Li-Hung Lin

Environmental genomics reveals a single-species ecosystem deep within earth

DNA from low-biodiversity fracture water collected at 2.8-kilometer depth in a South African gold mine was sequenced and assembled into a single, complete genome. This bacterium, Candidatus Desulforudis audaxviator, composes >99.9% of the microorganisms inhabiting the fluid phase of this particular fracture. Its genome indicates a motile, sporulating, sulfate-reducing, chemoautotrophic thermophile that can fix its own nitrogen and carbon by using machinery shared with archaea. Candidatus Desulforudis audaxviator is capable of an independent life-style well suited to long-term isolation from the photosphere deep within Earths crust and offers an example of a natural ecosystem that appears to have its biological component entirely encoded within a single genome.

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.

Dating ultra-deep mine waters with noble gases and 36Cl, Witwatersrand Basin, South Africa

Abstract Concentrations and isotopic ratios of dissolved noble gases, 36Cl, δD and δ18O in water samples from the ultra-deep gold mines (0.718 to 3.3 km below the surface) in the Witwatersrand Basin, South Africa, were investigated to quantify the dynamics of these ultra deep crustal fluids. The mining activity has a significant impact on the concentrations of dissolved gases, as the associated pressure release causes the degassing of the fissure water. The observed under saturation of the atmospheric noble gases in the fissure water samples (70–98%, normalized to ASW at 20°C and 1013 mbar) is reproduced by a model that considers diffusive degassing and solubility equilibration with a gas phase at sampling temperature. Corrections for degassing result in 4He concentrations as high as 1.55 · 10−1cm3STP4He g−1, 40Ar/36Ar ranging between 806 and 10331, and 134Xe/132Xe and 136Xe/132Xe ratios above 0.46 and 0.44, respectively. Corrected 134(136)Xe/132Xe and 134(136)Xe/4He-ratios are consistent with their production ratios, whereas the nucleogenic 4He/40Ar, and 134(136)Xe/40Ar ratios generally indicate that these gases are produced in an environment with an average [U + Th]/K-content 2–3 times above that of crustal average. In two scenarios, one considering only accumulation of in situ produced noble gases, the other additionally crustal flux components, the model ages for 14 individual water samples range from 13 to 168 Ma and from 1 to 23 Ma, respectively. The low 36Cl-ratios of (4–37) · 10−15 and comparatively high 36Cl-concentrations of (8–350) · 10−15 atoms 36Cl l−1 reflect subsurface production in secular equilibrium indicating an age in excess of 1.5 Ma or 5 times the half-life of 36Cl. In combination, the results suggest residence times of the fluids in fissures in this region (up to 3.3 km depth) are of the order of 1–100 Ma. We cannot exclude the possibility of mixing and that small quantities of younger water have been mixed with the very old bulk.

The Distribution of Microbial Taxa in the Subsurface Water of the Kalahari Shield, South Africa

Microbial communities within deep subsurface environments were analyzed by 16S rRNA gene cloning. Clone libraries from 27 borehole fluid, 7 mining-contaminated, and 5 rock samples were compared. Borehole fluids derived from deep fractures were populated by microbial communities with low diversity with an average of 11 and 5 bacterial and archaeal OTUs respectively. Low taxa richness was likely driven by limited biogeochemical reactions available for growth and not extreme parameters such as pH and temperature. Novel taxa of Firmicutes were discovered, commonly found in warm, slightly alkaline, anoxic fracture fluids. Highly divergent lineages of Archaea, unique to South African deep subsurface fracture fluids, are also described. Clone library clustering analyses based on LIBSHUFF phylogenetic relatedness revealed distinct groups of samples corresponding with sample source and geochemistry.

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.

Hydrogeologic Controls on Episodic H2 Release from Precambrian Fractured Rocks—Energy for Deep Subsurface Life on Earth and Mars

Dissolved H(2) concentrations up to the mM range and H(2) levels up to 9-58% by volume in the free gas phase are reported for groundwaters at sites in the Precambrian shields of Canada and Finland. Along with previously reported dissolved H(2) concentrations up to 7.4 mM for groundwaters from the Witwatersrand Basin, South Africa, these findings indicate that deep Precambrian Shield fracture waters contain some of the highest levels of dissolved H(2) ever reported and represent a potentially important energy-rich environment for subsurface microbial life. The delta (2)H isotope signatures of H(2) gas from Canada, Finland, and South Africa are consistent with a range of H(2)-producing water-rock reactions, depending on the geologic setting, which include both serpentinization and radiolysis. In Canada and Finland, several of the sites are in Archean greenstone belts characterized by ultramafic rocks that have under-gone serpentinization and may be ancient analogues for serpentinite-hosted gases recently reported at the Lost City Hydrothermal Field and other hydrothermal seafloor deposits. The hydrogeologically isolated nature of these fracture-controlled groundwater systems provides a mechanism whereby the products of water-rock interaction accumulate over geologic timescales, which produces correlations between high H(2) levels, abiogenic hydrocarbon signatures, and the high salinities and highly altered delta (18)O and delta (2)H values of these groundwaters. A conceptual model is presented that demonstrates how periodic opening of fractures and resultant mixing control the distribution and supply of H(2) and support a microbial community of H(2)-utilizing sulfate reducers and methanogens.

