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Project Progress

Task 5.1 Life in extreme environments
CoI Investigators Baross and Schrenk continue to explore the phylogeny, physiology, and biochemistry or microbial communities associated with deep-sea hydrothermal vent environments. CoI Baross and graduate student/postdoc Brazelton have focused on microbial communities associated with the Lost City Hydrothermal Field (LCHF), a serpentinization-influenced ecosystem located near the Mid-Atlantic Ridge. The LCHF is of interest because it may have been one of the most important settings for crucial steps required for the origins and early evolution of life. One of the hypotheses addressed is that the predominant groups of Bacteria and Archaea encountered at Lost City have metabolic pathways and other physiological characteristics that are relicts of ancient organisms. In particular the characteristics of hydrogen and methane metabolizing microbial biofilms in extreme vent environments may be analogues to the earliest microbial communities and thus could provide insight into the nature of the Last Universal Common Ancestor (LUCA).

Work by CoI’s Baross and Schrenk, and postdoc Brazelton established that microbial biofilms found at LCHF are dominated by a single, novel phylotype of Archaea affiliated with the order Methanosarcinales. “Deep sequencing” of the biofilms using 454 pyrotag sequencing revealed an unexpected cluster of rare sequences found in some chimneys that were different than the dominant archaeal phylotype and that some of the rare sequences became dominant in other chimneys of different ages (Brazelton et al., PNAS 2010; Brazelton et al., Environmental Microbiology Reports 2010). These are some of the first data that demonstrate a role for the now well-documented rare microbial biosphere found in most natural environments. Shifts within a cluster were observed to occur in time scales of less than 10 years, suggesting that species or strains pre-adapted to very specific geochemical regimes are able to quickly increase in abundance from rare to dominant when conditions allow. One of the conclusions from this study is that the Lost City biofilms, while dominated by a single phylotype, sustain multiple metabolisms including methanogenesis and methanotrophy. Metagenomic analyses substantiate this conclusion by documenting multiple genes for nitrogen fixation and genes for acetate utilization (Brazelton et al., in preparation). Furthermore, the metagenomic analyses of the Lost City carbonate chimney yielded most of the genes from a population related to Thiomicrospira an aerobic, sulfur-oxidizing autotrophic bacterium (Brazelton and Baross, PLoS One, 2010). We found that the Lost City Thiomicrospira had a similar genomic content to a similar but distinct species, Thiomicrospira crunogena XCL-2, isolated from a basalt-hosted hydrothermal environment along the East Pacific Rise. Despite inhabiting different types of hydrothermal systems in different oceans, both Thiomicrospira representatives share genes encoding functions that appear to be crucial for thriving in Lost City carbonate chimneys: the ability to aerobically oxidize reduced sulfur species and to concentrate CO₂ intracellularly. This genomic similarity likely reflects a recent evolutionary divergence and that both lineages inhabit niches where H₂S-containing hydrothermal fluids mix with oxygenated seawater.

CoI Schrenk along with Canadian Space Agency funded collaborator Morrill, CoI Steele, and postdocs Brazelton and Bower have expanded the study of serpentinite-hosted microbial communities to include terrestrial ultrabasic springs at the Tablelands Ophiolite in Newfoundland, Canada. Alkaline seeps of hydrogen and methane-rich, pH 11 fluid emanate from rocks that were emplaced along the continental margin more than 500 million years ago. Similar to the LCHF, highly reducing conditions coupled to the catalytic activities of ultramafic rocks are predicted to lead to the abiotic synthesis of small organic molecules- a potential precursor to biochemistry. Quinn Woodruff, a graduate student in Schrenk’s lab, mapped the abundance and diversity of microbial populations at the Tablelands relative to point sources of serpentinization, and isolated several new strains of alkalitolerant bacteria. They found that bacteria related to the Firmicutes, a clade of fermentative microorganisms, were abundant in the most reducing sites- not Archaea as in the marine systems. Postdoc Brazelton has continued this work with expeditions to the Tablelands in July and August 2010 to collect time series samples, and conduct both in situ and laboratory experiments to better understand microbial activities at these sites. Genomic analyses are being expanded from taxonomic studies to functional gene analyses to refine our understanding of terrestrial serpentinizing ecosystems.

