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12 Pages·2017·1.64 MB·English

university of copenhagen
Bacterial community structure in High-Arctic snow and freshwater as revealed by
pyrosequencing of 16S rRNA genes and cultivation
Møller, Annette K.; Søborg, Ditte A.; Abu Al-Soud, Waleed; Sørensen, Søren Johannes;
Kroer, Niels
Published in:
Polar Research
DOI:
10.3402/polar.v32i0.17390
Publication date:
2013
Document version
Publisher's PDF, also known as Version of record
Citation for published version (APA):
Møller, A. K., Søborg, D. A., Abu Al-Soud, W., Sørensen, S. J., & Kroer, N. (2013). Bacterial community
structure in High-Arctic snow and freshwater as revealed by pyrosequencing of 16S rRNA genes and cultivation.
Polar Research, 32, [17390]. https://doi.org/10.3402/polar.v32i0.17390
Download date: 22. Feb. 2023
RESEARCH/REVIEW ARTICLE
Bacterial community structure in High-Arctic snow and
freshwater as revealed by pyrosequencing of 16S rRNA
genes and cultivation
Annette K. Møller,1 Ditte A. Søborg,1 Waleed Abu Al-Soud,2 Søren J. Sørensen2 & Niels Kroer1
1 DepartmentofEnvironmentalScience,AarhusUniversity,Frederiksborgvej399,DK-4000Roskilde,Denmark
2 DepartmentofBiology,UniversityofCopenhagen,Sølvgade83H,DK-1307KCopenhagen,Denmark
Keywords Abstract
Taxonomicdiversity;microbial
The bacterial community structures in High-Arctic snow over sea ice and an
assemblages;bacterialdensity;DOC.
ice-covered freshwater lake were examined by pyrosequencing of 16S rRNA
Correspondence genes and 16S rRNA gene sequencing of cultivated isolates. Both the
NielsKroer, pyrosequenceandcultivationdataindicatedthatthephylogeneticcomposition
DepartmentofEnvironmentalScience, ofthemicrobialassemblageswasdifferentwithinthesnowlayersandbetween
AarhusUniversity,
snow and freshwater. The highest diversity was seen in snow. In the middle
Frederiksborgvej399,
and top snow layers, Proteobacteria, Bacteroidetes and Cyanobacteria dominated,
DK-4000Roskilde,Denmark.
although Actinobacteria and Firmicutes were relatively abundant also. High
E-mail:[email protected]
numbers of chloroplasts were also observed. In the deepest snow layer, large
percentages of Firmicutes and Fusobacteria were seen. In freshwater, Bacter-
oidetes,ActinobacteriaandVerrucomicrobiawerethemostabundantphylawhile
relatively few Proteobacteria and Cyanobacteria were present. Possibly, light
intensitycontrolledthedistributionoftheCyanobacteriaandalgaeinthesnow
while carbon and nitrogen fixed by these autotrophs in turn fed the
heterotrophic bacteria. In the lake, a probable lower light input relative to
snow resulted in low numbers of Cyanobacteria and chloroplasts and, hence,
limited input of organic carbon and nitrogen to the heterotrophic bacteria.
Thus, differences in the physicochemical conditions may play an important
role in the processes leading to distinctive bacterial community structures in
High-Arctic snow andfreshwater.
To access the supplementary material for this article, please see supple-
mentary filesunderArticle Tools online.
The Arctic region and the Antarctic continent constitute of cold shock (Cloutier et al. 1992) and antifreeze
upto14%ofthebiosphere(Priscu&Christner2004)and proteins (Gilbert et al. 2005) enable bacteria to survive
offer some of the coldest and most arid environments under cold conditions, and bacterial activity has been
on Earth. Snow is an important component of the polar detected at sub-zero temperatures in sea ice and snow
regions (Jones 1999) and recent reports suggest that (Carpenter et al. 2000; Junge et al. 2004; Panikov &
microorganisms may impact the dynamics, composition Sizova2006).
andabundanceofnutrients(Hodsonetal.2008)aswell Most studies on microbial diversity in polar environ-
as the surface albedoof snow(Thomas &Duval 1995). ments have focused on ice, permafrost and marine
Various physiological adaptations, such as increased environments,whilethemicrobialcommunitystructure
membranefluidity(Kumaretal.2002),synthesisofcold- in snow has only been scarcely examined. The few
adaptedenzymes(Groudievaetal.2004)andproduction available studies of snow have shown that Proteobacteria
1
PolarResearch2013.#2013A.K.Mølleretal.Thisisanopen-accessarticledistributedunderthetermsoftheCreativeCommonsAttribution-Noncommercial3.0
UnportedLicense(http://creativecommons.org/licenses/by-nc/3.0/),permittingallnon-commercialuse,distribution,andreproductioninanymedium,providedthe
originalworkisproperlycited. Citation:PolarResearch2013,32,17390,http://dx.doi.org/10.3402/polar.v32i0.17390
(pagenumbernotforcitationpurpose)
BacterialcommunitystructureinHigh-Arcticsnowandfreshwater A.K.Mølleretal.
