Thomas D. Niederberger

Microbial diversity of active layer and permafrost in an acidic wetland from the Canadian High Arctic

The abundance and structure of archaeal and bacterial communities from the active layer and the associated permafrost of a moderately acidic (pH < 5.0) High Arctic wetland (Axel Heiberg Island, Nunavut, Canada) were investigated using culture- and molecular-based methods. Aerobic viable cell counts from the active layer were ∼100-fold greater than those from the permafrost (2.5 × 10(5) CFU·(g soil dry mass)(-1)); however, a greater diversity of isolates were cultured from permafrost, as determined by 16S rRNA gene sequencing. Isolates from both layers demonstrated growth characteristics of a psychrotolerant, halotolerant, and acidotolerant community. Archaea constituted 0.1% of the total 16S rRNA gene copy number and, in the 16S rRNA gene clone library, predominantly (71% and 95%) consisted of Crenarchaeota related to Group I. 1b. In contrast, bacterial communities were diverse (Shannons diversity index, H = ∼4), with Acidobacteria constituting the largest division of active layer clones (30%) and Actinobacteria most abundant in permafrost (28%). Direct comparisons of 16S rRNA gene sequence data highlighted significant differences between the bacterial communities of each layer, with the greatest differences occurring within Actinobacteria. Comparisons of 16S rRNA gene sequences with those from other Arctic permafrost and cold-temperature wetlands revealed commonly occurring taxa within the phyla Chloroflexi, Acidobacteria, and Actinobacteria (families Intrasporangiaceae and Rubrobacteraceae).

Microbial characterization of a subzero, hypersaline methane seep in the Canadian High Arctic.

We report the first microbiological characterization of a terrestrial methane seep in a cryo-environment in the form of an Arctic hypersaline (∼24% salinity), subzero (−5 °C), perennial spring, arising through thick permafrost in an area with an average annual air temperature of −15 °C. Bacterial and archaeal 16S rRNA gene clone libraries indicated a relatively low diversity of phylotypes within the spring sediment (Shannon index values of 1.65 and 1.39, respectively). Bacterial phylotypes were related to microorganisms such as Loktanella, Gillisia, Halomonas and Marinobacter spp. previously recovered from cold, saline habitats. A proportion of the bacterial phylotypes were cultured, including Marinobacter and Halomonas, with all isolates capable of growth at the in situ temperature (−5 °C). Archaeal phylotypes were related to signatures from hypersaline deep-sea methane-seep sediments and were dominated by the anaerobic methane group 1a (ANME-1a) clade of anaerobic methane oxidizing archaea. CARD-FISH analyses indicated that cells within the spring sediment consisted of ∼84.0% bacterial and 3.8% archaeal cells with ANME-1 cells accounting for most of the archaeal cells. The major gas discharging from the spring was methane (∼50%) with the low CH4/C2+ ratio and hydrogen and carbon isotope signatures consistent with a thermogenic origin of the methane. Overall, this hypersaline, subzero environment supports a viable microbial community capable of activity at in situ temperature and where methane may behave as an energy and carbon source for sustaining anaerobic oxidation of methane-based microbial metabolism. This site also provides a model of how a methane seep can form in a cryo-environment as well as a mechanism for the hypothesized Martian methane plumes.

Fluorescence Microscopy as a Tool for In Situ Life Detection

The identification of extant and, in some cases, extinct bacterial life is most convincingly and efficiently performed with modern high-resolution microscopy. Epifluorescence microscopy of microbial autofluorescence or in conjunction with fluorescent dyes is among the most useful of these techniques. We explored fluorescent labeling and imaging of bacteria in rock and soil in the context of in situ life detection for planetary exploration. The goals were two-fold: to target non-Earth-centric biosignatures with the greatest possible sensitivity and to develop labeling procedures amenable to robotic implementation with technologies that are currently space qualified. A wide panel of commercially available dyes that target specific biosignature molecules was screened, and those with desirable properties (i.e., minimal binding to minerals, strong autofluorescence contrast, no need for wash steps) were identified. We also explored the potential of semiconductor quantum dots (QDs) as bacterial and space probes. A specific instrument for space implementation is suggested and discussed.

Tumebacillus permanentifrigoris gen. nov., sp. nov., an aerobic, spore-forming bacterium isolated from Canadian high Arctic permafrost

