Viability, diversity and composition of the bacterial community in a high Arctic permafrost soil from Spitsbergen, Northern Norway

Viability, diversity and composition of the bacterialcommunity in a high Arctic permafrost soil fromSpitsbergen, Northern Norway

Aviaja A. Hansen,1† Rodney A. Herbert,1,2

Karina Mikkelsen,1 Lars Liengård Jensen,1

Tommy Kristoffersen,1 James M. Tiedje,3

Bente Aa. Lomstein1* and Kai W. Finster1

1Department of Biological Sciences, Section forMicrobiology, University of Aarhus, Denmark.2Division of Environmental and Applied Biology,Institute of Biological Sciences, University of Dundee,Scotland, UK.3Center for Microbial Ecology, Michigan State University,East Lansing, MI, USA

Summary

The viable and non-viable fractions of the bacterialcommunity in a 2347-year-old permafrost soil fromSpitsbergen were subjected to a comprehensiveinvestigation using culture-independent and culture-dependent methods. LIVE/DEAD®BacLightTM stainingrevealed that 26% of the total number of bacterial cellswere viable. Quantitatively, aerobic microcolonies,aerobic colony-forming units and culturable anaero-bic bacteria comprised a minor fraction of the totalnumber of viable bacteria, which underlines thenecessity for alternative cultivation approaches inbacterial cryobiology. Sulfate reduction was detectedat temperatures between -2°C and 29°C while metha-nogenesis was not detected. Bacterial diversity washigh with 162 operational taxonomic units observedfrom 800 16S rDNA clone sequences. The 158 purecultures isolated from the permafrost soil affiliatedwith 29 different bacterial genera, the majority ofwhich have not previously been isolated from perma-frost habitats. Most of the strains isolated were affili-ated to the genera Cellulomonas and Arthrobacter andseveral of the pure cultures were closely related tobacteria reported from other cryohabitats. Character-ization of viable bacterial communities in permafrostsoils is important as it will enable identification offunctionally important groups together with the as yet

undescribed adaptations that bacteria have evolvedfor surviving subzero temperatures for millennia.

Introduction

Permafrost describes soil, sediment or rock with a tem-perature continuously below 0°C for 2 or more consecu-tive years (Péwé, 2006). Permafrost is found below theactive layer of cryogenic soils and occupies approximately25% of the terrestrial area of the Earth (Anisimov andNelson, 1996). Continuous zones of permafrost are pre-dominantly found in North America, Eurasia and Antarc-tica (Gilichinsky, 2002). In the Northern Hemisphere thedeepest permafrost zones are found in Northern Siberia,where they extend to a depth of 1500 metres (Péwé,2006) and are 3–4 million years old (Gilichinsky et al.,1995).

Over the past decade there has been an increasedinterest in permafrost biology due to the reservoir of viablemicroorganisms and bio-signatures present in the earliestpermafrost layers (Vorobyova et al., 1997; Willerslevet al., 2004). The stability of subzero temperatures com-bined with isolation from anthropogenic impacts makespermafrost habitats unique in the reconstruction ofpaleoenvironments (Gilichinsky and Wagener, 1995;Willerslev et al., 2003). Furthermore, viable bacteria in theoldest permafrost layers may possess as yet unknownsurvival mechanisms enabling them to survive subzerotemperatures over geological timescales (Vishnivetskayaet al., 2006).

Despite the widespread distribution of permafrost,almost all our present knowledge on permafrost microbi-ology has been obtained from investigations of Siberianpermafrost soils and in particular those from the Kolymalowlands in Eastern Siberia (e.g. Vorobyova et al., 1997;Rivkina et al., 2000; Bakermans et al., 2003). Research ofthe Siberian permafrost soils have revealed that the totalnumber of bacterial cells in permafrost samples rangefrom 107 to 109 cells [gram dry weight (gdw)]-1 independentof the age of the permafrost soil (Vorobyova et al., 1997;Vishnivetskaya et al., 2000; 2006). Likewise, the numberof viable anaerobic bacteria is independent of the geologi-cal age of the permafrost sample (Rivkina et al., 1998). Incontrast the number of viable aerobic bacteria decreases

Received 13 March, 2007; accepted 19 June, 2007. *Forcorrespondence. E-mail [email protected]; Tel. (+45)89423243; Fax (+45) 89422722. †Present address: Section forEnvironmental Engineering, Aalborg University, Denmark.

Environmental Microbiology (2007) 9(11), 2870–2884 doi:10.1111/j.1462-2920.2007.01403.x

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

with increasing age of permafrost soil (Vorobyova et al.,1997; Vishnivetskaya et al., 2000; 2006).

Many prokaryotic cells in permafrost soils exhibit anultrastructure resembling resting cysts with thick, multiplelayered cell walls, suggesting that they are in an anabioticstate (Dmitriev et al., 2004; Soina et al., 2004; Suzinaet al., 2004). However, rates of in situ substrate incorpo-ration and methanogenesis have been measured in per-mafrost samples at temperatures from -10 to -16°C(Rivkina et al., 2000; 2004), while methane consumptionhas been measured down to -5°C (Khmelenina et al.,2002). Furthermore, metabolic activity of bacterial perma-frost isolates has been observed down to -19°C (Panikovand Sizova, 2007). Bacterial cell division in permafrostsoils under in situ conditions has yet to be demonstratedalthough pure cultures from permafrost samples havebeen shown to grow at in situ temperatures (-10 to -17°C)(Bakermans et al., 2003; 2006; Panikov and Sizova,2007).

Non-spore-forming Gram-positive bacteria are thedominant permafrost bacteria isolated so far (Kochkinaet al., 2001; Bakermans et al., 2003; Vishnivetskayaet al., 2006), but endospore formers and Gram-negativebacteria have also been isolated (Bakermans et al., 2003;Vishnivetskaya et al., 2006; Steven et al., 2007). Thegrowing library of Siberian permafrost isolates are affili-ated to at least nine bacterial classes and more than35 genera (Shi et al., 1997; Vorobyova et al., 1997;Vishnivetskaya et al., 2000; 2006; Kochkina et al., 2001;Bakermans et al., 2003; Gilichinsky et al., 2003; Stevenet al., 2007). Evaluation of the bacterial permafrost com-munities has almost exclusively been culture-based andknowledge on the overall composition and viability of thebacterial communities is therefore limited. RecentlyVishnivetskaya and colleagues (2006) and Steven andcolleagues (2007) reported the first community character-izations of permafrost samples using culture-dependentand culture-independent methods.

In the present study, we specifically accessed the viablebacterial community in an Arctic permafrost soil fromSpitsbergen. We combined oxic and anoxic cultiva-tion and isolation techniques with culture-independentmethods and quantified the number of viable and deadbacteria by epifluorescence microscopy. The viable frac-tion of the bacterial community was further characterizedby construction of clone libraries from long-term incubatedsoil enriched under different conditions and by measure-ment of sulfate-reduction rates.

