a National Science Foundation Engineering Research Center in the MSU College of Engineering

Center for Biofilm Engineering

Cryoconite holes are miniature aquatic ecosystems on the surface

of glaciers worldwide (Figure 1). Cryoconites form due to

preferential melting around low-albedo aeolian particles, which

proceed to sink below the surface until finally reaching their

equilibrium depth in the ice. Organic windblown sediment called

cryoconite, meaning ‘cold dust,’ collects at the bottom of these

water-filled holes. Past work has shown that cryoconite holes can

support a diverse microbial ecosystem that sustains itself despite

the extreme environmental characteristics. The focus of this study

are the biologically active aggregations of microbes associated with

cryoconite found on the Canada Glacier of the McMurdo Dry

Valleys, Antarctica.

Figure 1. Image of McMurdo Dry Valleys cryoconite hole with inset image

showing the upper frozen water (A) and lower sediment containing

portions (B).

Figure 4. CLSM imaging of the post-stepwise thermo-gravimetric samples

demonstrates the selective binding of SYBR Green nucleic acid stain to

cellular material and not unspecific staining of sediment particles.

Figure 2. CLSM image of microbial community associated with cryoconite

sediments. Red= autofluorescent cells and green= SYBR Green stained

cells. Scale bar= 20mm.

• Determine metabolic processes used by cryoconite microbial

community members to obtain nitrogen. It is unknown if the

Cyanobacteria present in cryoconites have the genetic machinery

necessary to fix nitrogen or if they rely on the presence of organic

nitrogen. PCR methods will be used with specific primers designed to

target genes involved in different steps of the nitrogen cycle.

• Calculate carbon fixation and nitrogen uptake rates for Oscillatoriales

sp., as well as calculate nitrogen uptake rates for a dominant

microbial guild of Bacteroidetes sp..

Microbial Diversity and Ecophysiology of Cryoconite Sediments from the McMurdo Dry Valleys, Antarctica

A. Schmit1, H. Smith1, B. Pitts1, R. Foster2, C. Foreman1 1Montana State University, 2Max-Planck Institute for Marine Microbiology

METHODS

Cryoconite samples were collected during the austral summer of

2010 using a Kovacs Mark II coring system from the Canada

Glacier of the McMurdo Dry Valleys, Antarctica. Cores were placed

into sterile Whirl-Pak® bags and then frozen at -20ºC for shipment

to Montana State University.

A Leica confocal laser scanning microscope (CLSM) was used to

image the cryoconite sediments and the biotic material. Cryoconite

sediment was thawed and placed into a petri dish. SYBR Green

stain (final concentration 40X) was placed on the sample and

allowed to sit, covered from light, for 30 minutes. Using a sterile

pipette, the stain was removed and the sample rinsed with DI water.

A 60X water immersion objective was used to image the cryoconite

material. Images of SYBR Green stained microbes,

autofluorescence, and a reflection of the granule itself, were

overlaid to produce the images shown to the right.

Community composition was examined using a combination of 454

pyrosequencing and catalyzed reporter deposition (CARD)-

fluorescence in situ hybridization (FISH). Pyrosequencing was

performed using barcoded amplicons of 16s SSU rRNA. Analysis of

454 data was performed using Qiime. Quality control of sequences

was implemented using the Qiime workflow, with sequences

trimmed to a minimum length of 350bp. Reference sequences were

selected and aligned using the UCLUST algorithm via the Qiime

interface and were compared to the RDP database for closest

sequence identity. Standard protocols were followed for CARD-

FISH analysis (Pernthaler, et al. 2002), with samples dipped in

agarose (1%[wt/vol]) to avoid cell loss during permeabilization.

The sediments were analyzed using powder X-ray diffraction

(XRD). Spectral profiles of the sediments were used to determine

the identification of the crystalline material. Sediments were

subject to a stepwise thermo-gravimetric analysis. Three 500mg

sub-samples of cryoconite sediment were dried for three days at

38ºC. The samples were weighed and placed in a combustion oven

at four different temperatures (105ºC, 200ºC, 350ºC, and 520ºC) for

four hours each. The sediment was weighed after each

temperature step in order to determine the percentage of organic

matter remaining. The results were averaged between the three

samples.