Planktonic Microbial Communities Associated with Fracture-Derived Groundwater in a Deep Gold Mine of South Africa

The vertical distribution and function of terrestrial planktonic microbial communities at depths greater than 600 m remain poorly established. Culture-independent methods using 16S rRNA genes and geochemical approaches were employed to investigate the heterogeneity and potential function of microbial communities residing within fractures at 0.7 to 1.4 kilometers below land surface of Beatrix Au Mine, South Africa. The salinity (26 to 47 mM Cl−), temperature (33 to 40°C) and age (1 to 5 Ma) of these fracture water increased with depth. The δD and δ18O values of fracture water ranged from −44 to −39‰ and from −7 to −4‰ VSMOW, respectively, and exhibited a mixing trend with fracture water collected from the same mine in a previous study where isotopic signatures were indicative of hydrothermal origin. Fracture water from Beatrix Mine was distinct from the groundwater in the overlying Karoo sedimentary strata in terms of its Cl−, He and CH4 concentrations, and its δD and δ18O signatures and from Vaal River (source of service water) in terms of its δD and δ18O signatures. The differences constrain the maximum amount of mixing with service water or shallow groundwater to be less than 4%. The 16S rDNA analyses revealed diverse and numerous novel 16S rRNA genes affiliated with Proteobacteria, Firmicutes, Nitrospira, Chlorobi, Thermus, Candidate Division OP3 and Euryarchaeota. The proportion of each phylum in clone libraries varied markedly among samples and suggests km-scale, spatial heterogeneity in community structures. Potential metabolisms inferred from the presence of 16S rRNA genes are generally consistent with estimates of the available free energy.

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.

Microbial methane cycling in a terrestrial mud volcano in eastern Taiwan

Microbial communities responsible for methane cycling in mud volcanoes onshore are poorly characterized. This study analysed bubbling fluids and cored sediments retrieved from a mud volcano in eastern Taiwan. The pore water profiles revealed that methane concentrations generally increased with depth and changed dramatically at different depth intervals at different sites. The methane concentrations were inversely correlated with Fe(2+)/Mn(2+) concentrations and δ(13)C values of methane, marking iron/manganese-methane transition zones in the sediment cores. Archaeal communities were dominated by ANME-2a members and methylotrophic methanogens, whereas bacterial communities consisted primarily of Proteobacteria, Firmicutes and Bacteroidetes. The 16S rRNA gene copy numbers of ANME-2a and Desulfuromonas/Pelobacter populations varied by two to three orders of magnitude along the profile and exhibited a pattern comparable with those of Fe(2+) and δ(13)C values of methane. These lines of evidence suggest a coupling between anaerobic methanotrophy and metal reduction in the metal-methane transition zones under sulfate-deficient conditions, a metabolic scheme contrasting with that observed in marine cold seeps. Anaerobic methanotrophs proliferate by removing methane produced from in situ methanogenesis and originating from the deep source. Methane finally emitted into the atmosphere is quantitatively and isotopically altered by various microbial processes compartmentalized at different depth intervals.

Mitogenomic sequences effectively recover relationships within brush-footed butterflies (Lepidoptera: Nymphalidae).

BackgroundMitogenomic phylogenies have revealed well-supported relationships for many eukaryote groups. In the order Lepidoptera, 113 species mitogenomes had been sequenced (May 14, 2014). However, these data are restricted to ten of the forty-three recognised superfamilies, while it has been challenging to recover large numbers of mitogenomes due to the time and cost required for primer design and sequencing. Nuclear rather than mitochondrial genes have been preferred to reconstruct deep-level lepidopteran phylogenies, without seriously evaluating the potential of entire mitogenomes. Next-generation sequencing methods remove these limitations by providing efficiently massive amounts of sequence data. In the present study, we simultaneously obtained a large number of nymphalid butterfly mitogenomes to evaluate the utility of mitogenomic phylogenies by comparing reconstructions to the now quite well established phylogeny of Nymphalidae.ResultsWe newly obtained 30 nymphalid mitogenomes via pyrosequencing on the Roche 454 GS Junior system, and combined these sequences with publicly accessible data to provide a 70-taxa dataset covering 37 genes for a 15,495 bp alignment. Polymorphic sites were not homogeneously distributed across the gene. Two gene regions, nad6 and 3’ end of nad5, were most variable, whereas the cox1 and 5’ ends of rrnL were most conserved. Phylogenetic relationships inferred by two likelihood methods were congruent and strongly supported (>0.95 posterior probability; ML bootstrap >85%), across the majority of nodes for multiple partitioning strategies and substitution models. Bayes factor results showed that the most highly partitioned dataset is the preferred strategy among different partitioning schemes. The most striking phylogenetic findings were that the subfamily Danainae not Libytheinae was sister of the remaining brush-footed butterflies and that, within Limenitidini, the genus Athyma was clearly polyphyletic. None of the single-gene phylogenies recovered the highly supported topologies generated on the basis of the whole mitogenomic data.ConclusionsThirty mitogenomes were assembled with 89% completeness from the contigs of pyrosequencing-derived reads. Entire mitogenomes or higher-quality sequences could be obtained by increasing pyrosequencing read coverage or by additional Sanger sequencing. Our mitogenomic phylogenies provide robust nodal support at a range of levels, demonstrating that mitogenomes are both accurate and efficient molecular markers for inferring butterfly phylogeny.