(Alpha and Beta), Bacteroidetes (Flavobacteria and Sphingo- andslowlymeltedat5(cid:2)78Cinthedarknessforupto48h
bacteria) and Thermus(cid:2)Deinococcus appear to be frequent toavoid stressing thebacteria during melting.
when using culture-independent approaches, i.e. clone Freshwater samples were collected at a depth 70 cm
libraries(Carpenteretal.2000;Laroseetal.2010),while belowtheiceusingapump.Thehosefromthepumpwas
Proteobacteria, Firmicutes and Actinobacteria have been flushed with 3000 litres of water before 2 litres were
isolated by cultivation-based methods (Amato et al. collectedin sterileglassbottles.
2007).
Pyrosequencing has been applied to a few Arctic
Chemical analyses
environments, including glacial ice (Simon et al. 2009),
permafrost (Yergeau et al. 2010) and the Arctic Ocean Triplicate samples of 10(cid:2)15 ml snow meltwater were
(Kirchman etal.2010).No characterization ofmicrobial filteredthrough0.2mmMinisartsyringefilters(Sartorius,
communitiesinsnowandfreshwaterbypyrosequencing Go¨ttingen, Germany) into acid rinsed (10% HCl) glass
of 16SrRNA genesisavailable. scintillations vials and stored frozen at (cid:3)15 to (cid:3)188C.
The goal of thisstudy was to exploreand contrast the The syringe filters were rinsed in 5 ml sample water
bacterialcommunitystructureindifferentlayersofsnow before collecting the sample. The content of dissolved
over sea ice and an ice-covered freshwater lake in the organic carbon (DOC) was measured on a TOC 5000
High Arctic, and to possibly identify the sources of the analyser (Shimadzu Scientific Instruments, Columbia,
bacteria to the communities. To the best of our knowl- MD, USA; Kroer 1993). Prior to analysis, samples were
edge, this study is the first to assess the taxonomic acidified and purged with O for 5 min to remove
2
diversity of High-Arctic snow and freshwater microbial inorganiccarbon.
assemblages by analysis of pyrosequencing-derived data
sets. Tocomplement thisapproach,we usedcultivation-
Total bacterial abundance
dependent approaches, i.e., direct cultivation on rich
medium and pre-incubation under simulated natural Total bacterial abundance was determined by direct
conditions priorto platingonrichmedium. countswith aBH2microscope (Olympus, CenterValley,
PA, USA). Unfixed bacteria were collected on 0.2 mm
pore-size black Nucleopore polycarbonate membranes
Experimental procedures (Whatman, Maidstone, Kent, UK) and stained with
a 1:1000(cid:4)dilution of SYBR Gold (Invitrogen(cid:2)Life
Study area and sampling
Technologies, Carlsbad, CA, USA). Samples were ana-
Snow and freshwater were collected in May(cid:2)June lysed immediately after sampling or as soon as snow
2007 and May 2010 at Station Nord in north-eastern samplesweremelted.
Greenland. Snow over sea ice was sampled in Dagmar
Sund (81836.58?N; 16842.83?W) between Station Nord
Enumeration and isolation of cultivable bacteria
and Prinsesse Dagmar Island, while the freshwater
samples were taken from a small ice and snow covered Bacteria were isolated by two different procedures: i)
lake about 2 km south of the station (81834.48?N; direct plating and ii) pre-incubation under simulated
16837.46?W). The samples taken in 2010 were exclu- natural conditions using polycarbonate membranes
sively usedfor analysisof pHand salinity(see below). as a growth support before plating on rich medium
Tocollect the snow samples, a vertical snow profile of (Møller et al. 2011). The pre-incubation procedure was
120cmheightwasmadebydiggingwithasterileshovel. applied to enhance the culturability of the bacteria
Immediately prior to sampling, the outermost 1 cm of (Rasmussen et al. 2008). Briefly, subsamples of melted
snow was removed with a sterile knife. Sampling was snow or freshwater were plated onto 10% strength
done using a sterile Plexiglas corer (internal diameter: Tryptic Soy Agar (TSA) and incubated at 4(cid:2)108C and
14cm,length:25cm)thatwasinsertedhorizontallyinto the colony forming units were counted at successive
thesnowpackat31(cid:2)52cm(top),75(cid:2)90cm(middle)and intervals until a constant count was obtained. The pre-
96(cid:2)112 cm (bottom) depths. Up to 10 replicate cores incubation procedure involved filtration onto 0.2 mm
were taken at ca. 10 cm distance at each sampling pore-size polycarbonate membranes (25 mm diameter).
