Alison Murray

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Supporting Information

Murray et al. 10.1073/pnas.1208607109

SI Materials and Methods

Dissolved Inorganic Carbon Determinations. Dissolved inorganiccarbon(DIC) concentration for the 2005 Lake Vida brine (LVBr)

was determined by infrared gas chromatography on acid-spargedsamples following calibration with bicarbonate standards. DICstable carbon isotope samples for 2005 LVBr were acidified withphosphoric acid (15 mol·L −1) and the liberated CO2 was cryo-genically separated on a vacuum extraction line, with DIC con-centrations measured via digital manometer. Samples wereanalyzed on a dual inlet Thermo Finnigan Delta Plus XL massspectrometer. Samples for DIC concentration and isotope analysescollected in 2010 were analyzed using a method closely followingthat of Salata et al. (1). In this approach, 100 μL of LVBr wereinjected into evacuated 10-mL serum vials containing 100 μL of concentrated H3PO4 (∼100%). After > 24 h of equilibration, vials

were brought to atmospheric pressure with pure N2 and 150 μL of the headspace was analyzed using a TraceGas System interfaced toan Isoprime mass spectrometer. Values are reported in δ notation

relative to Vienna Pee Dee Belemnite (VPDB). The mass spec-trometer has an analytical precision of 0.1‰. No mineral phasefractionation factors were applied. C-isotope values are reported

with respect to the international standard, VPDB.

Inorganic Nitrogen and Sulfur Analyses. In addition to dissolvednutrient determinations described in the main text for samplescollectedinbothexpeditions,wedeterminedoxidizedintermediatesof sulfur and isotopic compositions of N and O (from nitrate), Nfrom ammonium, and S for the LVBr samples collected in 2010.The isotopic composition of nitrate was determined by steam dis-tillation and microbial reduction method (2). The isotopic com-position of ammonium was analyzed using the acidified disk method described by Holmes et al. (3). N and O isotope valuesdetermined using an Elementar Isoprime are reported with re-spect to the international standards Air and Vienna StandardMean Ocean Water (VSMOW), respectively.

Sulfur intermediate analyses were performed on samples pre-served in a gas-tight glass syringe that was flash-frozen and kept at−78 °C wrapped in foil until analysis on a Dionex Ion Chro-matograph System (IC-2000) and were separated using an IonPac

AS17-C column with KI eluent to separate different sulfur species.To determine sulfide levels, samples were measured using CdCl2

precipitation and sulfur (δ34S) isotope values measured on a Fin-nigan Mat 252. Specifically, LVBr was loaded into 60-mL syringes

with 10 mL of 1.5 M CdCl2 solution to precipitate any free sulfideas cadmium-sulfide (CdS). LVBr syringe samples were first fil-tered onto 0.4-μm glass fiber filters to trap any precipitated CdSand any particulates in the brine. Sulfate was recovered from the

LVBr filtrate by bringing the brine sample to pH ∼3 and addingan excess of 0.2 M Ba(NO3)2 solution to precipitate dissolvedsulfate as BaSO4. The BaSO4 was rinsed and centrifuged mul-tiple times with ultrapure water to remove chlorine, and thendried overnight at 80 °C before isotopic analysis. The filteredresidue from LVBr samples was first reacted with hot 6N HCl for3 h in a N2-purged flask, following methods of Brüchert and Pratt(4). Flowing N2 carried evolved H2S, from monosulfide decom-position, through a buffersolution (0.1 M citrate solution adjustedto pH 4) into a 0.1 M AgNO3 solution where it was precipitated as

Ag2S. The Ag2S precipitate was then filtered, rinsed, and driedonto a baked0.22-μm quartz-fiber filter that was saved for isotopicanalyses. Finally, the remaining acid-insoluble residue was reacted

with mixture of ∼40 mL of 12 N HCl and ∼40 mL of 1.0 M chro-mous chloride (CrCl2•6H2O) in a N2-purged extraction flask and

evolved H2S from any polysulfides was precipitated as Ag2S(see description above). No measurable sulfide was recoveredfrom either the mono- or polysulfide extractions in any of theLVBr samples.

Sulfur (δ34S) isotope values were measured (on a Vienna-Canyon Diablo Troilite scale) using a Carlo Erba Elemental

Analyzer (EA1110) coupled to a Finnigan Mat 252 isotope ratiomass spectrometer via a ConFlo II split interface in the StableIsotope Research Facility at Indiana University. Analytical repro-ducibility was better than ±0.13‰ (1 σ) for standards and du-plicate samples.

