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2010 Annual Science Report

Pennsylvania State University Reporting  |  SEP 2009 – AUG 2010

Biosignatures in Relevant Microbial Ecosystems

Project Summary

In this project, PSARC team members explore the isotope ratios, gene sequences, minerals, organic molecules, and other signatures of life in modern environments that have important similarities with early earth conditions, or with life that may be present elsewhere in the solar system and beyond. Many of these environments are “extreme” by human standards and/or have conditions that are at the limit for microbial life on Earth.

4 Institutions
3 Teams
8 Publications
6 Field Sites
Field Sites

Project Progress


Molecular signatures of microbial life in the Dead Sea House, Freeman, Macalady & graduate students M. Rhodes and K. Dawson (in collaboration with A. Oren & J. R. Spear)

The sub-project published a paper on metagenomics of the hypersaline Dead Sea, focusing on the amino acid content of proteins predicted by the genomic data collected. The work presents a remarkable relationship between predicted amino acid content of proteins (acidic to basic amino acids) and salinity at an environmental scale. By comparing the amino acid content of reads, their best-hit phyogenetic relationship, and the results of amplicon sequencing, we further differentiate DNA from endiginous species, lateral gene transferred material, and foreign DNA. The work shows evidence for lateral gene transfer between a halophilic archaeon and relatives of the thermophilic bacterial genus Thermotoga and also suggests the presence of indigenous Dead Sea representatives from 10 traditionally non-hyperhalophilic bacterial lineages. A second paper in preparation studies lateral gene transfer by comparing events that involve members of the Halobacteria with events that involve members of the Thermococcales. The work suggests that the mechanisms involved in lateral gene transfer in saline environments are different than in lateral gene transfer in thermophilic environments. We have also started proposed work on DNA fossilization processes involving saline environments using the Dead Sea samples. Graduate student Katherine Dawson is unraveling the contributions of archaeal membrane physiology and shifts in community composition to the organic biomarker record of salinity fluctuations in the Dead Sea.

Peer reviewed publications:
Rhodes, M.E., Fitz-Gibbon, S.T., Oren, A., House, C.H. 2010. Amino
Acid Signatures of Salinity on an Environmental Scale with a Focus on
the Dead Sea. Environmental Microbiology. Volume 12, Number 9,
September 2010 , pp. 2613-2623(11).

Rhodes, M.E., Fitz-Gibbon, S.T., Spear, J., Oren, A., House, C.H. in
prep. Lateral Gene Transfer in Thermal and Saline Environments.
Extermophiles (in preparation).

Abstracts presented:
Abscicon: April 26-29th, 2010 League City, Texas (Poster)
Rhodes M. E. Fitz-Gibbon S. T. Spear J. R. Oren A. House C. H.
Identifying and Categorizing Lateral Gene Transfer Events in
Hypersaline Environments

Impact cratering and subsurface microbial life Macalady, Shapiro & graduate student Kristine Korzow-Richter (in collaboration with C. Cockell, D. Vanko & M. Voytek)
Ph.D. student Kristine Korzow-Richter, co-advised by Beth Shapiro (primary) and Jenn Macalady, continues work on authenticated core samples from the ~2 km deep Chesapeake Bay impact structure. A research meeting with USGS collaborators including Mary Voytek and Greg Gohn (head of the USGS/ICDP drilling project) was held on Nov. 30, 2010 at the USGS in Reston to view the core lithologies and retrieve preserved microbiology/genomics samples from the drilling project archives. Kristine has taken classes that set the stage for her project and fulfill requirements of the Astrobiology Dual Ph.D. Degree program. Her achievements in the past year include a comprehensive evaluation of DNA extraction, purification and contamination control methods for low-biomass rock samples containing significant concentrations of PCR inhibitors. She is now poised to extract DNA from 10 authenticated drill core samples, and to evaluate the quantity and quality of DNA in preparation for metagenomic sequencing (planned for December 2010).

