Taylor Glacier Blood Falls Geomicrobiology

Sample Collection Samples were collected from subglacial outflow waters associated with Blood Falls discharge during austral summers from 1999-2003. Samples near the outlet of Blood Falls were collected for enumeration and bacterial activity in December of 2000 and 2001. A transect of samples (TransX) was collected in December (see Figure 1), 2002 to capture a profile of releases from Blood Falls into Lake Bonney, starting at the source of the outflow (Trans1) and out onto the moat ice surrounding the lake with increasing distance from the source (Trans2-5). Trans2 was collected from basal ice below the source, Trans3 was collected from waters at the shoreline where Blood Falls meets the moat ice, Trans4 was collected 5 m from the shoreline just below moat ice that melted and refroze daily, and Trans5 was collected under 0.4 m of moat ice approximately 70 m from the shoreline before the moat ice meets the permanent ice cover of Lake Bonney. Autoclaved metal spatulas were used to scrape ice samples into sterile plastic bags (Whirl-Pak) or sterile plastic conical tubes (Falcon). Subsequent manipulations were carried out aseptically in a positive flow hood. Subglacial flow water was collected in clean Nalgene bottles for geochemical analysis. All ice samples were melted slowly at 2-4 °C in sterile containers before analysis. Samples for analysis of dissolved organic carbon were collected in acid-washed, combusted (450 °C for > 4h) amber glass bottles. Dissolved inorganic carbon samples were collected in gas tight glass serum vials. Samples collected for bacterial enumeration were treated with formalin (2% final concentration) to prevent a change in bacterial numbers during storage (~ 2 weeks) before counting Geochemistry The pH was measured with a calibrated Beckman Φ12 pH meter. Eh was measured using a Hach ORP combination electrode (Hach Company, Loveland, CO) connected to a Beckman Φ12 digital voltmeter, with corrections for the reference electrode made in accordance with the ORP electrode instruction manual. Dissolved oxygen (DO) was determined by Winkler titration (Standard Methods, 1992). Dissolved inorganic carbon (DIC) was measured by infrared gas analysis on acid sparged samples. Dissolved organic carbon (DOC) was measured on filtered (25mm Whatman GF/F), acidified (HCl to pH~2-3) samples with a Shimadzu 5000 TOC analyzer. Nitrate (NO3-) and nitrite (NO2-) concentrations in water samples were measured with a Lachat autoanalyzer according to Standard Methods (1992). Nitrite levels were determined by diazotization, with sulfanilamide, and then coupled with a diamine to produce a pink dye, analyzed spectrophometrically on a Lachat autoanalyzer. Nitrate was reduced to nitrite by passing the sample through a copperized cadmium column and analyzing for nitrite, as above, giving a nitrate plus nitrite concentration. The nitrate concentration was determined by subtracting the nitrite concentration from the total (nitrate + nitrite) concentration. Ammonium (NH4+) concentrations were measured separately by reaction with alkaline phenol followed by sodium hypochlorite, forming indophenol blue. Sodium nitroprusside was added to enhance sensitivity and the sample was analyzed spectrophometrically on a Lachat autoanalyzer. Dissolved inorganic nitrogen (DIN) was reported as the summation of nitrate, nitrite and ammonium (DIN = NO3-+ NO2-+ NH4+). Soluble reactive phosphorus (SRP) was determined on 10 ml samples with the antimony- molybdate method (Standard Methods, 1992), absorbance was read in a 10 cm cell with a Beckman DU-640 spectrophotometer. Chlorophyll-a (CHL) was extracted into 90% acetone for 24 h at <0 °C in the dark from particles collected on a Whatman GF/F filter, and the subsequent concentration was determined fluorometrically Conductivity was measured at 1-meter depth intervals in the west lobe of Lake Bonney using a Seabird SBE-25 conductivity temperature depth (CTD) sensor (Sea-Bird Electronics, Inc., Bellevue, Washington). Salinity was calculated using the UNESCO salinity algorithm available in the Seabird data acquisition software package (Sea-Bird