Another line of research by CoI Baross and his students involves the characterization of the virus communities associated with hydrothermal vent environments and their possible role in horizontal gene transfer. They have expanded our studies of the microbial ecology of diffuse-flow vents at different hydrothermal vent environments to include the incidence, host range and genetic diversity of the viral community. Baross and colleagues have obtained a metagenome of a viral assemblage from 170 liters of diffuse-flow fluids from a hydrothermal vent on the Juan de Fuca Ridge in the Northeastern Pacific. In the initial analysis of the viral metagenome, bioinformatic analysis included targeting sequences associated with the CRISPR (clustered regularly interspaced short palindromic repeat) immune system. CRISPR spacer matches to the marine vent virome suggest that viruses infecting hosts from diverse taxonomic groups are present in this vent environment. Analyses showed that a high percentage of CRISPR spacers from vent isolates and from thermophiles in general have higher matches to the vent virome than to other marine or terrestrial hot spring viromes. The CRISPR spacers were significantly more abundant in thermophiles than in mesophiles or psychrophiles, suggesting that the virus-host relationship in thermophiles is distinct from other organisms. By combining metagenomics with CRISPR analyses, CoI Baross and his student Rika Anderson showed that the gradient-dominated hydrothermal vent environment hosts a viral assemblage that is extensive in the scope of its diversity. The long-term goals from these studies are to better understand the role of viruses as agents of horizontal gene transfer and particularly in the vent archaeal community and ultimately to obtain a thermophilic archaeal/viral host system.

CoI Schrenk and his graduate student Heather Blumenfeld have analyzed the genes associated with carbon assimilation within the walls of ‘black smoker’ hydrothermal vent chimneys. These environments likely harbor all six of the known pathways of carbon fixation, and potential contain novel pathways. Furthermore, conditions within the chimney walls overlap with those that favor abiotic organic syntheses. Detailed geochemical and taxonomic maps of the vent chimneys have been produced by CoI Schrenk and collaborators Kelley (U. of Washington) and Girguis (Harvard). Within the past year, graduate student Blumenfeld has documented the presence/absence of genes involved in the Calvin Cycle, the reverse TCA cycle, and the acetyl Co-A pathway within the vent chimney walls. Similar studies are being conducted by Schrenk and collaborators Amend and Meyer-Dombard in shallow-sea hydrothermal systems of the Aeolian Islands, Italy. In these systems, readily accessible gradients in temperature and chemistry occur at depths from the surface to 25 m below sea level. These systems also reflect the contributions of both photosynthetic and chemosynthetic carbon assimilation, and represent ideal systems to tease apart the two inputs to carbon biogeochemistry at high temperatures.

CoI Steele and graduate student Starke have continued their investigations of geothermal springs on Svalbard, near the Arctic Circle. The Svalbard hydrothermal ecosystems have extreme and variable conditions that place strong selective pressures on microorganisms. Conditions vary from the warm aqueous environment of the hydrothermal seeps to the cold desiccating environment of carbonate terrace structures that surround the springs. Analyses by Steele and colleagues have shown that Cyanobacteria at the geothermal springs appear to have adapted to a wide range of conditions, allowing them to survive in rock as endoliths, as well as in water as both planktonic cells and biofilms. Parallel-tagged 454 sequencing (pyrosequencing) was used for the partial analyses of microbial 16S ribosomal RNA genes. The comparison of data sets from planktonic populations filtered from the pool water and endolithic communities indicated that the pool and endolithic environments have fewer than 3% of phyla in common. In other words, the communities of free-floating organisms in the pool and endolithic organisms in the terraces are radically different taxonomically. These data indicate that endolithic cyanobacteria may be more closely related to the cyanobacteria in the biofilms than organisms found in the fluids. Further sequence-based analyses are being conducted to verify these observations. Documenting the diversity of microbial ecosystems like those found in Svalbard provides a basis to understanding the changes in microbial population structures relative to environmental gradients. Similar environmental gradients may be important in other settings of astrobiological significance and potentially support extraterrestrial life. Organisms that are adapted to the extreme conditions of Svalbard may be similar to those that could have existed (or could exist today) on Mars.

