2013 Annual Science Report

Pennsylvania State University Reporting  |  SEP 2012 – AUG 2013

Biosignatures in Relevant Microbial Ecosystems

Project Summary

PSARC is investigating microbial life in some of Earth’s most mission-relevant modern ecosystems. These environments include the Dead Sea, the Chesapeake Bay impact structure, methane seeps, ice sheets, and redox-stratified Precambrian ocean analogs. We target environments that, when studied, provide fundamental information that can serve as the basis for future solar system exploration. Combining our expertise in molecular biology, geochemistry, microbiology, and metagenomics, and in collaboration with some of the planet’s most extreme explorers, we are deciphering the microbiology, fossilization processes, and recoverable biosignatures from these mission-relevant environments.

PSARC Ph.D. (now postdoctoral researcher at Caltech) Katherine Dawson published a new paper documenting the anaerobic biodegradation of organic biosignature compounds pristane and phytane. PSARC Ph.D. Daniel Jones (now postdoctoral researcher at U. Minnesota) published a new paper that uses metagenomic data to show how sulfur oxidation in the deep subsurface environments may contribute to the formation of caves and the maintenence of deep subsurface microbial ecosystems. PSARC Ph.D. student Khadouja Harouaka published a new paper that represents some of the first available information about possible Ca isotope biosignatures. Lastly, the Macalady group published a paper showing how ecological models based on available energy resources can be used to predict the distribution of microbial populations in space and time.

4 Institutions
3 Teams
0 Publications
6 Field Sites
Field Sites

Project Progress

Impact cratering and subsurface microbial life
Macalady, Shapiro & graduate student Kristine Korzow-Richter, in collaboration with C. Cockell & M. Voytek

Additional personnel: Dr. Mathias Stiller (helped with library preparation of core DNA extracts)

Astrobiology dual-title graduate student Kristine Korzow-Richter, co-advised by Beth Shapiro (primary) and Jenn Macalady, continued work on authenticated core samples from the ~2 km deep Chesapeake Bay impact structure. Among the most challenging aspects of this project was the extremely low biomass expected within each of the sampled sediment layers. A highly efficient method of DNA recovery was therefore required. Previously we compared six DNA extraction protocols, including one developed specifically for this project, to determine the most appropriate method for extracting DNA from the core.

After performing a detailed quantitative analysis of DNA extraction methods, three methods were used to extract DNA from 0.5 to 5.0 g subsamples of the Eyreville core from six depths spanning from 160 to 850 meters. Each DNA extract was tested with several PCR primer sets targeting gene fragments between 100 bp and 1400 bp long. Samples with high clay content were purified with PVPP columns before PCR. Extracts from three sampling depths (150, 240, 840 meters) were sequenced with next-generation sequencing methods (Illumina) to analyze the community composition. Comparison with a negative extraction control and other deep subsurface samples revealed that each of the three layers is comprised by a unique microbial community whose composition correlates with the geochemistry for the layer. The lowest layer tested was subjected to the impact-induced transient hydrothermal system. We found bacterial and archaeal thermophiles in high concentration in this layer relative to the other layers. As it is unlikely that DNA sequences would survive the 34.5 million years since the impact, we hypothesize that the sequences identified as thermophiles are living remnants of the hydrothermal system that may have survived to the present day in a low metabolic state. A manuscript is in preparation detailing these results.

In addition to the laboratory work described above, we have been developing a computational test to determine the authenticity of any DNA sequence data generated from the ancient cores. As with any ancient DNA project, the possibility exists that the sample was contaminated at some point by modern bacteria, and that any data generated is not authentically ancient, but instead from modern, contaminating bacterial or archael sequences. One way to distinguish between these two possibilities is to take advantage of the molecular clock, a concept that defines a near-constant rate of evolutionary change within organisms over time. Assuming a molecular clock, we would expect authentically ancient DNA sequences to contain many fewer mutations than contaminating, modern sequences, when compared to other modern data. We can therefore verify the authenticity of ancient sequences by asking whether they behave as expected for ancient sequences (fewer mutations along external branches in a phylogeny than modern sequences), or simply fall within the distribution of rates of the modern data included in the test. Using simulated and real bacterial gene sequence alignments, we developed a bioinformatics approach to evaluating the distribution of evolutionary rates along external branches in a phylgeny, which we have implemented in the popular software package BEAST. This method will be used to evaluate the results of the sequencing data recovered from both the Illumina sequencing and the Sanger sequencing of the cultured bacterial colonies.

