2011 Annual Science Report

Pennsylvania State University Reporting  |  SEP 2010 – AUG 2011

Developing New Biosignatures

Project Summary

The development and experimental testing of potential indicators of life is essential for providing a critical scientific basis for the exploration of life in the cosmos. In microbial cultures, potential new biosignatures can be found among isotopic ratios, elemental compositions, and chemical changes to the growth media. Additionally, life can be detected and investigated in natural systems by directing cutting-edge instrumentation towards the investigation of microbial cells, microbial fossils, and microbial geochemical products. This project combines our geomicrobiological expertise and on-going field-based environmental investigations with a new generation of instruments capable of revealing diagnostic biosignatures. Our efforts are focused on creating innovative approaches for the analyses of cells and other organic material, finding ways in which metal abundances and isotope systems reflect life, and developing creative approaches for using environmental DNA to study present and past life.

Apatite-biomineralized Neoproterozoic protists Using specimens provided by N. J. Butterfield (Cambridge University) and P.A. Cohen (Harvard University), our studies by optical microscopy, confocal laser scanning electron microscopy, and Raman spectroscopy show that ~775-Ma-old protistan scale fossils (from Yukon Territory, Canada) were originally composed of apatite — a surprising discovery, given that the scales of extant protists are most commonly silica or calcite. The Yukon Territory fossils, the earliest example of biomineralization now known from the geological record, have recently been reported by our group in Geology Today; a more detailed manuscript reporting these findings was published in Geology.

Chert-permineralized Paleoproterozoic sulfuretum

Using samples originally provided by Martin Van Kranendonk, augmented by samples that Malcolm Walter and I collected in June 2011, we have discovered a ~2,300-Ma-old microbial ecosystem previously unknown in Precambrian deposits. This ecosystem, composed of sulfate-reducing and sulfur-oxidizing bacteria, is remarkably similar to extant sulfureta first described by Victor Gallardo and Carola Espinoza in 2007 from numerous core samples collected at the sediment-water interface in the sub-oxygen minimum zone off the western coast of South America. The stratigraphy and geologic setting indicates that the fossiliferous rocks were deposited at depth, well below wave base and most likely below the photic zone. Geochemical data are consistent with the presence of sulfate reducers. The major taxa of the abundant, diverse assemblage are morphologically distinct from fossil and modern cyanobacteria but are essentially identical to sulfur-oxidizing bacteria described by Gallardo and Espinoza. Similarly, the wispy cobweb fabric of the microbial assemblage differs distinctly from the laminated fabric of photoautotroph-dominated stromatolite/microbial mat communities but is indistinguishable from the fabric of modern sulfureta. When current SIMS work is completed, designed to measure the carbon isotopic composition of individual benthic and planktonic members of the assemblage (and based on the pioneering work of Christopher House, published in 2000), a paper reporting these new findings will be submitted to Nature.

Our studies of extant microorganisms (biology), fossil megascopic and microscopic organisms (paleontology), the stratigraphy, mineralogy and taphonomy of their preservation (geology), and their chemical and isotopic composition (geochemistry) are highly interdisciplinary, involving both the life sciences (biology, microbiology, evolutionary biology) and the physical sciences (geology and geochemistry).

In addition to the UCLA contributors noted above (J. William Schopf, Anatoliy B. Kudryavtsev, and Ian B. Foster), contributors to this research include: Jack D. Farmer (NAI Astrobiology Center, Arizona State University); Malcolm R. Walter and Martin Van Kranendonk (Australian Centre for Astrobiology, University of New South Wales, Sydney, Australia); Kliti Grice (Curtin University, Perth, Australia); John Valley and Ken Williford (NAI Astrobiology Center, University of Wisconsin); Victor Gallardo and Carola Espinoza (Universidad de Concepción, Chile); Phoebe A. Cohen (MIT); Francis Macdonald (Harvard University); and Nicolas J. Butterfield (Cambridge University)