Postdoc M. Glamoclija, A. Kish along with CoIs A. Steele and M. L. Fogel have studied microbial sulfur and nitrogen cycles and associated microbial diversity in modern sulfate mineral deposits. The White Sands National Monument (WSNM) in New Mexico is an excellent terrestrial analog to sulfate deposits found to be exposed to the surface on Mars and, as such, the WSNM represents a unique natural laboratory for studying arid evaporitic ecosystems and their relationship to microbial life. As gypsiferous aeolian settings have restricted nutrient supplies, understanding of the nitrogen and sulfur cycling may be more important in this kind of environment than in soils with an abundant carbon supply. Nearly uniform levels of ammonium nitrogen (~ 1.2 ±0.015 µg/g _sediment) were detected at 10 cm depth suggesting that ammonium may be delivered to the system by the groundwater. Whereas predominance of nitrite and nitrate over ammonium nitrogen (2:16:1) implies that nitrification processes might be important in this ecosystem. Microbial involvement in nitrogen and sulfur cycling tested by genetic assays of functional and 16S rRNA genes showed the presence of microbes capable of nitrogen and sulfur cycling. β-, γ-Proteobacteral and Archaeal ammonium oxidizers were detected in nearly all deposits including migrating dune sides, suggesting microbial involvement in nitrification. Additionally presence of anaerobic ammonium oxidizing (anammox) bacteria in most of the analyzed samples suggests active process of denitrification in many of the WSNM settings. Microbes with metabolic abilities for sulfur cycling were identified in all settings, except dune sites. Genetic assays encoding dissimilatory sulfite reductase were identified in the samples along with the presence of purple sulfur and green non-sulfur bacteria. These particular organisms have the ability to reduce sulfate, and in addition to re-oxidize reduced sulfur compounds in photosynthesis back to sulfate. Our data indicate that the active gypsum rich dune field provides a home to metabolically diverse microbial ecotypes capable of utilizing extremely limited nutrient resources. However, ongoing ARISA and 16S rRNA gene sequencing analyses will provide more precise information about microbial diversity at the WSNM. Additionally, coupled high-resolution microscopy and spectroscopy will be used to identify and characterize possible mineral biosignatures.

Task 5.2 Microbial Adaptations to Chemistry and Pressure at Life’s Extremes

Ongoing work by CoI Schrenk has investigated the effects of high hydrostatic pressure upon microbial physiology and activity. Work with collaborator Edwards using samples from 5,000 m water depths at Loihi Seamount, a volcano near Hawaii have yielded enrichment cultures adapted to in situ hydrostatic pressures. These cultures are closely related to other strains from the pelagic ocean, and are providing insights into microbial adaptations to extreme pressures.

This past year graduate student D. Smith successfully cultivated over 90 enrichment cultures of sulfate reducing, iron oxidizing, iron reducing, denitrifying, and nitrifying bacteria from Mono Lake, Hot Creek, and Travertine Hot Spring, CA along with a Dartmouth University undergraduate student. They explored the physiology of the bacterial communities and sequenced purified DNA extracts; they hope to have summarized results before the end of the year. Smith continued his biogeochemical exploration of Fayetteville Green Lake, NY by further examining the δ¹³C and δ¹⁵N, lipid extracts, and biomarkers of the sediments. Purple sulfur bacteria enrichments were cultivated from the chemocline of the lake for the purpose of lipid and fatty acid extraction, δ¹³C fractionation, and pigment analysis. A manuscript entitled “A Geochemical Assessment of the Water Column and Surficial Sediment of Fayetteville Green Lake, NY” has been sent to colleague Josef Werne at the Large Lakes Observatory at Minnesota Duluth for revision. In August, Smith joined the Carnegie NAI team working with Marilyn Fogel, George Cody, and Andrew Steele. He is culturing Chromatium and other photosynthethic microbes in order to understand pigment formation in these organisms.

