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Project Progress

Raman imaging of microfossils
Chert-permineralized Duck Creek (~1,800 Ma) sulfuretum, W. Australia
For evidently the first time, our work has affirmed the null hypothesis required of Darwinian evolution: if there is no change in the physical-biological environment of a well adapted ecosystem there will be no evolution of the form, function or metabolic requirements of its biotic components
Chert-permineralized Bambuí (~775 Ma) sulfur cycling bacteria, Brazil
We have now shown, evidently for the first time, that pyritized microbes of this (and no doubt many other Precambrian microbiotas, previously regarded as Bacteria Incertae Sedis), are anaerobic sulfur-circlers.
Biologic impact of the Great Oxidation Event
This work is the first to document the biotic response to the GOE, based on four lines of evidence; (1) the anaerobic to aerobic transition of metabolic and biosynthetic pathways; (2) rRNA phylogeny-correlated fossil evidence of O₂-protection in immediately post-GOE-preserved cyanobacteria; (3) the earliest fossil occurrences of obligately aerobic eukaryotes; and (4) recent discoveries of mid-Precambrian anaerobic microbial sulfuretums dependent on the GOE-promoted increase of required nutrients.

Biosigntures through Secondary Ion Mass Spectrometry (SIMS)
The ca. 3 Ga Farrel Quartzite (FQ, Western Australia) contains possible organic microfossils of unusual spindle-like morphology that are surprisingly large and complex, preserved along with spheroids. The unusual nature of the possible fossils, coupled with their antiquity, makes their interpretation as biogenic difficult and debatable. We published 32 in situ carbon isotopic analyses of 15 individual FQ specimens. The spheroids and the spindle-like forms have a weighted mean δ¹³C value of –37‰, an isotopic composition that is quite consistent with a biogenic origin. Both the spheroids and the spindle-like structures are isotopically distinct from the background organic matter in the same thin section (weighted mean δ¹³C value of –33‰), which shows that the preserved microstructures are not pseudofossils formed from physical reprocessing of the bulk sedimentary organic material. When considered along with published morphological and chemical studies, these results indicate that the FQ microstructures are bona fide microfossils, and support the interpretation that the spindles were planktonic. Our results also provide metabolic constraints that imply most of these preserved microorganisms were autotrophic. The existence of similar spindles in the ca. 3.4 Ga Strelley Pool Formation of Australia and the ca. 3.4 Ga Onverwacht Group of South Africa suggests that the spindle-containing microbiota may be one of the oldest, morphologically preserved examples of life. If this is the case, then the FQ structures represent the remains of a cosmopolitan biological experiment that appears to have lasted for several hundred million years, starting in the Paleoarchean.

F430: the Gateway for AOM
If AOM Archaea oxidize methane by reverse methanogenesis, coenzyme F430 likely catalyzes the first step. Culture studies of methanogenic Archaea that can carry out trace oxidation of methane provide supporting evidence for a reversed methanogenesis biochemical pathway. The methanogen Methanosarcina acetovorines was shown to oxidize trace amounts of methane to CO₂ (Moran et al., 2006) as documented by observations that ¹³C-labeled methane became incorporated into CO₂. Studies of Methanothermobacter marburgensis in pure culture demonstrated the last step in methanogenesis is also the first step in methane oxidation (Scheller et al., 2010) by the incorporation of ¹³C-labeled methane into methyl-coenzyme M (2-mercaptoethanesulfonate) catalyzed by coenzyme F430. Genetic evidence from environmental samples provides additional support for reverse methanogenesis during AOM. Hallam et al., (2004) found genes that code for the enzymes used in methanogenesis, including for the last step, in ANME-1 and ANME-2 dominated samples from the Eel River Basin. This suggests reverse methanogenesis capability is present among organisms in the sediment, and if the process takes place, the signature coenzyme F430 should also be present in the sediment.

We extracted, identified and quantified F430 from four samples of Hydrate Ridge sediment. The concentration of F430 covaried with the concentration of AOM signatures as indicated by cell counts, fatty acids, sulfide, sulfate and methane (Figure 2). The maximum in F430 concentrations (0.086 μg/g) in the 3-6 cm core section this coincides with maxima in ANME cell counts and fatty acid abundances. This section of the core also corresponds to a drop in methane and sulfate and an increase in sulfide concentration. Notably, F430 is more than 30 ‰ enriched in 13C relative to hydroxyarchaeol extracted from the same sediment (Figure 3), as measured using nano-EA/IRMS. From these intriguing results, we hypothesize CO₂ assimilation provides carbon for biosynthesis of F430, while methane-derived carbon is incorporated into lipids.