Subproject: Weathering and inorganic chemical biosignatures

Secondary minerals and regolith profiles on basaltic rocks

Based upon images taken during the 2003-2004 series of missions to Mars, several different weathering features appear to be present on the Mars surface including surface flaking, dirt cracking, exfoliation, honeycomb weathering and the formation of what appear to be weathering rinds (Thomas et al., 2005). However, the underlying mechanisms responsible for these features are difficult to ascertain from images alone. The goal of this study is to understand the role that different climatic conditions on Earth play in facilitating the weathering of basalts, which are thought to be the predominant lithology present on Mars. Basalts from three different sites are compared, two of which come from the temperate northeastern United States, while the third site is located in the dry, Arctic region of Svalbard and is thus considered to be a Mars analogue. Soil profiles from the two North American sites are much deeper than those from Svalbard and show clear evidence of chemical weathering in the bulk soil profile. While no evidence for chemical weathering is observed in bulk soils from Svalbard, the clay-sized fraction nevertheless shows clear evidence for the elemental depletion of K, Na, Ca and Mg. In contrast, Al and Fe appear to be immobile even in the finest size fractions of the Svalbard soil. Coupled with SEM images showing evidence of spalling on the outermost 100 μm of Svalbard rocks, this chemical evidence suggests that the process of spalling in cold climates may preferentially remove certain elements over others both on Earth as well as on Mars. Extractions with 0.5 N HCl, designed operationally to remove amorphous Fe oxides, indicate that the prevalence of amorphous Fe is greatest in the lowermost horizons of a Pennsylvania diabase, a finding that has been noted previously in a granodiorite-derived soil profile from Puerto Rico that is thought to contain a lithoautotrophic Fe-oxidizing community at depth (Buss et al., 2010). In further agreement with findings from Puerto Rico, Fe(II) concentrations in the lowermost soil of the Pennsylvania site decrease over a short distance as a results of rapid oxidation in the lowermost portion of the 4-m profile analyzed here. In contrast, no such change in extractable Fe concentrations or Fe(II) content is noted within the regolith of the Svalbard Mars analogue site, at which Fe-oxidizing bacteria are presumably absent.

DNA has been extracted from several of the sampling sites on Svalbard, and metagenomic sequencing carried out. Preliminary analysis of the sequence data shows that while Proteobacteria, Actinobacteria, Acidobacteria, and Bacteroidetes are the dominant phyla at all sites sequenced, there are significant differences in phylum relative abundance and community structure between sites.

Community structure and geochemistry of weathering in a Puerto Rican watershed (Note: Primary funding for this project is through DOE)

We have hypothesized that Fe oxidation may be important in weathering of intact bedrock to disaggregated regolith at depth (9.2 m) in the Bisley volcaniclastic watershed in the Luquillo Mountains of Puerto Rico. The regolith forms along a gradient between conditions of low oxygen and higher pH at depth, to higher oxygen and lower-pH waters at the surface. The active weathering front was observed to be ~ 8.3 m, as secondary clay minerals and Fe oxides are present from the surface down to this depth, while the primary minerals chlorite and feldspars were detected below 8.3 m. Consistent with an active weathering front at ~ 8.3 m, ammonium-acetate extracted Ca and Mg were present in higher concentrations below 7 m. There were also increases in both total and heterotrophic cell counts in this region, as well as increased concentrations of HCl-extracted Fe(II) and organic-bound Fe. As a consequence of these gradients in oxygen, pH, and chemical composition, we expect to see changes in microbial communities along the regolith. Metagenomics analysis revealed that sub-phylum groups containing known iron-oxidizing microorganisms were found in the deepest samples, near the regolith-bedrock interface, consistent with the hypothesis that chemolithoautotrophic bacteria play an important role in weathering and element cycling in the deep regolith. Rarefaction curves generated at a 97% similarity value indicated that the regolith was representatively sampled for determining community richness.

Subproject: Intermolecular Isotopic Patterns

F430 Isolation and isotopic characterization

Methyl-coenzyme M reductase, an enzyme that plays a key role in methanogenesis, has recently been linked also to the anaerobic oxidation of methane (Scheller, 2010). Cofactor F430 is a tetrapyrrole nickel complex contained within the active site of methyl-coenzyme M and appears to be used in both methanogenesis and methanotrophy (Scheller, 2010; Mayer et al., 2008). Our efforts have been focused on purifying F430 from natural samples in order to determine δ15N and δ13C values both at natural abundance and in isotopically enriched samples from tracer studies. F430 is isolated using multidimensional preparatory and high-performance chromatographic separations. Compound identity and purity are confirmed using molar C:N ratios, light absorbance and MSn detection and fragmentation of F430 isolated from pure cultures of Methanosarcina acetivorans. Our efforts to measure C isotope abundances using nano-EA/IRMS (Polissar et al., 2009) were challenged by the presence of co-eluting non-tetrapyrrole structures, and this has been addressed through refined HPLC separation on a hydrocarb column. Nitrogen isotopic measurements have been successful on standards and natural samples. F430 increases in abundance in environmental samples with active anaerobic methane oxidation in a low oxygen environment and provides support for an oxidation pathway that includes reversal of the enzymes involved in methanogenesis.