Task 5.3 Biosignatures

Research into Biosignatures by the CIW team encompasses several distinct avenues ranging from isotopic to mineralogical biosignatures.

CoI Fogel and Weifu Guo acquired a new elemental analyzer to conduct simultaneous N, C, and S isotope analysis of organics at the Geophysical Laboratory based upon a continuous flow IRMS method developed by Guo. Fogel also participated in two field trips (India and Svalbard) and finished the analysis of hydrogen isotopes in microbes as biosignatures.

Postdoc Dominic Papineau collected new carbon isotope data from dolomite, carbonate fluorapatite and organic matter in stromatolitic phosphorites from the Paleoproterozoic Jhamarkotra Formation of the Aravalli Supergroup (India) revealed distinct geochemical signatures. The most striking difference is the carbon isotope composition of carbonate rocks associated with phosphorites, which generally is between d¹³C_carb = -2 and +2‰ and typical for seawater, whereas the carbon isotope composition of carbonate rocks not associated with phosphorites often has excursions up to +10.8‰. These carbon isotope data may reflect high levels of primary productivity during phosphorite deposition in the Jhamarkotra embayment. No carbon isotope excursion was found in stratigraphically lower and higher dolomites in the embayment, but excursions up to +5‰ exist in contemporary carbonates from nearby localities. Similarly organic matter in carbonates and associated phosphorites had heavy d¹³C_org values of up -13‰. These carbon isotope data may reflect high levels of primary productivity during phosphorite deposition in the Jhamarkotra embayment. No carbon isotope excursion was found in stratigraphically lower and higher dolomites in the embayment, but excursions up to +5‰ exist in contemporary carbonates from nearby localities. In addition, carbonate fluorapatite from columnar stromatolites is systematically depleted in ¹³ C (-1.8 to -0.2‰) compared to intercolumnar dolomite (0.3 to +0.6‰). These results suggest late diagenetic substitution in carbonate fluorapatite by HCO₃ from the oxidation of organic matter. These differences appear systematic and reveal important clues on the underlying causes of the Paleoproterozoic Great Oxygenation Event, which are currently under consideration. Breana Hashmann, a B.Sc. student in Geological Sciences at Dickinson College (PA) worked with Papineau during summer 2010 on a project on the geochemistry of Paleoproterozoic carbonates from the Aravalli Supergroup. Hashmann prepared more than one hundred rock homogenates from Aravalli carbonates and phosphorites, which allowed her to develop a new approach to colorimetrically determine the phosphate concentration in rock powders. Her work showed that there is very little phosphate (less than 0.4%wt) in other rock-types associated with the phosphorites.

Additionally, Papineau, collaborator De Gregorio, and CoI’s Stroud, Steele and Fogel have investigated the isotopic geochemistry of graphite in Banded Iron Formations (BIFs). In BIFs metamorphosed at the upper amphibolite facies from Eoarchean-Hadean the Nuvvuagittuq Supracrustal Belt (NSB) in northern Québec, they found that around 20% of apatite grains are associated with carbonaceous material (CM). Raman micro-spectroscopic analyses of this CM associated with apatite revealed sharp and intense D- and G-bands, which points to poorly crystalline graphite. High-resolution Transmission Electron Microscopy of graphite targets associated with apatite revealed that these occur in microscopic mineral assemblages that formed from fluid-deposition under low-temperature hydrothermal conditions, late in the metamorphic history of the rock. While BIFs from the NSB have a large range of Δ13Corg values with an average around -22, not inconsistent with a biological origin, data suggest that fluid-deposition of graphite with apatite can obscure possible sources of CM and demonstrate an important alternative graphitization pathway for such mineral associations in highly metamorphosed Eoarchean sedimentary rocks.