We have initiated incubation studies of F430 in sediment core samples from Hydrate Ridge along with other metrics of ANME characterization, including FISH (florescent in situ hybridization) and DNA data (by collaborator V. Orphan at Caltech). Using different substrates, we will compare 13C-label incorporation into F430, and signature lipids of ANME (Orphan et al., 2001a; Bertram et al., 2013). We have set up an experimental matrix using Hydrate Ridge sediment samples with known ANME activity, incubated with 13C-labeled methane, bicarbonate and acetate with and without a methane head space. The experimental matrix is designed to test functional affiliation of F430 by following labeled carbon incorporation during methane production and methane oxidation.

Using Methanosarcina as a model for searching for archaeal biosignatures
The role of the multi-subunit sodium/proton antiporter (Mrp) of Methanosarcina acetivorans was investigated with a deletion mutant. Antiporter activity was five-fold greater in acetate- versus methanol-grown wild-type cells consistent with the previously published relative levels of mrp transcript. The rate, final absorbance, and dry weight/methane ratio decreased for the mutant versus wild-type when cultured with a growth-limiting concentration of acetate. All growth parameters of the mutant or wild-type were identical when grown with methanol in media containing growth-limiting 1.04 M Na⁺. The lag phase, growth rate and final absorbance for growth of the mutant were sub-optimal versus wild-type when cultured with acetate in media containing either 0.54 or 1.04 M Na⁺. ATP synthesis in resting cell suspensions driven by potassium diffusion potential was stimulated by the addition of 25 mM NaCl. ATP synthesis was greater in wild-type versus mutant cells grown with acetate, a trend that held for methanol-grown cells albeit less pronounced. Both sodium and proton ionophores reduced ATP synthesis in the wild-type grown with either substrate. The results indicate Mrp is important for efficient ATP synthesis during growth with acetate, particularly in the native habitat of M. acetivorans.

Metals and Metal isotopes as biosignatures
We published an article reporting experiments conducted to determine (i) cellular trace metal concentrations of the hyperthermophilic Archaea Methanococcus jannaschii and Pyrococcus furiosus, and (ii) a first estimate of the metallome for these hyperthermophilic species via ICP-MS. The metal contents of these cells were compared to parallel experiments using the mesophilic bacterium Escherichia coli grown under aerobic and anaerobic conditions. Fe and Zn were typically the most abundant metals in cells. Metal concentrations for E. coli grown aerobically decreased in the order Fe > Zn > Cu > Mo > Ni > W > Co. In contrast, M. jannaschii and P. furiosus show almost the reverse pattern with elevated Ni, Co, and W concentrations. Of the three organisms, a biosignature is potentially demonstrated for the methanogen M. jannaschii that may, in part, be related to the metallome requirements of methanogenesis. The bioavailability of trace metals more than likely has varied through time. If hyperthermophiles are very ancient, then the trace metal patterns observed here may begin to provide some insights regarding Earth’s earliest cells and in turn, early Earth chemistry.

DNA as a biosignature – ancient DNA
We are exploring the extent of DNA preservation in soil from various environments. We have collected soil sample for genetic analysis from sand, lake and sedimentary sites from three locations in the Arctic: the Pribilof Islands, Old Crow Flats, Yukon Territory, and the Klondike gold fields, Yukon Territory. The sites differ in sediment composition and in age. Pribilof is highly sandy and samples were taken from Holocene-age sites (less than 10ky old). Old Crow sites date to the Late Pleistocene and Holocene. Klondike sites span the Pleistocene, with the oldest sites ca 720,000 years old. We find that temperature is the best predictor of DNA survival; we are able to recover DNA from 700,000 year old sediment and from an associated horse metapodial. This is, to date, the oldest recovered DNA sequences produced. However, the fragments are very short and show a variety of chemical and physical damages, as expected for very old DNA. As a result of this work, we predict that DNA will not survive as a biomarker for longer than 1My, even in the most ideal environments.

DNA as a biosignature – marine subsurface
We have continued work on subsurface marine microbial ecosystems as a location for developing metagenomics as an astrobiological tool. In particular, we have new data showing microbial distributions subsurface along the Costa Rican margin and into hydroghermal mounds of the Okinawa Back-arc basin. Next-generation DNA sequencing is providing a remarkable opportunity to study these sites. In the Costa Rica samples, we see a striking inverse relationship between the MCG microbes and the Nitrospira. At Okinawa, there is a marked change in microbial groups as in situ temperatures increase.

Abiotic organics
We also contintued work on what organics might be expected to form abiotically as a necessary step to understanding organic biosigntures. This work resulted in two submitted papers in collaboration with the NASA Goddard team.

Hot water heater microbiology
We are continuing the work on the microbiology of water heaters, which started as a citizen science project. The samples collected are being studied using various molecular biological tools. Additionally, we have deployed a set of sealed stainless steel chambers containing sterile substrates and connected to the continuous, approximately 50 to 60°C condensed steam output feed at the Penn State Steam Generation Plant.