Publications by authors named "Susan M Pfiffner"

25 Publications

  • Page 1 of 1

Comparative Metagenomics of the Active Layer and Permafrost from Low-Carbon Soil in the Canadian High Arctic.

Environ Sci Technol 2021 09 2;55(18):12683-12693. Epub 2021 Sep 2.

Center for Environmental Biotechnology, University of Tennessee, Knoxville, Tennessee 37996, United States.

Approximately 87% of the Arctic consists of low-organic carbon mineral soil, but knowledge of microbial activity in low-carbon permafrost (PF) and active layer soils remains limited. This study investigated the taxonomic composition and genetic potential of microbial communities at contrasting depths of the active layer (5, 35, and 65 cm below surface, bls) and PF (80 cm bls). We showed microbial communities in PF to be taxonomically and functionally different from those in the active layer. 16S rRNA gene sequence analysis revealed higher biodiversity in the active layer than in PF, and biodiversity decreased significantly with depth. The reconstructed 91 metagenome-assembled genomes showed that PF was dominated by heterotrophic, fermenting Bacteroidota using nitrite as their main electron acceptor. Prevalent microbes identified in the active layer belonged to bacterial taxa, gaining energy via aerobic respiration. Gene abundance in metagenomes revealed enrichment of genes encoding the plant-derived polysaccharide degradation and metabolism of nitrate and sulfate in PF, whereas genes encoding methane/ammonia oxidation, cold-shock protein, and two-component systems were generally more abundant in the active layer, particularly at 5 cm bls. The results of this study deepen our understanding of the low-carbon Arctic soil microbiome and improve prediction of the impacts of thawing PF.
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http://dx.doi.org/10.1021/acs.est.1c00802DOI Listing
September 2021

Genomic reconstruction of fossil and living microorganisms in ancient Siberian permafrost.

Microbiome 2021 05 17;9(1):110. Epub 2021 May 17.

Princeton University, B88, Guyot Hall, Princeton, NJ, 08544, USA.

Background: Total DNA (intracellular, iDNA and extracellular, eDNA) from ancient permafrost records the mixed genetic repository of the past and present microbial populations through geological time. Given the exceptional preservation of eDNA under perennial frozen conditions, typical metagenomic sequencing of total DNA precludes the discrimination between fossil and living microorganisms in ancient cryogenic environments. DNA repair protocols were combined with high throughput sequencing (HTS) of separate iDNA and eDNA fraction to reconstruct metagenome-assembled genomes (MAGs) from ancient microbial DNA entrapped in Siberian coastal permafrost.

Results: Despite the severe DNA damage in ancient permafrost, the coupling of DNA repair and HTS resulted in a total of 52 MAGs from sediments across a chronosequence (26-120 kyr). These MAGs were compared with those derived from the same samples but without utilizing DNA repair protocols. The MAGs from the youngest stratum showed minimal DNA damage and thus likely originated from viable, active microbial species. Many MAGs from the older and deeper sediment appear related to past aerobic microbial populations that had died upon freezing. MAGs from anaerobic lineages, including Asgard archaea, however exhibited minimal DNA damage and likely represent extant living microorganisms that have become adapted to the cryogenic and anoxic environments. The integration of aspartic acid racemization modeling and metaproteomics further constrained the metabolic status of the living microbial populations. Collectively, combining DNA repair protocols with HTS unveiled the adaptive strategies of microbes to long-term survivability in ancient permafrost.

Conclusions: Our results indicated that coupling of DNA repair protocols with simultaneous sequencing of iDNA and eDNA fractions enabled the assembly of MAGs from past and living microorganisms in ancient permafrost. The genomic reconstruction from the past and extant microbial populations expanded our understanding about the microbial successions and biogeochemical alterations from the past paleoenvironment to the present-day frozen state. Furthermore, we provided genomic insights into long-term survival mechanisms of microorganisms under cryogenic conditions through geological time. The combined strategies in this study can be extrapolated to examine other ancient non-permafrost environments and constrain the search for past and extant extraterrestrial life in permafrost and ice deposits on Mars. Video abstract.
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http://dx.doi.org/10.1186/s40168-021-01057-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8130349PMC
May 2021

Detection of the deep biosphere in metamorphic rocks from the Chinese continental scientific drilling.

Geobiology 2021 05 9;19(3):278-291. Epub 2021 Feb 9.

State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China.