Active endolithic microorganisms and methane cycling in authigenic carbonates
Orphan, House, Ferry, Freeman, and McKeegan (in collaboration with J. Grotzinger)

Other team members: Jeffrey Marlow (Caltech graduate student), Katherine Dawson (PSARC postdoc at Caltech)

Methane derived authigenic carbonates have been previously shown to harbor lipid and DNA biosignatures affiliated with methanotrophic archaea and sulfate-reducing bacteria. While these biosignatures are often interpreted as ‘fossil’ remnants of microorganisms in the literature, it is also possible that they are derived from viable endolithic microorganisms. Using FISH combined with assays of methanotrophic activity (14CH4 and CH3D), we documented abundant methanotrophic archaea/ SRB consortia within the interiors of authigenic carbonates from a variety of deep-sea habitats (at the seabed within active seeps and inactive areas as well as sediment hosted carbonate nodules). The aggregate morphology and abundance of these organisms varied according to habitat, with the largest archaeal biomass recorded in seafloor carbonates and nodules in proximity to active CH4 venting and approximately half as many cells in carbonates from inactive areas. In support of these observations, methane oxidation rates in these samples (assessed by the biological conversion of CH3D to D2O) followed a similar trend, with the highest rates of methanotrophy affiliated with active carbonates and nodules relative to carbonates from dormant seep areas. Stable isotope incubation experiments with 15NH4 and CH4 coupled with FISH-nanoSIMS analysis were also used to demonstrate active growth of methanotrophic consortia within the interiors of carbonate nodules (Marlow et al. In Review).

Within the past year, graduate student Marlow and PSARC PI Orphan formed a collaboration within the new ‘life underground’ NAI team at USC to conduct predictive thermodynamic models of the potential for biologically mediated sulfate-based methane oxidation (AOM) on ancient Mars (Marlow et al., in review Astrobiology). In this study, seven distinct fluids representative of putative martian groundwater were used to calculate Gibbs energy values in the presence of dissolved methane under a range of atmospheric CO2 partial pressures. In all scenarios, AOM was exergonic, ranging from -31 to -135 kJ/mol CH4. A reaction transport model was constructed to examine how environmentally relevant parameters such as advection velocity, reactant concentrations, and biomass production rate affect the spatial and temporal dependences of AOM reaction rates. Two geologically supported models for ancient martian AOM are presented in the manuscript: a sulfate-rich groundwater with methane produced by serpentinization, and acid-sulfate fluids with methane from basalt alteration. The simulations presented in this study indicate that AOM could have been a feasible metabolism on ancient Mars, and fossil or isotopic evidence of this metabolic pathway may persist beneath the surface and in surface exposures of eroded ancient terrains.

Marlow has also been working on characterizing the diversity, activity, and biomass of archaea and bacteria associated within the pore spaces of methane-derived authigenic carbonates from active methane seeps and areas of low methane flux from Hydrate Ridge, Oregon and Mound 12 along the Costa Rica margin. Using molecular DNA-based diversity analyses, microscopy, stable isotope tracers coupled with FISH-nanoSIMS, and radiotracer rate measurements, we have demonstrated that the interior of carbonates are a viable habitat for anaerobic methanotrophs and support an abundant endolithic microbial community, which is capable of oxidizing methane in both active seepage areas as well as within carbonates recovered from dormant seep systems with little evidence of active methane flux at the seabed. This work represents the first 14C radiotracer measurements of methane oxidation in carbonates and documented methane-oxidation rates that, per volume, have the potential to represent a significant sink for methane in seep areas relative to unlithified marine sediments. A manuscript is currently being revised and prepared for submission to Nature Communications.