depth. Sampling depths were determined on the basis The polycarbonate membranes, with the bacterial cells
of observations of snow hardness and texture to insure facing upward, were placed on the fixed 0.22-mm
that different snow layers were sampled. The snow was Anopore disc of 25-mm Nunc tissue culture inserts, and
transferred to sterile plastic buckets covered with a lid the tissue culture inserts were placed in six-well plates
2
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A.K.Mølleretal. BacterialcommunitystructureinHigh-Arcticsnowandfreshwater
containing 1 ml of sample water supplemented with detached from the filters during storage (i.e., pellet
Tryptic Soy Broth (TSB) at concentrations of 0, 0.01 or obtained after centrifugation of the RNAlater). Extrac-
0.1%, respectively. The membranes were incubated at tions were performed with the Genomic Mini Kit (A&A
4(cid:2)108C and the growth medium replaced with fresh Biotechnology, Gdynia, Poland) according to the manu-
medium every seven days. Formation of micro-colonies facturer’s instructions with the following modifications:
wasfollowedbymicroscopy.After77daysofincubation, filters and pellets were combined in 1 ml extraction
bacteria from the membranes were extracted in salt buffer (50 mM Tris-HCl, 5 mM EDTA, 3% SDS)
buffer (KH PO [0.25 g litre(cid:3)1], MgSO (cid:2)7 H O [0.125 g and beadbeated for 30 sec on a Mini-Beadbeater (Glen
2 4 4 2
litre(cid:3)1], NaCl [0.125 g litre(cid:3)1], [NH ] SO [0.2 g Mills, Clifton, NJ, USA) and the supernatant added to
4 2 4
litre(cid:3)1]) by vigorous vortexing for 1 min. Appropriate 465 ml 5 M ammonium acetate. After centrifugation
dilutions were plated on 10% TSA plates and incubated at16000(cid:4)gfor10min,twovolumesof7MGuanidine-
at 68C until numbers of colonies became constant. HCl were added to the supernatant and the mixture
Between 140 and 200 colonies from each location were applied to the spin column provided in the kit and the
randomly picked. All isolates were re-streaked on TSA DNApurified following the manufacturer’s instructions.
at leasttwotimes toensure purity. A526bpDNAfragmentflankingtheV3andV4regions
of the 16S rRNA gene was amplified by PCR using
universal prokaryote primers modified after Yu et al.
Partial sequencing of 16S rRNA genes of isolated
(2005):341F(5’-CCTACGGGRBGCASCAG-3’)and806R
bacteria
(5’-GGACTACNNGGGTATCTAAT-3’).ThePCRamplifica-
DNAwas extracted from the isolated bacteria by boiling tion(25ml)weredonein1(cid:4)PhusionHFbuffer,0.2mM
(Frickeretal.2007).Forsomeoftheisolates,extraction dNTPmixture,0.5UPhusionHotStartDNAPolymerase
by boilingwas not applicable due to lowDNAyields.As (Finnzymes(cid:2)Thermo Scientific, Espoo, Finland), 0.5 mM
an alternative, the PowerMax DNA soil kit (Mo Bio ofeachprimerand1mlDNA.PCRconditionswere30sec
Laboratories,Carlsbad,CA,USA)wasusedfollowingthe at 988C followed by 30 cycles of 5 sec at 988C, 20 sec at
manufacturer’s instructions. All DNA preparations were 568Cand20secat728Cfollowedbyafinalextensionfor
storedat (cid:3)208C. 5minat728C.AfterthePCRamplification,sampleswere
The 16S rRNA gene of the bacteria was amplified by heldat708Cfor3minandthenmoveddirectlyontoiceto
polymerasechainreaction(PCR)withuniversalbacterial prevent hybridization between PCR products and short
primers 27f (5?-AGA GTT TGA TCM TGG CTC AG-3?) non-specificamplicons.ThePCRproductswereanalysed
and 519r (5?-GWA TTA CCG CGG CKG CTG-3?). The on 1% agarose gel and purified with QIAEX II Gel
PCRmixture(25ml)consistedof2mlDNAtemplate,1U Extraction Kit (Qiagen, Venlo, The Netherlands). The
Taq DNA polymerase (Fermentas(cid:2)Thermo Scientific, purified PCR products were tagged by another PCR (15
Waltham, MA, USA), 0.4 mM of each primer, 400 mM cycles) using primers 341F and 806R with adapters and
dNTPs and 0.5 mM MgCl . PCR incubation conditions barcode sequences, also known as tags (Supplementary
2
were2minat958Cfollowedby35cyclesof30secat958C, Table S1). The tagged PCR amplicons were gel purified
30 sec at 558C and 1 min at 728C followed by a final with Montage Gel Extraction Kit (Millipore, Billerica,
extension for 5 min at 728C. The PCR products were MA,USA)andthefragmentsquantifiedusingaQubitTM
analysedonagarosegelsstainedwithethidiumbromide. fluorometer(Invitrogen(cid:2)LifeTechnologies)andmixedin
Sequencing of the PCR products were performed by approximately equal amounts (4(cid:4)105 copies per ml) to
Macrogen Inc. (Amsterdam, The Netherlands) using ensureequalrepresentationofeachsample.DNAsamples
primer27f. weresequencedusingoneofthetwo-regionsofa70(cid:4)75
GS PicoTiterPlate (PTP) by using a GS FLX pyrosequen-
cing system (Roche Diagnostics, Basel, Switzerland) ac-
Pyrosequencing
cordingtothemanufacturer’sinstructions.
