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1.  Comparative Metagenomics of Two Microbial Mats at Cuatro Ciénegas Basin I: Ancient Lessons on How to Cope with an Environment Under Severe Nutrient Stress 
Astrobiology  2012;12(7):648-658.
The Cuatro Ciénegas Basin (CCB) is an oasis in the desert of Mexico characterized by low phosphorus availability and by its great diversity of microbial mats. We compared the metagenomes of two aquatic microbial mats from the CCB with different nutrient limitations. We observed that the red mat was P-limited and dominated by Pseudomonas, while the green mat was N-limited and had higher species richness, with Proteobacteria and Cyanobacteria as the most abundant phyla. From their gene content, we deduced that both mats were very metabolically diverse despite their use of different strategies to cope with their respective environments. The red mat was found to be mostly heterotrophic, while the green mat was more autotrophic. The red mat had a higher number of transporters in general, including transporters of cellobiose and osmoprotectants. We suggest that generalists with plastic genomes dominate the red mat, while specialists with minimal genomes dominate the green mat. Nutrient limitation was a common scenario on the early planet; despite this, biogeochemical cycles were performed, and as a result the planet changed. The metagenomes of microbial mats from the CCB show the different strategies a community can use to cope with oligotrophy and persist. Key Words: Microbial mats—Metagenomics—Metabolism. Astrobiology 12, 648–658.
PMCID: PMC3426886  PMID: 22920515
2.  Comparative Metagenomics of Two Microbial Mats at Cuatro Ciénegas Basin II: Community Structure and Composition in Oligotrophic Environments 
Astrobiology  2012;12(7):659-673.
Microbial mats are self-sustained, functionally complex ecosystems that make good models for the understanding of past and present microbial ecosystems as well as putative extraterrestrial ecosystems. Ecological theory suggests that the composition of these communities might be affected by nutrient availability and disturbance frequency. We characterized two microbial mats from two contrasting environments in the oligotrophic Cuatro Ciénegas Basin: a permanent green pool and a red desiccation pond. We analyzed their taxonomic structure and composition by means of 16S rRNA clone libraries and metagenomics and inferred their metabolic role by the analysis of functional traits in the most abundant organisms. Both mats showed a high diversity with metabolically diverse members and strongly differed in structure and composition. The green mat had a higher species richness and evenness than the red mat, which was dominated by a lineage of Pseudomonas. Autotrophs were abundant in the green mat, and heterotrophs were abundant in the red mat. When comparing with other mats and stromatolites, we found that taxonomic composition was not shared at species level but at order level, which suggests environmental filtering for phylogenetically conserved functional traits with random selection of particular organisms. The highest diversity and composition similarity was observed among systems from stable environments, which suggests that disturbance regimes might affect diversity more strongly than nutrient availability, since oligotrophy does not appear to prevent the establishment of complex and diverse microbial mat communities. These results are discussed in light of the search for extraterrestrial life. Key Words: Cuatro Ciénegas—Metagenomics—Microbial mats—Oligotrophic—Phosphorus limitation—Stromatolites. Astrobiology 12, 659–673.
PMCID: PMC3426889  PMID: 22920516
3.  The Cuatro Ciénegas Basin in Coahuila, Mexico: An Astrobiological Precambrian Park 
Astrobiology  2012;12(7):641-647.
The Cuatro Ciénegas Basin (CCB) is a rare oasis in the Chihuahuan Desert in the state of Coahuila, Mexico. It has a biological endemism similar to that of the Galapagos Islands, and its spring-fed ecosystems have very low nutrient content (nitrogen or phosphorous) and are dominated by diverse microbialites. Thus, it has proven to be a distinctive opportunity for the field of astrobiology, as the CCB can be seen as a proxy for an earlier time in Earth's history, in particular the late Precambrian, the biological frontier when prokaryotic life yielded at least partial dominance to eukaryotes and multicellular life. It is a kind of ecological time machine that provides abundant opportunities for collaborative investigations by geochemists, geologists, ecologists, and population biologists in the study of the evolutionary processes that structured Earth-based life, especially in the microbial realm. The CCB is an object of investigation for the identification of biosignatures of past and present biota that can be used in our search for extraterrestrial life. In this review, we summarize CCB research efforts that began with microbial ecology and population biology projects and have since been expanded into broader efforts that involve biogeochemistry, comparative genomics, and assessments of biosignatures. We also propose that, in the future, the CCB is sanctioned as a “Precambrian Park” for astrobiology. Key Words: Microbial mats—Stromatolites—Early Earth—Extremophilic microorganisms—Microbial ecology. Astrobiology 12, 641–647.
PMCID: PMC3426885  PMID: 22920514
4.  Characterization of Phototrophic Purple Nonsulfur Bacteria Forming Colored Microbial Mats in a Swine Wastewater Ditch 
The community structure of pink-colored microbial mats naturally occurring in a swine wastewater ditch was studied by culture-independent biomarker and molecular methods as well as by conventional cultivation methods. The wastewater in the ditch contained acetate and propionate as the major carbon nutrients. Thin-section electron microscopy revealed that the microbial mats were dominated by rod-shaped cells containing intracytoplasmic membranes of the lamellar type. Smaller numbers of oval cells with vesicular internal membranes were also found. Spectroscopic analyses of the cell extract from the biomats showed the presence of bacteriochlorophyll a and carotenoids of the spirilloxanthin series. Ubiquinone-10 was detected as the major quinone. A clone library of the photosynthetic gene, pufM, constructed from the bulk DNA of the biomats showed that all of the clones were derived from members of the genera Rhodobacter and Rhodopseudomonas. The dominant phototrophic bacteria from the microbial mats were isolated by cultivation methods and identified as being of the genera Rhodobacter and Rhodopseudomonas by studying 16S rRNA and pufM gene sequence information. Experiments of oxygen uptake with lower fatty acids revealed that the freshly collected microbial mats and the Rhodopseudomonas isolates had a wider spectrum of carbon utilization and a higher affinity for acetate than did the Rhodobacter isolates. These results demonstrate that the microbial mats were dominated by the purple nonsulfur bacteria of the genera Rhodobacter and Rhodopseudomonas, and the bioavailability of lower fatty acids in wastewater is a key factor allowing the formation of visible microbial mats with these phototrophs.
