,
contrast the massive microbial diversity generally
using denaturing gradient gel electrophoresis,
encountered in natural ecosystems (Sogin et al.,
Jackson et al. (2001) and later Lyautey et al. (2005)
2006; Newton et al., 2011). In numerous aquatic
studied successional changes in lake and river
ecosystems, surface-attached biofilms assemble
biofilms. Essentially, their findings suggest that
from the microbial diversity contained in the
biofilm assembly is not a random process, and that
overlying water. According to metacommunity
certain bacterial groups contribute more to biofilm
theory (Leibold et al., 2004; Holyoak et al., 2005),
formation than others. A conceptual model pro-
local (abiotic environment, biotic interactions) and
posed by Jackson et al. (2001) suggests elevated
regional (dispersal) processes regulate the assembly
bacterial diversity during initial biofilm forma-
tion and decreasing diversity as biofilm growth
progressed as a result of the combined effects
Correspondence: K Besemer, Department of Limnology, Vienna of niche availability and competition. Besemer
University,ViennaA-1090,Austria.
et al. (2007) compared community succession in
E-mail:[email protected]
stream biofilms and found consistent differences
Received29August2011;revised25November2011;accepted3
December2011;publishedonline12January2012 betweenthebiofilmandstreamwatercommunities,
Assemblyofstreambiofilmcommunities
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which indicate the existence of a specific biofilm Glassbeadswerepackedintonets(1mmmeshsize)
community. that were cased in perforated pipes (diameter: 5cm,
On the basis of these previous findings, we length:20cm).Triplicatepipeswereinstalledinthe
hypothesize that the assembly of a local biofilm thalweg (30cm above bottom) of the respective
community is not a mere reflection of the source stream parallel to the main flow direction to allow
community suspended in the overlying stream continuousflowthroughofthebeadpackages.During
water. The compositions of the biofilm and the the 3-week colonization period, we sampled stream
suspended communities are thus anticipated to water seven times for the analysis of the suspended
differ.Wearguethatstreamwatertransportsbacteria community.Sampleswerefilteredontosterile0.2mm
from multiple sources within the catchment, filters (GSWP filter, Millipore, Solna, Sweden) and
whereas biofilms, according to the species sorting frozen ((cid:2)801C). Beads with biofilms were sampled
perspective in metacommunity theory (Leibold after 3 weeks. Aliquots were suspended in sterile
etal.,2004; Holyoak etal., 2005),specifically select (autoclaved and 0.2mm filtered) water and sonicated
for certain taxonomic groups. We also hypothesized (10min, 40Woutput; Branson Sonifier, Danbury, CT,
that the diversity of the suspended community may USA) to detach cells. Suspended cells were concen-
exceed the diversity in biofilms, as various sources tratedonsterilefilters(0.2mmGSWPfilter,Millipore)
within the catchment continuously feed the com- and stored ((cid:2)801C) pending for further processing.
munitysuspendedinthestreamwater.Weareaware
that niche diversification could, nevertheless, sup-
portahighdiversityinbiofilms(Jacksonetal.,2001; Nucleic acid extraction and reverse transcription
Besemer et al., 2007). Fingerprinting methods as Nucleic acids were extracted from biofilms and
usedintheseearlierstudiesare,however,limitedin suspended communities using the PowerSoil DNA
their ability to detect and quantify rare species Isolation Kit (MoBio, Carlsbad, CA, USA) and the
(Blackwood et al., 2007; Bent and Forney, 2008). In Easy-DNA Kit (Invitrogen, Paisley, UK) omitting the
this study, we used a dual approach to explore RNase step (Logue and Lindstro¨m, 2010). Although
possible mechanisms of biofilm community assem- the PowerSoil DNA Isolation Kit is designed to
bly in three headwater streams within the same extract DNA only, the resulting DNA and RNA
catchment.Weappliedterminal-restrictionfragment yields were higher than those obtained with the
length polymorphism (T-RFLP), which we supple- Easy-DNA Kit; we therefore used the PowerSoil
mented with 454 pyrosequencing to gain deeper DNA Isolation Kit also for RNA.