Methane-derived isotopic & mineralogical biosignatures in cold seeps Orphan, Ferry, Freeman, and McKeegan (in collaboration with J. Grotzinger)

Other team members: Jake Bailey (postdoc), Benjamin Harrison (graduate student), Anne Dekas (graduate student), Jeffrey Marlow (graduate student), O. Mason (postdoc), Joshua Steele (postdoc), Grayson Chadwick (undergraduate), Tom Bristow (Caltech), Tim Raub (Caltech), Lisa Levin (SIO)

Microbial diversity associated with methane-derived authigenic carbonates
To characterize the similarity in diversity and carbon-13 isotopic signatures between carbonates and host seep sediments, molecular methods (T-RFLP and 16S rRNA clone libraries) were used in combination with statistical ordination analysis (PC-ORD). Based on our findings, it appears that AOM-induced authigenic carbonates form close to the seabed and generally record the in situ sediment associated microbial diversity, rather than being exclusively associated with specific methane-oxidizing lineages (Harrison et al, 2010, Mason et al, in prep). Parallel research examining microbial diversity in exhumed carbonate slabs with low d13C values (-45 to -60‰) both within and outside areas of active methane seepage revealed distinct differences in bacterial assemblages between active and inactive sites. In contrast, archaeal diversity was similar across both habitats, showing a dominance in methane-oxidizing ANME archaea. The shift in bacterial diversity between active and inactive carbonates likely reflects early stages of weathering and may have implications for the composition and isotopic values of organic biosignatures that are ultimately archived in ancient methane-seep deposits. This research was conducted by undergraduates and first year graduate students as part of the laboratory training exercise for the Introduction to Geobiology course taught by V. Orphan and J. Grotzinger. First year graduate student Jeffrey Marlow continued this research, specifically characterizing spatial variations in carbonate-associated microbial diversity on surfaces directly exposed to seawater vs. the interior carbonate matrix.

Mineral phases associated with methanotrophic consortia.
Sulfate-dependent anaerobic oxidation of methane increases the alkalinity of the local environment, stimulating carbonate precipitation. It is currently not known whether this phenomenon is strictly mediated via the alteration of pore water alkalinity from microbial respiration or if there is a more direct role for methane-oxidizing consortia, where the microbes themselves serve as nucleation points for carbonate precipitation. Surveys of methane oxidizing microbial consortia from methane seep sediments using epifluorescence and transmitted light microscopy revealed the frequent occurrence of a translucent mineral phase associated with the exterior of many of the methanotrophic aggregates. It was hypothesized that these micro-scale crusts were comprised of calcite or aragonite, however closer examination however revealed a texture unusual for carbonates that was unaltered upon treatment with acid. Energy-dispersive X-ray spectroscopy (EDS) of individual mineral coated aggregates was employed to determine the composition of the crust and revealed the presence of C, O, Si, Al, Mg, Na, and Fe, elements suggestive of organic material in combination with aluminosilicate clay. Analyses using a nanoSIMS 50L further revealed the close association of a Si-rich component on the exterior of the aggregate surface. Follow-up work will include Raman microscopy to better characterize the clay-like mineral phase and to determine how this exterior rind influences the activity and preservation/taphonomy of anaerobic methane oxidizing consortia.

Preservation of AOM body fossils in methane-derived authigenic carbonates
With the goal of determining whether methane seep endemic microbes are preserved as microfossils in authigenic seep precipitates, we undertook a survey of Quaternary authigenic carbonates from the Eel River Basin. Petrographic examination of carbonates revealed structures that bear close morphological resemblance to the bacterial and archaeal cell aggregates thought to mediate seep carbonate precipitation in surrounding sediments. However, additional analyses using SEM, EDS, Raman spectroscopy, and rock magnetic analyses suggest that these structures are not the fossilized remains of microorganisms at all, but rather the products of the diagenetic alteration of sulfide framboids (Bailey et al., 2010). These results are important because the ability to differentiate between cellular remains and acellular mineral matter is critical for life detection efforts on other planets, as well as for tracking the evolution of biogeochemical cycles on Earth.


Bailey JV, Raub TD, Meckler AN, Harrison BK, Raub TMD, Green AM, Orphan VJ (2010) Pseudofossils in relict methane seep carbonates resemble endemic microbial consortia. Palaeogeography, Palaeoclimatology, Palaeoecology, 285, 131-142.

Bailey, J.V., V.J. Orphan, S.M. Joye, F. A. Corsetti (2009) Chemotrophic microbial mats and their potential for preservation in the rock record. Astrobiology. 9: 843-859.

J. V. Bailey, V. Salman, G. W. Rouse, H. N. Schulz-Vogt, L. A. Levin, V. J. Orphan (in review) A dimorphic life cycle in methane seep dwelling ecotypes of the largest known bacteria. ISME J.

Newman D, V.J. Orphan, A.L. Reysenbach (In Review) Molecular biology’s contributions to Geobiology. In Fundamentals of Geobiology. Ed. Knoll, A. D. Canfield, K. Konhauser.