PMCID: PMC1563592  PMID: 16957249
5.  Biogeochemistry and Community Composition of Iron- and Sulfur-Precipitating Microbial Mats at the Chefren Mud Volcano (Nile Deep Sea Fan, Eastern Mediterranean)▿  
Applied and Environmental Microbiology  2008;74(10):3198-3215.
In this study we determined the composition and biogeochemistry of novel, brightly colored, white and orange microbial mats at the surface of a brine seep at the outer rim of the Chefren mud volcano. These mats were interspersed with one another, but their underlying sediment biogeochemistries differed considerably. Microscopy revealed that the white mats were granules composed of elemental S filaments, similar to those produced by the sulfide-oxidizing epsilonproteobacterium “Candidatus Arcobacter sulfidicus.” Fluorescence in situ hybridization indicated that microorganisms targeted by a “Ca. Arcobacter sulfidicus”-specific oligonucleotide probe constituted up to 24% of the total the cells within these mats. Several 16S rRNA gene sequences from organisms closely related to “Ca. Arcobacter sulfidicus” were identified. In contrast, the orange mat consisted mostly of bright orange flakes composed of empty Fe(III) (hydr)oxide-coated microbial sheaths, similar to those produced by the neutrophilic Fe(II)-oxidizing betaproteobacterium Leptothrix ochracea. None of the 16S rRNA gene sequences obtained from these samples were closely related to sequences of known neutrophilic aerobic Fe(II)-oxidizing bacteria. The sediments below both types of mats showed relatively high sulfate reduction rates (300 nmol·cm−3·day−1) partially fueled by the anaerobic oxidation of methane (10 to 20 nmol·cm−3·day−1). Free sulfide produced below the white mat was depleted by sulfide oxidation within the mat itself. Below the orange mat free Fe(II) reached the surface layer and was depleted in part by microbial Fe(II) oxidation. Both mats and the sediments underneath them hosted very diverse microbial communities and contained mineral precipitates, most likely due to differences in fluid flow patterns.
PMCID: PMC2394935  PMID: 18378658
6.  Characterization of air contaminants formed by the interaction of lava and sea water. 
Environmental Health Perspectives  1994;102(5):478-482.
We made environmental measurements to characterize contaminants generated when basaltic lava from Hawaii's Kilauea volcano enters sea water. This interaction of lava with sea water produces large clouds of mist (LAZE). Island winds occasionally directed the LAZE toward the adjacent village of Kalapana and the Hawaii Volcanos National Park, creating health concerns. Environmental samples were taken to measure airborne concentrations of respirable dust, crystalline silica and other mineral compounds, fibers, trace metals, inorganic acids, and organic and inorganic gases. The LAZE contained quantifiable concentrations of hydrochloric acid (HCl) and hydrofluoric acid (HF); HCl was predominant. HCl and HF concentrations were highest in dense plumes of LAZE near the sea. The HCl concentration at this sampling location averaged 7.1 ppm; this exceeds the current occupational exposure ceiling of 5 ppm. HF was detected in nearly half the samples, but all concentrations were <1 ppm Sulfur dioxide was detected in one of four short-term indicator tube samples at approximately 1.5 ppm. Airborne particulates were composed largely of chloride salts (predominantly sodium chloride). Crystalline silica concentrations were below detectable limits, less than approximately 0.03 mg/m3 of air. Settled dust samples showed a predominance of glass flakes and glass fibers. Airborne fibers were detected at quantifiable levels in 1 of 11 samples. These fibers were composed largely of hydrated calcium sulfate. These findings suggest that individuals should avoid concentrated plumes of LAZE near its origin to prevent over exposure to inorganic acids, specifically HCl.
PMCID: PMC1567144  PMID: 8593853
7.  Cultivation and Complete Genome Sequencing of Gloeobacter kilaueensis sp. nov., from a Lava Cave in Kīlauea Caldera, Hawai'i 
PLoS ONE  2013;8(10):e76376.