insightintocommunityassembly.Weanalyzedboth ReversetranscriptionofRNAintocomplementary
the 16S ribosomal RNA (rRNA; as a measure for the DNA was performed as described by Logue and
active fraction of a community) and the 16S rRNA Lindstro¨m (2010). Briefly, an aliquot of the nucleic
gene (for the bulk community) to test whether the acid extract was subjected to DNA digestion with
active members of the suspended microbial com- DNase I (Invitrogen) for 15min at room temperature
munity differ from the inactive members in their following the manufacturer’s recommendations.
ability to contribute to biofilm formation. Absence of DNA was verified by PCR of the DNA
digests as described below. RNAwas transcribed at
421C for 50min using SuperScript II reverse
Materials and methods transcriptase and random primer oligonucleotides
(Invitrogen), followed by an enzyme inactivation
Biofilm growth and sampling procedure step at 701C for 15min. Samples without reverse
Streambed (hyporheic) biofilms were grown on transcriptase served as negative controls.
initially sterile, sintered, borosilicate glass beads
(2mm diameter) deployed in three headwater
streams. Beads were exposed for colonization from T-RFLP analysis
the suspended microbial community for 3 weeks The PCR primers used for T-RFLP analysis were
during snowmelt in April, when terrestrial–aquatic the hexachlorofluorescein-labeled bacteria-specific
connectivity was high. The streams are located in primer 27F (50-AGRGTTTGATCMTGGCTCAG-30)
Fiby Urskog (N 591 530 700 E 171 200 4300), a protected and the universal primer 519R (50-GWATTACCGC
forest area close to Uppsala, Sweden. One of the GGCKGCTG-30). Each 50ml PCR mixture contained
streams (referred to as ‘outflow stream’ hereafter) is bothprimersat0.4mmoll(cid:2)1(Invitrogen),eachdeoxy-
theoutflowoflakeFibysjo¨n.Downstream,itmerges nucleosidetriphosphateat0.2mmoll(cid:2)1(Invitrogen),
withasmallhumic-richditch(referredtoas‘humic 75mgbovineserumalbumin(NewEnglandBioLabs,
stream’ hereafter) that drains a forest, into a conflu- Ipswich, UK), MgCl at 3.5mmoll(cid:2)1, 1.5U of
2
ence (referred to as ‘confluence’ hereafter). Water DyNAzyme II DNA polymerase and the recom-
chemistry was largely similar in all three streams, mended PCR buffer (Finnzymes, Espoo, Finland).
except for the concentration of dissolved organic The amplification protocol consisted of an initial
carbon, which was, on average, 75.2mgCL(cid:2)1 in the denaturation step of 941C for 3min, 25 cycles of
humic,34.0mgCL(cid:2)1intheoutflowand32.9mgCL(cid:2)1 denaturation at 941C for 45s, annealing at 501C for
in the confluence (Supplementary Table 1). 45s,extensionat721Cfor1minandafinalextension
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step at 721C for 10min. Each PCR was run in for chimeras with an intercept of a¼(cid:2)7.5 and
triplicates and subsequently pooled. PCR products coefficient of b¼0.5 (Quince et al., 2011). This
were cleaned applying the QIAquick PCR Purifica- procedurereducedtheoriginally229026flowgrams
tion kit (Qiagen, Hilden, Germany) and quantified to118612reads.Thedenoisedreadswereclustered
using agarose gel electrophoresis in combination to OTUs, with a complete linkage algorithm on a
with the Low DNA Mass Ladder (Invitrogen). 97% sequence identity level. The taxonomic affilia-
The fluorescently labeled PCR products were tion of the OTUs was determined using a naı¨ve
digested separately with the restriction enzymes Bayesian rRNA Classifier (Wang et al., 2007) and a
HaeIIIandHinfI(NewEnglandBioLabs).Restriction confidence threshold of 80%.