It is generally accepted that there is a vast, well-populated biosphere in the subsurface, but the depth limit of the terrestrial biosphere has yet to be determined, largely because of the lack of access to the subsurface. Here as part of the Chinese Continental Scientific Drilling (CCSD) project in eastern China, we acquired continuous rock cores and endeavored to probe the depth limit of the biosphere and the depth-dependent distribution of microorganisms at a geologically unique site, that is, a convergent plate boundary. Microbiological analyses of ultra-high-pressure metamorphic rock cores taken from the ground surface to 5,158-meter reveal that microbial distribution was continuous up to a depth of ~4,850 m, where temperature was estimated to be ~137°C. The metabolic state of these organisms at such great depth remains to be determined. Microbial abundance, ranging from 10 to 10  cells/g, was also related to porosity, but not to the depth and rock composition. In addition, microbial diversity systematically decreased with depth. Our results support the notion that temperature is a key factor in determining the lower limit of the biosphere in the continental subsurface.
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http://dx.doi.org/10.1111/gbi.12430DOI Listing
May 2021

Insights into community of photosynthetic microorganisms from permafrost.

FEMS Microbiol Ecol 2020 11;96(12)

Soil Cryology Laboratory, Institute of Physicochemical and Biological Problems in Soil Science, Institutskaya Street, Bldg. 2, Pushchino, Russia.

This work integrates cultivation studies of Siberian permafrost and analyses of metagenomes from different locations in the Arctic with the aim of obtaining insights into the community of photosynthetic microorganisms in perennially frozen deposits. Cyanobacteria and microalgae have been described in Arctic aquatic and surface soil environments, but their diversity and ability to withstand harsh conditions within the permafrost are still largely unknown. Community structure of photosynthetic organisms in permafrost sediments was explored using Arctic metagenomes available through the MG-RAST. Sequences affiliated with cyanobacteria represented from 0.25 to 3.03% of total sequences, followed by sequences affiliated with Streptophyta (algae and vascular plants) 0.01-0.45% and Chlorophyta (green algae) 0.01-0.1%. Enrichment and cultivation approaches revealed that cyanobacteria and green algae survive in permafrost and they could be revived during prolonged incubation at low light intensity. Among photosynthetic microorganisms isolated from permafrost, the filamentous Oscillatoria-like cyanobacteria and unicellular green algae of the genus Chlorella were dominant. Our findings suggest that permafrost cyanobacteria and green algae are expected to be effective members of the re-assembled community after permafrost thawing and soil collapse.
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http://dx.doi.org/10.1093/femsec/fiaa229DOI Listing
November 2020

Thaumarchaea Genome Sequences from a High Arctic Active Layer.

Microbiol Resour Announc 2020 May 21;9(21). Epub 2020 May 21.

Department of Geosciences, Princeton University, Princeton, New Jersey, USA

The role of archaeal ammonia oxidizers often exceeds that of bacterial ammonia oxidizers in marine and terrestrial environments but has been understudied in permafrost, where thawing has the potential to release ammonia. Here, three thaumarchaea genomes were assembled and annotated from metagenomic data sets from carbon-poor Canadian High Arctic active-layer cryosols.
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http://dx.doi.org/10.1128/MRA.00326-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7242670PMC
May 2020

Characterization of subsurface media from locations up- and down-gradient of a uranium-contaminated aquifer.

Chemosphere 2020 Sep 10;255:126951. Epub 2020 May 10.

University of Washington, Department of Civil and Environmental Engineering, Seattle, WA, USA.

The processing of sediment to accurately characterize the spatially-resolved depth profiles of geophysical and geochemical properties along with signatures of microbial density and activity remains a challenge especially in complex contaminated areas. This study processed cores from two sediment boreholes from background and contaminated core sediments and surrounding groundwater. Fresh core sediments were compared by depth to capture the changes in sediment structure, sediment minerals, biomass, and pore water geochemistry in terms of major and trace elements including pollutants, cations, anions, and organic acids. Soil porewater samples were matched to groundwater level, flow rate, and preferential flows and compared to homogenized groundwater-only samples from neighboring monitoring wells. Groundwater analysis of nearby wells only revealed high sulfate and nitrate concentrations while the same analysis using sediment pore water samples with depth was able to suggest areas high in sulfate- and nitrate-reducing bacteria based on their decreased concentration and production of reduced by-products that could not be seen in the groundwater samples. Positive correlations among porewater content, total organic carbon, trace metals and clay minerals revealed a more complicated relationship among contaminant, sediment texture, groundwater table, and biomass. The fluctuating capillary interface had high concentrations of Fe and Mn-oxides combined with trace elements including U, Th, Sr, Ba, Cu, and Co. This suggests the mobility of potentially hazardous elements, sediment structure, and biogeochemical factors are all linked together to impact microbial communities, emphasizing that solid interfaces play an important role in determining the abundance of bacteria in the sediments.
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http://dx.doi.org/10.1016/j.chemosphere.2020.126951DOI Listing
September 2020

Metagenome-Assembled Genome of USCα AHI, a Potential High-Affinity Methanotroph from Axel Heiberg Island, Canadian High Arctic.

Microbiol Resour Announc 2019 Nov 14;8(46). Epub 2019 Nov 14.