Tininoid Isotopic Analysis: Pilot Study

Tanja Bosak and her coworkers (Bosak et al., 2011) have recently documented well preserved microfossils in limestrones of Mongolia at 715-638 Ma. These small (~100 μm) organic-walled fossils morphologically resemble tintinnids, modern planktonic ciliates. Bosak has identified these unique microfossils in Cryogenian strata associated with the Tayshir isotopic excursion in carbonate and organic carbon. A new collaboration between Penn State and MIT researchers aims to perform isotopic analyses of these microfossils in order to provide insights to potential role(s) of tintinnoids in the carbon cycle of the ancient ocean. To date, test analyses have been conducted for pilot samples and reveal δ13C values that are consistent with those of modern planktonic organisms. Nitrogen isotopic abundances are slightly enriched relative to values in modern marine eukaryotes. Work is underway to scale up analyses from this pilot study and evaluate across the Mongolian section

Scytonemin: a biomarker for cyanobacterial in harsh environments

We have advanced our study of scytonemin, including new stable isotopic data to identify provenance, and strengthening interpretations based on its occurrence in ancient sediments. Scytonemin is found in extracellular polysaccharide sheaths of terrestrial or benthic cyanobacteria and is most abundant when cells are exposed to direct sunlight, such as in desert soil crusts and intertidal mats. Scytonemin is preserved in abundance in mid-Holocene sedimentary intervals in the Black Sea, a novel deep-sea occurrence that demonstrates that scytonemin may be preserved in other marine or lacustrine sapropel sediments. We report new results of both C and N isotopic compositions from modern settings and for the sedimentary compounds and these findings support the interpretation that scytonemin was derived from cyanobacteria in cryptobiotic desert soils, resisting degradation during erosion and transport. Its occurrence points toward the expansion of arid conditions during the Subboreal Phase (5.5-2 ka, during Unit IIa deposition) in the Black Sea region. Because it survives under harsh irradiative and oxidative conditions and during terrestrial transport processes, scytonemin has potential to serve as an important and diagnostic organic biomarker for tracing the evolution and expansion of cyanobacterial populations especially in association with elevated UV stress.

Subproject: Abiotic production of organic material

In collaboration with the NAI team at NASA Goddard, we have been looking at both pre-RNA chemistry and organics in meteorites. Graduate student Karen Smith published with the NASA Goddard group a paper in PNAS on purine diversity in primitive meteorites. The work shows non-biological purines in meteorites, and also reveals that the abundance of purines in meteorites is likely related to aqueous alteration. Karen is continuing work on the organics in meterorites, as well as her work on pre-RNA chemistry. Undergraduate student Danielle Gruen is continuing studies of HCN production under different atmospheric conditions.

Subproject: Using Methanosarcina to develop new biosignatures

Long chain poly-P is ubiquitous to microbial life on Earth (Proc. Natl. Acad. Sci. USA (2004) 101:16085), although few investigations have been reported in species from the Archaea domain. A role for poly-P in stress response has been suggested for the methane-producing archaeon Methanosarcina acetivorans (J. Proteome Res. (2007) 6:759). The probable ancient origin and widespread occurrence in diverse microbes, coupled with the role in coping with extreme environments, identifies poly-P as a facile biosignature for exploration of extraterrestrial life. We are developing poly-P for use as a biosignature by investigating poly-P metabolism in the domain Archaea with M. acetivorans for which the genome is annotated with genes encoding two putative poly-P kinases (Ppk1 and Ppk2) catalyzing synthesis of poly-P.

The levels of polyphosphate (poly-P) during methanol- or acetate-dependent growth of Methanosarcina acetivorans were measured to begin to assess the physiological role. The results (Fig. 1) show that levels increase with growth and then rapidly decline at the onset of stationary phase in methanol-grown cells, although well before stationary phase in acetate-grown cells suggesting different roles for poly-P depending on the growth substrate. Furthermore, the maximum level of poly-P accumulation at any time during growth with acetate was substantially lower compared to growth with methanol.

Beta-glucuronidase:polyphosphate kinase reporter gene fusions were constructed to assess expression of the kinase during growth with methanol vs. acetate. The results in Figure 4 show greater levels of expression during growth with acetate vs. methanol. The most parsimonious interpretation is that the lower levels of poly-P in acetate-grown cells are the consequence of increased utilization rather than synthesis indicating an important role for poly-P during growth with acetate.