digests were performed according to Logue and
Lindstro¨m (2010). The product was subjected to
capillary electrophoresis in an ABI 3730XL DNA Data analysis
Analyzer(UppsalaGenomeCenter,Uppsala,Sweden) Similarity matrices of community compositions
using the size marker GS 500 Rox (Applied Biosys- based on T-RFLP and 454 pyrosequencing data
tems, Foster City, CA, USA). The electropherograms were calculated using the presence/absence-based
were analyzed using the Peak Scanner software Sørensen index and the relative abundance-based
(Applied Biosystems). The relative contribution of Horn index. These similarity indices were chosen
the respective operational taxonomic units (OTUs) to because they are independent from alpha-diversity
thecommunitywasestimatedaspeakheightdivided and therefore consistent with valid beta-diversity
by the cumulative peak height of the given sample. indices (Jost, 2007). Nonmetric multidimensional
scaling (nMDS) analysis was performed on the
similarity matrices to visualize patterns of commu-
454 pyrosequencing nity composition. Similarity matrices obtained for
To reduce the number of samples for 454 pyro- therRNAgene-based(referredtoasbulkcommunity
sequencing, equal amounts of extracted or trans- hereafter) and the rRNA-based (referred to as active
cribed DNA of the suspended communities from community hereafter) communities were compared
the seven sampling dates were pooled to yield using Mantel’s matrix randomization test (Mantel,
time-integrated samples for each active and bulk 1967) with Pearson’s correlation and 999 permuta-
community from the three streams. Multiplex tions. Diversities were estimated applying indices
amplicon sequencing was then performed on the of the Hill family (Hill, 1973), namely, richness and
six biofilm samples and the six time-integrated the number equivalents of the Shannon entropy.
suspended community samples. The V3 and V4 Data analysis was performed with PAST (Hammer
regions of bacterial 16S rRNA genes were amplified et al., 2001) and R 2.13.0 (R Development Core
using the fusion primers 341F (50-CCTACGGGNGG Team, 2011).
CWGCAG-30) and 805R (50-GACTACHVGGGTATCT Usingthe454pyrosequencingdata,weperformed
AATCC-30), containing the 454 FLX adaptors and a random sampling procedure to estimate the
a sample-specific multiplex identifier (Andersson probability that a biofilm community represented
et al., 2008). Each 50mL PCR mixture contained a random subsample of the respective suspended
each primer at 0.5mmoll(cid:2)1, each deoxynucleoside source community in the stream water. Each tested
triphosphate at 0.25mmoll(cid:2)1 (Invitrogen), MgCl at sample pair consisted of a biofilm and a suspended
2
1.5mmoll(cid:2)1, 1.25U of Phusion High-Fidelity DNA community, either bulk or active, from the same
Polymerase and the recommended PCR buffer stream, respectively. OTUs were sampled from the
(Finnzymes). Triplicate PCR products for each suspended community with replacement until the
sample were pooled, purified using the QIAquick number of OTUs in this randomly assembled
Gel Extraction Kit(Qiagen) and quantified using gel community equaled the richness of the respective
electrophoresis and the Low DNA Mass Ladder biofilm community. This procedure was repeated to
(Invitrogen). Equal amounts of the barcoded yield 1000 random subsamples of each suspended
PCR products were mixed and submitted to the community. The probability of the biofilm commu-
KTH Biotechnology Sequencing Center (Stockholm, nity to fall within the distribution of these random
Sweden) for pyrosequencing on a 454 GS20 FLX subsamples was calculated as the percentage of
platform. the distances of the random subsamples to their
The obtained pyrosequencing data were denoised centroid, which were as high or higher than the
using the software package AmpliconNoiseV1.0 distance of the biofilm community to the centroid.
(Quince et al., 2011). Pyrosequencing flowgrams The biofilm community data set was reduced to
with an exact match to the primer and multiplex OTUs, which occurred also in the respective
identifiersequenceswerepreclusteredwithPyroNoise suspended community, thereby increasing the
(AmpliconNoiseV1.0)toremovepyrosequencingnoise. chance of the biofilm community to resemble the
PCR single base errors were corrected using SeqNoise suspended community. The estimated differences
(AmpliconNoiseV1.0), a sequence-based clustering between the biofilm community and random sub-
method, which performs the alignment of the samples of the suspended community can therefore
sequences.ThePerseusalgorithmwasusedtocheck be regarded as conservative.
