Susan M. Pfiffner

Alkaliphilus transvaalensis gen. nov., sp. nov., an extremely alkaliphilic bacterium isolated from a deep South African gold mine.

A novel extreme alkaliphile was isolated from a mine water containment dam at 3.2 km below land surface in an ultra-deep gold mine near Carletonville, South Africa. The cells of this bacterium were straight to slightly curved rods, motile by flagella and formed endospores. Growth was observed over the temperature range 20-50 degrees C (optimum 40 degrees C; 45 min doubling time) and pH range 8.5-12.5 (optimum pH 10.0). The novel isolate, one of the most alkaliphilic micro-organisms yet described, was a strictly anaerobic chemo-organotroph capable of utilizing proteinaceous substrates such as yeast extract, peptone, tryptone and casein. Elemental sulfur, thiosulfate or fumarate, when included as accessory electron acceptors, improved growth. The G+C content of genomic DNA was 36.4 mol %. Phylogenetic analysis based on the 16S rDNA sequence indicated that the isolate is a member of cluster XI within the low G+C gram-positive bacteria, but only distantly related to previously described members. On the basis of physiological and molecular properties, the isolate represents a novel species, for which the name Alkaliphilus transvaalensis gen. nov., sp. nov. is proposed (type strain SAGM1T = JCM 10712T = ATCC 700919T). The mechanism of generation of the highly alkaline microbial habitat and the possible source of the alkaliphile are discussed.

Comparison between geochemical and biological estimates of subsurface microbial activities

Geochemical and biological estimates of in situ microbial activities were compared from the aerobic and microaerophilic sediments of the Atlantic Coastal Plain. Radioisotope time-course experiments suggested oxidation rates greater than millimolar quantities per year for acetate and glucose. Geochemical analyses assessing oxygen consumption, soluble organic carbon utilization, sulfate reduction, and carbon dioxide production suggested organic oxidation rates of nano- to micromolar quantities per year. Radiotracer timecourse experiments appeared to overestimate rates of organic carbon oxidation, sulfate reduction, and biomass production by a factor of 103–106 greater than estimates calculated from groundwater analyses. Based on the geochemical evidence, in situ microbial metabolism was estimated to be in the nano- to micromolar range per year, and the average doubling time for the microbial community was estimated to be centuries.

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

ABSTRACT Alkaline, sulfidic, 54 to 60°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 SO42− 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 SO42− 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.

Methods for recovery of deep terrestrial subsurface sediments for microbiological studies

Methods for the aseptic recovery of sediments from the terrestrial deep subsurface for microbiological analyses are defined. Sediments were recovered from depths > 300 m by rotary drilling techniques using bentonite drilling techniques. Four sampling tools were successfully used and compared for their ability to retrieve different types of subsurface materials. Upon retrieval, sediments were pared and processed under anaerobic conditions in a glove bag. Materials were stored under N2 gas and shipped via overnight express to collaborating investigators. Six quality assurance protocols were incorporated to ensure that appropriate sediments were obtained and to monitor contamination from drilling fluid infringement. Two quality assurance protocols were field-applicable, and four were performed by independent laboratories. The quality assurance protocols provided multiple techniques for detecting 10 mg contamination from drilling fluids·kg−1 sediment. These techniques, which proved appropriate for different types of subsurface sediments, provided samples which were deemed acceptable for microbiological analyses.

Temporal Shifts in the Geochemistry and Microbial Community Structure of an Ultradeep Mine Borehole Following Isolation