Department of Geosciences, Princeton University, Princeton, New Jersey, USA

Metagenomic sequencing of active-layer cryosols from the Canadian High Arctic has yielded a nearly complete genome for an atmospheric CH-oxidizing bacterium belonging to upland soil cluster α (USCα). This genome contains genes involved in CH metabolism, H metabolism, and multiple carbon assimilation pathways.
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http://dx.doi.org/10.1128/MRA.01178-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6856278PMC
November 2019

Deep-Subsurface Pressure Stimulates Metabolic Plasticity in Shale-Colonizing spp.

Appl Environ Microbiol 2019 06 30;85(12). Epub 2019 May 30.

Department of Soil and Crop Sciences, Colorado State University, Fort Collins, Colorado, USA

Bacterial strains become the dominant persisting microbial community member in produced fluids across geographically distinct hydraulically fractured shales. is believed to be inadvertently introduced into this environment during the drilling and fracturing process and must therefore tolerate large changes in pressure, temperature, and salinity. Here, we used a strain isolated from a natural gas well in the Utica Point Pleasant formation to investigate metabolic and physiological responses to growth under high-pressure subsurface conditions. Laboratory incubations confirmed the ability of strain WG8 to grow under pressures representative of deep shale formations (21 to 48 MPa). Under these conditions, broad metabolic and physiological shifts were identified, including higher abundances of proteins associated with the production of extracellular polymeric substances. Confocal laser scanning microscopy indicated that extracellular polymeric substance (EPS) production was associated with greater cell aggregation when biomass was cultured at high pressure. Changes in central carbon metabolism under the same conditions were inferred from nuclear magnetic resonance (NMR) and gas chromatography measurements, revealing large per-cell increases in production of ethanol, acetate, and propanol and cessation of hydrogen production. These metabolic shifts were associated with carbon flux through 1,2-propanediol in response to slower fluxes of carbon through stage 3 of glycolysis. Together, these results reveal the potential for bioclogging and corrosion (via organic acid fermentation products) associated with persistent growth in deep, hydraulically fractured shale ecosystems, and offer new insights into cellular mechanisms that enable these strains to dominate deep-shale microbiomes. The hydraulic fracturing of deep-shale formations for hydrocarbon recovery accounts for approximately 60% of U.S. natural gas production. Microbial activity associated with this process is generally considered deleterious due to issues associated with sulfide production, microbially induced corrosion, and bioclogging in the subsurface. Here we demonstrate that a representative species, frequently the dominant microbial taxon in hydraulically fractured shales, responds to pressures characteristic of the deep subsurface by shifting its metabolism to generate more corrosive organic acids and produce more polymeric substances that cause "clumping" of biomass. While the potential for increased corrosion of steel infrastructure and clogging of pores and fractures in the subsurface may significantly impact hydrocarbon recovery, these data also offer new insights for microbial control in these ecosystems.
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http://dx.doi.org/10.1128/AEM.00018-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6544827PMC
June 2019

Microbial lipid biomarkers detected in deep subsurface black shales.

Environ Sci Process Impacts 2019 Feb;21(2):291-307

Department of Geology and Geography, West Virginia University, Morgantown, WV 26506, USA.

Evidence for microbes has been detected in extreme subsurface environments as deep as 2.5 km with temperatures as high as 90 °C, demonstrating that microbes can adapt and survive extreme environmental conditions. Deep subsurface shales are increasingly exploited for their energy applications, thus characterizing the prevalence and role of microbes in these ecosystems essential for understanding biogeochemical cycles and maximizing production from hydrocarbon-bearing formations. Here, we describe the distribution of bacterial ester-linked phospholipid fatty acids (PLFA) and diglyceride fatty acids (DGFA) in sidewall cores retrieved from three distinct geologic horizons collected to 2275 m below ground surface in a Marcellus Shale well, West Virginia, USA. We examined the abundance and variety of PLFA and DGFA prior to energy development within and above the Marcellus Shale Formation into the overlying Mahantango Formation of the Appalachian Basin. Lipid biomarkers in the cores suggest the presence of microbial communities comprising Gram (+), Gram (-) as well as stress indicative biomarkers. Microbial PLFA and DGFA degradation in the subsurface can be influenced by stressful environmental conditions associated with the subsurface. The PLFA concentration and variety were higher in the transition zone between the extremely low permeability Marcellus Shale Formation and the more permeable Mahantango Formation. In contrast to this distribution, more abundant and diverse DGFA membrane profiles were associated with the Mahantango Formation. The stress indicative biomarkers like the trans-membrane fatty acids, oxiranes, keto-, and dimethyl lipid fatty acids were present in all cores, potentially indicating that the bacterial communities had experienced physiological stress or nutrient deprivation during or after deposition. The DGFA profiles expressed more stress indicative biomarkers as opposed to the PLFA membrane profiles. These findings suggest the probable presence of indigenous microbial communities in the deep subsurface shale and also improves our understanding of microbial survival mechanisms in ancient deep subsurface environments.
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http://dx.doi.org/10.1039/c8em00444gDOI Listing
February 2019

Impacts of Methane on Carbon Dioxide Storage in Brine Formations.