Meltwater or freshwater (200(cid:2)500 ml) were filtered
through 0.2 mm polycarbonate filters until the filters
Sequence analyses
clogged. Filters were stored in 1 ml RNAlater (Ambion(cid:2)
LifeTechnologies,Austin,TX,USA)at (cid:3)208C.Atotalof Sequencesobtainedfrompyrosequencingweredenoised
10replicatefreshwaterfilterswerecollected,while10,9 toremovespurioussequencesandcheckedforchimeras
and6replicatefilterswerecollectedfromthetop,middle using the AmpliconNoise and ChimeraSlayer tools inte-
andbottomsnowlayers,respectively.DNAwasextracted gratedintheopen-sourceQIIMEsoftwarepackage(http://
fromthecellsonthefiltersaswellasfromcellsthathad qiime.org; Caporaso et al. 2010). The error correcting
3
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BacterialcommunitystructureinHigh-Arcticsnowandfreshwater A.K.Mølleretal.
procedure removed between 17% (snow-top) and 59% the density was two orders of magnitude higher. The
(freshwater) of the sequences. Adaptors, amplification cultivability of the bacteria was highly variable, ranging
primers and barcode sequences were trimmed from from 0.3%in freshwater to 12% in snow (Table 1). The
denoised data, and the sequences filtered based on highest cultivability was observed in the two deepest
a quality score ]25 and a length ]200 bp. Finally, snowlayers.Itshouldbenotedthatonlyonecolonywas
sequences were sorted on the basis of the barcode isolated from the top snow layer, making an estimate of
sequence using QIIME’s split_libraries.py tool. The Pyr- cultivabilityuncertain.
osequencingPipelineClassifieroftheRibosomalDatabase Cultivation of the snow and freshwater microorgan-
Project (RDP; http://rdp.cme.msu.edu; Cole et al. 2009) isms resulted in a total of 791 bacterial isolates.
wasusedtoassignthe16SrRNAgenesequencestohigher- About two-thirds of these (570) originated from the
orderbacterialtaxonomywithaconfidencethresholdof pre-incubation approach, in which the bacteria under
50%asrecommendedforsequencesshorterthan250bp. simulated natural conditions were pre-grown to micro-
Alignmentsandclustering(maximumdistanceof3%)of
colony size on polycarbonate membranes before being
the sequence libraries was done using the Aligner and
transferred to rich medium for further growth, while
Complete Linkage Clustering tool of the RDP’s Pyrose-
221 resulted from direct plating onrich medium. Partial
quencingPipeline.Diversityindexeswerecalculatedusing
sequencing of the 16S rRNA genes was done for
theShannonandChao1indexanalysistoolinRDP.Bray
all bacterial isolates; 22 poor-quality sequences were
Curtissimilarityanalysisbetweendifferentsamplesatthe
discarded. The total number of unique culturable OTUs
phylum-level was done with PRIMER 5 for Windows,
(]97% sequence similarity) among the remaining 769
version5.2.9(http://www.primer-e.com).
isolates was 32. In snow, the total number of unique
The quality of the partial 16S rRNA gene sequences
OTUs was slightly higher than in freshwater as 20
of the isolates was manually checked and sorted. All
unique OTUs were found in this environment while 17
analysisanddiversitycalculationswereperformedasfor
unique OTUs were observed in freshwater (Table 2).
the pyrosequences.
Five OTUs belonging to the Proteobacteria, Actinobacteria
and Bacteroidetes were found both in freshwater and
Nucleotide sequence accession numbers snow.
Cellsinsnowmeltwaterandfreshwaterwerecollected
16S rRNA gene sequences derived from pyrosequencing
on0.2mmpolycarbonatemembranesandtheir16rRNA
have been deposited at the National Center for Biotech-
genes partially pyrosequenced. When combining the up
nology Information (NCBI), in Bethesda, MD, USA,
to10replicatefiltersperenvironment,thetotalnumber
under accession number SRP003408. Partial 16S rRNA
of 16 rRNA gene fragments (tags) obtained for each
gene sequences of one representative of each of the
bacterial assemblage ranged between 34000 and 85000
32 different culturable operational taxonomic units
(OTUs) have been deposited at the NCBI under acces- (Table 3). The number of different bacterial OTUs
sion numbers JF280090-JF280117, and GU932934, (]97% sequence similarity) per assemblage varied
GU932935, GU932939, GU932940, GU932949 and between 2000 and 6000 (Table 3). Several chloroplasts
GU932975. (primarily Streptophyta but also some Chlorophyta) were
identified in snow and excluded from the analysis.
The highest number of chloroplast tags was observed
Results
in the middle snow layer (17620; ca. 28% of bacterial
Thebacterialabundanceinsnowrangedfrom8(cid:4)102to tags)followedbythetop(5035;ca.6%ofbacterialtags)
3(cid:4)103cellsml(cid:3)1of meltwater(Table1).In freshwater, and the bottom (2075; ca. 5% of bacterial tags) layers.
Table1 Chemicalandbiologicalcharacteristicsofthesamplingsites.