A borehole draining a water-bearing dyke fracture at 3.2-km depth in a South African Au mine was isolated from the open mine environment. Geochemical, stable isotopic, nucleic acid-based, and phospholipid fatty acid (PLFA) analyses were employed as culture-independent means for assessing shifts in the microbial community and habitat as the system equilibrated with the native rock-water environment. Over a two-month period, the pH increased from 5.5 to 7.4, concurrent with a drop in pe from −2 to −3. Whereas rDNAs related to Desulfotomaculum spp. represented the major clone type encountered throughout, lipid biomarker profiling along with 16S rDNA clone library and terminal restriction fragment length polymorphism (T-RFLP) analyses indicated the emergence of other Gram-positive and deeply-branching lineages in samples during the later stages of the equilibration period. A biofilm that formed on the mine wall below the borehole produced abundant rDNAs related to the α Proteobacteria. β- and γ −Proteobacteria appeared to transiently bloom in the borehole shortly after isolation. Chemical modeling and sulfur isotope analyses of the borehole effluent indicated that microbial sulfate reduction was the major terminal electron-accepting process shortly after isolation, whereas Fe+3 reduction dominated towards the end of the experiment. The persistence of Desulfotomaculum-like bacteria throughout suggests that these organisms adapted to changing geochemical conditions as the redox decreased and pH increased following the isolation of the borehole from the mine atmosphere. The restoration of anaerobic aquatic chemistry to this borehole environment may have allowed microbiota indigenous to the local basalt aquifer to become more dominant among the diverse collection of bacterial lineages present in the borehole.

The origin and age of biogeochemical trends in deep fracture water of the Witwatersrand Basin, South Africa

Water residing within crustal fractures encountered during mining at depths greater than 500 meters in the Witwatersrand basin of South Africa represents a mixture of paleo-meteoric water and 2.0–2.3 Ga hydrothermal fluid. The hydrothermal fluid is highly saline, contains abiogenic CH 4 and hydrocarbon, occasionally N 2 , originally formed at ∼ 250–300°C and during cooling isotopically exchanged O and H with minerals and accrued H 2 , 4 He and other radiogenic gases. The paleo-meteoric water ranges in age from ∼ 10 Ka to > 1.5 Ma, is of low salinity, falls along the global meteoric water line (GMWL) and is CO 2 and atmospheric noble gas-rich. The hydrothermal fluid, which should be completely sterile, has probably been mixing with paleo-meteoric water for at least the past ∼100 Myr, a process which inoculates previously sterile environments at depths > 2.0 to 2.5 km. Free energy flux calculations suggest that sulfate reduction is the dominant electron acceptor microbial process for the high salinity fracture water and that it is 10 7 times that normally required for cell maintenance in lab cultures. Flux calculations also indicate that the potential bioavailable chemical energy increases with salinity, but because the fluence of bioavailable C, N and P also increase with salinity, the environment remains energy-limited. The 4 He concentrations and theoretical calculations indicate that the H 2 that is sustaining the subsurface microbial communities (e.g. H 2 -utilizing SRB and methanogens) is produced by water radiolysis at a rate of ∼1 nM yr −1 . Microbial CH 4 mixes with abiogenic CH 4 to produce the observed isotopic signatures and indicates that the rate of methanogenesis diminishes with depth from ∼ 100 at < 1 kmbls, to < 0.01 nM yr −1 at > 3 kmbls. Microbial Fe(III) reduction is limited due to the elevated pH. The δ13C of dissolved inorganic carbon is consistent with heterotrophy rather than autotrophy dominating the deeper, more saline environments. One potential source of the organic carbon may be microfilms present on the mineral surfaces.

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

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.

Geochemically Generated, Energy-Rich Substrates and Indigenous Microorganisms in Deep, Ancient Groundwater

Recent studies have shown that the biosphere extends to depths that exceed 3 km, raising questions regarding the age of the microbes in these deep ecosystems and their sources of energy for metabolism. Abiogenic energy sources that are derived from in situ, purely geochemical sources and thus independent from photosynthesis have been suggested. We sampled saline fracture water emanating from a 3.1-km deep borehole in a Au mine in the Witwatersrand Basin of South Africa and characterized the chemical constituents (including stable isotopes), groundwater age, and indigenous microorganisms. Salinity data and ratios of dissolved noble gases indicate that extremely ancient (2.0 Ga) saline fracture water has mixed with meteoric water to yield an average subsurface residence time of 20–160 Ma, the oldest age of any waters collected to date in the Witwatersrand Basin. H2 isotope data suggest the water originated from a depth of 4 to 5 km. Sulfur isotope fractionation indicates biological sulfate reduction. Calculations of free energies and steady state energy fluxes based on water chemistry data also support sulfate reduction as the dominant terminal electron accepting process. Lipid and flow cytometry data indicate a sparse microbial community (103 cells ml−1), despite the presence of relatively high concentrations of energy-rich compounds (H2, CH4, CO, ethane, propane, butane, and acetate). The H2 can be explained by radiolysis of water. Stable isotopic signatures of the CH4 and short chain hydrocarbons indicate abiogenic synthesis. The persistence of energy-rich compounds suggests that other factors are limiting to microbial metabolism and growth, e.g., availability of an inorganic nutrient, such as Fe or phosphate.