Ground Water 2018 03 16;56(2):176-186. Epub 2018 Jan 16.

Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN.

In the context of geological carbon sequestration (GCS), carbon dioxide (CO ) is often injected into deep formations saturated with a brine that may contain dissolved light hydrocarbons, such as methane (CH ). In this multicomponent multiphase displacement process, CO competes with CH in terms of dissolution, and CH tends to exsolve from the aqueous into a gaseous phase. Because CH has a lower viscosity than injected CO , CH is swept up into a 'bank' of CH -rich gas ahead of the CO displacement front. On the one hand, this may provide a useful tracer signal of an approaching CO front. On the other hand, the emergence of gaseous CH is undesirable because it poses a leakage risk of a far more potent greenhouse gas than CO if the cap rock is compromised. Open fractures or faults and wells could result in CH contamination of overlying groundwater aquifers as well as surface emissions. We investigate this process through detailed numerical simulations for a large-scale GCS pilot project (near Cranfield, Mississippi) for which a rich set of field data is available. An accurate cubic-plus-association equation-of-state is used to describe the non-linear phase behavior of multiphase brine-CH -CO mixtures, and breakthrough curves in two observation wells are used to constrain transport processes. Both field data and simulations indeed show the development of an extensive plume of CH -rich (up to 90 mol%) gas as a consequence of CO injection, with important implications for the risk assessment of future GCS projects.
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http://dx.doi.org/10.1111/gwat.12633DOI Listing
March 2018

Corrigendum: Modified Lipid Extraction Methods for Deep Subsurface Shale.

Front Microbiol 2017 24;8:2141. Epub 2017 Oct 24.

Department of Geology and Geography, West Virginia University, Morgantown, WV, United States.

[This corrects the article on p. 1408 in vol. 8, PMID: 28790998.].
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http://dx.doi.org/10.3389/fmicb.2017.02141DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5660830PMC
October 2017

Modified Lipid Extraction Methods for Deep Subsurface Shale.

Front Microbiol 2017 25;8:1408. Epub 2017 Jul 25.

Department of Geology and Geography, West Virginia UniversityMorgantown, WV, United States.

Growing interest in the utilization of black shales for hydrocarbon development and environmental applications has spurred investigations of microbial functional diversity in the deep subsurface shale ecosystem. Lipid biomarker analyses including phospholipid fatty acids (PLFAs) and diglyceride fatty acids (DGFAs) represent sensitive tools for estimating biomass and characterizing the diversity of microbial communities. However, complex shale matrix properties create immense challenges for microbial lipid extraction procedures. Here, we test three different lipid extraction methods: modified Bligh and Dyer (mBD), Folch (FOL), and microwave assisted extraction (MAE), to examine their ability in the recovery and reproducibility of lipid biomarkers in deeply buried shales. The lipid biomarkers were analyzed as fatty acid methyl esters (FAMEs) with the GC-MS, and the average PL-FAME yield ranged from 67 to 400 pmol/g, while the average DG-FAME yield ranged from 600 to 3,000 pmol/g. The biomarker yields in the intact phospholipid Bligh and Dyer treatment (mBD + Phos + POPC), the Folch, the Bligh and Dyer citrate buffer (mBD-Cit), and the MAE treatments were all relatively higher and statistically similar compared to the other extraction treatments for both PLFAs and DGFAs. The biomarker yields were however highly variable within replicates for most extraction treatments, although the mBD + Phos + POPC treatment had relatively better reproducibility in the consistent fatty acid profiles. This variability across treatments which is associated with the highly complex nature of deeply buried shale matrix, further necessitates customized methodological developments for the improvement of lipid biomarker recovery.
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http://dx.doi.org/10.3389/fmicb.2017.01408DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5524817PMC
July 2017

The unique chemistry of Eastern Mediterranean water masses selects for distinct microbial communities by depth.

PLoS One 2015 25;10(3):e0120605. Epub 2015 Mar 25.

Department of Civil and Environmental Engineering, University of Tennessee, Knoxville, Tennessee, United States of America; Center for Environmental Biotechnology, University of Tennessee, Knoxville, Tennessee, United States of America; Department of Earth and Planetary Sciences, University of Tennessee, Knoxville, Tennessee, United States of America; Department of Microbiology, University of Tennessee, Knoxville, Tennessee, United States of America; Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, United States of America.

