Iron Corrosive Sulfate Reducing Bacteria Uptake Extracellular Electrons Via Outer Membrane C-Type Cytochromes
Authors: Deng Xiao , Hashimoto Kazuhito , Okamoto Akihiro 
Published: February 2020

Sulfate reducing bacteria (SRB) can oxidize a large variety of soluble organics or H2 and reduce sulfate to sulfide which is corrosive towards various materials, therefore have been recognized to play an important role in anaerobic iron corrosion which causes enormous economic losses through chemical reaction (Fe + H2S → FeS + H2). Recently, several novel SRB strains including Desulfovibrio ferrophilus IS5 were isolated using solid iron as a sole electron donor, and thus a new corrosion mechanism for SRB to promote corrosion via direct electron uptake from iron surface was proposed1,2. Previously, we showed that D. ferrophilus IS5 can extract electrons from an –0.4 V (vs. SHE)-poised indium tin-doped oxide (ITO) electrode surface without using H2 as an electron carrier3. However, unlike well-studied EET mechanism in iron-reducing bacteria, the EET mechanism for SRB remains ambiguous despite of its emerging importance. Here we examined our hypothesis that D. ferrophilus IS5 extracts electrons from an ITO electrode via outer-membrane (OM) c-type cytochromes by a combination of electrochemical, biochemical, and gene analysis.
Electrochemical measurements using intact microbes were conducted in a single-chamber anaerobic 3-electrode system with negative potential poised-ITO electrode served as a sole electron donor. By measuring differential pulse (DP) voltammetry, we detected biogenic reduction signals including one with a peak potential of –0.26 V (vs. SHE), which negatively shifted for more than 120 mV when the reactor was heated from 30 °C to 60 °C. This strong temperature dependency suggests that its assignment is not small redox molecule but redox protein localized at the cell/electrode interface, as the temperature change may alter the protein structure to affect its redox potential. To directly identify the genes coding for detected OM redox active proteins in D. ferrophilus IS5, we analyzed the OM extracted from cells. Diffuse-transmission UV/Vis absorption spectrum of OM fraction showed characteristic absorption peaks of oxidized c-type cytochromes. Heme staining analysis of OM proteins showed five significant bands at 71, 31, 24, 15, 13 kDa, which amino acid sequences were sequenced and identified as c-type cytochromes in the genome of D. ferrophilus IS5. Notably, it contained 95 genes coding for cytochromes, including 2 extracellular cytochromes. Next to these 2 genes, there were 5 periplasmic cytochromes, with 2 were detected in the OM heme staining gel. Therefore, we proposed an EET pathway of D. ferrophilus IS5 composing of OM-spanning c-type cytochromes. Furthermore, based on blastp search in the NCBI non-redundant protein database, microbial species of a wide range, especially the sulfur-metabolizing microbes, turned out to have proteins identical to the detected OM c-type cytochromes of D. ferrophilus IS5, suggesting that the electron extraction via OM c-type cytochromes may also be the corrosion mechanism by other SRB.
We will present our other biofilm voltammetric data, the whole genome analysis results, and transmission electron microscopic (TEM) visualization of cytochrome-stained bacterial OM. Our data suggest that microbial electron uptake was mediated via the c-type cytochromes located at cell surface of D. ferrophilus IS5.
1. Dinh H.T., Kuever J., Muβmann M., Hassel A. W., Stratmann M., and Widdel F. (2004) Iron Corrosion by Novel Anaerobic Microorganisms. Nature, 427, 829-832.
2. Enning D. and Garrelfs J. (2014) Corrosion of Iron by Sulfate-Reducing Bacteria: New Views of an Old Problem. Appl. Environ. Microbiol., 80 (4): 1226-1236.
3. Deng X., Nakamura R., Okamoto A., and Hashimoto K. (2015) Electron Extraction from an Extracellular Electrode by Desulfovibrio ferrophilus Strain IS5 Without Using Hydrogen as an Electron Carrier. Electrochemistry, 83 (7):529-531.

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