Subproject: Developing Secondary Ion Mass Spectrometry (SIMS) as a tool for Astrobiology

During the past year, we started to develop new samples and standards for the analysis of ancient microfossils and organic material using SIMS.

Subproject: Isotopic biosignatures in minerals

The generation and recording of isotopic biosignatures in minerals is the general focus of this subproject. We are specifically interested in determining if microbes interact with their surrounding environment in a manner that affects the isotopic composition of minerals. We begin this investigation in year 1 by collecting gypsum samples from sulfidic caves and measuring the calcium and sulfur isotopic composition of these samples. In year 2, we initiated abiotic experiments aimed at elucidating the abiotic isotope effects, thus serving as context for evaluating the previous measurements.

In year 3, we completed two main tasks. One, we completed the initial Ca isotope experiments and measured the Ca isotopic composition of the experiments at IFM-GEOMAR in Kiel, GER (Figure 5). This work is the foundation of Harouaka’s Masters thesis. She will graduate in December 2011 and plans to continue her studies in astrobiology working towards a PhD at PSU. Two, Fantle, Harouaka, and Gonzales traveled to the Frasassi caves and sampled atmospheric sulfide for S isotopic analysis and additional gypsum from the cave walls at Grotta Bella in order to characterize the system better.

We also initiated two preliminary studies with the help of undergraduate researchers. The first was a study of low temperature anhydrite formation, which was the basis of Present’s senior thesis. The second is an on-going study of laboratory cultures of A. thiooxidans, work that Mansor is conducting as an UGA. Manor is coadvised by Fantle and Macalady. The latter study is helping us characterize A. thiooxidans morphologically and geochemically and we hope to use this information to design isotope experiments over the next year.

Subproject: DNA as a biosignature

Ancient DNA and DNA preservation

Dr. Beth Shapiro (PSU) collected a series of new permafrost cores during the last field season, from which experiments about the rate and process of DNA decay is now underway. Her team is implementing a new drilling technique to spike samples with bacteria whose genomes contain the pET23a-gfp construct at the time of drilling. This enables extraordinary sensitivity with regard to permafrost thaw, DNA migration and contamination. In addition, this provides a means to monitor and evaluate DNA decay via irradiation. In addition, her work with fossil DNA has resulted in the publication of six peer-reviewed manuscripts, with five more in press.

Metagenomics as a tool for Astrobiology

The House research group participated in two IODP expeditions to gain new samples for the developing of metagenomics for Astrobiology. Leah Brandt and Chris House were shipboard participants in IODP Expedition 331 to study the Deep Hot Biosphere of the Okinawa Hydrothermal Mounds. Then, Mandi Martino was the shipboard microbiologist for IODP Expedition 334 to the Costa Rica margin. These new exciting locations will be very useful for our present work. We also published a paper on the metagenomics of the subsurface Brazos-Trinity Basin (IODP Site 1320), which provided a initial view of the subsurface microbiology at a sandy, low biomass continental margin site. Finally, we have submitted a paper on our newly-developed method for amplifying DNA from low biomass environmental samples. This paper provides a useful method for amplification when the work requires good negative blanks, often essential for applying metagenomics in astrobiology.

This reporting period say the initiation of our Citizen Science Project on thermophiles in domestic water heaters. During the reporting period, we developed the kits that will be sent to participants and recruited participants from across the United States. The kits will be sent out Fall 2011 and returned to our labs in early 2012. Additionally, we isolated a novel Halomonas from wastewater which can grow at a remarkable range of pH and salinity. It is also highly resistant to Asenate. Finally, we also initiated a new collaboration with astrobiologists in Colombian.

4 Institutions
3 Teams
29 Publications
0 Field Sites
Field Sites

Project Progress

Subproject: Raman imaging of kerogen and microfossils as a tool for Astrobiology

Fossil preservation in Miocene and Permian sulfates

By use of optical microscopy, confocal laser scanning microscopy, and Raman spectroscopy, we have recently discovered and documented the presence of abundant and diverse benthic and planktonic microorganisms (e.g., filamentous and coccoidal cyanobacteria, spheroidal green algae, pennate diatoms, etc.) permineralized in Miocene (Messinian, ~6-Ma-old) gypsum deposits of northern Italy and in Permian sulfate deposits of West Texas and southeastern New Mexico. These studies, carried out in collaboration with J. Farmer (Arizona State University), coupled with our recent discovery of gypsum-permineralized microorganisms in Recent sulfate deposits, have obvious relevance to the search for evidence of past life on Mars, where deposits of sulfate, including gypsum are abundant and widespread. These results were presented at the 2011 meeting of the Geological Society of America; a detailed manuscript reporting these findings, to be submitted to Astrobiology, is currently in preparation.