Results

Characteristics of the permafrost soil

Data presented in Table 1 summarize the major physico-chemical characteristics of the 2347-year-old permafrost Ta

ble

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Viable bacterial community in an Arctic permafrost soil 2871

© 2007 The AuthorsJournal compilation © 2007 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 9, 2870–2884

soil from Spitsbergen. These data show that the Spitsber-gen permafrost soil is slightly acidic (pH 5.01) and com-posed predominantly of silt and clay particles (79%). Theorganic carbon content (1.6% gdw-1) is in the same rangeas reported for other Arctic permafrost soils (Vorobyovaet al., 1997). The soil was slightly saline (equivalent to8.2 mM NaCl) while the sulfate concentration was rela-tively high (3.1 mM).

Sulfate reduction was detected in soil samples thatwere re-suspended in sterile phosphate-buffered salineand incubated at temperatures between -2 and 29°C.Highest rates were recorded at 20–25°C (see Supple-mentary material). Addition of nutrients (R2A broth) to thesoil samples stimulated sulfate reduction at temperatures> 4°C compared with unamended samples, but no activitywas detected below 1°C. No methane production wasobserved after 4 weeks of incubation at temperaturesbetween 4 and 30°C (data not shown).

Bacterial numbers

The total number of bacterial cells recorded in the perma-frost soil was 1.7 ¥ 109 cells gdw-1 (Table 2). Dual stainingwith the LIVE/DEAD® BacLight™ kit revealed that 26% ofthe cells had an intact cell membrane and were thereforeclassified as viable (Table 2). The highest number ofculturable aerobic heterotrophic bacteria was 1.4 ¥ 105

colony-forming units (cfu) gdw-1 on R2A agar, thusaccounting for 0.03% of the total viable cell count. Lowerrecoveries were obtained using the VL55-based growthmedia (medium II–IV; Table 2). Given that R2A mediumgave the highest cell recoveries this medium was usedin subsequent culture-based work. The number ofmicrocolony-forming units (cells dividing at least twice;mcfu) was one order of magnitude higher thanmacrocolony-forming units (Table 2). The number ofanaerobic bacteria in R2A broth was 7.3 ¥ 105 gdw-1,lower counts were obtained on solid R2A agar and VL55medium supplemented with sugars and amino acids(Table 2). The number of anaerobic endospore-formingbacteria in liquid broth was one order of magnitude higherthan the number of aerobic endospore formers on agarplates (Table 2). Under both oxic and anoxic conditionsthe endospore-forming bacteria accounted for a minorfraction of the culturable bacteria (0.9% and 1.4%respectively).

Growth, composition and diversity ofthe bacterial community

Growth was indirectly measured in the long-term perma-frost soil enrichments as an increase in DNA concentra-tion compared with the original permafrost soil (T0)(Fig. 1). The largest increase in DNA concentration was Ta

ble

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2872 A. A. Hansen et al.

© 2007 The AuthorsJournal compilation © 2007 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 9, 2870–2884

observed in enrichments where nutrients [0.3% w/wtryptic soy broth (TSB)] were added (N). The increasecorresponded to 2.5 ¥ 108 cells gdw-1 under oxic condi-tions (oN) and 1.2 ¥ 108 cells gdw-1 under anoxic condi-tions (anN) (Table 3), while the increase in enrichmentswithout supplements corresponded to 1.6 ¥ 108 cells-gdw-1 under oxic conditions (o–) and 9 ¥ 107 cells gdw-1

under anoxic conditions (an–) (Table 3). There was noincrease in the DNA concentration in enrichments at insitu water activity (S, NS) (Fig. 1).

Growth of the enriched permafrost soil communitiescoincided with a concomitant decrease in the numbers ofoperational taxonomic units (OTUs) in the clone libraries(Table 3). The observed bacterial diversity in the originalpermafrost soil was 41 OTUs, while 31, 20 and 32 OTUs

were observed in the oxic enrichment without nutrientaddition (o–), the oxic enrichment with added nutrients(oN) and the anoxic enrichment supplemented with nutri-ents (anN) respectively (Table 3). In the anoxic enrich-ments at in situ water activity (anS and anNS) and in theoxic enrichment at in situ water activity with nutrients(oNS) the observed diversity was 49–51 OTUs (Table 3).The slightly lower number of OTUs in the original perma-frost soil compared with these enrichments was mostlikely due to the different sizes of the clone libraries(Table 3). The observed diversity in the permafrost soilwas 162 OTUs, when calculated from all 800 sequences(all retrieved sequences pooled) (Table 3). The richnessestimate of 205 OTUs (Chao1) revealed that the numberof analysed clones did not exhaust the diversity of thepermafrost soil and it would have required more than1500 sequences to fully cover the diversity. The coverageof the libraries (sequences from triplicate pooled samples)was estimated to be 0.64–0.8 (CACE) and 0.66–0.9(Good’s C) (Table 3).

Taken together, sequences from all nine clone librariesaffiliated to 15 different bacterial classes and three can-didate divisions. Sequences not readily classified intoknown classes or phyla were denoted unclassified(Table 4). The bacterial classes identified in the originalpermafrost soil (T0) were also represented in the enrichedpermafrost samples (Table 4). Actinobacteria and TM7were observed in all nine samples (Table 4). Thermomi-crobia, Verrucomicrobiae, OD1, OP10 and unclassifiedBacteria were found in enrichment cultures, where noincrease in DNA concentration was detected (oSN, anS,anNS). Members of the Planctomycetacia were onlyobserved in the oxic enrichment with no nutrient supple-ments (o–). Alpha-, Beta- and Gammaproteobacteria,Actinobacteria, Acidobacteria, Flavobacteria, Sphingo-bacteria and TM7 were observed in the oxic enrichments

0.0

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1.5

2.0

2.5

3.0

o- oN oS oNS an- anN anS anNS

[DN

A]

µg

gd

w-1

T0

Fig. 1. DNA concentration in the original permafrost soil andpermafrost soil enrichments. (T0) original permafrost soil; (o) oxicenrichments; (an) anoxic enrichments. Enrichment conditions: (–)no supplement; (N) nutrient supplement; (S) sodium chloridesupplement; and (NS) supplement of both nutrients and sodiumchloride.

Table 3. Cell numbers in permafrost soil enrichments and clone library information [total number of sequences, observed OTUs, estimatedrichness (Chao1) and estimated coverage (CACE and Good’s C)].

