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

Center for Biofilm Engineering

On the surface of glaciers and the ablation zone of ice sheets

worldwide aquatic miniature ecosystems can be found in the form

of cryoconite holes. Cryoconite holes form when debris resting on

the frozen surface gradually melts to an equilibrium depth in the ice.

As such, they form a cylindrical depression filled with water and

substrates. Intensive research has shown that cryoconite holes

host a diverse mixture of autotrophic and heterotrophic community

members capable of carbon production and transformation, and

molecular evidence suggests the potential of a microbially mediated

nitrogen cycle. The current investigation focuses on the nitrogen

cycle within these aquatic ecosystems using culture dependent and

culture independent assays. Cryoconite samples were obtained

from Canada Glacier, McMurdo Dry Valleys, Antarctica, and

brought back to Montana State University. To enrich for microbes

involved in nitrogen fixation and nitrification, cryoconite sediments

were suspended in selective growth media and incubated at 4°C

and 15°C, respectively. Bacteria with 100% sequence similarity to

Mesorhizobium sp. were isolated, indicating the presence of highly

specific nitrogen-fixing bacteria in the cryoconite sample. Ammonia

oxidation and subsequent nitrate production was observed with

nitrate production rates estimated at approximately 0.15µM day-1.

PCR amplicons (nifH, narG, nirS, and amoA) migrated into a band

of the correct size in the agarose gel and verified by comparison

to a standard molecular weight DNA marker. Further sequence

analyses, however, are required for complete phylogenetic

characterization of the genes. Our biogeochemical, culture

dependent, and molecular evidence suggest the potential of an

active nitrogen cycle in cryoconite holes.

ABSTRACT

I would like to thank to the Foreman Lab Group for being excellent

to work with. Dr. Christine Foreman and the Undergraduate

Scholars Program for funding. The Center for Biofilm Engineering

for hosting me. Betsey Pitts for her expertise on the CLSM. My

mother, Laura Reckrodt.

Figure 1. Location of Canada

Glacier in the Taylor Valley,

Antarctica. A) Map of

Antarctica. Insert shows the

position of the Dry Valleys. B)

Canada Glacier. C) Cryoconite

hole.

Figure 5. The nitrogen cycle and associated phases of the cycle. Genetic

operons responsible for coding metabolic enzymes associated with the

nitrogen cycle are shown next to their respective steps. Genes that have

been PCR amplified are shown in green boxes.

Isolation of Nitrogen Fixing Organisms

A 700bp sequence had a 100% match to Mesorhizobium sp. based

on NCBI Blast search. The RDP Classifier and the SILVA database

confirmed this result. Mesorhizobium ciceri biovar biserrulae was

originally isolated from a pasture legume that forms a highly

specific nitrogen-fixing symbiotic interaction with Mesorhizobium.

Nitrogen Cycling in Cryoconites from the Canada Glacier

in the McMurdo Dry Valleys, Antarctica Amber Schmit1,2, Heidi Smith2,3, Markus Dieser2, Christine Foreman1,4

(1)Chemical and Biological Engineering (2) Center for Biofilm Engineering, (3) Department of Land Resources and Environmental Sciences

April 15, 2014

RESULTS DISCUSSION

CONCLUSIONS

ACKNOWLEDGMENTS

INTRODUCTION

Microorganisms play a crucial role in nutrient cycling in glacial

environments. Our study specifically addressed the cycling of

nitrogen within cryoconite holes. A nitrogen fixing bacterium in the

genus Mesorhizobium was isolated. Nitrate production

measurements provided direct evidence of nitrification.

Figure 5 shows a diagram of the nitrogen cycle and genes

associated with each step of the process (anaerobic ammonium

oxidation is not shown). Highlighted are the genes that were

targeted by PCR amplification:

• nifH: nitrogenase reductase gene subunit H

• amoA: ammonia monooxygenase gene subunit A

• narG: nitrate reductase gene subunit G

• nirS: nitrite reductase gene subunit S

These preliminary results are in agreement with previous findings

on Arctic and Antarctic cryoconite holes (Cameron 2012, Telling

2012). We hypothesize that the microbial community has the

genetic potential for maintaining nitrogen cycling in cryoconite holes

on the Canada Glacier of the McMurdo Dry Valleys, Antarctica.

Figure 2. A) Sketch of cryoconite formation modified from Bagshaw, et al

2013. B) Nutrient cycling within cryoconite holes modified from Stibal and

Tranter, 2007. C) A CLSM image of the microbial community associated with

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

stained cells. Scale bar= 20mm.

A

B

C

Figure 4. Nitrate production by ammonia oxidizing bacteria in the

cryoconite sediments.

