JoVE Logo
Faculty Resource Center

Sign In

Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Biology

Removal of Exogenous Materials from the Outer Portion of Frozen Cores to Investigate the Ancient Biological Communities Harbored Inside

Published: July 3rd, 2016

DOI:

10.3791/54091

1Biogeochemical Sciences Branch, Cold Regions Research and Engineering Laboratory, US Army Engineer Research & Development Center, Hanover, NH, 2Environmental Processes Branch, Environmental Laboratory, US Army Engineer Research & Development Center, Vicksburg, MS, 3Terrestrial and Cryospheric Scienes Branch, Cold Regions Research and Engineering Laboratory, US Army Engineer Research & Development Center, Hanover, NH, 4Biogeochemical Sciences Branch, Cold Regions Research and Engineering Laboratory, US Army Engineer Research & Development Center, Fairbanks, AK

The cryosphere offers access to preserved organisms that persisted under past environmental conditions. A protocol is presented to collect and decontaminate permafrost cores of soils and ice. The absence of exogenous colonies and DNA suggest that microorganisms detected represent the material, rather than contamination from drilling or processing.

The cryosphere offers access to preserved organisms that persisted under past environmental conditions. In fact, these frozen materials could reflect conditions over vast time periods and investigation of biological materials harbored inside could provide insight of ancient environments. To appropriately analyze these ecosystems and extract meaningful biological information from frozen soils and ice, proper collection and processing of the frozen samples is necessary. This is especially critical for microbial and DNA analyses since the communities present may be so uniquely different from modern ones. Here, a protocol is presented to successfully collect and decontaminate frozen cores. Both the absence of the colonies used to dope the outer surface and exogenous DNA suggest that we successfully decontaminated the frozen cores and that the microorganisms detected were from the material, rather than contamination from drilling or processing the cores.

The cryosphere (e.g., permafrost soils, ice features, glacial snow, firn, and ice) offers a glimpse into what types of organisms persisted under past environmental conditions. Since these substrates can be tens to hundreds of thousands of years old, their microbial communities, when preserved frozen since deposition, reflect ancient environmental conditions. To appropriately analyze these ecosystems and extract meaningful biological information from frozen soils and ice, proper collection and processing of the frozen samples is necessary. This is of utmost importance as climate projections for the 21st century indicate the potential for a pronounce....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

1. Equipment Preparation and Permafrost Core Collection

  1. Equipment preparation and field sample collection and preservation gear
    1. Assemble auger for sample collection by inserting the drive adapter into the top of the barrel and rotating the lever to lock it in place. Pin the adapter tube onto the drive adapter and pin the motor onto the adapter tube. Insert the cutters on the barrel.
    2. Wear light duty suits, nitrile gloves, and masks to reduce any contamination to the samples. Wear ear pro.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

The presented method could be used to decontaminate frozen samples collected from various cryosphere environments, from glacial ice to permafrost. Here, we present data specifically collected from ice and permafrost samples collected from the Engineering Research and Development Center - Cold Regions Research and Engineering Laboratory (ERDC-CRREL) Permafrost Tunnel located in Fox, AK (Figure 1A and 1B). The Permafrost Tunnel extends approximately 110 m i.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

The cryosphere offers access to preserved organisms that persisted under past environmental conditions. Though the recovered taxa may not represent the complete historic community, those recovered from analysis of glacial ice and permafrost samples can yield valuable historic information about select time periods15-16. For instance, meaningful biological information has been drawn from ice studies investigating anaerobic activity in the Greenland ice sheet20 and permafrost studies investigating carb.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

This work was funded through the U.S. Army Engineer Research and Development Center, Basic Research Program Office. Permission for publishing this information has been granted by the Chief of Engineers.