Papineau and CoI Hauri have also progressed with in situ analyses of multiple sulfur isotope compositions of sulfides using a beamsize of 15 × 15 microns with NanoSIMS, allowing the simultaneously measurement of ³²S, ³³S, and ³⁴S isotopes. They have achieved 2-sigma reproducibility of around ±0.45 and ±0.22‰ for d³⁴S and D³³S, respectively. More than 110 analyses on sulfides from banded iron formations and associated supracrustal rocks from BIF’s in the Eoarchean-Hadean Nuvvuagittuq Supracrustal Belt in northern Québec revealed a range larger than 20‰ in d³⁴S and a range of 5‰ in Δ³³S values. These data are consistent with a generally anoxic Eoarchean atmosphere and possibly with some microbial sulfur processing in the original depositional environment.

PI Cody and ORAU NAI postdoc Gupta in collaboration with Roger Summons of the MIT-NAI team investigated the preservation of chitin-protein complex in Paleozoic arthropod fossils. Employing high spatial resolution Soft X-ray absorption spectroscopic imaging they were able to show that greater than 50 % of the carbon in a Pennsylvanian scorpion fossil (320 Ma) and a Silurian eurypterid fossil (410 Ma) is derived from chitin-protein complex. They believe that esterification of fatty acids to the hydroxylated molecular remnants of chitin-protein complex explains how organic fossil preservation occurs over time spans of hundreds of millions of years. A manuscript discussing this research is in press in the journal Geology.

The availability of specific minerals in the earliest surface environments, where prebiotic chemistry took place, was likely a key factor for the emergence of life and was strongly influenced by Hadean-Eoarchean (4.56 to 3.60 billion years ago) rock-types. A mineralogical inventory of the oldest rocks could thus help to constrain the likeliest available minerals involved in the origin of life. Such a survey is important in the perspective of mineral evolution, as the emergence of life and subsequent global changes caused by organisms were responsible for more than half of the 4300 minerals on the modern Earth. CoInvestigator Hazen, collaborator Sverjensky, and postdoc Papineau completed a comprehensive evaluation of mineral assemblages over geologic time to develop a model of the co-evolution of Earth’s geo-sphere and the bio-sphere.

Postdoc Guo’s work during the past year focused mostly on sulfur isotope studies. He conducted series of low-pressure SO₂ UV photolysis experiments, and examined the mechanisms of mass independent sulfur isotope fractionations associated with it using the new continuous flow-IRMS method he developed in CoI Fogel’s laboratory. The preliminary results of this study were presented at the 2010 Goldschmidt meeting in Knoxville, TN. Collaborating with CoI Farquhar (University of Maryland), Guo also performed first principles theoretical calculations to predict the equilibrium sulfur isotope fractionations among different volatile organic sulfur compounds. These theoretical predictions provide the basic framework for interpreting the future isotope data on these compounds. Postdoc Guo also conducted collaborative research with Dr. Lance Christensen (Jet Propulsion Laboratory) to study in more detail the sulfur isotope fractionations associated with SO₂ photolysis using tunable laser spectroscopy, which was supported by a research scholarship through the NAI. Besides research on sulfur isotope geochemistry, Guo continued work on carbonate clumped isotope thermometry and presented those research results in 2009 AGU fall meeting, 2010 Ocean Sciences meeting, and 2010 clumped isotope workshop.