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Rarefactioncurvesforthe454pyrosequencingdata
were computed using the AmpliconNoise software
S∅rensen Horn
package. Rank-abundance curves were constructed
from relative OTUs abundances obtained form 454
pyrosequencingdata.Linearregressionmodelswere
2
fitted to each curve after log transformation of the s
xi
rank and abundance data. The slopes of these S a
regressionmodelswereusedasasimpledescriptive MD
statistic of community structure (Ager et al., 2010)
and were compared using Student’s t-test. The ‘true
richness’ of the communities was estimated by S = 0.178 S = 0.123
Bayesian fitting of the OTUs abundances obtained
by 454 pyrosequencing to the Sichel distribution
(Sichel,1974)usingtheDiversityEstimationsoftware
S∅rensen Horn
according to Quince et al. (2008). The Sichel distri-
butionwaschosenasthebestmodeltodescribeOTU
abundances based on deviance information criterion
2
calculation (Spiegelhalter et al., 2002). s
xi
a
S
D
M
Results
Community composition S = 0.095 S = 0.081
A total of 141 and 126 OTUs were found by T-RFLP
MDS axis 1 MDS axis 1
analysis with the enzymes HaeIII and HinfI, respec-
Figure 1 nMDS analysis of the microbial community composi-
tively. OTUs from both enzymatic digestions were
tionsestimatedbyT-RFLP(a,b)and454pyrosequencing(c,d),
combined for further analysis. nMDS analyses of calculatedfromthepresence/absence-basedSørensenindex(a,c)
both the presence/absence-based Sørensen and the andtheabundance-basedHornindex(b,d).Kruskal’sstandardized
relative abundance-based Horn similarity matrices stress values (S) below 0.2 indicated acceptable representation of
the calculated similarities. Circles represent the bulk (16S rRNA
revealed clear differences between biofilm and
gene based), crosses the active (16S rRNA-based) community
suspended communities (Figures 1a and b).
compositions,brownthebiofilmcommunityhumicstream,orange
Although considerable variation existed among the thebiofilmcommunityoutflowstream,redthebiofilmcommunity
suspended communities from all streams and confluencestream,greenthesuspendedcommunityhumicstream,
among the different sampling times, biofilm and bluethesuspendedcommunityoutflowstreamandturquoisethe
suspendedcommunityconfluencestream.
suspended communities did not overlap. Biofilm
communities from all three streams were similar,
and no relation with their respective suspended analyses on 454 pyrosequencing data yielded simi-
counterpart could be observed. These patterns were lar patterns of community compositions as T-RFLP
congruent for the bulk (16S rRNA gene based) and data, showing no resemblance between biofilm and
the active (16S rRNA based) communities, even suspended communities from the same stream
though differences in community compositions of (Figures 1c and d). Analysis of the 16S rRNA gene
bulk and active communities are apparent from the and16SrRNAgaveaccordantpatterns,asconfirmed
nMDS analysis. Mantel’s test confirmed significant by Mantel’s correlations (Sørensen index: r¼0.98,
correlations between the similarity matrices of the Po0.05, n¼6; Horn index: r¼0.93, Po0.01, n¼6).
bulk and the active communities (Sørensen index: Totestforspeciessortingasapossiblemechanism
r¼0.82, Po0.01, n¼23; Horn index: r¼0.79, of biofilm assembly, we compared the biofilm
Po0.01, n¼23). communities with random subsamples of the sus-
The denoised 454 pyrosequencing data set con- pended communities that might result from purely
sisted on average of 9884±1321 reads per sample, stochastic immigration to an empty habitat patch
which clustered into 7512 OTUs at a 97% sequence from a source community. The bulk and the active
similarity level. The sequence data are available at biofilm communities of all three streams differed
the NCBI Sequence Read Archive under the acces- significantly from the random assemblages pro-
sion number SRX099353. A total of 4899 (that is, duced (probability of the biofilm community to fall
65%) of the detected sequences were singletons. In within the distribution of the random subsamples,
all, 6270 OTUs occurred only in the suspended Po0.001; Figure 2).