The waters of the Eastern Mediterranean are characterized by unique physical and chemical properties within separate water masses occupying different depths. Distinct water masses are present throughout the oceans, which drive thermohaline circulation. These water masses may contain specific microbial assemblages. The goal of this study was to examine the effect of physical and geological phenomena on the microbial community of the Eastern Mediterranean water column. Chemical measurements were combined with phospholipid fatty acid (PLFA) analysis and high-throughput 16S rRNA sequencing to characterize the microbial community in the water column at five sites. We demonstrate that the chemistry and microbial community of the water column were stratified into three distinct water masses. The salinity and nutrient concentrations vary between these water masses. Nutrient concentrations increased with depth, and salinity was highest in the intermediate water mass. Our PLFA analysis indicated different lipid classes were abundant in each water mass, suggesting that distinct groups of microbes inhabit these water masses. 16S rRNA gene sequencing confirmed the presence of distinct microbial communities in each water mass. Taxa involved in autotrophic nitrogen cycling were enriched in the intermediate water mass suggesting that microbes in this water mass may be important to the nitrogen cycle of the Eastern Mediterranean. The Eastern Mediterranean also contains numerous active hydrocarbon seeps. We sampled above the North Alex Mud Volcano, in order to test the effect of these geological features on the microbial community in the adjacent water column. The community in the waters overlaying the mud volcano was distinct from other communities collected at similar depths and was enriched in known hydrocarbon degrading taxa. Our results demonstrate that physical phenomena such stratification as well as geological phenomena such as mud volcanoes strongly affect microbial community structure in the Eastern Mediterranean water column.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0120605PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4373936PMC
February 2016

Microbial community composition and diversity in Caspian Sea sediments.

FEMS Microbiol Ecol 2015 Jan 5;91(1):1-11. Epub 2014 Dec 5.

Department of Civil and Environmental Engineering, University of Tennessee, 37996-2313 Knoxville, TN BioSciences Division, Oak Ridge National Laboratory, 37831-6038 Oak Ridge, TN Center for Environmental Biotechnology, University of Tennessee, 37996-1605 Knoxville, TN Department of Earth and Planetary Sciences, University of Tennessee, 37996-1410 Knoxville, TN Department of Microbiology, University of Tennessee, 37996-0845 Knoxville, TN.

The Caspian Sea is heavily polluted due to industrial and agricultural effluents as well as extraction of oil and gas reserves. Microbial communities can influence the fate of contaminants and nutrients. However, insight into the microbial ecology of the Caspian Sea significantly lags behind other marine systems. Here we describe microbial biomass, diversity and composition in sediments collected from three sampling stations in the Caspian Sea. Illumina sequencing of 16S rRNA genes revealed the presence of a number of known bacterial and archaeal heterotrophs suggesting that organic carbon is a primary factor shaping microbial communities. Surface sediments collected from bottom waters with low oxygen levels were dominated by Gammaproteobacteria while surface sediments collected from bottom waters under hypoxic conditions were dominated by Deltaproteobacteria, specifically sulfate-reducing bacteria. Thaumarchaeota was dominant across all surface sediments indicating that nitrogen cycling in this system is strongly influenced by ammonia-oxidizing archaea. This study provides a baseline assessment that may serve as a point of reference as this system changes or as the efficacy of new remediation efforts are implemented.
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http://dx.doi.org/10.1093/femsec/fiu013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4399438PMC
January 2015

Metagenomes from thawing low-soil-organic-carbon mineral cryosols and permafrost of the canadian high arctic.

Genome Announc 2014 Nov 20;2(6). Epub 2014 Nov 20.

Microbial release of greenhouse gases from thawing permafrost is a global concern. Seventy-six metagenomes were generated from low-soil-organic-carbon mineral cryosols from Axel Heiberg Island, Nunavut, Canada, during a controlled thawing experiment. Permafrost thawing resulted in an increase in anaerobic fermenters and sulfate-reducing bacteria but not methanogens.
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http://dx.doi.org/10.1128/genomeA.01217-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4239366PMC
November 2014

Draft genome sequences of 10 strains of the genus exiguobacterium.

Genome Announc 2014 Oct 16;2(5). Epub 2014 Oct 16.

DOE Joint Genome Institute, Walnut Creek, California, USA.

High-quality draft genome sequences were determined for 10 Exiguobacterium strains in order to provide insight into their evolutionary strategies for speciation and environmental adaptation. The selected genomes include psychrotrophic and thermophilic species from a range of habitats, which will allow for a comparison of metabolic pathways and stress response genes.
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http://dx.doi.org/10.1128/genomeA.01058-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4200161PMC
October 2014

Trends and future challenges in sampling the deep terrestrial biosphere.

Front Microbiol 2014 12;5:481. Epub 2014 Sep 12.

Department of Geological Sciences, Michigan State University East Lansing, MI, USA.

