MCM LTER Routine Limnological Sampling during Polar Night season

Physical Parameters
ICE: Lake Ice Thickness and Piezometric Lake Depth

Biological Parameters
CHL: Chlorophyll-a Extraction
PHYTO: Phytoplankton Enumeration and Biomass
PPR: Primary Production Determined by 14C Incorporation
BAC: Bacteria Enumeration and Biomass
THY/LEU: Bacterial Production Determined by 3H Thymidine and Leucine
                  Incorporation

Chemical Parameters
PH: Hydrogen Ion Concentration
DO: Dissolved Oxygen by Winkler Titration
DIC: Dissolved Inorganic Carbon
CN: Particulate Organic Carbon and Nitrogen
PP: Total Particulate Phosphorus
NUT: Dissolved Nutrients (NO3, NO2, NH4, soluble reactive phosphorus)
DOC/TN: Dissolved Organic Carbon and Total Nitrogen
AN/CAT: Anions and Cations

Profiling Instruments
CTD: Seabird 25 Conductivity, Temperature, and Depth
Fluoroprobe: Underwater Spectral Fluorometer
PNF: Biospherical Profiling Natural Fluorescence
PAR Logged: Underwater Time Series -Phototsynthetically Active Radiation Logged
                      During Primary Production Experiment (dates noted below are ending
                     dates of 24 hour logging period)
PAR Profile: Underwater Profile - Phototsynthetically Active Radiation and
                    Attenuation

•  For a description of methods see "Limnological Methods" on Data page.

Physical, Biological and Chemical Parameters

Profiling Instruments

Principal Investigators

Dr. Mike Lizotte, University of Wisconsin Oshkosh

Photophysiological studies were performed in Dry Valley lakes to derive parameters for use in primary production models, and to determine physiological changes in the algae as they responded to decreasing light.

• PE: Photosynthesis-irradiance experiments
• ap: Phytoplankton absorption
• Chl: Chlorophyll
• Spectroradiometer: Measures the spectrum of light (300-900nm) underwater and at
   the surface.

Dr. Jill Mikucki, Dartmouth College

Chemoautotrophy during POLAR NIGHT
Genomic and substrate incorporation experiments were designed to address the hypothesis that chemoautotrophic bacterial production provides a source of new carbon to the ecosystem during polar night. These experiments included Stable Isotope Probing (SIP) using 13C substrates to look at active microbial populations, Microautoradiography-Flourescent In Situ Hybridiation (MAR_FISH) to explore structure function relationships of individual bacterial cells and their carbon substrate choice, and lastly the construction of functional gene clone libraries to look for the genes that mediate specific carbon assimilation pathways. Incubations were performed and samples were collected and preserved while in the field. Samples will be analyzed at Jill's home institution.

Experiments
• Stable isotope probing incubations using 13C substrates (acetate, glucose and bicarbonate) on samples from WLB (15m, 20m, 25m), ELB (13m, 20m) and FRX (9m, 11m)
• Bulk sample collection for RNA/DNA analyses from WLB (15m, 20m, 25m), ELB (13m, 20m) and FRX (9m, 11m) throughout season
• Sulfate samples (for S and O natural isotope abundance measurements); profiles collected from WLB, ELB, FRX, HOR, VAN
• Incubations for Microautoradiography/FISH (Light Dark –Bicarbonate and Dark – Glucose, Tdr, Leu, Acetate) on WLB 15m
• 14C-Bicarbonate incorporation experiments for WLB 15m (Light/Dark; + Phosphate; 3.0΅m filtered) at 72h and 96h.

Dr. Rachael Morgan-Kiss, Miami University

Collections
 

Date	Lake	Depth (m)	Process
24 Feb 08	West Bonney	5,13,15,20	Background filtration for pigments, lipids and molecular assays
25 Feb 08	East Bonney	5,13,18,20	Background filtration for pigments, lipids and molecular assays
2 Mar 08	West Bonney	6,13,15,20	Background filtration for pigments, lipids and molecular assays
2 Mar 08	East Bonney	6,13,18,20	Background filtration for pigments, lipids and molecular assays
9 Mar 08	West Bonney	6,13,15,20	Background filtration for pigments, lipids and molecular assays
9 Mar 08	East Bonney	6,13,18,20	Background filtration for pigments, lipids and molecular assays
16 Mar 08	West Bonney	6,13,15,20	Background filtration for pigments, lipids and molecular assays
16 Mar 08	East Bonney	6,13,18,20	Background filtration for pigments, lipids and molecular assays
24 Mar 08	West Bonney	6,13,15,20	Background filtration for pigments, lipids and molecular assays
24 Mar 08	East Bonney	6,13,18,20	Background filtration for pigments, lipids and molecular assays
29 Mar 08	East Bonney	6,13,18,20	Background filtration for pigments, lipids and molecular assays
30 Mar 08	West Bonney	6,13,15,20	Background filtration for pigments, lipids and molecular assays
10 Apr 08	East Bonney	6,13,18,20	Background filtration for pigments, lipids and molecular assays

Experiment

Dr. John Priscu, Montana State University

Nutrient Bioassay
A phytoplankton nutrient bioassay experiment was conducted on the shallow and deep chlorophyll layers in ELB and WLB and at 7 m in Fryxell. Samples were collected on 2 March 2008.

DOC Characterization
A submersible UV spectrofluorometer was used to characterize DOC in ELB and WLB on 25 and 26 February, respectively.

Don Juan Pond substrate incorporation
Experiments based on substrate incorporation were conducted on Don Juan Pond were processed in March 2008. The experiments were started in December 2007.