The initial collaboration between PIs Freeman and Orphan on isotopic measurements of F430 from methanotrophic archaea was a direct outgrowth of a PSARC annual meeting at Penn State. Through this PSARC project, Orphan hosted Penn State graduate student Lawrence Bird at Caltech to conduct stable isotope incubation experiments and conversely, Caltech postdoc Kat Dawson has visited Penn State to work with PSARC team members.
Peer reviewed publications: Marlow, J, D. Larowe, B. Ehlmann, J. Amend, and V. Orphan (in revision) The Potential for Biologically Catalyzed Anaerobic Methane Oxidation on Ancient Mars. Astrobiology.

Marlow, J, J. Steele, W. Ziebis, A Thurber, L Levin, V Orphan. (in revision) Carbonate hosted methanotrophy: an unrecognized methane sink in the deep sea. Nature Communications.

Marlow, J, J. A. Steele, D. H. Case, L. A. Levin, V. J. Orphan (in prep) Microbial Abundance and Diversity Associated with Sediments, Nodules, and Carbonates from the Hydrate Ridge, OR, USA, Methane Seep. Microbial Ecology.

Figure 1 PSARC 2013: A model for potential AOM on Mars, using the stratigraphy at NE Syrtis Major. Within a water-permeated section, serpentinization of the olivine-bearing unit produces hydrogen, which in turn generates methane in the abiotic reduction of CO2. This methane diffuses upward and is entrained in buoyant, heat-driven advective flow. In the overlying jarosite-bearing layer, dissolved sulfate and incoming methane provide the reactants for AOM, which produces carbonate species that may form magnesium carbonate minerals. Many of the minerals involved in this model have been observed from orbit, and calculations suggest that the AOM metabolism is energetically favorable given modeled fluid chemistries (Marlow et al. in review).

Life in Greenland glacial ice
Brenchley (PI), Miteva (Sr Res Assoc), and Kaitlyn Rinehold (technician) with undergraduate students C. Burlingame and A. Gifford, in collaboration with Todd Sowers

The goal of this research is to study the abundance, viability and diversity of the indigenous microbial populations from different depths/age of the recently NEEM Greenland core using high resolution techniques. Ice core samples originated from depths 100 m, 634 m, 1730 m and 2051m corresponding to 340, 3,220, 36,500 and 78,500 years before present. The authenticity of previously obtained glacial isolates from these decontaminated ice core samples was evaluated against a collection of contaminants isolated from the drilling fluid. A manuscript reporting these findings and addressing the problem of potential exogenous microbial contamination of non-aseptically drilled deep ice cores is ready for submission to the Special Issue on Polar and Alpine Microbiology, FEMS Microbiology Ecology. Further studies of the spatial and temporal variations in microbial abundance, viability and diversity in the studied NEEM Greenland ice samples included construction and comparative analyses of universal SSU rRNA gene clone libraries using both Sanger and Illumina next generation sequencing. Preparatory work for the paired-end Illumina sequencing included testing of specific universal primers targeting the V4-V5 SSU rRNA gene region on selected NEEM samples and a set of reference bacterial, archaeal, cyanobacterial and eukaryotic DNAs, plus the additional purification and precise quantification of each DNA before the construction of bar-coded and indexed rRNA libraries. Initial analysis of over 3 million Illumina reads of about 400 nucleotides showed that Firmicutes, beta-, gamma- and deltaproteobacteria were most abundant and each of the four studied samples had one dominant group (Fig. 1). The proportional distribution of phyla differed between samples but was relatively similar as revealed by both sequencing approaches. (manuscript in preparation).

Peer reviewed publications:
Miteva V., Birlingame C., Sowers T., Brenchley, J. (2013) Comparative evaluation of the indigenous microbial diversity versus drilling fluid contaminants in the NEEM Greenland ice core. (to be submitted to the Special Thematic Issue on Polar and Alpine Microbiology, FEMS Microbiology Ecology).

Miteva V., Rinehold K., Sowers T., Brenchley, J. (2013) Microbial abundance, viability and diversity at different depths of the NEEM Greenland ice core. (in preparation).

Presentations:
Birlingame C., Miteva V., Brenchley, J. (2013) Validation of indigenous microbiological isolate diversity from a 1730.5 m deep NEEM ice core, Greenland. Undergraduate Student Exhibition at Penn State, April 9-10, 2013.