The ancestor of Gloeobacter violaceus PCC 7421T is believed to have diverged from that of all known cyanobacteria before the evolution of thylakoid membranes and plant plastids. The long and largely independent evolutionary history of G. violaceus presents an organism retaining ancestral features of early oxygenic photoautotrophs, and in whom cyanobacteria evolution can be investigated. No other Gloeobacter species has been described since the genus was established in 1974 (Rippka et al., Arch Microbiol 100:435). Gloeobacter affiliated ribosomal gene sequences have been reported in environmental DNA libraries, but only the type strain's genome has been sequenced. However, we report here the cultivation of a new Gloeobacter species, G. kilaueensis JS1T, from an epilithic biofilm in a lava cave in Kīlauea Caldera, Hawai'i. The strain's genome was sequenced from an enriched culture resembling a low-complexity metagenomic sample, using 9 kb paired-end 454 pyrosequences and 400 bp paired-end Illumina reads. The JS1T and G. violaceus PCC 7421T genomes have little gene synteny despite sharing 2842 orthologous genes; comparing the genomes shows they do not belong to the same species. Our results support establishing a new species to accommodate JS1T, for which we propose the name Gloeobacter kilaueensis sp. nov. Strain JS1T has been deposited in the American Type Culture Collection (BAA-2537), the Scottish Marine Institute's Culture Collection of Algae and Protozoa (CCAP 1431/1), and the Belgian Coordinated Collections of Microorganisms (ULC0316). The G. kilaueensis holotype has been deposited in the Algal Collection of the US National Herbarium (US# 217948). The JS1T genome sequence has been deposited in GenBank under accession number CP003587. The G+C content of the genome is 60.54 mol%. The complete genome sequence of G. kilaueensis JS1T may further understanding of cyanobacteria evolution, and the shift from anoxygenic to oxygenic photosynthesis.
PMCID: PMC3806779  PMID: 24194836
8.  Bacterial Communities and the Nitrogen Cycle in the Gypsum Soils of Cuatro Ciénegas Basin, Coahuila: A Mars Analogue 
Astrobiology  2012;12(7):699-709.
The OMEGA/Mars Express hyperspectral imager identified gypsum at several sites on Mars in 2005. These minerals constitute a direct record of past aqueous activity and are important with regard to the search of extraterrestrial life. Gale Crater was chosen as Mars Science Laboratory Curiosity's landing site because it is rich in gypsum, as are some desert soils of the Cuatro Ciénegas Basin (CCB) (Chihuahuan Desert, Mexico). The gypsum of the CCB, which is overlain by minimal carbonate deposits, was the product of magmatic activity that occurred under the Tethys Sea. To examine this Mars analogue, we retrieved gypsum-rich soil samples from two contrasting sites with different humidity in the CCB. To characterize the site, we obtained nutrient data and analyzed the genes related to the N cycle (nifH, nirS, and nirK) and the bacterial community composition by using 16S rRNA clone libraries. As expected, the soil content for almost all measured forms of carbon, nitrogen, and phosphorus were higher at the more humid site than at the drier site. What was unexpected is the presence of a rich and divergent community at both sites, with higher taxonomic diversity at the humid site and almost no taxonomic overlap. Our results suggest that the gypsum-rich soils of the CCB host a unique microbial ecosystem that includes novel microbial assemblies. Key Words: Cuatro Ciénegas Basin—Gale Crater—Gypsum soil microbial diversity—Molecular ecology—Nitrogen cycle. Astrobiology 12, 699–709.
PMCID: PMC3426888  PMID: 22920518
9.  Spatially Resolved Genomic, Stable Isotopic, and Lipid Analyses of a Modern Freshwater Microbialite from Cuatro Ciénegas, Mexico 
Astrobiology  2012;12(7):685-698.
Microbialites are biologically mediated carbonate deposits found in diverse environments worldwide. To explore the organisms and processes involved in microbialite formation, this study integrated genomic, lipid, and both organic and inorganic stable isotopic analyses to examine five discrete depth horizons spanning the surface 25 mm of a modern freshwater microbialite from Cuatro Ciénegas, Mexico. Distinct bacterial communities and geochemical signatures were observed in each microbialite layer. Photoautotrophic organisms accounted for approximately 65% of the sequences in the surface community and produced biomass with distinctive lipid biomarker and isotopic (δ13C) signatures. This photoautotrophic biomass was efficiently degraded in the deeper layers by heterotrophic organisms, primarily sulfate-reducing proteobacteria. Two spatially distinct zones of carbonate precipitation were observed within the microbialite, with the first zone corresponding to the phototroph-dominated portion of the microbialite and the second zone associated with the presence of sulfate-reducing heterotrophs. The coupling of photoautotrophic production, heterotrophic decomposition, and remineralization of organic matter led to the incorporation of a characteristic biogenic signature into the inorganic CaCO3 matrix. Overall, spatially resolved multidisciplinary analyses of the microbialite enabled correlations to be made between the distribution of specific organisms, precipitation of carbonate, and preservation of unique lipid and isotopic geochemical signatures. These findings are critical for understanding the formation of modern microbialites and have implications for the interpretation of ancient microbialite records. Key Words: Microbial ecology—Microbe-mineral interactions—Microbial mats—Stromatolites—Genomics. Astrobiology 12, 685–698.
PMCID: PMC3426887  PMID: 22882001
10.  Olivine-Respiring Bacteria Isolated from the Rock-Ice Interface in a Lava-Tube Cave, a Mars Analog Environment 
Astrobiology  2012;12(1):9-18.
The boundary between ice and basalt on Earth is an analogue for some near-surface environments of Mars. We investigated neutrophilic iron-oxidizing microorganisms from the basalt-ice interface in a lava tube from the Oregon Cascades with perennial ice. One of the isolates (Pseudomonas sp. HerB) can use ferrous iron Fe(II) from the igneous mineral olivine as an electron donor and O2 as an electron acceptor. The optimum growth temperature is ∼12–14°C, but growth also occurs at 5°C. Bicarbonate is a facultative source of carbon. Growth of Pseudomonas sp. HerB as a chemolithotrophic iron oxidizer with olivine as the source of energy is favored in low O2 conditions (e.g., 1.6% O2). Most likely, microbial oxidation of olivine near pH 7 requires low O2 to offset the abiotic oxidation of iron. The metabolic capabilities of this bacterium would allow it to live in near-surface, icy, volcanic environments of Mars in the present or recent geological past and make this type of physiology a prime candidate in the search for life on Mars. Key Words: Extremophiles—Mars—Olivine—Iron-oxidizing bacteria—Redox. Astrobiology 12, 9–18.
