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Project Progress

Introduction:

A core focus for the research within the BioMars project has centered on the topic of microbial communities sustained by iron and sulfur cycling, as these elements are abundant at the Mars surface and exist in multiple redox states, as would be required if they play a metabolic role. Our team has investigated a number of potential Earth analog systems, with a view to understanding their geochemistry, microbiology, and potential biosignatures.

Iron oxidation at Yellowstone, Delaware Inland Bays, Kilo-Moana, and Tennyson, WI.

The Luther group has been looking at a variety of environments where Fe(II) oxidation occurs. In two areas sulfide is not present (creeks in VA and Chocolate Pots, Yellowstone National Park) and in two other locations sulfide is present [local Delaware Inland Bays and the hydrothermal vents at Kilo-Moana (20°3’S, 176°8’W), located on the East Lau Spreading Centre (ELSC), in the Lau Basin, SW Pacific Ocean].

The Luther group have continued to work with the Emerson group on measuring in situ Fe(II) oxidation — both abiotic and biotic. Ferrous iron (Fe²⁺) oxidation by microbial iron mat (MIM) samples, dominated by helical stalks of Gallionella ferruginea or sheaths of Leptothrix ochracea, was examined. Pseudo-first order oxidation rates for the microbial mat samples ranged from 0.029 ± 0.004 to 0.249 ± 0.042 min^-1 and correlated well with total iron content (R2 = 0.929). Rates for Na azide (1 mM) treated MIM samples measured autocatalytic oxidation by iron oxide stalks or sheaths, with values ranging from 0.016 ± 0.008 to 0.062 ± 0.006 min^-1. Fe²⁺ oxidation attributable to cellular activities (MIM oxidation rate minus azide treated rate) was variable with respect to sampling location and sampling time, with rates from 0.013 ± 0.005 to 0.187 ± 0.037 min^-1. Rates of oxidation on the same order of magnitude for cellular processes and autocatalysis suggested that bacteria harnessing Fe²⁺ as an energy source compete with their own byproducts for growth, not chemical oxidation (under conditions where aqueous oxygen concentrations are less than saturating). The use of cyclic voltammetry within this study for simultaneous measurement of Fe²⁺ and oxygen allowed collection of statistically meaningful and reproducible data, two items that have limited aerobic, circumneutral, Fe²⁺ oxidation rate studies. A manuscript is near ready for submission to Environmental Science and Technology.

Similar in situ Fe(II) oxidation work in the microbial mats at Chocolate Pots by the Luther group shows that Fe(II) oxidation is mainly caused by photosynthetic O₂ production and 10% or less by chloroflexus, a Fe(II) oxidizing organism. This work was done in collaboration with Dr. Beverley Pierson of the University of Puget Sound, who is a member of the University of Arizona NASA NAI. Figure 1 shows dark and light profiles indicating that cyanobacteria produce O₂ which oxidizes Fe(II).