community, 556 OTUs only in biofilm and 686
OTUs were shared by both communities. Applying
a confidence threshold of 80% to the Bayesian Microbial biodiversity
classifier, 99.86% of all reads were classified as ForT-RFLPdata,OTUrichnesswasgenerallyhigher
bacteria, 0.01% were classified as Archaea and in the suspended than in the biofilm communities,
0.12% failed to be classified to any domain. nMDS whereas the number equivalents of the Shannon
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40 30
Richness Shannon
30
s
s 20 nt
U e
T al
O v
of 20 qui
ber er e
m 10 b
u m
n 10 u
n
0 0
4000 1000
Richness Shannon
800
3000 s
mber of OTUs 2000 640000 mber equivalent
nu 1000 nu
200
0 0
80 40
Richness Shannon
Figure 2 nMDS analysis visualizing the results of a random
sampling procedure to estimate the probability that the biofilm 60 30 s
cssstahuaaonmmemsdpsppmebullnleeusudspnepeeoiadtncfiideurt.sshebodWeeurescrthpuhcoiersmeteepsmcseceuonuinmsrdntpceemlieddetniusedcnrsroea,iemdtnrpieedmrcdseoo.usmmtenrAnimiatstintauetogmhnstlaeiepwtlyltreeh;aorsenfehduoba1ofmis0oms0ifteci0hmlsemsurbitarblrcneesordaademmmosfompmpr(eluaeces,nstauiibcvotb)hyef-, mber of OTUs 40 20 mber equivalent
outflowstream(c,d),confluencestream(e,f),bulk(a,c,e)and nu 20 10 nu
active(b,d,f)communities.
entropy didnotshowanyclearpatterns(Figure3a). 0 0
The active fraction exhibited similar richness and hum out con hum out con
Shannonentropyestimatesasthebulkcommunities Figure3 Microbialdiversityinbiofilmandsuspendedcommu-
without showing a consistent difference. nity, as estimated by T-RFLP (a) and 454 pyrosequencing (b),
Bacterial OTU richness estimates by 454 pyrose- calculated as richness and the number equivalents of the
Shannonentropy. A thresholdof0.2%contribution to thecom-
quencing were 3–7 times higher in the suspended
munitywasappliedtothe454 pyrosequencingdatatocompare
than in the respective biofilm communities. The results from 454 pyrosequencing with T-RFLP analysis (c).
number equivalents of the Shannon entropy esti- con,confluencestream;hum,humicstream;out,outflowstream.
mateswere4–22timeshigherinthesuspendedthan Cross-hatched bars represent the biofilm community, solid bars
in the biofilm communities (Figure 3b). Both the suspended community, green bars the bulk community and
bluebarstheactivecommunity.
measures indicated higher diversity in the bulk
community than in the active community, with the
exception of the Shannon entropy of the biofilm in and diversity estimates were in the same order of
theoutflowstream. To assess the importance ofrare magnitude as values estimated by T-RFLP; on
species to the observed patterns of diversity and average, one T-RFLP-based OTU corresponded to
to compare results from 454 pyrosequencing with two OTUs as defined by 454 pyrosequencing
T-RFLPanalysis,athresholdof0.2%contributionto (Figure 3c). The reduced 454 pyrosequencing data
the community was applied to the 454 pyrosequen- set failed to show clear differences in diversity
cing data. The threshold was chosen because 0.2% between the suspended and biofilm communities
was the percentage, which was represented by the and between active and bulk community, respec-
lowest T-RFLP peaks considered. Obtained patterns tively.Instead,thereduced454pyrosequencingdata
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100
8000
10
%) s 6000
e ( 1 TU
c O
an of
nd 0.1 er 4000
u b
b m
a
u
n
0.01
2000
0.001
1 10 100 1000 10000 0
rank bulk communtiy active communtiy
Figure 4 Rank-abundance curves of biofilm and suspended Figure 5 ‘True diversity’ estimates (medians with 95% con-
communities for relative abundances obtainedfrom 454 pyrose- fidence interval) for the biofilm and suspended communities,
quencing data. Curves are displayed in log–log scale for clarity. calculatedbyfittingSicheldistributioncurvestotheabundance
ColorsaresameasinFigure1. distributionsobtainedfromthe454pyrosequencingdata.Colors
aresameasinFigure1.