....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

Name Company Catalog Number Comments
Auger Snow, Ice, and Permafrost Research Establishment (SIPRE), Fairbanks, AK N/A
70% Isopropanol Walmart 551116880
95% Ethyl Alcohol (denatured)  Fisher Scientific, Pittsburgh, PA A407-4
DNA decontamination solution, DNA Away Molecular Bio-Products, Inc., San Diego, CA 7010
RNase decontamination solution, RNase Away Molecular Bio-Products, Inc., San Diego, CA  7002
Light Duty Suits Kimberly-Clark Professional, Roswell, GA 10606
Nitrile Gloves Fisher Scientific, Pittsburgh, PA FFS-700
Antiviral Masks Curad, Walgreens CUR3845
Sterile Sample Bags  Nasco, Fort Atkinson, WI B01445
Steel Microtome Blade  B-Sharp Microknife, Wake Forest, NC N/A
Metal Rack Fabricated at CRREL, Hanover, NH N/A
Tray Handy Paint Products, Chanhassen, MN 7500-CC
Aluminum Foil Western Plastics, Temecula, CA N/A
500 ml Bottle with 0.22 μm Filter Corning, Corning, NY 430513
Serratia marcescens  ATCC, Manassas, VA 17991
Biosafety Hood NuAire, Plymouth, MN NU-425-400
Petri Dish Fisher Scientific, Pittsburgh, PA FB0875712
ATCC Agar 181- Tryptone Acros Organics, NJ 61184-5000
ATCC Agar 181- Glucose Fisher Scientific, Pittsburgh, PA BP381-500
ATCC Agar 181- Yeast Extract Fisher Scientific, Pittsburgh, PA BP1422-500
ATCC Agar 181- Dipotassium Phosphate JT Baker, Phillipsburg, NJ 3252-01
ATCC Agar 181- Agar Difco, Sparks, MD 214530
NanoDrop 2000 UV Vis Spectrophotometer Thermo Fisher Scientific, Wilmington, DE
Lightcycler 480 System Roche Molecular Systems, Inc., Indianapolis, IN
Halobacterium salinarum American Type Culture Collection (ATCC), Manassas, VA
Pseudomonas fluorescens  American Type Culture Collection (ATCC), Manassas, VA
Microbial DNA Isolation Kit MoBio Laboratories, Carlsbad, CA 12224-50
Ear Protection Elvex EP-201
Hard Hat N/A N/A
Kimwipes Kimberly-Clark Professional, Roswell, GA 34705
Glass Wool Pyrex 430330
Ruler N/A N/A
Weighing Tin  Fisher Scientific, Pittsburgh, PA 08-732-100
Sodium chloride Sigma Aldrich, St Louis, MO S-9625
Potassium chloride JT Baker, Phillipsburg, NJ 3040-04
Potassium phosphate, monobasic JT Baker, Phillipsburg, NJ  3246-01
Potassium phosphate, dibasic JT Baker, Phillipsburg, NJ 3252-01
Sodium phosphate dibasic, anhydrous Fisher Scientific, Pittsburgh, PA BP332-500
50 ml Centrifuge Tubes Corning, Corning, NY 4558
2 ml Microcentrifuge Tubes MoBio Laboratories, Carlsbad, CA 1200-250-T
2 ml Ceramic Bead Tubes (1.4 mm) MoBio Laboratories, Carlsbad, CA 13113-50
Scoopula Thermo Fisher Scientific, Wilmington, DE 1437520
Balance Ohaus, Parsippany, NJ E12130
Diethylpyrocarbonate (DEPC) Sigma Aldrich, St Louis, MO D5758
Hexadecyltrimethylammoniabromide (CTAB)  Acros Organics, NJ 22716-5000
Polyethylene glycol 8000  Sigma Aldrich, St Louis, MO P5413-1KG
Phenol-chloroform-isoamyl alcohol (25:24:1) (pH 8)  Fisher Scientific, Pittsburgh, PA BP1752-400
Centrifuge Eppendorf, Hauppauge, NY 5417R
Chloroform-isoamyl alcohol (24:1) Sigma Aldrich, St Louis, MO C0549-1PT
TE Buffer Ambion (Thermo Fisher), Wilmington, DE AM9860
Pipets Rainin, Woburn, MA Pipet Lite XLS, 2, 10, 200, 1nd 1000ul pipets
Pipet tips Rainin, Woburn, MA Rainin LTS presterilized, low retention, filtered tips, 10, 20, 200, 1000ul
Vortexor Scientific Industries, Bohemia, NY G-560
Vortex Adaptor MoBio Laboratories, Carlsbad, CA 13000-V1
Clear Bottle Corning, Corning, NY C1395500
Amber Bottle Corning, Corning, NY C5135250
Bottle Top Filters, 0.22um Corning, Corning, NY 430513
60 mL Syringe Becton, Dickenson and Company, Franklin Lakes, NJ BD 309653
Millex Syringe filters, 0.22 μm EMD Millipore, Billerica, MA SLGV033RB
70% Ethanol Fisher Scientific, Pittsburgh, PA BP2818-500 diluted & filter sterilized
Isotemp 100 L Oven Fisher Scientific, Pittsburgh, PA 151030511
Cell Spreader Fisher Scientific, Pittsburgh, PA 08-100-10
Disposable Inoculating Loops Fisher Scientific, Pittsburgh, PA 22-363-602