Elemental Analysis. All sample and standard preparation wasperformed under class 100 clean-room conditions using tracemetal clean techniques. The 2005 LVBr samples were acidified topH < 2 with ultra-pure nitric acid (HNO3), stored for > 2 wk,and diluted to a salinity of < 1‰ with 1% HNO3 (prepared

with > 18 mΩ ultrapure water) before analysis. Isobaric inter-

ferences were resolved using multiple resolution settings (nomi-nally m/ Δm ∼400, 4,000, and 10,000). The instrumental response was quantified, after normalization to an indium internalstandard, using external standards (NIST traceable). Multiplemethod blanks and test standards (Environmental Protection

Agency drinking water Quality Assurance Protocol solutions) were analyzed for quality control purposes. Brine samples col-lected in 2010 were analyzed for total and dissolved (<0.2 μm,Steriltech silver membrane) elements. Triplicate total samples

were prepared by nitric and hydrofluoric acid digestion usinga block heater and microwave digestion. Both total and dissolvedsamples were diluted with 1% HNO3 spiked with an indiuminternal standard. These samples were analyzed by high-resolu-tion inductively coupled plasma mass spectrometry at the Uni-

versity of Tasmania under similar conditions to the 2005 samples with the exception that the guard electrode was deactivated tominimize isobaric interferences on some isotopes (e.g., 95Mo16Oon 111Cd; following ref. 5). Quantitation of these samples wasalso by external calibration with NIST traceable standards (Gai-thersburg, MD). The method accuracy was verified through theanalysis of the NIST 1640 “Trace Elements in Natural Water”standard reference material. Samples analyzed by the “total re-coverable” acid digestion and dissolved analytical methods werecomparable. This result is likely because of the high fractionalsolubility of elements in the brine samples.

Volatile Hydrocarbon and Dissolved Gas (N2O and H2) Analyses. Theprocedures for analysis of the 2010 LVBr samples includedanalyses to detect a broader spectrum of gases than for the 2005

samples (including hydrogen) in addition to a different approachfor N2O determinations. A custom-designed gas chromatograph with FID/TCD detectors (analyses conducted at Microseeps) was used in determinations of CO2, CH4, C2H6, and C2H4. Thedetection sensitivity of CO2 was 0.11 mM and for hydrocarbonsdetected (CH4, C2H6, and C2H4) were 6.2, 0.83, and 0.89 nmol·L −1.The levels of C3H8, C3H6, C2H2, CO, C4H10 (iso and n-forms)

were below detection limits (0.05 μg/L −1 for all, except 0.5 μg/L −1

for C2H2 and 1.0 mg/L −1 for CO).The N2O concentration was determined based on the m/z 44

ion beam intensity produced within a mass spectrometer duringisotopic analysis (Elementar Isoprime). Before introduction onto the mass spectrometer, N2O was purified using a Trace Gassystem (Elementar) that involved quantitatively stripping of N2Ofrom 100 mL of LVBr under He flow, water, and CO2 removal

Murray et al. www.pnas.org/cgi/content/short/1208607109 1 of 14

http://www.pnas.org/cgi/content/short/1208607109




using chemical scrubbers (magnesium perchlorate and Carbo-sorb, respectively), then the gas was chromatographically sepa-rated on a Porapak Q column (6, 7).

For the H2 and N2 determinations ∼150 mL of LVBr wasintroduced to evacuated glass bottles (300 mL) and equilibratedfor 8 h at room temperature (8). Water was then removed by

vacuum and sample gases introduced to an evacuated 3-mL gassampling loop interfaced to a mass spectrometer (ElementarIsoprime) with magnesium perchlorate in-line for water vapor

removal. H2 and N2 was then chromatographically purified usinga molecular sieve 5 Å column under He flow and concentrationdetermined from the m/z 2 ion beam intensity for the H2; con-centrations were not determined for the N2 (9). The stable iso-tope values for N2 and H2 are in reference to air and VSMOW,respectively.

Dissolved Organic Carbon Methods. DOC in LVBr was quantifiedusing high temperature oxidation on a Shimadzu Total OrganicCarbon (TOC) analyzer with a nonpurgeable organic carbon(NPOC) method (10). Carbohydrate concentration of the 2010brine averaged over six replicates was measured after Dubois et al.(11)using pullulan (a soluble linear glucose polysaccharide; Sigma)as the standard.

To characterize various fractions of DOC, LVBr was serially diluted to the point in which the last two samples were below thechloride interference threshold (12) based on anion chemistry data. Average values for the last two samples were reported.Fractions of dissolved organic carbon (DOC) were then isolatedand characterized by acidifying two 1-L samples of LVBr to pH∼2, diluting the LVBr (factor of 40) followed by a two-step fil-tration procedure. Diluted brine was partitioned into four fractionson XAD-8 and XAD-4 resins (13), separating fulvic (FA) andtransphilic acid (TPIA) fractions, respectively followed by tan-gential flow ultra-filtration (TFUF; 1-kDa membrane) of thecolumn ef fluent to concentrate the > 1 kDa fraction, which alsoresults in a low molecular weight, permeate fraction (TFUFpermeate). The FA, TPIA, and > 1 kDa fractions were analyzed

with fluorescence spectroscopy and freeze-dried for downstream

analysis (14). δ13C-DOC and radiocarbon (14

C) ages of the six Lake Vida DOM fractions were prepared at the University of Colorado at Boulder’s radiocarbon laboratory (NSRL) and an-alyzed by accelerator mass spectrometry (AMS) at the University of Arizona AMS facility according to Stuiver and Polach (15).

