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This project is funded by NSF grant 0732966
Our overall objective is to reach an understanding of the subzero microbial processes and their impacts on permafrost dynamics, including thermal degradation. We will achieve this objective through an interdisciplinary systems study based on combination of mechanistic mathematical modeling with experimental studies using modern molecular-genetic techniques, environmental physics, and biogeochemistry.
N. Panikov (Stevens Institute of Technology) is PI; M. M. Häggblom (Rutgers University), L. J. Kerkhof (Rutgers University), and T. Zhang (University of Colorado) are Co-PIs.
The International Polar Year (March 2007 through March 2009) is considered a milestone event in exploring polar regions and improving our understanding of their critical role in global processes. Contrary to the previous three International Polar Years, this event coincides with dramatic climatic changes, such as the frightening speed of sea ice melt and permafrost thaw and degradation, which affect the functioning of regional human and biotic communities as well as the entire global system. That is why the current IPY foci include not only geophysical aspects of the polar processes, but also biological and human perspectives, as well as their interactions and feedbacks, including non-linear responses resulting in abrupt changes.
This proposal is focused on the first of three emphasis areas dealing with "Understanding Environmental Change in Polar Regions." Specifically, this proposal deals with the recently discovered ability of microorganisms to metabolize in deeply frozen soil and subsoils cooled down to -40 degrees Celsius. Conventional biological wisdom fails to explain this natural phenomenon. By recognizing the existence of subzero microbial activity, we should reconsider the validity of several basic principles and concepts of polar biology and soil science. Long-term traditions that refer to summer-thawed top soil as "active layer" (in contrast to the "inactive layer" of underlying frozen ground) should now be considered as fundamentally incorrect, or merely a rough approximation. Some microorganisms are able to sustain growth and metabolic activity within the frozen layer, although they do so with lower intensity. We will explore whether subzero microbial activity is significant for understanding natural polar systems, their function under stable cold conditions and transiently under global climatic changes.
In this proposal, we plan to:
We believe that our study will improve predictive capabilities of models simulating permafrost and winter tundra dynamics. Our results can be used for optimal engineering design in northern regions that are subjected to warming and thermokarst erosion. In addition, the isolated psychroactive organisms can be used in biotechnological applications.
Our research will build upon our previous microbiological studies of psychrophiles in Alaska (NSF Microbial Observatory Projects) and northern Finland and will combine mechanistic study at one particular site (Toolik Lake, Smith Lake) with systematic collection of phylogenetic and subzero metabolic activity data in various types of permafrost and surface soils in Alaska, Siberia, Polar Ural, and Greenland.
These biological data will be correlated with physical and chemical characteristic of frozen ground, such as content of unfrozen water, mineral composition, quantity and quality of organic matter, texture, and ice content, to find the most essential environmental drivers and proxies for prediction of subzero biological activity. Along with statistical data analysis, we will conduct simulation modeling of carbon and energy flow in the permafrost microbial community to investigate the impact of microbial activity on permafrost warming and possible thawing. Finally, in laboratory experiments with intact frozen cores, we will examine the effect of artificially enhanced microbial activity (by providing preferential energy source) on permafrost resistance to thaw and erosion.
We plan to test the following hypotheses:
Field Work and Data
To meet the major challenges and objectives of the International Polar Year, we designed our research plan so that it could be completed in two parts: 1) intensive laboratory studies with samples taken from Toolik Lake and Fairbanks (Smith Lake and Fox Tunnel), aimed at elucidating mechanisms behind the subzero microbial activity; and 2) extensive studies on circumpolar samples collected around the Arctic circle.
We selected the intensive sites was based on: 1) their accessibility; 2) the instability of permafrost near Toolik Lake, which makes it an especially urgent task to observe the early stages of permafrost degradation; and 3) the wealth of high quality data obtained earlier during the long-term ecological research at Toolik Lake and through the recently launched National Ecological Observatory Network (NEON) program with Toolik Lake as the focal point.
The Toolik Lake site (68°38'N, 149°36'W) is moist acidic tundra, at the Arctic Tundra Long Term Ecological Research site in the northern foothills of the Brooks Range. The Smith Lake site is small forest wetland patch within the gas flux site at the University of Alaska Fairbanks (64°52'N 147°51'W), and Fox Tunnel is a unique passageway constructed by the U.S. Army in 1961 that gives access to deep permafrost anaerobic layers aged from 4,000 to 40,000 years.
The Co-PI, Tingjun Zhang, from the University of Colorado at Boulder, will conduct sampling, including shipping in frozen state to Rutgers University in New Jersey, and will keep track of current changes in the state of permafrost. We are prepared for on-demand responses to any unexpected "interesting cases" (significant cryoturbation, permafrost erosion, anomalous precipitations, or drought) and will promptly perform any microbiological analysis.