Sample DOCa(mgClitre(cid:3)1) Totalcountsb(cellsml(cid:3)1)c Directplatingb(cfuml(cid:3)1)c
Freshwater Notdetermined 9.4(cid:4)10593.8(cid:4)104 2.5(cid:4)10397.1(cid:4)101
Snow:top 4.090.7 3.1(cid:4)10391.5(cid:4)103 2.0(cid:4)10092.0(cid:4)100
Snow:middle 3.890.8 1.4(cid:4)10391.1(cid:4)103 1.7(cid:4)10291.2(cid:4)102
Snow:bottom 1.390.02 8.5(cid:4)10299.0(cid:4)101 6.8(cid:4)10193.0(cid:4)101
aDissolvedorganiccarbon.
bStandarderrors(n(cid:5)3(cid:2)5).
cBacterialdensitiesinsnowarereportedasnumbersofcellspermlmeltwater.
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A.K.Mølleretal. BacterialcommunitystructureinHigh-Arcticsnowandfreshwater
Table 2 Summary of number of obtained 16S rRNA partial gene quencing and the cultivation approaches suggested
sequences and number of observed operational taxonomic units that the entire diversity of the communities was not
(OTUs; sequence similarity ]97%) among cultivated bacterial isolates captured, as the curves did not reach a plateau with
infreshwaterandsnow(differentsamplingdepthscombined).
increasing sample size. Based on the Chao1 species
Pre-incubation
richness estimator, an estimated 57(cid:2)61% of all OTUs
onfilters Directplating
were identified in snow while 61% were identified in
Sample Sequences OTUs Sequences OTUs Shannon-Weaver freshwater.
Freshwatera 249 13 70 6 1.34 Overall similarity of the microbial communities
Snowb 299 12 145 12 1.39 was examined at the phylum level (distribution of tags
aTwoOTUsinfreshwaterwerefoundbothbydirectplatingandbyfilterincubation. within each phylum compared between the four en-
Therefore,thetotalnumberofuniqueOTUsinfreshwaterwas17. vironments) by a x2-test and by the Bray(cid:2)Curtis
bFourOTUsinsnowwerefoundbothbydirectplatingandbyfilterincubation.
similarity measure (Fig. 2). Although the communities
Therefore,thetotalnumberofuniqueOTUsinsnowwas20.
were significantly different as determined by the x2-test
In freshwater, the number of observed chloroplast tags (PB0.001), the Bray(cid:2)Curtis similarity measure, based
waslow(11; ca.0.03%of bacterial tags). onthepyrosequencingdataandthesequencedatafrom
the cultivated bacteria obtained by the pre-incubation
technique, indicated that the three snow layers were
Structure of the microbial communities
more similar to each other (65(cid:2)80% similarity) than to
The structure of the bacterial communities at the three
snow depths and the freshwater lake was compared
by rarefaction analysis (Fig. 1) and other measures of
diversity (Table 3). Rarefaction curves indicated that
amongthesnowcommunities,thediversitywashighest
inthemiddlesnowlayer.However,diversityatallsnow
depths was higher than in the freshwater lake (Fig. 1a).
Rarefactionanalysisbasedontheculturablebacteriadid
not indicate notable differences between the snow and
lake communities (Fig. 1b). The Chao1 species richness
estimator and the Shannon-Weaver diversity index for
OTUs sharing ]97% sequence similarity supported the
conclusion that the middle snow layer showed the
highest diversity and that the snow microbial commu-
nities were more diverse than the freshwater commu-
nity (Table 3). The Shannon(cid:2)Weaver diversity index
calculated for the culturable bacteria (Table 2) did not
indicate a difference in the diversity between snow and
freshwater. The rarefaction curves of both the pyrose-
Table 3 Summary of pyrosequence (tags) numbers, numbers of
operational taxonomic units (OTUs; ]97% sequence similarity) and
diversity estimates. To allow comparison of the Shannon-Weaver
diversity index between samples, an equal number of sequences
(33882) from each community was randomly selected and used for
the analysis. Note that the data represent summed tags for replicate
sampleswithineachenvironment.Chloroplastswereexcludedfromthe
analysis.
Numberof
Sample sequences OTUs Chao1 Shannon-Weaver
Freshwater 33882 1957 3204 4.58
Fig.1 Rarefactioncurvesofoperationaltaxonomicunits(OTUs; ]97%
Snow:top 85456 4356 7576 5.06
sequencesimilarity)ofthe(a)16SrRNAgenemetagenomiccommunity
Snow:middle 62345 6084 9961 5.31
librariesand(b)cultivatedbacteria.Notethatin(b),OTUsofthethree
Snow:bottom 40279 3421 5986 5.60
snowdepthshavebeencombined.
5
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BacterialcommunitystructureinHigh-Arcticsnowandfreshwater A.K.Mølleretal.
Fig.2 DendrogramsbasedontheBray(cid:2)Curtissimilaritymeasureshowingtherelatedness ofthemicrobialcommunitiesinsnowandfreshwater:
(a) pyrosequencing; (b) cultivation/pre-incubation on filters; (c) cultivation/direct plating. Also shown is the distribution of the most frequent
phylogenetic groups within each community. Note that in (a), the data represents the combined results of several replicate samples within each
environment(seeSupplementaryFig.1Sfordataassociatedwithindividualsamples).
freshwater (55(cid:2)60%). Within the snow, the top and In snow, however, the most abundant phyla had more
middle layers were most related (80(cid:2)90%; Fig. 2a, b). OTUsthan freshwater.