Additional Microbial Activity Assays. Additional measures of het-erotrophic bacterial growth were attempted via an evaluation of thymidine and acetate incorporation and respiration at 0 and−12 °C with an ambient atmosphere headspace [incubating with20 nM 3H-labeled thymidine for 54.5 h, extraction with cold 5%tricarboxylic acid (TCA), and filtering (16) and 10 mM 14C-la-beled acetate for 65.5 h, extraction with cold 5% TCA, and fil-tering] in 2005. Although thymidine incorporation and acetate

values were higher than formalin-killed controls, the difference

was not significant (Mann–Whitney test). Similarly, acetate res-piration values were not different from formalin-killed controls.Thymidine incorporation assays conducted with the 2010 LVBrsamples were run in parallel with the leucine incubations (17)under N2 atm at 0 and −13.5 °C for 6–16 d, and again, significantlevels of incorporation indicating DNA biosynthesis were notdetected.

Microbial Cultivation. LVBr was transferred from anoxic bottles atthe Desert Research Institute under an N2 atmosphere to anambient atmosphere, where it was used to inoculate various mediaunder aerobic or anoxic and cold (0–3 °C) conditions. Halophilicbroth (HB) and R2A (a freshwater heterotrophic media) did notproduce evident growth after one month, whereas standard Ma-rine Broth (DIFCO) yielded growing mixed cultures. Isolates were

obtained by directly plating brine on Marine Agar (DIFCO) andincubating at 0–3 °C. Colonies (appearing after 2 wk) were iso-lated through subsequent streaking (3× per colony). DNA wasextracted from isolates following standard procedures (18), thesmall subunit (SSU) rRNA gene was PCR-amplified using bac-terial primers (27F and 1492R), purified (Qiaquick; Qiagen), andsequenced at the Nevada Genomics Center, Reno, NV. Repre-sentatives were archived in 10% glycerol.

Anaerobic enrichment media stock (10 mL per tube) was

prepared using a hungate station in which LVBr was diluteddeionized water (50% LVBr), degassed, and sterilized, and theheadspace was filled with N2 (19). Fifteen millimolar malt extract(final concentration) or 0.2% Casein extract (ICN Biomedicals)and 0.01 mL of Wollin’s Vitamin Solution (American Type Cul-ture Collection) were injected into sealed Balch tubes just beforeinoculation. This media was inoculated with 1.1 mL anoxic brineand incubated in the absence of light at −2 °C or 10 °C for 9 mo.SSU rRNA gene sequences (Enrichment-D2, B11, C3, C6, C10,and C12) were obtained from a clone library (method describedbelow) derived from DNA harvested from organisms grown withthe 15 mM malt extract. SSU rRNA gene sequence derived from−2 °C Enrichment-E3 was obtained from organisms grown inenrichment culture with 0.02% Casein at 10 °C. DNA was ex-tracted (DNeasy Blood and Tissue kit; Qiagen) from 2-mL en-richment culture aliquots that were concentrated to 30 μL (Vivaspin; Sartorius).

Molecular Analyses. Cells were harvested in the field in 2005 by filtering brine through 0.2-mm Sterivex filters (Millipore), thenfilters were storedfrozen in eithersucrose-Tris-EDTA buffer (500mmol L −1), 40 mmol L −1, and 0.73 mol L −1, respectively) untilDNA extraction or RNAlater (Ambion, Inc.) for RNA. Theresults presented here were conducted with nucleic acids ex-tracted from the 2005 LVBr following ref. 20. DNA was initially surveyed by PCR for amplification of the three domains of life.The primers used included: Archaea using Arch20F (20), 109F(21), and 958R (22); Bacteria using 27F and 1391R (23); andEukarya using Euk1A and 1520R (24). A strong band in the

bacterial amplifi

cation was detected, but no archaeal or eukaryalsignals were observed. The bacterial SSU rRNA gene library wasconstructed using the the products of eight PCR reactions am-plified with Bact27F and Univ1391R (25 cycles each) that werepooled then ligated into the PCR4TOPO vector (Invitrogen) andtransformed into Escherichia coli cells in LB with 100 mg/mL −1

ampicilin and kanamycin. Purified DNA (Millipore) with insertsof the correct size were sequenced bidirectionally on an AB 3100automated sequencer (Applied Biosystems). Sequences were as-sembled using Sequencher (GeneCodes). Sequences were checkedfor chimeras using three on-line tools Bellepheron (25) (http:// greengenes.lbl.gov/cgi-bin/nph-bel3_interface.cgi), CheckChimera(26), and pintail ( www.bioinformatics-toolkit.org/Web-Pintail/ ).

Comparative phylogenetic analyses were performed with SSUrRNA gene sequence datasets from polar lakes and streams,

brines, sea ice, and other sites with sequences in common to theLVBr community. Analyses presented here include 15 datasets where nearly full-length SSUrRNA gene sequences were available(TableS3).DatasetsweredownloadedfromGenBankandalignedusingthe nearest alignmentspacetermination alignmenttool (27).Distance matrices were generated individually for each library,and DOTUR (28) was run to generate nonredundant datasets foreach library with all sequences represented at a distance of 0.01.Overlapping regions between all libraries (460 sequences) pro-

vided an alignment with a minimum of 1,188 base pairs. Neighbor- joining trees using pair-wise deletion were calculated in MEGA (v4) (29). UniFrac (30) was used to calculate statistical com-parisons among the libraries.