There will be repetitive sampling to follow seasonal dynamics of frozen soil at Smith Lake. Samples will be taken every month during the warm season and every other month during winter and kept frozen (-80 degrees Celsius) before analysis. At Fox Tunnel and Toolik Lake, the cores will be taken during spring with column rotation drilling, packed into sterile disposable plastic containers in portable coolers with "blue ice" and delivered to the laboratory within 20 to 40 hours. The core temperatures will be continuously recorded. All samples will be stored in air-tight plastic wrap; samples from Fox Tunnel or other sites suspected of anaerobic processes will be kept at -80 degrees Celsius in desiccators under nitrogen (N2).
The sampling for the circumpolar survey will be done mainly by researchers at field stations located outside of the United States. Samples from these sites will be shipped frozen to Panikov, Häggblom, and Kerkhof. A USDA soil import permit has been acquired.
Laboratory incubation studies and mathematical simulations:
Handling of permafrost and frozen soil samples required specially designed procedure. The critical points are: 1) to avoid thawing during transportation and lab manipulations; 2) to homogenize the frozen cores into three to eight millimeter aggregates under continuous cooling, sterility, and nitrogen (N2) flow (in the case of anoxic samples); 3) to minimize even short-term warming of incubated frozen samples during kinetic measurements. To fulfill these requirements, we used high precision freezers and manufactured several of our own lab devices.
The mathematical simulation of microbial growth and heat transfer in the described experiments will be based on use of ordinary and partial differential equations. Examples of such models are numerous in soil physics and microbial biokinetics. Without going into details, we would like to stress that simulations will accompany lab experiments to organize data and make sure that we understand correctly all major processes in frozen media.
Culture-independent characterization and isolation of psychroactive microorganisms:
We are going to use the following molecular techniques: 1) Polymerase chain reaction Single-Strand Conformation Polymorphism (PCR-SSCP) for relatively fast characterization of microbial diversity in extensive studies of all sites; 2) PCR-amplification, cloning and sequencing of ribosomal genes for detailed phylogenetic characterization of communities at key sites; 3) Fluorescence in situ hybridization (FISH) probes to observe the actual abundance and morphology of the targeted species including unculturable organisms; and 4) Stable Isotope Probing (SIP) to identify species/operational taxonomic unit (OTU) responsible for consumption and transformation particular compounds added to permafrost.
Finally, we have the problem of differentiation between concurrently active psychroactive organisms and those fungi and bacteria which were active hundreds or thousand years ago. Two approaches will be tried to solve this problem: to isolate and characterize rRNA which correlates with growth rates, and to use the stable isotope Probing (SIP) technique as originally described and then modified in the laboratory of L. Kerkhof, which has been shown to detect very small amounts of carbon 13 (13C)-labeled DNA.
We already learned from our previous studies that successful isolation of psychrophiles takes many years and plenty of patience. We will continue isolation work with solid-state frozen media by testing those growth substrates and incubation conditions which will produce greatest stimulation of microbial activity in soil incubations. At least three to five new organisms will be described at the end of the proposed research and deposited in U.S. and European culture collections. We will also have developed storage conditions and performed all tests required for valid description. Both the Panikov and Häggblom laboratories have extensive experience in physiological and taxonomic description of novel bacteria.
Laboratory test of large permafrost cores
All experiments described above deal with aggregated samples of permafrost. In this series of experiments we will test performance of microorganisms in large cores that are ~10 centimeters in diameter and 10 to 20 centimeter in length without crushing. 10 to 15 replicate cores will be packed into plastic thermoinsulated columns placed in dry freezer at -5 to -20 degrees Celsius and incubated for several months. Microbial activity will be stimulated by delivery of methane and ethanol vapor by continuous passing gas mixture through half of replicate columns (the rest of control columns will be flushed by the same flow of purified air). The consumption of methane, ethanol, and carbon dioxide (CO2) production will be recorded as difference between input and output concentrations. Temperature and the content of unfrozen water (UW) will also be recorded. A home-made multiplexer based on solenoid valves will be used to run 10 channels simultaneously. Antifreeze proteins and temperature will be tested as described above to see changes in the state of cores.
Survey of permafrost and tundra samples
The basic analysis scheme includes measuring of dark 14CO2 fixation (DF); respiration rate (RR) as CO2 evolution rate above the mercuric chloride (HgCl2)-poisoned control) at +10, ~0 and 10 degrees Celsius; and PCR-SSCP fingerprint of the microbial community. Supplementary tests will include: alkalinity (pH), the ice/moisture content and bulk density by standard gravimetric methods, total and soluble organic carbon, and volatile compounds. Identification of individual compounds (for instance, fatty acids from formic to decanoic acids, alcohols, aldehydes, and ketones) will be done by mass-spectral interpretation. The Permafrost Laboratory at UAF, and our partners from the field sites outside the United States, will provide background site-specific information.
Progress Report from Dr. L. J. Kerkhof and Dr. M. M. Häggblom, Rutgers University (Microsoft Word document, 3.2 MB)
Progress Report from Dr. Nicolai Panikov, Dartmouth College (forthcoming)
Progress Report from Dr. Tingjun Zhang, University of Colorado at Boulder (Microsoft Word document, 9.5 MB)
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