Only one isolate belonging to the Burkholderiales was
obtained from the top layer of the snow by direct
Phylogeneticcompositionofmicrobialcommunities
plating. Hence, it was not possible to include this
layer in the Bray(cid:2)Curtis similarity measure, and the The phylogenetic composition of the microbial assem-
similarity analysis was consequently different from blageswasdifferentwithinthesnowlayersandbetween
the analyses based on the pyrosequencing data and snow and freshwater (Fig. 2). In snow, Proteobacteria,
the cultivated bacteria isolated from the pre-incubated Actinobacteria and Bacteroidetes dominated (frequencies of
filters (Fig. 2c). 31(cid:2)43%, 7(cid:2)8% and 12(cid:2)14%, respectively) although in
The most abundant phyla had more unique OTUs the top and middle and snow layers, Cyanobacteria also
than the rare phyla in both snow and freshwater, i.e., accounted for a substantial fraction (16(cid:2)27%) of the
abundant bacterial groups had higher within-group communities (Fig. 2a). Another major difference be-
diversity than rare groups (Fig. 3). The correlation tween the bottom and middle/top snow layer was the
between log [relative abundance] and log [number of presence of a large percentage of Firmicutes (24%) and
uniqueOTUs]washighforallcommunities(PB0.0001; Fusobacteria(8%)inthebottomlayer.Minorfrequencies
r(cid:5)0.97,0.97and0.92fortop,middleandbottomsnow, ofunclassifiedbacteria(3(cid:2)6%),Acidobacteria(3(cid:2)4%)and
respectively, and PB0.005; r(cid:5)0.78 for freshwater). Verrucomicrobia(1(cid:2)2%)wereobservedinallthreelayers.
6
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A.K.Mølleretal. BacterialcommunitystructureinHigh-Arcticsnowandfreshwater
about3%.Actinobacteria,whichconstituted 7(cid:2)8%ofthe
snowcommunities(Fig.2a)wererepresentedbyseveral
genera(SupplementaryTableS3)and,hence,onlyinthe
middle snow was the genus Illumatobacter among the 10
most abundant (]97%) phylo-types (Supplementary
Table S2), accounting for B2% of all sequences. Fuso-
bacteria, on the other hand, which constituted 8% of
the community of the bottom snow layer (Fig. 2a),
was exclusively represented by the genus Fusobacterium
(Supplementary Table S2). The most dominant genus*
by far*in the bottom snow layer was Streptococcus,
constituting 15% of all observed sequences. Streptococcus
wasalsorepresentedinthetopsnowlayer,where3%of
Fig. 3 Relative abundance of phyla as a function of the number of the sequences belonged to this genus. Also, among the
uniqueoperationaltaxonomicunits(OTUs). Firmicutes,staphylococciwereabundantespeciallyinthe
bottom snow layer (Supplementary Tables S2, S3). In
Severalotherphylawereobservedatfrequencies B1%, freshwater, the genera Flavobacterium and Illumatobacter
includingthecandidatedivisionTM7(50.3%),andthe were predominant, constituting 13% and 6%, respec-
Archaean phyla Euryarchaeota (50.5%) and Crenarch- tively, of all observed sequences in this environment
aeota (50.1%). In contrast to snow, the freshwater (Supplementary Table S2). Within the Planctomycetes,
community was characterized by a relatively large Isophaera completely dominated, comprising (cid:2)93% of
fraction of Planctomycetes (3%) and Actinobacteria (23%), allthePlanctomycetesfoundinfreshwater(Supplementary
several unclassified bacteria (16%) and a relatively TableS3).
infrequent population of Proteobacteria (9%; Fig. 2a). Among the cultivable bacteria, the dominance of
Thefrequency ofTM7and Archaeawas B0.05%. individual genera differed from what was observed by
Similar to the phyla observed by pyrosequencing of pyrosequencingofextractedDNAasGammaproteobacteria
extracted DNA, the cultivable fraction of the bacterial were, in particular, highly abundant (Supplementary
communities included Proteobacteria, Actinobacteria, Bac- Table S4). Gammaproteobacteria were dominant in fresh-
teroidetes and Firmicutes (Fig. 2b, c). Depending on water and in the bottom and middle snow layers,
cultivation method (direct vs. pre-incubation), the Pro- accounting for up to 90% of the isolates of the Proteo-
teobacteria accounted for 48(cid:2)100% of the snow commu- bacteria phylum (Supplementary Table S4). In top snow,
nitieswhiletheActinobacteriaconstituted0(cid:2)50%andthe on the other hand, alpha and beta classes each repre-
phylum Bacteroidetes 0(cid:2)4%. In contrast to snow, the sentedabout47(cid:2)50%oftheProteobacteria.Infreshwater
freshwater community contained relatively few Actino- and bottom snow, Actinobacteria were only observed by
bacteria (0(cid:2)9% depending on the cultivation method), direct plating. Micrococcaceae and Microbacteriaceae domi-
whereas the Bacteroidetes phylum was more abundant natedinfreshwater,whileArthrobacterweredominantin
(9(cid:2)13%). Also, Firmicutes was only seen in freshwater bottomsnow(SupplementaryTableS4).Inthetwoother
(0(cid:2)1%;Fig.2b,c).Whenpre-cultivatingthebacteriaon snowlayers,SalinibacterandKineococcusdominatedinthe
polycarbonate membranes before plating on rich med- middle snow layer, whereas Rhodococcus was only found
ium, the membranes were floated on sample water in top snow. Among the Bacteroidetes, which were
supplemented with TSB at concentrations of 0, 0.01 or primarily observed in freshwater (Fig. 2a), Flavobacteria
0.1%,respectively.Theseamendments,however,hadno constituted (cid:2)90% of the isolated bacteria within this
effect on the composition of the isolated bacteria (data phylum.