Conference Proceedings:

Orphan V. J. , Dekas A. E. ,Poretsky R. ,Amend J. 2010. Methane-oxidizing Archaea Fix Nitrogen in Cooperation with Sulfate-reducing Bacteria in Deep-Sea Methane Seeps. Program and Abstracts, AbSciCon10, Houston, TX.

A. E. Dekas, J. V. Bailey, T. F. Bristow, S. S. Walavalkar, V. J. Orphan. 2010. Clay Crust Surrounds Microorganisms Mediating the Anaerobic Oxidation of Methane in Marine Sediments. 4th annual Southern California Geobiology Symposium, Pasadena, CA.


Bailey J, Salman V, Rouse G, Orphan V, Schulz-Vogt H, Levin L (2010a) DIMORPHISM IN METHANE SEEP INHABITING THIOMARGARITA In: International Symposium of Microbial Ecology, Seattle, WA.

Life in Greenland glacial ice Brenchley, Miteva, and Loveland-Curtze with undergraduate students L. Seth and N. Lazar

1. Novel bacterial species isolated from Greenland glacial ice
We continue to develop novel cultivation strategies to recover isolates from Greenland glacial ice from different depths. Previously we reported about the characterization and description of two new Gram-negative bacterial species, Chryseobacterium greenlandense of the phylum, Bacteroidetes, and Herminiimonas glaciei of the phylum, Proteobacteria that are the only validly described species from Greenland glacial ice. Our article concerning C. greenlandense was published in Extremophiles in January 2010 and that citation was published in International Journal of Systematic and Evolutionary Microbiology (IJSEM) validation list #134 (July, 2010) so that the species is now officially recognized. A press release in June 2009 that relating to our IJSEM article about H. glaciei resulted in many news reports in the scientific and popular press. In the September 2009 issue of the ASM News Magazine, “Microbe” an article about extremophiles highlighted H. galciei was published:
DiGregorio, B. 2010. New extremes for earth habitats supporting microbial species Microbe 4:399-401.
Other events and programs in which some of our isolates from cold environments and glacial ice were highlighted include an exhibit at the Earth and Mineral Sciences Museum at Penn State University Park Campus, entitled “Beyond the Edge of the Sea: Diversity of Life in the Deep-Ocean Wilderness” from January 2010 to August 2010. A poster about “Life in Ice” featuring some of our isolates from cold environments and petri dishes containing living cultures of glacial isolates were displayed. In addition, a Brenchley lab psychrophilic isolate from Antarctica, Rhodoglobus vestalii was shown as an example of an extremophile in the program “Journey to an alien moon” on the National Geographic channel, that first aired April 20, 2010.

2. New method development
As a result of characterizing novel isolates from glacial ice we recognized the need for a more rapid and sensitive method for assessing hybridization of DNA from different organisms to estimate the relatedness of bacterial species. To demonstrate the reliability of our new method, we compared our calculated results to published values for pairs of bacterial species from diverse phyla. We concluded that the new technique is appropriate when describing a new species or for quickly screening uncharacterized isolates to determine their relatedness. We have submitted a manuscript about the evaluation of this new technique.

3. Diversity of Greenland GISP2 ice from the last glacier maximum (LGM)
Previous analyses of the microbial abundance and viability at different depth of the 3 km deep Greenland GISP2 core showed that microbial distribution and viability differed with depth. The “silty” ice below 3042 m contained large amounts of debris and showed abundant (107 –108 cells/ml) and viable (6–30%) populations including up to 15% ultra-small cells (<1 µm). Cell concentrations in the clear ice samples ranged from 103 to 105/ml with several peaks corresponding to concentrated dust layers and colder climates. Interestingly, these higher peaks in cell numbers correlated with higher Ca++ concentrations considered a proxy of dust concentrations. Recently we studied the microbial diversity in one of these peaks from 2010 m depth representing ice deposited during the last glacial maximum (about 25 ka ago). Cell concentrations and viability were very high – 106 –107 cells/ml and 55%, respectively. The constructed universal SSU rRNA gene clone library was dominated by Proteobacteria (27.1% Alpha and 66.7% Beta Proteobacteria) and had fewer Gram-positive sequences (6.25%). As shown on the phylogenetic tree the most closely related sequences originated from Asia considered to be a major source of dust deposited over Greenland and from other cold environments. The microbial diversity found in this sample differed from other clear ice samples deposited under different climate conditions and indicates that climate may have influenced the microbial records preserved in deep glacial ice.

