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 pronounced warming in Arctic and sub-Arctic regions1. Specifically, Interior Alaska and Greenland are expected to warm approximately 5 °C and 7 °C, respectively by 21002,3. This is expected to significantly impact soil and aquatic microbial communities, and therefore, related biogeochemical processes. The warmer temperatures and altered precipitation regime are expected to initiate permafrost degradation in many areas2-5 potentially leading to a thicker, seasonally thawed (active) layer6,7, the thawing of frozen soils, and the melting of massive ice bodies such as ground ice, ice wedges, and segregation ice8. This would dramatically change the biogeochemical attributes in addition to the biodiversity of plants and animals in these ecosystems.
Glacial ice and syngenetic permafrost sediment and ice features have trapped chemical and biological evidence of an environment representing what lived there at the time the features formed. For example, in Interior Alaska, both Illinoisan and Wisconsin aged permafrost are present and this permafrost in particular provides unique locations dating from modern to 150,000 years before the present (YBP) that contain biological and geochemical evidence of the impact of past climatic changes on biodiversity. As a result, these sediments provide a record of the biogeochemistry and biodiversity over many thousands of years. Since the area has low sedimentation rates and has never been glaciated, undisturbed samples are accessible for collection and analysis, either drilling vertically into the soil profile or drilling horizontally in tunnels. More importantly, extensive records exist that especially highlight the unique biogeochemical features of permafrost in this region9-14. Specifically, the application of DNA analysis to estimate presence and extent of biodiversity in both extant and ancient ice and permafrost samples enables exploration of the linkage of ancient environmental conditions and habitat to occupation by specific organisms.
Previous studies have identified climatic impacts on mammals, plants and microorganisms from samples dating to 50k YBP11, 15-19, though each study used a different methodology to collect and decontaminate the permafrost or ice cores. In some instances, the drilling cores were sterilized16, 20-21, though the specific methodology did not clarify whether foreign nucleic acids were also eliminated from the samples. In other studies, bacterial isolates15 (e.g., Serratia marcescens) as well as fluorescent microspheres22 have been used to measure the efficacy of decontamination procedures.
This experiment was part of a larger study investigating microbial communities from permafrost samples dating back to approximately 40k YBP. The specific objective of this portion of the study was to successfully decontaminate ice and permafrost cores. To our knowledge, no methodology has integrated the use of solutions designed to eliminate foreign nucleic acids and associated nucleases from the outer portion of the frozen cores. This is despite the fact that these solutions are commonly used to decontaminate laboratory equipment for molecular experiments.
Once the cores were decontaminated, genomic DNA was extracted using the protocols developed by Griffiths et al.23 and Töwe et al.24, quantified using a spectrophotometer, and diluted with sterile, DNA-free water to achieve 20 ng per reaction. Bacterial 16S rRNA genes were amplified with primers 331F and 797R and probe BacTaq25 and archaeal 16S rRNA genes were amplified with primers Arch 349F and Arch 806R and probe TM Arch 516F26 under the following conditions: 95 °C for 600 sec followed by 45 cycles of 95 °C for 30 sec, 57 °C for 60 sec, and 72 °C for 25 sec with final extension at 40 °C for 30 sec. All qPCR reactions were conducted in duplicate. The 20 µl reaction volumes included 20 ng DNA, 10 µM of primers, 5 µM of the probe, and 10 µl of the qPCR reaction mix. Standards for bacterial and archaeal qPCR were prepared using genomic DNA from Pseudomonas fluorescens and Halobacterium salinarum, respectively. Both were grown to log phase. Plate counts were conducted and DNA was isolated from the cultures. Genomic DNA was quantified with a spectrophotometer with the assumption of one and six copies of the 16S rRNA gene per genome for H. salinarum and P. fluorescens, respectively27-28. Copy numbers of the bacterial and archaeal genes were calculated based on the standard curve, log-transformed to account for unequal variances between treatments, and assessed by ANOVA.