Figure 1. ​Miocene (~6-Ma-old) gypsum-permineralized microfossils compared, in part, with a modern cyanobacterium and modern diatoms.

Apatite-biomineralized Neoproterozoic protists

Using specimens provided by N. J. Butterfield (Cambridge University) and P.A. Cohen (Harvard University), our studies by optical microscopy, confocal laser scanning electron microscopy, and Raman spectroscopy show that ~775-Ma-old protistan scale fossils (from Yukon Territory, Canada) were originally composed of apatite — a surprising discovery, given that the scales of extant protists are most commonly silica or calcite. The Yukon Territory fossils, the earliest example of biomineralization now known from the geological record, have recently been reported by our group in Geology Today; a more detailed manuscript reporting these findings was published in Geology.

Chert-permineralized Paleoproterozoic sulfuretum
Using samples originally provided by Martin Van Kranendonk, augmented by samples that Malcolm Walter and I collected in June 2011, we have discovered a ~2,300-Ma-old microbial ecosystem previously unknown in Precambrian deposits. This ecosystem, composed of sulfate-reducing and sulfur-oxidizing bacteria, is remarkably similar to extant sulfureta first described by Victor Gallardo and Carola Espinoza in 2007 from numerous core samples collected at the sediment-water interface in the sub-oxygen minimum zone off the western coast of South America. The stratigraphy and geologic setting indicates that the fossiliferous rocks were deposited at depth, well below wave base and most likely below the photic zone. Geochemical data are consistent with the presence of sulfate reducers. The major taxa of the abundant, diverse assemblage are morphologically distinct from fossil and modern cyanobacteria but are essentially identical to sulfur-oxidizing bacteria described by Gallardo and Espinoza. Similarly, the wispy cobweb fabric of the microbial assemblage differs distinctly from the laminated fabric of photoautotroph-dominated stromatolite/microbial mat communities but is indistinguishable from the fabric of modern sulfureta. When current SIMS work is completed, designed to measure the carbon isotopic composition of individual benthic and planktonic members of the assemblage (and based on the pioneering work of Christopher House, published in 2000), a paper reporting these new findings will be submitted to Nature.

Figure 2. ​Paleoproterozoic broad filaments compared with modern sulfur bacteria from off the western coast of South America.

Figure 3. ​Paleoproterozoic medium-diameter (left) and narrow-diameter (right) “bead cell filaments” compared with modern sulfur bacterial bead cell filaments from off the western coast of South America.

Our studies of extant microorganisms (biology), fossil megascopic and microscopic organisms (paleontology), the stratigraphy, mineralogy and taphonomy of their preservation (geology), and their chemical and isotopic composition (geochemistry) are highly interdisciplinary, involving both the life sciences (biology, microbiology, evolutionary biology) and the physical sciences (geology and geochemistry).

In addition to the UCLA contributors noted above (J. William Schopf, Anatoliy B. Kudryavtsev, and Ian B. Foster), contributors to this research include: Jack D. Farmer (NAI Astrobiology Center, Arizona State University); Malcolm R. Walter and Martin Van Kranendonk (Australian Centre for Astrobiology, University of New South Wales, Sydney, Australia); Kliti Grice (Curtin University, Perth, Australia); John Valley and Ken Williford (NAI Astrobiology Center, University of Wisconsin); Victor Gallardo and Carola Espinoza (Universidad de Concepción, Chile); Phoebe A. Cohen (MIT); Francis Macdonald (Harvard University); and Nicolas J. Butterfield (Cambridge University)