The evolutionary history of LVBr nearly full-length SSU rRNA gene sequences was inferred using the minimum evolution method


http://greengenes.lbl.gov/cgi-bin/nph-bel3_interface.cgi


http://www.bioinformatics-toolkit.org/Web-Pintail/



http://www.bioinformatics-toolkit.org/Web-Pintail/





(31) and 1,000 bootstrapped trees. Evolutionary distances werecomputed using the maximum composite likelihood method (32)and are in units of the number of base substitutions per site, thecomplete deletion option was used. There were a total of 1,020positions in the final dataset. Neighbor-joining analysis (29) wasused to generate the phylogenetic tree to characterize related-ness between crDNA and culture sequences, using MEGA4based on 209 aligned positions, in which 1,000 bootstrap values

were calculated.Four libraries with nearly full-length sequences and publishedevenness data [LVBr, Lake Vida ice cover at 4.8 and 15.9 m(LVI_4_8, LVI_15_9) and the surface outflow of Blood Fallsbrine] were compared using weighted abundances to furtherinvestigate specific associations between these environments withsimilar microbial communities (and physical proximity). Neigh-bor-joining trees were calculated using complete deletion (144unique sequences of 546 redundant sequences were compared

with 1,255 bases) (30).

RNAlater (Ambion) was added to brine samples filteredthrougheither 0.2-mm or 47-mm Supor or Sterivex filters (Millipore), thenstored at 4 °C overnight, frozen at −20 °C. RNA was extractedusing a Totally RNA kit (Ambion) following the manufacturer’sinstructions. Total RNA extracts were subjected to two rounds of DNase (Ambion) followed by purification with phenol:chloro-form following standard procedures. PCR-denaturing gradient gelelectrophoresis (DGGE) was used to compare reverse-transcribedSSU rRNA (crRNA) and SSU rRNA gene profiles (33) in which

crRNA was first reverse-transcribed with superscript III usinga 1:1 mixture of hexamers (300 ng/ μL −1) and Univ1391R. Nega-tive control PCR reactions were run with total RNA to check forDNA contamination in the RNA. One microliter of thecrRNA product was used in PCR with GC-clamped primersGC358F and 517R (10 pmol each). A crRNA clone library wasalso prepared with the same cDNA (1 μL) using primers 358F-1391R to amplify the crRNA, which was then cloned into TOPOPCR4 vector (Invitrogen) following standard conditions. Se-quencing proceeded as described above.

1. Salata GG, Roelke LA, Cifuentes LA (2000) A rapid and precise method for measuring

stable carbon isotope ratios of dissolved inorganic carbon. Mar Chem 69(1-2):153–161.

2. Casciotti KL, Böhlke JK, McIlvin MR, Mroczkowski SJ, Hannon JE (2007) Oxygen

isotopes in nitrite: Analysis, calibration, and equilibration. Anal Chem 79(6):2427–

2436.3. Holmes R, McClelland J, Sigman D, Fry B, Peterson B (1998) Measuring 15N-NH4

+ in

marine, estaurine and fresh waters: An adaptation of the ammonia diffusion method

for samples with low ammonium concentrations. Mar Chem 60(3-4):235–243.

4. Brüchert V, Pratt L (1996) Contemporaneous early diagenetic formation of organic

and inorganic sulfur in estuarine sediments from St Andrew Bay, Florida, USA.

Geochim Cosmochim Acta 60:2325–2332.

5. Townsend A (2000) The accurate determination of the first row transition metals in

water, urine, plant, tissue and rock samples by sector field ICP-MS. J Anal At Spectrom

15(4):307–314.

6. Sutka RL, Ostrom NE, Ostrom PH, Gandhi H, Breznak JA (2003) Nitrogen isotopomer

site preference of N2O produced by Nitrosomonas europaea and Methylococcus

capsulatus Bath. Rapid Commun Mass Spectrom 17(7):738–745.

7. Sutka RL, et al. (2006) Distinguishing nitrous oxide production from nitrification and

denitrification on the basis of isotopomer abundances. Appl Environ Microbiol 72(1):

638–644.

8. Emerson S, Stump C, Wilbur D, Quay P (1999) Accurate measurement of O2, N2, and Ar

gases in water and the solubility of N2. Mar Chem 64(4):337–347.

9. Yang H, et al. (2012) Using gas chromatography/isotope ratio mass spectrometry todetermine the fractionation factor for H2 production by hydrogenases. Rapid Commun

Mass Spectrom 26(1):61–68.

10. Suginura Y, Suzuki Y (1988) A high-temperature catalytic-oxidation method for the

determination of non-volatile dissolved organic carbon in seawater by direct injection

of a liquid sample. Mar Chem 24(2):105–131.