PMCID: PMC3264960  PMID: 22165996
11.  Calcite Biomineralization by Bacterial Isolates from the Recently Discovered Pristine Karstic Herrenberg Cave 
Karstic caves represent one of the most important subterranean carbon storages on Earth and provide windows into the subsurface. The recent discovery of the Herrenberg Cave, Germany, gave us the opportunity to investigate the diversity and potential role of bacteria in carbonate mineral formation. Calcite was the only mineral observed by Raman spectroscopy to precipitate as stalactites from seepage water. Bacterial cells were found on the surface and interior of stalactites by confocal laser scanning microscopy. Proteobacteria dominated the microbial communities inhabiting stalactites, representing more than 70% of total 16S rRNA gene clones. Proteobacteria formed 22 to 34% of the detected communities in fluvial sediments, and a large fraction of these bacteria were also metabolically active. A total of 9 isolates, belonging to the genera Arthrobacter, Flavobacterium, Pseudomonas, Rhodococcus, Serratia, and Stenotrophomonas, grew on alkaline carbonate-precipitating medium. Two cultures with the most intense precipitate formation, Arthrobacter sulfonivorans and Rhodococcus globerulus, grew as aggregates, produced extracellular polymeric substances (EPS), and formed mixtures of calcite, vaterite, and monohydrocalcite. R. globerulus formed idiomorphous crystals with rhombohedral morphology, whereas A. sulfonivorans formed xenomorphous globular crystals, evidence for taxon-specific crystal morphologies. The results of this study highlighted the importance of combining various techniques in order to understand the geomicrobiology of karstic caves, but further studies are needed to determine whether the mineralogical biosignatures found in nutrient-rich media can also be found in oligotrophic caves.
PMCID: PMC3273019  PMID: 22179248
12.  Ultra-diffuse hydrothermal venting supports Fe-oxidizing bacteria and massive umber deposition at 5000 m off Hawaii 
The ISME Journal  2011;5(11):1748-1758.
A novel hydrothermal field has been discovered at the base of Lōihi Seamount, Hawaii, at 5000 mbsl. Geochemical analyses demonstrate that ‘FeMO Deep', while only 0.2 °C above ambient seawater temperature, derives from a distal, ultra-diffuse hydrothermal source. FeMO Deep is expressed as regional seafloor seepage of gelatinous iron- and silica-rich deposits, pooling between and over basalt pillows, in places over a meter thick. The system is capped by mm to cm thick hydrothermally derived iron-oxyhydroxide- and manganese-oxide-layered crusts. We use molecular analyses (16S rDNA-based) of extant communities combined with fluorescent in situ hybridizations to demonstrate that FeMO Deep deposits contain living iron-oxidizing Zetaproteobacteria related to the recently isolated strain Mariprofundus ferroxydans. Bioenergetic calculations, based on in-situ electrochemical measurements and cell counts, indicate that reactions between iron and oxygen are important in supporting chemosynthesis in the mats, which we infer forms a trophic base of the mat ecosystem. We suggest that the biogenic FeMO Deep hydrothermal deposit represents a modern analog for one class of geological iron deposits known as ‘umbers' (for example, Troodos ophilolites, Cyprus) because of striking similarities in size, setting and internal structures.
PMCID: PMC3197161  PMID: 21544100
geomicrobiology; deep biosphere; hydrothermal; iron bacteria; iron oxidation
13.  Unexpected Diversity and Complexity of the Guerrero Negro Hypersaline Microbial Mat 
We applied nucleic acid-based molecular methods, combined with estimates of biomass (ATP), pigments, and microelectrode measurements of chemical gradients, to map microbial diversity vertically on a millimeter scale in a hypersaline microbial mat from Guerrero Negro, Baja California Sur, Mexico. To identify the constituents of the mat, small-subunit rRNA genes were amplified by PCR from community genomic DNA extracted from layers, cloned, and sequenced. Bacteria dominated the mat and displayed unexpected and unprecedented diversity. The majority (1,336) of the 1,586 bacterial 16S rRNA sequences generated were unique, representing 752 species (≥97% rRNA sequence identity) in 42 of the main bacterial phyla, including 15 novel candidate phyla. The diversity of the mat samples differentiated according to the chemical milieu defined by concentrations of O2 and H2S. Bacteria of the phylum Chloroflexi formed the majority of the biomass by percentage of bulk rRNA and of clones in rRNA gene libraries. This result contradicts the general belief that cyanobacteria dominate these communities. Although cyanobacteria constituted a large fraction of the biomass in the upper few millimeters (>80% of the total rRNA and photosynthetic pigments), Chloroflexi sequences were conspicuous throughout the mat. Filamentous Chloroflexi bacteria were identified by fluorescence in situ hybridization within the polysaccharide sheaths of the prominent cyanobacterium Microcoleus chthonoplastes, in addition to free living in the mat. The biological complexity of the mat far exceeds that observed in other polysaccharide-rich microbial ecosystems, such as the human and mouse distal guts, and suggests that positive feedbacks exist between chemical complexity and biological diversity.
PMCID: PMC1472358  PMID: 16672518
14.  Seasonal Pulses of Marburg Virus Circulation in Juvenile Rousettus aegyptiacus Bats Coincide with Periods of Increased Risk of Human Infection 
PLoS Pathogens  2012;8(10):e1002877.