Subproject: Weathering and inorganic chemical biosignatures

Secondary minerals and regolith profiles on basaltic rocks
Based upon images taken during the 2003-2004 series of missions to Mars, several different weathering features appear to be present on the Mars surface including surface flaking, dirt cracking, exfoliation, honeycomb weathering and the formation of what appear to be weathering rinds (Thomas et al., 2005). However, the underlying mechanisms responsible for these features are difficult to ascertain from images alone. The goal of this study is to understand the role that different climatic conditions on Earth play in facilitating the weathering of basalts, which are thought to be the predominant lithology present on Mars. Basalts from three different sites are compared, two of which come from the temperate northeastern United States, while the third site is located in the dry, Arctic region of Svalbard and is thus considered to be a Mars analogue. Soil profiles from the two North American sites are much deeper than those from Svalbard and show clear evidence of chemical weathering in the bulk soil profile. While no evidence for chemical weathering is observed in bulk soils from Svalbard, the clay-sized fraction nevertheless shows clear evidence for the elemental depletion of K, Na, Ca and Mg. In contrast, Al and Fe appear to be immobile even in the finest size fractions of the Svalbard soil. Coupled with SEM images showing evidence of spalling on the outermost 100 μm of Svalbard rocks, this chemical evidence suggests that the process of spalling in cold climates may preferentially remove certain elements over others both on Earth as well as on Mars. Extractions with 0.5 N HCl, designed operationally to remove amorphous Fe oxides, indicate that the prevalence of amorphous Fe is greatest in the lowermost horizons of a Pennsylvania diabase, a finding that has been noted previously in a granodiorite-derived soil profile from Puerto Rico that is thought to contain a lithoautotrophic Fe-oxidizing community at depth (Buss et al., 2010). In further agreement with findings from Puerto Rico, Fe(II) concentrations in the lowermost soil of the Pennsylvania site decrease over a short distance as a results of rapid oxidation in the lowermost portion of the 4-m profile analyzed here. In contrast, no such change in extractable Fe concentrations or Fe(II) content is noted within the regolith of the Svalbard Mars analogue site, at which Fe-oxidizing bacteria are presumably absent.

DNA has been extracted from several of the sampling sites on Svalbard, and metagenomic sequencing carried out. Preliminary analysis of the sequence data shows that while Proteobacteria, Actinobacteria, Acidobacteria, and Bacteroidetes are the dominant phyla at all sites sequenced, there are significant differences in phylum relative abundance and community structure between sites.

Community structure and geochemistry of weathering in a Puerto Rican watershed
(Note: Primary funding for this project is through DOE)

We have hypothesized that Fe oxidation may be important in weathering of intact bedrock to disaggregated regolith at depth (9.2 m) in the Bisley volcaniclastic watershed in the Luquillo Mountains of Puerto Rico. The regolith forms along a gradient between conditions of low oxygen and higher pH at depth, to higher oxygen and lower-pH waters at the surface. The active weathering front was observed to be ~ 8.3 m, as secondary clay minerals and Fe oxides are present from the surface down to this depth, while the primary minerals chlorite and feldspars were detected below 8.3 m. Consistent with an active weathering front at ~ 8.3 m, ammonium-acetate extracted Ca and Mg were present in higher concentrations below 7 m. There were also increases in both total and heterotrophic cell counts in this region, as well as increased concentrations of HCl-extracted Fe(II) and organic-bound Fe. As a consequence of these gradients in oxygen, pH, and chemical composition, we expect to see changes in microbial communities along the regolith. Metagenomics analysis revealed that sub-phylum groups containing known iron-oxidizing microorganisms were found in the deepest samples, near the regolith-bedrock interface, consistent with the hypothesis that chemolithoautotrophic bacteria play an important role in weathering and element cycling in the deep regolith. Rarefaction curves generated at a 97% similarity value indicated that the regolith was representatively sampled for determining community richness.

Subproject: Intermolecular Isotopic Patterns

F430 Isolation and isotopic characterization
Methyl-coenzyme M reductase, an enzyme that plays a key role in methanogenesis, has recently been linked also to the anaerobic oxidation of methane (Scheller, 2010). Cofactor F430 is a tetrapyrrole nickel complex contained within the active site of methyl-coenzyme M and appears to be used in both methanogenesis and methanotrophy (Scheller, 2010; Mayer et al., 2008). Our efforts have been focused on purifying F430 from natural samples in order to determine δ15N and δ13C values both at natural abundance and in isotopically enriched samples from tracer studies. F430 is isolated using multidimensional preparatory and high-performance chromatographic separations. Compound identity and purity are confirmed using molar C:N ratios, light absorbance and MSn detection and fragmentation of F430 isolated from pure cultures of Methanosarcina acetivorans. Our efforts to measure C isotope abundances using nano-EA/IRMS (Polissar et al., 2009) were challenged by the presence of co-eluting non-tetrapyrrole structures, and this has been addressed through refined HPLC separation on a hydrocarb column. Nitrogen isotopic measurements have been successful on standards and natural samples. F430 increases in abundance in environmental samples with active anaerobic methane oxidation in a low oxygen environment and provides support for an oxidation pathway that includes reversal of the enzymes involved in methanogenesis.