Miteva V., Birlingame C., Sowers T., Brenchley, J. (2013) Comparative evaluation of the indigenous microbial diversity versus drilling fluid contaminants in the NEEM Greenland ice core. 5th International Conference on Polar and Alpine Microbiology, Big Sky, Montana, USA, Sept 8-12, 2013.

Biosignatures of life in extremely energy-limited environments
Macalady, McCauley (PSU Ph.D. student), Jones (PSU Ph.D. student), in collaboration with A. Montanari, K. Broad, A. Crocetti, S. Mariani

Additional personnel: Dr. Dirk de Beer (Microsensor Group Head, Max Planck Institute for Marine Microbiology), Stefan Haeusler (Ph.D. student, MPIMM), Dr. Miriam Weber (HYDRA Institute and MPIMM), Dr. Lubos Polerecky (MPIMM)

Frasassi Cave System

With Macalady’s participation, major new discoveries were made this year at the Frasassi Cave System (Italy) ~600 m below the ground surface by cave diver Alejandro Crocetti and cave explorer Sandro Mariani. The discoveries included new understanding of the hydrologic controls on aquifer goechemistry and vertical mixing rates, and an expanded view of the extent and diversity of microbial life in the energy-limited anoxic zone of the aquifer. This expanded view suggests that the microbial ecosystem in the aquifer anoxic zone is pervasive and more diverse than previously thought.

Along with Macalady’s Hanse Fellowship funding, the developments in underwater exploration inspired a new collaboration with the Max Planck Institute for Marine Microbiology (MPIMM). With assistance from explorers Crocetti and Mariani, scientists de Beer (MPIMM), Macalady (PSU) and Haeusler (MPIMM) conducted in situ radioisotopic incubation experiments documenting extremely slow rates of carbon fixation and sulfate reduction in pyrite-precipitating microbial biofilms deep within the anoxic zone of the aquifer. Microsensor measurements of biofilm activity are planned for the upcoming year with the participation of technical and science diver Miram Weber from the HYDRA Institute. Weber has previously developed the only available diver-operated microsensing equipment, which we deployed successfully to study Frasassi cave biofilms at the air-water interface. The planned work represents a quantum leap in the challenge to both divers and scientists and will require significant instrument development effort in coordination with cave divers and MPIMM personnel.

PSU Astrobiology Dual Title Ph.D. student Rebecca McCauley continues to characterize microbial life in the anoxic aquifer water using molecular methods, including new metagenomic sequences from the pyrite-precipitating deep subsurface biofilm community (currently in assembly and annotation stage). Ongoing analyses from two previously sequenced anoxic zone biofilm metagenomes and 8 Illumina sequence tag runs are currently being analyzed, and suggest a microbial world dominated by Deltaproteobacterial sulfate reducers (~20%), anaerobic organoheterotrophic Chloroflexi (~15%), Caldithrix/KSB1 (0-10%), and Planctomyces (~8%). The remaining bacteria and archaea have extremely high species richness, and include primarily taxa from uncultivated clades. Thermodynamic calculations based on in situ geochemistry indicate that sulfate reduction, sulfur-dependent ammonia oxidation (annamox), and methanogenesis are among the most favorable energy metabolisms. Neither methanogenic archaea nor anaerobic ammonia oxidizers are present in sequence tag or metagenomic libraries, suggesting that novel taxa may perform these metabolisms, with implications for biosignature production.

The project attracted the attention of the BBC “How to Build a Universe” documentary film team. Macalady, Crocetti, and Mariani participated in the filming of a documentary segment focused on astrobiology and the central importance of liquid water (even in the absence of light or abundant chemical energy) for the proliferation of life. The 2-hour documentary recently aired on BBC1 and the Science Channel, and can be viewed on the web at http://www.dailymotion.com/video/x16eb5t_how-to-build-a-planet-ep-2_creation. This project requires the close cooperation of cave divers and explorers (Broad, Crocetti, Mariani), geologists (Montanari) and microbial geochemists (Macalady, McCauley). McCauley’s dissertation research encompasses both aqueous geochemistry (field and laboratory analyses, thermodynamic calculations) and metagenomic DNA sequence analysis using bioinformatics programming and data mining.