Marburg virus (family Filoviridae) causes sporadic outbreaks of severe hemorrhagic disease in sub-Saharan Africa. Bats have been implicated as likely natural reservoir hosts based most recently on an investigation of cases among miners infected in 2007 at the Kitaka mine, Uganda, which contained a large population of Marburg virus-infected Rousettus aegyptiacus fruit bats. Described here is an ecologic investigation of Python Cave, Uganda, where an American and a Dutch tourist acquired Marburg virus infection in December 2007 and July 2008. More than 40,000 R. aegyptiacus were found in the cave and were the sole bat species present. Between August 2008 and November 2009, 1,622 bats were captured and tested for Marburg virus. Q-RT-PCR analysis of bat liver/spleen tissues indicated ∼2.5% of the bats were actively infected, seven of which yielded Marburg virus isolates. Moreover, Q-RT-PCR-positive lung, kidney, colon and reproductive tissues were found, consistent with potential for oral, urine, fecal or sexual transmission. The combined data for R. aegyptiacus tested from Python Cave and Kitaka mine indicate low level horizontal transmission throughout the year. However, Q-RT-PCR data show distinct pulses of virus infection in older juvenile bats (∼six months of age) that temporarily coincide with the peak twice-yearly birthing seasons. Retrospective analysis of historical human infections suspected to have been the result of discrete spillover events directly from nature found 83% (54/65) events occurred during these seasonal pulses in virus circulation, perhaps demonstrating periods of increased risk of human infection. The discovery of two tags at Python Cave from bats marked at Kitaka mine, together with the close genetic linkages evident between viruses detected in geographically distant locations, are consistent with R. aegyptiacus bats existing as a large meta-population with associated virus circulation over broad geographic ranges. These findings provide a basis for developing Marburg hemorrhagic fever risk reduction strategies.
Author Summary
Marburg virus, like its close relative Ebola virus, can cause large outbreaks of hemorrhagic fever with case fatalities nearing 90%. For decades the identity of the natural reservoir was unknown. However, in 2007 Marburg viruses were isolated directly from Egyptian fruit bats (Rousettus aegyptiacus) that inhabited a Ugandan gold mine where miners were previously infected. Soon after, two tourists became infected with Marburg virus after visiting nearby Python Cave, a popular attraction in Queen Elizabeth National Park, Uganda. This cave also contained R. aegyptiacus bats (∼40,000 animals). These events prompted a long-term investigation of Python Cave to determine if, 1) R. aegyptiacus in the cave carried infectious Marburg virus genetically similar to that found in the tourists, and 2) what ecological factors might influence virus spillover to humans. In the study, we found that, 1) approximately 2.5% of the bat colony is actively infected at any one time and that virus isolates from bats are genetically similar to those from infected tourists, and 2) specific age groups of bats (juveniles∼six months of age) are particularly likely to be infected at specific times of the year that roughly coincide with historical dates of Marburg virus spillover into humans.
PMCID: PMC3464226  PMID: 23055920
15.  Microbially Induced Sedimentary Structures Recording an Ancient Ecosystem in the ca. 3.48 Billion-Year-Old Dresser Formation, Pilbara, Western Australia 
Astrobiology  2013;13(12):1103-1124.
Microbially induced sedimentary structures (MISS) result from the response of microbial mats to physical sediment dynamics. MISS are cosmopolitan and found in many modern environments, including shelves, tidal flats, lagoons, riverine shores, lakes, interdune areas, and sabkhas. The structures record highly diverse communities of microbial mats and have been reported from numerous intervals in the geological record up to 3.2 billion years (Ga) old. This contribution describes a suite of MISS from some of the oldest well-preserved sedimentary rocks in the geological record, the early Archean (ca. 3.48 Ga) Dresser Formation, Western Australia. Outcrop mapping at the meter to millimeter scale defined five sub-environments characteristic of an ancient coastal sabkha. These sub-environments contain associations of distinct macroscopic and microscopic MISS. Macroscopic MISS include polygonal oscillation cracks and gas domes, erosional remnants and pockets, and mat chips. Microscopic MISS comprise tufts, sinoidal structures, and laminae fabrics; the microscopic laminae are composed of primary carbonaceous matter, pyrite, and hematite, plus trapped and bound grains. Identical suites of MISS occur in equivalent environmental settings through the entire subsequent history of Earth including the present time. This work extends the geological record of MISS by almost 300 million years. Complex mat-forming microbial communities likely existed almost 3.5 billion years ago. Key Words: Archean—Biofilms—Microbial mats—Early Earth—Evolution. Astrobiology 13, 1103–1124.