Tininoid Isotopic Analysis: Pilot Study
Tanja Bosak and her coworkers (Bosak et al., 2011) have recently documented well preserved microfossils in limestrones of Mongolia at 715-638 Ma. These small (~100 μm) organic-walled fossils morphologically resemble tintinnids, modern planktonic ciliates. Bosak has identified these unique microfossils in Cryogenian strata associated with the Tayshir isotopic excursion in carbonate and organic carbon. A new collaboration between Penn State and MIT researchers aims to perform isotopic analyses of these microfossils in order to provide insights to potential role(s) of tintinnoids in the carbon cycle of the ancient ocean. To date, test analyses have been conducted for pilot samples and reveal δ13C values that are consistent with those of modern planktonic organisms. Nitrogen isotopic abundances are slightly enriched relative to values in modern marine eukaryotes. Work is underway to scale up analyses from this pilot study and evaluate across the Mongolian section.

Scytonemin: a biomarker for cyanobacterial in harsh environments
We have advanced our study of scytonemin, including new stable isotopic data to identify provenance, and strengthening interpretations based on its occurrence in ancient sediments. Scytonemin is found in extracellular polysaccharide sheaths of terrestrial or benthic cyanobacteria and is most abundant when cells are exposed to direct sunlight, such as in desert soil crusts and intertidal mats. Scytonemin is preserved in abundance in mid-Holocene sedimentary intervals in the Black Sea, a novel deep-sea occurrence that demonstrates that scytonemin may be preserved in other marine or lacustrine sapropel sediments. We report new results of both C and N isotopic compositions from modern settings and for the sedimentary compounds and these findings support the interpretation that scytonemin was derived from cyanobacteria in cryptobiotic desert soils, resisting degradation during erosion and transport. Its occurrence points toward the expansion of arid conditions during the Subboreal Phase (5.5-2 ka, during Unit IIa deposition) in the Black Sea region. Because it survives under harsh irradiative and oxidative conditions and during terrestrial transport processes, scytonemin has potential to serve as an important and diagnostic organic biomarker for tracing the evolution and expansion of cyanobacterial populations especially in association with elevated UV stress.

Subproject: Abiotic production of organic material
In collaboration with the NAI team at NASA Goddard, we have been looking at both pre-RNA chemistry and organics in meteorites. Graduate student Karen Smith published with the NASA Goddard group a paper in PNAS on purine diversity in primitive meteorites. The work shows non-biological purines in meteorites, and also reveals that the abundance of purines in meteorites is likely related to aqueous alteration. Karen is continuing work on the organics in meterorites, as well as her work on pre-RNA chemistry. Undergraduate student Danielle Gruen is continuing studies of HCN production under different atmospheric conditions.

Subproject: Using Methanosarcina to develop new biosignatures

Long chain poly-P is ubiquitous to microbial life on Earth (Proc. Natl. Acad. Sci. USA (2004) 101:16085), although few investigations have been reported in species from the Archaea domain. A role for poly-P in stress response has been suggested for the methane-producing archaeon Methanosarcina acetivorans (J. Proteome Res. (2007) 6:759). The probable ancient origin and widespread occurrence in diverse microbes, coupled with the role in coping with extreme environments, identifies poly-P as a facile biosignature for exploration of extraterrestrial life. We are developing poly-P for use as a biosignature by investigating poly-P metabolism in the domain Archaea with M. acetivorans for which the genome is annotated with genes encoding two putative poly-P kinases (Ppk1 and Ppk2) catalyzing synthesis of poly-P.

The levels of polyphosphate (poly-P) during methanol- or acetate-dependent growth of Methanosarcina acetivorans were measured to begin to assess the physiological role. The results (Fig. 1) show that levels increase with growth and then rapidly decline at the onset of stationary phase in methanol-grown cells, although well before stationary phase in acetate-grown cells suggesting different roles for poly-P depending on the growth substrate. Furthermore, the maximum level of poly-P accumulation at any time during growth with acetate was substantially lower compared to growth with methanol.

Beta-glucuronidase:polyphosphate kinase reporter gene fusions were constructed to assess expression of the kinase during growth with methanol vs. acetate. The results in Figure 4 show greater levels of expression during growth with acetate vs. methanol. The most parsimonious interpretation is that the lower levels of poly-P in acetate-grown cells are the consequence of increased utilization rather than synthesis indicating an important role for poly-P during growth with acetate.