Samplea Bacterial numbersb (¥107 gdw-1) Sequences OTUsc Chao1c CACE Good’s C

T0 4 77 41 64 0.70 0.70o– 20 87 31 46 0.79 0.83oN 29 91 20 27 0.73 0.90oS 2 91 39 114 0.64 0.72oNS 2 89 49 71 0.70 0.70an– 13 90 38 59 0.78 0.78anN 16 88 32 44 0.80 0.83anS 2 92 49 107 0.66 0.66anNS 3 95 51 93 0.67 0.67Total 800 162 205 0.86 0.93

a. (T0) original permafrost soil; (o) oxic enrichments; (an) anoxic enrichments. Enrichment conditions: (–) no supplement; (N) nutrient supplement;(S) sodium chloride supplement; and (NS) supplement of both nutrients and sodium chloride.b. Calculated from the DNA yield and the median of the minimum and maximum estimate of cellular DNA content of soil bacteria in literature [6.5 fgcell-1 (median), 1.6 fg cell-1 (min) as reported by Bakken and Olsen (1989) and 11.4 fg cell-1 (max) as reported by Sandaa and colleagues (1998)].c. OTUs and Chao1 were defined by using a distance level of 3%.

Viable bacterial community in an Arctic permafrost soil 2873

© 2007 The AuthorsJournal compilation © 2007 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 9, 2870–2884

with growth (o–, oN) (Table 4). Beta- and Deltaproteobac-teria, Clostridia, Mollicutes, Actinobacteria, Bacteroidetesand TM7 were observed in the anoxic enrichments withgrowth (an–, anN) (Table 4). Moreover, sequences fromsulfate-reducing bacteria affiliated with the genera Des-ulfocapsa were observed in all samples, except from theoxic enrichments. Sequences affiliating with the sulfate-reducing groups Desulfobacula and Desulfosporosinuswere only observed in the original permafrost soil and thesamples where no increase in DNA concentration wasdetected.

Identification of pure cultures and anoxic cultures

In total 158 colonies belonging to six different bacterialclasses were isolated from oxic agar plates. The majorityof the pure cultures were Gram-positive (85%) of which5% were endospore formers (Table 5). The dominantgroup was Actinobacteria (128 isolates) which affiliatedto 19 genera. Arthrobacter and Cellulomonas were thedominant genera accounting for 55% of all isolates(Table 5). The Gram-negative pure cultures accounted for15% of the isolates and were related to Alpha-, Beta- andGammaproteobacteria and Sphingobacteria. The majorityof the pure cultures were recovered from R2A agar andonly 14 isolates were recovered exclusively from VL55-based growth media compared with 76 isolates recoveredsolely from R2A agar (Table 5). All isolates were relativelyclosely related to validly described strains with similaritiesranging from 89% to 100% (Table 5).

The community structures of the anoxic most probablenumber (MPN) enrichments were analysed by denaturantgradient gel electrophoresis (DGGE) fingerprinting of the16S rDNA genes and selected bands were identified bysequencing (Fig. 2). Thirteen different bands all affiliatedwith Gram-positive bacteria were identified in the commu-nity fingerprint (Fig. 2). The bands from the fingerprints ofthe highest dilutions affiliated to the genus Cellulomonas.Nine different bands in fingerprints from the lower dilu-tions affiliated with Clostridium and one with Bacilli(Fig. 2). Members of the genera Clostridium and Cellu-lomonas were also identified in the community fingerprintof the original permafrost soil (T0) where sequences affili-ated to the sulfate-reducing groups Desulfobacula andDesulfocapsa were observed.

Bacteria related to members of the sulfate-reducinggenera Desulfocapsa and Desulfobacterium wereretrieved from incubations enriching for sulfur-disproportionating bacteria.

Discussion

Numbers of viable and dead cells in the soil

The total bacterial count of 1.7 ¥ 109 cells gdw-1 in theSpitsbergen soil samples was in the upper range ofprevious reports for permafrost soils (Vorobyova et al.,1997; Vishnivetskaya et al., 2000; 2006). LIVE/DEAD®

BacLight™ staining revealed that only 26% of the cellswere viable (see Results for definition of viable). To our

Table 4. Phylogenetic affiliation of 16S rDNA clones of the Spitsbergen permafrost community.

Phylogenetic classificationa T0 o– oN oS oSN an– anN anS anNS

Thermomicrobia + +Alphaproteobacteria + + +Betaproteobacteria + + + + + + +Gammaproteobacteria + + + + + + +Deltaproteobacteria + + + + + + +Clostridia + + + + + + +Mollicutes + + +Actinobacteria + + + + + + + + +Planctomycetacia +Spirochaetes + + +Acidobacteria + +Unclassified Bacteroidetes + + + + + + + +Bacteroidetes + + + + + + + +Flavobacteria + +Sphingobacteria + + + +Verrucomicrobiae + +OD1b + + +OP10b + +TM7b + + + + + + + + +Unclassified Bacteria + +

a. Samples: (T0) original permafrost soil; (o) oxic enrichments; (an) anoxic enrichments. Enrichment conditions: (–) no supplement; (N) nutrientsupplement; (S) sodium chloride supplement; and (NS) supplement of both nutrient and sodium chloride.b. Candidate division, known by sequence only.+, presence of clone sequences affiliated with the phylogenetic division.

2874 A. A. Hansen et al.

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knowledge this is the first time this method has been usedto investigate a microbial permafrost soil communityalthough it has been applied to soil, marine and freshwater sediment communities (Janssen et al., 2002;Haglund et al., 2003; Quéric et al., 2004). The proportion

of viable cells recorded in our study is not dissimilar to thatreported for deep-sea surficial sediments (20–60%)(Quéric et al., 2004), which is perhaps surprising giventhat the Spitsbergen permafrost soil has been in a frozenstate for more than 1000 years. These data suggest that

Table 5. Phylogenetic affiliation of permafrost isolates on the basis of 16S rDNA sequence similarity by use of the BLAST search tool in theGenBank database (Altschul et al., 1997).

Taxonomic affiliation No. of isolatesa Isolation mediab Closest relativec Accession No.c Similarityd (%) Length (bp)

ProteobacteriaAlphaproteobacteria

Kaistobacter e 1 I K. koreensis AY785128 96 1317Porphyrobacter e 1 I P. neustonensis AF465838 97 830

BetaproteobacteriaBurkholderia e 2 I B. fungorum AJ544691 100 878–1318Rhodoferax e 7 I R. ferrireducens AF435948 98 857–1330Variovorax e 1 III V. paradoxus AF209469 97 820

GammaproteobacteriaPseudomonas 4 I, III, IV P. fluorescens AY653221 99 818–826

1 I P. reactans AY747594 99 13251 I P. veronii AY081814 99 1295

FirmicutesBacilli f

Bacillus 1 I B. psychrodurans AJ277984 99 1332Paenibacillus 4 I P. antarcticus AJ605292 93–99 812–1347

1 III P. mendelii AF537343 92 13281 I P. maqueriensis AB073193 97 820

ActinobacteriaActinobacteria

Aeromicrobium e 3 I A. marinum AY166703 97 821–1287Arthrobacter 19 I A. oxidans AJ243423 97–99 806–1300