Figure 3. Phylogenetic analysis of 16S rRNA gene sequences by

Maximum Likelihood method using MEGA6 with the Jukes-Cantor

correction model. Bootstrap values ≥50% using 1000 replications are

shown. Scale bar represents 2% sequence divergence. Cryoconite

isolate is indicated by a solid circle(●) and bold lettering. Accession

numbers are shown in parentheses. Tree was rooted to Nitrosomonas

communis.

INTRODUCTION

METHODS

Isolation of Nitrogen Fixing Organisms

• Cryoconite sample (100 µL) was spread on agar plates

containing rhizobium media and incubated at 4°C

• After two months colonies appeared which were transferred onto

new media plates for further isolation

• DNA was extracted (MoBio Power Soil Extraction Kit) from

isolates and amplified using 16S rRNA gene primers (9F and

1492R)

• 16S amplicon sequenced unidirectional through Functional

Biosciences

• Sequences were processed using BioEdit. Closest neighbors

were identified in NCBI Blast. Sina Aligner was used to align the

sequences. Alignments were trimmed and phylogenetic trees

were constructed using MEGA6.

Nitrate Production

• Cryoconite sediments (200mg) were enriched in R2A media at

15°C for one week

• Ammonia oxidizing media was inoculated with 1mL of R2A

cryoconite enrichment. An autoclaved cryoconite sample in

ammonia oxidizing media served as an abiotic control.

• Subsamples of 1 mL were taken weekly and stored at -80°C

• NO3- concentrations were analyzed on a ion chromatograph

PCR Amplification

• MoBio Power Soil Extraction kit was used to extract DNA from

cryoconite sediment samples (~250mg)

• PCR amplification was performed targeting the following genes

involved in the N-Cycle: amoA, nifH, nirS, and narG.

• PCR reactions contained the 5’ Master Mix Taq polymerase and

10pmol of each primer. Annealing temperatures were optimized

for each primer.

• PCR amplicons were visualized by electrophoresis on a 1%

agarose gel in a 1X TAE buffer stained with gel red.

• The isolation of Mesorhizobium suggests that organisms other

than cyanobacteria (heterocyst) are involved in nitrogen fixation

within cryoconite holes. However, the genus Mesorhizobium are

known to fix nitrogen by forming symbiotic relationships with root

nodules; thus, further phenotypical characterization is required.

• Observed nitrate production and amplification of the amoA gene

suggests that nitrification occurs in cryoconite communities.

• Amplifcation of genes involved in nitrogen fixation, nitrification,

and denitrification suggests the potential for complete nitrogen

cycling within the cryoconite.

Nitrate Production

At t=0 NO3- concentrations of both the cryoconite sample and

control were 0.07mg L-1. After ~50 days of incubation at 15°C the

NO3- concentration in the cryoconite sample increased to 0.54 mg

L-1 and were significantly different from the control (one way

ANOVA, p-value <0.001). NO3- production increased linearly

(R2=0.967) and a rate of 0.15 µM NO3- day-1 was calculated.

PCR Amplification

The expected base pair size of PCR amplicons was verified by

comparison to a standard molecular weight DNA marker. All four

gene amplicons (nifH, amoA, narG, nirS) migrated into a band of

the correct size in the agarose gel. Further sequence analyses are

required for complete phylogenetic characterization of the genes.

A

B

C

The Dry Valleys are the largest relatively ice-free regions of

Antarctica (~2%) with a mean annual temperature of -19°C. The

Dry Valleys are a polar desert with an annual mean precipitation of

~10 cm and strong katabatic winds (up to 320 km/h). Perennially

ice covered lakes, seasonal meltwater streams, soils, and glaciers

provide habitats for microorganisms with a roundworm (nematode)

at the top of the food chain in soils.

Cryoconite holes are one type of seasonal habitats and are local

hotspots for biological activity. Based on previous research, Figure

2B shows potential major element cycles within cryoconite holes.

Carbon is cycled through photosynthesis and respiration. Direct

metabolic evidence supports the presence of nitrogen fixation and

ammonification, and molecular analyses have identified functional

genes involved in the nitrogen cycle. A metagenomic study also

revealed genes associated with sulfur, iron, and phosphorus cycles.

Bagshaw E, et al. 2014. Do Cryoconite Holes have the Potential to be Significant Sources of C, N, and P to

Downstream Depauperate Ecosystems of Taylor Valley, Antarctica? Arctic, Antarctic, and Alpine Research,

45(4):440-454.

Cameron K, et al. 2012. Carbon and nitrogen biogeochemical cycling potentials of supraglacial cryoconite

communities. Polar Biology 35:1375–1393

Telling J, et al. 2012. Microbial nitrogen cycling on the Greenland Ice Sheet. Biogeosciences 9:2431–2442

Stibal M, Tranter M. 2007. Laboratory investigation of inorganic carbon uptake by cryoconite debris from

Werenskioldbreen, Svalbard. Journal of Geophysical Research 112:G04S33. Pg 1-9

REFERENCES

Time (Days)

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Cryoconite Sediment

Control