not shown).
Among the Proteobacteria, Sphingomonas was the most
Discussion
frequent genus in snow, accounting for 7(cid:2)11% of all
sequencesinthetopandmiddlesnowlayersand4%in The structure of High Arctic snow and freshwater
the bottom layer based on the pyrosequencing data bacterial communities was assessed by pyrosequencing
(Supplementary Table S2). GpI Cyanobacteria were also of extracted DNA and by sequencing of DNA from
abundantinthetopandmiddlesnowlayersconstituting bacteriaisolatedbytwodifferentcultivationapproaches.
13% and 17% of all sequences, respectively. In the As expected, the composition of the culturable bacteria
bottom layer, the frequency of GpI Cyanobacteria was didnotreflectthecompositionfoundbypyrosequencing,
7
Citation:PolarResearch2013,32,17390,http://dx.doi.org/10.3402/polar.v32i0.17390
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BacterialcommunitystructureinHigh-Arcticsnowandfreshwater A.K.Mølleretal.
illustrating the well-known discrepancy between mole- Proteobacteria (alpha, beta and gamma), Actinobacteria and
cular and cultivation based techniques. However, den- Bacteriodetes (especially Flavobacteria and Sphingobacteria)
drograms constructed on the basis of the Bray(cid:2)Curtis arecommonly foundinpolar snow.
similarity measure of the pyrosequencing data and the Streptococciwerehighlyfrequentinthedeepestsnow
pre-incubation procedure both indicated that the three layer. Streptococcus is recognized as a potential pathogen
snow layers were more similar to each other than to and most commonly associated with a host organism
the freshwater lake. This was not the case for direct or environments influenced by faecal contamination
cultivationapproach.Thus,thepre-incubationtechnique (Lopez-Benavides et al. 2007). Another unexpected
seemed to better represent the community structure genusinthedeepestsnowlayerwasFusobacterium,which
obtained bypyrosequencing. is also considered an animal and human pathogen.
Total bacterial numbers were ca. 1(cid:4)103 cells ml(cid:3)1 in WithinthephylumFusobacteria,amarinepsychrotrophic
snow and ca. 1(cid:4)106 cells ml-1 in freshwater (Table 1). Psychrilyobacter atlanticus has recently been isolated from
Since 200(cid:2)500 ml of sample was collected for pyrose- theArcticOcean(Zhaoetal.2009)andclonesthatwere
quencing,aconservativeestimateofthenumberofcells 99(cid:2)100% identical to Ilyobacter psychrophilus have been
in thesamples is (cid:3)2(cid:4)105 cellsforthe snow layersand identified in Arctic sediment (Tian et al. 2009). This
ca. 2(cid:4)108 cells for freshwater. By comparison to the suggests the occurrence of environmental Fusobacteria.
Chao1 species richness estimator, a rough calculation However, wildlife droppings could also account for
suggeststhattheaveragepopulationdensityofeachOTU the presence of these bacteria in this context. Further
was around 25 cells in snow and 6.2(cid:4)104 cells in sampling will be required to determine if Streptococcus
freshwater. Thus, the snow microbial communities ap- and Fusobacteria are widespread members of the snow
peared to consist of a highly diverse assemblage of microbial communities.
differentbacteria,eachatalowpopulationdensity,while In freshwater, Actinobacteria, Bacteroidetes and Verruco-
thefreshwatercommunitiesconsistedofOTUswithlarge microbia were particularly abundant but Proteobacteria,
population densities butthese werefew innumber. Acidobacteria and Planctomycetes also occurred at frequen-
The abundance of the bacterial phyla appeared to be cies between 3 and 9%. Previous investigations of
relatedtotheirdiversityastheabundantphylahadlarge microbialcommunitiesinArcticandAntarcticfreshwater
numbers of unique OTUs rather than few highly abun- lakes have revealed Proteobacteria, Bacteroidetes, and
dant ones (Fig. 3). A similar observation has been made Actinobacteria to be the major phyla (Pearce et al. 2003;
in the Arctic Ocean (Kirchman et al. 2010), suggesting Crump et al. 2007). Cyanobacteria are often common in
thattheecologicalsuccessofabacteriallineagedepends freshwater (Zwart et al. 2002) and have been identified
upon its diversity rather than superior competiveness of inanAntarcticfreshwaterlake(Ellis-Evans1996).Inthe
a few phylotypes(Kirchman etal. 2010). freshwater lake at Station Nord, Cyanobacteria were only
Becauseourstudyincludedsamplesfromjustonelake detected at a very low frequency. However, since the
and one snow site, our findings cannot be considered lake had been covered with ice and snow for at least
representative of High-Arctic snow and freshwater lakes 22 months prior to sampling, the resulting low light
in general. However, the composition of the bacterial intensity probably was not suitable for growth of these
assemblages resembled the bacterial composition found microorganisms.