4 I, IV A. stackebrandtii AJ640198 96–97 820–13732 I A. psychrolactophilus AF134183 97 861–884

Blastococcus e 1 I B. saxobsidens AJ316571 98 1303Brachybacterium 1 I B. paraconglomeratum AJ415377 99 824Cellulomonas 51 I, III, IV C. cellasea X83804 94–98 623–1304

11 I C. fermentans X83805 94–97 816–1318Cryobacterium 6 I, III, IV C. psychrophilum AJ544063 96–99 810–1305Cryocola 3 I C. antiquus AF505513 96–97 820–1356Kocuria 1 IV K. carniphila AJ622907 99 828Leifsonia 2 III L. shinshuensis DQ232614 98–99 814–816Micrococcus 2 I M. luteus AJ536198 99 817–826Modestobacter e 2 I M. multiseptatus AJ871304 94–97 826–872Mycobacterium e 3 I, III M. wolinsky AY457083 96–97 724–832

1 I M. aichiense AF498656 97 13371 I M. hodleri X93184 98 828

Nocardioides 1 I N. kribbensis AY83592 95 1311Pimelobacter e 1 I P. simplex NSJ78212 93 806Rhodococcus 2 I R. opacus X80631 95 845–1298

1 I R. rhodnii X81935 95 572Sporichthya e 1 I S. polymorpha AB025317 93 1295Streptacidiphilus e 1 III S. albus AF074415 98 822Streptomyces 2 I S. aureofaciens AY207608 98 820–875

3 III, IV S. cremeus AY999744 97 818–8261 I S. turgidiscabies AY207583 97 1337

Terrabacter e 2 IV T. tumescens X83812 97 815–820

BacteroidetesSphingobacteria

Pedobacter e 3 IV P. caeni AJ786798 96 806–828Spirosoma e 2 I S. escalantus AY279981 89–92 824–826

a. Number of isolates affiliated to same relative.b. Isolation media: (I) R2A, (III) VL55+sugars and (IV) VL55+sugars+amino acids.c. Closest culture with the highest bit score is listed.d. Range illustrates different similarities of isolates to the closest relative.e. Genera not previously isolated from permafrost samples.f. Endospore formers.

Viable bacterial community in an Arctic permafrost soil 2875

© 2007 The AuthorsJournal compilation © 2007 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 9, 2870–2884

bacteria in permafrost, which in most cases do not formspores, either have evolved hitherto undescribed survivalmechanisms or are metabolically active even under thefrozen conditions of this habitat. In a study of Arctic andAntarctic permafrost soils, Soina and colleagues (2004)demonstrated the predominance of cyst-like cells in situwhich they proposed enabled the bacterial cells to survivelong periods in an anabiotic state. At the present timeformation of cyst-like cells has only been reported for alimited number of bacteria although significantly thesedo include Arthrobacter and Micrococcus spp. whichare commonly found in permafrost soils as revealed byboth culture-dependent as well as culture-independentmethods (Kochkina et al., 2001; Vishnivetskaya et al.,2006).

Repeated observations from studies on different habi-tats have shown that bacterial populations contain largeand in our study dominant fractions of dead nucleoid-containing cells (Mason et al., 1995; Luna et al., 2002;Schumann et al., 2003; Manini and Danovaro, 2006). Thishas major implications for the interpretation of clonelibrary data and evaluation of the cultured fraction of themicrobial community because the dead cells contribute tothe structure but not the function of the microbialcommunity. Thus, when clone library data alone are used

to establish ecological functions this may lead to invalidconclusions as dead cells may be ascribed functions thatthey are no longer able to perform. In the worst casescenario, depending on the efficiency of the DNA extrac-tion protocol, more DNA may be retrieved from dead thanliving cells and consequently the clone library will bebiased towards the non-functional part of the microbialcommunity. This possibility needs to be taken into con-sideration when studying permanently frozen environ-ments where DNA is preserved for long periods of time(Willerslev et al., 2004; Hebsgaard et al., 2005). Further-more, the proportion of viable cells rather than total cellnumbers must be factored into the calculation whendetermining the efficiency of cultivation procedures. Thus,in our study the cultivation efficiency increased by a factorof 4 when viable instead of total cell numbers were takeninto consideration.

DNA recovery from permafrost soil

The quantity of DNA extracted from the Spitsbergen soilsamples was equivalent to 1–9% of the total amount ofDNA in the soil sample estimated from the total number ofcells and a per cell DNA content of 1.6–11.4 fg (Bakkenand Olsen, 1989; Sandaa et al., 1998). The difference

T0 10-1 10-2 10-3 10-4 10-5 10-6

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1: Clostridium sp. (tunisiense)2: Clostridium sp. (tunisiense)3: Clostridium akagii4: Clostridium sp. (tunisiense)5: Clostridium bowmanii 6: Clostridium bowmanii 7: Clostridium estertheticum8: Clostridium vincentii 9: Clostridium vincentii10: Paenibacillus wynnii/Bacillus circulans11: Cellulomonas sp. (fermentans)12 : Cellulomonas sp. (fermentans)13: Cellulomonas cellasea

a : Bacteroidetes sp.b : Cytophaga sp.c: Desulfobacula sp.d: Clostridium sp. (termitidis/cellobioparum)e: Clostridium sp. (alkalicellum/straminisolvens)f: Desulfocapsa sp.g: Desulfocapsa sp. (thiozymogenes)h: Actinobacteria sp.i: Cellulomonas sp. (fermentans)j: Cellulomonas fermentans

II I II

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Fig. 2. Denaturant gradient gel electrophoresis (DGGE) community fingerprint of 16S rDNA amplified from 10-fold diluted anoxic MPNenrichments (10-1-10-6) of the permafrost soil. Band pattern of the original permafrost soil serves as reference (T0). Bands marked with whitecircles were excised and sequenced. Letters and numbers refer to the phylogenetic affiliation of each band obtained by the BLAST search toolin the GenBank database (Altschul et al., 1997). sp. is given when the closest relative was uncultured, the closest cultured relative(s) is thengiven in parenthesis. I and II are given when the banding patterns were different among the two replicates (n = 2).