Electronics, 1998). Modifications for the high salinity concentrations found below the halocline of Lake Bonney are described in Spigel and Priscu (1996). Total iron was determined using the ferrozine assay (Fulton et al., 2004). Microbial Activity Heterotrophic bacterial activity was measured using 3H-thymidine (20 nM) and 3H-leucine (20 nM) incorporation according to the methods described in Takacs and Priscu (1998). Formalin (5% final concentration)-treated replicates served as killed controls. Samples were incubated in the dark at 0-1 °C. Incubation was terminated by the addition of cold trichloroacetic acid (TCA) to a final concentration of 5%. The effect of temperature on the productivity of the natural bacterial assemblages from Blood Falls outflow was determined by incubating triplicate samples (10 mL) with 10 nM 3H-leucine for 24 h at varying temperatures using the methods described above. Doubling rates were calculated from the 3H-leucine incorporation data. Culture work Samples were collected for culture work during the austral summers (October – January) between 2001 and 2004 (Table 4.1). Outflow was collected directly into 74 mL serum vials by submerging the vials in the flow and crimp sealing them with a butyl rubber stopper with no head space. Serum vials were kept in the dark below 4 °C until return to McMurdo Station for incubation in selective media (approx 5-10 days). Media included R2A agar (Difco), Marine agar (Bacto) and thiosulfate-oxidizing agar (15%). Media are described in detail in Appendix C. Outflow sample (100 μl) was removed from the sealed serum vials with a 1 ml syringe, inoculated onto agar plates (15 g L-1), spread with a sterile cotton swab and incubated at 2-4o C until colonies appeared (about 3-4 weeks). Enrichments for autotrophic microbes were done in selective medium using the Hungate method (Hungate, 1969). Enrichments for iron-reduction were prepared by inoculating 0.5 ml of outflow collected as described above, into 4.5 ml mineral media that was prepared under CO2 gas and supplemented with Fe-OOH sludge (2 ml L-1), (Lovley and Phillips, 1986) and pressurized with H2 gas (30 psi), to a 0.7 M NaCl final concentration. Samples were serially diluted three times to a 10-3 extinction. DNA ExtractionSamples for environmental DNA extraction were collected directly from Blood Falls outflow during the 1999-00 austral summer (Table 4.1). Samples (1L) were kept chilled (below 4 °C) and filtered immediately upon returning to the field camp (~ 4 h). Cells were collected onto a 90 mm membrane filter, placed in a sterile plastic bag; heat sealed and stored at -20° C until extraction. Filters were cut into pieces using flame sterilized scissors and tweezers and then processed according to the manufacturer’s protocol (Mo Bio Laboratories, Inc. UltraClean Soil DNA Isolation Kit). The alternative protocol for maximum yields was followed. DNA extraction on a single isolated colony from each pure culture used the same methodology described above. 16S rDNA Clone Library ConstructionThe microbial 16S rDNA genes were amplified from extracted DNA using oligonucleotide primers 4F and 9F with 1492R (Table 4.2). Primers were purchased from Macromolecular Resources (Colorado State University). The 50-μl PCR reaction contained 5 μl DNA template, dNTPs nucleotide mix (Fisher Scientific, 200μM final concentration), bovine serum albumin (BSA) (0.5 μg/ μl), 0.5 μM final concentration of each primer, 10x Taq Buffer (1.5 mM Mg2+), 5x TaqMaster (a proprietary PCR enchancer, Eppendorf), and 1.25 units of Taq DNA polymerase (Eppendorf MasterTaq Kit). The reaction mixture was PCR amplified in an Eppendorf Mastercycler Gradient Thermal Cycler under conditions that included 15 sec of denaturation at 95o C, 45 sec of annealing at 55o C, and 1.5 min of primer extension at 72o C. These steps were repeated for 30 cycles. PCR products were purified using the QIAquick PCR purification kit (Qiagen) to remove primers, nucleotides, polymerases and salts. Purified PCR products were cloned using the pGEM-T Easy Vector System cloning kit with the pGEM-T Easy vector (Promega) according to the manufacturer’s protocol.