Community composition was determined by sequencing the 16S rRNA gene using flow cells and bridge amplification technologies and analyzing the communities with 'quantitative insights into microbial ecology' (QIIME)29. Forward and reverse reads were joined together and then sequences were filtered, indexed, and high quality representatives were selected for de novo operational taxonomic units (OTU) assignment through sequence alignment with a reference database. Aligned sequences were compared to a separate reference database for taxonomic assignment. A phylum level OTU table was created to determine general community composition.
1. Equipment Preparation and Permafrost Core Collection
2. Permafrost and Ice Core Processing
3. Obtain Subsample for Nucleic Acid Extraction from Ice Cores and Permafrost
4. Extract Nucleic Acids from Permafrost and Ice Cores
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 into the side of Goldstream valley and provides access to ice rich silt and alluvium30-31. Samples from an ice wedge and frozen soil were carefully collected in triplicate from the walls of the tunnel on 24 October 2014. At the time of sampling, the temperature of the permafrost wall was -2.9 °C. Samples were collected from an ice wedge feature approximately 27 m from the portal of the tunnel, and frozen soil at 35 m and 60 m from the portal of the tunnel (Figure 1C and 1D). Special care was taken during sample collection to limit contamination of the ice and permafrost cores (Figure 1E). The frozen cores were handled according to our decontamination protocol (Figure 2) and transported on dry ice to the CRREL soil microbiology laboratory in Hanover, NH for processing.
All cores were processed using the protocol described in Section 2 using sterile microtome blades and solutions (Figure 3A, B, and C). The goal of this study was to successfully extract endogenous nucleic acids from the frozen samples to determine the microbial communities present at the time that the material was deposited. The decontamination protocol was assessed by doping the cores with a dilute culture of Serratia marcescens and then examining the Petri plates for growth after the cores were treated31. The absence of S. marcescens colonies indicated that the exogenous microorganisms were properly removed from the outer portion of the core. However, the absence of colonies would not indicate whether exogenous DNA was removed from the outer portion of the core. Therefore, we sequenced samples from the ice core to check for DNA from Serratia sp. Because microbial abundance in the ice wedge was likely significantly lower than in the permafrost, we used the ice cores to determine if DNA from S. marcescens would be present following the decontamination protocol. If the cores were not properly decontaminated, then sequences related to S. marcescens would be expected to be present in the samples. Figure 4 shows the low bacterial diversity within the ice core from 16S rRNA gene sequencing results. Sequences related to Pseudomonas sp., of the phylum Gammaproteobacteria, dominated the ice samples. Members related to Planctomycetia were also present in the ice, but to a lesser extent. Of particular note is the absence of Serratia sp. in the sequencing results, suggesting that the decontamination protocol sufficiently removed exogenous DNA.
Furthermore, there is an additional risk of contamination during the extraction of nucleic acids. Negative extraction blanks were used to reveal contamination from exogenous nucleic acids. The DNA from the samples and negative controls were amplified using qPCR. qPCR data showed that the permafrost harbored bacteria, but archaea were not detected in the permafrost (Table 1). Successful decontamination was further evidenced by the lack of bacterial or archaeal amplicons in extraction blanks, in conjunction with positive amplification of the controls, Pseudomonas fluorescens and Halobacterium salinarum (Table 1). The abundance measurements revealed that there were no significant differences in bacterial abundance between the cores collected at 35 m from the portal as compared to the 60 m cores (Table 1). Ongoing research is being conducted to determine whether microbial community composition was different between the cores.
Figure 1. Sample sites near Fairbanks, AK. (A) Map of Fairbanks, AK (http://www.volunteer.noaa.gov/alaska.html), (B) ERDC-CRREL Permafrost Tunnel, (C) collection of permafrost cores using the SIPRE auger, (D) view of auger entering permafrost wall, and (E) preparing the core for archival. Please click here to view a larger version of this figure.