Biosignatures of life in Archaean-analog anoxic biofilms Macalady & graduate students McCauley, Jones (incollaboration with A. Montanari, A. Zerkle, J. Farquhar)

A rare window into an anoxic subsurface aquifer is accessible in lakes deep with the sulfidic Frasassi cave complex in central Italy, where cave divers exploring the system discovered unusual, rope-like microbial biofilms. Previous work suggested that little redox energy is available for life in the water hosting the biofilm, consistent with low signal from domain-specific FISH probes, and that most of the populations are relatives of known sulfur reducers or affiliated with uncultivated or novel prokaryotic lineages. These microorganisms and their potentially novel metabolic strategies are relevant for understanding biogeochemistry and biosignatures of non-photosynthetic, energy-limited environments on the modern and ancient Earth and elsewhere in the solar system. A new biofilm sample collected in September 2009 by Macalady, Jones, Italian cave explorers, and cave diver Kenneth Broad (Rosenstiel School of Marine and Atmospheric Sciences, University of Miami) was interrogated using enrichment culturing and metagenomics. Astrobiology Dual Title Ph.D. student Rebecca McCauley is currently characterizing the resulting bacterial and archaeal cultures and annotating a metagenomic dataset. Taxonomic marker genes in the metagenome indicate that archaea make up a minor percentage (10%) of the biofilm, and that both Deltaproteobacteria (likely sulfur reducers) and Chloroflexi (unknown metabolisms) are important populations. Further metagenomic sequencing has been approved by the DOE Joint Genome Institute and will commence in late fall 2010. The major and minor sulfur isotope systematics of the cave ecosystem are being investigated by Zerkle and Farquhar (Univ. of Maryland).

Abstracts presented:
Zerkle, A., Macalady, J. L., and Farquhar, J. 2009. S isotope investigation of sulfur cycling in the Frasassi cave system. Sulfidic Karst Ecosystems, September 10-14, 2009, Genga, Italy.
Jones, D. S., Tobler, R., Schaperdoth, I., Galdenzi, S., Mainiero, M., and Macalady, J. L. 2009. Sulfur oxidizing extremophiles from the caves of Acquasanta Terme, Italy. Sulfidic Karst Ecosystems, September 10-14, 2009, Genga, Italy.

Macalady, J. L. [invited], Jones, D. S., McCauley, R., Schaperdoth, I., Bloom, D., and Mariani, S. 2009. Frasassi’s Microbial Sulfur World: Progress and Prospects. Sulfidic Karst Ecosystems, September 10-14, 2009, Genga, Italy.

Biosignatures of life in ancient stratified ocean analogs Macalady, Freeman, & Kump with graduate students J. Fulton, K. Meyer & R. McCauley

Instigated by Macalady, Kump and National Geographic Explorer Kenneth Broad (University of Miami), this new project investigates biosignatures of life in modern analogs for stratified ancient and/or extraterrestrial oceans. Seed funding provided by National Geographic/NOVA resulted in a preliminary dataset for stratified sinkholes in the Bahamas as well as a 1-hour television documentary that aired on NOVA and National Geographic International in Spring 2010 and a National Geographic cover story in August 2010. A field workshop at stratified Fayetteville Green Lake sponsored by NAI, Agouron Institute, and CIFAR was attended by 50 national and international participants. A new website monitoring the activities of an informal working group on Early Earth Photosynthesis is maintained by Macalady (see

Peer reviewed publications:
Meyer, K. , Freeman, K. H. , Macalady, J. L. , Fulton, J., Schaperdoth, I., and Kump, L. 2011. Benthic okenone production in Fayetteville Green Lake (NY). In review, Geobiology.
Gonzalez, B. C., Iliffe, T. M., Macalady, J. L., Schaperdoth, I., and Kakuk, B. 2011. Biogeochemical diversity of anchilaline blue holes, The Bahamas. In revision, Hydrobiologia (special issue).
Macalady, J. L., Schaperdoth, I., Iliffe, T. M., and Broad, K. Chlorobi dominate the shallow chemocline of Sawmill Sink, Bahamas. In preparation for Applied and Environmental Microbiology.

Abstracts presented:
Macalady, J. L., Schaperdoth, I., Fulton, J. M., Freeman, K. H., and Hanson, T. E. 2010. Microbial biogeochemistry of a meromictic blue hole. Geochimica et Cosmochimica Acta 74 (12, Supplement 1 June 2010): 1496.
Macalady, J. L. [invited]. 2010. Molecular biogeochemistry of microbial ecosystems, Gordon Research Conference on Organic Geochemistry, August 1-6, New Hampshire, USA.