Redox cycling

BioMARS-funded research in the Roden subproject is focused on studies of microbial Fe redox cycling in a neutral-pH groundwater Fe seep environment in Tuscaloosa, AL, and in unsaturated Triassic-age weathered basalt materials from Box Canyon, ID. We have also evaluated the composition and function of an anaerobic, nitrate-dependent Fe(II)-oxidizing enrichment culture obtained from Bernhard Schink and colleagues at the University of Konstanz in Germany, together with a variety of other Fe/N redox cycling microbial systems. The goal of these studies is to gain insight into the biogeochemical organization of Fe-based life systems that may have existed on the early Earth and that may exist elsewhere within and beyond our solar system. Culture-based studies in the Fe seep material and weathered basalt have revealed significant numbers of both Fe(III)-reducing and Fe(II)-oxidizing microorganisms, which suggested the potential for microbially-catalyzed Fe redox cycling. Several highly-purified Fe(III)-reducing and Fe(II)-oxidizing cultures have been obtained and are currently being physiologically and phylogenetically characterized. A 16S rRNA gene clone library indicated the presence of a variety of lithotrophic ammonium- and Fe(II)-oxidizing phylotypes in the Fe seep community, and additional clone libraries are being constructed for both the Fe seep and weathered basalt communities. Incubation of amorphous Fe(III) oxide-rich seep material under anaerobic conditions demonstrated the potential for rapid Fe(III) oxide reduction. These results are conceptually consistent with those from the experimental cocultures of Fe(III)-reducing and Fe(II)-oxidizing bacteria, and suggest that tight coupling of microbial Fe oxidation and reduction takes place in the seep materials. Similar results were obtained with the weathered basalt materials, which are unique in that they contain magnetic Fe(III) oxide phases (presumably maghemite), which may or may not be converted to the magnetite during microbial reduction. The end-products of microbial reduction of the Fe seep and Box Canyon materials are currently being analyzed by XRD and Mössbauer spectroscopy. The Fe seep and weathered basalt systems provide models for how microbially-catalyzed Fe redox cycling could take place in subsurface Martian environments where reduced fluids/solids contact oxygen-bearing water or water vapor. Simultaneous operation of Fe(III) oxide reduction and Fe(II) oxidation reactions could in principle support a self-sustaining Fe redox cycle-based microbial life system that could be sustainable over geological time scales. An analogous long-term cycling of Fe through microbial metabolism has also been demonstrated in systems where nitrate replaces oxygen as the main oxidant for Fe(II), and the results collectively suggest that coupled Fe/N redox metabolism could provide an alternative means for sustained microbial life in extraterrestrial environments where inorganic N is abundant.

As part of an ongoing collaboration between co-Is Roden and Emerson, a proposal was submitted (along with another collaborator, Dianne Newman at Caltech) to the DOE’s Joint Genome Institute to obtain whole genome sequences of 6 freshwater Fe-oxidizing bacteria. This proposal was selected for funding, and the results promise to provide critical information on the mechanisms of microbial Fe(II) oxidation at circumneutral pH. The organisms include 3 Fe-oxidizers, two obligate lithotrophs and one heterotroph, and strain TW-2, a mixotrophic Fe-oxidizer. This will provide a great deal of information and will also provide data that will be interesting to compare with Dr. Banfield’s ongoing genomic projects at Iron Mountain.

Mineral spectroscopy in Fe-cycling microbial environments:

The Bishop group are working on the identification of minerals associated with water (and possibly life) on Mars using spectral remote sensing; emphasis on S- and Fe- bearing minerals.
We have characterized VNIR and other spectral properties in the lab of minerals found in Mars analog rocks. Coordinated analyses with colleagues studying Mössbauer, transmittance, and emittance spectra and in some cases XRD, SEM and TEM has enabled more accurate mineral identification in natural samples and has enabled more accurate band assignments for some previously poorly assigned spectral features. These include altered basaltic ash from Haleakala, Maui, and solfataric sites near Kilaueau, Hawaii. We have found alunite and jarosite in altered tephra near cinder cones at Haleakala in association with phyllosilicates and iron oxides/oxyhydroxides. Magnetite and in some cases maghemite were also found in these soils. A similar process could be occurring on Mars in order to form these magnetic minerals in combination with jarosite in the soils there. Studies of solfataric sites at the southern region of the Kilauea crater have shown the presence of jarosite and hydrated silica. The combination of these two phases in altered volcanic material could also help explain the chemistry observed in the Martian soils and is under further study this year.

Because of the importance of understanding the spectral properties of jarosite and alunite on Mars, we have been studying the spectral properties of a larger collection of jarosite and alunite samples to expland on the paper published last year. This new study includes a suite of 19 jarosites with variable Na, K and H₃O cations as well as several natural jarosites and alunites. We are coordinating visible, infrared and Mössbauer spectra of these samples in order to understand the influence of the monovalent and trivalent cations on the spectral properties and in order to develop techniques for coordinated identification and characterization of these minerals by the Pancam, Mössbauer and Mini-TES data of Mars. We have also expanded our collection of unusual sulfate minerals and are continuing to collect visible-infrared reflectance spectra in conjunction with Mössbauer spectra from collaborator Dyar and emittance spectra from collaborator Lane.