correlatedwiththeT-RFLPdata(richness:Pearson’s community, respectively. Actinobacteria, Sphingo-
r¼0.81, Po0.01; Shannon entropy: Pearson’s bacteria and Alphaproteobacteria contributed
r¼0.67, Po0.05). similarly to communities, whereas Flavobacteria,
Rarefaction curves did not reach an asymptote, Gammaproteobacteria and Bacilli were relatively
indicating a significant amount of undetected more abundant in biofilms than in the suspended
diversity,especiallyforthesuspendedcommunities communities. Chlamydiae, Deltaproteobacteria and
(Supplementary Figure 1). The rank-abundance members of the OD1 group were relatively more
distributions showed a strong dominance of a few abundant in the suspended than in the biofilm
OTUs and a long tail of rare OTUs (Figure 4). The communities;anumberofchloroplastsofeukaryotic
dominance of the most abundant OTUs was higher algae were found in the suspended communities
in the biofilms, and the number of rare OTUs was (Figure 6).
higher inthe suspended communities. Accordingly, Generally, bulk and active communities showed
theslopesoftheregressionmodelsfittedtotherank- similar taxonomic compositions. Alpha-, Beta-,
abundancecurves(r240.95,Po0.001forallmodels; GammaproteobacteriaandBacillioccurredathigher
Supplementary Table 2) differed significantly be- relative abundance in the active than in the bulk
tween suspended and biofilm communities (t-test, biofilm community, Flavobacteria, Sphingobacteria
Po0.001, n¼6). Rank-abundance curves of bulk and Actinobacteria at lower relative abundance. In
and active communities exhibited no significant the suspended communities, Alphaproteobacteria
difference. Computed values of ‘true richness’ constituted a higher percentage of the active com-
ranged from 526 to 1347 in biofilms and from 2854 munity, whereas Flavobacteria, Actinobacteria,
to 6512 in the suspended communities (Figure 5). Chlamydiae and the OD1 group were less abundant
Richness of the bulk community was consistently in the active than in the bulk community.
higher than of the respective active fraction. In total, 1606 OTUs (21% of all OTUs) were
classifiedtothegenuslevel,representing52%ofall
reads. The three most common genera were Acid-
Taxonomic composition ovorax (1 OTU), Flavobacterium (56 OTUs) and
Overall, 3603 OTUs (that is, 48% of all OTUs), Polynucleobacter (7 OTUs) in both the biofilm and
representing79%ofallreads,couldbeassignedtoa suspended communities, although in different order.
class at a confidence threshold of 80%. Biofilm Together,theycontributedbetween33%and41%to
OTUs were allocated to 29 classes belonging to 14 the individual biofilm communities and between
phyla; OTUs of the suspended community were 13% and 21% to the suspended communities
allocated to 48 classes of 24 phyla. Those classes (Table 1). A nMDS analysis of a Horn similarity
contributing most to the observed diversity were matrix, including only the OTUs of these three
present in the biofilm and suspended communities, most common genera, revealed patterns similar
although in several cases the distribution of their to those obtained from the whole communities,
relativeabundanceindicatedapreferenceforoneof showing a clear separation of biofilm and suspen-
the two life forms (Figure 6). Betaproteobacteria ded communities, as well as bulk and active
accounted for more than one-third and one-fourth communities (Supplementary Figure 2). Fewgenera
of the reads in the biofilm and the suspended showed different abundances inthe bulkandactive
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Bulk biofilm community Bulk suspended community anddiversityestimatesforthemicrobialcommunities
in streams and rivers remain scarce (Vishnivetskaya
et al., 2011). Our estimates derived from 454
pyrosequencing data are the first to provide com-
prehensive insights into the microbial diversity
contained in stream water and streambed biofilms.
The ‘true richness’ estimates for the suspended
communities are comparable to reports from soils,
whereas the lower richness of the biofilms are
similar to values from the ocean, as computed by
Quince et al. (2008).