  1. Solomon, S., et al. . Climate Change 2007: The Physical Science Basis. , (2007).
  2. Marchenko, S., Romanovsky, V., Tipenko, G. Numerical Modeling of Spatial Permafrost Dynamics in Alaska. Proc. Ninth Int. Conferen. Permafr. 29, 1125-1130 (2008).
  3. Pachauri, R. K., Meyer, L. A. . Climate Change 2014: Synthesis Report. Contributions of Working Groups I, II, and III to the Fifth Assessment Report of the Intergovernmental Panel on Climate Change. , (2007).
  4. Osterkamp, T. E., Romanovsky, V. E. Evidence for warming and thawing of discontinuous permafrost in Alaska. Permafr. Periglac. Process. 10 (1), 17-37 (1999).
  5. Wolken, J. M., et al. Evidence and implications of recent and projected climate change in Alaska's forest ecosystems. Ecosphere. 2 (11), 1-35 (2011).
  6. Hinzman, L. D., Kane, D. L., Gieck, R. E., Everett, K. R. Hydrologic and thermal properties of the active layer in the Alaskan Arctic. Cold Reg. Sci. Technol. 19 (2), 95-110 (1991).
  7. Hinzman, L. D., Goering, D. J., Kane, D. L. A distributed thermal model for calculating temperature profiles and depth of thaw in permafrost regions. J. Geophys. Res.: Atmos. 103 (D22), 28975-28991 (1998).
  8. Osterkamp, T. E., Jorgenson, J. C. Warming of Permafrost in the Arctic National Wildlife Refuge. Alaska. Permafr. Periglac. Process. 17, 65-69 (2006).
  9. Petrone, K. C., Jones, J. B., Hinzman, L. D., Boone, R. D. Seasonal export of carbon, nitrogen, and major solutes from Alaskan catchments with discontinuous permafrost. J. Geophys. Res. 111, G02020 (2006).
  10. Guo, L., Ping, C. -. L., Macdonald, R. W. Mobilization pathways of organic carbon from permafrost to arctic rivers in a changing climate. Geophys. Res. Lett. 34 (13), L13603 (2007).
  11. Katayama, T., et al. Phylogenetic analysis of bacteria preserved in a permafrost ice wedge for 25,000 years. Appl. Environ. Microbiol. 73 (7), 2360-2363 (2007).
  12. Katayama, T., et al. Glaciibacter superstes gen. nov., sp. nov., a novel member of the family Microbacteriaceae isolated from a permafrost ice wedge. Int. J. Syst. Evol. Microbiol. 59, 482-486 (2009).
  13. Waldrop, M. P., White, R., Douglas, T. A. Isolation and identification of cold-adapted fungi in the Fox Permafrost Tunnel, Alaska. Proc. Ninth Int. Conferen. Permafr. , 1887-1891 (2008).
  14. Douglas, T. A., et al. Biogeochemical and geocryological characteristics of wedge and thermokarst-cave ice in the CRREL Permafrost Tunnel. Alaska Permafr. Periglac. Process. 21 (2), 120-128 (2011).
  15. Willerslev, E., et al. Diverse plant and animal genetic records from Holocene and Pleistocene sediments. Science. 300 (5620), 791-795 (2003).
  16. Bellemain, E., et al. Fungal palaeodiversity revealed using high-throughput metabarcoding of ancient DNA from Arctic permafrost. Environ. Microbiol. 15 (4), 1176-1189 (2013).
  17. Steven, B., Pollard, W. H., Greer, C. W., Whyte, L. G. Microbial diversity and activity through a permafrost/ground ice core profile from the Canadian high Arctic. Environ. Microbiol. 10 (12), 3388-3403 (2008).
  18. Lorenzen, E. D., et al. Species-specific responses of Late Quaternary megafauna to climate and humans. Nature. 479 (7373), 359-364 (2011).
  19. Wilhelm, R. C., Radtke, K., Mykytczuk, N. C. S., Greer, C. W., Whyte, L. G. Life at the wedge: The activity and diversity of Arctic ice wedge microbial communities. Astrobiol. 12 (4), 347-360 (2012).
  20. Sheriden, P. P., Miteva, V. I., Brenchley, J. E. Phylogenetic analysis of anaerobic psychrophilic enrichment cultures obtained from a Greenland glacier ice core. Appl. Environ. Microbiol. 69 (4), 2153-2160 (2003).