Figure 5. CARD-FISH image of sediments using probes CF319a//CF319b

targeting Bacteroidetes sp. associated with a filamentous Oscillatoriales

sp.

Figure 3. CLSM image of microbial community associated with

cryoconite sediments. Red= autofluorescent cells and green= SYBR

Green stained cells. Scale bar= 20mm.

• CLSM of individual sediment grains confirmed the association of

microbial populations with sediment surfaces. Bacterial cell abundance

was 7.26x105 cells ml-1 of sediment slurry. Based on 454

Pyrosequencing the dominant microbial assemblages for the analyzed

Canada Glacier cryoconite are from the Cyanobacteria, Actinobacteria,

Bacteroidetes, Betaproteobacteria, and Alphaproteobacteria lineages.

• Bulk organic matter content and composition determined using step-

wise thermogravimetric analysis found organic matter was 7.7% of the

dry weight, with the greatest mass loss (4.3%) occurring between

200°C and 350°C, indicating that the majority of organic matter

present is thermolabile and dominated by carbohydrates.

• Powder XRD analysis determined that granule composition is relatively

homogenous with a dominance of silica oxides (primarily quartz).

• Cryoconite sediments are important reservoirs of organic carbon,

nutrients, and microbial activity on glacial surfaces, where resources

are sparse.

• Cryoconites accumulate windblown sediment, which promotes the

aggregation of microorganisms to granule surfaces providing a refuge

for microbial life in these harsh environments.

RESULTS INTRODUCTION

CONCLUSIONS

FUTURE WORK

ACKNOWLEDGEMENTS

1. SiO2 - Quartz

2. KAl2(AlSi3O10)(F,OH)2- Muscovite

3. KAlSi3O8 - Orthoclase

4. CaCO3 – Calcite

5. Mg2Al4Si5O18 – Cordierite

1.

1.

1.

1.

1.

2.

3.

3. 3. 2.

4.

4.

4. 4.

2.

5.

5. 5. 1.

1.

2.

3.

2θ (degrees)

counts

per

second

Figure 7. Powder XRD spectral profile of cryoconite sediments, used to

illustrate the interpretation of present constituents. Cryoconite sediment

diffraction patterns suggest a dominance of silicate materials, specifically

quartz and corresponding weathering products.

Funding for this project came from the National Science Foundation

(OPP-0838970 and DGE 0654336). Logistical support was provided by

Raytheon Polar Services and Petroleum Helicopters Incorporated. We

would to thank ICAL for use of the SEM and P. Dirckx for her graphics

assistance.

Table 1. Triplicate thermogravimetric analysis of organic matter (as a

percentage of dry weight) from cryoconite sediments, and characterization

of organic matter present (Kristensen, 1990). Total organic matter

accounted for 7.7% of the cryoconite dry weight.

Figure 6. Comparison of the relative abundance of dominant cryoconite organisms using different methods. A.) CARD-FISH using probes ALF968/ALF1B,

BETA42a, GAM42a, CF319a/CF319b B.) 454 Pyrosequencing. Both methods show similarities in the dominant community members, except for

Bacteroidetes, which was identified in greater abundance using CARD-FISH.

A. B.

Temperature Sample Average of

% Dry Weight Classification Description

38-105°C 0.49 ± 0.73

Loss of Water

Crystalline lattice water,

and hygroscopic water

of salts and organic

matter 105-200°C 4.3 ± 2.08

200-350°C 2.19 ± 1.02 Thermolabile Dominated by

carbohydrates

350-520°C 0.76 ± 0.80 Stable Organic Matter

Oxidation of aromatic

groups (lignin, humic

substances, kerogens)

and char

References Cited:

Kristensen, E. 1990. Biogeochemistry

Pernthaler, A., et al. 2002. Applied and Environmental Microbiology