PMCID: PMC3870916  PMID: 24205812
16.  Microbial communities at the borehole observatory on the Costa Rica Rift flank (Ocean Drilling Program Hole 896A) 
The microbiology of subsurface, hydrothermally influenced basaltic crust flanking mid-ocean ridges has remained understudied, due to the difficulty in accessing the subsurface environment. The instrumented boreholes resulting from scientific ocean drilling offer access to samples of the formation fluids circulating through oceanic crust. We analyzed the phylogenetic diversity of bacterial communities of fluid and microbial mat samples collected in situ from the observatory at Ocean Drilling Program Hole 896A, drilled into ~6.5 million-year-old basaltic crust on the flank of the Costa Rica Rift in the equatorial Pacific Ocean. Bacterial 16S rRNA gene sequences recovered from borehole fluid and from a microbial mat coating the outer surface of the fluid port revealed both unique and shared phylotypes. The dominant bacterial clones from both samples were related to the autotrophic, sulfur-oxidizing genus Thiomicrospira. Both samples yielded diverse gamma- and alphaproteobacterial phylotypes, as well as members of the Bacteroidetes, Planctomycetes, and Verrucomicrobia. Analysis of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) genes (cbbL and cbbM) from the sampling port mat and from the borehole fluid demonstrated autotrophic carbon assimilation potential for in situ microbial communities; most cbbL genes were related to those of the sulfur-oxidizing genera Thioalkalivibrio and Thiomicrospira, and cbbM genes were affiliated with uncultured phylotypes from hydrothermal vent plumes and marine sediments. Several 16S rRNA gene phylotypes from the 896A observatory grouped with phylotypes recovered from seawater-exposed basalts and sulfide deposits at inactive hydrothermal vents, but there is little overlap with hydrothermally influenced basaltic boreholes 1026B and U1301A on the Juan de Fuca Ridge flank, suggesting that site-specific characteristics of Hole 896A (i.e., seawater mixing into borehole fluids) affect the microbial community composition.
PMCID: PMC3386569  PMID: 22754551
basalt; chemolithoautotrophic bacteria; CORKs; Costa Rica rift; formation fluids; ocean drilling program; subsurface; thiomicrospira
17.  Fermentation couples Chloroflexi and sulfate-reducing bacteria to Cyanobacteria in hypersaline microbial mats 
Past studies of hydrogen cycling in hypersaline microbial mats have shown an active nighttime cycle, with production largely from Cyanobacteria and consumption from sulfate-reducing bacteria (SRB). However, the mechanisms and magnitude of hydrogen cycling have not been extensively studied. Two mats types near Guerrero Negro, Mexico—permanently submerged Microcoleus microbial mat (GN-S), and intertidal Lyngbya microbial mat (GN-I)—were used in microcosm diel manipulation experiments with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), molybdate, ammonium addition, and physical disruption to understand the processes responsible for hydrogen cycling between mat microbes. Across microcosms, H2 production occurred under dark anoxic conditions with simultaneous production of a suite of organic acids. H2 production was not significantly affected by inhibition of nitrogen fixation, but rather appears to result from constitutive fermentation of photosynthetic storage products by oxygenic phototrophs. Comparison to accumulated glycogen and to CO2 flux indicated that, in the GN-I mat, fermentation released almost all of the carbon fixed via photosynthesis during the preceding day, primarily as organic acids. Across mats, although oxygenic and anoxygenic phototrophs were detected, cyanobacterial [NiFe]-hydrogenase transcripts predominated. Molybdate inhibition experiments indicated that SRBs from a wide distribution of DsrA phylotypes were responsible for H2 consumption. Incubation with 13C-acetate and NanoSIMS (secondary ion mass-spectrometry) indicated higher uptake in both Chloroflexi and SRBs relative to other filamentous bacteria. These manipulations and diel incubations confirm that Cyanobacteria were the main fermenters in Guerrero Negro mats and that the net flux of nighttime fermentation byproducts (not only hydrogen) was largely regulated by the interplay between Cyanobacteria, SRBs, and Chloroflexi.
PMCID: PMC3935151  PMID: 24616716
microbial mats; hydrogen; fermentation; Guerrero Negro; NanoSIMS
18.  Phototrophs in High-Iron-Concentration Microbial Mats: Physiological Ecology of Phototrophs in an Iron-Depositing Hot Spring 
Applied and Environmental Microbiology  1999;65(12):5474-5483.
At Chocolate Pots Hot Springs in Yellowstone National Park the source waters have a pH near neutral, contain high concentrations of reduced iron, and lack sulfide. An iron formation that is associated with cyanobacterial mats is actively deposited. The uptake of [14C]bicarbonate was used to assess the impact of ferrous iron on photosynthesis in this environment. Photoautotrophy in some of the mats was stimulated by ferrous iron (1.0 mM). Microelectrodes were used to determine the impact of photosynthetic activity on the oxygen content and the pH in the mat and sediment microenvironments. Photosynthesis increased the oxygen concentration to 200% of air saturation levels in the top millimeter of the mats. The oxygen concentration decreased with depth and in the dark. Light-dependent increases in pH were observed. The penetration of light in the mats and in the sediments was determined. Visible radiation was rapidly attenuated in the top 2 mm of the iron-rich mats. Near-infrared radiation penetrated deeper. Iron was totally oxidized in the top few millimeters, but reduced iron was detected at greater depths. By increasing the pH and the oxygen concentration in the surface sediments, the cyanobacteria could potentially increase the rate of iron oxidation in situ. This high-iron-content hot spring provides a suitable model for studying the interactions of microbial photosynthesis and iron deposition and the role of photosynthesis in microbial iron cycling. This model may help clarify the potential role of photosynthesis in the deposition of Precambrian banded iron formations.
PMCID: PMC91746  PMID: 10584006
19.  Arsenic species in weathering mine tailings and biogenic solids at the Lava Cap Mine Superfund Site, Nevada City, CA 
A realistic estimation of the health risk of human exposure to solid-phase arsenic (As) derived from historic mining operations is a major challenge to redevelopment of California's famed "Mother Lode" region. Arsenic, a known carcinogen, occurs in multiple solid forms that vary in bioaccessibility. X-ray absorption fine-structure spectroscopy (XAFS) was used to identify and quantify the forms of As in mine wastes and biogenic solids at the Lava Cap Mine Superfund (LCMS) site, a historic "Mother Lode" gold mine. Principal component analysis (PCA) was used to assess variance within water chemistry, solids chemistry, and XAFS spectral datasets. Linear combination, least-squares fits constrained in part by PCA results were then used to quantify arsenic speciation in XAFS spectra of tailings and biogenic solids.