A Gram-positive, aerobic, rod-shaped bacterium (strain Eur1 9.5(T)) was isolated from a 9-m-deep permafrost sample from the Canadian high Arctic. Strain Eur1 9.5(T) could not be cultivated in liquid medium and grew over the temperature range 5-37 degrees C; no growth was observed at 42 degrees C and only slow growth was observed at 5 degrees C following 1 month of incubation. Eur1 9.5(T) grew over the pH range 5.5-8.9 and tolerated NaCl concentrations of 0-0.5 % (w/v). Eur1 9.5(T) grew heterotrophically on complex carbon substrates and chemolithoautotrophically on inorganic sulfur compounds, as demonstrated by growth on sodium thiosulfate and sulfite as sole electron donors. Eur1 9.5(T) contained iso-C(15 : 0) as the major cellular fatty acid and menaquinone 7 (MK-7) as the major respiratory quinone. The cell-wall peptidoglycan was of type A1gamma. The DNA G+C content was 53.1 mol%. The 16S rRNA gene sequence of strain Eur1 9.5(T) was only distantly related (<or=87 % sequence similarity over 1407 bp) to any recognized bacterial species. Based on physiological and phylogenetic analyses, strain Eur1 9.5(T) is suggested to represent a novel species of a new genus, for which the name Tumebacillus permanentifrigoris gen. nov., sp. nov. is proposed. The type strain of Tumebacillus permanentifrigoris is Eur1 9.5(T) (=DSM 18773(T) =JCM 14557(T)).

Diverse and highly active diazotrophic assemblages inhabit ephemerally wetted soils of the Antarctic Dry Valleys

Eolian transport of biomass from ephemerally wetted soils, associated with summer glacial meltwater runoffs and lake edges, to low-productivity areas of the Antarctic Dry Valleys (DV) has been postulated to be an important source of organic matter (fixed nitrogen and fixed carbon) to the entire DV ecosystem. However, descriptions and identification of the microbial members responsible for N(2) fixation within these wetted sites are limited. In this study, N(2) fixers from wetted soils were identified by direct nifH gene sequencing and their in situ N(2) fixation activities documented via acetylene reduction and RNA-based quantitative PCR assays. Shannon-index nifH diversity levels ranged between 1.8 and 2.6 and included the expected cyanobacterial signatures and a large number of phylotypes related to the gamma-, beta-, alpha-, and delta-proteobacteria. N(2) fixation rates ranged between approximately 0.5 and 6 nmol N cm(-3) h(-1) with measurements indicating that approximately 50% of this activity was linked with sulfate reduction at some sites. Comparisons with proximal dry soils also suggested that these communities are not ubiquitously distributed, and conditions unrelated to moisture content may define the composition, diversity, or habitat suitability of the microbial communities within wetted soils of the DVs.

Bacterial and Archaeal Diversity in Permafrost

Microorganisms in permafrost survive in an extreme environment characterized by constant subzero temperatures, low water and nutrient availability, and prolonged exposure to background radiation. Despite the harsh conditions, considerable abundance and diversity of microorganisms inhabit permafrost. Pioneering studies focusing on permafrost microbiology simply attempted to determine if permafrost harbored viable microorganisms. For example, microorganisms cultured from Canadian (James and Sutherland 1942), Alaskan (Boyd and Boyd 1964) and Antarctic (Cameron and Morelli 1974) permafrost samples were generally poorly characterized, and the studies were hampered by an inability to demonstrate that drilling and sample handling were performed aseptically. Recent developments using fluid-less drilling (Gilichinsky et al. 1989; Khlebnikova et al. 1990; Juck et al. 2005), tracer microorganisms (Christner et al. 2005; Juck et al. 2005), nucleic acid stains (Christner et al. 2005) and fluorescent microspheres as microbial surrogates (Juck et al. 2005) have greatly improved our ability to recover intact permafrost samples and to monitor exogenous microbiological contamination of pristine permafrost samples. Permafrost also contains various other geomorphological structures including massive ground ice, cryopegs, and ice wedges (Steven et al. 2006) that harbor microbial populations. The description of the abundance, diversity, activity and distribution of microorganisms in permafrost and associated environments will be fundamental to our understanding of how microorganisms survive in permafrost, and how they will respond to future climatic warming and permafrost thawing. Lastly, permafrost microorganisms and microbial ecosystems are considered significant terrestrial analogs for similar organisms that may inhabit permafrost environments that exist beyond the Earth, especially in light of the recent evidence of massive amounts of shallow ground ice near the surface of Mars (Gilichinsky 2002a; Gilichinsky et al. 2007).

Virgibacillus arcticus sp. nov., a moderately halophilic, endospore-forming bacterium from permafrost in the Canadian high Arctic

A novel, moderately halophilic, endospore-forming bacterial strain, designated Hal 1T, was isolated from a permafrost core collected from the Canadian high Arctic. The temperature for growth of strain Hal 1T was 0-30 degrees C with no growth observed at either -5 or 37 degrees C (optimum growth at about 25 degrees C). Strain Hal 1T was able to grow at NaCl concentrations of 0-20% (w/v) and did not have an absolute NaCl requirement for growth; optimal growth was at 5% (w/v) NaCl. The level of 16S rRNA gene sequence similarity between strain Hal 1T and the type strains of Virgibacillus carmonensis and Virgibacillus necropolis was 98.2%; values with respect to the type strains of other recognized Virgibacillus species were below 96.0%. The DNA G+C content of strain Hal 1T was 38.2 mol%. Levels of DNA-DNA relatedness between strain Hal 1T and the type strains of V. carmonensis and V. necropolis were 14.0 and 21.0%, respectively. The major fatty acid of strain Hal 1T was anteiso-C15:0, consistent with species of the genus Virgibacillus. The cell-wall peptidoglycan of strain Hal 1T was type A1alpha and the major respiratory quinone was MK-7. On the basis of genotypic and physiological results, strain Hal 1T (=DSM 19574T=JCM 14839T) is proposed as the type strain of a novel species of the genus Virgibacillus, namely Virgibacillus arcticus sp. nov.