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between the measured and calculated DNA content of thesoil can be explained by a number of factors, which mayact alone or in combination. First, the low DNA recoverymay be due to the high clay content (12%) of the Spits-bergen permafrost soil. Clay minerals have been shownto readily adsorb DNA (Ogram et al., 1988; Cai et al.,2006) and inhibit cell lysis and hence DNA extraction(Zhou et al., 1996). Second, the staining procedure mayoverestimate the number of DNA-containing cells as thedye may bind non-specifically to cell components otherthan DNA. Studying the viable fraction of water columnbacteria Zweifel and Hagström (1995) demonstrated thatby incorporating a washing step in the staining procedure,only 2–30% of the cells contained a nucleoid andthus DNA. Similar results were reported by Manini andDanovaro (2006) who determined the number ofnucleoid-containing bacterial cells in sediments. Further-more, these authors demonstrated that the number ofcells containing a nucleoid was dependent on the fluores-cent DNA stain used and ranged from 5% to 32% for cellsstained with propidium iodide and from 30% to 47% whenethidium homodimer-2 was used. These data indicate thatnumbers of nucleoid-containing cells depend on the prop-erties of the DNA stain and therefore cannot be consid-ered as absolute values. We are confident that while thequantity of DNA extracted from the Spitsbergen soilsamples was low these data are realistic as confirmed bythe low standard deviation values obtained for the tripli-cate DNA extractions from both the original permafrostsoil and the incubations without community growth. Weattribute the discrepancy between the measured and thecalculated DNA content of the permafrost samples to bedue to a combination of matrix (clay) effects and an over-estimation of the number of DNA containing cells. Therelative importance of these two factors needs to beinvestigated in future studies.

Clone libraries from different soil incubations

The high proportion of non-viable DNA-containing bacte-rial cells in the permafrost soil is probably also reflected inthe composition of the clone library constructed from theoriginal permafrost soil. As an alternative, clone librariesconstructed from long-term incubations of permafrost soilsamples can be used to discriminate between viable andnon-viable bacteria by comparing the clone libraries. Cellproliferation was measured indirectly as an increase inDNA concentration, which remained unchanged in incu-bations with low water activity despite the relatively highincubation temperature (10°C). As the DNA concentrationincreased substantially in the other incubations, we pos-tulate that water activity rather than temperature regulatesgrowth in permafrost soils. The net increase in extractablebacterial DNA (240–691%) observed in oxic and anoxic

incubations with and without nutrient addition was accom-panied by a decrease in the genetic diversity recoveredfrom the clone libraries. This was probably due to theselective enrichment of a viable subpopulation of the bac-terial community. Thus, the bacterial groups present in theclone libraries constructed from incubations with growthwere most likely components of the viable fraction of thebacterial permafrost soil community. However, it must bestressed that cell proliferation of these bacterial classes isonly indicative as the presence of sequences fromdormant or even dead bacteria in the clone libraries ispossible.

In total 162 OTUs were recovered from the Spitsbergenpermafrost soil, which to our knowledge is the highestdiversity reported from permafrost samples to date. Forcomparison, 17–31 OTUs were observed in the Siberianpermafrost samples studied by Vishnivetskaya andcolleagues (2006) and 42 OTUs were observed in theCanadian permafrost samples studied by Steven andcolleagues (2007). A possible explanation of this differ-ence is that amplified ribosomal DNA restriction analysis(ARDRA) was used for clone screening and identificationof OTUs in the previous permafrost studies (Vishniv-etskaya et al., 2006; Steven et al., 2007). ARDRA has alower taxonomic resolution when compared with thesequence-based approach applied in our study. More-over, in our study 800 sequences were obtained from onesampling site, which is much higher than the maximum of101 clones analysed in the previous studies (Vishniv-etskaya et al., 2006; Steven et al., 2007). This large dif-ference in the number of analysed clones should per seresult in large differences in recovered OTUs, assumingthe in situ diversity is also large. When comparing thenumber of OTUs of our sublibraries (20–51 OTUs) withthe numbers obtained by Vishnivetskaya and colleagues(2006) and Steven and colleagues (2007), they aresimilar. Interestingly, the number of OTUs in the Vishniv-etskaya study appears to be unaffected by the age of thesample, which may reflect either the survival capacities ofthe permafrost bacteria or the capacity of subzero habi-tats to preserve DNA.

Despite its limited coverage the clone library revealsthat the Spitsbergen permafrost soil community is geneti-cally diverse including 15 different bacterial classes dis-tributed over nine bacterial phyla. The bacterial groupsThermomicrobia, Mollicutes, Spirochaetes, Acidobacteria,Bacteroidetes, Verrucomicrobiae and the three candidatedivisions have not previously been reported from perma-frost samples and two sequences could not be affiliated toany presently established phyla. The latter illustrates thatthe Spitsbergen permafrost soil may harbour hithertounknown bacterial lineages and that more comprehensivestudies are needed if an exhaustive recovery of thebacterial diversity is required. Alternative experimental

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approaches such as the recently described parallel tagsequencing strategy used by Sogin and colleagues(2006), which allows the rapid analysis of thousands ofpolymerase chain reaction (PCR) amplicons, may bemore appropriate for a detailed evaluation of the bacterialdiversity of these highly complex communities.

Number of cells recovered by cultivation from theSpitsbergen permafrost soil

In order to evaluate the fraction of cells recovered bycultivation from the Spitsbergen permafrost soil enumera-tion of both micro- and macrocolony forming units wascarried out. Microcolony-forming bacteria accounted for0.01% of the total bacterial cell number, which is a minorfraction compared with the 11% reported for a temperatesurface soil (Winding et al., 1994). This may be explainedby a large fraction of non-viable, dormant and metaboli-cally inactive bacteria in the permafrost soil. However, theshort incubation period of 7 days for mcfu retrieval mayalso explain the low recovery of microcolonies. It haspreviously been proposed that the difference in thenumber between microcolony-forming bacteria and thenumber of macrocolony-forming bacteria, which in thisstudy is 89% of the mcfu, is indicative of the presence ofa viable but non-culturable subpopulation (Binnerup et al.,1993; Winding et al., 1994). We do not agree with thisinterpretation and find the term ‘viable but non-culturable’unhelpful. The difference in number between culturedcells and living cells reflects our lack of knowledge on thegrowth and nutritional requirements of the uncultured frac-tion rather than an immanent character of unculturedmicrobes.

From the macrocolonies, 158 isolates were obtained inpure culture, which represented 9% of the 800 clonesequences. This is a relatively high proportion consideringthat only 0.01% of the total cell count could be recoveredas mcfu. The recovery of a significant fraction of thegenetic diversity may be attributed to the enrichment pro-cedure we applied prior to DNA extraction and construc-tion of clone libraries, i.e. addition of nutrients may havefavoured the same bacterial groups as those on the iso-lation media.

Cultures obtained from the permafrost soil

Consistent with the data reported by other investigatorswe found that members of the Actinobacteria were thedominant group present in the Spitsbergen permafrostsoil samples (Kochkina et al., 2001; Bakermans et al.,2003; Vishnivetskaya et al., 2006). The phylogeneticrelatedness of bacterial strains isolated from cryohabitatssuggests that the applied culturing methods are biasedtowards a specific recoverable fraction of the indigenous

bacterial communities. To assess this culture bias weinvestigated the quantitative and qualitative recovery ofbacteria on four different cultivation media: R2A agar(Reasoner and Geldreich, 1985), a cultivation mediumused in several permafrost studies (e.g. Vishnivetskayaet al., 2000; Bakermans et al., 2003; Gilichinsky et al.,2005), and three VL55-based growth media supple-mented with xylan, sugars and/or amino acids (Sait et al.,2002). The number of colonies obtained on the differentVL55 media increased with increasing medium richness.However, even when supplemented with sugars andamino acids the number of colonies recovered was con-sistently lower than on R2A agar from which 80% of theisolates were recovered. Thus, as reported by Reasonerand Geldreich (1985) and Porter and colleagues (1997),we found the highest bacterial recovery with R2A agar,which probably is due to its nutritional complexity.

