Cultivation of an Obligate Fe(II)-Oxidizing Lithoautotrophic Bacterium Using Electrodes

Abstract

Fe(II)-oxidizing aerobic bacteria are poorly understood, due in part to the difficulties involved in laboratory cultivation. Specific challenges include (i) providing a steady supply of electrons as Fe(II) while (ii) managing rapid formation of insoluble Fe(III) oxide precipitates and (iii) maintaining oxygen concentrations in the micromolar range to minimize abiotic Fe(II) oxidation. Electrochemical approaches offer an opportunity to study bacteria that require problematic electron donors or acceptors in their respiration. In the case of Fe(II)-oxidizing bacteria, if the electron transport machinery is able to oxidize metals at the outer cell surface, electrodes poised at potentials near those of natural substrates could serve as electron donors, eliminating concentration issues, side reactions, and mineral end products associated with metal oxidation. To test this hypothesis, the marine isolate Mariprofundus ferrooxydans PV-1, a neutrophilic obligate Fe(II)-oxidizing autotroph, was cultured using a poised electrode as the sole energy source. When cells grown in Fe(II)-containing medium were transferred into a three-electrode electrochemical cell, a cathodic (negative) current representing electron uptake by bacteria was detected, and it increased over a period of weeks. Cultures scraped from a portion of the electrode and transferred into sterile reactors consumed electrons at a similar rate. After three transfers in the absence of Fe(II), electrode-grown biofilms were studied to determine the relationship between donor redox potential and respiration rate. Electron microscopy revealed that under these conditions, M. ferrooxydans PV-1 attaches to electrodes and does not produce characteristic iron oxide stalks but still appears to exhibit bifurcate cell division.

A Previously Uncharacterized, Nonphotosynthetic Member of the Chromatiaceae Is the Primary CO 2 -Fixing Constituent in a Self-Regenerating Biocathode

“…Electrode-associated cell biomass increases over time as the magnitude of the current increases while no other electron donor is provided other than the poised electrode and no other source of carbon is intentionally provided other than CO 2 . The rates of cellnormalized EET are at least as high as that previously noted for M. ferrooxydans growing on a cathode (13), suggesting that the num- 3 oxidase. The proton motive force is used to generate ATP and to power the reductive branch, which produces NADH to provide reducing equivalents for CO 2 fixation.…”

“…This estimate is ca. 4 to 40 times greater than the 0.075 pmol electrons h Ϫ1 cell Ϫ1 reported for M. ferrooxydans cathodes (13). This could be due to a real increase in the rate of EET for our biocathode community or to an underestimation of the number of electrodeassociated cells by an order of magnitude or more due to challenges in disaggregating the biofilm for flow cytometry.…”

“…A suite of consensus genetic markers is not known for cathode EET. Based on the identification and relative abundance of Marinobacter bacteria (a known FeOB) from our previous work (22), as well as recent demonstrations of cathode EET by other FeOB (13,14), we hypothesized that our biocathode consortium utilizes iron oxidation pathways for biocathode EET. We explored the biocathode metagenome for genetic evidence of known or predicted iron oxidation pathways from all characterized FeOB (43).…”

“…Although biocathode EET has been demonstrated for a variety of microorganisms, including acetogens (5) and a methanogenic archaeon (6), studies aimed at identifying EET conduits from the electrode to cells have mostly been confined to the model organisms Geobacter (8) and Shewanella (9), due to the massive effort put forth to understand how these iron-reducing bacteria are able to catalyze EET at bioanodes (10-12). The ability of iron-reducing bacteria to reduce anodes led to the hypothesis that iron-oxidizing bacteria (FeOB) would be able to perform EET with cathodes, which has been demonstrated for at least two FeOB, Mariprofundus ferrooxydans and Rhodopseudomonas palustris (13,14). While many bacteria have been shown to attach to electrodes and "consume" electrons, electrode-dependent growth has thus far been demonstrated only for M. ferrooxydans (13).…”

“…The ability of iron-reducing bacteria to reduce anodes led to the hypothesis that iron-oxidizing bacteria (FeOB) would be able to perform EET with cathodes, which has been demonstrated for at least two FeOB, Mariprofundus ferrooxydans and Rhodopseudomonas palustris (13,14). While many bacteria have been shown to attach to electrodes and "consume" electrons, electrode-dependent growth has thus far been demonstrated only for M. ferrooxydans (13). Furthermore, most cathode EET processes studied to date rely on a fairly negative electrode potential (between 0 and Ϫ0.400 V standard hydrogen electrode [SHE]) to catalyze CO 2 or O 2 reduction.…”