11. Dubois M, Gilles K, Hamilton J, Reber P, Smith F (1956) Colorimetric methods for

determination of sugars and related substances. Anal Chem 28(3):350–356.

12. Aiken G (1992) Chloride interference in the analysis of dissolved organic carbon by

the wet oxidation method. Environ Sci Technol 26(12):2435–2439.

13. Aiken G, McKnight DM, Thorn KA, Thurman EM (1992) Isolation of hydropholic arganic-

acids from water using nonionic macroporous resins. Org Geochem 18(4):567–573.

14. Rees C, Jenkins WJ, Monster J (1978) The sulphur isotopic composition of ocean water

sulphate. Geochim Cosmochim Acta 42(4):377–381.

15. Stuiver M, Polach H (1977) Discussion: Reporting of 14C data. Radiocarbon 19(3):

355–363.

16. Kirchman DL, Keil RG, Simon M, Welschmeyer NA (1993) Biomass and production of

heterotrophic bacterioplankton in the oceanic subarctic Pacific. Deep Sea Res Part I

Oceanogr Res Pap 40(5):967–988.

17. Smith DC, Azam F (1992) A simple, economical method for measuring bacterial

protein synthesis rates in seawater using 3H-leucine. Mar Microb Food Webs 6(2):

107–114.

18. Sambrook J, Russell D (2001) Molecular Cloning: A Laboratory Manual (CSHL, New

York), 3rd Ed.

19. Sowers KR, Schreier HJ, DasSarma S, Fleischmann EM (1995) Methanogens:

Growth and identification. Archaea: A Laboratory Manual, Methanogens, eds

Robb KRSFT, Schreier HJ, Place AR (Cold Spring Harbor Lab Press, Plainview, NY),pp 24–31.

20. Massana R, Murray AE, Preston CM, DeLong EF (1997) Vertical distribution and

phylogenetic characterization of marine planktonic Archaea in the Santa Barbara

Channel. Appl Environ Microbiol 63(1):50–56.

21. Galand PE, Lovejoy C, Vincent WF (2006) Remarkably diverse and contrasting archaeal

communities in a large arctic river and the coastal Arctic Ocean. Aquat Microb Ecol 44

(2):115–126.

22. DeLong EF (1992) Archaea in coastal marine environments. Proc Natl Acad Sci USA 89

(12):5685–5689.

23. Lane DJ (1991) 16S/23S rRNA sequencing. Nucleic Acid Techniques in Bacterial

Systematics, eds Stackebrandt E, Goodfellow M (Wiley, New York), pp 115–175.

24. Díez B, Pedrós-Alió C, Marsh TL, Massana R (2001) Application of denaturing gradient

gel electrophoresis (DGGE) to study the diversity of marine picoeukaryotic assemblages

and comparison of DGGE with other molecular techniques. Appl Environ Microbiol 67

(7):2942–2951.

25. Huber T, Faulkner G, Hugenholtz P (2004) Bellerophon: A program to detect chimeric

sequences in multiple sequence alignments. Bioinformatics 20(14):2317–2319.

26. Cole JR, et al.; Ribosomal Database Project (2003) The Ribosomal Database Project(RDP-II): Previewing a new autoaligner that allows regular updates and the new

prokaryotic taxonomy. Nucleic Acids Res 31(1):442–443.

27. DeSantis TZ, Jr., et al. (2006) NAST: A multiple sequence alignment server for

comparative analysis of 16S rRNA genes. Nucleic Acids Res 34(Web Server issue):W394-9.

28. Schloss PD, Handelsman J (2005) Introducing DOTUR, a computer program for defining

operational taxonomic units and estimating species richness. Appl Environ Microbiol 71

(3):1501–1506.

29. Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: Molecular Evolutionary Genetics

Analysis (MEGA) software version 4.0. Mol Biol Evol 24(8):1596–1599.

30. Lozupone C, Hamady M, Knight R (2006) UniFrac—An online tool for comparing

microbial community diversity in a phylogenetic context. BMC Bioinformatics 7

(August):371.

31. Rzhetsky A, Nei M (1992) A simple method for estimating and testing minimum

evolution trees. Mol Biol Evol 9(5):945–967.

32. Tamura K, Nei M, Kumar S (2004) Prospects for inferring very large phylogenies

by using the neighbor-joining method. Proc Natl Acad Sci USA 101(30):11030–

11035.

33. Mosier AC, Murray AE, Fritsen CH (2007) Microbiota within the perennial ice cover of

Lake Vida, Antarctica. FEMS Microbiol Ecol 59(2):274–288.






Fig. S1. ( A) Map of Antarctica showing the location of the McMurdo Dry Valleys. (B) National Aeronautics and Space Administration’s (NASA) Earth Ob-

servatory, Landsat 7 satellite image of a part of McMurdo Dry Valleys showing the location of Lake Vida (Vi) and Lake Vanda (Va), Fryxell (F), Hoare (H), Bonney

(B), and Blood Falls (BF). The filled circle at the center of Lake Vida indicates the location of the borehole. The white rectangle indicates the area covered by the

geologic map (Fig. S3). The location of sampling in 2005 and 2010 is indicated by a dot.