The dominance of Actinobacteria in permafrost soil hasbeen attributed to their ability to form resting stages, theso-called cysts. While the presence of cysts as the domi-nant cell structure has been demonstrated by scanningand transmission electron microscopy in permafrostsamples from Siberia and Antarctica (Dmitriev et al.,2004; Soina et al., 2004; Suzina et al., 2004) we have yetto determine whether any of our isolates produce similardormant resting stages.

Isolates belonging to the genera Arthrobacter and Cel-lulomonas were repeatedly recovered from the Spitsber-gen permafrost soil. While strains affiliated to the genusArthrobacter have been commonly isolated from perma-frost soils, the isolation of Cellulomonas-affiliated strainshas only rarely been reported (Vorobyova et al., 1997).In our study, strains that were affiliated to the speciesCellulomonas cellasea and Cellulomonas fermentansaccounted for 39% of all the pure cultures and were alsodominant in the anoxic MPN cultures where they outnum-bered cells related to the genus Clostridium by severalorders of magnitude. The fact that Cellulomonas-relatedsequences could be recovered from the anoxic 106 MPNdilutions confirms the observation that Cellulomonas wasthe most abundant group among the isolates and showsthat their predominance was not due to culture retrievalbias. The dominance of Cellulomonas in the Spitsbergenpermafrost soil may be attributed to the relatively highamount of plant residues in the soil (~10% v/v), which mayselect for bacteria with cellulolytic capabilities such asCellulomonas (Stackebrandt et al., 2005).

In contrast to other soil types (e.g. Hansen et al., 2005)aerobic and anaerobic endospore-forming bacteria wereonly present in low numbers, which is consistent withresults from Siberian permafrost soil studies (Vishniv-etskaya et al., 2000; Soina et al., 2004). As yet we haveno explanation for the apparent low incidence of spore-forming bacteria in permafrost soils. However, the recent

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report by Steven and colleagues (2007) that endospore-forming bacteria comprised a substantial fraction (69%) ofthe isolates retrieved from a Canadian permafrost soilindicates the need for a comprehensive study to quanti-tatively determine their distribution in permafrost soilsfrom different geographical regions.

Cosmopolitan bacteria in the Spitsbergenpermafrost soil

One-third of the Spitsbergen pure cultures had highsequence similarities (" 95%) with sequences obtainedfrom Siberian permafrost isolates (Bakermans et al., 2003;Vishnivetskaya et al., 2006). Furthermore, 41% of theSpitsbergen isolates had close relatives found in othercryohabitats, e.g. Antarctic lakes, Arctic ocean and icecores. These data suggest that these species are cosmo-politan and their presence in cryohabitats distant in bothspace and time implies that similar selection mechanismsmay operate in cryohabitats independent of habitat type,age and geographical location. For example, three of ourisolates are affiliated to the species Cryocola antiquus,which originally was isolated from a 1.8 M- to 3 M-year-oldSiberian permafrost sample (Kochkina et al., 2001).Another cosmopolitan bacterium found in the Spitsbergenpermafrost soil was Cryobacterium psychrophilum, whichoriginally was isolated from an Antarctic soil (Suzuki et al.,1997) and later found in other cryohabitats such as an icecore from China (Xiang et al., 2005), an ice core fromGreenland (Miteva and Brenchley, 2005) and mountainsnow from Japan (Segawa et al., 2005). However, as theaffiliation is only based on the 16S rDNA gene, moredetailed genetic and physiological investigations areneeded to determine how similar these strains actually are.

Conclusions

Our study of a Spitsbergen bacterial permafrost soil com-munity has demonstrated that while a large fraction (74%)of the cells contained DNA they should be consideredphysiologically dead as their plasma membranes nolonger function. This observation has important conse-quences for the interpretation of results obtained solelyusing DNA-based methods as the diversity recovered bythese techniques may include a high proportion of dys-functional cells which no longer have any ecologicalsignificance. The preservation of dead cells may be afeature of permanently cold habitats where cells and DNAcan resist degradation on a geological timescale. Thisstudy has also demonstrated that when determining cul-tivation efficiencies they should be based on the numberof viable cells rather than the total number of cells thatstain with a DNA dye. It has also highlighted the urgentneed for new and improved cultivation techniques to

isolate functionally important members of the bacterialpermafrost communities and to identify the properties bywhich they survive the harsh physicochemical conditionsof permafrost.

Experimental procedures

Sampling and characteristics of the permafrost soil

The permafrost soil samples were obtained from the Advent-dalen valley, Spitsbergen (78°12!N, 15°50!E), NorthernNorway. To reach the permafrost layer, samples wereacquired by digging laterally into an eroded river bank.Samples were then collected aseptically from the uppermost10 cm of the permafrost horizon (~1.4–1.6 m below soilsurface). The soil was kept frozen during transportation andstored at -20°C until used. Sample preparation was carriedout under aseptic conditions at 4°C.

The age of the permafrost soil was determined by 14C-dating of both seed pods and unidentified plant materialpresent in the soil (~10% v/v). The organic material wassubjected to a standard base-acid-base treatment to removepossible contaminants (e.g. carbonates and humic sub-stances) and combusted with CuO in sealed combustiontubes at 950°C. A sample of the resulting CO2 gas wasanalysed according to Vogel and colleagues (1984) and theage was calculated according to Andersen and colleagues(1989). The seed pods were determined to be2295 ! 42 years old and the unidentified plant material2398 ! 43 years old, resulting in an average age of2347 years.

The water content and dry weight of the soil was deter-mined as the weight loss of fresh soil dried at 105°C for 24 h(n = 5). The salinity of the pore water was determined from aNaCl standard curve with a conductivity meter (EG-1 GradientMonitor, Bio-Rad) and the pH was determined directly in thepore water with a Ag/AgCl2 glass electrode (Radiometer). Thecontribution of the smallest particle size fractions (< 250 mm)to the soil matrix was determined by laser diffraction (n = 4;Heuer and Leschonski, 1985), while the contribution of thelargest size fractions (> 250 mm) was determined by sieving(n = 1). Concentrations of total organic carbon (TOC) and totalnitrogen (TN) were determined on H2SO3-treated, homog-enized dried soil (n = 3) using a Carlo-Erba NA 1500 CHNanalyser (Carlo-Erba Instruments). The concentration oftotal hydrolysable amino acids (THAA) was determined byhigh-performance liquid chromatography (HPLC; WatersChromatographic System) on o-phthaldialdehyde-derivatizedproducts (Lindroth and Mopper, 1979). In short, the THAAsamples (0.5 gdw; n = 3) were hydrolysed with 10 ml 6 N HClat 105°C for 24 h under N2 atmosphere. Hydrolysates (200 ml)were dried in a vacuum desiccator (24 h) and the sampleresidues were dissolved in Milli-Q water, dried again andanalysed after re-suspension in 0.2 ml of Milli-Q water. Theiron concentration of the permafrost soil was determined byflame mode atom absorption spectrophotometry (n = 2;Perkin Elmer 5100 PC) (Mehra and Jackson, 1960). Sulfateconcentration in the pore water was measured by ion chro-matography (n = 1; ICS2500, Dionex).