The highest dissolved arsenic concentrations were found in Lost Lake porewater and in a groundwater-fed pond in the tailings deposition area. Iron, dissolved oxygen, alkalinity, specific conductivity, and As were the major variables in the water chemistry PCA. Arsenic was, on average, 14 times more concentrated in biologically-produced iron (hydr)oxide than in mine tailings. Phosphorous, manganese, calcium, aluminum, and As were the major variables in the solids chemistry PCA. Linear combination fits to XAFS spectra indicate that arsenopyrite (FeAsS), the dominant form of As in ore material, remains abundant (average: 65%) in minimally-weathered ore samples and water-saturated tailings at the bottom of Lost Lake. However, tailings that underwent drying and wetting cycles contain an average of only 30% arsenopyrite. The predominant products of arsenopyrite weathering were identified by XAFS to be As-bearing Fe (hydr)oxide and arseniosiderite (Ca2Fe(AsO4)3O3•3H2O). Existence of the former species is not in question, but the presence of the latter species was not confirmed by additional measurements, so its identification is less certain. The linear combination, least-squares fits totals of several samples deviate by more than ± 20% from 100%, suggesting that additional phases may be present that were not identified or evaluated in this study.
Sub- to anoxic conditions minimize dissolution of arsenopyrite at the LCMS site, but may accelerate the dissolution of As-bearing secondary iron phases such as Fe3+-oxyhydroxides and arseniosiderite, if sufficient organic matter is present to spur anaerobic microbial activity. Oxidizing, dry conditions favor the stabilization of secondary phases, while promoting oxidative breakdown of the primary sulfides. The stability of both primary and secondary As phases is likely to be at a minimum under cyclic wet-dry conditions. Biogenic iron (hydr)oxide flocs can sequester significant amounts of arsenic; this property may be useful for treatment of perpetual sources of As such as mine adit water, but the fate of As associated with natural accumulations of floc material needs to be assessed.
PMCID: PMC3037876  PMID: 21261983
20.  An interlaboratory comparison of 16S rRNA gene-based terminal restriction fragment length polymorphism and sequencing methods for assessing microbial diversity of seafloor basalts 
Environmental Microbiology  2009;11(7):1728-1735.
We present an interlaboratory comparison between full-length 16S rRNA gene sequence analysis and terminal restriction fragment length polymorphism (TRFLP) for microbial communities hosted on seafloor basaltic lavas, with the goal of evaluating how similarly these two different DNA-based methods used in two independent labs would estimate the microbial diversity of the same basalt samples. Two samples were selected for these analyses based on differences detected in the overall levels of microbial diversity between them. Richness estimators indicate that TRFLP analysis significantly underestimates the richness of the relatively high-diversity seafloor basalt microbial community: at least 50% of species from the high-diversity site are missed by TRFLP. However, both methods reveal similar dominant species from the samples, and they predict similar levels of relative diversity between the two samples. Importantly, these results suggest that DNA-extraction or PCR-related bias between the two laboratories is minimal. We conclude that TRFLP may be useful for relative comparisons of diversity between basalt samples, for identifying dominant species, and for estimating the richness and evenness of low-diversity, skewed populations of seafloor basalt microbial communities, but that TRFLP may miss a majority of species in relatively highly diverse samples.
PMCID: PMC2784043  PMID: 19508561
21.  Filamentous “Epsilonproteobacteria” Dominate Microbial Mats from Sulfidic Cave Springs 
Hydrogen sulfide-rich groundwater discharges from springs into Lower Kane Cave, Wyoming, where microbial mats dominated by filamentous morphotypes are found. The full-cycle rRNA approach, including 16S rRNA gene retrieval and fluorescence in situ hybridization (FISH), was used to identify these filaments. The majority of the obtained 16S rRNA gene clones from the mats were affiliated with the “Epsilonproteobacteria” and formed two distinct clusters, designated LKC group I and LKC group II, within this class. Group I was closely related to uncultured environmental clones from petroleum-contaminated groundwater, sulfidic springs, and sulfidic caves (97 to 99% sequence similarity), while group II formed a novel clade moderately related to deep-sea hydrothermal vent symbionts (90 to 94% sequence similarity). FISH with newly designed probes for both groups specifically stained filamentous bacteria within the mats. FISH-based quantification of the two filament groups in six different microbial mat samples from Lower Kane Cave showed that LKC group II dominated five of the six mat communities. This study further expands our perceptions of the diversity and geographic distribution of “Epsilonproteobacteria” in extreme environments and demonstrates their biogeochemical importance in subterranean ecosystems.
PMCID: PMC194925  PMID: 12957939
22.  Repeatedly Evolved Host-Specific Ectosymbioses between Sulfur-Oxidizing Bacteria and Amphipods Living in a Cave Ecosystem 
PLoS ONE  2012;7(11):e50254.