Fig. S2. Geological description of Lake Vida basin and Victoria Valley. ( A) View of the southern and southwestern shores of Lake Vida showing two large

Ferrar dolerite sills (darker layers) cross cutting granite of the Orestes Pluton. The lake is 349 m above sea level; the marked summit is ∼1,450 m high. The arrow

points to the dark dolerite-dominated Bull Drift. ( B) View of the southern shore of Lake Vida showing two large Ferrar dolerite sills (darker layers) crosscutting

granite (lighter in color). The marked summit is Mt. Cerberus (1,766 m). The arrow points to the camp site in the center of the lake. (C ) View toward the

northeastern shore of Lake Vida. The darker layers are made of Ferrar dolerite the lighter layers are granite. The marked summit is above 1,200 m. The white

arrow points to aeolian sand deposits dominated by Fe.Mg silicates and Ca.Fe.Mg silicates (pyroxenes). The black arrow points to camp in the center of Lake

Vida. The rocks at the forefront are granite ventifacts.






Fig. S3. Simplified geological map of the Victoria Valley around Lake Vida adapted from Turnbull et al. (1). The location of the area covered by this map is

shown in Fig. S1. Adapted with permission from the Institute of Geological and Nuclear Science.

1. Turnbull IM, Allibone AH, Forsyth PJ, Heron DW (1994) Geology of the Bull Pass- St Johns Range area, southern Victoria Land, Antarctica, 1/50000. Institute of Geological & Nuclear

Sciences Geological Map 14 (Institute of Geological & Nuclear Sciences Limited, Lower Hutt, New Zealand), 1 sheet and 52 pp.






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Fig. S4. PCR-DGGE analysis of Lake Vida DNA, reverse-transcribed RNA (crRNA), and cloned bacterial SSU rRNA gene sequences. The crRNA, an indicator of

biologically active organisms, was PCR-amplified with the same primers as for the DNA (lane 5). Selected cloned SSU rRNA genes from Lake Vida brine wereincluded in the analysis to assign identities to comigrating bands, (identified with symbols: lane 1 clone LVBR10cG04, an uncultivated Lentisphaera-related

bacterium (same as LVBR10aD09, GQ167314.1); lane 2 clone LVBR10cE03, related to uncultivated Marinobacter clone ELB25-092, DQ015769.1, # (same

as LVBR10aG09 GQ167320.1, and LVBR10cA05, GQ167307.1); lane 3 clone LVBR10cF06, a Sphaerochaeta-related strain,^

(same as LVBR10bB02, GQ167322.1);

lane 11 clone LVBR10cD03, an uncultivated Psychrobacter -related bacterium, * (same as LVBR10cG07 GQ167313.1); lane 12 clone LVBR10cA08 (GQ167319.1), an

uncultivated epsilonproteobacterium, >; and lane 13 clone LVBR10cA04, related to an uncultivated flavobacterium,< (same as LVBR10cF01, GQ167312.1). DNA

extracted from four brine samples was analyzed in independent lanes (6, 8–10). As a negative control for the RT-PCR, RNA that was not reverse-transcribed was

amplified in the same manner as the other reactions to verify that amplification in the RT-PCR product was from cDNA and not contaminating DNA (lane 4).

Lane 7 is empty. Bands excised and sequenced along with nearest neighboring sequence are indicated with letters: “a,” LVBR2ERNA-4, an uncultivated Sul-

furovum-related epsilonproteobacterium, GQ167343; “b,” LVBR2B-2, uncultured Psychrofl exus bacterium, GQ167344.1.






Isolate

Enrichment culture

SSU rRNA gene clone

SSU crRNA clone

DGGE band clone Psychrobacter -group

Marinobacter -group

Bacteriodetes-phylum

Sulfurovum-group

Isolate LVBR 79

Enrichment-D2

Enrichment-B11

LVBRrRNAA1

Enrichment-C3

Enrichment-C6

Enrichment-C10

LVBR10cG07

LVBR10cC08

Enrichment-E3

LVBR10cA05

LVBRrRNAH2

Isolate LVBR 90

LVBRrRNAD1

LVBR10cE12

LVBRrRNAB2

LVBRrRNAF1

LVBR10aF06

LVBR10cF05

LVBR10aH12

DGGE-LVBR2B-2

LVBR10cE09

LVBR10aF03

LVBR10cE02

LVBR10aA11

LVBR10cA11

LVBR10cD02

LVBR10aC08

LVBRrRNAG5

LVBR10cA08

DGGE-LVBR2ERNA-4

LVBR10aG04

LVBR10aF05

LVBR10cB02

LVBR10bB06

LVBR10aA04

LVBR10cC03

LVBR10aH06

LVBR10cH06

LVBR10aC07

LVBR10aD09

LVBR10bA03

36

42

58

49

46

100

91

71

62

76

89

61

100

100

99

78

65

69

57

52

51

34

34

20

13

21

17

30

64

89

27

21

15

42

34

93

93

0.02

*

*

**

*

Fig. S5. Neighbor-joining phylogenetic tree of Lake Vida SSU rRNA and rRNA gene sequences derived from cultivated isolates, anaerobic enrichments, cloned

crRNA, cloned DGGE bands (Fig. S4), and cloned rRNA gene sequences. Symbols indicate source of the sequence. The Marinobacter and Psychrobacter genera

both were well-represented in cultivation-based, crRNA and PCR-amplification approaches suggesting that they are active in LVBr. An asterisk indicates se-

quence with comigrating crRNA-derived DGGE band.