Sulfate-reduction rates were measured in permafrost soilsuspensions (34% w/v) in anoxic phosphate-buffered saline

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solution or R2A broth, using test tubes that were sealed withbutyl-rubber stoppers. The gas phase of the test tubes con-tained oxygen-free N2 gas. Radiolabelled 35S-sulfate wasinjected into each tube followed by vortex mixing (finalactivity = 1 Mbq). Prior to incubation ZnAc solution (1 ml,20% w/v) was added to two tubes from each series to stopmicrobial activity (controls). The tubes were incubated in atemperature gradient block at temperatures from -5.1 to32.7°C (intervals of 2–4°C) for 13 or 31 days (n = 2). Incu-bations were terminated by injection of 20% (w/v) ZnAcsolution. Radiolabelled reduced sulfur compounds werequantified as described in Isaksen and Finster (1996) andthe ratio between radiolabelled sulfide and radiolabelledsulfate was determined as described by Fossing and Jør-gensen (1989).

Methane production was measured in anoxic saline sus-pensions of permafrost soil (5 cm3 in 5 ml) under a gas phaseof oxygen-free N2 gas at 4, 10, 15, 20 and 25°C for 4 weeks(n = 2). Subsamples of the headspace (0.3 ml) were analy-sed for methane at weekly intervals on a gas chromatograph(GC) equipped with an FID detector using the following set-tings: injection port 100°C, oven 80°C and detector 100°C.Separation was performed on a Poropak Q column with N2 ascarrier gas (20 ml min-1).

Bacterial numbers

The total number of bacterial cells in the permafrost soil wasdetermined by epifluorescence microscopy (Zeiss Axiovert200 M) after staining the cells with either SYBR-Gold(Molecular Probes, Invitrogen) for total counts or with fluoro-chromes from the LIVE/DEAD® BacLightTM kit (MolecularProbes, Invitrogen) (Boulos et al., 1999) for differentiationbetween viable and dead cells (n = 5). Sample preparationprior to staining was: suspension of 1 g of permafrost soil inNa pyrophosphate (10 mM) followed by vigorous vortexmixing for 15 min and sonication in an ultrasonic bath(2 ¥ 20 s, 42 kHz, Branson 5510).

The number of aerobic cfu was evaluated on four differentcultivation media: (I) R2A (Difco) (Reasoner and Geldreich,1985), (II) VL55 (Sait et al., 2002), (III) VL55+sugars(containing 2 mM of each of the following sugars: D-xylose, D-glucose, D-arabinose and D-galactose) and (IV)VL55+sugars+amino acids [0.05% (w/v) casamino acids].The VL55-based media were gelled with either 1.5% (w/v)agar or 1.0% (w/v) gellan (Phytagel, Sigma). Nystatin(50 mg l-1) was added to the growth media to prevent fungalgrowth. Prior to inoculation on agar plates permafrost soilwas added to 8 ml of sterile phosphate-buffered saline and1 ml of 2 mM Na pyrophosphate, vigorously vortex mixed for15 min and finally sonicated as described previously. Serialdecimal dilutions (1:600–1:20 000) were then prepared inphosphate-buffered saline and aliquot volumes (0.1 ml) ofeach dilution plated out in triplicate on the different agarmedia. The number of endospore-forming bacteria wasevaluated on a pasteurized inoculum (80°C for 10 min). Agarplates were incubated at 15°C for 30 days (R2A medium) or12 weeks (VL55-based media).

Microcolony-forming units were counted as described byBinnerup and colleagues (1993) after incubation on R2A agarplates at 15°C for 7 days.

The number of anaerobic bacteria was estimated from10-fold serial dilutions (10-1-10-6) in anoxic R2A broth (n = 3)after 16 weeks of incubation at 15°C. Most probable numberswere calculated using index tables (Taras et al., 1971).

Long-term enrichment of the permafrost soil

Eight different long-term soil enrichments (n = 3) were madefrom 4.6 gdw permafrost soil supplemented with either: (i)nutrients (N), 1/10 strength (0.3% w/w) of TSB (Difco), (ii)sodium chloride (S), 2.69 molal NaCl (16% w/w), (iii) nutrientsand sodium chloride (NS), 1/10 TSB and 2.69 molal NaCl,and (iv) no supplements (–), 7 ml of MilliQ water gdw-1 soilequivalent to the volume added to the enrichments supple-mented with 1/10 strength TSB and/or NaCl. The enrichmentprocedure followed specification presented in a study byVishnivetskaya and colleagues (2000).

The water activity (aw) of the enrichments supplementedwith sodium chloride was simulating in situ water activity(aw = 0.91). The amount of sodium chloride added to obtainthe desired water activity was calculated from water activitytables (Robinson and Stokes, 1965; Mazur, 1980; Andraskiand Scanlon, 2002). The enrichments (n = 3) were incubatedunder both oxic (o) and anoxic (an) conditions for 11 weeks at10°C.

By the end of incubation DNA was extracted (n = 3) fromthe enriched and the original permafrost soil (T0) using theFastDNA spin kit for soil (Bio 101, CA) with the followingmodifications: (i) samples were thoroughly ground prior toDNA extraction and bead beaten at medium speed for 30 s(Mini-Beadbeater-8, BioSpec Products, OK), (ii) the centrifu-gation time after bead beating was 10 min, (iii) samples con-taining the DNA binding matrix were shaken for 1 h, and (iv)the mixture of DNA binding matrix and DES incubated for5 min followed by vortex mixing. DNA yield was quantifiedfluoremetrically (TD-700, Turner BioSystems, CA) after stain-ing with PicoGreen (Molecular Probes, Invitrogen) (Sandaaet al., 1998).