Ectosymbioses between invertebrates and sulfur-oxidizing bacteria are widespread in sulfidic marine environments and have evolved independently in several invertebrate phyla. The first example from a freshwater habitat, involving Niphargus ictus amphipods and filamentous Thiothrix ectosymbionts, was recently reported from the sulfide-rich Frasassi caves in Italy. Subsequently, two new Niphargus species, N. frasassianus and N. montanarius, were discovered within Frasassi and found to co-occur with N. ictus. Using a variety of microscopic and molecular techniques, we found that all three Frasassi-dwelling Niphargus species harbor Thiothrix ectosymbionts, which belong to three distinct phylogenetic clades (named T1, T2, and T3). T1 and T3 Thiothrix dominate the N. frasassianus ectosymbiont community, whereas T2 and T3 are prevalent on N. ictus and N. montanarius. Relative distribution patterns of the three ectosymbionts are host species-specific and consistent over different sampling locations and collection years. Free-living counterparts of T1–T3 are rare or absent in Frasassi cave microbial mats, suggesting that ectosymbiont transmission among Niphargus occurs primarily through inter- or intraspecific inoculations. Phylogenetic analyses indicate that the Niphargus-Thiothrix association has evolved independently at least two times. While ectosymbioses with T1 and T2 may have been established within Frasassi, T3 ectosymbionts seem to have been introduced to the cave system by Niphargus.
PMCID: PMC3510229  PMID: 23209690
23.  Analysis of Facultative Lithotroph Distribution and Diversity on Volcanic Deposits by Use of the Large Subunit of Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase†  
A 492- to 495-bp fragment of the gene coding for the large subunit of the form I ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) (rbcL) was amplified by PCR from facultatively lithotrophic aerobic CO-oxidizing bacteria, colorless and purple sulfide-oxidizing microbial mats, and genomic DNA extracts from tephra and ash deposits from Kilauea volcano, for which atmospheric CO and hydrogen have been previously documented as important substrates. PCR products from the mats and volcanic sites were used to construct rbcL clone libraries. Phylogenetic analyses showed that the rbcL sequences from all isolates clustered with form IC rbcL sequences derived from facultative lithotrophs. In contrast, the microbial mat clone sequences clustered with sequences from obligate lithotrophs representative of form IA rbcL. Clone sequences from volcanic sites fell within the form IC clade, suggesting that these sites were dominated by facultative lithotrophs, an observation consistent with biogeochemical patterns at the sites. Based on phylogenetic and statistical analyses, clone libraries differed significantly among volcanic sites, indicating that they support distinct lithotrophic assemblages. Although some of the clone sequences were similar to known rbcL sequences, most were novel. Based on nucleotide diversity and average pairwise difference, a forested site and an 1894 lava flow were found to support the most diverse and least diverse lithotrophic populations, respectively. These indices of diversity were not correlated with rates of atmospheric CO and hydrogen uptake but were correlated with estimates of respiration and microbial biomass.
PMCID: PMC383153  PMID: 15066819
24.  Colonization of subsurface microbial observatories deployed in young ocean crust 
The ISME Journal  2010;5(4):692-703.
Oceanic crust comprises the largest hydrogeologic reservoir on Earth, containing fluids in thermodynamic disequilibrium with the basaltic crust. Little is known about microbial ecosystems that inhabit this vast realm and exploit chemically favorable conditions for metabolic activities. Crustal samples recovered from ocean drilling operations are often compromised for microbiological assays, hampering efforts to resolve the extent and functioning of a subsurface biosphere. We report results from the first in situ experimental observatory systems that have been used to study subseafloor life. Experiments deployed for 4 years in young (3.5 Ma) basaltic crust on the eastern flank of the Juan de Fuca Ridge record a dynamic, post-drilling response of crustal microbial ecosystems to changing physical and chemical conditions. Twisted stalks exhibiting a biogenic iron oxyhydroxide signature coated the surface of mineral substrates in the observatories; these are biosignatures indicating colonization by iron oxidizing bacteria during an initial phase of cool, oxic, iron-rich conditions following observatory installation. Following thermal and chemical recovery to warmer, reducing conditions, the in situ microbial structure in the observatory shifted, becoming representative of natural conditions in regional crustal fluids. Firmicutes, metabolic potential of which is unknown but may involve N or S cycling, dominated the post-rebound bacterial community. The archaeal community exhibited an extremely low diversity. Our experiment documented in situ conditions within a natural hydrological system that can pervade over millennia, exemplifying the power of observatory experiments for exploring the subsurface basaltic biosphere, the largest but most poorly understood biotope on Earth.
PMCID: PMC3217339  PMID: 21107442
geomicrobiology; deep biosphere; oceanic crust; observatory; hydrothermal
25.  A Brief Survey to Identify Priorities for Improving Clinician Recruitment and Retention: Results from Hawai‘i Island Physicians 
In light of the documented physician shortage on Hawai‘i Island, the Hawai‘i Clinician Recruitment and Retention survey was designed and implemented to assess perceptions of quality of life and the work environment among clinicians on Hawai‘i Island and to identify aspects of the environment on Hawai‘i Island that predict responses to questions regarding recruitment and retention.
The respondents were 127 Hawai‘i Island clinicians, specifically 96 physicians, 15 nurses, five pharmacists, four physician assistants, two social workers, and five “other” healthcare workers. The internal reliability of the survey was high (alpha=.91) and its convergent validity was supported by the significant correlation of item total scores with anchor items that measured overall ratings of the environment and likelihood of recruitment and retention. Given the small number of non-physician clinicians responding, descriptive analyses included only physicians. Physicians who indicated they plan to retire within 5 years were excluded from the correlation analyses to focus on patterns within the target group for retention.
Overall, results indicate that, while the majority of physicians who relocated to Hawai‘i Island did so primarily for the quality of life, the best predictors of retention are financial sustainability, professional opportunities, community support, and access to good K-12 schools. Survey results also indicate that Hawai‘i Island will lose 32% of its current physicians within the next five years due to retirement or other causes.
These findings indicate that increased urgency to find solutions is warranted.
PMCID: PMC3347737  PMID: 22737642

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