Research in the deep terrestrial biosphere is driven by interest in novel biodiversity and metabolisms, biogeochemical cycling, and the impact of human activities on this ecosystem. As this interest continues to grow, it is important to ensure that when subsurface investigations are proposed, materials recovered from the subsurface are sampled and preserved in an appropriate manner to limit contamination and ensure preservation of accurate microbial, geochemical, and mineralogical signatures. On February 20th, 2014, a workshop on "Trends and Future Challenges in Sampling The Deep Subsurface" was coordinated in Columbus, Ohio by The Ohio State University and West Virginia University faculty, and sponsored by The Ohio State University and the Sloan Foundation's Deep Carbon Observatory. The workshop aims were to identify and develop best practices for the collection, preservation, and analysis of terrestrial deep rock samples. This document summarizes the information shared during this workshop.
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http://dx.doi.org/10.3389/fmicb.2014.00481DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4162470PMC
October 2014

Commercial DNA extraction kits impact observed microbial community composition in permafrost samples.

FEMS Microbiol Ecol 2014 Jan 17;87(1):217-30. Epub 2013 Oct 17.

University of Tennessee, Knoxville, TN, USA.

The total community genomic DNA (gDNA) from permafrost was extracted using four commercial DNA extraction kits. The gDNAs were compared using quantitative real-time PCR (qPCR) targeting 16S rRNA genes and bacterial diversity analyses obtained via 454 pyrosequencing of the 16S rRNA (V3 region) amplified in single or nested PCR. The FastDNA(®) SPIN (FDS) Kit provided the highest gDNA yields and 16S rRNA gene concentrations, followed by MoBio PowerSoil(®) (PS) and MoBio PowerLyzer™ (PL) kits. The lowest gDNA yields and 16S rRNA gene concentrations were from the Meta-G-Nome™ (MGN) DNA Isolation Kit. Bacterial phyla identified in all DNA extracts were similar to that found in other soils and were dominated by Actinobacteria, Firmicutes, Gemmatimonadetes, Proteobacteria, and Acidobacteria. Weighted UniFrac and statistical analyses indicated that bacterial community compositions derived from FDS, PS, and PL extracts were similar to each other. However, the bacterial community structure from the MGN extracts differed from other kits exhibiting higher proportions of easily lysed β- and γ-Proteobacteria and lower proportions of Actinobacteria and Methylocystaceae important in carbon cycling. These results indicate that gDNA yields differ between the extraction kits, but reproducible bacterial community structure analysis may be accomplished using gDNAs from the three bead-beating lysis extraction kits.
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http://dx.doi.org/10.1111/1574-6941.12219DOI Listing
January 2014

Comparative c-type cytochrome expression analysis in Shewanella oneidensis strain MR-1 and Anaeromyxobacter dehalogenans strain 2CP-C grown with soluble and insoluble oxidized metal electron acceptors.

Biochem Soc Trans 2012 Dec;40(6):1204-10

Graduate School of Genome Science and Technology, University of Tennessee, Knoxville, TN 37919, USA.

The genomes of Shewanella oneidensis strain MR-1 and Anaeromyxobacter dehalogenans strain 2CP-C encode 40 and 69 putative c-type cytochrome genes respectively. Deletion mutant and biochemical studies have assigned specific functions to a few c-type cytochromes involved in electron transfer to oxidized metals in S. oneidensis strain MR-1. Although promising, the genetic approach is limited to gene deletions that produce a distinct phenotype and to an organism for which a genetic system is available. To investigate and compare c-type cytochrome expression in S. oneidensis strain MR-1 and Anaeromyxobacter dehalogenans strain 2CP-C more comprehensively, proteomic measurements were used to characterize lysates of cells grown with soluble Fe(III) (as ferric citrate) and insoluble Mn(IV) (as MnO2) as electron acceptors. Strain MR-1 expressed 19 and 20, and strain 2CP-C expressed 27 and 25, c-type cytochromes when grown with Fe(III) and Mn(IV) respectively. The majority of c-type cytochromes (77% for strain MR-1 and 63% for strain 2CP-C) were expressed under both growth conditions; however, the analysis also revealed unique c-type cytochromes that were specifically expressed in cells grown with soluble Fe(III) or insoluble Mn(IV). Proteomic characterization proved to be a promising approach for determining the c-type cytochrome complement expressed under different growth conditions, and will help to elucidate the specific functions of more c-type cytochromes that are the basis for Shewanella and Anaeromyxobacter respiratory versatility.
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http://dx.doi.org/10.1042/BST20120182DOI Listing
December 2012

Microbial communities in subpermafrost saline fracture water at the Lupin Au mine, Nunavut, Canada.

Microb Ecol 2009 Nov 1;58(4):786-807. Epub 2009 Jul 1.

Department of Geosciences, Princeton University, Princeton, 08544, NJ 08544, USA.