Collaborating Scientists

Dr. Elanor Bell, Dunstaffnage Marine Laboratory

Viral studies in Dry Valley Lakes
Viral and bacterial abundance and productivity at 4 depths in the East and West lobes of Lake Bonney were followed to determine the response of viral-bacterial interactions to decreasing light.

•  SYBR Gold nucleic acid staining of viral and bacterial cells and enumeration under epifluorescence microscopy
•  Viral lysogeny determined during 24 hour incubations of lake water. Induction using Mitomycin C.
•  Viral lysis determined during 24 hour incubations of concentrated bacteria in virus-free lake water

The primary objective of this work was to monitor protist abundance and grazing within the lakes throughout the transition period from 24 hour light to 24 hour darkness. Of particular interest was the phytoflagellate community. It was hypothesized that grazing by protist groups capable of mixotrophy, such as the cryptophytes, would be up-regulated as the light was lost. Grazing was monitored through use of heat-killed bacteria, live bacteria and uptake of fluorescent dextrans.

Classic Experiments
• Abundance/Biovolume:
        Bacterial abundance (SYBR gold stained)
        Bacterial biovolume (DAPI stained)
        PNAN(Photosynthetic Nanoflagellate)/HNAN (Heterotrophic Nanoflagellate)
        abundance (DAPI stained)
        PNAN/HNAN biovolume (DAPI stained)
        PNAN/HNAN identification (Glut fixed for SEM)
        Ciliate identification (Lugols Iodine fixed)

• DTAF grazing:
        Grazing experiments using DTAF bacteria at 20% indigenous bacterial
        concentration. Performed at 2^oC and at relevant PAR value from field
        data. Fixed at 5, 10, 20, 30, 40, 50 and 60 min. Average No of prey per
        PNANs and HNANs over time to yield grazing rates.

• Dextran uptake:
        Dextrans (4, 9, 38 and 580Kda) added at 1% final concentration and
        fixed after 60 min. % population of PNAN and HNAN taking them up.

• Prey selection:
        Live GMOs (genetically modified organisms) added in couples – Blue and
       Green (each at 10% indigenous bacterial conc.) and Blue and Yellow (each
       at 10% indigenous bacterial conc.). Performed at 2^oC and at relevant PAR
       value from field data. Fixed at 5, 10, 20, 30, 40 min. Average No of each
       coloured prey per PNANs and HNANs over time to yield grazing rates.
       Grazing rates on these live bacteria compared to dead DTAF bacteria also
        possible.

 • Algal diversity:
        1L water aseptically filtered onto 47mm SuporR200 filters with a pore size of
        0.2΅m. Filters dried in desiccator, placed in eppendorfs and stored frozen (
       -20^oC). Transport to Centre of Ecology and Hydrology at 4-10^oC.  Molecular
        analysis of algal community using DGGE.

• Phytoplankton isolation:
        Water diluted and incubated at different light intensities and temperatures. All
        collected at the end of the period, transferred to eppendorfs for transport to
        Lancaster.

Dr. Steve Jepsen, NASA Jet Propulsion Laboratory (JPL)

Lake Fryxell Ice Sediment Melt Experiment
A 1 cm thick layer of sediment was put onto the surface of Lake Fryxell in a 1.2 x 0.9 m plot. The sediment was allowed to melt into the ice, and measurements of sediment depth and pieziometric lake water level relative to the upper ice table were taken every 2-4 weeks between 10 Nov 07 and mid-Jan 08.

Graduate and Post-Graduate Students

Chao Tang, PhD student, University of California, Riverside & Microbial Observatory (MO)

Lake water samples were collected at both MCM-LTER and Microbial Observatory depths and included:
DNA/RNA filters: supor-200 0.2΅m filters
FISH filters: 47mm 0.2΅m polycarbonate filters
DAPI filters: 25mm 0.2΅m polycarbonate filters black
Frozen stock: 4.5ml+500ml DMSO

Microbial Observatory depths in above chart include:
Fryxell: 6, 9, 12, 18 m
East Bonney: 6, 13, 25, 37 m
West Bonney: 6, 13, 18, 38 m
Vanda: 4.5, 45 m

• 1 sediment core was collected from Don Juan Pond, dissected and stored at -80 C.

Suni Shah, Post-Doc, Harvard University

Archaeal lipids in lake water samples
Lake water samples were collected from 3 lakes and filtered onto 47mm GFF filters to be extracted for archaeal lipids at Harvard University. 

Tristy Vick, PhD student, University of Nevada, Las Vegas

Amino acids in lake water samples
Lake water samples were collected from 4 lakes for amino acid analysis. 40 ml of sample were filtered through 0.2 ΅m 25 mm nylon membrane filters. Filters were dried and stored at room temperature. Filtrate was stored at -20C. All were shipped to the Desert Research Institute in Las Vegas at -20C.

MIRADA: Microbial Inventory Research Across Diverse Aquatic LTERs. 
Samples were collected for the MIRADA project as part of their Microbial Biodiversity Survey and Inventory across all the major aquatic (marine and freshwater) Long Term Ecological Research (LTER) sites.  Sampling involved sterile collection of 1 or more liters of water followed by processing of the samples onto 0.22 ΅m sterivex filters for DNA extraction, filtration of a portion of the sample onto membrane filters to provide an archive sample for future Fluorescence In Situ Hybridization (FISH), and archival cryopreservation of a portion of the samples in both DMSO and glycerol for future culturing.

IPY Polar Night 2008 Project Page