elsewhere. For instance, we observed that the most A number of physico-chemical parameters within
abundant phyla in snow included Proteobacteria (Alpha, a snowpack, and between snow and freshwater, may
Beta, Gamma and Epsilon), Actinobacteria, Bacteroidetes, confront microbial communities with different environ-
Cyanobacteria, Firmicutes and Fusobacteria. Larose et al. mental challenges. In snow, for instance, the light
(2010)establishedclonelibrariesofDNAextractedfrom intensity decreases with depth (Rowland & Grannas
snow and a meltwater river in Svalbard and found 2011), and the temperature becomes less variable
Proteobacteria (Alpha, Beta and Gamma), Bacteroidetes and extreme (Liston & Winther 2005). Also, the deeper
(Sphingomonas and Flavobacteria), Cyanobacteria and eu- layers in snow on sea ice may be affected by seawater
karyotic chloroplasts to be dominant. In Antarctic snow, penetrating through cracks in the ice. Contrary to the
clones belonging to Deinococcus, beta-Proteobacteria and variable conditions in snow, the freshwater lake can be
Bacteroideteshavebeenidentified(Carpenteretal.2000). characterized by a relative constant temperature regime
Amato et al. (2007) cultivated bacteria directly from around08C,andmostlikelybylimitedlightintensitydue
snow in Svalbard and isolates belonging to alpha-, beta- tothe snow andicecoverage.
and gamma-Proteobacteria, Actinobacteria and Firmicutes Bacteria may be transported over long distances
were found. Our data and the literature indicate that with dust particles (Kellogg & Griffin 2006), and have
8
Citation:PolarResearch2013,32,17390,http://dx.doi.org/10.3402/polar.v32i0.17390
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A.K.Mølleretal. BacterialcommunitystructureinHigh-Arcticsnowandfreshwater
been demonstrated to be metabolically active in icy limited. The environmental conditions in the lake,
super-cooled cloud droplets (Sattler et al. 2001). therefore, can be considered more constant and less
Atmospheric and aeolian deposition may, therefore, extreme than in snow and are probably the reason for
be significant as sources of bacteria to the snow. The the relatively low observed bacterial diversity.
highly diverse bacterial assemblages in snow, with each In conclusion, our investigations suggested that the
group having a low population density, support this diversitywashigherinsnowascomparedtofreshwater,
notion. The fact that the freshwater assemblage showed which may reflect that the climatic conditions of the
the opposite trend further supports this idea. freshwater ecosystem were less extreme and that the
Cyanobacteria and chloroplasts werealmost exclusively lake received less light. Regardless, a strong overlap
found in the top and middle layers with the highest between the genera found in snow and freshwater
density in the middle layer. This shift in Cyanobacteria indicated that snow meltwater may have been a
density with depth from high, to very high, to low, significant source of microorganisms to the freshwater
suggests that light intensity controlled the distribution lake. The phylogenetic composition in the three snow
of the Cyanobacteria and the algae in the snow pack layers was significantly different, yet the two upper
with optimal light intensity in the middle layer. We snowlayersweremoresimilartoeachotherthantothe
primarily found GpI Cyanobacteria that are known to deepest snow layer. For instance, the two top layers
include species capable of fixing nitrogen. A likely were inhabited by large numbers of Cyanobacteria
scenario for explaining the heterotrophic community and chloroplasts, which were probably feeding the
heterotrophic members of the microbial communities.
structureinthedifferentsnowlayersisthatsomeofthe
This suggests that the microbial communities within
nitrogen fixed by the Cyanobacteria stimulated the pri-
snowaremetabolicallyactiveandthat,fromamicrobial
mary production of the algae, while the carbon and
perspective, snow is a heterogeneous and structured
nitrogen produced by the autotrophs were feeding the
habitat.
heterotrophic bacteria. Our DOC data (Table 1) con-
firmed this hypothesis sincethe DOC concentrationwas
about three times higher in the top and middle snow Acknowledgements
layers as compared to the bottom layer. Other sources
This work has been funded by the Danish Agency for
ofcarbon tothebacterial communities,especially inthe
Science, Technology and Innovation (grant no. 645-06-
toplayers,couldbeaerosolsfromtheatmosphere(Bauer
0233).
etal.2002).Thedeepestsnowlayerdidnotseemheavily
affected by the autotrophs, as their densities and the
DOC concentration were relatively low. However, as
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Citation:PolarResearch2013,32,17390,http://dx.doi.org/10.3402/polar.v32i0.17390
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