  21. Rivkina, E., et al. Biogeochemistry of methane and methanogenic archaea in permafrost. FEMS Microbiol. Ecol. 61 (1), 1-15 (2007).
  22. Juck, D. F., et al. Utilization of fluorescent microspheres and a green fluorescent protein-marked strain for assessment of microbiological contamination of permafrost and ground ice core samples from the Canadian High Arctic. Appl. Environ. Microbiol. 71 (2), 1035-1041 (2005).
  23. Griffiths, R. I., Whiteley, A. S., O'Donnell, A. G., Bailey, M. J. Rapid method for coextraction of DNA and RNA from natural environments for analysis of ribosomal DNA- and rRNA-based microbial community composition. Appl. Environ. Microbiol. 66 (12), 5488-5491 (2000).
  24. Töwe, S., et al. Improved protocol for the simultaneous extraction and column-based separation of DNA and RNA from different soils. J. Microbiol. Methods. 84 (3), 406-412 (2011).
  25. Nadkarni, M. A., Martin, F. E., Jacques, N. A., Hunter, N. Determination of bacterial load by real-time PCR using a broad range (universal) probe and primers set. Microbiol. 148, 257-266 (2002).
  26. Takai, K., Horikoshi, K. Rapid detection and quantification of members of the archaeal community by quantitative PCR using fluorogenic probes. Appl. Environ. Microbiol. 66 (11), 5066-5072 (2000).
  27. Fogel, G. B., Collins, C. R., Brunk, C. F. Prokaryotic genome size and SSU rDNA copy number: Estimation of microbial relative abundance from a mixed population. Microb. Ecol. 38, 93-113 (1999).
  28. Bodilis, J., Nsigue-Meilo, S., Besaury, L., Quillet, L. Variable copy number, intra-genomic heterogeneities and later transfers of the 16S rRNA gene in Pseudomonas. PLOS One. 7, e35647 (2012).
  29. Caporaso, J. G., et al. QIIME allows analysis of high-throughput community sequencing data. Nature Methods. 7, 335-336 (2010).
  30. Sellmann, P. V. . Geology of the USA CRREL permafrost tunnel, Fairbanks, Alaska. US Army Cold Reg. Res. Eng. Lab. Technical Rep. 199. , (1967).
  31. Sellmann, P. V. . Additional information on the geology and properties of materials exposed in the USA CRREL permafrost tunnel. US Army CRREL Special Rep. , (1972).
  32. Christner, B. C., Mikucki, J. A., Foreman, C. M., Denson, J., Priscu, J. C. Glacial ice cores: A model system for developing extraterrestrial decontamination protocols. Icarus. 174 (2), 572-584 (2005).
  33. Mackelprang, R., et al. Metagenomic analysis of a permafrost microbial community reveals a rapid response to thaw. Nature. 480 (7377), 368-371 (2011).
  34. Champlot, S., et al. An efficient multistrategy DNA decontamination of PCR reagents for hyper sensitive PCR applications. PLoS One. 5 (9), e13042 (2010).
  35. Yergeau, E., Hogues, H., Whyte, L. G., Greer, C. W. The functional potential of high Arctic permafrost revealed by metagenomic sequencing, qPCR, and microarray analyses. The ISME J. 4 (9), 1206-1214 (2010).
  36. Welzl, G., Schloter, M. Bacterial community structure in soils of the Tibetan Plateau affected by discontinuous permafrost or seasonal freezing. Biol. Fertil. Soils. 50 (3), 555-559 (2014).
  37. Vishnivetskaya, T. A., et al. Commercial DNA extraction kits impact observed microbial community composition in permafrost samples. FEMS Microbiol. Ecol. 87 (1), 217-230 (2014).
  38. Wagner, D., Kobabe, S., Liebner, S. Bacterial community structure and carbon turnover in permafrost-affected soils of the Lena Delta, northeastern Siberia. Can. J. Microbiol. 55 (1), 73-83 (2009).
  39. Jiang, N., et al. Characteristic microbial communities in the continuous permafrost beside the bitumen in Qinghai-Tibetan Plateau. Environ. Earth Sci. 74, 1343-1352 (2015).

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2024 MyJoVE Corporation. All rights reserved