We report the first investigation of a deep subpermafrost microbial ecosystem, a terrestrial analog for the Martian subsurface. Our multidisciplinary team analyzed fracture water collected at 890 and 1,130 m depths beneath a 540-m-thick permafrost layer at the Lupin Au mine (Nunavut, Canada). 14C, 3H, and noble gas isotope analyses suggest that the Na-Ca-Cl, suboxic, fracture water represents a mixture of geologically ancient brine, approximately25-kyr-old, meteoric water and a minor modern talik-water component. Microbial planktonic concentrations were approximately10(3) cells mL(-1). Analysis of the 16S rRNA gene from extracted DNA and enrichment cultures revealed 42 unique operational taxonomic units in 11 genera with Desulfosporosinus, Halothiobacillus, and Pseudomonas representing the most prominent phylotypes and failed to detect Archaea. The abundance of terminally branched and midchain-branched saturated fatty acids (5 to 15 mol%) was consistent with the abundance of Gram-positive bacteria in the clone libraries. Geochemical data, the ubiquinone (UQ) abundance (3 to 11 mol%), and the presence of both aerobic and anaerobic bacteria indicated that the environment was suboxic, not anoxic. Stable sulfur isotope analyses of the fracture water detected the presence of microbial sulfate reduction, and analyses of the vein-filling pyrite indicated that it was in isotopic equilibrium with the dissolved sulfide. Free energy calculations revealed that sulfate reduction and sulfide oxidation via denitrification and not methanogenesis were the most thermodynamically viable consistent with the principal metabolisms inferred from the 16S rRNA community composition and with CH4 isotopic compositions. The sulfate-reducing bacteria most likely colonized the subsurface during the Pleistocene or earlier, whereas aerobic bacteria may have entered the fracture water networks either during deglaciation prior to permafrost formation 9,000 years ago or from the nearby talik through the hydrologic gradient created during mine dewatering. Although the absence of methanogens from this subsurface ecosystem is somewhat surprising, it may be attributable to an energy bottleneck that restricts their migration from surface permafrost deposits where they are frequently reported. These results have implications for the biological origin of CH4 on Mars.
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http://dx.doi.org/10.1007/s00248-009-9553-5DOI Listing
November 2009

Sulfur isotope enrichment during maintenance metabolism in the thermophilic sulfate-reducing bacterium Desulfotomaculum putei.

Appl Environ Microbiol 2009 Sep 26;75(17):5621-30. Epub 2009 Jun 26.

Department of Geosciences, Princeton University, Princeton, NJ 08544, USA.

Values of Delta(34)S (=delta(34)S(HS)-delta(34)S(SO(4)), where delta(34)S(HS) and delta(34)S(SO(4)) indicate the differences in the isotopic compositions of the HS(-) and SO(4)(2-) in the eluent, respectively) for many modern marine sediments are in the range of -55 to -75 per thousand, much greater than the -2 to -46 per thousand epsilon(34)S (kinetic isotope enrichment) values commonly observed for microbial sulfate reduction in laboratory batch culture and chemostat experiments. It has been proposed that at extremely low sulfate reduction rates under hypersulfidic conditions with a nonlimited supply of sulfate, isotopic enrichment in laboratory culture experiments should increase to the levels recorded in nature. We examined the effect of extremely low sulfate reduction rates and electron donor limitation on S isotope fractionation by culturing a thermophilic, sulfate-reducing bacterium, Desulfotomaculum putei, in a biomass-recycling culture vessel, or "retentostat." The cell-specific rate of sulfate reduction and the specific growth rate decreased progressively from the exponential phase to the maintenance phase, yielding average maintenance coefficients of 10(-16) to 10(-18) mol of SO(4) cell(-1) h(-1) toward the end of the experiments. Overall S mass and isotopic balance were conserved during the experiment. The differences in the delta(34)S values of the sulfate and sulfide eluting from the retentostat were significantly larger, attaining a maximum Delta(34)S of -20.9 per thousand, than the -9.7 per thousand observed during the batch culture experiment, but differences did not attain the values observed in marine sediments.
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http://dx.doi.org/10.1128/AEM.02948-08DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2737900PMC
September 2009

Use of gene probes to assess the impact and effectiveness of aerobic in situ bioremediation of TCE.

Arch Microbiol 2009 Mar 26;191(3):221-32. Epub 2008 Nov 26.

Lawrence Berkeley National Laboratory, Center for Environmental Biotechnology, MS 70A-3317, One Cyclotron Rd, Berkeley, CA 94720, USA.