Postdoc Bower conducted experimental investigations on the effects of diagenesis and low-grade metamorphism on the preservation of organic matter and formation of Fe- and Ti- oxides in microfossils. This project involves culturing and growing mat building microbes under simulated natural conditions, as well as designing simple diagenetic experiments. The analytical methods include high-resolution microscopic imaging, X-ray diffraction (XRD), scanning electron microscopy (SEM), and micro Raman spectroscopy. The goals are to understand the role that microbes play in mineral phase changes during or just before diagenesis, as well as to establish mineralogic biosignatures that can be used for life detection in ancient rocks on Earth, as well as on Mars or other planets. Preliminary results were presented at AbSciCon in April 2010.

Bower is also using micro Raman spectroscopy to fully characterize microfossiliferous cherts from the ~2.0 Gunflint Formation. Although the exact environment of the chert formation is still enigmatic, the Gunflint cherts contain structures that have been established as bona-fide microfossils, so they are an excellent standard for comparison to older possible fossil-bearing cherts of questionable origin. Bower is conducting further comparisons between the carbonaceous material in the older fossiliferous cherts and hydrocarbons in a set of well-characterized meteorites using Gunflint samples as a reference point. In the process of uncovering the origins of carbon compounds in a suite of rock types, she is also trying to perfect Raman spectral mapping techniques in order to tease out the relationships between carbonaceous materials and morphological features, as well as subtle differences in associated mineral phases. This work aids in establishing biosignatures using a method that can be deployable on future Mars Rover missions. The results representing each of these aspects have been presented at the October 2009 Annual Geological Society of America meeting, 41^st Lunar and Planetary Science Conference March 2010, and the Goldschmidt conference in June 2010. Two manuscripts are presently in preparation for submission next month.

During 2009-2010, NAI funds provided partial support for several projects undertaken by the Farquhar Group at the University of Maryland. (1) The NAI funds helped to support research by Harry Oduro (a Ph.D. student) to develop and apply techniques for the isolation and analysis of organic sulfur compounds. A manuscript describing the techniques was submitted to Marine Chemistry and is currently in review. A goal of this work is to understand the isotope effects associated with the assimilatory pathways for sulfur compounds in marine macro and microalgae, and to develop criteria that can be used to interpret the geologic record of sulfur isotopes. Work by Oduro has also focused on using this approach to trace the mechanisms and pathways for the formation of organic sulfur compounds (specifically volatile methylated sulfur compounds) in stratified sulfidic lake systems such as Green Lake (NY) that is under study as an analog for sulfidic environments early in Earth history. Oduro has also used this approach to trace the pathways for the formation of dimethylsulfoniopropionate (a precursor to dimethylsulfide- the most abundant biogenic sulfur gas) in water columns and sediments. Work is underway to prepare manuscripts on these projects. The work on organic sulfur compounds extends work that we are doing to understand the isotope effects associated with the dissimilatory (rather than assimilatory) sulfate reduction metabolism that is used by prokaryotes. (2) This second direction for research is being undertaken by Brian Harms (a Ph.D. student) and has focused on performing multiple sulfur isotope analyses for the products of a set of experiments using batch cultures of Archaeaglobus fulgidus and a temperature block. This study aims to evaluate a hypothesis about the metabolic response to different temperature conditions presented by our collaborators in a publication (Mitchell et al., 2009) using the minor sulfur isotopes. Mitchell et al. (2009) argue that the isotopic fractionations produced by A. fulgidus vary as a function of temperature-dependent changes in enzymatic activity and transport of sulfur across the cell wall. The specific hypothesis carries implications for the evolution of the minor sulfur isotopes that can be tested. Harms is focusing on testing this hypothesis, and therefore testing our understanding of how the metabolism operates. (3) A third research focus that is partially supported by the NAI funds is an effort by Daniel Eldridge (a Ph.D. student) to study the isotope effects associated with abiological oxidation pathways of S(IV) and sulfide in aqueous solutions. This work has just begun, and aims to provide data that can be directly compared with our recent and ongoing experiments with organisms that oxidize sulfide via a variety of metabolic pathways (work of A. Zerkle – a research scientist). The aim of this work is to provide criteria for distinguishing between systems that are subject to biological and abiological oxidation.