Our research revolves around identifying iron oxidizing bacteria (FeOB), tracking their populations in various environments, and understanding how they affect iron, carbon, nitrogen and other element cycles at Loihi. The major purpose of the FeMO project is to elucidate the full physiological, phylogenetic and biochemical diversity of environmentally relevant and ecological important neutrophilic FeOB. Below follows an introduction to the specific intrigues of Fe cycling that inspire this project as well as the approaches we use.

FeS gradient tube culture
of FeOB (right) and
control (left)
[credit: Clara Chan]

Phase contrast micrograph of
two FeOB cells on
a branched stalk
[credit: Dave Emerson]

Increasingly, Fe containing compounds are being recognized as important energy sources for chemoautotrophy both in terrestrial and marine environments, although the extent and magnitude of Fe-driven systems has been largely unexplored. Our group uses a variety of methods to ascertain the contribution of FeOB to primary production at Loihi. Short term incubations of Loihi samples with 14C-labeled carbon dioxide will indicate the rate of primary production, as the labeled carbon dioxide is converted into biomass. Similar production experiments will be conducted with isolated FeOB to correlate with field experiments. The presence of RuBisCo, a key protein in carbon fixation pathways, is being evaluated using quantitative PCR. The various groups of microorganisms in Loihi samples including the FeOB are being measured by molecular ecological techniques such as quantitative PCR and fluorescence in situ hybridization.

Phylogenetic tree showing placement of Loihi
FeOB isolate Mariprofundus ferrooxydans
PV-1, as part of a candidate novel
group Zetaproteobacteria
[credit: Dave Emerson]

Confocal image of clusters of
Syto9-stained microbes (green)
in pits in basalt (red)
[credit: Erin Banning]

We hypothesize that there is a wide range of physiological diversity of FeOB, which will include (in addition to the already recognized obligate, mesophilic, microaerophilic group) thermophilic, psychrophilic, anaerobic and mixotrophic FeOB at Lo`ihi. These groups are likely to exhibit differences in growth rates and yields and substrate utilization. We also hypothesize that the phylogenetic diversity of FeOB varies with temperature, O2, CO2, and form of Fe (e.g. aqueous versus solid). The biogeographical distribution of FeOB is being systematically addressed by explicit collection of samples from a range of habitat types as well as from gridded trans-habitat survey collections. To deepen our knowledge of physiology, a variety of culturing techniques and conditions are being employed in an attempt to isolate and enrich for novel FeOB. Phylogenetic diversity will be assessed using taxonomic (i.e. 16S) and functional gene-targeted methods, such as clone library construction, quantitative PCR, terminal restriction fragment length polymorphism, and fluorescence in situ hybridization.

Rock and mineral incubation chambers
deployed on pillow basalts on the seafloor

UH-developed tripod housing a micro-
manipulator (AIS, Inc.) for collecting
microprofiles of the mat-water
interface using gold-amalgam
electrodes [credit: Brian Glazer]

We hypothesize that FeOB control the rates of both Fe oxidation and basalt glass weathering. At Loihi we predict that FeOB rapidly colonize fresh basalt and harness Fe released during weathering for growth. In addition, we hypothesize that the oxidation state of Fe in the basalt is a principal driver in colonization by FeOB and that basalt with low reduced iron/total iron ratios will not support growth. Basalt-hosted communities will contrast with hydrothermal vent FeOB communities, which receive relatively constant supplies of reduced iron over a given spatial scale.

Polished thick sections of
rock mounted on glass
slides, in incubation
chamber, prior to deploy-
ment on the seafloor
[credit: Katrina Edwards]

Specific microbe/mineral interactions are being evaluated using multiple complimentary techniques including colonization experiments on deployed mineral surfaces and flow-through experiments on collected materials for parallel mass balances of Fe and other compounds over time. Detailed geochemical surveys of habitats are being conducted in and ex situ by microelectrodes and fluid sampling while the corresponding mineralogy is being studied by synchrotron-based x-ray microscopy (e.g. STXM, micro-XRF, micro-EXAFS). Mineral x-ray absorbance spectra are being tested as a tool for evaluating biotic versus abiotic signatures of mineral formation. Rates of mineral dissolution and secondary product formation are also being assessed using stable iron isotope ratios. Incubation experiments with 15N-labeled nitrogen containing compounds such as ammonium and nitrate will ascertain nitrogen transformation, fixation, and loss in the environment.

TEM image of a Maripro-
fundus cell (rod shape in
upper left) attached to an
iron-mineralized stalk
[credit: Clara Chan]

We aim to understand the overall pathway of iron oxidation, from the ferrous iron sources (basalt and vent fluids) to oxidized mineral products, including the proteins and ligands (e.g. siderophores) involved. We are working to isolate and characterize Fe oxidases from cultured FeOB and environmental samples, especially since almost nothing is known about the iron oxidation pathways in neutrophilic FeOB. We study the wide variety of morphologies found in the microbial mats, and try to correlate these with structures formed by isolated FeOB. We conduct electron and x-ray microscopy studies to determine the spatial relationships and composition of extracellular polymers and minerals.