Magical Sink (Bahamas)

A second field expedition to stratified Bahamian blue hole Magical Sink was led by Macalady assisted by postdoc Hamilton (PSU), and graduate student Sebastian Haas (MPIMM). Measurements of photosynthetically active radiation (PAR) collected earlier in collaboration with technical diver and cave explorer Brian Kakuk confirmed that anoxygenic phototrophs in the sinkhole grow using light levels similar to or lower than those at the bottom of the Black Sea chemocline, thus making the microbial species involved candidates for the earth’s most extreme low-light phototrophs. Microsensor measurements of sulfide dynamics and stable isotopic tracer measurements of carbon fixation and sulfate reduction were made in situ on diver-collected biofilm samples. A diver-operated underwater science instrumentation package (LUSCA) was developed and deployed at water depths up to 60 meters in the sinkhole in order to record simultaneous light quantity and quality, depth, and geochemistry measurements. A masters thesis will be submitted to MPIMM by Haas in early March 2014, and a mansucript is in preparation.

Figure 2 PSARC 2013: LUSCA (Light Underwater Sensing Cave Array), diver-operated instrument package developed to make simultaneous light quantity, light quality, and geochemical measurments at depths of up to 60 m in flooded cave environments.

Peer reviewed publications:
Macalady, J. L., Hamilton, T. L., Grettenberger, C. L., Jones, D. S., Tsao, L. E. and Burgos, W. D. 2013. Energy, ecology, and the distribution of microbial life. Philosophical Transactions Royal Society B 368: 20120383.

Dawson, K. S., Schaperdoth, I., Freeman, K. H., and Macalady, J. L. 2013. Anaerobic biodegradation of the isoprenoid biomarker analogues pristane and phytane. Organic Geochemistry 65:118-126.

Jones, D. S., Schaperdoth, I. and Macalady, J.L. 2014. Metagenomic evidence for sulfide oxidation in extremely acidic cave biofilms, Geomicrobiology Journal in press.

Jones, D.S., Polerecky, L., Galdenzi, S., Dempsey, B. and Macalady, J.L. 2014. Fate of sulfide in limestone aquifers and implications for sulfuric acid speleogenesis (SAS), in review at Geology.

Biosignatures of life in ancient stratified ocean analogs
Macalady, Freeman, & Kump with T. Hamilton (PSU postdoc), J. Havig (PSU postdoc), J. Fulton, K. Meyer & R. McCauley (PSU Ph.D. students), B. McClure & T. Bergen (PSU undergraduate students)

Additional personnel: Dr. M. McCormick (Hamilton College), Dr. P. Welander (Stanford), Dr. R. Summons (MIT), Dr. D. Newman (Caltech), Brian Kakuk (Bahamas Caves Research Foundation), Dr. Dirk de Beer (Microsensor Group Head, Max Planck Institute for Marine Microbiology), Dr. Miriam Weber (HYDRA Institute and MPIMM)

Instigated by Macalady and Kump in 2010, this project investigates biosignatures of life in modern analogs for stratified ancient and/or extraterrestrial oceans. A website monitoring the activities of an informal working group on Early Earth Photosynthesis is maintained by Macalady (http://www.geosc.psu.edu/~jlm80/EEP.html).

Little Salt Spring
A new Proterozoic-analog microbial ecosystem was discovered at Little Salt Spring (FL) in 2011 and has unique potential to help us understand biosignatures of earth evolution, specifically the long delay in the continued oxidation of the surface earth after the initial rise at the Great Oxidation Event (GOE, ~2.4 Ga). The sinkhole is weakly stratified and has low concentrations of oxygen, sulfide and sulfate throughout the water column, similar to the geochemistry hypothesized for Proterozoic oceans. The sinkhole hosts a mixed oxygenic/anoxygenic community of microbial phototrophs. This site thus provides an opportunity to investigate biogeochemical and microbial processes in a Proterozoic ocean analog, including the ecological and geochemical controls on oxygen production and organic matter preservation. In addition, the microbial ecosystem at the site produces large quantitites of hopanoids, an important class of organic biomarkers that can be preserved in rocks for billions of years.

