This study investigated the nitrogen cycle within cryoconite holes on the Canada Glacier in Antarctica. A bacterium capable of nitrogen fixation was isolated and identified as Mesorhizobium sp. Incubation of cryoconite sediments showed ammonia oxidation and nitrate production, providing evidence of nitrification. PCR amplification verified the presence of genes involved in nitrogen fixation, nitrification, and denitrification, suggesting the potential for a complete nitrogen cycle within cryoconite holes.

1. 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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NO3Concentration(mg/L)
0.0
0.1
0.2
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0.5
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Cryoconite Sediment
Control