Gene probe hybridization was used to determine distribution and expression of co-metabolic genes at a contaminated site as it underwent in situ methanotrophic bioremediation of trichloroethylene (TCE). The bioremediation strategies tested included a series of air, air:methane, and air:methane:nutrient pulses of the test plot using horizontal injection wells. During the test period, the levels of TCE reduced drastically in almost all test samples. Sediment core samples (n=367) taken from 0 m (surface)-43 m depth were probed for gene coding for methanotrophic soluble methane monooxygenase (sMMO) and heterotrophic toluene dioxygenase (TOD), which are known to co-metabolize TCE. The same sediment samples were also probed for genes coding for methanol dehydrogenase (MDH) (catalyzing the oxidation of methanol to formaldehyde) to assess specifically changes in methylotrophic bacterial populations in the site. Gene hybridization results showed that the frequency of detection of sMMO genes were stimulated approximately 250% following 1% methane:air (v/v) injection. Subsequent injection of 4% methane:air (v/v) resulted in an 85% decline probably due to nutrient limitations, since addition of nutrients (gaseous nitrogen and phosphorus) thereafter caused an increase in the frequency of detection of sMMO genes. Detection of TOD genes declined during the process, and eventually they were non-detectable by the final treatment, suggesting that methanotrophs displaced the TOD gene containing heterotrophs. Active transcription of sMMO and TOD was evidenced by hybridization to mRNA. These analyses combined with results showing the concomitant decline in TCE concentrations, increases in chloride concentration and increases in methanotroph viable counts, provide multiple lines of evidence that TCE remediation was caused specifically by methanotrophs. Our results suggest that sMMO genes are responsible for most, if not all, of the observed biodegradation of TCE. This study demonstrates that the use of nucleic acid analytical methods provided a gene specific assessment of the effects of in situ treatment technologies.
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http://dx.doi.org/10.1007/s00203-008-0445-8DOI Listing
March 2009

Application of nonlinear analysis methods for identifying relationships between microbial community structure and groundwater geochemistry.

Microb Ecol 2006 Feb 31;51(2):177-88. Epub 2006 Jan 31.

Computational Sciences and Engineering Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA.

The relationship between groundwater geochemistry and microbial community structure can be complex and difficult to assess. We applied nonlinear and generalized linear data analysis methods to relate microbial biomarkers (phospholipids fatty acids, PLFA) to groundwater geochemical characteristics at the Shiprock uranium mill tailings disposal site that is primarily contaminated by uranium, sulfate, and nitrate. First, predictive models were constructed using feedforward artificial neural networks (NN) to predict PLFA classes from geochemistry. To reduce the danger of overfitting, parsimonious NN architectures were selected based on pruning of hidden nodes and elimination of redundant predictor (geochemical) variables. The resulting NN models greatly outperformed the generalized linear models. Sensitivity analysis indicated that tritium, which was indicative of riverine influences, and uranium were important in predicting the distributions of the PLFA classes. In contrast, nitrate concentration and inorganic carbon were least important, and total ionic strength was of intermediate importance. Second, nonlinear principal components (NPC) were extracted from the PLFA data using a variant of the feedforward NN. The NPC grouped the samples according to similar geochemistry. PLFA indicators of Gram-negative bacteria and eukaryotes were associated with the groups of wells with lower levels of contamination. The more contaminated samples contained microbial communities that were predominated by terminally branched saturates and branched monounsaturates that are indicative of metal reducers, actinomycetes, and Gram-positive bacteria. These results indicate that the microbial community at the site is coupled to the geochemistry and knowledge of the geochemistry allows prediction of the community composition.
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http://dx.doi.org/10.1007/s00248-004-0137-0DOI Listing
February 2006

Desulfotomaculum and Methanobacterium spp. dominate a 4- to 5-kilometer-deep fault.

Appl Environ Microbiol 2005 Dec;71(12):8773-83

Environmental Microbiology Group, Pacific Northwest National Laboratory, Richland, Washington 99352, USA.

Alkaline, sulfidic, 54 to 60 degrees C, 4 to 53 million-year-old meteoric water emanating from a borehole intersecting quartzite-hosted fractures >3.3 km beneath the surface supported a microbial community dominated by a bacterial species affiliated with Desulfotomaculum spp. and an archaeal species related to Methanobacterium spp. The geochemical homogeneity over the 650-m length of the borehole, the lack of dividing cells, and the absence of these microorganisms in mine service water support an indigenous origin for the microbial community. The coexistence of these two microorganisms is consistent with a limiting flux of inorganic carbon and SO4(2-) in the presence of high pH, high concentrations of H2 and CH4, and minimal free energy for autotrophic methanogenesis. Sulfide isotopic compositions were highly enriched, consistent with microbial SO4(2-) reduction under hydrologic isolation. An analogous microbial couple and similar abiogenic gas chemistry have been reported recently for hydrothermal carbonate vents of the Lost City near the Mid-Atlantic Ridge (D. S. Kelly et al., Science 307:1428-1434, 2005), suggesting that these features may be common to deep subsurface habitats (continental and marine) bearing this geochemical signature. The geochemical setting and microbial communities described here are notably different from microbial ecosystems reported for shallower continental subsurface environments.
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http://dx.doi.org/10.1128/AEM.71.12.8773-8783.2005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1317344PMC
December 2005
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