A

BF

LVBR

LVI 15 9

LVI 4 8

0.05

64.6

100.0

100.0

B

Fig. S6. LVBr microbial diversity and comparisons to other environmental SSU rRNA gene libraries. ( A) Principle components analysis of 15 environmental SSU

rRNA bacterial clone libraries based on sequence composition (richness, not evenness) of each library. Designations of libraries are as follows: Lake Vida Brine,

LVBr; Lake Vida Ice, LVI; East and West Lobe Lake Bonney, ELB and WLB, respectively; oil sands tailings pond, OSTP; Romanian oil-contaminated soil, PIT;Canadian biodegraded oil reservoir, PL; Svalbard marine sediments, SVA; Blood Falls glacial outflow, BF; Arctic cold springs Color Peak, CP and Gypsum Hill, GH;

ARC-1 and ANT-1, Arctic sea ice Antarctic sea ice, respectively; ANT-P, seawater of Arthur Harbor (Antarctica); ACE-S, BURT-S, and ORG-S, sediments of Ace,

Burton, and Organic lakes, respectively (Vestfold Hills, Antarctica); ABBB, BBBB, DBBB, and UBBB are Mediterranean deep anoxic brines of L’Atalante, Bannock,

Discovery, and Urania basins, respectively (see Table S3 for accompanying sample and library data). The first and second principle components explain 10.53%

and 8.04% of the variance, respectively. (B) Closely related libraries (BF, LVBr, and LVI surface at 4.8 m and deep ice at 15.9 m) were clustered with weighted

abundances (evenness), branches were normalized to account for different rates and compared using the jackknife statistic. Values at the nodes are per-

centages of 1,000 iterations.







http://www.mcmlter.org/



Table S2. Characterization of LVBr dissolved organic material fractions

DOM fraction % Carbon FI δ13C-DOC ‰14C Fraction modern 14C Fraction modern error 14C age (y BP) 14C age error (y BP)

Filtered water 1 — 1.78 — — — — —

Filtered water 2 — 1.79 — — — — —

LV-FA 1 9.3 1.56 −17.7 0.6226 ±0.0011 3,805 ±15

LV-FA 2 10 1.59 −19.5 0.6021 ±0.0012 4,075 ±20

LV-TPIA 1 7.4 1.79 −17.9 0.6118 ±0.0012 3,945 ±20

LVTPIA 2 7.9 1.84 −16.8 0.5964 ±0.0011 4,150 ±20

LV > 1 kDa 1 48.6 1.46 −15.3 0.6922 ±0.0013 2,955 ±15LV > 1 kDa 2 54.2 1.58 −14.6 0.6399 ±0.0015 3,585 ±20

TFUF permeate 1 24.4 1.78 — — — — —

TFUF permeate 2 25.8 1.75 — — — — —

Neutrals 3–6.5 — — — — — —

Initial determinations were conducted with 0.45 μm filtered brine before column chromatography and TFUF. DOC was partitioned into FA, and TPIA

fractions, followed by TFUF of the column effluent which yielded a > 1 kDa retentate fraction and a < 1 kDa permeate fraction. SUVA254 is the carbon

normalized UV absorbance at 254 nm, which is an indicator of aromaticity. The percent carbon of each fraction was determined by mass balance using

dissolved organic materials (DOM) concentrations and fraction volumes. The fluorescence index (FI) is the ratio of the emission intensities at 470 nm and 520 nm

when excited at 370 nm and is an indicator of DOM origin. 14C data were derived using the prepared DOM fractions.