Microbial community composition of transiently wetted Antarctic Dry Valley soils

During the summer months, wet (hyporheic) soils associated with ephemeral streams and lake edges in the Antarctic Dry Valleys (DVs) become hotspots of biological activity and are hypothesized to be an important source of carbon and nitrogen for arid DV soils. Recent research in the DV has focused on the geochemistry and microbial ecology of lakes and arid soils, with substantially less information being available on hyporheic soils. Here, we determined the unique properties of hyporheic microbial communities, resolved their relationship to environmental parameters and compared them to archetypal arid DV soils. Generally, pH increased and chlorophyll a concentrations decreased along transects from wet to arid soils (9.0 to ~7.0 for pH and ~0.8 to ~5 μg/cm3 for chlorophyll a, respectively). Soil water content decreased to below ~3% in the arid soils. Community fingerprinting-based principle component analyses revealed that bacterial communities formed distinct clusters specific to arid and wet soils; however, eukaryotic communities that clustered together did not have similar soil moisture content nor did they group together based on sampling location. Collectively, rRNA pyrosequencing indicated a considerably higher abundance of Cyanobacteria in wet soils and a higher abundance of Acidobacterial, Actinobacterial, Deinococcus/Thermus, Bacteroidetes, Firmicutes, Gemmatimonadetes, Nitrospira, and Planctomycetes in arid soils. The two most significant differences at the genus level were Gillisia signatures present in arid soils and chloroplast signatures related to Streptophyta that were common in wet soils. Fungal dominance was observed in arid soils and Viridiplantae were more common in wet soils. This research represents an in-depth characterization of microbial communities inhabiting wet DV soils. Results indicate that the repeated wetting of hyporheic zones has a profound impact on the bacterial and eukaryotic communities inhabiting in these areas.

Novel sulfur-oxidizing streamers thriving in perennial cold saline springs of the Canadian high Arctic.

The perennial springs at Gypsum Hill (GH) and Colour Peak (CP), situated at nearly 80 degrees N on Axel Heiberg Island in the Canadian high Arctic, are one of the few known examples of cold springs in thick permafrost on Earth. The springs emanate from deep saline aquifers and discharge cold anoxic brines rich in both sulfide and sulfate. Grey-coloured microbial streamers form during the winter months in snow-covered regions of the GH spring run-off channels (-1.3 degrees C to 6.9 degrees C, approximately 7.5% NaCl, 0-20 p.p.m. dissolved sulfide, 1 p.p.m. dissolved oxygen) but disappear during the Arctic summer. Culture- and molecular-based analyses of the 16S rRNA gene (FISH, DGGE and clone libraries) indicated that the streamers were uniquely dominated by chemolithoautotrophic sulfur-oxidizing Thiomicrospira species. The streamers oxidized both sulfide and thiosulfate and fixed CO(2) under in situ conditions and a Thiomicrospira strain isolated from the streamers also actively oxidized sulfide and thiosulfate and fixed CO(2) under cold, saline conditions. Overall, the snow-covered spring channels appear to represent a unique polar saline microhabitat that protects and allows Thiomicrospira streamers to form and flourish via chemolithoautrophic, phototrophic-independent metabolism in a high Arctic winter environment characterized by air temperatures commonly below -40 degrees C and with an annual average air temperature of -15 degrees C. These results broaden our knowledge of the physical and chemical boundaries that define life on Earth and have astrobiological implications for the possibility of life existing under similar Martian conditions.

Microbial diversity and activity in hypersaline high Arctic spring channels.

Lost Hammer (LH) spring is a unique hypersaline, subzero, perennial high Arctic spring arising through thick permafrost. In the present study, the microbial and geochemical characteristics of the LH outflow channels, which remain unfrozen at ≥−18°C and are more aerobic/less reducing than the spring source were examined and compared to the previously characterized spring source environment. LH channel sediments contained greater microbial biomass (~100-fold) and greater microbial diversity reflected by the 16S rRNA clone libraries. Phylotypes related to methanogenesis, methanotrophy, sulfur reduction and oxidation were detected in the bacterial clone libraries while the archaeal community was dominated by phylotypes most closely related to THE ammonia-oxidizing Thaumarchaeota. The cumulative percent recovery of 14C-acetate mineralization in channel sediment microcosms exceeded ~30% and ~10% at 5 and −5°C, respectively, but sharply decreased at −10°C (≤1%). Most bacterial isolates (Marinobacter, Planococcus, and Nesterenkonia spp.) were psychrotrophic, halotolerant, and capable of growth at −5°C. Overall, the hypersaline, subzero LH spring channel has higher microbial diversity and activity than the source, and supports a variety of niches reflecting the more dynamic and heterogeneous channel environment.