Figure 2. Schematic of decontaminating frozen material. (A) The frozen material (e.g., ice core or permafrost core) is processed in a cold room. (B) It is purposefully contaminated with a bacterial culture. (C) The core is shaved with a sterile microtome blade to remove the outer 5 mm portion. (D) A series of solutions are applied to further decontaminate the outer portion of the sample. DDS indicates DNA decontamination solution and RDS indicates RNase decontamination solution. (E) By placing the core in a sterile biohood held at RT, the outer 2-5 mm of the material thaws. The liquid that drips from the ice or permafrost is collected in Petri plates and the core is swabbed and plated to check that the core was properly decontaminated. (F and G) If bacterial growth is not detected, then the core is thawed at 4 °C in a sterile container. Please click here to view a larger version of this figure.
Figure 3. Decontamination of permafrost cores in cold room under sterile conditions. (A) Remove the outer layer of the permafrost core with a metal microtome blade by scraping. (B) A closer view of a scrape. (C) Solution rinse to decontaminate the outer layer of the core. Please click here to view a larger version of this figure.
Figure 4. Microbial sequences from the ice wedge samples. Bar chart showing the average relative abundance of bacterial phyla present in the ice wedge samples by percentage. Please click here to view a larger version of this figure.
Table 1. Bacterial abundance in permafrost. qPCR results showing the abundance of bacteria in the permafrost samples and associated extraction blanks. Values in table are reported as mean ± standard error. n is the number of samples. ND indicates not detected.
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 carbon cycling processes as a result of thaw33 and have provided insight into fungal diversity from permafrost in Beringia16. Proper sample collection and decontamination must occur before conducting downstream analyses to investigate biological communities within these frozen materials. Even though these steps have significant implications on interpreting the data, many studies do not offer specific details on how the samples were collected and processed or images showing how to handle the frozen materials. For instance, previous studies have doped the outer portion of cores with fluorescent microspheres or known bacteria15, though the exact concentrations of these materials were not described. Other studies have described decontamination protocols in detail, but have not applied high throughput analyses to test the efficacy of the methodology15, 32. Therefore, a detailed screening of intact bacteria and exogenous DNA through growth on Petri plates and qPCR was used to determine whether the sample collection, preservation, and analytical procedures were sterile enough to identify microbial communities present in the frozen materials.
This protocol has been modified to successfully isolate genetic material of interest from ancient cores. Care such as wearing specific gear and decontaminating supplies must be taken during the collection, processing, and extraction from the cores since contamination could occur at any step. Furthermore, shipping the cores with dry ice or a comparable cooling agent is imperative because if the cores thaw during transit, then there is a higher propensity that contaminants from the outer region of the core may migrate to the inner portion of the core. To further minimize the potential for contamination, consider collecting larger cores with a motorized auger rather than a push core. We found that the push cores were too small to properly decontaminate in the scraping step. Alternatively, a larger volume of sample could be extracted from the larger cores, which is especially important for samples with a low abundance of biological material. Additionally, the larger volume affords more room for error such that if Serratia marcescens colonies are detected on plates, then the core is large enough to be processed again.
Various supplies were tested to determine the most efficient method to decontaminate the cores. For instance, sterile aluminum foil, rather than glass dishes or plastic materials, allowed for the core to be placed on a sterile surface quickly and easily. Also, non-traditional supplies such as a metal rack and a tray proved to be beneficial to allow the scraped outer materials to accumulate away from the core, decreasing the risk of re-contaminating the cores. In earlier iterations of the protocol, the scraping occurred on a flat surface, which increased the risk of contamination. Finally, sterile bags, rather than glass dishes, were found to be conducive to mixing and storing the samples.