Table S3. List of studies used for rRNAgene library comparisons

Source Location

Library

designation

No. of sequence

OTUs

No. of

clones

in library

Salinity/

conductivity Temp °C H’ S Chao1 S Ace Coverage % Source

Anoxic brine Lake Vida, Victoria

Valley

LVBR 32 155 188 psu −13.4 2.87 82 112 NA Present

study

Lake water

column

East Lake Bonney,

Taylor Valley

ELB-16 14 95 1 S/m 6 2.7 32 40 NA 1

Lake watercolumn

East Lake Bonney,Taylor Valley

ELB-19 20 114 10 S/m 5.5 2.6 38 40 NA 1

Lake water

column

East Lake Bonney,

Taylor Valley

ELB-25 4 144 11 S/m 4 2.0 27 20 NA 1

Lake water

column

West Lake Bonney,

Taylor Valley

WLB-13 9 28 1 S/m 3 2.1 15 24 NA 1

Lake water

column

West Lake Bonney,

Taylor Valley

WLB-16 16 57 8 S/m 0 2.4 104 52 NA 1

Glacier ice Blood Falls, Taylor

Valley

BF 16 81 NA NA 1.75 30 27 NA 2

Lake ice Lake Vida, Victoria

Valley

LVI-4–8 62 135 NA −25 NA 133 NA 63 3

Lake ice Lake Vida, Victoria

Valley

LVI-15_9 34 181 NA −11.6 NA 48 NA 83 3

Lake sediment Organic Lake,

Vestfold Hills

ORG-S 11 NA 21 psu −6 - -8 1.01 32 NA 91 4

Lake sediment Burton Lake,

Vestfold Hills

BUR-S 51 NA 43 psu −1.8 NA NA NA NA 5

Lake sediment Ace Lake,

Vestfold Hills

ACE-S 37 NA 35 psu 2.5 NA NA NA NA 5

Sea ice Arctic sea ice ARK-I_1 40 106 NA NA 1.09 NA NA 74 6

Sea ice Antarctic sea ice,

Weddell Sea

ANT-I 16 103 NA NA 0.84 NA NA 85 6

Marine sediment Svalbard SVA 68 353 NA 3 NA NA NA 72 7

Spring water Gypsum Hill,

Canadian Arctic

GH 46 155 7.9 psu 6.3 3.17 71 NA 84 8

Spring water Color Peak,

Canadian Arctic

CP 30 174 15.5 psu 5.7 2.16 50 NA 91 8

Oil sand tailings

pond

Canadian arctic OSTP 42 NA NA NA NA NA NA NA —

Biodegraded oil

reservoir

Canadian arctic PL 53 NA 3 psu 20 NA NA NA NA 9

Oil contaminated

soil

Romania PIT 99 NA NA NA NA NA NA NA —

Deep anoxic

brine

L’Atalante Basin,

Mediterreanean

ABBB 41 NA 358 psu 13.9 3.26 NA NA NA 10

Deep anoxic

brine

Bannok Basin,

Mediterranean

BBBB 43 NA 322 psu 14.7 3.2 NA NA NA 10

Deep anoxic

brine

Discovery Basin,

Mediterreanean

DBBB 49 NA 470 psu 14.5 3.5 NA NA NA 10

Deep anoxic

brine

Urania Basin,

Mediterranean

UBBB 17 NA 237 psu 16.7 2.17 NA NA NA 10

Seawater Arthur Harbor,

Antarctic

Peninsula

ANT-P 52 NA 33.5 psu −1 N/A NA NA NA 11

Select environmental and diversity-related parameters are shown. NA, not analyzed. GenBank accession numbers for studies not published include OSTP:

EF420191.1–EF420233.1; EU517535.1–EU517561.1. and PIT: DQ366084.1–DQ378273.1.






1. Glatz RE, Lepp PW, Ward BB, Francis CA (2006) Planktonic microbial community composition across steep physical/chemical gradients in permanently ice-covered Lake Bonney,

Antarctica. Geobiology 4(5):53–67.

2. Mikucki JA, Priscu JC (2007) Bacterial diversity associated with Blood Falls, a subglacial outflow from the Taylor Glacier, Antarctica. Appl Environ Microbiol 73(12):4029–4039.

3. Mosier AC, Murray AE, Fritsen CH (2007) Microbiota within the perennial ice cover of Lake Vida, Antarctica. FEMS Microbiol Ecol 59(2):274–288.

4. Bowman JP, McCammon SA, Rea SM, McMeekin TA (2000) The microbial composition of three limnologically disparate hypersaline Antarctic lakes. FEMS Microbiol Lett 183(1):81–88.

5. Bowman JP, Rea SM, McCammon SA, McMeekin TA (2000) Diversity and community structure within anoxic sediment from marine salinity meromictic lakes and a coastal meromictic

marine basin, Vestfold Hilds, Eastern Antarctica. Environ Microbiol 2(2):227–237.

6. Brinkmeyer R, et al. (2003) Diversity and structure of bacterial communities in Arctic versus Antarctic pack ice. Appl Environ Microbiol 69(11):6610–6619.

7. Ravenschlag K, Sahm K, Pernthaler J, Amann R (1999) High bacterial diversity in permanently cold marine sediments. Appl Environ Microbiol 65(9):3982–3989.

8. Perreault NN, Andersen DT, Pollard WH, Greer CW, Whyte LG (2007) Characterization of the prokaryotic diversity in cold saline perennial springs of the Canadian high Arctic. Appl

Environ Microbiol 73(5):1532–1543.

9. Grabowski A, Blanchet D, Jeanthon C (2005) Characterization of long-chain fatty-acid-degrading syntrophic associations from a biodegraded oil reservoir. Res Microbiol 156(7):

814–821.

10. van der Wielen PW, et al.; BioDeep Scientific Party (2005) The enigma of prokaryotic life in deep hypersaline anoxic basins. Science 307(5706):121–123.

11. Murray AE, Grzymski JJ (2007) Diversity and genomics of Antarctic marine micro-organisms. Philos Trans R Soc Lond B Biol Sci 362(1488):2259–2271.




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