Anaerobic carbon monoxide (CO) oxidation was likely a significant mode of metabolism on the early Earth. Hydrothermal settings today may provide a modern analogue for the study of this ancient microbial energy source and its relation to carbon fluxes on the early Earth. CO is a common trace gas in volcanic emissions and associated hot springs. CO is toxic to numerous microbes because of its strong affinity for the active sites of many metalloenzymes. Nevertheless, several hot spring bacteria have been isolated that are capable of chemolithoautotrophic growth via anaerobic oxidation of CO (CO + H2O = CO2 + H2) in cultures supported by headspace CO partial pressures exceeding 1 atm. Uzon Caldera and Geyser Valley on the Kamchatka Peninsula in far eastern Siberia are the source regions for several anaerobic carboxydotrophic species. Curiously, the concentrations of CO in the isolation locales are much lower than those in which these carboxydotrophic microbes thrive when cultured. Furthermore, CO consumption rates observed in the anoxic muds of the hot spring outflow channels are far higher than can be supported by the CO supplied by venting reduced waters. These observations imply that in situ production of CO, presumably by other microbes and resulting in locally high CO concentrations, sustains the thriving carboxydotrophic communities. Before the rise of oxygen in the Paleoproterozoic, outgassing of a small portion of this CO produced in anaerobic mats could have been a significant source of CO to the atmosphere.

Intellectual Merit. This study will provide the first detailed field and laboratory examination of CO production and consumption in hot spring sediments. We will target hot springs in Uzon Caldera, Kamchatka, and analogous springs in Devil?s Kitchen, Lassen Volcanic National Park, California. Fine scale chemical gradients in spring sediments will be assessed using scanning voltammetry microelectrodes. CO consumption rates and products will be determined by in situ 14CO tracer incubations. Couplings between CO cycling and various metabolic modes (i.e., sulfate, FeIII, and MnIV reduction as inferred in the field from microelectrode profiles) will be tested using redox-gradient reactors in the lab. Cryosections of sediment mini-cores will be used for DNA extraction to profile microbial communities in these redox gradients. We will also make preliminary assessments of CO exchange with the atmosphere through measuring CO concentrations in vadose zone gases in relation to dissolved concentrations in the underlying saturated zone. As a result, this work will improve our understanding of the role that carboxydotrophy may have played as a standalone metabolism and as a means of mediating CO fluxes to and from the atmosphere on the early Earth.

This work will also pioneer the use of fluorescent reporters in genetically engineered thermophiles as an analytical tool for laboratory microcosm experiments. Localized hot spots of CO production are not resolvable using current methods. The carboxydotrophs with recombinant fluorescent reporters will serve as visual indicators of zones with high dissolved CO concentrations, and will demonstrate more broadly the utility of biosensors in geochemistry.

Broader Impacts. This proposal provides partial support for one graduate student at University of Chicago and one at U. Maryland Biotechnology Inst., with additional travel and logistical support for a summer undergraduate intern at U. Chicago to participate in both field seasons. This work will extend our existing international collaboration with members of the Kamchatka Institute of Volcanology and Seismology and the Russian Academy of Sciences in Moscow. Finally, the proposed research will be shared with the general public through the extension of a nationally recognized public outreach collaboration on Kamchatka extremophiles with the San Francisco Exploratorium. A web exhibit will be developed in both English and Russian to describe the research approach and major findings from this biogeochemistry study.

Project Report

This project involved the isolation, genomic and biochemical analysis of novel carbon monoxide utilizing extremophiles from Far Eastern Russia and Lassen Volcanic Park. Thorough analysis of microbial genomes led to further documentation of widespread CO oxidation as both energy and carbon sources for microbial growth. The analysis led to the identification of many bacteria and archaea that were not previously characterized as CO-utilizing, and the first reported instance of a "smoking gun" finding for intergeneric lateral gene transfer of a 10 gene CO locus for anaerobic CO oxidation and hydrogen production. Molecular studies on the regulation of gene expression and the thresholds for CO detection led to the discovery of the internal metabolic pacing needed for adaptaion to a "feast or famine" of CO, further supporting the assumption that, at times, CO is locally abundant in anaerobic sediments (Techtmann et al, 2011). A new chaperonin,( protein remodeling enzyme) was purified and characterized by biochemical, structural and modeling experiments (Techtmann, SM and Robb FT PNAS, 2010).Genomic studies also led to the discovery a phylogenetically unique protein chaperonin class in the genomes of six CO-oxidizing bacteria, representing the first report of archaeal type chaperonins in bacteria. The project also supported geochemical measurements on hot spring gas chemistry in Lassen Volcanic National Park and Uzon Caldera, Kamchatka, Russia. A comparison between free phase gas concentrations (ascending bubbles in hot springs) with the predicted equilibrium concentrations of those gases in the dissolved phase in the springs revealed a striking difference between the behavior of CO and the behavior of H2 and CH4. CO was found to be consistently three orders of magnitude or more supersaturated in the dissolved state. Geochemical equation of state modeling coupled with a simple 1-D flow model revealed that the findings for CO could not be explained by inheritance of equilibrium conditions from deeper depths. Rather, a proximal source of CO was implied. These findings (He et al., 2012) are in press in Geochim. Cosmochim. Acta. Within the last three years, Dr. Robb has advised three underrepresented minority undergraduates and one female minority high school student, Toby Fadiran, on projects related to this proposal, and will continue to engage undergraduate students and high school students in interdisciplinary research themes. Ms. Fadiran worked with the Robb lab on fuel cell technology and has recently applied successfully to several high ranked colleges including Stanford University. Undergraduate research assistants William Plum and Katharine Chen learned the ropes of anaerobic microbiology and biochemistry in the Robb lab and are continuing in medical and engineering studies, respectively. Benefits to Society The Co PI, Frank Robb, is the author of a Provisional Patent filed 07/11/2012, based on the discovery that the CO sensor/gene regulator CooA binds to Nitric Oxide (NO) as well as it does to CO. The patent is named "Nitric Oxide Regulation of Recombinant Gene Expression" and has attracted interest from Merck Pharmaceuticals. Thus, the fundamental knowledge created by this project may facilitate creation of a new suite of biotechnology products, enhancing United States competitiveness and onshore job creation.

National Science Foundation (NSF)
Division of Earth Sciences (EAR)
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Enriqueta Barrera
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University of Maryland Baltimore
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