Deep Subsurface Microbial Biomass and Community Structure in Witwatersrand Basin Mines

The extreme environments of South Africa mines were investigated to determine microbial community structure and biomass in the deep subsurface. These community parameters were determined using phospholipid fatty acid (PLFA) technique. Air, water and rock samples were collected from several levels and shafts in eight different mines. Biomass estimates ranged over nine orders of magnitude. Biofilm samples exhibited the highest biomass with quantities ranging from 10 3 to 10 7 pmol PLFA g −1 . Rock samples had biomass ranging from 10 3 to 10 6 pmol PLFA g −1 . Mine service waters and rock fracture waters had biomass estimates ranging from 10 0 to 10 6 pmol PLFA L −1 . Air samples biomass values ranged from 10 −2 to 10 0 pmol PLFA L −1 . The biomass estimates were similar to those estimates for other deep subsurface sites. Redundancy analysis of the PLFA profiles distinguished between the sample types, where signature lipid biomarkers for aerobic and anaerobic prokaryotes, sulfate-and metal-reducing bacteria were associated with biofilms. Rock samples were enriched in 18:1 ω 9 c , 18:2 ω 6, br17:1s and br18:1s, which are indicative of microeukaryotes and metal- reducing bacteria. Air samples were enriched with 22:0, 17:1, 18:1, and a polyunsaturated fatty acid. Service waters had monounsaturated fatty acids. Fracture waters contained i17:0 and 10Me18:0 which indicated gram-positive and other anaerobic bacteria. When the fracture and service water sample PLFA responses to changes in environmental parameters of temperature, pH, and anion concentrations were analyzed, service waters correlated with higher nitrate and sulfate concentrations and the PLFAs 18:1 ω 7 c and 16:1 ω 7 c . Dreifontein shaft 5 samples correlated with chloride concentrations and terminally branched saturated fatty acids and branched monounsaturated fatty acids. Kloof, Tau Tona, and Merriespruit fracture waters aligned with temperature and pH vectors and 18:0, 20:0 and 22:6 ω 3. The redundancy analysis provided a robust method to understand the PLFA responses to changes in environmental parameters.

An active atmospheric methane sink in high Arctic mineral cryosols

Methane (CH4) emission by carbon-rich cryosols at the high latitudes in Northern Hemisphere has been studied extensively. In contrast, data on the CH4 emission potential of carbon-poor cryosols is limited, despite their spatial predominance. This work employs CH4 flux measurements in the field and under laboratory conditions to show that the mineral cryosols at Axel Heiberg Island in the Canadian high Arctic consistently consume atmospheric CH4. Omics analyses present the first molecular evidence of active atmospheric CH4-oxidizing bacteria (atmMOB) in permafrost-affected cryosols, with the prevalent atmMOB genotype in our acidic mineral cryosols being closely related to Upland Soil Cluster α. The atmospheric (atm) CH4 uptake at the study site increases with ground temperature between 0 °C and 18 °C. Consequently, the atm CH4 sink strength is predicted to increase by a factor of 5–30 as the Arctic warms by 5–15 °C over a century. We demonstrate that acidic mineral cryosols are a previously unrecognized potential of CH4 sink that requires further investigation to determine its potential impact on larger scales. This study also calls attention to the poleward distribution of atmMOB, as well as to the potential influence of microbial atm CH4 oxidation, in the context of regional CH4 flux models and global warming.