Figure 4. ​Time course of β-glucuronidase activity in cell extracts of methanol-grown (left panel) or acetate-grown (right panel) Methanosarcina acetivorans strains harboring β-glucuronidase:polyphosphate kinase gene fusions. Blue = growth; red = β-glucuronidase activity measured with p-nitro-phenol-glucuronide as substrate.

Subproject: Developing Secondary Ion Mass Spectrometry (SIMS) as a tool for Astrobiology

During the past year, we started to develop new samples and standards for the analysis of ancient microfossils and organic material using SIMS.

Subproject: Isotopic biosignatures in minerals

The generation and recording of isotopic biosignatures in minerals is the general focus of this subproject. We are specifically interested in determining if microbes interact with their surrounding environment in a manner that affects the isotopic composition of minerals. We begin this investigation in year 1 by collecting gypsum samples from sulfidic caves and measuring the calcium and sulfur isotopic composition of these samples. In year 2, we initiated abiotic experiments aimed at elucidating the abiotic isotope effects, thus serving as context for evaluating the previous measurements.

In year 3, we completed two main tasks. One, we completed the initial Ca isotope experiments and measured the Ca isotopic composition of the experiments at IFM-GEOMAR in Kiel, GER (Figure 5). This work is the foundation of Harouaka’s Masters thesis. She will graduate in December 2011 and plans to continue her studies in astrobiology working towards a PhD at PSU. Two, Fantle, Harouaka, and Gonzales traveled to the Frasassi caves and sampled atmospheric sulfide for S isotopic analysis and additional gypsum from the cave walls at Grotta Bella in order to characterize the system better.

We also initiated two preliminary studies with the help of undergraduate researchers. The first was a study of low temperature anhydrite formation, which was the basis of Present’s senior thesis. The second is an on-going study of laboratory cultures of A. thiooxidans, work that Mansor is conducting as an UGA. Manor is coadvised by Fantle and Macalady. The latter study is helping us characterize A. thiooxidans morphologically and geochemically and we hope to use this information to design isotope experiments over the next year.

Figure 5. ​Comparison of this study’s results for Ca isotopic fractionation (Δij = δi-δj) during gypsum precipitation (yellow circles) and previously published Ca isotopic fractionation during calcite precipitation. Rate is the BET surface area normalized precipitation rate.

Subproject: DNA as a biosignature

Ancient DNA and DNA preservation
Dr. Beth Shapiro (PSU) collected a series of new permafrost cores during the last field season, from which experiments about the rate and process of DNA decay is now underway. Her team is implementing a new drilling technique to spike samples with bacteria whose genomes contain the pET23a-gfp construct at the time of drilling. This enables extraordinary sensitivity with regard to permafrost thaw, DNA migration and contamination. In addition, this provides a means to monitor and evaluate DNA decay via irradiation. In addition, her work with fossil DNA has resulted in the publication of six peer-reviewed manuscripts, with five more in press.

Metagenomics as a tool for Astrobiology

The House research group participated in two IODP expeditions to gain new samples for the developing of metagenomics for Astrobiology. Leah Brandt and Chris House were shipboard participants in IODP Expedition 331 to study the Deep Hot Biosphere of the Okinawa Hydrothermal Mounds. Then, Mandi Martino was the shipboard microbiologist for IODP Expedition 334 to the Costa Rica margin. These new exciting locations will be very useful for our present work. We also published a paper on the metagenomics of the subsurface Brazos-Trinity Basin (IODP Site 1320), which provided a initial view of the subsurface microbiology at a sandy, low biomass continental margin site. Finally, we have submitted a paper on our newly-developed method for amplifying DNA from low biomass environmental samples. This paper provides a useful method for amplification when the work requires good negative blanks, often essential for applying metagenomics in astrobiology.

This reporting period say the initiation of our Citizen Science Project on thermophiles in domestic water heaters. During the reporting period, we developed the kits that will be sent to participants and recruited participants from across the United States. The kits will be sent out Fall 2011 and returned to our labs in early 2012. Additionally, we isolated a novel Halomonas from wastewater which can grow at a remarkable range of pH and salinity. It is also highly resistant to Asenate. Finally, we also initiated a new collaboration with astrobiologists in Colombian.

Figure 6. ​Phylogenetic associations of all BLAST hits (nr database) (a) and only single copy protein-encoding genes (b) for the Brazos-Trinity Basin and Peru Margin datasets. mbsf = meters below seafloor.