Following a 2012 field expedition, PSARC postdoc Trinity Hamilton isolated the abundant oxygenic phototroph from the pinnacle mats resulting in an axenic culture of a model cyanobacterium. The isolate is metabolically diverse, capable of both oxygenic and anoxygenic photosynthesis. Freeman lab Astrobiology Dual-title Ph.D. student Laurence Bird confirmed that the axenic culture produces the organic biomarkers 2-methyl hopanoids. As a result of the 2012 field expedition and an ambitious field campaign in the Fall of 2013, light-dependent primary productivity was measured in situ. With assistance from Paula Welander and Dianne Newman’s lab, the isolate and mat samples were analyzed, confirming the cyanobacterium in pure culture is a major source of the important biomarker 2-methyl hopanoid in the biofilm. The MIT and Caltech collaborations are a direct result of NAI connections.

Figure 3 PSARC 2013: Little Salt Spring cyanobacterium in axenic culture (left), the hopanoids synthesized by the pure culture (center), and results of tracer experiment documenting the anoxygenic photosynthetic activity of the isolate (right).

Fayetteville Green Lake

Fayetteville Green Lake (NY) is a meromictic lake with a chemocline at 15 m that separates an oxygenated and well mixed upper layer from an euxinic lower layer, and supports a bacterial plate at 21 m made up of anoxygenic phototrophs and sulfate reducers. The goal of the current project is to create a high-resolution geochemical characterization with which to formulate and test predictions of microbial community structure and function that can then be linked to the rock record.

Water and sediment samples were collected in November of 2012 by JRH, Trinity Hamilton, Brianna McClure, and the McCormick Lab in a collaborative effort with the McCormick Lab at Hamilton College, resulting in over 70 water, sediment, and molecular samples from the entire 50+ m water column through to the lake floor sediments in order to create a comprehensive and high-spatial-resolution geochemical characterization of the site. In situ measurements were made using a YSI SONDE for temperature, pH, conductivity, turbidity, and dissolved oxygen. Water samples were analyzed for major cations (Ca, Mg, Na, and K), trace elements (V, Mn, Fe, Co, Ni, Cu, Zn, and Mo), dissolved inorganic carbon concentrations and δ13C values, dissolved organic carbon concentrations and δ13C values, and concentration of CH4, NO3-, NH4+, and sulfide. Sediment incubations testing for CH4 production were set up. Sampling was conducted in June, 2013 by JRH and Brianna McClure to collect water samples and to collect biomass and water for setting up water column incubations testing for CH4 production. A second major sampling trip was conducted with the McCormick Lab in July, 2013 to repeat the sampling conducted in November for comparison, and to collect samples for methane δ13C analysis, as well as set up a series of coupled water column and sediment incubations looking for CH4 production.

Analysis of November samples is complete, as well as CH4 δ13C analysis of samples from July. Incubations set up from samples collected in November (sediments), June (water column), and July (water column plus sediments) have been analyzed for CH4. Water geochemistry samples from June and July are queued for analysis. Abstract was submitted by Brianna McClure to AGU (2013), reporting results of geochemistry and incubations related to methane production and consumption at Green Lake. Data from previous Green Lake work have been compiled, as well as geochemistry from eight similar meromictic field sites into a comprehensive geochemical dataset.

Peer reviewed publications:
Hunter, S., Kump, L., Macalady, J.L. and Freeman, K. H. 2014. Spatial and temporal variability in an anoxygenic phototrophic chemocline ecosystem: Fayetteville Green Lake, New York (USA), in review at FEMS Microbiology Ecology.

Hamilton, T. L., Bovee, R. J., Sattin, S.R., Mohr, W., Schaperdoth, I., Gilhooly, W. P. III, Lyons, T. W., Pearson, A., and Macalady, J. L. 2014. Sulfur dynamo in the phototrohic plate of a meromictic lake, planned submission to Geobiology Dec. 2013.