We are also undertaking a spectral study of Pancam data, with emphasis on Paso Robles bright region soils that appear to have unique spectral properties and high S levels. The Bishop group is using statistical analyses of the images containing these soils in order to differentiate the typical Martian soil from the unusual pixels in each scene. A collection of about a hundred lab spectra of minerals and analogs have been resampled to the Pancam channels for comparison of these lab spectra with the Martian spectra. Preliminary analyses suggest that minerals such as ferricopiapite, coquimbite, and/or fibroferrite may be present in these unusual bright region soils in Gusev Crater.

The biology of neutrophilic iron oxidizing bacteria:

Work in Emerson’s lab has continued to focus on the biology of Fe-oxidizing bacteria that grow at neutral pH under sub-oxic conditions. A good deal of work has been done on completing characterization of strains already in hand including a revised phylogenetic analysis that indicates this group of microorganisms is quite unique. An analysis of 6 freshwater isolates reveals they all belong to the betaproteobacteria and include two new genera and 4 new species; one of the new genera represents the first cultured isolate of a novel order of betaproteobacteria. We have shared this data with Dr. Eric Roden, who is in the process of carrying out a community analysis of environmental Fe-oxidizing communities by doing clone libraries. His postdoctoral student has found a number of clones from these communities that are related to our isolates suggesting they maybe quite universally distributed in freshwater habitats where Fe-cycling is occurring.

Another aspect of our work has focused on field studies to determine the presence and importance of FeOB in local Fe-rich springs where microbial mats of Fe-oxidizing bacteria occur. Our approach has been to closely document physical parameters of one site in particular, measuring temperature, pH, Fe(II) concentrations, and O₂ concentrations as well as collecting samples for molecular analysis of the populations that are present. This is done at approximately weekly intervals. In addition, we are deploying in-situ colonization chambers that allow us to follow the development of the community. This approach has proven quite interesting, in addition to morphologically unique Fe-oxidizing bacteria that have previously been described including Gallionella spp. and Leptothrix spp. we have discovered a previously undescribed sheath-forming organisms that is a putative Fe-oxidizer. This organism is illustrated in Fig 1 and forms an S-shaped sheath structure composed of Fe-oxides. What is remarkable about it, is that it only appears during the first 24-48 hours of colonization, and then there are only 1 or 2 cells inside each sheath, these can be seen in Fig 1b. It appears the organism begins to grow and extrude an Fe-oxide sheath that can be 10s of micrometers long, it divides once or twice and then abandons the sheath and presumably initiates the process again. FISH analysis indicates this is a Bacteria, but we have not identified it beyond that. We are in the process of following up on these studies.

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Another activity of the Emerson lab has been determining in-situ Fe-oxidation rates for bacteria both in the field and in the laboratory. Emerson spent several days last summer working in the lab of Dr. Greg Druschel at the University of Vermont (a former postdoc of Dr. Luther’s) to complete the data-collection for measuring Fe-oxidation rates of pure cultures using voltametric microelectrodes. These measurements were successful and showed that the pure cultures of Fe-oxidizing bacteria contribute substantially to the direct Fe-oxidation rates. They also point out the challenges of distinguishing between the biologically catalyzed rates of Fe-oxidation and auto-oxidation of Fe(II) by pre-formed Fe-oxides. We are currently in the finishing stages of putting together a manuscript for submission on this project.

Coupled iron and sulfur cycling and ultra-small archaeal lineages

The Luther group have continued to work on metal sulfide clusters, MSaq, as a source of reduced metals including Fe(II) that may be used by microorganisms instead of the aqueous Fe(II) cation. Two papers were published on this topic in the last year (Luther and Rickard, 2005; Luther, 2005). Mn(II) can also occupy Fe sites in FeS_aq clusters. The FeS_aq clusters are excellent catalysts (Fe/S catalytic cycle, Figure 2) in mediating sulfide oxidation by molecular oxygen. The subsequent oxidation of sulfide produces polysulfides, elemental sulfur and thiosulfate, which can be used by organisms to fix carbon via chemosynthesis.