This protocol was targeted to eliminate microorganisms and nucleic acids on the outer portion of the frozen cores. While isopropanol and ethanol are effective disinfectants, they do not remove nucleic acids. Therefore, solutions that remove nucleic acids and associated enzymes from the outer portion of the cores were used. These solutions are commonly used to remove nucleic acids and associated nucleases from laboratory supplies and equipment. When testing their efficacy on laboratory surfaces, RNA decontamination solution removed more exogenous materials than the DNA decontamination solution34. Using nucleases to decontaminate the outer portion of the core might not always be the preferred method because genetic materials of interest may also be removed from samples with a low abundance of a particular organism. In particular, genetic material stored in permafrost and ice for extended periods of time undergo degradation. Because microorganisms are in a high abundance in these Alaskan samples, the concern that the solutions would remove a high proportion of endogenous genetic materials was greatly reduced. However, caution should be taken when using solutions that contain nucleases to ensure that the nucleic acids that have been severely degraded or are from organisms in low abundance are not removed by the nucleases.
The absence of S. marcescens colonies on the Petri plates provided confidence that the intact cells were removed from the outer portion of the permafrost cores. Furthermore, analysis of ice cores that underwent the same protocol showed no detectable sequences related to S. marcescens. Sequence results revealed that only Pseudomonas were detected in these samples, even though both Pseudomonas sp. and Serratia sp. are within the class Gammaproteobacteria. Together, the absence of the S. marcescens colonies on Petri plates and the absence of DNA amplicons from qPCR suggest that the frozen cores were successfully decontaminated. Therefore, the microorganisms detected were likely embedded in the frozen material, rather than contamination from drilling or processing the cores. Other drilling techniques include hydraulic drilling to penetrate soil or ice at greater depths. Though this method would be beneficial to investigate low microbial abundance in these oligotrophic environments, it does not reveal whether drilling fluids would be successfully removed. Furthermore, if high throughput sequencing is not part of the downstream analysis, specific PCR reactions targeting S. marcescens should be used to amplify the extracted DNA.
The results from our example dataset showed that intact genomic DNA was successfully extracted from the frozen materials. Furthermore, both the permafrost and ice wedge harbored bacteria, as evidenced by qPCR and sequencing, respectively. The Alaskan discontinuous permafrost from this study contained similar bacterial numbers as permafrost in the Canadian high Arctic35, though the Alaskan samples contained an order of magnitude fewer bacteria than permafrost from the Tibetan Plateau in Qinghai province, China36 and active layer/permafrost soils from Nunavut, Canada37. Similar to an ice wedge collected in Nunavut, Canada37, sequences related to Gammaproteobacteria, specifically Pseudomonas, which are common to soils and metabolically diverse, dominated the Alaskan ice wedge in this study. In fact, Katayama et al.11 isolated bacteria within the phyla Actinobacteria, Bacilli, and Gammaproteobacteria from one of the ice wedges from the ERDC-CRREL Permafrost Tunnel. These studies corroborate the detection of Gammaproteobacteria in this study. Planctomycetia, which are common to aquatic sample, were also detected in the ice wedge. These organisms have been found in active layer soils above permafrost in northeastern Siberia38, in addition to permafrost in the Tibetan Plateau in Qinghai, China39.
Many studies have investigated the presence of archaea, particularly methanogens, in permafrost, with the notion that as permafrost thaws, methanogens will likely become more active, contributing to the efflux of methane to the atmosphere21, 33-34. Surprisingly, archaea were not detected in the Alaskan ice wedge or permafrost samples even though both Euryarchaeota and Crenarchaeota were found in permafrost from the Canadian high Arctic17 and methanogens were found in permafrost samples from the Arctic tundra in Russia21.
Frozen materials harbor ancient microorganisms, providing a record of biogeochemical processes that occurred many thousands of years ago. This biodiversity is of great interest under the current climate warming regime because microorganisms that were once restricted in the cryosphere environment may become liberated when the frozen materials thaw. In order to confidently identify the biodiversity harbored in these frozen materials, the frozen soils and ice samples must be properly handled and decontaminated. Here, we present a protocol to remove foreign cells and DNA from frozen samples to ensure that the microorganisms detected were from the material, rather than contamination from drilling or processing the cores.
The authors have nothing to disclose.
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.
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 |