Diversity, as derived by 454 pyrosequencing, was
consistently lower in the biofilm communities than
Active biofilm community Active suspended community
inthesuspendedcommunities.Thisagreeswiththe
generallylowerslopesoftherank-abundancecurves
forthesuspendedthanforthebiofilmcommunities.
Pommier et al. (2010) suggested that low-slope rank
abundance distributions for bacterial communities
in coastal waters resulted from the mixing of
terrestrial and deep-water taxa. This would be in
accordancewithourhypothesisthatvarioussources
of bacterial species within the catchment support a
diverse community suspended in the stream water.
The occurrence of typical soil bacteria, such as
members of the Deltaproteobacteria and OD1 divi-
sion (Spring et al., 2000; Harris et al., 2004;
Betaproteobacteria Elshahed et al., 2005), in the suspended commu-
Flavobacteria nities supports this notion. This is also in line with
Actinobacteria results from a recent meta-analysis of published
Sphingobacteria environmental sequences showing that the taxo-
nomic profiles of freshwater and terrestrial habitats
Gammaproteobacteria
widelyoverlap(Tamamesetal.,2010),whichmakes
Chloroplasts and Cyanobacteria
particularlysense forheadwaters where theintegra-
Alphaproteobacteria
tion with the landscape is most pronounced (Battin
Bacilli
et al., 2008). Headwater streams might thus be
OD1
considered as important terrestrial–aquatic links
Chlamydiae that collect bacterial diversity from the surrounding
Deltaproteobacteria landscape into a source community that potentially
Opitutae seeds the benthic biofilms.
other classes and not classified We integrated the samples of the suspended
stream water communities over time for 454 pyr-
Figure 6 Relative abundances of the most important phyloge- osequencing analysis to represent the full diversity
neticclassesinthebiofilmandsuspendedcommunities.Eachpie of the microbes, potentially seeding the biofilm.
chart represents the pooled data from all the three investigated Considering the short residence time of the stream
streams.
water with the suspended bacteria and assuming
that the temporal dynamics of the suspended
community was higher than captured by our
communities. Arcicella constituted 5% of the bulk sampling scheme, we likely missed some of the
biofilm community but was not among the 10 most suspended diversity. Accordingly, the finding of
abundant genera in the active community. Pseudo- higher diversity in the suspended community as
monaswasamongthemostabundantgeneraonlyin compared with the biofilms may be conservative.
the active biofilm community. Two of the most However, we studied relatively young rather than
common genera in the suspended community were mature biofilms, where diversity may not have
identified as chloroplasts of eukaryotic algae. reached its maximum yet (Jackson et al. 2001).
ThedominanceofrelativelyfewOTUsandalong
tail of rare OTUs is typical for rank-abundance
Discussion curvesofmicrobialcommunities(Schwalbachetal.,
2004; Pommier et al., 2010). Such rank-abundance
Methodological constraints have until recently curves have been postulated to be composed of
hindered the accurate measurement of microbial a set of abundant taxa, performing most ecosystem
diversity (Lunn et al., 2004; Quince et al., 2008), functions, and of a seed bank containing rare taxa
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Table1 Listofthetenmostcommongenerainbiofilmandsuspendedcommunities
Bulkbiofilm Activebiofilm Bulksuspended Activesuspended
community(%) community(%) community(%) community(%)
Flavobacterium(16.3) Acidovorax(21.9) Polynucleobacter(9.3) Acidovorax(9.3)
Acidovorax(12.0) Polynucleobacter(8.1) Acidovorax(4.6) Flavobacterium(5.7)
Polynucleobacter(9.5) Flavobacterium(7.5) Flavobacterium(4.0) Polynucleobacter(4.0)
Arcicella(5.2) Polaromonas(6.7) OD1generaincertaesedis(2.9) Cryptomonadaceaechloroplast(3.6)
Alkanindiges(3.6) Alkanindiges(5.6) Sediminibacterium(2.5) Bacillariophytachloroplast(2.8)
Polaromonas(3.6) Pseudomonas(2.1) Cryptomonadaceaechloroplast(1.5) Sediminibacterium(2.3)
Sediminibacterium(2.2) Sediminibacterium(1.7) Bacillariophytachloroplast(1.4) Duganella(0.7)
Methylophilus(1.8) Beijerinckia(1.2) Arcicella(1.0) Verrucomicrobiasubdivision3
generaincertaesedis(0.7)
Pedobacter(0.9) Comamonas(1.1) Methylophilus(0.8) Methylophilus(0.6)
Fluviicola(0.8) Herbaspirillum(1.0) TM7generaincertaesedis(0.6) Arcicella(0.6)
Numbersindicatetheaveragerelativeabundanceofthegenusinthecommunitiesfromallthethreestreams.