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The Fe/S catalytic cycle was investigated in two environments by the Luther group. One site is a local Inland Bay area where several deep anoxic holes occur and the other area is the hydrothermal vent sites at Lau Bain off Fiji. At the oxic-anoxic interface of the Inland Bay, FeSaq clusters as well as dissolved Fe(II) and H₂S interact with molecular oxygen leading to sulfide oxidation and up to 30 micromolar elemental sulfur. Several hydrothermal sites at Lau Basin off Fiji were investigated which showed significant differences in Fe,S chemistry that correlated with macrofauna (Figure 3) and their endosymbiotic microbes (still under investigation). Certain locations at a given site showed only FeS as the dominant redox chemical species. However, thiosulfate, polysulfides and Fe(III) were also detected indicating that FeS is being oxidized (the energy from this oxidation should also be beneficial for life forms). The partially oxidized sulfur species are produced as sulfide from a diffuse flow area penetrates the basalt substrate and reduces Fe(III) in the solid phase.

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The Banfield group have continued to study microbial communities associated with iron and sulfur cycling at low pH. Community genomic and PCR-derived gene sequence data are combined with cultivation-based studies to understand organism metabolism and microbial community structure. Co-culture-based studies indicate that novel (not yet isolated) archaea contribute to sulfur oxidation at low pH. Analysis of genomic sequence has revealed the presence of a group of deeply branching lineages within the Euryarchaeaota with no cultivated representatives. The 16S rRNA genes of these organisms were previously undetected in PCR-based surveys due to multiple mismatches with commonly used primers. The genes from two of the three new lineages contain intron-like protein-coding sequences within the 16S rRNA genes. We have reconstructed large genomic fragments from two of the lineages using community genomic sequence anchored by the 16S rRNA genes. The phylogeny of all gene sequences is consistent with the placement of the lineage within a new archaeal phylum.

Fluorescent probes designed to hybridize to the rRNA and based on the gene sequences, as well as PCR surveys with newly designed primers to the 16S rRNA genes and an arsenate reductase gene, indicate that the lineage is widespread within the Richmond Mine field site, though organisms typically comprise only a few percent of the community. Probes also indicated an extremely small cell size. Thus, organisms were targeted for physical concentration by filtration.

PCR studies of the filtrate derived from a biofilm sample demonstrate the only detectable genes are those of the targeted organisms. The filtrate was characterized by transmission electron microscopy via collaboration with Richard Webb, of the microscopy center at the University of Queensland, Australia. Results indicate that the cells have an archaea-like cell wall, with an S-layer, and one or two folded membrane protrusions of unknown function. Most importantly, the organisms from the novel archaeal lineages are exceptionally small, in fact they are smaller in volume than any previously described organism by a factor of > 3x (including nanoarchaea that are parasitic on other archaea). Current indications are that the cells are smaller than the size widely accepted as the minimum for cellular life forms. A sample of genomic DNA was obtained from one filtrate and is currently being used for library construction and sequencing at the Joint Genome Institute (Brett Baker, a scientist in the Banfield lab and supported by this project, is the PI for the funded sequencing proposal). Future work will be focused toward analysis of genomic sequence as well as cultivation attempts, with the goal of determining the metabolic basis for these organisms, to determine if they are free-living or parasitic on other organisms.

At another site, Lake Tyrrell, Australia, we are studying the coupling of iron and sulfur redox cycling through study of processes occurring within the lake and associated sediments (see the report on the hypersaline lake “Microbial adaptation to salinity: molecular mechanisms and the biosignature record”). As shown in Figure 2, iron concretions at the south end of the lake localize accumulation of organic materials, leading to localized zones of iron and sulfate reduction and metal sulfide precipitation. In some places, very large amounts of black sediment are formed (Figure 3). Associated iron concretions are laminated, and may constitute part of the biosignature record.