Hamilton, T.L., Freeman, K. H., Fulton, J. M., Schaperdoth, I., White, T. S. and Macalady, J. L. 2014. Microbial communities and organic biomarkers in a Proterozoic-analog sinkhole, planned submission to Geobiology Dec. 2013

McClure, B., Havig, J. R., Sowers, T., McCormick, M., Hamilton, T., and Kump, L. (2013) Dynamics of the methane profile through the water column of meromictic Fayetteville Green Lake, N.Y. Submitted to 2013 Fall Meeting, AGU, San Francisco, Calif., 9-13 Dec., 2013

Ca isotopic biosignatures (project began in 2012)
Faculty: Matthew Fantle (PSU), Jenn Macalady (PSU)
External NAI members: Marilyn Fogel (CIW)
External faculty: Anton Eisenhauer (GEOMAR)
Staff: Irene Shaperdoth (PSU); Ana Kolevica (GEOMAR)
Students: Khadouja Harouaka, PhD student, Geosciences (PSU); Matthew Gonzales, PhD student, Geosciences (PSU); Amanda Fobes, Geosciences undergraduate (PSU); Muammar Mansor, Ph.D. student Geosciences (PSU).

The overall goal of the research is to constrain the potential for microbes to affect the Ca isotopic composition of minerals in nature. We are testing this experimentally by characterizing abiotic Ca isotope fractionation during gypsum precipitation as a function of precipitation rate, saturation state, and Ca:SO4 ratio. Natural samples of gypsum are then collected and measured for Ca and S isotopes, particle size, and particle morphology to determine if Ca isotopes might serve as a biosignature in the environment. This work will lead to a greater fundamental understanding of the controls on Ca isotopic fractionation in nature, which enables an evaluation of its general utility as a biosignature.

Harouaka’s Masters work involved abiotic calibration of Ca isotopic fractionation during gypsum precipitation, as well as S isotopic analysis of experimental products. This Ca work is in revision at GCA and should be in press soon. Harouaka and Mansor recently collaborated on the precipitation of gypsum under biotic conditions, in cultures of Acidithiobacillus thiooxidans at low pH. They successfully precipitated gypsum, analyzed solution chemistry of replicate cultures, and measured the Ca isotopic composition of crystals in both the biotic and abiotic control experiments. The Ca isotope measurements, a difficult measurement, were conducted at Penn State using the new Thermo Neptune Plus. These are the first Ca isotope data produced with the Neptune at Penn State, and truly represent a huge achievement. Harouaka presented the results of this study at the Goldschmidt conference in Florence, Italy in 2013.

In the summer of 2013, Fantle and Mansor worked in the Frasassi cave system in Italy with Macalady, for which Mansor received a Lewis and Clark Fellowship to conduct field work. Gypsum samples were collected in Ramo Sulfureo and Mansor subsequently imaged the samples by SEM. Mansor and Gonzales conducted an in situ precipitation experiment in a flow-through reactor designed by Gonzales, and viewed the precipitation in real time at high magnification. The goal of this work is to understand how aqueous geochemical conditions affect the crystal morphology and the isotopic composition of the gypsum precipitates. Mansor has also been working with Fantle and Macalady to culture organisms that precipitate intracellular calcite, and has attempted to isolate and culture such organisms with the ultimate goal of evaluating isotopic effects associated with biomineralization.

Finally, Fobes has worked with Harouaka and Fantle to conduct a senior thesis study of gypsum precipitation rates and morphology in the presence of biomolecules. This work has involved the addition of biomolecules to oversaturated gypsum solutions in batch reactors, monitoring of Ca concentations to determine rate, and, ultimately, collection of crystals for isotopic analysis. Fobes is focusing on the former two goals, while Harouaka will build on this work to meet the third objective.

This project brings together an isotope geochemist (Fantle) and a microbial geochemist (Macalady). The collaboration extends to the students involved in the project. Harouaka (Geosciences) collaborated with Mansor, an undergraduate researcher (B.S. Biotechnology) to precipitate gypsum in the presence of Acidithiobacillus thiooxidans.

The project spawned collaborations between Fantle and European colleagues (Eisenhauer, GEOMAR, Kiel, Germany) and colleagues at the Carnegie Institution of Washington (Fogel). The presence of NAI led directly to these specific collaborations, and the NAI link between PSU and CIW definitely facilitated the S isotope work.

Peer reviewed publications:
Harouaka K., Eisenhauer A., Fantle M. (2014) Experimental investigation of Ca isotopic fractionation during abiotic gypsum precipitation. Geochimica et Cosmochimica Acta DOI 10.1016/j.gca.2013.12.004 (in press).