(Pedro´s-Alio´, 2006). This rare biosphere has since presence/absence of rare taxa, rather than the
beenreportedtocontainalargeproportionofactive complete replacement of the dominant groups. The
taxa(JonesandLennon,2010)andtobesubjectedto observationthat the most abundantgenera occurred
environmental controls (Andersson et al., 2010; in both suspended and biofilm communities is
Campbelletal.,2011).Otherstudiesfoundthatrare surprising, given the clear separation of the two
phylotypes tend to stay rare, arguing against the groups in the nMDS analysis. This apparent contra-
seedbankhypothesis(Galandetal.,2009;Kirchman diction can be partly explained by our finding that
et al., 2010). If the abundant OTUs were actively variance in the dominant genera Acidovorax spp.,
growing while a large part of rare OTUs was Flavobacterium spp. and Polynucleobacter spp.
inactive, we would expect steeper slopes in the alone yielded similar community composition pat-
rank-abundance curves of the active community ternsasderivedfromthecompletecommunity.This
than of the bulk community. However, the rank- indicates that community diversification below the
abundance curves of bulk and active communities genuslevelcontributestotheobservedseparationof
wereindistinguishableinthepresentstudy,indicat- the suspended and biofilm communities.
ing that at least a certain fraction of the rare OTUs Bulk and active populations, although clearly
wasactive.Theserarebutactivepopulationsmaybe different from each other, generated comparable
controlled by top–down forces or competition; patterns of community composition among the
however, they have the potential to increase in biofilms and the suspended communities. This
abundance, which supports the idea that microbial suggeststhatsimilarmechanismscontroltheassem-
rank-abundance curves may be highly dynamic bly of these populations. Although the most abun-
(Jones and Lennon, 2010). dant genera were active in both the biofilm and
The fact that the composition of the suspended suspended communities, others showed opposing
communities differed to some extent among the patterns. For instance, Proteobacteria occurred in
three investigated streams while the biofilm com- the active fraction either in similar or in higher
munities were similar is evidence that biofilm percentages than in the bulk community, whereas
assembly did not simply reflect differences in the members of the Bacteroidetes phylum and Actino-
source communities. Furthermore, simulated bio- bacteria contributed less to the active community.
film communities from random sampling of the Similar differences in the distribution of active taxa
respective suspended community demonstrate that have been reported from lakes (Jones and Lennon,
stochasticdispersalfromthesourcecommunitywas 2010). We found no evidence that the apparently
unlikely to shape the observed community compo- active populations in the suspended community
sition of the biofilms. This supports our hypothesis contributed more to biofilm formation than the less
that species sorting has a certain role in the active populations. For instance, Alphaproteobac-
assembly of the biofilm community. Previous work teria, which were active in the suspended commu-
showed that sorting, as induced by fine-scale nity, were less abundant in the biofilms than in the
hydrodynamic niche differentiation, rather than suspended community.
mass effects, was a potential mechanism of stream To initiate biofilm formation, bacteria need to
biofilm community assembly (Besemer et al., 2009). be able to attach to surfaces or to co-aggregate
The interplay of niche availability and competi- (Rickard et al., 2003, 2004). This ability might
tion has been suggested to drive the patterns of have favored the proliferation of certain groups of
bacterial biodiversity in biofilms (Jackson et al., Betaproteobacteria, which were found to dominate
2001) and may induce species sorting. Our results biofilm communities in this as well as in earlier
suggest that species sorting resulted in different studies (Schweitzer et al., 2001; Araya et al., 2003).
relative abundances of dominant taxa and in the Interestingly, the most abundant genus Acidovorax
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