Clone library construction and sequencing

Clone libraries (16S rDNA) were constructed from the originalpermafrost soil samples (n = 3) and from each of the threereplicates of the eight different long-term soil enrichments.Polymerase chain reaction amplification (20 cycles)with the primers 27F (Lane, 1991) and 1392R(5!-ACGGGCGGTGTGTACA-3!; C.L. Moyer unpublished)followed procedures described in Hansen and colleagues(2005). Three PCR products were pooled by purificationbefore cloning (eight PCR products were pooled when theDNA concentration of the DNA extract was < 0.5 mg gdw-1).Similar cloning conditions were created by ligating the sameamount of PCR product from each sample (10 ng) into thepCR 4-TOPO vector (TOPO TA cloning® Kit for sequencing,Invitrogen). The cloning procedure followed the instructionsfrom the manufacturer with the following modifications: liga-tion for 1.5 h at room temperature and heat shocking ofcompetent cells for 1 min at 42°C. Sequencing of 32 clonesfrom each library was carried out by Macrogen (Seoul, Korea;http://www.macrogen.com) using the 27F primer and the

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BigDye™ terminator cycle sequencing kit on an ABI3730XLsequencer (Applied Biosystems). Reverse reads with the1392R primer were performed when longer sequences weredesired. Sequences from replica libraries (3 ¥ 32 sequences)were pooled and analysed as one sample in the sequenceanalysis.

Sequence analysis

The sequences were trimmed by the software program LUCY

(Chou and Holmes, 2001) using the RDP pipeline myRDP(Cole et al., 2005) and analysed for the presence of chimerasusing the CHIMERA_CHECK ver. 2.7 (Cole et al., 2003). In total800 sequences with an average length of 752 bp werealigned and a distance matrix was generated for each sampleusing myRDP (Cole et al., 2005). Rarefaction curves,numbers of OTUs and Chao1 richness estimates were cal-culated from the uncorrected distance matrix using the com-puter program DOTUR (Schloss and Handelsman, 2005). Thesequences were classified phylogenetically with the RDPclassifier (Cole et al., 2005). Sequences not readily classifiedinto known phyla were aligned and classified using the ARB

software package (Ludwig et al., 2004). Clone libraries werecompared with the RDP library compare (Cole et al., 2005).Community coverage was calculated with the two non-parametric estimators Good (Good, 1953) and CACE (Chaoet al., 1993) as described in Kemp and Aller (2004).

Identification of cultures

Pure cultures were obtained from 158 randomly picked colo-nies from the agar plates described previously. Amplificationof 16S rDNA gene fragments from the pure cultures wascarried out with the bacterial primer 26F (Hicks et al., 1992)and the universal primer 1390R (Zheng et al., 1996) followingprocedures described in Hansen and colleagues (2005).Partial 16S rDNA sequences (~800 bp) from the pure cultureswere obtained as previously described using the 26F primer.Reverse reads were performed with the 1390R primer.Sequences were compared with the current database ofrRNA gene sequences from GenBank using the BLAST searchtool (Altschul et al., 1997). Pure culture sequences werecompared with the clone sequences using stand-alone BLAST

(Altschul et al., 1997) with a cut-off value of "97%.Enrichments of sulfur-disproportionating bacteria in the

permafrost soil were set up as described in Finster and col-leagues (1998). Positive enrichment cultures were identifiedby formation of FeS after 2 weeks of incubation. Identificationof the bacteria in the enrichment cultures was performed asdescribed in the next section DGGE.

Denaturant gradient gel electrophoresis (DGGE)

The community composition of the anoxic MPN enrichmentswas analysed by DGGE. DNA was extracted from eachenrichment culture using the FastDNA spin kit for soil withmodifications ii–iv described previously. DGGE was carriedout according to Muyzer and colleagues (1998) on 16S rDNAfragments amplified with the primers 341F-GC (Muyzer et al.,1993) and 907R (Lane, 1991) following procedures described

in Hansen and colleagues (2005). Selected bands wereexcised from the gel, reamplified with 341F-GC and 907Rand repeatedly run on DGGE gels until clean. Clean bandswere reamplified with 341F (without GC clamp) and 907Rprimers and sequenced. Sequences were compared with thecurrent database of rRNA gene sequences from GenBankusing the BLAST search tool (Altschul et al., 1997).

Nucleotide sequence accession numbers

Partial 16S rDNA sequences generated in the present studywere deposited in GenBank with Accession No. EF034156–EF034955 for the clones, EF034956–EF035006 for theDGGE bands and EF451631–EF451787 for the culturedisolates.

Acknowledgements

The present study was supported by the Danish NationalScience Research Council Grant 21-03-0557 and throughNASA’s Astrobiology Institute at Michigan State Universityduring Aviaja Hansen’s stay there. The Faculty of Science,University of Aarhus, financed part of Aviaja A. Hansen’s PhDgrant. We thank Ole Humlum for guidance on sample siteselection, Monica Ponder and Jens Risbo for their helpfulcontribution to the experimental design, Jim Cole, Benli Chai,Qiong Wang and Kasper Urup Kjeldsen for their invaluablehelp with sequence analysis and Tove Wiegers, BrittaPoulsen, Rikke Holm for skilful technical assistance. Wethank Jan Heinemeier for carrying out the 14C-dating mea-surements and Birte Eriksen and Anne Thoisen for measure-ments of the iron content in the soil.

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Supplementary material

The following supplementary material is available for thisarticle online:Fig. S1. Fraction of 35SO4

2– sulfate tracer reduced in slurrieswith permafrost soil from Spitsbergen. The permafrost soilwas suspended either in saline water (A and B) or in R2Abroth (C and D). The incubations were stopped by injection ofa 20% ZnAcetate (ZnAc) solution after an incubation periodof 13 (circles) or 31 days (squares). Samples that served ascontrols (ù) were stopped immediately after preparation byinjection of 1 ml of ZnAc (20%) into the test tube and kept at15°C in the temperature block for 31 days. Samples wereconsidered positive when the fraction of reduced 35S-Sulfatewas larger than the fraction obtained in the ZnAc-treatedcontrols. (A) (saline water) and (C) (R2A broth) present dataobtained within the entire temperature range. (B) (salinewater) and (D) (R2A broth) show data obtained in the ‘low’temperature range. In saline water sulfate reduction wasdetectable at temperatures higher than 0°C in samplesstopped after 13 days of incubation and at temperaturehigher than -2°C in samples stopped after 31 days ofincubation. In R2A broth, sulfate reduction was detectableabove 10°C in samples stopped after 13 days of incubationand above 2°C in samples stopped after 31 days ofincubation.Fig. S2. Observed diversity in clone libraries illustrated asrarefaction curves. Black squares indicate original permafrostsoil (T0), open symbols indicate oxic enrichments (o) andgrey symbols indicate anoxic enrichments (an). Enrichmentconditions: (–) no supplement; (N) nutrient supplement; (S)sodium chloride supplement; and (NS) supplement of bothnutrients and sodium chloride. Rarefaction curves were cal-culated from a uncorrected distance matrix using the com-puter program DOTUR (Schloss and Handelsman, 2005).Operational taxonomic units were defined by using a distancelevel of 3%.

This material is available as part of the online article fromhttp://www.blackwell-synergy.com

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